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Medically Assisted Conception: An Agenda for Research (1989)

Chapter: Responses to Gonadotropins in Domestic Animals

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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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Suggested Citation:"Responses to Gonadotropins in Domestic Animals." Institute of Medicine. 1989. Medically Assisted Conception: An Agenda for Research. Washington, DC: The National Academies Press. doi: 10.17226/1433.
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RESPONSES TO GONADOTROPINS IN DOMESTIC ANIMALS J.E. Fortune INTRODUCTION Superovulation and embryo transfer in domestic animals and human in vitro fertilization (I9F) programs have in common the use of large doses of gonadotropins to "persuade" additional follicles to grow to preovulatory size and to produce fertilizable oocytes that will develop into normal young. It has been known since the 1940's that both pituitary gonadotropins, LH and FSH, interact with the developing preovulatory follicle and are essential to its growth and differentiation (Fevold, 1941; creep et al., 1942; Fraenkel-Conrat et al., 1943~. Hence, protocols used for superovulation of animals and hyperstimulation of women in IVF programs are based on the idea that abnormally high doses of gonadotropins will overcome the normal restraints imposed by each species on the number of ovulations, and will recruit and select "extra" follicles for ovulation. In the first part of this manuscript I will review the effects of ovarian hyperstimu~ation with exogenous gonadotropins (or "superovulation") in cattle, sheep and goats, horses, and pigs. In the second part I will make suggestions for future directions for research with animal models, directions that may generate new information to improve protocols for superovulation in both domestic animals and women. Superovulation In Domestic Animals Much of the impetus for the use of gonadotropins to stimulate superovulation in domestic animals comes from the desire to exploit the genetic potential of superior females. Artificial insemination, especially in the cattle industry, has made possible a very wide dissemination of the genes of superior males. It is estimated that popular bulls may sire around 50,000 calves per year (Seidel, 1981~. Since the use of frozen semen allows reproduction by males even after death, the genes of a highly desirable male can be disseminated widely through a population. However, the genetic improvement of species or breeds is still limited by the small number of offspring that an individual female can produce in her lifetime. Repeated superovulation of superior females and the collection and transfer of their embryos to less valuable recipients greatly enhances the reproductive potential of these females and makes it 130 -

possible to disseminate their genes more widely than could be achieved through natural pregnancies and more directly than through ~natural" male offspring. Among the domestic animals, superovulation and embryo transfer have had the greatest impact on the cattle industry. As techniques for ovarian stimulation and for non-surgical embryo collection and transfer were developed and improved this industry has boomed. Therefore, will concentrate the discussion of superovulation in domestic animals primarily on cattle; other domestic species will be discussed more briefly in subsequent sections. Superovulation of cattle ~ How are cattle superovulated? The bovine estrous cycle averages 21 days in length (Day 0 = day of estrus behavior). Ovulation occurs on Day 1 and is followed by a luteal phase of about 18 days and then a follicular phase of 2-4 days. The first experimental induction of superovulation in farm animals was reported by Casida et al. (1943) who used pituitary gonadotropins to superovulate cattle (for a review of the early experimental work see Willet, 1953~. Current superovu~atory protocols involve the injection of exogenous gonadotropin during mid-luteal phase (Day 8-14) to recruit extra follicles near the end of the luteal phase for ovulation following the next follicular phase. Several gonadotropin preparations have been used in commercial superovulation programs. In the 1970's pregnant mare's serum gonadotropin (PMSG, also called equine chorionic gonadotropin or eCG) was widely used for superovulating cattle. This gonado- tropin is produced by the equine conceptus and can be obtained from the blood of pregnant mares. Although it has only LH-like action in horses, PMSG has the interesting feature of binding to both AH and FSH receptors in other mammalian species (Stewart and Allen, 1979) and exhibiting both LH-like and FSH-like activity in bioassays (PapRoff, 1981~. Therefore, its effects as a superovu- lating treatment are probably due to its capacity to mimic the actions of both pituitary gonadotropins. The advantage of PMSG in superovulation of domestic animals is that it can be given as a single injection to produce multiple ovulations several days later. This is because the higher sialic acid content of PMSG confers on it a much longer half-life (the half-life appears to have two components - a shorter one of 40-50 h and a longer of 118-123 h; Schams et al., 1978) than the pituitary gonadotropins. However, the long half-life of PMSG is also a disadvantage, since the gonadotropin continues to stimulate the ovaries over a long period of time and this can lead to asynchronous ovulations and large unovulated follicles on the ovaries after ovulation. Although attempts have been made to attenuate PMSG's long half-life by injecting anti-PMSG just before ovulation (see 131 -

