ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

VIContentsChapter 9Evaluation of a New Method andDiagnostic Test in Semen Analysis 131Petra ZrimšekChapter 10 Particularities of Bovine Artificial Insemination 153Antônio Nelson Lima da Costa, Airton Alencar de Araujoand José Valmir FeitosaChapter 11 Management Factors Affecting Fertility in Sheep 167Pilar Santolaria, Inmaculada Palacin and Jesús YánizChapter 12Chapter 13Chapter 14Chapter 15Chapter 16Effect of Cryopreservation onSperm Quality and Fertility 191Alemayehu LemmaEffect of Fatty Acids on ReproductivePerformance of Ruminants 217José Herrera-Camacho, Alejandra Soberano-Martínez,Karlos Edmundo Orozco Durán, Carlos Aguilar-Pérezand Juan Carlos Ku-VeraMechanical and Pharmacologic Applicationsof Artificial Insemination in Ewes 243Faruk Aral, Füsun Temamoğulları and Semra Sezen AralRelationship Between IFN- Production byBovines Embryos Derived Ex Vivo andCompletely Produced In Vitro 255Jorge Alberto Neira, Daniel Tainturier,René L’Haridon and Jacques MartalReproductive Endocrinology Diseases: HormoneReplacement and Therapy for Peri/Menopause 269Zoe Roupa, Greta Wozniak, Konstantinos Tsiprasand Penelope Sotiropoulou

PrefaceAs we look back over the millennium, it is difficult to imagine man’s evolution in theabsence of domesticated livestock. Likewise, domesticated animals are so dependentupon man that in his absence their very existence would be jeopardized to the pointwhere they would not thrive and some would fail to survive. Artificial insemination(AI) - one of the most important techniques ever devised for the genetic improvementof farm animals - is a widely used tool for livestock breeding and managementprograms and is a process by which sperm are collected from the male, processed,stored and artificially introduced into the female reproductive tract for the purpose ofconception. A male animal produces millions of sperms daily. Theoretically, it caninseminate females regularly and produce several offsprings.Artificial insemination is used instead of natural mating for reproduction purposes.This is when a male animal, for example, a bull, is kept with a herd of cows and‘covers’ (copulates with) them when they are ready to mate (in oestrus) so the bull’ssemen fertilizes the cow’s eggs to produce calves. Fertilization can take place awayfrom the bull and the two animals do not even meet! Although AI (in the form ofintrauterine insemination) is not frequently used in human patients, it is the mostcommonly used method of breeding food production animals in developed countries,with more than 90% pigs and almost the same proportion of dairy cattle bred by thismethod in the European Union and North America. In the actual procedure used,semen is obtained from a male animal and, after being diluted, is deep-frozen, afterwhich it can be stored for long periods of time without losing its fertility. For use, thesemen is thawed and then introduced into the genital tract of a female animal.The first successful experiment with artificial insemination in animals was performedby an Italian physiologist Lazzaro Spallanzani, who in 1780, while investigating animalreproduction, developed a technique for artificial insemination in dogs. This approachwas refined in the 1930s in Russia, and the subsequent development of methods forthe cryopreservation (preservation through freezing) of semen led to the widespreaduse of AI in animals.There are many advantages to artificial insemination (AI) in domesticated and zooanimals, such as smaller chance of injury to either partner during the mating process,

XPrefaceless stress to the female, who is often the one that has to be transported to and fromthe home of the male, but one should keep in the mind that the system of reproductionis perfect, including artificial insemination. The chief priority of artificial inseminationis that the desirable characteristics of a bull or other male livestock animal can bepassed on more quickly and to more progeny than if that animal is mated with femalesin a natural fashion. Ten thousand or more calves are produced annually from a singlebull through the use of artificial insemination.Artificial insemination has been most widely used for breeding dairy cattle and pigsand has made bulls of high genetic merit available to all. It has been used to facilitatethe reproductive success and conservation of threatened or endangered animals.Although AI (in the form of intrauterine insemination) is not frequently used inhuman patients, it is the most commonly used method of breeding food productionanimals in developed countries, with more than 90% pigs and almost the sameproportion of dairy cattle bred by this method in the European Union and NorthAmerica. Examples of wild animals that have been successfully impregnated throughartificial insemination include big cats (e.g., the tiger, the puma, the cheetah, and theclouded leopard), the white rhinoceros (Ceratotherium simum) and the onager (Equusonager).This book contains under one cover 16 chapters of concise, up-to-date information onartificial insemination. AI in buffalos, ewes, pigs, swine, sheep, goats, pigs and dogswill be detailed in different chapters. Cryopreservation effect on sperm quality andfertility, new method and diagnostic test in semen analysis, management factorsaffecting fertility after cervical insemination, factors of non-infectious nature affectingthe fertility, fatty acids effects on reproductive performance of ruminants,particularities of bovine artificial insemination, sperm preparation techniques andreproductive endocrinology diseases will be described in these chapters.The purpose of this book is to provide, as both a college text book and a referencesource, a comprehensive text that contains current information on artificialinsemination. This book is not a presentation of concepts of artificial insemination withan extensive list of references, but rather a consensus of important information withkey references to allow the reader to further explore the artificial insemination field.This book will deal with the use of artificial insemination (AI) in animals, currentlyand in the future, with particular emphasis on comparative aspects between species.This book will explain the advantages and disadvantages of using AI, the variousmethodologies used in different species, and how AI can be used to improvereproductive efficiency in farm animals.I hope this book will be used worldwide as a college textbook and authoritativereference book for research and extension specialists, AI practitioners, teachers andstudents.

1Artificial Insemination:Current and Future TrendsJane M. MorrellSwedish University of Agricultural Sciences, Uppsala,Sweden1. IntroductionThe chapter will deal with the use of artificial insemination (AI) in animals and humans,both currently and in the future, with particular emphasis on comparative aspects betweenspecies. Although AI (in the form of intrauterine insemination) is not frequently used inhuman patients, it is the most commonly used method of breeding food production animalsin developed countries, with more than 90% pigs and almost the same proportion of dairycattle bred by this method in the European Union and North America. This chapter willexplain the advantages and disadvantages of using AI, the various methodologies used indifferent species, and how AI can be used to improve reproductive efficiency in farmanimals, sport animals, and human patients. To finish, some speculation is made aboutfuture trends for this biotechnology.1.1 What is artificial insemination (AI)?Artificial insemination (AI) is the manual placement of semen in the reproductive tract ofthe female by a method other than natural mating. It is one of a group of technologiescommonly known as “assisted reproduction technologies” (ART), whereby offspring aregenerated by facilitating the meeting of gametes (spermatozoa and oocytes). ART may alsoinvolve the transfer of the products of conception to a female, for instance if fertilization hastaken place in vitro or in another female. Other techniques encompassed by ART include thefollowing: in vitro fertilization (IVF) where fertilization takes place outside the body;intracytoplasmic sperm injection (ICSI) where a single spermatozoon is caught and injectedinto an oocyte; embryo transfer (ET) where embryos that have been derived either in vivo orin vitro are transferred to a recipient female to establish a pregnancy; gamete intrafallopiantransfer (GIFT) where spermatozoa are injected into the oviduct to be close to the site offertilization in vivo; and cryopreservation, where spermatozoa or embryos, or occasionallyoocytes, are cryopreserved in liquid nitrogen for use at a later stage.AI has been used in the majority of domestic species, including bees, and also in humanbeings. It is the most commonly used ART in livestock, revolutionising the animal breedingindustry during the 20 th century. In contrast to medical use, where intra-uterineinsemination (IUI) is used only occasionally in human fertility treatment, AI is by far themost common method of breeding intensively kept domestic livestock, such as dairy cattle(approximately 80% in Europe and North America), pigs (more than 90% in Europe andNorth America) and turkeys (almost 100% in intensive production). AI is increasing in

2Artificial Insemination in Farm Animalshorses, beef cattle and sheep, and has been reported in other domestic species such as dogs,goats, deer and buffalo. It has also been used occasionally in conservation breeding of rareor endangered species, for example, primates, elephants and wild felids. The other ARTs inanimals are generally confined to specialist applications or for research purposes, since thecost would be prohibitive for normal livestock breeding. In contrast, IUI is used less often inhuman fertility treatments than IVF or ICSI.1.2 Advantages and disadvantages of artificial inseminationAI in animals was originally developed to control the spread of disease, by avoiding thetransport of animals with potential pathogens to other animal units for mating and byavoiding physical contact between individuals. The use of semen extenders containingantibiotics also helped to prevent the transmission of bacterial diseases. The advantages anddisadvantages of AI are as follows:Advantages: AI helps prevents the spread of infectious or contagious diseases, that can be passed onwhen animals are in close contact or share the same environment; The rate of genetic development and production gain can be increased, by using semenfrom males of high genetic merit for superior females; It enables breeding between animals in different geographic locations, or at differenttimes (even after the male´s death); Breeding can occur in the event of physical, physiological or behavioural abnormalities; AI is a powerful tool when linked to other reproductive biotechnologies such as spermcryopreservation, sperm sexing; AI can be used in conservation of rare breeds or endangered species.Disadvantages: Some males shed virus in semen without clinical signs of disease (“shedders”). Some bacterial pathogens are resistant to the antibiotics in semen extenders or canavoid their effects by forming bio-films; There has been a decline in fertility in dairy cattle and horses associated with anincrease in AI; The focus on certain individuals may result in loss of genetic variation.1.2.1 Viruses in semenCryopreserved semen doses can be “quarantined” until the male is shown to have been freeof disease at the time of semen collection. In contrast, the short shelf-life of fresh semendoses means that they must be inseminated into the female before the disease-free status ofthe male has been established. Breeding sires used for semen collection are tested routinelyfor the presence of antibodies in serum as being indicative of past infection, but someviruses, e.g. equine arteritis virus, may be shed in semen for several weeks before there isevidence of sero-conversion. In other cases, usually of congenital infection, individuals maybe permanent virus “shedders” without ever developing antibodies. Semen from theseindividuals represents a source of pathogens for disease transmission to naive females.1.2.2 Bacteria in semenNormally, in a healthy male, the ejaculate itself does not contain microorganisms, butcontamination occurs at semen collection from the prepuce and foreskin, the male´s

Artificial Insemination: Current and Future Trends 3abdomen and the environment. Semen processing from livestock usually takes placewithout access to a laminar air flow hood, resulting in potential contamination from thelaboratory environment. Antibiotics are added to semen extenders to limit the growth ofthese contaminants and prevent disease in the inseminated female. Although the femalereproductive tract has well-developed physiological mechanisms for dealing withcontamination introduced during mating, these can be overwhelmed by bacteriamultiplying in semen extenders or where semen is deposited in a non-physiologicallocation.1.2.3 Antibiotics in semen extendersThe addition of antibiotics to semen extenders is controlled by government directives, bothnationally and internationally, which state the types of antibiotic to be used and also theirconcentrations. In general, there is a tendency to use broad spectrum, highly potentantibiotics in various combinations to reduce sperm toxicity. However, these antibiotics mayexacerbate the development of resistance, both for the people handling the semen extendersand in the environment during the disposal of unused extenders or semen doses. The scaleof the problem becomes apparent if one considers that approximately four million liters ofboar semen extender containing antibiotics are used in Europe alone per year.2. Pre-requisites for AIPre-requisites for AI include a supply of semen, reliable methods for oestrus detection in thefemale and a means of inserting the semen into the female reproductive tract.2.1 Collection of semenIn most domestic animals, semen is collected by means of an artificial vagina, for example,bull, ram, stallion, after allowing the male to mount either an oestrous female or a phantom.The artificial vagina consists of a lubricated liner inserted into an outer jacket, the spacebetween the two being filled with warm water. The pressure can be increased by adding air.The ejaculate is deposited into an insulated collecting vessel attached to one end of the liner.Boar and dog semen is usually collected by manual stimulation.In some species that are accustomed to being handled, it is possible to obtain semen byvaginal washing after natural mating, for example, dogs and marmoset monkeys. However,in this case the spermatozoa have already been exposed to vaginal secretions which may bedetrimental to sperm survival. Human males can usually supply a sample by masturbation,except in the case of spinal injury when electroejaculation may be necessary. Some otherprimates can be trained to supply a semen sample on request in the same manner. For otherspecies, for example, most non-domestic species, electroejaculation represents the onlypossibility for obtaining a semen sample. The problem with electroejaculation is that thesecretions of the accessory glands may not be present in the usual proportions, which mayhave a detrimental effect on sperm survival.2.1.1 Constituents of semenSemen consists of spermatozoa contained in a watery fluid known as seminal plasma thatrepresents the combined secretions of the different accessory glands, such as the seminalvesicles, bulbourethral gland and prostate. The relative contributions of these different

4Artificial Insemination in Farm Animalsglands vary between species. In some species, such a most primates, the semen coagulatesimmediately after ejaculation and then liquefies over a period of approximately 30 minutes.In most other species, the ejaculate remains liquid, the exception being in camelids wherethe seminal plasma is highly viscous and does not liquefy readily in vitro. The addition ofenzymes has been suggested as a means of liquefying primate or camelid semen. However,all the enzymes tested thus far (collagenase, fibrinolysin, hyaluronidase and trypsin) havebeen seen to cause acrosomal damage in spermatozoa (Wani et al., 2007) and are contraindicatedif the spermatozoa are to be used for AI. Recent advances have shown thatcamelid semen, extended 1:1 volume to volume, will liquefy in 60-90 min at 37°C.Seminal plasma contains an energy source (often fructose), proteins and various ions such ascalcium, magnesium, zinc and bicarbonate. Seminal plasma not only activates thespermatozoa, which have been maintained in a quiescent state in the epididymis, but alsofunctions as a transport medium to convey the spermatozoa into the female reproductivetract and to stimulate the latter to allow spermatozoa to swim to the site of fertilization. Ithas been suggested that seminal plasma, at least in horses, is also a modulator of sperminducedinflammation, which is thought to play an important role in sperm eliminationfrom the female reproductive tract (Troedsson et al., 2001). Various proteins in the seminalplasma, such as spermadhesins and the so-called CRISP proteins (CRISP = cysteine-richsecretory proteins) are thought to be associated with sperm fertility. It is likely that theseproteins bind to spermatozoa immediately, setting in motion a sequence of intracellularevents via a second-messenger pathway. In some species, small membrane-bound vesicleshave also been identified in seminal plasma, apparently originating from different accessoryglands in various species. These vesicles, variously named prostasomes, vesiculosomes, orepididysomes depending on their origin, fuse with the sperm outer membrane, increasingmotility and possibly being involved in sperm capacitation and acquisition of fertilizingability. However, their exact mechanism of action has yet to be elucidated.Seminal factors promote sperm survival in the female reproductive tract, modulate thefemale immune response tolerate the conceptus, and to condition the uterine environmentfor embryo development and the endometrium for implantation (Robertson, 2005). Themechanism of action in the endometrium is via the recruitment and activation ofmacrophages and granulocytes, and also dendritic re-modelling, that improve endometrialreceptivity to the implanting embryo. Cytokine release has embryotrophic properties andmay also influence tissues outside the reproductive tract.Exposure to semen induces cytokine activation into the uterine luminal fluid and epithelialglycocalyx lining the luminal space. These cytokines interact with the developing embryoas it traverses the oviduct and uterus prior to implantation. Several cytokines are thought tobe involved, for example granulocyte-macrophage colony stimulating factor (GM-CSF), aprinciple cytokine in the post-mating inflammatory response, targets the pre-implantationembryo to promote blastocyst formation, increasing the number of viable blastomeres byinhibiting apoptosis and facilitating glucose uptake (Robertson et al., 2001). Interleukin-6(IL-6) and leukocyte inhibitory factor (LIF) are similarly induced after exposure to semen(Gutsche et al., 2003; Robertson et al., 1992).Clinical studies in humans showed acute and cumulative benefits of exposure to seminalfluid but also a partner-specific route of action. Live birth rates in couples undergoingfertility treatments are improved if women engage in intercourse close to embryo transfer(Bellinge et al., 1986; Tremellen et al., 2000). The use of seminal plasma pessaries by womensuffering from recurrent spontaneous abortion is reported to improve pregnancy success

Artificial Insemination: Current and Future Trends 5(Coulam and Stern, 1993, cited in Robertson, 2005). Partner-specificity of the response issuggested by increased rates of preeclampsia in pregnancies from donor oocytes or semenwhen prior exposure to the donor sperm or conceptus antigens has not occurred (Salha etal., 1999).2.1.1.2 Semen processingAlthough seminal plasma plays such an important role in activating spermatozoa and in thefemale reproductive tract, it is detrimental to long-term sperm survival outside the body.Under physiological conditions, spermatozoa are activated by seminal plasma at ejaculationand then swim away from the site of semen deposition in the female. It is only during invitro storage that spermatozoa become exposed to seminal plasma long-term. Thus it iscustomary to add a semen extender to the semen, to dilute toxic elements in seminal plasma,to provide nutrients for the spermatozoa during in vitro storage and to buffer their metabolicby-products. The addition of extender also permits the semen to be divided into severalsemen doses, each containing a specific number of spermatozoa that has been determined tobe optimal for good fertility in inseminated females.2.1.2 Semen preservationSemen is used either immediately after collection (“fresh”) for example turkeys, humanbeings; after storage at a reduced temperature (“stored”) for example horses, pigs, dogs; orafter freezing and thawing (“cryopreservation”) for example, bulls.2.1.2.1 Fresh semenIn contrast to animal species, human semen is not extended prior to processing (seeprevious section) and is not usually kept for more than a few hours before use. Poultrysemen cannot be extended as much as is customary for other species since the spermatozoaare adversely affected by increased dilution. Goat semen cannot be kept at 37°C because anenzymatic component of the bulbo-urethral gland secretion hydrolyses milk triglyceridesinto free fatty acids, which adversely affects the motility and membrane integrity of buckspermatozoa (Pellicer-Rubio and Combarnous, 1998). For liquid preservation, goat semencan be stored at 4°C although fertility is retained for only 12-24h. The rate of extension usedfor stallion semen varies between countries but rates of 1:2, 1:3 or even 1:4 (v/v)semen:extender are common. The standard practice in some countries is to have 500 millionor one billion progressively motile stallion spermatozoa for fresh or cooled semen dosesrespectively. Boar semen doses contain three billion progressively motile spermatozoa.2.1.2.2 Stored semenStoring extended semen at reduced temperature helps to extend sperm life by slowing theirmetabolism as well as by inhibiting bacterial growth. Bacteria grow by utilizing thenutrients in semen extenders, thus competing with spermatozoa for these limited resources,and release metabolic byproducts, thus creating an environment that is not conducive tomaintaining viable spermatozoa. Furthermore, as bacteria die, they may release endotoxinsthat are toxic to spermatozoa. However, cooled stored semen is the method of choice forbreeding horses and pigs, enabling the semen dose to be transported to different locationsfor insemination. Stallion semen is stored at approximately 6°C while boar semen is storedbetween 16 and 18°C.Most boar semen doses are sold as cooled doses. In contrast, some stallions producespermatozoa that do not tolerate cooling, rapidly losing progressive motility. In such cases,

6Artificial Insemination in Farm Animalsthe only option currently is to use fresh semen doses for AI immediately after semencollection, although a new method of processing, centrifugation through a single layer ofcolloid, has been shown to solve the problem, as discussed later.2.1.2.3 CryopreservationSemen is most useful for AI if it can be cryopreserved, since this method of preservationideally enables the semen to be stored for an unlimited period without loss of quality untilneeded for AI. Since the frozen semen does not deteriorate, it can be quarantined until themale has been shown to be free from disease at the time of semen collection. However, thespermatozoa of various species differ in their ability to withstand cryopreservation:ruminant spermatozoa survive well whereas poultry spermatozoa do not, with less than 2%retaining their fertilizing ability on thawing (Wishart, 1985). For farm animal breeding, thecost of cryopreservation and the likelihood of a successful outcome following AI must beconsidered when deciding whether to use fresh, cooled or frozen sperm doses.The spermatozoa are mixed with a protective solution containing lipoproteins, sugars and acryoprotectant such as glycerol. These constituents help to preserve membrane integrityduring the processes of cooling and re-warming. However, sperm motility must also bemaintained, so that the thawed spermatozoa can reach the oocytes after insemination andfertilize them. In most species, the seminal plasma is removed by centrifugation beforemixing with the cryoextender, for example, stallion, boar, goat and human semen. Theextended semen is packed in straws and frozen in liquid nitrogen vapour before plunginginto liquid nitrogen for long-term storage. There is considerable variation in the success ofsperm cryopreservation between different species, despite intensive research into theconstituents of cryoextenders and the rates of cooling and re-warming. Human spermatozoacan be frozen relatively successfully using commercially available cryoextenders andprogrammable freezing machines.2.2 Oestrus detection and ovulationSuccessful AI also depends on depositing the semen in the female tract at around the time ofovulation. Like human beings, some domestic animals breed throughout the year, forexample cattle and pigs, but others show a defined period of reproductive activity known asthe breeding season, for example sheep and horses. The onset of the breeding season iscontrolled by photoperiod. Both of these patterns of reproductive behaviour arecharacterised by waves of ovarian activity, culminating in ovulation. However, in someother species ovulation occurs in response to the stimulus of mating, for example, cats,rabbits and camels. In spontaneously ovulating species, ovulation occurs at some timeduring, or shortly after, oestrus, which is the period of time when the female is receptive tothe male. Since a successful outcome for AI depends on the deposition of spermatozoa at asuitable time relative to ovulation, oestrus detection is crucial if the female is to beinseminated at the correct time. Males of the same species are, of course, very good atdetecting oestrus females, but since many livestock breeding units that practice AI do nothave male animals in the vicinity, it is essential that husbandry personnel become good atrecognising oestrous behaviour.Although some domestic animals may show well-developed oestrous behaviour, e.g. dairycows, others may not. Behavioural signs of oestrus in cows include restlessness or increasedactivity, vocalization, chin resting, swelling of the vulva, vaginal discharge and mountingother cows, although there are breed differences in the frequency and intensity of these

Artificial Insemination: Current and Future Trends 7signs. In sheep and goats, vulval swelling and vaginal discharge may be seen, and there isusually pronounced male-seeking behaviour. When AI is to be used in sheep, it is usual tosynchronize oestrus with hormones: intravaginal sponges impregnated with progestagensare inserted to suppress the ewe´s natural ovarian cycle for 12 days. On sponge removal,pregnant mare serum gonadotrophin is administered, with AI taking place at a set timethereafter. Alternatively, a vasectomised ram wearing a marker can be run with the females.When the females are in oestrus, the vasectomised ram marks them as he mounts, thusenabling them to be identified for AI. Oestrous sows and mares can be identified by thebehaviour exhibited towards teaser males.2.2.1 Induced ovulationWhen AI is performed in species that are normally induced ovulators, such as rabbits, catsand camels, it is necessary to stimulate ovulation. The easiest way to achieve thisstimulation is to mate the female with a vasectomised male, but this practice is not desirablefrom the point of view of disease control and necessitates having vasectomized malesavailable. The most acceptable alternative is to administer luteinising hormone , usually inthe form of human chorionic gonadotrophin. However, the major disadvantage is thatrepeated injections of this foreign protein may cause the female to develop antibodies, thusinactivating subsequent doses.2.2.2 Artificially induced ovulationHormones may be administered to spontaneous ovulators to ensure that ovulation occurs atthe correct time relative to AI. However, since 2006, the use of hormones in food-producinganimals has been forbidden in the European Union, and local regulations may also apply inother parts of the world. Previously most dairy goats in France were inseminated out of thebreeding season with deep frozen semen, after induction of oestrus and ovulation byhormonal treatments. This protocol provided a kidding rate of approximately 65% (Leboeufet al., 2008). As an alternative to administering artificial hormones, out-of season breedingmay be induced by altering the photoperiod or by introducing a buck to the herd. Thispractice is also widespread in intensive sheep flocks.2.3 Deposition of semen in the femaleThere are differences between species in the site of semen deposition during natural mating.In ruminants and primates, semen is deposited in the vagina whereas in pigs, dogs, camelsand horses, semen deposition is intrauterine. In most species, it is possible to pass aninsemination catheter through the cervix, thus enabling semen to be deposited in the uterusduring AI. Exceptions are sheep and goats, where the tightly folded nature of the cervixdoes not permit easy passage of an insemination catheter. The advantages of depositing thesemen in the uterus are that the spermatozoa have less far to travel to reach the oviducts andfewer spermatozoa are lost through back-flow. A smaller volume of semen can be used perinsemination dose than for intravaginal deposition, thus permitting an ejaculate to bedivided into several AI doses, and the cervix, which can act as a barrier to the passage ofspermatozoa, is bypassed. A disadvantage, particularly for human IUI, is that seminalplasma is also introduced into the uterus, unless specific steps are taken to separate thespermatozoa from seminal plasma before IUI.

8Artificial Insemination in Farm Animals3. Species differences in the use of AIDespite the fact that the basic principles of AI are the same in all species, there is widevariation in the uptake of this biotechnology in different species.3.1 AI in cattleIn cattle, frozen semen doses are used most widely in Europe and North America, sincethere are well-established protocols for cryopreserving bull semen. Semen doses typicallycontain approximately 15 million motile spermatozoa. In New Zealand, however, freshsemen doses are used instead, with AI occurring within 24h of semen collection.3.2 AI in pigsThe porcine AI industry uses liquid semen that has been stored for one to several days at 16-18C. In contrast, AI with cryopreserved boar spermatozoa results in lower farrowing ratesand litter sizes than with cooled, stored spermatozoa, making the use of frozen-thawedsperm doses unattractive for commercial pig breeders. Exceptions to this rule are whensemen is transported over long distances, which creates problems in temperature regulation,and in instances where it is vital that the boars can be shown to be free of disease at the timeof semen collection. The ability of boar spermatozoa to survive cool storage so well isattributed to low levels of reactive oxygen species (ROS) in semen or to the efficientscavenging of ROS by anti-oxidative components in seminal plasma.3.3 AI in horsesAI has increased in horses in the last 25 years. Initially, fresh semen was used for AI shortlyafter semen collection, but nowadays the use of cooled semen has largely replaced freshsemen in Europe and North America. The extended semen is cooled to approximately 5°,and transported in insulated containers, together with a cold pack. The fertility of the cooledsemen is maintained for approximately 24h. Frozen semen doses are used infrequently,although this trend may change with the development of better freezing protocols.However, with the increased use of cooled semen, a concomitant decrease in foaling rate hasbeen observed in several countries, such as Finland and Sweden, although the reason forthis apparent decline in fertility is unknown. Unlike bulls and boars, which are selected fortheir semen quality as well as for their potential “genetic merit” in production characteristics(body composition, weight gain, milk production etc), the choice of stallions as breedingsires is based solely on their performance in competition. Thus, considerable variation insemen quality exists between stallions. This variation, coupled with increased use of a widerrange of stallions, may be contributing to the observed decline in foaling rate. Otherimportant considerations are the lack of established standard methods for cooling andfreezing of stallion spermatozoa, for the sperm concentration in the insemination dose, orfor quality control of raw or frozen/thawed spermatozoa.3.4 AI in sheepRam semen differs from stallion and boar semen in consisting of a small volume (a few mL)of seminal plasma containing a very high concentration of spermatozoa. In Europe,reproductive research in livestock has tended to focus on cattle and pigs rather than onsmall ruminants, with the result that sperm handling and cryopreservation for AI is less

Artificial Insemination: Current and Future Trends 9advanced in the latter species. In addition, the anatomy of the female reproductive tract inthese species presents more of a barrier to successful insemination than in cattle, since thecervix is tightly folded, making insertion of the insemination catheter difficult. Productivityin sheep and goats could be increased, by improving the quality of the spermatozoaassigned for use in AI, and improving the AI techniques in these species. Recent innovationsin sheep breeding include the development of a flexible catheter at the National Center forGenetic Resource Preservation, Fort Collins, Colorado, that can be inserted through theovine cervix, thus overcoming the barrier to effective AI in this species.AI in sheep and goats is traditionally performed with fresh or cooled spermatozoa, withacceptable fertility results. However, use of foreign breeds, genetic improvement and theuse of “safe” semen from other countries requires the use of frozen semen, to enableanalyses for contaminants or diseases in the “donor” male to be completed before the semendoses are used for AI. Although the post-thaw motility of frozen semen from goats andsheep is usually considered acceptable, low fertility has been associated with its use in AI,mainly owing to a shortened lifespan of the spermatozoa.3.5 Intrauterine insemination in human fertility treatmentIt is estimated that 10-20% of couples wanting to conceive are unable to do so without someassistance. In 40% of cases, sub-fertility is due to female factors, with a further 40% beingdue to male factors. The remaining cases may be multifactorial or idiopathic in origin. Theuse of IUI is generally contraindicated in male factor infertility, with IVF or ICSI being thetreatments of choice. Since spermatozoa must be able to reach the site of fertilization and theproducts of conception must be able to reach the uterus for implantation, female factorinfertility due to blockage of the oviducts is better treated by IVF or ICSI than by IUI. Thepatient´s own semen or donor semen may be utilized for these fertility treatments.4. AI - State of the artAI can help to improve reproductive efficiency in animals for food production or sport. Weare living in a world of scarce resources where there is constant competition for water, food,land and energy. Since protein of animal origin continues to be one of the most importantforms of nourishment for human beings, animals are an essential part of the ecosystem andmust be husbanded in a sustainable fashion. Animal production not only “competes” withhuman beings for the aforementioned resources, but also produces large amounts of effluentand gaseous emissions which can affect the environment. Therefore, it is vital for thesurvival of the planet that all aspects of animal production are justified and optimized.Through grazing or browsing and the recycling of nutrients, animals also contribute tomaintaining the landscape in a productive state.The production of food of animal origin is based on breeding offspring to enter varioushusbandry systems. Therefore, one of the first points for optimization is in increasingreproductive efficiency, using an holistic approach. Females should be bred for the first timeat an appropriate age to ensure the birth of healthy offspring and optimum lactation,without compromising the health of the female. Subsequent breeding attempts should alsobe timed appropriately to balance the metabolic requirements of lactation and earlypregnancy. Females not conceiving or showing early embryonic loss should be identified atan early stage for re-breeding or culling. However, optimizing female reproductiondemands a supply of spermatozoa. The spermatozoa must be readily available (i.e. can be

10Artificial Insemination in Farm Animalsstored), robust, and capable of fertilization, initiation of early embryonic development andregulation of placental formation, and there must be a means of delivery to an appropriatesite in the female.5. AI in other speciesAI in non-domestic species presents several new challenges compared with domesticspecies. In many cases little is known about the reproductive biology of the species inquestion, and handling the animals may cause them stress, with the attendant risk of injury.The animals must be managed correctly for the establishment and maintenance ofpregnancy. There are reports of successful AI in deer, buffalo and camelids.6. Future trends in AIIt is highly probable that the use of AI in livestock will continue to increase. AI not onlyfacilitates more effective and efficient livestock production, but can also be coupled to otherdeveloping biotechnologies, such as cryopreservation, selection of robust spermatozoa bysingle layer centrifugation, and sperm sex selection.6.1 AI in increasing the efficiency of livestock productionApart from some specialist sheep or goat units focussing on milk production for cheese andintensive meat production, farming of these species tends to be confined to marginal landthat is unsuitable for crop production or grazing for dairy cattle. There has been limitedselection for production traits. However, there is a resurgence of interest in them now indeveloped countries because of growing awareness that small ruminants could representbetter utilization of scare resources than larger ones, such as cattle, while producing lessmethane and effluent. In many developing countries, sheep and goats are better suited tothe climate than cattle, and it is culturally acceptable to eat their meat and milk products.Thus it is likely that there will be an upsurge in the use of AI in sheep and goats in thefuture, with an emphasis on improving production traits by the introduction of superiorgenes. However, it is essential that any A.I. scheme aimed at large scale improvement of thenational herd must be supported by improved animal husbandry and animal health,otherwise the pregnancies resulting from AI will not go to term, and the offspring will eithernot survive or will fail to thrive. Many of the advanced ART are of little help in areas wherebasic husbandry skills are inadequate.6.2 Biomimetic sperm selectionOne potential disadvantage of AI is that the natural selection mechanisms within the femalereproductive tract to select the best spermatozoa for fertilization may be bypassed when AIis utilized. Biomimetics is the use of technologies and/or processes that mimic a naturallyoccurring event. Several in vitro procedures have been suggested that could be used tomimic selection of good quality spermatozoa in the female reproductive tract and thus fitthe definition of biomimetics in ART. These include sperm processing procedures such asswim-up, sperm migration, filtration and colloid centrifugation (reviewed by Morrell &Rodriguez-Martinez, 2009). Of these methods, the one that is most applicable to livestockand human spermatozoa is colloid centrifugation.

Artificial Insemination: Current and Future Trends 116.2.1 Density gradient centrifugationHuman spermatozoa for fertility treatment are usually processed to remove the seminalplasma and to select those of better quality. In most cases, this is achieved either by spermmigration, in which the more motile spermatozoa are separated from the rest of theejaculate, or by density gradient centrifugation, where the most robust spermatozoa areselected. The benefits of density gradient centrifugation are as follows (Morrell, 2006):i. Poorly motile and abnormal spermatozoa are removed,ii. Sources of ROS (cell debris, leukocytes, epithelial cells and dead or dying spermatozoa)are removed;iii. Sperm survival is improved during frozen and non-frozen storage;iv. Bacterial contamination is controlled without antibiotics.6.2.2 Single layer centrifugationDensity gradient centrifugation is seldom used when processing animal semen because of thelimited volume of semen that can be processed at one time and the time taken to prepare thedifferent layers. A novel sperm preparation technique, Single Layer Centrifugation (SLC)through a colloid, was developed at the Swedish University of Agricultural Sciences (SLU) toselect the most robust spermatozoa from ejaculates. This method is similar to density gradientcentrifugation (DGC), but is better suited for animal semen since it has been scaled-up toprocess whole ejaculates. The major applications for SLC-selection are similar to DGC endhave been reviewed extensively by Morrell & Rodriguez-Martinez (2010)6.3 Sex selectionFor many centuries, animal breeders and researchers have endeavoured to control the sex ofthe offspring born, for various reasons. Initially male offspring were preferred for meatproduction, because of the better feed conversion efficiency and lean-to-fat ratio of males,whereas females were preferred for dairy purposes, except that some males of high geneticmerit were still required as sires. Couples may want a child of a specific sex to avoid theexpression of sex-linked disorders.Many methods have been proposed for separating X- and Y-chromosome bearingspermatozoa, based on physical properties, e.g. size of the sperm head, or functionalproperties e.g. swimming speed. However, the only method which has been shown to workreliably is that of selection and separation of spermatozoa whose DNA is stained with a bisbenzimidazoledye, H33342, using the sorting capacity of a flow cytometer (Morrell et al.,1988; Johnson et al., 1989). This method functions because the X chromosome is larger thanthe Y, therefore taking up more of the DNA-specific stain and showing a higher fluorescencewhen the spermatozoa are passed through a laser beam. In bulls, for example, the differencein DNA content between the X and Y- chromosome is approximately 4.2%. However, theprocess of sorting sufficient numbers for an insemination dose in the flow cytometer takestoo long, since the stained spermatozoa must pass one at a time through a laser beam fordetection of their DNA content. Moreover, the pregnancy rate after insemination of sexedbull spermatozoa is lower than with unsexed spermatozoa, making the procedure inefficientand expensive. Experience has shown that the staining profiles are highly individual, withthe result that it is not possible to separate the X- and Y-chromosome bearing spermatozoaefficiently from all males.Alternative methods of sex selection are also being investigated. A company in Wales,Ovasort, has identified sex-specific proteins on the sperm surface and have raised antibodies

12Artificial Insemination in Farm Animalsto them. It is intended to use the antibodies to aggregate spermatozoa bearing a specific sexchromosome, thus enabling them to be removed from the general population.A combination of ARTs would also be relevant for sperm sexing. Thus, the speed of flowsorting can be increased by first removing the dead and dying spermatozoa from thepopulation, for example by density gradient centrifugation or single layer centrifugation.Such a combination may increase the “sortability” of sperm samples. Sufficient sexedspermatozoa may be obtained from flow sorting for IVF, thus generating embryos orblastocysts for subsequent transfer. However, methods of speeding up the selection processare needed if flow cytometry is to become useful for species other than the bovine.6.4 Sperm cryopreservationAs previously mentioned, the ability of cryopreserved spermatozoa to retain their fertilizingability varies widely between species. New cryoextenders and new protocols are beingdeveloped constantly in an effort to address this issue. One recent advance has been theintroduction of dimethylsulphoxide and the amides formamide and dimethylformamide ascryoprotectants, in place of glycerol. These molecules seem to function better than glycerolfor some individuals whose spermatozoa do not freeze well, for example, some stallions.One explanation for this observation is that these molecules are smaller than glycerol andtherefore may cause less damage when they penetrate the sperm membrane. However, nomethod appears to be universally successful within one species. As far as turkeyspermatozoa are concerned, it seems that the development of a successful freezing methodwill require more than new cryoprotectants and additives (Holt, 2000).6.5 Removal of viruses from ejaculatesViral infectivity can be removed from the semen of patients with viral infections such asHIV and hepatitis, by a sequential method of sperm preparation i.e. centrifugation on adensity gradient followed by a “swim-up” (reviewed by Englert et al., 2004). Spermatozoafrom virally infected men prepared by this method have been used in assisted reproductionattempts, apparently without sero-conversion of mothers or children. However, somestudies with HIV report that density gradient centrifugation alone will not remove all viralinfectivity (Politch et al., 2004). Since spermatozoa may function as vectors for viruses (Chanet al., 2004), further work is required to investigate how closely different viral particles areassociated with the sperm membrane with putative carry-over during processing. Thedouble method of processing has also been successful in removing equine arteritis virusfrom an infected stallion ejaculate in a preliminary study (Morrell & Geraghty, 2006). SLCtogether with swim-up was used to reduce viral infectivity from boar semen spiked withporcine circo virus 2 (Blomqvist et al., 2011).6.5 AI in conservation biologyIt has been suggested that AI and other forms of ART could be useful for geneticconservation and preservation of rare breeds. Many of these technologies have beensuccessful to some degree in a research setting, but none have produced results sufficient toeffect population-wide improvements in genetic management (Morrow et al., 2009).Cryopreservation of semen has been the most widely applied ART in this respect, but muchof the frozen semen in so-called gene banks has never been tested for fertility. A lack ofsuitable females or dearth of knowledge about the reproductive biology of the species

Artificial Insemination: Current and Future Trends 13involved may contribute to this deficit. However, long-term storage of frozen gametes ofunknown fertility is not a sustainable policy for the conservation of rare breeds andendangered species. The development of in vitro methods of testing sperm fertility wouldcontribute considerably to conservation efforts. Since the semen quality in these animalsmay be poor (Gamboa et al., 2009), techniques such as SLC of samples prior to AI could beof considerable benefit in conservation breeding.7. ConclusionAI revolutionized animal breeding in the 20th century, particularly in combination withsperm cryopreservation. The AI industry has developed dramatically in most domesticspecies in the last few decades and its use is now widespread in intensive animalproduction. The development of other associated technologies, such as sperm selection andsex selection, are predicted to create powerful tools for the future, both for domesticlivestock breeding and for the purposes of conservation. AI will continue to play a role infertility treatment for human patients, although it may be superseded by IVF or ICSI. It hasbeen suggested that AI (in animals) is entering a new era where it will be used for theefficient application of current and new sperm technologies (Roca, 2006). Excitingpossibilities are offered by emerging techniques, such as Single Layer Centrifugation, forimproving sperm quality in AI doses as well as for increasing sperm survival duringcryopreservation.8. ReferencesBellinge, B.S., Copeland, C.M., Thomas, T.D., Mazzucchelli, R.E., O’Neil, G., Cohen M.J.(1986). The influence of patient insemination on the implantation rate in an in vitrofertilization and embryo transfer program. Fertil Steril,, Vol. 46, 2523–2526.Chan, P.J., Su, BC., Kalugdan, T., Seraj, J.M., Tredway, D.R., King, A. (1994). Humanpapillomavirus gene sequences in washed human sperm deoxyribonucelic acid.Fertil Steril., Vol. 61, pp. 982-985.Englert, Y., Lesage, B., van Vooren, J.P, Liesnard, C., Place, I., Vannin, A.S., Emiliani, S.,Delbaere, A. (2004). Medically assisted reproduction in the presence of chronic viraldisease. Hum. Reprod. Update, Vol. 10, pp. 149-162.Gamboa, S., Machado-Faria M,, Ramalho-Santos J. (2009). Seminal traits, suitability forsemen preservation and fertility in the native Portuguese horse breeds Puro SangueLusitano and Sorraia: Implications for stallion classification and assistedreproduction. Anim Reprod Sci.Vol.113, pp.102-113.Gutsche, S., Wolff, M., von Strowitzki, T., Thaler CJ. (2003). Seminal plasma induces mRNAexpression of IL-1β, IL-6 and LIF in endometrial epithelial cells in vitro. Mol HumReprod, Vol. 9, pp. 785–791.Holt, WV. (2000). Basic aspects of frozen storage of semen. Anim. Reprod. Sci., Vol. 62, 3–22.Leboeuf, B., Delgadillo, JA., Manfredi, E., Piacere, A., Clement, V., Martin, P., Pellicer, M.,Boue, P., de Cremoux, R. (2008) Management of Goat Reproduction andInsemination for Genetic Improvement in France Reprod Dom Anim, Vol. 43 (Suppl.2), pp. 379–385

14Artificial Insemination in Farm AnimalsJohnson, L.A., Flook, J.P. and Hawk, H.W. (1989). Sex preselection in rabbits: live birthsfrom X and Y sperm separated by DNA and cell sorting. Biol Reprod. Vol. 41,pp.199-203.Morrell, J.M., Keeler, K.D., Noakes, D.E., MacKenzie, N.M. & Dresser, D.W. (1988). Sexing ofsperm by flow cytometry. Veterinary Record, Vol. 122, pp. 322-324.Morrell, J.M. (2006). Update on semen technologies for animal breeding. Reproduction inDomestic Animals, Vol. 41, pp. 63-67.Morrell, J.M. & Geraghty, R.J. (2006). Effective removal of equine arteritis virus from stallionsemen. Equine Veterinary Journal. Vol.38, pp. 224-229.Morrell, J.M., Rodriguez-Martinez, H. (2009). Biomimetic techniques for improving spermquality in animal breeding: a review. The Open Andrology Journal, Vol. 1, pp. 1-9.Morrell, J.M., Rodriguez-Martinez, H. (2010). Practical applications of sperm selectiontechniques as a tool for improving reproductive efficiency. Veterinary MedicineInternational, doi:10.4061/2011/2984767Morrow, C.J., Penfold, L.M,, Wolfe, B.A.(2009). Artificial insemination in deer and nondomesticbovids. Theriogenology. Vol. 71, pp. 149-65Pellicer-Rubio, M., Combarnous, Y. (1998). Deterioration of goat spermatozoa in skimmedmilk-based extenders as a result of oleic acid released by the bulbourethral lipaseBUSgp60. J Reprod Fertil, Vol. 112, pp. 95–105.Politch, J.A., Xu, C., Tucker, L., Anderson, D.J. (2004). Separation of humanimmunodeficiency virus type 1 from motile sperm by the double tube gradeintmethod versus other methods. Fertil. Steril., Vol. 81, pp. 440-447.Robertson, S.A., Mayerhofer, G., Seamark, R.F. (1992). Uterine epithelial cells synthesizegranulocyte-macrophage colony-stimulating factor and interleukin-6 in pregnantand nonpregnant mice. Biol Reprod, Vol. 46, pp. 1069–1079Robertson, S.A., Sjoblom, C., Jasper, M.J., Norman, R.J., Seamark, R.F. (2001). Granulocytemacrophagecolony-stimulating factor promotes glucose transport and blastomereviability in murine preimplantation embryos. Biol Reprod Vol. 64, pp. 1206–1215Robertson, S.A. (2005). Seminal plasma and male factor signalling in the femalereproductive tract. Cell Tissue Res, Vol. 322, pp. 43–52Roca, J., Vazquez, J.M., Gil, M.A., Cuello, C., Parrilla, I., Martinez, E.A. (2006). Challenges inPig Artificial Insemination. Reprod Dom Anim, Vol. 41 (Suppl. 2), pp. 43–53.Salha, O., Sharma, V., Dada, T., Nugent, D., Rutherford, A.J., Tomlinson, A.J., Philips, S.,Allgar, V., Walker, J.J. (1999). The influence of donated gametes on the incidence ofhypertensive disorders of pregnancy. Hum Reprod, Vol. 14, pp. 2268–2273.Tremellen, K.P., Valbuena, D., Landera, S. J., Ballesteros, A., Martinez, J., Mendoza, S.,Norman, R.J., Robertson, S.A., Simon, C. (2000). The effect of intercourse onpregnancy rates during assisted human reproduction. Hum Reprod, Vol. 15, pp.2653–2658.Troedsson, M.H., Loset, K., Alghamdi, A.M., Dahms, B., Crabo, B.G. (2001). Interactionbetween equine semen and the endometrium: the inflammatory response to semen.Anim Reprod Sci , Vol. 68, pp. 273–278.Wani, N.A., Billah, M., Skidmore, J.A. (2007). Studies on liquefaction and storage ofejaculated dromedary camel (Camelus dromedarius) semen. Anim Reprod Sci, Vol.109, 309-318.Wishart, G.J. (1985). Quantitation of the fertilising ability of fresh compared with frozen andthawed fowl spermatozoa. British Poultry Science, Vol. 26, pp. 375–380.

2Artificial Insemination atFixed Time in BufalloesGustavo Ángel Crudeli 1 and Rodolfo Luzbel de la Sota 21 Professor of Theriogenology, Faculty of Veterinary Sciences,Nor Eastern National University, Corrientes,2 Professor of Theriogenology, Faculty of Veterinary Sciences,National University of La Plata, La Plata,Argentina1. IntroductionTo maintain a calving interval of 13-14 month in buffaloes, successful breeding must takeplace within 85-115 days (d) after calving. Complete uterine involution and resumption ofovarian activity and heat expression usually takes place around 20-50 d post partum (dpp);therefore, there is a window of 35-95 d to rebreed a cow and get her pregnant to maintainthe desired calving interval. Although artificial insemination (AI) has the potential to make asignificant contribution to genetic improvement in buffaloes, its practical application hasbeen difficult because poor estrus expression by cows and poor estrus detection by humans,a variable duration of estrus and the difficulty to predict time of ovulation. More recently,the development of protocols for synchronization of ovulation and fixed timed insemination(TAI) in buffaloes have been used to overcome these constrains and be able to use moreextensively AI in commercial herds. Nevertheless, resynchronization of ovulation and TAIstill remains a problem herds managed under extensive conditions for similar reasonsabovementioned.Very recently, we did four field trials to study the efficacy of different protocols thatcombined use of GnRH, or estradiol benzoate (EB), prostaglandin (PGF) and intravaginalprogesterone (P 4 ) releasing device (PIVD) or norgestomet ear implant (NOR) toresynchronize estrus and ovulation at day 18 post AI in buffalo cows under commercialconditions.2. Materials and methods2.1 First trialIn the first field trial, we assessed with ultrasonography the ovarian follicular dynamics tostudy the efficacy of a combined treatment of GnRH, PGF and NOR to synchronize andresynchronize ovulations in TAI programs. Eighteen Mediterranean buffalo cows with abody condition score (BCS) of 2.70±0.26 (scale 1-5) from a farm in northeastern CorrientesArgentina (27 ◦ 20’ 33” S and 58 ◦ 08’ 27” W) were used in the study. Cows were randomlyassigned to one of 3 treatments (TRT, Figure 1): 1) TRT1 (n=6); synchronization: day (d) -10,

16Artificial Insemination in Farm Animals8 ug GnRH (buserelin, Receptal ® , Intervet SA, Argentina); d -3, 150 ug PGF (cloprostenol,Preloban ® , Intervet SA, Argentina); resynchronization: d18 8 ug GnRH; d 25, 150 ug PGF; 2)TRT2 (n=6); synchronization: d -10, 8 ug GnRH and ½ ear implant for 7 days (norgestomet,Crestar ® , Intervet SA, Argentina); d -3, 150 ug PGF; d -1 8 ug GnRH; resynchronization: d 18,8 ug GnRH and ½ NOR ear implant for 7 days; d 25, 150 ug PGF; d 27 8 ug GnRH; 3) TRT3(n=6): same protocol as TRT2 but without ear implant during synchronization andresynchronization (Figure 1). Daily ultrasounds and blood samples were taken from day -3to day 2 during synchronization and from day 18 to day 30 during resynchronization(Figure 1). Blood samples were stored at -20 C ◦ until P4 concentrations were analyzed byRIA (Count-A-Count®, DPC, Los Angeles, USA; intra-assay CV, 3.78%; Inter-assay CV,9.28%).Fig. 1. Experimental design for studying follicular dynamics, time of ovulation, and fertilityafter synchronization and resynchronization of estrus and ovulation in buffaloes in fieldtrials 1 and 2.

Artificial Insemination at Fixed Time in Bufalloes 172.2 Results and discussionDominant follicle diameter prior to ovulation tended to be bigger in TRT1 compared tothe TRT2 and TRT3 (12.58±0.67 vs. 10.97±0.74 mm; P

18Artificial Insemination in Farm Animals181614TRT2TRT1TRT3Diameter DF (mm)12108642086ATRT2TRT1TRT3Diameter SF (mm)4201210BTRT2TRT1TRT3Dominance (mm)8642C0-3 -2 -1 0 1 2Day of protocolFig. 2. Follicular dynamics by day of protocol during synchronization: diameter of thedominant follicle (A), diameter of the subordinate follicle (B), and dominance (C). TRT1: 8ug GnRH (d-10), 150 ug PGF (d-3), heat detection every 12 h; TRT2: ½ Crestar ear implant(d-10 al -3), 8 ug de GnRH (d-10), 150 ug PGF (d-3), 8 ug GnRH (d -1); TRT3: 8 ug GnRH (d -10), 150 ug PGF (d -3), 8 ug GnRH (d -1).

Artificial Insemination at Fixed Time in Bufalloes 19181614TRT2TRT1TRT3Diameter DF (mm)12108642086TRT2TRT1TRT3ADiameter SF (mm)4201210TRT2TRT1TRT3BDominance (mm)8642019 20 21 22 23 24 25 26 27 28 29 30 31 32 33Day of protocolFig. 3. Follicular dynamics by day of protocol during resynchronization: diameter of thedominant follicle (A), diameter of the subordinate follicle (B), and dominance (C). TRT1: 8ug GnRH (d-10), 150 ug PGF (d-3), heat detection every 12 h; TRT2: ½ Crestar ear implant(d-10 al -3), 8 ug de GnRH (d-10), 150 ug PGF (d-3), 8 ug GnRH (d -1); TRT3: 8 ug GnRH (d -10), 150 ug PGF (d -3), 8 ug GnRH (d -1).C

20Artificial Insemination in Farm Animals3. Material and methods3.1 Second trialIn the second field trial, we assessed the fertility obtained with protocols used in theprevious experiment in a commercial farm. We used 57 Mediterranean buffalo with a BCS of4.41±0.12 (scale 1-5) from a farm in northeastern Corrientes Argentina (29 ◦ 42’ 20” S and 59 ◦23’ 17” W). Cows that were randomly assigned to one of three TRT (Figure 4): 1) TRT1(n=20); 2) TRT2 (n=18); 3) TRT3 (n=19).65ATRT2TRT1TRT34P 4(ng/ml)3210-3 -2 -1 0 1 265BDay of protocolTRT2TRT1TRT34P 4(ng/ml)321019 20 21 22 23 24 25 26 27 28 29 30 31 32 33Day del protocolFig. 4. Plasma P 4 concentrations by day of protocol during synchronization (A), and duringresynchronization (B). Synchronization, TRT1: 8 ug GnRH (d-10), 150 ug PGF (d-3), heatdetection every 12 h; TRT2: ½ Crestar ear implant (d-10 al -3), 8 ug GnRH (d-10), 150 ug PGF(d-3), 8 ug GnRH (d -1); and TRT3: 8 ug GnRH (d -10), 150 ug PGF (d -3), 8 ug GnRH (d -1).Resynchronization, TRT1: 8 ug GnRH (d 18), 150 ug PGF (d 25), heat detection every 12 h;TRT2: ½ Crestar ear implant (d 18 al 25), 8 ug GnRH (d 18), 150 ug PGF (d 25), 8 ug GnRH (d27); and TRT3: 8 ug GnRH (d 18), 150 ug PGF (d 25), 8 ug GnRH (d 27).

Artificial Insemination at Fixed Time in Bufalloes 213.2 Results and discussionAt synchronization, the percentage of cows AI was lower for the HDAI group compared tothe TAI groups (80% vs. 100%, P

22Artificial Insemination in Farm AnimalsPGF; d 27, 8 ug GnRH; d 28 TAI), and 2) TRT2 (n=44; synchronization: d -9, 2 mg BE (BE ® ,Syntex, Argentina) and 1 g PIVD (Triu-B ® , Biogenesis-Bagó, Argentina) for 7 days; d -2, 150ug PGF; d -1 1 mg BE; d 0 TAI; resynchronization: d 19, 1 mg BE and 1 g PIVD for 7 days; d26, ultrasound pregnancy diagnosis, open 150 ug PGF; d 27, 1 mg BE; d 28 TAI). Only 61cows finished the experiment (Table 2). Synchronization pregnancy rate was higher in TRT2group compared to TRT1 group (68% vs. 44%, P

Artificial Insemination at Fixed Time in Bufalloes 23Fig. 5. Experimental design for studying fertility after synchronization andresynchronization of estrus and ovulation in buffaloes in field trial 3.TRT1 1 TRT2 2 TotalAI 1 37 44 81NPD 1 3 3 6PD 1 44% (15/34) A 68% (28/41) B 57% (43/75)NAI 2 6 3 9NPD 3 5 5PD2 75% (6/8) 80% (8/10) 78% (14/18)PREG AI 91% (21/23) 95% (36/38) 93% (57/61)EL 50 13% (2/15) 11% (3/28) 12% (5/43)PREG Final 83% (19/23) 87% (33/38) 85% (52/61)PD: pregnancy diagnosis, EL: Embryo losses, NPD1: did not come to PD1, NAI2: did not come to AI2,NPD2: did not come to PD2;A different form B, P

24Artificial Insemination in Farm AnimalsFig. 6. Experimental design for studying fertility after synchronization andresynchronization of estrus and ovulation in buffaloes in field trial 4.

Artificial Insemination at Fixed Time in Bufalloes 25TRT1 TRT2 TRT3 TRT4 TotalSYN100100 (16/16) 100 (39/39) 100 (44/44) 100 (20/20)(119/119)PD1 38 (6/16) 36 (14/39) 48 (21/44) 40 (8/20) 41 (49/119)NAI2 2 5 3 0 10RESYN 100 (8/8) A 100 (20/20) A 100 (20/20) A 67 (8/12) B 93 (56/60)PD2 88 (7/8) 45 (9/20) 65 (13/20) 42 (5/12) 57 (34/60)PREGAI 93 (13/14) 68 (23/34) 83 (34/41) 65 (13/20) 76 (85/109)NS 100 (1/1) 100 (9/9) 100 (6/6) 100 (7/7) 100 (23/23)PD3 0 (0/1) 22 (2/9) 33 (2/6) 43 (3/7) 30 (7/23)NPD3 0 4 1 0 5PREGT 93 (13/14) 77 (23/30) 90 (36/40) 80 (16/20) 85 (88/104)SYN: synchronization, RESYN: resynchronization, PD: pregnancy diagnosis, NAI2: did not come toresynchronization, NPD3: did not come to PD3;A different from B, P

26Artificial Insemination in Farm AnimalsAwasthi Mk, Kavani FS, Siddiquee GM, Sarvaiya NP, Terashri HJ. 2007. Is slow folliculargrowth the cows of silent estrus in water buffaloes? Anim Reprod Sci. 99:258-268.Barkawi AH, Hafez YM, Ibrahim SA, Ashour G, El Asheeri AK, Ghanem N. 2009.Characteristics of ovarian follicular dynamics throughout the estrous cycle ofEgyptian buffaloes. Anim Reprod Sci. 110:326-334.Campanile G, Neglia G, Gasparrini B, Galiero G, Prandi J, Di Palo R, Dócchio Mj, ZicarelliL. 2005. Embryonic mortality in buffaloes synchronized and mated by artificialinsemination during the seasonal decline in reproductive function. Theriogenology.63:2334-2340.Campanile G, Di Palo R, Neglia G, Vecchio D, Gasparrini B, Prandi A, Galiero G, Dócchio,MJ. 2007. Corpus luteum function and embryonic mortality in buffaloes treatedwith a GnRH agonist, hCG and progesterone. Theriogenology. 67:1393-1398.Chauhan FS, Sharma RD, Singh GB. 1983. Serum progesterone concentrations in normalcycling and sub oestrus buffaloes. Indian J Dairy Sci. 36:28-33.De Araujo Berber RCA, Madureira EH, Baruselli PS. 2002. Comparison of two Ovsynchprotocols (GnRH vs. LH) for fixed time insemination in buffalo (Bubalus bubalis).Theriogenology. 57:1421-1430.De Rensis F, Ronci J. 2005. Conception rate after fixed time insemination following Ovsynchprotocol with and without progesterone supplementation in cyclic and no-cyclicMediterranean Italian buffaloes (Bubalus bubalis). Theriogenology. 63:1824-1831.Kumar R, Jindal ED, Rattan PJS. 1991. Plasma hormonal profile during oestrus cycle ofMurrah buffalo heifer. Indian J Anim Sci. 61:382-385.Paul V, Prakash BS. 2005. Efficacy of the ovsynch protocols for synchronization of ovulationand fixed time artificial insemination in Murrah buffaloes (bubalus bubalis).Theriogenology. 64:1049-1060.Presicce G, Parmegiani A, Senatore E, Estecco R, Barile VL, De Mauro G, De Santis G,Terzano G. 2003. Hormonal dymanics and follicular turnover in prepuberalMediterranean buffaloes (bubalus bubalis). Theriogenology. 60:485-493.Presicce G, Senatore E, Bella A, De Santis G, Barile VL, De Mauro G, Terzano G, Estecco R,Parmegiani, A. 2004. Ovarian follicular dynamics and hormonal profiles in heifersand mixed parity Mediterranean buffaloes (bubalus bubalis) following and estrussynchronization protocols. Theriogenology. 61:1343-1355.Presicce GA, Senatore EM, De Santis G, Bella A. 2005. Follicle turnover and pregnancy ratesfollowing oestrus synchronization protocols in Mediterranean buffaloes (Bubalusbubalis). Reprod Dom Anim. 40:443-447.Ronci G, De Rensis F. 2005. Comparison between Ovsynch protocols plus GnRH for fixedtime artificial insemination in Buffalo cows. Proc 1º European Buffalo Congress,Salerno, Italia. p 248.Vale WG, Ohashi, OM, Souza JS, Ribeiro HFL, Silva AOA, Nanba SY. 1989. Morteembrionária e fetal em búfalos (bubalus bubalis). Rev Bras Reprod Anim. 13:157-165.Warriach HM, Channa AA, Ahmad N. 2008. Effect of oestrus synchronization methods onoestrus behaviour, timing of ovulation and pregnancy rate during the breeding andlow breeding seasons in Nili-Ravi buffaloes. Anim Reprod Sci. 107:62-67.

Artificial Insemination of Sheep –Possibilities, Realities and Techniquesat the Farm Level3Sándor Kukovics 1 , Erzsébet Gyökér 2 ,Tímea Németh 1 and Elemér Gergátz 21 Research Institute for Animal Breeding and Nutrition2 Pharmagene-Farm Ltd, Biotechnical Research StationHungary1. Introduction1.1 History of artificial insemination over the last 50 yearsThe state of artificial insemination in the sheep and goat industries has developeddifferently in Europe over the last couple of decades. The number of artificial inseminationsin the sheep industry and the ratio of inseminated ewes increased sharply in East Europe,especially in the eastern part of Mid-Europe, during the 1950s and 1960s. The main reasonfor this increase could be due to the planned economy and certain central pressure from thestate. The presence and the ratio of use of this method were much lower in other parts ofEurope, and its development was rather slower.Because of unfavourable economical circumstances, the profitability of the sheep industryfell in the eastern part of Europe and the number of sheep kept on big state and cooperativefarms declined during the 1970s and the second half of the 1980s. With the changingeconomy in the early 1990s, the decline in sheep number continued. In Hungary, inparticular, during the preparation period prior to accession to the EU, there was a sharpincrease in sheep number, with the increasing trend lasting until the end of 2005. The trendhas reversed since then, with a gradual and intensive reduction.As the consequences of the use of artificial insemination (AI) with semen from importedbreeding rams, wool production traits (fibre diameter, shearing, greasy wool weight andstaple length among others) have steadily and gradually increased in Hungarian Merinoflock. Artificial insemination centres were founded by the state between early 1950s and theend of the 1970s. Some regional sub-stations belonging to each county AI centres weresupplying flocks from state and cooperative farms. Over this period, the state helpedimprove sheep breeding with the operation of AI centres. The number of inseminated ewesreached its peak in the mid 1960s, when 63% of ewes in the national flock were artificiallyinseminated with a relatively wide range, but from the end of this decade, the use of AIstarted to go back. In the Hajdu-Bihar County (east of the country) for instance, the numberof inseminated ewes exceeded 85%, even in mid 1970s’ (Kukovics, 1974; Jávor et al., 2006;Kukovics & Gergátz, 2009). From the mid 1970s, the state-owned artificial inseminationcentres started to close down, the number of rams kept for semen collection was reducedand the breeding animals were sold to various farms.

28Artificial Insemination in Farm AnimalsAfter this period privately-owned self-owned ram and artificial insemination units wereestablished and took advantage of the sheep breeding state and cooperative farms.Meanwhile, artificial insemination started to be more intensively used in Western Europe.The number of inseminated ewes and their ratio increased in breeding programmes whererapid genetic development was essential. One of these programmes was the French dairyLacaune breeding system, where more than 82% of the nucleus part of the population (about160 000 out of the 750 000 heads) were artificially inseminated by 1993 with semen mainlytransported from several AI centres. During the previous thirty years, average milkproduction increased from 50 to 70 litres to 300 litres per ewe annually (Barillet et al., 1993).This trend did not change and the system expanded to other breeds in France, Spain andItaly (Jávor et al., 2006).Since the beginning of the 1980s, the number of inseminated ewes has decreased noticeablyin Hungary. As the whole economy of the country was reorganised and privatised from theearly 1990s, the number of farms utilising AI as the breeding method has almostdisappeared. Nowadays, less than 2 % of breeding ewes are inseminated artificially onabout 15 to 20 farms out of the registered 6,900 sheep farms. Indeed, the relatively smallsize of flocks (about 150 heads of adult females) has an important role in the development ofthis situation. Almost twenty breeds are bred in the country, but AI is only used in limitednumbers. The Merinos are the dominant breed in the country; however, very few farmersbreeding Merino sheep use AI.It was quite well known many years ago and even today that AI can not be carried outwithout special skills. Several hundred people were educated on artificial insemination (inthe 1950s and 1960s up to the mid 1970s) in order to use this modern breeding method in thecountry.The education of shepherds practically decreased in Hungary, and no one received evenminimal skills in the AI of sheep and goats between 1986 and 1999. On behalf of theHungarian Goat Breeders Association and the Hungarian Sheep Dairying Association, aseries of indoor courses were organised for sheep and goat breeders in 1999 and 2000. Thecourses were carried out in the Biotechnical Research Station University of WesternHungary, in Mosonmagyaróvár. More than 60 people (shepherds and goat breeders)finished the three courses and took successful theoretical and practical examinations,receiving a certificate for their knowledge. Unfortunately, the organisation of furthercourses had to be stopped because of a shortage of funds needed to cover the costs of thecourses. However, a couple of years later, special official courses were announced by thestate in sheep and goat AI, but there was no interest until now.At present, only a limited number of breeders are convinced about the importance and thevalue of AI. Most of the sheep and goat keepers have several numbers of breeding males formating.Until 2008, two officially certificated artificial insemination stations (Pharmagene-Farm Ltd,Mosonmagyaróvár, and Bakonszegi Awassi Corporation, Bakonszeg) were operating in thecountry; however, some research centres (universities and research institutes) had completelaboratories ready to offer services to various farms. In 2011, only one AI station remainedactive in Mosonmagyaróvár, and there was a new embryo transfer station officially certifiedin Budapest.Unfortunately, not only is there a shortage of state-organised shepherds as well as a lack ofeducating inseminators (cattle and pig excluded), but there is also an absence of interest ofthe breeders association in forcing farmers to get knowledge and use artificial insemination.

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 29The lack of interest, insufficient knowledge and education, absence of organisation, laziness,shortage of labour and low profitability can be found behind this symptom.The director of Hőgyész State Farm, Béla Szüllő (Hungary), who supervised the breedingand production of more than 4,000 sheep stated in 1972, believed that the profitability ofmeat sheep farming could be achieved when 1.5 lambs could be utilised after one eweannually (Kukovics, 1974). This level could be attained with the use of frequent lambing,utilisation of artificial insemination, and with the application of breeds with high prolificacyin crossbreeding programmes. That result was later confirmed by Kukovics & Jávor (2002)in their study.1.2 The main factors affecting AIThere are several factors that could modify the effectiveness of artificial insemination andsome of them are mentioned below.1.2.1 The breedIn many publications, the ewe breed has been found to have a large effect on the pregnancyrate after artificial insemination. According to Hill et al. (1998), the wool type (strong wool –67.6%; fine wool – 71.7%; fine medium and medium 73%) of Australian Merino affected theaverage pregnancy rate. In a Greek study (Karagiannidis et al., 2001), the conception raterank of ewes was Chios, Vlachiki and (Vlachiki x Chios), with a significant differencebetween them. The pregnancy rate of Suffolk ewes was much lower (12%) than that ofFinnish landrace (65%) in Irish studies (Donovan et al., 2001 and 2004), while the breed ofthe ram also had a significant effect on prolificacy after AI (Perkins et al., 1996; Donovan etal., 2001 and 2004; Anel et al., 2005).1.2.2 Age of the eweThe fertility rate in laparoscopic insemination gradually decreases from the age of 1.5 to 2.5years (Anel et al., 2005), while it increases until 3.5 to 4.5 years of age in vaginalinsemination.1.2.3 The season of inseminationThe season of insemination could have a strong effect on the results. According to Hill et al.(1998), the month of insemination exerted highly significant differences: it was 71.5% inMarch, April, or May and 67.6% in November, December, January or February. In the studyof Anel et al. (2005), the season modified the conception rate after both laparoscopic andvaginal insemination (September-January 46.88 vs. 35.53%; February-June 43.96 vs. 29.79 %;July-August 38.95 vs. 22.72%), but the data of the first one were always higher.1.2.4 The use of fresh, cooled, chilled, frozen semenIt is generally stated that the fertility of the semen decreases with cooling temperature. Theuse of freshly diluted semen could give the best result: 70 to 82 % (Donovan et al., 2001 and2004) and 82.2% (Hill et al., 1998; Ehling et al., 2003). A similar level could be reached withcooled and stored semen /74-76% (Gergátz&Gyökér, 1997) and 56.7% (Fernandez-Abella etal., 2003) as well as with chilled and stored semen /between 37.5% (Fernandez-Abella et al.,2003) and 64.2-73.33% (Stefanov et al., 2006)/. The conception rate frozen-thawed semenwas the lowest in all of the publications: 38 to 46% (Gergátz & Gyökér, 1997), 69.5 to 71.6%

30Artificial Insemination in Farm Animals(Hill et al., 1998), 29 to 52% (Donovan et al., 2001, 2004), and 42.86 to 53.33 % (Stefanov et al.,2006).1.2.5 The labourIn the study of Anel et al. (2005), the results of laparoscopic (from 40.60 to 51.54%) andvaginal (from 23.85 to 43.16%) insemination highly depended on the technician who carriedit out.1.2.6 The yearAnel et al. (2005) reported that the level of pregnancy rate decreased for both techniques(from 62 to 44% for laparoscopic AI and from 38 to 31% for vaginal AI) between 1990 and1997.1.2.7 Time of insemination after oestrus synchronisationThe time of AI is especially important in the case of oestrus synchronisation. The best timeof insemination could be 46 (Fernandez-Abella et al., 2003), 48 to 72 (Karagiannidis et al.,2001), and 58 to 63 (Donovan et al., 2001, 2004) hours after the pessary removal.1.2.8 Dose of PMSG usedHill et al. (1998) reported that the type and dosage of PMSG (Pregnant Mare Serum Globulin- 200 IU – 62.4%; 250 IU – 72.9%; 300 IU - 79.1%; 375 IU and above – 69.4%) had a significanteffect on the conception rate in artificial insemination.1.2.9 The extender usedThe main aim of diluting is to enlarge the fertility and storing ability of ram semen (Mucsi,1997; Sarlós, 1999; Gergátz, 2007) with additional energy. There are many kinds of extendersused for this reason (skimmed milk, Ivanov, Salamon, improved versions of them, etc.(Kukovics, 1974; Becze, 1982, Gergátz & Gyökér, 1997; Sarlós, 1999; Fernandez-Abella et al.,2003; Jávor et al., 2006), and most of the authors suggest materials. The semen used in anykind of artificial insemination is mainly diluted, and dilution is particularly important in thecase of cooling, chilling and deep freezing of semen. The most common dilution rate is the1:1 – 1:2 – 1:3 – 1:4, but in practice, a ratio higher than 1:8 is not really used (Fernandez-Abella et al., 2003; Gergátz, 2007).1.2.10 Dose of inseminated semenIn general, the suggested dose of semen is 0.1 to 0.2 ml with about 50 to 100 million activespermatozoa. About 50 to 100 million spermatozoa is needed in one dose to vaginalinsemination, but 25 to 50 million are suggested for cervical insemination and 15 to 20million is enough for laparoscopic AI (Kukovics, 1974; Jávor et al., 2006; Gergátz, 2007).However, several scientists have used much higher numbers: 106 (Fernandez-Abella et al.,2003), 50 to 300 (Ehling et al., 2003) and 400 (King et al., 2004) million spermatozoa in onedose of semen.1.2.11 The method (vaginal, cervical, cervico-uterinal or laparoscopic) usedThe simplest method is vaginal insemination, which could result in the lowest conceptionrate. The use of cervical and trans-cervical methods could give much higher pregnancy

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 31rates, but laparoscopic AI is the most effective one. However, it is also the most expensiveand complicated method. Apart from the general trends, the publications show quite a widerange of rates: 31.25% (Anel et al., 2005) in vaginal, 18 (Yamaki et al., 2003); 42 (King et al.,2004); and 65-75% (Salamon & Maxwell, 1995; Donovan et al., 2001, 2004; Stefanov et al.,2006) in cervical; 69.6 to 76.4% (Szabados, 2006) in cevico-uterinal; and 44.89 (Anel et al.,2005), 64 (Yamaki et al., 2003), and 69 % (King et al., 2004) in laparoscopic insemination. Itshould be noted that these results are affected according to the semen used (fresh, cooled,chilled or frozen (Perkins et al., 1996; Moses et al., 1997).1.3 Detection of ewes on heat and the number on inseminationsOne of the key questions of artificial insemination is the detection of ewes on heat, the timeof insemination following the selection, and the number of inseminations made. The maindetection systems could be summarised as follows (Kukovics, 1974; Jávor et al., 2006):I. Selection once a day (24 hours) in the morning (or in the afternoon)(A) first insemination made immediately(a) insemination every 24 hours1. no further insemination2. second insemination made without further selection3. second insemination only in the case of when heat is detected4. further insemination in the case that the ewe is still on heat4.1. no more than three inseminations4.2. further inseminations until the animal is on heat(b) insemination every 8 to 12 hourssub-points are the same as in point (a)(B) the first insemination made 3 to 4 hours after selection or latersub-points are the same as in point (A/a)II. Selection every 8 to 12 hours(A) insemination made right after detection(a) no further insemination made(b) further inseminations every 8 to 12 hourssub-points are the same as in point I. (A/a)(B) first insemination made 3 to 4 hours after detection or latersub-points are the same as in point I. (A/a)Since heat in ewes lasts 24 hours, in general, and is shorter in the case of yearlings, the first(I) method could have several limitations and so, the second (II) method is proposed andcould be more effective.Concerning the daily distribution of real heats, adjusted to the possibilities of practical life,the ewes on heat are detected between 6 and 7 hours in the morning as part of the so called“Mosonmagyaróvár insemination technique” (Gergátz, 2007). The first insemination iscarried out between 10 and 11 am, and the second one between 3 and 4 pm. Because of theonce daily detection, about 5% of the yearlings will be omitted from insemination at first,but 95% of them will be inseminated in the next heat.Many opponents of artificial insemination declare that using this method, the level ofpregnancy obtained naturally cannot be performed. The results of studies from the lastdecades have proved that the pregnancy rate of ewes selected and inseminated only oncecould reach 60 to 65%. The pregnancy rate of the ewes selected and inseminated twiceduring the same cycle could exceed 75% and reach 80 to 85%. With the use of three

32Artificial Insemination in Farm Animalsinseminations, 90 to 95% of pregnancy could be performed (Kukovics, 1974; Jávor et al.,2006; Gergátz, 2007).Fig. 1. Collection of semen at a farm (Photo: Németh, A.)1.4 The costs of artificial inseminationIt is rather difficult to estimate the costs of artificial insemination per average ewe on a farm.The following jobs and movements are involved: preparation of rams and ewes for AI,preparing equipments and tools for collecting semen and artificial insemination, collecting,qualifying, diluting and dosing semen, detecting and selecting ewes on heat, fixing animalsto carry the AI, and finally performing the insemination, and cleaning the tools andequipments. These basically cover the expenditures of buying tools (and feeds) and the costsof labour. The use of oestrus synchronisation is an additional cost, even if it consists ofseveral parts.The cost of artificial insemination increased from 1.2 to 3.2 HUF/head to 2.5 to 4.5HUF/head between 1966 and 1971 in the abovementioned Hajdú-Bihar County (Kukovics,1974). This increase was the consequence of the intensive development of feed prices(especially the crops and concentrates). In order to evaluate current values (2010), thesenumbers should be multiplied by 100 to 500 (in 2011 1 Euro = 270 HUF approx.)The estimated costs of artificial insemination change according to the method utilised, theplaces of semen collection, the labour/service company that is performing the job, thepresence of oestrus synchronisation, and the handling of rams before the season, among

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 33others. Laparoscopic AI is the most expensive because it needs veterinary service and help.At the same time, vaginal, cervical or even trans-cervical insemination can be carried out bywell-skilled shepherds, at a much lower level of costs, without significant losses inefficiency.Due to reorganisation of the national economies of Mid-East European countries, stateownedAI centres have disappeared (Jávor et al, 2006) and most of the remaining sheepfarms use rams for natural services. Shepherds educated on performing AI have either diedor have left the sheep industry and the remaining ones do not want to work with it as itmeans extra work, attention and accuracy. AI is only used on very few farms nowadays andavailable data on expenditures of AI are rather limited or not published at all.In Western Europe and the Western world, artificial insemination became a special serviceof companies, not really based on the skills of sheep owners and shepherds. At the sametime, laparoscopic AI (LAI) has become the most popular, which can be carried out byveterinarians. Cervical and trans-cervical AI can also be provided by different veterinaryand genetic companies.Donovan et al. (2001) stated that cervical insemination was much cheaper than laparoscopicAI, but exact cost values were not given in the study. After out-of-season mating (May) ofpurebred and crossbred Lacaune ewes with oestrus synchronisation and induction, Gulyáset al. (2007) reported that the extra costs of using biotechnological methods were refundedwith the good profits raised by selling lambs during Christmas time. According to anAmerican presentation for sheep farmers, cervical and trans-cervical methods are easy tolearn and use. Each sheep takes about two minutes to inseminate at a cost of $1.29, making itsignificantly faster and less expensive than laparoscopic surgical insemination(http://www.farmanddairy.com/news/ultrasound-and-artificial-insemination-techniques).The service charges of these companies are quite different, and most of them concerningonly LAI do not cover all the costs. The service cost of LAI could be, for instance, GBP 10 to15 per ewe (http://www.innovis.org) or GBP 10 to 25 per ewe plus indirect cost, whichmeans the total costs could be around GBP 20 to 35 per ewe(http://www.trialanderroracres.com); or AU$ 15 to 30 per ewe (Genstock Animal BreedingServices, Australia – http://genstock.com.au) ; or NZ$ 15 to 30 (Genetic Gains Ltd andPremier Genetics Ltd, New-Zealand – http://genetic-gains.co.nz) , or the prepared semencould be ordered at the price of US$ 15 to 135 per straw (Super Sire Ltd – Pathway toGenetic Improvement – http://www.topRams.com). Apart from these, limited data areavailable about the real costs of AI. The service reports and offers discuss pregnancy ratelevels and the charges.2. Materials and methodsAs mentioned above, less than 2% of the ewes kept in Hungary were artificially inseminatedbetween 2000 and 2010; however, more than 63% of ewes were artificially inseminated in1963. Examining the main characteristics of more than 6,900 sheep farms and about 500 goatfarms, we observed that AI was utilised only on a limited number of sheep farms, and nogoat farm practically used this breeding method.2.1 The farms and the breeds keptIn order to study artificial insemination at the farm level, 11 sheep farms out of the less than20 farms with available data were selected from 2003 to 2010. Twelve breeds and genotypes

34Artificial Insemination in Farm Animalsof sheep were breed on the studied farms. The breeds kept on the various farms and thenumber of years when artificial insemination was utilised are summarised in Table 1.On most farms, only one breed was kept, but three different breeds were bred on farm No.2, 9 and 10, and two breeds on farm No. 11.In the first farm, which was a corporation, a breeding project has been carried out since 1990and purebred Awassi and crossbred Awassi (F 1 , R 1 , R 2 , R 3 , R 4 ) flocks have been reared. Themajority of female sheep was artificially inseminated during 2003 to 2007, but because oflow AI efficiency, natural insemination with breeding rams was used to get betterpregnancy results. The farm was finally reorganised and sold in 2009, so the use of AI endedfrom 2008.A breeding project was performed on the second farm, which was a state farm aiming tocreate a new synthetic breed and started operating from early 1970s. The Bábolna Tetrasheep breed was finally developed from the crossings of Hungarian Merino as well as 5lines of Finnish Landrace and 3 lines of Romanov breeds, and it was officially accepted as anew breed in 1995. In addition to this, Ile the France and American Suffolk nucleus flockswere also developed on the farm, starting in 1988. The reorganisation and final selling of thefarm and its sheep happened in 2009 and 2010, and it is currently a much smaller privatefarm.FarmcodeNo. and name of breeds keptNo. of years1 1: Awassi and Awassi crossbreds 5 (2003-2007)2 3. Bábolna Tetra, Ile de France, Suffolk 8 (2003-2010)3 1: Hungarian Merino 4 (2003-2006)4 1: German Mutton Merino 6 (2003-2008)5 1: Hungarian Merino 6 (2003-2008)6 1: Lacaune 2 (2009-2010)7 1: Lacaune 7 (2003-2010)8 1: Lacaune 2 (2003-2004)93: Hungarian Merino, German Mutton Merino,German Blackheaded Mutton Sheep8 (2003-2010)10 3: British Milksheep, Charollais, Hungarian Merino 5 (2005-2010)11 2: British Milksheep, British Milksheep Crossbreds 8 (2003-2010)Table 1. Number of breeds and the studied years on the farmsThe other private farms were founded in the 1990s, when most of the cooperative and statefarms were demolished, reorganised and the sheep were sold from them. The onlyexception was farm No. 7, which was a research station belonging to the University of WestHungary founded in 1982, and it is still a research station.The use of AI stopped on farm No. 3 in 2007, and in 2009 in the case of farms No. 4 and 5because of labour problems. On farm No. 8, the labour problem together with the lowquality of AI made with transported semen led to the replacement of AI with the use ofextra breeding rams.On farm No. 10, only a small population of the sheep (purebred imported breeds) wereartificially inseminated, and the AI started to be utilised in the nucleus part of the Merinoflock only in 2007. On farm No. 12, the British Milksheep crossbred sheep (Hungarian

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 35Merino x British Milksheep) were handled separately since 2007. On farm No.6, the use ofAI was only started in 2009.The results of AI were not possible to evaluate for year 2010 in the case of farms No. 6, 7, 10and partly 11 because the lambing of artificially inseminated ewes started only in 2011 andwas not finished before the time of data collection.2.2 Data collectionTwo surveys were carried out in late 2007 and at the beginning of 2011 in order to collectdata of artificial insemination on the selected farms. Each farm was visited and the data wascollected based on a questionnaire covering the following information:- number of sheep kept,- number of artificially inseminated ewes per breed and per year,- number of progeny born and weaned after AI per breed and per year,- method of selection and time searching ewes on heat,- time and number of insemination,- method of collecting and qualifying semen to use,- level of dilution, kind of extender and the size of inseminating dose,- method and use of oestrus synchronisation,- method of insemination,- fixing method of ewes during insemination,- control of pregnancy and use of ram service after insemination,- number of labour used over AI,- cost of AI.2.3 Processing of dataThe methods of AI used on various farms were evaluated in details. The results of AI werestudied on farm levels because of the farm difference in the number of breeds kept.Pregnancy rate (P%) =Lambing rate (L%) =Weaning rate (W%) =lambed ewesinseminated ewesborn lambslambed ewesweaned lambsborn lambsThe pregnancy rate (P%), lambing rate (L%) and the weaning rate (W%) were calculated byfarms and by breeds in every studied year. In the case of those farms where more than onebreed was kept, the effects of breed and year were studied. In the case of farms where onlyone breed was kept, the effects of year were examined.Chi-square test of SPSS 10.0 was applied to compare the breeds to each other and tocompare the years by breed. Significant differences between breeds and years weredetermined at P≤0.05.

