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Bovine Embryo Transfer
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Table of Contents
· Donor Selection
· General Procedural Steps
· Physiology and Endocrinology of the Normal Bovine Estrous Cycle
· Manipulation of the Follicular Wave for Superstimulation
· Superstimulation for Ovum Pick-up (OPU)
· Estrus Synchronization for Embryo Transfer
· Fixed-time Embryo Transfer
· Embryo Recovery
· Embryo Evaluation
· Embryo Transfer
· Embryo Cryopreservation
· In vitro Embryo Production
· Adoption of New Technologies
· Summary and Conclusions
· Identification, Certification and Registration of Offspring
The commercial bovine embryo transfer industry arose in North America in the early 1970's [Betteridge, 1981; 2003]. Continental breeds of cattle imported into Canada were very valuable and relatively scarce because of international health and trade restrictions. Embryo transfer offered a means by which their numbers could be multiplied rapidly. However, it was private veterinary practitioners and small commercial embryo transfer companies that developed the technology for commercial use; they took techniques from the laboratory to the field. These pioneers encountered countless practical difficulties and founded the International Embryo Transfer Society (IETS) to facilitate open discussion which was considered necessary for progress to be made. For a historical perspective, the reader is referred to a comprehensive review of farm animal embryo transfer and its associated technologies by Keith Betteridge .
The IETS was founded in 1974, with 82 Charter Members, representing researchers, academics and veterinary practitioners from around the world [Carmichael, 1980; Schultz, 1980]. The IETS became the main forum for scientific and regulatory exchange and discussion in the areas of embryo transfer and associated technologies. The Proceedings of the Annual Meeting of the IETS, which were published as the first issue each year of the journal, Theriogenology, served as a yard-stick with which to measure changes in emphasis and intensity of activity in embryo transfer. More recently, the IETS Proceedings have been published in the first issue each year of the journal, Reproduction Fertility and Development.
Although many IETS members are now basic researchers representing government, industrial or academic institutions, including human medicine [Hasler, 2003], the IETS has played an important role in the dissemination of basic and applied information, allowing for the rapid growth of the embryo transfer industry. In particular, the Import/Export Committee of the IETS (now referred to as the Health and Safety Advisory Committee; HASAC) has been instrumental in gathering and disseminating scientific information on the potential for disease control with bovine embryo transfer. The Manual of the International Embryo Transfer Society "A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures" has become the reference source for sanitary procedures used in embryo export protocols [IETS Manual, 4th Edition, 2010].
In 1982, the American Embryo Transfer Association (AETA) was formed to unite and organize the commercial embryo transfer industry in the USA, and in 1984, the Canadian Embryo Transfer Association (CETA) was formed. Stated objectives included the establishment of standards of performance and conduct, and a liaison with Federal agencies for both domestic and international embryo transfer. These associations also interact directly with breed associations, producer groups and international groups such as the IETS. They established standards of practice to provide confidence within each country, and internationally, for the utilization of embryo transfer technology. In this regard, their Certification Programs are integral in ensuring that Embryo Transfer Practitioners are technically and ethically competent in the handling of embryos used in international trade [Mapletoft and Hasler, 2005]. In South America, the Brazilian Embryo Technology Society (SBTE) was founded in Brasilia in 1985 [Rubin, 2005] to "promote the science of animal embryo technology by encouraging effective research, disseminating scientific and educational information, maintaining high standards of ethics and cooperating with other organizations with similar objectives". Several other countries and regions e.g., Argentina (SATE), Japan and the European Union (AETE) have now established their own embryo transfer organizations with similar goals and objectives.
The embryo transfer industry in North America grew rapidly in the late 1970s, both in terms of the number of practitioners and in the number of donors. Seidel  reported that more than 17,000 pregnancies resulted from the transfer of bovine embryos in North America in 1979. More recently, Perry  reported that 464,582 in vivo-derived bovine embryos were transferred world-wide in 2014, of which 56% were frozen/thawed. In addition, 364,727 in vitro-produced bovine embryos were transferred, 69% of which were in South America; North America transferred 92,930 in vitro-produced embryos of which 23% were frozen/thawed. North America continued to lead in commercial embryo transfer activity in 2014 with collection of 58,934 donor cows and the transfer of more than 271,346 in vivo-produced embryos (58.4% of all transfers).
Very briefly, bovine embryo transfer technology involves the selection, management and treatment of donors and recipients, and the collection and transfer of embryos within a narrow window of time following estrus. This technology has been incorporated into dairy and beef cattle operations, and often requires the participation of herd veterinarians. The following review draws heavily on material contained in prior reviews, extensive literature of primary research on the topic, including reviews and research reports from the authors’ laboratories.
Over the years, techniques associated with embryo transfer have had many uses, especially in research. The widespread use of this technology in animal breeding schemes, however, is relatively recent. Genetic engineering and related new technologies will only increase its utilization [Gibson and Smith, 1989]. For example, several research laboratories have used in vitro fertilization (IVF) techniques to study the fertilizing capacity of sperm and almost all of the so-called "new technologies" utilize IVF techniques in one way or another. A few of the more common uses of embryo transfer technology in animal production follow.
With the development of commercial embryo transfer in the 1970s, its most common use in animal production programs was the proliferation of so-called desirable phenotypes. However, in 1987, the University of Guelph introduced the concept of MOET (multiple ovulation and embryo transfer) [Smith, 1988]. They showed that MOET programs could result in increased selection intensity and reduced generation intervals, resulting in increased genetic gains. The establishment of nucleus herds and "Juvenile MOET" in heifer offspring was shown to result in genetic gains that approached twice those achieved with traditional progeny test schemes [Smith 1988]. It is noteworthy that prior to this research, most embryo transfer done in Canada was in beef cattle, whereas most done today involves dairy cattle. Interestingly, a larger proportion of embryo transfer work in the USA involves beef cattle [Perry, 2015].
Embryo transfer is now commonly used to produce AI sires from the top producing cows and proven bulls for the dairy industry [Teepker and Keller, 1989]. In addition, new genomic techniques are being used increasingly to select embryo donors and genomic analysis has become essential for the selection of bull dams to be used in embryo transfer [Seidel, 2010]. Although economics would seem to preclude the use of embryo transfer techniques for anything but seed-stock production, the commercial cattle industry has benefited from the use of commercial bulls produced through well designed MOET programs [Christensen, 1991].
By far the most common use of embryo transfer in animal production programs has been the proliferation of so-called desirable phenotypes. As AI has permitted the widespread dissemination of a male's genetic potential, embryo transfer has provided the opportunity to disseminate the genetics of proven, elite females. Embryo transfer also has permitted the development of herds of genetically valuable females, most of which may be sibs if not full-sibs. As AI has led to the very valuable bull, embryo transfer has resulted in the very valuable female [Betteridge, 1981]. Many breeders have identified individual females whose offspring are most saleable and used them exclusively in embryo transfer. Embryo transfer has also been used to expand a limited gene pool rapidly. The dramatic rise of the embryo transfer industry in Canada in the early 1970's was a direct result of the introduction of European breeds of cattle, which were then in short supply. The production of AI bulls through embryo transfer is currently the most common application of planned mating [Teepker and Keller, 1989; Lohuis, 1995].
The success of MOET programs has now led to the use of this technology to genetically test AI sires. The Canadian Association of Animal Breeders developed a program for the production and testing of the next generation of AI sires utilizing embryo transfer technology [Lohuis 1995; Smith and Ruane 1987; Teepker and Keller 1989]. Bull mothers were selected, super-stimulated and inseminated to the most highly proven bulls available. Male offspring were placed in waiting while female offspring were placed in production. Bulls were then proven by their sisters' production records rather than waiting for their daughters' records. With this approach, it was possible to genetically test a bull in 3.5 years as opposed to 5.5 years using traditional progeny testing. The use of genomic analysis is now facilitating this process even further.
Several large studies have shown that in vivo-derived bovine embryos do not transmit infectious diseases. In fact, the IETS has categorized disease agents based on the risk of transmission by in vivo-derived bovine embryos [Stringfellow and Givens, 2000; Mapletoft and Hasler, 2005; IETS Manual 2010]. Category 1 diseases include disease agents for which sufficient evidence has accrued to show that the risk of transmission is negligible, provided that embryos are handled properly between collection and transfer. This includes inspection of the zona pellucida at >50X magnification and washing/trypsin treatment procedures. Category 1 diseases include Enzootic bovine leukosis, Foot and mouth disease (cattle), Bluetongue (cattle), Brucella abortus (cattle), Infectious bovine rhinotracheitis, Pseudorabies in swine and Bovine spongiform encephalopathy. Category 2, 3 and 4 diseases are those for which less research information has been generated. However, it is noteworthy that none of the infectious diseases studied have been transmitted by in vivo-derived bovine embryos. Consequently, it has been suggested that embryo transfer be used to salvage genetics in the face of a disease outbreak [Wrathall et al., 2004]. For example, breeders have used embryo transfer techniques to establish disease-free herds to be used strictly for export purposes [Stringfellow et al., 2004; Wrathall, 2004].
Import and Export
The ability to utilize embryos in preventing the transmission of infectious disease makes them ideal for the international movement of animal germ-plasm [Mapletoft 1987; Wrathall et al., 2004]. The intercontinental transport of live animals costs thousands of dollars, whereas an entire herd can be transported, in the form of frozen embryos, for less than the price of a single plane fare. Additional benefits of embryos for the international movement of animal genetics include reduced risk of disease transmission, reduced quarantine costs, a wider genetic base from which to select, the retention of the original genetics within the exporting country, and adaptation. Over the last 10 years, embryo import regulations for many countries have been simplified. In 2014, 26,457 embryos were frozen in North America for export purposes, and 10,920 embryos were exported from Canada alone [Perry, 2015].
Several potential problems must be overcome in order to make the international movement of embryos commonplace. Firstly, widespread use is dependent on the production of inexpensive embryos, and as IVF holds the greatest promise in this regard, successful cryopreservation of IVF embryos is necessary [Hasler, 1998; 2003; Hasler et al., 1997]. Secondly, the inadvertent introduction of disease into a herd and/or country with or within the embryo is of great concern to regulatory officials. Although well-defined methods of collection, handling and washing in vivo-derived embryos have been developed to ensure that disease transmission is avoided, the in vitro-produced embryo may be more difficult to deal with [Stringfellow and Givens, 2000; Stringfellow et al., 2004]. The zona pellucida of in vitro-produced bovine embryos differs from that of in vivo-derived embryos [Stringfellow and Givens, 2000], and it has been shown that pathogens are more likely to remain associated with the zona pellucida of in vitro-produced embryos following washing procedures. This has potentially serious ramifications and export protocols must be revised accordingly. Obviously, correct handling procedures are the key [Stringfellow, 2010]. Finally, the international movement of embryos is heavily dependent on technology transfer as personnel within the importing country must be able to successfully thaw and transfer embryos, much as they are currently doing with semen and artificial insemination (AI).
Embryo transfer techniques have proven to be a very useful research tool. In fact, many technical developments in embryo transfer before 1970 were directed toward research purposes rather than multiplication of superior livestock [Betteridge, 1981; 2003]. These studies included natural limitations to twin pregnancies, uterine capacity, endocrine control of uterine environment, maternal recognition of pregnancy, embryo-endometrium interactions, and the endocrinology of pregnancy. Newer techniques have added an entirely new perspective to the utilization of embryo transfer technology for research. The production of identical twins, clones, chimeras, to mention a few, will certainly advance many of these sciences. As alluded to earlier, IVF techniques are being used to study the fertilizing capacity of semen and IVF techniques are of immense value in the study of oocyte competence and embryo metabolism, and are being utilized in almost all of the emerging technologies.
Selection criteria for donor animals are very likely to differ depending on the reason for doing embryo transfer; however, most are economically driven. Until recently, embryo transfer has been feasible for only very valuable cattle, and the costs associated with embryo transfer have tended to be reduced, relatively speaking, as economic and genetic value increase [Mapletoft, 1986]. Non-surgical techniques of recovery and transfer of embryos and improved pregnancy rates from frozen-thawed embryos, including Direct Transfer of frozen embryos have also reduced costs [Hasler, 2003; Mapletoft, 1985]. Under these circumstances, the top 10% of a purebred herd could be used as donors and then inseminated to conceive naturally to maintain close to a yearly calving interval, while the lower 90% of the herd could be used as recipients.
Although scarcity and promotion have tended to influence value, true genetic value, the ability to transmit desirable traits, must be the most important long-range consideration. Selection should be based on three criteria: genetic superiority, reproductive ability and market value of the progeny [Mapletoft, 1985]. When selecting genetically superior beef donors, objective traits such as calving ease, milk production, weaning and yearling weights and carcass value should be considered. As beef bulls can now be evaluated for genetic merit quite accurately, the selection of the service sire is extremely important. The selection of dairy donors is already well established [Ruane and Smith, 1989] and improved upon with the use of genomic testing [Seidel, 2010].
As optimal results will also reduce costs, donor selection may involve a previous history of success in embryo transfer [Keller and Teepker, 1990; Moor et al., 1984]. In addition, daughters from cows that have been used successfully in embryo transfer are also likely to be successful. It has been suggested that the potential donor animal be at its prime reproductive age, have a history of a high level of fertility and demonstrated superiority in traits of economic importance. Strict selection criteria also include antral follicle counts and circulating levels of anti-mullerian hormone (AMH) [Ireland et al., 2008; 2011; Monniaux et al., 2013; Singh et al., 2004; Souza et al., 2014].
General Procedural Steps
Although the applications and techniques associated with bovine embryo transfer have been reviewed extensively [Mapletoft, 1985; 1987], a brief historical perspective may be useful. Early investigators described non-surgical embryo recovery techniques [Rowson and Dowling, 1949], but these were not successful, and all embryo recoveries and transfers in the early 1970s were performed surgically [Betteridge, 1981; 2003]. The first commercial embryo transfer programs relied on mid-ventral surgical exposure of the uterus and ovaries with the donor under general anesthesia. This necessitated surgical facilities and limited the use of the technology in the dairy industry because the udder of dairy cows hindered mid-ventral access to the reproductive tract. It was not until 1976 that nonsurgical embryo recovery was developed sufficiently to be used in practice [Drost et al., 1976; Elsden et al., 1976; Rowe et al., 1976]. In the late 1970s, nonsurgical embryo transfer techniques [Rowe et al., 1980] were also described, allowing for on farm embryo transfer.
Although there has been no appreciable increase in the number of embryos produced per superovulated donor cow over the past 40 years, the importance of follicle wave dynamics [Adams, 1994] and methods for the synchronization of follicular wave emergence [Bó et al., 1995; 2002], have simplified the means by which superovulation might be achieved, resulting in increased embryo production per unit time. Donor cows are being super-stimulated more frequently than in the past (often every 30 days), and more embryos are being produced per year with no change in the actual superstimulation protocol [Bó and Mapletoft, 2014]. Sartori et al.  have discussed various factors affecting viable embryo production in dairy cows.
