REVIEW Changes in Equine Reproduction: Have They Been Good or Bad for the Horse Industry? E.L. Squires, MS, PhD, ACT (Hon) ABSTRACT In the past four decades there have been tremendous changes in equine reproduction. Most breeds now allow the use of artificial insemination with fresh, cooled and frozen semen. Artificial insemination has many advantages for the breeder, in particular the control of bacteria through the use of semen extenders containing antibiotics. Deposition of sperm in small volumes onto the uterotubal junction has allowed the use of relatively low numbers of sperm. Intracytoplasmic injection of sperm into oocytes allows older, subfertile stallions to be used as breeding stallions. Advances in mare reproduction have included developing tools for hastening the onset of the breeding season. Other advances include embryo transfer, oocyte collection and transfer, and cloning. The acceptance of reproductive technology depends on the success of the technology, the attitude of the breeders/veterinarians, and the cost/benefit ratio to the industry and breed registry. Keywords: Mare; Stallion; Technology; Semen; Embryo INTRODUCTION This is one man’s view and opinion regarding changes that have occurred in equine reproduction since the 1970s. Obviously others will have a different perception of these changes and particularly will have different views as to whether these changes have been good or bad for the equine industry. There is no doubt that in the last four decades there have been tremendous advances and discoveries in equine reproduction. A lot of the studies in the 1970s and 1980s were essentially whole animal studies, but more recently, studies have looked at cellular mechanisms of the reproductive process. The new generation of equine scientists must certainly be knowledgeable and skilled in molecular biology to provide new information. Ideally From the University of Kentucky, Gluck Equine Research Foundation, Lexington, KY. Reprint requests: E. L. Squires, MS, PhD, ACT (Hon), University of Kentucky, Gluck Equine Research Foundation, 108 Gluck Equine Research Center, Lexington, KY 40546-0099. 0737-0806/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2009.04.184 268 researchers should have these new molecular techniques as well as an appreciation for the whole horse. The acceptance of reproductive technology depends on the success of the technology, the attitude of the breeders and veterinarians, and the cost/benefit ratio to the industry and the breed associations. Some breed associations such as the Thoroughbred industry, for example, have accepted very few of the reproductive technologies that have been developed in the last 20 years and thus have changed their breeding habits very little over the years. In contrast, the Quarter Horse industry has allowed nearly all of the new technologies to be incorporated into their breeding practices and have changed their ways of breeding mares and handling semen dramatically. STALLIONS AND SEMEN Most breeds allow the use of artificial insemination with fresh, cooled, stored semen, and some of the breeds allow the use of frozen/thawed semen. The one exception is the Thoroughbred industry, which allows only natural mating. Obviously, this breed has avoided the ‘‘slippery slope’’ of technology. Presented in Table 1 is the number of foals registered per year by breed. As one can see, the American Quarter Horse Association is the largest breed, followed by the Paints and the Thoroughbred industry. By opposing the use of artificial insemination, the Thoroughbred industry had hoped to limit the number of mares that are bred to a given stallion and thus control the production of foals. In years past, a full book of mares to a Thoroughbred stallion would have been 50 to 60; however, by breeding stallions three to four times a day, the number of mares bred to some Thoroughbred stallions has increased to well above 100 mares and sometimes 200 mares per year. This obviously puts stress on the stallion, requiring him to mate several mares in a given day throughout the breeding season. In addition, many of these stallions are bred during the breeding season in North America and then shipped to Australia, New Zealand, or South America. One of the major limiting factors as to how many mares a stallion can breed in a given day is his sex drive. Generally stallions with normal-sized testicles have the ability to provide enough sperm to impregnate several mares a day, but their willingness to breed multiple times each day throughout the breeding season is sometimes limited. Studies