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Estrous Synchronization
CHAPTER
3
Estrous Synchronization E T TA A G A N B R A D E C A M P
A
s a prelude to this chapter, it is necessary to make a few comments about estrous synchronization as it pertains to the mare and how this varies from estrous synchronization in the cow. Practitioners who are familiar with estrous synchronization in cattle and the number of different synchronization programs available must realize that estrous synchronization in the mare is much more relative and does not yield as reliable results as estrous synchronization in cattle. In the bovine industry a large number of cows can be synchronized to reliably display estrus and ovulate within a narrow window of time, and there are several programs described to achieve the desired results. Unfortunately, the mare’s reproductive cycle does not lend itself to such a tight synchronization among a group of mares, and currently there is only one protocol that consistently provides reliable results. The length of estrus in the mare is 5 to 7 days, and the time of ovulation relative to the end of estrus varies with ovulation occurring on the last 2 days of estrus in 69% of mares and after the end of estrus in 14% of mares.1 These factors make the development of a synchronization program that allows for a single breeding at a predetermined time difficult. In equine reproduction the most common use of estrous synchronization is in embryo transfer programs. If a large recipient herd providing mares that have ovulated in synchrony with the donor is not available, then a small group of recipient mares must be synchronized with the donor mare. To be suitable recipients, these mares must ovulate from 1 day before to 3 days after the date of ovulation of the donor mare. There is even one report in the literature that recipient mares that ovulated up to 5 days after the donor have been used successfully in a large embryo transfer program.2 A second less common use of estrous synchronization in the mare is to synchronize a group of mares for breeding due to limited availability of a stallion, for example due to a show schedule. In addition to synchronization of estrus in a group of mares, pharmacologic agents can be used to induce ovulation to allow for the synchronization of ovulation. When breeding with both frozen and cooled shipped semen it is often desirable to use only one dose of semen, and therefore it is essential that the ovulation be synchronized within 36 to 48 hours of breeding. This is also important where natural service of mares is performed and the stallion has a full book of mares, thereby limiting the number of times a mare can be bred during a cycle. There are four basic methods used to achieve estrous synchronization in the mare: termination of the luteal 22
phase, lengthening of the luteal phase, induction of ovulation, and inhibition of the follicular phase. The pharmacologic agents most commonly used to accomplish these methods are prostaglandins, progestins, human chorionic gonadotropin (hCG) (Chorulon, Intervet Inc., Millsboro, Del.) or deslorelin acetate (Ovuplant, Peptech Limited, NSW, Australia), and estradiol-17b. The most reliable and successful estrous synchronization programs are achieved when a combination of these methods and pharmacologic agents are used. Prostaglandin F2a (PGF2a) is used to terminate the luteal phase by causing lysis of the corpus luteum (CL), thereby allowing the return to estrus. The most common prostaglandins used are dinoprost (Lutalyse, Pfizer, New York) and cloprostenol (Estrumate, Schering-Plough Animal Health Corporation, Union, N.J.). Dinoprost is a naturally occurring PGF2a, and cloprostenol is a synthetic prostaglandin analogue. For prostaglandins to effectively terminate the luteal phase a mature CL must be present that is responsive to prostaglandin. The equine CL exhibits incomplete sensitivity to PGF2a until approximately day 5 postovulation in most mares.3 The standard recommended dose of dinoprost is 9 mg/kg (5 to 10 mg per 1000 lb/454 kg horse). The recommended dose of cloprostenol is 250 mg per 1000 lb/454 kg horse (0.55 mg/kg). More recent research has shown that much lower doses of prostaglandin are just as effective at lysing the CL with fewer negative side effects. Douglas and Ginther demonstrated that doses of prostaglandin as low as 1.25 mg were as effective at inducing luteolysis as more standard doses.4 Nie et al showed that a microdose of cloprostenol (25 mg) was just as effective at lysing the equine CL as the larger standard dose.5 On average, mares return to estrus 5 to 7 days after administration of prostaglandin in the presence of a mature CL, and ovulation occurs 9 to 11 days after administration. However, because the follicular phase plays a greater role in controlling the total length of the estrous cycle in the mare, the length of time from administration of prostaglandin to onset of estrus and ovulation can be quite variable depending on the size and status of follicles present on the ovaries at the time of administration. The time of ovulation after PGF2a-induced estrus may vary as much as 2 to 15 days post treatment.6 If prostaglandin is administered when there is a large follicle present, the mare may return to estrus and ovulate in 2 to 3 days. Some of these mares never develop overt signs of standing heat, endometrial folds, or relaxation of the cervix before ovulation. If a large follicle destined for atresia is present on the ovary at the time of prostaglandin administration, the follicle may not