The donor may be inseminated naturally or artificially and embryos are normally collected non-surgically 6 to 8 days after breeding. Following collection, embryos must be identified, evaluated and maintained in a suitable medium prior to transfer. At this point, they may also be subjected to manipulations, such as splitting and sexing, and may be cooled or frozen for longer periods of storage [Hasler 2003]. The discussion of donor superovulation, recipient synchronization and embryo transfer must begin with a review of recent information on estrous cycle physiology.
Physiology and Endocrinology of the Normal Bovine Estrous Cycle
The endogenous control of the bovine estrous cycle involves the interrelated secretion of a number of hormones from the hypothalamus, anterior pituitary gland, ovaries and uterus [reviewed in Senger, 2003]. These include gonadotropin releasing hormone (GnRH) from the hypothalamus, follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary gland, estrogen, progesterone and inhibin from the ovary and prostaglandin F2a (PGF) from the uterus. The primary timing mechanism of the bovine estrous cycle is ovulation at which time the first follicular wave emerges [Adams, 1994]. The corpus luteum (CL) forms over the ensuing days and in the absence of pregnancy, regresses around Day 16 or 17 of the cycle [Senger, 2003]. The simplest hypothesis for regression of the CL is that the non-pregnant uterus secretes a luteolytic agent into the uterine venous blood. This material is transferred through a local veno-arterial pathway to the ovarian artery whereby it reaches the ovary and causes luteolysis [reviewed in Senger, 2003]. PGF has been proposed as the natural luteolytic agent, although definitive proof and details of the mechanism(s) of action are unclear. Regression of the CL results in a rapid fall in serum progesterone concentrations to values less than 1 ng/ml, LH pulse frequency increases and follicular growth is stimulated further. The growth and maturation of the preovulatory follicle results in increasing secretion of estradiol, which causes estrogenic changes in the oviduct and uterus, behavioral estrus, and a preovulatory release of LH (around the time of onset of estrus). The preovulatory LH peak results in resumption of oocyte meiosis, ovulation 24 to 32 hours later and luteinization of the ovulated follicle to form a secreting corpus hemorrhagicum. Growth and development of the corpus hemorrhagicum into a fully functional CL results in progestational changes in the oviduct and uterus that are conducive to embryonic development and establishment of pregnancy. Should pregnancy not occur, the cycle will begin again with the demise of the CL.
It has now been shown by ultrasonography that follicles in cattle develop in a wave-like fashion [Pierson and Ginther, 1984]. Bovine estrous cycles are composed of 2 or 3 waves of follicular development. A follicular wave has been defined as a group of growing antral follicles 3 - 5 mm in diameter from which a dominant follicle is selected while the remaining follicles become subordinate and undergo atresia (Fig. 1) [Adams, 1994; 1998; Ginther et al., 1989; Pierson and Ginther, 1987]. In both 2- and 3-wave estrous cycles, emergence of the first follicular wave occurs on the day of ovulation (Day 0) while the second wave emerges 9 or 10 days after ovulation (Days 10 or 11 of the cycle) in 2-wave cycles, and 8 or 9 days after ovulation (Days 9 or 10 of the cycle) in 3-wave cycles, with a third wave emerging on Days 15 or 16. Duration of the estrous cycle is approximately 20 days in 2-wave cycles and 22 days in 3-wave cycles. The dominant follicle present at the time of luteolysis will become the ovulatory follicle, and emergence of the next wave is delayed until the ensuing ovulation. The proportion of animals with 2- vs. 3-wave cycles are probably more or less equally distributed, and follicular waves have been reported in heifers before puberty, and postpartum cows before the first ovulation [Adams, 1998]. Follicle waves persist in pregnant animals until approximately the last 3 weeks before parturition.
Recruitment of follicular waves and selection of a dominant follicle is based on differential responsiveness to FSH and LH [Adams et al., 1992a; 1992b; 1993; Ginther et al., 1996]. Surges in plasma FSH are responsible for eliciting the emergence of a follicular wave (Fig. 1), while FSH is subsequently suppressed by products of the growing follicles (e.g., estradiol and inhibin). In each wave, the dominant follicle acquires LH receptors while subordinates which are dependent on FSH undergo atresia. Suppression of LH as a consequence of progesterone secretion by the CL causes the dominant follicle to eventually cease its metabolic functions and it begins to regress. This leads to FSH release and emergence of a new follicular wave. Luteal regression allows LH pulse frequency to increase, the dominant follicle increases its growth and dramatically higher concentrations of estradiol result in a positive feedback on the hypothalamo-pituitary axis and a surge of LH followed by ovulation.
Figure 1. Bovine ovarian follicular wave dynamics during 2-wave and 3-wave estrous cycles (OV=ovulation).
The top panel shows the relationship between progesterone secretion and luteinizing hormone (LH) release. The shaded area represents progesterone secretion in ng/ml (left axis). The dashed line represents typical serum concentrations of LH in ng/ml (right axis). Episodic pulses of LH are schematic.
The middle panel shows the relationship between follicle stimulating hormone (FSH) and follicle wave status. The dashed line represents concentrations of FSH in ng/ml (right axis). The solid line represents changes in amounts of various regulatory factors produced by follicles, due to multiple follicles early in the wave and then from the dominant follicle during its late growing-early static phase of development.
The bottom panel shows diameters of follicles within follicle waves (in mm on the left axis) as seen with serial ultrasound examinations of the ovaries. The first follicle wave emerges on the day of ovulation, while the second follicle wave emerges 9 or 10 days later. [Modified from Adams, 1994].
Estrus synchronization and superovulation are critical components of an embryo transfer program. These techniques involve the manipulation of the basic endocrine patterns outlined above [Wiltbank, 1997]. The key to successful estrus synchronization is synchronous growth and ovulation of a viable dominant follicle and closely synchronized, rapid declines in circulating progesterone to values <1 ng/ml [Adams, 1994; 1998]. If properly implemented, within the physiological constraints of their mechanism of action, current techniques for synchronization of estrus and ovulation are highly successful [Mapletoft et al., 2003]. However, variation in ovarian follicular wave dynamics makes it difficult to control the time of estrus and ovulation precisely.
The objective of superstimulation treatments in the cow is to obtain the maximum number of fertilized and transferable embryos with a high probability of producing pregnancies. However, wide ranges in superovulatory response and embryo yield have been detailed in several reviews of commercial embryo transfer records. In a report of 2048 beef donor collections, a mean of 11.5 ova/embryos with 6.2 good or transferable embryos were collected from each donor [Looney, 1986]. However, variability was great; 24% of the collections did not produce viable embryos, 64% of donors produced fewer than average numbers of transferable embryos and 30% of the collections yielded 70% of the embryos. In another study of 987 dairy cows, embryo recovery yielded slightly fewer ova/embryos, on average, but there was similar variability [Hasler et al., 1983]. These reports demonstrate the high degree of unpredictability in superovulatory response that creates problems affecting both the efficiency and profitability of embryo transfer programs.
Variability in ovarian response has been related to differences in super-stimulatory treatments such as gonadotropin preparation [Alkemade et al., 1993; Murphy et al., 1984], batch of gonadotropin, duration of treatment, timing of treatment with respect to the estrous cycle, total dose of gonadotropin and the use of additional hormones in the superstimulation protocol [Bó et al., 1995; Foote, 1986; Lerner et al., 1986; Mapletoft et al., 2002; Monniaux et al., 1983; Seidel, 1981]. Additional, perhaps more important sources of variability are factors inherent to the animal and its environment. These factors may include nutritional status [Santos et al., 2008; Velazquez, 2011], reproductive history, age, season, breed, ovarian status at the time of treatment and perhaps most importantly, inherent numbers of antral follicles [Ireland et al., 2007; Singh et al., 2004]. While considerable recent progress has been made in the study of bovine reproductive physiology, factors inherent to the donor animal that affect superovulatory response are only partially understood [Bó et al., 2002; Mapletoft et al., 2002].
Superovulation-inducing treatments are usually initiated between Days 8 and 12 of the estrous cycle (estrus = Day 0) [Mapletoft, 1985; 1986; Mapletoft et al., 2002]. We demonstrated a greater superovulatory response when gonadotropin treatments were initiated on Day 9 of the estrous cycle (Day 8 post-ovulation) as compared to Days 3, 6 or 12 [Lindsell et al., 1986]. This observation has been supported by ultrasonographic evidence showing the second follicle wave beginning 8.5 days post-ovulation (Day 9.5 of the cycle) in 3-wave cows and 9.5 days post-ovulation (Day 10.5 of the cycle) in 2-wave cows [Adams, 1994; Ginther et al 1989; Pierson and Ginther 1987]. In a practical sense, it is noteworthy that two-wave cows tended to have shorter cycles (18 to 20 days) than three-wave cows (21 to 23 days). Therefore, the length of the previous estrous cycle can provide a clue as to the optimal time to initiate super-stimulatory treatments, however, it must also be recognized that some cows change from one wave type to the other. Traditional superovulation protocols are illustrated in Table 1.
Table 1. Super-stimulatory protocols with a pituitary FSH extract initiated around the time of emergence of the second follicular wave in both 2- and 3-wave cattle
Superstimulation on the CL of the Cycle
Superstimulation on the CL Plus a Progestin Device
Day 0 – Estrus
Day 0 - Estrus
Day 10 – FSH Bid
Day 7 – Insert progestin device
Day 11 – FSH Bid
Day 10 – FSH Bid
Day 12 – FSH & PGF Bid
Day 11 – FSH Bid
Day 13 – FSH Bid
Day 12 – FSH & PGF Bid; remove progestin in PM
Day 14 AM – Estrus
Day 13 – FSH Bid
PM – AI
Day 14 AM - Estrus
Day 15 AM – AI
PM - AI
Day 15 AM – AI
The first and most obvious way to increase the number of embryos that are produced during a superstimulation procedure is to increase the number of ovulations that occur. Cows differ dramatically in ovarian follicular reserve i.e., the number of small follicles that are available for superstimulation [Ireland et al., 2011]. Moor et al.  suggested that both ovulation rate and number of viable embryos produced are relatively consistent within individual cows; animals that responded poorly in one trial did so in subsequent trials, and animals that responded well initially continued to do so. Although there was considerable variability between cows, the number of follicles was shown to be similar between ovaries in the same cow. Further, the number of follicles >1.7 mm in diameter in an ovary was positively correlated with ovulatory response to gonadotropin treatments.
Follicle Growth and Maturation Patterns
During a normal follicular wave, subordinate follicles regress because of decreasing concentrations of FSH, caused by the secretion of estradiol and inhibin by follicles in the cohort, and especially of the dominant follicle. Small follicles require FSH to continue their growth [Adams et al., 1993; 2008]. We have shown that exogenous FSH will cause these follicles to continue to grow (superstimulation) and if given sufficient time, reach a size and maturational status to ovulate [Garcia-Guerra et al., 2015]. Assuming a growth rate of 1 to 2 mm per day, these follicles could be utilized by adding 2 to 3 days to the superstimulation treatment protocol [Garcia Guerra et al., 2012]. Indeed, we have successfully super-stimulated donors at random stages of the estrous cycle, without regard to the presence of a dominant follicle, using this approach [Bó et al., 2008]. Alternatively, the 2 days of Folltropin-V pretreatment might be replaced with a single injection of 500 IU of equine chorionic gonadotropin (eCG) 2 days before initiating FSH treatments [Caccia et al., 1999]. In addition, we have shown that pretreatment of poor responding donors with 400 IU of eCG 2 days before initiating FSH treatments resulted in an improved superovulatory response over that achieved previously without the use of eCG. More recently, we have shown that lengthening the super-stimulatory treatment protocol from the traditional 4 days to 7 days, without increasing the total amount of Folltropin-V administered, increased the percentage of follicles that ovulated, the number of ovulations and the synchrony of ovulations, and tended to increase the mean numbers of total ova/embryos, fertilized ova, and transferable embryos [Garcia Guerra et al., 2012]. In other words, the lengthened superstimulatory treatment protocol resulted in more follicles reaching an ovulatory size and acquiring the capacity to ovulate. It was concluded that prolonged FSH treatment protocols may be an effective strategy to rescue small follicles that would otherwise become atretic. These results also suggest that traditional 4-day superstimulatory treatment protocols may not provide adequate time for all follicles to acquire the capacity to ovulate. The application of lengthened FSH treatment protocols may be preferred in cows with low follicle numbers (i.e., less than 15 follicles per wave) than in those with high follicle numbers. However, more research is required to confirm these observations.
One way to predict super-stimulatory response is to use ultrasonography to count the numbers of antral follicles in the ovary [Jaswal et al., 2004]. The numbers of small antral follicles counted by ultrasonography at the time of initiating gonadotrophin treatments has been shown to be correlated with subsequent super-stimulatory response in cattle [Ireland et al., 2007; Singh et al., 2004]. In humans, circulating anti-mullerian hormone (AMH) concentrations have been found to be the most informative serum marker for ovarian follicle reserve largely replacing other serum markers such as basal FSH testing [Broekmans et al., 2006; Fanchin et al., 2003; Toner and Seifer, 2013]. Information is also accumulating that measurement of circulating AMH concentrations may be the most reliable method for predicting antral follicle numbers in cattle [Batista et al., 2014; Ireland et al., 2011; Monniaux et al., 2013; Rico et al., 2012]. There is a high repeatability of AMH measurements across different phases of the estrous cycle, days in milk, levels of milk production, and parities [Ireland et al., 2011; Monniaux et al., 2013] and this makes AMH determinations particularly useful as a diagnostic tool to predict superovulatory response [Souza et al., 2014].
Collectively, these data suggest that some of the variability in superovulatory response resides in genetic or physiological makeup of the animal rather than in exogenous factors. Indeed, cows and heifers selected for a high incidence of twinning had higher superovulatory responses than unselected controls [reviewed in Mapletoft et al., 2002; Mapletoft and Bo, 2015].
Ultrasonographic examination of the ovaries has provided indications for the most propitious moment to initiate treatments to obtain maximal super-stimulatory responses from individual donors. In this regard, the initiation of superstimulatory treatments in the presence of a dominant follicle resulted in a 40 to 50% decrease in superovulatory response [Bungartz and Niemann, 1994; Kim et al., 2001; Shaw and Good, 2000]. During the mid-part of an ovarian follicular wave, the dominant follicle, acting locally or systemically, induces atresia in other developing follicles [Adams, 1994; Adams et al., 1993]. Collectively, these data suggest that the presence of an active dominant follicle at the time super-stimulatory treatments are initiated may be expected to depress superovulatory response [Guilbault et al., 1991], and the presence of actively growing follicles in the range of 3 to 6 mm in diameter (follicle wave emergence) maybe associated with an improved superovulatory response. It is difficult with a single ultrasonographic examination under field conditions to determine whether a large follicle is functionally dominant and whether smaller follicles are actively growing or becoming atretic [Bungartz and Niemann, 1994]. However, the presence of a large number of follicles 3 to 6 mm in diameter 8 to 10 days after ovulation, in the presence of a large follicle, provides strong evidence for the beginning of a new follicle wave [Singh et al., 2004]. In this regard, it has been shown clearly that superovulatory response was greater when superstimulatory treatments were initiated at the time of follicle wave emergence; as little as 1 day asynchrony significantly reduced the superovulatory response compared to initiating treatments on the day of wave emergence [Nasser et al., 1993].