36Artificial Insemination in Farm Animals3. ResultsThe total number of sheep kept on various farms is summarised in Table 2. The size of flocksbelonging to various breeds per farm changed over the studied period, the number of ewesmainly decreasing over the years. The numbers of artificially inseminated ewes per breedand per farm are presented in Table 3.3.1 The tools of artificial inseminationThe most important factors in artificial insemination are well-skilled labour (mainlyshepherds or inseminators), well-prepared rams of good quality, ewes with good bodycondition, and the necessary equipments and tools.Based on our study, at least one person per farm with proper skill in artificial inseminationwas working, and he was primarily the owner shepherd. There were technicians on farmsNo. 1 and 7. The animals were prepared (details presented later on) on each farm before theseason.The most important equipments and tools necessary for performing artificial inseminationon farms are shown in Figure 2: artificial vagina, semen collecting glass with double wall(and warm water between the walls), warm water bath, thermometer, vaginal speculum(with different size for adult ewes and ewe hoggets), lamp for providing light into thevagina, pipettes, catheter, syringe and vaginal sponges or implants for oestrussynchronization. A microscope with relatively smaller capacity is also needed with a tableand sheet object heater.Farmcode2003 2004 2005 2006 2007 2008 2009 20101 1960 2120 2140 2200 2080 - - -2 3138 2905 2753 2103 2251 2170 971 5883 312 359 411 461 450 - - -4 700 700 750 700 840 834 - -5 820 900 950 980 1050 960 - -6 - - - - - - 1100 9807 239 204 177 206 216 237 295 2908 588 456 - - - - - -9 1160 1168 1183 1157 988 1060 968 94010 - - 430 450 470 460 460 45011 420 390 350 380 380 350 345 340Table 2. The total number of ewes kept on the studied farms between 2003 and 2010

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 37Breed,farmcode,yearHungarian MerinoGermanMuttonMerinoGermanBlackheadedMutton SheepBritishMilksheepBritishMilksheepCrossbreds9 10 3 5 9 4 9 10 11 112003 446 - 274 160 438 640 174 - 420 -2004 458 - 350 200 461 650 159 - 390 -2005 477 - 385 220 476 690 171 33 350 -2006 440 - 317 350 462 600 179 41 280 -2007 522 118 - 165 319 650 147 31 280 702008 563 150 - 130 335 670 162 28 240 602009 498 150 - - 288 - 182 41 235 602010 475 150* - - 310 - 155 70* 240* 65*Breed,farmcode,yearCharollais Lacaune AwassiBábolnaTetraIle deFranceSuffolk10 7 8 6 1 2 2 22003 - 212 198 - 1960 1717 937 1952004 - 196 127 - 2120 1379 996 2452005 - 165 - - 1760 1222 863 2402006 - 151 - - 370 1147 732 2242007 11 167 - - 50 1014 866 2142008 8 127 - - - 1043 784 3042009 16 245 - 297 - 311 310 2422010 18* 255* - 369* - 286 186 107* lambing after AI made in 2010 started in 2011Table 3. Distribution of the number of inseminated ewes by breed and farms

38Artificial Insemination in Farm AnimalsBased on the results of our survey (visiting the eleven farms), all necessary tools andequipments were available. Moreover, officially-accepted artificial insemination stationswere operating on farms No. 1 and 7, from where semen could be bought by other farms.Fig. 2. The equipments and tools for artificial insemination (Photo: Kukovics, S.)3.2 Preparing the animal for the mating (inseminating) periodThe preparation of rams and ewes for the mating (inseminating) period was a commonpractice on all farms. In the case of rams, the level of nutrition started to improve at leastfour weeks before the planned season. Parallel to this, the training of the rams was alsoinitiated and semen was collected at least two times a week. The quality of the semen wasstudied.3.2.1 Preparing ewes and using oestrus synchronisationOestrus synchronisation and induction are highly recommended in the case of usingartificial insemination. Yet these methods were not commonly utilised on the studied farmsduring the period under study. The nutritional method (flushing started four weeks beforeAI) was used on every farm in order to prepare the ewes for the mating period. The rameffect was not really used. However, on some farms, vasectomised rams were introduced tothe flock of ewes, but it was not correctly planned. No other method (like reducing thelength of light hours) was used on these farms.Oestrus synchronisation and induction were used on only five farms (No. 1, 5, 6, 7 and 10).These methods were used during the main season and in spring time on farms No. 1 and 10(only in the case of Merinos), only in spring on farms No. 5 and 7, and only in winter onfarm No. 6.During the first four years, the most popular product for oestrus synchronization was the“Eazy-bred” vaginal implant (produced in New Zealand), but since 2005 new purchaseswere not possible because of EU regulations. The vaginal sponge (Chrono-Gest) was usedon the farms over the last 5 years of the studied period. There were significant differencesamong the farms in the size of PMSG dose. For example, on farm No. 1, 750 IU wasadministered in the first two years and 600 IU in the following years. 500 IU PMSG dose was

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 39used on farms No. 5, 6 and 7. On farm No. 10, 550 IU was utilised during the first threeyears, but because of too strong effects the dose was reduced to 425 IU over the last fouryears of the studied period.3.3 Collecting and examining the semenThe semen was collected locally on most of the farms by the shepherd with appropriateskills, except for farm No. 8 where transported semen was used. Artificial insemination wascarried out by the skilled shepherd, except for farm No. 1, where it was done by aveterinarian in the first two years and by a technician during the following years (Figure 1).Visual examination of the semen was performed before use on every farm, but three(No. 2, 6 and 11) out of the 11 farms used only this method. Following the visual study,microscopic and morphological examinations of the semen were also carried out on farmsNo. 1 and 7. Microscopic examination of semen together with the visual study wasperformed on the other six farms.3.4 Diluting of semenSeveral kinds of extenders are available for every day use, and in many studies, it has beendemonstrated that the use of diluting liquids could help the survival of spermatozoa in thesemen. Nevertheless, most shepherds thought that using un-diluted semen produced betterresults and was safer. Six (No. 2, 3, 4, 5, 9 and 11) out of the eleven farms did not use anykind of extender to dilute the semen before insemination. The 1:2 and 1:4 diluting ratioswere used on the first farm and in the first three years on farm No. 10, where a 1:3-dilutingratio was used during the following three years. The 1:4 ratio was used on farm No. 5 (in2003) and 1:8 on farms No. 6 and 7.In the 1950s and 60s, one of the most popular extender was skimmed cow milk on farms.However, the Ivanov and the Salamon kinds as well as their improved versions wereavailable after the late 60s and most of the shepherds carrying out the inseminations knewall of them and heard about several other ones. It was particularly interesting because morethan half of the shepherds successfully participated in the AI courses mentioned above, anda few were even performing AI in cattle.Three of the farms (6, 7 and 8) employed a special extender developed by the researchstation (farm No. 7). Semen diluted with this extender and cooled and kept at 2 to 4°C couldbe used successfully for 72 hours after collection. The improved Salamon kind of extenderwas used on farms No. 1 and 10.3.5 Semen dose for inseminationIn general, 0.2 ml was the most common dose of inseminating semen. It was used on seven(No. 1, 2, 3, 4, 5, 7 and 8) out of the 11 farms. Doses of 0.1 and 0.3 ml were used on farms No.9 and 11, as well as No. 6 and 10, respectively.3.6 Detecting ewes on heat for inseminationAs the one of the most important factors of successful artificial insemination is selecting theewes on heat, morning and afternoon selections were used on most farms (No. 1, 3, 4, 5 and8). The selection was either only performed in the morning (No. 2, 7 and 9) or in theafternoon (No. 6 and 10) on the other farms. Midday was the selection time on farm No. 11for the first 4 years, which was changed to the morning system during the following years.

40Artificial Insemination in Farm AnimalsThe selection lasted half an hour to one hour during each part of the day. Vasectomised ramswere used on farms No. 1 and 7 over the whole studied period. Entire rams with apron wereused as teasing rams on five farms (No. 3, 4, 6, 9 and 10) and both kinds of rams were used ononly one farm (No. 5). On farm No 2 and 8, vasectomised rams were used in the first fouryears, and entire rams with apron during the second four years, while on farm No. 11, theorder was the reverse: the vasectomised rams were used in the second four years of the period.3.7 The time and number of inseminationTwo inseminations were used on most of the farms (No. 1, 2, 3, 4, 5, 6, 7 and 8) about 8 to 10hours apart (morning-afternoon or afternoon-morning), but only one insemination wasutilised on two farms (No. 9 in the morning and No. 11 in the afternoon). On farm No. 10,three inseminations (morning – afternoon - morning or afternoon – morning – afternoon)were carried out each year.3.8 Performing inseminationFor successful insemination, ewes have to be fixed and the rear part of their body should belifted up. The rear legs of the ewes were lifted up and fixed by one labourer on the top of thebarrier (Figure 3) in almost all of the studied farms. Farms No. 6 and 8 were the exceptions,where the labourer had to lift up the rear part of the ewes and hold them during the time ofinsemination.This operation needs more than one labourer, therefore, one catcher and one inseminatorshould be used for this job as a minimum. On most of the farms, the inseminations wereperformed by the owner shepherd with one or two labourers to help him. On farms No. 1and 7, technicians conducted the inseminations. There was only one catcher helping theinseminator on farms No. 5, 7, 9, 10 and 11. Two labourers caught and held the ewes onfour farms (No. 3, 4, 6 and 8). On the first farm, 5 catchers helped the work of 2 inseminatorsduring the first three years, and in the following two years only one inseminator with twolabourers performed the job. On farm No. 2, the number of catchers decreased from two toone during the last three years and only one inseminator worked there.Fig. 3. The fixing of ewe for insemination (Photo: Kukovics, S.)

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 413.9 The place of semen depositionThe place of semen deposition is the other rather critical point of insemination. Traditionalvaginal insemination was used on only one farm (No. 9) and only in the first three years ofthe studied period. They then changed to cervical deposition. Cervico-uterinal inseminationwas performed on farms No. 6, 7 and 10 (and sometimes on 11), while cervical inseminationwas used on other farms (Figure 4). Laparoscopic insemination was only used at theexperimental level in the country. It was too expensive for farm practice.Inseminating pipettes were available on all farms. The special catheter (Figure 1) for transcervicalinsemination developed by Tassy and Gergatz (Kukovics, 1974) were also used,which was utilised by most inseminators during the studied period. This catheter has aspecial bent tip that allows passage through the cervix and is made in different sizes foradult ewes and yearlings.3.10 The pregnancy controlThe results of lambing were too late to determine the effectiveness of artificial insemination.“State-of-the-art” pregnancy tests to determine the results of AI are important for profitablesheep farming. Yet, the level of pregnancy control in the case of inseminated ewes wasrather low on the studied farms. The most up-to-date trans-rectal ultrasonography was usedonly on farm No. 1. Ultrasonography was performed within 60 days of AI in every studiedyear on farm No. 10, in the first 4 years on farm No. 7 and only in the first two years on farmNo. 5.The commonly used method to reach and keep pregnancy at the highest possible level wasthe post-mating with entire rams that started one cycle after the AI and lasted for two cycles.Farm No. 4 was an exception, where no post-mating was utilised. An interesting thinghappened in the case of farm No. 7, where post-mating was not used during the second fouryears of the studied period.Fig. 4. The insemination (Photo: Kukovics, S.)

42Artificial Insemination in Farm Animals3.11 The results of artificial inseminationThere were significant differences found among the studied farms and among the variousbreeds kept on various farms and also between the breeds within the farms. The pregnancyrate in general exceeded 80% and results over 90% were not exceptional at all. However,effects of farm, breed and year were observed on the results.At least two or three breeds were kept on four farms (No. 2, 8, 10 and 11) and only one breedwas bred on the other seven farms. Due to this, the results found in the case of four farmswere evaluated separately, and the findings concerning the other seven farms were pooledtogether.The pregnancy, lambing and weaning rate of the lambs were evaluated and the results arepresented in the Tables from 4 to 10.3.11.1 Farms breeding more than one breedThe pregnancy rate (P%) on farm No. 2 was different among breeds in 2004, where Suffolkewes had a significantly lower value than the other two breeds (Table 4). In 2006, thepregnancy rate of Ile de France ewes was significantly higher than that of the other breeds.In 2005, 2008 and 2009, there were significant differences among all three breeds. In theBábolna Tetra breed, the highest P% was measured in 2009, which was significantly higherthan in the other years (except 2005 and 2010). The P% in 2008 was significantly lower thanin the other years. In the Ile de France breed, the lowest P% in 2009 was different from theother values, like the values in 2007 and 2008. In the Suffolk breed, the P% of 2009 wassignificantly lower than in the other years (Table 4).Significant effects of year were observed within the breeds in this trait, more than a 16%-range was found between the smallest and highest values in all three breeds; however, thebiggest deviations were in the case of the Suffolk breed.The lambing rate (L%) in the Bábolna Tetra breed varied between 1.6 and 2.0, while in Ile deFrance, the interval was narrower (between 1.2 and 1.4). In the case of the Suffolk breed, theL% changed every year (Table 4). It meant that the effect of the year was stronger in theBábolna Tetra and Suffolk breeds than in the Ile the France. However, the differencesreached the significant level (P

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 43breed, the data of 2006 differed from that of 2003, 2005 and 2007. In the GermanBlackheaded Mutton breed, there were no significant differences between 2003 and 2006 andfrom 2005 to 2010 (Table 5).Breed,trait,yearBábolna Tetra Ile de France SuffolkP% L% W% P% L% W% P% L% W%2003 82.2 aA 1.7 82.2 aA 81.4 aA 1.2 85.8 bcA 76.9 aA 1.4 86.4 acA2004 86.1 aB 1.8 86.0 aB 83.3 aA 1.3 84.3 acAC 73.9 bAB 1.7 79.9 bcA2005 87.3 aBE 1.7 94.2 aC 90.4 bB 1.3 91.1 bcB 66.3 cB 1.5 93.2 acB2006 79.4 aC 1.7 83.7 aA 92.2 bB 1.2 88.7 bcAB 77.4 aA 1.4 85.4 acA2007 79.7 aAC 1.6 88.5 aD 79.6 aA 1.3 87.1 aA 80.4 aA 1.2 68.4 bC2008 73.1 aD 1.7 84.4 aAB 94.6 bC 1.3 83.3 aAC 79.6 cA 1.4 81.8 aA2009 90.7 aE 2.0 97.8 aDE 84.7 bD 1.2 87.9 aAD 85.7 C 1.3 86.9 aAD2010 86.7 aABE 1.9 82.3 aA 90.3 aB 1.4 81.8 aA 83.2 aA 1.5 82.1 aAThe different small letters in rows and the different upper case letters in columns mean significant differences(P≤0.05) per trait (pregnancy, weaning) among breeds and years.Table 4. The result of AI on farm No. 2The lambing rate (%) in Hungarian Merino varied between 1.5 and 1.7, while it was between1.6 and 1.8 in German Mutton Merino. In German Blackheaded Mutton sheep, the lowestrate was 1.5 in 2009, while the highest 1.8 value was found four times (Table 5). The value ofthis trait exceeded the national average by 0.3 to 0.4 lambs in the case of Hungarian Merino.Additionally, German Mutton Merino and German Blackheaded Mutton sheep had a 0.2 to0.3 advantage per lambing over the national average in the country.Breed,trait,yearHungarian Merino German Mutton Merino German BlackheadedMutton SheepP% L% W% P% L% W% P% L% W%2003 89.5 aA 1.6 96.4 aA 92.2 aA 1.7 95.9 aA 87.9 aA 1.8 96.7 aA2004 92.4 aAC 1.6 97.4 aA 3.9 aAB 1.6 96.7 aA 85.5 bA 1.7 95.4 aA2005 90.4 aAC 1.7 97.2 aA 93.1 aA 1.7 96.6 aA 91.8 aAC 1.8 95.3 aA2006 97.5 aB 1.5 96.6 aA 96.3 aB 1.7 95.8 aA 89.9 bAC 1.8 94.9 aA2007 91.4 aAC 1.7 4.9 aAB 92.8 aA 1.6 95.0 aA 95.2 aBC 1.8 93.0 aA2008 93.1 aC 1.7 96.3 aA 93.7 aA 1.6 95.4 aA 94.4 aBC 1.7 94.3 aA2009 94.6 aACD 1.7 96.6 aA 93.4 aA 1.8 95.3 acA 90.7 aAC 1.5 93.3 bcA2010 96.4 aB 1.6 95.1 aAB 93.5 aA 1.7 95.3 aA 92.9 aAC 1.7 94.6 aAThe different small letters in rows and the different upper case letters in columns mean significant differences(P≤0.05) per trait (pregnancy, weaning) among breeds and years.Table 5. The results of AI on farm No. 9

44Artificial Insemination in Farm AnimalsAs this farm was one of the best, these data were very close to each other and their levelswere close to the maximum of the genetic possibilities, yet the differences found amongthem originated from the breed characteristics. Of course, the year effects were alsoobserved among these data, but the differences reached the significant level (P

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 45The lambing rate in purebred was higher than that in the crossbred population, varyingbetween 2.0 and 2.4, while in crossbreds, it was between 1.9 and 2.4 (Table 7). In the case ofcrossbreds, the year 2009 was almost exceptional (P

46Artificial Insemination in Farm AnimalsFarmcodebreed / year 2003 2004 2005 2006 2007 2008 20091Awassi+Awassicrossbreds37.3 a 35.0 a 45.0 b 56.8 c 80.0 d - -3 Hungarian Merino 83.2 a 93.4 b 67.0 c 83.0 a - - -5 Hungarian Merino 75.0 a 67.5 ac 78.6 ab 62.9 bc 66.7 a 61.5 acd -4German MuttonMerino84.4 a 83.8 a 87.0 ab 86.7 a 84.3 a 81.0 ac -6 Lacaune* - - - - - - 88.27 Lacaune* 95.3 a 85.2 b 81.8 b 96.7 a 97.6 a 82.7 b 79.6 b8 Lacaune 58.6 a 66.1 a - - - - -The different small letters in rows indicate significant differences (P≤0.05).*The lambing after AI made in 2010 started in 2011Table 8. The pregnancy rate (%) on farms with one breedFarmcodebreed / year 2003 2004 2005 2006 2007 2008 20091Awassi, Awassicrossbreds1.3 1.3 1.3 1.3 1.3 - -3 Hungarian Merino 1.4 1.3 1.3 1.4 - - -5 Hungarian Merino 1.5 1.5 1.5 1.4 1.2 1.2 -4German MuttonMerino1.4 1.4 1.4 1.4 1.5 1.5 -6 Lacaune* - - - - - - 1.57 Lacaune* 1.4 1.6 1.5 1.6 1.5 1.4 1.68 Lacaune 1.6 1.6 - - - - -*The lambing after AI made in 2010 started in 2011Table 9. The lambing rate (%) on farms with one breedFarmcodebreed / year 2003 2004 2005 2006 2007 2008 20091Awassi, Awassicrossbreds94.5 a 95.3 a 94.8 a 95.6 a 96.2 a - -3 Hungarian Merino 96.6 a 91.3 bd 86.5 cd 90.5 d - - -5 Hungarian Merino 88.0 a 89.8 a 95.6 b 97.4 b 84.1 ac 90.9 abc -4German MuttonMerino89.3 a 89.0 a 89.3 ab 90.9 a 87.0 ac 88.3 a -6 Lacaune* - - - - - - 95.27 Lacaune* 53.0 a 86.5 b 84.8 b 80.3 b 85.5 b 79.7 bc 71.2 c8 Lacaune 89.8 a 93.9 a - - - - -The different small letters in rows indicate significant differences (P≤0.05).*The lambing after AI made in 2010 started in 2011Table 10. The weaning rate (%) on farms with one breed

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 47In the Awassi breed, there were no significant differences among years in the weaning rate.On farm No. 3, the W% was the highest in 2003, which was significantly different from theother studied years. However, the ratio in 2006 was close to the values found in 2004 and2005. On farm No. 5, 2006 was the year most different from the others. On farm No. 4, theonly difference observed was between 2006 and 2007, while on No. 7, the two terminal years(2003 and 2009) differed from the other years. On farm No. 8, there was no significantdifference among years in the weaning rate (Table 10).3.12 The cost of artificial inseminationRather big differences were observed among the studied farms (Table 11). In most cases,these data were calculated by the owner of the farm and mainly covered the direct cost ofsemen collection and insemination, while other costs were not included. On some farms, thesame level was calculated every year, while on others, the annual costs increased with theyears. Apart from these, the use of oestrus synchronisation increased the costs by about 7 orabove 8 euros per ewe.In general, the average direct costs of artificial insemination per ewe could reach 0.4 to 0.5euros, and in the case of oestrus synchronisation, the total costs could exceed 7 to 8 eurosunder present Hungarian circumstances.Farmcode2003 2004 2005 2006 2007 2008 2009 20101 3.70* 3.70** 3.70** 3.70** 5.56** - - -2 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.743 1.48 1.67 1.85 1.85 - - - -4 0.37* 0.37* 0.37* 0.37* 0.37* 0.37* - -5 0.74 0.74 0.93 1.11 1.11 1.31 - -6 - - - - - - 0.37* 0.37*7 3.70 3.70 3.70 3.70* 3.70* 3.70** 3.70** 3.70**8 0.93 1.11 1.30 1.30 1.30 1.85 1.85 1.859 1.85 1.85 - - - - - -10 - - 0.37* 0.37* 0.37* 0.37* 0.37* 0.37*11 0.37 0.37 0.37 0.37 0.37 0.37 0.37It was calculated on the changing rate of 1 euro = 270 HUFIn the case of oestrus synchronisation, the costs reached seven (*) or eight euros (**) per ewe.Table 11. The estimated costs of artificial insemination (euro/ewe)4. ConclusionsApart from the fact that artificial insemination is used only on a limited number ofHungarian sheep farms, the effectiveness of this method was quite reasonable. Based on theresults, the following conclusions can be drawn. Apart from the lack of officially-organised education of shepherds, there are somesheep owners and shepherds who can operate with the method of artificialinsemination at a very good level (Kukovics & Gergazt, 2009).

48Artificial Insemination in Farm Animals Artificial insemination of ewes can be performed with very high effectiveness on farmsin every day practice mainly by shepherds. The reality is that AI does not needveterinary assistance, but maintaining the health of ewes needs veterinary control. The procedure of artificial insemination requires well-skilled shepherds with goodpractice and enough support. The breed of ewe has a significant effect on the pregnancy rate, which is consistent withthe results of Hill et al. (1998), Perkins et al. (1996), Donovan et al. (2001 and 2004),Karagiannidis et al. (2001) and Anel et al. (2005). However, the results could bepositively modified by the interest of the sheep owner. The dose of PMSG affected conception rate, similar to the findings of Hill et al. (1998).Thus, in some cases, the dose should be reduced in order to avoid too many lambsbeing born and higher lamb loss originating from the weakness of the lambs at birth. Most shepherds use fresh, locally collected, un-diluted semen with good results, andthe importance of dilution is only realised by a small number of the shepherds. The year had a strong effect on the results of artificial insemination, in accordance withthe results of Anel et al. (2005), but no trends could be discovered in the data. Theenvironmental circumstances (available pasture and feed for instance) had strongerimportance. The cost of artificial insemination depends on the farm, but in general, the direct costwas less than one euro per ewe. Of course, in the case of oestrus synchronisation andinduction, these costs could reach 7 to 8 euros per ewe. The results of artificial insemination could easily be controlled by usingultrasonography (Egerszegi at al., 2008) at an early stage of pregnancy, but this methodwas only used on a small number of farms.5. AcknowledgementsThe authors would like to thank the owners and the representatives of the sheep farms fortheir excellent technical help in collecting the data and analysing the information needed forfinal evaluation. They are in alphabetic order: Attila Harcsa (Abbod Farm, Szendrő), AntalKádas (Túrkeve), István Nagy and Imre Nagy (Ganna), László Nagy (Eger), Sándor Nagy(Bakonszeg Awassi Corporation, Bakonszeg), Zoltán Nagy (Harkakötöny), Mihály Sebők(Törtel), Ede Sipos (Móriczhida), Károly Szabó (Hajdúbagos) and Zoltán Szabó (Karcag).The study was made by K-OVI-CAP BT within the framework of University of DebrecenCentre of Agriculture and Economic Sciences with the help of the Jedlik Ányos projectsupported by National Office of Research and Technology (NKTH)6. ReferencesAnel, L., M. Kaabi, B. Abroug, M. Alvarez, E. Anel, J.C. Boixo, L.F. de la Fuente, P. De Paz(2005). Factors influencing the success of vaginal and laparoscopic artificialinsemination in Churra ewes: a field assay, Theriogenology, 63, 1235-1247.Barillet, F., S., Sanna, D., Boichard, J.M., Astruc, A., Carta, S., Casu (1993). Geneticevaluation of the Lacaune, Manech and Sarda dairy sheep with Animal Model,Proceedings of 5th International Symposium on Machine Milking of SmallRuminants /Ed.: Kukovics, S./, Hungary, May 1993, 289-303.

Artificial Insemination of Sheep –Possibilities, Realities and Techniques at the Farm Level 49Becze, J. (1982). Tanulmányok haszonállatok szaporításáról. (Studies on the reproduction ofdomestic animal breeds); Mezőgazdasági Kiadó, ISBN 963-231-114-0, BudapestDonovan, A. , J.P. Hanrahan, T. Lally, M.P. Boland, G. P. Lonergan, D.J. O’Neil (2001). AIfor sheep using frozen-thawed semen, ARMIS 4047 Project report, under theResearch Stimulus Fund; OPARDF measure 5b, ISBN 1 84170 152 1Donovan, A. , J.P. Hanrahan, E. Kummen, P. Duffy, M.P. Boland (2004). Fertility of the ewefollowing cervical insemination with fresh or frozen-thawed semen at natural orsynchronised oestrus, Animal Reproduction Science, 84, 359-368.Egerszegi, I., A., Molnár, P., Sarlós, F., Soós, J., Rátky (2008). Investigation of the folliculardevelopment and early Pregnancy in Hungarian Black Racka ewes by meansultrasonography – preliminary study (A tüszőnövekedés és korai vemhességultrahangos vizsgálata fekete racka juhokban – előkísérlet) AWETH 4. (2.) 311-318.2008 I. Gödöllői Állattenyésztési Tudományos Napok, Gödöllő 12-13, (inHungarian)Ehling, C., P. Wirth, L. Schindler, K.-G. Hadeler, H.-H. Döpke, E. Lemme, D. Herrmann, H.Niemann (2003). Laparoscopical intrauterine insemination with different doses offresh, conserved, and frozen-thawed semen for the production of ovine zygotes,Theriogenology, 60, 777-787.Fernandez-Abella, D., M.O. Preve, N. Villegas (2003). Insemination time and diluting rate ofcooled and chilled ram semen affects fertility, Theriogenology, 60, 21-26.Gergátz, E. (2007). A juhok mesterséges termékenyítése; in: Házi emlősállatok mesterségestermékenyítése. (The artificial insemination in sheep; in Artificial insemination indomestic mammals); Szerk. (Edited) Tamás Pécsi; Pécsi Tamás, MezőgazdaKiadó,Bp. ISBN 978-963-286-237-8Gergátz, E., E., Gyökér (1997). Cervicouterinal Insemination method with cooled and deepfrozen ram semen; 48. Annual Meeting of EAAP,Vienna, Book of Abstract, 319-325.Gulyás, L., E., Gergátz, J., Végh, A., Németh (2007). A fogyasztási csúcsokhoz igazodóbárányelőállítás lehetőségei biotechnikai módszerek felhasználásával. (Possibilitiesof lamb meat production adjusted to consuming peaks using biotechnicalmethods). Acta Agronomica Óváriensis 49. 1.-29-42.Hill, J. R., J. A., Thomson, N.R., Perkins (1998). Factors affecting pregnancy rates followinglaparoscopic insemination of 28,447 Merino ewes under commercial conditions: asurvey, Theriogenology, 49, 697-709.Jávor, A., S., Kukovics, Gy. Molnár (2006). Juhtenyésztés A-tól Z-ig (Sheep breeding to A toZ), Mezőgazda Kiadó, ISBN 963 286 275 9, Budapest.Karagiannidis, A., S. Varsakeli, G. Karatzas, C. Brozos (2001). Effect of time of artificialinsemination on fertility of progestegen and PMSG treated indigenous Greekewes, during non-breeding season, Small Ruminant Research, 39, 67-71.King, M.E., W.A.C. McKelvey, W.S. Dingwall, K.P. Matthews, F.E. Gebbie, M.J.A. Mylne, E.Stewart, J.J. Robinson (2004). Lambing rates and litter sizes following intrauterineor cervical insemination of frozen-thawed semen with or without oxytocinadministration, Theriogenology, 62, 1236-1244.Kukovics, S. (1974). A mesterséges termékenyítés eredményeinek értékelése, valamint a juhszaporaságának és gazdaságosságának néhány kérdése (The evalation of theresults of artificial insemination and some questions prolificacy and profitability in

50Artificial Insemination in Farm Animalssheep); Állattenyésztési Kutatóintézet (Research Institute for Animal Production)Herceghalom, research study report made for Ministry of Agriculture, p. 120.Kukovics, S., E. Gergatz (2009). A juh mesterséges termékenyítése üzemekben (Artificialinsemination of sheep on farm), In: Magyar Állatorvosok Lapja (HungarianVeterinary Journal), 131, 21-26.Kukovics, S., A., Jávor (2002). A világfajták szerepe a juh árutermelésben. (The role of worldsheep breeds in commodity production), Magyar Juhászat + Kecsketenyésztés(Hungarian Sheep Farming + Goat Breeding), Vol. 11, No. 11, pp. 4-8.Moses, D., A.G., Martinez, G., Iorio, A., Valcárcel, A., Ham, H., Pessi, R., Castanon, A.,Maciá, M.A. de las Heras (1997). A large-scale program in laparoscopic intrauterineinsemination with frozen-thawed semen in Australian Merino sheep in ArgentinePatagonia, Theriogeneology, 48, 651-657.Mucsi, I. (1997). Juhtenyésztés és – tartás (Sheep breeding and keeping), Mezőgazda Kiadó,ISBN 963912124, Budapest.Perkins, N.R., J.R. Hill, R.G. Pedrana (1996). Laparoscopic insemination of frozen-thawedsemen into one or bothuterine horns without regard to ovulation site insynchronised Merino ewes, Theriogenology, 46, 541-545.Salamon, S., W.M.C. Maxwell (1995). Frozen storage of ram semen. I. Processing, freezing,thawing and fertility after cervical insemination, Animal Reproduction Science, 37,185-249.Sarlós, P. (1999). Kosspermatermelés, kosspermaminőség (Production of ram semen, qualityof ram semen), Állattenyésztési és Takarmányozási Kutatóintézet, Herceghalom,Retrieved from Stefanov, R., E., Krumova, M., Dolashka, W., Voelter, Z., Zachariev (2006). Artificialinsemination of sheep and cow with semen treated by Cu/Zn-superoxidedismutase from fungal Humicola lutea 103, World Journal of Zoology, 1 (1), 36-39.Szabados, T. (2006). A cervikouterinális inszeminálás eredményességének vizsgálatajuhászatokban /Study of effectiveness in cervico-uterinal insemination of ewes/;PhD thesis, University of Western Hungary, Faculty of Agriculture and FoodSciences, Mosonmagyaróvár, Hungary.Yamaki, K., M. Morisawa, A. Ribadulla, J. Kojima (2003). Sheep semen characteristics andartificial insemination by laparoscopy, Tohoku Journal of Agricultural Research,54(1-2), 17-26.http://www.farmanddairy.com/news/ultrasound-and-artificial-insemination-techniqueshttp://genetic-gains.co.nzhttp://genstock.com.auhttp://www.innovis.orghttp://www.trialanderroracres.comhttp://www.topRams.com

4Artificial Insemination in DogsRita Payan-Carreira 1 , Sónia Miranda 2 and Wojciech Niżański 31 CECAV – Univ. of Trás-os-Montes and Alto Douro,2 Escola Universitária Vasco da Gama,3 Univ. Environmental and Life Sciences, Wrocław,1,2 Portugal3 Poland1. IntroductionIn Artificial Insemination (AI) the semen is collected manually from a stud male andthereafter deposited (inseminated) in the female so that fertilization can occur in the absenceof natural mating. Artificial Insemination, one of the earliest techniques for assistedreproduction in animals and humans, took longer to be implemented in dogs due to speciesspecificparticularities. In past decades, progresses in the knowledge of canine physiologyand new advances in canine semen technology allowed these services to become availableworldwide. Hence, subsequent to the increase in the artificial insemination demand amongdog breeders and owners and the broaden of the AI to preserved semen as a managementtool in canine breeding, as through international exchange of frozen semen, inbreedingwithin breeds can be reduced. Therefore, with spread of canine AI dog, breeders now mayselect stud dogs from all over the world to improve their kennel´ genetics, withouttransport-associated stress to the animals. Also, it is possible to save semen from valuabledogs into sperm bank to be used in next generations, after their death or the peak ofreproductive age. In addition, breeders also are aware of the sanitary benefits associatedwith AI. Avoiding direct contact between the male and female, AI also prevents the spreadof sexually transmitted diseases, as those originated by Brucella canis or Herpes virus(Farstad, 2010; Linde Forsberg, 2005a).Although the first reports on AI in dogs subsequent to the Spallanzani experiments (in lateXVIII century) appeared by the end of the fifties, reporting the use of fresh semen, or in thesixties, the use of frozen semen, only in the nineties this technique was introduced into dogbreeding practice, particularly in USA and Nordic countries (Foote, 2002; England & Millar,2008). The reproductive physiology of this species and unfavourable response of the dogsperm to freezing were the two major constraints to the initial efforts to improve the AItechnique in dogs (Linde Forsberg, 2005a). A lot of research was performed in those areas,especially in the northern Europe, to overcome these issues, generating a large amount ofinformation and allowing technical development, in particular in the canine sementechnology. Nowadays, as a consequence of the demand for reproductive technologies, inparticular the AI with fresh or refrigerate semen, this is a current service offered in the smallanimal veterinary practice.

52Artificial Insemination in Farm AnimalsAccording to Linde-Forsberg (2001, 2005a), from all the AI in dogs performed byveterinarians today in Europe, about 50-55% is done with fresh semen, collected at the clinic,10% with chilled semen and around 35-40% with frozen semen. However, at least inPortugal, the use of imported chilled semen is far most frequent than the use of frozensemen when compared to other countries in Northern Europe.Research on AI in the domestic dog, along with other reproductive technologies, proceedworldwide, particularly on sperm survival at freezing and the identification of deleteriouscomponents to spermatozoa or fertilization, providing important information for thepreservation of wild canidae semen that are currently threatened or endangered.2. Indications for artificial inseminationSeveral main indications exist to perform AI in the dog (Linde Forsberg, 2005a; England &Millar, 2008; Farstad, 2010). In parallel, some ethical conditions must be discussed whenfacing the different interests of specific groups, namely dogs, breeders, owners andveterinarians.Main indications for AI in dogs include both medical and breeding-management reasons(Table 1). As major potential advantage, AI may allow to reduce physical distances, the useof genetically valuable stud dog semen all over the word, fighting the stress oftransportation of animals and inbreeding (Johnston et al., 2001; Linde-Forsberg, 2005a). It isalso an important technique whenever physical and behavioural abnormalities in the maleor female preventing natural mating (Table 2).3. The ethics and role of artificial insemination in canine breeding programsPerforming canine AI may raise some ethical concerns, mostly to central institutions like theNational Kennel Clubs or Veterinarian Orders or equivalent, in particular on what concernsthe use of frozen semen and the need for intra-uterine insemination, mainly those involvingsurgical procedures. In fact, several countries (such as Norway, Sweden and the UnitedKingdom) refer to welfare concerns and discourage or even forbid the use of surgicalprocedures to obtain intra-uterine insemination (England & Millar, 2008; Linde-Forsberg,2005b).Ethical issues are seldom associated with the non-surgical process of artificial inseminationper se. Most procedures used for semen deposition are neither detrimental to the bitch, norinterfere with animal welfare, and even allow protection against certain diseases. However,some attention may be given to the inbreeding of animals that may compromise health offollowing generations (England & Millar, 2008).Restrictions to the use of AI in animals that never matted despite all physiologicalconditions met together to guarantee a successful mate, may respond to the ethical issuethat demands for ruling out clinical reasons for AI, as an underlying unaware problem(congenital or behavioural) may exists. This concern is in fact previewed in the FédérationCynologique International (FCI) breeding rules (http://www.fci.be/circulaires/102-2010-annex-fr.pdf). According to those rules, AI should not be performed in animals not havingat least one previous litter registered from natural service. Furthermore, AI to be arecognisable breeding technique must be performed by veterinarian or a specificallyrecognisable technician, which skills will avoid complications or adverse effects, as well asstress or risks of welfare infringements towards the animals, in particular the female.

Artificial Insemination in Dogs 53Potential benefits- Decrease stress, infectious deseasestransmission, travel expenses- Semen collection without interruption of themale activity (show or training)- Splitting of an ejaculate to bred more females- Reduction of the costs with maintenance of studdogs in a colony- Worldwide availability of the semen of a givendog- Allow early castration of working dogs whilemaintaining availability of their genes- Evaluation of semen quality prior to AI- Early detection of male reproductivepathologies- Semen preservation, so genetic material may beavailable in the future- Overcome problems associated with the refusalto breed (psychological or physical reasons,precocious ejaculation), inexperienced males- May overcome quarantine restrictionsTable 1. Main advantages and inconveniences for canine AI.Potential weakness- Induction of physical or psychologicaltrauma during the AI process- Risk of performing AI for inappropriatereasons- Failure in careful clinical examinationof the breeding animals- Potential risk for maintaining somedisorders in a particular genetic line(hip dysplasia or anatomicalabnormality of the reproductive tract)- Potential risk for introduction ofinherited diseases or abnormalities- Potential overuse of a given malewithin a programme or breed- May allow confusion of parentageFactorFemale-dependentMale-dependentAssociated to bothmale and femaleCause- Male rejection- Aggression- Congenital abnormalities (e.g. presence of a vaginal septum;genital tract strictures, small vulva and vagina)- Reduced libido (due to local or systemic diseases, age, deficientbreeding management, drugs)- Pain- Physical deficiency (such as inability to mount or to obtainpenile erection, lumbar muscle problems, congenitalabnormalities)- Inexperience- Male to female disproportion- Social and behavioural problems (dominant female, inversion ofthe social hierarchy)Table 2. Main causes for refusal of natural mating.The competence of the operator to perform the procedures is essential to avoid alltechnique-related ethical constraints to the use of AI in dogs. Before offering canine AIservices, practitioners ought to specialised themselves, acquiring profound knowledge ofthe reproductive physiology and pathology of the species and the skills to collect semen andto inseminate the female without risking animal health or welfare.

54Artificial Insemination in Farm Animals4. Semen collection and evaluation4.1 Semen collection in the dogSemen collection in the dog is a relatively easy procedure, although requiring some trainingfor optimization of the technique. Semen collection and evaluation is necessary to obtaingood results in canine AI. Although practitioners are often asked to collect semen andperform AI without detailed semen analysis, every sample of semen collected should beevaluated (at least progressive forward motility, total sperm count and morphology) beforeit is used for artificial insemination or cryopreservation. Semen evaluation prior toinsemination warrants the male potential fertility and consequently may predict the fertilitypotential for the AI. In addition, when preparing semen preservation, fertility certificatemay be needed. In such cases andrological evaluation of the stud dog (breeding soundnessevaluation or BSE) has to be performed. Semen collection should be performed before thephysical exam or any stressful procedures on the stud, or can be booked to another day(Freshman, 2002; Johnston et al, 2001).Semen can be collected from most dogs in the absence of a teaser, in a quiet and isolatedroom, where interruptions should be prevented, although the presence of a bitch wouldallow better ejaculates. In reluctant males, stimulating estrus scent can be provided by thepresence of a female in estrus or by using frozen-thawed swabs or gauze sponges takenfrom vaginal secretions of estrus bitches (Freshman, 2002; Kutzler, 2005; Olson & Husted,1986). Although possible, not everyone achieves the use of a chemical pheromone (methyl p-hydroxybenzoate, Aldrich Chemical, Milwaukee, WI) swabbed on the perineal area and tailof an anestrus teaser (Johnston et al., 2001; Kutzler, 2005).Collection of semen should be prepared in advance, and interval between collections orbetween the natural mating and collection, should be registered, if the male is regularly used.Ideal intervals between collections are 2 to 5 days, whilst intervals longer than 10 days mayresult in an increased number of morphological abnormalities and decreased motility(Freshman, 2002; Johnston et al., 2001). In longer periods, it is advisable to perform oneprevious collection, if semen is to be chilled or frozen for shipment. If semen preservation isplanned, semen extender should be prepared before the arrival of the animal (Freshman, 2002).The most common method for semen collection in the dog is by digital manipulation, in thepresence of a female. However, bitch presence, although desirable as it facilitatesprocedures, is not essential to accomplish the collection (Farstad, 2010; Linde Forsberg,2005a). It should be noticed that when the collection is achieved in the presence of the bitchejaculates present higher concentration.The use of manual massage is the most commonly used technique (Farstad, 2010; Johnstonet al., 2001; Linde Forsberg, 2005a), although in the past semen was collected from dogsusing an artificial vagina. Nowadays, semen collection into a tube is commonlyaccomplished by penile massage and the use of a cone or plastic sleeve, a funnel or a specialcollecting vial (Linde Forsberg, 2005a). Briefly, the process is started with a massage of thedog prepuce at the level of the bulbus glandis until developing partial erection, followed bythe quick retraction of the prepuce and penile expose. If the collector is right-handed, semenmust be collected from the dog’s left side, with the operator holding the dog’s penis with theright hand and the collection container in the left hand. During pelvic thrusting, rigid vialsshould be kept at a distance from the penis, to avoid trauma. When pelvic movements arefinished and the dog lifts its rear leg, a 180º backward rotation of the penis should beobtained and the erectile penis should then be directed into the collection cone or the funnel.

Artificial Insemination in Dogs 55Some pressure may be applied with the thumb on the apex of the glans penis, at the level ofthe urethral process, to stimulate ejaculation. When a crystal clear fluid (prostatic fluid)begins to flow into the collection tube, you can gently slide the collection cone off the penis.Watch for semen to flow in the collection tube (Farstad, 2010; Linde Forsberg, 2005a).Canine ejaculate consists of 3 fractions, with the first and third fraction consisting ofprostatic fluid and the second being rich in spermatozoa (England et al., 1990) (Table 3). Thefirst fraction, the presperm portion, is emitted in 0.5 to 1 minute and is colourless, with avolume range of 1-5 ml. It is expelled during first stage of erection, at the moment of thepresence of evident copulatory movement of male. The second fraction, the sperm-richportion, is also rapidly completed (1-2 minutes), and is grayish-white in colour, with avolume of 1-3 ml. It is expelled when thrusting movement of the male ceases and fullerection is observed. The third fraction comes from the prostate and may be up to 30-40 ml;it may take up from 5 to 30 minutes to be completed (Günzel-Apel, 1994; Johnston et al.,2001).Characteristics 1 st fraction 2 nd Fraction 3 rd FractionVolume0.1-2 mL(average 0.33 mL)0.1-3 mL(average 1.17 mL)Sometimes larger volume1-2 to >20 mLQuite variable dependingon the animal.Colour clear or opaquegreyish-white, white,milky-whiteclear, transparentConsistency watery watery-milky, milky wateryCharacterprostatesecretion withadmixture ofepithelial cells,urine, bacteria andsperm cellssperm cells suspended inseminal plasmapH (average) 6.37 6.10 7.20Duration5-90 sec.(average 13.5 sec)5-300 sec.(average 52.4 sec.)Table 3. Main characteristics of the different fractions of the dog ejaculate.Size of the dog< 20 kg> 20 kgVolume of the ejaculate1-22.5 mL(average 5.38 mL)2-45 mL(average 12.75 mL)prostate gland secretion60 sec-20 min.(average 6 min. 55 sec.)Table 4. Variation on the volume of the ejaculate with the size of the dog (Dubiel, 2004)In the dog, the volume of whole ejaculate varies between breeds (Table 4) mainly withanimal size and is partially dependent on the volume of the third fraction collected, whichconstitute about 95% of the volume of the ejaculate in dogs (Farstad, 2010).In most dogs, semen can be collected twice at 30 minutes interval (Farstad, 2010), althoughthe second sample is usually slightly diluted.

56Artificial Insemination in Farm AnimalsMost often, artificial insemination with freshly collected semen is performed withoutfractioning the ejaculate, although for artificial insemination, only the second fraction is ofinterest (Thomassen & Farstadt 2009; Root Kustritz, 2003). Furthermore, it has beendemonstrated the existence of detrimental effects on fertility when this fraction is notseparated from the second one, particularly if semen will be processed as chilled or frozen.Consequently, ejaculate fractioning should always be accomplished, particularly separationof the third fraction. If the ejaculate has a very small volume, it may be diluted with semenextender, to facilitate its handling during insemination procedures.4.2 Semen assessmentSemen assessment is an important part of the evaluation of fertility in males and it should beperformed as routine element of prebreeding examination. Furthermore, semen evaluationought to be completed before artificial insemination or sperm preservation. Semen shouldbe assessed immediately after collection and it has to be handled carefully during all theprocedures. Rapid changes of environmental temperature may be deleterious forspermatozoal motility and structure, and may also artifactually influence the results ofexamination. Any delay in semen assessment may decrease the percentage of motile spermand simultaneously increase the percentage of dead sperm. It is advisable to keep allequipment necessary for semen collection and evaluation at the temperature near 37ºC(Christiansen, 1984; Feldman & Nelson, 1996; Linde-Forsberg, 1991).On table 5, the most frequent indications for routine semen evaluation are presented. Semenevaluation is also frequently performed in the absence of known reproductive pathology,upon request of the owner. In addition, it can be performed at a predetermined momentafter the diagnosis of a clinical disease that may have negative reflects on the potentialfertility of a male dog.It should be notice that reliable in vitro estimation of the real fertilizing ability of sperm cellsis not always possible. Usually, in males with aspermic (no ejaculate), azoospermic (nospermatozoa), or necrospermic (no motile spermatozoa) semen, the fertilizing potential maybe excluded. When the quality of semen in a dog with history of unsuccessful matings islow, premises exist to exclude such male from the breeding programme. However, it shouldalways be remembered that the semen characteristics should be recheck 2-3 times at 1-2weeks intervals, to confirm the male infertility. On the other hand, good in vitro semenquality does not always prove the fertilizing potential of a particular dog.Most frequent indications- Semen evaluation before artificialinsemination- Semen evaluation before/after chilling orcryopreservation- Clinical signs of a disorder of male genitalorgans- Whenever infertility or subfertility of amale is suspectedTable 5. Common reasons for canine semen assessmentOther situations, on request- New stud dog introduced to the breedingcolony- Evaluation of young stud dog before firstmating- In cases of serial unsuccessful matings ofparticular dog- Pathological lesions of male genital tractobserved by the owner- Re-evaluation after the treatment ofdiseases of male genital tract

Artificial Insemination in Dogs 57The semen assessment performed once is not always reliable, because: Frequent matings or semen collections may temporary result in a decreased semenquality; After a prolonged sexual rest dogs may ejaculate many dead, immotile spermatozoa ofabnormal morphology; In young inexperienced males and dogs which mated earlier only naturally, withoutexperience on semen collection, the obtained semen sample may contain only the partof sperm-rich fraction.4.2.1 Conventional assessment of semenDifferent approaches are available to assess the quality of the dog semen that can begrouped in conventional and advanced techniques. The later, usually requires moresophisticated means for the semen assessment and the support of a technical equipment,while the former may be performed in an inhouse lab.The conventional approaches to semen evaluation include macroscopical evaluation of thesemen (volume and colour), but also the microscopical assessment, which will give theconcentration and the number of viable cells in the ejaculate.4.2.1.1 Macroscopic evaluationVolume. The volume of the ejaculate may be assessed in the calibrated tubes used for semencollection. It mainly dependends on the size of the dog, the size of the prostate gland, theanimal age, the frequency of semen collection, the level of erotisation, and the volume of 3 rdfraction collected. A decrease of semen volume is observed in cases of benign prostatichyperplasia, prostatic cysts, inflammatory lesions of prostate and testicles, inflammation ofepididymis, vas deferens or urethra and at weak libido.Colour. The colour of whole ejaculate depends on the volume of third fraction of ejaculatecollected, on the concentration of spermatozoa per mL and the potential presence of nongermcells in the ejaculate. When analysing the colour, one should be aware of the methodof collection, as colour varies with the fraction to be analysed and the fact that analysis maybeen performed on the whole semen or on fractioned semen. The normal colour of wholeejaculate is greyish-white. Pathological colours include: green-greyish typical for thepresence of the pus in semen; red or pink-specific for erythrocytes contamination(haemorrhages from urethra or corpora cavernosa, prostatitis); yellow specific for urinecontamination; and brown, if in the presence of blood.Any kind of semen contamination, such as hair or mud, exclude the specimen from furtherprocedures including artificial insemination or semen preservation. It is therefore importantto check the region of praeputial opening before semen collection and to clean it.The presence of sediment consisting of sperm cells at the bottom of the tube is a normalfeature if the semen is left for several minutes.4.2.1.2 Microscopic evaluationMotility. One of the most important step of conventional semen assessment is the subjectiveevaluation of progressively motile spermatozoa (Spz) under contrast-phase microscope. Theoptimal temperature for assessment of dog sperm cell motility is 39ºC. A small drop ofabout 20 µL of semen is placed on in a pre-warmed slide and cover by the coverslip. Theevaluation is performed under the objective of x20 to x40. If the highly concentrated sperm-

58Artificial Insemination in Farm Animalsrich fraction is collected separately, the semen should be extended with saline or Tris-bufferto a concentration allowing the observation of particular, single sperm cells. The assessmentis based on the evaluation of the average percentage of progressively motile spermatozoa ina few different fields of the specimen. The normal dog semen contains at least 70% ofprogressively motile spermatozoa (Feldman & Nelson, 1996; Günzel-Apel, 1994).A decrease in the percentage of motile spermatozoa may results from temperature shock,contamination with water, urine, blood or lubricants but also from long sexual abstinenceand systemic or infectious diseases, such as brucellosis. Sperm agglutination is alwayspathological and is frequently found in cases of infectious diseases.Concentration and total sperm count. The sperm concentration in whole canine normalejaculate usually exceeds 80 x10 6 Spz/mL. If the second fraction of ejaculate is collectedseparately, the sperm cells concentration in sperm-rich fraction varies usually between 200-600 x 10 6 Spz/mL. It is generally assumed that the number of motile spermatozoa necessaryfor successful AI should be >150 x10 6 (Linde-Forsberg, 1991). Therefore, under normalconditions, the dog´s ejaculate contains far more sperm cells than those needed for a seminaldose, although sometimes, especially in miniature or toy breeds, ejaculate volume and thetotal number of sperm cells are relatively low (

Artificial Insemination in Dogs 59originating from abnormalities of semen maturation, transit through the ductal system andspecimen preparation. According to another classification sperm abnormalities may bedivided into major defects, negatively correlated with fertility, and minor defects,unassociated with fertility (Table 6) (Oettle, 1993).HeadNeckMidpieceTailPrimary spermatozoa defectsMacrocephalus, microcephalus,double, pointed, indented headsThickened, eccentric insertionThickened, thinned, coiled,kinked, double midpieceThin, double, triple tail.Secondary spermatozoa defectsFree, bent heads, swollen acrosomes,detaching acrosomesBent midpiece, extraneous materialsurrounding midpiece, proximal, midand distal cytoplasmatic dropletsCoiled, looped, kinked, folded,detached tail.Table 6. Main defects of the dog spermatozoaThe acrosomal status, which is frequently assessed for the estimation of the quality of thefrozen-thawed semen, may be evaluated with the use of eosin-nigrosin, Giemsa, Trypanblue, Bismarck brown, Rose Bengal or Spermac® stainings (Dahlbom et al. ,1997; Dott &Foster, 1972; Watson, 1975). When a spermatozoon presents more than one abnormality, itshould be classified according to the most important abnormality or with the most prevalentone, if they have equal significance (Oettle, 1993).‘Live-dead’ spermatozoa. The assessment of the percentage of live and dead spermatozoa isbased on the assumption that dead spermatozoa possess disintegrated plasma membraneallowing eosin penetration. Thus the percentage of eosin positive cells stained with nigrosineosinstain is considered as percentage of dead cells. The normal dog semen consists ofmaximal percentage of 30% of dead sperm cells. The evaluation of the percentage of live anddead spermatozoa and the percentage of morphological defects may be performed on thesame nigrosin-eosin stained slides.4.2.2 Advanced semen assessmentIn the past 2 to 3 decades, several strategies were developed to escape the subjectivity in thesemen evaluation, related to the experience and skills of the observer, the method ofspecimen preparation, staining technique and number of cells evaluated, and wich isparticularly important when the fertility potential of preserved sperm cells has to beascertain. It is well documented that variations in results of the conventional evaluation ofthe same semen samples obtained by different observers and laboratories may reach 30-60%(Coetzee et al., 1999; Davis & Katz, 1992). Moreover, implementation of such methodologies,not routinely usable in the small to median veterinary clinics due to their costs, allowsaccurate comparisons between laboratories worlwide and minimizes occurence of largeerrors. Furthermore, advanced semen assessment is essential whenever the semen has to bepreserved, in particular for freezing. Advanced semen assessment techniques are sumarizedon table 7. In general, the results obtained with these methods are better correlated with theAI outcome than the results of traditional semen evaluation.

60Artificial Insemination in Farm AnimalsTests Aims Procedure and Analysis ReferencesHypo-osmoticswelling test.Spz membraneintegrity(indirectmethod)Computer Objectiveassisted sperm evaluation ofanalysis sperm cell[CASA] motilityZonapellucidabinding assayFluorescentprobes andflowcytometryAssessment ofspermfertilizingpotentialMembraneintegrity andevaluation oflive and deadcellsCapacitationstatusSperm incubation with hypo-osmoticsolutions for 30 minutes at 37ºCSpz with intact plasmalemma becomeswolled and show coiled tailsDetermination of motility parametersfor individual spzCharacterization of Spz movementaccording to the average velocity, thetrajectory, the amplitude of movementand beat cross frequency. It allowsidentification of Spz subpopulationsEngland &Plummer, 1993;Kumi-Diaka, 1993Verstegen et al.,2001;Rijsselaere et al.,2003;Niżański et al.,2009- ZP-binding assay ( ZBA) using intact Hermansson ethomologous oocytesal., 2006;- hemizona binding assay (HZA) using Kawakami et al.,bisected hemizonae1998;The number of spermatozoa bound to Rijsselaere et al.,ZP is counted with contrast-phase 2005;microscopy. The number of bound Spz Ström-Holst et al.,reflects its fertilizing potential 2001Combined use of several fluorescentdyes (i.e, propidium iodide PI andcarboxyfluorescein diacetate, SYBR-14/PI) allow the identification of livecellsLive cells activate fluorescence(deacylation) which is maintainedintracellularly in intact membranecells. Dead Spz are stained red due tothe influx of PI through damagedplasma membrane.Fluorescent antibioticchlorotetracycline (CTC), when boundto free calcium ions, is fluorescent.Combined with Hoechst 33258 allowsalso assessment of percentage of livecells and capacitation statusThree classes of sperm cells may beassessed: uncapacitated and acrosomeintact (F-pattern), capacitated andacrosome intact (B-pattern) andcapacitated and acrosome reacted (ARpattern)Hewitt &England, 1998;Peña et al., 1998;Rijsselaere et al,2005;P.F. Silva &Gadella, 2006Guérin et al.,1999;Hewitt &England, 1998;Petrunkina et al.,2004;Rota et al. 1999b

Artificial Insemination in Dogs 61Tests Aims Procedure and Analysis ReferencesAcrosomalstatusMitochondriaIntegrity ofDNA structureLectins conjugated with fluoresceinisothiocyanate, such as PeanutAgglutinin (FITC-PNA) or PisumSativum Agglutinin (FITC-PSA). PNAlabelling is specific for the outeracrosomal membrane whereas PSA islabelling acrosomal matrix.The absence of the fluorescence of theliving sperm indicates an intactacrosome, whereas the presence of thefluorescence is indicative for acrosomedisruption or acrosome reactionRhodamine 123 (R123) is apotentiometric membrane dye used forthe selective staining of functionalmitochondria.It fluoresces only when the protongradient over the inner mitochondrialmembrane (IMM) is built up- Sperm chromatin structure assay(SCSA) with acridine orange (AO).- Terminal deoxynucleotidyltransferase-mediated nick end labeling(TUNEL)The SCSA is a flow cytometric methodfor identification of changes in theDNA status. AO shows greenfluorescence when DNA is intact andred fluorescence when DNA isdenaturatedKawakami et al.,2002;Peña et al., 2001;Sirivaidyapong etal., 2000;P.F. Silva &Gadella, 2006Garner et al.,1997;Gravance et al.,2001Chohan et al.,2006;Bochenek et al.,2001;Garcia-Macis etal, 2006Table 7. Concise description of the available advanced methods for sperm qualityassessment.5. Success rates for artificial inseminationThe key-issues to obtain good results by using canine artificial insemination are: Proper timing of the insemination The use of adequate number of viable sperm cells per dose Good semen preparation and handling Adequate deposition of semen in the female reproductive tractOn next sections the major issues on timing the AI and available techniques of semendeposition on the bitch genital tract will be discussed.

62Artificial Insemination in Farm Animals5.1 Timing the moment for inseminationObtaining successful pregnancies and adequate number of offspring per litter depends uponthe correct timing for mating, as well as for insemination, particularly because bitches aremono-estrous, presenting usually one to two reproductive cycles per year. Althoughrelationship among behavioral, hormonal and physiological events for the average bitchexists, considerable individual variation are also currently found on what concerns theduration of the estrogenic and early luteal stages (proestrus and estrus) and of the anestrus(Concannon et al., 1977; Concannon, 2004). The bitch usually presents a relatively longfollicular phase and considerable variability exists in the onset of estrous behavior andacceptance of the male, making it difficult to determine occurrence of the LH surge andonset of ovulation in this species unless specific methods for timing the ovulation andestimating the fertile period are used (Linde-Forsberg, 1991). Furthermore, in this species,ovulation of immature oocytes (primary oocytes, before extrusion of the first polar body)determines the need for a maturation period in distal oviducts that may last for 96-108 hours(Concannon, 2004, 2010; Tsuitsui, 1989; Tsutsui et al., 2009); for most bitches, the secondaryoocytes present a life span of 24-48h (Tsuitsui, 1989). Those particularities in thereproductive physiology may explain why the major cause for infertility in the bitch is theinappropriate breeding management (Goodman, 2001; Linde-Forsberg, 1991). Consequently,careful planning of mating time by timing ovulation is a key step in canine artificialinsemination.5.2 Vaginal cytology and progesterone blood levelsDetermination of blood progesterone and the vaginal cell cornification on cytologicalspecimens are the most widely used techniques (Linde Forsberg, 2003), to which recentlyhas been added the vaginal endoscopy (that replaces the vaginoscopic exam) and theultrasonographic follow-up of the follicular development and ovulation (England &Concannon, 2002; Hewitt & England, 2000; Fontbonne & Malandain, 2006; Levy &Fontbonne, 2007). These evaluations should be performed in sequence and with 2-3 daysintervals for the majority of females (if the bitch has been reported to present short heatperiod, of about 6 to 9 days, is possible that daily evaluations may be needed).On the vaginal cytology, epithelial cells of the vagina change their form in response toestrogen impregnation, and passes from small round cells with a clearly visible cytoplasm innon-estrogenic stages, to larger, cornified, angular shaped-cells with small pyknotic nucleus,almost to the point of disappearing, under the influence of estrogens (Figure 1). Atbeginning of estrus, vaginal cytology presents its maximum cornification index (>70%). Bythat time, serial blood sampling for progesterone determination should start to detect theinitial progesterone rise (2-3 ng/mL) which correlates with LH surge, which in turn triggersovulation within 2 days. On the day of ovulation (day “0” of the cycle) progesteroneconcentrations may vary between 4 and 10 ng/mL. The sudden increase in the number ofround-shaped cells and of neutrophils reflects the onset of diestrus (Fontbonne &Malandain, 2006).Progesterone semi-quantitative immunoenzymatic assays are available for clinical routines,but although rapids, these test lack accuracy. They give progesterone concentrationaccording to a colorimetric scale for values corresponding to basal progesterone levels (0-1ng/mL), intermediate levels corresponding to the LH surge (around 1-2.5ng/mL) and theovulation periods (2.5 – 8ng/mL), and high progesterone levels (more than 8 or 10,

Artificial Insemination in Dogs 63depending on the kit). A recent study showed that, in dogs, semi-quantitative methods forprogesterone determination are less accurate than the quantitative methods, in particular atintermediate plasma progesterone concentrations (Moxon et al., 2010). According to thisstudy, the tested semi-quantitative assay estimated higher progesterone concentration thanRIA (radioimmunoassay), which could suggest that the fertilization period had commencedearlier than it was actually the case. In addition to those assays, quantitative radio orchemiluminescent assays can also be used, even if not always available in the house lab,since cross-reactivity exist to the molecule between different species, for example withhuman progesterone.Fig. 1. Schematic representation of the major morphological changes of the predominantepithelial cell in the vaginal cytology during the dog estrous cycle. On the bottom, images ofthe vaginal péri-estrus cytological preparations stained with Harris-Shorr.5.2.1 Ultrasound examinationAlthough ovarian ultrasound examination is a reliable and accurate method to determineovulation in most domestic females, in the bitch fat accumulation in ovarian bursa thatencloses the ovaries may difficult the value of the technique. In addition, several studiesdemonstrated that ultrasound images of the ovaries around ovulation are more difficult toanalyze, due to the fact that ovarian follicles do not differ much in the immediate pre- andpost-ovulatory period (England & Concannon, 2002), as not all dog follicles collapse atovulation (Yeager & Concannon, 1996) and also because non-ovulated follicles frequentlyremain in the ovary (Wallace et al. 1992).Consequently, follicular dynamics evaluation through ultrasonography (US) in dogs is stillexperimental and must follow a very precise protocol, which accuracy increase with the useof frequent examinations. In a recent study, Fontbonne (2008) reported that US was accurateenough to detect the occurrence of ovulation and obtain comparable numbers of ovarianstructures between US examinations and macroscopic visual count on the surface of the

64Artificial Insemination in Farm Animalsovaries after surgical removal, even if only one daily examination was performed. However,that author accepts that features of ovulation may be difficult to visualize in large breedsand in overweight animals. Pre-ovulatory follicles may present different aspects at US.Usually they appear as round to slightly triangular anechoic structures, sometimes slightlyflattened, giving a honeycomb aspect to the ovary (Figure 2). At ovulation, different degreesof follicular collapse can be found in the US images, and usually a clear change of theovarian echogenicity has been detected in a large number of bitches, giving the ovary amore homogeneous aspect (Fontbonne, 2008; Fontbonne & Malandain, 2006). Persistence ofnon-ovulatory follicular structures was perceived in US images after ovulation. Also, in theimmediate post-ovulation period, until 24 hours after US changes of the ovaries atovulation, hypoechoic structures were observed in most cases (Figure 2). These structureswere very similar to the pre-ovulatory follicles, although slightly smaller, and tending toincrease in echogenicity (from the border to the interior of the structure) with time(Fontbonne, 2008; Fontbonne & Malandain, 2006).Fig. 2. Ultrasonographic scans of canine ovaries before and after LH surge and ovulation. USare compared with images of longitudinal sections of canine ovaries of similar stages offollicle developement.5.2.2 Vaginal endoscopyIt is possible to use vaginal endoscopy to determine the fertile period although it does notallow accurate timing of ovulation. However, this method requires expensive equipment.Nevertheless, it may give a huge contribution to the vaginal evaluation and detection ofanatomical abnormalities that may impair proper reproductive performance.The fluctuation of estrogen and progesterone concentrations in the blood at consecutivestages of estrous cycle in the bitch results in specific morphologic changes of the vaginalmucosa. Analysis of these changes allows for exact assessment of the stage of the estrouscycle and for determination of the optimal insemination time (Goodman, 2001; Jeffcoate &Lindsay, 1989; Lindsay 1983). The observation of the cranial part of the vagina is performedfor this purpose. The deep introduction of the tip of endoscope into the narrow part of thevagina close to the cervix (dorsal median postcervical fold) or paracervix, is of lessdiagnostic value (Pineda et al., 1973).Vaginoscopic examination is performed using a rigid endoscope 3-4 mm in diameter, withdiagnostic sheath and a length of 30-33 cm or longer. The examination should be done onthe standing animal. Usually there is no need of administration of sedatives. The tip of theendosope is introduced at the beginning at angle of 45-60º cranially and dorsally. When thetip of the optics reaches the vagina it should be repositioned at horizontal axis.

Artificial Insemination in Dogs 65During proestrus the increase of the estrogen concentration results in the oedema of thevaginal mucosa. Vaginoscopy reveals rounded folds in the vagina. The mucosa of the foldsis turgid, pink in colour and with a smooth surface. Normally the bloody discharge is alsovisible in the vagina. Sometimes, periodic blood outflow from the cervix, through theparacervix may be observed. The lumen of the vagina is narrow, which can be appreciatedwhen the endoscope is advanced cranially. At the last days of proestrus and at beginning ofestrus, the decrease of estrogen concentration and increase of progesterone (P 4 ) level isnoted. It results in the collapse of vaginal folds. Formerly turgid and smooth, the mucosa,becomes wrinkled and shrunked. Vaginal folds become smaller. Maximal intensity ofshrinkage of vaginal mucosa is observed between 3 and 7-8 days of estrous cycle. This timethe loss of fluid from the tissue of vaginal mucosa and submucosa is great and the shape ofvaginal folds become angulated with sharp angles at the top of folds. As the result, thelumen of the vagina is wider in comparison to proestrus. During diestrus vaginal foldsbecome flat and round. The mucosa is red and small petechia may be visible at placestouched by the tip of the endoscope. This is due to the fact that epithelium of the vagina isthin and consists of only 2-3 cell layers in diestrus and anestrus. An opaque, thick mucus issometimes visible on the surface of epithelium (Figure 3).5.2.3 Proposed alternative methodsOther methods has been proposed to monitor the bitch oestrous cycle, such as serial readingof the electrical resistance of the vaginal mucus around the time of ovulation, using probesinserted into the vagina during the heat period (Fontbonne, 2008), or the crystallizationpatterns in anterior vaginal fluids (England & Allen, 1989) or in saliva (Pardo-Carmona etal., 2010), which have been found up not to present an acceptable reliabily in theidentification of the canine ovulation.Fig. 3. Vaginal endoscopy of the bitch. [From left to right] Aspect of the vaginal folds at earlyproestrus, proestrus, estrus and diestrus.5.3 The moment for inseminationIn the bitch, when timing the day of ovulation as accurately as possible is essential toguarantee adequate fertility in natural mating systems, it becomes even more important todetermine precisely when to inseminate bitches according to the sort of semen to be used(fresh, chilled or frozen semen), as usually semen longevity and sperm cells survivaldecreases with time. In addition, in frozen/thawed semen sperm cell capacitation is shorterdue to secondary effects of the frozen procedure.When fresh or chilled semen is used, insemination should be performed on the day ofovulation, and a second insemination must be schedule for 2 days later. On the contrary,when frozen/thawed semen is used, and considering the need of canine oocytes to maturein the oviducts, insemination should be performed 2 days after ovulation, and the second

66Artificial Insemination in Farm Animalsinsemination 48h later (Figure 4). However, scheduling for the artificial inseminations maybe slightly adjusted according to the experience of the operator, the place for semendeposition and the limitation on the number of inseminations. Consequently, regimes forcanine AI may vary with authors (Root Kustritz, 2003). Table 8 condenses the availableinformation on the AI schedules for fresh, chilled and frozen semen.Fig. 4. Graphic representation of the fertile period and the ideal moment for canine AIaccording to the type of semen.SemenDosisExpectedspzsurvivalInsemination scheduleExpectedfertilityFresh150-200x10 6spz/mL(extended)4-6 days- Every other day, when P 4rise above 4ng/mL, up to3 times.- Day 1 to 4 post-ovulation- P 4 levels between 8 and15ng/mL- 80-90%(either withtranscervicalor vaginaldeposition)Chilled150 - 200x10 6spz/mL(extended)24-72hrs- Breeding once or twice 2-4 days post ovulation(P 4 = 4 -10ng/mL).- Day 2 to 4 post-ovulation- P 4 levels between 8 and15ng/mL- 80-90%(either withtranscervicalor vaginaldeposition)Frozen50 - 300x10 6spz/mL(extended)12-24hrs.- Twice, at P 4 levels above8ng/mL and estrusvaginal cytology- Day 5 to 7 post-ovulation- P 4 levels between 18 and28 ng/mL- 45% if vaginaldeposition- 67 -84% iftranscervicalor intrauterineTable 8. Artificial insemination schedules for dogs, according to the type of semen used.

