Hormonal Manipulation of the Mare: A Review

REFEREED

ORIGINAL RESEARCH

Hormonal Manipulation of the Mare: A Review Edward L. Squires, PhD

Keywords: Cycling; Hormones; Mares; Transitional

TRANSITIONAL MARES Progestins Production of foals early in the year is essential for performance in the show ring and maximizing prices obtained for yearlings. It is now commonplace to expose mares to increasing photoperiod beginning approximately December 1 in order to hasten the onset of the breeding season. Even though artificial photoperiod has been shown to be quite effective in hastening the first ovulation of the year, mares still experience a transition period from winter anestrus to normal cyclicity. The combination of artificial photoperiod and a 12- to 15-day exposure to exogenous progesterone has been used in combination to synchronize the first ovulation of the year.1 This approach has been called ‘‘programming’’ the mare, such that the time of breeding during the transition can be somewhat controlled. Generally, one waits until the mare has been exposed to artificial photoperiod for approximately 30 to 60 days, and then administers progestins after some follicular development on the ovary has been induced. There is some controversy as to whether exogenous progesterone during the transition period will actually hasten or possibly just synchronize the first ovulation of the year. Generally, either oral altrenogest is administered or progesterone in oil injected on a daily basis for 10 to 15 days. More recently, long-acting progesterone compounds have been made available from compounding pharmacies which allow the administration of progestins only once. These long-acting compounds are designed to release progesterone over a period of 7 to 10 days.2 Regardless of the type of progesterone, it has been shown numerous times that only mares in middle to late transition respond favorably to progestin treatment. This is generally defined as mares having R25 mm follicles. One of the advantages of using progestins for programming mares for breeding is that progesterone can be administered variable lengths of time from 10 to 20 days without any potential From theAnimal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO. Reprint requests: Edward L. Squires, PhD, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683. 0737-0806/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2008.10.010

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adverse effects. This would allow one to control the number of mares that need to be bred on a given day. Other Hormonal Methods Another approach used to hasten the onset of the breeding season is twice-daily injections of gonadotropin-releasing hormone (GnRH) agonist. Studies have shown that twice-daily injections of buserelin will, in fact, hasten the first ovulation of the year.3 The disadvantage of this approach is the need to handle the mares twice a day for several days in order to induce ovulation. Equine follicle stimulating hormone (eFSH) also has been shown to hasten the first ovulation of the year. Administration of eFSH twice daily for 5 to 7 days resulted in ovulation about 7 days after the onset of treatment compared to over 30 days for nontreated control mares.4 The disadvantage, however, is the expense of eFSH and the need to administer eFSH twice daily for several days. Oral administration of the dopamine antagonist, domperidone, has been shown by Brendemuehl and Cross5 to hasten the first ovulation of the year by 78 days. A similar antagonist, sulpiride, also has been shown to hasten ovulation by 33 days.6 However, both of these treatments appear to be somewhat dependent upon environmental temperatures. The mechanism of action for these dopamine antagonists is via stimulation of pituitary prolactin secretion. Kelley et al7 investigated the effect of pretreating transitional mares with estradiol to enhance prolactin secretion and ovarian response to daily injections of sulpiride. In experiment 1, 18 mares on January 11 were randomly assigned to either estradiol treatment or a control group. Treated mares received 11 mg of intramuscular estradiol benzoate injected every other day for a total of 10 injections. Beginning on day 11, all mares received daily subcutaneous injections of 250 mg of sulpiride. Sulpiride injections were continued until mare ovulated or through day 45, whichever occurred first. Plasma prolactin concentrations were higher in mares receiving estradiol than in controls from days 12 through 36. Plasma luteinizing hormone (LH) concentrations also were increased by estradiol treatment. Mean day of first ovulation was 74 for control mares and 29 for estradiol-treated mares. These researchers concluded that estradiol treatment greatly enhanced prolactin secretion in response to sulpiride and increased LH secretion in seasonally anovulatory mares. Experiment 2 was performed in normally cycling mares. Treatments were similar to experiment 1 except that domperidone was used instead of sulpiride and the

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domperidone was administered as a single 3-g domperidone, microparticle injection on day 11. Prolactin concentrations in cycling mares also were increased by estradiol treatment. They concluded that the single injection of domperidone possibly could replace daily sulpiride injections. This study is interesting in that ovarian activity and ovulation were induced in seasonally anovulatory mares through the use of estradiol and sulpiride. This appears to be one of the few treatments that is successful in stimulating follicular development and ovulation in anovulatory mares. Most other treatments only are effective when administered to transitional mares.