are needed to define the factors Journal of Equine Veterinary Science  Vol 29, No 5 (2009) EL Squires  Vol 29, No 5 (2009) 269 Table 1. Number of Foals Registered in Major Breeds per Year AQHA Paint Horse Thoroughbred Miniature Standardbred Walking Horses Arabians 165,000 55,000 45,000 20,000 16,000 12,000 8,000 Table 2. Method of Breeding Artificially (%) Fresh Cooled Frozen 50 60 30 45 5 10 that control sexual behavior and to develop schemes of enhancing sexual behavior. It is my estimate that, of mares bred artificially, 50% to 60% are bred with fresh semen that is collected on the farm and inseminated into the mares within 1 hour, 30% to 40% are inseminated with cooled transported semen in which mares are inseminated generally 24 hours after collection, and 5% to 10% of mares are inseminated with frozen/thawed semen (Table 2). Pregnancy rates per cycle with fresh semen are in the range of 65% to 70% per cycle; with cooled semen, 50% to 60% per cycle; and with frozen semen, 40% to 50% per cycle (Table 3). Certainly one of the major changes in the horse industry is the widespread acceptance of cooled transported semen. Each year the number of mares bred with cooled transported semen increases slightly. The major advantage of this technology is that the mare remains at home; thus, there is less possibility of injury or disease to the mare or foal. Furthermore, there is access to a greater number of stallions. The major disadvantage of cooled semen is the increased cost for veterinary care in preparing the mare for breeding, the cost of collecting and shipping semen, the reduced fertility of some stallions, and the limited lifespan of sperm to approximately 24 to 48 hours. There is certainly an opportunity for scientists to develop semen extenders that would maintain the viability of equine sperm for longer than 48 hours. I have often asked breeders and veterinarians, if one could have equal fertility with frozen semen and cooled semen, which would they prefer? In essentially all cases, the breeder would prefer frozen semen, because of the increased advantages attributed to frozen semen. The added advantage is that the semen can be ordered many days in advance and stored at the farm, allowing the mare to be bred at the optimal time for maximum fertility. Theoretically, the semen could be shipped only one time in the breeding season, many days in advance of when the mare needs to be bred. Frozen semen, if maintained in liquid nitrogen, will last essentially indefinitely. Thus, frozen semen has the advantage of being able to be shipped both nationally and internationally. This potentially can result in increased sales of stud fees and an increase in the number of mares bred to a given stallion. Obviously, the disadvantage is the slightly lower pregnancy rate for frozen semen and the fact that not all semen from all stallions will freeze. Other disadvantages include the increased veterinary costs for mare management and the poor correlation between sperm motility and fertility with frozen/thawed semen. The quality of frozen/thawed semen varies depending on the technician that is involved in the production and handling of the frozen semen. Thirty percent of stallions have semen with a freezability rating of ‘‘good,’’ 40% of stallions have semen that freezes ‘‘satisfactorily,’’ and 30% of stallions have semen that freezes ‘‘very poorly.’’1 Studies have been conducted to identify components of the seminal plasma or sperm that may be useful in predicting the freezability of stallion semen.2 Areas of potential research with frozen/thawed semen include determining biomarkers for fertility, developing cheaper devices for shipping semen, developing improved extenders, and developing tests for assessment of sperm damage. To develop a strategy for minimizing the amount of veterinary care needed to inseminate a mare with frozen/thawed semen, Loomis and Squires3 developed a fixed-time breeding schedule for insemination of mares (Fig. 1). Once mares have acquired a large preovulatory follicle, they are administered an ovulatory agent such as human chorionic gonadotropin (hCG). They are then bred with either a full dose or a half dose of frozen/thawed sperm 24 hours after hCG administration. A second dose is inseminated 40 hours after hCG administration. With this timed insemination protocol, any mare that ovulates between 18 and 52 hours after hCG administration will have semen in her uterus 0 to 12 hours before ovulation or up to 6 hours after ovulation. This has been shown to be the window of maximum fertility with frozen/thawed