Estrous Synchronization ovulate and instead regress as a new wave of follicles develops.7 In cattle the administration of two doses of prostaglandin 11 to 12 days apart results in approximately 80% exhibiting estrus within 2 to 5 days after the second injection. Although the administration of two doses of prostaglandin 14 days apart has been described in the literature as a protocol for estrous synchronization in mares, it is not a very reliable method of ensuring that a recipient mare will be synchronized with a donor.6 Research has shown that after two injections of prostaglandin, on average ovulation occurs between 7 and 10 days after the second injection,6,8,9 with ovulation potentially occurring anywhere between 0 and 17 days.8,9 In a review of the literature it was stated that when this protocol was administered, one would need to treat at least 10 recipient mares to have an 80% chance that at least 1 recipient would ovulate within 24 hours of the donor.10 Obviously, this is not ideal, especially when in most small embryo transfer programs there are only a few mares available to be used as recipients. Due to the large variation in time of ovulation from administration of prostaglandin, it is not very useful when used alone in the synchronization of estrous cycles in mares. However, if examination via transrectal ultrasonography is performed first to eliminate those mares that will not respond appropriately to the administration of prostaglandin, tightening of date of ovulation can be achieved with this protocol. Exogenous progestins are commonly used in estrous synchronization programs to lengthen the luteal phase. The two most common forms of progestins used are progesterone in oil and altrenogest (allyl trenbolone, Regumate, Intervet Inc., Millsboro, Del.), a synthetic progestin. Progesterone or a synthetic progestin administered alone or in combination with estradiol-17b will effectively lengthen the luteal phase indefinitely until their administration is discontinued. Progesterone has an inhibitory effect on LH release from the anterior pituitary; therefore the rationale is if it is administered for a long enough period of time (15 to 18 days), the CL will regress and the only source of progesterone to suppress estrus is the exogenous progesterone. However, even the endogenous level of progesterone present during the midluteal phase is not sufficient to suppress gonadotropin release, follicular development, and ovulation.11 Studies have also shown that the synthetic progestin, altrenogest, administered at 0.044 mg/kg orally once a day fails to suppress follicular development.12 When using progestins for estrous synchronization it is recommended that either altrenogest (0.044 mg/kg PO q24h) or progesterone in oil (150 mg/day IM) be administered for 10 days combined with the administration of PGF2a on the last day of progestin therapy to lyse any CLs present. To improve estrous synchronization in randomly cycling mares, a combination of progesterone and estrogen, rather than progesterone alone, is administered. The combined effect of the two hormones provides a more profound negative feedback on gonadotropin release than does progesterone alone, which results in a more uniform inhibition of follicular development. When the exogenous progesterone/estrogen therapy is discontin-
23
ued, there is less diversity in follicular maturation and the date ovulation occurs can be more closely synchronized. The recommended protocol involves intramuscular injections of progesterone (150 mg) combined with estradiol-17b (10 mg) in oil for 10 consecutive days with a single injection of PGF2a administered on the tenth day of treatment. To further tighten the synchronization of ovulation, hCG (5 IU/kg IV) or deslorelin acetate is administered when a 35-mm follicle is present. With this protocol, greater than 70% of mares ovulate between 10 and 12 days after the last steroid injection with a range of ovulations occurring from 8 to 17 days. Currently, there is no commercial preparation containing progesterone and estradiol-17b combined; therefore this formulation must be ordered from a compounding pharmacy.13 Induction of ovulation is also an important part of an estrous synchronization program. The two most common pharmacologic agents used to induce ovulation are hCG and deslorelin acetate. Human chorionic gonadotropin is a protein consisting of two peptide chains that, although chemically different from pituitary luteinizing hormone (LH), has primarily luteinizing-like activity. hCG is produced by cytotrophoblasts of the chorionic villi of the human placenta and appears in the urine of pregnant women a few weeks after conception. Deslorelin acetate is a potent synthetic gonadotropinreleasing hormone (GnRH) analogue. Deslorelin is a small peptide that is administered subcutaneously in the form of an implant (Ovuplant) containing 2.1 mg of the active ingredient. The advantage to using deslorelin over hCG to induce ovulation is that its smaller molecular weight makes it less antigenic, thereby reducing the chance that antibodies will be developed against it after multiple administrations in a season. However, it must be noted that administration of deslorelin implants in a small percentage of mares does result in a prolonged interovulatory interval due to down-regulation of the pituitary by the continued release of deslorelin from the implant after ovulation has occurred.14-16 This down-regulation is most pronounced when prostaglandins are administered early in diestrus after the administration of deslorelin, compared with mares that are allowed to return to estrus naturally. This side effect can be avoided by removing the implant after ovulation has occurred.16 Currently, Ovuplant is not available in the United States; however, an injectable formulation of deslorelin, BioRelease Deslorelin Injection (BET Pharm, Lexington, Ky.), has become available. This deslorelin product is administered intramuscularly at the recommended dose of 1.5 mg when a 35-mm or greater follicle is present and the mare is in estrus. Based on the results of recent research, this injectable formulation of deslorelin does not cause the prolonged interovulatory interval seen with the administration of deslorelin implants.17 Administration of hCG when a 35-mm or greater diameter follicle is present in an estrual mare results in ovulation in 36 ± 4 hours and deslorelin acetate administration with the presence of a 35-mm or greater follicle in an estrual mare results in ovulation in 41 ± 3 hours.18 hCG and deslorelin are most commonly used to induce ovulation to occur within 48 hours post breeding.