Figure 2. Two pairs of super-stimulated bovine ovaries. The pair of ovaries on top, removed 7 days after ovulation, show the newly formed corpora lutea that are pinkish in color. Many have an obvious depression that is likely the region of the ovulation stigma (pore). The bottom pair of ovaries was removed several days later and have multiple corpora lutea that are more yellow in color, and a few dark follicles that appear to have been only partially luteinized and may have failed to ovulate
New methods of actively recruiting follicles for the purpose of superovulation may be directed at the sources of variability identified above. In other words, it may be possible to recruit a large cohort of responsive follicles by stimulating early antral (or perhaps even pre-antral) follicles, so that a larger, more uniformly responsive group is available when gonadotropin treatments are initiated [Bó et al., 2005a]. Alternatively, it is possible to mimic the effects of the dominant follicle and suppress the development of all antral follicles; gonadotropin treatments initiated at selected times after the termination of follicle suppressing treatments could be expected to catch a cohort of responsive follicles as they begin to grow [Bó et al., 1995; 2002].
Hormone Profiles in Superovulated Cattle
Low progesterone concentrations at the time of initiation of gonadotropin treatment have been shown to be related to a reduced superovulatory response indicating the importance of a functional CL [Mapletoft et al., 2002]. It has also been shown that fertilization rates and embryo quality may be compromised when superstimulation is conducted in the face of low circulating progesterone values [Lonergan, 2011; Nasser et al., 2011; Rivera et al., 2011]. However, the influence of progesterone on superovulatory response and ova/embryo quality still requires further study. Progesterone has been shown to increase within 24 h after treatment with eCG or a crude pituitary extract suggesting a luteotrophic action of these gonadotropins [Alkemade et al., 1993; Murphy et al., 1983]. This did not occur with more highly purified pituitary extracts [Alkemade et al., 1993]. Normally, the decline in progesterone is rapid after treatment with PGF, dropping to less than 1 ng/ml of serum within 10 to 32 h [Beal, 1996; Kastelic et al., 1990; Seguin, 1987]. However, as complete luteolysis (i.e., < 0.4 ng/ml) is required to ensure optimal fertilization rates, two injections of PGF are normally recommended. Ovulation occurs approximately 72 hours after the first PGF treatment in super-stimulated cows [Bó et al., 2005a; 2005b; 2006]. Elevated progesterone concentrations at the time of estrus may affect LH release and sperm transport and capacitation. At the onset of estrus, progesterone concentrations were lower in super-stimulated cows that yielded high quality embryos than in cows that yielded unfertilized ova [Wiltbank et al., 2014]. After ovulation, the increase in serum progesterone concentration occurs earlier and the slope of the progesterone curve is steeper as the number of CL increases in superovulated cattle [Callesen et al., 1986; Greve et al., 1983].
Manipulation of the Follicular Wave for Superstimulation
The conventional protocol of initiating ovarian superstimulation during mid-cycle was originally based on anecdotal and experimental information in which a greater superovulatory response was reported when super-stimulatory treatments were initiated 8 to 12 days after estrus [reviewed in Mapletoft et al., 2002]. However, none of these early studies evaluated follicular status at the time that gonadotropin treatments were initiated. Although it is now known that 8 to 12 days after estrus (equivalent to Days 7 to 11 after ovulation) would be the approximate time of emergence of the second follicular wave, the day of emergence of the second follicular wave differs among individuals within wave type and is 1 or 2 days later in 2- than 3-wave cycles. In addition, the necessity of waiting until mid-cycle to initiate superstimulatory treatments implies monitoring estrus and an obligatory delay making it very difficult to group large numbers of donors for superstimulation at the same time. To obviate these problems, an alternative approach is to initiate superstimulation treatments subsequent to the synchronization of follicular wave emergence. Basically, there are three methods of synchronizing follicle wave emergence for superstimulation.
The most efficacious approach to the synchronization of follicle wave emergence involves transvaginal ultrasound-guided ablation of all follicles ≥5 mm, regardless of stage of the estrous cycle [Bergfelt et al., 1994; Garcia and Salaheddine, 1998]. This removes the suppressive effects of follicle products (estradiol and inhibin) on FSH release, resulting in an FSH surge and emergence of a new follicular wave 1 day later. Super-stimulatory treatments are then administered, beginning 1 day after ablation, and PGF is administered 48 or 72 h later [Bergfelt et al., 1997]. It is also noteworthy that the timing of estrus was more synchronous when a progestin device was inserted for the period of superstimulation and two injections of PGF were administered on the day of progestin removal. Transvaginal ultrasound-guided follicle ablation of all follicles [Bergfelt et al., 1997] or just the dominant follicle [Bungartz and Niemann, 1994; Shaw and Good, 2000] during mid-diestrus, followed in 2 days by superstimulation, also resulted in a higher superovulatory response than when the dominant follicle was not ablated. Conversely, in a retrospective analysis of superovulatory responses of lactating dairy cows, follicle ablation resulted in a significantly higher number of ova/embryos, but a comparable number of transferable embryos as cows super-stimulated 7 to 13 days after estrus [Kim et al., 2001]. We have now shown that ablation of the two largest follicles at random stages of the estrous cycle was as efficacious as ablating all follicles ≥5 mm in synchronizing follicular wave emergence for superstimulation [Baracaldo et al., 2000]. However, it is advised that a progestin device always be used during superstimulation and that two injections of PGF be used to ensure complete luteolysis (Table 2). Unfortunately, follicle ablation is difficult to utilize under field conditions because distances and the need for sophisticated equipment.
Table 2. Superstimulation following follicle ablation
Day 0 – Ablate the two largest follicles and insert a progestin device
Administration of Estradiol and Progesterone
Traditionally, estradiol has been administered near the beginning of progestin treatment for estrus synchronization to induce luteolysis and allow for shortened progestin treatment protocols [Bó et al., 1995; Odde, 1990; Wiltbank, 1997]. We have also shown that the benefit of estradiol in shortened progestin treatment protocols may be associated with the regression of small antral follicles [Bó et al., 1995; 1996]. The mechanism involves suppression of FSH and possibly LH which results in regression of FSH- and LH-dependent follicles. Once follicle regression begins and the exogenously administered estradiol is metabolized, FSH surges and a new follicle wave emerges 1 day later. The use of a short acting estradiol-17β in progestin-implanted cows was followed by the emergence of a new wave, approximately 3 to 5 days later regardless of the stage of follicular growth at the time of treatment [Bó et al., 1995; 1996]. Estradiol-17β is normally injected with 50 to 100 mg of progesterone at the same time as placement of a progestin device to prevent an estrogen-induced LH surge in those animals that do not have a functional CL [Bó et al., 1995; 1996; Mapletoft et al., 2003]. A superstimulation protocol using estradiol to synchronize follicle wave emergence is shown in Table 3.
Table 3. Superstimulation following treatment with estradiol and progesterone
Day 0 – Insert progestin device and inject estradiol and progesterone
Our preferred approach for synchronization of follicular wave emergence prior to superstimulation is an injection of 5 mg estradiol-17β + 100 mg progesterone at the time of insertion of progestin releasing device, with FSH treatments beginning 4 days later [Bó et al., 1995; 1996]. Data from experimental [Bó et al., 1996] and commercial [Bó et al., 2002] superovulation programs have shown that the superovulatory response of donors given estradiol-17β and progesterone at unknown stages of the estrous cycle was comparable to that of donors super-stimulated 8 to 12 days after observed estrus (Table 4).
Table 4. Superovulatory response in beef and dairy cattle super-stimulated 8 to 12 days after estrus (traditional) or 4 days after treatment with estradiol-17β, progesterone and a progestogen/progesterone releasing device (P4 + E-17β) [Adapted from Bó et al., 2002]
12.8 ± 0.3
6.6 ± 0.2
8.9 ± 0.4
5.1 ± 0.3
P4 + E-17β
12.1 ± 0.9
6.3 ± 0.6
10.3 ± 0.5
6.0 ± 0.4
Means did not differ (P>0.2).
Many practitioners are now utilizing estradiol-17β and progesterone along with one of the many progestin-releasing devices that are available to synchronize follicle wave emergence for superstimulation of donors [reviewed in Mapletoft et al., 2002]. On Day 0 (random stages of the estrous cycle; try to avoid the last 2 or 3 days of the cycle), donors receive a progestin device and an injection of 5 mg of estradiol-17β and 100 mg progesterone. On Day 4, FSH treatments are initiated. On Day 6 or 7 cows receive two injections of PGF and the progestin device is removed with the second injection. Estrus is expected to occur approximately 36 to 48 hours after the first injection of PGF. Artificial insemination is normally done 12 and 24 hours after onset of estrus, or 60 and 72 hours after the first injection of PGF. In this way, the full extent of the estrous cycle is available for superstimulation and the need for detecting estrus or ovulation and waiting 8 to 12 days to initiate treatment is eliminated.
This is a fairly robust protocol; for example, it is possible to use 2.5 mg estradiol-17β and 50 mg progesterone with no apparent effect on results. Furthermore, FSH is often given for 3 days before PGF is administered and several practitioners remove the progestin device 24 hours after PGF treatment to avoid early expression of estrus. In addition, FSH is often not administered on the last day of the protocol i.e., Day 7 above.
Unfortunately, estradiol-17β is not readily available for commercial use in many countries [Lane et al., 2008]. Therefore, we investigated the possibility of using other commercially available estrogen esters (i.e., estradiol benzoate or estradiol valerate). Treatment with 2.5 mg estradiol benzoate + 50 mg progesterone given at the time of progestin device insertion, resulted in synchronous emergence of a new follicular wave 3 to 4 days later [Bó et al., 2002]. Superstimulatory treatments initiated 4 days after estradiol benzoate and progesterone treatment resulted in superovulatory responses comparable to those initiated 4 days after treatment with 5 mg estradiol-17β + 50 mg progesterone or 2.5 mg estradiol-17β + 50 mg progesterone or those initiated 8 to 12 days after estrus. Treatment with 5 mg estradiol valerate and 3 mg norgestomet resulted in less synchronous emergence of a follicular wave and a lower superovulatory response than 5 mg estradiol-17β + 100 mg progesterone [Colazo et al., 2005]. However, a dose of 1.0 or 2.0 mg estradiol valerate was shown to result in follicular wave emergence in 3.2 and 3.4 days, respectively, with little variability, which was suitable for superstimulation [reviewed in Mapletoft et al., 2002].
Administration of GnRH
Another method of synchronizing follicular wave emergence for superstimulation involves the use of GnRH or porcine LH (pLH) to induce ovulation of a dominant follicle which is followed by emergence of a new follicle wave 2 days later [Macmillan and Thatcher, 1991; Martinez et al., 1999; Pursley et al., 1995; Thatcher et al., 1993]. However, the administration of GnRH or pLH does not always induce ovulation, and if ovulation does not occur, follicle wave emergence will not occur (or be synchronized) [Martinez et al., 1999]. Therefore, the reported asynchrony in follicular wave emergence (range, 3 days before treatment to 5 days after treatment) suggests that GnRH-based approaches may not be feasible for superstimulation [Martinez et al., 1999]. In a report involving three different experiments [Deyo et al., 2001], GnRH or pLH treatments consistently resulted in fewer embryos than when follicular wave emergence was synchronized with other methods.
More recently, retrospective analysis of commercial embryo transfer data has revealed no differences in the numbers of transferable embryos between donors super-stimulated 4 days after treatment with estradiol and those super-stimulated 2 days after treatment with GnRH [Hinshaw, 1999; Steel and Hasler, 2009; Wock et al., 2008]. It is noteworthy that Hinshaw administered GnRH 2 days after insertion of a progestin device. The improved superovulatory responses may have been a consequence of progestin-induced development of an unovulated dominant follicle that arose under the influence of the progestin device and which would be more responsive to treatment with GnRH [Mapletoft et al., 2003]. Obviously, controlled studies with the use of GnRH must be conducted to validate these promising results.
Most fixed-time AI (FTAI) protocols utilizing GnRH to synchronize follicle wave emergence employ a form of pre-synchronization to improve the ovulatory response to the first injection of GnRH [Mapletoft and Bo, 2012; Souza et al., 2008]. Bó et al.  recently reported on a series of experiments with the overall objective of developing a protocol for superstimulation following ovulation induced synchronization of follicle wave emergence 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 at the same time as the insertion of a progestin device 7 to 10 days before the administration of GnRH [Small et al., 2009]. In that study, ovulation and follicle wave emergence occurred 1 to 2 days after the administration of GnRH in >90% of cows, indicating that this approach could be used in groups of randomly cycling donors. Recently, we have examined superovulatory responses comparing the administration of GnRH 2 vs 7 days after insertion of a vaginal device and found no significant difference in ova/embryo production [Hinshaw et al., 2015]. Thus, either approach would appear to be efficacious.
The recommended 7-day superstimulation protocol is presented schematically in Fig. 3. 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 one 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 [Bó and Mapletoft, 2014].
Figure 3. Treatment schedule for superstimulation of donors following GnRH-induced ovulation of a retained dominant follicle
Progestin device & PGF
Start FSH (PM)
FSH, Progestin removal,
Traditionally, donors have been subjected to embryo collections at 2-month intervals. However, the elective synchronization of follicular wave emergence has resulted in cows being super-stimulated successfully every 25 to 30 days, without regard to expression of estrus [Bó et al., 2002]. Once multiple CL have been induced to regress by the administration of PGF, and the cow ovulates, normal follicular wave patterns are reestablished and the cow can be superstimulated again. The following protocol shows how GnRH 2 days after insertion of a progestin device can be used to super-stimulate cows every 30 days, without the need for estrus detection and without compromising results (Table 5).
Table 5. Superstimulation of donors at 30 day intervals utilizing GnRH to synchronize follicle wave emergence
Day 0 – Insert Progestin device
Collectively, these studies demonstrate that exogenous control of follicle wave emergence offers the advantage of initiating super-stimulatory treatments at a time that is optimal for follicle recruitment, regardless of the stage of the estrous cycle and convenient for the embryo transfer practitioner. The treatment is practical, easy to follow by farm personnel and, more importantly, it eliminates the need for detecting estrus or ovulation and waiting 8 to 12 days to initiate gonadotrophin treatments. Synchronization of follicular wave emergence by follicle ablation, GnRH or pLH treatments or estradiol + progesterone treatments has resulted in comparable superovulatory responses. Furthermore, the use of estradiol and progesterone for the synchronization of follicle wave emergence for superstimulation makes it possible to superstimulate cows that are not cycling or have abnormal ovarian function [reviewed in Mapletoft et al., 2002].