below), the use of pituitary gonadotropins has gradually displaced PMSG for superovulation of cattle. The most widely used are commercially available preparations of FSH extracted from pig (or other domestic animal) pituitaries, referred to as ~FSH-P". These preparations usually contain high and variable levels of contaminating AH and are typically given as a series of 8 injections of either equal or decreasing strength for 4 days, beginning around Day 10 of the cycle. Superovulation with pituitary gonadotropins was reported to produce more transferrable embryos (Critser et al., 1980) and pregnancies {Elsden et al., 1978) than superovulation with PMSG. In a few studies human menopausal gonadotropin (HMO) has been used successfully to produce multiple ovulations in cattle (Laurie et al., 1982a,b), but it is not used commercially. Early in the history of bovine superovulation, it became apparent that the variable length of the estrous cycle in cattle makes it difficult to control the application of gonadotropins relative to the time of luteolysis. Normal estrous cycles can vary in length from around 18-26 days and, in contrast to the situation in humans, it is the luteal phase that is the most variable. Therefore, current superovulatory regimens with PMSG or pituitary gonadotropins include the injection of prostaglandin F2a (PGF;a) to regress the corpus luteum (CL) and reduce the variability in the interval from gonadotropin injection to estrus (Betteridge, 1977; Elsden et al., 1978; Seidel, 19813. The use of PGF<a to regress the CL also makes superovulation more economical, since it allows the cycles of recipients to be synchronized with that of the donor female. Many more potential recipients are needed if their cycles are not synchronized with that of the donor. With recently developed techniques for freezing embryos, synchronized recipients are not quite as critical. - Superovulatory regimens for cattle do not usually include the injection of hCG or any other gonadotropin to trigger ovulation. With the protocols used ovulation occurs spontaneously and injection of hCG does not seem to improve the outcome. A. How successful are superovulatory treatments in cattle? The protocols currently used to stimulate bovine follicular development - i.e., a single dose of PMSG or multiple doses of porcine pituitary gonadotropins, with regression of the CL by PGF2~ - are successful in producing multiple ovulations and transferrable embryos (see Table 1~. The endpoints that have been used to look at the success of superovulation have varied - number of corpora lutea, number of ova/embryos recovered, nether of "good" or ~transferrable" embryos recovered, or number of young born to recipients. It is generally agreed that, since 132 -

numbers of CLs or ova/embryos recovered can give misleading ideas of success if the ova/embryos are either not recovered or not developing properly, the important endpoint is number of transferrable embryos. Hence, it is important to look at success in terms of transferrable embryos, rather than numbers of ovulations. The success of superovulation depends partially on when the embryos are collected. It was found that collection of embryos on Day 6-8 after estrus is optimal, since they can be recovered non-surgically, and in good condition, from the uterus at that time (Hasler et al., 1983, 1987; Seidel, 1981) The major point that should be made about the success of superovulation in cattle is that it is highly variable. While it may be very successful, yielding many good embryos from one donor, it may fad] miserably with others. A number of recent reviews on superovulation in cattle have emphasized this lack of consistency as a major problem (Bindon et al., 1986; Foote and Ellington, 1988; Monniaux et al., 1983; Moor et al., 1984; Murphy et al., lg84; Seidel, 1981). There is variability both among animals and also within the same animal from one treatment cycle to the next. The variability that one finds in surveying the literature has a number of sources. Season, breed, gonadotropin preparation, number of previous superovulations, and dose and timing of gonadotropin preparations may all contribute to variability of response. The literature on these sources of variation will be reviewed briefly. It has been reported that different breeds of cattle treated with the superovulatory regimens respond differently (Betteridge, 1977; Saumande, et at., 1978). There are conflicting reports on effects of season of the year on the superovulatory response, with some authors finding differences with season (Hasler et al., 1983) and others reporting no difference (Critser et al., 1980; Hasler et al., 1987; see Betteridge, 1977 for review of earlier literature). The potential existence of variation due to breed and/or season makes comparisons among studies much more difficult. Since the goal of bovine superovulation is to reproduce a valuable female as many times as possible, a critical question is the number of times a female can be successfully superovulated and whether repeated superovulation leads to refractoriness or a lowered response. It seems possible that animals treated repeatedly with heterologous gonadotropins could develop antibodies towards them that would lessen or prevent the response. Hasler et al. (1983) have reported that ovulation rate remained the same over the course of ten superovulations, but the number of fertilized eggs declined. Donaldson and Perry (1983) also found that transferrable embryos decreased with repeated