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CHAPTER 3
In addition to their application in programs using fresh and cooled semen, these pharmacologic agents are used extensively in frozen semen programs. Due to the decreased longevity of frozen semen, insemination must occur within the time frame of 12 hours before to 6 hours after ovulation. If two doses of semen are available, a timed insemination program can be used. In this program, once a 35-mm follicle is detected in an estrual mare, deslorelin is administered at 7 PM that day. The mare is then examined via transrectal ultrasonography and inseminated at 24 and 41 hours. If hCG is used, then the second dose is inseminated at hCG + 36 hours. If only one dose of semen is available, the same basic plan is followed, except the mare is not inseminated at the 24-hour examination and is then examined via transrectal ultrasonography every 6 hours until ovulation is detected. Once ovulation is detected, the mare is inseminated. In addition to the pharmacologic agents previously described, there are other products and estrous synchronization programs described in the literature that warrant discussion. Numerous reports in the literature discuss the use of an intravaginal progesterone-releasing device originally designed for cattle (CIDR-B) in mares to synchronize estrus. These devices have been used with and without the administration of estradiol (10 mg, IM or intravaginal) on the day of CIDR-B placement. Results of these studies were similar to those with progesterone in oil or Regumate. Without the addition of the estradiol, CIDR-B administration resulted in synchronization of estrus but not of ovulations.19-21 The CIDR was first produced in New Zealand and was approved in May 2002 for use in the United States, where it is marketed under the name Eazi-Breed CIDR Cattle Insert (Pharmacia Animal Health, Kalamazoo, Mich.) and contains 1.38 g of progesterone. In Canada this product contains 1.9 g of progesterone. These products are not labeled for use in horses. A similar intravaginal progesterone-releasing device designed for mares, Cue-Mare (Duirs PfarmAg, Hamilton, New Zealand), has been described for the use of estrous synchronization in transitional mares.22 After a 10-day application of the Cue-Mare in 38 transitional mares, all of the mares exhibited signs of estrus 3 days following removal of the devices. Further research is needed to determine the value of this product in estrous synchronization programs.22 One advantage to intravaginal progesterone-releasing devices is that they do not require the labor associated with daily administration of progesterone in oil or Regumate. However, mild to moderate vaginitis is observed in most mares that have an intravaginal device administered. The use of ultrasound-guided transvaginal follicle ablation for synchronizing ovarian function in the mare has been described. In one study, on day 0 all follicles greater than or equal to 10 mm were ablated via ultrasoundguided transvaginal follicular aspiration. Four days later two doses of cloprostenol (250 mg IM) were given 12 hours apart followed by hCG administration 6 days after that. Results of this study showed that 96% (22/23) mares ovulated within 48 hours of hCG treatment. Compared with conventional steroid regimens where the interval from beginning of treatment to ovulation can be up to 20 to 25 days with23 or without24 hCG administration,
this protocol represents an approximate 50% reduction in the interval from follicle ablation to ovulation. In addition, use of this procedure avoids the labor associated with administration of steroids daily. However, more research is needed to assess the effects of this procedure on pregnancy rates and its practicality in a practice setting.25 As mentioned previously, one of the most common uses of estrous synchronization is in an embryo transfer program. To achieve the best results, at least three recipient mares should be synchronized with the donor mare. Administration of a progestin or progesterone/estradiol combination should be in the donor and recipients at the same time. Discontinuation of the progestin therapy and administration of prostaglandin should also occur on the same day among all of the mares. Realizing that it is ideal for the recipient to ovulate from 1 day before to 3 days after the donor, hCG or deslorelin should be administered to induce ovulations to occur preferably 1 to 3 days after the donor. It is important to realize that all of the recipients may not synchronize with the donor, nor may they be ideal recipients at the time of transfer due to presence of uterine fluid or edema, poor cervical tone, or inadequate CL formation. For this reason, it is important to synchronize at least three recipients with each donor to maximize the chances of having a suitable recipient at the time of transfer. Synchronization of estrus and, more importantly, ovulation plays an important role in equine breeding management programs. The use of a combination of pharmacologic agents, most commonly progesterone and estradiol-17b, followed by the administration of PGF2a and hCG or deslorelin, yields the most reliable results. However, there is ongoing research in this field, and current knowledge of the latest results will afford the veterinarian the best options when developing an estrous synchronization program.
