Superovulation in Cattle

Chapter 75
Superovulation in Cattle

Reuben J. Mapletoft1 and Gabriel A. Bó2

1 Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

2 Instituto de Reproducción Animal Córdoba (IRAC), Cno. General Paz – Paraje Pozo del Tigre- Estación General Paz, CP 5145, Córdoba, Argentina


The objective of superovulation in cattle is to maximize the number of fertilized and transferable embryos with a high probability of producing pregnancies.1 However, wide ranges in superovulatory responses and embryo yield have been reported in reviews of commercial embryo transfer records. In 2048 beef donor collections, a mean of 11.5 ova/embryos with 6.2 transferable embryos was recorded.2 Variability was great, both in the superovulatory response and embryo quality; 24% of the collections did not yield viable embryos, 64% produced fewer than average numbers of transferable embryos, and 30% yielded 70% of the embryos. Embryo recovery from 987 dairy cows yielded slightly fewer ova/embryos but there was similar variability.3 The high degree of unpredictability in superovulatory response creates problems that affect both the efficiency and profitability of embryo transfer programs.4

Variability in ovarian response has been related to differences in superstimulatory treatments, such as gonadotropin preparation, batch and total dose of gonadotropins, duration and timing of treatments, and the use of additional hormones. Additional factors, which may be more important, are inherent to the animal and its environment. These may include nutritional status, reproductive history, age, season, breed, effects of repeated superstimulation, and ovarian status at the time of treatment. While considerable progress has been made in the understanding of bovine reproductive physiology, factors inherent to the donor animal are only partially understood.

Gonadotropins and superovulation

Factors affecting superovulatory response associated with the administration of exogenous gonadotropins include source, batch, and biological activity.5 Three different types of gonadotropin preparations have been used to induce superovulation in the cow: gonadotropins from extracts of porcine or other domestic animal pituitaries, equine chorionic gonadotropin (eCG), and human menopausal gonadotropin.5,6 Human menopausal gonadotropin did not offer any advantages in cattle7 and has not been used commercially; therefore it will not be discussed further.

Pituitary extracts contain follicle-stimulating hormone (FSH). The biological half-life of FSH in the cow has been estimated to be 5 hours or less,8 so it must be injected twice daily to successfully induce superovulation.9,10 The usual treatment regimen is twice-daily intramuscular treatments with FSH for 4 or 5 days, with a total dose of 28–50 mg (Armour) of a crude pituitary extract, or 260–400 mg NIH-FSH-PI of a partially purified pituitary extract (Folltropin®-V, Bioniche Animal Health Inc., Belleville, Ontario, Canada). At 48 or 72 hours after initiation of treatment, prostaglandin (PG)F is administered to induce luteolysis. Estrus occurs in 36–48 hours, with ovulations beginning 24–36 hours later.11,12

Equine chorionic gonadotropin is a complex glycoprotein with both FSH and luteinizing hormone (LH) activity.13 It has been shown to have a half-life of 40 hours in the cow and persists for up to 10 days in the bovine circulation; thus it is normally injected intramuscularly once followed by a PGF injection 48 hours later.14 The long half-life of eCG causes continued ovarian stimulation, unovulated follicles, abnormal endocrine profiles, and reduced embryo quality.15–18 These problems have been largely overcome by the intravenous injection of antibodies to eCG at the time of the first insemination, 12–18 hours after the onset of estrus.14,19 Recommended doses of eCG range from 1500 to 3000 IU, with 2500 IU by intramuscular injection commonly chosen.

Monniaux et al.10 treated groups of cows with either 2500 IU eCG or 50 mg (Armour) pituitary FSH and observed that ovulation rate and the percentage of cows with more than three transferable embryos was slightly higher with FSH than eCG. Although these results were in agreement with those of Elsden et al.,20 others have found no differences between pituitary extracts containing FSH and eCG.21,22 Endocrine studies have revealed that eCG-treated animals more frequently had abnormal profiles of LH and progesterone,15,23 which were associated with reductions in both ovulation and fertilization rates24 as compared with FSH-treated cows.