semen.4 I would argue that artificial insemination with fresh, cooled, or frozen/thawed semen has been a good thing for the horse industry. Artificial insemination has allowed the breeder or owner to select from a wider range of stallions, thus potentially increasing the genetic pool. It has also provided the opportunity to breed mares to stallions not just in the United States, but around the world. It would also appear that artificial insemination using appropriate extenders with antibiotics may be a way of controlling venereal disease. This was recently demonstrated in the outbreak of contagious equine metritis (CEM) in 2008. Because mares were inseminated with extended semen, only 3 of 527 mares were shown to be positive for the CEM bacteria. If these mares had been bred by 270 EL Squires  Vol 29, No 5 (2009) Table 3. Pregnancy Rates/Cycle (%) Fresh Cooled Frozen 65 70 50 60 40 50 natural mating, it is likely that a much higher percentage of them would be positive for CEM. One of the more recent trends in artificial insemination is the use of much lower sperm numbers, that is, low-dose insemination.5 Instead of inseminating a half billion or one billion spermatozoa into the body of the uterus, there are many breeders and veterinarians who are now inseminating anywhere from 5 to 100 million sperm at the tip of the uterine horn in the area of the uterotubal junction (UTJ). This is done in an attempt to minimize the amount of semen that is used, particularly from older stallions that have limited sperm, and frozen/thawed semen from stallions that have died. Another use is poor-quality semen that has been centrifuged through a gradient to enrich the population of morphologically normal sperm.6 Unfortunately, this centrifugation procedure generally results in a low number of sperm recovered and thus a limited number for insemination. It is now fairly common for mares to be inseminated with flexible catheters that allow the technician or veterinarian to deposit the semen on the tip of the UTJ. The only possible disadvantage of this is that some breeders have automatically assumed that they can lower the number of frozen/thawed sperm used for insemination without knowing the fertility of the stallion before insemination. Other trends include the increased use of intracytoplasmic sperm injection for obtaining pregnancy from a subfertile stallion.7,8 Oocytes are retrieved from the donor mare 24 hours after hCG administration. Those oocytes are then injected with one sperm inducing fertilization. Approximately 65% to 70% of the oocytes that are injected fertilize, and if these fertilized embryos are transferred, approximately 50% of those result in a pregnancy. This has allowed continual production from stallions that have very low sperm numbers or stallions that have died and have limited frozen/thawed sperm available. Potential disadvantages are the high cost of sperm injection and the apparent increased embryonic loss. Furthermore, questions remain as to the viability and economic value of these foals, as well as the question of whether we are perpetuating infertility by using such techniques. In the last several years, the number of cows inseminated with sex-sorted frozen/thawed bull sperm has increased dramatically. Nearly 8 million doses of sex-sorted frozenthawed bull sperm will be used for insemination of cows in 2009.9 Although foals have been produced from sexsorted frozen/thawed stallion sperm, the fertility was extremely low.10 Use of sex-sorted sperm in the horse industry is extremely limited because only fresh or cooled semen can be used. Valuable stallions are too busy in the breeding season to provide ejaculates for sex sorting. The procedure does not appear to work on all stallions and requires that the stallion, mare, and sorting equipment be in near proximity for sexed semen to be incorporated in the horse industry. Semen needs to be collected in the fall, sorted, and frozen, and result in reasonable fertility when inseminated before sexed semen is routinely used. This is certainly an opportunity for scientists to improve frozen/thawed sexed semen and potentially develop other cheaper methods for sexing semen. MARES The January 1 calendar date for registering foals has forced the breeder to initiate the breeding season by February 15 in order to obtain foals born early in the year. Use of an extended photoperiod is the primary means of hastening the first ovulation of the year. Studies would indicate that adding the light in the afternoon and evening is much more effective than providing light in the morning. Providing