References 1. Ginther OJ: Reproductive Biology of the Mare: Basic and Applied Aspects, 2nd edition, pp 173-232, Cross Plains, Wis, Equiservices, 1993. 2. Foss R, Wirth N, Schiltz P et al: Nonsurgical embryo transfer in a private practice (1998). Proceedings of the 45th Annual Convention of the American Association of Equine Practitioners, pp 210-212, 1999. 3. Allen WR, Rowson LEA: Control of the mare’s oestrous cycle by prostaglandins. J Reprod Fertil 1973; 33:539-543. 4. Douglas RH, Ginther OJ: Route of prostaglandin F2a injection and luteolysis in mares. Proc Soc Biol Med 1975; 148:263-269. 5. Nie GJ, Goodin AN, Braden TD et al: Luteal and clinical response following administration of dinoprost tromethamine or cloprostenol at standard intramuscular sites or at the lumbosacral acupuncture points in mares. Am J Vet Rev 2001; 62:1285-1289. 6. Bristol F: Studies on estrous synchronization in mares. Proceedings of the Annual Meeting of the Society of Theriogenology, p 258, 1981. 7. Asbury AC: The use of prostaglandins in broodmare practice. Proceedings of the 34th Annual Convention of the American Association of Equine Practitioners, pp 191-196, 1988.
Estrous Synchronization 8. Holtan DW, Douglas RH, Ginther OJ: Estrus, ovulation and conception following synchronization with progesterone, prostaglandin F2a and human chorionic gonadotropin in pony mares. J Anim Sci 1977; 44:431. 9. Voss JL, Wallace RA, Squires EL et al: Effects of synchronization and frequency of insemination on fertility. J Reprod Fertil Suppl 1979; 27:256. 10. Irvine CHG: Endocrinology of the estrous cycle of the mare: applications to the embryo transfer. Theriogenology 1981; 15:895. 11. Meyers PJ: Control and synchronization of the estrous cycle and ovulation. In Youngvist RS (ed): Current Therapy in Large Animal Theriogenology, pp 97-102, Philadelphia, WB Saunders, 1997. 12. Lofstedt RM, Patel JH: Evaluation of the ability of altrenogest to control the equine estrous cycle. J Am Vet Med Assoc 1989; 194:361. 13. Blanchard TL, Varner DD, Schumacher J: Manual of Equine Reproduction, pp 22-23, St Louis, Mosby, 1998. 14. Farquhar VJ, McCue PM, Nett TM et al: Effect of deslorelin acetate on gonadotropin secretion and ovarian follicle development in cycling mares. J Am Vet Med Assoc 2001; 218:749-752. 15. Farquhar VJ, McCue PM, Carnevale EM et al: Deslorelin acetate (Ovuplant™) therapy in cycling mares: effect of implant removal on FSH secretion and ovarian function. Equine Vet J 2002; 34:417-420. 16. McCue PM, Farquhar VJ, Carnevale EM et al: Removal of deslorelin (Ovuplant™) implant 48 hours after administration results in normal interovulatory intervals in mares. Theriogenology 2002; 58:865-870.
25
17. Stich KL, Wendt KM, Blanchard TL et al: Effects of a new injectable short-term release deslorelin in foal-heat mares. Theriogenology 2004; 62:831-836. 18. McKinnon AO, Perriam WJ, Lescun TB et al: Effect of a GnRH analogue (Ovuplant), hCG and dexamethasone on time to ovulation in cycling mares. World Equine Vet Rev 1997; 2:16-18. 19. Lübbecke M, Klug E, Hoppen HO et al: Attempts to synchronize estrus and ovulation in mares using progesterone (CIDR-B) and GnRH-analog deslorelin. Reprod Domest Anim 1994; 29:305-314. 20. Arbeiter K, Barth U, Jöchle W: Observations on the use of progesterone intravaginally and of deslorelin Sti in acyclic mares for induction of ovulation. J Equine Vet Sci 1994; 14:21-25. 21. Klug E, Jöchle W: Advances in synchronizing estrus and ovulations in the mare: a mini review. J Equine Vet Sci 2001; 21:474-479. 22. Grimmett JB, Hanlon DW, Duirs GF et al: A new intravaginal progesterone-releasing device (Cue-Mare) for controlling the estrous cycle in mares. Theriogenology 2002; 58:585-587. 23. Varner DD, Blanchard TL, Brinsko SP: Estrogen, oxytocin and ergot alkaloids: uses in reproductive management of mares. Proceedings of the 34th Annual Conference of the American Association of Equine Practitioners, 1998, pp 219-241. 24. Loy RG, Pemstein R, O’Canna D et al: Control of ovulation in cycling mares with ovarian steroids and prostaglandin. Theriogenology 1981; 15:191-197. 25. Bergfelt DR, Adams GP: Ovulation synchrony after follicle ablation in mares. J Reprod Fertil Suppl 2000; 56:257-269.