Although folliculogenesis in mammals requires both FSH and LH, there is considerable variability in FSH and LH content of crude gonadotropin preparations.5 Radioreceptor assays and in vitro bioassays have revealed variability in both the FSH and LH activity of eCG, not only among pregnant mares but also within the same mare at different times during gestation.13 The effects of the FSH/LH ratio of eCG on superovulatory responses have been examined and there was a positive correlation between the ratio of FSH/LH activity and superovulatory response. Lower ratios of FSH/LH activity reduced ovulatory response in immature rats and LH added to eCG reduced superovulatory response in cows.5,13

Purified pituitary extracts with low LH contamination have been reported to improve superovulatory response in cattle. Chupin et al.25 superstimulated three groups of dairy cows with an equivalent amount of 450 μg pure porcine (p)FSH and varying amounts of LH and showed that the mean ovulation rate and the number of total and transferable embryos increased as the dose of LH decreased. It has been suggested that high levels of LH during superstimulation cause premature activation of the oocyte.16 Several experiments with an LH-reduced pituitary extract26 utilizing several different total doses, ranging from 100 to 900 mg of NIH-FSH-PI, revealed no evidence of detrimental effects of dose on embryo quality.6,27 On the other hand, doubling the dose of crude pituitary extracts containing both FSH and LH resulted in significantly reduced fertilization rates and percentages of transferable embryos.6 Collectively, data support the hypothesis that the detrimental effects of high doses of pituitary gonadotropins on ova/embryo quality is due to an excess of LH.

Although it is generally believed that some LH is required for successful superovulation, endogenous LH levels may be adequate. Looney et al.28 reported that recombinantly produced bovine (b)FSH induced high superovulatory responses without the addition of exogenous LH. These data suggest that LH is not needed in superstimulatory preparations and that embryo quality may be superior with pure FSH. The very high fertilization rates and transferable embryo rates in the absence of exogenous LH suggest that administration of LH in superstimulation protocols, at any dose, may be detrimental to embryo quality. Further, these results and more recent results with recombinant bFSH29 indicate that future progress lies in the use of recombinantly derived gonadotropins.

The effects of LH on superovulatory response has also been demonstrated in an experiment involving Brahman-cross (Bos indicus) heifers superstimulated with 400 mg NIH-FSH-PI containing 100%, 16% or 2% LH.30 Although the more purified preparations in this experiment caused higher superovulatory responses, there were obvious seasonal effects; responses with the most purified and intermediate preparations were superior to the least purified preparation during summer months, but only the most purified preparation was highly efficacious during winter months. These results would appear to contradict the findings of Page et al.31 who reported that superovulatory response and embryo quality in Holstein heifers was not affected by LH levels in cool weather, but that during heat stress a more purified preparation yielded more corpora lutea and significantly more fertilized ova and transferable embryos. It becomes apparent that stress is the common factor; Bos taurus breeds likely find summer heat stressful, whereas Bos indicus breeds likely find winter temperatures stressful. In either case, the more purified extracts resulted in greater superovulatory responses during conditions of environmental stress.

Follicle wave dynamics and superstimulation

It has been reported that superstimulatory response is greater if treatment is initiated before selection of a dominant follicle. In an early study recombinant bFSH given to heifers on day 1 of the cycle (ovulation = day 0), before the time of selection of the dominant follicle of wave 1, resulted in more ovulations than that given on day 5, after the time of selection.32 A subsequent study was done to determine if exogenous FSH given at the expected time of the endogenous wave-eliciting FSH surge had a positive effect on the superstimulatory response.33 As the endogenous surge of FSH was expected to peak 1 day before wave emergence,34 superstimulatory treatments were initiated on the day before, the day of, or 1 or 2 days after wave emergence. Significantly more follicles were recruited when treatments were initiated on the day of, or the day before, follicular wave emergence and more ovulations were detected when treatment was initiated on the day of wave emergence rather than the day before or 1 or 2 days after wave emergence. A subsequent study indicated that superovulation was induced with equal efficacy when treatments were initiated during the first or second follicular waves and that the superstimulatory response was optimized when treatment was initiated at the time of follicle wave emergence.35