Artificial Insemination in Dogs 67If in the AI with fresh semen the success of the procedure is strongly related to the quality ofsemen used and the moment for AI (Table 9), when using chilled semen both the quality ofsemen and the site of semen deposition are important factors for success, whilst in the AIwith frozen semen, the intra-uterine semen deposition is critical (Table 10).PregnancyratesWhelpingratesLength ofpregnancyNumberper group2AI, 48h apartDay 9-13 after bloodyvaginal dischargeEosinophilicindex >80%Multiple AIEosinophilic index>80%2AI, day 3 and 5post LH surgeProgesteronemeasurementsn % n % n % n %29 55.8 a 33 66.0 ab 37 78.7 b 37 80.4 b27 51.9 a 31 62.0 a 36 76.6 b 36 78.3 b63.71.4 a 64.52.6 b 64.42.4 b 63.11.3 c52 50 47 46a,b,c – different superscript mean significant differences (P

68Artificial Insemination in Farm Animals(fresh, chilled or frozen) and sperm quality (total and progressive motility and spermspeed), besides some individual variations (Rijsselaere et al., 2004). However, despite theinfluence of the intrauterine vs. vaginal insemination on the success of the procedure, onceintrauterine insemination is achieve, the exact place of semen deposition is not of the mainimportance for the sperm distribution within the uterus (England et al., 2006; Rijsselaere etal., 2004). Consequently, no potential differences or advantages exist between the vaginalendoscopic approach and laparoscopy when the intra-uterine insemination is intended, asno differences were found in the deposition of the semen in the uterine body or the cranialtip of the uterine horns (Fukushima et al., 2010). Nevertheless, abdominal laparoscopy orsurgery is strongly discouraged on the basis of animal welfare issues, as non-healthy relatedinvading procedure that should be avoided.Based on the last two years inseminations performed at the clinic (with a global success rateof around 75%), using a comercial kit for progesterone determination, fresh semen andintra-vaginal deposition (2 AI, 48h apart), it was found that when inseminations wereperformed with progesterone levels above 8 ng/mL a higher success rate and a closer topredicted whelping dates were achieved (Table 11), comparing to inseminations at lowerlevels of progesterone (2.5-8 ng/mL). No diferences were found in the litter size betweenthese groups, which were very similar in age and parity of the bitches.SemenFresh 39Number ofanimalsChilled 421812Range ofage (mean)1-6 years(2.94 years)1-6 years(3.23 years)1-6 years(3.6 years)Progesteroneat AI (ng/ml)2.5 to 8ng/mLDays fromlast IA towhelpingSuccess rate(nº femaleswhelped/inseminated)Liter size(± SD)63 66.6 6.33± 2.84> 8 ng/mL 61 80.95 6.29 ± 2.26>8 ng/mL 61 73.8 6.1 ±2.51Table 11. Results for the AI procedures with fresh and chilled semen and 2 AI per animal,48h apart.Independently of the place for semen deposition, repeating the AI at 24-48 hrs intervalsresults in a significantly higher fertility: for fresh semen both the pregnancy rate and littersize present a significantly increase when multiple AI are performed (Linde-Forsberg &Forsberg, 1993), whilst for frozen semen the differences on the pregnancy rates are notsignificant, although litter size tended to increase with the number of inseminations (Linde-Forsberg, 2000, 2002a).5.4.1 Deep vaginal inseminationDeep vaginal insemination is probably the widestly used method for insemination with freshsemen when the technique is performed by the breeder or in small budget clinics. For vaginalAI a simple plastic catheter of proper length may be used, to which a plastic disposablesyringe containing the semen is attached. Or a commercial catheter in flexible latex tubepresenting an inflatable balloon at the tip, like the Osiris gun, may be used; when inflated, this

Artificial Insemination in Dogs 69kind of device has the advantage of increasing the probability for intrauterine transport of thesemen and of preventing semen backflow (Farstadt, 2010; Linde Forsberg, 2005a).Before AI procedures start, cleaning of the perineal area, in particular the peri-vulvar area, isneeded. As transabdominal palpation is usually used to guide or ascertain the vaginalcatheter position, the owner of the female should be instructed to bring the animal with anempty stomach, which facilitates the procedure (Linde Forsberg, 2005a).The bitch is placed in a standing position on an examination table or on the floor (accordingto the size of the female). To avoid catheterization of the urethra (the urethral opening in thebitch is located at the pelvic brim), particular attention should be paid not to unintentionallyintroduce into the urinary bladder. The insemination catheter is carefully introduced in thevagina of the bitch, first steeply upwards until the pelvic brim has been passed, and then ina horizontal angle, when it is carefully pushed further ahead (Farstad, 2010). In alternative,the vulva may be elevated to just below the anus (as the bitch does when stimulated by themale dog) (Linde Forsberg, 2005a). At this point, the position of the AI catheter must belearn by palpation, and orientated. If the catheter is in the urinary bladder, the cranial partof the vagina and the cervix may be palpable above the catheter and also the tip of thecatheter stands out more clearly, due to the thinner walls of the urinary bladder incomparison to those of the vagina (Linde Forsberg, 2005a). After certification that thecatheter is correctly placed, it is moved onward through the cranial portion of the vaginadelimited by the dorsal medial folds. In smaller or primiparous bitches this point can bedifficult to overcome, and may not be possible to pass the catheter into the cervical fornix.Except for those females, the AI catheter should be further introduced until it reaches theparacervical area, which can be palpated as a 1- to 2-cm-long, firm structure that ends at thecervix (a firm, rounded to ovoid structure, freely movable). The semen is deposited once thecatheter has been located in the paracervical area, close to the external cervical os.During AI the bitch is held with the hindquarters up and head down, in an angle of 45-60º.This position facilitates transabdominal palpation of the cervix and ensures that the semenwill not be expelled through backflow. According to earlier reports, the bitch should bemaintained in the same position up to period of time varying from 5 to 20 min after AI.However, reducing the interval of elevated hindquarters to 1 min seems not affect fertility(Pinto et al., 1998). Also, feathering or stroking of the vulvar or perineal region is reportedby several authors as form of stimulating the semen transport into the uterus, in an attemptto mimic the vaginal stimulation by the thrusting movements of the dog during naturalmount. However, the contribution of such procedures to the exit of the technique has notbeen proven yet.5.4.2 Intrauterine inseminationIntrauterine insemination may be performed by using non-surgical transcervicalcatheterisation (Linde-Forsberg, 1991; Linde-Forsberg and Forsberg, 1989, 1993; Linde-Forsberg et al., 1999) or by surgical semen deposition by laparotomy (Brittain et al., 1995;Günzel-Apel & Thiet, 1990) or laparoscopy (L.D.M Silva et al., 1995, 1996). The majority ofEuropean centers working on small animal reproduction prefer transcervical intrauterineinsemination (TCI) due to reasons associated with animal welfare. However, catheterisationof uterine cervix in the bitch is a difficult procedure and demand skill and experience. Thesemen of lower quality, such as frozen-thawed or that collected from subfertile dogs have tobe deposited intrauterine to assure satisfactory results of artificial insemination (Linde-

70Artificial Insemination in Farm AnimalsForsberg et al., 1999; Thomassen et al., 2006). The conception rates after intravaginalinsemination with frozen-thawed semen are significantly lower when compared with theresults of intrauterine insemination.5.4.2.1 The Norwegian or Scandinavian techniqueThe method of non-surgical transcervical intrauterine insemination was first time describedin 1975 (Andersen, 1975). The technique has been adapted from the artificial inseminationperformed in foxes. Two catheters are used in this method - the outer plastic catheter andinner metal thin catheter. There are 3 sizes of the catheters, for small, medium and largebreeds. The catheterisation should be made on standing animal. Sometimes there is no needfor administration of sedatives, but usually a small dose of alpha-mimetic, such asmedetomidine, is advisable for abdominal muscles relaxation. The outer plastic cathetershould be introduced into the vagina. It should be advanced as far as is possible. In manybitches, especially those of larger breeds, the tip of the catheter passes into the cranialnarrow part of vagina. However, in some smaller bitches the introduction of the outercatheter through the paracervix is difficult. It is necessary to palpate the end of the catheterand the cervix through the abdominal wall. The cervix is palpable at estrus as solid, ovoidstructure. It is advisable to move the tip of the catheter ventrally towards the ventral regionof abdominal wall. This procedure is helpful in palpation of the cervix. The inner metalcatheter should be introduced through the plastic catheter. The cervix should be fixedbetween the thumb and other fingers and tilted to horizontal axis. The metal catheter isintroduced into the cervical canal under the control of the position of the cervix by palpationthrough the abdominal wall (Andersen, 1975; Linde-Forsberg, 1991). This techniquedemands skill and experience. It is harder to perform uterine catheterisation in obese ornervous bitches and in giant breeds.The scandinavian method of uterine catheterization is recommended for routineinsemination of bitches (Ferguson et al., 1989; Günzel-Apel 1994; Linde-Forsberg, 1991,1995). The use of this technique of insemination is especially advisable in cases when usingof semen of lower quality due to male subfertility or sperm cryopreservation. Linde-Forsberg and Forsberg (1989) obtained 83.9% and 69.3% of pregnant bitches (data correctedfor the stage of estrus) after insemination with fresh and frozen-thawed semen, respectively.The litter sizes were lower by 23.3%, when frozen-thawed semen was used in comparison tofresh semen. Rota et al. (1999a) reported 25% higher pregnancy rate after intrauterine semendeposition when using scandinavian technique than after vaginal semen deposition. On thebasis of analysis of 327 inseminations Linde-Forsberg et al. (1999) concluded that successrate of scandinavian method and vaginal insemination with frozen-thawed semen was84.4% and 58.9, respectively. Niżański (2006) proved that results of vaginal inseminationwith frozen-thawed semen are significantly lower in comparison with fresh semen, in spiteof the use of modification of the technique of vaginal semen deposition, plasma additionand adjustment of the number of spermatozoa.5.4.2.2 Endoscope-assisted vaginoscopic method (New Zealand method)Intrauterine insemination of the bitch under the visual control of endoscopic equipment wasfirst time described by Wilson (Wilson, 1993, 2001), using a rigid endoscope – cystouretroscopeof the length 29 cm with diagnostic external sheath. The procedure is performedon the standing animal. Uterine catheterisation is made with the use of flexible catheterintroduced into the working channel of the endoscope. The endoscope is introduced into the

Artificial Insemination in Dogs 71cranial narrow part of the vagina, while a flexible catheter is introduced cranio-dorsal intothe external orifice of the cervical canal under the visual control performed through theendoscope. Usually it is not necessary to administer any sedatives.The results for the intrauterine deposition of frozen-thawed semen when using thistechnique are quite satisfactory (Table 12). Wilson (1993), with the use of frozen semen,refers a pregnancy rate and litter size 83.3% and 7.5 puppies per litter, respectively. Niżański(2005) obtained whelping rates of 68.7% and 27.8%, when frozen-thawed semen wasdeposited by intrauterine vaginoscopic method and by vaginal insemination, respectively.Results obtained by Linde-Forsberg et al. (1999) were poorer in comparison with theScandinavian method. However, vaginoscopic intrauterine insemination is currentlyconsidered as the practical, modern and useful tool in assisted reproductive techniques indogs which may become in the future the routine method of insemination.InseminationtechniqueIntravaginal,using aninfusion pipeteIntrauterine,using theendoscopen1816Whelping rate(nº femaleswhelped/inseminated)27.8 a(5/18)68.7 b(11/16)Litter sizeat birth(range)3.0 ±1.2 a(2-5)4.9 ±1.7 b(3-8)Litter sizeat weaning(range)2.6 ±0.9 a(2-4)4.6 ±1.7 b(2-8)Different superscripts in the same column indicate significant difference (p

72Artificial Insemination in Farm Animalsthe skill of the operator and on the anatomical features of the vagina, and varies usuallybetween 0.5-3 minutes, but it may be longer. In nervous bitches the administration of smalldoses of sedatives, such medetomidine, is advisable, although in not too high doses, as thecatheterisation should be done on standing bitch.Currently the vaginoscopic method of intrauterine insemination appears to be advantageousand useful technique of semen deposition in the uterine lumen in bitches. The techniquedemands skill but it is practical and quick to perform for experienced operators. The visualcontrol of introduction of the catheter into the uterus is the important advantage of thetechnique. The observation of the moments of semen deposition and control if there is nosemen backflow is therefore possible. Moreover, it allows also uterine sampling when afemale is suspected of infertility due to uterine disease (Thomassen & Farstad, 2009). Forthese reasons this method of intrauterine insemination is becoming more popular.5.4.3 Surgical techniqueSurgical insemination technique have been proposed once for frozen semen or when thebitch presents an anatomical obstruction that prevents the insertion of the catheter orendoscope. Both the laparoscopic approach and the laparotomy requires anaesthesia andgood surgical skills. The semen is introduced into the uterus by puncture of uterine wall orincision with a scapel and passage of a tom cat catheter (Farstad, 2010; Thomassen &Farstad, 2009). However, in such methods, semen deposition is performed only once.Some restrictions may exists to application of this method in different countries, that maycompromise the registration of litters obtained without fulfilment of the legal requirements,such as previous evaluation of the situation or previous authorization of the local KennelClub for the procedure. Furthermore, some ethical constraints have been raised regardingthe use of surgical techniques for AI in dogs. Surgery is an invasive procedure, so it isunlikely to carry it out in the best interest of the animal, and the possibility of transmissionof an undesirable trait in a particular animal genetic line should be kept in mind.6. Rules and regulations concerning the import or export of spermBefore implementing canine artificial insemination the owner and the clinician should beaware of the national or international regulations on semen import, if applicable, and of thelocal Kennel Club requirements respecting the use of canine AI and litter registration. Inaddition, procedures may differ between the use of national or imported semen.Consequently, attention should be paid well in advance to this matter.In the absence of a specific national regulation, most Kennel Clubs follows FCI (FédérationCynologique Internacionale) determinations for AI, transposed to the FCI InternationalRegulation for Breeding (http://www.fci.be/circulaires/102-2010-annex-fr.pdf). To ensurethat ethical issues are minimised, FCI recommends that AI should only be done in healthydogs with proven fertility (article number 13). In addition, in the introductory section of thisregulation, FCI specifically limit the use of dogs presenting diseases possible to betransmitted to following generations and those presenting major, eliminatory defects inregard to the breed standard. Furthermore, it presumes that the AI is performed by aVeterinarian, which should certify the quality of the dog semen (either for the fresh and theprocessed semen, the later being certified in a standard document to be released uponsemen collection and preservation) and also to attest the Kennel club to the occurrence of anAI for a specific female. In both documents, correct identification of the animals (either the

Artificial Insemination in Dogs 73male or female) is mandatory, and can be obtained through the use of a tattoo or amicrochip. The time to submit the AI certificate may differ between national Kennel Clubs.In some countries the Club must be informed of the AI procedure within the first 2 weeks,whilst in others, only when the litter is to be registered.Besides regulations on performing AI to a bitch, attention must be paid to issues concerningthe semen collection and use. Besides the use of a recognisable male, with a certifiedpedigree, particular requirements may exist from national Kennel Clubs or the OfficialAgriculture entities, which may vary for chilled and frozen semen. In some situations thepermit to import dog semen is required, which may or may not need to be accompanied bya DNA sample, and a health certificate that may include blood testing against the mostimportant infectious or congenital canine diseases. Awareness of the latest officialrequirements is essential when considering semen international shipment. Additionalinformation on the shipment regulations may be obtained throught the references LindeForsberg, 2001, 2005b.7. ConclusionDemands for canine artificial insemination is growing worldwile together with an increaserequest for semen preservation in sperm banks. Furthermore, a tendency exists to increasethe demands for the use of frozen/thawed semen over fresh semen AI, as part of breedingtools for genetic improvement. Nowadays is possible to achieve adequate whelping ratesand litter sizes regardless of the type of semen used, as long as proper timing of AI andproper semen deposition are used. Client education and technical councelling mustcomplete the AI services to be offered by specialized practicioners, in particular whenbreeding a problematic bitch.8. AcknowledgmentsThe authors are greatly indebted to Dr Malgorzata Ochota for her valuable cooperationand thoughtful comments during language revision of this manuscript.9. ReferencesAmann R.P. (1986) Reproductive physiology and endocrinology of the dog, In Currenttherapy in theriogenology. 2 nd edition, Morrow D.A. (ed), 523-538, W.B. SaundersComp., ISBN 978-0721665801, Philadelphia.Andersen K. (1975) Insemination with frozen dog semen based on a new inseminationtechnique. Zuchthygiene, 10, 1-4.Bochenek M., Smorąg Z., & Pilch J. (2001) Sperm chromatin structure assay of bulls qualifiedfor artificial insemination. Theriogenology, 56, 557-567.Brittain D., Concannon P.W., Flanders J.A., Flahive W.J., Lewis B.L., Meyers-Wallen V., &Moise N.S. (1995) Use of surgical intrauterine insemination to manage infertility ina colony of research German Shepherd dogs. Lab. Anim. Sci., 45, 404-407.Chohan K.R., Griffin J.T., Lafromboise M., De Jonge C.J. & Carrell D.T. (2006) Comparison ofchromatin assays for DNA fragmentation evaluation in human sperm. J. Androl., 27,53-59.Christiansen I.J. (1984) Reproduction in the Dog & Cat. Bailliere Tindall, ISBN 978-0702009181,London.

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78Artificial Insemination in Farm AnimalsRota A., Iguer-Ouada M., Verstegen J. & Linde-Forberg C. (1999a) Fertility after vaginal oruterine deposition of dog semen frozen in a TRIS extender with of without EquexSTM Paste. Theriogenology 51, 1045-1058.Rota A., Peña A.I., Linde-Forsberg C. & Rodriguez-Martinez H. (1999b) In vitro capacitationof fresh, chilled and frozen-thawed dog spermatozoa assessed by thechloretetracycline assay and changes in motility patterns. Anim Reprod Sci. 57, 199-215.Silva L.D.M., Onclin K., Snaps F. & Verstegen J. (1995) Laparoscopic intrauterineinsemination in the bitch. Theriogenology 43, 615-623.Silva L.D.M., Onclin K., Lejeune B. & Verstegen J.P. (1996) Comparisons of intravaginal andintrauterine insemination of bitches with fresh or frozen semen. Vet. Rec. 17, 154-157.Silva P.F. & Gadella B.M. (2006) Detection of damage in mammalian sperm cells.Theriogenology 65, 958-978.Sirivaidyapong S., Cheng F.P., Marks A., Voorhout W.F., Bevers M.M. & Colenbrander B.(2000) Effect of sperm diluents on the acrosome reaction in canine sperm.Theriogenology 53, 789-802.Ström-Holst B., Larsson B., Rodriguez-Martinez H., Lagerstedt A.-S. & Linde-Forsberg C.(2001). Zona pellucida binding assay- a method for evaluation of caninespermatozoa. J. Reprod. Fertil. Suppl 57: 137-140Thomassen R. & Farstad W. (2009) Artificial insemination in canids: a useful tool in breedingand conservation. Theriogenology, 71, 190-199.Thomassen R., Sanson G., Krogenæs A., Fougner J.A., Andersen Berg K. & Farstad W. (2006)Artificial insemination with frozen semen in dogs: A retrospective study of 10 yearsusing a non-surgical approach. Theriogenology 66, 1645-1650.Tsutsui T. (1989) Gamete physiology and timing of ovulation and fertilization in dogs. JReprod Fertil Suppl. 39, 269-75.Tsutsui T., Takahashi F., Hori T., Kawakami E. & Concannon P.W. (2009) Prolongedduration of fertility of dog ova. Reprod Domest Anim. 44 Suppl 2, 230-3.Verstegen J., Iguer-Ouada M. & Onclin K. (2001) Computer assisted semen analyzers inandrology research and veterinary practice. Theriogenology 57, 149-179.Wallace S.S., Mahaffey M.B., Miller D.M. Thompson F.N. & Chakraborty P.K. (1992)Ultrasonographic appearance of the ovaries of dogs during the follicular and lutealphases of the estrous cycle. Am J Vet Res 53(2): 209-215.Watson P.F. (1975) Use of Giemsa stain to detect changes in acrosomes of frozen ramspermatozoa. Vet. Rec. 97, 12-15.Wilson M.S. (1993) Non-surgical intrauterine artificial insemination in bitches using frozensemen. J. Reprod. Fert. Suppl. 47, 307-311.Wilson M.S. (2001) Transcervical insemination techniques in the bitch. Vet. Clin. North. Am.(Small Anim. Pract.) 31, 291-304.Yeager A.E. & Concannon P.W. (1996) Ovaries. In: Small animal ultrasound, Green RW (Ed.),Philadelphia: Lippincott Raven, 293-303.

5Artificial Insemination in PigsMaes Dominiek, López Rodríguez Alfonso, Rijsselaere Tom,Vyt Philip and Van Soom AnnGhent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 MerelbekeBelgium1. IntroductionArtificial insemination (AI) of swine is widely practiced in countries with intensive pigproduction. In Western Europe, more than 90% of the sows have been bred by AI for morethan two decades (Gerrits et al., 2005; Vyt, 2007). When compared with natural mating, AI isa very useful tool to introduce superior genes into sow herds, with a minimal risk of disease(Maes et al., 2008). The outcome of AI largely depends on the semen quality and theinsemination procedure. In practice, fresh diluted semen for intracervical insemination ismostly used in pigs. Semen is obtained from boars on farms or from specialised AI-centres.The latter offer a diversity of breeds and genetic lines and distribute ready-to-use semendoses of constant quality to different sow herds.Three important aspects should be considered. Firstly, only semen from healthy boarsshould be used, as diseased boars may ejaculate semen that is contaminated with pathogens.The semen from commercial AI-centres is shipped to a large number of sow farms.Contaminated semen could therefore lead to a rapid transmission of pathogens and todisease outbreaks in many different sow herds. Strict regulations and guidelines to preventdisease spreading are therefore implemented on porcine AI-centres. The second importantaspect is the fertilizing capacity of the produced semen doses. The fertilizing potential of asemen dose is inherently linked to the quality of the spermatozoa itself (Tsakmakidis et al.,2010). Examination of the ejaculates is therefore necessary. A third important aspect of AIcentresis the semen processing procedure (Waberski et al., 2008). This is not only importantto guarantee a low microbial presence but even more to obtain high quality sperm, namelyviable spermatozoa in ready-to-use semen doses that can be used for several days. Thedilution procedure and semen handling, the properties of the extender and the microenvironmentfor the sperm cells influence the survival and longevity of the spermatozoa.The present chapter will review and critically discuss the different steps during the entire AIprocedure in pigs, starting from the semen collection, dilution and processing, methods andtechnologies used to assess the semen quality, the storage conditions and the characteristicsof the semen extenders that are required to maintain semen quality. A last part will focus onthe different AI strategies.2. Collection of semen, dilution and processingAlthough automated semen collection systems have been developed (Barrabes Aneas et al.,2008), semen is mostly collected by the gloved hand technique from a boar trained to mount

80Artificial Insemination in Farm Animalsa dummy sow. Dummy sows should be solid in construction without sharp edges, andlocated in a quiet designated semen collection room with a non-slippery floor. A prewarmed(38°C) collection container is used. The top of the container is covered withcheesecloth to filter out gel portion of the semen. The end of the penis is grabbed firmly witha gloved hand and the collection process is initiated with firm pressure to the spiral end ofpenis with the hand so that the penis cannot rotate. This process imitates the pressureapplied by the corkscrew shape of the sow’s vagina. Polyvinyl gloves can be used, not latexgloves as these are toxic for the semen (Ko et al., 1989). The first part of the ejaculate (presperm)should be discarded. It is clear, watery fluid and does not contain sperm (~25 ml),but it may have a high bacterial count. The sperm-rich fraction should be collected (40-100ml). It is very chalky in appearance and contains 80-90% of all sperm cells in the ejaculate.Once the sperm-rich fraction is complete, the remainder of the ejaculate is again more clear,watery fluid, and should not be collected (70-300 ml). After collection, the filter with gelshould be discarded, and the collection container should be placed in warm water. Thesemen should be extended within 15 min. after collection. The ejaculation lasts up to 5 to 8min, but may continue up to 15 min. About 100 to 300 ml of semen is collected. Semencollection from boars in AI-centres is performed approximately 2 times per week (Vyt et al.,2007).The extension process should be done in a warm room with clean and sterile equipment.The extender is added to the semen, and cold shock should be avoided by diminishing thetemperature gradually. A normal ejaculate usually contains enough sperm to inseminate 15to 25 sows using conventional AI. Each dose should contain 2-3 billion spermatozoa in 80-100 ml.3. Semen quality assessment3.1 Assessment of the concentration of spermatozoa in the ejaculateThe number of spermatozoa in a semen dose is important for the fertilization process. Onthe other hand, AI-centres tend to dilute the ejaculates as much as possible to maximizesemen dose production. Variation in the number of spermatozoa in an ejaculate has beendescribed between different pig breeds e.g. Landrace, Duroc and Yorkshire (Kommisrud etal., 2002), which is a first factor influencing semen dose production. Not only differences insperm number but also in sperm volume, ranging from 100 to 300 ml (Kondracki, 2003),influence sperm concentration. Individual variation within a breed is also very important(Johnson et al., 2000). Xu et al. (1998) demonstrated a difference in litter size of 0.09 to 1.88piglets when inseminating sows either with 2*10 9 or 3*10 9 spermatozoa. Differences in littersize between both semen doses were largely dependent on individual variations betweenboars. Another study (Alm et al., 2006) using 2*10 9 spermatozoa per dose mentioned notonly a smaller litter size but also a lower farrowing rate at lower semen dose. In addition,several studies (Alm et al., 2006; Xu al., 1998) described lower fertility results when lowersemen doses were used in boars with suboptimal semen quality. A general guideline for thenumber of good quality spermatozoa (Table 1) in a semen dose was set at 3*10 9 spermatozoaper dose. According to the morphological or motility characteristics, the number ofspermatozoa should be adapted (Martin-Rillo et al., 1996). By multiplying the total volumeof the gel-free ejaculate (ml) times the sperm concentration per ml, the total sperm number iscalculated. The volume is routinely measured by weighing the ejaculate considering 1 gramequal to 1 ml and the obtained total sperm numbers is a good indicator to evaluate

Artificial Insemination in Pigs 81spermatogenesis (Amann, 2009).The data above clearly indicate the importance of anaccurate determination of the concentration of spermatozoa in the ejaculate.3.1.1 Inspection of the raw ejaculateVisual evaluation of the opacity of the ejaculate gives a rough idea on the spermconcentration. However, this method is crude and very subjective and therefore not suitablefor AI-centres with large semen production.3.1.2 Counting chambersDifferent glass chambers are described to count cells in a known volume. Haemocytometers,such as the Neubauer, Thoma and Bürker chamber are reusable glass chambers with fixedvolume used for counting immobilized spermatozoa in a grit. Other reusable glass chambersas the Mackler chamber are used for assessing concentration as well as motility (Tomlinsonet al., 2001). Disposable chambers (Microcell R , Leja R ) are commonly used in ComputerAssisted Semen Analysis (CASA) since their small depth limits movement in the thirddimension (Z-axis) when the sperm path is analysed (Verstegen et al., 2002).Haemocytometers are considered as the standard method for determining spermconcentration and have a lower coefficient of variation than disposable chambers(Christensen et al., 2005; Tomlinson et al., 2001). The concentration determined by thehaemotocytometer however, was generally higher than the concentration determined usingother chambers, especially with increasing concentration. Makler chambers were describedas having higher standard deviations and more inconsistent results compared with thehaemocytometer (Christensen et al., 2005; Tomlinson et al., 2001). Disposable chambers onthe other hand, although they are also used for counting live cells, were reported to be moreconsistent and accurate (Mahmoud et al., 1997). The accuracy of different counting chambersis also dependent on the manner in which the chamber is filled. Thin, capillary-filled,disposable chambers are generally found to underestimate sperm concentration due to theSegre-Silberberg effect (Kuster, 2005). However, the variations between chambers whenanalysing sperm concentration seems to be technician and laboratory dependent(Christensen et al., 2005).3.1.3 PhotometryPhotometers (single wavelength) or spectrophotometers (multiple wavelengths) measurethe optical density, i.e. the relative absorption and scattering of a light beam that is sentthrough a semen sample. The absorption and scattering is proportional to the spermconcentration. Next to the concentration of spermatozoa, the absorbance is also influencedby gel particles in the seminal plasma or the extender, by the quality of the sample cuvette,and the dilution of the sample (Knox, 2004). Photometry is commonly used in practicebecause it is fast and easy to perform (Woelders, 1991). Accurate dilution and a correctcalibration curve are imperative to obtain proper results.3.1.4 Flow cytometrySeveral studies use fluorescent dyes that stain intact or damaged spermatozoa differently,and measure the distribution of dyes in the sperm cell population by a flow cytometer(Christensen et al., 2004; Ericsson et al., 1993). In that way, viability as determined by thepercentage of intact cells as well as the concentration of spermatozoa in an ejaculate can be

82Artificial Insemination in Farm Animalsdetermined by using fluorescent microspheres. Since this technology can discriminateinterference from gel particles, it has a low coefficient of variation (3.3%) (Christensen et al.,2004). However, the high costs and the dependence on qualified personnel make flowcytometry not the most suitable method for use in practice.3.1.5 Other methodsCoulter counters, determining the number of particles within a known volume, can be usedto assess spermatozoa concentration but discrimination of other particles with comparablesize within the sample is difficult, resulting in a lower accuracy (Woelders, 1991). Othersystems, e.g. computer assisted semen analysis (CASA) use image analysis to determinesperm concentration within counting chambers (Prathalingam et al., 2006; Verstegen et al.,2002). The accuracy of these systems depends not only on the optical properties and thesoftware settings, but also on the kind of counting chamber that is used (Kuster, 2005).Nucleocounters are also used for determining sperm concentration, and they providesimilar counts as those obtained with photometers (Camus et al., 2011; Hànsen andHedeboe, 2003). In these devices, DNA is fluorescently labelled and counted by imageanalysis resulting in an accurate determination of sperm concentration.The concern to obtain a correct estimate of sperm concentration led to a discussion on theaccuracy of the different systems. Maes et al. (2010) did not find major differences betweentwo types of colorimeters, the Bürker counting chamber, and the Hamilton Thorne Analyzer(Ceros 12.1) using two Leja chambers. Every system has its advantages and limitations. Theyconcluded that in commercial porcine AI-centres, economic considerations such as purchaseprices, labour, and high sample throughput are also important in the choice for one methodor the other.3.2 Morphology and viability assessmentThe microscopic appearance of spermatozoa can give information on morphologicalabnormalities, cell membrane integrity and the acrosome. These are three importantparameters that contribute to the fertilizing capacity of the sperm cells. Morphologicalabnormalities give an indication of aberrations in the spermatogenesis. Some malformationscompromise the function of the cells and cannot be compensated for, therefore leading toculling of the boar. Abnormal shape of the head which carries the genetic material orabnormalities of the mitochondrial sheet which is important for the function of the flagella,are therefore called primary abnormalities. Remainders of cytoplasm, proximal or distaldroplets, and small tail abnormalities are called secondary abnormalities and can becompensated for by the semen dose (Donadeu, 2004). Additionally, morphologicalanomalies (e.g. coiled tails) acquired by inappropriate handling of semen are called tertiaryabnormalities.Morphology can be assessed by staining techniques that do not require highly qualifiedpersonnel (Shipley, 1999). Normal morphology is correlated with fertility (Alm et al., 2006; Xu.et al., 1998), and should therefore be performed routinely in porcine AI-centres. Criteria for themaximum percentage of primary and secondary abnormalities in commercial porcine AIcentreswere determined as 10% and 20%, respectively (Shipley, 1999). The percentage ofspermatozoa with normal morphology should be at least 70% (Shipley, 1999). An overview ofthe criteria for use of porcine semen in artificial insemination is shown in Table 1.

Artificial Insemination in Pigs 83Semen parameter Requirement (%)Kuster andAlthouse, 1999Martin-Rilloet al., 1996*Shipley,1999Britt etal., 1999Motility > 70 60-100 > 70 >60Abnormal morphology < 20 < 20Normal acrosomes

84Artificial Insemination in Farm AnimalsFig. 1. Sperm morphology: spermatozoa with normal morphology, abnormal (narrow) head(primary defect) and proximal droplet (secondary defect) (arrows)3.3 Motility assessmentMotility of spermatozoa has always been considered a primary requirement to fertilize eggs.Although the spermatozoa are brought to the fertilization site mainly by uterinecontractions (Langendijk et al., 2002), sperm motility is required for penetration of the zonapellucida. Motility is known to be an important characteristic in predicting the fertilizingpotential of an ejaculate (Gadea, 2005). Therefore, several methods have been used formotility assessment.3.3.1 Visual motility estimationThe simplest way to evaluate sperm motility is by estimating the number of motilespermatozoa under a light microscope or using phase contrast microscopy. This method issubjective since it depends on the interpretation by an individual (Vyt et al., 2004b). It ishowever a cheap method and facilitates a high sample throughput which makes it popularin commercial AI-centres.3.3.2 Computer assisted semen analysis (CASA)Using digital image analysis, sperm cell tracks are analysed in different components(Rijsselaere et al., 2003; Verstegen al., 2002; Vyt et al., 2004b). CASA has major advantages:the method is objective, independent of the interpretation of the technician and givesdetailed information on the sperm movement. This way, different motility patterns can beobserved, e.g. progressive movement versus hyperactivity and even differentsubpopulations of spermatozoa within an ejaculate can be demonstrated (Peña et al., 2005;Rijsselaere et al., 2005; Verstegen et al., 2002). The detailed information given by the CASAsystemsrenders them also very susceptible to external influences on sperm movement, suchas operator variability, semen handling and system settings are causes of inter-laboratorydifferences (Rijsselaere et al., 2003; Verstegen al., 2002). At the moment, CASA instrumentshave been validated for many animal species (Holt et al., 1994, 1996; Rijsselaere et al., 2003;Wilson-Leedy and Ingermann, 2007) which makes the method available for use inveterinary practice or commercial AI-centres. The high cost of the equipment compared tothe alternative visual motility determination, is a restraint to the use of CASA in practice.3.3.3 Sperm Quality analyzer (SQA)The SQA systems convert variations in optical density into electrical signals to determinesperm concentration and motility. These electronic signals are analyzed by the SQAsoftware algorithms and translated into sperm quality parameters. The effectiveness ofdifferent SQA systems for sperm analysis has been studied both in humans and animals,

Artificial Insemination in Pigs 85and different algorithms are needed for each species. A previous version of the SQA namelythe SQA-IIC was consistent and suitable for the estimation of boar semen quality. There wasa good correlation between the sperm motility index (SMI) obtained by SQA-IIC and severalCASA parameters, especially with the percentage of motile sperm and with straight linevelocity (VSL). However, the SQA-IIC is based on an old technology meant for humansperm analysis and the SMI values are based on overall information of the quality of thesperm, and do not discriminate between concentration, morphology and motilityparameters. Recently, the SQA-Vp was introduced as an SQA device specifically designedfor boars in which the sperm movement can be visualized on a screen and motility is givenas percentage of motile sperm (López et al., 2011).In pigs, a motility score of 60% motile cells, independent of the method of assessment, isrequired to be considered as a fertile ejaculate (Donadeu, 2004). Above 60% motilespermatozoa, no differences in farrowing rate and litter size were recorded (Donadeu, 2004).Apart from morphology, several attempts were made to correlate motility with fertilityoutcome. When using adequate numbers of spermatozoa per insemination dose (3*10 9 ),correlation with fertility outcome was hard to establish (Gadea al., 2004). At lower semendose, motility was well-correlated with fertility parameters. In most studies involving pigs,the predictive effect of motility was evaluated using visual motility assessment. To increasethe discriminating power of the motility estimation, objective motility assessment by CASAmeasurements(Holt et al., 1997; Vyt et al., 2008) or motility of spermatozoa subjected to apercoll gradient (Popwell and Flowers, 2004) were used. These studies found motility to bepositively correlated with fertility, especially with litter size.3.3.4 Other sperm examination techniquesSperm examination techniques requiring specialized knowledge and expensive equipmentare not frequently used in commercial AI-centres. They are however used for research onporcine sperm. DNA fragmentation tests can be used to identify subfertile boars, but thestudy results are contradictory (Waberski et al., 2011; Boe Hansen et al., 2008). Somemetabolic responses of sperm like resistance to oxidative stress (López et al., 2010) and invitro fertilisation assays are also used for research purposes. The practical relevance of thesetechniques is limited due to the fact that most of the research on porcine semen is based onthe semen from good performing boars (López et al., 2010). Subfertile or nonfertile boars arerapidly culled because of economic considerations, and therefore, there is a lack ofinformation regarding sperm quality of infertile boars.4. Storage of liquid semenFrozen storage of boar semen still yields inferior fertility due to the loss of membraneintegrity during freezing and thawing. Consequently, freshly diluted semen (liquid semen)is widely used for AI on the day of collection or in the following days. For storage of liquidboar semen, two factors are very important: the temperature of collection and storage, andthe composition of the storage medium (Johnson et al., 2000).4.1 Temperature of collection, transport and storageA different composition of the phospholipids in the membrane of boar spermatozoacompared to bull spermatozoa, a low cholesterol/phospholipid ratio and an asymmetrical

86Artificial Insemination in Farm Animalsdistribution of cholesterol within the membrane render boar spermatozoa very susceptibleto cold temperatures resulting in increased permeability and loss of controlled membraneprocesses (De Leeuw et al., 1990). Hence, rapid cooling of ejaculates to 15°C or coolingbelow 15°C results in loss of viability or cold shock (Johnson et al., 2000). To avoid this coldshock, prediluted ejaculates are better left at temperatures above 15°C for several hours toinduce cold resistance. In practice, semen is collected in isolated cans to avoid contact withcolder surfaces and subsequent dilution is done in a manner in which temperature isdiminished gradually. Two different protocols are normally used for this purpose: 1) Onestep dilution with either preheated diluter (~33°C) or diluter at room temperature or 2) atwo steps dilution with first a 1:1 dilution with preheated diluter (~33°C), followed by asecond dilution in either a preheated diluter or a diluter kept at room temperature(Waberski, 2009). After the final dilution, filling of commercial doses is done and the semenis allowed to cool down gradually to 17°C. When semen doses are to be transported, specialprecautions are taken to avoid temperature fluctuations (Green and Watson, 2002). Furtherstorage of diluted semen is done at 17°C, at which temperature semen metabolism isreduced (Althouse et al., 1998), a condition necessary to extend storage time.4.2 Storage mediumThe storage media for liquid boar semen aim to prolong sperm survival, to provide energyto the cells, to buffer the pH of the suspension and to avoid the growth of bacteria (Vyt et al.,2004a). Therefore, porcine semen extenders contain ions to maintain the osmotic pressure ofthe medium, glucose as energy source, buffering systems to stabilize the pH of the extenderand EDTA and antibiotics to prevent bacterial overgrowth (Johnson et al., 2000). Thepresence of glucose as the only energy source and the low oxygen content in the recipient inwhich diluted semen is stored stimulates the glycolytic metabolism. Consequently, theintracellular pH of spermatozoa is lowered which reduces their motility and enables them tosurvive several days at ambient temperature (Henning et al., 2009). Glucose also contributeslargely to the osmotic equilibrium. The ions in the media for liquid boar semen are merelysodium bicarbonate and sodium citrate and are simultaneously used as buffer. In BTSextender, also KCl is added to prevent the potassium loss from inside the cells, andsubsequent loss of motility due to Na-K pump inefficacy. Porcine spermatozoa are rathertolerant to minor changes in osmolality of the extender (Johnson et al., 2000). Iso-osmoticand slightly hyper-osmotic media are preferred for optimal preservation of fertilizingcapacity (Weitze, 1990). Incubation in media below 250 mOsm and above 300 mOsmrendered irreversible damage to the membranes and subsequent loss of motility.EDTA is added for its chelating properties. When Ca-ions are captured, the initiation ofcapacitation is inhibited (Watson, 1995). As a consequence, the fertilizing capacity of thespermatozoa is preserved. Depending on the composition of the extender, semen can bestored for 2 to 3 days in short-term extenders and up to five days or longer in long-termextenders (Johnson et al., 2000). Long-term extenders differ from short-term extendersmainly by the use of complex buffering systems (HEPES, Tris), mostly in addition to thebicarbonate buffering system, and by the presence of Bovine Serum Albumin (BSA). Thelatter has a positive influence on sperm survival due to the absorption of metabolic bacterialproducts from the extender. Cysteine is used as a membrane stabilizer (Johnson et al., 2000)inhibiting capacitation.To prevent bacterial proliferation during storage, antibiotics are added to the extender.Bacteria originate mostly from the prepuce, thus depending on the semen collection

Artificial Insemination in Pigs 87technique, from semen manipulation or from the water used in the extender preparation(Althouse and Lu, 2005). Depending on the species, bacteria have deleterious effects onsemen quality, namely depressed motility, cell death and agglutination (Althouse al., 2000),either by direct effect on the spermatozoa or by acidifying the environment. Europeanlegislation prescribes an antibiotic combination equivalent to 500 IU/ml penicillin, 500IU/ml streptomycin, 150 mg/ml lincomycin and 300 mg/ml spectinomycin, for having abroad antibacterial spectrum and activity towards leptospira. In practice most commercialextenders use aminoglycosides, especially gentamycin (Althouse and Lu, 2005; Vyt et al.,2007). However, bacterial contamination should be first minimized by good hygiene andgeneral sanitation by personnel (Althouse, 2008).The extender-concentrates are diluted in distilled or de-ionized water. Next to the bacterialquality of the water, the electrolyte content, especially the absence of calcium ions, is animportant characteristic for the water used to make the extender.The comparison of different semen extenders has been subject of two kinds of studies:studies comparing different extenders in vitro, focusing on quality of semen after storage(De Ambrogi et al., 2006; Vyt et al., 2004a) and studies comparing fertility in vivo afterinsemination of semen stored for several days or stored in different extenders (Haugan al.,2007; Kuster and Althouse, 1999).In vitro experiments showed no differences in cell viability between short-term and longtermextenders during 9-day storage (De Ambrogi et al., 2006). Motility remainedunchanged within the first 72 hours, even in BTS-extenders, the most widely used shorttermextender. Based on in vivo results, differences were noticed between different extendersbut it was not always possible to relate these differences to the type of extender. Differencesbetween long-term extenders were observed from day 4 of storage onwards (Kuster andAlthouse, 1999). The limited number of extenders compared in each study makes it difficultto set up a ranking of the semen preserving quality of long-term semen extenders.4.3 Storage of porcine semen in frozen stateAs mentioned above, porcine spermatozoa are particularly sensible to low temperatures andto rapid cooling due to the specific composition of the cell membrane (De Leeuw al., 1990).Cold shock can be solved technically by inducing cold resistance, namely incubating spermat ambient temperature for several hours (Watson, 1995), by contact with seminal plasmathat has a protective effect on spermatozoa (Centurion et al., 2006), together with controlledfreezing protocols. The variation in freezability of individual boar’s semen is however moredifficult to solve.Semen extenders for frozen boar semen are completely different from extenders for liquidsemen. The presence of egg-yolk, containing low density lipoproteins and cholesterol, has aprotective effect on sperm membrane during cooling (Bathgate et al., 2006). Cryoprotectants,especially glycerol are added in low concentration to the medium in order to diminishmembrane damage by freezing. Additionally, sugars and synthetic detergents are added,the latter having a modifying effect on the egg yolk inducing a better membrane stability ofthe cell membrane (Johnson et al., 2000). Thawing of the semen dose has been another pointof concern. Thawing has to be fast in order to maintain sperm motility and acrosomeintegrity afterwards. Both processes i.e. freezing and thawing result in plasma membranechanges, explaining the variety of protocols available.The fertility results with frozen semen have improved: cervical insemination results in a75% farrowing rate and a litter size of 9.6 (Roca et al., 2003). Nevertheless, freezing and

88Artificial Insemination in Farm Animalsthawing are time consuming processes, restricting the use of frozen semen for specificindications, such as long transport times and conservation of valuable genetic material.5. Insemination strategiesThe management of AI is very important to determine the success of the procedure and thereproductive performance of the sows. Estrus control, timing and number of inseminations,the technique of AI, semen storage on farm and the use of new AI technologies, all require aspecialized knowledge of pig reproductive physiology. The following measures could betaken to optimize the efficiency of AI on pig herds.5.1 Boar stimuliCorrect timing of insemination requires careful detection of oestrus at regular intervals. Boarstimuli are important in promoting follicular development and expression of oestrusbehaviour (Langendijk et al., 2006). Additionally, a high level of boar stimuli increases thefrequency of uterine contractions, indicating a supportive role for passive sperm transportthrough the long uterine horns at the time of insemination. This effect can only be partiallymimicked by a robot teaser boar which emits olfactory, acoustic and visual boar cues(Gerritsen al., 2005). Increase of oxytocin concentrations in peripheral blood plasma occursin immediate response to boar presence and lasts for approximately 10 min (Langendijk etal., 2003). Therefore, exposure of sows to a boar during both back pressure testing andinsemination is crucial.5.2 Timing of inseminationMany studies have investigated time-relationships between oestrus, ovulation, inseminationand fertilization using ultrasound testing. The key observation is that ovulation occurs at thebeginning of the last third of oestrus regardless of the overall duration of oestrus. Preciseprediction of the time of spontaneous ovulation in individual pigs has not yet beenachieved. However, prediction of oestrus duration by observing the onset of oestrus afterweaning has found broad acceptance in AI practice for calculation of the expected time ofovulation (Weitze et al., 1994). AI should be timed as close as possible to ovulation,preferably within 12 to 24 h before ovulation. The benefit of ultrasound testing of ovarianmorphology for pig fertility management has been shown in practice (de Jong et al., 2009).Determination of the time of ovulation in relation to oestrus behaviour and AI managementin representative numbers of sows on consecutive days has a great potential to provideshort cuts in AI timing and to develop farm-specific strategies for improvement of AImanagement.5.3 Use of new AI technologiesThe development of techniques to inseminate with low numbers of spermatozoa in a smallvolume has increased insemination efficiency. This is particularly interesting when usingspermatozoa of high value that are impaired, e.g. by freezing and thawing or sex-sorting.Post-cervical or intrauterine insemination with several devices has been developed totraverse the cervix and deposit sperm in the uterine body or posterior horn of multiparoussows. Compared to standard transcervical AI, post-cervical AI allows a threefold reductionin the numbers of spermatozoa to be inseminated, whereas deep intrauterine AI allows a 5

Artificial Insemination in Pigs 89to 20 fold reduction (Vazquez et al., 2008). The use of post-cervical insemination variesamong and within countries. Limits may arise from the use in sows only, skills needed forcatheter handling, and the possibility of damaging cervical or uterine tissue. Semenencapsulation in a barium alginate membrane has been demonstrated to allow a singleinsemination (Vigo et al., 2009). Laparoscopy offers the possibility of inseminating a verylow number of spermatozoa (i.e. 0.3 x 10 6 ) into the oviduct in anaesthetized pigs. However,the risk of polyspermic fertilization is substantial. Due to surgical intervention, its use is notappropriate in practice.6. ConclusionsAI of swine is widely practiced and is a very useful tool to introduce superior genes intosow herds, with minimal risk for disease transmission. In practice, fresh diluted semen (3billion spermatozoa in 80-100 ml) is mostly used for intracervical insemination .The successof AI is largely determined by the semen quality and the insemination procedure. Differentparameters and techniques can be used to assess semen quality. Although more advancedtechnologies offer more accurate information, in commercial AI centres, semen quality isassessed based predominantly on concentration, morphology and motility using simple,cheap and practically easy-to-perform techniques. Critical issues for AI involve oestrusdetection in the sow, timing of insemination and applying strict hygiene measures. Futuredevelopments will focus on new technologies to better assess semen quality in practice, topreserve semen for a longer time and to inseminate sows successfully using a lower numberof spermatozoa using new AI techniques.7. ReferencesAlm, K.; Peltoniemi, O.; Koskinen, E. & Andersson, M. (2006). Porcine field fertility with twodifferent insemination doses and the effect of sperm morphology. Reproduction inDomestic Animals, Vol.41, pp. 210-213, ISSN 0936-6768Althouse, G. (2008). Sanitary procedures for the production of extended semen. Reproductionin Domestic Animals, Vol.43, pp. 374-378, ISSN 0936-6768Althouse, G. & Hopkins, S. (1995). Assessment of boar sperm viability using a combinationof two fluorophores. Theriogenology, Vol.43, pp. 595-603, ISSN 0093-691XAlthouse, G.; Kuster, C.; Clark, S. & Weisiger, R. (2000). Field investigations of bacterialcontaminants and their effects on extended porcine semen. Theriogenology, Vol.53,pp. 1167-1176, ISSN 0093-691XAlthouse, G. & Lu, K. (2005). Bacteriospermia in extended porcine semen. Theriogenology,Vol.63, pp. 573-584, ISSN 0093-691XAlthouse, G.; Wilson, M.; Kuster, C. & Parsley, M. (1998). Characterisation of lowertemperature storage limitations of fresh-extended porcine semen. Theriogenology,Vol.50, pp. 535-543, ISSN 0093-691XAmann, R. (2009). Considerations in evaluating human spermatogenesis on the basis of totalsperm per ejaculate. Journal of Andrology, Vol.30, pp. 626-641, ISSN 0196-3635Barrabes Aneas, S.; Gary, B. & Bouvier, B. (2008). Collectis® automated boar collectiontechnology. Theriogenology, Vol.70, pp. 1368–1373, ISSN 0093-691XBathgate, R.; Maxwell, W. & Evans, G. (2006). Studies on the effect of supplementing boarsemen cryopreservation media with different avian egg yolk types on in vitro post-

90Artificial Insemination in Farm Animalsthaw sperm quality. Reproduction in Domestic Animals, Vol.41, pp. 68-73, ISSN 0936-6768Boe-Hansen, G., Christensen, P., Vibjerg, D., Nielsen, M. & Hedeboe, A. (2008). Spermchromatin structure integrity in liquid stored boar semen and its relationships withfield fertility. Theriogenology, Vol.69, pp. 728-736, ISSN 0093-691XBritt, J.H.; Almond, G.W. & Flowers, W.L. (1999). Diseases of the reproductive system. In:Straw B, D’Allaire S, Mengeling W, Taylor D (Editors), Diseases of Swine, 8thEdition, Blackwell Science Ltd, Oxford, pp. 903.Camus, A.; Camugli, S.; Lévêque, C.; Schmitt, E. & Staub, C. (2011). Is photometry anaccurate and reliable method to assess boar semen concentration?. Theriogenology,Vol.75, pp.577-583, ISSN 0093-691XCenturion, F.; Echeverria, S.; Canul, N.; Ake, R.; Alfaro, M.; Santos, R. & Sarmiento, L.(2006). Influence of seminal plasma added to highly diluted semen on spermmotility of young boar feed with selenium and vitamin E. Reproduction in DomesticAnimals, Vol.41, pp. 103, ISSN 0936-6768Christensen, P.; Stryhn, H. & Hansen, C. (2005). Discrepancies in the determination of spermconcentration using Bürker-Türk, Thoma and Makler counting chambers.Theriogenology, Vol.63, pp. 992–1003, ISSN 0093-691XChristensen, P.; Stenvang, J. & Godfrey, W. (2004). A flow cytometric method for rapiddetermination of sperm concentration and viability in mammalian and aviansemen. Journal of Andrology, Vol.25, pp. 255-264, ISSN 0196-3635De Ambrogi, M.; Ballester, J.; Saravia, F.; Caballero, I.; Johannisson, A.; Wallgren, M.;Andersson M. & Rodriguez-Martinez H. (2006). Effect of storage in short- and longtermcommercial semen extenders on the motility, plasma membrane andchromatin integrity of boar spermatozoa. International Journal ofAndrology, Vol.29,pp. 43-552, ISSN 0196-3635de Andrade, A.; de Arruda, R.; Celeghini, E.; Nascimento, J.; Martins, S.; Raphael, C. &Moretti, A. (2007). Fluorescent stain method for the simultaneous determination ofmitochondrial potential and integrity of plasma and acrosomal membranes in boarsperm. Reproduction in Domestic Animals, Vol.42, pp. 190-194, ISSN 0936-6768de Jong, E.; Vanderhaeghe, C.; Beek, J.; Van Soom, A.; de Kruif, A. & Maes, D. (2009).Ultrasonography of the ovaries in the sow: a helpful tool to determine the time ofinsemination. Vlaams Diergeneeskundig Tijdschrift, Vol.78, pp. 276-281, ISSN 0303-9021De Leeuw, F.; Colenbrander, B. & Verkleij, A. (1990). The role membrane damage plays incold shock and freezing injury. Reproduction in Domestic Animals Supplement, Vol.1,pp. 95-104, ISSN 0936-6768Donadeu, M. (2004). Advances in male swine artificial insemination (AI) techniques. The PigJournal, Vol.54, pp. 110-122, ISSN 1352-9749Ericsson, S.; Garner, D.; Thomas, C.; Downing, T. & Marshall, C. (1993). Interrelationshipsamong fluorometric analyses of spermatozoal function, classical semen qualityparameters and the fertility of frozen-thawed bovine spermatozoa. Theriogenology,Vol.39, 1009-1024, ISSN 0093-691XGadea, J. (2005). Sperm factors related to in vitro and in vivo porcine fertility. Theriogenology,Vol.63, pp. 431-444, ISSN 0093-691X

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92Artificial Insemination in Farm AnimalsKuster, C (2005). Sperm concentration determination between hemacytometric and CASAsystems: Why they can be different? Theriogenology, Vol.64, pp. 614–617, ISSN 0093-691XKuster, C.E. & Althouse, G.C. (1999). The fecundity of porcine semen stored for 2 to 6 daysin Androhep R and X-cell TM extenders. Theriogenology, Vol.52, pp. 365–376, ISSN0093-691X0Langendijk, P.; Bouwman, E.; Kidson, A.; Kirkwood, R.; Soede, N. & Kemp, B. (2002). Roleof myometrial activity in sperm transport through the genital tract and infertilization in sows. Reproduction, Vol.123, pp. 683-690, ISSN 1470-1626Langendijk, P.; Bouwman, E.G.; Schams, D.; Soede, N.M. & Kemp, B. (2003). Effects ofdifferent sexual stimuli on oxytocin release, uterine activity and receptive behaviorin estrous sows. Theriogenology, Vol.59, pp. 849-61, ISSN 0093-691XLangendijk, P.; Soede, N.M. & Kemp, B. (2006). Effects of boar stimuli on the follicular phaseand on oestrus behaviour in sows. Society for Reproduction and Fertility Supplement,Vol.62, pp. 219-30, ISSN 1470-1626López, A.; Rijsselaere, T.; Van Soom, A.; Leroy, J.L.; De Clercq, J.B.; Bols, P.E. & Maes, D.(2010). Effect of organic selenium in the diet on sperm quality of boars. Reproductionin Domestic Animals, Vol.45, pp. 297-305, ISSN 0936-6768López, A.; Rijsselaere, T.; Bijttebier, J.; Vyt, Ph.; Van Soom, A. & Maes, D. (2011).Effectiveness of the sperm quality analyzer (SQA-Vp) for porcine semen analysis.Theriogenology, Vol.75, pp. 972-977, ISSN 0093-691XMaes, D.; Nauwynck, H.; Rijsselaere, T.; Mateusen, B.; Vyt, Ph.; de Kruif, A. & Van Soom, A.(2008). AI transmitted diseases in swine: an overview. Theriogenology, Vol.70, pp.1337-1345, ISSN 0093-691XMaes, D.; Rijsselaere T.; Vyt, Ph.; Sokolowska, A.; Deley, W. & Van Soom, A. (2010).Comparison of five different methods to assess the concentration of boar semen.Vlaams Diergeneeskundig Tijdschrift, Vol.79, pp. 42-48, ISSN 0303-9021Mahmoud, A.; Depoorter, B.; Piens, N. & Comhaire, F. (1997). The performance of 10different methods for the estimation of sperm concentration. Fertility and Sterility,Vol.68, pp. 340–345, ISSN 1556-5653Martin-Rillo, S.; Martinez, E.; Garcia C.;Artiga C. & De Alba, C. (1996). Boar semenevaluation in practise. Reproduction in Domestic Animals, Vol.31, pp. 519-526, ISSN0936-6768Peña, F.; Saravia, F.; Garcia-Herreros, M.; Nunes-Martinez, I.; Tapia, J.; Johannisson, A.;Wallgren, M. & Rodriquez-Martinzez, H. (2005). Identification of spermmorphometric subpopulations in two different portions of the boar ejaculate and itsrelation to post-thaw quality. Journal of Andrology, Vol.26, pp. 716-723, ISSN 0196-3635Pérez-Llano, B.; Lorenzo, J.; Yenes, P.; Trejo, A. & Garcia-Casado, P. (2001). A short hypoosmoticswelling test for the prediction of boar sperm fertility. Theriogenology,Vol.56, pp. 387-398, ISSN 0093-691XPetrunkina, A.; Gröpper, B.; Günzel-Apel, A. & Töpfer-Petersen, E. (2004). Functionalsignificance of the cell volume for detecting sperm membrane changes andpredicting freezability in dog semen. Reproduction, Vol.128, pp. 829-842, ISSN 1470-1626

Artificial Insemination in Pigs 93Popwell, J. & Flowers, W. (2004). Variability in relationships between semen quality andestimates of in vivo and in vitro fertility in boars. Animal Reproduction Science, Vol.81,pp. 97-113, ISSN 0378-4320Rijsselaere, T.; Van Soom, A.; Maes, D. & de Kruif, A. (2002). Use of the Sperm QualityAnalyzer (SQA II-C) for the assessment of dog sperm quality. Reproduction inDomestic Animals, Vol.37, pp. 158–163, ISSN 0936-6768Rijsselaere, T.; Van Soom, A.; Maes, D. & de Kruif, A. (2003). Effect of technical settings oncanine semen motility parameters measured by the Hamilton–Thorne analyzer.Theriogenology, Vol.60, pp. 1553–1568, ISSN 0093-691XRijsselaere, T.; Van Soom, A.; Tanghe, S.; Coryn, M.; Maes, D. & de Kruif, A. (2005). Newtechniques for the assessment of canine semen quality: A review. Theriogenology,Vol.64, pp. 706-719, ISSN 0093-691XRoca, J.; Carvajal, G.; Lucas, X.; Vasquez, J. & Martinez, E. (2003). Fertility of weaned sowsafter deep intrauterine insemination with a reduced number of frozen-thawedspermatozoa. Theriogenology, Vol.60, pp. 77-87, ISSN 0093-691XShipley, C. (1999). Breeding soundness examination in the boar. Swine Health and Production,Vol.7, pp. 117–120, ISSN 1066-4963Tomlinson, M.; Turner, J.; Powell, G. & Sakkas, D. (2001). One-step disposable chambers forsperm concentration and motility assessment: how do they compare with theWorld Health Organization's recommended methods? Human Reproduction, Vol.16,pp. 121-124, ISSN 0268-1161Tsakmakidis, I.; Lymberopoulos, A. & Khalifa, T. (2010). Relationship between spermquality traits and field-fertility of porcine semen. Journal of Veterinary Science,Vol.11, pp. 151-154, ISSN 1229-845XVazquez, J.M.; Roca, J.; Gil, M.A.; Cuello, C.; Parilla, I.; Vazquez, J.L. & Martínez, E.A. (2008).New developments in low-dose insemination technology. Theriogenology, Vol.70,pp. 1216-24, ISSN 0093-691XVerstegen, J.; Iguer-ouada, M. & Onclin, K. (2002). Computer assisted semen analyzers inandrology research and veterinary practice. Theriogenology, Vol.57, pp. 149–179,ISSN 0093-691XVigo, D.; Faustini, M.; Villani, S.; Orsini, F.; Bucco, M.; Chlapanidas, T.; Conte, U.; Ellis, K. &Torre, M.L. (2009). Semen controlled-release capsules allow a single artificialinsemination in sows. Theriogenology, Vol.72, pp. 439–444, ISSN 0093-691XVyt, Ph. (2007). Examination and storage of liquid porcine semen. PhD thesis, GhentUniversity, pp. 156, ISBN 978-90-586-4121-2Vyt, Ph.; Maes, D.; Dejonckheere, E.; Castryck, F. & Van Soom, A. (2004a). Comparativestudy on five different commercial extenders for boar semen. Reproduction inDomestic Animals, Vol.39, pp. 1-5, ISSN 0936-6768Vyt, Ph.; Maes, D.; Quinten, C.; Rijsselaere, T.; Deley, W.; Aarts, M.; de Kruif, A. & VanSoom, A. (2008). Detailed motility evaluation of boar semen and its predictive valuefor reproductive performance in sows. Vlaams Diergeneeskundig Tijdschrift, Vol.77,pp. 291-299, ISSN 0303-9021Vyt, Ph.; Maes, D.; Rijsselaere, T.; Dejonckheere, E.; Castryck, F. & Van Soom, A. (2004b).Motility assessment of porcine spermatozoa: a comparison of methods.Reproduction in Domestic Animals, Vol.39, pp. 447-453, ISSN 0936-6768

94Artificial Insemination in Farm AnimalsVyt, Ph.; Maes, D.; Rijsselaere, T.; Dewulf, J.; de Kruif, A. & Van Soom, A. (2007). Semenhandling in porcine artificial insemination centres: the Belgian situation. VlaamsDiergeneeskundig Tijdschrift, Vol.76, pp. 195-200, ISSN 0303-9021Waberski, D. (2009). Critical steps from semen collection to insemination. Proceedings of theAnnual Meeting of the EU-AI-Vets, September 2009, Gent Belgium, pp. 66-69Waberski, D.; Petrunkina A. & Töpfer-Petersen E. (2008). Can external quality controlimprove pig AI efficiency? Theriogenology, Vol.70, pp. 1346-1351, ISSN 0093-691XWaberski, D., Schapmann, E., Henning, H., Riesenbeck, A. & Brandt, H. (2011). Spermchromatin structural integrity in normospermic boars is not related to semenstorage and fertility after routine AI. Theriogenology, Vol.75, pp. 337-345, ISSN 0093-691XWatson, P. (1995). Cooling of spermatozoa and fertilizing capacity. Reproduction in DomesticAnimals, Vol.31, pp. 135-140, ISSN 0936-6768Weitze, K. (1990). The use of long-term extenders in pig AI - a view of the internationalsituation. Pig News and Information, Vol.11, pp. 23-26, ISSN 0143-9014Weitze, K.F.; Wagner-Rietschel, H.; Waberski, D.; Richter, L. & Krieter, J. (1994). The onset ofoestrus after weaning, heat duration, and ovulation as major factors in AI timing insows. Reproduction in Domestic Animals, Vol.29, pp. 433-443, ISSN 0936-6768Wilson-Leedy, J. & Ingermann, R. (2007). Development of a novel CASA system based onopen source software for characterization of zebrafish sperm motility parameters.Theriogenology, Vol.67, pp. 661-672, ISSN 0093-691XWoelders, H. (1991). Overview of in vitro methods for evaluation of semen quality,Proceedings of the second International Conference on Boar Semen Preservation, KFWeitze and B Colenbrander (Editors), Paul Parey Scientific Publishers, Berlin andHamberg, pp. 145-165Xu, X.; Pommier, S.; Arbov, T.; Hutchings, B.; Sotto, W. & Foxcroft, G. (1998). In vitromaturation and fertilization techniques for assessment of semen quality and boarfertility. Journal of Animal Science, Vol.76, pp. 3079-3089, ISSN 0021-8812

6Artificial Insemination in SwineEduardo Paulino da Costa 1 , Aurea Helena Assis da Costa 2,3 ,Gustavo Guerino Macedo 3 and Emílio César Martins Pereira 31 Federal University of Viçosa2 Germovet3 Post-graduation students from the first authorBrazil1. IntroductionThe world population is 6.4 billion people approximately and is constantly growing. In thiscontext, there is the expectation that it will reach 8.1 billion in 2030 and nine billion in 2050.In the next 25 years, this population growth will demand some 50% increase in foodproduction. So, the world will be required some 53% increase in meat production, thereforeelevating from 367 to 562 million tons. This will be necessary due the growth of thepopulation and the increase of the per capita consumption, which is foreseen to reach19.1Kg swine meat for inhabitant in 2030. So, the production of swine meat should present agrowth around 20%, therefore reaching 155 million tons (Roppa, 2006). This growth is reallyhappening, as considering that the world production of swine meat in 2010 reached 101million tons, with projection of 133 million for 2019 (ABIPECS, 2011).The increase of the productivity in the world swine confinement is happening along the lastdecades. According to data from ABIPECS (2011), China leads the world ranking byannually producing about 50 thousand tons of meat, as followed by the European Union,United States and Brazil (22,250, 10,052 and 3,170, respectively). This high productionbasically occurred by development and adoption of new technologies in practically all areas,such as genetics, nutrition, management, sanity and reproduction. Undoubtedly, in thereproduction area, the artificial insemination (AI) represents an enormous progress inproduction of swine. Since the beginning of the 70-ies, this technique provoked a greatimpact on increment of the swine production, especially in Europe and more recently inUSA (Gerrits et al., 2005).Initially, AI appeared in order to provide the genetic improvement of the animals and tosolve sanitary problems. However, a significant improvement in both productive andeconomical aspects were later observed, as making possible an acceleration in diffusion ofthe desirable characteristics of the reproducers with high genetic value. This occurred due toAI great potential in making possible the use of biotechnologies such as those related totechnology of the semen, preservation of embryos, and others.The intracervical insemination (ICAI) is most used in the technified farms. Under thepractical viewpoint, it is a simple and easily accomplished procedure. In this technique, thesemen is deposited in cervix and the spermatozoids are transported until the ampulla of theuterine tube, the place where fecundation occurs (Rath, 2002).

96Artificial Insemination in Farm AnimalsMore recently, new AI techniques in which the deposition of the semen is accomplished inuterus or in the uterine tube have been developed. So, there are intrauterine artificialinsemination (IUAI), deep intrauterine artificial insemination (IUPAI) and the intratubalartificial insemination (IOAI) through laparoscopy. These new techniques are used in orderto reduce the number of spermatozoids and the volume of the insemination dose. Manystudies have been developed toward the improvement of those techniques, so that thespermatic concentration and the semen volume are maximally reduced, without negativelyinterfering in the reproductive efficiency. So, those techniques would make possible anincrement in the genetic gain for reducing the cost of the dose and maximizing the use of thegenetically superior males.However, in spite of the relative simplicity of the AI in swine, there are several factors thatdirect or indirectly affect negatively the reproductive efficiency of inseminated sows. It isimportant to emphasize that many of those factors also interfere into reproductive efficiencyof the sows submitted to natural mating.In this chapter, the objective is to discuss the advantages, limitations and procedures of theAI. Besides, some important factors that direct or indirectly affect the reproductive efficiencyof the swine herds.2. The artificial inseminationAccording to the first reports, the use of AI in swine occurred in Russia and Japan (Ivanow,1907; Nishikawa, 1964). Later, the AI diffusion was gradually happening in severalcountries. It is probable that the natural prolificacy of the swine species has delayed thedevelopment of the reproduction biotechnologies. However, the needs for genetic exchangeand the sanitary pressures constituted a strong impulse for AI development.In many countries, the AI growth is linked to expansion of the swine production atindustrial scale. Considering the AI advantages, compared with the natural mating, theimplantation of this one substantially facilitates the reproductive management of herds withhigh number of sows.Most countries of the European Union adopt the AI at least in 60% of their females. In thelast two decades, more than 90% swine females in the European west were artificiallyinseminated (Gerrits et al., 2005). In Holland, for instance, more than 98% sows areartificially inseminated (Feitsma, 2009).The AI use contributes for a larger sanitary control and hygienic cares in the matings. It alsomakes possible a better control of the semen quality due to rejection of inappropriateejaculates. Besides those advantages, AI facilitates the management by the reduction of bothtime and work for mating. Another important aspect is the reproductive performance can beequal or superior to that obtained with the use of the natural mating.This reproduction method presents great advantages compared to natural mating. In thiscontext, the following advantages are distinguished: the genetic gains with the use of thegenetically superior males, the reduction of the covering costs by female; and the decrease inthe number of males in the farm. This last condition optimizes the use of the facilities.Today, it is still possible to observe less technified farms that use the natural mating, whichrequires higher amount of males in a herd. This occurs because the male/femalerelationship for this condition to be a male for each 20 or 25 females, approximately. So, theproducer will have higher expenses with facilities, feeding and medicines.

Artificial Insemination in Swine 97Despite all those advantages, however, the AI has some limitations in swine. In closed AIprograms, in which the collection and processing of the semen are accomplished at the ownfarm, the investments in constructions and equipments are necessary for the installation ofone semen production unit. In open programs, in which the doses are acquired from thecenters external to farms, the main limitations are related to communication and to dosetransports (Hansen, 2004).Other limitations are also common in both programs, such as the need for maintaining thedoses at temperature from 15 o C to 18 o C and the short storage period of the cooled doses(usually up to 72 hours). The reduced survival of the spermatozoids in the female genitalorgans is also a limiting factor. Besides those aspects, there are other factors such as thegreat variability in duration of the oestrum (from 12 to more than 96 hours) and at themoment of the ovulation among the swine females. However, the range of advantagesobtained with AI undoubtedly overcomes the disadvantages of the same one.2.1 Intracervical artificial inseminationThe artificial intracervical insemination (ICAI) technique is the most used in technifiedfarms. On the practical viewpoint, it is a simple and easily executed technology. Theapplication of this technique optimizes the use of the males, which can supply up to 2,000doses/year when under good management conditions (Bennemann et al., 2003).In spite of this simplicity, a careful training of the employees and their understanding withreference to this technology are fundamentally important. Another relevant aspect is the wayto implant the insemination technique in a farm. Since the year 1995, our work group alreadyimplanted ICAI in 69 farms at the states of Minas Gerais and Espírito Santo - Brazil. For thisprocedure, a transition period in the change of the natural mating to ICAI was defined. Oursuggestion is the implantation to be partial, along approximately six months (transition phase).So, natural mating and inseminations occur weekly in the farm during this phase.This condition makes possible to compare monthly the estrus replication between bothmethods. As soon the parturitions begin, the size of the litter will also be monitored. So,after approximately six months under evaluation, the decision for total implantation of theinsemination is made. For this decision, the reproductive efficiency of the inseminated sowsmust be the same or superior to that of the sows submitted to natural mating.The spermatic concentration required for ICAI are three billion spermatozoids. Thisconcentration is important, as taking into account that in ICAI the semen is deposited in thecervix and its great part stays retained in the protuberances and cervical crypts. Then, thesestructures work as the first physical barriers to spermatic transport.The swine specie is the only ones in which the volume of the insemination dose is asimportant as the spermatic concentration. In the other domestic species, the average or thinpallets (0,5 and 0,25mL, respectively) are generally used as containers for semenconditioning. In sow, the volume of the insemination dose used for ICAI is 80 to 100 mL. So,the semen recipients must have capacity to condition this volume. Very reduced volumesfor ICAI can increase the rate of the estrus replication and/or to reduce the average numberof pigs born by litter.When the spermatozoids are deposited in the cervix, they are transported until the ampullaof the uterine tuba, where fecundation happens (Rath, 2002). For this condition, thespermatozoids find other physical barrier that is the uterutubal junction, which also worksas spermatic reservoir (Langendijk et al., 2005).

98Artificial Insemination in Farm AnimalsBesides the barriers to be broken for the spermatozoid to reach the place of fecundation,other inconvenience of the insemination is the occurrence of seminal reflux. Theinseminator’ ability is fundamental to minimize the amount of reflux. However, only thisability has no effect, in case the inseminator is impatient. Besides, the time required foraccomplishment of the insemination is an important factor. This makes sense, as consideringthat in the natural mating the penis introduced into female provokes the oxytocin liberationand, consequently, contributes to spermatic transport (Hafez, 2000). According toLangendijk et al. (2005), the IA pipette should remain in the animal’cervix during enoughtime for liberation of the oxytocin.In this context, the recommendation by our work group is the insemination to beaccomplished slowly, as maintaining the pipette fixed in cervix for approximately 10minutes. Nowadays there are many available supports in the market, which are placed onthe back of the sow for elevation and fixation of the pipette segment that is external tovagina, whereas the same one is fixed in the cervix. This way, the inseminator caninseminate other animals without the need for awaiting 10 minutes in each animal and laterto begin IA in another animal.In the ICAI technique, the semen is deposited in the first centimeters of the cervix. Due toanatomical characteristics of this structure, it acts as a natural barrier that hinders the arrivalof the semen into uterus, therefore facilitating the occurrence of reflux through vagina.The occurrence of reflux in the swine species is very common and it was observed in 100%animals inseminated by Steverink et al. (1998). According to those authors, the refluxpresents differences in volume and in the spermatic concentration, according to eachinseminated animal. However, some authors consider the reflux to be a physiologic event inthe swine species. According to them, this reflux could only influence the fertility rate whenthe concentration of the insemination dose is equal or inferior to one billion spermatozoids,in 80 mL volume (Steverink et al. ,1998).This spermatic concentration effect on fertility can be evidenced in the work by Watson &Behan (2002), When inseminating the females, those authors used three different spermaticconcentrations (three, two and one billion spermatozoids) by ICAI and they concluded thatthe females inseminated with one billion spermatozoids presented low number of newbornpiglets.In a work carried out by our team, 120 females were inseminated by ICAI (Araújo et al.,2009), as being the semen reflux found in 100% females. The animals were observed up to120 minutes after insemination. Some 100mL doses containing 3x10 9 spermatozoids wereused. The average volume of the reflux was 85.8mL, with a loss of 782.4 millionspermatozoids by each IA. In this work, a relevant aspect is that insemination was carefullyaccomplished during a period of 10 minutes, by people highly expert in insemination.Surprisingly, animals with more than 105% reflux were observed, despite the carespreviously mentioned, as indicating that secretions from the genital organs also constitutethe volume reflowed. The reflux volume varied from 50 to 105%.The ICAI allows for using the fresh, refrigerated or frozen semen. Concerning to freshsemen, it must be used immediately after its processing, without previous cooling. Thecooling at temperature from 15 o to 18 o C is more used in both farm routines andinsemination centers. It allows the maintenance of the spermatic viability for a period up to72 hours.Concerning to frozen semen, it was firstly used on the beginning of the 70-ies, as firstly withthe insemination into uterine tuba and later with ICAI. There were progresses in using the

Artificial Insemination in Swine 99frozen semen, due to researches accomplished with different cryoprotectors, conditioningpackages, diluents, and freezing and defrosting curves. However, the use of the frozensemen in ICAI is still associated with the reduction from 10% to 20% in the parturition rateand from one to two piglets by litter, when compared to the use of refrigerated semen(Bernardi et al., 2005).2.2 Intrauterine artificial inseminationIn order to reduce the number of the spermatozoids/female/year, new techniques forartificial insemination were recently presented. Among them, the intrauterine artificialinsemination (IUAI) through the use of the post-cervical probe is distinguished. Thistechnique consists of deposition of the semen doses directly into body of the sow’ uterus,from which the length is five to ten centimeters. The IUAI technique optimizes the semenproduction, as using low spermatic concentration by dose. This condition increases two tothree times the number of doses by ejaculate.The cost in maintenance of the semen donors includes the costs of the male acquisition cost,its depreciation, medicines, feeding and facilities. These expenses can represent 30 to 50%the total cost of the semen dose. In this sense, as higher is the number of the doses producedby each housed male, the higher will be the efficiency and lower the cost (Bennemann et al.,2003; Weber et al., 2003; Hansen, 2004). Whereas the traditional insemination requires oneboar for each 100 to 150 female, one boar can attend up to 450 females in the intrauterineinsemination, approximately.Taking into account the better use of the ejaculate in IUAI, it is also distinguished thepossibility to increase the selection intensity in the females production, by using thegenetically superior males. Evidently, this condition would not be applied at commercialfarms, from which the purpose is the production of animals for slaughter. In addition, this isa very useful technique for the researches with swine frozen semen, as taking into accountthat the IUAI, the spermatic volume and concentration are more reduced than in ICAI.At first, IUAI is a higher perilous technique, as taking into account the impossibility to fixthe cervix by hand, such as in cow or even retracting towards the outside of the vagina, asperformed on goats and sheep. So, many technicians consider its implantation to be difficultin commercial farms. However, a work carried out by our group (Araújo et al., 2009)demonstrated the opposite. In this work, the ICAI techniques were compared to IUAI. ICAIwas performed using a Melrose (Minitub ® ) pipette. IUAI was performed using anintrauterine catheter “Verona” (Minitub ® ). Despite the difficult passage of the pipette in4.6% females submitted to IUAI, 100% of those females were inseminated (Table 1).DescriptionInsemination techniqueIntracervical IntrauterineNumber of inseminations 120 480Difficulty to introduce (n) 0 a 22 bTable 1. Degree of dificulty to introduce the pipete in females for intracervical insemination(n=120) and intrauterine (n=480). A difference (P

100Artificial Insemination in Farm Animalshappened successfully in all of the animals. However, Diehl et al. (2006) observed to beimpossible the introduction of the catheter into uterus of 4.5% females. This difficultyprobably occurred due to the short insemination time used by the authors (average: 2.3minutes/insemination), especially in primiparous females, where there was higher numberof animals in which there was difficulty in introduction of the catheter. In those cases, whilethe catheter is introduced, either inseminator’s patience and the constant stimulation of thefemale by massage on lumbar area allow the success of the technique.It is important to emphasize the possibility for occurrence of bleeding during theintroduction of the insemination pipette, a condition verified by Watson & Behan (2002).This is due to factors such as the technical ability of the person responsible for inseminationas well as the pipette type. Another interfering factor is the speed in introduction of thepipette, since as higher is the insemination speed as higher will be the bleeding possibility(Diehl et al., 2006). Besides those factors, the females with higher parturition number presentlarger development of the genital organs than the primiparas or nulliparas. Thus, it is easierthe introduction of the catheter into cervix, therefore reducing the incidence of lesions.However, the occurrence of bleeding during insemination does not affect the reproductiveefficiency. This condition was verified by our work group, as the ICAI was compared withIUAI (Table 2). The presence of blood was observed in 1.6 and 7.7% of the animalsinseminated via ICAI and IUAI, respectively. Nevertheless, this bleeding did not influencethe estrum replication rate neither the total newborns by litter. Those results corroborate theby Watson and Behan (2002), who did not observe any deficit in the reproductive efficiencyof the sows that presented bleeding after IUAI.InseminationtechniqueIntracervicalIntrauterinePresence ofbloodNumber ofInseminationsNumber ofsowsReturn toestrus rate 1Litter size perparityWithout 118 58 4.2 11.7 ± 3.2With 2 02 02 0.0 9.5 ± 9.1Without 443 211 4.5 11.6 ± 3.1With 37 29 5.4 10.8 ± 4.3Table 2. Return to estrus rate and litter size per parity with inseminations in the presenceand absence of blood. Adapted from Araújo et al. (2009). No differences (P>0.05) foundbetween the insemination techniques by the Chi-square test for return to estrus rate. Nodifferences (P>0.05) between the insemination techniques (Duncan Test) for litter size meanper parity. 1 Return to estrus rate percentage of total number of inseminated sows for eachinsemination technique. 2 Not evaluated statistically due to the reduced number ofoccurrences.The ICAI is known as technique presenting considerable vulvar reflux of the semen after AI.However, our work group (Araújo et al., 2009) verified that such a fact also happens withIUAI. In this experiment, we verified the semen reflux to occur in practically all animals,independent of the technique used (100 and 98% for ICAI and IUAI, respectively (Table 3).On the other hand, some works do not mention the presence of reflux in IUAI, perhapsbecause they only observed the first instants after AI (Benneman et al., 2004; Mezalira et al.,2005), differently of our work, in that the animals were observed until 120 minutes postIUAI, once the reflux does not occur right after insemination.