3
Estrous Synchronization E T TA A G A N B R A D E C A M P
A
s a prelude to this chapter, it is necessary to make a few comments about estrous synchronization as it pertains to the mare and how this varies from estrous synchronization in the cow. Practitioners who are familiar with estrous synchronization in cattle and the number of different synchronization programs available must realize that estrous synchronization in the mare is much more relative and does not yield as reliable results as estrous synchronization in cattle. In the bovine industry a large number of cows can be synchronized to reliably display estrus and ovulate within a narrow window of time, and there are several programs described to achieve the desired results. Unfortunately, the mare’s reproductive cycle does not lend itself to such a tight synchronization among a group of mares, and currently there is only one protocol that consistently provides reliable results. The length of estrus in the mare is 5 to 7 days, and the time of ovulation relative to the end of estrus varies with ovulation occurring on the last 2 days of estrus in 69% of mares and after the end of estrus in 14% of mares.1 These factors make the development of a synchronization program that allows for a single breeding at a predetermined time difficult. In equine reproduction the most common use of estrous synchronization is in embryo transfer programs. If a large recipient herd providing mares that have ovulated in synchrony with the donor is not available, then a small group of recipient mares must be synchronized with the donor mare. To be suitable recipients, these mares must ovulate from 1 day before to 3 days after the date of ovulation of the donor mare. There is even one report in the literature that recipient mares that ovulated up to 5 days after the donor have been used successfully in a large embryo transfer program.2 A second less common use of estrous synchronization in the mare is to synchronize a group of mares for breeding due to limited availability of a stallion, for example due to a show schedule. In addition to synchronization of estrus in a group of mares, pharmacologic agents can be used to induce ovulation to allow for the synchronization of ovulation. When breeding with both frozen and cooled shipped semen it is often desirable to use only one dose of semen, and therefore it is essential that the ovulation be synchronized within 36 to 48 hours of breeding. This is also important where natural service of mares is performed and the stallion has a full book of mares, thereby limiting the number of times a mare can be bred during a cycle. There are four basic methods used to achieve estrous synchronization in the mare: termination of the luteal 22
phase, lengthening of the luteal phase, induction of ovulation, and inhibition of the follicular phase. The pharmacologic agents most commonly used to accomplish these methods are prostaglandins, progestins, human chorionic gonadotropin (hCG) (Chorulon, Intervet Inc., Millsboro, Del.) or deslorelin acetate (Ovuplant, Peptech Limited, NSW, Australia), and estradiol-17b. The most reliable and successful estrous synchronization programs are achieved when a combination of these methods and pharmacologic agents are used. Prostaglandin F2a (PGF2a) is used to terminate the luteal phase by causing lysis of the corpus luteum (CL), thereby allowing the return to estrus. The most common prostaglandins used are dinoprost (Lutalyse, Pfizer, New York) and cloprostenol (Estrumate, Schering-Plough Animal Health Corporation, Union, N.J.). Dinoprost is a naturally occurring PGF2a, and cloprostenol is a synthetic prostaglandin analogue. For prostaglandins to effectively terminate the luteal phase a mature CL must be present that is responsive to prostaglandin. The equine CL exhibits incomplete sensitivity to PGF2a until approximately day 5 postovulation in most mares.3 The standard recommended dose of dinoprost is 9 mg/kg (5 to 10 mg per 1000 lb/454 kg horse). The recommended dose of cloprostenol is 250 mg per 1000 lb/454 kg horse (0.55 mg/kg). More recent research has shown that much lower doses of prostaglandin are just as effective at lysing the CL with fewer negative side effects. Douglas and Ginther demonstrated that doses of prostaglandin as low as 1.25 mg were as effective at inducing luteolysis as more standard doses.4 Nie et al showed that a microdose of cloprostenol (25 mg) was just as effective at lysing the equine CL as the larger standard dose.5 On average, mares return to estrus 5 to 7 days after administration of prostaglandin in the presence of a mature CL, and ovulation occurs 9 to 11 days after administration. However, because the follicular phase plays a greater role in controlling the total length of the estrous cycle in the mare, the length of time from administration of prostaglandin to onset of estrus and ovulation can be quite variable depending on the size and status of follicles present on the ovaries at the time of administration. The time of ovulation after PGF2a-induced estrus may vary as much as 2 to 15 days post treatment.6 If prostaglandin is administered when there is a large follicle present, the mare may return to estrus and ovulate in 2 to 3 days. Some of these mares never develop overt signs of standing heat, endometrial folds, or relaxation of the cervix before ovulation. If a large follicle destined for atresia is present on the ovary at the time of prostaglandin administration, the follicle may not
