Animal Reproduction Science 99 (2007) 18

Review article

Superovulation in mares
E.L. Squires , P.M. McCue
Animal Reproduction and Biotechnology Laboratory, ARBL Building, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683, USA Received 4 April 2005; accepted 25 April 2006 Available online 12 June 2006

Abstract Embryo recovery from single ovulating mares is approximately 50% per estrous cycle. Superovulation could be used to increase embryo recovery and provide extra embryos for embryo freezing. This review addresses some historical approaches to superovulation, as well as examines factors that affect the response of mares to equine FSH. eCG, GnRH and inhibin vaccines have been of limited success in stimulating multiple ovulation. Numerous studies have shown that injection of equine pituitary extract (EPE) will result in three to four ovulations per estrous cycle and two embryos. A puried, standardized EPE preparation (eFSH) also results in a similar response to EPE. Factors affecting the response to EPE and eFSH include day of initial treatment, size of largest follicle at initial treatment and frequency of injection. Embryos from single ovulating, untreated mares and eFSH-treated mares provide similar pregnancy rates upon nonsurgical transfer. Five to 7 days of eFSH treatment also has been shown to hasten the rst ovulation of the breeding season. Potential problems after eFSH injections include anovulatory or luteinized follicles and overstimulation. Studies are needed to further evaluate the criteria for initiation of treatment and to determine how to increase ovulation rate without decreasing embryo recovery per ovulation. 2006 Elsevier B.V. All rights reserved.
Keywords: Horsessuperovulation; EPE; eFSH; Embryo recovery

1. Introduction The majority of mares in an equine embryo transfer program are spontaneous, single-ovulating mares. Embryo recovery from single-ovulating mares is approximately 50% per estrous cycle. The pregnancy rate upon transfer of an embryo from a single-ovulating mare is approximately

Corresponding author. Tel.: +1 970 491 8409; fax: +1 970 491 3557. E-mail addresses: esquires@colostate.edu, Edward.Squires@ColoState.edu (E.L. Squires).

0378-4320/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.04.054

E.L. Squires, P.M. McCue / Animal Reproduction Science 99 (2007) 18

75%. Thus, on any given estrous cycle, the client has approximately a 35% chance of obtaining a pregnancy. Mares that spontaneously double ovulate, such as thoroughbreds, warmbloods and draft horses, have a greater embryo recovery rate per estrous cycle and are more productive in an embryo transfer program. There appear to be many misconceptions about superovulation in the mare. Some say that superovulation cannot occur because of the anatomical conguration of the mares ovary. That is, the ovary is inside out and only a small portion of the ovary has germinal epithelium that will accommodate ovulation, termed the ovulation fossa. Others have reported that embryo recovery per ovulation is lower in superovulated mares than single-ovulating mares and that embryos obtained from superovulated mares have a lower chance of fertility. This review will address some of the historical approaches to superovulation of the mare, as well as examine the factors that affect the response of mares to equine FSH. The review also presents potential problems with superovulation, as well as addresses many of the challenges that need to be examined before superovulation becomes a standard procedure in the equine embryo transfer industry. Potential benets of superovulation include: increased embryo recovery, increased oocyte collection, availability of extra embryos for embryo freezing, enhancement of fertility in subfertile mares and advancement of the rst ovulation. 2. Approaches to superovulation 2.1. Treatments There have been numerous approaches to superovulation of the mare, including injection of porcine FSH, inhibin vaccines, equine chorionic gonadotropin (eCG) and GnRH. eCG injected even in large amounts has not been successful in inducing superovulation in mares (Allen, 1982). Several investigators have shown that injections of GnRH or GnRH agonist into transitional mares resulted in multiple ovulation (Ginther and Bergfelt, 1990). However, twice daily injections of GnRH into mares having estrous cycles at regular intervals did not result in multiple ovulations (Squires et al., 1989). Secretion of inhibin from the dominant ovarian follicle suppresses FSH secretion (Ginther et al., 2001). Therefore, inhibin antibodies have been used to decrease inhibin concentrations and increase concentrations of FSH. Active immunization of mares against inhibin resulted in a doubling of ovulation rate in two studies (McKinnon et al., 1992; McCue et al., 1993). Multiple inoculations over several weeks were required. Occasional adverse reactions at the injection site have prevented the commercial use of inhibin vaccines for superovulation. 2.2. Porcine FSH Several studies have examined the use of porcine FSH for induction of multiple ovulations in mares. Squires et al. (1986) compared the response of mares to injection of equine pituitary extract (EPE; primarily FSH and LH) to that of porcine FSH (6.4 and 6.8 days of treatment, respectively). Mare were given 150 mg of porcine FSH and only 1.6 ovulations were obtained compared to 2.2 ovulations for mares given EPE. Fortune and Kimmich (1983) evaluated various doses of porcine FSH for induction of multiple ovulation in mares. Mares were given 8, 16 or 32 mg of porcine FSH twice daily for approximately 6 days. The ovulation rate ranged from 1.5 to 1.8 and the percent of mares with double ovulations was 5083%. The variation in concentrations of porcine FSH needed to induce multiple ovulation in mares was extreme and, thus, quite costly. Furthermore, the superovulatory response was less with injection of porcine FSH.

