In: IVIS Reviews in Veterinary Medicine, I.V.I.S. (Ed.). International Veterinary Information Service, Ithaca NY (www.ivis.org), Last updated: 17-Nov-2006; R0104.1106

Bovine Embryo Transfer

R. J. Mapletoft

Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 5B4.

Table of Contents

Introduction Bovine embryo transfer technology involves the selection and management, both physical and pharmacological, of donor and recipient animals, and the collection and transfer of embryos within a narrow window of time following estrus. This technology has been incorporated into large dairy and beef cattle operations, and often requires the participation of herd veterinarians. The following review is based extensively on handout material for a pre-conference seminar presented by the author for the annual meeting of the American Association of Bovine Practitioners (AABP). It also draws heavily on material contained in prior reviews, extensive literature of primary research on the topic, including reviews and reports from the author's laboratory.

The commercial embryo transfer industry in North America developed in the early 1970's with the introduction of continental breeds of cattle [11,12]. The use of embryo transfer technology in cattle breeding has continued to increase (especially within the dairy industry) over the past 30 years with the movement toward genetic improvement as opposed to the production of desirable phenotypes [29,75]. In Canada, approximately 70% of the embryo transfer work is now being done on dairy cattle, and approximately 15,000 embryos are being frozen annually for export (Canadian Embryo Transfer Association Statistics, www.ceta.ca). Throughout the world over the past year, more than 100,000 donor cows were superstimulated and more than 500,000 bovine embryos were transferred [83]. This technology is influencing the direction of cattle breeding industries; the numbers are small but the impact is high. Commercial cattle breeders must recognize that they can benefit from well-designed embryo transfer programs providing selection criteria are appropriate for their environment and individual breeding objectives [35,71].

Applications 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 [29]. For example, several research laboratories are presently using in vitro fertilization (IVF) techniques to study the fertilizing capacity of sperm. A few of the more common uses of embryo transfer technology in animal production follow.

Genetic Improvement Genetic progress has generally been considered to be slower with embryo transfer than with conventional artificial

Introduction

Applications

Donor Selection

General Procedural Steps

Physiology and Endocrinology of the Normal Bovine Estrous Cycle

Superovulation

Embryo Recovery

Embryo Handling

Embryo Transfer

Estrus Detection

Estrus Synchronization for Embryo Transfer

Fixed-time Embryo Transfer

Embryo Freezing

Identification, Certification and Registration of Offspring

insemination (AI), especially on a national herd basis. However, with increased selection intensity and shortened generation intervals, i.e., transferring female offspring, genetic gain can be made on a within-herd basis [18,20,75]. This has resulted in the term MOET (multiple ovulation and embryo transfer) [69,75,76]. In several countries around the world nucleus herds are now being developed and heifer offspring are being subjected to "Juvenile MOET", while male offspring are being selected for the next generation of AI bulls [77,81]. In this way, it has been estimated that genetic gains can be doubled. On the other hand, it has been estimated that the production of about six offspring per donor cow could double selection intensity and the rate of response to genetic selection for traits such as growth that can be measured in both sexes [77]. This would be especially worthwhile in improving elite herds, the genetics of which could be spread over a large population using AI [47,81]. Embryo transfer is now commonly used to produce AI sires from proven donor cows and bulls in AI service. Although economics would not at this time support the use of embryo transfer techniques for anything but seed stock production, the commercial cattle industry can benefit by the use of bulls produced through well-designed MOET programs.

Planned Mating By far the most common use of embryo transfer in animal production programs is the proliferation of so-called desirable phenotypes. As AI has permitted the widespread dissemination of a male's genetic potential, embryo transfer provides the opportunity to disseminate the genetics of proven, elite females. Embryo transfer also permits 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 [11]. 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 rapidly expand a limited gene pool. 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 the most common application of planned mating [81].

Genetic Testing The success of MOET programs has now led to the use of this technology to genetically test AI sires [47,81]. The Canadian Association of Animal Breeders developed a program for the production and testing of the next generation of AI sires [47,77,81]. Selected donor cows were superstimulated 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 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. Although accuracy may have been sacrificed, the shorter generation intervals can result in greater overall genetic gains.

Disease Control None of the infectious diseases studied in the bovine species have been transmitted by in vivo-produced embryos, providing embryo handling procedures were done correctly [73,78,80]. Several large studies have now shown that the zona-intact, washed, bovine embryo will not transmit infectious diseases. Consequently, it has been suggested that embryo transfer be used to salvage genetics in the face of a disease outbreak. For example, this may be a useful alternative in the establishing herds that are free of Bovine Leukosis, as this virus was not transmitted with embryos. Breeders are now using embryo transfer techniques to establish disease free herds to be used strictly for export purposes [80,87].

Import and Export The intercontinental transport of a live animal may cost several 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. However, the reduced risk of infectious disease transmission is the overwhelming benefit for using embryos in international trade [53,87]. This may be the single most important potential application of embryo transfer. Additional benefits of the export of embryos over that of live animals include a wider genetic base from which to select, the retention of genetics within the exporting country and adaptation. This is particularly true of tropical and subtropical climates where the embryo would have the opportunity to adapt first in the uterus, and then by suckling a recipient cow indigenous to the area.