Estrous Synchronization ovulate and instead regress as a new wave of follicles develops.7 In cattle the administration of two doses of prostaglandin 11 to 12 days apart results in approximately 80% exhibiting estrus within 2 to 5 days after the second injection. Although the administration of two doses of prostaglandin 14 days apart has been described in the literature as a protocol for estrous synchronization in mares, it is not a very reliable method of ensuring that a recipient mare will be synchronized with a donor.6 Research has shown that after two injections of prostaglandin, on average ovulation occurs between 7 and 10 days after the second injection,6,8,9 with ovulation potentially occurring anywhere between 0 and 17 days.8,9 In a review of the literature it was stated that when this protocol was administered, one would need to treat at least 10 recipient mares to have an 80% chance that at least 1 recipient would ovulate within 24 hours of the donor.10 Obviously, this is not ideal, especially when in most small embryo transfer programs there are only a few mares available to be used as recipients. Due to the large variation in time of ovulation from administration of prostaglandin, it is not very useful when used alone in the synchronization of estrous cycles in mares. However, if examination via transrectal ultrasonography is performed first to eliminate those mares that will not respond appropriately to the administration of prostaglandin, tightening of date of ovulation can be achieved with this protocol. Exogenous progestins are commonly used in estrous synchronization programs to lengthen the luteal phase. The two most common forms of progestins used are progesterone in oil and altrenogest (allyl trenbolone, Regumate, Intervet Inc., Millsboro, Del.), a synthetic progestin. Progesterone or a synthetic progestin administered alone or in combination with estradiol-17b will effectively lengthen the luteal phase indefinitely until their administration is discontinued. Progesterone has an inhibitory effect on LH release from the anterior pituitary; therefore the rationale is if it is administered for a long enough period of time (15 to 18 days), the CL will regress and the only source of progesterone to suppress estrus is the exogenous progesterone. However, even the endogenous level of progesterone present during the midluteal phase is not sufficient to suppress gonadotropin release, follicular development, and ovulation.11 Studies have also shown that the synthetic progestin, altrenogest, administered at 0.044 mg/kg orally once a day fails to suppress follicular development.12 When using progestins for estrous synchronization it is recommended that either altrenogest (0.044 mg/kg PO q24h) or progesterone in oil (150 mg/day IM) be administered for 10 days combined with the administration of PGF2a on the last day of progestin therapy to lyse any CLs present. To improve estrous synchronization in randomly cycling mares, a combination of progesterone and estrogen, rather than progesterone alone, is administered. The combined effect of the two hormones provides a more profound negative feedback on gonadotropin release than does progesterone alone, which results in a more uniform inhibition of follicular development. When the exogenous progesterone/estrogen therapy is discontin-
23
ued, there is less diversity in follicular maturation and the date ovulation occurs can be more closely synchronized. The recommended protocol involves intramuscular injections of progesterone (150 mg) combined with estradiol-17b (10 mg) in oil for 10 consecutive days with a single injection of PGF2a administered on the tenth day of treatment. To further tighten the synchronization of ovulation, hCG (5 IU/kg IV) or deslorelin acetate is administered when a 35-mm follicle is present. With this protocol, greater than 70% of mares ovulate between 10 and 12 days after the last steroid injection with a range of ovulations occurring from 8 to 17 days. Currently, there is no commercial preparation containing progesterone and estradiol-17b combined; therefore this formulation must be ordered from a compounding pharmacy.13 Induction of ovulation is also an important part of an estrous synchronization program. The two most common pharmacologic agents used to induce ovulation are hCG and deslorelin acetate. Human chorionic gonadotropin is a protein consisting of two peptide chains that, although chemically different from pituitary luteinizing hormone (LH), has primarily luteinizing-like activity. hCG is produced by cytotrophoblasts of the chorionic villi of the human placenta and appears in the urine of pregnant women a few weeks after conception. Deslorelin acetate is a potent synthetic gonadotropinreleasing hormone (GnRH) analogue. Deslorelin is a small peptide that is administered subcutaneously in the form of an implant (Ovuplant) containing 2.1 mg of the active ingredient. The advantage to using deslorelin over hCG to induce ovulation is that its smaller molecular weight makes it less antigenic, thereby reducing the chance that antibodies will be developed against it after multiple administrations in a season. However, it must be noted that administration of deslorelin implants in a small percentage of mares does result in a prolonged interovulatory interval due to down-regulation of the pituitary by the continued release of deslorelin from the implant after ovulation has occurred.14-16 This down-regulation is most pronounced when prostaglandins are administered early in diestrus after the administration of deslorelin, compared with mares that are allowed to return to estrus naturally. This side effect can be avoided by removing the implant after ovulation has occurred.16 Currently, Ovuplant is not available in the United States; however, an injectable formulation of deslorelin, BioRelease Deslorelin Injection (BET Pharm, Lexington, Ky.), has become available. This deslorelin product is administered intramuscularly at the recommended dose of 1.5 mg when a 35-mm or greater follicle is present and the mare is in estrus. Based on the results of recent research, this injectable formulation of deslorelin does not cause the prolonged interovulatory interval seen with the administration of deslorelin implants.17 Administration of hCG when a 35-mm or greater diameter follicle is present in an estrual mare results in ovulation in 36 ± 4 hours and deslorelin acetate administration with the presence of a 35-mm or greater follicle in an estrual mare results in ovulation in 41 ± 3 hours.18 hCG and deslorelin are most commonly used to induce ovulation to occur within 48 hours post breeding.