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2.3. Equine pituitary extract (EPE) The majority of studies on superovulation in the mare have utilized EPE prepared according to the procedures of Braselton and McShan (1970) and later by Guillou and Combarnous (1983). This later procedure yielded approximately 6 g of crude pituitary extract/kg of pituitaries. EPE contained approximately 610% LH and 24% FSH. Many of these studies were performed on pony mares, which are quite resistant to spontaneous natural double ovulation. Therefore, the ovulation response in the light horse mare generally is greater than in pony mares. The initial studies on using EPE for induction of ovulation were conducted in pony mares in Wisconsin during both the anestrous season and physiologic breeding season. Douglas (1979) reported a 2.3 ovulation rate for mares given EPE once daily for 7 days compared with 1.0 ovulation for untreated controls. Woods and Ginther (1983) reported an average of 3.0 ovulations per mare in 112 mares treated with EPE. Seventy percent of the mares experienced two or more ovulations. Squires et al. (1987) reported 3.8 ovulations for EPE-treated mares and 2.0 embryos recovered per mare. This compared to 1.2 ovulations in untreated control mares and 0.65 embryos per cycle. The embryo recovery per ovulation was similar between treated and control mares (55%). Summarized over numerous studies conducted at Colorado State University, the ovulation rate after EPE treatment ranged from a minimum of 2.3 to a maximum of 3.9 ovulations per mare. Of 170 mares treated with EPE at Colorado State University, an average of 3.2 ovulations was detected and 1.96 embryos were recovered per mare compared to 0.65 embryo recovered from untreated control mares. These data would certainly indicate that the mare is responsive to EPE and will provide two to four ovulations and approximately two embryos recovered per estrous cycle. 3. Factors affecting response to EPE 3.1. Day of initial treatment Similar to cattle treated with porcine FSH, there is extreme variation in the response of mares to EPE or equine FSH. Many of the initial studies with EPE were conducted prior to the knowledge regarding ovarian follicular waves in the mare. Most mares experience one follicular wave and, thus, it is important to initiate treatment at the beginning of the follicular wave prior to the emergence of a dominant follicle. Lapin and Ginther (1977) compared the response to EPE when given on day 11 of diestrus compared with day 1 of estrus. They reported no difference in ovulation rate. However, the number of mares in each treatment group (n = 7) was quite small and the ovulatory response was minimal (1.7). Woods and Ginther (1983) reported that treatment on days 1519 (day 0 = day of ovulation) was more effective than on days 1923 (2.9 ovulations per mare versus 1.3 ovulations per mare). They also reported no difference when treatment was started on day 11 or 15. Dippert et al. (1992) in our laboratory compared the response of mares to treatment initiated on day 5 compared with day 12. Number of ovulations was greater for mares given treatment initially on day 5 (2.9) compared with those in which treatment was delayed until day 12 (1.1). Thus, in the majority of our subsequent studies, treatment was initiated 57 days after ovulation, around the time of the rst follicular wave. Pierson and Ginther (1990) examined the effect of follicle size at the initiation of EPE treatment on the superovulatory response. Mares were given progesterone and estradiol for 10 days and then prostaglandins on the last day of treatment. The EPE treatment was initiated when mares obtained a follicle of 15, 20, 25 or 30 mm. Number of large preovulatory follicles that developed in response to EPE was much greater when treatment was initiated at a follicle size of 15 or 20 mm, compared