additional lighting for several months in the springtime is quite costly and labor intensive. There certainly is a need to develop other methods of hastening the first ovulation of the year. Although many hormonal treatments have been attempted, nothing practical or inexpensive has been developed for replacing the use of artificial lights. Mares experience a transition period during the months of February, March, and early April in which they experience long, erratic estrous periods not always accompanied by ovulation. Progestin treatments (oral Altrenogest or injectable progesterone) are commonly used as a means of synchronizing the first ovulation of the year.11 In the Thoroughbred industry, the breeders and veterinarians call this ‘‘programming the mare’’ for natural mating. This involves placing the mare under lights December 1 and then exposing the mare to oral Altrenogest or injectable progesterone for 10 to 20 days after 60 days of artificial light. Once the mare is taken off the progesterone treatment, they can be scheduled for breeding in approximately 7 to 10 days. Most breeding strategies have incorporated the use of progestins, prostaglandins, and some sort of an ovulatory agent.12 Progestins are used for estrous suppression, estrous synchronization, maintenance of pregnancy, high-risk pregnancy, embryo transfer, postsurgery, and during stressful situations.13 Progestins can be administered orally (Altrenogest) or given as daily injections or one injection every 7 to 10 days using a slow-release formulation.14 Prostaglandins are used for induction of estrous, estrous synchronization, treatment of a mare with EL Squires  Vol 29, No 5 (2009) 271 First AI Second AI HCG 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 Hrs after injection Mares ovulating during this period will have been inseminated within 6 hours after or within 12 hours prior to ovulation. Figure. 1. Timed insemination protocol. a maintained corpus luteum, evacuation of uterine fluid, induction of abortion, and to hasten ovulation.13 Most mares are administered some hormonal agent to more accurately time breeding or insemination with ovulation. The two main products used for induction of ovulation in mares are the gonadotropin-releasing hormone (GnRH) agonist (deslorelin) and hCG. Human chorionic gonadotropin is a human hormone that, if injected several times in a breeding season, can cause mares to become unresponsive.13 In addition, mares older than 16 years of age have been shown to be insensitive to hCG. Therefore, the trend is for most mares to receive the GnRH agonist deslorelin as a means of inducing ovulation. One of the commonly asked questions is: Are we using too many hormones that may ultimately be detrimental to the health of the mare? Recent evidence would suggest that the administration of prostaglandins or hCG increases the incidence of twins.15 It is also of some concern that the treatment of mares with altrenogest during gestation may result in a population of broodmares that are dependent on altrenogest for production of a live foal. Hormones are essential breeding managements tools but should be used with discretion. The advent of the use of ultrasonography for evaluation of the mare’s genitalia certainly revolutionized the breeding industry. This has become a powerful tool for evaluating follicular development, ovulation, corpus lutea development, uterine health, and detection of pregnancy.16 More recent advances have been made in Doppler ultrasound for evaluation of blood flow to the reproductive organs.17 One controversial technique that has been developed and ingrained in the horse industry is embryo transfer. The acceptance of embryo transfer by major breed associations has resulted in a major change in management of broodmares. Initially the technique was accepted for mares that had reproductive problems or could not carry a foal to term and was restricted to mares who were 16 years of age or older. This was quickly changed, and most breed registries have no restrictions on the age or reproductive status of the mare that is enrolled in an embryo transfer program. In addition, one of the major changes was the unlimited foal registrations from embryo transfers. This allows multiple foals to be registered from a given donor mare in 1 year. There are those that would argue that multiple foal registration allows a valuable genetic mare to dominate the breed. This seems like a nonsensical argument because stallions are allowed to impregnate several hundred mares per year, and a mare may have only three or four foals