Artificial Insemination in Swine 101InseminationtechniqueNumber of inseminations Semen backflow rate (n)Intracervical 120 100% (120)Intrauterine 480 98% (471)Table 3. Semen backflow in the inseminations according to the different inseminationtechniques. No differences (P>0.05) found between insemination techniques by the Chisquaretest. Adapted from Araújo et al. (2009).The occurrence of the semen reflux can have negative effects on the reproductive efficiency,such as the losses of spermatozoids. This condition is based on the fact that here is aminimum number of spermatozoids by dose, for the maximum reproductive efficiency.However, despite the high occurrence of semen reflux found by our work group, nonegative effects occur in the return rate to estrum and in litter size (table 4).InseminationNumber of Return to estrus Litter size perBackflowtechniqueinseminations rateparityIntracervical With 120 5.0% 11.56 ± 3.4Intrauterine With 471 4.0% 11.48 ± 3.3Table 4. Return to estrus rate and litter size per parity in inseminations with backflowaccording to the different insemination techniques. No differences (P>0.05) found betweenthe insemination techniques by the Chi-square test for the return to estrus rate. Nodifferences (P>0.05) found between the insemination techniques by the F test for litter sizeper parity (Adapted from Araújo et al., 2009).It is evident the spermatozoids number and the insemination dose volume are decisivefactors for the volume reflux to interfere in the reproductive efficiency. In the experimentcarried out by our group (Araújo et al.,2009), IUAI was compared with ICAI, as confrontingtwo insemination volumes (100 vs. 50mL) and different concentrations of spermatozoids.Although the volume of the semen reflux has been similar among the treatments (P>0.05),the amount of spermatozoids of the reflux in females receiving IUAI was smaller (Table 5).InseminationtechniqueNumber ofspermatozoidsBackflowvolume in mL(% 1 )Total of backflowsptz in millions(% 1 )Number ofbackflowscollectedIntracervical 3x10 9 /100 mL 85.8 (85.8%) 782.4 (26.0%) a 23Intrauterine 1x10 9 /100 mL 83.2 (83.2%) 164.0 (16.4%) b 25Intrauterine 1x10 9 /50 mL 41.5 (83.0%) 111.4 (11.1%) b 25Intrauterine 5x10 8 /100 mL 87.8 (87.8%) 80.5 (16.1%) b 28Intrauterine 5x10 8 /50 mL 45.3 (90.6%) 58.0 (11.6%) b 30Table 5. Total number of spermatozoa during backflow in millions and number of backflowscollected using the different insemination techniques. 1 Correspond to percentage in thereflux, as considering the volume or the total number of spermatozoids of the inseminationdose. No differences (P>0.05) occurred between the insemination techniques by the Kruskal- Wallis test, concerning to the collected volume. There was difference (P

102Artificial Insemination in Farm AnimalsThis occurred because the semen is deposited at the third initial/medium of the uterus, asprobably facilitating the fast progression of the spermatozoids toward the spermaticreservations, therefore allowing a high retention of cells in the genital organs (Dallanora etal., 2004).Taking into account the advantages of IUAI, many researchers have been accomplished inthe last years, in order to define the spermatic concentration and the ideal inseminationvolume for maximization of the results by using this technique. So, Dollanora et al. (2004)compared the use of ICAI (three billion spermatozoids at 90mL doses) with IUAI (1.5 billionspermatozoids at 60mL doses). Those authors obtained no differences between bothtreatments for the adjusted childbirth rate and total number piglets born.When comparing ICAI (three billion spermatozoids in 100ML doses) with IUAI (1 billionspermatozoids in 50mL), Sumransap et al. (2007) verified there were no differences amongthe total number of spermatic cells in different segments of the genital organs from the mostcaudal area of the uterus until the ampulla of the uterine tuba. Thus, even with the reducednumber of spermatozoids in the dose, IUAI provides the same number of spermatic cells inthe spermatic reservoirs.However, highly reduced concentrations of spermatozoids in the insemination dose (250million) can reduce the size of the litter, by reducing the spermatic reserves (Mezalira et al.,2005).The volume of the insemination dose is also a decisive factor in the reproductive efficiencyof the herd. In this context, some works report that IUAI accomplished with highly reducedvolume endangers the reproductive efficiency of the herd. This is evident in the work byBennemann et al. (2005) who used IUAI with 500 million spermatozoids by dose, in volumeof 20 mL (154 sows), as comparing with ICAI with three billion spermatozoids in 90 mL (144sows). The farrowing rate did not differ between treatments. When using IUAI, however, asignificant reduction occurred in the total number of born pigs.The experience of our work group (Araújo et al., 2009) shows that the use of 5x10 8spermatozoids in 50mL can adequately substitute the traditional technique (ICAI) withoutendangering the reproductive efficiency of the inseminated animals (Table 6). It is probablethat the use of the oxytocin in semen has contributed to those positive results. In worksaccomplished by our research group, the addition of 2.5 UI oxytocin at the inseminationdose of 100 mL does not interfere in the physical parameters of the semen andmorphological ones of the spermatozoids (Podda et al., 1999), as well as it does notendanger the replication rate of estrous. Additionally, the oxytocin in this preconized doseincreases the size of the litter (Costa et al., 1999). With the physiologic role to promoting thecontraction of the flat musculature of the uterus (Bevan, 1979), the oxytocin can facilitate theascension of higher number of spermatozoids until the fecundation site, taking into accountthat only a small proportion of the spermatozoids deposited during natural mating orinsemination reach the distal portion of the uterine tuba.In spite of those positive results found in this experiment, in which the insemination wasaccomplished by the same employees who performed the insemination routine, we still didnot implant the IUAI with 5x10 8 spermatozoids/50mL in the routine of commercial farms.However, Since the year 2007, our work group implanted the IUAI with 1x10 9espermatozoids/100mL, by using two inseminations (at zero and 24 hours after thebeginning of the estrus) in 100% primiparous and pluriparous females at four farms (total of2.500 females). No nuliparous females exist in those farms. The primiparas are proceedingfrom other farm of the same company. It is important to emphasize that the inseminations

Artificial Insemination in Swine 103are accomplished by employees of the farms. This condition reinforces our position that theIUAI technique can be accomplished at commercial farms by the own employeesresponsible for the gestation sector.InseminationtechniqueSpermatozoidnumberFarrowing rate( 1 n)Estrusrepetition rateNumber of newbornsby farrowingIntracervical 3x10 9 /100 mL 90.0 (54) 10.0 11.5 ± 3,4Intrauterine 1x10 9 /100 mL 93.3 (56) 6.7 11.7 ± 3,4Intrauterine 1x10 9 /50 mL 86.7 (52) 13.3 11.4 ± 3,2Intrauterine 5x10 8 /100 mL 93.3 (56) 6.7 11.8 ± 3,0Intrauterine 5x10 8 /50 mL 90.0 (54) 10.0 11.4 ± 3,6Table 6. Farrowing, estrus repetition rates and total piglets born per farrowing in eachinsemination technique (60 females per treatment). 1 n: Number of animals which gave birthaccording to each insemination technique. No differences (P>0.05) occurred between theinsemination techniques for farrowing and estrus repetition rates by the chi-square test. Nodifferences occurred (P>0.05) between the insemination techniques for number of newbornsby farrowing using Kruskal – Wallis test. Adapted from Araújo et al. (2009).2.3 Deep intrauterine artificial and intratubal InseminationsIn the last years, many researches concerning to the deep intrauterine insemination (IUPAI)have been accomplished. In this technique, a low insemination volume (5mL) is used as wellas reduced concentration of spermatozoids (200 million), without the need for surgicalintervention (Vazquez et al., 2000). The objective of the researches accomplished until themoment is to turn this technique applicable (not endanger the reproductive efficiency of theherd, so that it can be commercially implanted at large scale. In addition, the use of reducedvolume and low concentration of the sperm in IUPAI will favor the use of frozen semenand/or sexed.The reduction of the semen volume used in IUPAI rather guarantees the optimization of theboar, as providing economical advantages to the farms. The possibility for using thistechnique is in line with the needs imposed by modern swine raise, which looks forreducing the insemination dose under use. This would provide a reduction in the malebreeding stock and even in the frequency of using these ones.It is considered that a great number of the spermatozoids are lost in ICAI process (Martínezet al. 2001). This occurs due to the semen reflux as well as to spermatozoid phagocytosis bythe polymorphonuclear leucocytes. It is believed that approximately 1/3 of thespermatozoids by backflow in 2 hours after AI, due to those physiologic processes (Viring &Einarsson, 1981). After overcoming those obstacles, approximately 1 X 10 3 spermatozoidscan be rescued at the caudal portion of the isthmus, a place where the spermatic cell staysuntil ovulation to occur (Mburu et al., 1996).In this context, the IUPAI objective is the reduction of the spermatic flow inside the uterus,as reducing the seminal reflux and the phagocytosis rate on those cells (Vazquez et al.,2008). In addition, some physical barriers are transposed as the cervical folds andendometrial crypts. Thus, the insemination dose under use could be significantly reduced.So, Martínez et al. (2002) verified that IUPAI with 5 x 10 7 spermatozoids by dose (5mL)presents no differences in the gestation and parturition rates neither in the size of the litter,when compared with ICAI by using 3 x 10 9 spermatozoids (100mL). However, it is

104Artificial Insemination in Farm Animalsimportant to emphasize that the control group (n=147) presented low rate for eitherparturition (83%) and for those born by litter (9.97), although those researchers used a highnumber of animals by treatment. Other aspect to be considered is that the estrum of thefemales submitted to IUPAI was induced, whereas the estrum of the control group was not.The IUPAI technique consists of using a special pipette, which is fixed into cervix as in ICAI.Successively, a flexible catheter with 1.8m length is inserted through pipette along thecervical canal until reaching the final portion of the uterine horn. This technique providesthe deposition of the semen in one of the uterine horns near the fertilization place.The main IUPAI obstacle is the anatomical complexity of the sow´s genital organs. Thecervical channel is characterized by presence of the cervical folds and uterine horns due tolong length and naturally rolled. These characteristics delayed the development of a catheterfor nonsurgical insertion in the uterine horns. So, Vazquez et al. (2005) report that, in 1999,they developed a nonsurgical catheterization technique for access to the uterus, by using amodified endoscope provided of flexible 1.35m optic fiber. Those researchers report thesuccess in accomplishing this procedure.Thus, the first accomplished IUPAI were based on the use of an endoscope at extremity ofthe insemination pipette, therefore allowing the visualization of either cervical channel anduterine horns. This technique associated with induction of the hormonal ovulation in sowshas been making possible the deep deposition of the spermatozoid into uterus. It wasdemonstrated that the passage of the pipette associated with endoscope, along the cervicalchannel and uterine horn, is a simple process to be accomplished, as lasting 4.1 minutes onaverage (Martínez et al. 2001). Those authors show that the endoscopic IUPAI generatesinteresting results, such as parturition rates of 86.6%, 88.9% and 92.3%, by using 100, 20 or 5x 10 7 spermatozoids in 5 mL of diluter, respectively. The average size of the litter was 9.41.Those data do not differ from ICAI (n=48) with 3 x 10 9 in 100mL. However, it is important toemphasize that the authors used a small number of animals (15, 18 and 13 females,respectively) for IUPAI. Besides, those animals were submitted to hormonal synchronizationprocedures, what could make unfeasible the use of this technique routinely in thecommercial farms due to high cost.The use of the endoscope represented a great progress in the procedure of the artificialinsemination in swine. Due to deposition of the semen at proximities of the fecundationplace, the IUPAI technique makes possible the use of the processed and weakenedspermatozoids proceeding from cooling, freezing or sexing (Vazquez et al., 2005). However,the limitation of this technique is the cost of the equipment and its fragility. Thus, its usewould not be applicable at field (Vazquez et al., 2005). From this verification, a number ofresearches were developed in order to eliminate the use of the endoscope in this procedure.This situation required the development of new IUPAI pipettes.The proof of the IAIUP efficiency at field, without using the laparoscopy, was laterconfirmed by Martínez et al., 2005b. This author demonstrated that the fertilization rate ofthe sows inseminated with 150 x 10 6 spermatozoids diluted into 5ml BTS did not differ fromthat when the animals were inseminated with 3 x 10 9 spermatozoids diluted into 100ml ofthe same diluent through IAIC. However, some 10.9 reduction in size of the litter wereobserved in the conventional IA for 9.8 piglets in IAIUP. Based on these results, the authorsverified the IAIUP application in commercial farms to depend on the proof that thistechnique will not endanger the number of the piglets born by parturition.With the progress of the researches, the number of spermatozoids used in IUPAI weretwenty times reduced for refrigerated semen and up to six times for frozen semen, in

Artificial Insemination in Swine 105comparison with ICAI. In relation to volume, the decrease resulting from the use of IUPAIwas 8 to 20 times lower compared to ICAI (Vazquez et al., 2008).However, the recurring concern of the researchers refers to unilateral fertilization. Althoughsmall semen doses are only deposited in an uterine horn, the bilateral fertilization wasproven in approximately 100% of the cases, according to either Martínez et al. (2002) whoused the IUPAI with endoscopic catheter and Tummaruck et al. (2007) by using IUPAI withcatheter without endoscope. Those authors did not find significant difference in the numberof embryos found on each side. For this evaluation, they slaughtered the sows atapproximately 60 hours after IUPAI. Then, they evaluated the washed of the uterine tubaand extremity of the horns.However, a detailed study by Martínez et al. (2006) showed that, in sows ovulatingspontaneously (without induced ovulation), the bilateral fertilization that is, in both uterinehorns, is not 100% effective. For this confirmation, those authors used IUPAI with doses of0.15x10 9 spermatozoids/ 20 mL. So, those researchers verified that 21% sows submitted toIUPAI presented unilateral fertilization. In addition, 15.8% sows presented partial bilateralfertilization. Those researchers also found differences regarding to the rate of normalembryos in the horn with less embryos, when comparing IUPAI with ICAI (2.95x10 9spermatozoids/95mL). Corroborating with those authors, Buranaamnuay et al. (2011)demonstrated that, from a total of five inseminated animals, three presented unilateralfertilization. The animals were submitted to IUPAI procedure without laparoscopy, as using1 x 10 9 defrosted spermatozoids.Those contradictory discoveries suggest the mechanism in which the spermatozoid reachesthe counterlateral horn still stays obscure and needs to be more studied, in spite of theevidences for trans-uterine and trans-peritoneal migration (Martínez et al. 2005a;Tummaruck et al. 2007).The IUPAI will represent a great economical advantage to the farm, since it will reduce thecosts with acquisition of males, ration, medicines, vaccines, management. Besides, it wouldguarantee a larger uniformization of both herd and litter. However, the high cost of thepipette for this procedure and the difficulties in execution of the technique still representimpediments for its implantation in commercial farms. To these factors are added the resultsstill inferior in reproductive efficiency, when compared with ICAI and IUAI.Thus, it is clear that IUPAI represents a technique with a promising future to becommercially used at the farms, since the costs are decreased and the reproductiveefficiency is not endangered.Another developed technique is the artificial intratubal insemination through laparoscopy(ITAI). This new technology attends the premise to use doses at much reduced spermaticconcentrations and at small volumes. Above all in specific situations, when the use offrozen, sexed semen or the genetically modified semen is proposed, the low number ofviable spermatozoids can be compensated by deposition of the close semen or the semeninside the uterine tuba, then obtaining satisfactory fertilization rates.The ITAI allows the inseminator, with the aid of a laparoscope, to determine the time andthe ideal place for deposition of the semen, as reducing the exhibition of the spermatic cellsto adversities and positioning them close to the uterus-tubaric junction.For execution of this technique (Vazquez et al., 2008), initially the animal is placed inTrendelemburg position (that is, dorsal decubitus with the head side lightly sloping) at 20°angle with the horizontal. Successively, an incision close to 1.5cm near the navel isaccomplished. The borders of the incision are tractioned and a optiview trocar associated

106Artificial Insemination in Farm Animalswith a laparoscope is inserted into incision, as this being removed later. So, the access to theabdominal cavity is possible with laparoscope. The abdominal cavity is inflated with CO 2and two lateral openings are accomplished in the hemi-abdomen for the access of theForceps tweezers. Those tweezers aid in the manipulation and fixation of the uterine hornsand uterine tubas for the introduction of the insemination needle. After accomplishing theprocedure, the tweezers are removed and a small suture is necessary. The procedure takesapproximately 15 minutes.Laparoscopy is considered a less invasive technique than laparatomy for introduction of thesemen into uterus or in the uterine tuba (Vazquez et al., 2008), since laparatomy can causeeither higher stress to the animal and adherences at the postoperative period, thereforeprejudicing the future inseminations (Fantinati et al., 2005).The insemination by ITAI makes possible the use of doses as low as five millionspermatozoids in 0.5mL (refrigerated semen) in each uterine horn, without affecting theefficiency of the fertilization and production of piglets, when associated with laparoscopy(Fantinati et al., 2005). According this author, however, this technique should be obligatorilyaccomplished in both horns, since the low concentration, the small volume and thedeposition in a precise place impede the spermatozoid to migrate and reach the collateralhorn.Due to high number of spermatozoids introduced into uterine tuba (3-6 x 10 5 ) during ITAI,special attention should be taken with regard to polyspermy. The polyspermy is affected bythe spermatozoid: oocytes proportion and by the insemination moment, as this one isrelated to modifications in the environment of the uterine tuba. In this context, Vazquez etal. (2008) verified the polyspermy incidence to be very low, when spermatic concentrationsof 3x10 5 and 5x10 5 are used by dose or when the sows are inseminated before ovulation.Otherwise, when those animals are inseminated with 1 x 10 6 spermatozoids/dose or duringthe preovulatory period, the possibilities for polyspermy are increased. Thus, lowspermatozoid concentrations (3x10 5 ) were shown to be effective when used before ovulationin ITAI, as opening possibility for use of the sexed and frozen semen.When working with the dose of 5 x 10 6 spermatozoids/0.5mL in each horn in thelaparoscopic IAITU and 3 x 10 9 in ICAI, Fantinati et al. (2005) obtained high fertilizationrates of oocytes and developmental competence of the embryos, which were collected andcultivated in vitro. However, it is worth to emphasize that the females had the estruminduced with eCG and hCG.Although this technique was commercially applied in sheep (Anel et al., 2006), in swine it isstill limited to experimental assays. The highest difficulties for its commercialaccomplishment are the need for personal training, structure and specialized equipments.2.4 Some factors affecting the reproductive performance of inseminated femalesSeveral aspects are able to interfere into results of the AI programs. However, it is importantto emphasize that many of those factors also interfere in the reproductive efficiency of sowssubmitted to natural mating. The objective of this topic is to distinguish some importantaspects in this context.In the last 25 years, the AI in swine was expressively desenvolved, as contributing to geneticimprovement of the herd and increase in production. During this period, a reduction in thenumber of spermatozoids by each dose, that passed from 6x10 9 to up 5x10 4 . At the sametime, the useful life of the semen increased considerably, as turning more flexible andpracticable the process (Waberski et al., 2008).

Artificial Insemination in Swine 107The use of reduced spermatic concentrations in the insemination doses comes to encounterthe premises of the swine confinement of the XXI century. To attend those requirements,however, it is necessary a high-qualified semen, therefore guaranteeing a high reproductiveefficiency. In addition, to assure this condition, it is necessary an effective quality controland the monitorship of several factors that can interfere into results.In this context, several factors are very important such as: the action of the pathogenicmicroorganisms, nutritional conditions, the age of the first mating, the lactation period, theseasonal influence and the management in detecting the estrus detection.The reproductive efficiency of the artificially inseminated sows is extremely affected when acontaminated semen is used. The microbial contamination of the semen can result intoreduced reproductive efficiency due to low seminal quality, precocious embryonic death,and endometritis (Guerín & Pozzi, 2005).The forms of the semen contamination can be classified as being from animal origin or not.The contamination from animal origin is due to infection of the boar, proceeding from thetesticles or other segments of the genital organs. In addition, a number of contaminationsmay occur by breathing secretions or feces, which happen during the collection process andsemen processing. Otherwise, the contamination not arising from the boars can occur, inmost cases inadvertently during manipulation of the semen by the person responsible forthe collect. Another responsible factor would be the excessive and mistaken exposure of thematerial collected at the air, skin and breathing secretions. Besides those aspects, the semencan be also contaminated by the quality of the water used during the processing, theventilation system, sinks and drains (Maes et al., 2008).A second factor affecting the reproductive performance of the animals is feeding. Thesupply of a ration that is in perfect balance of nutrients is essential. For this reason, it isfundamental the producer to receive technical orientation of the professionals specialized inthe animal nutrition area.The age of the animal at the first natural mating is also an important factor that should betaken into account. Usually, the sows present the first estrus at the age of five or six months.However, it is not indicated those animals to be inseminated before the seventh or eighthmonth. On this occasion, they will be weighing approximately 130 to 140kg on average, asdepending on the female’ genetics, and they will be presenting the third estrus. This fact isbased on verification by Martín Rillo et al. (2001) who confirmed that the length of thefemale’ genital organs during the first natural mating is directly proportional to dimensionof the animal. Additionally, those authors found correlation between the length of thevagina and the length of the uterus. They still verified a larger size of the litter in animalspresenting more developed genital organs at the action of insemination.Another factor affecting the reproductive efficiency of the inseminated female is the nursingperiod. In the past, it was consensus that a shorter nursing period would increase thenumber of piglet births by year and, consequently, larger number of parturition by year andconsequently higher piglets/sow/year. However, this theory was mistaken, because basedon survey of 79,729 parturitions, our work group observed that as longer is the length of thelactational period as larger is the size of the following litter (Table 8). Thus, lactationalperiods from 22 to 25 days result in approximately one more pig in the following litter,compared with periods from 8 to 13 days. This condition was verified either in primiparousand pluriparous sows (Costa et al., 2004).

108Artificial Insemination in Farm AnimalsLactation length (days)PrimiparousPluriparousN LS N LS8 to 13 1,074 10.34 ± 0.9 a 3,513 10.70 ± 0.5 a14 and 15 2,911 10.41 ± 0.5 a 13,951 11.16 ± 0.2 b16 and 17 2,249 10.46 ± 0.6 a 16,095 11.15 ± 0.2 b18 to 21 2,987 10.68 ± 0.5 b 24,069 11.34 ± 0.1 c22 to 25 1,186 11.43 ± 0.8 c 8,692 11.87 ± 0.3 dTotal 10,867 10.86 ± 0.3 68,862 11.44 ± 0.1Table 8. Average of litter size (LS) of primiparous and pluriparous sows submitted todifferent lactation lengths and respective parity number (N). Averages with different lettersin the same column differ (P

Artificial Insemination in Swine 109Another aspect that should be observed in artificial insemination programs is the thermalcondition of the environment to which the animal is submitted. The high temperaturesreduce the efficiency of the heat loss, as making the animal to enter a hyperthermal state.This condition leads to embryonic mortality at the initial gestation stage. A surveyaccomplished with 100,934 parturitions in a tropical climate area shows the size of the litterto be significantly smaller in the hottest months of the year (Table 10). In the same way, intemperate area (Mediterranean conditions), the efficiency of the IA (parturition rate andlitter size) is lower in summer-autumn season. Additionally, the administration ofexogenous hormones (eCG and hCG) in the attempt to improve the ovulation rate proved tohave no effects during this period (Bolarín et al., 2009).However, when the environmental temperature becomes a restrictive factor for theembryonic viability, there are some alternatives to minimize the thermal effect. Theventilation, the floor cooling and the use of nebulization could partially reduce the adverseeffect of the temperature on sow. This practice is applicable, mainly in tropical countrieswhere the hangars for animals are open and exposed to adverse effects of the climate. Adultanimals can have their critical temperature increased, that is, their resistance to heat isincreased up to 2 o C when they are submitted to ventilation from fans within facilities (Nääs,2000).Another aspect to be considered in AI is the efficiency in detecting the estrus. For theobtainment of indexes compatible with the goals established by the reproductive program, itis necessary to observe the IA ideal moment, as considering both estrus and ovulation. Thiscondition is important, since a long IA - ovulation interval reduces the gestation rate, theembryonic survival and the litter size (Spencer et al., 2010).The insemination protocol (AI moment) is defined as a function of the estrus beginning.Thus, more important than to find a sow under estrus is to detect the beginning of the sameone. However, even with a good management in detection of the estrus, many times thebeginning of this one is not characterized, taking into account that it might have happenedduring night. This fact can be the responsible for the highest incidence of the estrus detectedat the beginning of the morning and not during afternoon. In a study conducted by ourwork group (Pinheiro, 2000), we verified that 16.66% of the estrus were initially detected at15:30 hours. However, at 7:30 and 23:30 the estrus were detected in 44.44 and 38.88% of theanimals. Therefore, 83.24% initial detections of the estrus occur in the morning, taking intoaccount there is no routine at the farms for night detection.Considering the importance of the initial detection of the estrus, it is worth to emphasizethat some animals do not accept promptly the natural mating even when they are in estrus.However, when insisting with detection incentive, the animal presents the immobilityreflex. So, it is very important a careful detection, mainly in females that already presentmodifications such as the vulva edema.When the gilts are housed in stalls, the introduction of the teaser is recommended in thosestalls, on the beginning of the morning and on the end of the afternoon, in order to detectthe females in estrus. Those females presenting immobility to natural mating, a behaviorknown as reflex of tolerance to male (RTM), are considered to be in estrus. However, specialattention should be given when RTM does not occur in females that already presentmodifications such as the vulva edema. In this case, we recommend to take the female tostall of the teaser it can be carefully evaluated.Though, the management of the estrus detection in primiparous and pluriparous femalesare differentiated. After weaning of the litter, the females are housed in collective stalls or

110Artificial Insemination in Farm Animalsindividual cages. Usually, the beginning of the estrus happens from the third and fourth dayafter weaning. However, RTM should be made already at the following day after weaning,at the beginning of the morning and final of the afternoon. This procedure is important,taking into account that some sows can advance the beginning of the estrus, in other words,at the first or second day after weaning.When the females are housed in cages, the teaser is conducted in the corridor of the hangar, sohe has contact with the sow. At this moment, an employee stimulates the sow by pressuringthe back or even mounting on the same one. From the third day from weaning, the femalespresenting no characteristic behavior of estrus should be individually taken to the stall of theteaser. For standardization either in gilts and primiparous and pluriparous, the zero hour ofthe estrus is the moment at which the female presents RTM for the first time.Another procedure used in practice for detection of the estrus is the reflex of tolerance to theman (RTH). This reflex is the result of the man-animal interaction, without the presence of amale. However, the results are various and inconsistent. A study carried out by our workteam (Pinheiro, 2000) showed that 23% sows in estrus do not present RTH. For this study,the estrus was confirmed by RTM, besides the occurrence of ovulation that was confirmedby ultrasonography. The detection of the estrus was accomplished at 8hrs intervals (7:30,15:30 and 23:30). The RTH reflex was accomplished before RTM, as considering that manysows in estrus can present positive RTH after they were previously sensitized by the contactwith male.We also verified that 44% females, which were in estrus, presented very short RTH (lessthan 16 hours). Those considerations were corroborated by DIAS et al. (1999), who found avery varied RTH period. Those researchers observed that 11% animals presented no RTHand 26.5% presented it for a period lower than 16 hours. Also SOEDE (1996) found that 18%animals presented RTH for 16 hours or less and many animals presented a discontinuous orvery short symptomatology. Thus, considering the mentioned aspects, RTH should not beconsidered as an efficient procedure in the estrus detection.3. ConclusionThe artificial intrauterine insemination (IUAI) allows for better use of the ejaculates,compared with the intracervical artificial insemination ICAI). This condition is possible, astaking into account that a lower spermatic concentration can be used in the inseminationdose. The IUAI technique can be used at commercial farms in substitution to ICAI withoutendangering the reproductive efficiency.In spite of the progresses and refinement of the different artificial insemination techniques,the deep intrauterine insemination (IUPAI) is a promising procedure. In this context, thepossibility for using the insemination doses with small volume and reduced spermaticconcentration will optimize the use of the males, as providing economical advantages to thefarms. In addition, the use of the reduced volume and low spermatic concentration inIUPAI will allow progresses in the use of frozen semen.However, the high cost of the pipette for this procedure and the difficulties in execution ofthe technique still represent impediments for its implantation in commercial farms. Theresults still inferior in the reproductive efficiency, when compared with ICAI and IUPAI, areadded to those factors. Thus, it is evident that IUPAI is a technique with promising future tobe used commercially at the farms, since the costs are decreased and the reproductiveefficiency is not endangered.

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114Artificial Insemination in Farm AnimalsPodda, M. C. A.; Costa, E.P.; Pinheiro, R.W.; Vilela, C.G.; Paiva, F.P.; Carvalho, F.F.; Costa,A.H.A. Oxitocina no sêmen diluído de varrões. I – Influência nos aspectos físicos dosêmen e morfológicos dos espermatozóides Proceedings of Congresso Brasileiro deVeterinários Especialistas em Suínos (ABRAVES). pp. 343-344. Belo Horizonte, MinasGerais, Brasil, outubro, 1999.Rath, D. Low dose insemination in the sow – A Review. (2002). Reproduction of DomesticAnimals. v.37, pp. 201 – 205.Roppa, L. (September/October 2006). Perspectivas da produção mundial de carnes, 2006 a2030. Revista Pork World, Editora Paulínia, n. 34, pp. 16 – 27 , São Paulo, SP.Soede, N.M., Kemp, B. (1996). Timing of AI and ovulation in sows. Proceedings ofInternational Conference On Boar Semen Preservation,1996Spencer, K.W.; Purdy, P.H.; Blackburn, H.D.; Spiller, S.F.; Stewart, T.S.; Knox, R.V. (2010).Effect of number of motile, frozen-twawed boar sperm and number of fixed-timeinseminations on fertility in estrous-synchronized gilts. Animal Reproduction Science,Vol. 121, (July 2010), pp.259-266, doi:10.1016/j.anireprosci.2010.07.002.Steverink, D.W.; Soede, N.M., Bouwman, E.G. (1998). Semen backflow after inseminationand its effect on fertilization in sows. Journal of Reproduction and Fertility, v.111,p.165-171, 1998.Sumransap, P.; Tummaruck P.; Kunavongkrit, A. (2007). Sperm distribution in thereproductive tract of sows after intrauterine insemination. Reproduction DomesticAnimals, vol. 42, No.2, (2007), pp.113-117, ISSN 0936-6768.Tummaruk, P.; Sumransap, P.; Techakumphu, M.; Kunavongkrit, A. (2007). Distribution ofSpermatozoa and Embryos in the Female Reproductive Tract after Unilateral DeepIntra Uterine Insemination in the Pig. Reproduction Domestic Animals, vol. 42, No.6,(December 2007), pp.603-609, ISSN 0936-6768.Vazquez, J. L.; Martínez, E. A.; Vazquez, J. M.; Lucas, X.; Gil, M. A.; Parrilla, I.; Roca, J.(2000). Development of a non-surgical deep intrauterine insemination technique.Proceedings of International Conference on Sêmen Preservation, Beltsville, 2000.Vazquez, J.M.; Martinez, E.A.; Roca, J.; Gil, M.A.; Parilla, I.; Cuello, C.; Carvajal, G.; Lucas,X.; Vazquez, J.L. (2005). Improving the efficiency os sperm Technologies in pigs: thevalue of deep intrauterine insemination. Theriogenology. Vol. 63, No.2 (January2005), pp.563-547. doi:10.1016Vazquez, J.M., Martinez, E.A., Roca, J., Gil, M.A., Parrilla, I., Cuello, C., Carvajal, G., Lucas,X., Vazquez, J.L., (2005). Improving the efficiency of sperm technologies in pigs: thevalue of deep intrauterine insemination. Theriogenology, Vol. 63, pp. 536–547.Vazquez, J.M.; Roca, J.; Gil, M.A.; Cuello, C.; Parrilla, I.; Vazquez, J.L.; Martínez, E.A.(2008). New developments in low-dose insemination technology. Theriogenology.Vol. 70, No. 8, (November 2008), pp. 1216-1224, DOI: 10.1016.Viring, S.; Einarsson, S. (1981) Sperm distribuition within the genital tract of naturallyinseminated gilts. Nordisch Veterinarian Medicine, v.33, p.145-149.Waberski D, Petrunkina AM, Töpfer-Petersen E. (2008). Can external quality controlimprove pig AI efficiency? Theriogenology. Vol. 70, No. 8, November, pp. 1346-51.Watson, P. F. & Behan, J. R. (2002). Intrauterine insemination of sow with reduced spermnumbers: results of a commercially based field trial. Theriogenology. Vol. 57, pp.1683 – 1693.Weber, D.; Bennemann, P.E.; Wentz, I.; Bortolozzo, F.P. (2003). Avaliação do Custo de DosesInseminantes Produzidas em Centrais de Inseminação Artificial de Suínos emSistema Fechado. Proceedings of XI Congresso Brasileiro de Veterinários Especialistas emSuínos (ABRAVES), Goiânia - GO, outubro, 2003.

Sperm Preparation Techniques forArtificial Insemination - Comparison ofSperm Washing, Swim Up, andDensity Gradient Centrifugation Methods7Ilaria NataliSterility Center, Gynecology and Obstetrics Unit,S.S. Cosma and Damiano Hospital, PistoiaItaly1. IntroductionThe Artificial Insemination (AI) is the first option treatment for infertile couples withcervical factor subfertility, mild-moderate male subfertility and unexplained infertility. Withthe exception of cases in which the use of in vitro fertilization (IVF or ICSI) is strictly due asa consequence of a severe male or female factor, the artificial insemination must be part of agradual approach to the techniques of artificial insemination. This is particularly the casesince the AI is a valid low-cost method, minimally invasive and easily acceptable for thefemale’s hormone treatment (Aribarg & Sukcharoen, 1995).The AI, as other assisted reproductive techniques, needs a selection of the ejaculatedspermatozoa before the performance of the treatment. In fact, some components of theseminal fluid may become an obstacle to the fertilization when the in vitro fertilization orthe intrauterine insemination are performed (Bjorndahl et al., 2005). Spermatozoa andleukocytes produce many oxygen radicals that alter the possibility of the sperm-oocytefusion after repeated centrifugations. So, the selection of the sperms from the othercomponents with methods like the swim up technique or the gradient density centrifugationmust be preferred (Aitken & Clarkson, 1988).Some different techniques are used to prepare the spermatozoa for the AI, but the choicestrongly depend on the quality of the semen, that is on the concentration, motility andmorphology, in order to obtain the higher number of good spermatozoa, even from thepoorest semens.The principle techniques of sperm preparation consist of migration, density gradientcentrifugation and filtration techniques. While for the migration the method is based onmovement of the spermatozoa, for density gradient centrifugation and filtration techniquesthe method is based on a combination of the motility and the retention at phase borders andadherence to filtration matrices, respectively (Henkel & Schill, 2003).The main techniques used for the AI are the sperm washing, the swim-up technique, and thedensity gradient centrifugation and they will be described as follow. The aim of the presentchapter is to shed light on the key principles and the best method for sperm selection inorder to obtain higher pregnancy rate.

116Artificial Insemination in Farm Animals2. Sperm preparation techniques2.1 Semen collectionThe semen consists of a suspension of spermatozoa stored in the epididymes that, at themoment of the ejaculation, is mixed with the secretions of the accessory glands. Theseglands are mainly the prostate and the seminal vesicles, while the bulbourethral glands andthe epididymes represent only the minor contribution of the ejaculate.Two main fractions are present in the seminal fluid; the first one is prostatic, rich inspermatozoa. The last fraction of the semen consists of vesicular fraction, less rich inspermatozoa (Bjorndahl & Kvist, 2003).During ejaculation, it is very important to collect the entire volume of the sample: if the firstfraction (rich in spermatozoa) is lost, the assessment of the semen features will be moredifficult. In case of the AI, the semen sample will not contain the best portion of thespermatozoa.For these reasons, the first step throughout the sperm preparation, is the correct spermcollection.The semen collection is strongly recommended after an abstinence period of 2-3 days(Jurema et al., 2005; Marshburn et al., 2010) to maximize the conception rate. A sterilecontainer (non-toxic for the spermatozoa) will be used and the collection of the semen willoccur in a private room very close to the laboratory. All of these elements are mandatory forthe therapeutic use. After the collection, the name of the couple should be clearly written onthe container.2.2 Choice of the techniqueThe techniques for the selection of the most efficient spermatozoa are very important forclinical practice. The choice of the best technique for semen preparation, before the AI,strictly depends on the quality of the sample (Canale et al., 1994). So, if we have a samplewith normal count, motility and morphology of sperms we choose a sperm washing or aswim up method. By contrast, with a suboptimal quality sample we usually prefer a densitygradient centrifugation. With the first methods, we obtain good quality sperms; while thedensity gradient centrifugation is usually preferred for the greater number of mobilespermatozoa selected from poor characteristics samples (low number, motility andmorphology samples). Each technique can be changed or improved with simple changes, inorder to optimize the recovery of the sperms.The efficiency of the sperm selection is expressed as the concentration of spermatozoa withnormal motility (that is progressively motile spermatozoa, according to the definition of theWorld Health Organization Manual of 2010) (WHO Manual, 2010).Glass-wool columns are reported to be as effective as density gradient for the separation ofspermatozoa also with intact acrosome from semen with suboptimal characteristics(Rhemrev et al., 1989; Sterzik et al., 1998), but this technique is less used.The swim up method and the density gradient centrifugation produce different levels ofcontamination in the sample in the final preparation. In fact, the swim-up techniqueproduces an higher level of non-sperm components (e.g. debris, bacteria) and the diffusionof other substances (e.g. the prostatic zinc) from the semen into the overlaying mediumrespect of the density-gradient centrifugation (Bjorndahl et al., 2005). Some differences alsoexist in the presence and the production of the Reactive Oxygen Species (ROS) and the

Sperm Preparation Techniques for Artificial Insemination -Comparison of Sperm Washing, Swim Up, and Density Gradient Centrifugation Methods 117sperm DNA damage, associated with high levels of ROS, after the application of the twomain techniques (Irvine et al, 2000; Zini et al, 1993, 2009).The final volume of the preparation depends on the technique performed. If the IntraUterineInsemination (IUI) is performed, 0,3-0,4 milliliters (ml) of spermatozoa resuspended insterile medium is required. If the case of the Fallopian Tube Sperm Perfusion (FSP), thevolume of the suspension must be 4 ml, because it must perfuse the uterus and the bothtubes. Because of its simplicity the first technique is the most used, even if some authors,comparing the IUI versus the FSP, demonstrate the superiority of the FSP technique aboutthe pregnancy rate in stimulated cycles (Fanchin, 1995).3. The sperm countBefore and after the treatment of the seminal fluid, the following parameters must beevaluated in line with the WHO Manual 2010. Volume (ml) Concentration (millions/ml) Motility (Progressive motility) Morphology (%normal sperms)In addition, it is very important to establish the concentration of spermatozoa withprogressive motility in the final preparation. The concentration of the progressivespermatozoa is calculated by multiplying the percentage (%) of the progressive sperms forthe concentration of the sperms in the final preparation.[PS] = %PS X [S] f1(1)The total number of the progressive spermatozoa is calculated by multiplying theconcentration of the progressive sperms for the final volume of the suspension.TPS = [PS] X V f2(2)The total number of the progressive sperms in the preparation before the AI may be definedas a threshold value in predicting outcome in AI. This threshold is not absolute and mayvary from study to study, even if some authors have identified this value in 10 millionsperms (Miller et al., 2002; Van Voorhis et al., 2001).4. Sperm washingFor the best quality samples (number and motility of sperms) the sperm washing is oftenperformed (Boomsma et al., 2004) for the AI. The procedure simply consists in the washingof the semen with a sterile medium added with human albumin. After the fluidification ofthe sample, the entire volume is divided in fractions of not more than 2 ml into centrifugetubes. The sterile medium of the equal volume (e.g. for the volume of the sample of 2 ml themedium added is 2 ml) is added in each tube and gently mix with a sterile pipette. After1 P=Progressive; S=Spermatozoa; [PS]=Concentration of Progressive Spermatozoa; [S]f=Concentrationof the sperms of the final preparation.2 TPS=Total Number of the Progressive Spermatozoa; [PS]=Concentration of Progressive Spermatozoa;Vf =Final volume of the preparation.

118Artificial Insemination in Farm Animalsthat, the samples are centrifuged at 300g (the rpm must be calculated for the centrifuge ineach laboratory) for 10 min and than the supernatant is very carefully removed with a sterilepipette. The pellet is resuspended in 1 ml of the medium, gently mixed and centrifugedagain for 5 min at 300g. The supernatant is removed again and the final pellet isresuspended in sterile medium for the AI.It is very important to determine the count and the motility of the final preparation beforethe insemination.In spite of the simplicity and velocity of the method, it must be reminded that the repeatedcentrifugations without the separation of the good sperms from leukocytes and dead spermscan produce many oxidative species and the damage of the sperms function (Aitken &Clarkson, 1988).5. Swim up methodThe swim up is the most common technique used in IVF laboratories and is preferred if thesemen sample has a normal number of good sperms (normozoospermia). By this technique,the sperms are selected on their motility and the capability to swim out of the seminalplasma.If the “direct swim up” is performed, after the fluidification of the sample, the entire volume(well mixed) is divided in fractions of 1 ml into centrifuge tubes (round bottom is preferred).1,3 ml of culture medium is placed over the semen with extreme attention in each tube. Thetubes must be put in the incubator, inclined at an angle around 45° and incubated at 37°Cfor 30-60 min. By inclining the tubes at 45°, we increase the surface between the mediumand the semen and we improve the capability of the sperms to swim out of the semen and toreach the medium. After that, the tube must be returned in the vertical position and 1 ml ofthe supernatant of each tube can be gently removed, aspirating the sperms from the uppermeniscus downwards with a sterile pipette (Henkel et al., 2003).In alternative, the culture medium can be placed in each tubes and the semen can bestratified under the medium, in order to obtain a much cleaner surface between the semenand the medium. In addition, the recovery of the sperms can be optimized by increasing thenumber of the tubes and decreasing the volume of the semen in each tube. 2 ml of mediumare added to the supernatant of each tubes and than centrifugated at 300g for 10 minutes.The supernatant is removed again and the pellet is resuspended in the sterile medium forthe AI.The “not direct” swim up from pellet is performed with the centrifugation of the semenfollowed by the stratification of the medium over the resuspended pellet. The liquefiedsemen is divided in fractions of 1 ml into each tubes, the medium is added (1:1) and after thecentrifugation the supernatant is gently removed. Over the resuspended pellet, 1,3 ml ofmedium is replaced with caution and the tubes is put into the incubator from 30 to 60 min at37°C (inclined at 45°); after the migration of the sperms, the volume of the semen for the AIis removed and the sperm count and motility are assessed.The centrifugation for the direct swim up occurs after the migration of the sperms, that is,after the separation of the good sperms from the leukocytes and dead sperms. These species,usually produce the reactive oxygen species after the centrifugation (Irvine et al, 2000; Ziniet al, 1993, 2009) so the direct swim up is the preferred method respect to the “not direct”swim up to select sperms for the AI.

Sperm Preparation Techniques for Artificial Insemination -Comparison of Sperm Washing, Swim Up, and Density Gradient Centrifugation Methods 1196. Density gradient centrifugationThis is the preferred technique to select the greater number of motile spermatozoa in casesof severe oligozoospermia, teratozoospermia or asthenozoospermia. In this method, goodquality sperms can be separated from dead sperms, leukocytes and the other components ofthe seminal plasma by a density discontinuous gradient. Cells with different density andmotility can be selected during the centrifugation by the colloidal silica coated with silane ofthe gradient; the sperms with high motility and good morphology are at the bottom of thetube, finally free from dead spermatozoa, leukocytes, bacteria and debris.The most applied discontinuous density-gradient is a two layers density-gradient, formedby a top layer of 40% (v/v) and a lower layer of 80% (v/v). Density gradient media areavailable in commerce ready to use or ready to make the different density layers; the toplayer phase (40%) is prepared by adding 4 ml of density gradient medium to 6 ml isotonicsterile medium (BWW, Earle, Ham F-10 or HTF) supplemented with HAS (Human SerumAlbumin); the lower layer phase (80%) is prepared by adding 8 ml of density gradientmedium to 2 ml of isotonic sterile medium. The density gradient is prepared by layering 1ml of 40% medium over the 80% medium, or by layering the 80% medium under the 40%medium in a conical centrifuge tube (not the round bottom tube!). The number of the tubesdepends on the volume of the semen sample, but the total volume could be divided in notmore of 1 ml of semen per tube.After the fluidification, 1 ml of the semen is layered over the upper layer (40%) andcentrifuged at 300g for 15 minutes. If the volume of each layer is reduced (

120Artificial Insemination in Farm Animalsejaculate as soon as possible, first because some components of the ejaculate contrast withthe fertilizing capability of the spermatozoa (Bjorndahl et al., 2005; Mortimer et al., 1998).Then, because spermatozoa and leukocytes produce many oxygen radicals that cannegatively influence the fertilizing sperm function (Agarwal & Sekhon, 2010; Aitken et al.,1998; De Jonge, 2002; Shamsi et al., 2008; Sharma & Agarwal, 1996; Zini & Sigman, 2009). So,the methods who separates the functional sperms from the other cells must be preferred(Aitken & Clarkson, 1988). The choice of the best method to select the functionallycompetent sperms depends on the features of the samples.The swim up technique and the density gradient centrifugation have different efficiency inseparating the sperms: the sperms isolated with the swim up are clean and motile, butdamaged by the ROS and with higher DNA integrity; the sperms isolated with the densitygradient centrifugation are not damaged by the ROS but with low DNA integrity.When we compare the pregnancy rate after artificial insemination obtained with the spermwashing, the advanced sperm preparation methods (swim up and density gradientcentrifugation) offer the higher rate of pregnancies (Carrell et al., 1998). These data indicatethat the correct choice of the method of sperm selection can represent a good chance ofpregnancy, after ovarian stimulation, in the artificial insemination.Finally, the threshold value of 10 million sperms in the final preparation for the IUI has apredictive value for the pregnancy rate in IUI. Some authors demonstrate that when thetotal count of the progressive sperms is less than 10 millions the pregnancy rate decreased,even if, in practice, a pregnancy is also possible with an inferior total sperm count (Miller etal., 2002; van Weert et al., 2004; Van Voorhis et al., 2001).If the total sperm count is very low, and in presence of a severe male factor, other alternativemust be considered, like the In Vitro Fertilization (IVF).8. AcknowledgmentsI want to thank the book editor Milad Manafi for comments and support.9. ReferencesAgarwal, A. & Sekhon, L.H. (2010). The role of antioxidant therapy in the treatment of maleinfertility. Human Fertility, Vol.13, No.4, (December 2010), pp.217–225, ISSN 1464-7273Aitken, R.J. & Clarkson, J.S. (1988). Significance of Reactive Oxygen Species andAntioxidants in Defining the Efficacy of Sperm Preparation Techniques. J of Androl,Vol.9, No.6, (November/December 1988), pp.367-76Aitken, J.R.; Gordon, E., Harkiss, D., Twigg, J.P., Milne, P., Jennings, Z. & Irvine, D.S. (1998).Relative Impact of Oxidative Stress on the Functional Competence and GenomicIntegrity of Human Spermatozoa. Biol of Reprod, Vol.59, (1998), pp.1037–1046Aribarg, A. & Sukcharoen, N. (1995). Intrauterine insemination of washed spermatozoa fortreatment of oligozoospermia. Int J Androl, Vol. 18, No. 1, (1995), pp.62-6Bjorndahl, L. & Kvist, U. (2003). Sequence of ejaculation affects the spermatozoon as acarrier and its message. Reprod Biomed Online, Vol.7, No. 4, (October/November2003), pp.440-8

Sperm Preparation Techniques for Artificial Insemination -Comparison of Sperm Washing, Swim Up, and Density Gradient Centrifugation Methods 121Bjorndahl, L.; Mohammadieh, M., Pourian, M., Soderlund, I. & Kvist, U. (2005).Contamination by seminal plasma factors during sperm selection. J of Androl, Vol.2,(December 2005), pp.170-73Boomsma, C.M.; Heineman, M.J., Cohlen, B.J. & Farquhar, C. (2004). Semen preparationtechniques for intrauterine insemination. Cochrane Database of Systematic Reviews2004; (3):CD004507Canale, D.; Giorgi, P.M., Gasperini, M., Pucci, E., Barletta, D., Gasperi, M. & Martino, E.(1994). Inter and intra-individual variability of sperm morphology after selectionwith three different techniques: layering, swim up from pellet and percoll. JEndocrinol Invest, Vol. 17, No. 9, (October 1994), pp.729-32Carrell, D.T.; Kuneck, P.H., Peterson, C.M., Hatasaka, H.H., Jones, K.P. & Campbell, B.F.(1998). A randomized, prospective analysis five sperm preparation techniquesbefore intrauterine insemination of husband sperm. Fertil Steril, Vol. 69, No. 1,(January 1998), pp. 122-26De Jonge, C. (2002). The clinical value of sperm nuclear DNA assessment. Hum Fertil, Vol. 5,No. 2, (May 2002), pp.51-3Evenson, D.P. & Wixon, R. Data analysis of two in vivo fertility studies using SpermChromatine Structure Assay-derived DNA fragmentation index vs. pregnancyoutcome. Fertil Steril, Vol. 90, No. 2, (October 2008), pp.1229-31Fanchin, R.; Olivennes, F., Righini, C., Hazout, A., Schwab, B. & Frydman, R. (1995). A newsystem for fallopian tube sperm perfusion leads to pregnancy rates as high asstandard intrauterine insemination. Fertil Steril, Vol. 64, No. 3, (September 1995),pp. 505-510Henkel, R.R. & Schill, W-B. (2003). Sperm preparation for ART. Reproductive Biology andEndocrinology, Vol. 1, (2003), p.108Irvine, D.S.; Twigg, J.P., Gordon, E.L., Fulton, N., Milne, P.A. & Aitken, R.J. (2000). DNAintegrity in human spermatozoa: relationships with semen quality. J Androl, Vol.21, No. 1, (January/February 2000), pp.33-44Jurema, M.W.; Vieira, A.D., Bankowski, B., Petrella, C., Zhao,Y., Wallach, E. & Zacur, H.(2005). Effect of ejaculatory abstinence period on the pregnancy rate afterintrauterine insemination. Fertil Steril, Vol. 84, No. 3, (September 2005), pp.678-81Loft, S.; Kold-Jensen, T., Hjollund, N.H., Giwercman, A., Guyllemborg, J., Ernest, E., Olsen,J., Scheike, T., Poulsen, H.E. & Bonde, J.P. (2003). Oxidative DNA damage in humansperm influences time to pregnancy. Hum Reprod, Vol. 18, (2003), pp.1265-72Marshburn, P.B.; Alanis, M., Matthews, M.L., Usadi, R., Papadakis, M.H., Kullstam, S. &Hurst, B.S. (2010). A short period of ejaculatory abstinence before intrauterineinsemination is associated with higher pregnancy rates. Fertil Steril, Vol. 93, No. 1,(January 2010), pp. 286-88Miller, D.C.; Hollenbeck, B. K., Smith, G.D., Randolph, J.R., Christman, G.M., Smith, Y.R.,Lebovic, D.I. & Ohl, D.A. (2002). Processed total motile sperm count correlates withpregnancy out come after intrauterine insemination. Urology, Vol. 60, (2002), pp.497–501Mortimer, S.T.; Swan, M.A. & Mortimer, D. (1998). Effect of seminal plasma on capacitationand hyperactivation in human spermatozoa. Hum Reprod, Vol. 13, No. 8, (1998),pp.2139–2146

122Artificial Insemination in Farm AnimalsRhemrev, J.; Yejendran, R.S., Vermeiden, J.P. & Zaneveld, L.J. (1989). Human spermselection by glass wool filtration and two-layer, discontinuous Percoll gradientcentrifugation. Fertil Steril, Vol. 51, No. 4, (April 1989), pp.685-90Shamsi, M.B.; Kumar, R. & Dada, R. (2008). Evaluation of nuclear DNA damage in humanspermatozoa in men opting for assisted reproduction. Indian J Med Res, Vol. 127,(2008), pp.115-123Sharma, R.K. & Agarwal, A. (1996). Role of reactive oxygen species in male infertility.Urology, Vol. 48, No. 6, (December 1996), pp.835-50Spano, M.; Bonde, J.P., Hjollund, H.I., Kolstad, H.A., Cordeli, E. & Leter, G. (2000). Spermchromatine damage impairs human fertility. Fertil Steril, Vol. 73, (2000), pp.43-50Sterzik, K.; De Santo, M., Uhlich, S., Gagsteiger, F. & Strehler, E. (1998). Glass wool filtrationleads to a higher percentage of spermatozoa with intact acrosomes: anultrastructural analysis. Hum Reprod, Vol. 13, No. 9, (September 1998), pp.2506-11Van Voorhis, B.J.; Barnett, M., Sparks, A.E.T., Syrop, C.H., Rosenthal, G., Dawson, J. (2001).Effect of the total motile sperm count on the efficacy and cost-effectiveness ofintrauterine insemination and in vitro fertilization. Fertil Steril, Vol. 75, No. 4, (April2001), pp. 661-8van Weert, J-M; Repping, S, Van Voorhis, van der Veen, F, Bossuyt, P.M.M. & Mol, B.V.J.(2004). Performance of the postwash total motile sperm count as a predictor ofpregnancy at the time of intrauterine insemination: a meta-analysis. Fertil Steril,Vol. 82, No. 3, (September 2004), pp.612-20World Health Organization. (2010). WHO laboratory manual for the examination andprocessing of human semen. Fifth edition. 2010, GenevaZini, A.; de Lamirande, E. & Gagnon, C. (1993). Reactive oxygen species in semen ofinfertile patients: levels of superoxide dismutase- and catalase-like activities inseminal plasma and spermatozoa. Int J Androl, Vol. 16, No. 3, (June 1993), pp.183-8Zini, A.; Mark, V., Phang, D. & Jarvi, K. (1999). Potential adverse effect of semen processingon human sperm deoxyribonucleic acid integrity. Fertil Steril, Vol. 72, No. 3,(September 1999), pp.496-9Zini, A.; Finelli, A., Phang, D. & Jarvi, K. (2000). Influence of semen processing technique onhuman sperm integrity. Urology, Vol. 56, No. 6, (December 2000), pp.1081-4Zini, A. & Sigman, M. (2009). Are tests of sperm DNA damage clinically useful? J of Androl,Vol. 30, No. 3, (May/June 2009), pp.219-29

8Effect of Vitamin E onthe Development of Testis in SheepHailing Luo, Suyun Ge, Dubing Yue, Leyan Yan,Xu Xu, Kun Liu and Fei YuanState Key Laboratory of Animal Nutrition, College of Animal Science and Technology,China Agricultural University, Beijing,PR China1. IntroductionVitamin E (VE) is a term that encompasses a group of potent, lipid-soluble, chain-breakingantioxidants. Structural analyses have revealed that molecules having VE antioxidantactivity include four tocopherols (α-, β-, γ-, and δ-tocopherols) and four tocotrienols (α-, β-,γ-, and δ-tocotrienols). One form, a-tocopherol, is the most abundant form in nature , has thehighest biological activity. As a naturally occurring antioxidant, VE is located in biologicalmembranes where it acts to protect the membrane PUFA—polyunsaturated fatty acid(PUFA) from oxidation and attenuate oxidative damage to the cellular membranes(Sugiyama, 1992). Tappel (1962), Burton and Ingold (1986) and Esterbauer et al. (1991) foundVE was effective in preventing lipid peroxidation and other radical-driven oxidative events.VE was first isolated from green leafy vegetables by Herbert Evans and Katherine Bishop,two prominent researchers from Berkeley and described as a fertility factor in 1922, thenwas named tocopherol in 1924 and synthesized in 1938 (Sen et al., 2007). The role of VE inreproductive performance was shown up that supplementing VE increased total spermoutput and sperm concentration in boars (Brzezinska-Slebodzinska et al., 1995), rabbits(Yousef et al., 2003) and rams (Luo et al., 2004; Yue et al., 2010).Impairment of mammalian fertility has also been attributed to VE deficiency. The crucialrole of VE in animal reproduction has been recognized since 1922 (Evans and Bishop, 1922).To date, there are approximately 100 publications on this topic, which highlight thebeneficial effects of this antioxidant on viability, membrane integrity and motility ofspermatozoa of different species. The protective effects of VE against oxidative damage ofsperm cells become even more significant when hygienic conditions are poorly controlled,as they frequently occur in field. Such conditions are associated with increased incidence ofinfections/inflammations of reproductive apparatus. During inflammation, the antioxidantdefence of reproductive system downplays and generates an oxidative stress (Potts andPasqualotto, 2003), which may impair testis function and affect negatively semencharacteristics (O’Bryan et al., 2000). Because of high content of polyunsaturated membranelipids, testicular tissue becomes one of the targets for oxidative stress (Mishra and Acharya,2004).VE supplementation in diet can protect the cell membrane from oxidation and improve thesurvival rates of cells. Adding VE in diet also increased activity of total anti-oxidation

124Artificial Insemination in Farm Animalscompetence (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX),decreased content of nitric oxide (NO), malondialdehyde (MDA) and activity of nitric oxidesynthase (NOS) in testis in Boer goat (Zhu et al., 2010). In Aohan fine-wool sheep,supplementing VE have a positive role in reducing MDA level and improving the activitiesof SOD and GSH-PX in testicular cell membrane and mitochondria (Yue et al, 2010) andimproving testicular marker enzyme, such as ATPase, lactate dehydrogenase (LDH),sorbitol dehydrogenase (SDH), and alkaline phosphatase (ALP) activity (Yan et al, 2010).Some reports proved that VE had effects on reproductive organs. Marin-Guzman et al.(1997) found the length and width of testis in boar were larger in group of VEsupplementation at 220 IU/kg in diet than in group without VE supplementation (P>0.05).Soleimani et al. (2009) found testis volume of rat in VE treated group (100 mg/kg per day)was bigger than in Control (P>0.05), the thickness of germinal epithelium and diameter ofseminiferous tubules in VE treated group was increased compared with Control (P

Effect of Vitamin E on the Development of Testis in Sheep 125Ingredients % Nutrient contents of DMForage 60 CP (%) 4.86Concentrate 40 EE (%) 8.97NDF (%) 31.23ADF (%) 22.33Vitamin E (IU/Kg) 3.42The composition and nutrient level of the concentrateIngredients % Nutrient content of DMCorn 62 CP (%) 18.68Soybean meal 26 EE (%) 14.41Wheat bran 8 NDF (%) 10.14CaHPO 4 2 ADF (%) 6.52Salt 1 Vitamin E (IU/Kg) 7.56Additives 1CP: crude protein; EE: ether extract; NDF: neutral detergent fiber; ADF: acid detergent fiberTable 1. The composition and nutrient level of the diet2.3 Statistical analysesData were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s newmultiple range tests with the SAS 9.1 software program to determine the level ofsignificance among mean values. Results were expressed as a mean and standard error.Values of P0.05), the order was Group 2>Group 3>Group 1>Group 4>Control.Item Control Group 1 Group 2 Group 3 Group 4Width (cm) 5.59±0.65 5.90±1.35 6.69±0.40 6.23±0.78 5.89±1.12Length (cm) 8.64±0.82 9.56±2.09 10.51±0.63 10.14±1.60 9.32±1.48Transverse girth(cm)13.42±1.85 14.85±2.77 16.90±0.28 15.58±1.52 14.63±2.79Vertical girth (cm) 19.63±2.01 21.03±3.54 24.22±1.71 22.67±3.04 20.93±3.96Volume ( cm 3 ) 289.63±98.12 383.85±255.42 495.17±81.67 426.81±156.44 358.20±172.70Values in same row with same superscript differ insignificantly (P>0.05), with different superscriptsdiffer significantly (P

126Artificial Insemination in Farm AnimalsItem Control Group 1 Group 2 Group 3 Group 4Density of sertoli cell(number per seminiferoustubule)Density of spermatogenic cell(number per seminiferoustubule)Density of leydig cell(number/mm2)Thickness of germinalepithelium (μm)Diameter of seminiferoustubule (μm)10.03±2.08 b 11.20±2.13 ab 13.57±0.56 a 13.40±1.13 a 13.33±1.27 a60.13±25.18 b 93.03±44.90 ab 145.10±52.79 a 137.35±19.59 ab 113.80±36.05 ab285.14±14.82 299.74±18.30 325.87±15.69 320.49±6.52 313.57±38.1075.05±17.21 b 91.31±36.23 ab 143.72±28.17 a 116.02±15.24 ab 141.28±29.48 a303.93±51.41 c 334.02±57.55 bc 465.78±56.16 a 374.32±16.73 abc 443.66±64.45 abValues in same row with same superscript differ insignificantly (P>0.05), with different superscriptsdiffer significantly (P

Effect of Vitamin E on the Development of Testis in Sheep 127Compared with the Control, supplementing VE in diets increased the diameter ofseminiferous tubule, it was significantly wider in Group 2 (at the level of 200 IU sheep -1 d -1 )than in Control and Group 1 (P0.05).Feeding with dietary VE tended to have a higher density of sertoli cell, it was significantlyincreased in Group 2, Group 3 and Group 4 compared with Control (P0.05). Similar results were observed in thepresent study, the width, length, transverse girth, vertical girth and volume of testis in VEtreated groups were higher than Control, but no significant differences were observedbetween Control and treatment groups (P>0.05).This study proved that for Aohan fine-wool sheep, the thickness of germinal epithelium,diameter of seminiferous tubule and the density of spermatogenic cell, sertoli cell and leydigcell in VE treatment groups were larger than in Control. Similar results have been proved inBoer goats in our previous research (Zhu et al., 2009), which showed that diameters ofseminiferous tubules and numeric density of spermatogenic cells tended to be larger in80 IU kid −1 d −1 VE supplemented group compared to the control group (P0.05).Sertoli cells are the nurse cells for the spermatogonium in the seminiferous tubules and canthus influence the development of the sperm precursor cells and the subsequent number ofspermatids. This effect may be accomplished through their production of various factorssuch as plasminogen activator, transferrin, and sertoli cell growth factor (Lacroix et al., 1977;Feig et al., 1980; Skinner and Griswold, 1980). Besides, sertoli cells can maintain highconcentrations of androgens in seminiferous tubules and epididymis (Lacroix et al., 1977),they can also transport testosterone from the testis into epididymis (Krishnamoorthy et al.,2005). Our research showed supplementing VE in diet increased the density of sertoli cell,which in Group 2, Group 3 and Group 4 was significantly higher than in Controlrespectively (P

128Artificial Insemination in Farm AnimalsTestosterone secretion is critical for male secondary sexual differentiation and leydig cellsare the principal source of testosterone production in the males (Ren-Shan et al., 2007). ROScan be produced in leydig cells through mitochondrial respiration (Chen et al., 2001) as wellas through the cytochrome P450 enzymes of the steroidogenic pathway (Hornsby, 1989;Peltola et al., 1996). VE’s function as an antioxidant, can quench lipid peroxidation andeliminate the ROS to protect the leydig cells from damage. Mather et al. (1983) reported thatVE could prolong the survival and function of porcine leydig cells cultured in vitro. In ourstudy, the density of leydig cell in treatment groups was larger than in Control, but nosignificant differences were observed among Control and treatment groups (P>0.05).VE supplementation in diet increased the thickness of germinal epithelium. Significantdifferences were observed between Group 2, Group 4 and Control (P

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9Evaluation of a New Method andDiagnostic Test in Semen AnalysisPetra ZrimšekClinic for Reproduction and Horses,Veterinary Faculty,University of Ljubljana,Slovenia1. IntroductionSperm concentration is an important parameter affecting fertility. Animal species ofagricultural interest are mainly produced by artificial insemination (AI) which contributeshighly to the development of worldwide swine production, making the impact of the malein reproductive efficiency of the pig herds more crucial (Jounala et al., 1998).The efficiency of AI (fertility rate and prolificacy) is directly dependent on the quality ofsemen doses and on the number of spermatozoa used for insemination (Camus et al., 2011).In commercial farms, routine examination of boar semen is performed aiming to predict themale’s fertility. Evaluation of concentration is crucial to adapt dilution rate and to optimizesperm concentration which will directly impact fertility performance. In the first part of apresent chapter we address the basic concepts of a method comparison study and present anexample of a method comparison experiment concerning determination of spermconcentration.Various laboratory methods techniques are used to evaluate sperm quality, such as spermconcentration, motility, viability, and morphology. However, there is no single semen assaythat provides complete information about semen quality (Holt & Medrano, 1997; Johnson etal., 2000; Liu & Baker, 2002). Studies in domestic animals showed that these semencharacteristics were often not significantly correlated to fertility, while the most validassessment of boar semen quality is to obtain viable pregnancies and normal offspringfollowing AI (Tsakmakidis et al., 2010). Since fertilization is a complex process involving ahuge number of events, fertility research must not only device more predictive laboratorytests, but also properly combine different assays aiming to predict male fertilizing ability, asspermatozoa should satisfy many requirements for successful fertilization (Quintero-Moreno et al., 2004). Assessment of metabolic status of spermatozoa could provide a usefultool for evaluation of semen quality, because sufficient metabolism for energy production isone of the several attributes that a sperm must posses to fertilize an oocyte. In the secondpart of this chapter developing and diagnostic evaluation of a spectrophotometricapplication of the resazurin reduction assay will be presented.Learning objectives of a chapter are to: Investigate repeatability in continuous data

132Artificial Insemination in Farm AnimalsPerform method agreementConstruct Bland-Altman plotsExplain limits of agreement between two methodsChose an appropriate regression analysis used in the interpretation of comparing dataDefine the diagnostic parameters: specificity, sensitivity, accuracy, predictive values ofa testRecognize the validity and usefulness of the testEvaluate the performance of a diagnostic test using ROC (receiver operatingcharacteristic) analysisConstruct and compare ROC curvesDetermine optimal cut-off point for a testExplain the developing of a new method in semen evaluation2. Method agreement for determining sperm concentrationSemen samples, which often contain a variety of cells (immature germ cells, blood cells,epithelial cells, and cellular debris) in addition to spermatozoa, differ markedly from bloodsamples because of their heterogeneity. There is also no specific standard available forsperm cells of each species. It is therefore important to compare a new, more appropriate oradditional method to a conventional one. The counting chamber technique for estimatingsperm count appears to be adequate because of its simplicity, low cost and reproducibility.However, photometers are widely used routinely for determining sperm concentration bymany AI organisations, for bulls and boars as well as other species (Woelders, 1991). Theyneed to be evaluated before use, because accurate concentration measurement is the firstand crucial step of the semen preparation process for production of semen doses (Camus etal., 2011). Correct assessment of sperm concentration is essential to ensure that the numberof sperm per insemination dose meets requirements and that the maximal number of dosescan be produced per ejaculate.The increasing use of AI in swine emphasizes the need for the distribution of good qualitysperm by the AI centres (Vyt et al., 2004). Boar sperm quality is routinely assessed bymeasuring concentration, morphology and motility of spermatozoa (Johnson et al., 2000).Determination of sperm concentration is essential in evaluating fertility, whether in vivo orin vitro. However, there is no agreed method for use as a standard. Knuth et al. (1989)showed that introduction of an unevaluated laboratory method, without appropriate qualitycontrol, can cause a bias in semen analysis. However, the methodology of semen evaluationis complex, and standardization is difficult (Brazil et al., 2004). For example, the first largescale, nation-wide proficiency testing program for clinical andrology laboratories in theUnited States reported that the inter-laboratory coefficient of variation for manual spermconcentration determination was 80%, with a range for a single semen specimen of 3 – 492 x10 6 /ml (Keel et al., 2000). The accuracy, reliability and repeatability of different instrumentsthat evaluate sperm concentration of raw semen have already been compared in severalprevious studies (Christensen et al., 2004; Hansen et al., 2006; Prathalingam et al., 2006;Anzar et al., 2009; Camus et al., 2011). Variation in the results from different laboratoriescould be due to the lack of standardisation of methods between laboratories (Maatson,1995).The reason for comparing methods is often that a quicker, more convenient and moreeconomical adaptation has been made to an existing method. Studies comparing a new

Evaluation of a New Method and Diagnostic Test in Semen Analysis 133method with an established method are performed to assess whether the newmeasurements are comparable with existing ones (Jensen & Kjelgaard-Hansen, 2006).2.1 Precision of the evaluated methodsIt is necessary to establish that a method is repeatable before comparing two measurementsfor reproducibility (Petrie & Watson, 1999). Repeatability of boar semen concentrationassessment depends on instruments and procedures, for example CV for instruments FACS,HEMO, Photo C254, SpermVision, UltiMate and SP-100 were 2.7, 7.1, 10.4, 8.1, 5.4 and 3.1%,respectively (Hansen et al., 2006). Imade et al. (1993) reported similar overall precision(5.9%) for the Makler chamber, whereas CV for sperm counts in sperm suspensions can behigher, for example 18.6% (Christensen et al., 2005) or even 26.3% (Mahmoud et al, 1997). Itis generally admitted that intra-observer CVs are often greater than 10%. Althoughguidelines for standardizing the procedure have been proposed, relatively importantdegrees of intra- and inter- technician or inter-laboratory variability have been reported. Inthe external quality assessment (EQA) reported by Neuwinger and coworkers (1990), whichinvolved 10 experienced German laboratories in the evaluation of 8 sperm samples, themean CV was 37.5%. From the data of the external quality control obtained under the BritishFertility Society and reported by Matson (1995), the calculated inter-individual CV forsperm concentration was 64.7% for 24 semen samples collected by technicians from 20laboratories.2.2 Method agreementAccording to the literature, a very common way of investigating method agreement is toperform a paired t-test or to calculate a correlation coefficient to provide a measure ofagreement. However, in this instance, neither method is appropriate for the followingreasons (Petrie & Watson, 1999). The paired t-test tests the null hypothesis that thedifference is zero. If the differences between pairs are large – indicating that the methods donot agree – but are evenly scattered around zero, then the result is non-significant. We canonly conclude that there is no bias, not that the methods agree. Correlation is a statisticalmethod used to quantify any association between two continuous variables (Ma & Smith,2003). The correlation coefficient provides a measure of the linear association between themeasurements obtained by the two methods. It provides an indication of how close theobservations in the scatter diagram are to a straight line. R measures the strength of arelation between two variables, not the agreement between them (Bland and Altman, 1999).For example, the Pearson correlation coefficient gives no information of value in methodcomparison studies, because R can be highly significant even when there is an obvious biasbetween the two methods. It measures the strength of association, rather than agreement,although in the literature it has been used in many studies, such as comparison betweendifferent methods to determine sperm concentration (Prathalingam et al., 2006). R was alsoused to evaluate agreement between assessments within lab technician in sperm analysis(Christensen et al., 2005). In order to assess agreement, it is necessary to know how close thepoints are to the line of equality, i.e. the 45 line (Petrie & Watson, 1999). Therefore, in thestudy of Sokol et al. (2000), comparison of two methods for measuring sperm concentrationusing only Wilcoxon signed rank test and F-test, appears to be insufficient.Scatter plots and absolute and relative bias plots give the best overview of comparisons ofdata (Twormey, 2004; Twormey, 2005). Absolute bias plots are also called Bland and Altman

134Artificial Insemination in Farm Animalsplots, usually used for method comparison (Bland and Altman, 1999). In absolute bias plots,the biases are plotted against their average value for each sample. The mean of thesedifferences ( d ) is an estimate of the average bias of one method relative to that of the other.If this value is zero, then the two measurements agree on average. However, this does notimply that they agree for each individual measurement.In order to assess how well the paired measurements agreed with each other, limits ofagreement have to be determined. The upper and lower limits of agreement are calculatedasd ± 2s difff (1)where d is the mean of differences for all the samples (average bias) and s diff is the standarddeviation of the differences; 2s diff is also referred to as the British Standard Institutionrepeatability (or, reproducibility, as appropriate) coefficient and indicates the maximumdifference likely to occur between two measurements. This coefficient is the value belowwhich the bias between paired results may be expected to lie (Petrie & Watson, 1999).We performed method agreement between two clinical laboratory methods for determiningboar sperm concentration using the statistical programme Analyse-it, General + ClinicalLaboratory statistics, version 1.71, where linear regression, Deming regression and PassingBablok regression can be applied in the evaluation. We chose Deming regression, because itis appropriate for describing the relationship between two variables, both measured witherror. In the case of observed increasing imprecision, i.e. where a proportional bias betweenmethods is detected, the Passing Bablock regression procedure is more accurate thanDeming’s method. When the assumption that the independent variable is determinedwithout error is satisfied, linear regression should be used to describe the agreementbetween two methods (Jones & Payne, 1997). The intercept is calculated, as in conventionalleast squares regression, as the mean of y minus the product of the slope and the mean of x.The standard error (SE) of the intercept defines how much the line might vary in the ydirection, and SE of the slope defines how much the line might pivot about the central pointthrough the means of x and y. Thus, SEs allow calculation of the confidence intervals of theslope and the intercept (Jones & Payne, 1997).2.3 Experiment: Agreement between two methods of sperm concentrationmeasurementIn the present study we compared two clinical laboratory methods for determining boarsperm concentration, the Makler chamber and the photometer (Photometer SDM5, Minitüb,Germany) (Mrkun et al., 2007). Prior to method comparison, precision of each method wasassessed. Scatter plots with fitted regression line, and absolute and relative bias plots wereused to get the best overview of comparative data (Twormey, 2004; Twormey, 2005).Deming regression was applied to describe the relationship between variables bothmeasured with error by proposing that the sum of the squares of the deviations from a lineshould be minimised in both the x and the y directions at the same time, thus taking accountof the analytical imprecision of each method (Jones & Payne, 1997). The purpose of thisstudy was to compare the two methods and to assess method agreement together with theappropriate regression analysis used in the interpretation of the data.

Evaluation of a New Method and Diagnostic Test in Semen Analysis 1352.3.1 Semen samplesTwenty-three semen samples were obtained from eight 12 to 24 month old boars of variousbreeds. Each semen sample was collected with gloved hand using a clean semen collectingflask that filters out gel, dust and bristles, while the boar mounted a dummy sow. Semensamples were diluted 1:2 with BTS semen extender (Beltsville Thawing Solution, Truadeco,Netherlands) and delivered to the laboratory.2.3.2 Counting with the Makler chamberImmediately before each semen aliquot was analysed, the entire semen specimen wasvortexed. To render the spermatozoa immotile and to prepare the semen samples for theMakler chamber (Sefi Medical Instruments, Israel), semen samples were diluted 1:2 withdistilled water. 6 parallel dilutions of each semen sample were prepared and the average ofthe measurements on each used as the representative value.Following dilution, sperm suspensions were again vortexed and an aliquot of 5 µl wasloaded into the Makler chamber. The next step was to assess whether sperm were evenlydistributed or whether there were movements in the fluid in the counting chamber. If eitherof these problems was observed; the chamber was cleaned and refilled. The fields werechosen according to a prescribed pattern: 10 fields spaced left to right and 10 fields spacedtop to bottom. Chosen fields formed a plus sign centred in the middle of the chamber,excluding the areas 2-3 mm from the chamber edges. Only recognizable spermatozoa,including lost heads, were counted, while other cells and lost tails were ignored. Theconcentration in the original semen sample was calculated from the total number of spermin the counting area.2.3.3 Counting with a photometerSperm concentration was determined by measuring the sample opacity, as the percentagetransmittance of light through a sample, using a photometer (Photometer SDM5, MiniTüb,Germany). Boar ejaculates are normally too opaque, so a small semen sample was dilutedwith an isotonic solution before measuring. A blank tube was loaded with 3.5 ml 0.9% NaCland a sample tube with 70 µl semen sample added to 0.9% NaCl. Sperm concentration wasdetermined from a previous calibration of the spectrophotometer, performed by themanufacturer (Photometer SDM5, MiniTüb, Germany). Six measurements were made foreach semen dilution.2.3.4 Precision of the evaluated methodsThe precision of each method was determined by making 6 measurements of each of 23semen samples. Coefficients of variation (CV) were calculated for each method and scattergraphs of CV versus average sperm count for each semen sample were constructed. In ourstudy CVs were calculated to be 6.6 3.5 % and 1.6 0.6 % for Makler chamber andphotometer, respectively. Both methods yielded acceptable precision (Christensen et al.,2005), although the precision of the Makler chamber was significantly poorer.In a diagram of the CV plotted against the average for each sperm concentration, the scatterof the points is random for the photometer (Fig.1). In contrast, for the Makler chamber, thesize of CV is related to the size of the sperm concentration, shown by the higher CVs forlower sperm counts (Fig.2).