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CHAPTER 3
In addition to their application in programs using fresh and cooled semen, these pharmacologic agents are used extensively in frozen semen programs. Due to the decreased longevity of frozen semen, insemination must occur within the time frame of 12 hours before to 6 hours after ovulation. If two doses of semen are available, a timed insemination program can be used. In this program, once a 35-mm follicle is detected in an estrual mare, deslorelin is administered at 7 PM that day. The mare is then examined via transrectal ultrasonography and inseminated at 24 and 41 hours. If hCG is used, then the second dose is inseminated at hCG + 36 hours. If only one dose of semen is available, the same basic plan is followed, except the mare is not inseminated at the 24-hour examination and is then examined via transrectal ultrasonography every 6 hours until ovulation is detected. Once ovulation is detected, the mare is inseminated. In addition to the pharmacologic agents previously described, there are other products and estrous synchronization programs described in the literature that warrant discussion. Numerous reports in the literature discuss the use of an intravaginal progesterone-releasing device originally designed for cattle (CIDR-B) in mares to synchronize estrus. These devices have been used with and without the administration of estradiol (10 mg, IM or intravaginal) on the day of CIDR-B placement. Results of these studies were similar to those with progesterone in oil or Regumate. Without the addition of the estradiol, CIDR-B administration resulted in synchronization of estrus but not of ovulations.19-21 The CIDR was first produced in New Zealand and was approved in May 2002 for use in the United States, where it is marketed under the name Eazi-Breed CIDR Cattle Insert (Pharmacia Animal Health, Kalamazoo, Mich.) and contains 1.38 g of progesterone. In Canada this product contains 1.9 g of progesterone. These products are not labeled for use in horses. A similar intravaginal progesterone-releasing device designed for mares, Cue-Mare (Duirs PfarmAg, Hamilton, New Zealand), has been described for the use of estrous synchronization in transitional mares.22 After a 10-day application of the Cue-Mare in 38 transitional mares, all of the mares exhibited signs of estrus 3 days following removal of the devices. Further research is needed to determine the value of this product in estrous synchronization programs.22 One advantage to intravaginal progesterone-releasing devices is that they do not require the labor associated with daily administration of progesterone in oil or Regumate. However, mild to moderate vaginitis is observed in most mares that have an intravaginal device administered. The use of ultrasound-guided transvaginal follicle ablation for synchronizing ovarian function in the mare has been described. In one study, on day 0 all follicles greater than or equal to 10 mm were ablated via ultrasoundguided transvaginal follicular aspiration. Four days later two doses of cloprostenol (250 mg IM) were given 12 hours apart followed by hCG administration 6 days after that. Results of this study showed that 96% (22/23) mares ovulated within 48 hours of hCG treatment. Compared with conventional steroid regimens where the interval from beginning of treatment to ovulation can be up to 20 to 25 days with23 or without24 hCG administration,
this protocol represents an approximate 50% reduction in the interval from follicle ablation to ovulation. In addition, use of this procedure avoids the labor associated with administration of steroids daily. However, more research is needed to assess the effects of this procedure on pregnancy rates and its practicality in a practice setting.25 As mentioned previously, one of the most common uses of estrous synchronization is in an embryo transfer program. To achieve the best results, at least three recipient mares should be synchronized with the donor mare. Administration of a progestin or progesterone/estradiol combination should be in the donor and recipients at the same time. Discontinuation of the progestin therapy and administration of prostaglandin should also occur on the same day among all of the mares. Realizing that it is ideal for the recipient to ovulate from 1 day before to 3 days after the donor, hCG or deslorelin should be administered to induce ovulations to occur preferably 1 to 3 days after the donor. It is important to realize that all of the recipients may not synchronize with the donor, nor may they be ideal recipients at the time of transfer due to presence of uterine fluid or edema, poor cervical tone, or inadequate CL formation. For this reason, it is important to synchronize at least three recipients with each donor to maximize the chances of having a suitable recipient at the time of transfer. Synchronization of estrus and, more importantly, ovulation plays an important role in equine breeding management programs. The use of a combination of pharmacologic agents, most commonly progesterone and estradiol-17b, followed by the administration of PGF2a and hCG or deslorelin, yields the most reliable results. However, there is ongoing research in this field, and current knowledge of the latest results will afford the veterinarian the best options when developing an estrous synchronization program.