Superstimulation of Donors with Abnormal Ovarian Function
Cows with abnormal ovarian function are difficult to super-stimulate because they usually do not have a functional CL, or they come into estrus at unpredictable times. In addition, it is almost impossible to predict the stage of follicle development in these cows. Progestin inserts and elective synchronization of follicle wave emergence have been used in superstimulation protocols for donor cattle with abnormal ovarian function. In fact, it was the need to super-stimulate cows with abnormal ovarian function that led to the use of estradiol prior to the administration of FSH [Johnson, 1986]. In a retrospective study, embryo production did not differ between 190 problem cows super-stimulated 7 days after the receiving a norgestomet implant and an injection of norgestomet and estradiol valerate and 260 Control cows super-stimulated between Days 8 and 12 of the cycle [Mapletoft et al., 1984]. Although this protocol was developed to provide an artificial CL with the norgestomet implant, it was subsequently shown that estradiol valerate played a role by synchronizing emergence of a new follicle wave [Bó et al., 1995]. Benefits probably accrued from both the implant (artificial CL) and the estradiol valerate (induction of follicle wave emergence). Follicle ablation may also be used to synchronize follicular wave emergence in problem cows that receive a progestin device. The use of progestin devices by themselves have not been critically evaluated in problem cows.
In cows with a history of poor superovulatory response due to anovulation, the progestin/estradiol treatment protocol has been followed with the administration of pLH at the time of first AI to induce ovulation [Nigro et al., 1996]. Administration of 25 mg pLH at the time of first AI in problem cows increased the number of transferable embryos as compared to the previous superstimulation without pLH. However, administration of pLH at the time of the first insemination did not improve embryo production in normal cows.
Gonadotrophins and Superovulation
Three different types of gonadotropins have been used to induce superovulation in the cow: gonadotropins from extracts of domestic animal pituitaries (FSH), equine chorionic gonadotropin (eCG) and human menopausal gonadotropin (hMG) [Alkemade et al., 1993; Kelly et al., 1997; Mapletoft et al., 2002]. Equine chorionic gonadotropin is a complex glycoprotein with both FSH and LH activity and it has been shown to have a half-life of approximately 40 hours in the cow [Schams et al., 1977], persisting for up to 10 days in the animal's circulation; thus it is normally injected once followed by PGF 48 hours later [Murphy and Martinuk, 1991]. Recommended doses of eCG range from 1500 to 3000 IU/animal with 2500 IU by intramuscular injection commonly chosen. The long half-life of eCG also causes protracted ovarian stimulation, abnormal endocrine profiles, large unovulated follicles present at the time of ova/embryo recovery and reduced embryo quality [Mikel-Jenson et al., 1982; Saumande et al., 1978]. These problems have been overcome by the intravenous injection of antibodies to eCG at the time of the first insemination, 12 to 18 hours after the onset of estrus [Dieleman et al., 1993; Gonzalez et al., 1994a; 1994b]. However, antibodies to eCG are not available commercially, and so eCG is seldom used to super-stimulate cattle. Similarly, human menopausal gonadotropin has not gained favor in bovine embryo transfer because of cost and no greater efficacy [Alkemade et al., 1993; McGowan et al., 1985].
Pituitary extracts are most commonly used to super-stimulate cattle. As the biological half-life of pituitary FSH in the cow has been estimated to be 5 hours or less [Laster, 1972], it must be injected intramuscularly twice daily to induce superovulation [Monniaux et al., 1983; Walsh et al., 1993]. The usual regimen is 4 or 5 days of twice daily treatments of FSH in decreasing doses. Forty-eight or 72 hours after initiation of treatment, PGF is injected to induce luteolysis. Estrus and preovulatory LH release occurs within 36 to 48 hours, with subsequent ovulation 24 to 36 hours later. Purified pituitary extracts (LH removed) are available in most countries today [Armstrong and Opavsky, 1986; Armstrong, 1993]. One product, Folltropin-V (Vetoquinol) is a porcine pituitary extract with approximately 84% of the LH content removed [Gonzalez-Reyna et al., 1990; Mapletoft et al., 2002]. It is available in bottles containing the equivalence of 400 mg NIH-FSH-P1 (equivalent to 700 IU FSH). It has been administered in a constant or decreasing dose schedule with PGF given either 48 or 72 h after initiation of treatment with no significant change in superovulatory response [Alkemade et al., 1993].
Individual animal variability has been an overriding factor in all superovulation studies. Breed may also be a factor. In one study, Holsteins required a higher proportion of FSH, whereas Charolais required a higher proportion of LH for maximal superovulation [Chupin et al., 1984; reviewed in Mapletoft et al., 2002]. It has also been reported that the purified pituitary extracts are more efficacious than crude pituitary extracts under conditions of heat-stress [Page et al., 1985], whereas there was no difference during more moderate environmental temperatures. In yet another study involving Bos indicus heifers in Argentina, all pituitary extracts were efficacious in the summer months, but a very highly purified pituitary extract was most efficacious during winter months suggesting that winter temperatures may be stressful to Bos indicus cattle [Tribulo et al., 1991]. It would appear that stress is the overwhelming consideration [Edwards et al., 1987; Stoebel and Moberg, 1982] and that under stressful conditions, purified pituitary extracts should be used [Mapletoft et al., 2002].
Recombinantly-produced bovine FSH (rbFSH) has also been used to induce superovulation in cattle. Wilson et al.  reported high superovulatory responses in cattle following twice daily administration of rbFSH, and more recently, Carvalho et al.  reported the successful superstimulation of Holstein heifers with a single administration of a long-acting rbFSH. Although there are currently no products available commercially for use in cattle, rhFSH is used commonly in human medicine suggesting that recombinant gonadotropins are likely to be used in cattle, provided they gain registration and are affordable.
Reducing the Need for Multiple Treatments with FSH
Because the half-life of pituitary FSH is short in the cow [Laster, 1972], traditional superstimulatory treatment protocols have consisted of twice daily intramuscular injections over 4 or 5 days [Bó et al., 1994]. This requires frequent attention by farm-personnel and increases the possibility of failures due to non-compliance. In addition, twice daily treatments may cause undue stress in donors with a subsequent decreased superovulatory response and/or altered preovulatory LH surge [Edwards et al., 1987; Stoebel and Moberg, 1982]. Therefore, simplified protocols may be expected to reduce donor handling and improve response, particularly in less tractable animals.
A single subcutaneous administration of FSH behind the shoulder 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 of >3 out of 5 [Bó et al., 1994], but results were not repeatable in Holstein cows, presumably because of 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 [Lovie et al., 1994].
An alternative in 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 which are biodegradable and non-reactive in the tissues facilitating use in animals [Sutherland, 1991]. In a series of experiments, FSH was diluted in a 2% hyaluronan solution and 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 protocol [Tribulo et al., 2011]. 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 [Tribulo et al., 2012].
The split intramuscular treatment protocol consists of diluting the FSH lyophilized powder with 10 mlL of a 1% or 0.5% hyaluronan solution and the intramuscular administration of two-thirds of the total dosage of FSH on the first day, followed by administration of the remaining one-third 48 hours later, when PGF is normally administered. When compared to the twice daily treatment protocol (Controls) in 29 beef cows super-stimulated three times in a cross-over design, the numbers of transferable embryos with the split-injection protocol did not differ from Controls. Data suggested that splitting the FSH dose in reduced concentrations of hyaluronan into two intramuscular injections 48 hours apart would result in a comparable superovulatory response to the traditional twice-daily intramuscular injection protocol in beef cattle (Table 6).
Furthermore, the less concentrated solutions of hyaluronan were not difficult to mix with FSH, even under field conditions. A recent report derived from commercial embryo transfer data has now confirmed these results in beef cattle in North America [Hasler and Hockley, 2012]. However, the single or split-dose of FSH in hyaluronan is not recommended for lactating dairy cattle; superovulatory results have been variable and inconsistent in dairy cattle and the reason is still unknown.
Table 6. Superovulatory response (mean ± SEM) of beef cows treated with 300 mg Folltropin-V given by twice daily I.M. injections over 4 days (control) or diluted in 10 mg/ml (1%) hyaluronan or 5 mg/ml (0.5%) hyaluronan and given by two I.M. injections 48 hours apart
12.0 ± 1.3a
10.2 ± 1.8a
6.7 ± 1.3
4.0 ± 0.8
15.1 ± 1.0b
14.4 ± 2.0b
8.9 ± 1.4
5.0 ± 0.9
15.3 ± 1.1b
14.3 ± 2.1b
9.3 ± 1.9
6.1 ± 1.3
ab Means within columns with different superscripts differed (P<0.05).
Fixed-time AI of Superstimulated Donors
Barros and Nogueira [2005; Nogueira and Barros, 2003] 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 super-stimulatory 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, fixed-time AI is scheduled 12 and 24 hours administration of pLH, eliminating the need for estrus detection.
In a series of experiments in which the timings of ovulations were monitored ultrasonically, Bó et al.  developed a protocol for fixed-time AI in Bos taurus beef 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 AM and PM and the progestin device was removed on Day 7 in the AM (24 hours after the first administration of PGF). On Day 8 AM (24 hours after the removal of the progestin device), GnRH or pLH was administered and fixed-time AI were done 12 and 24 hours later (Table 7). Delaying the removal of the progestin device to Day 7 AM resulted in a higher number of ova/embryos and fertilized ova than removal on Day 6 PM. 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 [Larkin et al., 2006].
Table 7. Superstimulation following treatment with estradiol/progestin and the use of fixed-time AI
Day 0 : Insert progestin device and inject estradiol and progesterone
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., Day 7 PM; P36) followed by GnRH or pLH 24 hours later i.e., Day 8 PM [Martins et al., 2012]. In Bos indicus breeds, Baruselli et al.,  also reported that it was preferable to remove the progestin device on Day 7 PM (P36), followed by GnRH 12 hours later (i.e., Day 8 AM). Although donors are typically inseminated twice, 12 and 24 hours after administration of pLH or GnRH, it is possible to use a single insemination with high quality semen 16 hours after pLH. This protocol has also worked well with sex-selected semen except that inseminations are delayed by 6 hours to coincide more closely with timing of ovulation [Soares et al., 2011].
Semen and Semen Quality in Super-stimulated Donors
We indicated above that donors are normally inseminated 12 and 24 hours after onset of estrus (around 60 and 72 hours after injection of PGF), and this is based on studies conducted several years ago which suggested that the 24 hour insemination was probably the more important breeding [Schiewe et al., 1987]. However, no mention was made of sperm numbers or sperm quality. Many practitioners use two straws of semen at each breeding, and in 1988, Hawk et al.  provided data that supported this notion; insemination of super-stimulated animals with 4.4 billion fresh sperm resulted in a greater number of fertilized ova (15.3 vs 9.1) and fertilization rates (93% vs 53%) than 70 million frozen-thawed sperm. However, this was not consistent with our experience that a single straw of frozen/thawed semen at each insemination time resulted in acceptable fertilization and transferable embryo rates. In order to examine this further, we selected three bulls with normal spermiograms and cryopreserved their semen in insemination doses of 20 million, 50 million and 100 million sperm in 0.25 ml or 0.5 ml straws. When used as a single straw at 12 and 24 hours after onset of estrus in super-stimulated heifers, there were no differences in fertilization rates (overall 88.2%; range 72.0 – 93.5%) among bulls or any of the sperm dosages [Garcia et al., 1994]. The key would appear to be normal spermiogram when dosages of at least 10 million motile sperm are used.
Superstimulation places extraordinary pressure on the capacity of frozen/thawed semen to fertilize multiple oocytes. As ovulation rate increases, the number of accessory sperm decreases, and it has been observed that unfertilized oocytes from superovulated cattle seldom had sperm attached to their zona pellucida [DeJarnette et al., 1992]. Clearly sperm transport in super-stimulated cattle is compromised. However, viability may also be compromised. Saacke et al.  have shown that the number of viable sperm in the lower isthmus of the oviduct is less for a shorter period of time in super-stimulated than in single ovulating cattle. Thus the two inseminations would appear to be warranted.
There is strong evidence that examining semen before use in super-stimulated cattle pays dividends. Miller et al.  reported a fertilization rate of 72% when so called "satisfactory" semen was used to inseminate super-stimulated heifers and 46% when "questionable" semen was used. Nigro et al.  reported a fertilization rate 76.1% (n=66) in superovulated cattle inseminated with semen that had been previously evaluated and 59.3% (n=37) when semen that had not been previously evaluated was used. However, MacDonald [unpublished] also showed that there are differences among so called "normal" bulls with transferable embryo rates ranging from a low of 40% (n= 132) to a high of 60% (n=393) in bulls that had exceeded minimum quality control standards for processed semen. This difference was accounted for, in part, by season (and heat stress) of semen collection. Miller et al.  and Witt et al.  have also reported different fertilization rates in super-stimulated heifers inseminated with semen from the same bull, but with different percentages of sperm with the diadem (nuclear vacuoles) defect. Sperm morphology is clearly important in super-stimulated cattle.
Semen used in super-stimulated cattle must exceed the minimum standards established by the American Society for Theriogenology for frozen/thawed semen [Barth, 1993]. Briefly, these are a minimum of 70% morphologically normal sperm, and immediately after thawing 25% directional motility with a rate of 3/5 and a minimum of 60% intact acrosomes. After a 2 hour stress test, directional motility must exceed 15% (rate 2) and percentage of intact acrosomes must exceed 40%. Super-stimulated cattle should be inseminated at 12 and 24 hours after onset of estrus with a minimum of 10 million normal, directionally motile sperm in each insemination.
Donor cows must be a minimum of 50 days postpartum, cycling normally, and cows with low body condition scores should be on an increasing plane of nutrition with no specific nutritional deficiencies. However, donors with high body condition scores (4 to 5 in the 1 to 5 scale) have been reported to have a lower superovulatory response than those with body condition scores of 2 and 3 [Garcia Guerra et al., 2007]. Trace mineral supplementation is often recommended before superstimulation, and although there are no supporting data, beyond clinical impressions, the use of chelated minerals is recommended to improve superovulatory response and embryo yield. There should be no history or physical evidence of infertility. It is noteworthy that cows (or daughters of cows) with a previous history of superovulatory success or of twinning are likely to be most responsive. Similarly, donors with high circulating AMH concentrations or high antral follicle counts are likely to have high superovulatory responses.
Super-stimulatory treatments are normally initiated on Days 8, 9 or 10 of the estrous cycle. If a cow is not cycling normally or the day of estrous cycle is unknown, the use of a progestin releasing device along with an injection of estradiol-17β (or estradiol benzoate) and progesterone can be used to synchronize follicular wave emergence for superstimulation. Super-stimulatory treatments are usually initiated in 4 days and the progesterone releasing device is removed 72 to 84 hours later (24 to 36 hours after PGF treatment). Similarly, super-stimulatory treatments can be initiated 24 to 36 hours after follicle ablation or 48 to 60 hours after induction of ovulation with GnRH or pLH.