superovulations. In contrast a decline in the number of viable embryos was not observed in studies in which animals were superovulated fewer times (Moor et al., 1984; Lubbadeh et al., 1980). Since the timing of injections of gonadotropins, relative to luteolysis and relative to changes in follicular populations, may be critical to the success of superovulation, several investigators have examined the effects of injecting gonadotropins at different times during the cycle. Donaldson (1984) reported that there was no difference in the nether of embryos or transferrable embryos when FSH-P was administered on Day 9, 10, 11, 12, or 13 of the cycle (with PGF2a given on the third day of treatment). Using a similar protocol, Hasler et al. (1983) reported similar responses to treatment begun on Days 8- 13, except that the responses of infertile cows were lower on Days 8 and 13. However, Lerner et al.~1986) observed that greater numbers of embryos plus ova were recovered when treatment began on Day 10 or 11, as compared with Day 7-9 or Day 12-14. The effects of varying the dose of gonadotropin on the success of superovulation have been examined. With older donors increasing doses of FSH-P were associated with increases in the recovery of ova plus embryos, whereas increasing doses of FSH had the opposite effect on younger donors (Lerner et al., 1986~. Variability among gonadotropin preparations, even from the same supplier, may explain part of the variability observed in different superovulation trials. Murphy et al (1984) reported that the amount of AH vs. FSH immuno- or big-activity varies considerably among different preparations of PMSG and FSH-P. They found that preparations with lower ratios of FSH/LH are less successful in producing ovulations. Donaldson et al. (1986) removed most of the contaminating LH from FSH-P and found that their FSH preparation, which had no detectable AH activity, did not increase the total number of embryos, but did increase the number and percent transferrable embryos. A subsequent field study further confirmed the efficacy of the purified FSH preparation (Donaldson and Ward, 1986~. Even when the factors mentioned above are controlled for - i.e. when animals of the same breed are given the same dose of the same gonadotropin preparation at the same time of the cycle and the same time of year, and number of previous superovulations is controlled for, there is still tremendous variability in the response among, and within, animals. Although part of this - 134

variability may be explained by the treatment of donors inadvertently at inappropriate times of the cycle (Britt and Holt, 1988), it also seems clear that there is much unexplained variability. It seems logical to conclude that this unexplained variability comes from individual genetic differences among animals that determine how they respond to supra-physiological doses of gonadotropins. The next section addresses the question of what is currently being done to try to increase the uniformity of the response. c. Current strategies to improve the success of superovulation. One goal is to determine an optimal gonadotropin treatment. This has not been easy because of the variability of the preparations that have been marketed. Several investigators have concluded that preparations with low ratios of FSH to LH give poorer superovulatory responses. Several investigators have tested this idea by using highly purified FSH and adding known amounts of AH to it in attempts to determine the optimum FSH/LH ratio (Chupin et al., 1987; Donaldson and Ward, 1986; Murphy et al., 1984~. Another recent suggestion is that it may be helpful to prime the animals with injections of FSH early in the cycle to recruit more small follicles to grow prior to the superovulatory treatment in mid-cycle (Rajamahendran et al., 1987; Ware et al., 1988~. PMSG is attractive as a superovulatory gonadotropin because it need only be injected once, as opposed to 8 injections of FSH-P. Therefore, several groups have tried to improve the response to PMSG by injecting an antiserum to PMSG around the time of ovulation to effectively shorten its biologically active life in the animals and to limit the effects of PMSG on the ovary to the follicular phase (Dieleman et al., 1987; Saumande et al., 1984; Wang et al., 1987~. Some investigators have attempted to analyze what may be going wrong when superovulation fails by comparing endocrine profiles and follicular characteristics of superovulated cattle with normal cattle. As would be expected, estradiol is higher prior to ovulation and progesterone is higher following ovulation in superovulated cattle as compared with controls (Henricks et al., 1973~. Plasma levels of estradiol before ovulation and progesterone after ovulation are positively correlated with the number of ovulations (Saumande, 1980; Saumande and Batra, 1985~. Very high levels of estradiol after superovulation with PMSG are probably caused by the presence of large, unovulated follicles (Booth et al., 1975) e Although the magnitude of the AH and FSH surges appears to be normal in superovulated animals, in some cases their timing is abnormal and they may not be coincident (Saumande, 1980; Donaldson, 1985~. Further evidence of lack of normal coordination of endocrine events in superovulated animals comes from studies of individual follicles. Profiles of steroids - 135 -

in the follicular fluid varied from those seen in control animals at the same time relative to LH surge and there was considerable variability among superovulated animals (Fortune and Hansel, 198S). Callesen et al. (1986) found that in superovulated animals relatively no ~ al plasma levels of progesterone and LR were associated with normal follicular steroid levels, but that animals with deviating patterns of AH and progesterone during the pre- and pert-ovulatory periods had abnormal progesterone: estradiol ratios in follicular fluid and prematurely activated or meiotically arrested oocytes. Moor et al. (1984) concluded that premature activation of oocytes during superovulation protocols causes significant loss of potential embryos. d. Summary of superovulation in cattle. Superovulation of cattle using a combination of gonadotropin treatment and PGF2a is ~ multi-million dollar industry in the United States. Yet it is clear that the variability of the ovarian response in terms of numbers of ovulations, but more importantly in terms of numbers of good embryos, is a definite limitation to its successful use. There are no obvious solutions to this problem and the approaches being taken now represent a fine-tuning of the system that may lead to only slight improvements, rather than more radical approaches that might lead to more substantive improvements. Recommendations for research that might provide new insight into this problem are discussed in the last section of this paper. Superovulation of Sheep and Goats Sheep and goats have also been successfully superovulated for embryo transfer. Induction of multiple pregnancies with gonadotropin treatment was reported as early as 1944 (Casida et al., 1944~. A number of studies since that time have demonstrated that ewes can be synchronized by treatment with progestagens, and multiple ovulations readily induced by a single injection of PMSG given one day before the end of progestagen treatment or by multiple injections of pituitary extracts (see Wright et al., 1981 for references). Australian scientists have been especially active in this area of research (see Shelton et al., 1982 for reviews). Since most breeds of sheep are seasonal breeders, the induction of ovulation during the anestrous season is of interest. The use of exogenous gonadotropins for this purpose has been explored, beginning with the successful induction of ovulation by Cole and Miller (1933~. An interesting aspect of research on superovulation in sheep is that some approaches have bypassed gonadotropin treatment entirely, using more indirect means to raise endogenous gonadotropin levels and thereby recruit excess follicles. It is believed that inhibin made by the ovulatory follicle suppresses basal levels of FSH in the blood during the follicular phase and - 136 -