per year and possibly 30 to 40 in her lifetime. Techniques have been devised for cooling and storing an embryo for 24 hours without a reduction in viability.18 This allows embryos to be collected on one farm and shipped across the country to a centralized recipient station. More recently, studies have demonstrated that embryos can be frozen and thawed without a major reduction in viability.19 This opens the possibility for embryos to be exported around the world, similar to what is done with cattle embryos. There is no question that embryo transfer has impacted the economics of the horse industry. If one overproduces from a given mare, more than likely the price of the foals will decrease. However, in contrast, if one has a very valuable mare that can be used as an embryo donor, then the price of that mare can increase dramatically. Embryo transfer in a horse is a relatively inefficient process compared with that in cattle. Although superovulation can be induced in mares, generally only approximately two embryos per donor are collected after superovulation compared with approximately six transferable embryos coming from a donor cow.20 Furthermore, there are currently no superovulatory drugs commercially available for superovulation in the mare. In the past 10 years, there has been an explosion in development of techniques for assisted reproduction in mares.7,8 Techniques include oocyte collection and transfer and intracytoplasmic sperm injection. Oocyte 272 collection has the advantage of allowing one to go directly to the ovary, collect the oocyte, and transfer the oocyte into a healthy inseminated mare.21 Most of the mares that end up in an oocyte program are those that can no longer provide an embryo into the uterus because of many acquired reproductive problems. This might include oviductal pathologic conditions, cervical adhesions or tears, and chronic uterine infection. Oocyte retrieval from older mares can be as high as 80%, and those oocytes, when transferred into young recipients, can result in a 40% to 50% pregnancy rate.21 There are some that would praise this technology as a means of obtaining foals from older valuable subfertile mares. Others would say that this type of technology is being used to propagate subfertility. One other controversial technique is intracytoplasmic sperm injection (ICSI). This is generally a treatment for a subfertile stallion, whereas oocyte transfer is a treatment for the subfertile mare. Stallions that have undergone testicular degeneration because of age or stallions that have died, leaving a limited supply of frozen semen, can be used in an ICSI program. Oocytes are obtained from both normal and subfertile mares and then a single sperm is injected into the oocyte to induce fertilization. These oocytes are then transferred at the two-cell stage into the oviduct of the recipient or, in some recent cases, the fertilized egg is incubated and transferred nonsurgically once it has become an embryo.21 The success of sperm injection is similar to that of oocyte transfer. Both techniques are too new to determine the long-term effects. Our initial impression is that the foals that are born from these advanced reproductive technologies are quite normal, with no higher incidence of developmental problems. One of the most controversial techniques that has been developed in the last few years is that of cloning. The first successful clone of equids was a mule born in May 2003 at the University of Idaho.22 The first cloned horse foal was born in 2003 in Italy.23 Since that time, there have been numerous clones that have been born and, according to Viagen (the only commercial company), there will be approximately 50 cloned foals born in 2009. The American Quarter Horse Association is seriously considering whether to register cloned foals. There are those that would argue that this is just one more step in technology that should be used in the horse industry. Others would say that this technology is not needed, and cloning is only a mechanism of helping the rich that have extremely valuable horses. Regardless of one’s stance, it is certain that a clone is not an identical replacement for the donor. Even though horses may have identical DNA, there will be differences in gene expression between the donor and the clone. Female clones will inherit some mitochondrial DNA from the oocyte donor, and because all clones are being carried by recipients, EL Squires  Vol 29, No 5 (2009) there will be some environmental influence from the recipient. Thus, is it likely that using cloning to replace a great