PGF may be administered on either the third or fourth day of FSH treatment; i.e., 48 or 72 hours after initiating treatment and a two doses of PGF are normally divided over two FSH treatments to ensure complete luteolysis. Cows are normally in estrus 36 to 48 hours after the first PGF and are inseminated with a single straw of high fertility semen at 12 and 24 hours after the onset of estrus or 60 and 72 hours after PGF. Cows of Bos indicus breeding may require a lower dose FSH, whereas older or highly stressed (lactating) cows of Bos taurus breeding may require a higher dose of FSH. If the superovulatory response is poor, it may be necessary to increase the dose of gonadotropins in subsequent treatment protocols, or attempt to more closely synchronize treatments to the time follicular wave emergence. If embryo quality is poor in the face of a high superovulatory response, it may be necessary to reduce the FSH dose or to initiate treatments earlier in the follicle wave. In either case, it may be advisable to use a purified pituitary extract.
Superstimulation for Ovum Pick-up (OPU)
With the exception of indicus breeds of cattle which have high antral follicle counts, most breeds are super-stimulated with a half dose of Folltropin prior to oocyte aspiration. The common approach is to synchronize follicle wave emergence and administer four or six intramuscular injections of Folltropin over 2 or 3 days. Following a "coasting" period of approximately 40 hours (with no treatments), oocytes for in vitro maturation and fertilization are recovered from antral follicles by ultrasound-guided oocyte aspiration [Blondin et al., 2002]. This has resulted in significant increases in blastocyst production in taurus breeds of cattle [Vieria et al., 2014]. Folltropin diluted in hyaluronan and administered as a single injection has been shown to be equally efficacious in the superstimulation of donors for oocyte aspiration [Vieria et al., 2015]. On the day of follicle wave emergence, a half-dose of Folltropin is administered intramuscularly in 0.5% hyaluronan. Seventy-two hours later oocytes are recovered by transvaginal ultrasound-guided oocyte aspiration with excellent results.
Several gonadotropin preparations are available for use. Within optimal dose ranges, they all work. Evidence indicates that gonadotrophins must be administered by deep intramuscular injection. Avoid fat deposits unless the purpose is to administer a single subcutaneous injection. The single subcutaneous injection of a pituitary extract may be advisable if stress of handling could be an impediment to successful superstimulation, however, the injection must be into a fat pad. Similarly, split intramuscular injections of FSH in a 0.5% solution of hyaluronan may be used to induce superovulation. Regardless of whether a single, double or multiple intramuscular injections are administered, stress and overdosing must be avoided. If all else fails, one may consider single embryo collections at 10 or 21 day intervals. One high quality embryo is superior to any number of poor quality embryos.
Estrus Synchronization for Embryo Transfer
Acceptable pregnancy rates following embryo transfer are partially dependent upon the onset of estrus in the recipient being within 24 hours of synchrony with that of the embryo donor [Hasler et al., 1987]. Recipients can be selected for an embryo transfer program by detection of natural estrus in untreated animals or by detection after drug-induced estrus synchronization. Regardless of the method of synchronization used, timing and critical attention to estrus detection are important. Recipients synchronized with PGF must be treated 12 to 24 hours before donor cows because PGF-induced estrus will occur in recipients in 60 to 72 hours [Kastelic et al., 1990] and in super-stimulated donors in 36 to 48 hours [Bó et al., 2002; 2005a; Mapletoft et al., 2002; 2003]. Although pregnancy rates do not seem to differ in recipients with natural or PGF-induced estrus, pregnancy rates were higher in PGF-synchronized recipients in at least one study [Hasler et al., 1987], probably because of improved estrus detection. It must be remembered that exogenous steroid hormones may induce estrus and even ovulation in post-partum cows and prepubertal heifers that are not cyclic [Mapletoft et al., 2003]. Therefore, postpartum interval, nutrition and body condition in cows, and age, weight and body condition in heifers must be closely monitored. A prospective recipient can be culled prior to embryo transfer because of one or more of these factors.
The success of estrus synchronization programs is dependent on an understanding of three general areas: 1) estrous cycle physiology (described earlier), 2) pharmacological agents and their effects on the estrous cycle, and 3) herd management factors that reduce anestrus and increase conception rates. The normal bovine estrous cycle was described earlier; the use of pharmacological agents for the synchronization of estrus follows.
PGF has become the most commonly used treatment for estrus synchronization in cattle [Folman et al., 1990, Larson and Ball, 1992, Odde, 1990, Seguin, 1987]. PGF is not effective in inducing luteolysis in the first 5 or 6 days following estrus and when luteolysis is effectively induced by PGF, the ensuing estrus is distributed over a 6-day period [Kastelic et al., 1990]. This is due to follicular status at the time of treatment. In a two-dose PGF synchronization scheme, an interval of 10 or 11 days between treatments has been used because it represents the mid-point of the estrous cycle and theoretically, all animals should have a PGF-responsive CL at the time of the second treatment. However, a higher conception rate has been reported with a 14-day interval [Foman et al., 1990], probably because a growing dominant follicle is more likely to be present 14 days after an initial treatment with PGF. Stage of the cycle during which PGF treatment is given affects fertility; pregnancy rates are usually higher when cattle are treated with PGF after mid-cycle (e.g., after Day 12) compared to early in the cycle (e.g., Day 7 or 8).
Various progestins (progesterone and progesterone-like compounds) have been utilized for estrus synchronization [Mapletoft et al., 2003]. Progestin treatment for >14 days will synchronize estrus, but fertility at the induced estrus will be reduced due to the development of a persistent follicle [Mapletoft et al., 2003, Revah and Butler, 1996]. These effects are transitory, and fertility in the following cycle is normal.
Progesterone alters ovarian function in cattle by suppressing estrus and preventing ovulation. It also suppresses LH pulse frequency, which in turn causes suppression of the growth of LH-dependent follicles (i.e., dominant follicle) in a dose-dependent fashion; but it does not suppress FSH secretion [Adams, 1998, Adams et al., 1992b]. Thus, follicular waves continue to emerge in the presence of a functional CL. Progestins given for longer than the CL life-span (i.e., for 14 days or more) result in synchronous estrus upon withdrawal, but fertility is low [Revah and Butler, 1996] because the types and dosages of progestins used to control the estrous cycle in cattle have relatively less suppressive effects on LH secretion than the CL-secreted progesterone and are associated with high LH pulse frequency and development of "persistent" follicles, which contain aged oocytes [Mapletoft et al., 2003, Revah and Butler, 1996]. Ovulation of an aged oocyte results in poor fertility. However, it has been reported that the CL resulting from the ovulation of a persistent follicle is capable of supporting a pregnancy after embryo transfer [Wehrman et al., 1997]. More recently, Mantovani et al.  reported reduced pregnancy rates in recipients with a CL resulting from persistent follicles.
The progesterone-impregnated CIDR-B (controlled internal drug release) intravaginal device is approved in Canada and USA for synchronization of estrus in cattle [Mapletoft et al., 2003]. Label directions for artificial insemination state that the device should be in the vagina for 7 days; PGF is given 24 hours before device removal and estrus detection begins 48 hours later. Because of the short treatment period (7 days), persistent follicles do not form. There are several other progesterone-releasing vaginal devices available in other countries such as New Zealand, Australia, Argentina and Brazil, and it is only a matter of time before they become available in North America. Progesterone releasing vaginal devices are well suited to various approaches used to synchronize follicular development and ovulation [Mapletoft et al., 2003].
Figure 4. A CIDR-B vaginal insert. This is a progesterone releasing device that can be inserted into the vagina and used to mimic luteal function during estrus synchronization or ovarian superstimulation protocols. The removal of the CIDR-B device and injection of prostaglandin results in progesterone withdrawal intended to mimic natural luteolysis and initiate mechanisms responsible for the maturation and ovulation of the growing dominant follicle(s).
Figure 5. Various progestin-releasing devices used to control the estrous cycle in cattle available around the world.
The estrous cycle in cattle averages 21 days, with 84% lasting from 18 to 24 days. Behavioral estrus lasts approximately 12 to 16 hours; ovulation normally occurs 24 to 36 hours after the onset of estrus [Kastelic, 2001]. Estrous behavior waxes and wanes, but nearly all cattle will be detected in estrus if observation is continuous. Therefore, the incidence of true silent estrus is negligible. Causes of anestrus (lack of observed estrus) include pregnancy, cystic ovaries, ovarian atrophy, pyometra, embryonic death, free-martinism and white-heifer disease. Most anestrous dairy cows that are non-pregnant are cycling and have a normal genital tract. Dairy heifers and postpartum suckled beef cattle often have a prolonged interval of anestrus due to ovarian inactivity. Increasing energy intake and/or a 7 to 10 day treatment with progestins will hasten resumption of ovarian activity in most anestrous animals.
The primary sign of estrus is a cow standing firmly when being mounted. Secondary signs of estrus include mounting other cows, mucus discharge, swollen vulva, hyperactivity, and bellowing.
Figure 6. The primary and most definitive sign of estrus in the cow is standing firm when mounted.
The two principal problems with estrus-detection are missed estrus and estrus detection errors. Indicators of missed estrus include prolonged intervals from calving to breeding, prolonged intervals between breedings, and <50% of potential estrous periods detected. Several factors can contribute to missed estrus. Often the observer does not spend adequate time observing cattle for estrus or tries to combine estrus detection with other farm activities (e.g., feeding or milking). If many cattle are in estrus at the same time, they will congregate and form a "sexually active group", which facilitates estrus detection. However, if only a single animal is in estrus, mounting activity will be much less frequent. Slippery or hard surfaces will also reduce mounting activity. Indicators of estrous detection errors include high concentrations of progesterone in milk or blood at the time of breeding and interbreeding intervals <17 d or >25 d. Up to 20% of cattle have been shown to have high progesterone concentrations at the time of breeding, and therefore were not in estrus. Factors contributing to estrus detection errors include misinterpretation of signs of estrus, misinterpretation or misuse of estrus detection aids, and standing estrus in pregnant cows. Means by which estrus detection can be improved include allocating adequate time for observation, using estrus detection aids, predicting the next estrus and inducing estrus pharmacologically [Kastelic, 2001].
Figure 7. Estrus detection is the most important factor affecting widespread use of AI and an impediment to the successful use of embryo transfer.
Estrus detection aids include heat-mount detectors, tail-head chalk or paint, pedometers, androgen-treated marker animals and electronic estrus detection systems [Kastelic, 2001; O’Connor and Senger, 1997]. These methods should be utilized in addition to, and not as a substitute for, visual observation of estous behavior. Marker animals are typically given several treatments with testosterone to initiate mounting activity, followed by periodic treatments to maintain activity. It has been reported that freemartin heifers implanted with Synovex-H (four implants in each ear) were effective marker animals. The duration of effectiveness of the implants was approximately 3 months. This is an extra-label use of these implants and the appropriate withdrawal period prior to slaughter is unknown.
Figure 8. Kamar Heatmount Detectors are valuable heat detection aids that assist in identifying cattle that are in estrus. The detector is a pressure sensitive device with a built-in timing mechanism designed to be activated by pressure. Glued onto the sacrum (tail head), pressure from the brisket of a mounting animal requires approximately 3 seconds to turn the detector from white to red. This timing mechanism helps distinguish between true standing estrus versus false mounting activity.
Figure 9. The Kamar Heatmount Detector is glued onto the sacrum (tail head) of the cow. Cows must be observed twice daily to determine when the detector turns from white to red.
Figure 10. Heat detection using tail-chalk. Freshly applied chalk is positioned on the tail head. When cows are mounted, the chalk is removed (rubbed off), with more and longer mounts resulting in the removal of more chalk. (Courtesy of Dr. Glen Selk).
Fixed-time Embryo Transfer (FTET)
Embryo transfer techniques can be further refined to eliminate the need of estrus detection in either donors or recipients [Bó et al., 2005a; 2005b; 2012; 2013]. In recipients it is possible to eliminate the need for estrus detection by taking advantage of protocols that have been developed for fixed-time AI in cattle [Mapletoft et al., 2003]. The use of progestin devices and the synchronization of follicular wave emergence were described earlier. Basically, two approaches have been used: the so-called Ovsynch or Cosynch protocols utilizing GnRH [Pursley et al., 1995, Wiltbank 1997] or pLH [Martinez et al., 1999] to synchronize dominant follicle growth and ovulation, with or without a progestin-releasing device [Lamb et al., 2001, Mapletoft et al., 2003, Martinez et al., 2000; 2002], and estradiol to synchronize follicle wave emergence and ovulation in progestin-treated animals [Bó et al., 2005b, Mapletoft et al., 2003].
Gonadotropin releasing hormone (GnRH) became available in the 1970's as a treatment for follicular cysts [Drost and Thatcher, 1992]. However, treatment of a cow with a growing dominant follicle with GnRH will also induce ovulation [Macmillan and Thatcher, 1991, Thatcher et al., 1993] with emergence of a new follicular wave approximately 2 days later [Mapletoft et al., 2003, Martinez et al., 1999]. Treatment with PGF 7 days after GnRH resulted in ovulation of the new dominant follicle, especially when a second GnRH injection was given 36 to 48 hours after the PGF [Pursley et al., 1995; Thatcher et al., 1993; Wiltbank, 1997]. An ovulation synchronization scheme utilizing GnRH for fixed-time AI called "Ovsynch '' was developed by Pursley et al. . The first injection of GnRH is followed 7 days later with an injection of PGF followed in 56 hours by a second injection of GnRH; fixed-time AI is performed 16 hours later. The Ovsynch protocol has been more efficacious in lactating dairy cows than in heifers [Wiltbank, 1997]. The cause for this variability is not known, but ovulation to the first injection of GnRH occurred in 85% of cows and only 54% of heifers [Pursley et al., 1995]. In addition, 19% of heifers showed estrus before the injection of PGF making fixed-time AI impossible [Wiltbank, 1997]. Results from our laboratory confirm that a first dose of GnRH does not always result in ovulation of the dominant follicle in heifers (56%) and, hence, it does not consistently induce the emergence of a new follicular wave [Martinez et al., 1999]. However, the addition of a CIDR to a 7-day GnRH-based protocol improved pregnancy rates after fixed-time AI in heifers [Martinez et al., 2000], and improved pregnancy rates in non-cycling, lactating cows [Lamb et al., 2001, Martinez et al., 2000].
GnRH-based protocols have also been used to synchronize ovulation in recipients that received in vivo- [Baruselli et al., 2000, 2010; Hinshaw et al., 1999] or in vitro- [Ambrose et al., 1999] derived embryos. In these studies, more recipients received embryos because the GnRH-based protocol was not dependent on estrus detection; although conception rates were often lower than in controls, pregnancy rates were higher. Recent studies have shown that the first GnRH resulted in ovulation in 44 to 54% of dairy cows [Bello et al., 2006; Colazo et al., 2009], 56% of beef heifers [Martinez et al., 1999] and 60% of beef cows [Small et al. 2009], and the emergence of a new follicular wave was synchronized only when treatment caused ovulation [Martinez et al., 1999]. If the first GnRH does not synchronize follicular wave emergence, ovulation following the second GnRH may be poorly synchronized [Martinez et al., 2002], and recipients may be asynchronous to the stage of embryo development at the time of transfer. Prevention of the early ovulations by addition of a progestin-releasing device to a 7-day GnRH-based protocol has improved pregnancy rates in heifers after fixed-time AI [Martinez et al., 2002], and the addition of a norgestomet implant to a GnRH-based protocol for FTET resulted in pregnancy rates that were similar to observed estrus [Hinshaw 1999]. The same investigators treated 1637 recipients with GnRH plus a progestin-releasing vaginal device without estrus detection with an overall pregnancy rate of 59.9%. In summary, results indicate that acceptable pregnancy rates can be achieved when embryos are transferred to recipients that have been treated with a GnRH and progestin device protocol to synchronize ovulation, without the necessity of estrus detection.