CYCLING MARES The focus of this section will center on hormonal manipulation of follicular development and manipulation of the lifespan of the corpus luteum. The increased pressure placed on the veterinarian and stallion manager to breed a larger number of mares to a given stallion and to obtain multiple pregnancies from embryo donors has increased the use of hormonal manipulation in the cycling mares. Progestins Progestins are a broad category that includes natural progesterone and synthetic progestins, such as altrenogest. Progestins can be administered either orally, in multiple injections, as single injections of slow-release formulations, or as intravaginal devices. Probably the most common use of progestins is for suppression of estrus in performance mares and maintenance of pregnancy. There are some occasions where estrus synchronization may be beneficial to the veterinarian and horse breeder. This may include manipulation of the mare’s cycle such that mares are in estrus at the time that the stallion is available. Alternatively, synchronization of estrus may be used as a means of maximizing the number of mares in estrus at a given time such that the entire ejaculate can be used from the stallion. There is some controversy as to how effective progesterone is, when given alone, in controlling ovulation. There is no doubt that progestins effectively suppress estrus, but they generally have a minimal suppressive effect on FSH secretion. Therefore, mares can, in fact, develop large follicles during the period of progestin administration. The options for estrus synchronization include progestin administered daily for 8 or 10 days as an oral preparation (altrenogest) or as a daily injection of 150 mg of progesterone in oil. More recently, progesterone in slow-release formulations have allowed administration of a single injection of progesterone that will maintain elevated blood levels of progesterone for approximately 7 days.8 Many practitioners still utilize a combination of progesterone and estradiol as a means of synchronizing estrus

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and ovulation. Estradiol has a very strong inhibitory effect on FSH secretion. A combination of both steroids effectively suppresses estrus and follicular development. Unfortunately, this combination must be given daily for 8 to 10 days. In cycling mares, it is best to administer prostaglandin F2a on the last day of progestin treatment (ie, either on day 8 or 10, depending on the duration of progestin treatment). After the prostaglandin injection, mares generally begin to exhibit estrus in 3 to 4 days and ovulation occurs 7 to 10 days after the prostaglandin injection.9 Storer et al10 evaluated the ability of a sustained-release altrenogest formulation in delaying return to estrus. Thirty-one randomly selected, cycling light-horse mares were used for this study. Ovulation was confirmed in all mares, and 6 days later mares were treated with 10 mg of prostaglandin F2a and then randomly assigned to five treatment groups. These included: controls, medroxyprogesterone acetate, altrenogest LA150 (1.5 mL solution, 225 mg), altrenogest LA150 (3.0 mL solution, 450 mg), and altrenogest MP500 as lactide-glycolide microparticles (500 mg). Days to estrus was similar for control mares and those given medroxyprogesterone (3.9 and 6.2 days, respectively). The MP 500 altrenogest extended days to estrus until 33 days, whereas the LA 150 (1.5 mL) extended days to estrus to 12.7 days and LA 150 (3.0 mL) extended days to estrus for 15.8 days. They concluded that all altrenogest formulations extended the interovulatory interval and delayed estrus with MP 500 formulation having the greatest inhibitory effect. By far the greatest use of progestins in broodmare reproduction is for maintenance of pregnancy. Unfortunately, much of the rationale for deciding whether mares should be administered exogenous progestins during early gestation is based on tradition and not scientific information. Generally, mares with a history of losing pregnancies during early gestation become candidates for exogenous progesterone. Timing as to when progestins should be initiated in early gestation and when they should be stopped appears to be up to the veterinarian. Generally, progestin treatment is initiated at the time of the first pregnancy examination (days 12–16) and then continued until approximately 120 to 150 days, a time when the placental secretion of progestins is available. However, there are some occasions where mares are placed on progestins beginning 1 to 2 days after ovulation and progestin treatment is continued until nearly the time of foaling. Progestins also are commonly given to embryo transfer recipients as a means of ensuring high levels of progesterone during the critical time of early gestation. In a survey of six major embryo transfer facilities within the United States, some form of progestin treatment was administered to recipient mares in five of the six facilities.11 Recently, at the 7th International Symposium on Equine Embryo Transfer, a series of studies was presented on the effect of