References 1. Ginther OJ: Reproductive Biology of the Mare: Basic and Applied Aspects, 2nd edition, pp 173-232, Cross Plains, Wis, Equiservices, 1993. 2. Foss R, Wirth N, Schiltz P et al: Nonsurgical embryo transfer in a private practice (1998). Proceedings of the 45th Annual Convention of the American Association of Equine Practitioners, pp 210-212, 1999. 3. Allen WR, Rowson LEA: Control of the mare’s oestrous cycle by prostaglandins. J Reprod Fertil 1973; 33:539-543. 4. Douglas RH, Ginther OJ: Route of prostaglandin F2a injection and luteolysis in mares. Proc Soc Biol Med 1975; 148:263-269. 5. Nie GJ, Goodin AN, Braden TD et al: Luteal and clinical response following administration of dinoprost tromethamine or cloprostenol at standard intramuscular sites or at the lumbosacral acupuncture points in mares. Am J Vet Rev 2001; 62:1285-1289. 6. Bristol F: Studies on estrous synchronization in mares. Proceedings of the Annual Meeting of the Society of Theriogenology, p 258, 1981. 7. Asbury AC: The use of prostaglandins in broodmare practice. Proceedings of the 34th Annual Convention of the American Association of Equine Practitioners, pp 191-196, 1988.
Estrous Synchronization 8. Holtan DW, Douglas RH, Ginther OJ: Estrus, ovulation and conception following synchronization with progesterone, prostaglandin F2a and human chorionic gonadotropin in pony mares. J Anim Sci 1977; 44:431. 9. Voss JL, Wallace RA, Squires EL et al: Effects of synchronization and frequency of insemination on fertility. J Reprod Fertil Suppl 1979; 27:256. 10. Irvine CHG: Endocrinology of the estrous cycle of the mare: applications to the embryo transfer. Theriogenology 1981; 15:895. 11. Meyers PJ: Control and synchronization of the estrous cycle and ovulation. In Youngvist RS (ed): Current Therapy in Large Animal Theriogenology, pp 97-102, Philadelphia, WB Saunders, 1997. 12. Lofstedt RM, Patel JH: Evaluation of the ability of altrenogest to control the equine estrous cycle. J Am Vet Med Assoc 1989; 194:361. 13. Blanchard TL, Varner DD, Schumacher J: Manual of Equine Reproduction, pp 22-23, St Louis, Mosby, 1998. 14. Farquhar VJ, McCue PM, Nett TM et al: Effect of deslorelin acetate on gonadotropin secretion and ovarian follicle development in cycling mares. J Am Vet Med Assoc 2001; 218:749-752. 15. Farquhar VJ, McCue PM, Carnevale EM et al: Deslorelin acetate (Ovuplant™) therapy in cycling mares: effect of implant removal on FSH secretion and ovarian function. Equine Vet J 2002; 34:417-420. 16. McCue PM, Farquhar VJ, Carnevale EM et al: Removal of deslorelin (Ovuplant™) implant 48 hours after administration results in normal interovulatory intervals in mares. Theriogenology 2002; 58:865-870.
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17. Stich KL, Wendt KM, Blanchard TL et al: Effects of a new injectable short-term release deslorelin in foal-heat mares. Theriogenology 2004; 62:831-836. 18. McKinnon AO, Perriam WJ, Lescun TB et al: Effect of a GnRH analogue (Ovuplant), hCG and dexamethasone on time to ovulation in cycling mares. World Equine Vet Rev 1997; 2:16-18. 19. Lübbecke M, Klug E, Hoppen HO et al: Attempts to synchronize estrus and ovulation in mares using progesterone (CIDR-B) and GnRH-analog deslorelin. Reprod Domest Anim 1994; 29:305-314. 20. Arbeiter K, Barth U, Jöchle W: Observations on the use of progesterone intravaginally and of deslorelin Sti in acyclic mares for induction of ovulation. J Equine Vet Sci 1994; 14:21-25. 21. Klug E, Jöchle W: Advances in synchronizing estrus and ovulations in the mare: a mini review. J Equine Vet Sci 2001; 21:474-479. 22. Grimmett JB, Hanlon DW, Duirs GF et al: A new intravaginal progesterone-releasing device (Cue-Mare) for controlling the estrous cycle in mares. Theriogenology 2002; 58:585-587. 23. Varner DD, Blanchard TL, Brinsko SP: Estrogen, oxytocin and ergot alkaloids: uses in reproductive management of mares. Proceedings of the 34th Annual Conference of the American Association of Equine Practitioners, 1998, pp 219-241. 24. Loy RG, Pemstein R, O’Canna D et al: Control of ovulation in cycling mares with ovarian steroids and prostaglandin. Theriogenology 1981; 15:191-197. 25. Bergfelt DR, Adams GP: Ovulation synchrony after follicle ablation in mares. J Reprod Fertil Suppl 2000; 56:257-269.