136Artificial Insemination in Farm Animals4.03.53.02.5CV (%)2.01.51.00.50.00 50 100 150 200 250 300 350mean c (M/ml) - photometerFig. 1. Coefficient of variation (CV) versus mean sperm count for the photometer methodMean counts were calculated as the average of six parallel counts for each sampleCV (%)1816141210864200 50 100 150 200 250 300 350mean c (M/ml) - Makler chamberFig. 2. Coefficient of variation (CV) versus mean sperm count determined by the Maklerchamber methodMean counts were calculated as the average of six parallel counts for each sample

Evaluation of a New Method and Diagnostic Test in Semen Analysis 1372.3.5 Method agreement between Mackler chamber and photometerWe were interested in assessing the similarity between sperm counts measured with Maklerchamber and photometer, so we compared pairs of measurements. For this purpose, wecalculated the differences between pairs of measurements of sperm counts – by Maklerchamber and photometer – obtained by each method for each sperm sample.The mean percentage bias between methods was – 0.6 6.9 %. The scatter of the points liesin the interval –15 to 15 % (Fig. 3), which is in the range of satisfactory between-runreproducibility of the assay. From the absolute bias plot (Fig. 4) it is also evident that thescatter is random, indicating that the size of the difference between sperm counts obtainedby two methods is not related to the size of the counts. Thus, no proportional bias has beendetected. Average absolute bias was close to zero (–1.092 15.237 M/ml). Sperm countsobtained with Makler chamber and photometer agree; 90% of the differences lie within thelimits of agreement (Fig. 4), confirming that the level of agreement between the methodswas satisfactory. Therefore, measurements of sperm concentration with photometer andcounting chamber techniques are equally appropriate for estimating sperm counts.Using scatter diagrams with regression lines fitted, we established that the pairedmeasurements, sperm counts obtained with Makler chamber and with photometer, wereclose to the line of equality. Deming regression was used to solve the problem of describingthe relationship between sperm counting with methods, both measured with error.Deming’s method gives only a single regression line, whether x or y is used as the‘’independent variable’’ (Fig. 5).The estimated intercept for the regression line, 4.7069 M/ml, does not differ much fromzero. The estimated regression equation indicates that the points are close to the line ofequality, i.e. the 45 line and SE of the slope (0.0600) indicates that there is almost nopivoting of the line about central point through the means of x and y.4530perentage bias (%)150-15-30-450 50 100 150 200 250 300 350mean c(M/ml) Makler - photometerFig. 3. Relative bias plot of sperm concentration obtained by Makler chamber versusconcentration obtained by photometer

138Artificial Insemination in Farm Animalsabsolute bias c (M/ml) Makler - photometer5040upper limit of agreement3020100average bias-10-20-30lower limit of agreement-40-500 50 100 150 200 250 300 350 400mean c (M/ml) Makler - photometerFig. 4. Absolute bias plot of sperm concentrations obtained by Makler chamber versusconcentrations obtained by photometer showing average bias and limits of agreement350300c (M/ml) - photometer250200150100Identity lineY = Xy = 0,9706x + 4,70695000 50 100 150 200 250 300 350c (M/ml) - Makler chamberFig. 5. Scatter diagram of sperm concentration obtained by photometer versus spermconcentration obtained with Makler chamber, with Deming regression line fitted- - - - - : line of equality (Y=X)______ : Deming regression line:c (photometer) = 4.7069 + 0.9706 x c (Makler chamber)

Evaluation of a New Method and Diagnostic Test in Semen Analysis 1393. Development and diagnostic evaluation of spectrophotometric applicationof the resazurin reduction assay to evaluate boar sperm qualityThere are several attributes that a sperm must posses to fertilize an oocyte, includingmotility, normal morphology, sufficient metabolism for energy production, and membraneintegrity. Although various analytical techniques have been developed to evaluate spermquality, including sperm concentration, motility, viability and morphology, there is nosingle method that provides complete information about semen quality (Holt & Medrano,1997; Johnson et al., 2000). Due to the complexity of the fertilization process, single tests arenot able to predict fertility. Instead, a set of semen tests has to be selected with highrelevance for important sperm traits and low redundancy of assay results (Petrunkina et al.,2007). Moreover, particularly in pig industry, the choice of semen test has considered costeffectiveness. Routine testing of fresh boar sperm predominantly aims to identify subfertileboar ejaculates. In number of countries, liquid preserved boar semen is used after severaldays of in vitro storage. It’s well known that boars differ in their capacity to maintain spermfunction during preservation in vitro. These differences can only be partially visualized bystandard sperm parameters, such as loss of motility and membrane integrity (Waberski etal., 2011). However, the battery of diagnostic methods used by the industry is as yetrestricted (Tejerina et al., 2008). A reliable, simple, cost effective and rapid method ofassessing the quality of boar spermatozoa would be of benefit to livestock producers andveterinary practitioners (Dart et al., 1994). Reproductive performance depends on metabolicprocesses; therefore assessment of metabolic status of spermatozoa could provide valuableinformation for predicting sperm fertilizing capacity. The resazurin reduction assay, one ofthe methods for evaluating the metabolic status of spermatozoa, depends on the ability ofmetabolically active spermatozoa to reduce the resazurin redox dye to resorufin.Dehydrogenase activity of spermatozoa is manifested as a visual colour change from blue(resazurin) to pink (resorufin) and further to white (dihydroresorufin) (Glass et al., 1991;Fuse et al., 1993; Reddy Venkata Rami et al., 1997). The resazurin reduction assay usingvisual detection of colour change is quite subjective and varies between evaluators (Wang etal., 1998). The colour change of resazurin is usually matched with a colour chart, consistingof a spectrum of colours from blue to pink, varying between investigators. The possibility ofhuman error therefore, has led to the spectrophotometric modification of the resazurinreduction test. It has been mostly used for the evaluation of human semen (Mahmoud et al.,1994, Rahman & Kula, 1997; Zalata et al., 1998; Reddy Venkata Rami & Bordekar; 1999) but,to our knowledge, in veterinary medicine only for evaluating ram (Wang et al., 1998) andboar semen quality (Zrimšek et al., 2004). The visual assay has been used for evaluatingstallion (Carter & Ericsson, 1998), bull (Dart et al., 1994), sheep (Cooper et al., 1996; Martin etal., 1999) and boar (Mesta et al., 1995) semen. Spectrophotometric measurement of resazurinreduction provides a quantitative and objective method.The aim of the present study was to develop and evaluate diagnostically thespectrophotometric application of the resazurin reduction test for evaluating boar spermquality (Zrimšek et al, 2004; Zrimšek et al., 2006). Following Zalata et al. (1998), who developeda spectrophotometric method of resazurin reduction to evaluate human semen we extractedthe developed colour after the assay with boar semen with butanol and measured theabsorbance in the clear upper layer of butanol, eliminating the problem of sample turbidity.The optimisation and developing of the test included several steps as follows:- determination of specific absorbance wavelength, used for analysis on the basis ofabsorbance spectra of resazurin and resorufin

140Artificial Insemination in Farm Animals- optimisation of the test procedure- determination of repeatability of the assay- correlations between resazurin reduction assay and various semen parameters;Spearman rank correlation analysis- relationship between resazurin reduction and concentration of motile spermatozoa andsperm index; linear regression analysis- statistical comparison of the results obtained between the groups of satisfactory andunsatisfactory semen; Mann-Whitney U-test- diagnostic evaluation of the assay; ROC analysis- stability of butanol extracts in terms of A 610 ; measuring agreementIn this study, receiver operating characteristics (ROC) was used to determine the optimalcut-off value and diagnostic accuracy of the resazurin reduction assay. A complete pictureof test accuracy is presented by the ROC plot, which provides a view of the whole spectrumof sensitivities and specificities as functions of selected cut-off values (Greiner et al., 2000). Aglobal summary statistic of the diagnostic accuracy of the assay was quantified by the areaunder the ROC curve. Likelihood ratios were used to revise the probability of the semenstatus in individual samples (Greiner et al., 1995).3.1 Development of resazurin reduction assay3.1.1 Semen samples and analysisForty-one semen samples from eight 12-24-month-old boars of various breeds were includedin the study. Semen was collected with a glove hand using a clean semen collecting flaskthat filters out gel, dust and bristles, while the boar mounted a dummy sow. Semen waskept at the temperature collected and analyzed within 1 h. Sperm concentration and motilitycharacteristics were determined by computer-assisted semen analysis (Hamilton ThorneIVOS 10.2; Hamilton Thorne Research, MA, USA) with a Makler counting chamber (SefiMedical Instruments, Haifa, Israel). Sperm morphology was examined on Giemsa-stainedsamples (Hafez, 1993). Sperm index (SI) was calculated by multiplying sperm concentrationby the square root of percentage sperm motility multiplied by the percentage of normalsperm morphology (Mahmoud et al., 1994). Combining concentration, motility andmorphology in sperm index allows the concentration of active spermatozoa to bedetermined, and may provide a better means of evaluating semen quality than assessing thecharacteristics, mentioned above, independently.3.1.2 Determination of specific absorbance wavelengths of resazurin and resorufinBefore developing the assay, specific absorbance wavelengths of resazurin and resorufinwere determined. Ten μl 1.8 mM resazurin (Sigma, Steinheim, Germany) in physiologicalsaline was added to 1 ml of 1:2 dilution of semen sample in BTS and incubated at 37˚C in awater bath. After the semen sample completely turned to pink, the developed dye(resorufin) was extracted from the solution by adding n-butyl alcohol (Sigma, Germany) andfast vortexing. The control sample (blue colour solution) was prepared by adding butanolimmediately after the resazurin. After centrifugation, the blue (resazurin) and pink(resorufin) solutions were separated from the clear upper layers of butyl alcohol and werescanned in the range from 400 to 850 nm, using a scanning spectrophotometer (UV/VISSpectrometer Lambda 12, Perkin Elmer). Resazurin exhibits an absorption peak at 610 nm,

Evaluation of a New Method and Diagnostic Test in Semen Analysis 141while that of resorufin is at 575 nm (Fig. 6). There was minimal overlapping betweenabsorption peaks of resazurin and resorufin at 610 nm; therefore the absorbance at 610 nmwas used in further analysis.Fig. 6. Specific absorbance wavelengths of resazurin () and resorufin ()3.1.3 Resazurin reduction assay and correlation with semen parametersThe resazurin reduction assay was performed within 1 h after semen collection. Briefly,30µL of 1.8mmol/L resazurin (Sigma, Steinheim, Germany) diluted in physiological salinewas added to 3mL of semen sample diluted 1: 2 with Beltsville thawing solution semenextender (Beltsville Thawing Solution, Truadeco, the Netherlands) and incubated at 37°C ina water bath for 10min. After incubation, two sub-samples of 1mL were added to 1.5 mL ofbutanol (Merc, Germany). After rapid vortexing, samples were centrifuged at 3 000×g for10min. Absorbance in the clear upper layer of butanol was measured at 610mm (UV/VISSpectrometer Lambda 12; Perkin Elmer Corp., Analytical Instruments, Norwalk, CT, USA).The within-run coefficient of variation, calculated as 7.79±4.06 %, confirmed satisfactoryrepeatability of the assay. Spearman rank correlation analysis was used to determine thecorrelation between resazurin reduction assay and semen parameters such as sperm density,morphology, motile sperm concentration, viable sperm concentration and sperm index. Weobserved the highest correlations of resazurin reduction with sperm concentration followedby motile sperm concentration and viable sperm concentration. Inverse correlations indicatethat better values of seminal parameters are correlated with a lower level of absorbance,indicating a stronger reducing capacity of the dye (resazurin). There were correlations(P

142Artificial Insemination in Farm Animals0.50.40.3A 6100.20.10.00 50 100 150 200 250 300conc motile spermatozoa (x 10 6 / ml)Fig. 7. Relationship between the reductions of resazurin expressed as absorbance and theconcentration of motile spermatozoaRegression equation: C (motile spermatozoa) = 258.345 – 0.325 x A 6100.50.40.3A 6100.20.10.00 50 100 150 200 250 300SI (x 10 6 /ml)Fig. 8. Relationship between the reductions of resazurin expressed as absorbance and spermindexRegression equation: SI = 250.546 – 0.339 x A 6103.2 Diagnostic evaluation of resazurin reduction assaySemen samples were divided into satisfactory (SAT) and unsatisfactory (UNSAT) accordingto various criteria. Criteria considering the concentration of motile sperm included preselectedminimums of 160, 200 and 240×10 6 sperm/mL. Criteria considering theconcentration of motile, normal sperm (SI) included pre-selected minimums of 140, 180 and220×10 6 sperm/mL. There was a significant difference between the absorbance values in

Evaluation of a New Method and Diagnostic Test in Semen Analysis 143groups of satisfactory and unsatisfactory semen samples (P

144Artificial Insemination in Farm AnimalsSe = TP/(TP+FN) (2)Sp = (TN)/(TN + FP) (3)Semen samples statusTest resultUnsatisfactoryTotalSatisfactory (SAT)(UNSAT)Positive (T+) True positive (TP) False positive (FP) TP + FPNegative (T-) False negative (FN) True negative (TN) FN + TNTotal TP + FN FP + TNTable 1. Complete 2x2 tableROC curves plotted all sensitivity versus 1-specificity for the complete range of cut-offpoints (Greiner et al., 2000; Yuan et al., 2004). Sensitivity and specificity were estimated at 39cut-off values. A diagonal line in a plot corresponds to a test that is positive or negative justby chance.All possible combinations of sensitivity and specificity that can be achieved by changing thetest’s cut-off value were summarized by a single parameter; that is, AUC (Greiner et al.,2000). The slope of the ROC curve represents the LR for a diagnostic test, and the tangent ata point on the ROC curve corresponds to the LR for a single test value represented by thatpoint (Choi et al., 1998).LR=Se/(1-Sp) (4)The optimal cut-off values were selected based on the best balance of sensitivity, specificityand Youden index (J) along with larger increases in LR for each criterion value (Weiss et al.,2003-2004).J =Se+Sp-1 (5)The diagnostic potential of resazurin reduction assay according to motile spermconcentration and SI was not different on the basis of AUC. The AUC was the same forcriteria of 200×10 6 motile sperm/mL and 180×10 6 motile, normal sperm/mL (AUC=0.92;standard error for ROC curve (SE)=0.047 and 0.048, respectively; P

Evaluation of a New Method and Diagnostic Test in Semen Analysis 145A1.00.8sensitivity0.60.40.2c=160 M/ml, AUC=0.960c=200 M/ml, AUC=0.922c=240 M/ml, AUC=0.8850.00.0 0.2 0.4 0.6 0.8 1.01 - specificity (false positive rate)B1.00.8sensitivity0.60.40.2SI=140 M/ml, AUC=0.903SI=180 M/ml, AUC=0.922SI=220 M/ml, AUC=0.9480.00.0 0.2 0.4 0.6 0.8 1.01 - specificityFig. 10 (A, B). ROC plots of resazurin reduction assay for identifying semen samples withpre-selected minimum concentration of motile sperm concentration (A) and motile andnormal sperm (B).However, in clinical use of the test, it is often important to 100% correctly identifysatisfactory or unsatisfactory samples. Therefore, a cut-off value of A 610 at 0.342 was selectedto enable 100% correct identification of unsatisfactory semen samples. For both criteria, thetest is 100% sensitive at A 610 of 0.342. A cut-off value at A 610 of 0.121 gives 100% specificityfor motile sperm concentration and 95.8% specificity for SI. For pre-selected minimumconcentration of motile sperm concentration of 160×10 6 sperm/mL and SI of140×10 6 sperm/mL, 100% specificity was obtained at the optimal cut-off value of A 610 at0.254, whereas only moderate levels of sensitivity were observed (80.6% and 73.5%,respectively; Figures 11A and 12A). In contrast, at the highest criteria values 100%sensitivity corresponded to only moderate levels of specificity (Figures 11C and 12C). Incontrast, semen samples with A 610 below 0.121 in the resazurin reduction assay were 100%

146Artificial Insemination in Farm AnimalsAvalidity estimate1.00.80.60.40.2c = 160 M/mlsensitivityspecificityYouden index0.00.0 0.1 0.2 0.3 0.4 0.5cut-off at A 610Bvalidity estimate1.00.80.60.40.2c = 200 M/mlsensitivityspecificityYouden index0.00.0 0.1 0.2 0.3 0.4 0.5cut-off at A 610CCvalidity estimate1.00.80.60.40.2c = 240 M/mlsensitivityspecificityYouden index0.00.0 0.1 0.2 0.3 0.4 0.5cut-off at A 610Fig. 11 (A, B, C). Plot of the diagnostic specificity, sensitivity and Youden index of resazurinreduction assay according to motile sperm concentration as a function of the cut-off value at610nm.

Evaluation of a New Method and Diagnostic Test in Semen Analysis 147Avalidity estimate1.00.80.60.40.2SI = 140 M/mlsensitivityspecificityYouden index0.00.0 0.1 0.2 0.3 0.4 0.5cut-off at A 610Bvalidity estimate1.00.80.60.40.2SI = 180 M/mlsensitivityspecificityYouden index0.00.0 0.1 0.2 0.3 0.4 0.5cut-off at A 610Cvalidity estimate1.00.80.60.40.2SI = 220 M/mlsensitivityspecificityYouden index0.00.0 0.1 0.2 0.3 0.4 0.5cut-off at A 610Fig. 12 (A, B, C). Plot of the diagnostic specificity, sensitivity and Youden index of resazurinreduction assay according to sperm index as a function of the cut-off value at 610nm.

148Artificial Insemination in Farm Animalsand 95.8% correctly identified as satisfactory according to the criteria of 200×10 6 motilesperm/mL or 180×10 6 motile, normal sperm/mL, respectively. In our quantitative test, themaximum overall accuracy of 92.9% confirmed the high discrimination power for boarsemen according to a criterion value of SI at 180×10 6 sperm/mL.3.3 Stability of butanol extracts in terms of A610After developing the assay, we wondered if it was possible to measure the absorbance at alater date, i.e. within a day or even a week of the assay. A satisfactory level of agreementwould indicate that the modification was successful, which in turn would greatly enhancethe usefulness of the assay as it could then be performed even if a spectrophotometer wasnot immediately available.We measured the A 610 of each butanol extract of 112 samples on days 0, 1 and 7 after storageat 4C. The differences were obtained between A 610 at day 0 (A0) and day 1 (A1) andbetween days 0 (A0) and 7 (A7).The limits of agreement were calculated as follows: limits = d 2sdiff , where d is themean of differences for all the samples, and sdiff is the standard deviation of the differences.2sdiff is also named the reproducibility coefficient. Differences between absorbances (A1 -A0) were plotted against their average value ((A1 + A0)/2) for each sample. Satisfactoryagreement is achieved when minimum 95% of the absolute differences are less than thereproducibility coefficients (Petrie & Watson, 1999).It is necessary to establish that a method is repeatable before comparing two measurementsfor reproducibility (Petrie & Watson, 1999). The within-run coefficient of variation,calculated as 7.79 4.06 %, confirmed satisfactory repeatability of the method, therefore thepairs of measurement of A610 were allowed to compare. The differences betweenmeasurements (A 610 ) immediately after centrifugation (day 0) and after 7 days were plottedagainst the average of these values. 95.54 % of differences lie within the limits of agreement(Fig. 13).4030difference in A 61020100-10-20upper limit of agreementaverage of differenceslower limit of agreement-300 100 200 300 400 500 600average A 610Fig. 13. A 610 values as a function of time of measurementMeasurements obtained on the day of performing the test and the measurements after 24hours also agree; 99.1 % of the differences lie within the limits of agreement (data notshown). The results obtained leading to the conclusion that we can measure A 610 of butanol

Evaluation of a New Method and Diagnostic Test in Semen Analysis 149extracts within 7 days from the day of test performing, confirming a great practical value ofthe method.In a diagram of the differences between absorbances plotted against their average, thescatter of the points is random (Fig. 13) indicating, that the size of the discrepancy betweenthe two absorbance is not related to the size of the absorbance. More than 95 % of absolutedifferences were less than the reproducibility coefficients in both cases of testing the stabilityof butanol extracts. This is a satisfactory agreement, therefore we can measure theabsorbance immediately after performing the test or within 7 days of that time. Thereforethe test is useful even if spectrophotometer is not available at the location of semenevaluation. The results obtained leading to the conclusion that we can measure A 610 ofbutanol extracts within 7 days from the day of test performing, confirming a great practicalvalue of the method.4. ConclusionsThe usefulness of sperm counting is greatly enhanced by the simplicity of determination byphotometer (Photometer SDM5, MiniTüb, Germany) in on-farm AI laboratories. The use ofphotometer for determining sperm concentration would, therefore, be of benefit also tolivestock producers in evaluating the quality of boar semen.The resazurin reduction assay was shown to be a reliable, easy-to-perform test that requiresno sophisticated equipment. It was demonstrated that the results of the assay can be used toselect semen samples with minimum requirements of sperm concentration, motility andnormal morphology, which are all combined in sperm index. Because reproductiveperformance depends on metabolic processes, the assessment of metabolic rates ofspermatozoa could provide even better or more complete information about semen qualitythan other tests. It allows the concentration of active spermatozoa to be determined, andmay provide a better means of evaluating semen quality than assessing the characteristics,mentioned above, independently. Expressing the latter in semen evaluation is complex,although fertility results from insemination with evaluated semen could provide a goldstandard of fertilizing capacity. Additional research is required for relevant and validinformation about replacing or updating the methodology of semen evaluation.5. AcknowledgementsThis work was supported by the Slovenian Ministry of Higher Education, Science andTechnology, programme group ''Endocrine, immune, nervous and enzyme responses inhealthy and sick animals'' (P4-0053).Special thanks go to author's collegues who contributed to the research work, presented inthis chapter: Janko Mrkun, DVM, PhD, Marjan Kosec, DVM, PhD, Janez Kunc, DVM, MSc,Maja Zakošek Pipan, DVM.6. ReferencesAnzar, M., Kroetsch, T., Buhr, M.M. (2009). Comparison of different method for assessmentof sperm concentration and membrane integrity with bull semen. J Androl, 30, 6, pp.(661-668)

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10Particularities of Bovine Artificial InseminationAntônio Nelson Lima da Costa, Airton Alencar de Araujoand José Valmir FeitosaFederal University of CearaBrazil1. IntroductionIn beef production systems a good reproductive performance is essential for the efficientmanagement and production. The production of cattle can be divided into two sectors: dairyand meat production. In many European countries and developing countries, even the cattleis used as a source of meat and milk and are called dual fitness. Unlike in countries such asAustralia, Brazil and the United States, the functions of production of meat and milk wereseparated and the creation of selective breeding is directed to a single feature.The productive and reproductive performance of cattle herd is directly related to the serviceperiod, calving interval, number of service per conception and number of calves weaned.Artificial insemination (AI) has been proven worldwide and has proven to be a viabletechnical and economical to increase the genetic gain and increase efficiency, especially inproduction systems for meat and milk.In cattle, AI despite presenting a series of known proven advantages, gradually beingreplaced by fixed-time artificial insemination (FTAI) because of lack of skilled labor,logistical problems in large AI programs, in failures detection of estrus; costs forimplementing the program, no optimization of herd reproductive efficiency, and thedifficulty of practical application in field conditions.In this chapter discuss techniques to improve conception rates using artificial insemination,such as: improved detection of estrus, a reduction in calving interval, implementation ofFTAI; calving and breeding seasons, care with the semen, body condition score of females,the female gynecological evaluation, diagnosis of pregnancy, parturition rate.After the reading of these topics, many barriers bovine artificial insemination will beelucidated, and the artificial insemination technique is best applied in various conditions ofmanagement of cattle herds worldwide.2. Improvement in detection of estrusWorldwide there are reports that indicate low rates of service in artificially inseminatedcattle, mainly due to problems in the detection of estrus. While few cows are detected inheat losses occur in significant herd reproductive efficiency, and commitment of the artificialinsemination program. This commitment is even higher in Bos indicus cattle, whose breedingbehavior has special features - heat of short duration with a high percentage of expressionduring the night (Galina et al., 1996, Pinheiro et al., 1998). This feature was confirmed with

154Artificial Insemination in Farm Animalsthe radiotelemetry system (Heat-watch) in Nelore, Angus x Nelore and Angus to the terrainunder the same management (Mizuta, 2003). The results are indicative that the heat ofNelore (Bos indicus) and Nellore x Angus is about 4 hours less than the duration of estrusAngus cows (Bos taurus).The secretion of estrogen, a manifestation of oestrus LH surge and ovulation are closelyrelated and well known. With follicular growth, the amount of estrogen secreted increasesto a peak serum concentration, triggering a preovulatory LH surge, follicular maturationand ovulation, lasting 27 hours. The goal of increased concentrations of estrogen istriggering hormonal cascade of events that includes the LH surge and a series of changesthat promote follicular ovulation, and sexual behaviors associated with acceptance ofmounts.The main characteristic of estrus is the posture of immobility assumed by acceptance of thecows and ride. High producing cows milk manifest estrus of shorter duration than cowswith lower production (Lopez et al., 2004). Females of childbearing age are pregnant or inthe luteal phase of the cycle (under the domain of progesterone) are less likely to mountother females are in estrus. Almost 86% of females who ride other females are in estrus andproestrous (under the domain of the estrogen). Consequently, it should be kept open orbarren females with other similarly to occur as much sexual behavioral interactions (Helmerand Britt, 1985). The type of flooring is essential for the expression of mounts andimmobility. When you can choose, cows in estrus spend 73% of time on the ground and noton the concrete and assemble increases by 15 times in paddocks of land in relation to theconcrete. Moreover, the duration of estrus and immobility also increases on gravel (Vail &Britt, 1990).Over the years various devices have been developed to detect estrus and they were effectiveas an aid in visual identification, as Bos indicus tend to present turnover night (Baruselli etal., 2004). These devices range from the tail to paint the most sophisticated pedometers(figure 1) and tags that track electronic activity detectors and electronic pressure"HeatWatch. The pedometers have been used to measure activity or movement of the cowthrough a microprocessor chip and miniature device is fixed to a collar or bracelet. A cow inestrus walks about 4 times more than another that is not in estrus. These devices can beaccessed manually or automatically when the cow enters the milking parlor or throughreceiving antennas mounted in the stalls. The information is sent to a computer andcompared with individual basal activity of the same cow over a similar time interval of 2 or3 days earlier. If the activity has increased significantly this cow, this cow is identified by awarning light that flashes on the ankle or on the computer, generating a warning report tothe responsible to verify whether it is in heat and should be inseminated (Stevenson, 2001).Using these devices in heifers, it was found that they are effective in identifying animalswith short periods of estrus and few events of immobility. When compared the accuracy ofheat identification of 49 heifers with synchronized estrus, with pedestrian detection by thefarm, it failed to comply with estrus in 13 of 49 heifers (26%), while the electronic deviceidentified all foodstuffs (Stevenson et al., 1999). Remembering that the aids can be used toincrease the efficiency of our detection of estrus, but not to replace it.Intensive detection of estrus was defined as 2 h of heat detection in the mornings andafternoons and one additional hour of estrus detection around noon. The detection wasdefined as a casual observation of the cows in the mornings and afternoons for 30 minutes.In the same herd, cows of both groups underwent intensive observation and casual, were

Particularities of Bovine Artificial Insemination 155Fig. 1. Cow with pedometer in previous membersynchronized with the same protocol and inseminated by the same technician. In animalswith the intensive detection of estrus, the percentage of cows observed in estrus increased in30% conception rate in 20% and pregnancy rate doubled in this group. The highestconception rates among cows intensively observed may have been the result of a moreappropriate time of artificial insemination relative to ovulation (Geary et al., 2000).Whenever is observed a cow in heat, it should be removed from the herd as soon as possiblebecause it diverts attention from other cows are in heat in two ways. First, when are makingthe detection of estrus, each time a cow is in a position to accept mounts noted his number.If the cow is being mounted, has been identified previously, then get distracted and fail tosee other cows. Second, there is always regarded as timid cows to avoid the pushing ofestrous behavior and estrous pass through undetected. These cows may show signs of estruswhen the cow in estrus ruling was removed. The only time when one considers thepossibility of letting the cows are in heat in the pasture to help identify other cows in heat iswhen you do not make use of estrus synchronization and when it is expected that less than5% of cows in the herd come into estrus per day (Geary et al., 2005).All of estrus detection devices should be used cautiously and their results interpreted bytrained people, since people are the most important component in any program of estrusdetection and artificial insemination. The ability to detect estrus in the interpretation andcommon sense are key to the success of an accurate detection of estrus and this increases therate of submission to artificial insemination.3. Reducing the gap between birthsCalving interval (CI) covers the period between two consecutive deliveries from a cow and ayearly calving interval is a common goal for both beef producers and for the milk. Longintervals are uneconomical because cows with extended intervals produce little milk andfewer calves per year. It is one of the main parameters to measure the reproductiveefficiency of cattle.The CI can be divided into two components: the interval from calving to conception andduring pregnancy. The first covers the period from calving to the establishment of a newpregnancy and is the main determinant of CI being the parameter usually manipulated to

156Artificial Insemination in Farm Animalsachieve a desirable CI. The second component comprises an average of 285 days, varyingonly according to the genetics of both the matrix and the player and can only be reduced byinducing premature parturition.To get an CI for 365 days, the interval between delivery and conception should not be longerthan 80-85 days. A key factor for achieving these goals is good nutrition. A study conductedin Florida stressed the importance of dry matter intake for cows in early cyclicity. Cows withhigher dry matter intake returned to cycle faster and lost less weight postpartum (Staples etal., 1998). Care of the dry matter intake and body condition is essential and should beginwell before birth in the last 100 days of lactation. So there will be enough time to adjust bodycondition and prepare all the cows in their dry period and lactation next breeding season.The relationship between energy balance and ovulation is directly related to the pattern ofLH secretion, which is the gonadotropin responsible for follicular maturation and ovulation.After delivery, there is limited secretion of LH, but as approach the time of first ovulation,the basal concentration of LH secretion increases and the pattern becomes more pulsatile.The LH pulse frequency should occur at hourly to support follicular maturation andovulation. The first ovulation occurs two weeks after the increased frequency of LH pulses.Thus, it should provide a well balanced diet and palatable to cows to meet their metabolicneeds as soon as possible after birth, stimulating consumption fractionated into four meals aday until the cows to produce milk and ovulate early, reducing the CI (Butler and Smith,1989).Another factor that must be considered is the diagnosis of pregnancy, IE this should be doneup to 30 days after artificial insemination, and the empty livestock referred for proceduressuch as fixed-time artificial insemination. Resynchronization of repeat breeder cows is alsoan alternative to reduce the CI, because prior to the diagnosis of pregnancy can identifyanimals that are empty and reinseminated them, improving conception rates and shorteningthe CI (Chebel et al., 2006). Furthermore can mention other factors that influence theduration of the CI as appropriate handling and deposition of semen, the ability of artificialinsemination, animal care not to subject him to stress management, such as abuse, alsocontribute a good mineralization for the appearance of fertile estrus, improvement in thedesign and decrease the CI.4. Application of fixed-time artificial inseminationThe fixed-time artificial insemination (FTAI) has become very popular because of reducedmanpower and independence of estrus detection, since there are many difficulties to detectcows in heat, especially at night. The rates of detection of estrus and submission to artificialinsemination are limiting factors due to the type of installation, which limit the expression ofestrus, or lack of skilled labor for estrus detection. For artificial insemination programs aresuccessful in herds not using hormonal intervention, the rate of estrus detection shouldexceed 70%. Most programs result in FTAI pregnancy rates similar to those obtainedfollowing the detection of natural estrus.The inefficiency of reproductive cows causes great frustration and losses for farmers. Evenunder ideal conditions, the reproductive process is far from perfect, considering the myriadfactors involved in producing a live calf. The reproductive efficiency involves not onlyproper management of cows, but also management of carers and inseminate. Theconception rates of lactating dairy cows declined from the 50's in the USA, while the annualyield per cow increased 3.3 times (Lucy, 2001). Given the inverse relationship of milk yield

Particularities of Bovine Artificial Insemination 157and fertility, there is a genetic antagonism between milk yield and reproductive traitsmanifested primarily in first lactation cows, but good management practices such as use ofFTAI, can overcome this inverse correlation and allowed its acceptable levels ofreproductive efficiency.The programmed insemination is a method that plans and manages a program of artificialinsemination of a herd. This lineup of estrous cycles has some advantages such asconvenience of scheduling tasks and use of manpower, control the occurrence of estrus,ovulation, or both, and knowledge of the estrous cycle and reproductive status of females inthe herd. The categories of reproductive status are scheduled to open cows at first artificialinsemination, cows inseminated to be subjected to pregnancy diagnosis, cows scheduled toopen re-insemination; open cows destined for disposal (will not be inseminated) and cowsconfirmed pregnant. Thus, the programmed insemination can be applied in two distinctgroups: cows that are scheduled for their first insemination and cows confirmed to beopened after diagnosis of pregnancy and scheduled for re-insemination. The programmedinsemination involves hormonal timing of estrus, ovulation, or both. In the USA, FTAIprograms are used in 90% of herds, with 86% of synchronizing first services, 77% of resynchronizationof repeaters and 59% of treatments for cystic cows in anovulatory oranestrus (Caraviello et al., 2006).The best program is the simplest and easiest to apply, varying with the level of training ofofficials of the farm. In this case, simplicity means lower number of manipulations in cowsduring the implementation of a protocol. Employees should be trained to take a disciplinedapproach and comprehensive programs work, i.e., the implementation of the protocolsshould be approximately 100% to achieve a greater number of pregnant cows and fewerdischarges of pregnant cows.Some studies have evaluated the cost-effectiveness of fixed-time artificial inseminationprotocols. One study estimated the value of a pregnancy in a flock where half of the cowswere inseminated only after the detection of natural estrus, compared to the application ofFTAI the other half of the cows. In herds with low estrus detection, the cost of pregnancywas significantly reduced with the use of FTAI compared detection only. In herds with highrates of detection of estrus, the cost of pregnancy was higher with the use of FTAI, but therewas a better reproductive performance of cows treated. The highest costs of pregnancy wereassociated with damage resulting from longer periods opened and account for up to 83% oftotal costs (Tenhagen et al., 2004). Thus, the use of a system of FTAI is considered aprofitable alternative for large commercial flocks in the estrus detection rates are low.5. Breeding seasons and calvingThe breeding season or insemination consists of a period of the year in which breedingfemales are exposed to males or are inseminated, and calving season is closely correlated,because the births are concentrated in a pre-determined by the breeding season orinsemination. It is a strategy for increasing productivity in terms of number of weanedcalves, zero cost, rationalizing the reproductive activity of animals with the concentration ofdeliveries, facilitating and regulating the management of livestock.The most widely used method of breeding in central Brazil is one where the bull stays withthe herd throughout the year. As a result, births are distributed throughout the year, despitea higher concentration during the months from July to September. The occurrence of birthsat times inappropriate affect the calves, due to higher incidence of diseases and parasites, or

158Artificial Insemination in Farm Animalsthe lower availability of pasture for sows during the lactation period. The biggest drawback,however, that limits the use of rides throughout the year, concerns the difficulty ofcontrolling the health of livestock and livestock due to lack of uniformity (age and weight)of animals. These factors ultimately affect the selection of cattle for increased reproductivepotential, rather than female fertility (Embrapa, 2011).The advantages in adopting the breeding season are many and are related to the calves, thematrices and the production system. With regard to calves, they will be born in morefavorable time of year with a lower incidence of ectoparasites and endoparasites, withproper nutrition will have a greater supply of fodder, assist in the formation of moreuniformity, reduced mortality, increased weaning weight and easy of recreating.Concerning the matrices, the mating season coincides with the increased availability offorage providing suitable conditions for the restoration of reproductive activity with higherpregnancy rates, select arrays to better reproductive efficiency after the breeding season anddiagnosis gestation selecting the best females, and lactating occurring over a period of goodsupply of food. The advantages to the production system is characterized in rationalizationof manpower, purchase of inputs less frequently, and much lower price, and ease ofadoption of other practices such as early weaning, supplementation of calves, estroussynchronization and artificial insemination of matrices (Santos, 2003).The choice of the breeding season depends on several factors such as climatic conditions,availability of pasture land, labor, adequate time for the birth of calves and purpose ofproduction, i.e., commercial or purebred animals. Based on these facts, it is much easier towork towards the establishment of a nature to breeding season in the property, aiming tostreamline the reproductive activity in both the biological and practical.When the producer chooses to adopt the breeding season of short duration (2-3 months),replacing the system where there insemination of cows throughout the year, we recommenda gradual reduction in the period of insemination, eliminating every year one to two monthsto reach the optimal duration, so there is no reduction in fertility. For heifers, it isrecommended to advance the breeding season in 30 days than cows, so they have more timeto restore their reproductive activity when they become primiparous. Adequate proteinsupplementation of females during the breeding and supply of mineral throughout the yearcontribute to elevation of the reproduction of the herd.6. Care of semenThe canister is an insulated container with vacuum insulation, for the preservation ofsemen, and for that he should receive liquid nitrogen, which preserves the doses of semenfrozen at a temperature of -196ºC (one hundred ninety-six degrees Celsius) indefinitely,provided they maintain a certain level, supplying them periodically. It should be handledwith the utmost care to avoid damage that may result in losses. To lessen the risks to thecanister, it is advisable to build a wooden box for packaging, avoiding shocks, movementsare too fast, and overturn spilling its entire contents.The liquid nitrogen evaporates constantly, and the inseminator is alert to prevent loss ofsemen due to lack of nitrogen. To do so, you should always measure your level withappropriate meter; never letting the level below 15 cm. High consumption of nitrogen canindicate problems with the canister as well as the formation of frost or condensate on anyexternal surface may also indicate defects.

Particularities of Bovine Artificial Insemination 159The thawing of semen at appropriate temperatures, according to the recommendations of thesupplier is required to maximize post-thaw survival and motility of sperm. The cold shock canbe avoided by maintaining the temperature, because when the blade is exposed for 30 secondsat room temperature, the temperature drops to 35ºC to 23ºC. Heating the artificialinsemination pipette and maintenance of the temperature of the semen to its insertion into thevagina prevents thermal shock. Only straws should be thawed enough to inseminate cows in10 minutes. All equipment must be kept artificial insemination extremely clean.The deposition of semen in inadequate reproductive tract may be a limiting factor when thecoach is not sure of the location of the pipette tip. Surveys show that less motile spermreaching the oviduct when the semen is deposited in the cervix. Insemination, the goal is toreach the body of the uterus. When in doubt, it is better to deposit the semen in one or bothuterine horns and fertility will be less compromised than if the semen is deposited only inthe cervix. As 85 to 90% of the semen is expelled from the female reproductive tract byretrograde flow, it is essential that the total dose is deposited in the uterus.The use of sexed semen has become common, but it is important to remember that it isdifferent from the conventional. To achieve 90% purity of a specific sex, sperm are treatedwith fluorescent dyes and X and Y chromosome sperm are separated by a cell separator(flow cytometry) based on fluorescence intensity after exposure to the laser beam. There aremany data Dairy Heifers, describing a design with sexed average, around 70% to 80% of thedesign of conventional semen used in the first service. The specific reason for this drop infertility in artificial insemination with sexed semen, as compared to conventional, is stillunknown. Nevertheless, given the potentially negative effects of the procedures for sexing,of course it is very important to the careful handling of sexed semen to optimize fertility.Sexed semen for commercial use, is currently stored in straws thin (0.25 mL), containing 2.1million sperm. Although 0.25 mL straws were handled similarly to 0.5 mL, the smallerdiameter makes them more sensitive to errors in handling semen. The deposition of sexedsemen in the uterus of the heifer must be as fast as possible, not exceeding 5 minutes.Fertility variations found after the use of sexed semen are quite large and are determined byseveral factors, including error handling and storage of semen. Handle carefully sexed andconsider the ongoing evaluation of procedures, because every successful artificialinsemination program starts with good handling practices Semen.7. Body condition score of femalesThe estimate of the nutritional status of ruminant livestock of interest by assessing bodycondition (BC) is a subjective measure based on the classification of animals with thecoverage of the muscle and fat mass. Therefore, the body condition score (BCS) estimates thenutritional status of animals by means of visual assessment and / or tactile and representsan important tool of management. The method is fast, convenient and cheap; it reflects theenergy reserves of the animals and serves as an aid in the identification of practices to beadopted in the nutritional management of the herd.The assessment of body condition or its variation to estimate body reserves is moreappropriate than the measurements of body weight, for its analysis independent of the sizeand physiological status of the animal. The importance of body assessment scores stemsfrom the knowledge about the partition of nutrients according to the priority needs of theanimal. The premise is to maintain life and then to preserve the species. Thus, Adams andShort (1988) proposed the following order of partition of energy nutrients: 1. basalmetabolism, 2. mechanical activities, 3. growth, 4. set of basic bodily reserves of energy, 5.

160Artificial Insemination in Farm Animalsongoing maintenance of pregnancy, 6. lactation, 7. extra reserves of energy, 8. estrouscyclicity, ovulation and early pregnancy, and 9. excess reserves. Therefore, the reproductivefunctions, in terms of partition of nutrients, are not priorities for the animal economy(Wright & Russel, 1984).Knowledge of body condition score herd contributes to decisions on measures of impact onproduction and costs of livestock development. In fact, you can set times to wean the calvesor to define when and how to supplement the diet of breeders, aiming to reduce the periodof postpartum anestrus (Moraes et al., 2007). Furthermore, knowing the body conditionscore is useful even in the prediction of productive performance (Short et al., 1996) andreproductive performance (Dunn & Moss, 1992).The score is obtained by the visual and tactile (palpation) of the animal by a trainedprofessional. There are scores of different scales, which vary in concept, the topology of thepoints of observation and animal species for which they are applied. The notes are given toanimals in accordance with the amount of tissue reserves, especially fat and muscle incertain areas of the body, often associated with specific anatomic landmarks, such as certainbony protrusions, ribs, spinous processes of the spine, processes transverse spine, flank, tipof the ileum, above the tail, sacrum and lumbar vertebrae. Extreme scores 1 and 5 (obese andcachectic) are undesirable in any scale and in any animal species studied (figure 2).The monitoring of changes in the body condition score and body weight providesinformation on the reproductive potential of the cows (Dunn & Moss, 1992), which isScore 1Score 2

Particularities of Bovine Artificial Insemination 161Score 3Score 4Score 5Fig. 2. Body condition score of dairy cows.directly related to nutrition in the pre-delivery and postpartum period. Kunkle et al. (1994)found that body condition score at calving and during breeding season is closely related tothe interval between births, the proportion of cows not pregnant at the end of the breedingseason, milk production and cow weight at weaning. Cows with body condition at calvingcycle more rapidly than those with body condition score.In cows of high milk production is expected high demand for nutrients and thusmobilization of reserves in the first three to five weeks postpartum. This phenomenon is

162Artificial Insemination in Farm Animalsaccompanied by rapid weight loss and BCS, which submits the ovarian follicles to largemetabolic changes. Such variations affect the normal development of follicles and lowerlevels of progesterone. This scenario is associated with reduced fertility (Butler and Smith,1989). Indeed, Walters (2000) found that the decrease in BCS after delivery decreased by 42%the quality of oocytes collected by follicular aspiration from Holstein cows.Vizcarra et al. (1998) inferred that the nutritional status influence postpartum luteal activityand concentrations of glucose, insulin and saturated fatty acids, which are high in cows withhigh body condition score at calving. According to Walters (2000), this framework explainswhy the delay of first ovulation in postpartum cows with negative energy balances. In fact,low plasma levels of glucose, insulin, non-esterified fatty acids and growth factor type 1insulin are associated with inhibition of pulse frequency of luteinizing hormone andestradiol production by the dominant follicle (Walters, 2000). In cows that consumeadequate dry matter during this period, follicular development is apparently normal(Staples et al., 1991). Already decreased by 1.0 point in the body condition score in these firstfive weeks postpartum resulted in lower fertility at first service (Britt, 1992). In contrast,over-conditioning, ie very high body condition score cows at the end of the pre-deliverycaused an increase in service period and embryonic mortality (Flipot et al., 1988).The productivity and profitability of farms are closely related to achieving highreproductive rates, which are only achieved through the adoption of certain managementpractices. Among these, science-based nutrition should provide the matrix of metabolicconditions ideal to meet certain strategic moments of the production cycle, such as thebreeding season, the season of birth and lactation season. In this context, the body conditionscore is a useful tool in assessing the nutritional status of the animal and therefore hasstrategic application in reproductive management of herds that are artificially inseminated.8. Evaluation of female gynecologicalAll females of reproductive age in a herd must be submitted to gynecological examinationfor selection of suitable animals for artificial insemination program. This is an internalexamination by rectal palpation, ultrasound and vaginoscopy and can be complemented bylaparoscopy and biopsy. On rectal palpation and ultrasonography are checked the size,consistency and contraction of the uterus, uterine horns and symmetry. In the ovaries areobserved consistent form and size of follicles, cysts and persistent corpus luteum.Vaginoscopy complements rectal palpation and ultrasonography, because it turns out theshape of the vaginal portion of cervix, the opening degree of the cervical canal, mucosacolor, moisture content and characteristic vaginal and cervical mucus.The pelvic examination should be thorough, seeking to know and understand the bestpossible animal's reproductive status, either for a simple confirmation of pregnancy or toidentify diseases or reflection of management that is harming their reproductive efficiency.Gynecological examination involves a complete evaluation of all components of the externaland internal genitalia, with emphasis on the ovaries, combining the findings of theexamination with a score of animal body, with its history and with the herd.For purposes of gynecological evaluation should consider the following groups of animals:from 20 to 30 days postpartum, postpartum with abnormal vaginal discharge, irregularestrous cycles, not seen in estrus 60 days postpartum; covered or inseminated by two ormore times, and that return to estrus 45 days after artificial insemination. In the historicalsurvey of the animal should always consider the age, number of childbirths and theirconditions and cyclicality observed, as these factors may reflect the ovarian function and

Particularities of Bovine Artificial Insemination 163reproductive efficiency. Are essential, too, about the coverage, treatment for retainedplacenta and uterine infection, drugs and dosages, treatment outcome, nutritional programfor the animals used in pre-natal and post partum, body condition at calving, stage lactation,milk production and herd health program.It is crucial at the end of gynecological examination, classify the animals examinedaccording to their reproductive status in pregnant and not pregnant, the latest beingidentified as normal or with any individual or reproductive problems reflecting amanagement problem. The results of gynecological examination of different groups ofanimals held in a coherent and detailed by a veterinarian, provides a satisfactoryunderstanding of the breeding herd during the exam, allowing them to be defined andadopted measures to keep the reproductive efficiency of flock.9. Diagnosis of pregnancyPregnancy diagnosis is an important tool for the management of rural property. This mustbe done by a veterinarian trained at around 28-30 days after artificial insemination by rectalpalpation and / or ultrasonography. There are other methods that can be used for diagnosisof pregnancy such as: no return to service, measurement of progesterone, pregnancyspecificproteins, estrone sulfate, breast enlargement and abdominal distension. Theaccurate diagnosis of pregnancy is important in establishing and maintaining optimalreproductive performance. The producer should know as early as possible if the female iscovered or not pregnant for it to be inseminated again.A cow is diagnosed not pregnant if she has been observed in estrus approximately 21 daysafter artificial insemination. The percentage of cows not observed in estrus around thisperiod is known as the rate of no return and is not a method to estimate pregnancy rates. Asthe percentage of cows ovulating truly seen in estrus is often low, the rate of return notoverestimate the rates of pregnancy. If a cow is in estrus detected three weeks after artificialinsemination, it can still be pregnant and her artificial insemination can cause miscarriage.One must be careful to confirm that it is true and not in heat pregnant through othermethods of pregnancy diagnosis.The detection of pregnancy by measurement of hormones, especially if they occur in milkhas advantages when interference is minimal with the cow and risk-free pregnancy. Themeasurement of progesterone to verify pregnancy also offers the possibility of diagnosingthe twenty-first day. In pregnant cows, progesterone concentrations in milk and bloodremain high between the twenty-first and twenty-fourth day after ovulation, when theywould be basal in the animal not pregnant. The simplest procedure is to obtain a sample ofmilk from the cow 21 days after artificial insemination. If the progesterone level is low, thecow is not pregnant. If the progesterone level is high, the cow may be pregnant.The factor of early pregnancy is a gestation dependent protein complex that has beendetected in the serum of various species. It is detected using an immunological technique,the rosette inhibition test. It is expected that this substance appear in the serum soon afterconception and disappear very quickly after embryonic death. Two additional proteins,protein B pregnancy specific bovine and bovine pregnancy associated glycoprotein, can alsobe measured during early pregnancy in the cow.The availability of ultrasound has been a major advance in the diagnosis and monitoring ofpregnancy reproductive taking the advantage of not being invasive. The baby begins to fillthe uterine horn near the seventeenth day of gestation and can be seen as an non-echogenicarea. In the nineteenth day, the amniotic sac has expanded considerably and the lumen of

164Artificial Insemination in Farm Animalsthe uterus can be observed. In the twenty-second day you can hear the heartbeat with thethirtieth day embryo and fetus is very visible.Rectal palpation of the fetus relies on the ability to detect the presence of a fetus growing inone of the uterine horns by inserting the arm into the rectum of the cow. This can be adangerous procedure, since trauma can be generated in both the cow and the fetus and musttherefore be done by a trained examiner. In non pregnant animal and the animal in earlypregnancy, the uterine horns can be felt in size and approximately equal diameters. It ispossible to detect a difference in the size of the two uterine horns from 35-40 days ofgestation forward.The identification of components produced by the fetus, rather than the mother, hasadvantages in the diagnosis of pregnancy. Estrogens are produced by the bovine fetus andthe concentrations of estrone sulfate in maternal plasma increases from the seventeenth dayof gestation. The content of estrone sulfate in milk reflects the plasma and estrone sulfatetest positive at 15 weeks of gestation provides 100% effective diagnosis of pregnancy. Inmost cases, detection of non-pregnancy at this late stage is of little value in reproductivemanagement, but can be used as a confirmatory test after diagnosis of early pregnancy.The mammogensis or mammary gland development as a result of pregnancy can bedetected in heifers as early as four months of pregnancy. Promoters like growth steroids canstimulate similar changes which may be a confounding factor. It is only during the last daysof gestation, when the udder is distended with colostrum, the breast development may beregarded as accurate diagnosis of pregnancy.The abdomen of the pregnant animal is getting distended around the seventh month ofpregnancy. If a hand is pushed firmly against the right side of the abdomen, the fetus cansometimes be felt rebounding against the hand. It is not a reliable indicator of pregnancy.Pregnancy diagnosis is a vital aid for reproductive management and must be sought thebest combination of earliness and accuracy.10. Calving rateCows get pregnant has always been and will remain the major challenge of cattle. Over theyears many methods evaluation were developed, but, unfortunately, with the interval betweendata calving, days in milk at first AI, percentage of pregnant cows on visit the veterinarian andthe first AI conception do not tell the whole story, that is, do not reflect reality.Averages can leave much to be desired, especially when analyzing calving interval. Twoherds may have intervals of 13 months births, but in the herd A the cows were pregnant inthe early part and the remaining lactation and late lactation and few between. While in herdB pregnancies are distributed during lactation, and the vast majority of cows becomespregnant at the beginning of lactation and the rest evenly distributed during lactation. HerdB has a performance reproductive better, but the average interval between births of the twoherds is equal. The most reliable measure that reflects what is happening in the herd and hasresulted in the birth of calves is the calving rate.The calving rate is the percentage of cows calved in the total of pregnant cows at thebeginning of the breeding season. Even under ideal conditions with 100% of normal cowsand 100% efficiency in detection of estrus, farrowing rates will fail to reach 100%. Only 60-70% of inseminated cows produce a calf born alive and the great majority of failures occurbefore the second half of pregnancy. This is partly due to the failure of design and partly ofembryonic or fetal death. The proportions of embryonic or fetal death are far greater thanthe failures in the design and the vast majority of these occurred probably by genetic

Particularities of Bovine Artificial Insemination 165abnormalities in embryos, but this hypothesis has never been proven. The cause is probablymultifactorial, involving interactions between genetics, environment and management.11. ConclusionThe importance of reproductive efficiency in cattle production systems is directly tied to thesuccess of a program of insemination with calving and breeding seasons predetermined.The records of fertility are complex, but must be done and constantly updated so that allsteps of the breeding program of the property are met and at the end of the breeding season,is to obtain good rates with many calves born alive. This chapter dealt with the main stepsfor the success of a program of artificial insemination, and if they are properly followed, thebreeding season will be profitable.12. ReferencesBaruselli, P.S.; Reis, E.L.; Carvalho, N.A.T. & Carvalho, J.B.P. ECG increase ovulation rateand plasmatic progesterone concentration in Nelore (Bos indicus) heifers treatedwith progesterone releasing device. In: International Congress on AnimalReproduction, 2004.Britt, J. H. Impacts of early postpartum metabolism on follicular development and fertility.Bovine Proceedings, vol. 24, pp. 39-43, 1992.Butler, W.R. & Smith, R.D. Interrelationships between energy balance and postpartumreproductive function in dairy cattle. Journal of Dairy Science. vol. 72, pp. 767-783,1989. ISSN 0022-0302.Caraviello, D.Z.; Weigel, K.A.; Fricke, P.M.; Wiltbank, M.C.; Florent, M.J.; Cook, N.B.;Nordlund, K.V.; Zwald, N.R. & Rawson. C.L. Survey of management practices onreproductive performance of dairy cattle on large U.S. commercial farms. Journal ofDairy Science. vol. 89, pp. 4723-4735, 2006. ISSN 0022-0302.Chebel, R.C., Santos, J.E.P.; Cerri, R.L.A.; Rutigliano, H.M. & Bruno, R.G.S. Reproduction indairy cows following progesterone insert presynchronization and resynchronizationprotocols. Journal of Dairy Science. vol. 89, pp. 4205-4219, 2006. ISSN 0022-0302.Dunn, T.G. & Moss, G.E. Effects of nutrient deficiencies and excesses on reproductive efficiencyof livestock. Journal of Animal Science. vol. 70, pp. 1580-1593, 1992. ISSN 0021-8812.Embrapa. Planejamento Sanitário de Gado de Corte. Available inhttp://www.cnpgc.embrapa.br. Access in: 07 de Abril de 2011.Flipot, P.M.; Roy, G.L. & Dufour, J.J. Effect of peripartum energy concentration onproduction performance of Holstein cows. Journal of Dairy Science, vol. 71, pp. 1840-50, 1988. ISSN 0022-0302.Galina, C.S.; Orihuela, A. & Bubio, I. Behavioural trends affecting oestrus detection in Zebucattle. Animal Reproduction Science. vol. 42, pp. 465-470, 1996. ISBN 0378-4320.Geary, T.W.; Ansotegui, R.P.; Roberts, A.J.; Waterman, R.C. Macneil, M.D.; Grings, E.E.;Thompson, B.D. & Lipsey, R.J. Effects of Flunixin Meglumini on pregnancyestablishment in beef cattle. Proc. West. Sec. Am. Soc. Journal of Animal Science. vol.56, pp.309-311, 2005. ISBN 0021-8812.Geary, T.W.; Downing, E.R.; Bruemmer, J.E. & Whitthier, J.C. Ovarian and estrous responseof suckled beef cows to the Select Synch estrous synchronization protocol. Prof.Journal of Animal Science. vol. 16, pp. 1-5, 2000. ISBN 0021-8812.Helmer, SD & Britt, JH. Mounting behavior as affected by stage of Estrous cycle in Holsteinheifers. Journal of Dairy Science. vol. 68, pp. 1290-1296, 1985. ISSN 0022-0302.

166Artificial Insemination in Farm AnimalsKunkle, W.E.; Sand, R.S. & Era, D.O. Effect of body condition on productivity in beef cattle. In:Fields, M.J. & Sands, R.S. (Ed.). Factors affecting calf crop. Boca Raton: CRC Press, 1994.Lopez, H.Z.W.; Satter, L.D & Wiltbank, M.C. Effect of dietary phosphorus concentration onestrous behavior of lactating dairy cows. Theriogenology, vol. 61 pp. 437-445, 2004.ISSN 0093-691X.Lucy, M.C. Reproductive loss in high-producing dairy cattle: Where will it end Journal ofDairy Science. vol. 84, pp. 1277-1293, 2001. ISSN 0022-0302.Mizuta, K. Estudo comparativo dos aspectos comportamentais do estro e dos teoresplasmáticos de LH, FSH, progesterona e estradiol que precedem a ovulação emfêmeas bovinas Nelore (Bos taurus indicus), Angus (Bos taurus taurus) e Nelore XAngus (Bos taurus indicus X Bos taurus taurus). 2003. 98 f. Tese (Doutorado emReprodução Animal) – Faculdade de Medicina Veterinária e Zootecnia,Universidade e São Paulo, São Paulo, 2003.Moraes, J.C.F.; Jaume, C.M. & Souza, C.J.H. Reproductive management of beef cow. Journalof Animal Reproduction, vol. 31, n. 2, pp. 160-166, 2007.Pinheiro, O.L.; Barros, C.M.; Figueredo, R.A.; Valle, E.R.; Encarnação, R.O. & Padovani, C.R.Estrous behavior and the estrus-to-ovulation interval in Nelore cattle (Bos indicus)with natural estrus or estrus induced with prostaglandin F2 alpha or norgestometand estradiol valerate. Theriogenology, vol. 49, pp. 667-81, 1998. ISSN 0093-691X.Santos, K.J.G. Estação de monta: Técnica para Melhorar e Eficiência Reprodutiva. Jornal dasCidades. São Luis Montes Belos-GO. 2003.Short, R.E. & Adams, D.C. Nutritional and hormonal interrelationships in beef cattlereproduction. Journal of Animal Science, vol. 68, pp. 29-39, 1988. ISSN 0021-8812.Short, R.E.; Grings, E.E.; MacNeill, M.D.; Heitschimidt, R.K. & Haferkamp, M.R. Effects ofsupplement, and sire breed of calf during fall grazing period on cow and calfperformance. Journal of Animal Science, vol. 74, pp. 1701-1710, 1996. ISSN 0021-8812.Staples, C.R.; Burke, J.M. & Tatcher, W.W. Influence of supplemental fats on reproductivetissues and performance of lactating cows. Journal of Dairy Science. 81: 856-871. 1998.ISSN 0022-0302.Staples, C.R.; Lucy, F.M.; Michael, M.C. & Thatcher, W.W. Energy balance and size andnumber of ovarian follicles detected by ultrasonography in early postpartum cows.Journal of Dairy Science, vol. 74, pp. 473-482, 1991. ISSN 0022-0302.Stevenson, J.S. Reproductive management of dairy cows in high milk-producing herds.Journal of Dairy Science. vol. 84, pp. 128-143, 2001. ISSN 0022-0302.Stevenson, J.S.; Kobayashi, Y. & Thompson, K.E. Reproductive performance of dairy cows invarious programmed breeding systems including Ovsynch and combination ofgonadrotopin-releasing hormone and prostaglandin F2. Journal of Dairy Science. vol.82, pp. 506-515, 1999. ISSN 0022-0302.Tenhagen, B.A.; Drillich, M.; Surholt, R. & Heuwieser, W. Comparison of timed AI aftersynchronized ovulation to AI at estrus: Reproductive and economic considerations.Journal of Dairy Science. vol. 87, pp. 85-94, 2004. ISSN 0022-0302.Vailes, L.D., & Britt, J.H. Influence of footing surface on mounting and other sexualbehaviors of estrual Holstein cows. Journal of Animal Science. vol. 68, pp. 2333-2339,1990. ISSN 0021-8812.Walters, A.H. Analysis of early lactation reproductive characteristics in holstein cows. 2000.83 p. Thesis (M. Sc. in Dairy Science) − Virginia Polytechnic Institute and StateUniversity, Blacksburg, Virginia.Wright, I.A. & Russel, A.J.F. Partition of fat, body composition and body condition score inmature cows. Animal Production, Edinburgh, vol. 38, pp. 23-32, 1984.

11Management Factors Affecting Fertility in SheepPilar Santolaria, Inmaculada Palacin and Jesús YánizInstituto Universitario de Ciencias Ambientales y Departamento de Producción Animal yCiencia de los Alimentos. Escuela Politécnica Superior, HuescaUniversidad de ZaragozaSpain1. IntroductionAn Arab horse breeder in the early 13th century carried out the first insemination reported,by trapping stallion semen in wool placed in the vagina of a mare and transferring this tothe vagina of another mare (Heape, 1898). Later, in 1780, an Italian priest and physiologistnamed Lazzaro Spallanzani performed artificial insemination with dog semen, andrevolutionised the way scientists thought. Since then, scientist and farmers have striven toimprove this technology, motivated by the benefits that could be achieved. Sheep is oneof the species subsequently linked to this technology and in which many questions stillremain to be resolved to improve fertility. However, the potential impact of this techniqueon the genetic progress of sheep is high and further studies are needed to improve itsefficiency.In Spain, artificial insemination programmes in sheep are linked to the genetic selectionschemes of the breeds, but it has not been successfully integrated with reproductivetechnology on farms as happen in sows or cows. The technical difficulty and weak fertility,ranged between 15 to 60 % for pregnancy rate, limits its application. In Spain, mostcommercial programmes use refrigerated semen (15 degrees C) by superficial intracervicaldeposition (cervical), whereas the use of frozen-thawed semen by intrauterine deposition(laparoscopy) is more restricted. Cervical insemination with fresh semen is the main methodchosen due to its simplicity and satisfactory results. With the aim of improving its efficiency,this paper focuses on identifying the main management effects affecting AI results whenthis technology is applied.2. Female associated factorsManagement factors associated with artificial insemination in the ewe can modify fertility.In reproductive planning, intervals between lambings, season, age of ewe, heat stress,nutrition state or breed are some of the factors which have a great effect on fertility results.David et al. (2008), using a joint model combining two main traits, one relative to female andthe other relative to the male, reported that the main variation factors of AI success wererelative to non-sex-specific effects and to female effect, suggesting that choosing females toinseminate might slightly improve the AI results.

168Artificial Insemination in Farm Animals2.1 SeasonSeasonal variations are described as a limiting factor in sheep reproduction. In naturalconditions, seasonality, which is mediated by photoperiod, modifies hormonal balance andcauses seasonal reproductive variations in sheep (Karsch, et al. 1984; Yeates, 1949), givingrise to a decrease in reproductive activity during long days (anoestrous season).Photoperiodic information is translated into neuroendocrine changes through variations inmelatonin secretion from the pineal gland (Bittman, et al., 1983). Melatonin, secreted inpineal gland, triggers variations in the secretion of luteinising hormone-releasing hormone(GnRH), luteinising hormone (LH) and follicle stimulating hormone (FSH) (Arendt, et al.,1983, Karsch, et al., 1984). In any case, seasonal changes in reproductive activity are clearlydefined in sheep breeds from high latitudes (>40º)(Pelletier, et al., 1987), where thedifferences in daylight duration between short days and long days are more notable.As in natural mating, season affects fertility after AI, although hormonal treatment is usedto synchronise and induce oestrus. Windsor (1995) reported low cervical AI fertility rates innon-breeding season in Merino ewes, a shallow seasonal breed. According to this, Anel et al.(2005) found a season effect on the AI fertility in Churra ewes, which was more important incervical than laparoscopic artificial insemination. In cervical AI, semen is deposited in theexternal portion of the cervix and the sperm transport is affected by cervical mucus quality.Theses authors suggest that photoperiod could alter progestagens and so cervical mucuscharacteristics, making it scarcer and more viscous. In consequence, sperm transport in thecervix can be interfered with. It is important to note that seasonality affects the ramreproductive parameters in the same way and changes in seminal quality during anoestrousseason may decrease the fertility results after AI.Moreover, subcutaneous melatonin implants are widely used to bring the breeding seasonforward and improve reproductive performance in non-breeding season (Chemineau, et al.,1991; Haresign, et al., 1990). Melatonin treatments act by mimicking a short-day-likeresponse (O'Callaghan, et al., 1991) and induce oestrus during the non-breeding season. Notonly an increase in the percentage of pregnant ewes (fertility) has been described aftermelatonin implant treatment in anoestrous season, but also the number of lambs born perewe (litter size) (Abecia, et al., 2007; Arrebola, et al., 2009; Chemineau, et al., 1992). Thisimprovement could be due to a higher rate of embryonic survival, an improvement in lutealfunction or a reduction in the antiluteolytic mechanisms (Abecia, et al., 2008). In AI, afertility rate increase has been reported with melatonin implant treatments (Laliotis, et al.,1998; Legaz, et al., 2000) after cervical AI.2.2 AgeAnother important factor affecting fertility after cervical AI is ewe age. In comparison withadult ewes, young and maiden ewes have lower fertility, probably due to impaired spermtransport combined with low mucus production in the cervical canals during oestrus(Selaive-Villarroel & Kennedy, 1983a, b). After that, most studies have described a decreasein AI success with increasing female age. Shackell et al. (1990), predicted 2-3% fertilityreduction per year of age for different breeds. Esmailizadeh et al., (2009) reported that as theage of the ewes increased from 2 to 7 years, the proportion of barren ewes significantlydecreased from 29 to 5%. In a more recent study in the Spanish Churra dairy breed, Anel etal. (2005), described how, after 1.5 years of age, the lambing rate decreased by 1.74% peryear for cervical AI. The highest rates of fertility declining with age were described in theLacaune breed by Colas et al. (1973), who reported a drop in fertility of 15% per year.

Management Factors Affecting Fertility in Sheep 169Paulenz et al. (2007), observed that the age of the ewes had a significant effect on the nonreturnrate, but not on lambing rate, whereas in Fukui et al. (2010), both the pregnancy andlambing rates in the ewes significantly declined as age increased. The detrimental effect ofincreased fertility age could be explained by the fact that aged ewes have increased risks ofreproductive disorders and decreased ovulation rates with quality ovulated oocytescompared with younger ewes.The question is: what is the optimal age for cervical AI? Alabart et al. (2002) , studied theinfluence of age on fertility in a total of 3819 Rasa Aragonesa ewes aged 1 to 12 years. In thisstudy, maximum fertility (56.7%) was observed at 3 years, and ewes aged from 2 to 5 years(79.5% of the inseminated ewes) had mean fertility values above 50%. These results partiallyconfirm the observations by Colas et al. (1973), who reported a significant decrease in thefertility of ewes inseminated when over 3.5 years old, and Gabiña and Folch (1987), whoobserved a strong fall in the fertility of ewes inseminated at 4 or more years of age. Anel etal. (2005) recorded the best fertility rates in ewes aged between 1.5 and 4.5 years; beyondthis age, fertility declined remarkably. Fertility also decreased depending on the number ofprevious parturitions. In other studies, the maximum fertility was obtained at around 2years of age, with a progressive fall afterwards (Fantova, et al., 1998). It may be concludedthat insemination groups should be made up of 2 to 5 year old ewes while younger andolder ewes should be used for natural mating.2.3 Lambing-AI intervalThe need for a resting period for the ewe after lambing to allow uterine involution is wellknown. However, sometimes the increasing reproductive rate imposed by the demandingproduction system involves short resting periods from lambing to AI, which affects fertility ina negative way. According to Bodin et al. (1999), reducing the lambing-AI interval to below 40-50 days induces a significant decrease in fertility, even after natural mating. Most authorsrecommend not inseminating ewes any sooner than 50 days post-partum (Anel, et al., 2005).2.4 BreedEwe breed is also a significant source of variation in fertility after AI (Donovan, et al., 2004;Fukui, et al., 2007; Papadopoulos, et al., 2005; Salamon & Maxwell, 1995). Differences in themean time of ovulation and ovulation rates in different breeds of ewes at differenceslocations may explain the variation in fertility (Salamon & Maxwell, 1995).Alternatively, variation may be due to differences in the morphometric characteristics of thecervix (Eppleston, et al., 1994). In this sense, Kaabi et al. (2006), carried out a morphometricstudy in four ovine breeds (Assaf, Churra, Castellana and Merino) showing importantdifferences in length, width, number of folds and distance between folds, which originatesbreed variations in the depth of catheter penetration into the cervix during AI. In this study,the breeds yielding lower fertility after AI resulted in higher cervical complexity, andachieved a lesser degree of cervical penetration of the catheter during cervical AI. Asexplained below, different studies have found a positive correlation between cervical AIdepth and fertility (Kaabi, et al., 2006).2.5 Body weight and body condition scoreFor an adequate response in a breeding programme, ewes must be suitably nourished andmaintained in good body condition. Clearly, ewes with a good nutrient intake respond most

170Artificial Insemination in Farm Animalsrapidly to the onset of the breeding season and continue to respond with an increase inovulation rate (Keisler & Buckrell, 1997). Flushing is understood as the rapid increase inovulation rate of ewes receiving a nutrient supplementation before mating. Under harshernutritional conditions in the semi-arid southern Mediterranean region, where regular foodsupply is not guaranteed, lambing and twinning rates were shown to be boosted followingnutritional flushing (Younis, et al., 1978; Landau & Molle, 1997; Branca, et al., 2000) or whenthe live weights of the ewes were higher at mating (Gunn & Doney, 1979, Thomson &Bahhady, 1988).However, Lassoued et al. (2004), showed important interactions between genotype and levelof nutrition. In this sense, in highly prolific ewes like D’Man breed, higher levels of nutritionprior to and during mating were associated with improved reproductive performance, butin low prolific breeds such as Queue Fine de l'Ouest, neither ovulation rate nor lambing ratewere affected by the dietary treatment. In a recent work by Fukui et al. (2010) body weightdid not significantly affect fertility.Body condition score (BCS) has proved useful as a management tool for subjectivelyassessing the nutritional status of ewes. In this way, body condition directly affectshypothalamic activity and GnRH secretion, but not pituitary sensitivity to GnRH, and theseeffects on reproductive performance are also mediated through changes in ovarianhormones or in hypothalamo-pituitary sensitivity to ovarian hormones (Rhind, et al., 1989).The effect of body condition on the ovulation rate of ewes has been extensively reported(Ducker & Boyd, 1977; Morley, et al., 1978; Adalsteinsson, 1979; Gunn & Doney, 1979;Gonzalez, et al., 1997). High body condition score has been associated with an increase ofovulation (Rhind, et al., 1989; Xu, et al., 1989), especially in Mediterranean breeds at thebeginning of the breeding season (Forcada, et al., 1992). Most authors recommend a BCS of2.5 to 3.0 either for natural or artificially breeding (Contreras-Solis, et al., 2009; Husein &Ababneh, 2008). In a study carried out in inseminated Rasa Aragonesa ewes (Bru, et al.,1995), the lowest pregnancy rates (32,7 %) were obtained in sheep with a BCS3.The importance of BCS in fertility has been also reported in Spanish Manchega breed(Montoro, 1995).Fukui et al. (2010), , concluded that body nutritional condition is an important factor, next toewe age, influencing the fertility of ewes after AI regardless of body weight. Nulliparousewes less than 3 years old and with a BCS of more than 3.0 are expected to have higherfertility than other types of ewes.2.6 Farm/HerdDifferent ewe farms have different management practices and this may have an impact onfertility after AI. Reproductive planning (intervals between lambings, season, age of firstmating, AI technique, etc.) and animal handling (feeding, health, preparation of AI lots, etc.)have a great effect on fertility results (Anel, et al., 2005). The significant effect of the farm hasbeen described in several studies (Anel, et al., 2005, Fantova, et al., 1999, Paulenz, et al.,2002).Thus, in order to improve fertility results, handling conditions on farms must beimproved, along with more widespread use of AI techniques.The geographical area where the farm is located may also have an influence on the successof AI. In a recent study carried out in north-eastern Spain (Palacin et al., 2011), data from18.528 AI in Rasa Aragonesa ewes belonging to a selection scheme with similar management

Management Factors Affecting Fertility in Sheep 171were recorded in order to analyse the effect of farm geographical location and thetheoretical time distance between the farm and insemination centre on the fertility aftercervical AI. An average fertility of 54.3% was observed, with significant differences amongthe 14 regions studied. Fertility rates higher than 60% were found in the northern regionsnear the Pyrenees Mountains and the lowest results (38.5-48.3%) were obtained in thesouthern regions. The average time distance of these regions did not differ. The regionsnearest to the insemination centre, with similar climatic conditions, showed medium fertilityrate (54.0-57.8%). These results showed a huge variability after insemination, taking intoaccount the geographical location of the farm.2.7 Heat stressIt has been reported than in tropical and sub-tropical areas the local sheep show restrictedsexual activity in the summer months (Marai et al., 2004). Marai et al., 2007 reviewed howexposure to high ambient temperature causes impairment of reproductive functions insheep. The heat effect is aggravated when heat stress is accompanied with high ambienthumidity (Marai et al., 2000, 2004, 2006, 2007). Heat stress evokes a series of drastic changesin animal biological functions, which include a decrease in feed intake efficiency and use,disturbances in the metabolism of water, protein, energy and mineral balances, enzymaticreactions, hormonal secretions and blood metabolites. (Shelton, 2000; Marai et al., 2006).2.8 Synchronization treatmentOestrous synchronisation and ovulation induction treatments are widely spread in AI inorder to control the optimum insemination time. Oestrous behaviour in small ruminants isnot clearly shown, so treatments are needed to prevent an asynchrony between ovulationand insemination time, which may be the commonest cause of failure of artificialinsemination programmes (Jabbour & Evans, 1991).Synchronisation treatments are a useful tool not only for AI programmes but also in naturalmating, particularly to ensure lambing in anoestrous season. However, many studies reportthat synchronisation causes reduced fertility after cervical AI (Robinson, et al., 1970; Hawk,1971) and after natural mating (Quinliva.Td & Robinson, 1969; Hawk & Conley, 1975;Allison & Kelly, 1978). Different hormonal treatments have been used in the control of sheepreproduction, but progestagen analogues (fluorogestone acetate and medroxyprogesteroneacetate) are the most commonly used to induce and synchronise oestrus in natural mating orAI of small ruminants (Lunstra & Christenson, 1981; Pearce & Robinson, 1985; Langford, etal., 1982; Baril, et al., 1993, Greyling, et al., 1997). Progestagens produce a mimetic effect ofthe luteal phase of the oestrous cycle and a sudden progestagen removal followed byadministration of an equine chorionic gonadotropin (eCG) dose (FSH- and LH-likestimulation) induces oestrous activity. Currently, intravaginal progestagen-impregnateddevices (sponges or CIDR) for 12-14days followed by administration of 250-500IU eCG hasbeen proposed (Abecia, et al., 2011) as a synchronised treatment in sheep, in whichinsemination can be performed from 47h (intrauterine) to 55 (cervical) hours after deviceremoval.Although long-term progestagen treatment results in efficient oestrous synchronisation,high variability has been reported in fertility (Vinoles, et al., 2001; Menchaca & Rubianes,2004;). Progestagen treatment appears to result in an asynchrony between oestrus andovulation (Scaramuzzi, et al., 1988; Sirois & Fortune, 1990) and reduce sperm transport

172Artificial Insemination in Farm Animalsthrough the cervix (Killen & Caffery, 1982; Armstrong & Evans, 1984; Pearce & Robinson,1985). Other studies have reported lower fertility rate caused by the negative effect of longtermprogesterone treatment on oocyte development (Vinoles, et al., 2001; Menchaca &Rubianes, 2004) related to subluteal progesterone levels.According to this, recent research efforts are focused on shortening synchronisationtreatments. Shortening treatment (5-6days) with different progestagen devices seems to beenough to achieve efficient oestrous synchronisation in natural mounting both during theanoestrous season (Ungerfeld & Rubianes, 1999) and in breeding season (Vinoles, et al.,1999; Ustuner, et al., 2007) with a similar fertility rate to long-term treatment. Nevertheless,the interval between progestagen device withdrawal and the onset of oestrus was shortened(Ungerfeld & Rubianes, 1999; Vinoles, et al., 2001; Zeleke, et al., 2005; Ustuner, et al., 2007)due to a delay in corpus luteus regression in cyclic ewes (Menchaca & Rubianes, 2004). Thisasynchrony could decrease the success of artificial insemination programmes, so a treatmentthat ensures an acceptable luteolysis seems to be necessary to enhance the oestroussynchronisation (Ustuner, et al., 2007).The luteolytic effect of prostaglandin F 2α (PGF 2α ) (McCracke, et al., 1972) and its analogueshas been used to control corpus luteus activity. In goats, the use of a short-term progestagenprotocol combined with PGF 2α administered at progestagen sponge insertion time wassuccessful in artificial insemination with frozen-thawed semen (Corteel, et al., 1988). Insheep, Beck et al (1993) reported acceptable oestrous synchronisation and fertility results innatural mating using PGF 2α treatment combined with short-term progestagen. Nowadays,PGF 2α treatment use in small ruminant reproduction control has increased because ofnarrow restrictions on the use of progestagens in animal production both in the UnitedStates and the European Union (Martin, et al., 2004; Menchaca & Rubianes, 2004). PGF 2α isquickly metabolised with a minimum residue level (Light, et al., 1994). In some SouthAmerican countries, short progestagen treatment with PGF 2α administration is used in goatmanagement (Menchaca & Rubianes, 2004). However, the effectiveness of PGF 2α or itsanalogues depends on the ovary status (Houghton, et al., 1995) and is not very useful duringthe anoestrous season (Acritopoulou & Haresign, 1980). Moreover, a varied response andlow pregnancy rates in AI have been reported with single PGF 2α treatment or progestagencombined (Boland, et al., 1978; Hackett, et al., 1981; Olivera-Muzante, et al., 2011), so withthe current methods it is not an appropriate synchronisation treatment for AI unlessprevious oestrous detection is carried out.Another factor affecting fertility after cervical AI related with hormonal synchronisation isthe development of anti-eCG antibodies. Females involved in repeated treatmentthroughout their reproductive life, particularly those involved in AI genetic programmes orintensive lambing systems, develop anti-eCG antibodies (Baril, et al., 1996; Bodin, et al.,1997; Roy, et al., 1999). These antibodies are associated with low reproductive rates,especially in fixed time AI in sheep (Maurel, et al., 2003) and goats (Baril, et al., 1996), andin multiple ovulated ewes (Forcada, et al., 2011). This is less pronounced when repeatedtreatment is combined with natural mating. High concentrations of anti-eCG antibodies arereported with a lack or delay in oestrus and pre-ovulatory LH surge (Baril, et al., 1993; Roy,et al., 1999; Maurel, et al., 2003), decreasing the fertility after AI.Alternatives for reproductive activity control that ensure optimum oestrous synchronisationand successful AI results are needed in sheep, in line with current demands in public andanimal welfare.

Management Factors Affecting Fertility in Sheep 1733. Male associated factorsThe ram may greatly influence fertility results after cervical AI. It has been reported thatvariation in fertility of ram ejaculates exists independently of the sperm quality (Choudhry,et al., 1995; Paulenz, et al., 2002). Variations in the fertility of rams have been reported aftercervical inseminations with fresh semen (Anel, et al., 2005; Paulenz, et al., 2002), with frozensemen (Colas, 1979; Windsor, 1997; Soderquist, et al., 1999; Paulenz, et al., 2005, 2007) andafter laparoscopic inseminations with frozen semen (Eppleston et al., 1986; Maxwell, 1986;Eppleston, et al., 1991; Eppleston & Maxwell, 1995). In a large scale epidemiological study,Anel et al. (2005) observed that the male factor significantly influenced fertility. Despite therestrictions in the choice of ejaculates, the authors found important differences in fertilityamong rams, particularly when cervical AI with cooled semen was used. Salamon andMaxwell (1995) proposed that ram differences in fertility could be both genetic andenvironmental, whereas ejaculate differences are probably due to nutrition, managementand previous frequency of ejaculation.Whereas differences in fertility have been demonstrated among fertile males in differentspecies, the causes of these differences remain unclear (Ostermeier, et al., 2001). Saacke et al.(1988, 1994) have suggested in bulls that factors associated with semen quality which affectfertility can be classified as either compensable or non-compensable. It was suggested thatthe effects of compensable factors on fertility might be sensitive to the number of sperminseminated, whereas those of non-compensable factors were not. As the number of sperminseminated increases, fertility increases until a plateau is reached (den Daas, 1992). At thispoint, compensable factors no longer have an effect on fertility. Commercial insemination ofovine in Mediterranean Countries provides at least the plateau number of sperm in aninsemination dose. It is thus the non-compensable factors that contribute most to the fertilitylevel of a ram. A non-compensable defect in sperm would be one in which a sperm reachesthe fertilisation site and initiates the egg activation process, but fails to sustain zygotic,embryonic, or foetal development (Ostermeier, et al., 2001). Evidence of such defects insperm has been described in bulls with fertility differences (Eid, et al., 1994). Likelycandidates for non-compensable factors would be incorrectly assembled chromatin ordamaged DNA within the sperm nucleus. It seems logical to assume that the transfer of acomplete and intact DNA molecule from sperm to ovum is crucial to obtain fertilisationwith certain prospects of success. It is well-known that the presence of defects in the geneticmaterial, such as anomalies in chromatin condensation related with the sperm maturationprocess, the integrity of the DNA molecule associated with the presence of breaks both ofsingle and double DNA strands, or the presence of chromosomal anomalies, are closelyassociated with infertility (Aravindan, et al., 1997).SeasonAlthough seasonality is less marked in male than in female, changes in testicular volume,hormonal profiles, sexual behaviour and semen quality that affect the reproductiveperformance of rams have been reported (Casao, et al., 2010a). In this sense, the treatment oframs with slow release implants of melatonin during the non-breeding season accounted forincreased scrotal diameter and improved the reproductive performance of ewes inseminatedduring anoestrus with semen from these melatonin-implanted males. A direct beneficial actionof melatonin on sperm motility (Casao, et al., 2010c) and on other ram sperm characteristicsduring the non-breeding season has recently been demonstrated, with decreased apoptoticlikechanges and modulating capacitation and fertilisation rates (Casao, et al., 2010b).