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to mares in which treatment was delayed until follicles had reached a size of 2530 mm. Thus, treatment with EPE should be initiated when follicles are <25 mm. Ginther et al. (2001) has reported that the follicle of the pony mare becomes dominant at approximately 23 mm. 3.2. Frequency of injection Frequency of injection is another factor that will affect the response to EPE. In initial studies, EPE was given twice daily (Douglas et al., 1974). However, in subsequent studies, EPE was given only once per day (Lapin and Ginther, 1977; Douglas, 1979). Alvarenga et al. (2001) compared once with twice daily injections of EPE. Twice daily injection of EPE in mares having estrous cycles at regular intervals resulted in a much greater ovulation and embryo recovery rate per mare (7.1; 3.5) than once daily (2.4; 1.6) injections. It was not known, from that study, whether the increased response was due to the frequency of injection or the total amount of hormone injected. Therefore, in a subsequent study (Scoggin et al., 2002), mares were given either 25 mg of EPE once or twice daily, or 12.5 mg of EPE once or twice daily. The number of ovulations per mare was greatest in mares given 25 mg of EPE twice daily (4.7). However, the number of embryos recovered per mare was greatest from mares given 12.5 mg of EPE twice daily (2.6). Based on these studies, it is recommended that equine FSH be given twice per day. 3.3. GnRH treatment prior to EPE Protocols for superstimulation of follicles in women generally involve the use of a GnRH agonist for suppression of endogenous LH. Two studies have been conducted in our laboratory (Dippert et al., 1992; Scoggin et al., 2002) in which a GnRH agonist was given prior to administration of EPE. In neither of these studies was response to EPE enhanced by administration of a GnRH agonist. 3.4. Puried EPE The amount of LH in the EPE varies considerably and, in many studies, the exact FSH to LH ratio has not been reported. However, in cattle, too much LH in the FSH preparation generally results in a poor ovulatory response. Thus, two studies were conducted in which EPE was puried further to provide a lesser contamination of LH in the FSH preparation. Hofferer et al. (1993) compared the ovulatory response of mares given 25 mg of equine pituitary extract (1.2 mg eFSH CY1368 + 2 mg eLH CY60) to that of mares given a 10 mg fraction of an enriched FSH preparation containing less LH contamination (1.25 mg eFSH CY1368 + 0.3 mg eLH CY760). No greater ovulatory response for mares administered the enriched FSH preparation was found. A subsequent study (Rosas et al., 1998) also reported a similar ovulatory response for mares administered EPE compared with those given an enriched FSH preparation. 4. Viability of embryos from superovulated mares Early studies by Woods and Ginther (1983) suggested that embryos collected from superovulated mares were less viable than those from single-ovulating mares. However, subsequent studies did not support this nding. Squires et al. (1987) transferred embryos from single-ovulating mares and multiple-ovulating mares and reported similar pregnancy rates between the groups. Furthermore, Dippert et al. (1994) in our laboratory recovered oviductal embryos from superovulated and

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single-ovulating mares and cultured the embryos for 10 days. Similar development occurred in culture between embryos recovered from superovulated mares and single-ovulating mares. Mares (n = 136) on a commercial breeding farm in Texas (Landers, unpublished data) were administered a commercial equine FSH product to induce superovulation. Embryos were recovered from these mares and transferred into synchronized recipients. Pregnancy rates from transferred embryos from superovulated mares were similar to those for embryos collected from non-treated, singleovulating mares. Thus, it would appear that the viability of embryos from superovulated mares was not different than from single-ovulating mares. 5. eFSH Recently, a commercial puried equine pituitary extract product (eFSH) has been made available from Bioniche Animal Health (Athens, GA). Several recent studies have been conducted using this commercially available eFSH product. Niswender et al. (2003) evaluated two doses of eFSH: 12.5 and 25 mg twice per day. The dose of 25 mg twice per day resulted in a greater number of preovulatory follicles. However, there was not ovulation from many of these follicles. The 12 mg dose of eFSH given twice daily followed by hCG after mares developed preovulatory follicles, resulted in 3.4 ovulations per mare and, at 14 days after insemination, 1.8 pregnancies per mare compared with 0.6 pregnancies for untreated controls. Thus, a dose of 12.5 mg given twice per day was chosen for subsequent studies and commercial use. In Brazil, 16 mares were used during two estrous cycles (Niswender et al., 2003). The rst estrous cycle was considered a control cycle. In the second estrous cycle, mares were given eFSH (12.5 mg bid). Embryo recovery was attempted 7 days after ovulation. In the control estrous cycle, mares ovulated one follicle per mare and embryo recovery was 0.5 embryos per mare. In contrast, mares given eFSH had 3.6 ovulations and 1.9 embryos recovered per mare. In a current study at Colorado State University, light horse cycling mares were assigned to one of four treatments: (1) 12.5 mg of eFSH bid and hCG once follicles had obtained a size of 35 mm; (2) 12.5 mg of eFSH bid and hCG 30 h after mares had obtained a follicle of 32 mm; (3) 12.5 mg of eFSH bid for 3.5 days, then 12.5 mg of eFSH plus eLH added for 3 or 4 days and then hCG once a 35 mm follicle had been obtained; (4) 25 mg of eFSH once daily and hCG 30 h after a 35 mm follicle had been obtained. Number of ovulations was greater for mares given 12.5 mg (4.2) compared with those given 25 mg once a day (2.8). Furthermore, the number of embryos recovered was 1.9, 2.5, 1.9 and 1.8 for groups 14, respectively.