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using progestins as a means of treating noncycling mares for use as embryo transfer recipients. In most studies, it appeared that the use of noncycling, progesterone-treated mares for embryo transfer recipients provided a slightly lower pregnancy rate than that of normal cycling, synchronized recipients.12 Vaginal Devices There are at least three vaginal devices that have been used to control the estrous cycle of mares. These include CIDR devices, PRID13 devices, and, more recently, the CueMare device (Bioniche Animal Health, Melbourne, Australia).14 These vaginal devices have been shown to successfully suppress estrus, but the use of this device in the United States is limited by its availability. There are none currently approved for use in the horse in the United States. Furthermore, the majority of these devices do result in a high percentage of mares having mild to moderate vaginitis. This seems to be more of a concern because of the unsightliness of the vaginitis versus any potential adverse effects on fertility. Grimmett et al14 first described the use of the Cue-Mare device. This device consists of two main components, the carrier body, called a ‘‘wishbone,’’ and two treatment pods attached to the arms of the wishbone, which carry progesterone formulated into a silicone matrix. This vaginal device currently is available in Australia, but not in the United States. Recently we conducted a study in Australia (Squires, unpublished) using the Cue-Mare device as a means of synchronizing a group of experimental mares. Although there were no untreated controls in this particular study, pregnancy rates on the first cycle after withdrawal of the CueMare was more than 60% per cycle and the majority of mares ovulated 7 to 10 days after withdrawing the vaginal device. Gonadotropin-Releasing Hormone Vaccine GnRH vaccines have been used to suppress reproductive activity in mares.15 This involves the injection of GnRH conjugated to a foreign protein and combined with and adjuvant to induce anti-GnRH antibody formation. These antibodies bind to endogenous GnRH and prevent it from binding to receptors on the pituitary gonadotropes, thus removing the stimulus for gonadotropin secretion. Studies have demonstrated that a commercial GnRH vaccine was effective in long-term suppression of estrus in mares. However, there have been anecdotal reports that some mares do not cycle normally for one or more years after vaccination. Elhay et al16 reported that after administration of the commercially available GnRH vaccine (Equity; Pfizer Animal Health, Australia), estrous related behavior was suppressed for a minimum of 3 months and correlated with the initial levels of the antibody response. Imboden et al17 reported on a second GnRH vaccine available in