Superstimulation: the traditional approach

In the very early days of bovine embryo transfer, treatment with eCG was made to coincide with natural regression of the corpus luteum (i.e., about day 16 of the cycle).12 With the introduction of PGF in the 1970s, it became possible to initiate gonadotropin treatments at other times during the estrous cycle and most practitioners began treating with FSH during mid-cycle (i.e., 8–12 days after estrus).36,37 Although this was initially based on anecdotal evidence, and then some experimental data,38 it is now known that this encompasses the time of emergence of the second follicular wave in cattle exhibiting two- or three-wave cycles.39,40

Many practitioners prefer decreasing FSH dose schedules and treating with PGF on the third day of the treatment protocol, while others prefer to treat with PGF on the fourth day, and many do not treat with FSH on the day after the administration of PGF. Although no differences have been found between 4- and 5-day treatment protocols, recent experiments have shown that ovulation rate can be improved in some donors if FSH treatments are prolonged to 6 or 7 days.41,42 Regardless, most superstimulation protocols have been successful in inducing superovulation under most circumstances.12 Still others incorporate a progestin insert into the protocol which ensures that donors do not come into estrus early, especially if it is not possible to confirm the presence of a corpus luteum before initiating FSH treatments. In all cases inseminations are normally done 12 and 24 hours after the onset of estrus.11,37

Although the initiation of superstimulatory treatments during mid-cycle has served the embryo transfer industry over the years, conventional treatment protocols have two drawbacks: (i) the requirement to have trained personnel dedicated to the detection of estrus, both before and after initiating treatments; and (ii) the necessity to have all donors in estrus at the same time in order to begin the superstimulatory treatments at the most appropriate time in groups of cows (i.e., mid-cycle). To obviate these problems, protocols that facilitate superstimulation subsequent to elective induction of follicular wave emergence have been developed.

Synchronization of follicle wave emergence for superovulation

Estradiol and progesterone

The ability to electively induce follicular wave emergence permits initiation of superstimulation without regard to the stage of the estrous cycle and eliminates the need for estrus detection or waiting 8–12 days to initiate gonadotropin treatments.37 In the 1990s the use of progestins and estradiol to induce synchronous emergence of a new follicular wave was reported36 and its use in superstimulation protocols has been reviewed extensively.37,43 The estradiol treatment causes suppression of FSH release and follicle atresia. Once the estradiol has been metabolized, FSH surges and a new follicular wave emerges on average 4 days after treatment.36,44

The most common protocol involves the administration of 5 mg estradiol-17β or 2.5 mg estradiol benzoate, plus 100 or 50 mg progesterone by intramuscular injections at the time of insertion of an intravaginal progestin device (day 0).37,43 Twice-daily intramuscular FSH treatments are then initiated on day 4. On day 6, PGF is injected in the morning and evening and the progestin device is removed in the evening. Estrus normally occurs on day 8 (approximately 48 hours after the first PGF injection) and inseminations are done 12 and 24 hours later.

Unfortunately, estradiol cannot be used in many countries around the world because of concerns about the effects of estrogenic substances in the food chain.45 This restriction leaves many embryo transfer practitioners with a serious dilemma and created the need to develop treatments that do not involve the use of estradiol.

Follicle ablation

An alternative to the use of estradiol in superstimulation protocols is to eliminate the suppressive effect of the dominant follicle by ultrasound-guided follicle aspiration of all follicles of 5 mm or more.46 Follicle wave emergence occurs very consistently 24–36 hours later and superstimulatory treatments are initiated at that time. This approach to the synchronization of follicle wave emergence for superstimulation is very efficacious and results in superovulatory responses that do not differ from the use of estradiol.46 In addition it has been found that it is necessary to ablate only the two largest follicles to effectively synchronize follicle wave emergence.47 The protocol involves the ablation of the two largest follicles at random stages of the estrous cycle and the insertion of a progestin device; FSH treatments are initiated 24–48 hours later and the remainder of the protocol is as described above. The disadvantage of ultrasound-guided follicle aspiration is that it requires ultrasound equipment and trained personnel, making it appropriate only when donors are held in an embryo production facility; it is very difficult to apply in the field.