thereby helps ensure the dominance of the dominant (ovulatory) follicle. It is known that one of the prolific sheep breeds, the Booroola, which typically ovulate about five oocytes, has lower levels of inhibin than less prolific breeds of sheep (Bindon et al, 1986~. Injections of antibodies against inhibin have the potential of increasing FSH levels and recruiting additional follicles to ovulate (Bindon et al., 1986~. Another approach to increasing ovulation rate is immunization against steroids. Scaramuzzi et al. (1977) first reported an increase in ovulation rate from I.1 to 2.1 in sheep immunized against androstenedione. Induction of superovulation with gonadotropin preparations has been particularly successful in goats (Armstrong and Evans, 1983~. The ability to reproduce genetically superior animals is especially important for valuable breeds, such as Angora goats. A purebred Angora female is very expensive, but through superovulation and embryo transfer, she can potentially produce many embryos that can be transferred to non-Angora recipients. Results of experiments performed in Australia and Canada by Armstrong et al. (1983a,b) showed that injection of porcine FSH in decreasing doses from day 12-15 of the estrous cycle resulted in a higher mean ovulation rate (17.6 ~ 5.5/ewe) than injection of PMSG on day 12 (10.1 + 3.0) of the cycle in feral goats (Armstrong et al., 1983a). Application of these two gonadotropin treatments to Angora females in a second study gave almost identical results (Armstrong et al., 1983b). In the second study transfer of the embryos to recipients resulted in equal rates of survival for embryos from FSH vs. PMSG-treated females. However, these authors found that survival was better when recipients had 2 or 3 corpora lutea (CL) as compared with 1 CL (63 and 75% vs. 52%) and that transfer of two embryos to recipients increased their chances of survival (65%) as compared with transfers of single embryos (65% vs. 48% survival). Effects of Exogenous Gonadotropins on Horses There have also been attempts to superovulate mares, with a view towards embryo transfer. Horses are seasonally polyestrus; their estrous cycles average 21-22 days in length and consist of a period of diestrus for about 15 days following ovulation, followed by an estrous period that averages about 7 days (Ginther, 1979~. The mare is unique among the domestic animals in having an estrous period that is long and variable in length, lasting 2-12 days (Ginther, 1979~. AH rises progressively during the estrous period, but does not peak until after ovulation. In contrast, FSH is low during most of the estrous period, but there appears to be a surge of FSH coincident with the LH peak following ovulation and a second rise in FSH at about Day 10 of the cycle (reviewed by Irvine, 19811. 137 -

In contrast to cattle, where the effect of exogenous gonadotropins is to dramatically increase the number of ovulatory follicles, injections of gonadotropins have not produced large numbers of synchronous ovulations in mares. PMSG, even in large doses, is not effective in inducing multiple ovulations (Ginther, 1979~. Its lack of effectiveness in horses, contrary to the marked response observed when cattle are injected with PMSG, is probably due to the fact that PMSG is an DH-like hormone in horses. It binds with only about 4% the affinity of AH to equine LEI receptors and binds not at all to equine FSH receptors (Stewart and Allen, 1979~. This is in contrast to the actions of PMSG in other species, in which it binds to both AH and FSH receptors. It appears that its lack of FSH action in mares makes PMSG ineffective and unsuitable for inducing multiple ovulation in this species. Treatment of mares with other gonadotropic preparations has been effective in inducing ovulation in seasonally anestrus mares and in increasing the ovulation rate in mares treated during the breeding season. In these studies pony mares or horse mares have been treated with either a crude extract of equine pituitary glands, an acetone extract of equine pituitary glands, or a commercially available preparation of porcine FSH (FSH-P, which probably also contains some LH). In some studies hCG has been injected at or towards the end of the treatment with ESH. Such treatments have been effective in inducing a high percentage of treated mares to ovulate outside the breeding season (>90%) and a number of these mares had multiple ovulations (overall average for mares ovulating = 2.1; Douglas et al., 1974; Lapin and Ginther, 1977; Woods et al., 1982~. Treatment with these gonadotropin preparations for 5 to 7 days during the breeding season has also induced multiple ovulations. Treatment with FSH- P in two studies resulted in ovulation rates of 1.6 and 1.7 ovulations per mare (Irvine, 1981; Squires et al., 1986), whereas ovulation rates of 2-3 ovulations per mare were observed when equine pituitary extract was injected (Lapin and Ginther, 1977; Douglas, 1979; Woods and Ginther, 1983a; Squires et al., 1986~. Ovulation rates for controls were always one, or very close to one, per mare. The inclusion of an injection of hCG near the end of treatment with FSH produced of rations that were more synchronous and treatments that began on Day 11-15 produced more ovulations than treatments beginning on Day 19 (Woods and Ginther, 1983a). In summary, the use of gonadotropins to stimulate ovulation in mares has progressed much less than with cattle and sheep and there is currently no commercially available preparation for superovulating horses. There appear to be several reasons for lack of progress with this species. First, the ovaries of mares seem much more resistant than ovine and bovine ovaries to superovulatory treatments. There may be tighter intrinsic control over ovulation rate in horses than in species like · 138 -