athlete will result in disappointment. However, using cloning to produce a stallion from a gelding or to replace a stallion may be of some value. Some would argue that this takes the challenge out of horse breeding and that cloning a stallion over and over would result in narrowing the gene pool and the population from a specific genetic line. This would be detrimental if the cloned stallion was a carrier for genetic diseases. Similar to any other technology, those that have been developed for the breeding industry can either be extremely helpful or can be abused and result in detrimental effects. In general, I think that most of the reproductive technologies that have been developed in the past several decades have been beneficial to the horse industry. However, only time will tell us how detrimental or beneficial these techniques have been. Sponsored by: Colorado State University Animal Reproduction and Biotechnology Laboratory, Ft. Collins, CO. REFERENCES 1. Vidament M. French field results (1985–2005) on factors affecting fertility results of frozen stallion semen. Anim Reprod Sci 2005;89: 115–136. 2. Zahn FS, Papa FO, Melo CM. Blood serum, seminal plasma and sperm membrane protein profiles in stallions: are they correlated to semen freezability? Anim Reprod Sci 2006;94:64–66. 3. Loomis PR, Squires EL. Frozen semen management and equine breeding program. Theriogenology 2004;64:480–491. 4. Squires E, Barbacini S, Matthews P, Byers W, Schwenzer K, Steiner J, et al. Retrospective study of factors affecting fertility of fresh, cooled and frozen semen. Equine Vet Educ 2006;18:296–299. 5. Brinsko S. Insemination doses: how low can we go? Theriogenology 2006;66:543–550. 6. Varner DD. Developments in stallion semen evaluation. Theriogenology 2008;70:448–462. 7. Squires EL. Integration of future biotechnologies into the equine industry. Anim Reprod Sci 2005;89:187–198. 8. Carnevale EM, Maclellan LJ. Collection, evaluation, and use of oocytes in equine assisted reproduction. Vet Clin North Am Equine Pract 2006;22:843–856. 9. Hutchison JL, Norman HD. Characterization and usage of sexed semen from US field data. Theriogenology 2009;71:48. 10. Lindsey AC, Schenk JL, Graham JK, Bruemmer JE, Squires EL. Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa. Equine Vet J 2002;34:121–127. 11. Squires EL, Shideler RK, Voss JL, Webel SK. Clinical applications of progestin in mares: Compendium on Continuing Education for the Practicing Veterinarian 1983;5:S16–S22. EL Squires  Vol 29, No 5 (2009) 273 12. Blanchard TL, Varner DD, Burns PJ, Everett KA, Brinsko SP, 18. Carnevale EM, Squires EL, McKinnon AO. Comparison of Ham’s Boehnke L. Regulation of estrus and ovulation in mares with proges- F10 with CO2 or Hepes buffer for storage of equine embryos at 5 terone or progesterone and estradiol biodegradable microspheres with or without PGF(2)alpha. Theriogenology 1992;38:1091– 1106. C for 24 H. J Anim Sci 1987;65:1775–1781. 19. Hudson J, McCue PM, Carnevale EM, Welch S, Squires EL. The effects of cooling and vitrification of embryos from mares treated with 13. Squires E. Hormonal manipulation of the mare: a review. J Equine equine follicle-stimulating hormone on pregnancy rates after nonsur- Vet Sci 2008;28:627–634. 14. Storer WA, Thompson DL Jr, Gilley RM, Burns PJ. Evaluation of gical transfer. J Equine Vet Sci 2006;26:51–54. 20. Squires EL, McCue PM. Superovulation in mares. Anim Reprod Sci injectable sustained release progestin formulations for suppression of estrus and ovulation in mares. J Equine Vet Sci 2009;29: 33–36. 15. Ginther OJ. Increased frequency of double ovulation following induction of luteolysis with exogenous prostaglandin Fa. J Equine Vet Sci 2009; In press. 2007;99:1–8. 21. Carnevale EM, Coutinho da Silva MA, Panzani D, Stokes JE, Squires EL. Factors affecting the success of oocyte transfer in a clinical program for subfertile mares. Theriogenology 2005;64:519–527. 22. Vanderwall DK, Woods GL, Roser JF, Schlafer DH, Sellon DC, Tester DF, et al. Equine cloning: applications and outcomes. Reprod 16. Ginther OJ, ed. Ultrasonic imaging and animal reproduction: horses. Book 2. Cross Plains, WI: Equiservices Publishing; 1998. Fertil Dev 2006;18:91–98. 23. Lagutina I, Lazzari G, Duchi R, Colleoni S, Ponderato N, Turini P, 17. Ginther OJ, ed. Ultrasonic imaging and animal reproduction: color et al. Somatic cell nuclear transfer in horses: effect of oocyte morphol- doppler ultrasonagraphy. Book 4. Cross Plains, WI: Equiservices ogy, embryo reconstruction method and donor cell type. 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