Recent studies have shown that reducing the period of follicle dominance (by removing the progestin device 5 days after insertion) and increasing the time from progestin device removal to the second GnRH treatment and FTAI may improve pregnancy per AI in beef and dairy cattle treated GnRH-based protocols [Bridges et al., 2008; Lima et al., 2011; Santos et al., 2010]. When Bridges et al.  compared a 7-day Cosynch protocol plus progestin device with FTAI at 60 hours and a 5-day Cosynch protocol with FTAI at 72 hours in postpartum beef cows, pregnancy rates were 11% higher with the 5-day protocol. Santos et al.  reported similar findings in dairy cattle. The hypothesis proposed was that the 5-day protocol provided for a longer proestrus with increasing estradiol concentrations due to continuous gonadotropin support for the dominant follicle. The ovulatory follicle of cows in the 5-day program benefited from this extra time and additional gonadotropin support. However, due to a shorter interval between the first GnRH and induction of luteolysis in the 5-day protocol, two injections of PGF 6 to 8 hours apart were necessary to induce complete regression of the GnRH-induced CL. More recently, Colazo et al.  showed that pregnancy per FTAI did not differ between 5-day and 7-day Cosynch protocols with a single administration of PGF in dairy heifers. In that study, the use of the first GnRH in the 5-day Cosynch protocol also did not seem to be necessary as pregnancy rates did not differ when it was not used. Lima et al.  observed an increased pregnancy rate in dairy heifers receiving the final GnRH concurrent with AI at 72 hours after PGF compared 16 hours before AI, however, they also showed no benefit of a first GnRH.
We have preliminary information indicating that the 5-day GnRH protocol results in a comparable proportion of recipients receiving an embryo and becoming pregnant per embryo transfer when compared to the estradiol/progestin FTET protocol (see next section). Similarly, Sala et al.  have recently reported similar pregnancy rates with in vitro-produced embryos into recipients that were synchronized with two PGF treatments 14-days apart and transferred 6 to 8 days after observed estrus and those synchronized with the 5-day Cosynch (without GnRH on Day 0) and transferred at a fixed time 6 to 8 days after GnRH injection.
Estradiol and Progesterone
As indicated earlier, treatment with progestins and estradiol has been used for several years to synchronize estrus, but Bó et al.  demonstrated that estradiol treatment affected follicle development. In a series of studies, estradiol treatment was found to suppress antral follicle growth and suppression was found to be more profound when it was given with a progestin. The mechanism responsible for estrogen-induced suppression of follicular growth appears to involve suppression of FSH through a systemic pathway. Once the estradiol is metabolized, FSH surges and a new follicular wave emerges. Following the treatment of progestin-treated heifers with estradiol-17β, emergence of a new follicular wave occurred 3 to 5 days later, regardless of the stage of follicular growth at the time of treatment [Bó et al., 1995]. For estrus synchronization, progesterone need not be injected with estradiol, but a progestin device must be in place [Bó et al., 2013, Mapletoft et al., 2003]. In estrus synchronization programs, a second, lower dose of estradiol is given 24 hours after PGF treatment and progestin device removal to induce LH release, which occurs approximately 16 to 18 hours later, synchronizing ovulation for fixed-time AI approximately 24 hours after the estradiol treatment [Mapletoft et al., 2003]. Pregnancy rates to fixed-time AI have been high with this protocol.
Estradiol treatments are the most commonly used treatment to synchronize follicle wave emergence and ovulation in beef and dairy recipients in South America [Baruselli et al., 2010; 2011]. The protocol consists of insertion of a progestin-releasing device and the administration of 2 mg estradiol benzoate on Day 0 (to synchronize follicular wave emergence), and PGF either 5 days later, or at the time of insertion and removal of the progestin device (to ensure luteolysis). The progestin device is usually removed on Day 8 and ovulation is induced by the administration of 0.5 or 1 mg of estradiol cypionate at the time of progestin device removal, or injection of 1 mg of EB 24 hours after progestin removal, or GnRH 48 to 54 hours after progestin removal [reviewed in Bó et al., 2002; Baruselli et al., 2010; 2011]. As estrus detection is usually not preformed, Day 9 is considered to be the day of estrus. All recipients with an apparently functional CL on Day 17 receive an embryo and conception rates are comparable to those obtained with embryo transfer 7 days after observed estrus [Bó et al., 2002].
Use of eCG in Estradiol/Progestin-based Protocols
The most common strategy used to increase pregnancy rates in pasture-managed beef cattle in South America is the addition of 400 IU of eCG on either Day 5 or Day 8 of the estradiol/progestin treatment protocol. Overall, 75 to 85% of the recipients treated with this protocol receive an embryo because of the presence of a functional CL (compared to 50% or less with simple PGF synchronization), progesterone concentrations at the time of embryo transfer were high, and conception rates exceeded 50% [reviewed in Baruselli et al., 2010; 2011; Bó et al., 2002].
The FTET treatment protocol utilizing EB, progestin and eCG has been evaluated in different parts of the world. In a commercial embryo transfer program in Argentina, 1309 (84.9%) of 1542 recipients were suitable for embryo transfer (because of the presence of a CL on the day of embryo transfer) and 692 (44.9%) became pregnant following Direct Transfer of frozen-thawed embryos [Bó et al., 2005]. In a commercial embryo transfer program in Brazil, Nasser et al.  obtained 46.1% pregnancies at 30 days and 41.7% at 60 days of gestation following the transfer of 12,580 in-vitro-produced fresh embryos. In another study involving 988 recipients of in vitro-produced, frozen-thawed embryos in China [Remillard et al., 2006], overall pregnancy rates were significantly higher in eCG-treated animals because of the higher percentage of recipients receiving embryos. In a study in Mexico with 949 Brahman-influenced recipients, treatment with eCG increased the number of recipients receiving an embryo, with similar conception rates, resulting in higher pregnancy rates [Looney et al., 2010]. In summary, results indicate that the administration of eCG in an estradiol/progestin-based synchronization protocol for FTET results in increased pregnancy rates, especially in recipients that were nutritionally stressed or with Bos indicus influence. Considering that feeding recipients until they become pregnant is one of the most costly items in an embryo transfer program [Hinshaw, 1999], a protocol that increases the number of pregnant recipients per synchronization treatment seems cost-effective, especially considering that this treatment also avoids the necessity of estrus detection [Looney et al., 2006].
Use of eCG in GnRH-based Protocols
As estradiol is not available in many countries, GnRH has been used to synchronize follicle wave emergence and ovulation in fixed-time protocols. In a Canadian study designed to evaluate the potential use of eCG in beef cattle recipients synchronized with GnRH/progestin for FTET [Small et al., 2007], recipient selection rates did not differ whether cows did or did not receive a progestin device or eCG. In addition, pregnancy rates did not differ whether cows did or did not receive a progestin device or eCG. On the other hand, eCG significantly increased pregnancy rates in a Colombian study [Mayor et al., 2008]. Bos indicus cross heifers were randomly allocated in one of three treatment groups. Heifers in the control group received a progestin device and 2 mg of EB on Day 0 and PGF plus 400 IU eCG on Day 5. Progestin devices were removed on Day 8 and 1 mg of EB was administered on Day 9. Heifers in the GnRH treatment group received a progestin device and GnRH on Day 0, PGF at progestin removal on Day 7 and GnRH on Day 9. Heifers in the GnRH+eCG group were treated similarly except that they also received 400 IU eCG on Day 3. All heifers with a CL >16 mm in diameter received a frozen-thawed embryo by Direct Transfer 7 days after GnRH or 8 days after EB. The number of recipients selected/treated was higher in the EB+eCG and the GnRH+eCG groups than in the GnRH group. Conception and pregnancy rates were also significantly higher in recipients in the EB+eCG and GnRH+eCG groups than in the GnRH group. In summary, the addition of eCG to estradiol- or GnRH-based protocols which included the use of progestin devices resulted in increased pregnancy rates depending on the type and body condition of the recipients. However, treatment with eCG may not improve pregnancy rates in Bos taurus beef recipients managed under more optimal conditions.
Other Treatments Designed to Increase Pregnancy Rates in Recipients
There have been several studies investigating the relationship between circulating progesterone concentrations and pregnancy rates in recipients [reviewed in Baruselli et al., 2010; Carter et al., 2008]. However, the use of supplementary progesterone has resulted in inconsistent effects on pregnancy rates. An alternative strategy to increase circulating progesterone concentrations in recipients is to create an accessory CL by induction of ovulation of the first wave dominant follicle around the time of embryo transfer [reviewed in Thatcher et al., 2001]. Again, results have not been entirely clear. In Bos indicus recipients, treatment with human chorionic gonadotropin (hCG) on Day 7 (day of embryo transfer) increased progesterone concentrations [Marques et al., 2002] and in another study, treatment with GnRH, hCG, pLH or a progestin device at the time of embryo transfer resulted in increased pregnancy rates [Marques et al., 2003]. However, pregnancy rate in non-treated (control) recipients was lower than normally expected in these studies. This result was confirmed in another experiment [Rodrigues et al., 2003] involving the induction of an accessory CL with GnRH at the time of embryo transfer in Bos indicus cross recipients. However, in another study involving Bos indicus-cross recipients synchronized with the progestin/estradiol plus eCG protocol [Tribulo et al., 2005], pregnancy rates were not affected by treatment with hCG or GnRH at the time of FTET. Small et al.  were also unable to improve pregnancy rates in Bos taurus recipients treated with GnRH or pLH on Days 5 or 7 after estrus. In a very recent study [Wallace et al., 2011], 719 beef recipients alternately received 1,000 IU hCG or saline (control) at the time of embryo transfer. Serum progesterone concentrations in pregnant cows with lower body condition score (i.e. <5 in the 1 to 9 scale) at pregnancy diagnoses were higher after hCG treatment than in controls, whereas no differences were detected in recipients with high body condition score (>5). The authors concluded that giving hCG at embryo transfer increased the incidence of accessory CL and serum progesterone resulting in increased pregnancy rates in recipients with lower body condition scores. Finally, lower embryonic losses in recipients that received GnRH two days prior to the transfer of in vitro-produced embryos were reported more recently [Garcia Guerra et al., 2016]. With the exception of the last report, the beneficial effects of increasing circulating concentrations of progesterone seem to be evident when pregnancy rates in control (not treated) recipients were lower than expected.
Resynchronization of Non-pregnant Recipients
Progestin devices have also been used to resynchronize cattle that previously received an embryo [Bó et al., 2005b; Mapletoft et al., 2003; Colazo et al., 2007]. In one approach, animals receive a new or used progestin device at the time of embryo transfer, or on Days 12 or 13 after estrus. When devices were removed on Day 21, 50 to 60% of the non-pregnant animals were detected in estrus on Days 20 to 25 [Bó et al., 2005b]. Recipients not detected in estrus were presumed pregnant, whereas those in estrus were examined by ultrasonography 7 days later and if found not to be pregnant were reused for embryo transfer. In any case, embryo transfer programs can be designed, utilizing these approaches, or variations on these approaches, to minimize the interval between a diagnosis of non-pregnancy and transfer of another embryo. Normally, a recipient is removed from an embryo transfer program after being given two and sometimes three opportunities to become pregnant.
The two management factors that determine the success or failure of an estrus synchronization program are nutrition and post-partum interval. If cows lose weight during pregnancy, the onset of estrous cycles after calving will be delayed. Cows that are fed adequately during pregnancy but fail to gain weight between calving and breeding will cycle but have reduced conception rates and may also have reduced pregnancy rates after receiving a viable embryo by embryo transfer [Bó et al., 2005b]. In a field study, recipients were body condition scored at the time of embryo transfer on a scale of 1 (thin) to 5 (obese). Pregnancy rates were significantly higher in recipients scoring 3 and 4 than in those scoring 1, 2 or 5 [reviewed in Mapletoft, 1986]. Therefore, the nutritional status of recipients must be evaluated before setting up an embryo transfer program. Other nutrients important to reproductive efficiency are phosphorus and trace minerals. Although effects of mineral deficiencies can be profound in affecting reproductive function, a much more common and dramatic effect on reproduction occurs with energy deficiencies.
In the early days of commercial bovine embryo transfer, embryos were collected surgically around Day 4 after estrus [Betteridge, 1981; 2003]. Three methods of non-surgical embryo recovery were described in 1976 [Drost et al., 1976; Elsden et al., 1976; Rowe et al., 1976]. Non-surgical techniques are preferred as they are not damaging to the reproductive tract, are repeatable and can be performed on the farm [Mapletoft, 1985; 1986]. Briefly, the donor cow is placed in a squeeze chute and the rectum is evacuated of feces and air. The number of CL is usually estimated at this time or just prior to ova/embryo recovery. The perineal region and vulvar labia are washed thoroughly and dried, and the tail is tied out of the way. Embryo recovery is not attempted until a satisfactory epidural anesthetic is completed. It is also important to avoid ballooning of the rectum with air as sensitivity of palpation and manipulation is compromised; collection efficiency will be poor if air is not expelled.
Non-surgical techniques involve the passage of a cuffed catheter through the cervix and into one of the uterine horns on Days 6 to 8 after estrus [Mapletoft, 1985; 1986]. Once the catheter is in place, the cuff is inflated with saline or flushing medium. Care must be taken not to over-distend the cuff as the endometrium may split causing loss of collection medium and embryos into the broad ligament. There are two basic types of catheters used for non-surgical embryo collection. Original reports were on the use of two-way and three-way Foley catheters [Rowe et al., 1976]. Many groups still use the Foley catheter as it is inexpensive and readily available. However, the rubber is soft and the catheter is short and difficult to thread into the uterine horn. Furthermore, the distance from the cuff to the catheter tip is short. The two-way Rusch catheter has been preferred by many [Schneider, 1979]. It is 67 cm long, 14- or 18-gauge (O.D.) and has Luer-Lok fittings. The tip in front of the cuff measures 5.5 cm and has four holes. The catheter is stiffened for passage through the cervix by a stainless steel stilette, which locks into the Luer-Lok fittings. It is long enough for large cows and is stiff enough that it can be easily threaded down the uterine lumen. Many other catheters are now available from embryo transfer suppliers, but they are really modifications of the above two types. The more important consideration today is whether the catheter can be autoclaved or it must be considered disposable. Silicon catheters are most commonly used nowadays.