cattle, sheep, and humans. Although natural double ovulations are not uncommon (Woods and Ginther, 1983b), the birth of twins is rare in horses, indicating that there are mechanisms for preventing the development of twins when two follicles ovulate (Ginther et al., 1982; Woods and Ginther, 1983b; Ginther 1984~. Second, the variable length of estrus makes embryo transfer logistically more difficult than in other species, since it is harder to synchronize the donor and recipients. Finally, many purebred organizations are conservative and stud-book regulations prohibit most artificial techniques for reproduction. However, there is a need to understand better the regulation of follicular development in mares in order to develop better treatments for infertility and because the development of methods for superovulating mares could be of particular benefit in preserving and increasing numbers of individuals in endangered equine species, through transfer of embryos to recipients of similar but non-endangered species. Ovulation Induction in Pigs The commercial applications of superovulation and embryo transfer techniques are more limited for swine than for the other domestic species discussed above. Since sows give birth to litters and have a relatively short gestation period (about 110 days) , each fertile female is naturally capable of producing numerous progeny. However, superovulation and embryo transfer could still be used, theoretically, to increase the rate of reproduction of genetically superior animals. In the 1960's several research groups successfully ovulated or superovulated pigs with exogenous gonadotropins (Day et al., 1967; Pope et al., 1968; Bazer et al., 1969~. James and Reeser (1979) showed that ovulation could be induced and embryos collected (surgically) repeatedly in the same sows. In their study follicular development was ensured by the injection of PMSG on Day 16 of the estrous cycle, at 15-20 days of lactation, or at weaning 21 to 35 days post-partum. Ovulation was timed by injecting hCG 72-96 h after PMSG injection. This treatment was applied and embryos were recovered an average of 4.S times per donor with an average recovery rate of 15.6 transferable embryos per surgery. It should be noted that the gonadotropin treatments were used more to time follicle development and ovulation, than to ~superovulate~ the animals, since pigs would be expected normally to ovulate around 15 oocytes. However, these results were encouraging because they showed that embryos could be surgically collected repeatedly at 3 to 6 week intervals without the formation of extensive adhesions or any other apparent impairments of reproductive function. Other studies have shown that females can be superovulated with a single dose of PMSG on Day 15 or 16 of the cycle. Doses of 500, 750, 1000, 1250, and 1500 i.~. resulted in mean numbers of CLs of 15, 25, 26, 25, and 38, respectively (Hunter, 1980~. Despite these positive results, - 139

gonadotropin treatments and embryo transfer are not much used to increase the genetic potential of superior female gilts or sows. In 1981 only 8 of 13,000 purebred seedstock producers in the U.S. used embryo transfer primarily to exploit genetically superior females (Martin, 1983~. One reason for this is the difficulty of identifying superior sows. Therefore, the use of superovulation and/or embryo transfer for genetic improvement is not a high priority in the swine industry. embryo transfer in swine has advantages other than potential improvement of the gene pool. Disease is a major concern in the swine industry. Herds have increased in size and they are frequently confined to environmentally controlled buildings. Both these conditions encourage the spread of disease and some herds that are disease-free try to maintain that condition by becoming ~closed" (i.e. not allowing new animals to be added to the herd). Embryo transfer offers a means by which new genetic material can be introduced into closed herds, without the risk of also introducing disease vectors (Martin, 1983~. Embryo transfer also provides a means of repopulating diseased herds and of exporting animals to other countries. Finally, embryo transfer has proven to be a useful tool in research on pigs, especially in studies of maternal-fetal interactions (e.g., Pope et al., 1972; Rampacek et al., 1975~. In summary, embryo transfer in swine has not been exploited to increase the reproductive potential of superior females, as it has in cattle, sheep, and goats. The applications of this technique to swine center more on prevention of the spread of disease and on research applications. Although gonadotropin treatments have been used to ensure and time follicular development and ovulation, they have not been used to significantly enhance the already high ovulation rate of pigs. Recommendations For Future Directions In Research It is clear that while superovulation of domestic animals, especially cattle, has produced some spectacular successes, the current techniques have the important limitation of variable and unpredictable response. Superovulation is currently the weak link in the reproduction of genetically superior animals through superovulation and embryo transfer. Techniques for non-surgical collection and transfer of embryos have been well developed and major advances have been made in the cryopreservation, splitting, and sexing of embryos. However, in the last decade there has been little improvement in the success of superovulation in cattle. What kinds of research might lead to substantive improvements in the regulation of female reproduction in cattle? ~ believe that several approaches should be supported simultaneously. - 140 -