174Artificial Insemination in Farm AnimalsNutritionSeveral studies on nutrition in rams have demonstrated that diet may have an effect ontestis size and sperm production (Brown, 1994). It also has been described that specificcomponents of the diet, such as Vitamin E, may have a positive effect in increasing semenquality and quantity (Yue, et al., 2010). The effect of diet of the rams on the reproductivesuccess of ewes after AI remains to be determined.4. Artificial insemination-associated techniquesInadequate semen preservation and difficulty in passing through the cervix during AI arethe major obstacles to the extensive use of cooled or cryopreserved semen in sheep AIprogrammes (Yaniz, et al., 2010, 2011). Exo-cervical deposition of diluted liquid ram semen,preserved at 15◦ C for less than 8 h, is currently the AI technique predominantly used in theMediterranean countries (Lopez-Saez, et al., 2000; Yaniz, et al., 2010, 2011).4.1 Semen collection4.1.1 Semen collection frequencySemen collection frequency may have an impact on sperm quality. Long abstinence periods(Pascual, 1993) and successive ejaculations (Ollero et al., 1994) have been associated withmembrane alterations of spermatozoa. A decrease in semen volume and spermconcentration with successive ejaculations has been reported in several studies (Ollero et al.,1996; Kaya et al., 2002). In the study by Ollero et al. (1996), the maximum proportion ofviable cells was obtained in the second ejaculate after an abstinence period of 3 days. Theauthors concluded that the use of the second and/or a mixture of second and thirdejaculates would improve the results in artificial insemination. The general recommendationis to establish a routine of semen collection, for example of two-three times per week (twocollections per day/per ram) on different and non-consecutive days, independently of theuse of the semen obtained. Increased semen collection frequency may have an effect onsperm quality and the composition of the seminal plasma (Kaya et al., 2002), although itremains to be determined whether this has an impact on field fertility. In this sense, theprocedure of taking one or two collections per day from each ram during the working week(Monday-Friday), with a 2-day rest period during the weekend, has been described inIreland (Gordon, 1997).4.1.2 Hygienic conditionsSemen collection in farm animal species is not a sterile procedure, and some degree ofcontamination with bacteria cannot be avoided (Clément et al., 1995; Varner et al., 1998;Althouse et al., 2000; Thibier and Guerin, 2000; Althouse and Lu, 2005; Aurich and Spergser,2007; Bielanski, 2007; Yániz et al., 2010). In rams, semen is usually collected with an openendedartificial vagina, which may be contaminated with bacteria from the surface of thepenis and prepuce, collection area, equipment and people. As a consequence, bacteria mightcompromise semen quality during storage and contaminate the female’s reproductive tract.We have recently described that ram semen is normally colonised by a variety ofmicroorganisms that may reduce semen preservation and fertility (Yániz et al., 2010). Inparticular, the contamination of ram semen with enterobacterial species reduced spermquality during storage at 15 ◦C in a concentration-dependent manner.

Management Factors Affecting Fertility in Sheep 175Different strategies may be taken to minimise the effects of bacterial contamination onextended semen, as the bacterial concentration remains below a threshold level, so fertility isnot affected (Althouse et al., 2000). The first and most viable option is to enhance the hygienicmeasures during semen collection and processing. Dilution of the ejaculates with sterilediluents will further decrease the concentration of contaminants (Bielanski, 2007), althoughthis aspect has low influence in ovine because of the high sperm concentration employed forAI. Finally, control of bacterial growth is usually performed by the use of semen extenderscontaining antibiotics with broad-spectrum bactericidal or bacteriostatic activity (Maxwell andSalamon, 1993; Salamon and Maxwell, 2000). Perhaps too much reliance is often placed on thismethod of bacterial control in ovine semen. In this species, necessarily short storage periodsfor semen determine that the control of bacterial multiplication may be less important than inother animal species in which successful long-life semen extenders have been developed.Interestingly, in a recent study (Yániz et al., 2010), it was observed that 13% of identifiedbacteria were simultaneously resistant to penicillin and streptomycin, the most commonpreservative antibiotic combination used in ovine semen extenders, whereas E. coli , thebacteria with the highest impact on sperm quality, was frequently resistant to both antibiotics(31.7 %, 13/41). Antibiotics with higher antimicrobial activities were gentamycin and ceftiofur,and their inclusion in ram semen diluents should be considered.4.2 Semen evaluationSemen evaluation is a useful tool in the selection of males and ejaculates for assistedreproduction. Traditional evaluation techniques, based on the subjective assessment ofparameters such as sperm motility and morphology, semen volume or concentration, havelong been employed in the diagnosis of male subfertility and sterility (Verstegen et al., 2002).An in vitro system that could accurately predict field fertility would facilitate stricterselection of AI rams with regard to the semen quality and would provide a valuable tool forincreasing conception rate (Donovan et al., 2004). However, finding a laboratory test reliableenough to predict the potential fertility of a given semen sample or a given sire for AI is stillconsidered utopian, as indicated by the modest correlations seen between results obtainedin vitro and field fertility (Rodríguez-Martínez, 2003). Male fertility is complex, anddepends upon a heterogeneous population of spermatozoa interacting at various levels ofthe female genital tract, the vestments of the oocyte, and the oocyte itself (Rodríguez-Martínez, 2003). For this reason, laboratory assessment of semen must include the testing ofas many relevant sperm attributes for fertilisation and embryo development as possible, notonly in individual spermatozoa but also within a large sperm population (Rodríguez-Martínez, 2003). In practice, routine sperm analysis requires fast, objective and accessiblemethods of assessing different aspects of sperm viability (Yániz et al., 2008). In this sense,the common use of computer-assisted sperm analysis (CASA) methods for sperm motility,of image analysis for the evaluation of membrane integrity (Yániz et al., 2008), of DNAfragmentation with sperm chromatin diffusion techniques (SCD), (Gosalvez et al., 2008;López-Fernández et al., 2008), or fluorimetry, etc., would theoretically improve thepredictive capacity of semen analysis, although more studies are needed to determine theutility of these techniques in the practical use of AI.4.3 Sperm number per AI doseThe difficulty in passing through the cervix during AI due to the type of cervical canalfound in this species determines that semen can only be deposited inside the cervix, usually

176Artificial Insemination in Farm Animalsin the external portion. The retention capacity of the ovine cervix is very low (0,1-0,2 ml)(Gordon , 1997), whereas a large sperm number per dose is required to compensate the hugebarrier effect of the cervix (around 400 x 10 6 sperm/dose in Spanish AI).It is important to determine the minimal sperm dose per insemination in order to maximisethe genetic diffusion of males, without decreasing AI success. In the study by Langford andMarcus (1982), fertility after insemination of 400 or 200 x 10 6 spermatozoa was similar to thatobserved after natural service at progestagen-induced oestrus. However, when less than orequal to 100 x 10 6 spermatozoa were inseminated, fertility fell markedly and the number oflambs per ewe inseminated decreased. These data indicate that insemination of 200 x 10 6spermatozoa, i.e. less than 10% of the number in a single ram ejaculate, allows normalconception rates in progestagen-treated ewes. It seems that the minimal sperm dose may bebetween more than 100 and 200 x 10 6 (120 million sperm in Australian works, Gordon,1997), although a breed-effect should not be discarded, as differences in the cervix anatomyhave been described.4.4 Semen preservation4.4.1 DiluentsDespite numerous past efforts to improve semen diluents, few new additives have beenintroduced in the extender composition for ram semen (Yániz et al., 2005). Biologicalcomponents such as milk or egg yolk in the diluent have not really been effective in theapplied use for AI. So, for example, skimmed milk, a complex and variable biologicalcomponent, is still the main diluent used to preserve sheep semen at 15 ◦C for AI innumerous countries (López-Sáez et al., 2000; Yániz et al., 2011). The basic components ofsemi-synthetic diluents for the liquid storage of ram semen (buffers combined with sugarsand egg yolk), have changed little since those first used in the early 20th century (Maxwelland Salamon, 1993).The predominant empirical approach of most of the studies could partially explain the slowadvances made in the development of chemically defined extenders for ram semen storage.An individual study on the effect of each extender component on the viability of the spermcell could greatly contribute to the development of a more rational synthetic diluent. Forexample, in a recent work (Yániz et al., 2011) we studied the effect of different buffersystems included in the composition of well-defined and synthetic diluents (in vitro), onsperm quality parameters during storage at 15ºC. TRIS caused drastic modifications ofcertain sperm kinematic parameters during storage at 15ºC although, along with citrate, it isone of the main buffers included in the composition of semi-synthetic ram semen diluents(López et al.,1999; López-Sáez et al., 2000; Salamon and Maxwell, 2000; Paulenz et al., 2003;Martí et al., 2003; Yániz et al., 2008).With the available diluents, semen preservation in the non-frozen state is limited to 6-8hwithout reducing fertility (Maxwell and Salamon (1993); Salamon and Maxwell, 2000).Irrespective of the diluent, dilution rate, storage temperature or conditions, the spermdeteriorated as the storage duration increased. The main changes that occurred duringstorage included reduction in motility and morphological integrity of sperm. These changesmay be attributed to the accumulation of the toxic products of metabolism, mainly ofreactive oxygen species (ROS) that cause lipid peroxidation of the sperm plasmamembranes. The above events are accompanied by a decline in transport and survival ofspermatozoa in the female reproductive tract and reduction in fertility (Salamon and

Management Factors Affecting Fertility in Sheep 177Maxwell, 2000). When longer period of storage are required, the use of reduced temperature(about 4ºC) and egg-yolk-based media is recommended (Gordon, 1997), although in thiscase the insemination time should also be considered (Fernandez-Abella et al., 2003)4.4.2 Cold shockSevere changes in temperature are a common feature of semen storage protocols for assistedreproduction, but are not biological traits to which species have become adapted (López-Fernández et al., 2008). In the ram, as in other mammals, there is a loss of semen qualitywhen cooled semen samples are used for assisted reproduction. Cooled semen undergoes adecrease in sperm quality, which includes reduction in motility, destabilisation of spermmembranes and DNA integrity impairment of sperm function (López-Fernández et al., 2008;Muiño-Blanco et al., 2008). It is well known that ram spermatozoa are more sensitive to coldshockstress than those of other species (Muiño-Blanco et al., 2008). In fact, the ram exhibits afaster DNA degradation under similar conditions than other species studied (Gosálvez et al.,2007). Temperature excursion episodes in spermatozoa are associated with oxidative stressinduced by the generation of reactive oxygen species, which promote DNA fragmentation(López-Fernández et al., 2008). Reactive oxygen species, produced by dead spermatozoaduring a sperm temperature reduction, give rise to sperm membrane alterations with thesubsequent release into the media of free active enzymes. Then, the accumulation of toxicmetabolic products and active free enzymes, such as those contained in the acrosome, ishigher in the media as spermatozoa disintegrate. This could facilitate intact spermdegradation in an exponential fashion (López-Fernández et al., 2008). This loss of quality,accompanied by a decline in sperm survival in the female reproductive tract, gives rise to areduction in fertility and increased embryonic loss (Paulenz et al, 2002). In ovine species,ovulation may take place several hours post-insemination (Cheminau et al., 1992; Romano,2004) and, consequently, the time that a spermatozoon is able to survive after AI is of criticalimportance to achieve pregnancy. Consequently, obtained semen samples must be used asquickly as possible with the diluents currently available.4.4.3 Time from semen recovery to dilutionThe role of seminal plasma (SP) in mammalian sperm function remains largely a matter ofspeculation as both inhibitory and stimulating effects have been found (Muiño-Blanco et al.,2008). It has been reported that exposure of ram spermatozoa to seminal plasma causes areduction of fertility (Dott et al., 1979), although some components of the seminal plasma,such as certain proteins, seem to have the important function of maintaining the stability ofthe membrane up to the process of capacitation, and are able to protect and repair the coldshockdamage to sperm (Muiño-Blanco et al., 2008). However, in practice, semen dilution inthe ram has to be done as soon as possible after recovery to avoid the inhibitory effects ofseminal plasma.4.5 Insemination technique4.5.1 Semen deposition siteIn mammals, establishing an adequate sperm reservoir in the caudal isthmus and uterotubaricjunction is very important after mounting or AI, as spermatozoa may ascend to thefertilisation site from this reservoir. In ovine species, the cervix is the main anatomical andphysiological barrier to the ascent of spermatozoa after mounting or AI. This is particularly

178Artificial Insemination in Farm Animalsrelevant after cervical insemination as, in comparison to fresh spermatozoa, a relativelysmall proportion of the stored cells penetrates the cervical canal and migrates through theuterus of the ewe to the oviducts (Salamon and Maxwell, 2000). Spermatozoa functionallyaffected during liquid storage may not migrate, or may migrate slowly, and their survival inthe female tract is also reduced to about half that of fresh spermatozoa (Salamon andMaxwell, 2000). Attempts to improve the transport of spermatozoa from the posterior cervixto the oviducts of oestrous ewes by prostaglandins added to stored semen have givenconflicting results (Maxwell and Salamon, 1993).In the non-pregnant ewe, the funnel-shaped rings of the cervix, which average around fivein number, are not concentrically aligned, and their openings are constricted in mostinstances to less than 3 mm (King et al, 2004). As explained above, breed is an importantdeterminant of the morphology of the cervix, and that could at least partially explaindifferences in fertility after cervical AI (Kaabi et al., 2006). Many studies have found apositive correlation between the depth of cervical AI and fertility (Kaabi et al., 2006). Inconsequence, numerous efforts have been made to develop new methods to deposit thesemen as deep as possible into the uterus. Studies based on the use of modified pipettes, orhormones such as oxytocin to dilate the cervical canal, have shown that cervical penetrationcan be improved. However, fertility results have been very variable (Kaabi, et al., 2006).Special care should be taken to avoid cervical trauma with the catheter during AI, as it hasbeen associated with reductions in pregnancy and lambing rates (Kaabi et al., 2006).Secondary effects of oxytocin may also have an adverse effect on fertility (King, 2004).4.5.2 TechnicianThe ability of the inseminator may be another important source of variation of the outcomeof sheep AI (Gordon, 1997; Anel et al., 2005). Cervical penetration rates are influenced byoperator skill (Eppleston and Maxwell, 1993), and the establishment of training programmesis highly recommended.4.5.3 Stress around AIThere is some evidence that nutritional or management stress inflicted upon the ewe aroundAI can markedly reduce fertility by interfering with fertilisation or by increasing earlyembryo mortality rates (Gordon, 1997). Ewes and rams should be handled with theminimum of disturbance and receive good nutrition around oestrus and the first weeks afterAI.5. AcknowledgementsThe authors thank Neil Macowan for assistance with the English translation. This work wassupported by the Araid Foundation (grant OTRI 2010-0464), Spanish MICINN (grant IPT-010000-2010-33) and OVIARAGON S.C.L. (grant OTRI OTRI 2010-0465).6. ReferencesAbecia, J.A., Forcada, F. & Gonzalez-Bulnes, A. (2011). Pharmaceutical Control ofReproduction in Sheep and Goats. Veterinary Clinics of North America-Food AnimalPractice, Vol.27, No.1, (Mar), pp. 67-+, 0749-0720

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Management Factors Affecting Fertility in Sheep 189Soderquist, L., Lundeheim, N. & Nilsson, B. (1999). Assessment of fertility after usingdifferent procedures to thaw ram spermatozoa frozen in mini straws. Reproductionin Domestic Animals, Vol.34, No.2, (May), pp. 61-66, 0936-6768Thibier, M. & Guerin, B. (2000). Hygienic aspects of storage and use of semen for artificialinsemination. Animal Reproduction Science, Vol.62, pp. 233-251, 0378-4320Thiéry, J.C. , Chemineau, P., Hernandez, X., Migaud, M. & Malpaux, B. (2002).Neuroendocrine interactions and seasonality. Domestic Animal Endocrinology,Vol.23, No.1-2, pp 87-100Thomson, E.F. & Bahhady, F.A. (1988). A note on the effect of live weight at mating onfertility of Awassi ewes in semi-arid northwest Syria. Animal Production, Vol.47,(Dec), pp. 505-508, 0003-3561Ungerfeld, R. & Rubianes, E. (1999). Effectiveness of short-term progestogen primings forthe induction of fertile oestrus with eCG in ewes during late seasonal anoestrus.Animal Science, Vol.68, (Apr), pp. 349-353, 1357-7298Ustuner, B., Gunay, U., Nur, Z. & Ustuner, H. (2007). Effects of long and short-termprogestagen treatments combined with PMSG on oestrus synchronization andfertility in awassi ewes during the breeding season. Acta Veterinaria Brno, Vol.76,No.3, (Sep), pp. 391-397, 0001-7213Vázquez, M.I., Abecia, J.A., Forcada, F. & Casao, A. (2010). Effects of exogenous melatoninon in vivo embryo viability and oocyte competence of undernourished ewes afterweaning during the seasonal anestrus. Theriogenology, Vol. 74, pp. 618-26, 0093-691XVerstegen, J., Iguar-Ouada, M. & Onclin, K. (2002). Computer assisted semen analyzers inandrology research and veterinary practice. Theriogenology. Vol.57, pp. 49-79, 0093-691XVinoles, C., Forsberg, M., Banchero, G. & Rubianes, E. (2001). Effect of long-term and shorttermprogestagen treatment on follicular development and pregnancy rate in cyclicewes. Theriogenology, Vol.55, No.4, (Mar), pp. 993-1004, 0093-691XVinoles, C., Meikle, A., Forsberg, M. & Rubianes, E. (1999). The effect of subluteal levels ofexogenous progesterone on follicular dynamics and endocrine patterns during theearly luteal phase of the ewe. Theriogenology, Vol.51, No.7, (May), pp. 1351-1361,0093-691XWindsor, D.P. (1995). Factors influencing the success of transcervical insemination in merinoewes. Theriogenology, Vol.43, No.6, (Apr), pp. 1009-1018, 0093-691XWindsor, D.P. (1997). Variation between ejaculates in the fertility of frozen ram semen usedfor cervical insemination of Merino ewes. Animal Reproduction Science, Vol.47, No.1-2, (May), pp. 21-29, 0378-4320Xu, Z., McDonald, M.F. & McCutcheon, S.N. (1989). The effects of nutritionally-inducedliveweight differences on follicular development, ovulation rate, oestrus activityand plasma follicle-stimulating hormone levels in the ewe. Animal ReproductionScience, Vol.19, pp 67-78, 0378-4320Yániz, J., Marti, J.I., Silvestre, M.A., Folch, J., Santolaria, P., Alabart, J.L. & Lopez-Gatius, F.(2005). Effects of solid storage of sheep spermatozoa at 15 degrees C on theirsurvival and penetrating capacity. Theriogenology, Vol 64, pp. 1844-1851, 0093-691XYániz, J.L., Santolaria, P., Marco-Aguado, M.A. & López-Gatius, F. (2008). Use of imageanalysis to assess the plasma membrane integrity of ram spermatozoa in differentdiluents. Theriogenology Vol.15, No.70(2), pp.192-8, 0093-691X

190Artificial Insemination in Farm AnimalsYaniz, J.L., Marco-Aguado, M.A., Mateos, J.A. & Santolaria, P. (2010). Bacterialcontamination of ram semen, antibiotic sensitivities, and effects on sperm qualityduring storage at 15 degrees C. Animal Reproduction Science, Vol.122, No.1-2, (Oct),pp. 142-149, 0378-4320Yaniz, J.L., Mateos, J.A. & Santolaria, P. (2011). Zwitterionic buffers preserve ram semenquality more efficiently than TRIS during storage at 15 degrees C. Small RuminantResearch, Vol.95, No.1, (Jan), pp. 54-60, 0921-4488Yeates, N.T.M. (1949). The breeding season of the sheep with particular reference to itsmodification by artificial means using light. Journal of Agricultural Science, Vol.39,No.1, pp. 1-&, 0021-8596Younis, A.A., Alkamali, A.A. & Eltawil, E.A. (1978). Effect of flushing on fertility of Awassiand Hamdani ewes. World Review of Animal Production, Vol.14, No.2, pp. 41-48,0043-8979Yue, D., Yan, L., Luo, H., Xu, X. & Jin, X. (2010). Effect of Vitamin E supplementation onsemen quality and the testicular cell membranal and mitochondrial antioxidantabilities in Aohan fine-wool sheep. Animal Reproduction Science, Vol.118, No.2-4, pp.217-222, 0378-4320Zeleke, M., Greyling, J.P.C., Schwalbach, L.M.J., Muller, T. & Erasmus, J.A. (2005). Effect ofprogestagen and PMSG on oestrous synchronization and fertility in Dorper ewesduring the transition period. Small Ruminant Research, Vol.56, No.1-3, (Jan), pp. 47-53, 0921-4488

12Effect of Cryopreservation onSperm Quality and FertilityAlemayehu LemmaAddis Ababa University, School of Veterinary Medicine, Debre Zeit,Ethiopia1. IntroductionGeographical barriers to breeding animals have long been reduced because of possibilitiesof semen transportation. In modern cattle breeding, where artificial insemination (AI) is themost widely applied tool facilitating extensive utilization of frozen semen from geneticallysuperior sires, cryopreservation has been an invaluable technique. In order to extend thetime span of the viability of spermatozoa, their metabolic rate has to be slowed downthereby reducing the rate at which substrates are used and toxins are produced. As a generalrule cooling of spermatozoa is the simplest method that can successfully depressspermatozoal metabolic rate and therefore, prolong sperm survival. The use of carbondioxide and other metabolic inhibitors like proteinase inhibitors are also known to producea similar but less successful effect (Colenbrander et al, 2003; Cremades et al, 2005; Curry et al,2000).Semen stored after cooling to 5-8°C will survive for 24-48 h without a significant decline inmotility and even up to 96 h without a significant drop in fertilization rates. Though chillingsemen provides an efficient and successful means of short-term storage, it has yet someadverse effects on the spermatozoa manifested as a depression in viability rate, structuralintegrity, depressed motility and conception rates (Batellier et al, 2001; Medeiros et al, 2002;Watson, 2000). Preferably, the spermatozoa of many species can now be stored indefinitelyat -196°C in liquid nitrogen for future use, while still retaining acceptable fertilization ratespostthaw. The techniques for successful cryopreservation of spermatozoa have also slowlyprogressed over the past several decades (Hammerstedt et al, 1990) and are now fairlystandardized. However, cryopreservation is also known to be detrimental to sperm functionand fertility even with the most up to date techniques. Generally, sperm viability isdecreased by 50%, whereas fertilizing capacity is affected by a factor of sevenfold aftercryopreservation (Lessard et al, 2000).The effects of cryopreservation on sperm function and fertility have been widely studied,particularly in bovine. Various sperm organelles have been known to be affected due to thedetrimental effects of cryopreservation. Induction of premature acrosomal reaction, alteredmitochondrial function, reduction of motility and failure of chromatin decondensation, all ofwhich influence the viability and fertility of the sperm cells have been reported by differentauthors (Chaveiro et al. 2006; Cooter et al, 2005; Watson, 2000; Wongtawan et al, 2006).Cooling is a major stressor, as a result of which membrane bound phospholipids reorientthemselves into a different configuration that disrupt membrane function and permeability

192Artificial Insemination in Farm Animals(Amman and Graham, 1993; Lessard et al, 2000). The stress response shown by spermatozoaas a reaction to a drop in temperature is referred as cold shock. Generally, cold shockdamage manifests itself as a decline in cell metabolism, altered membrane permeability, lossof intracellular components, irreversible loss of spermatozoan motility and an increase inthe number of dead spermatozoa. The damage to the cellular membranes is of mostsignificance because it has a carry-over effect on other cellular structures and functions.The severity of the cold shock depends upon the final temperature and the rate oftemperature drop. The cellular damage resulting from cooling or freezing affecting both thestructure and function of the cells can be categorized as direct or indirect (Amann andPickett, 1987; Watson, 1990). Direct damage is more definable and is the type usuallyassociated with cold shock evident shortly after the drop in temperature and is affected bythe rate of cooling. Indirect or latent damage is more difficult to quantify and may not beinitially apparent; it tends not to be dependent upon the rate of cooling. Addition ofcryoprotectant agent (CPA) such as glycerol, or of other components such as egg yolk, milk,bovine serum albumin, polyvinyl alcohol and liposomes in extenders have been used in anattempt to providing some protection to spermatozoa and minimizing the adverse effects ofcryopreservation (Katila, 1997). Direct cellular damage is irreversible and is usuallyapparent at later stage during postthaw analysis particularly in samples that are to rapidlyfrozen (Bildeau et al, 2000; Pommer et al, 2002; Samper, 2001). Hence, the ability to predictpostthaw sperm quality and fertility from a routine sperm function assay would be greatlybeneficial to the success of cryopreservation.2. Cryopreservation of semenIn order to realize many of the potential advantages of AI, long-term storage of semen isnecessary. This is only possible by freezing, a system which halts the metabolic processes ofthe spermatozoa, allowing indefinite storage without a significant loss of fertility. Thediscovery of the cryoprotectant properties of glycerol has made cryopreservation possible.Among the many benefits resulting from the process of cryopreservation are the geneticimprovement of important farm species and control of diseases affecting them both of whichhave a highly significant impact on the sustainability of the agri-food industry (Bailey et al,2000). Cryopreservation has been widely used in the modern cattle industry and AI hasbeen the most widely applied tool in facilitating the extensive utilization of frozen semen.Today, cryopreservation has enabled producers to have the ability to access superiorgenetics for a fraction of the price of buying a bull. Increased breeding efficiency andexploitation of sires through AI programs has become widespread in both the dairy andequine industry. However, the development of a reliable method to cryopreserve sperm isextremely important for preservation of superior genes from valuable animals. Themaximum time allowed from semen collection to insemination varies considerably, rangingfrom immediate insemination requirements to an indefinite in vitro semen storage period.As a result of the growing popularity of these artificial breeding programs, the need tomaintain fertility of sperm after varying periods of storage has become increasinglyimportant. In addition to this cryopreservation has enabled the storage of animal genetics tokeep allele variation and keep hope for endangered species. Genome resource banking topreserve the biodiversity of rare and endangered species or valuable transgenic lines wouldalso benefit from sperm cryopreservation. Moreover, reproductive research with nondomesticanimals particularly those involving cryopreservation of sperm, oocytes, and

Effect of Cryopreservation on Sperm Quality and Fertility 193embryos can also provide insight and direction into establishing more effective genetic andconservation management programs.The success of cryopreservation depends upon many other factors, including interactionsbetween cryoprotectant, type of extender, cooling rate, thawing rate and packaging, as wellas the individual animal variation (Andrabi, 2007; Clulow et al, 2008; Cooter et al, 2005).Some loss in spermatozoan viability is inevitable due to the processing procedures prior tofreezing as well as during the actual freezing process. Research reports of the success ofcryopreserved semen vary significantly often affected by the method of experimentationand recording, which is unstandardized in many reproductive researches. Informationabout pregnancy rate to a single insemination, timing of insemination, the number ofspermatozoa inseminated, volume of inseminate used or type of extender used are stillincomplete for some farm animals. Moreover, motility of spermatozoa has proven to be aneven poorer indicator of fertility in frozen–thawed samples (Samper et al, 1991). Regardlessof all these considerations, for cryopreservation to be considered a success the processshould enable a spematozoon to retain its fertilizing capacity at postthaw. To achieve this itmust retain its ability to produce energy via metabolism; to maintain normal plasmamembrane configuration and integrity; retain its motility; and enzymes, such as acrosin,within the acrosome to allow penetration of the ova. Disruption of any of these functions orabilities will significantly affect the spermatozoon’s ability to achieve fertilization. Thegreatest risk to the maintenance of these attributes is presented by the formation of icecrystals and the resultant movement of water up osmotic gradients during the process ofcryopreservation.During the process of freezing, several biophysical changes are evident within the semensample. As the temperature drops to below freezing the sample undergoes supercooling. Asthe temperature drops further beyond supercooling, extracellular ice crystals begin to formfrom the water within the surrounding medium. This ice formation increases theconcentration of solutes, such as sugars, salts and proteins. In response to this newlydeveloped osmotic pressure gradient and the fact that water within the spermatozoon isslower to form ice crystals than the water in the surrounding medium, water passes out ofthe spermatozoa, particularly from the spermatozoon head, across the semi-permeableplasma membrane. Consequently, the spermatozoon becomes increasingly dehydrated(Andrabi, 2007; Watson, 2000; Woelders, 1997). The rate of efflux of water from thespermatozoa also depends upon the speed of temperature drop: the slower the drop, thegreater would be the time needed for the efflux of water, and hence a much greaterdehydration. This does reduce the chance of ice crystal formation within the spermatozoon,which could cause considerable physical damage (Amann and Pickett, 1987; Hammerstedtet al, 1990), but an even greater damage occur due to increased intracellular dehydration andsolute concentration. On the other hand, if the cooling rate is rapid, water has little time tomove out of the spermatozoon and hence large intracellular ice crystals form, causingphysical damage to cell membranes and other intracellular components. However, theproblems of dehydration and solute concentration are less evident with rapid cooling. Asuccessful cryopreservation should, therefore, aim at arriving at an optimum cooling ratethat will provide a compromise between all these factors.There are two main temperature ranges of concern regarding damage to spermatozoaduring freezing: the period of supercooling (0°C to -5°C) and the formation of ice crystals (-6°C to -15°C) (Woelders, 1997). Excessive supercooling results in a rapid ice formation, with

194Artificial Insemination in Farm Animalsthe possibility of physical damage. In samples other than semen, this problem can beovercome by a technique termed seeding, which is designed to induce ice formation moregradually over a greater temperature range. However, there is little or no evidence thatseeding a semen sample during the freezing process has any advantageous effects. Thesecond area of concern is known to have a significant effect on spermatozoan function postthaw. In an attempt to overcome some of these problems, the use of CPA has beeninvestigated. Cryoprotectants may be divided into either penetrating or non-penetratingdepending on their action. Penetrating cryoprotectants are able to penetrate the plasmamembrane of the spermatozoa and, therefore, act intracellularly as well as extracellularly.The second type of CPA is non-penetrating and can only act extracellularly.Glycerol remains to be one of the most favored CPA, especially with bovine semen. It is apenetrating cryoprotectant, acting as a solvent and readily taken up by spermatozoa,entering the cell within one minute of addition to the surrounding medium (Pickett andAmann, 1993). Its presence, both intra- and extracellularly, acts to lower the freezing pointof the medium to a temperature much lower than that of water. This in turn reduces theproportion of the medium which is frozen at any one time, reducing the effect of lowtemperature on solute concentrations and hence on osmotic pressure differences (Amannand Pickett, 1987; Medeiros et al, 2002; Watson and Duncan, 1988). It also provides channelsof unfrozen medium, between ice crystals, in which spermatozoa may exist while at lowtemperatures. A further effect of glycerol may be a salt buffering action. Other penetratingCPAs include dimethyl sulphoxide (DMSO) and propylene glycol. Non-penetrating CPAsinclude sugars like lactose, mannose, raffinose, trehalose and proteins, such as egg yolklipoprotein. These CPAs are believed to act by increasing the osmotic pressure of theextracellular fluid and hence drawing water out of the spermatozoa, thereby decreasing therisk of formation of ice crystals and hence physical damage. However, they do not alleviate,and may even exacerbate the problem of dehydration and increases in solute concentration(Steinmann, 1996).Other alternatives have been used as CPA, including Orvus ES paste, a mix of anionicdetergents, and the synthetic detergent OEP, an amino-sodium lauryl sulphate. OEPapparently alters the composition of egg yolk, improving its cryprotectant properties. Itsinclusion was initially tried in extenders for use with boar semen with some success. It wasused in an extender containing 5% egg yolk, 2.5% glycerol at 0.4% proportion of OEP, alongwith lactose, fructose, glucose, ethylenediamine tetraacetic acid (EDTA), sodium citrate andsodium bicarbonate (Christanelli et al, 1984).Both penetrating and non-penetrating CPAs themselves were later on known to cause someform of damage to the spermatozoa (Fiser et al, 1991). This was believed to be either due tophysical damage as a result of the changes in osmotic pressure gradients or to biochemicaldisruption of subcellular components. The addition of cryoprotectant such as glycerol has amore adverse effect on motility than on mortality or fertility (Blach et al, 1989). A study wascarried out in stallion semen sample (n=41) to compare two freezing techniques (cooling at+4°C for 2.5 hrs, suspending the straws on a rack in a Styrofoam box 3cm above liquidnitrogen for 7 minutes followed by plunging the straws in to the liquid nitrogen; andprogrammable freezer; IceCube 14S, Vers. 1.30, SY-LAB Gerate GmbH, Austria) using anextender containing egg yolk and glycerol (Gent extender, Minitube Int., Germany). Themean (±SD) total motility for samples frozen in liquid nitrogen was 32.8 ±13% (range=5-55%) while the mean (±SD) live percent was 43.3 ±13% (range=14-63%). Total motility andlive sperm percent were also highly correlated (Fig.-1) in the samples frozen by liquid

Effect of Cryopreservation on Sperm Quality and Fertility 195nitrogen. Though greater numbers of sperms were live, not all were found to be motile inboth methods of freezing. Conversely, other studies indicate that the use of motility as anindication of viability is not a very accurate assessment in post–thaw samples. This isbelieved to be because of a greater detrimental effect of cryopreservation agent on themitochondria than on the acrosome region of the spermatozoan head. The detrimental effectof glycerol on such function is apparently more evident in stallions than in other speciessuch as bull.6050Total motility [%]40302010R Sq Linear = 0.542095% confindence10203040Live/Dead percent [%]Fig. 1. Correlation between total motility and live/dead percent of stallion semen preservedin Gent extender containing egg yolk and glycerol preserved using liquid nitrogenA further detrimental effect of the use of glycerol is the rapid efflux of glycerol from thespermatozoa into the glycerol-free secretions of the female’s tract at insemination. This rapidexit across the plasma membrane is also believed to cause damage to the spermatozoa and,hence, loss of sperm function (Pickett and Amann, 1993). This accounts in part for theapparent poor positive correlation between motility and fertilization rates in post thawequine spermatozoa. Computer-automated sperm head morphometry has been used todetermine the effects of cryopreservation on bovine sperm head. The protocol for the use ofcryoprotectants is ultimately a compromise between the advantages and detrimental effectsof their incorporation. Consequently, the exact protocol may vary with individual breedingmales in order to obtain optimal results. However, such individual tailoring is not practicalin a commercial situation and hence further compromise is normally required.506070

196Artificial Insemination in Farm Animals3. Effect of cryopreservation on spermatozoaSpermatozoa continuously change and develop from their origins as somatic cells until theirdestination as highly specialized cells capable of fertilization. They have basically threefunctional regions comprising a head that contain the condensed nuclear material, a midpiece serving as a powerhouse and a tail which is the propulsive region. Subsequentmaturation occur within the epididymis, followed by further development induced first bycontact with seminal plasma and then by the secretions of the female tract (Varner andJohnson, 2007). The final stages of spermatozoon development are induced by theimmediate environment of the oocyte and its zona. In the process, most of the organelles arelost together with the cytoplasm, and the spermatozoal chromatin is remodeled. Thisspecialization, though, is achieved at a cost, reducing the spermatozoon’s ability to repairitself leading to a greater susceptibility to environmental change. Hence, even under idealconditions, it is inevitable that some damage will occur to spermatozoa during the freezingprocess (Andrabi, 2007).3.1 Effect on spermatozoal metabolismThe structural changes produced in the postthaw sperm cells membrane are primarilylinked to altered abilities for energy sourcing. This would later on influence both cellularmetabolism and other sperm functions such as motility (Cerolini et al, 2001; Dziekonska et al,2009; Gillan et al, 2004). A spermatozoon is one of the smallest cells in the body specificallydesigned for propagation of genetic material achieved through fertilization. Like many othercells in the body, it requires a constant supply of energy for maintenance of cellular orderand functions needed for survival and accomplishment of its task. This energy requirementincreases significantly with the onset of activated motility, and becomes even morepronounced when hyperactivated motility is initiated (Granish and Suarez, 2002; Varnerand Johnson, 2007). Plenty of exogenously derived nutrients are required by thespermatozoa to gain the strength needed for the long journey from the epididymis to theovum in the female reproductive tract. These nutrients are metabolized intracellularly,resulting in the release of useable energy available for cellular processes primarily in theform of ATP. Like many metabolically active body cells, spermatozoa possess the metabolicmachinery required for glycolysis, the citric acid cycle, and oxidative physphorylation. ATPfor spermatozoa is mainly derived either by glycolysis in the cytoplasm or throughoxidative phosphorylation in the mitochondria (Dziekonska et al, 2009; Januskauskas, andZillinskas, 2002). The relative contributions of the two processes to ATP generation are asyet unclear. A gradual reduction in the metabolic activity of spermatozoa during storage atcold shock temperature could limit the production of detrimental by-products, which mightcompromise sperm function but metabolic activity altered in this way does also influenceessential sperm functions such as motility. Among the different alterations of activity of theintracellular enzymes, glucose-6-phosphate-dehydrogenase is the first enzyme which leavesthe cell when the cellular membrane is damaged during cold shock. Generally, theintracellular concentration of ATP is decreased or lost and the AMP/ADP-rate is increasedby the cryopreservation.Spermatozoal motility, considered to be one of the most frequently used characteristics forevaluating the fertility potential of ejaculated spermatozoa, is known to be dependent onmitochondrial function. The ATP generated by oxidative phosphorylation in the innermitochondrial membrane is transferred to the microtubules to drive motility. Hence reduced

Effect of Cryopreservation on Sperm Quality and Fertility 197sperm motility induced by cryopreservation is believed to be mainly associated withmitochondrial damage (Januskauskas, and Zillinskas, 2002; Ruiz-Pesini et al, 2001). Inhuman spermatozoa, mitochondrial enzymatic activities were shown to be correlated withspermatozoal motility (Ruiz-Pesini et al, 2001). Male infertility can result from a significantdecrease in the number of motile forms and/or from movement quality disorders. Somestudies reported a significant correlation between computer assessed spermatozoal motilityand field fertility (Januskauskas et al, 2003) however, in a more recent study (Garcia-Maciaset al, 2007), spermatozoal total and progressive motilities and velocity parameters wereknown to have no correlation with fertility though the authors noticed that velocityparameters were highest in the high-fertility group in their study. The decrease incorrelation between motility and fertility has been suggested to be due to a difference in thechange of permeability of the acrosome and mitochondrial membranes to calcium ions. Theacrosome membrane suffers most from cold shock which accounts for most of the fertilityfailure (Deneke N., Lemma A., and Yilma T., 2010, Unpublished information). Vesiculationof the acrosomal and plasma membranes occurs during sperm cell death which is termed asfalse acrosomal reaction. A true acrosome reaction, which precedes fertilization, occurs onlyin live, intact spermatozoa. Thus, it is important not only to analyze semen for spermviability but also to determine the alteration of acrosomal integrity simultaneously. HOSTand acrosomal integrity tests were employed to evaluate sperm membrane integrity inHolstein Friesian AI bulls belonging to the National Artificial Insemination Centre(Ethiopia). The proportion of sperm cells that reacted to HOST (n=36) was 60.6±9.2% and theproportion of sperm with altered acrosome in the same sample was 50.6%. HOST reactionwas highly correlated (r = 0.82, p

198Artificial Insemination in Farm Animals8075HOST reaction in frozen thawed semen [%]70656055504540353020 30 40 50 60 70 80 90Alteration of acrosome in frozen thawed bull semen [%]Fig. 2. Correlation between HOST reaction and alteration of acrosomal integrity in frozenthawedbull semen (n = 36; r= -0.45; p

Effect of Cryopreservation on Sperm Quality and Fertility 199cholesterol in which higher concentration of phospholipids results in a more fluidmembrane, and the temperature of the membrane. As membranes are cooled, lipidsundergo transition from their normal fluid state to a liquid crystalline state, in which thefatty acyl chains become disordered (Medeiros et al, 2002; Parks and Lynch, 1992; Watson,2000). During freezing this liquid crystalline state is transformed to a gel state where thefatty acyl chains become re-ordered in a parallel fashion producing a rigid structure. Thephase transition temperature for these changes varies with different lipids and dependsupon their structures. In general, the longer the fatty acyl chains, the higher is the phasetransition temperature. As each lipid class within a membrane reaches its phase transitiontemperature it conforms to the gel configuration and tends to aggregate together with othersimilarly conformed lipids within the membrane (Parks and Graham, 1992). The remainderof the lipids within the membrane may still be fluid, so areas of gel membrane can beidentified within a mainly fluid structure. In addition, the junction areas between the geland the other lipid and protein fractions become areas of weakness, subject to fusion andrupture as well as being permeable to ions (Hammerstedt et al, 1990).The peak phase transition temperature for phospholipids within the membrane of boar’s,bull’s and stallion’s spermatozoa are 24.0°C, 25.4°C and 20.70°C, respectively. Similarly, thepeak transition temperature for glycolipids in stallion spermatozoon membranes is 33.4°C,compared with 36.2°C and 42.8°C for boars and bulls, respectively (Parks and Lynch, 1992).These differences in peak transition temperatures account for the variable tolerance to coldshock exhibited by spermatozoa from these species of domestic animals. The membraneconfiguration has a roughly even distribution of phospholipids in both the outer and innerlayers for reasons of stability. The major phospholipids within the spermatozoon plasmamembrane namely phosphatildylcholine, sphingomyelin and phosphatidylethanolaminehave differing positions within the membrane bilayer. Phosphatidylcholine andsphingomyelin are associated with the outer layer of the bilayer, whereasphosphatidylethanolamine has an affinity for the inner, cytosolic layer. These affinities arenot normally evident except when the membrane is under stress. Hence, cold shock causeschanges to the distribution of the phospholipids across the bilayer which results in alteredmembrane function (Amann and Pickett, 1987; Hammerstedt et al, 1990). Generally, changesin plasma membrane integrity and motility are both indicators of sperm viability andmetabolic intactness. In this regard boar spermatozoa are known to suffer extensivemembrane and tail damage during freezing and thawing, and those spermatozoa thatsurvive suffer from a shortened lifespan, requiring AI to be carried out with large numbersof spermatozoa closely timed to the moment of ovulation (Wongtawan et al, 2006).Another major component of the spermatozoon membrane is protein. The protein–lipidinteractions are critical for the efficient functioning of the membrane. It is important toensure even distribution and molding of proteins into the bilayer, thus eliminating poresand membrane faults. These interactions may also be required for the efficient functioningof these proteins as enzymes, receptors or channels for the movement of ions like calciumions. These configurations of the membrane and interactions between its componentsfunction ideally at a normal body temperature. Hence, freezing beyond the transition phasetemperature results in a change to the gel state and a gradual aggregation of specific lipidswithin the membrane. Consequently, these protein-lipid interactions are disrupted andtherefore, the proteins no longer act efficiently as enzymes, receptors or ionic channels(Medeiros et al, 2002). The membrane as a whole loses some of its structural and functionalintegrity. Disruption of the membrane configuration also interferes with the function of the

200Artificial Insemination in Farm Animalsglycocalyx components, peripheral proteins known to confer stability to the spermatozoa intheir passage through the female system. This will influence peripheral protein attachment,causing them to aggregate in the areas of membrane still in the fluid state once gel formationhas begun (Andrabi, 2007; Housley and Stanley, 1982). Many of these changes to membraneconfiguration involving lipids are known to be irreversible and subsequent warming of thespermatozoa does not restore the original membrane configuration.Another sperm alteration linked to freezing is related to the transfer of proteins through thecell, which is modulated by the distribution of lipids along the membrane, altering theresponse to induction of capacitation and the acrosome reaction of frozen/thawedspermatozoa during fertilization (Guthrie and Welch, 2005). Dislocation of proteins in theplasma membrane, such as those belonging to the glucose transporter (GLUT) family havealso been reported as major problem related to cryopreservation. These GLUT proteins aremainly responsible for the transport of hexose across mammalian sperm membranes (Kokket al, 2005). GLUT proteins have been detected in the spermatozoa membrane of dog (Rigauet al, 2002), human (Kokk et al, 2005) and boar (Medrano et al., 2006), highlighting theirimportant role in the regulation of sperm glucose and fructose metabolism.Ultrastructural studies have shown the presence of detrimental effects of cryopreservationon various sperm organelles such as altered spermatozoal mitochondria (Nishizono et al,2004). Microscopic examination of stallion spermatozoa indicates that the function of themitochondrial cristae is also affected by cold shock. This damage to mitochondrial structureand hence its function within the spermatozoa is likely to account for the decrease inmotility observed after freezing (Ruiz-Pesini et al, 2001). Moreover, swelling of theacrosomal area was observed to be a consequence of cold shock, which indicates a loss ofintegrity as membranes are normally unable to stretch. Freezing is also known to changemotion characteristics of spermatozoa due to the irreversible changes to the mid-piece andcoiling of the tail (Watson, 1990). Particularly in donkeys, spermatozoa show an increasedincidence of backward motion because of an over-bending of the tail area (Ayalew E. andLemma A., 2010; Tsega A. and Lemma A., 2009; Unpublished observation).Cryopreservation has also been shown to induce the acrosome reaction in spermatozoa.DNA integrity of sperm is essential for accurate transmission of paternal geneticinformation. Normal condensation and stabilization of sperm chromatin in the nucleusfollowed by decondensation after sperm penetration and injection into the cytoplasm of theoocyte are pre-requisites for fertilization. However, sperm chromatin structure and DNA areknown to be altered or damaged during cryopreservation (Donnelly et al, 2001; Fraser andStrzezek 2004; Hammadeh et al, 2001; Peris et al, 2004). It is reported that thecryopreservation process of freezing and thawing can increase abnormal chromatincondensation in human (Donnelly et al, 2001; Hammadeh et al, 2001; Royere et al, 1991), boar(Fraser and Strzezek, 2004), and ram (Peris et al, 2004) sperm. Normal chromatin packagingis also known to significantly decrease after the freeze-thawing procedure in human sperm.The chromatin structure of a mature spermatozoon is normally highly condensed, makingabout 5-10% in volume of that of a somatic cell. This packing is a result of a markedalteration in the composition of nucleoproteins that occurs during epididymal transit. Thespermatid genome encodes for protamines, a unique type of spermatozoal protein, whichpredominate as nucleoproteins during spermatozoal maturation in the epididymis. Thecysteine residues of this protein establish intramolecular and intermolecular disulfidelinkages that result in compaction and stabilization of the associated DNA. This design isbelieved to provide protection to the chromosomes during their transport within the female

Effect of Cryopreservation on Sperm Quality and Fertility 201reproductive tract (Varner and Johnson, 2007). It has been postulated that a reduction ofsperm surface area due to alteration of sperm chromatin may ultimately be manifested inabnormal morphology of the sperm head. A decrease in the percentage of normal spermheads in the ejaculate has been correlated with lowered fertility in bulls and overcondensationof chromatin appears to be associated with reduced fertility in men (Royere etal, 1991). Moreover, cryopreservation appears to reduce the ability of sperm chromatin todecondense. The adverse effects of cryopreservation on sperm chromatin and headmorphology may be responsible for lowered fertility of spermatozoa observed aftercryopreservation.3.3 Cpacitaion-like effect of cryopreservationOnce spermatozoa reach the site of fertilization, there appear to be highly coordinatedcellular and molecular events that should happen before the actual fertilization. The spermcells first attach temporarily to oviductal epithelial cells, a process that requires specific cellto cell attachment, possibly mediated through spermatozoal surface carbohydrate-bindingproteins, termed lectins (Varner and Johnson, 2007). They then undergo capacitation andhyperactivation, bind to the oocyte zona pellucida, undergo the acrosome reaction,penetrate the zona, and finally fuse with and penetrate the oolemma. Intracellular calciumlevels increase in sperm during capacitation, hyperactivation and the zona pellucidainducedacrosome reaction. Increased concentrations of calcium ions are believed to triggeran intracellular signalling way associated with capacitation. Capacitaion andcryopreservation induce several similar changes to the sperm including calcium influx in tothe cells (Bailey et al, 2000). However, during cryopreservation sperm cells fail to properlymoderate normal internal calcium levels. Restructured membranes and distorted lipidproteinassociations are believed to favour further calcium ion influx duringcryopreservation. Disruption of the normal capacitation and/or the acrosome reaction dueto abnormal concentrations of calcium ion would severely compromise the fertilizingpotential of spermatozoa post-thaw. On the other hand, cryopreserved sperm cells exhibit acapacitaion-like behaviour and appear to be in a partially capacitated state due to thecryopreservation-induced membrane changes that makes the cells to be more active to theirenvironment after thawing. As demonstrated by different authors, capacitaion normallycreates a state of destabilization with which the sperm cell acquires the fertilizing capacitywhile remaining susceptible to membrane degeneration and spontaneous acrosomalreaction when fertilization fails (Bailey et al, 2000). Cryopreservation creates a subpopulationof killed and partially or fully capaciatated sperm thereby reducing the heterogeneity of thesperm population. This will produce a sperm subpopulation with a shortened lifespan invivo and whose fertilization potential has been severely compromised reducing the fertilityof the semen sample as a whole.Capacitaion like effect or ‘Cryo-capacitation’ is one of the major factors associated withreduced longevity and poor survivability of cryopreserved spermatozoa in femalereproductive tract (Bailey et al, 2000; Watson, 2000), resulting in reduced fertility of frozenthawedsemen. At present, it is generally accepted that poor survival of spermatozoa in thefemale reproductive tract is among the most important consequences of sperm cryoinjurycaused by cryopreservation. This concept of premature capacitation and reduced longevityof sperm cells in the female reproductive tract has led to the routine use of oviductalinsemination by laparscopy rather than vaginal or even transcervical insemination indifferent animals (Bailey et al, 2000). The capacitation-like changes have been demonstrated

202Artificial Insemination in Farm Animalsby greater proportion of chlortetracycline fluorescent pattern “B” due to freezing thawing inbull (Cormier et al, 1997), boar (Maxwell, and Johnson, 1997), equine spermatozoa (Thomas,et al, 2006), and in buffalo bull semen (Kadirvel et al, 2009). Impaired sperm membranefunction due to cryopreservation inevitably diminishes the successful union of the oocyteand spermatozoa during in vivo fertilization. The structural reorganization of the spermhead plasma membranes after cryopreservation appears to disrupt the ability of the spermto interact normally with cells of the female genital tract (Lessard et al, 2000; Medeiros et al,2002; Watson, 2000). Poorly motile spermatozoa are also less likely to arrive at the site offertilization in vivo or to penetrate the zona. Moreover, the proportion of motile spermpopulation itself is adversely affected by cryopreservation (Cerolini et al, 2001; Gillan et al,2004). Reduced sperm binding is likely a result of membrane injury, possibly by structuraldamage to the sperm receptors or by incomplete receptor aggregation.4. Evaluation of post-thaw semen qualityThe semen quality and its relationship with fertility have great importance in animalproduction. Hence, in vitro tests are frequently applied to determine the quality of semenfor its approval and use in both AI and other biotechnology procedures. Conventionallaboratory tests for assessment of semen quality include light microscopic study ofspermatozoal morphology, and estimation of spermatozoal motility which in turnencompass percentages of motile and progressively motile sperm; velocity of spermatozoalmovement; and longevity following in vitro storage. Other features of semen quality includeconcentration, volume, detection of the presence of urine, blood, or potentially pathogenicbacteria and functional integrity tests. The choice of adequate parameters by reproducible,fast and sensitive methods is of increasing concern. This is because the predictive value ofthe standard seminal parameters is limited or insufficient for the identification of subfertileindividuals (Clement, 2001; Love et al, 2000).The nucleus, acrosome, the flagellum, mitochondria, and the plasma membrane are the mostimportant regions of the spermatozoa that need to be assessed during postthaw semenevaluation. A series of laboratory tests devised to evaluate these various compartments willaid in the improved localization of spermatozoal dysfunction thereby improving thepredictive values of laboratory-based semen evaluation to a relatively more accurate level.More specific techniques such as testing the mitochondrial function, flagellar substructure,and plasma membrane integrity are already available (Graham and Moce, 2005; Gravance etal, 2001; Thomas et al, 1998). A variety of laboratory procedures used today in theassessment of the integrity of the plasma membrane are important components of thepostthaw semen evaluation. Among them is the evaluation of the ability of spermatozoa toexclude extracellular dyes, such as eosin, which are non permeable when the membrane isintact. Another approach is the hypo-osmotic swelling test (HOST) in which thespermatozoa are exposed to a hypotonic media (50 to 100-mOsm range) to test theirosmoregulatory function (Davies-Morel, 1999; Neild et al, 1999). Dyes that can traverse themembrane and those that are membrane-impermeable can be combined in a solution beforespermatozoal exposure to provide a more accurate reflection of membrane integrity. Forinstance, fluorescent plasma-membrane dyes can be combined with mitochondrial dyes oracrosomal dyes to provide more thorough coverage of the functional regions in the assay(Garner et al, 1994; Kavak et al, 2003; Love et al, 2003). More recently, a computer assistedsemen analysis (CASA) is in use and gives extensive information about the kinetic property

Effect of Cryopreservation on Sperm Quality and Fertility 203of the ejaculate based on measurements of the individual sperm cells. Using CASA, motilityand movement characteristics of spermatozoa have been correlated to in vivo fertility. Still,CASA-assessed motility is done on a rather limited number of spermatozoa and ispredisposed to a certain degree of human bias.An understanding of how molecular and ultra-structural basis of spermatozoal function,spermatozoa-oviductal interactions, and gamete engagement are influenced bycryopreservation will undoubtedly lead to many practical applications in semen evaluation.From this may arise possibilities for detailed laboratory tests to assess spermatozoalfunction, to introduce improved methods of semen preservation, options for applications ofassisted reproductive technologies and even treatment options for subfertile animals. Thechromatin structure assay (SCSA) tests, for instance, is a flow cytometric procedure that usesthe metachromatic fluorochrome to test the denaturability of spermatozoal chromatin that isnot normally monitored by conventional methods in various species (Evenson et al, 1995;Love 2005; Makhlouf and Niederberger, 2006). Chromatin susceptibility to denaturation iscorrelated with the level of actual DNA strand breaks and might be indicative of geneticallydefective spermatozoa (Evenson et al, 1995). Several types of defective sperm organelles andDNA can be detected in large number of sperm by immunochemical assays and flowcytometry. Microarray profiling of sperm mRNA has been shown to indicate geneexpression associated with both fertile and infertile males (Evenson et al, 2002; Thomas et al,1998). More importantly, spermatozoa affected by such damage might show no apparentlydetectable alteration in motility or membrane integrity, but may induce embryonic failureafter fertilization (Fatehi et al, 2006). Further DNA damages are not evident until the time offertilization making the chromatin defect clinically significant as it represents a potentialnon repairable defect. This becomes quite important clinically, because affectedspermatozoa in an ejaculate may not be impaired from fertilization, and hence performingrepeated insemination may not increase pregnancy rate.The search for the identification of biochemical markers of spermatozoal function is stillongoing. Such finding will improve the efficiency of laboratory-based detection of infertilityinduced by the process of freezing/thawing by targeting specific cellular components.However, incorporation of detailed tests such as SCSA for semen evaluation should notreplace or reduce the value of the conventional methods of spermatozoal motility ormorphology tests. More recently, much attention has been given to the test of capacitationprocess, as an immediate precursor to fertilization. However, on the path to fertilizationthere are many preliminary steps prior to capacitation leading to the need for spermevaluation involving tests of spermatozoal response to particular environmental conditionsrelated to the overall fertilization process (Petrunkina et al, 2007).5. Fertility of cryopreserved spermatozoaThe process of cryopreservation represents an artificial interruption of the progress of thespermatozoon towards post-ejaculation maturation and fertilization. Even with the bestpreservation techniques to date, cryopreservation process still causes harmful damage to thespermatozoa. As it has been discussed earlier in this chapter, cryopreservation affectsfertility by virtue of its effect on sperm membranes, cytoskeleton, motile apparatus andnucleus, and cell metabolism. Moreover, freezing and subsequent thawing proceduresrender the remaining surviving spermatozoa physiologically different from spermatozoabefore cryopreservation. Sprmatozoa become very sensitive to any form of stress in their

204Artificial Insemination in Farm Animalsenvironment in vivo as well as in vitro. As a result, fertility from frozen thawed semen ispoorer than that obtained from fresh semen. For this reason, proper evaluation of the postthawquality of spermatozoa is of utmost interest for AI industry to obtain information onthe fertilizing capacity of the cryopreserved semen.Many tests of sperm motility, morphology, acrosomal status, defective sperm organelles andDNA, and metabolism have been correlated with fertility (Evenson et al, 2002; Larsson andRodriquez-Martinez, 2000; Muller, 2000; Saacke et al, 2000; Thomas et al, 1998). All of thesespermatozoal attributes have been shown to be either directly or indirectly affected bycryopreservation or the thawing process. The correlation between fertility and percentage ofmotile sperm in a semen sample has already been demonstrated. In one study, afterinsemination of 55 cows with frozen semen a 30.9% (17 cows) pregnancy rate with anaverage number of services per conception of 2.7 was found. Conception rate to first servicewas only 7.2%. The mean (±SD) alteration of acrosome and positive reaction to HOST forsuccessful (pregnant) and failed insemination (non pregnant) were 47.6 ± 9.9% and 64.7 ±3.0%, and 62.7 ± 7.3% and 42.1 ± 3.9%, respectively with a highly significant (p

Effect of Cryopreservation on Sperm Quality and Fertility 2056. Extenders used in freezing semenThe addition of a cryoprotectant in to the semen sample is needed in order to protectspermatozoa from cold shock. A large variety of extenders combining various components(sugars, electrolytes, buffers, egg yolk, milk and milk products), have been proposed andused for extending sperm. Milk and milk-based extenders are known to be practical andefficient in protecting spermatozoa of various species (Batellier et al, 2001; Varner et al, 1989).Based on the composition and dynamics of the spermatic membranes, some substances suchas lipids, fatty acids and proteins have been incorporated to the semen with the goal ofdecreasing sperm damages related to cryopreservation. Glycerol and egg yolk extenders areamongst the first to be used for freezing semen (Curry, 2000; Garner et al, 1999; Holt, 2000;Medeiros et al., 2002), and today many extenders use glycerol as the major cryoprotectant.Glycerol is used at a relatively high concentration which can be detrimental tospermatozoan viability at higher temperatures hence it is added after the semen has beencooled (Fahy, 1986). The deleterious effects are due to osmotic stress, changes in membraneorganization, fluidity and permeability, as well changes in the lipid composition. Thus, acompromise has to be reached with regards to the concentration of glycerol and the lengthof time that the glycerol is in contact with the spermatozoa prior to freezing, in order tomaximize the beneficial effects of glycerol as a cryoprotectant but minimize its toxic effects.In one study, the inclusion rate of 4% glycerol in an extender containing 20% egg yolk wasfound to be superior to 2% or 6% glycerol as regards to progressive motility (Cochran et al,1984). In addition, the efficiency of glycerol may be affected by the diluents to which it isadded, as well as the method of storage.The cryoprotectant nature of many other substances, including sugars and liposomes hasalso been demonstrated. In equines, it is a common practice today that the preparation ofsemen for cryopreservation involves the use of two extenders: a primary extender for initialdilution, which is aspirated off after centrifugation, prior to the addition of a secondaryextender for freezing. Numerous extenders have been used as primary or secondaryextenders. Examples of extenders for freezing include egg yolk and those based on skimmedmilk with egg yolk (Pickett and Amann, 1993). Good success has been reported with the useof trehalose as a cryoprotectant within a skimmed milk–egg yolk extender. It is suggestedthat trehalose has a stabilizing effect on the spermatozoon plasma membrane (Steinmann,1996).Unlike the bull and the ram in which fructose is the major energy source, most extenders useglucose as the major source of energy for metabolic activity and movement of spermatozoain equines (Katila, et al, 2001). In this regard, three extenders were evaluated for theirefficiency of sustaining the viability of jack sperm as measured by motility characteristics(Ayalew E. and Lemma A., 2010; Unpublished observation). The first extender was a heatedskimmed milk (95°C for 10 minutes and cooled to 37°C before use). The second extenderwas prepared from glucose (4gm), glycerol (4%), and crystalline penicillin (150,000 IU)diluted in heated skimmed milk to make up 100ml extender (represented as SMGLU). Thethird extender contained 4% glycerol in skimmed milk (represented as SMGLY). SM wasused for storage in Equitainer (Agtech Inc, Manhattan, USA) while SMGLU and SMGLYwere used for liquid nitrogen storage after a 2.5hr optimization at +4°C, suspension overliquid nitrogen vapour for 7 minutes and plunging the straws immediately into the liquidnitrogen. SMGLU preserved sperm showed superior results (p

206Artificial Insemination in Farm AnimalsSemen parameterSM[Equitainer]n=15SMGLY[Liquid nitrogen]n=32SMGLU[Liquid nitrogen]n=13Live sperm after 24hr [%] 36.2±24.3 10.2±11.7 34.3±7.5Total Motility after 24hr [%] 24 ±17.4 8.8 ±9.6 30±10.6Progressive motility at 24hr [%] 19.3 ±14.5 7.2±7 24.6±10.5Table 1. Mean (±SD) of semen parameters for three different extenders used to dilute jacksemenIn another study, insemination of 34 animals (17 mares and 17 jennys) with jack semenextended in heated skimmed milk and stored in Equitainer (Agtech Inc, Manhattan, USA)for 24 hrs resulted in 38.2% pregnancy rate (Tsega A. and Lemma A., 2009; Unpublishedobservation). Addition of trehalose to bull semen extenders is known to provide a modestimprovement in fertility when used in combination with glycerol, which remains thecryoprotective agent of choice. Egg yolk phospholipids can also lessen chilling injury on bullsperm by binding to low density lipoproteins of the membrane and by increasing thepermeability of the membrane, although they do not alter intrinsic membrane compositionand/or physical properties (Holt, 2000). The addition of concanavalin-A to the freezingdiluent, a substance that has the ability to coat and thus protect spermatozoa membrane, hasbeen suggested to provide additional protection for acrosome membranes and help topreserve motility post freezing and during thawing (Koskinen et al, 1989). The inclusion ofliposomes, which have proved successful in bulls, has been tried with some success inequine semen (Heitland et al, 1995).7. Freezing and thawing ratesStorage of semen at ambient temperature does not in itself significantly reduce thespermatozoan metabolic rate, thus limiting the potential length of storage and demandingcryopreservation at -196°C in liquid nitrogen for long term storage. The cooling/freezingrate in the critical temperature range is of considerable importance during cryopreservationprocess because this determines whether the spermatozoa will remain in equilibrium withtheir extracellular environment or become progressively supercooled with the increasingpossibility of intracellular ice formation (Kumar et al, 2003). During slow cooling, thedehydration of the spermatozoa can proceed to the point of osmotic equilibrium betweenintracellular and extracellular space with maximal, often detrimental, cellular dehydration.However, raising the cooling rate too much will not prevent the formation of intracellularice because of the slow dehydration. Therefore, the survivability of the spermatozoadepends upon the optimum cooling rate. Optimal cooling rate will reduce the excessiveconcentration of intracellular solutes and intracellular dehydration thereby reducingexcessive shrinkage of the sperm cells. However, even at optimum cooling rates,spermatozoa remain vulnerable to the unfavorable conditions for a shorter period of time(Woelders, 1997).The type of extender used and the speed of temperature drop are known to have an effecton susceptibility of spermatozoa to cold shock and the success rate of freezing semen.Moreover, the freezing rate depends on the method of processing and of storage. Thecooling of straws can be conveniently done by either initial suspension in racks over a tankof liquid nitrogen or a computer-controlled programmable freezer followed by plunging

Effect of Cryopreservation on Sperm Quality and Fertility 207into liquid nitrogen for long-term storage (Clulow et al, 2008). The extent of damage to aspermatozoon as a result of cold shock depends not only on the drop in temperature butalso the speed with which this drop is attained. The rate of temperature drop was found tobe most critical over the specific temperature range of 0-5°C when motility was evaluatedlater. In general, the faster the rate of cooling, the more severe is the damage (Kayser, 1990).There is further evidence which suggests that the rate of temperature drop also determinesthe subsequent active life of the spermatozoa (Andrabi, 2007).Frozen spermatozoa are further injured during the thawing process, which has beenregarded as being due to re-crystallization of ultra-microscopic ice crystals to formcomparatively large ice crystals (Watson, 2000; Woelders, 1997). The warming damageoccurs when the spermatozoa pass through the critical temperature zone of -5°C to -15°C(Kumar et al, 2003). Water bath temperatures between 4°C and 75°C can be used tosuccessfully thaw semen however, the temperature chosen for the water bath depends onthe desired rate of thawing. During fast thawing (optimum; at 37°C for at least 45 sec) thetime for re-crystallization to occur is limited and this increases the survivability ofspermatozoa. However, when the duration of thawing is insufficient for the out-flow ofexcess cryoprotectant from the cell it suffers osmotic stress and the spermatozoa swells andlyses as the medium becomes abruptly diluted by the melting of extracellular ice (Pegg,2002). The thawing rate can be influenced by factors such as the temperature and nature ofthe environment (air or water bath) and the thermal conductivity of the packaging as relatedto the diameter of the lumen of the packing. Some semen thawing protocols involve theaddition of warmed extender to aid the process of thawing which will also increase thevolume of the inseminate and aid preservation of spermatozoan viability. Thawingextenders may be used for semen stored in pellets, vials or straws, and are added as part ofthe thawing process.8. Packaging for frozen semenTo maximally utilize the genetics of desired sires on a commercial basis, attempts are madeto package a minimal number of spermatozoa per insemination unit without sacrificingfertility (Foote and Parks, 1993; Shannon and Vishwanath, 1995). Ultimately, the number ofmotile spermatozoa per insemination is determined by the postthaw motility evaluationsand non return to estrus rates from a large number of inseminations. The ability to predictpostthaw sperm quality and fertility from a routine sperm function assay is beneficial whenone considers the extended period of progeny testing.Several methods are available for the packaging of spermatozoa for freezing in differentspecies. They include glass ampoules or vials, polypropylene, polyvinyl or plastic round orflat straws (usually 0.5-1.0 ml in volume), flat aluminium packets (10-15 ml); pellets (0.1-0.2ml), and macrotubes (Heitland et al, 1996; Kneissl, 1993; Park et al, 1995). Both ampoules andstraws are traditionally frozen by suspension over liquid nitrogen, followed by plunginginto liquid nitrogen at -196°C. Subsequent work investigating the effect of the rate offreezing led to the current application of the use of computer-controlled programmablefreezers at different packaging sizes (Clulow et al, 2008). Although pellets have theadvantage of allowing a rapid drop in temperature to be achieved, they are not suited foreasy identification after freezing. In addition, the re-use of the carbon dioxide block or metalplate carries the potential risk of cross contamination with spermatozoa from the previousfreezing batch. On the other hand, the use of vials or straws readily allows the accurate

208Artificial Insemination in Farm Animalsidentification of samples and considerably reduces the risk of cross-contamination duringcryopreservation.As different methods of storage have been used, the question of whether the means ofstorage has any effect on the success rate of cryopreservation has been raised. In this regard,different authors have compared spermatozoa stored in different packages (Heitland et al,1996; Kneissl 1993; Park et al, 1995). Their results showed an effect on spermatozoa qualitymanifested through reduced motility and conception rate. The reports further stressed theroles of different extenders used, the interaction between extender, and means of packaging.However, the reasons for these discrepancies was not fully explained, and it was also notclear in all work how the dimensions of the straws change with volume, in addition towhich different extenders and concentration of spermatozoa were used. On the other hand,a more recent work in stallion demonstrated that stallion spermatozoa can be frozen at aconcentration as low as 40×106 mL −1 in 0.25mL straws without a negative effect on spermmotility, morphology or acrosome integrity (Clulow et al, 2008).9. Recent advances in cryopreservationThe application of frozen-thawed semen technology is currently increasing worldwide.Several studies have focused on identifying damages during freezing and thawing, tests toscreen sperm quality post-thaw, evaluation of alternative cryoprotectants and otheradditives, and freezing procedures to improve sperm viability and fertility (Clulow et al,2008; Goolsby et al, 2004; Medeiros et al, 2002; Squires et al, 2004;). Most of the progress inimproving survival of frozen-thawed spermatozoa centers on minimizing the oxidativedamage and decreasing the osmotic stress on spermatozoa. Equine sperm are particularlyknown to be susceptible to oxidative stress, relative to other species, because of their highcontent of unsaturated fatty acids. In addition to membrane effects, lipid peroxidation canalso damage DNA. The addition of antioxidants to extenders has been used as a method todecrease lipid peroxidation and oxidative stress associated with cryopreservation (Bilodaeuet al, 2001; Peña et al, 2003; Roca et al, 2004). Different amides, compounds with lowermolecular weight than glycerol and penetrate the sperm plasma membrane more readily,have been evaluated as alternative cryoprotectants to glycerol in different animals (Bianchiet al, 2008; Medeiros et al, 2002; Squires et al, 2004;). These compounds include methylformamide (MF), dimethyl formamide (DMF) or ethylene glycol (EG) and dimethylacetamide. They were known to provide greater post-thaw motility when used at differentconcentrations. Particularly, MF and DMF or EG have been used as alternativecryoprotectants for individual males whose sperm has lesser post-thaw motility whenfrozen in glycerol (Bianchi et al, 2008; Squires et al, 2004). The use of low-density lipoproteins(LDLs), most often isolated from egg-yolk from different species, as additive has provenbeneficial for sperm function post-thaw, particularly for DNA-integrity(Rodriguez-Martinezand Wallgren, 2011). Attempts to minimize osmotic stress during cryopreservation haveincluded step-wise dilution of cryoprotectants, by incorporating cholesterol-loadedcyclodextrins (CLC) in freezing diluents (Wessel and Ball, 2004). As an alternative to addingCLC to extenders provision of polyunsaturated fatty acids in the feed as a means of alteringthe sperm-lipid membrane profile has been tried with some success in boars and stallions(Brinsko et al, 2005; Purdy and Graham, 2004).Different kinds of freezing procedures have also been reported in the last several years in anattempt to controlling the rates of cooling. Recent results indicate that the cryopreservation

Effect of Cryopreservation on Sperm Quality and Fertility 209of bull, stallion and boar semen could be improved by using a programmable freezer(Bianchi et al, 2008; Clulow et al, 2008; Woelders and Chaveiro, 2004). An interactionbetween glycerol concentration and cooling rate has been described for boar semen. Currentcryopreservation methods based on optimal combinations of glycerol and cooling rate hasallowed consistent sperm survival in the frozen semen, with acceptable variation amongindividuals. Another method, termed multi-thermal gradient (MTG), that aims to overcomethe problems of conventional freezing protocol has also been reported (Arav et al, 2002). Thisfreezing technology is based on directional freezing in which the spermatozoa are movedthrough a linear temperature gradient so that, theoretically, the cooling rate and ice frontpropagation are precisely controlled. Thus, the spermatozoa are preserved gently betweenhorizontal columns of ice thereby avoiding the damaging effects of the random ice crystalformation observed in conventional freezing. The technique is also known to allow theincorporation of controlled seeding into the freezing process and prevent the dehydration ofsperm commonly seen in conventional freezing while halving the level of glycerol required(Arav et al, 2002). A slightly different technique, termed unique freezing technology (UFT)which was originally designed to freeze foodstuffs, has been recently tested for semencryopreservation (Goolsby et al, 2004). The UFT involves placing extended samples in a baththat contains an organic fluid with a heat capacity similar to water with a freezing rate of -6.1°C /min. Similar results of post-thaw motilities with sperm frozen in traditional liquidnitrogen procedures have been reported for four UFT treatments (Goolsby et al, 2004).Another front of investigation in the last decade has been the development of methods ofexamining sperm ultrastructural characteristics and alterations. Amongst these are spermkinematics assessed by computer-assisted motility analysis, osmotic resistance tests, plasmamembrane integrity evaluation with fluorescent membrane-impermeable dyes, evaluationof acrosomal status with fluorescein isothiocyanate-conjugated lectins, investigation of DNAintegrity using the SCSA, or assessment of membrane architectural status (Gillan et al, 2004;2005). Most methods require the application of fluorescence microscopy and/or flowcytometric techniques. Because of their highly quantitative, repeatable, and sensitive nature,the techniques are already getting their place in many modern semen laboratories.10. ConclusionCryopreservation continues to be one the most frequently employed technique for use inmodern animal production. Commercial AI will inevitably use this technique to preserveand transport semen over a wider area around the world. However, even with the most upto date procedure cryopreservation still causes detrimental effect on sperm compartmentsand their function. To ensure that semen used for AI are of a relatively uniform, and of highquality, artificial breeding organizations should discard ejaculates based on seminal qualitytests immediately after ejaculation and after freezing and thawing. There are evidencesresulting from different investigation that different sperm compartments are interrelated,the defect in one will invariably affect the other compartment. The success of sperm cell inits ability to fertilize is also affected at different level during its course from its origin until itreaches the ovum. Cryopreservation would be an additional artificial interruption in thisjourney. Therefore, the knowledge of the biochemical basis of the detrimental effects ofcryopreservation and the means to detect these changes easily, cheaply and accuratelyduring semen evaluation would be of great significance. The use of combination of tests,rather than the employment of a single test, would give superior and more complete

210Artificial Insemination in Farm Animalsinformation about the status of the spermatozoa. There are already successes in improvingthe methods of detection of high quality sperm with good fertility using combination of testsaddressing different functions and compartments of the sperm. Tests addressing integrity ofsperm chromatine structure are able to identify sperm defects not normally detected duringconventional semen analysis. They are highly useful in that their application can avoidcarryover problems reflected in the embryo after fertilization has taken place. While most ofthe investigations were carried out in bovine and equine, the lack of complete informationfor other species has led to extrapolation of knowledge which in some instances has notgiven acceptable results. For this reason, the evaluation of different extender combinations,freezing techniques, and developing new methods of semen evaluation should beundertaken for various species of animals worldwide.11. ReferenceAitken, RJ and MA Baker, 2004. Oxygene stress and male reproductive biology. Reprod.Fertil. Dev.; 16:581 - 588Amann RP, 2005. Weaknesses in reports of ‘fertility’ for horses and other species.Theriogenol.; 63:698-715Amann RP and JK Graham, 1993. Spermatozoal function. In: DiRienzi D, ed. Equinereproduction. Philadelphia: Lea and Febiger; 715-745Amann RP and BW Pickett, 1987. Principals of cryopreservation and a review ofcryopreservation of stallion spermatozoa. J. Equine Vet. Sci. 7:145-173.Andrabi SMH, 2007. Fundamental principles of cryopreservation of Bos taurus and Bosindicus bull spermatozoa. Mini review. Int. J. Agri. and Biol.; 9:367-369Arav, A., S. Yavin, Y. Zeron, D. Natan, I. Dekel and H. Gacitu, 2002. New trends in gamete’scryopreservation. Mol. Cell. Endocrinol.; 187:77-81Ayalew E and A Lemma, 2010. Evaluation of cryopreservation methods and extender typesfor storage of donkey semen. As part of DVM thesis, Faculty of VeterinaryMedicine, Addis Ababa UniversityBailey JL, JF Bilodeau and N Cormier, 2000. Semen cryopreservation in domestic animals: Adamaging and capacitating phenomenon. J. Androl.; 21:1-7Bailey JL, A Morrie and N Cormier, 2003. Semen cryopreservation: success and persistent infarm species. Canadian J. Anim. Sci.; 83:393-401Ball BA, and A Vo, 1999. Reactive oxygene species generation by equine spermatozoa. Biol.Reprod.; 60:136-137Batellier F, M Vidament, J Fauquant, G Duchamp, G Arnaud, JM Yvon, and M Magistrini,2001. Advances in cooled semen technology. Anim. Reprod. Sci.; 68:181-190.Bianchi I, K Calderam, and K Calderam, 2008. Evaluation of amides and centrifugationtemperature in boar semen cryopreservation. Theriogenol.; 69:632-638.Bilodeau, JF, S. Blanchette, C. Gangnon, and MA Sirard, 2001. Thiols prevent H 2 O 2 -mediatedloss of sperm motility in cryopreserved bull semen. Theriogenol.; 56:275-286.Blach, EL, RP Amann, RA Bowen, and D Frantz, 1989. Changes in quality of stallionspermatozoa during cryopreservation: plasma membrane integrity and motioncharacteristics. Theriogenol.; 31:283–298.Brinsko SP, DD Varner, CC Love, TL Blanchard, BC Day and ME Wilson, 2005. Effect offeeding a DHA-enriched nutriceutical on the quality of fresh, cooled and frozenstallion semen. Theriogenol.; 63:1519-1527

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214Artificial Insemination in Farm AnimalsKoskinen E, M Junnila, T Katila and H Soini, 1989. A preliminary study on the use of betaineas a cryoprotective agent in deep freezing of stallion semen. J. Vet. Med.-A, 36: 110-114.Kumar S, JD Millar and PF Watson, 2003. The effect of cooling rate on the survival ofcryopreserved bull, ram and boar spermatozoa: a comparison of two controlledratecooling machines. Cryobiology; 46:246-53Larsson, B and H Rodriquez-Martinez, 2000. Can we use in vitro fertilization tests to predictsemen fertility? Anim. Reprod. Sci.; 60: 327-336.Lessard C, S Parent, P Leclerc, JL Bailey, and R Sullivan, 2000. Cryopreservation Alters theLevels of the Bull Sperm Surface Protein P25b. J Androl., 21:700-707Love CC, 2005. The sperm chromatin structure assay: a review of clinical applications.Anim. Reprod. Sci; 89:39-45.Love CC, JA Thompson, SP Brinsko, SL Rigby, TL Blanchard, VK Lowry, and DD Varner,2003 Relationship between stallion sperm motility and viability as detected by twofluorescence staining techniques using flow cytometry. Theriogenol.; 60:1127-1138.Love CC, DD Varner, JA Thompson, 2000. Intra- and interstallion variation in spermmorphology and their relationship with fertility. J. Reprod. Fertil. Suppl; 56:93-100.Makhlouf AA, and C Niederberger, 2006. DNA integrity tests in clinical practice: it is not asimple matter of black and white (or red and green). J. Androl.; 27:316-323.Mammoto A, N Masumoto, M Tahara, Y Ikebuchi, M Ochmichi, K Tasaka, and A Miyake,1996. Reactive oxygen species block sperm-egg fusion via oxidation of spermsulhydryl protein in mice. Biol. Reprod. 55:1063-68Maxwell, WM and LA Johnson, 1997. Chlortetracycline analysis of boar spermatozoa afterincubation; flow cytometric sorting, cooling, or cryopreservation. Mol. Reprod.Dev.; 46: 408-418Mazur P, N Katkova, and JK Critser, 2000. The enhancement of the ability of mouse spermto survive freezing and thawing by the use of high concentrations glycerol and thepresence of an Escherichia coli membrane preparation (Oxyrase) to lower theoxygen concentration. Cryobiology; 40:187-209Medeiros CMO, F Forell, ATD Oliveria, and JL Rodrigues, 2002. Current status of spermcryopreservation: why isn’t it better? Theriogenol.; 57: 327-44Medrano A, Garcia-Gil N, Ramió L, Rivera MM, Fernández-Novell JM, Ramírez A, Peña A,Briz MD, Pinart E, Concha II, Bonet S, Rigau T and Rodríguez-Gil JE, 2006. Hexosespecificityof hexokinase and ADP-dependence of pyruvate kinase play importantroles in the control of monosaccharide utilization in freshly diluted boarspermatozoa. Mol. Reprod. and Dev.; 73:1179-1194Muller Z, 1987. Practicalities of insemination of mares with deep frozen semen. J. Reprod.Fertil., Suppl.; 35:121-125.Muller CH, 2000. Rationale, interpretation, validation, and uses of sperm function tests. J.Androl.; 21:10-30.Neild D, G Chaves, M Flores, N Mora, M Beconi, A Aguero, 1999. Hypoosmotic test inequine spermatozoa. Theriogenol.; 51:721-727Nishizono H, M Shioda, T Takeo, T Irie and N Nakagata, 2004. Decrease of fertilizing abilityof mouse spermatozoa after freezing and thawing is related to cellular injury. Biol.Reprod.; 71: 973-978

Effect of Cryopreservation on Sperm Quality and Fertility 215Park NK, WY Oh, SS Lee, CA Oh, SW Kang, SB Ko, MS Kang and HS Kim, 1995. Studies onsemen freezing in Cheju native stallions. J. Agri. Sci., 37: 459-463.Parks JE, and JK Graham, 1992. Effects of cryopreservation procedures on spermmembranes. Theriogenol.; 38:210-223.Parks JE and DV Lynch, 1992. Lipid composition and thermotropic phase behavior of boar,bull, stallion and rooster sperm membrane. Cryobiology; 29:255-266.Pegg DE, 2002. The history and principles of cryopreservation. Semi. Reprod. Med.; 20: 5-13.Peña FJ, A Johannisson, M Wallgren, and H Rodriguez-Martinez, 2003. Antioxidantsupplementation in vitro improves boar sperm motility and mitochondrialmembrane potential after cryopreservation of different fractions of the ejaculate.Animal Reprod. Sci.; 78:85–98Peris SI, A Morrier, M Dufour and JL Bailey, 2004. Cryopreservation of ram semen facilitatessperm DNA damage: relationship between sperm andrological parameters and thesperm chromatin structure assay. J. Androl.; 25: 224-233.Petrunkina AM, D Waberski, AR Gu¨nzel-Apel, and E To¨pfer-Petersen, 2007. Focus ondeterminants of male fertility: Determinants of sperm quality and fertility indomestic animals. Reproduction; 134:3-17Pickett BW and RP Amann, 1987. Extension and storage of stallion spermatozoa. A review.Equine Veterinary Science; 7:289-302.Pickett BW and RP Amann, 1993. Cryopreservation of semen. In: McKinnon, AO and Voss,EL (eds) Equine Reproduction. Lea and Febiger, Philadelphia, pp. 768–789.Pommer AC, JJ Linfor and SA Mayers, 2002. Capacitation and acrosomal exocytosis areenhanced by incubation of stallion of spermatozoa in commercial semen extender.Theriogenol.; 57:1493-1501Purdy, PH and JK Graham, 2004. Effect of cholesterol-loaded cyclodextrin on thecryosurvival of bull sperm. Cryobiology; 48:36-45Rigau T, M Rivera, MJ Palomo, JM Fernandez-Novell, T Mogas, J Ballester, A Penã, PJOtaegui, JJ Guinovart and JE Rodriguez-Gil, 2002. Differential effects of glucoseand fructose on hexose metabolism in dog spermatozoa. Reproduction; 123: 579-591.Roca J, MA Gil, M Hernandez, I Parrilla, JM Vazquez, EA Martinez, 2004. Survival andfertility of boar spermatozoa after freeze-thawing in extender supplemented withbutylated hydroxytoluene. J Androl; 25:397-405Rodriguez-Martinez H and M Wallgren, 2011. Advances in Boar Semen CryopreservationReview article; Vet. Med. Int.; 2011:1-5Royere D, S Hamamah, JC Nicolle and J Lansac, 1991. Chromatin alterations induced byfreeze-thawing influence the fertilizing ability of human sperm. Int. J. Androl.;14:328-332Ruiz-Pesini E, E Alvarez, J Enriquez, and M Lopez-Perez, 2001. Association between seminalplasma carnitine and sperm mitochondrial enzymatic activities. Int. J. And.; 24:335-340.Saacke RG, JC Dalton, S Nadir, RL Nebel, JH Bame, 2000. Relationship of seminal traits andinsemination time to fertilization rate and embryo quality. Anim. Reprod. Sci.; 60:663-677.Samper JC, JC Hellander and BG Crabo, 1991. Relationship between the fertility of fresh andfrozen stallion semen and semen quality. J. Reprod. Fertil. Suppl.; 44:107-114.