Basically, there are two methods of embryo collection [Mapletoft, 1986]: the continuous or interrupted flow, closed-circuit system and the interrupted-syringe technique. However, any combination of these two techniques is possible. It must be recognized that each system has advantages and disadvantages relative to the other. With the closed system, it is easier to maintain sterility and there is less chance of losing medium and consequently, embryos. However, it is cumbersome and the extra tubing provides extra potential for contamination by either microbes or chemicals. Again, embryo transfer suppliers now provide disposable equipment for closed-circuit systems. With the interrupted syringe method, it is possible to use fully-disposable equipment, with the exception of catheters, and to search for embryos while the collection is in progress. However, syringe exchanges provide more opportunities for fecal contamination and loss of medium/embryos.
The embryo recovery medium is prepared before preparation of the cow. Dulbecco's phosphate-buffered saline (PBS) or other basic salt solutions can be prepared in 500 to 1000 ml bottles and kept refrigerated ready for use. In addition, quantities of heat-inactivated fetal calf serum (FCS), and of an antibiotic/antimycotic solution may be kept frozen so that a single quantity of each is required for each volume of PBS collection medium. Collection medium will then contain 1 to 2 % FCS and the appropriate concentration of antibiotics (normally, 100 IU of penicillin, 100 μg of streptomycin and 25 μg of Fungizone per ml). The holding medium, containing higher concentration of serum (∼10%), is normally held in a "plastic on plastic" syringe before use, (an antioxidant on the rubber in plastic syringes has been shown to be toxic to embryos) [Hasler, 2003]. Similarly, if syringes are used in the collection procedure, it is recommended that those with rubber plungers be washed and/or heat sterilized before use. Holding medium is normally passed through a disposable 0.22 μm Millipore filter prior to use, but the first 4 to 5 ml should be discarded as it may also affect embryo survival. Nowadays, ready-made embryo collection and holding media are available commercially; they are ready for use and have been filtered previously. However, if they contain animal products, e.g., serum or BSA, they must be refrigerated. Very recently, collection and holding media that do not contain animal products (contain polyvinyl alcohol or pluronate as a surfactant) have become available making refrigeration unnecessary [Hasler, 2010].
Temperature does not seem to be critical to embryo survival, provided extremes are avoided. Room temperature seems satisfactory. Similarly, sterility is not possible, but every attempt should be made to be as clean as possible. Sterilization with chemicals is more likely to kill embryos than microbial contaminants. Thorough washing of in vivo-derived embryos with sterile medium has been shown to remove all infectious agents [Singh, 1985]. As a routine, embryos should be passed through 10 washes of fresh, sterile medium prior to transfer or freezing. Certain infectious agents such as the Bovine Herpes virus, have been shown to "stick" to the zona pellucida. If these agents are of concern, two trypsin treatments after wash number 5 is recommended to dissociate the agent from the zona pellucida. Then, the final 5 washes are done [IETS Manual].
Embryos are located with a stereoscopic dissecting microscope at 10 X magnification after filtering the collection medium through a filter with pores that are approximately 50 to 70 μm in diameter [Mapletoft, 1986]. Although embryos are usually transferred as soon as possible after collection, it is possible to maintain embryos in holding medium for several hours at room temperature. It is also possible to cool bovine embryos in holding medium and to maintain them in the refrigerator for as long as 2 or 3 days. As a final alternative, embryos may be frozen for use at a later date.
Embryos are normally held in the same or a similar medium to that in which they were collected. Media must be buffered to maintain a pH of 7.2 to 7.6 and have an osmolarity around 300 mOs. Dulbecco's PBS or more complex media with the Hepes buffer and enriched with FCS and antibiotics are normally used in the field. More complex media with a carbonate buffer generally yield superior results for long term culture of bovine embryos in a laboratory setting. As indicated earlier, embryo collection, holding and freezing media that are free of animal products have become available recently, avoiding the need for refrigeration and at the same time, increasing biosecurity.
Evaluation of bovine embryos must be done at 50 to 100 X magnification, with the embryo in a small culture dish. The International Embryo Transfer Society (IETS) has a numerical system for classification of embryo developmental stage, ranging from 1 (single cell zygote) to 8 (hatched blastocyst) and quality from 1 (good and excellent) to 4 (dead). It is important to be able to recognize the various stages of development and to compare these with the developmental stage that the embryo should be based on the days from estrus. Often a decision as to whether an embryo is worthy of transfer will depend on the availability of recipients. Fair quality embryos should be transferred fresh, if recipients are available, while good and excellent quality embryos have a high probability of surviving cryopreservation. The IETS considers the export of poor and fair quality embryos to be improper [IETS Manual]. Bovine embryo evaluation has been reviewed recently [Bó and Mapletoft, 2013], but the IETS Manual has the most complete library of embryo pictures.
Figure 11. Evaluation of bovine embryos must be done at 50 to 100 X magnification, with embryos in a small culture dish. A and B are IETS quality code 1 (good and excellent) morulae while C is an IETS quality code 1 early blastocyst [IETS Manual].
Embryos are classified and evaluated by morphological examination at 50 to 100 X magnification according to the Manual of the International Embryo Transfer Society [IETS Manual]. The overall diameter of the bovine embryo is 150 to 190 um, including a zona pellucida thickness of 12 to 15 mm. The overall diameter of the embryo remains virtually unchanged from the one-cell stage to blastocyst stage. The best predictor of an embryo's viability is its stage of development relative to what it should be on a given day after ovulation. An ideal embryo is compact and spherical. The blastomeres should be of similar size with a homogenous color and texture. The cytoplasm should not be granular or vesiculated. The perivitelline space should be clear and contain no cellular debris. The zona pellucida should be uniform, neither cracked nor misshapen and should not contain debris on its surface. Embryos of good and excellent quality (quality code 1) and at the developmental stages of late morula to blastocyst yield the highest pregnancy rates. It is advisable to select the stage of embryo development to correspond to the stage of the cycle in the recipient (synchrony).
Stages of Embryo Development
- Morula: A mass of at least 16 cells. Individual blastomeres are difficult to discern from one another. The cellular mass of the embryo occupies most of the perivitelline space.
- Compact Morula: Individual blastomeres have coalesced, forming a compact mass. The embryo mass occupies 60 to 70% of the perivitelline space.
- Early Blastocyst: An embryo that has formed a fluid-filled cavity or blastocoele and gives a general appearance of a signet ring. The embryo occupies 70 to 80% of the perivitelline space. Early in this stage of development , as the blastocoele begins to form, the embryo may appear of questionable quality.
- Blastocyst: Pronounced differentiation of the outer trophoblast layer and of the darker, more compact inner cell mass is evident. The blastocoele is prominent, with the embryo occupying most of the perivitelline space. Visual differentiation between the trophoblast and the inner cell mass is possible at this stage of development.
- Expanded Blastocyst: The overall diameter of the embryo dramatically increases, with a concurrent thinning of the zona pellucida to approximately one-third of its original thickness. The zona pellucida may be seen to crack just prior to hatching.
- Hatched Blastocyst: Embryos recovered at this developmental stage can be undergoing the process of hatching or may have shed the zona pellucida completely. Hatched blastocysts may be spherical with a well-defined blastocoele or may be collapsed. Identification of an embryo at this stage can be difficult unless it re-expands.
Figure 12. A bovine embryo with about 16 cells, as it would appear in the uterus of a cow about 4 to 5 days after ovulation. The diameter of this embryo (about 0.15 mm) has likely changed little from that immediately after fertilization. (Courtesy of Harold Hafs).
Figure 13. A bovine morula with a mass of at least 32 cells. Individual blastomeres are difficult to discern from one another. The cellular mass of the embryo occupies most of the perivitelline space.
Figure 14. Expanded, hatching and hatched blastocysts produced by in vitro fertilization with frozen-thawed semen following in vitro maturation. One blastocyst has hatched from the zona pellucida and a second has begun to hatch; note that the zona pellucida is very thin. (Courtesy of Sanjay Khanna and John Parks).
Figure 15. A bovine blastocyst hatching through a crack in the zona pelucida. Note that the inner cell mass and some of the trophoblast are outside the zona pellucida. (Courtesy of Dr. John K. Thibodeaux).
- Excellent: An ideal embryo, spherical, symmetrical and with cells of uniform size, color and texture.
- Good: Small imperfections such as a few extruded blastomeres, irregular shape and a few vesicles.
- Fair: Problems that are more definite are seen, including presence of extruded blastomeres, vesiculation, and a few degenerated cells.
- Poor: Severe problems, numerous extruded blastomeres, degenerated cells, cells of varying sizes, large and numerous vesicles but an apparently viable embryo mass. These are generally considered to be not of transferable quality.
Recommended Quality Codes [IETS Manual]
The IETS recommended codes for embryo quality range from "1" to "4" as follows:
- Code 1: Excellent or good. Symmetrical and spherical embryo mass with individual blastomeres (cells) that are uniform in size, color and density. This embryo is consistent with its expected stage of development. Irregularities are relatively minor and at least 85% of the cellular material is an intact, viable embryo mass. This judgment should be based on the percentage of embryo cells represented by the extruded material in the perivitelline space. The zona pellucida should be smooth and have no concave or flat surfaces that might cause the embryo to adhere to a Petri dish or a straw.
- Code 2: Fair. Moderate irregularities in overall shape of the embryo mass or size, color and density of individual cells. At least 50% of the cellular material is an intact, viable embryo mass.
- Code 3: Poor. Major irregularities in shape of the embryo mass or size, color and density of individual cells. At least 25% of the cellular material is an intact, viable embryo mass.
- Code 4: Dead or degenerating. Degenerated embryos, oocytes or 1-cell embryos; non-viable.
The Manual of the International Embryo Transfer Society states, "It should be recognized that visual evaluation of embryos is a subjective evaluation of a biological system and is not an exact science. Furthermore, there are other factors such as environmental conditions, recipient quality and technician capability that play important roles in obtaining pregnancies from transferred embryos. It is also recognized that many different systems are used for "grading" embryos and that some are more sophisticated than others. The criteria for assigning a "quality code" in the standardized forms were simplified to be "user friendly". Generally, unless otherwise specified, only Code 1 embryos should be utilized in international commerce".
In the superovulated cow, there is likely to be a considerable range of embryo stages on any given day during development. On Day 7 after estrus, there may be morulae and blastocysts (or even hatching blastocysts) within the same collection. At the same time, there may be embryos of excellent quality and unfertilized and degenerate embryos. Generally, wide variations in embryo quality and stages of development are signals that normal-appearing embryos maybe stressed or compromised and that pregnancy rates may be disappointing. Embryos of excellent and good quality (quality code 1), at the developmental stages of compact morula (stage code 4) to blastocyst (stage codes 5 or 6) yield the highest pregnancy rates, especially after cryopreservation. Fair and poor quality embryos yield poor pregnancy rates after cryopreservation and should be transferred fresh. It is advisable to select the stage of embryo development for the synchrony of the recipient. Fair and poor quality embryos are most likely to survive transfer if they are placed in the most synchronous recipients.
Transfer of embryos in cattle will result in a high pregnancy rate providing the preceding estrus in the donor and recipient occurred within 24 hours of each other [Hasler et al., 1987]. Alternately, recipients must be synchronous with the stage of development of embryos that had been previously cryopreserved. Recipients can be made available by maintaining a large herd to obtain natural synchrony or by estrus synchronization, which is much more economical. Today most recipients are synchronized regardless of whether or not embryos are transferred "on farm".
Initially, embryo transfers in the cow were done surgically, whereas most are done today using non-surgical methods [Betteridge, 1981; 2003]. Surgical transfers were first done by way of a midline incision, which necessitated the use of general anesthesia and rather elaborate surgical facilities. During the mid to late 1970's, this gave way to a standing flank approach, an approach that could be done more quickly and because of lesser requirements in facilities made "on farm" embryo transfer possible. Most recently, the use of non-surgical embryo transfers has increased the utilization of embryo transfer on farm [Rowe et al., 1980; Wright, 1981].
Non-surgical embryo transfer techniques involve the use of specialized embryo transfer pipettes. After confirming the synchrony of estrus, the recipient is restrained and the rectum is evacuated of feces. At the same time, the presence and side of a functional CL is confirmed. Care is taken to prevent ballooning of the rectum with air. An epidural anesthetic is administered and the vulva is washed with water (no soap) and dried with a paper towel. The embryo is loaded in 0.25 ml straw between at least two air bubbles and two columns of medium and the straw is loaded in the embryo transfer pipette. Care is taken to ensure that the straw engages the sheath tightly so as to avoid leakage. The sheath is coated with sterile, non-toxic obstetrical lubricant and the sheathed pipette is passed through the vulvar labia while avoiding contamination. The embryo is placed in the uterine horn adjacent to the ovary bearing the CL by passing the pipette through the cervix, very similar to artificial insemination. However, an attempt is usually made to pass the transfer pipette at least half-way down the uterine horn. The uterine lumen should be lined-up prior to passage so as to prevent trauma to the endometrium. The embryo is deposited slowly and firmly while the tip of the transfer pipette is withdrawn slightly. Practice and dexterity seem to improve one's ability to achieve high pregnancy rates suggesting that trauma to the endometrium may be a limiting factor with this method of embryo transfer. Stimulation of the cervix or inadvertent introduction of bacterial contaminants do not seem to be major determinants of pregnancy rates under normal circumstances. With practice and attention to detail, pregnancy rates with non-surgical transfers can equal those of surgical transfers.
With existing technology, an average of 8 to 10 ova/embryos will be collected from each super-stimulated donor cow and 6 or 7 embryos will be transferred, resulting in 3 to 4 pregnancies. It must be emphasized that very few donors are average. Pregnancy rates are generally around 60% with fresh embryos and range from 50% to 60% with frozen/thawed embryos. One can anticipate a death loss of 10% from pregnancy diagnosis until the calf is six months of age. It is worthy of note that this is not different from that of experienced with natural service or AI and that embryo transfer procedures have been shown to result in no increase in calf abnormalities.
The development of effective methods of cryopreservation of embryos [Leibo and Mazur, 1978; Wilmut and Rowson, 1973] made bovine embryo transfer a much more efficient technology, no longer depending on the immediate availability of suitable recipients. Pregnancy rates following the use of frozen/thawed embryos are only slightly less than those achieved with fresh embryos [Leibo and Mapletoft, 1998]. Recently, the use of highly permeating cryoprotectants, such as ethylene glycol, has allowed the Direct Transfer of bovine embryos [Hasler et al., 1997; Voelkel and Hu, 1992] with no need for dilution of the cryoprotectant or post-thaw evaluation. In a study of the North American embryo transfer industry, more than 95% of frozen-thawed embryos were transferred by Direct Transfer and pregnancy rates from Direct Transfer embryos frozen in ethylene glycol were comparable to those achieved with glycerol and serial dilution prior to transfer [Leibo and Mapletoft, 1998]. In addition, a growing number of Direct-Transfer embryos are being transferred by technicians with experience in AI.