Basic Research on Bovine Ovarian Function. Current techniques for superovulating domestic animals are based on a very minimal understanding of the regulation of follicular growth and differentiation. They depend on the knowledge that AH and FSH are important for the development of ovulatory follicles and that PGF2a will cause regression of the corpus luteum. Although we do know something about the effects of gonadotropins on cultured theca and granulosa from the preovulatory follicle (McNatty et al., 1984a,b; Fortune, 1986, Fortune and Quirk, 1988; Fortune et al., 1988), we know little about the specific require- ments for gonadotropin support during the course of development of ovulatory follicles. Hence, doses and times of injection of gonadotropins in superovulatory regimes have been developed empirically. Basic research on the regulation of luteal function led to the discovery that PGF2a is involved in the regression of the corpus luteum. This led to the use of PGF2a and its analogues to synchronize the estrous cycles of cattle, a development that has had an enormous impact on superovulation and embryo transfer. I do not believe that more than small improvements in the success - of superovulation will be made until our knowledge of the mechanisms that regulate the development and regression of follicles during normal estrous cycles is increased. One exciting and promising recent development is the use of intra- rectal ultrasonography to visualize bovine ovaries. This technique was pioneered by Pierson and Ginther (1984) who showed that it was possible to see and count follicles on bovine ovaries. My laboratory has developed the use of ultrasonography to follow the development and/or regression of individual follicles over time by videotaping daily ultrasound exams (Quirk et al., 1986~. The tapes can be reviewed and analyzed to reproduce the patterns of growth and regression of individual follicles. ~ en we used these techniques to determine the pattern of follicular development through the course of the estrous cycle, the results were surprising in that they showed in all ten heifers examined a regular pattern of waves of follicuiar development that begin about every seven days (Figure 1; Sirois and Fortune, 1988), with one follicle in each wave growing larger, while the others regress. The regularity of these waves is exciting because it suggests a number of ways in which the development and regression of follicles can be studied experimentally. We can ask questions about~what governs the timing of these waves. Why do the dominant follicles of the first and second waves not ovulate and what would stimulate them to ovulate? Knowledge of the pattern of these waves may also be directly applicable to superovulation protocols. For example, is there any relationship between the nor her of follicles in the waves and the superovulatory responses of individual animals? 141 ~

Can gonadotropin injections be better timed to take advantage of the waves? When would the application of gonadotropins encourage more follicles to join a wave or overcome the mechanisms by which the largest follicle exerts dominance? Through research such as outlined above we can gain insight into why superovulation so frequently fails to produce the desired large numbers of embryos. However, the number of offspring produced by a species has evolved after millions of years of selection. We are trying to subvert biological mechanisms that are very important to a species when we attempt to superovulate. With cattle there seems to be much variability among animals in terms of the extent to which these biological control mechanisms can be overcome. It may not be possible in the near future to reliably superovulate all, or even most, individuals in a domestic species. Therefore, it might make more sense to develop subtler approaches to hyper-stimulation - i.e., through better knowledge of critical times for gonadotropin action during follicular development, a protocol might be developed that could reliably produce 5-6 good embryos per treatment. Currently, large doses of gonadotropins are given in the hope that large numbers of ovulations will result. The animals must then be allowed several cycles to recover from the perturbations that result. If gentler, but more specific treatments were applied, animals might be superovulated for many cycles consecutively, leading to higher yields of embryos over a given period of time. A better understanding of follicular dynamics and their regulation by gonadotropins might also lead to an ability to fine-tune superovulation to the extent that animals would reliably o w late two oocytes, which they could then carry to term to produce twins. This could increase the reproductive capacity of less valuable animals that were not candidates for supero w ration. Although twin births cause more problems at parturition and half of female twins would be freemartins, there would still be advantages to twinning, especially in the beef industry. Other Approaches To Increasing Reproductive Capacity. In the paragraphs above ~ have discussed potentially fruitful directions for basic research that could lead to improvements in the use of gonadotropins to superovulate domestic animals. At the same time, ~ believe that other approaches that do not involve gonadotropins should be explored. First, the recently isolated gonadal hormone inhibin has great potential as a regulator of fertility. This protein hormone is believed to be secreted by granulosa cells and feeds back on the pituitary to regulate selectively the secretion of FSH. It has been proposed that one follicle becomes dominant (ovulatory) because it is at T42 -