Gonadotropin-releasing hormone

Another alternative for the synchronization of follicle wave emergence is to induce ovulation of the dominant follicle by treatment with gonadotropin-releasing hormone (GnRH),48,49 which is followed by wave emergence 1–2 days later.50 However, emergence of the new follicular wave is synchronized only when treatment causes ovulation, and when administered at random stages of the estrous cycle GnRH results in ovulation in less than 60% of animals.50,51 Not surprisingly, treatment with GnRH prior to initiating superstimulatory treatments at random stages of the estrous cycle resulted in lower superovulatory responses than treatments initiated after follicular ablation or estradiol treatment.52

More recently, retrospective analysis of commercial embryo transfer data has revealed no differences in the numbers of transferable embryos between donors superstimulated 4 days after treatment with estradiol and those superstimulated 2 days after treatment with GnRH (Randall Hinshaw, personal communication).53,54 It is noteworthy that in each of these reports GnRH was administered 2 days after insertion of a progestin device. The improved responses may have been a consequence of progestin-induced development of a persistent dominant follicle that was more responsive to treatment with GnRH.55 Obviously, controlled studies with the use of GnRH must be conducted to validate these promising results.

Improving the ovulatory response to GnRH

Most fixed-time artificial insemination (AI) protocols utilizing GnRH to synchronize follicle wave emergence employ a form of presynchronization to improve the ovulatory response to the first injection of GnRH.55,56 Another alternative is to synchronize ovulation and then initiate FSH treatments at the time of emergence of the first follicular wave.33 To avoid the need to detect estrus and ovulation in Nelore (Bos indicus) donors, Nasser et al.57 induced synchronous ovulation with an estradiol-based protocol designed for fixed-time AI. Gonadotropin treatments were then initiated at the expected time of ovulation (and emergence of the first follicular wave). Superovulatory response did not differ from a contemporary group superstimulated 4 days after treatment with estradiol. However, the number of transferable embryos was reduced in cows superstimulated during the first follicular wave unless accompanied by the use of a progestin device. Similar results were obtained by Rivera et al.58 In this study Holstein cows superstimulated during the first follicular wave also produced a greater number of viable embryos following the addition of a progestin device.

et al.59 recently reported on a series of experiments with the overall objective of developing a protocol for superstimulation following ovulation induced synchronously by the administration of GnRH. This approach was based on a previous study in which ovulatory response was increased by causing a persistent follicle to develop with the administration of PGF and the insertion of a progestin device 7–10 days before the administration of GnRH.55 In that study, ovulation and follicle wave emergence occurred 1–2 days after the administration of GnRH, indicating that this approach could be used in groups of randomly cycling donors.

The recommended superstimulation protocol is schematically presented in Figure 75.1. It consists of the administration of PGF at the time of insertion of a progestin device. Seven days later (with the progestin device still in place) GnRH is administered to induce ovulation of the persistent follicle and follicle wave emergence; FSH treatments are initiated 36 hours after the administration of GnRH. Although this protocol was designed for 4 days of FSH treatments, a 5-day superstimulation protocol can be accomplished by simply delaying the removal of the progestin device by 1 day. Overall in this series of experiments, more than 95% of animals ovulated to the first GnRH administration and superovulatory response and ova/embryo numbers and quality were similar to that obtained when estradiol was used to synchronize follicular wave emergence.59


Figure 75.1 Treatment schedule for superovulation of donor cows during the first follicular wave after GnRH-induced ovulation. Donors receive a progestin device along with PGF followed by GnRH 7 days later. On day (D)0 (36 hours after GnRH), superstimulation with FSH is initiated (twice-daily decreasing doses over 4 days). PGF is administered with the last two FSH injections and the progestin device is removed with the last FSH injection. Ovulation is induced with GnRH 24 hours after progestin removal, donors are fixed-time inseminated (AI) 12 and 24 hours later, and ova/embryos are collected 7 days later. From Bó G, Carballo Guerrero D, Tríbulo A et al. New approaches to superovulation in the cow. Reprod Fertil Dev 2010;22:106–112.