Freezing and thawing procedures are time-consuming and require the use of biological freezers. Complicated embryo freezing procedures may be replaced by another relatively simple procedure called vitrification [Rall and Fehy, 1985]. With vitrification, the embryo in a high concentration of cryoprotectants is placed directly into liquid nitrogen. As a result of the high concentration of cryoprotectants and the ultra-rapid rate of freezing, ice crystals do not form; instead the frozen solution forms a "glass". Since ice crystal formation is one of the most damaging processes in freezing, vitrification has much to offer in the cryopreservation of oocytes and in vitro-produced embryos. However, its greatest advantage is its simplicity. Although bovine embryos have been vitrified successfully in 0.25 ml straws for direct transfer [van Wagtendonk et al., 1997], vitrification with smaller volume containers seem to result in higher embryo survival rates, especially with in vitro-produced embryos, because of faster cooling rates [Vajta and Kuwayama, 2006]. Despite the simplicity of vitrification and the need of only a few minutes to cryopreserve a single embryo, large numbers of embryos actually require considerably more time with vitrification than with slow, controlled freezing procedures.
The freezing of a living cell constitutes a complex physicochemical process of heat and water transport between the cell and its surrounding medium. There exists an optimum cooling-rate for each type of cell. It is dependent on the size of the cell, its surface to volume ratio, its permeability to water, and the temperature coefficient of that permeability [Leibo and Mazer, 1978, Palasz and Mapletoft, 1996].
Normally, the medium that contains the embryo cools below its freezing point without ice crystal formation, a phenomenon referred to as super-cooling. Then, at some lower temperature, ice nucleation occurs, followed by a rapid rise in temperature due to the release of latent heat of fusion. To avoid extensive super-cooling, ice crystallization is induced in the extracellular medium some 2°C below its freezing point (-4 to -7°C) by seeding the medium with an ice crystal [Palasz and Mapletoft, 1996]. Water in the cells of the embryo and between the ice crystals outside the embryo does not freeze at this temperature because of solutes lowering its freezing point. During further cooling and enlargement of ice crystals due to freezing pure water, the solute concentration rises and the embryo responds osmotically by losing water into the unfrozen extracellular medium.
Cells are injured during freezing and thawing primarily by solution effects and intracellular ice formation [Leibo and Mazur, 1978; Palasz and Mapletoft, 1996]. The latter is especially detrimental when relatively large amounts of large ice crystals form. To avoid intracellular ice crystal formation, embryos must be cooled at a rate of <1°C/min. However, very slow cooling rates can also damage cells by what has been referred to as the solution effect. This is especially harmful if cells are not allowed to rehydrate during very rapid thawing [Palasz and Mapletoft, 1996].
The required thawing rate depends on the freezing regimen used. When embryos are cooled slowly to temperatures between -27 and -40°C and then rapidly to -196°C (liquid nitrogen), thawing must be rapid, e.g., about 200°C/min. Cells treated in this way may contain some intracellular ice, and thawing has to be rapid to prevent injury from the recrystallization of that ice. On the other hand, if embryos are cooled slowly to temperatures below -60°C before transfer to liquid nitrogen, thawing is then normally done slowly at about 20°C/min [Leibo and Mazur, 1978]. Although both systems result in similar rates of embryo survival, more rapid techniques of freezing and thawing are preferred in the field.
Embryos are normally stored in liquid nitrogen at -196°C. The only reactions that occur at -196°C are direct ionizations from background radiation. Consequently, storage times of more than 200 years are unlikely to produce any detectable reduction in survival or cause genetic change of frozen embryos.
Cryoprotectants such as glycerol in concentrations ranging from 1.0 to 2.0 M are required to ensure embryo survival after freezing. It is thought that cryoprotectants act by reducing the amount of ice present at any temperature during freezing, thereby moderating the changes in solute concentration. Recommended criteria for a cryoprotectant include high solubility, low toxicity at high concentrations, and a low molecular weight both for easier permeation and to exert a maximum colligative effect [Palasz and Mapletoft, 1996]. Glycerol has been most often used for the protection of embryos during cryopreservation, but more recently, more highly permeating cryoprotectants such as ethylene glycol, have been preferred because they can be used with "Direct Transfer" i.e., transfer into a recipient without the need for removal of the cryoprotectant before transfer [Leibo and Mapletoft, 1998, Voelkel and Hu, 1992].
During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes resulting in swelling or contraction [Palasz and Mapletoft, 1996]. Consequently, if the addition, or particularly the dilution, is carried out inappropriately, the viability of cells can be compromised. Glycerol can be added to embryos in a single step, but there is clear evidence that the rate of glycerol removal is more critical. The standard empirical method of glycerol removal is to dilute it by the "stepwise" addition of isotonic medium or to pipette the embryos into decreasing concentrations of glycerol, e.g., 0.25 M steps [Palasz and Mapletoft, 1996]. However, Leibo and Mazur  suggested a modification in the procedure of cryoprotectant removal by including non-permeable solutes like sucrose into the dilution medium. The sucrose acts as an osmotic counterforce to restrict water movement across the membranes. As water and the cryoprotectant leaves the embryo, in response to the extracellular hypertonic dilution medium it will shrink. It regains its normal volume by taking in water when at the end of the process the embryo is placed in normal isotonic culture medium. Using this information, practical methods of quickly removing glycerol from thawed embryos have been devised. As a result, a "one-step straw" was developed so that embryos could be thawed, solutions mixed within the straw and transfer to the recipient done non-surgically, all in the field. In one field study, 476 frozen embryos were thawed and processed in sucrose in the straw prior to transfer, without microscopic evaluation, resulting in a 42.4% pregnancy rate [Leibo, 1984]. More recently, this method has given way to "Direct Transfer" utilizing highly permeating cryoprotectants, such as ethylene glycol, which do not harm the embryo osmotically if not removed prior to transfer. Pregnancy rates for "Direct Transfer" in Canada, with more than 19,000 bovine embryos, were not different from those achieved with glycerol and cryoprotectant removal prior to transfer [Leibo and Mapletoft, 1998].
The following protocol has been shown to be successful for the cryopreservation of Day 7 bovine embryos in Dulbeccos PBS supplemented with 10 to 20% FCS and 1.0 to 1.5 M glycerol [Mapletoft, 1985]. Embryos are pipetted into the freezing medium at room temperature (20°C) and left for eight to 10 minutes to permit the glycerol to enter and equilibrate within the embryo cells. During this equilibration period the embryo(s) are transferred in volumes of 0.25 or 0.5 ml of freezing medium into French straws (between air bubbles) that are then securely sealed. The samples can be immediately transferred into the freezing chamber at -6 or -7°C and held for 5 min. Ice crystallization (seeding) of the extracellular medium is initiated by touching the outside wall of the straw with a forceps, rod or Q-tip pre-cooled in liquid nitrogen (do not touch the column of medium that contains the embryo(s). The samples are held at the seeding temperature for an additional 10 min to allow the ice crystallization in the extracellular medium to progress to equilibrium (check to be sure that ice crystals are growing in the straw). Then, the straws are cooled at 0.3 to 0.8°C/min to a temperature between -30 and -40°C, at which time they are immersed directly in liquid nitrogen (-196°C) and stored.
Thawing is carried out by placing the straw into a water-bath at a temperature between 20 and 35°C; the thaw rate should be around 200°C/minute. It has been reported that the incidence of cracked zona pellucida was reduced in an air-thaw or when straws were thawed in air for 10 to 15 seconds prior to being submerged into a 35°C water bath. This is important if dilutions of glycerol must be done before transfer the embryo without a zona pellucida is difficult to locate for dilution), but is of no consequence with Direct Transfer [Leibo and Mapletoft, 1998].
When glycerol is used as the cryoprotectant, it must be removed without causing osmotic damage. The method of choice is the use of sucrose solution between 1.0 M and 0.5 M in a single step for 10 min or 0.3 M sucrose in a 3-step dilution of 5 min each (0.75 M glycerol and 0.3 M sucrose; 0.375 M glycerol and 0.3 M sucrose; 0.3 M sucrose) [Mapletoft, 1985]. The embryos are then transferred back into holding medium, washed and evaluated prior to transfer.
The use of highly permeating cryoprotectants such as ethylene glycol has allowed the Direct Transfer of bovine embryos without the necessity of microscope examination and cryoprotectant removal [Leibo and Mapletoft, 1998; Voelkel and Hu 1992]. With this approach, the embryo straw is thawed in a water-bath, much like semen, and the contents of the straw are deposited into the uterus of the recipient, much like artificial insemination. There is no need of a microscope or complicated dilution procedures. The cryoprotectant leaves the embryo in the uterus. As indicated earlier, the Direct Transfer of bovine embryos in Canada resulted in overall pregnancy rates which did not differ from that achieved by regular cryoprotectant dilution techniques with glycerol. The transfer of frozen/thawed bovine embryos is now becoming very similar to the use of frozen/thawed semen in AI.
In vitro Embryo Production
Bovine in vitro embryo production (IVP) is now a well-established and efficient procedure [Brackett and Zuelke, 1993]. Moreover, ovum pick-up (OPU) at frequent intervals, in combination with in vitro fertilization (IVF), has improved and increased the yield of embryos from designated donors [Bols et al., 1996; Garcia and Salaheddine, 1998; Looney et al 1994]. In vitro fertilization has also been used to produce the thousands of embryos needed for scientific research, including efforts to produce embryonic stem cells; the constituent oocyte maturation and embryo culture techniques are integral parts of the procedures for cloning and transgenesis [Campbell et al., 1996; Niemann and Kues, 2003]. A few laboratories have also reported very modest successes in producing pregnancies with IVP embryos from calves [Baruselli et al., 2015; Duby et al., 1996; Fry et al., 1998; Taneja et al., 2000;], which offers the potential for decreasing generation intervals [Betteridge et al., 1989]. In addition, OPU has shown to be safe and very successful in pregnant cattle [Eikelmann et al., 2000].
Several authors have addressed the question of using IVP as a substitute for in vivo embryo production [Bousquet et al., 1999; Gordon and Lu 1990; Hasler, 1998; Hasler et al., 1995; Sinclair et al., 1995]. Although IVP appears to be more expensive than conventional superovulation under commercial conditions in North America, its use is increasing rapidly. For many breeders, this technology has been an advantage for extremely valuable cows which are infertile or fail to produce embryos after superstimulation. Indeed, the number of IVP embryos produced in North America in 2014 was 39.5% higher than 2013. Brazil continues to account for most of the world’s total IVP, but other countries are increasing their numbers of IVP embryos very rapidly [Perry, 2015]. In 2014, 69,140 OPU sessions were performed in Brazil, yielding an average of 12 oocytes and 5 embryos per session. As a result, IVP embryo numbers have surpassed that of in vivo embryo production in Brazil; it will be interesting to see if the trend continues for other countries in the world.
The efficiency of frozen IVP embryos will likely determine the acceptance of IVP technology by other countries [Hasler et al., 1995]. So far, the majority of the IVP embryos in Brazil have been transferred fresh, not frozen. However, approximately 18% of the IVP embryos transferred worldwide in 2014 were frozen-thawed which is double that reported for the previous year; results are improving, especially in Brazil where Bos indicus breeds are used predominantly [Perry, 2015].
Adoption of New Technologies
Prenatal determination of sex potentially has great economic impact [Seidel, 2003] and the use of the polymerase chain reaction (PCR) to determine the sex of bovine embryos is a service offered by some embryo transfer practitioners [Thibier and Nibart, 1995]. However, embryo biopsy requires a high level of operator skill, and is an invasive technique resulting in disruption of the integrity of the zona pellucida and some reduction in the viability of the embryo, especially after cryopreservation. More recently, the use of sex selected semen has tended to replace embryo sexing as a means of producing altered sex ratios in embryo transfer offspring (see below). However, PCR assays to identify other traits of economic importance will no doubt become available [Bishop et al., 1995]. Marker-assisted selection (MAS), based on identifying genetic markers for unknown alleles of valuable traits, probably has a similar future [Georges and Massey, 1991]. Like genotyping of specific alleles, MAS can be applied to embryo biopsies if sufficiently valuable markers can be identified. A PCR assay currently exists for simultaneous detection of the bovine leucocyte adhesion deficiency gene and the sex of embryo biopsies [Hasler, 2003]. It is probable that PCR techniques will be developed that permit the analysis of a large number of markers from one biopsy leading to the concept of "embryo diagnostics' '. It has also been reported that genomic testing of embryos with SNP technology can be done with accuracy, again utilizing embryo biopsies and PCR technology [Ponsart et al., 2014; Seidel, 2010].
The flow cytometric technology used to separate X- and Y-bearing sperm into live fractions has improved over the last 20 years [Johnson et al., 1994; Johnson, 2000]. Approximately 10 million live sperm of each sex can be sorted per hour [Seidel, 2003], with a resulting purity rate of >90%. In AI field trials, pregnancy rates following insemination with 1 million sexed, frozen sperm were reported to be 70% to 90% that of unsexed controls inseminated with 20 to 40 million sperm [Seidel et al., 1999]. A study which compared 574 calves produced from sex-sorted sperm with 385 control calves concluded that there were no differences in gestation, neonatal deaths, ease of calving, birth weight or survival rate to weaning [Tubman et al., 2003]. The disadvantages of flow cytometry are the slow speed of sorting, the decreased sperm viability (pregnancy rates), especially in superovulated donors, the cost of the semen, and the availability of semen from specific bulls [Amann, 1999]. It is likely that sex-selected semen will have the greatest use in IVP of bovine embryos in the near future, but sex-selected sperm is also being used in superstimulated females at an insemination dose of 5 million sperm at 18 and 30 hours after the onset of estrus [Baruselli et al., 2015].
Summary and Conclusions
Commercial embryo transfer in cattle has become a well-established industry. Although a relatively small number of offspring are produced on an annual basis, its impact is large because of the quality of animals being produced. Embryo transfer is now being used for real genetic gain, especially in the dairy industry, and most semen used today comes from bulls that have been produced by embryo transfer. An even greater benefit of bovine embryo transfer may be that in vivo-derived embryos can be made specified pathogen-free by washing procedures, making this an ideal process for disease control programs or in the international movement of animal genetics. Techniques have improved over the past 40 years so that frozen-thawed embryos can be transferred to suitable recipients as easily and simply as artificial insemination is normally done. In vitro embryo production and embryo and semen sexing are also successful. A combination of embryo transfer using proven cows inseminated with semen from proven bulls, followed by industry-wide artificial insemination appears to be the most common use of bovine embryo transfer.
Identification, Certification and Registration of Offspring
Records for the accurate identification of parentage and of embryo transfer offspring is of vital importance for both domestic and international use of embryo transfer technology. The IETS has developed three forms for certification of embryo recovery, freezing and transfer, respectively. In addition, a fourth form (Certificate D) is recommended for use in embryo exports [IETS Manual]. The IETS also allocates embryo-freezing codes to practitioners that must appear on all embryo containers and all documentation so that the organization freezing embryos can be identified. Finally, standard procedures for labeling embryo freezing containers are also recommended by the IETS e.g., embryos frozen for Direct Transfer are to be frozen in yellow straws and placed in yellow goblets. Examples of the above forms and specific instructions on their use, the labeling of embryo freezing containers and the identification of embryo developmental stages and quality grades are available in the Manual of the International Embryo Transfer Society.
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Affiliation of the authors at the time of publication
1Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. 2Instituto de Reproducción Animal Córdoba (IRAC), Zona Rural General Paz, Córdoba, Argentina.