the right stage of its development to benefit from increases in plasma FSH. It then begins secreting estradiol and inhibin which feed back on the hypothalamus-pituitary to suppress FSH secretion, thereby depriving slightly smaller follicles of the critical amount of FSH needed to continue development towards ovulation. One approach, discussed above, is to identify critical times when the application of extra FSH will lead to development of additional follicles that are fairly synchronous with the ovulatory follicle. Another approach would be passive or active immunization against inhibin. It has been found that the Booroola breed of sheep, a natural superovulator, has lower levels of inhibin than less 1986). ~ prolific breeds (Bindon et al., Although inhibin injections selectively suppress FSH, the ~rebound" of FSH after the injections can cause an increase in ovulation rate (McNeilly and Wallace, 1987~. Research with inhibin is now hampered by lack of availability of purified inhibin to most researchers. Availability of purified inhibin would greatly stimulate research on this potential regulator of fertility. - Second, studies on the phenomenon of follicular dominance should be encouraged. We know little about the mechanisms by which one follicle suppresses others in its cohort, yet it is these mechanisms that we try to subvert in superovulation. The production of inhibin is one potential mechanism, but there is also evidence for the production of other substances by dominant follicles, such as follicle growth inhibitor (Bindon et al., 1986) and follicle regulatory protein (Ono et al., 1986) that may act more directly to suppress the development of other follicles. Third, approaches that do not involve attempts to manipulate the endocrine milieu should be explored. Especially promising is the idea of maturing and fertilizing bovine oocytes in vitro for transfer to foster mothers. Bovine ovaries contain around 150,000 primordial follicles at birth and thousands of them are maintained in a healthy state until about 4-6 years of age (Erickson, 1966), even though only a small number ever ovulate. Most of these oocytes reside in preantral follicles and have not acquired the ability to undergo spontaneous maturation if released from their follicles (meiotic competence). However, John Eppig's group at the Jackson Laboratory has been successful in isolating oocytes in preantral mouse ~follicles, growing them in vitro to meiotic competence, and producing live young (Eppig, personal communication). Adaptation of these techniques to cattle ovaries could be very exciting. -For example, one or both ovaries could be removed from a valuable heifer calf and preantral follicles obtained, grown to meiotic competence, and fertilized in vitro. The embryos could then be frozen and transferred to recipients as desired. In this way information could be gained about the characteristics of a female's progeny before she herself had even reached puberty. A fourth potentially productive area is research on genetic selection for 143 -

twinning in cattle or superfecundity in other domestic species (Bindon et al.,1986~. SCARY The use of commercially available gonadotropin preparations, coupled with the use of PGF2a to control the time of regression of the corpus luteum, has resulted in the ability to produce excess ovulations in cattle, sheep, pigs, and mares. These techniques have been most widely and successfully used with cattle and are key to the success of the wider dissemination of genes of valuable bovine females through embryo transfer. However, the variability in the response to exogenous gonadotropins is a major stumbling block to improvements in the success of superovulation in cattle and other domestic species. ~ have suggested a number of approaches to solving this problem. These include: 1) basic research on follicular dynamics and the roles of pituitary gonadotropins in the regulation of follicular growth and differentiation, 2) basic research on the role of inhibin in ovarian regulation in cattle and other species, 3) continued research on other mechanisms by which follicles may exert dominance, 4) the adaptation of techniques for growing mouse oocytes to meiotic competence in vitro to domestic species, 5) continuation of studies on genetic selection for multiple ovulation. I believe that we need a broad range of approaches for several reasons. First, this field is at a stalemate right now and it is hard to predict where the next big breakthrough might come. Second, it seems desirable to think in terms of different approaches for different types of animals - meiotic maturation and in vitro fertilization of thousands of oocytes from the most valuable cows, better or more reliable techniques for using gonadotropins to superovulate valuable cows, and endocrine or genetic methods for producing twins in less valuable animals. The cattle industry would benefit from having more available options. Also, a wider range of options increases the chance that some of them would also be useful in developing countries, where farmers may be unable to benefit from some techniques that work well for American producers. It seems well to remember that a high percentage of the worId's domestic animals live in developing countries and that increases in their reproduction are very crucial to health and nutrition worId- wide. Finally, cattle are an excellent model for humans and a wide variety of approaches to studying the regulation of follicle development in cattle could well generate new ideas and approaches f or regulating f ertility in women. 144 -