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The eFSH given to 74 light horse cycling mares on a commercial breeding farm (Joe Landers, unpublished data) resulted in 3.1 ovulations and 1.5 embryos recovered per mare, compared to 1.0 ovulations and 0.73 embryos per cycle in untreated mares. In the second year, 62 mares having estrous cycles at regular intervals were treated with eFSH and 2.5 ovulations were obtained and 1.37 embryos were recovered from each mare. The ovulation and embryo recovery rates were slightly less in year 2 compared with year 1, primarily due to a greater number of mares given eFSH once per day in year 2, instead of twice per day. Based on all the studies, it would appear that eFSH results in a similar ovulation rate and embryo recovery rate to that of mares given EPE. Treatment with eFSH has the potential of increasing embryo recovery by four-fold and, thus, can be used very effectively in a commercial embryo transfer program. 6. Transitional mares A majority of mares have inactive ovaries and are anovulatory from the fall until late winter or early spring. Mares go through a transition period in the spring, during which waves of ovarian follicular growth and regression occur. Horse owners want to have their mares initiating estrous cycles as early in the year as possible. The EPE (Coy et al., 1999) and eFSH (Niswender et al., 2004) have been used to induce multiple ovulation in transitional mares. A greater percentage of transitional mares (eight of nine; 89.9%) ovulated in response to EPE administration (Coy et al., 1999) as compared with mares in deep anestrus (two of nine; 22.2%). Niswender et al. (2004) randomly assigned mares to one of two treatment groups at the onset of transition (largest follicle 25 mm): untreated controls and treated mares given 12.5 mg eFSH twice daily for a maximum 15 days. Interval to ovulation was shorter for treated (7.6 days) than control mares (39.5 days). The number of ovulations was 1.0 and 2.5 for treated and control mares, respectively. Thus, eFSH can be used to hasten the rst ovulation of the year and induce multiple ovulations. 7. Potential problems There are occasionally some potential problems with eFSH treatment and some challenges for the future. It is our clinical impression that young, normal, mares that are having estrous cycles at regular intervals respond consistently to eFSH, whereas older mares have a much more variable response. Similar to cattle, there is tremendous variation in individual donors in response to eFSH treatment. Determining the appropriate dose for the given donor is one of the major challenges. If the dose is too great, then the ovary becomes overstimulated and many of the follicles do not ovulate but become luteinized or develop a follicular cyst. In a recent paper from France (Briant et al., 2004), a study was conducted to determine the dosage regimen that was able to reproduce physiological FSH concentrations. They reported that if a dose of eFSH was too great, progesterone concentrations greater than 1 ng/ml were detected in treated mares prior to ovulation. This premature progesterone secretion was due to elevated LH, which resulted in premature ovulation or luteinization of follicles. They suggested that the reason for the great variability in response to eFSH was due to the individual sensitivity of the mare to the amount of the eFSH injected. If a mare did not respond to eFSH then the dose was too small. Generally, if a mare has greater than ve ovulations, then embryo recovery per ovulation is less. Ideally, one would like to have at least a 50% embryo recovery per ovulation. Mares having two to four ovulations generally have a 50% embryo recovery per ovulation. In our most recent study,

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embryo recovery/ovulation in 76 cycles was 58%. Mares having greater than ve preovulatory follicles fail to ovulate or have a lesser embryo recovery per ovulation when ovulation does occur. The lesser embryo recovery per ovulation could be due to failure of fertilization, high embryonic loss in the oviduct or, perhaps, because of the tremendous size of the ovary, ovum pick-up into the oviduct is impaired. 8. Challenges for the future Some of the challenges in the future include determining the correct dose and FSH to LH ratio, clearly dening the criteria for initiation of treatment, determine the cause of anovulatory luteinized follicles and determine how to increase ovulation rate without decreasing embryo recovery per ovulation. Some of the ways to enhance response to eFSH may include: (1) selecting mares with the appropriate number of follicles and not initiating treatment of mares that already have a dominant ovarian follicle; (2) use of progesterone and estradiol to suppress follicular population prior to treatment and initiation of a new follicular wave; (3) perhaps stopping eFSH treatment earlier (3032 mm follicle) to prevent luteinization of follicles; (4) decreasing the dose for mares that have been shown to be extremely sensitive to eFSH. In summary, eFSH can be a very valuable tool in an equine embryo transfer program. By increasing the ovulation and embryo recovery rates, the embryo transfer procedure can become more efcient and the cost can be decreased. Availability of multiple embryos in a given embryo recovery ushing of the uterus also provides the opportunity for embryos to be frozen for subsequent transfer when recipient mares are available. References
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