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13Effect of Fatty Acids on ReproductivePerformance of RuminantsHerrera-Camacho, José 1 , Soberano-Martínez, Alejandra 1 , Orozco Durán,Karlos Edmundo 2 , Aguilar-Pérez, Carlos 2 and Ku-Vera, Juan Carlos 21 Instituto de Investigaciones Agropecuarias y ForestalesUniversidad Michoacana de San Nicolás de Hidalgo2 Campus de Ciencias Biológicas y AgropecuariasUniversidad Autónoma de YucatánMéxico1. IntroductionDifferent types of fats have been utilized in an attempt to improve reproductive function inruminant animals. Fatty acids derived from plants and oil seeds have exerted a majorimpact on reproductive performance, some of the most common sources include sunflower,linseed, cottonseed, rapeseed and soyabean. Animal fat (tallow) and calcium salts ofsaturated fatty acids may escape in a significant percentage rumen hydrogenation to beincorporated into adipose tissue and milk. Fish by-products contain a high proportion ofpolyunsaturated fatty acids (PUFAs) and pass without being altered in the rumen exertingno effects on rumen fermentation. Each dietary source of fat varies regarding composition ofspecific fatty acids (Table 1).Early studies of the effect of fat in the ration on reproductive performance were carried outby Burr & Burr (1930), who observed that fat deficiency in the ration of growing ratsinduced alterations in ovulation rate and on the onset of oestrus, while lipidsupplementation reestablished reproductive performance of the females, coining theconcept of essential fatty acids. In later studies, research was aimed at evaluating the effectof fat supplementation in different animal species both ruminant and non-ruminant, onreproductive aspects such as the establishment of puberty (Smith et al., 1989), semenproduction (Castellano et al., 2010), maternal recognition of pregnancy (Abayasekara &Wathes, 1999, Filley et al., 2000, Lopes et al., 2009) by means of the suppression of luteolyticsignals (Mattos et al., 2000), restart of ovarian activity after parturition (de Fries et al., 1998),follicle development, quality of oocytes (Staples & Thatcher, 2005; Bilby et al., 2006c), and ofthe embryo (Cerri et al., 2009), modification in the mechanism of synthesis and secretion ofhormones involved in reproductive processes (Staples et al., 1998) and on productionaspects such as quality of milk (Rego et al., 2004; Bernal et al., 2010) or meat (Wood et al.,2003). Due to the fact that some fatty acids (FA) are essential for mammals and to the role offatty acids on reproductive processes, it is possible that cattle reproduction will beinfluenced more by the type of lipids consumed than for the total lipid intake. This isparticularly important since ruminants hydrogenate PUFAs in the rumen, limiting theamount of PUFAs that are absorbed from the small intestine (Thatcher & Staples, 2007,

218Artificial Insemination in Farm AnimalsSantos et al., 2008, Doreau et al., 2011). However, it is possible that some specific PUFAsmay pass intact the reticulo-rumen and be absorbed from the small intestine, allowing inthis way the improvement of reproductive efficiency directly on the target tissue of thereproductive system of the female (autocrine or paracrine) or by an indirect effect mediatedby the endocrine system (Staples & Thatcher, 2005).Fat sourceFatty acidC14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3Myristic PalmiticPalmitoleicStearic Oleic Linoleic LinolenicTallow 3 25 3 18 43 3.8

Effect of Fatty Acids on Reproductive Performance of Ruminants 219al., 2000). Potential mechanisms may include increment of energy density of the ration(Ferguson et al., 1990), even when for some workers (Williams & Amstalden, 2010), theeffect of fat supplementation on reproduction is independent of the energy density of rationor of changes in live weight of animals. Considering all the above described, the aim of thisreview is to examine some of the reproductive processes in the bovine and ovine femaleswhich could be regulated or modified by the inclusion of lipids in the ration.2. Effect of lipid supplementation on pregnancy rateIncorporation of lipids in rations for dairy cattle usually increases energy density of rationand improves lactation and reproductive performance (Funston, 2004). However, when theyare supplied in early lactation, frequently there is a reduction in feed intake due to areduction in dry matter digestibility and to an increase in energy of greater availability, sowhen lipids are supplied in the early postpartum period, there is little alteration in theenergy status of the animal even when a higher energy density ration is consumed (Santoset al., 2008).Then, if dietary fat does not alter the energy status of dairy cows, reproductive responseresults more from the supply of some fatty acids, than from the effect of the energy supplyper se (Staples et al., 1998, Williams & Amstaldem, 2010).There are several studies that report a better reproductive performance in cows fedsupplementary lipids. In this respect, Staples et al. (1998), showed that lipid consumptionexerted a positive effect on reproductive aspects in dairy cows (Table 2). In beef cattle, thesame trend has been observed. It is in this context that, de Fries et al. (1998) reported thatBrahman cows consuming 5.2% lipids in the ration showed a trend towards an increase inpregnancy rate than those cows which consumed only 3.7% lipids in the ration. Ferguson etal. (1990) observed a 2.2 times increase in the possibility of pregnancy in lactating cowsconsuming 0.5 kg lipids per day. In another study, it was demonstrated that grazing cowssupplemented with fat, pregnancy rate at first service was 16% higher than in cows whichdid not receive fat in the ration (Bader et al., 2000).Bellows et al., (2001) observed that supplementation with safflower seed, soyabeans, orsunflower seed (4.7, 3.8 and 5.1% fat in the ration, respectively) for the last 65 days beforecalving increased subsequent pregnancy rates (94%, 90% and 91%, respectively) of first-calfbeef heifers compared with the control (79 %) that received only 2.4 % fat in the ration. Inanother study Bellows et al., (2001), using good quality forage and a higher amount of fat inthe ration (6.5%) during 68 days before calving, was unable to improve pregnancy ratesrelative to a control ration (2.2% fat), this result indicates that when adequate nutrients areavailable, the effect of supplemental fat may be masked.Grazing Holstein cows which were supplemented for 103 days, as from day 10 post-partum,with two sources of bypass fat Megalac plus 3% (MP; 0.4 kg/day, containing Ca salts ofpalm fatty acids and Ca salts of methionine hydroxy analogue) and Megapro Gold (MPG;1.5 kg/day, containing Ca salt of palm fatty acids, extracted rapeseed meal and wheypermeate), MPG increased (P

220Artificial Insemination in Farm Animalsweeks in-calf rate. Comparing the combined fat treatments to CG resulted in a higher(P

Effect of Fatty Acids on Reproductive Performance of Ruminants 221effect of fat on fertility may not be due to improvement in energy balance of the cows butrather to the specific effect of some dietary fatty acids on the physiology of the hypothalamushypophysis-ovaryaxis and even the uterus.In a review of previous studies in which conjugated linoleic acids (CLA) were supplementedto dairy cows during early lactation, de Veth et al. (2009) demonstrated that the probabilityof pregnancy increases in 26% when CLA are increased in the ration and that the optimumCLA amount is 10.0 g d -1 , after which the beneficial effects are reduced. It is possible that thepositive effect of lipid supplementation may be due to specific fatty acids (Staples &Thatcher, 2005), and the absorption of unsaturated FA in ruminants is limited due microbialbiohydrogenation in the rumen (Lopes et al., 2009). Some studies have evaluated thepossibility that unsaturated FA intake, particularly those of the n-6 (linoleic acid) and n-3 (αlinolenic,eicosapentaenoic, docosahexaenoic acids) families, may have some influence onreproduction in cows, even when reports in the literature are not always consistent (Santoset al., 2008). In this respect, when cows were fed 0.75 kg of linseed rich in -linolenic acid (n-3), or sunflower rich in linoleic acid (n-6), pregnancy rate tended to increase in cows of thefirst treatment (Ambrose et al., 2006a).In other studies, no response was observed with linseed (Fuentes et al., 2008). Similarly,feeding n-3 fatty acids from fish oil in the form of Ca-LCFA did not improve pregnancy ratepostpartum at first service in beef cows when compared to supplementation with beeftallow (Juchem, 2007) or with Ca-LCFA from palm oil (Silvestre, quoted by Santos et al.,2008), even when pregnancy rate at second service postpartum was higher in cows fed n-3fatty acids (Silvestre, quoted by Santos et al., 2008). In grazing F 1 (Bos taurus x Bos indicus)cows, Aranda-Ávila et al. (2010) observed a 15.4% increase in pregnancy rate when cowswere supplemented with corn oil, relative to a control group (54.5 vs 41.7 % respectively)after 35 day supplementation; however, differences were not statistically significant. It ispossible that the poor response observed in those studies may be due to an increase in milkproduction along with a loss of body weight, which occurs in greater or lesser degree incows during the early postpartum period (Sklan et al., 1994).3. Effect of lipid supplementation on the hypothalamus-hypophysis-ovaryaxisThe major objective of cow-calf enterprises is to produce one calf per cow annually. Thus,management strategies that enhance reproductive performance of milk and beef cows arebeneficial to the productivity of cow-calf operations. Previous studies reported thatutilization of dietary fat as a nutraceutical, particularly PUFAs, positively influencedreproductive function in both milk and beef cows (Williams & Stanko, 2000). Furthermore,these positive effects were independent of the additional energy contribution from thePUFAs sources (Funston, 2004). Different mechanisms have been proposed by means ofwhich fat supplementation may affect functioning of the hypothalamus-hypophysis-ovaryaxis. Early work in this respect suggested that fat supplementation may affect secretion ofreproductive and metabolic hormones and further research demonstrated that fat additionto the ration modified ovarian activity in heifers and adult cows postpartum.The mechanism (or mechanisms) by which dietary fat improves reproductive performancehas not been elucidated. Several hypotheses have been proposed: 1) an amelioration of anegative energetic balance, thus leading to an earlier return to oestrus postpartum and,therefore, improved fertility; 2) an increase in steroidogenesis favorable to improved

222Artificial Insemination in Farm Animalsfertility; 3) manipulation of insulin so as to stimulate ovarian follicle development; and 4) astimulation or inhibition of the production and release of PGF 2α , which influences thepersistence of the corpus luteum (Staples et al., 1998)3.1 Hormonal secretion and lipid metabolitesSome studies showed that dietary fat supplementation in dairy heifers increased circulatingconcentrations of progesterone (Talavera et al., 1985), and enhanced lifespan of inducedcorpus luteum during early postpartum in beef cows (Williams, 1989; Ryan et al., 1995).Other studies suggest that when lipids are included in the ration of cows to increase energydensity, caloric balance is improved which directly influences hypophysis-gonadal activitypostpartum (Harrison et al., 1995), increasing, in principle, the amplitude and frequency ofsecretion of luteinizing hormone (LH) in animals (Sklan et al., 1994). In this respect, de Lunaet al. (1982), reported an increase in the secretion of luteinizing hormone in ovariectomizedcows treated with GnRH and supplemented with beef tallow. In sheep, secretion ofluteinizing hormone in response to the injection of GnRH at day 10 of the oestrus cycle wasgreater in Pelibuey sheep supplemented with Ca-LCFA from palm oil during 30 days thanin the control group (Espinoza et al., 1997).Other studies, using isocaloric and isonitrogenous diets in cows of poor body conditionindicated that the increase in dietary fat consumption augmented the number of follicles ofmedium-size by 1.5- to 5-fold within 3 to 7 weeks and these changes occurred coincidentwith changes in serum insulin, growth hormone and intraovarian insulin-like growth factor(IGF-1) (Wehrman et al., 1991; Ryan et al., 1992; Thomas et al., 1997). Table 3 summarizes theeffects of dietary fat supplementation on follicular physiology and growth as observed indifferent studies.ReferenceWehrman et al., 1991; Ryan et al., 1992;Hightshoe et al., 1991; Lucy et al., 1991;Thomas & Williams, 1996; Thomas et al., 1997;Lammoglia et al., 1996; Stanko et al., 1997; deFries et al., 1998Lucy et al., 1989, 1991Wehrman et al., 1991; Ryan et al., 1992Lopes et al., 2009, Salas-Razo et al., 2011Ryan et al., 1992; Thomas & Williams, 1996de Fries et al., 1998; Bilby et al., 2006a;Garnsworthy et al., 2008EffectIncreased number of medium-sizedfollicles (polyunsaturated fat >saturated and highly polyunsaturatedfat effects)Milk cows supplemented with Ca-LCFA palm oil, the basal level of LHwas increaseIncreased granullosa cell progesteroneproduction in vitro, increased follicularfluid progesteroneCows supplemented with rumen inertpolyunsaturated fat had greater meanserum progesterone concentrationscompared with controlNo effect on superovulation rateIncreased number of large follicles;increased size of largest follicleTable 3. Summary of the effect of dietary fat supplementation in cattle on ovarian folliculargrowth and steroid production. (Modified from Williams & Amstalden, 2010)

Effect of Fatty Acids on Reproductive Performance of Ruminants 223On the other hand, it has been shown that hiperlipidic rations supplied both to dairy as wellas to beef cows, induced and increase in the levels of blood cholesterol, as it was observedby Hightshoe et al. (1991) in cows supplemented postpartum with Ca-LCFA from palm oil.Similar results, were reported in Angus and Hereford cows which consumed a supplementwhich contained 125 g of Ca-LCFA from palm oil (Espinoza et al., 1995), in Chinampas (Bostaurus) cows consuming Ca-LCFA from palm oil or beef tallow (Espinoza-Villavicencio etal., 2010); this is particularly important since cholesterol is the main precursor forprogesterone synthesis in the corpus luteum as well as of other steroid hormones at thefollicular level (Childs et al., 2008c). Thus, when supplementation with protected fats fromrumen biohydrogenation is augmented, it is possible to increase concentration of plasmaprogesterone in cows (Lopes et al., 2009), which is associated positively with pregnancy rate(Santos et al., 2008). When endometrial secretions are modified (Gray et al., 2001) changesare induced in endometrial architecture which are fundamental for the appropriatedevelopment of the embryo (Wang et al., 2007). Nonetheless, in other studies, no responsehas been observed of fat supplementation on progesterone synthesis. In this respect,Robinson et al. (2002), using cows fed a ration rich in linoleic acid showed a reduction of upto 50% in the serum levels of progesterone between days 4 and 8 of the oestrus cycle, whilethose cows consuming a ration rich in linolenic acid showed a lower concentration of thishormone, but in days 4 and 15 of the oestrus cycle (Robinson et al., 2002). Recent work(Huante, 2010), demonstrated that secretion of progesterone in Brahman cows with lowbody condition at calving and during the postpartum (2.0 points, in the scale 1-5) wasunaffected by supplementation with oilseeds with 60% of unsaturated fatty acids, relative tothe control group (0.065±0.013 vs 0.054±0.013 ng/ml, respectively). These results suggestthat due to the low body condition, the cow uses feed energy for its maintenance and not forreproductive processes. Those results suggest that the above mentioned fatty acid, exertdifferent effects in the synthesis of ovarian steroids (Hinckley et al., 1996). Childs et al.(2008b) fed beef heifers a ration enriched with fish oil (n-3) and even when no increase inserum progesterone was achieved at day 7 of the oestrus cycle, they postulated that therewas evidence of a greater synthesis of progesterone during the whole oestrus cycle due tothe increase in the serum concentration of cholesterol and to the greater size of the corpusluteum.While in sheep, concentration of progesterone in the follicular fluid was greater than insheep which consumed the ration enriched with n-3 fatty acids, than those which were fedwith a greater amount of n-6 fatty acids (Wonnacott et al., 2010). Some in vitro studies havedemonstrated that progesterone metabolism can be inhibited by high concentrations (300M) of -linolenic acid in the culture media (Piccinato et al., 2010). This is perhaps related tothe fact that certain PUFAs may also increase serum concentration of insulin and this inturn, reduce hepatic expression of some enzymes of the cythocrome P 450 complex whichcatabolize progesterone (Lemley et al., 2008). Oestradiol, another hormone derived fromcholesterol has stimulatory effects on the uterine secretion of PGF 2α (Knickerbocker et al.1986) and can increase sensibility of the corpus luteum to prostaglandins by intensifying itsregression (Howard et al., 1990). Previous studies demonstrate that intra-abomasal infusionof yellow fat induced a reduction in blood levels of oestradiol between days 15 and 20 of theoestrus cycle, compared to cows which were infused with glucose intra-abomasally. Therelationship between dietary fatty acids and synthesis of steroid hormones may be directthrough a direct effect on steroidogenesis (acute regulatory protein of steroids; STAR,cythocrome P450 protein, family 11; subfamily A polypeptide enzyme 1; CYP11A1) etc. or

224Artificial Insemination in Farm Animalsindirectly through prostaglandins. STAR plays a fundamental role in the synthesis of steroidhormones (Stocco et al., 2005). In previous work, it has been observed that in beef heifersthere was a reduction in the concentration of arachidonic acid in the endometrial tissuewhen they were fed n-3 PUFAs (Childs et al., 2008a; Childs et al., 2008b). On the other hand,there is evidence that supplementation with linoleic acid (n-6) increased uterine secretion ofPGF 2α (Cullens, 2005), which could derive in a greater availability of arachidonic acid in theendometrium. Under such conditions it could be speculated that a possible way to act of then-3 and n-6 fatty acids in steroidogenesis is by reducing or increasing the availability ofarachidonic acid, respectively and as a consequence, the expression of RNAm for STARsynthesis.3.2 Lipids on ovarian activityThese results suggest that another of the mechanisms by means dietary lipids may improvereproductive performance of cattle is influencing follicular development and ovulation. Inthis, respect, Lucy et al. (1991), replaced corn with Ca-LCFA from palm oil in the ration ofdairy cows at calving, and increased the number of medium size follicles (6-9 mm) and offollicles greater than 15 mm within 25 days postpartum. Furthermore, the diameter of thegreatest follicle was superior in cows fed Ca-LCFA from palm oil (18.2 vs 12.4 mm). Whenthis study was repeated with isocaloric diet, observed effects were similar (Lucy et al. 1993).The greatest increase in medium follicle populations occurred in response to plant oilconsumption, which is likely a direct result of the effects of high levels of linoleic acid in therumen. Maximum follicular growth responses to plant oil supplementation have occurredwhen plant oils were fed at 4 to 6% of dietary dry matter, with lesser increases observed atlower levels of added fat. Animal tallow, calcium salts of saturated fatty acids or fish oilhave been shown to have less clear effects on follicular growth than plant-derived oils.Moreover, postpartum beef cows which calved in a very poor body condition (BCS of 3; 1-9scale) were unable to develop medium or large follicles at a rate equal to those with a bodycondition score of 4 or greater after 3 weeks of fat consumption (Ryan et al., 1994).The number of medium size follicles (5 to 10 mm) was higher in beef cows which consumedfeed with a greater content of PUFAs (Thomas et al., 1997) and in dairy cows whichconsumed a diet enriched with 5% n-3 fatty acids derived from fish oil (Heravi-Moussavi etal., 2007). Similar results were observed in cows fed with diets enriched with n-3 or n-6 fattyacids (Robinson et al., 2002). Staples & Thatcher (2005) summarized the effect of increasinglipids in the ration on the size of the dominant follicle: on average, the size of the dominantfollicle was 3.2 mm greater than in females fed with some source of fat, which represents a23% increase. Other studies (Bilby et al., 2006d; Garnsworthy et al., 2008) showed that thesize of the dominant follicle is increased in cows fed diets rich in PUFAs. In sheep, the use ofCa-LCFA from palm oil (El-Shahat & Abo-El maaty, 2010) or from corn oil (Herrera et al.,2008) in the feed improved the number and size of the preovulatory follicles, as well as rateof ovulation and the superovulatory response in Pelibuey sheep (Herrera et al., 2008). Theseevidences demonstrate that consumption of lipids accelerate follicular growth (de Fries etal., 1998), which may influence the restart of ovulation postpartum, as it was shown byMarín-Aguilar et al. (2007) who observed that Holstein cows supplemented with plant oil(60% PUFAs) reduced by 7 days the restart of ovarian activity relative to a control group. deVeth et al. (2009) observed that time at first ovulation in dairy cows was reduced by 8 dayswhen they were supplemented with 8 g/d of trans-10, cis-12 CLA.

Effect of Fatty Acids on Reproductive Performance of Ruminants 2254. Lipids and its effect on endometrial secretion of prostaglandinsStudies in a variety of species have shown that dietary PUFAs can modulate prostaglandinsynthesis and metabolism. Eicosanoids, comprising prostaglandins, thromboxanes,leukotrienes and lipoxins, are all synthesized from C20 fatty acids (Mattos et al., 2000). Themost biologically active two series prostaglandins are derived from arachidonic acid, but theless active three series prostaglandins can be produced from eicosapentaenoic acid by theaction of the same enzymes (Robinson et al., 2002).Prostaglandins play an important role in reestablishing oestrus cycles both immediatelyafter parturition and thereafter until conception. Prostaglandin F 2α (PGF 2α ) is responsible foruterine involution after parturition. The uterus releases PGF 2α during each oestrus to regresseach new corpus luteum if the cow is not pregnant and initiate a new oestrus cycle. Duringthe period of corpus luteum regression, concentrations of PGF 2α and progesterone areinversely related. If the cow does conceive, release of PGF 2α from the uterus is prevented inorder to preserve the corpus luteum and maintain pregnancy (Funston & Filley, 2002).Linoleic acid is a substrate for the synthesis of PGF 2α . Linoleic acid can be desaturated andelongated to arachidonic acid (C20:4, n-6), which is a precursor for PGF 2α . Regulatoryenzymes for this conversion include delta 6 desaturase and cyclooxygenase. Linoleic acidcan inhibit prostaglandin synthesis by competitive inhibition with these key enzymes.Arachidonic, and two fatty acids found in fishmeal, eicosapentaenoic (C20:5) anddocosahexanoic (C22:6), have been shown to inhibit cyclooxygenase activity as well. It isimportant to note that linolenic acid (C18:3) was also present in the endometrialprostaglandin synthesis inhibitor isolated by Thatcher et al. (1994), and that linolenic acidhas been shown to be a strong inhibitor of prostaglandin synthesis (Mattos et al., 2000). Theamount and probably type of particular fatty acids reaching the target tissues likelyinfluence if prostaglandin synthesis is going to be stimulated or inhibited (Thatcher &Staples, 2000).Figure 1 shows the schematic metabolic pathway of dietary n-6 and n-3 PUFAs andpotential mechanisms for regulation of PGF 2α secretion. Absorbed PUFAs are desaturatedand elongated in organs such as the mammary gland, adipose tissue, testis, brain, placentaand the liver (of non-ruminants). Dietary PUFAs and their desaturation and elongationproducts are incorporated into phospholipids of the plasma membrane. The amount of eachfatty acid incorporated depends on the amount of precursor present in the diet. Externalstimuli such as the binding of oxytocin (OT) to the oxytocin receptor (OTr) stimulates theactivity of phospholipase A2 (PLA2) and phospholipase C (PLC), which cleavephospholipids from the plasma membrane and ultimately increase availability ofdiacylglycerol (DAG) and fatty acids for processing by prostaglandin H synthetase (PGHS).Eicosapentaenoic acid (EPA; C20:5, n-3) is processed by PGHS to generate prostaglandins ofthe 3 series. Arachidonic acid (AA; C20:4 n-6) can be processed by PGHS, epoxygenase andlipoxygenase to generate prostaglandins of the 2 series, epoxyeicosatrienoic acids (EETs),leukotrienes and hydroxyeicosatetraenoic acids (di-HETEs), respectively (Mattos et al.,2000).In vitro studies have demonstrated that some n-3 (eicosapentaenoic and docosahexaenoic)fatty acids reduce biosynthesis of prostaglandins of the series 2 in cells and tissues (Mattoset al., 2003). Similarly, some isomers from conjugated linoleic acid inhibited synthesis ofPGF 2α and the effect was independent of the concentration of linolenic acid and the ratio n-6:n-3 (Harris et al., 2001). The above results are interesting due to the fact that most

226Artificial Insemination in Farm AnimalsFig. 1. Schematic representation of the metabolism of dietary n-6 and n-3 polyunsaturatedfatty acids and potential mechanisms for regulation of PGF 2α secretion (Mattos et al., 2000).embryonic losses in cattle occur during days 8-16 after artificial insemination (Sreenan et al.,2001), which leads to believe that some embryos may not reach the appropriate size at thatmoment to inhibit synthesis of PGF 2α for luteolysis to occur (Thatcher et al., 1994), showingthe inability to inhibit luteolytic action by PGF 2α during the critical period of maternalrecognition of pregnancy (Childs et al., 2008a). In this context, inhibition of the synthesis ofPGF 2α could increase the rates of embryo survival and pregnancy (Binelli et al., 2001).PUFAs (n-3) such as eicosapentaenoic acid may inhibit uterine synthesis of PGF 2α bycompeting with arachidonic acid by means of COX, or in the case of docosahexaenoic acid,by competence with arachidonic acid by phospholipase A2 enzymes (Mattos et al., 2000).Fish meal has relatively high concentrations of eicosapentaenoic and docosahexaenoic acids,in such a way that their incorporation in the ration of cattle may reduce the synthesis ofPGF 2α and delay regression of the corpus luteum, improving embryo survival and herdfertility (Staples et al., 1998)Previous studies showed that the infusion of a fat source rich in linoleic acid (17%) into theabomasum of lactating dairy cows resulted in a significant reduction in the release of PGFM,as measured in peripheral plasma, in response to an injection of oxytocin on day 15 of asynchronized oestrous cycle (Oldick et al., 1997). These results indicate that highconcentrations of PUFAs in the diet can decrease endometrial secretion of prostaglandins.In this respect, in cows, fed with n-3 fatty acids derived from fish meal, a reduction wasobserved in endometrial secretion of arachidonic acid, increasing in this same tissue thelevels of eicosapentaenoic acid and total n-3 fatty acids (Burns et al., 2003; Bilby et al.,2006b). Similar effects were observed in dairy cows which consumed increasing amounts of

Effect of Fatty Acids on Reproductive Performance of Ruminants 227fish meal or Ca-LCFA from fish oil (Bilby et al. 2006a). Due to the addition of n-3 and n-6 inthe phospholipid component of endometrium, it is possible that changes in the content offatty acids in the endometrial tissue may modulate secretion of PGF 2α in cows (Santos et al.,2008). In further studies, Burns et al. (2003) reported an increase in eicosapentaenoic acid atday 18 postpartum, as well as a reduction in arachidonic acid in the caruncules of beef cowsfed fish meal. In another study, supplementing the diet with fish oil during theperiparturient period reduced uterine secretion of PGF 2α in lactating dairy cows. In thissense, beef heifers fed n-3 PUFAs reduced production of arachidonic acid in endometrialtissue, apparently due to the fact that linoleic acid must elongate and desaturate to formarachidonic acid using the same enzymes than for the synthesis of eicosapentaenoic acid,starting from α-linolenic acid (Coyne et al., 2008).There is evidence that during the prepartum period, lipid supplementation with 30% fattyacids as linoleic acid (n-6) increased uterine secretion of PGF 2α (Cullens, 2005). Increase inthe synthesis of PGF 2α when cows are fed n-6 fatty acids during the transition period beforecalving or during the early postpartum (puerperium), can increase the potential of theuterus and of the immune system to secrete eicosanoids, which have influence inpostpartum uterine health and in the inmunocompetence of the cow (Santos et al., 2008).Childs et al. (2008b) fed heifers with a diet rich in n-6 fatty acids (entire soyabeans) or n-3fatty acids (fish oil) and observed that on day 15 of the oestrous cycle, serum concentrationof PGFM metabolite was higher than in the first group. These results are interesting if it isconsidered that by stimulating secretion of PGF 2α during early postpartum uterineinvolution can be favored (Mattos et al., 2004) and reproductive efficiency is improved, sincewhen cows were supplemented before calving with calcium salts rich in n-6 fatty acids theoccurrence of postpartum diseases such as membrane retention, metritis and mastitis, wasreduced (Cullens, 2005). Thus, both arachidonic acid, but as well precursors of this fattyacid, such as linoleic acid increase the production of series 2 prostaglandin, while n-3 fattyacids compete with arachidonic acid and therefore reduce synthesis of such prostaglandin.In studies carried out in vitro, addition of eicosapentaenoic acid to the culture media ofendometrial cells of cows, reduced production of endometrial PGF 2α from 88 to 40%, but thiswas reverted as the ratio of n-6:n-3 in the culture media was increased from 0 to 19%(Caldari-Torres et al., 2006). This observation is consistent with previous reports and suggestthat the net inhibition of uterine PGF 2α synthesis by n-3 fatty acids may depend on theamount of n-6 fatty acids that reach the secretory tissue (Achard et al., 1997).However, recent studies (Meier et al., 2009) showed that the bovine endometrial andtrophoblastic tissues during short-term culture, incubated in a media supplemented withfatty acids: eicosapentaenoic (20:5-3; EPA), docosahexaenoic acids (22:6-3; DHA) or linoleicacids (C18:2-6; LIN), the release of PGE 2 from ‘pregnant’ endometrium was higher (P=0.094)than from ‘non-pregnant’ endometrium, while PGF 2α concentrations were similar.Treatment with fatty acids had no effect on PGF 2α or PGE 2 release from either pregnant ornon-pregnant endometrium. The individual fatty acid treatments had no effect on the ratioof PGF 2α to PGE 2 from trophoblast tissues, but when the data from the three fatty acidtreatments were combined (EPA, DHA and LIN treatment groups) the ratio of PGF 2α toPGE 2 was reduced (P=0.026) when compared to the medium only. This result indicated thatthe ability of exogenous fatty acids to modify embryonic prostaglandin release needs to beexamined in the context of supplementing dairy cows with different sources of fats.On other hand, the dynamics of bovine corpus luteum regression in response to exogenousPGF 2α can also be altered by dietary fish meal. In this respect, Burke et al., (1997) fed cows

228Artificial Insemination in Farm Animals(n=56) with fish meal at either 0 or 2.8% of ration dry matter from 24 to109 days postpartum.On day 58 postpartum, all cows were injected with a luteolytic dose of PGF 2α . Twodays after injection of PGF 2α , the proportion of cows with plasma concentrations ofprogesterone that were >1 ng ml -1 was greater when fish meal was included in the rationthan when a control ration was fed (29.0 vs 4%). Thus, it is possible that fatty acids presentin fish oil reduce the sensitivity of the corpus luteum to PGF 2α .5. Lipids their effect on embryo developmentEstablishment of pregnancy in the ruminant requires the ovulation of a competent oocyte, ofinsemination at the appropriate time and of a correct pattern of secretion of oestradiol andprogesterone during the follicular and luteal phase of oestrus. The embryo must develop inan appropriate way and avoid luteolysis producing enough interferon which stimulatesthe expression of genes in the endometrium to inhibit the synthesis of oxytocin receptorsand consequently final production of PGF 2 , allowing the establishment of a corpus luteum(Bott et al., 2010). In dairy cows there is a significant loss of embryos during this period, it isconsidered that only 40% of cows remain pregnant at day 28 after artificial insemination(Santos et al., 2008). There is evidence that such events can be influenced by PUFAsconsumed in the ration (Wathes et al., 2007). Fatty acids play an important role in themodification of the biophysical properties and in the activity of biological membranes,including fluidity and cell proliferation (Bilby et al., 2006d). The competence and quality ofthe ovocyte and of the embryo are related to the type of fatty acid, specifically, with thecontent of particular fatty acids en the phospholipids of cell membrane which play a role indevelopment and during and after fertilization (Santos et al., 2008).The amount of lipids in the ovocyte of ruminants is about 76 ng approximately and hasaround 58% triglycerides, 20% phospholipids, 20% cholesterol and 10% free fatty acids(McEvoy et al., 2000). Fatty acids found in greater amounts in the phospholipid fraction ofthe membrane of cattle ovocyte are palmitic (16:0) and oleic (18:1) acids. PUFAs representless than 20% of the total, being linoleic acid the most abundant of them (Santos et al., 2008).Marei et al. (2010) pointed out that linoleic acid (n-6) is the most abundant fatty acid infollicular fluid of cattle and has an important role in the regulation of the process ofmaturation of the ovocyte, since when the cell complex of the cumulus and ovocyte weretreated with linoleic acid there was a delay in the growth of the latter. Some studies haveshown the fatty acid profiles in follicular fluid are affected by the estrogenic activity of thefollicle (Renaville et al., 2010). Table 4 shows saturated and unsaturated fatty acids inplasma and in some reproductive tissues.Ratio of saturated fatty acids to PUFA in granulose cells (Adamiak et al., 2005) and in theovocyte (Wonnacott et al., 2010) is greater than in plasma. This suggests the presence of amechanism of selective uptake in the ovarian follicles or de novo synthesis of saturated fattyacids from acetate (Wonnacott et al., 2010). Fatty acids can be oxidized as an energy sourceduring maturation of the ovocyte and during early embryo development beforeimplantation, in such a way that cattle ovocytes exposed to metal palmoxirate to blockoxidation of fatty acids, showed a lower ability to form blastocysts after in vitro fertilization(Ferguson & Leese, 2006). Cetica et al. (2002) reported a significant increment in the activityof the enzyme lipase during maturation of cattle ovocyte, which releases fatty acids fromtriglycerides for later oxidation. Apart from the role of fatty acids, particularly thosesaturated, as sources of energy, the content of PUFAs in the ovocyte may affect maturation,

Effect of Fatty Acids on Reproductive Performance of Ruminants 229Fatty acid groupPlasma (µg/ml)Granulosa cell(µg/pellet)Oocytes(ng/oocyte)Dietary treatment abn-3 n-6 n-3 n-6 n-3 n-6Saturated 30.9 42.1 39.1 39.7 75.7 71.1Unsaturated 59.3 49.4 49.7 52.1 20.9 25.8Monounsaturated fattyacids21.5 18.0 20.9 23.9 7.8 12.9Polyunsaturated fatty acids 37.8 31.4 28.9 28.2 13.0 12.9n-6 PUFA 25.4 28.9 8.5 c 24.1 5.2 10.9n-3 PUFA 12.4 2.5 20.4 4.1 7.8 2.1Ratio n6:n3 2.1 11.5 0.4 6.2 0.7 8.3Table 4. Saturated and unsaturated fatty acids in plasma and some reproductive tissues.Adapted from Wonnacott et al. (2010) and Palmquist (2010). a 6.4 % oil in the dietsupplemented from linseed oil, b 5.7% oil in the diet supplemented from sunflower oil.cImportant effects due dietary fatty acids are in bold.cryopreservation and its capacity for further development (Wathes et al., 2007). From the n-3fatty acids, linolenic acid has been implicated in the growth and differentiation of theovocyte, regulation of meiotic arrest during the germinal vesicle stage and in avoiding thebreaking of this structure (Kim et al., 2001).In sheep, Zeron et al. (2002) showed that supplementation with Ca-LCFA from fish oilduring 13 weeks, resulted in better quality ovocytes and better integrity of their membrane,compared to that of sheep which were not fed lipid supplements (74.3% and 57.0%,respectively), which increased the ratio of long chain fatty acids in plasma of cells from thecumulus, although these changes were not observed in the ovocytes, suggesting selectiveuptake by the ovocyte or a highly regulated uptake, which could limit potential impact ofcow nutrition on the proportion of fatty acids in their gametes. While in beef cattle, Fouladi-Nashta et al. (2007) fed cows with 200 or 800 g per day of Ca-LCFA from palm oil, whichresulted in a greater percentage of ovum which developed up to the blastocyst stage andhad a greater amount of cells due to an increment in the number of cells of thetrophectoderm. By influencing the molecular mechanisms which control nucleus maturationof the ovocyte, in vitro treatment of the cumulus-oocyte complex with linolenic acid (n-3),increased the percentage of gametes which reached the meiotic second division, increasedthe division of the embryo as well as the rate of blastocyst (Marei et al., 2009).When a group of lactating superovulated cows were fed with rich sources of saturated fattyacids (n-6 or n-3), rate of fertilization and the number of transferable embryos was notdifferent; however, embryo development was increased in cows which consumedunsaturated fatty acids, compared to the cows which consumed saturated fatty acids(Thangavelu et al., 2007). Cerri et al. (2009) supplemented dairy cows with Ca-LCFA frompalm oil or linoleic + octadecaenoic acids between days 25 prepartum and 80 dayspostpartum observing in the second group a greater proportion of excellent and goodquality embryos, apart from a higher number of blastomere.In hair ewes, Herrera et al. (2008) showed that PUFAs in the ration increased superovulatoryresponse, registering increased (P

230Artificial Insemination in Farm Animalsembryos (6.72±1.78 vs 3.09±1.36) in the PUFAs treatment than in the control treatment,respectively.On the contrary, Childs et al. (2008b) fed cows with a ration enriched with n-3 PUFAs anddid not observe any effect on the number of normal embryos nor in the amount of goodquality embryos (grade 1 and 2); however, these cows showed a lower number ofdegenerated embryos. Similarly, Bilby et al. (2006d) did not find an effect of fatty acids inthe ration of dairy cows on the quality of embryos after maturation and in vitro fertilization.In a similar way, Thangavelu et al. (2007) did not establish a difference in the total numberof transferable embryos of dairy cows supplemented with either PUFAs or saturated fat.Marques et al. (2007) did not observe any effect of the addition of arachidonic oreicosapentaenoic acids to the culture media for the in vitro maturation of ovocytes on thesubsequent embryo development. This agrees with observations by Lawson et al. (2007)who added increasing amounts of eicosapentaenoic acid to the culture media for thematuration of ovocytes in vitro. In a recent study, Wakefield et al. (2007) suggested thatcontrary to the possible beneficial effects, supplementing a ration with n-3 PUFAs duringthe period just before or immediately after conception, may reduce normal development ofthe embryo, since this seems to disturb mitochondrial metabolism. Leroy et al. (2010)cultivated bovine zygotes in media supplemented with serum from heifers fed rations witha high content of lipids protected from rumen biohydrogenation and observed a lowerproduction of blastocysts compared to a control treatment, noting that in the first there wasa greater expression of genes related to apoptosis.Even when in vivo and in vitro studies have shown a better embryo development withrations supplemented with lipids, results are not consistent and it is important to ascertainin particular, which fatty acids are the most beneficial for embryo survival (Santos et al.,2008). Oocytes of all mammals contain an endogenous lipid reserve. This feature reflectstheir common ancestral origin, the yolk-rich amniote egg. However, lipids are speciesspecificin terms of their apparent abundance and utilization. Despite the significant role ofthe lipid reserves in cell structure and function, very few studies have provided detaileddescriptions of its nature and composition in mammalian oocytes. Table 5 gives the fattyacid composition of total lipid extracted from zone-intact oocytes of cattle and sheep.6. ConclusionData reviewed shows that supplementation with different sources of lipids and fatty acidsimprove reproductive performance of the female ruminant. However, it is important toconsider that the optimum response will be achieved when undernutrition status of thefemale is not extremely sever. A nutrient balance (protein:energy) in the ration consumed bythe animal is fundamental to obtain maximum benefit from supplementation with fat, sincefatty acids do not supply nitrogen for amino acid synthesis and consequently for the correctfunctioning of the hypothalamus-hypophysis axis. Improvements in reproductiveperformance may be a result of increased energy density of the ration or of the direct effectsof specific fatty acids on reproductive processes. As is the case for any technology ormanagement strategy that improves specific aspects of ovarian physiology and cyclicactivity, actual improvements in pregnancy rate or total weight of calf weaned aredependent on a variety of management practices and environmental conditions. Until theseinterrelationships are better understood, livestock producers are recommended to attemptto formulate low cost/balanced rations. If a source of supplemental fat is available locally

Effect of Fatty Acids on Reproductive Performance of Ruminants 231Mean (± SEM) distribution of fatty acids (%, w/w) aName Formula Cattle (n = 3) b Sheep (n = 2)Lauric 12:0 0.23 ± 0.15 ndMyristic 14:0 2.48 ± 1.02 0.39±0.032PalmiticPalmitoleic16:016:1 n-732.0 ± 1.642.24 ± 0.4524.7±0.744.38±0.20Heptadecanoic 17:0 0.76 ± 0.14 0.41±0.407StearicOleicVaccenicLinoleicγ-Linolenicα-LinolenicStearidonicArachidicEicosenoicEicosadienoicEicosatrienoicArachidonicEicosapentaenoicBehenicErucicDocosatetraenoicDocosapentaenoicDocosahexaenoic18:018:1 n-918:1 n-718:2 n-618:3 n-618:3 n-318:4 n-320:020:1 n-920:2 n-620:3 n-620:4 n-620:5 n-322:022:1n-922:4 n-622:5 n-322:6 n-314.2±2.4725. ±1.753.71± 0.125.17± 0.120.75 ± 0.160.49±0.09nd1.35±0.700.27±0.130.54±0.200.27±0.151.13±0.571.15±1.151.23±0.630.20±0.100.27±0.150.88±0.280.50±0.2516.2±0.3026.2±0.233.64±0.326.98±0.101.01±0.062.01±0.411.68±0.303.11±0.450.19±0.1870.91±0.4760.52±0.0701.50±0.603.03±1.093.03±1.09ndnd1.41±0.331.74±0.06Lignoceric 24:0 2.30±1.21 ndTable 5. Fatty acid composition of total lipid extracted from zone-intact oocytes of cattle andsheep (adapted from McEvoy et al., 2000). Each sample (n = 2–4) represents 1000 oocytes.aPercentage (w/w) of the total fatty acids in oocyte lipid. b One of the four original cattleoocyte samples was excluded because of uncertainty about the validity of the assay resultinvolving cholesterol ester component of total lipid. nd: not detected.and can be incorporated with little or no change in the cost of the ration, it would be wisefor farmers to do so. Research studying the role of fat supplementation on reproductiveresponses has not been that consistent, therefore, adding fat to the ration would be advisedwhen the risk of low reproductive performance (young, growing animals and limitingnutrients [protein, energy] in the basal ration) is the greatest.7. ReferencesAbayasekara, D.R.E. & Wathes, C.D. (1999). Effects of altering dietary fatty acid compositionon prostaglandin synthesis and fertility. Prostaglandins, Leukotrienes and EssentialFatty Acids, Vol.61, No.5, (November 1999), pp. 275-287. ISSN 0952-3278Achard, D.; Gilbert, M.; Benistant, C.; Slama, S.B.; DeWitt, D.L.; Smith, W.L. & Lagarde, M.(1997). Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1

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242Artificial Insemination in Farm AnimalsWood, J.D.; Richardson, R.I.; Nute, G.R.; Fisher, A.V.; Campo, M.M.; Kasapidou, E.; Sheard,P.R. & Enser, M. (2003). Effects of fatty acids on meat quality: a review. MeatScience, Vol.66, No.1 (January 2004), pp. 21-32. ISSN 0309-1740Zeron, Y.; Sklan, D. & Arav, A. (2002). Effect of polyunsaturated fatty acid supplementationon biophysical parameters and chilling sensitivity of ewe oocytes. MolecularReproduction and Development, Vol.61, No.2 (February 2002), pp. 271-278. ISSN 1098-2795

14Mechanical and Pharmacologic Applications ofArtificial Insemination in EwesFaruk Aral 1 , Füsun Temamoğulları 2 and Semra Sezen Aral 31 Nigde University, Bor Higher School for Business, Department of Veterinary, Nigde,2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine,Harran University, Sanliurfa3 Agriculture and Rural Affairs Ministry, KonyaTurkey1. IntroductionArtificial insemination (AI) is the keys to the efficient transfer of genetical knowledge fromrams to ewes and to ensure proper productive traits of theirs. The efficient insemination ofhigh-quality semen is very important in an artificial insemination of ewe. Artificialinsemination is also part of the biotechnological studies. The insemination process itselfmay, in fact, cause of poor fertility or reduce of frozen semen quality.Insemination process is regulated by a complex interplay of inseminator, semen, and genitaltract structure of ewe. Although there is general agreement that cervix is of critical importancein the initiation and improvement of semen in mammals during insemination, the role ofcervical canal in the regulation of frozen sperm transport in the ewes is stil problematic.Because of the size and shape of the external os and the tortuous structure of the cervicalcanal, intrauterine insemination of ewes generally carries out with frozen sperm.Transcervical AI is applied using specially designed inseminating equipment andmanipulation of the cervix using forceps. The number of reports in which transcervicaldeposition of semen has been achieved is relatively low and there are concerns about thepotential trauma involved. The role of cervix in the transport of frozen sperm in the ewe isnot entirely clear. Determination of the causes for the generally low fertility obtainedfollowing cervical deposition of frozen semen is still an important subject for AI procedurefor achieving acceptable pregnancy rates in ewes.A previous work observed that oxytocin treatment induced cervical dilation and decreasedthe difficulty of passing a catheter through the cervix and into the uterus. It decreases infertility have been associated with cervical manipulation.For a long time, the standard procedure with fresh semen when inseminating ewes hasbeen to deposit the semen in the external os of the cervical canal. However, recently severalgroups have reported differences in pregnancy rates when ewes were bred with artificialinsemination supported by air pomp, oxytocin, transcervical, etc.There are many methods of artificial insemination of sheep. Specialization is concentratingor limiting one’s focus to part of the whole methods of artificial insemination.Studies are guiding to the acquisition of new practice knowledge and skills. Artificialinsemination is a powerful tool that provides common genetic information and deep insightinto the insemination process that is at the heart of every embryo formation.

244Artificial Insemination in Farm AnimalsThe earliest idea of artificial insemination definitely was a transfer of sperm from male tofemale. That is, based on the sperm form of fresh or chilled. In time, researchers observedthat the fertility changes that produced physiologic reactions in ewes also are formedchanges in the computable traits of sperm. Among these traits are the volume of a sperm,the number of sperm at a moment of fertilization, the physiological changes of a sperm inthe female genitalia, and the transfer way of a sperm to female. Each of these traitscan generate the basis for an effective action of an artificial insemination (Khalifa et al., 1992;Sayre and Lewis, 1996; Wulster-Radcliffe and Lewis, 2002; Candappa et al., 2009).Donovan et al. (2004) reported higher pregnancy rates using fresh compared to frozen–thawed semen but found no differences in pregnancy rate following natural orsynchronized estrus. The reason for the variation in fertility among ewe breeds followingcervical AI with frozen–thawed semen may be due to differences in sperm transportthrough the cervix and uterus or due to early embryo mortality (Fair et al., 2005).The place of deposition of frozen–thawed semen has a key effect on fertilization rate. Inconsequence, considerably advanced fertility is usually achieved with laparoscopicAI via frozen– thawed ram semen after transcervical or cervical insemination (Fair et al., 2005).There are two obvious methods for struggle with the physical characteristics of the ovinecervix: set straight the servix and increase the diameter of the cervical lumen; or redesign TCAI(Trans-Cervical Artificial Insemination) equipment, or modify of embryo transfer catheter toinvent the tight, convolute configuration of the cervix. Methods for straightening (such as,attaching a hemostat to the external cervical os and retracting the cervix) and dilating thecervix (chemically with PGE2 or oxytocin, etc., or mechanically) are effective (Khalifa et al.,1992; Sayre and Lewis, 1996; Wulster-Radcliffe et al., 1999; Wulster-Radcliffe and Lewis, 2002;Candappa et al., 2009; Gunduz et al., 2010).Thus it is tried to give information about the last development of artificial insemination inthe ewes.2. Cervix antomy and functionThe ewe cervix is a long and fibrous tubular organ. It contains connective tissue with an outerserosal layer and inner luminal epithelium. Because the lumen is the presence of 4-7 cervicalrings that caudal opening of its provide a physical barrier to external contaminants andconvolute and tortuous structure catheter entrance is somewhat more difficult than in the cow.These cervical rings constitute the greatest obstacle against TCA. The first, second and thirdring in lumens does not take place in same line, ensuing in the inseminating pipette beingmisdirected apart from the central lumen. In addition, the first ring is the most difficult willbe further than 1 cm, can practically insemination pipette. Cervical canal length rangesbetween 2.5 and 10.5 cm according to breed, age, parity and physiological state. These majorchanges in length of the channel affect the success of in TCAI. The mean number of cervicalrings is approximately 5 with a range of two–seven rings per cervix. (Halbert et al., 1990;Campbell et al., 1996; Wulster-Radcliffe and Lewis, 2002; Kaabi et al., 2006).The external cervical os between ewes has in different location in the vagina. The cervicalos in sheep makes to protrude into the vagina and in some animals completely obscureFive types of external os were identified in the vagina (Halbert et al., 1990). (Fig. 1):1. The Duckbill: two opposing folds of cervical tissue protruding into the vagina with acentral horizontal slit like external os.2. The Slit: no protrusions into the anterior vagina with a slit like opening at the os of thecervix giving entry to the cervical canal.

Mechanical and Pharmacologic Applications of Artificial Insemination in Ewes 2453. The Rose: a cluster of cervical folds protruding into the anterior vagina obscuring theexternal os.4. The Papilla: a papilla protruding into the anterior vagina with the external os at its apex5. The Flap: one-fold of cervical tissue protruding into the anterior vagina and completelyor partially overlaying the external os creating the appearance of a flap.Kershaw et al. (2005) a found that the spreading of external os types differed with age. Inparticular, the rose type os is more common in adult ewes than ewe lambs and the reverse istrue for the papilla type os.Fig. 1. The classification of the appearance of the external os of the ewe (a) duckbill, (b) slit, (c)rose, (d) papilla, and (e) flap. from Kershaw et al., 2005 .Cervical penetration measuring is evaluated the penetration of the insemination pipette asshallow, middle and deep without being informed about the group to which theinseminated ewe belonged (shallow: 20mm, using thecolored tip of the plastic sheath as reference (Gunduz et al., 2010).3. Transcervical AI methodThere generally are 3 AI techniques 1) vaginal insemination, 2) the laparoscopic intrauterineinsemination 3) cervical insemination that have been used in the sheep industry and newlydeveloped fourth technique, trans-cervical artificial insemination (Leethongdee, 2010).Transcervical AI (TCAI) is seemed as a potential alternative to laparoscopic AI. The basis ofthis technique, a AI catheter is passed through cervix for sperm to leave the uterus. Thistechnique is used in other animals, but they are not used due to the difference of the sheepcervix. There is as a degree of natural cervical relaxation in oestrus and trans-cervical

246Artificial Insemination in Farm AnimalsFig. 2. The Guelph TCAI System: (a) fetal-like positioning of ewes in dorsal recumbency; (b)a plexiglass speculum with light source is inserted into the vagina and forceps are used tograb the cervix near the os and retract it. Subsequently, a bent-tipped and preloadedinsemination gun is introduced and manipulated through the cervix to deposit semen. FromCandappa et al., 2009.

Mechanical and Pharmacologic Applications of Artificial Insemination in Ewes 247penetration in low rate in multiparous ewes seems to be possible. The cervical relaxation isdue to affect of peri-ovulatory hormones such as oxytocin, estradiol and progesterone on thecervix. In Cows, the potential effect of oxytocin on cervical relaxation, leads to local growthof cyclooxygenase-2 (COX-2) after that, COX-2 causes an increase in the synthesis ofprostaglandin E2 (PGE2) (Zhang et al., 2007).3.1 The Guelph System for TCAIThe Guelph System of AI equipment fetal-like positioning of ewes in dorsal recumbency(Fig. 2). A speculum with light source is inserted into the vagina, and upon apparition of thecervix. A bent-tipped and preloaded insemination gun is lead into the cervix to depositsemen (Wulster- Radcliffe and Lewis, 2002; Candappa et al., 2009).3.2 Application of TCAI catheterSome studies were used an insemination catheter with a blunt and angled end, someflexibility, and a diameter that is smaller than the narrowest point of the cervical lumen inthe ewe (Halbert et al., 1990; Campbell et al., 1996; Wulster-Radcliffe Lewis, 2002; Kaabi etal., 2006; Candappa et al., 2009).The catheter used for embryo transfer. Later, he modified for sheep transcervical AI. Thecatheter is depicted as the actual size (17.5 cm long, 1.47 mm o.d, 1.07 mm i.d.) (Wulster-Radcliffe et al., 1999).Ewes are held back in a chute used for sheep in a dorsal recumbent position. The perinealarea is cleaned with an antiseptic soap and rinsed with water. Excess water and antisepticare removed with paper towel. A outside layer of obstetrics lubricant is applied to aspeculum, and the speculum is inserted into the vagina and pressed against tissue nearbythe cervix. The TC-AI catheter is located at the external cervical os and manually controlfrom beginning to end of the cervix. A TC-AI catheter used for cervical manipulation intranscervical AI (Fig 3) is equal to the TC embryo transfer apparatus described previously(Wulster-Radcliffe et al., 1999).Fig. 3. Modified embryo transfer catheter. A machined brass zero-volume fitting was used tosecure the catheter. From Wulster-Radcliffee al., 1999.During penetration of a pipette through the cervix the sheep position affects the success ofthe transition. Pipette passage is more easily with the standing ewe than over-the rail ewe.Rear side of the light (approximately 15 cm) elevated ewe with standing is stated as in avery convenient position (leethongdee, 2010). In this position, uterine penetration isachieved in 82.0 %.Transcervical AI is applied using specially designed inseminating equipment (Wulster-Radcliffe et al., 2002; Wulster-Radcliffe et al., 2004) and manipulation of the cervix usingforceps (Halbert et al., 1990). The number of reports in which position of semen has beenachieved is relatively low and there are concerns about the potential trauma involved.

248Artificial Insemination in Farm Animals4. Application of Air Pressure with Cervical Artificial Insemination (APCAI)Cervical artificial insemination (CAI) is a less expensive and invasive method in comparisonto the transcervical and intrauterine methods, and has been widely used for the artificialinsemination of ewes. The site of deposition of frozen-thawed semen in ewes has a majoreffect on fertilization rate (Fair et al., 2005).Whilst pregnancy rates in excess of 60% can be achieved with a single artificial inseminationof fresh semen deposited at the external cervical opening , corresponding rates for frozenthawedsemen occasionally exceed 45%, with values less than 17% not rare (O’Meara et a1.,2005).The insemination device in Air Pressure with Cervical Artificial Insemination method(APCAI) is modified from a stainless steel outer pipette sheath of a cattle AI pipette. The airpump (2 liter/min capacity; aerator, portable battery pump) is attached to the bluntrounded end of the pipette by means of a rubber pipe with an internal diameter of 2.6 mm(Fig 4). A speculum was introduced into the vagina so that the external opening of the cervixcould be seen in the light of the speculum lamp. Subsequently, 0.1 ml of semen is drawn intothe pipette sheath through a plastic syringe, and the pipette sheath is connected to the airpump. The pipette tip is placed at the external opening of the cervix and the air pump is runto spray semen into the cervical canal. For each ewe, a different pipette sheath is used (Aralet al., 2010).Fig. 4. Artificial insemination equipment of APCAI method; (A) Air pump, (B) Rubber pipe,(C) a stainless steel outer pipette sheath used in cattle AI. From Aral et al., 2010.

Mechanical and Pharmacologic Applications of Artificial Insemination in Ewes 249The pregnancy rate in this method was found to be significantly high the APCAI group thanin the CAI group (80.0% versus 46.7%) Table 1.ParametersNumber of ewesCAI(n=30)APCAI(n=30)Pregnancy rate (n/n) % (14/30) 46.7 a (24/30) 80.0 bLambing rate (n/n) % (14/30) 46.7 c (21/30) 70.0 dProlificacy (n/n) (16/14) 1.14 (23/21) 1.09Table 1. Pregnancy, lambing rate, prolificacy, following different AI methods in Awassiewes. From Aral et al., 2010.5. Pharmacologic cervical dilatation5.1 Prostaglandin E analogues5.1.1 CervidilCervical ripening involves enzymatic corruption of the connective tissues. It causes arelaxation of its smooth muscle fibers. Cervical ripening encourage with chemical matter.Thus, penetration of the cervix during TCAI may be achieved with effortlessness (Candappaet al., 2009). Cervidil® contains 10 mg of dinoprostone and have a vaginal form. Hormonereleases a constant rate of 0.3 mg/h over a 12-h period in a women vagina. For cervicalripening and induction of labour in women is a safe agent (Sánchez-Partida et al., 1999;Lambers et al., 2001). Adapting the use of Cervidil® to sheep was performed by Candappaet al., 2009.Cervidil® is inserted 12 h prior to insemination in ewes. Cervidil®, containing 10 mgdinoprostone (prostaglandin E2), introduce into the vagina in the immediacy of the cervix.After the 12-h priming with Cervidil, transcervical semen deposition is possible in all ewes(Candappa et al., 2009).5.1.2 Ovagen and misoprostolCervical relaxation and penetration were examined in Ovagen and misoprostol-treatedsheep in a previous study. Ovine FSH ( 2 mg; Ovagen; 1 CP bio (UK) limited, Wiltshire, UK)was administered at a dose of 2 mg dissolved in 0.5 ml of 50 % Gum acacia (Sigma-AldrichCo.,). A prostaglandin E 1 analogue, Misoprostol (1 mg; Misoprostol; Sigma-Aldrich Co.,Dorset England) was administered at a dose of 1 mg dissolved in 0,5 ml of 30 % gelatine(Sigma-Aldrich Co.,). External opening of the cervix treated sheep Ovagen and misoprostolsignificantly loosened. It is easier penetration of the cervix in these sheep (Leethongdee etal., 2007).5.2 HyaluronanThe cervix is relatively relaxed at oestrus. These are both a high-hyaluronan (HA) content ofthe cervix and the related increase in its water content. When aqueous 0.5 ml of the HAsuspension (2 mg low molecular weight (LMW) HA) and (25 mg high molecular weight(HMW) HA) is applied to an intra-cervical, it promotes cervical relaxation in oestrus ewes.

250Artificial Insemination in Farm AnimalsLMW HA has the greatest impact on vascularization, leading to the collection of leukocytes.In addition, it stimulates the biochemical changes in the cervix during softening (Perry et al.,2010).5.3 OxytocinOxytocin treatment caused relaxation of cervix and uterine catheter through the cervix havedemonstrated that reduced the difficulty of the transition (Khalifa et al., 1992; Sayre andLewis, 1996). The effect of oxytocin as a cervical dilator is different on the reproductiveoutcome in ewe. Sayre and Lewis (1997) observed no undesirable effect of oxytocin on ovumfertilisation rate. Stellflug et al., (2001) show a negative effect of oxytocin but not of thetranscervical insemination procedure. Fertilization rate decreases in the treatment ofoxytocin-cervical manipulations. However, the oxytocin treatment does not affect ovulationrate. Atraumatic cervical manipulation, does not affect the time of ovulation, fertilizationrate, early embryonic development and rate of lambing. Thus, oxytocin is used to softeningthe cervix decreases the ease of transition to a transcervical AI instrument, the fertilizationrate, pregnancy rate and lambing rate.For transcervical AI, different (50 to 400 USP units in Table 2 and 3; 10 IU of oxytocin) dose ofoxytocin can be given to ewes via intravenously 30 min. before AI to dilate the cervix. Whenoxytocin is given intramuscularly 15–30 min before insemination with frozen/thawed semen,it produced an impressive reduction (10% versus 42%) in the lambing rate of ewesinseminated cervically. However, oxytocin makes possible intrauterine insemination via thecervix, its undesirable effect on lambing rate may be less significant oxytocin has a small andnon-significant damaging outcome on litter size (King et al., 2004; Table 2, 3).oxytocin. USP unitsItem 0 200 400 600 SECervical penetration, cmOveralloxytocineBefore oxytocin 2.6 2.2d 1.0d .9d .3 1.5dAfter oxytocin 2.9 5.6e 6.1e 5.1e .3 5.7eTime to deepest cervicalpenetrationf, min10.0g 6.9h 5.6h 6.2h .4 6.4Uterine entries/no. Of ewes 0/15g 15/19h 10/12h 8/12h - 33/43(%) 0 79 83 67 77*Values are means or proportions.bSE are standard errors from analyses of variance models used to analyze the data.Values with different superscripts differ (P < .01).Table 2. Effects of intravenous oxytocin injection 52 hours after removal of progestogenatedpessaries from ewes. From Khalifa et al., 1992.Oxytocin injections dilate the cervix in some ewes. However, oxytocin administration 12 hafter 100 or 200 pg of estradiol-17 ((0, 100, or 200 pg in 5 mL of 1:1 saline ethanol) are themajority successful at dilating the cervix and permitting acceptance of a stainless steel rod

Mechanical and Pharmacologic Applications of Artificial Insemination in Ewes 251into the uterus. The 100-µg dose of estradiol-17 seem to be as a valuable dose (Khalifa et al.,1992; Wulster-Radcliffe et al., 1999).ItemSignificance oflaparoscopicCervicaltreatment effectsControl Oxytocin Control OxytocinNumber of ewes 49 50 100 99Number lambing (%) 34(69a) 29(58 c) 42(42 b) 10 (10 d) a>b** c>d***Mean litter size 1.91 a 1.83 c 1.52 b 1.40 d a>b* c>dzZ: P < 0.10.* P < 0.05.** P < 0.01.*** P < 0.001.Table 3. Data for lambing rates and litter sizes in relation to treatments and week ofinsemination. From King et al., 2004.These observations, together with those from the present experiment, suggest that anyadverse effect of oxytocin on lamb production when ewes are subjected to laparoscopicintrauterine insemination with frozen/thawed semen is likely to be small. Thus, had theoxytocin in the present experiment. Oxytocin make possible intrauterine insemination viathe cervix, its undesirable effect on lambing rate may have been less significant.The cervical manipulation seems to unfavorable an effect on fertility after AI, because theoxytocin treatment is not harmful. It likely to affect the sperm survival in the reproductivetract of sheep, or some issues of sperm capacitation (Stellflug et al., 2001). Perhaps,manipulation of the cervix may affect sperm transport within the reproductive tract, or astressed cervix may produce a spermicidal compound (Hawk et al., 1981).5.4 Human interleukin 8 (huIL-8)Human interleukin 8 (huIL-8) was applied to use in sheep to stimulate the cervicalrelaxation. This cytokine causes a neutrophil recruitment and an increase in collagenases inthe cervix during the peripartum period in mammals. However, its administration is failedto induce cervical dilatation in ewesAfter estrus synchronization protocol is applied, and a wax suppository (Witepsol as thewax formulation, mm x 3 mm in size) containing either 5 pg huIL-8a (derived from largescale human fibroblast cell culture) with an estimated total release of 0.6 pg huIL-8 isinserted into the anterior vagina near the cervical os (Croy et al., 1999).5.5 CarazololWhen animals are exposed to stress during artificial insemination, their bodies react byraising the adrenaline amount. The beta 2 adrenoreceptors in the myometrium is affected byadrenalin. After that, uterine contractions occurred by oxytocin is destroyed by it, which inturn produce a long time to get ahead of the genital canal for the spermatozoa. Thus,artificial insemination with aged spermatozoa leads to decrease of fertility in ewes (Kırsanet al., 1998; Gunduz et al., 2010).

252Artificial Insemination in Farm AnimalsIt eliminates the effect of oxytocin, stimulating uterine contractions. Carazolol (Suacron,Divasa, Farmavic, Spain) intramuscularly for each sheep is 0.5 mg administered 30 minutesbefore insemination. It increases the number of ewes inseminated with deep. In contrast, itdoes not have a significant effect on the pregnancy rate (85% for control, 95% for carazolol)(Gündüz et al., 2010). The middle and deep penetration rates are high but was found nonsignificant(for control 82 % and carazolol 85%).6. References[1] Aral F, Yavuzer U, Zonturlu AK. 2010. The effect of air pressure with cervical artificialinsemination on the fertility of Awassi ewes synchronized with PGF2α . KafkasUniv Vet Fak Derg., 16 (1), 37-41.[2] Campbell JW, Harvey TJ, McDonald MF, Sparksman RI. 1996. Transcervicalinsemination in sheep: an anatomical and histological evaluation. Theriogenology,45, 1535–1544.[3] Candappa IBR, Bainbridge HC, Price NT, Hourigan KR, Bartlewski PMA. 2009.Preliminary study on the suitability of Cervidil to induce cervical dilation forartificial insemination in ewes. Research in Veterinary Science, 87, 204–206.[4] Croy BA, Prudencio J, Minhas K, Ashkar AA, Galligan C, Foster RA, Buckrell B,Coomber BL. 1999. A preliminary study on the usefulness of huIL-8 in cervicalrelaxation of the ewe for artificial insemination and for embryo transfer.Theriogenology, 52, 271–287.[5] Donovan A, Hanrahan JP, Kummen E, Duffy P, Boland MP. 2004. Fertility in the ewefollowing cervical insemination with fresh or frozen–thawed semen at a natural orsynchronised oestrus. Anim Reprod Sci, 84, 359–68.[6] Fair S, Hanrahan JP, O’Meara CM, Duffy P, Rizos D, Wade M, Donovan A, Boland MP,Lonergan P, Evans AC. 2005. Differences between Belclare and Suffolk ewes infertilization rate, embryo quality and accessory sperm number after cervical orlaparoscopic artificial insemination.Theriogenology, 63, 1995–2005.[7] Gunduz MC, Turna Ö, Cirit Ü, Uçmak M, Tek Ç, Sabuncu A, Bacınoğlu S. 2010.Lambing rates and litter size following carazolol administration prior toinsemination in Kivircik ewes. Animal Reproduction Science, 118, 32–36.[8] Halbert G, Dobson H, Walton J, Buckrell B. 1990. The structure of the cervical canal ofthe ewe. Theriogenology, 33, 977–992.[9] Hawk HW, Cooper BS, Purse1 VG. 1981. Increased sperm death in the cervix and uterusof estrous ewes after synchronization of estrus with prostaglandin or progestagen. JAnim Sci, 52, 601-610.[10] Kaabi M, Alvarez M, Anel E, Chamorro CA, Boixo JC, de Paz P, Anel L. 2006. Influenceof breed and age on morphometry and depth of inseminating catheter penetrationin the ewe cervix: a postmortem study. Theriogenology, 66, 1876– 1883.[11] Kershaw CM, Khalid M, McGowan MR, Ingram K, Leethongdee S, Wax G, ScaramuzziRJ. 2005. The anatomy of the sheep cervix and its influence on the transcervicalpassage of an inseminating pipette into the uterine lumen. Theriogenology, 64(5),1225-1235.

Mechanical and Pharmacologic Applications of Artificial Insemination in Ewes 253[12] Khalifa RME, Sayre BL, Lewis GS. 1992. Exogenous oxytocin dilates the cervix in ewes. JAnim Sci, 70(1), 38–42.[13] Kırsan I, Alkan S, Baran A, Ozturkler Y, Senunver A, Ileri K. 1998. Sıgırlarda sunitohumlama anında uygulanan beta-2 blokoru carazololun uterus tonusu ve gebekalma oranı üzerine etkisi. I U Veteriner Fakultesi Dergisi, 24 (1), 89–97.[14] King ME, McKelvey WAC, Dingwall WS, Matthews KP, Gebbie FE, Mylne MJA,Stewart E, Robinson JJ. 2004.Lambing rates and litter sizes following intrauterine orcervical insemination of frozen/thawed semen with or without oxytocinadministration. Theriogenology, 62, 1236–1244.[15] Lambers D, Khoury J, Ventolini G. 2001. A meta-analysis of the safety and efficacy ofCervidil (dinoprostone): a vaginal insert for cervical ripening. American Journal ofObstetrics and Gynecology, 185, 203.[16] Leethongdee S, Khalid M, Bhatti A, Ponglowhapan S, Kershaw CM, ScaramuzziRJ.2007.The effects of the prostaglandin E analogue Misoprostol and folliclestimulatinghormone on cervical penetrability in ewes during the peri-ovulatoryperiod. Theriogenology, 67(4), 767-777.[17] Leethongdee S. 2010. Development of trans-cervical artificial Insemination in sheep withspecial reference to anatomy of cervix. Suranaree J Sci Technol, 17, 157-169.[18] O’Meara CM, Hanrahan JP, Donovan A, Fair S, Rizos D, Wade M, Boland MP, EvansACO, Lonergan P. 2005. Relationship between in vitro fertilization of ewe oocytesand the fertility of ewes following cervical artificial insemination with frozenthawedram semen. Theriogenology, 64, 1797-1808.[19] Perry K, Haresign W, Wathes DC, Khalid M. 2010. Intracervical application ofhyaluronan improves cervical relaxation in the ewe. Theriogenology , 1685-1690.[20] Sánchez-Partida LG, Windsor DP, Eppleston J, Setchell BP, Maxwell WMC. 1999.Fertility and its relationship to motility characteristics of spermatozoa in ewes aftercervical, transcervical, and intrauterine insemination with frozenthawed ramsemen. Journal of Andrology, 20, 280–288.[21] Sayre BL, Lewis GS.1997. Fertility and ovum fertilization rate after laparoscopic ortranscervical intrauterine artificial insemination of oxytocin-treated ewes.Theriogenology, 48,267–275.[22] Sayre BL, Lewis GS. 1996. Cervical dilation with exogenous oxytocin does not affectsperm movement into the oviducts in ewes. Theriogenology, 45, 1523-1533.[23] Stellflug JN, Wulster-Radcliffe MC, Hensley EL, Cowardin EA, Seals RC and Lewis GS.2001. Oxytocin-induced cervical dilation and cervical manipulation in sheep:effects on laparoscopic artificial insemination. J Anim Sci, 79, 568-573.[24] Zhang Q, Collins V, Chakrabarty K, Rose JC, Wu WX.2007. Regulation of theprostaglandin enzymatic system by estradiol and progesterone in nonpregnantsheep cervix. Reproduction,133, 1027–1034.[25] Wulster-Radcliffe MC, Wang S, Lewis GS. 2004.Transcervical artificial insemination insheep: effects of a new transcervical artificial insemination instrument andtraversing the cervix on pregnancy and lambing rates. Theriogenology, 62,990–1002.

254Artificial Insemination in Farm Animals[26] Wulster-Radcliffe MC, Costine BA, Lewis GS. 1999. Estradiol- 17β-oxytocin-inducedcervical dilation in sheep: Application to transcervical embryo transfer. J. Anim.Sci, 77, 2587–2593.[27] Wulster-Radcliffe MC, Lewis GS. 2002. Development of a new transcervical artificialinsemination method for sheep: effects of a new transcervical artificial inseminationcatheter and traversing the cervix on semen quality and fertility. Theriogenology,58, 1361–1371.