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Shelton, J. N., A. 0. Trounson, N. W. Moore, and J. W. James (eds.~. 1982. Embryo Transfer in Cattle, Sheep and Goats. Aust. Soc. Reproductive Biology Publication. Treasurer, ASRB, University of Sydney Farms, Camden, Australia, 2570. Sirois, J. and J. E. Fortune. 1988. Follicular dynamics during the estrous cycle in heifer" monitored by real-time ultrasonography. Biology of Reproduction 39:308-317. Squires, E. L., R. H. Garcia, 0. J. Ginther, J. L. Voss, and G. E. Seidel, Jr. 1986. Comparison of equine pituitary extract and follicle stimulating hormone for superovulating mares. Theriogenology 26:661-670. Stewart, F. and W. R. Allen. 1979. The binding of FSH, AH and PMSG to equine gonadal tissues. J. Reprod. Fert. Suppl. 27:431-440. ~ Wang, H., M. Wu, X. Xu, W. C. Hagele, and R. J. Mapletoft. 1987. Control of superovulation in the cow with a PMSG antiserum. Theriogenology 27:291. Ware, C. B., D. L. Northey, M. P. Boland, and N. L. First. 1988. Early cycle FSH-p priming as a prelude to superovulating gonadotropin administration in ewes and heifers. Animal Reproduction Science 16:97-105. Willet, E. L. 1953. Egg transfer and superovulation in fat animals. Iowa State College Journal of Science 28:83-100. Woods, G. L., and O. J. Ginther. 1983a. Induction of multiple ovulations during the ovulatory season in mares. Theriogenology 20:347-355. Woods, G. L., and O. J. Ginther. 1983b. Recent studies relating to the collection of multiple embryos in mares. Theriogenology 19:101-108. Woods, G. L., S. T. Scraba and O. J. Ginther. 1982. Prospects for induction of multiple ovulations and collection of multiple embryos in the mare. Theriogenology 17:61-72. Wright, R. W., Jr., K. Bondioli, J. Grammer, F. Kuzan, and A. Menino, Jr. 1981. FSH or FSH plus LH superovulation in ewes following estrus synchronization with medoxyprogesterone acetate pessaries. J. Anim. Sci. 52:115-118. - 151 -

TABLE 1. Superovulation An Cattle: Results fro Too Co crcial Ego Transfer Program: Hasler et al., 1983a Donaldson, 1984b # Animals 984 1,263 Mean Ova/Donor S.9 10.1 <0-70) He an Embryos/Donor . 5.1 . -- (0-38) Good Embryos/Donor 4.6 4.5 (0-37) No Embryos (' Donors) 36% 15% (32.4% - no good embryos) Reported in Theriogenology 19: 83-99 sported in Theriogenology 21: 517-524 - 152 8

. TABLE 2. Di~;~u=~m of pre~:tes frog tar ems. Prey cold by palpation though he rectal ·811 between day'; 90 Id 100 of ~eseation. (Seidel, Science 211: 354, !981.) Donors Pregnancies from S - exhalation (no. - Age (years) Breed First Second Third 1 Submental 4 7 ~ 1 Simmental S ~ 0 9 4 Cousin 0 0 1 2 Hereford S l 4 2 Pinzgauer 6 1 0 2 Pinzgauer 4 8 20 13 Angus . 4 2 8 lo Hereford 2 0 5 9 Hereford 9 3 7 1 Simmen"1 7 O O 1 Submental 2 1 0 4 Charolais 7 9 0 7 Brangus 2 0 1 2 S1mmen"l 5 11 4 6 Hereford 1 11 S 8 Hereford 2 0 ~ 14 Hereford 12 0 1 12 Hereford 0 3 2 2 Hereford 3 1 4 4 Angus - 3 0 2 7 Hereford 4 7 2 5 Angus 4 2 0 5 Angus 4 3 0 5 Angus 4 9 0 4 Submental ~ ]. 0 5 Angus 0 7 2 4 Hereford 0 4 ~ 10 Angus 0 6 0 1 Si~enta1 0 2 O 5 Angus 3 6 O 3 Simmental 3 4 14 Average 3 4 3 5 3 4 - ~ 5 3 -

17 15 _% E 33- _ . Il..! ~ 1 :E C) w _ . ., o S3 -7 -6 -5 0 -4 ~ ;D O Z -3 / / 1 / N; I ~ - * l / ~ - \ f ` \ / , rim Am_' ~ ~ \ ~ \ / 1~. m\\ 17 19 0 1 ~ 5 ~ 9 11 13 15 17 19 0 2 DAY OF CYCLE - 2 1 O Figure I. Pattern of growth and regression of follicles (solid lines) during a complete cstrous cycle. T.ne asterisk indicates the last day on - ich the ovulatory follicle was observed and the dashes line shows progesterone concentrations arotmd the the of luteolysis. - 154 -

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This book results from a study by a committee of the Institute of Medicine and the National Research Council's Board on Agriculture. The committee examined the scientific foundations of medically assisted conception and developed an agenda for basic research in reproductive and developmental biology that would contribute to advances in the clinical and agricultural practice of in vitro fertilization and embryo transfer. The volume also discusses some barriers to progress in research and ways of lowering them, and explains the scientific issues important to ethical decision making.

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