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Australia (Improvac, CSL Limited, Australia). In their study, the vaccine was administered intramuscularly 4 weeks apart. Ovaries and uteri of all mares were examined weekly by ultrasonography from 3 weeks before and up to 60 weeks after the first immunization. Thereafter, vaccinated mares were evaluated monthly until 100 weeks after the first vaccination. All vaccinated mares ceased reproductive cyclicity within 8 weeks after the first injection and ovarian activity remained suppressed for a minimum of 23 weeks. Of the nine mares, five mares resumed cyclicity while three mares showed only follicular activity and one mare remained completely suppressed for the entire duration of the study. These investigators concluded that the timing of resumption of cyclicity is highly variable. Usefulness of this method for suppression of estrus may be limited if a controlled return to estrus is required after the termination of treatment. Prostaglandins Prostaglandin is one of the most commonly used hormones on an equine breeding farm. Uses include induction of estrus, estrous synchronization, treatment of mares with a maintained corpus luteum (CL), induction of uterine contraction for evacuation of fluid, and induction of abortion. It has been known for years that the best response to prostaglandin is when given 5 to 10 days after ovulation.18 It also has been reported previously that the interval to estrus and ovulation is dependent upon the size of the largest follicle at the time of administration of prostaglandins. Mares having follicles >30 mm at the time of prostaglandin administration return to estrus quickly and ovulate early in the cycle.19 Recent information has demonstrated that administration of prostaglandins within the first few days after ovulation results in altered progesterone secretion.20 This has prompted the recommendation that if prostaglandins are being used for evacuation of uterine fluid, they are only used before ovulation and, after ovulation, that oxytocin be used for the induction of uterine contractions.20 Induction of Ovulation Hormonal induction of ovulation is a means of closely timing breeding/insemination with ovulation. This may in fact enhance fertility but, more importantly, it assists in minimizing the number of times the mare needs to be inseminated/bred during the estrous cycle. Because of the increasing demand for semen from very popular stallions, there is a need for minimizing the number of times a mare is bred in a given cycle. Furthermore, with the use of cooled and frozen semen, the amount of semen available during a given estrous cycle is limited and, thus, having to breed the mare only one time becomes quite important. The three hormonal agents that can be used for induction of ovulation in mares include: human chorionic

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gonadotropin (hCG), a GnRH agonist (commonly deslorelin), and recombinant equine LH (reLH). hCG has been used for several decades for the induction of ovulation in mares. This is an LH-like hormone with a relatively long half-life, which makes it very efficacious in inducing ovulation. The advantages of hCG are the low cost of administration and the efficacy. Although data are limited, the clinical impression is that hCG works better early in the year than the GnRH agonist deslorelin. The rationale behind this is that hCG works directly on the ovary, whereas deslorelin stimulates the pituitary to release LH. Studies have shown that pituitary stores of LH are somewhat limited early in the year21; mares may not respond as well to deslorelin as they do to hCG. There are, however, disadvantages to the use of hCG, which include potential diminished response of mares to hCG after repetitive use during the breeding season and a slight insensitivity of the ovaries of older mares to hCG. Barbacini et al22 reported that the percentage of mares ovulating within 48 hours after hCG was lower for barren mares and mares older than 16 years of age. In addition, they reported that mares given hCG in May, June, and July were more likely to ovulate within 24 hours of treatment than mares administered hCG in February, March, and April. Interestingly, they reported that repeated use of hCG did not decrease the ovulatory response. However, McCue et al23 examined the records of 797 mares administered a total of 1321 doses of hCG. They reported a linear decline in efficacy at inducing ovulation at a predictable time (24–48 hrs) as the number of hCG treatments increased during a given season. The efficacy of hCG in this study also decreased with an increase in the mare’s age. Overall percentage of mares induced to ovulate with hCG within 48 hr in this study was 78.4%. This is lower than the 91% reported by Barbacini et al.22 McCue et al23 concluded that hCG should not be used more than once or twice within a single breeding season. Green et al24 examined the relationship between uteroovarian parameters and the ovulatory response to hCG in mares. They performed a retrospective review of reproductive records from 353 light-horse and draft-cross mares that received hCG during the 2002 to 2005 breeding seasons. Mares were examined by transrectal palpation and ultrasonography at least once on the day of hCG administration and at least once daily thereafter until ovulation or for 96 hours after treatment. They observed a significant difference between the endometrial edema score response at 24 hours after hCG and ovulation within 48 hours of hCG administration. A decrease in uterine edema from time 0 to 24 hours was significantly associated with ovulation within the 24- to 48-hour interval from hCG administration. These authors also saw a decrease in percentage of mares ovulating within 48 hours of hCG administration after successive administrations.

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hCG generally is given to light-horse mares once they acquire a follicle R35 mm. Ovulation then occurs at a mean interval of approximately 36 hours. A study was conducted in our laboratory to determine whether follicles