Fixed-time AI of donors

Barros and Nogueira60 have developed a superstimulatory protocol for Bos indicus cattle that they refer to as the P-36 protocol. In this protocol the progestin device that is inserted prior to the initiation of superstimulatory treatments is left in place for 36 hours after PGF administration and ovulation is induced by the administration of pLH 12 hours after withdrawal of the progestin device (i.e., 48 hours after PGF administration). Since ovulation occurs between 24 and 36 hours after pLH administration,61 fixed-time AI is scheduled 12 and 24 hours later, eliminating the need for estrus detection.

In a series of experiments in which the timings of ovulations were monitored ultrasonically, Bó et al.11 developed a protocol for fixed-time AI in Bos taurus donors without the need for estrus detection and without compromising results. Basically, the time of progestin device removal was delayed to prevent early ovulations and allow late-developing follicles to “catch up,” followed by induction of ovulation with GnRH or pLH. In this protocol follicular wave emergence was synchronized with estradiol and a progestin device on day 0 and FSH treatments were initiated on day 4. On day 6, PGF was administered in the morning and evening and the progestin device was removed on the morning of day 7 (24 hours after the first administration of PGF). On the morning of day 8 (24 hours after the removal of the progestin device), GnRH or pLH was administered and fixed-time AI was done 12 and 24 hours later. Delaying the removal of the progestin device to the morning of day 7 resulted in a higher number of ova/embryos and fertilized ova than removal on the morning of day 6 (P. Chesta, MSc thesis, National University of Cordoba, Argentina, 2010). From a practical perspective, fixed-time AI of donors has been shown to be useful in eliminating the need for estrus detection for busy embryo transfer practitioners.62

Studies in high-producing Holstein cows (Bos taurus) in Brazil have indicated that it is preferable to allow an additional 12 hours before removing the progestin device (i.e., evening day 7; P36) followed by GnRH or pLH 24 hours later (i.e., evening day 8).63 In Bos indicus breeds, Baruselli et al.64 confimed that it was preferable to remove the progestin device on the evening of day 7 (P36), followed by GnRH 12 hours later (i.e., morning day 8). Although donors are typically inseminated twice, 12 and 24 hours after administration of pLH or GnRH,11 it is possible to use a single insemination with high-quality semen 16 hours after pLH.64

Reducing the need for multiple treatments with FSH

Because the half-life of pituitary FSH is short in the cow,8 traditional superstimulatory treatment protocols consist of twice daily intramuscular injections over 4 or 5 days.9 This requires frequent attention by farm personnel and increases the possibility of failures due to noncompliance. In addition, twice-daily treatments may cause undue stress in donors with a subsequent decreased superovulatory response and/or altered preovulatory LH surge.65,66 Simplified protocols may be expected to reduce donor handling and improve response, particularly in less tractable animals.

A single subcutaneous administration of FSH has been shown to induce a superovulatory response equivalent to the traditional twice-daily treatment protocols in beef cows in high body condition (i.e., body condition score >3 on scale of 5)67 but results were not repeatable in Holstein cows which had less adipose tissue. However, superovulatory responses were improved in Holstein cows when the single injection was split into two; 75% of the FSH dose was administered subcutaneously on the first day of treatment and the remaining 25% was administered 48 hours later when PGF is normally administered.68

An alternative for inducing a consistent superovulatory response with a single injection of FSH is to combine the pituitary extract with agents that cause the hormone to be released slowly over several days. These agents are commonly referred to as polymers that are biodegradable and nonreactive in the tissues, facilitating use in animals.69 In a series of experiments in which FSH diluted in a 2% hyaluronan solution was administered as a single intramuscular injection (to avoid the effects of body condition), a similar number of ova/embryos was produced as in the traditional twice-daily FSH protocol70 (Table 75.1). However, 2% hyaluronan was viscous and difficult to mix with FSH, especially in the field. Although more dilute preparations of hyaluronan were less efficacious as a single administration, their use was improved by splitting them into two injections 48 hours apart as was done with subcutaneous injections of FSH.

Table 75.1 Mean (±SEM) ova/embryo production in beef donors treated with Folltropin-V given by twice-daily intramuscular injections over 4 days (control) or diluted in 2% hyaluronan and given by a single intramuscular injection.

Source: Bó G, Carballo Guerrero D, Tríbulo A et al. New approaches to superovulation in the cow. Reprod Fertil Dev 2010;22:106–112.

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