15Relationship between IFN- Production byBovines Embryos Derived Ex Vivo andCompletely Produced In VitroJorge Alberto Neira 1,2 , Daniel Tainturier 2 ,René L’Haridon 3 and Jacques Martal 41 Laboratoire de Pathologie de la Reproduction et Biotechnologie animale, Ecole NationaleVétérinaire, Agroalimentaire et de l’Alimentation Nantes-Atlantique: ONIRIS2 Centro de Reproducción animal CRIA de la Corporación Colombiana de InvestigaciónAgropecuaria CORPOICA, Grupo Biología del Desarrollo, Bogotá DC,3 Laboratoire de Virologie et Immunologie Moléculaire, INRA Jouy-en-Josas cedex4 Station de Physiologie Animale, UMR-BDR, INRA. Jouy-en-Josas cedex1,3,4 France2 Colombia1. IntroductionInterferon-tau (IFN-) is secreted by the mononuclear cells of the primitive extra-embryonictrophoblast (Farin et al. 1989; Guillomot et al. 1990) which will become the main part of thefuture placenta, in ruminants. This cytokine is constitutively produced during the shortperiod of the conceptus periimplantation (Martal et al. 1979; Godkin et al. 1982; Hansen etal. 1988; Charlier et al. 1989). It plays an essential role for maternal recognition of pregnancy,particularly allowing the maintenance of the corpus luteum and its progesterone secretion(Spencer et al. 2004). Indeed, intrauterine injections of recombinant IFN- extend theprogesterone secretion by inhibiting pulsatile uterine secretion of luteolytic prostaglandin F-2 (Martal et al. 1990, 1998; Ott et al. 1993; Meyer et al. 1995). IFN- downregulates theexpression of endometrial oxytocin receptors concentrations (Spencer and Bazer 1996; Mannet al. 1999). In early pregnancy, IFN- constitutes therefore a major signal in ruminants.Thus, it plays a role of both cytokine and reproductive paracrine hormone (Roberts et al.1992; Bazer et al. 1994; Martal et al. 1997). In addition, IFN- exhibits potent antiviral andantiproliferative activities (Fillon et al. 1991; Pontzer et al. 1991, 1997; Bazer et al. 1994;Derreuddre et al. 1996).The regulation of IFN- secretion remains poorly understood (Martal et al. 1998; Yamagushiet al. 2001; Demmers et al. 2001; Ezashi et al. 2001; Stewart et al. 2002; Spencer and Bazer2002). Some growth factors and cytokines are implicated in the positive control of IFN-secretion such as Insulin-like Growth Factor (IGF-I ) and IGF-II (Ko et al. 1991), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukine-3 (Imakawa et al. 1993,1995; Emond et al. 2004). Indeed, these factors are secreted by trophoblast and endometriumin early pregnancy (Mathialagan et al. 1992; Vogiagis et al. 1997; Martal et al. 1997, 2002).

256Artificial Insemination in Farm AnimalsThe induction of IFN- expression appeared genetically determined because IFN- secretionhas been evidenced from hatched blastocysts completely produced in vitro (Hernández-Ledezma et al. 1992, 1993; Stojkovic et al. 1995). Therefore, the uterine environment is notnecessary for the induction of IFN- expression but it could play an essential role in thecontrol of IFN- secretion. Paternal genotype was a significant determinant of the embryo’sability to develop the blastocyst stage and of subsequent IFN- secretion (Kubish et al.2001a). Besides, there is some evidence that the presence of other blastocysts could increaseIFN- secretion (Larson and Kubish 1999).Generally, the evaluation of the development potential of embryos today still depends onsubjective morphological examination (Lindner and Wright, 1983; Buttler and Biggers 1989;Shamsuddin et al. 1992, Massip et al. 1995). However, the development stage and quality ofthe embryos significantly influence the IFN- secretion. Some authors have proposed thatthe produced amount of IFN- could be a useful objective indicator of embryo quality(Hernandez-Ledezma et al. 1992, 1993). But others, considering that the age of blastocystsformation in vitro exhibits significant effects on the IFN- production, have suggested anegative relationship between early IFN- production and blastocysts competence forembryonic development (Kubish et al. 1998, 2004).The aim of this study was therefore to compare the IFN- secretion after hatching in bovineblastocysts of good homogeneous quality wether they are produced in vivo or completely invitro.2. Materials and methods2.1 Production of in vitro bovine blastocysts2.1.1 In vitro maturation of oocytesUnless otherwise indicated, all chemicals in this study were purchased from Sigma-Aldrich,(Saint Quentin. Fallavier, France). Ovaries from Prim Holstein cows were quickly collectedafter death in a local slaughter-house and transferred to the laboratory into a saline solutionwith 0.9% (w/v) NaCl at approximately 35°C. The largest interval between animal killingand oocyte conditioning was 3 hours. Cumulus-oocyte complexes (COCs) were recoveredby aspiration of follicles of 2-8 mm in diameter using a 18 gauge needle under vacuumpressure of approximately 50 mm Hg. The COCs were collected into Hepes-buffered tissueculture medium 199 (TCM 199 ref. M-7528) supplemented with 0.4% (w/v) BSA (CohnFraction V, ref. 9647). Before in vitro maturation, COCs were assessed morphologically: onlythose which displaid a compact and non-atretic cumulus oophorus-corona radiata with anoocyte exhibiting homogeneous cytoplasm were chosen for further in vitro culture. SelectedCOCs were washed thoroughly in TCM 199 (ref. M-4530) plus 10% (v/v) fetal calf serum(FCS, Life Technologies, Inc., Grand Island, NY USA). The maturation of about 60 COCsbatches were achieved in 500µl TCM199 (ref. M-4530) supplemented with 10% FCS, and10ng per ml EGF (ref. E-4127) with 100 µg/ml gentamicin (ref. G-1264), for 24 h at 39°Cunder humidified 5% CO 2 in air.2.1.2 In vitro fertilizationSpermatozoa were prepared from frozen-thawed semen of a sole bull (Prim Holstein breed,electronic number: 44 13 835058) that had been characterized as suitable for in vitrofertilization in our laboratory. The contents of two 0.25 ml straws (each containingapproximately 10 6 spermatozoa per ml) were layered upon a Percoll discontinuous gradient

Relationship between IFN- Production byBovines Embryos Derived Ex Vivo and Completely Produced In Vitro 257(Hasler et al. 1995). Motile spermatozoa were collected after centrifugation at approximately700g for 30 min, at room temperature. Then they washed in Hepes-buffered Tyrode’salbumin lactate pyruvate medium (Talp) (Parrish et al. 1986) and pelleted by centrifugationat approximately 200g for 10 min at room temperature. Meanwhile, COCs were transferredto another four-wells dish containing 250µl of in vitro fertilization Talp supplemented with0.01 mmol/l heparin (ref. H-3149), 0.2 mmol/l penicillamine (ref. P-4875), 0.1 mmol/lhypotaurine, 0.1 mmol/l epinephrine (ref. E-4250) and 6mg/ml fatty acid-free BSA. (ref. A-8806) Insemination was performed to get the final concentration of 2 x 10 6 spermatozoa/ml tofertilize the oocytes. Plates were incubated 5% CO 2 in humidified air at 39°C.2.1.3 In vitro cultureAfter 20 hours, the cumulus cells were removed from the presumptive zygotes byintermittent gentle shaking for 2 min. Presumptive zygotes were then washed 4 times insynthetic oviduct fluid medium SOF (Tervit et al. 1972) according to Holm et al. (1999)containing 0.7 mM Na-pyruvate, 4.2 mM Na-lactate, 2.8 mM myo-inositol, 0.2 mMglutamine, 0.3 mM citrate, 30 ml/l essential amino acids mixture, 10 ml/l non-essentialamino acids, 50 µg/ml gentamycin and 10% (v/v) fetal calf serum (SOF-FCS). All mediawere passed through a 0.2 µm membrane filter and were equilibrated overnight in anincubator at 39°C in 5% O 2 in humidified air. Presumptive zygotes were cultured in groupsof 10-12 in 20µl droplets of SOF-FCS, culture was performed in a humidified atmosphere of5% CO 2 , 5% O 2 and 90% N 2 at 39 C. Cleavage was examined once between days 2 and 3,embryo development rate was evaluated on day 7 post insemination. On day 7, excellentand good quality blastocysts of grade 1 (according to the International Embryo TransferSociety standards-IETS, Stringfellow and Seidel, 1990) were selected for this study.2.2 Production of in vivo bovine blastocystsThe oestrous cycles of french Prim Holstein donor cows were synchronised with an implantof 3mg Norgestomet-3.8 mg valerate oestradiol (Crestar INTERVET S.A.- Holland) for 9days. The oestrous reference was observed 48-68 hours after removing the implant. Thecows were superovulated 8 to 12 days after oestrous with p-FSH (Stimufol Merial SAS -France). A total dose of 450µg p-FSH was administered (8 i.m. injections with decreasingdoses for 4 days) and an analogue of PGF 2 (500 µg i.m.; Cloprostenol Estrumate ScheringPlough Vet. S.A.) was injected at the moment of the 5th p-FSH injection. Two artificialinseminations (IA) were performed 12 and 24 hours after starting oestrous (day 0 ). Thefrozen-thawed semen for IA of embryos donor cows was the same that used for theproduction of in vitro bovine blastocysts. Generally, the in vivo embryos have a smallchronological delay in their development in comparison with the in vitro produced embryos,because of the lack of precision in the moment of fertilization. For this reason the cows werecollected at day seven and a half after IA. Embryos were recovered by uterine flushing withPBS (phosphate buffer saline) supplemented with 0.04% (w/v) BSA fraction V (sameprevious reference). Then embryos were washed 4 times in PBS supplemented with 0.4%(w/v) BSA fraction V. The viability of embryos was estimated according to IETS. Onlyexcellent and good quality blastocysts grade I were selected for this study.2.3 Determination of IFN- antiviral activityEmbryo culture media were assayed for antiviral activity by use of a cytopathic effecttitration assay that used a bovine kidney cell line designated Madin and Darby Bovine

258Artificial Insemination in Farm AnimalsKidney (MDBK cells) challenged with vesicular stomatitis virus. The extent of cell protectionagainst viral lysis was compared with that of a diluted human INF-α (Alpha therapeuticsCorporation Los Angeles, CA) used as a standard. This standard possesses 8.8 InternationalReference Units of activity per laboratory unit (u) when compared to human INF-α(Leukocyte IFN standard: GA-23-902-530 reference sample, obtained from NIH BethesdaMD, USA.)The sensitivity of the IFN- assay was 0.012 u /100µl. Titrated IFN were expressed inlaboratory units/embryo/24h, where one unit was equivalent to that amount of IFN thatprotected 50% of the MDBK cells monolayer from lysis upon exposure to the cytopathiceffects of the virus. Immunoneutralization assays were performed with polyclonal antiseraraised against IFN-. Briefly, IFN samples were serially diluted in a given dilution of anti-INF antisera (L’Haridon 1991). The excess of immunoneutralization results was determinedfrom the IFN- activity compared with that of untreated controls.2.4 Determination of the numbers of inner mass (ICM) and trophectoderm (TE) cells ofblastocysts produced in vitro and ex vivo.Equal numbers of hatched blastocysts (per treatment group, completely in vitro and ex vivoorigin’s) on day 10 after fertilizations (72h-cultured in individual droplets), were subjectedto differential cell staining with fluorocrome using a modification of the proceduredescribed by Stojkovic et al. (1997). Briefly, hatched blastocysts were washed several timesin 0.1M phosphate-buffered saline (PBS, pH 7.2) containing 0,2% BSA. Hatched blastocystswere incubated in a 1:10 dilution in PBS of antiserum raised in rabbit against recombinantovine IFN-τ (Martal et al. 1998) for 45 min at 39ºC in a humidified atmosphere of 5% CO 2 inair. Subsequently, embryos were washed five times in PBS supplemented with 5% (v/v)guinea-pig complement (ref. 72122 Bio-Mérieux S.A.) and 50µg/ml propidium iodide (ref.P4170) for 45 min at 39ºC in a humidified atmosphere of 5% CO 2 . Blastocysts were thenwashed in PBS and placed in cold absolute ethanol containing 25µg/ml fluorochromebisbenzimide (ref. B-2883) 30 min at 4°C. Finally embryos were washed in absolute ethanol,mounted in undiluted glycerol. Specific fluorescence was examined by confocal microscope(Zeiss, Paris, France) with mercury lamp under transmittance illumination and an UVexitation filter of 365 nm and a barrier filter of 420nm.2.5 Experimental designThe aim of this experiment was to compare IFN- production of blastocysts in vitro culturefrom days 7 to 12 according to their origin (from completely in vitro and in vivo) and theirquality. For this purpose, excellent and good quality blastocysts, developed over a stage of 7days of in vitro and in vivo origin were sorted and cultured in individual droppers in 50µl ofSOF- FCS for 5 days under paraffin oil. Plates were incubated in 5% CO 2 , 5% O 2 and 90% N 2 at39 °C in humidified air at 39°C. During the time of in vitro culture each equal volume of freshmedium was then added back to each culture every 24 hours and kept refrigerated at 4°C, andthe blastocysts morphologically evaluated as indicated by Lindner and Wright (1983). Briefly,embryos were evaluated and graded by morphological criteria as follows: excellent (spherical,symmetrical with cells of uniform size, color and texture), good (a few imperfections such asfew extruded blastomeres, irregular shape and presence of vesicles, >50% of extruded darkcells, cells of different size), fair (several imperfections, several extruded blastomeres, highpercentage of extruded dark cells, too many cells of different size.)

Relationship between IFN- Production byBovines Embryos Derived Ex Vivo and Completely Produced In Vitro 2592.6 Statistical analysisData on embryos origin (in vitro and ex vivo) and the quantity of IFN- released in a 24-hculture period and quality embryos, from day 7( Day 1 was the day of insemination ) today12, were evaluated in a 3 X 3 factorial arrangement in a complete randomized block design.The general linear models procedure (SAS Institute Inc.) was used by analysis of varianceand regression coefficient least squares means. Differences were considered significant atp

260Artificial Insemination in Farm Animalsn=9) and those of group B (13%, n=5) both secreted

Relationship between IFN- Production byBovines Embryos Derived Ex Vivo and Completely Produced In Vitro 261Between days 7 to 11 (96 hours), the production of IFN- increased in 1100±20 pM IFN-(n=29) for group A versus 670±117 pM IFN- (n=24) for group B, (p0.05); 1040±216 pM IFN- (n=17); 870±158 pM (n=33) and 507±262 pM (n=5),respectively.But the average IFN- production from days 7 to 12 (120h-culture) in relationship withembryo quality shows significant differences between excellent quality (1815±453 pM, n=10)or good quality (1356±200 pM , n=29) with fair quality (360±188 pM, n=4), (p

262Artificial Insemination in Farm AnimalsThe logistic regression analysis of the relationship between the embryo quality and IFN-production showed predicted probabilities from observed responses: Concordance: 60.3%and Discordance: 23.9%.EmbryoqualityQ1(Ex.)Time of culture (hours)0 h 72 h. 96 h. 120 h.n(%)81(70)n(%)24(21)IFN-concentrationmean ± sem (pM)534 ± 138an(%)20(17)IFN-concentrationmean ± sem (pM)1040 ± 216an(%)10(12)IFN- concentrationmean ± sem (pM)1815 ± 453 aQ1(G.)19(16)34(29)389 ± 101a38(33)870 ± 158a29(34)1356 ± 200abQ II -6(5) 328 ± 108a6(5)507± 262a4(5)360 ± 188 bQI (Ex.): excellent quality grade I; QI (G.): good quality grade I; QII: fair quality grade II. Least SquaresMeans indicate values with different superscripts (a vs b) are significantly different (p

Relationship between IFN- Production byBovines Embryos Derived Ex Vivo and Completely Produced In Vitro 263Bar corresponds to 50µm.Photograph 2. Blastocyst of completely in vitro origin, was cultured 10 days 7 individuallyin droplet. Staining was by immunochemistry is like in photograph 1.4. Discussion and conclusionOur average results for producing bovine blastocysts in vitro can be compared with differentreports, (Greve et al, 1993 Brackett and Zuelke 1993; Holm et al, 1999; Thompson 1997).Nowadays, after years of development, these techniques have reached a level of stability.The analysis of IFN- production by in vitro blastocysts confirms earlier studies (Kubish etal. 2004, 1998; Hernández-Ledezma et al. 1993; Stojkovic et al. 1995, 1999) and stronglysuggests that expression of IFN- is constitutive and partly non dependent on factors fromthe uterine environment. Nevertheless, it is known that IFN- expression and secretion levelmight be modulated in vivo by growth factors and cytokines (Ko et al. 1991; Imakawa et al.1993; Martal et al. 1998; Spencer and Bazer 2002; Miyazaki et al. 2002; Emond et al. 2004).The IFN- production on day 8, after 24 hours of in vitro culture, was below 54pM andtherefore undetectable in both ex vivo and completely produced in vitro; The IFN- is firstproduced by bovine conceptuses soon after the blastocysts expand and just prior to the timethat the zona pellucida ruptures and hatching occurs (Hernández –Ledezma et al. 1992). It wasonly detectable from day 9 in both cases. These results have no relationship with the resultsreported by Kubish et al. (1998). In our study we only used very homogeneous blastocysts ofgood quality. We reported the production generated every 24 hours from day 7 to 12.Nevertheless, the accumulated production for each embryo agrees with values obtained byKubish et al., (1998, 2004); Stojkovic et al., (1999); Hernandez- Ledezma et al., (1993).To compare well the IFN- production of ex vivo and completely produced in vitro embryosin in vitro culture from day 7 to 12, in the present study only excellent and good quality

264Artificial Insemination in Farm Animalsexpanded blastocysts were chosen on day 7 after fertilization. Thus, a homogeneousproduction would be expected, however, significant differences in the average productionof IFN- were found in relationship with embryo origin, ex vivo and completely produced invitro, between day 8 and 9, after 48h of culture and in IFN- production for embryoaccumulated, which showed that the IFN- production was greater for completely producedin vitro embryos even on day 9.In vitro, modulation of the IFN- production could be induced on one hand by the conditionsof the culture associated to the presence of biological factors contained in the fetal calf serum,and in the other hand by intrinsic causes of the embryo. Those reasons could explain theindividual variability of IFN- production between the embryos. Our data showed high rangesof standard deviation for in vitro and ex vivo embryos and were in agreement with the resultsof Kubish et al. (1998) who also obtained ample ranges in the standard deviation; they alsoobserved that the amount of IFN- produced was independent of the quality score of embryosreceived. Larson and Kubisch (1999) report that the presence of more blastocysts in the samemedium can increase the IFN- secretion, nevertheless the nature of most of these factors ofvariation are badly known. In in-vivo conditions, the production of IFN- is modulated by theuterine environment. This production seems to be strongly correlated to the progesterone sericconcentration, which would suggest that more elevated rates of progesterone would befavorable to the conceptus environment (Kerbler et al. 1997; Spencer et al. 2004). Indeed,progesterone stimulates the production of many others uterine factors than the IFN (Mann etal. 1999; Martal, 2002.). Several growth factors or citokynes have been involved in the secretionof IFN: IGF-I and IGF II (Ko et al. 1991), GM-CSF (Imakawa et al. 1995; Emond et al. 2004), thisone is known as a powerful growth factor for the trophoblastic cells. It is possible that thevariations in IFN- production between blastocysts at any of the stages of development is theresult of genetic factors; others have previously reported that the fact sire genotype appears toinfluence IFN- secretion (Kubish et al. 2001); for this study, the frozen-thawed semen for IA ofdonor cows was the same as production of in vitro bovine. More over, according to Kubish etal. (2001), the batch of ovaries for production of in vitro bovine blastocysts, does not take intoaccount the composition of breeds or ages of cows slaughter, these conditions may influencethe variation in IFN- production.However, their early-forming blastocysts were generally considered more developmentallycompetent than those which formed late, and these last authors suggested a possiblenegative relationship between early IFN- production and competence. In fact, thishypothesis has not been verified in the present study, possibly because of the homogeneityof the embryos chosen on the good appearance on day 7 according to the usualmorphological criteria. Embryo quality is usually based on series of subjective visualassessments of morphologic parameters evaluation which may include embryo shape, size,cellular integrity, appearance of the cytoplasm and nucleus and other often intangiblecriteria (Lindner and Wright, 1983; Shamsuddin et al. 1992, Massip et al. 1995). In this study,the embryos which maintained in good and excellent quality had a better production ofIFN- compared with those which turned to fair quality. This can be explained by a highernumber of degenerating cells; in others the present study shows that the embryo quality isassociated with IFN- production and confirms earlier studies (Kubish et al. 1998;Hernández-Ledezma et al; 1993; Stojkovic et al. 1995 and 1999).Finally this study leads to the conclusion that significant differences in the production ofIFN- were found in relationship with embryo origin, ex vivo and completely produced in

Relationship between IFN- Production byBovines Embryos Derived Ex Vivo and Completely Produced In Vitro 265vitro. The detectable interferon production in the precocious stage on days 7 to 8 reflects thedegree of embryonic development, but the amount of produced interferon has neither apositive nor negative effect on the future of the embryo viability9. References[1] Brackett B. and Zuelke K.A. (1993) Analysis of factors involved in the in vitro productionof bovine embryos. Theriogenology 39, pp 43-64.[2] Bazer F.W., Ot T.L., and Spencer TE, (1994) Analysis Pregnancy recognition inruminants, pigs and horses: signals from the trophoblast. Theriogenology 41, pp.79-94.[3] Butler J.E. and Biggers J.D. (1989) Assessing the viability of preimplantation embryos invitro. Theriogenology 3, pp. 115-126.[4] Chalier M., Hue D., Boisnasr M., Martal J. and Gaye P. (1989) Cloning and expression ofcDNA encoding ovine trophoblastin: its idently with a class-II alpha interferon.Gene 77, pp. 341-348.[5] Derreuddre-Bosquet N., Clayette P., Martin M., Mabondzo A., Frétier P., Gas G., MartalJ. , and Dormon D. (1996) Anti-HIV potential of a new interferon, interferon-τ(trophoblastin). J. AIDS Human Retrovi. vol. 11, pp. 241-246,.[6] Demmers, K. Derecka K. and Flint A. (2001) Trophoblast interferon and pregnancy.Reproduction vol. 121, pp. 41-49.[7] Emond V., MacLaren L.A., Kimminis S., Arosh J.A., Fortie M.A. and R.D. (2004)Expression of cyclooxygenase-2 and granulocyte-macrophage-colony-stimulatingfactor in the endometrial epithelium of the cow is up-regulated during earlypregnancy and in response to intrauterine infusions of interferon-tau. Biol. Reprod.vol. 70, pp. 54-64,.[8] Ezashi T., Ghosh D. and Roberts R.M. (2001) Repression of Ets-2-induced transactivationof the tau interferon promoter by Oct-4. Mol Cell Biology vol. 21, pp. 7883-7891.[9] Farin C.E., Imakawa K. and Roberts R.M. (1989) In situ localization of mRNA for theinterferon, ovine trophoblast protein-1 during early embryonic development of thesheep. Mol Endocrinology vol. 3, pp. 1099-1107.[10] Fillon C., Chauat G., Reianud P., Charpigny G., and Martal J. (1991) Immunoregulatoryeffects of trophoblastin (oTP): all 5 isoforms are immunosuppressive of PHA drivenlymphocyte proliferation. J Reprod Immunol vol. 19, pp. 237-249,.[11] Guillomot M., Michel C., Gaye P., Charlier M., Thojan J., and Martal J. (1990) Cellularlocalization of an embryonic interferon, ovine trophoblastin and its in-rna in sheepembryos during early pregnancy. Biol Cell vol. 68, pp. 205-211.[12] Godkin J.D., Bazer F.W., Moffat J., Session F. and Roberts M. (1982) Purification andproperties of a major, low molecular weight protein released by the trophoblaste ofsheep blastocysts at day 13-21 J Reprod Fertil vol. 65, pp. 141-150,.[13] Greve T., Avery B. and Callesen H. (1993) Viability of in vivo and in vitro producedbovine embryos. Reprod Dom Anim vol. 28, pp. 645-654.[14] Hasler J.F., Henderson W.B., Hurtgen P.J., Jin Z.Q, McCauley A.D., Mower S.A., NeelyB., Shuey L.S., Stokes J.E. and Trimmer S.A. (1995) Production, freezing andtransfer of bovine IVF embryos and subsequent calving results. Theriogenologyvol. 33, pp. 644-645,.

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16Reproductive Endocrinology Diseases:Hormone Replacement andTherapy for Peri/MenopauseZoe Roupa 1 , Greta Wozniak 2 , Konstantinos Tsipras 3and Penelope Sotiropoulou 41 European University, Nicosia,2 Medical School, University of Thessaly, Larissa,3 Endocrinology Primary Health Care Center, Athens4 Technological Education Institute of Athens,1 Cyprus2,3,4 Greece1. IntroductionThere are many female causes for infertility but the incidence of infertility increases withadvancing age. Colombat de L'Isere in a chapter on ‘Change of Life' in his “Treatise on thediseases and special hygiene of females” (1845) stated: Compelled to yield to the power oftime, women now cease to exist for the species, and hence forward live only for themselves”(Colombat de L’Isere M., 1945). Fortunately, this pessimistic outlook on life after menopausehas become outdated. Aiming to standardize terminology The World Health Organization(WHO) assembled in 1996 and the Council of Affiliated Menopause Societies (CAMS) in1999 (WHO Scientific Group: Research on Menopause in the 1990s, Utian WH, 1999).Commonly accepted terms, including pre-menopause, peri-menopause, menopausaltransition, and climacteric, were thought to be too vague to be useful. In July of 2001, theStages of Reproductive Aging Workshop (STRAW) was held to address the absence of arelevant staging system for female reproductive aging, and to discuss the confusing currentnomenclature for the pre-menopause (Soules MR, et al., 2001).The average age of menopause is 51 and less than 1% of women experience it before the ageof 40. Some women undergo premature menopause at a very early age affecting their abilityto have children. As more and more women delay child bearing, this life altering conditionhas become more prevalent. Population aging must be added to population growth as veryimportant social problems. Women in our society get married and have children later inlife. Therefore, evaluation of ovarian reserve is critical to understanding a patient’sreproductive potential.Ovulatory disorders are a common cause of infertility. Ovulation is controlled by complexinteractions between numerous endocrine hormones including FSH, LH, estradiol,progesterone and others. Menopause is the cessation of the primary functions of the humanovaries, with associated changes in pituitary gonadotropin secretion occurring secondary to

270Artificial Insemination in Farm Animalsthe decline in ovarian sex steroid and protein production. However, increasing evidencesuggests that aging is associated with dynamic changes in the hypothalamic and pituitarycomponents of the reproductive axis that are independent of changes in gonadal hormonesecretion (Hall JE, et al., 2000). Imbalances in these hormones, or alterations in the “feedbackmechanism”, can prevent ovulation, or cause it to be irregular.2. Epidemiology of menopauseMost estimates of age at natural menopause are based on samples of Caucasian women inWestern societies. In one large, comprehensive, prospective cohort study of mid-aged,Caucasian U.S. women (the Massachusetts Women's Health Study [MWHS]) the age atnatural menopause occurred at 51.3 years, (Gold EB, et al., 2001) confirming prior reports.The Study of Women's Health Across the Nation (SWAN), a multicenter, multiethnic,community-based cohort study of women and the menopausal transition, reported theoverall median age at natural menopause to be 51.4 years, after adjustment for other factors(Gold EB, et al., 2001). Studies performed outside the United States suggest that Africans,African Americans, (Bromberger JT, et al., 1997) and Hispanics of Mexican descentexperience menopause at an earlier age than Caucasian women, as opposed to Japanese(Tamada T & Iwasake H., 1995) and Malaysian (Ismael NN., 1994) women, who report asimilar median age of menopause to women of European descent.Lower educational attainment and unemployment have been independently associated withearlier age at menopause (Gold EB, et al., 2001; Cramer DW, 1994 et al) and may be markersfor elevated bio-psychosocial stress. Women who are separated, divorced, or widowed havebeen shown to have an earlier menopause than women who are married (Gold EB, et al.,2001). Age at natural menopause for parous women has been reported to occur significantlylater than for nulliparous women. (Gold EB, et al., 2001; Anasti JN., 1998; Tibilette MG, et al.,1999; Weel AE, , et al., 1999). Gold et al. and Cramer et al. observed a trend of increasingage at menopause with increasing number of life births, and that prior use of oralcontraceptives was associated with earlier age at natural menopause however, a slightprolongation of the reproductive life-span has been associated with oral contraceptive use(Cramer DW, et al., 1994).The proposed mechanism by which parity and use of oral contraceptives may result in laterage at natural menopause involves reducing ovulatory cycles earlier in life and thuspreserving oocytes longer, resulting in later menopause (Gold EB, et al., 2001). Some studiesshow that women with a lower body mass index (BMI) experience an earlier menopause;other studies have not confirmed this finding (Zapantis G & Santoro N., 2002).Environmental toxicants may play a role in early menopause. A large body of literatureshows that current smokers tend to experience menopause at an earlier age (1 to 2 years)than non-smokers (Reynaud K,, et al, 2001, Hreinsson JG, et al., 2002, Loffler KA, et al.,2003, Burger HG, et al., 2002) and may have a shorter menopausal transition (Gold EB, 2001,et al).It has been shown that polycyclic hydrocarbons in cigarette smoke are toxic to ovarianfollicles and may lead to their loss and thus an earlier menopause in smokers. Harlow et al.observed that women with a history of medically treated depression had a 20% increasedrate of entering peri-menopause sooner than women with no depression history, afteradjustment for age, parity, age at menarche, education, cigarette smoking, and BMI.Epidemiology gives answers about populations, whereas clinical medicine deals with

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 271patient samples and individuals. For example, in population-based studies, (Gold EB, et al.,2001) no global increased prevalence of depression has been associated with the menopausetransition, whereas in clinical samples, depression around menopause has reportedlyincreased. Furthermore, symptoms vary among women, and the distinction betweenpopulations versus individuals must be made when one is evaluating epidemiologic factorsrelated to menopause.3. Menopause pathogenesisThe basis of reproductive senescence in women is oocyte/follicle depletion in the ovary.Developmentally, a woman attains her peak oocyte complement at 20 weeks’ gestationalage. Between 20 and 40 weeks’ gestation, two thirds of a woman's oocyte complement islost, and total oocyte counts drop from a mean of about 6 to 8 million to 1 to 2 million(Zapantis G & Santoro N., 2002).The most massive wave of atresia (rate of follicle loss) that a woman ever experienceshappens before she is born. At the onset of puberty, germ cell mass has been reduced to300,000 to 500,000 units. Subsequent reproductive aging consists of a steady loss of oocytesthrough atresia or ovulation and does not necessarily occur at a constant rate. Atresia is anapoptotic process. During the 35 to 40 years of reproductive life, 400 to 500 oocytes will beselected for ovulation. By menopause, only a few hundred follicles remain (Speroff L, et al.,1999).The relatively wide age range (42 to 58 years) for menopause in normal women seems toindicate that women may be endowed with a highly variable number of oocytes, or that therate of oocyte loss varies greatly (Soules MR, et al., 2001). Concurrent with the loss ofovarian follicles as a woman transitions to menopause are hormonal changes in thehypothalamic-pituitary-ovarian axis. Follicle-stimulating hormone (FSH) is an establishedindirect marker of follicular activity; as follicle numbers decline, FSH levels increase (BurgerHG, et al., 2002). An elevated level is often the first clinically measurable sign ofreproductive aging. Large cross-sectional studies have reported a progressive, quantitativerise in FSH with age (Ahmed-Ebbiary NA, et al., 1994). In the late reproductive years, initialelevations in FSH are most prominent in the early follicular phase of the menstrual cycle butare intermittent and do not occur in every cycle (Klein NA, Soules MR., 1998). This increaseis first detectable some years before any clinical indications of approaching menopause areevident (Burger HG, et al., 2002).The rise in FSH appears to be the result of a decline in inhibin -B, a dimeric protein that reflectsthe fall in ovarian follicle numbers. Is a small pleomorphic peptide made within the ovariangranulosa and luteinized granulosa cells, which, although assay specificity and sensitivity inovarian physiology (Burger HG 1993). Inhibin may be an intraovarian regulator although otherpeptides such as activin and follistatin are more likely paracrine factors (Findlay JK, et al.,1990) but one of its more important functions appears to be feedback suppression of FSHproduction (Seifer DB, et al., 1996). In reproductive life, inhibin serves to selectively inhibitFSH by binding to receptors on the anterior pituitary (Robertson DM & Burger HG., 2002).Estradiol is stable or even elevated during the earlier menopause transition; closer to the finalmenstrual period, a decline is clearly observed (Longscope C, et al., 1986).Findings from the Melbourne Women's Midlife Health Project, a cohort of women followedthrough the menopause transition, confirm that a decline in inhibin B precedes the increasein FSH and the decline in estradiol that occur later in the transition (Burger HG, et al., 2007).

272Artificial Insemination in Farm AnimalsThe remaining follicles are less likely to function normally, which may lead to erraticfollicular development and dysregulation of folliculogenesis (Whiteman MK, et al., 2003,Schwingl PJ, et al., 1994). Although FSH and estradiol vary near menopause, steroidogenicenzymes appear to be completely absent in the postmenopausal ovary after all functionalfollicles are lost (Couzinet B, et al., 2001). Between the ages of 20 and 40 years,concentrations of total testosterone have been reported to fall by about 50% (Zumoff B, et al.,1995). This age-related decline does not change further during the transition years (BurgerHG, et al., 2000). Similarly, dehydroepiandosterone (DHEA) and its sulphate, DHEAS,decline with age (Rossmanith W et al., 1991., Santoro N, et al., 1998). Because circulating sexhormone–binding globulin (SHBG) decreases across the menopausal transition, freeandrogen levels actually rise, as indicated by a small increase in free androgen index (T ÷SHBG × 100) (Burger HG, et al., 2000). Androstenedione, which remains relatively stableduring the transition, is converted to estrone in extra-glandular tissue. This accounts foralmost all the estrogen in circulation after menopause.When ovulation stops, concurrent with a woman's FMP, serum progesterone levels areinvariably low (Rannevik G, et al., 1995). Luteinizing hormone (LH) eventually increases,although at a slower rate than FSH. Despite the epidemiologic trend toward elevated FSHand decreased estradiol with progression through the transition, measurement of FSH,inhibin, and estradiol provides at best an unreliable guide to the menopausal status of anindividual woman (El-Hage G, et al., 2007; Braunstein GD, et al., 2005). A more rationalapproach to diagnosing menopause would include an assessment of the longitudinalsymptoms of a woman who presents with peri-menopausal complaints (Lobo RA., 1999).Hormone profiles correlate well with symptoms and cycle features (Burger HD, et al., 1995).Thus, if a woman is >45 years old and has had a recent disruption in her menstrual patternand symptoms suggestive of transient hypo-estrogenemia, it is likely that she has enteredher menopausal transition (Santoro N., 2002). That being said, the clinician should take careto rule out other pathologies that can be masked by common complaints associated with themenopausal transition. At minimum, a screening TSH level should be performed, asmenstrual irregularity may be the only manifestation of thyroid dysfunction.4. Regulation of gonadotropins and control of ovarian steroid productionPituitary gonadotropes synthesize and secrete both LH and FSH. They account for 7% to15% of anterior pituitary cells. Gonadotropin subunit gene expression is regulated by thefrequency of GnRH signal input to pituitary gonadotropes (Haisenleder DJ, et al., 1991).During the menstrual cycle, LH pulse frequency is approximately every 90 minutes in theearly follicular phase, 60 to 70 minutes during the late follicular phase, 100 minutes duringthe early luteal phase, and 200 minutes during the late luteal phase. This variation reflectschanges in GnRH pulse frequency, which regulates relative FSH and LH secretion; this, inturn, determines follicle recruitment, development, and ovulation. More rapid GnRH pulsefrequencies promote LH secretion, and slower frequencies promote FSH secretion. Althoughgood evidence indicates that changes in GnRH pulse frequency determines differential LHand FSH secretion (Marshall JC, et al., 1993) it is apparent that ovarian steroids and peptidehormones have a major role. Women with hypothalamic stalk section regain theircharacteristic cycle length when administered an unchanging frequency of exogenousGnRH pulses every 90 minutes.

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 273This indicates that intrinsic rates of follicle development and regression of the corpusluteum, along with their phasing of steroid and peptide hormone production, are majordeterminants of LH and FSH responses to GnRH. Gonadotropins control the growth anddifferentiation of the steroid hormone–secreting cells of the ovary, intrinsically linking formand function. A defined sequence of gonadotropin action propels the growth of follicles andthe production of steroid hormones. Positive feedback on the pituitary by highconcentrations of estrogens leads to the ovulatory surge of LH, which in turn triggers adramatic differentiation event, resulting in structural reorganization of the pre-ovulatoryfollicle, release of the ovum, and striking changes in the steroidogenic capacity of theluteinizing cells. Follicular growth, which culminates in ovulation and corpus luteumformation, requires both FSH and LH. Steroidogenic competence of the ovarian follicle is notachieved in the absence of FSH, even if LH is present in abundance. FSH promotesproliferation of the granulosa cells and induces the expression of genes involved in estradiolbiosynthesis (Haisenleder DJ, et al., 1991; Kaiser UB, et al., 1997). During the last phase offollicular maturation, when granulosa cells acquire LH receptors, LH is then able to sustainfollicular estradiol synthesis. This LH substitution is thought to compensate for thediminished levels of FSH of the late follicular phase consequent to negative-feedback actionof estradiol and inhibin. LH action on the granulosa thus rescues the dominant LHexpressingfollicle from the fate of atresia. LH stimulation is indispensable for normalovarian hormone production not only before but also after ovulation. Suppression of LHrelease leads to a prompt decline in progesterone levels that precede changes in theabundance of mRNAs encoding steroidogenic enzymes or structural changes in the corpusluteum (King JA & Millar RP., 1982).This acute regulation of ovarian progesterone secretion is controlled by LH via theexpression of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) andprotein, present in both theca-lutein and granulosa-lutein cells throughout the luteal phaseare highly expressed in early and midluteal phase, whereas declining StAR mRNA andprotein levels are characteristic of late luteal phase. Moreover, StAR protein levels in thecorpus luteum are highly correlated with plasma progesterone levels; suppression of LHlevels during the midluteal phase markedly decreases plasma progesterone levels andabundance of StAR mRNA transcripts in the corpus luteum (Millar RP, et al., 2004).4.1 Intra-ovarian control mechanismsThe growth of follicles and the function of the corpus luteum, while under the primarydirection of the pituitary, are highly influenced by intra-ovarian factors that modulate theaction of gonadotropins. These intra-ovarian factors most likely account for gonadotropinindependentfollicular growth, observed differences in the rate and extent of developmentof ovarian follicles, arrest and initiation of meiosis, dominant follicle selection, andluteolysis. The list of potential paracrine factors that can influence steroid production bytheca and granulosa cells is long and diverse. The previous theca cells lacked the aromataseenzyme that is necessary to produce estrogen (Schwanzel-Fukuda M, & Pfaff DW, 1984) sothe production of estrogen in granulosa cells indicates presence of aromatase. It includesvarious growth factors, cytokines, peptide hormones, and steroids such as epidermalgrowth factor, transforming growth factor β (TGF-β), platelet-derived growth factor,fibroblast growth factors, transforming growth factor α (TGF-α), activins, inhibins, Anti-Müllerian hormone, insulin-like growth factors, estradiol, progesterone, and GnRH (MillarR: 2005; King JA, et al., 2002)

274Artificial Insemination in Farm Animals5. The peri-menopausal transitionThere is only one marker, menstrual irregularity that can be used to objectively define andestablish what is called the peri-menopausal transition. This irregularity will be perceivedby patients as skipped menstrual periods or longer durations (about 40 to 60 days) betweenperiods (Harlow SD, et al., 2008) There is no universal pattern; each woman will perceive achange that is her own individual characteristic alteration. Literally means “about or aroundthe menopause.” Generally speaking, the term “menopausal transition” is preferred overperi-menopause and climacteric.The menopause is that point in time when permanent cessation of menstruation occursfollowing the loss of ovarian activity. Menopause is derived from the Greek words men(month) and pausis (cessation). The years prior to menopause that encompass the changefrom normal ovulatory cycles to cessation of menses are known as the peri-menopausaltransitional years, marked by irregularity of menstrual cycles. Climacteric, an older, moregeneral, and less precise term, indicates the period of time when a woman passes from thereproductive stage of life through the peri-menopausal transition and the menopause to thepostmenopausal years (Treloar AE, et al., 1967). Menarche is followed by approximately 5–7years of relatively long cycles at first, and then there is increasing regularity as cyclesshorten to reach the usual reproductive age pattern. In the 40s, cycles begin to lengthenagain. The highest incidence of anovulatory cycles is under age 20 and over age 40 (CollettME, et al., 1954). At age 25, over 40% of cycles are between 25 and 28 days in length; from 25to 35, over 60% are between 25 and 28 days. The perfect 28-day cycle is indeed the mostcommon mode, but it totalled only 12.4% of Vollman’s study cycles. Overall, approximately15% of reproductive-age cycles are 28 days in length. Only 0.5% of women experience acycle less than 21 days long, and only 0.9% a cycle greater than 35 days (Munster K, et al.,1992).Most women have cycles that last from 24 to 35 days, but at least 20% of women experienceirregular cycles (Belsey EM & Pinol APY, 1997). When women are in their 40s, anovulationbecomes more prevalent, and prior to anovulation, menstrual cycle length increases,beginning 2 to 8 years before menopause (Treloar AE, et al., 1967). Cycles greater than 40days in length are prevalent in the year before menopause (Ferrell RJ, et al) The duration ofthe follicular phase is the major determinant of cycle length (Sherman BM, et al) Thismenstrual cycle change prior to menopause is marked by elevated follicle-stimulatinghormone (FSH) levels and decreased levels of inhibin, but normal levels of luteinizinghormone (LH) and slightly elevated levels of estradiol (Buckler HM, et al., 1991;MacNaughton J, et al., 1992; Hee J, et al., 1993; Burger HG, et al., 2000,2008). In the averagewoman, continuing follicular depletion and declining fertility begin at age 37–38, andmenopause follows approximately 13 years later (average age 51). However, inepidemiologic studies approximately 10% of women in the general population becomemenopausal by the age of 45, probably because they were born with a smaller than normalovarian follicular pool that is functionally depleted at an earlier age. Menopause occurswhen the number of remaining follicles falls below a critical threshold, about 1,000,regardless of age (Treloar AE, 1981).Recent longitudinal studies of women as they pass through the peri-menopausal transitionreveal that estrogen levels do not begin a major decline until about a year beforemenopause. (Burger HG, et al., 2008; Lasley BL, et al., 2002). Indeed, women experiencingthe peri-menopausal transition actually have higher overall estrogen levels, a response that

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 275is logically explained by an increased ovarian follicular reaction to the increase in FSHsecretion during these years (Santoro N, et al). Variability in estrogen levels is characteristicof the peri-menopausal transition, with greater variability observed in menstrual cycles thatdisplay greater irregularity (Meyer PM, et al). As noted, most women experience a 2- to 8-year period of time prior to menopause when anovulation becomes common (Treloar AE, etal., 1996). During this period of time ovarian follicles continue their rate of loss untileventually the supply of follicles is finally depleted (Gougeon A, et al., 1994). The agerelatedchanges in the endocrine characteristics of the menstrual cycle that result fromprogressive follicular depletion correlate with a measurable decrease in ovarian volume andin the number of antral follicles observed by trans-vaginal ultrasonography during the earlyfollicular phase (Lass A, et al., 1997; Yong PY, et al., 2003; Frattarelli JL, et al., 2000; DumesicDA, et al., 2001; Bancsi LF, et al., 2002; Kupesic S, et., 2003). The inverse and tightrelationship between FSH and inhibin indicates that inhibin is a sensitive marker of ovarianfollicular competence and, in turn, that FSH measurement is a clinical assessment of inhibin(MacNaughton J, et al., 1992; Hee J, et al., 1993). The decrease in inhibin secretion by theovarian follicles begins early (around age 35), but accelerates after 40 years of age. This isreflected in the decrease in fecundity that occurs with aging. The major decrease in estradiollevels began about 2 years before menopause (Sowers MR, , et al., 2008). Declining levels ofinhibin-B and Anti-Müllerian Hormone (AMH) reached a low to non-detectable point about5 years before menopause (Sowers MR, et al., 2008).Although the inhibin-B and AMH results are in general agreement with other reports, theexactness of the timing is limited by the fact that the blood samples were obtained from only50 women in the study. Nevertheless, the Michigan study confirms the validity of AMH as amarker for the ovarian reserve of follicles. Unlike inhibin-B, AMH is not a participant in thefeedback relationship between the ovary and the pituitary gonadotropins, rather AMH, aproduct of granulosa cells, reflects the number of follicles present in the ovaries awaitingFSH stimulation (Visser JA, et al). The variability in these measurements from individual toindividual, however, precludes the practical use of these tests to predict with accuracy thefuture rate of menopause.The peri-menopausal years are a time period during whichpostmenopausal levels of FSH (greater than 20 IU/L) can be seen despite continuedmenstrual bleeding, while LH levels still remain in the normal range. Occasionally, corpusluteum formation and function occur, and the peri-menopausal woman is not safely beyondthe risk of an unplanned and unexpected pregnancy until elevated levels of both FSH (>20IU/L) and LH (>30 IU/L) can be demonstrated. The median age for the onset of thistransition was 47.5 years. Only 10% of women ceased men-struating abruptly with noperiod of prolonged irregularity. The peri-menopausal transition from reproductive to postreproductivestatus was, for most women, approximately 4 years in duration. In the studyby Treloar, the average age for entry into the peri-menopausal transition was 45.1, and theage range that included 95% of the women was 39–51 (Treloar AE, 1996). The mean durationof the peri-menopausal transition was 5.0 years, with a range of 2 to 8 years.5.1 Endocrine activity of the peri and post-menopausal ovaryAs reviewed above, mean estradiol levels are normal or high in peri-menopausal women,and FSH levels are often not suppressed despite these high estradiol levels. These aspects ofpituitary-ovarian relationships are contrary to expected physiology. It is proposed that,decreasing ovarian production of inhibin plays a role in the high average estrogen levelsdocumented during the peri-menopause. More specifically, the B subtype of inhibin, a small

276Artificial Insemination in Farm Animalspeptide made in ovarian granulosa cells, which is known to be stimulated by FSH and, inturn, to suppress FSH, may play a role in the altered physiology of the perimenopause(Klein NA, et al., 1996, 1998). Increasing evidence suggests that ovarianinhibin plays a role in ovarian folliculogenesis (McLachlan RI, et al., 1986; Hughes EG,,et al.,1992), therefore, new information about inhibin levels and their functional relationshipsin women in there forties and fifties becomes important.The peri and post-menopausal ovary contains two different populations of cells withsteroidogenic capacity: hilar cells and cortico stromal cells that may represent residual thecalelements (De Roux N, et al., 1999). In vitro studies suggest that the post-menopausal ovaryhas some steroidogenic potential. Incubation of post-menopausal ovarian stromal slices withpregnenolone yielded progesterone, dehydroepiandrosterone, and testosterone. Incubationof strips of ovarian hilar tissue from postmenopausal women revealed a steroidogenicpattern similar to that of the postmenopausal ovarian stroma. However, the overall amountof steroids produced was substantially greater compared with stroma. Measurable in vitroformation of estradiol by postmenopausal cortical stroma and hilar cells has also beenreported (Bertherat J 1998: Ulloa-Aguirre A, et al., 1998).With increasing age, the adrenal contribution of precursors for estrogen production provesinadequate. In this final stage of estrogen availability, levels are insufficient to sustainsecondary sex tissues. Estrogens in peri and post-menopausal women appear to arise almostexclusively from extra-glandular aromatization of androstenedione (Arora KK, et al., 1997).Oophorectomy results in no significant reduction in urinary estrogen excretion by postmenopausalwomen. However, adrenalectomy after oophorectomy virtually eliminatesmeasurable estrogens from the urine. In vitro studies concluded that the postmenopausalovarian stroma is unable to aromatize androgens (Everest HM, et al., 2001). However,others have suggested that the post-menopausal ovary may synthesize limited amounts ofestrogens, because the concentrations of estradiol and estrone are two times higher inovarian venous blood than in peripheral blood of post-menopausal women (Illing N, et al.,1999).There is some evidence that ovarian androgen production in post-menopausal women canbe gonadotropin dependent. Administration of hCG to postmenopausal women results in asmall increase in the circulating levels of testosterone (Sun YM, et al). Daily injection of hCGcauses hyperplasia of the ovarian hilar cells and histochemical evidence suggestive of activesteroidogenesis (Tensen C, et al., 1997). Administration of hCG, but not ACTH, resulted inincreased androgen but not estrogen production by the ovaries (Wang L, et al., 2001).Binding sites for both LH and FSH were identified in the cortical stroma and in hilar cells(Davidson JS, et al., 1994). Addition of hCG to hilar cells results in increased cAMPformation and steroid biosynthesis, indicating preserved responsiveness to gonadotropins.Taken together, these observations suggest that ovarian androgen biosynthesis of the postreproductiveovary is at least partially gonadotropin dependent.The post-menopausal ovary is occasionally involved in pathologic endocrine activity.Stromal hyperplasia can occur, with the ovary enlarging with hyperplastic stromal nodulesconsisting of lipid-rich luteinized cells that resemble theca interna. The ovaries with stromalhyperplasia produce large amounts of androstenedione, resulting in hirsutism andvirilisation (Vrecl M, et al., 1998). Hilar cells can give rise to functional hilar cell tumors,which produce excess amounts of androgens, leading to virilisation (Pawson AJ, et al., 1998;Blomenrohr M, et al., 1999; Heding A, et al., 2000). Signs and symptoms of estrogen excessmay also be evident in circumstances of significant peripheral aromatization.

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 2776. Treatment of anovulationIf no primary pathology is apparent, or if the primary pathology has been treatedappropriately without restoration of normal endocrinology, treatment options lie betweenestrogen replacement (or the oral contraceptive in a younger woman) to preventosteoporosis and ovulation induction to restore fertility. Estrogen antagonists usually areineffective in inducing ovulation in progestogen-negative women, and treatment withpulsatile GnRH or gonadotropin treatment is normally required. (Elizur SE, et al., 2005).Treatment with pulsatile GnRH involves the woman carrying a small portable pump buthas the important advantage of a lower multiple pregnancy rates than is seen withgonadotropin treatment. Women with a low LH concentration (

278Artificial Insemination in Farm Animalsmultiple pregnancies and the costs of IVF have sparked renewed interest in natural cycleIVF and mild stimulation regimens (Nargund G, et al., 2001)7. Natural cycleThe first birth resulting from IVF derived from a single oocyte collected in a naturalovulatory cycle (Steptoe PC, & Edwards RG, 1978). Compared to stimulated IVF cycles,natural cycle IVF offers a number of attractive advantages. Natural cycle IVF involves onlymonitoring the spontaneous cycle and retrieving a single oocyte before the midcycle LHsurge occurs. It is physically less demanding, requires little or no medication, decreasescosts by 75–80%, (Aboulghar MA, et al., 1995; Nargund G, et al., 2001) and all but eliminatesrisks for multiple pregnancy and ovarian hyper-stimulation syndrome (OHSS). The chiefdisadvantages of natural cycle IVF are high cancellation rates due to premature LH surgesand ovulation, and the comparatively low success rate, which is approximately 7% (PelinckMJ, et al., 2002). When oocyte retrieval is based on detection of the mid-cycle rise in LH,careful and frequent monitoring is required and procedures are difficult to scheduleefficiently.Alternatively, exogenous human chorionic gonadotropin (hCG) can be administered whenthe lead follicle reaches a size consistent with maturity, thereby better defining the optimumtime for oocyte retrieval (Nargund G, et al., 2001). Adjuvant treatment with a GnRHantagonist also can be used to prevent a premature LH surge, but requires “add-back”treatment with exogenous FSH, and success rates are still quite low, ranging up to 14% percycle in non-randomized trials (Castelo-Branco A, et al., 2004; Kolibianakis E, et al., 2002,2003; Weghofer A, et al., 2004; Elizur SE, et al., 2005). In one large cohort study involving 844treatment cycles in 350 good prognosis patients, the cancellation rate was 13%, thepregnancy rate was 8% per cycle and the cumulative pregnancy rate after three “modifiednatural IVF cycles” was 21% (Pelinck MJ, et al., 2002). In a cohort of infertile couples withmale factor infertility, success rates in modified natural cycles have reached as high as 13%per cycle, with a cumulative pregnancy rate of 44% after six treatment cycles (Verberg MF,et al., 2006).8. Clomiphene citrateClomiphene citrate (CC) was the first method of ovarian stimulation used in IVF, (QuigleyMM, et al., 1984) but now has been almost entirely replaced by more effective stimulationregimens using human menopausal gonadotropins (hMG) or FSH, in combination with aGnRH agonist or antagonist (Macklon NS, et al., 2006). Clomiphene (100 mg daily) usuallyis administered for 5–8 days, beginning on cycle day 3, and induces development of two ormore follicles in most normally ovulating women, (Dickey RP, et al., 1998; Messinis IE &Milingos SD 1998; Ingerslev HJ, et al., 2001) although egg yields 1–3, are only slightlygreater than in un-stimulated cycles and substantially lower than in cycles stimulated withexogenous gonadotropins (Ingerslev HJ, et al., 2001; Branigan EF & Estes MA 2000;MacDougall MJ, Tan SL, Hall V, et al., 1996).Cycle cancellation rates are somewhat lower than in natural cycles and the numbers ofoocytes retrieved, embryos transferred, and pregnancy rates are greater. As in naturalstimulates multi-follicular development more effectively than treatment with Clomiphenealone (Corfman RS, et al., 1993; Dor J, et al., 1992). Drug costs and monitoring requirements

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 279are moderately cycles, exogenous hCG is administered when the lead follicle reaches maturesize and a GnRH antagonist can be used to prevent a premature endogenous LH surge.Sequential treatment with clomiphene (100 mg daily for 5 days) and modest doses ofexogenous gonadotropins (150–225 IU daily beginning on the last day of clomiphenetreatment or the day after) higher, but still substantially less than in standard stimulationregimens involving higher dose gonadotropin treatment after down-regulation with a longactingGnRH agonist (described below) (Weigert M, et al., 2002; Dhont M, et al., 1995). Inone comparative trial, higher cancellation rates and lower pregnancy rates were observed insequential clomiphene/gonadotropin cycles (Dhont M, et al., 1995). In another, thesequential stimulation regimen yielded fewer oocytes and embryos, but pregnancy rateswere similar and the risks of ovarian hyper-stimulation syndrome (OHSS) were lower(Weigert M, et al., 2002).In a randomized trial, sequential clomiphene/gonadotropin stimulation and GnRHantagonist treatment yielded a pregnancy rate comparable to that achieved with a moreaggressive standard treatment protocol, (Lin YH, et al., 2006) confirming the results of twoearlier retrospective studies, (Fiedler K & Ludwig M 2003; Williams SC, et al., 2002), butcontrasting with those of another observing lower pregnancy rates (Mansour R, et al., 2003).9. GnRH agonist “flare” gonadotropins stimulation protocolThe “short” or “flare” protocol is an alternative stimulation regimen designed to exploitboth the brief initial agonistic phase of response to a GnRH agonist and the suppression thatresults from longer-term treatment (Padilla SL, et al., 1996; Garcia JE, et al., 1990). In atypical standard short protocol, leuprolide acetate (1.0 mg daily) is administered on cycledays 2–4, continuing thereafter at a reduced dose (0.5 mg daily), and gonadotropinstimulation (225–450 IU daily) begins on cycle day 3. Later adjustments in the dose ofgonadotropin stimulation, if needed, are based on response and indications for hCGadministration are the same as in the long protocol (described above). An early metaanalysisincluding seven clinical trials comparing the short and long GnRH agonisttreatment regimens determined that the two protocols yielded similar cancellation andpregnancy rates: (Hughes EG, et al., 1992).A 2000 systematic review including 22 trials concluded that pregnancy rates achieved withthe long protocol were superior to those using the flare regimen (OR=1.27, CI=1.04–1.56)overall, (Daya S., 2000), but the analysis did not control for diagnosis and other prognosticfactors and results may not apply to all women, or to poor responders in particular.Whereas some have observed improved follicular response and lower cycle cancellationrates in poor responders treated with a flare protocol, pregnancy and live birth ratesremained low (Karande V, et al., 1997; Karacan M, et al., 2001). Decreased scheduling flareexibility is a distinct disadvantage of the flare is protocol, unless the onset of menses iscontrolled by preliminary treatment with an OC. The regimen also can result in a significantincrease in serum progesterone and androgen levels, presumably resulting from late corpusluteum rescue, (San Roman GA, et al., 1992) which may adversely affect oocyte quality andfertilization and pregnancy rates (Loumaye E, et al., 1989). The “OC micro-dose GnRHagonist flare” stimulation regimen is a variation of the standard short protocol involving 14–21 days of preliminary ovarian suppression with an OC (one pill daily), followed by microdoseleuprolide treatment (40 μg twice daily) beginning 3 days after discontinuation of OCtreatment, and high-dose gonadotropin stimulation (300–450 IU daily) starting on day 3 of

280Artificial Insemination in Farm Animalsleuprolide therapy. Indications for later gonadotropin dose adjustments and hCGadministration are the same as in other stimulation regimens. Its primary advantage overthe standard short protocol is that it does not induce any increases in serum progesterone orandrogen concentrations, possibly because the doses of GnRH agonist administered aremuch lower, but likely also because preliminary OC treatment all but eliminates thepossibility there may be a corpus luteum left to respond (Gonen Y, et al., 1990; Cedrin-Durnerin et al., 1996). The OC-micro-dose GnRH agonist flare protocol may be useful inprevious poor responders, in whom it can stimulate increased endogenous FSH release andmay yield lower cancellation rates and higher peak serum estradiol levels, transfer rates andpregnancy rates (Surrey ES, et al., 1998; Scott RT et al., 1994) GnRH AntagonistGonadotropin Stimulation Protocol.The introduction of GnRH antagonists into clinical practice provided another option forovarian stimulation in ART. In contrast to the long-acting agonists, which first stimulate andlater inhibit pituitary gonadotropin secretion by desensitizing gonadotropes to GnRH viareceptor down-regulation, the antagonists block the GnRH receptor in a dose-dependentcompetitive fashion and have no similar flare effect (Matikainen T, et al., 1992; Reissmann T,et al., 1974,1995) gonadotropin suppression is almost immediate. GnRH antagonists offerseveral potential advantages over agonists. First, the duration of treatment for an antagonistis substantially shorter than for an agonist. Since its only purpose is to prevent a prematureendogenous LH surge and its effects are immediate, antagonist treatment can be postponeduntil later in follicular development (after 5–6 days of gonadotropin stimulation), afterestradiol levels are already elevated, thereby eliminating the estrogen deficiency symptomsthat may emerge in women treated with an agonist (Olivennes F, et al., 2002).Second, because any suppressive effects that agonists may exert on the ovarian response togonadotropin stimulation also are eliminated, the total dose and duration of gonadotropinstimulation required is decreased (Olivennes F, et al., 2002; Albano C, et al., 2000). For thesame reason, GnRH antagonist stimulation protocols may benefit women who are poorresponders when treated with a standard long protocol (Olivennes F, et al., 2003; AkmanMA, et al., 2001). Third, by eliminating the flare effect of agonists; GnRH antagonists avoidthe risk of stimulating development of a follicular cyst. Finally, the risk of severe OHSSassociated with use of antagonists also appears lower than with agonists. GnRH antagonistshave some potential disadvantages. When administered in small daily doses, strictcompliance with the prescribed treatment regimen is essential (Olivennes F, et al., 2002).Antagonists suppress endogenous gonadotropin secretion more completely than agonists.Whereas the low levels of LH observed during agonist treatment are usually sufficient tosupport normal follicular steroid-genesis during stimulation with uFSH or rFSH, the evenlower concentrations in women treated with an antagonist may not be. Indeed, serumestradiol levels may plateau or fall when antagonist treatment begins (Olivennes F, et al2002;, de Jong D, et al., 2001). Although follicular growth appears unaffected, most prefer toadd or substitute a low dose of hMG (75 IU) at the same time if it was not already part of thestimulation regimen. Evidence also suggests that pregnancy rates in antagonist treatmentcycles may be modestly lower than in cycles using agonists in the long protocol (Al-Inany etal., 2006).The two GnRH antagonists available for clinical use, ganirelix and cetrorelix, are equallypotent and effective. For both, the minimum effective dose to prevent a premature LH surgeis 0.25 mg daily, administered sub-cutaneously (Albano C, et al., 1997). Either can beadministered in a series of small daily doses (0.25 mg). The treatment protocol may be fixed

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 281and begin after 5–6 days of gonadotropin stimulation, (Albano C, et al., 1997; Diedrich K, etal., 1994), or tailored to the response of the individual, starting treatment when the leadfollicle reaches approximately 13–14 mm in diameter. The individualized treatment regimengenerally requires fewer total doses and may yield better overall results (Ludwig M, et al.,2002). Alternatively, a single larger dose of cetrorelix (3.0 mg) will effectively prevent an LHsurge for 96 hours. If given on day 6–7 of stimulation, the interval of effective suppressionwill encompass the day of hCG administration in most women (75–90%); the remainder mayreceive additional daily doses (0.25 mg) as needed, ending on the day of hCG treatment(Olivennes F, et al 1995; Olivennes F, et al., 2000; Olivennes F, et al., 2003). The single doseantagonist treatment regimen also can be withheld until the lead follicle reaches 13–14 mmin diameter (Fanchin R, et al., 2003,2005).A common variation of the antagonist stimulation regimen uses preliminary treatment withan OC to control the onset of menses, typically ending approximately 5 days before thescheduled start, which also may help to synchronize the follicular cohort before stimulationbegins. Another variation advocated for poor responders uses micronized estradiol (2 mgtwice daily, administered orally, beginning on day 21 of the preceding cycle) to suppressFSH during the late luteal phase for the same purpose, ending on the day beforegonadotropins stimulation begins, (Fanchin R, et al 2003, 2005), or continuing through thefirst 3 days of gonadotropin stimulation (Hill MJ, et al., 2009). The improved folliculardynamics observed are similar to those achieved by down-regulation with a GnRH agonistin the long protocol. The rebound increase in endogenous FSH levels that follows thediscontinuation of estradiol treatment also may synergize with exogenous gonadotropins topromote multi-follicular development (de Ziegler D, et al., 1998; Fanchin R, et al., 2003).Results of a number of early trials comparing a fixed antagonist treatment protocol to thestandard long protocol suggested that the two stimulation regimens yielded similarpregnancy rates (Albano C, et al., 2000; Olivennes F, et al., 2000); The European Middle EastOrgalutran Study Group, 2001; Fluker M, et al., 2001). However, a 2006 systematic reviewand meta-analysis including 27 trials comparing different antagonist stimulation protocolswith the long GnRH agonist protocol observed a significantly lower clinical pregnancy rate(OR=0.84, CI=0.72–0.97) and ongoing pregnancy/live birth rate (OR=0.82, CI=0.69–0.98).Overall, the total dose and duration of gonadotropin stimulation required, peak serumestradiol levels, and the number of follicles and oocytes were lower in antagonist cycles.The explanation for the modestly lower pregnancy rates observed in antagonist treatmentcycles is not clear. It is possible, but unlikely, that GnRH antagonists may have adverseeffects on oocytes, embryos, or the endometrium (Hernandez ER 200; Ortmann O, et al.,2001). It is far more likely that early results reflected inexperience and improved with timeand further refinements in the treatment regimen like those described above. Many of theadvantages originally envisioned for GnRH antagonists already have been realized.Whether antagonists ultimately will replace agonists and become the standard ovarianstimulation regimen in ART cycles remains to be seen, but their place in the therapeuticarsenal already is firmly established. Whereas a single bolus injection of an agonist(leuprolide 0.5 mg, triptorelin 0.2 mg) triggers a physiologic LH surge that lasts less than 24hours, hCG levels remain elevated for several days and stimulate markedly higher estradioland progesterone concentrations (Fauser BC, et al., 2002).The antagonist treatment regimens currently in use have potential disadvantages for womenwith PCOS. Their tonically elevated LH levels will remain high until antagonist treatmentbegins. Consequently, LH levels may rise prematurely, particularly if antagonist treatment

282Artificial Insemination in Farm Animalsis withheld until the lead follicle reaches 14 mm or more. Moreover, evidence indicates thatincreased LH exposure during early follicular development may be detrimental andpredispose to lower pregnancy rates (Kolibianakis E, et al., 2002; Kolibianakis EM, et al.,2003; Kolibianakis EM, et al., 2003; Kolibianakis E, et al., 2003). In theory, pre-treatment withan OC might prove quite useful by suppressing LH and androgen levels before stimulationbegins, decreasing exposure during early follicular development and the risk of rising LHlevels before antagonist treatment starts. Preliminary OC suppression and later antagonisttreatment may help to limit the follicular response to gonadotropin stimulation whilepreserving the option to use an agonist to trigger final oocyte maturation. Theseconsiderations simply serve to illustrate that GnRH antagonists are not a panacea and arenot necessarily the best choice even for women with PCOS. Antagonist stimulationprotocols are advocated for poor responders, primarily because they avoid the suppressiveeffects that agonists can have on follicular response and can prevent the premature LHsurges observed commonly in women stimulated with gonadotropins alone (Surrey ES &Schoolcraft WB., 2000). However, evidence is insufficient to indicate they yield resultsconsistently better than other stimulation regimens (Pandian Z, et al., 2010; Centers forDisease Control and Prevention, Atlanta, GA, 2009).10. Ovarian reserveThe concept of ovarian reserve, generally defined as the size and quality of the remainingovarian follicular pool, and the various methods for its measurement. The total number ofoocytes in any given women is genetically determined and inexorably declines throughoutlife, from approximately 1–2 million at birth, to about 300,000 at puberty, 25,000 at age 40,and fewer than 1,000 at menopause (Battaglia DE, et al., 1996; Faddy MJ & Gosden RG,1996). The rate of follicular depletion is not constant, but increases gradually as the numberof follicles remaining decreases (Nilsson E, et al., 2007; Adhikari D & Liu K, 2009; Da Silva-Buttkus P, et al., 2009; Coxworth JE, & Hawkes K, 2010). As the size of the remainingfollicular pool decreases, circulating inhibin-B levels (derived primarily from smaller antralfollicles) decrease, resulting in lower levels of feedback inhibition and a progressive increasein serum follicle-stimulating hormone (FSH) levels, most noticeably during the earlyfollicular phase (Klein NA, et al., 1996; Welt CK, McNicholl DJ, Taylor AE, et al; Hale GE, etal., 2007 ; Knauff EA, et al., 2009; Burger HG, et al., 2008). Increasing inter-cycle FSHconcentrations stimulate earlier follicular recruitment, resulting in advanced folliculardevelopment early in the cycle, an earlier rise in serum estradiol levels, a shorter follicularphase, and decreasing overall cycle length (Klein NA, et al., 2002; de Koning CH, et al.,2008).The physiology of reproductive aging provides the foundation for all contemporary tests ofovarian reserve. In clinical practice, the basal early follicular phase (cycle day 2–4) FSH levelis the most common test, but antimüllerian hormone (AMH) and antral follicle count arealternatives having significant potential advantages. As basal FSH levels increase, peakestradiol levels during stimulation, the number of oocytes retrieved, and the probability forpregnancy or live birth decline steadily (Pearlstone AC, et al., 1992; Scott Jr RT & HofmannGE, 1995; Bukman A, & Heineman MJ, 2001). With current assays (using IRP 78/549), FSHlevels greater than 10 IU/L (10–20 IU/L) have high specificity (80–100%) for predicting poorresponse to stimulation, but their sensitivity for identifying such women is generally low(10–30%) and decreases with the threshold value (Broekmans FJ, et al., 2006) . Although

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 283most women who are tested have a normal result, including those with a diminishedovarian reserve (DOR), the test is still useful because those with abnormal results are verylikely to have DOR. In a 2008 study, an FSH concentration above 18 IU/L had 100%specificity for failure to achieve a live birth (Scott Jr RT, et al., 2008). The basal serumestradiol concentration, by itself, has little value as an ovarian reserve test (Hazout A, et al.,2004; Eldar-Geva T, et al., 2005; McIlveen M, et al., 2007), but can provide additionalinformation that helps in the interpretation of the basal FSH level. An early elevation inserum estradiol reflects advanced follicular development and early selection of a dominantfollicle (as classically observed in women with advanced reproductive aging), and willsuppress FSH concentrations, thereby possibly masking an otherwise obviously high FSHlevel indicating DOR. When the basal FSH is normal and the estradiol concentration iselevated (>60–80 pg/mL), the likelihood of poor response to stimulation is increased andthe chance for pregnancy is decreased (Evers JL, et al., 1998; Buyalos RP, al., 1997). Whenboth FSH and estradiol are elevated, ovarian response to stimulation is likely to be verypoor. Antimüllerian hormone (AMH) derives from pre-antral and small antral follicles.Levels are gonadotropin-independent and vary little within and between cycles (Fanchin R,et al., 2005; Tsepelidis S, et al., 2007; Hehenkamp WJ, et al., 2006). The number of smallantral follicles correlates with the size of the residual follicular pool and AMH levels declineprogressively with age, becoming undetectable near the menopause (Sowers MR, et al.,2008; van Rooij IA, et al., 2004; van Rooij IA, et al., 2005).10.1 Oocyte and ovarian tissue cryopreservationEach year, cancer occurs in approximately 100 per 100,000 women under age 50 in theUnited States. Chemotherapy and radiation therapy for malignant and non-malignantsystemic disease very often results in ovarian failure. Women with cancer and other seriousillnesses requiring treatments that pose a serious threat to their future fertility haverelatively few options. In some cases, the ovaries may be moved out of the radiation field.Treatment with GnRH agonists has been suggested as a way to protect the gonads from theinsult of chemotherapy, but there is no convincing evidence for its efficacy. Althoughembryo banking is effective, the time required for stimulation and retrieval are oftenprohibitive. With recent advances in cryobiology, oocyte and ovarian tissuecryopreservation hold promise as methods to preserve reproductive potential (Shaw JM, etal., 2000).10.2 Oocyte cryopreservationAlthough the first pregnancy resulting from oocyte cryopreservation was reported in 1986,(Chen C, 1986), success rates achieved with the technology were historically very low, andonly recently improving. The primary obstacle was the poor survival of oocytes, which arefragile due to their size, high water content, and chromosomal arrangement; the meioticspindle is easily damaged by intracellular ice formation during freezing or thawing (ShawJM, et al., 2000). Germinal vesicle stage oocytes are hardier, (Boiso I, et al., 2002), butprogress with in vitro maturation of immature oocytes has been slow. Another obstacle washardening of the zona pellucida, which interfered with normal fertilization. The improvedsurvival of cryopreserved oocytes today relates primarily to modifications in the sucroseand sodium concentrations in traditional “slow-freeze” protocols, (Fabbri R, et al., 2001;Stachecki JJ & Willadsen SM, 2000; Bianchi V, et al., 2007; De Santis L, et al., 2007), changes.,

284Artificial Insemination in Farm Animalsin the initial temperature of the cryoprotectant, and-seeding temperature (Trad FS, et al.,1999). Survival rates have been further improved with vitrification, a technique that useshigh concentrations of cryoprotectant and rapid freezing by immersion in liquid nitrogen,preserving oocytes in a solid glass-like state without ice formation (Loutradi KE, et al., 2008;Oktay K, et al., 1997). With the use of intra-cytoplasmic sperm injection (ICSI), the hardenedzona is not a barrier to fertilization (Polak de Fried E, et al., 1998). Survival, fertilization, andpregnancy rates achieved with cryopreserved oocytes are rap-idly improving andapproaching those achieved with fresh oocytes (Grifo JA, & Noyes N, 2010; Nagy ZP, et al.,2009). A randomized comparison of results achieved with slow-freeze and vitrificationobserved that vitrification resulted in better oocyte survival (81% vs. 67%), fertilization (77%vs. 67%), and clinical pregnancy rates per thawed oocyte (5.2% vs. 1.7%).A study examining outcomes achieved with vitrified donor oocytes observed 87% thawsurvival, 87% fertilization, and 68% blastocyst formation, with 15/20 recipients (75%)achieving pregnancy after embryo transfer (Cobo A, Kuwayama M, Perez S, et al). Anotherusing both slow-frozen and vitrified oocytes observed 92% survival, 79% fertilization, 42%implantation, and a 57% on going pregnancy rate (Grifo JA & Noyes N, 2010). Although thenumber of pregnancies and deliveries resulting from oocyte cryopreservation is stillsomewhat small, the number is rapidly increasing, and early perinatal outcomes data arereassuring. The incidence of chromosomal abnormalities in human embryos derived fromcryopreserved oocytes is no different from that observed in control embryos derived fromfresh oocytes (Gook DA, et al., 1994; Cobo A, et al., 2001). A study comparing outcomes in200 infants derived from vitrified oocytes and in infants resulting from conventional freshIVF found no differences in birth weight or in the incidence of birth defects (Chian RC, et al.,2008). A review of over 900 live births resulting from IVF of cryopreserved oocytes alsoobserved no increase in the prevalence of congenital anomalies compared to that in thegeneral population (Noyes N, et al., 2009). Oocyte cryopreservation is a viable fertilitypreservation strategy for women without partners seeking to preserve their fertility.Unfortunately few cancer patients have sufficient time to undergo ovarian stimulationbefore their treatment begins. The technology also holds enormous promise as a means tosimplify oocyte donation, via egg banking, and is rapidly emerging as an elective fertilitypreservation strategy for women anticipating delayed childbearing and concerned abouttheir future fertility. Currently, elective oocyte cryopreservation to defer reproductive agingis controversial, primarily because the great majority of outcomes data have come fromexperience with cryopreserved oocytes obtained from healthy young oocyte donors andcannot be extrapolated to older women who represent the majority of those expressinginterest in elective oocyte cryopreservation (Rybak EA & Lieman HJ 2009; ASRM PracticeCommittee). However, when age-stratified outcomes data become available, allowingwomen to be accurately informed about their prognosis for success, elective oocytecryopreservation may realistically offer women the means to set their “biological clock.”10.2.1 Ovarian tissue cryopreservationAt least in theory, ovarian tissue cryopreservation offers the means to freeze thousands ofprimordial follicles for later in vitro maturation or to store tissue for xenografting into ananimal host or later auto transplantation (Jeruss JS, Woodruff TK., 2009). Currently,autologous transplantation of ovarian tissue seems the most practical and effective approach

Reproductive Endocrinology Diseases: Hormone Replacement and Therapy for Peri/Menopause 285because the technique has successfully restored fertility to women with ovarian failureresulting from cancer chemotherapy (Andersen CY,, et al., 2008; Demeestere I, 2006,2010; etal; Silber SJ 2009). Ovarian tissue is removed surgically via laparoscopy or laparotomy andfrozen using either a slow-cool or vitrification technique, before the insult expected to resultin ovarian failure. Later, it can be thawed and transplanted back into the patient in or nearits original location (orthotopic transplantation) or to another site, such as the forearm orabdominal wall (heterotopic transplantation). The advantage of orthotopic transplantation isthat pregnancy might be achieved without assistance, whereas heterotopic transplantationrequires IVF (Jeruss JS & Woodruff TK. 2009). Live births have been achieved aftertransplantation of frozen-thawed ovarian tissue in sheep, (Candy CJ et al., 2000) and the firstlive birth in a primate after a fresh heterotopic ovarian transplantation has been reported(Lee DM, et al). Human oocytes have been obtained from heterotopic transplants andfertilized in vitro to yield embryos for transfer, resulting in a biochemical pregnancy(Rosendahl M, et al., 2006). The only human pregnancy achieved after heterotopictransplantation was achieved without assistance, indicating that the oocyte from which itarose came from the patient’s existing ovary rather than from the transplant. Orthotopictransplantation has been successfully achieved in humans.A number of live births have been reported after autologous orthotopic transplantation ofcryopreserved ovarian tissue. Frozen ovarian tissue also has been transplanted successfullybetween monozygotic twin sisters after the receiving twin developed premature ovarianfailure (Silber SJ, & Gosden RG, 2007). A 2008 systematic review identified 25 reportsdescribing a total of 46 cases of ovarian tissue transplantation for treatment of prematureovarian failure or infertility, although most involved transplantation of fresh rather thanfrozen ovarian tissue (Bedaiwy MA, et al., 2008). The mean time to return of ovarianfunction was 120 days (range 60–244 days) and data were insufficient to evaluate functionbeyond 1 year. Fresh grafts were more likely to succeed, and in 25 women who soughtpregnancy, eight conceived nine pregnancies. At least one potential risk of ovarian tissuecryopreservation and auto-transplantation is reseeding of tumor cells in women withmalignancies. Future research focusing on defining patient suitability, tissue collectionmethods, and cryopreservation protocols is certainly warranted, but until effectivetechniques and the possibility for success can be defined, ovarian tissue cryopreservationwill remain investigational and cannot be justified solely for the purpose of future use inhealthy women.11. ReferencesAboulghar MA, Mansour RT, Serour GA, Amin YM, Sattar MA, Ramzy AM, In vitrofertilization in a spontaneous cycle: a successful simple protocol, J Obstet Gynaecol(Tokyo 1995) 21:337, 1995.Adhikari D, Liu K, Molecular mechanisms underlying the activation of mammalianprimordial follicles, Endocr Rev; 30:438, 2009.Ahmed-Ebbiary NA, Lenton EA, Cooke ID: Hypothalamic-pituitary ageing: progressiveincreases in FSH and LH concentrations throughout the reproductive life inregularly menstruating women. Clin Endocrinol; 41:199-206. 1994.Akman MA, Erden HF, Tosun SB, Bayazit N, Aksoy E, Bahceci M, Comparison of agonisticfl are-up-protocol and antagonistic multiple dose protocol in ovarian stimulation of

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