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 freezing of IVF embryos is necessary [34-36]. 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-produced embryos have been developed to ensure that disease transmission is avoided, the in vitro-produced embryo may be more difficult to deal with [79,80]. Obviously, correct handling procedures are the key. 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 do with semen today.

Research 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 for the propagation of superior livestock [11,12]. 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. Studies that were originally planned to answer basic physiological questions are now being used to improve and increase the utilization of embryo transfer. Newer techniques have added an entirely new perspective to the utilization of embryo transfer for research purposes. 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.

Donor Selection Selection criteria for donor animals are very likely to differ depending on the reason for doing embryo transfer; however, reasons are most often economically motivated. Until recently, embryo transfer has been feasible for only very valuable cattle, and the costs associated with embryo transfer tend to be reduced, relatively speaking, as economic and genetic value increase [52]. Complete non-surgical techniques of recovery and transfer of embryos and improved pregnancy rates from frozen-thawed embryos, and direct transfer of frozen embryos have also reduced costs [35,51]. Under these circumstances, the top 10% of a purebred herd could be used as donors and then bred 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 [51]. When selecting genetically superior beef donors, objective traits such as calving ease, milk production, weaning and yearling weight and carcass value should be considered. As beef bulls can now be evaluated quite accurately for genetic merit, the selection of the service sire is also extremely important. The selection of dairy donors is already well established [69].

As optimal results will also reduce costs, donor selection may involve a previous history of success in embryo transfer [39,58]. In addition, daughters from cows that have been successfully used 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 should ensure genetic superiority and a high level of success, thereby making the procedure more economical.

General Procedural Steps The donor may be inseminated naturally or artificially and embryos will be collected non-surgically six to eight 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 [35]. The discussion of donor superovulation, recipient synchronization and embryo transfer must begin with a review of 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, ovaries and uterus. These include gonadotrophin releasing hormone (GnRH) from the hypothalamus, follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland, estrogen, progesterone and inhibin from the ovary and prostaglandin F2 (PGF) from the uterus. The primary timing mechanism of the bovine estrous cycle is the demise of the corpus luteum (CL), which occurs about Day 17 - 18 in the normal cycling, non-pregnant cow. 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. 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 further stimulated. 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. The preovulatory LH peak results in resumption of oocyte meiosis, ovulation 24 - 32 h later (the LH peak occurs around the onset of estrus) 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 on about Day 17 - 18 following ovulation.

It has now been shown by ultrasonography that follicles in cattle develop in a wave-like fashion [62]. Bovine estrous cycles are composed of 2 or 3 waves of follicular development. A follicular wave consists of a group of growing antral follicles 3 - 6 mm in diameter from which a dominant follicle is selected while the remaining follicles become subordinate and undergo atresia (Figure 1) [1,2,30,63]. 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 in 2-wave cycles, and on 8 or 9 days after ovulation in 3-wave cycles, with a third wave emerging on Day 15 or 16. Duration of the estrous cycle is approximately 20 days in 2-wave cycles and 23 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 [2], and postpartum cows before the first ovulation [2]. 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 [3,4,5,31]. Surges in plasma FSH are responsible for eliciting the emergence of a follicular wave (Figure 1). FSH is subsequently suppressed by products of the growing follicles (e.g., estradiol and inhibin). In each wave, the follicle that first acquires LH receptors becomes the dominant follicle while subordinates 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.

Estrus synchronization and superovulation are critical components of an embryo transfer program. These techniques involve the manipulation of the basic endocrine patterns outlined above [86]. The key to successful estrus synchronization is obtaining closely synchronized, rapid declines in circulating progesterone to values

physiology, factors inherent to the donor animal that affect superovulatory response are only partially understood [15,54].

Superovulation-inducing treatments are usually initiated between Days 8 and 12 of the estrous cycle (estrus = Day 0) [51,52,54]. These times were originally based on the theory that a wave of follicles in the ovary was maturing at that time. However, we clearly demonstrated a greater superovulatory response when gonadotrophin treatments were initiated on Day 9 of the estrous cycle (Day 8 post-ovulation) as compared to Days 3, 6 or 12 [46]. This observation has been supported by more recent 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 [1,30,63]. In a practical sense, it is noteworthy that two-wave cows tended to have shorter cycles (18 - 20 days) than three-wave cows (21 - 23 days). Therefore, the length of the previous estrous cycle can provide a clue as to the best time to initiate superstimulatory treatments.

Moor et al., [58] 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 gonadotrophin treatments. More recently, Singh et al., [74] showed that the numbers of follicles present at follicular wave emergence was predictive of superstimulatory response. Collectively, these data may be interpreted to suggest that some of the variability resides in genetic or physiologic 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 [54]).

Ultrasonographic scanning of ovaries holds the promise of improving superovulation by increasing an understanding of the sources of individual animal variability. During the mid-part of an ovarian follicular wave, the dominant follicle, acting locally or systemically, induces atresia in other developing follicles [1,5]. Ultrasonographic examination of the ovaries may provide indications for the most propitious moment to initiate superstimulatory treatments to obtain maximal responses from individual cows. In this regard, the initiation of superstimulatory treatments in the presence of a dominant follicle resulted in a 40 - 50% decrease in superovulatory response [19,40,72]. Similarly, a high correlation between the numbers of small follicles at the start of gonadotrophin treatments and superstimulatory response has been demonstrated [74]. Collectively, these data may be interpreted to suggest that the presence of an active dominant follicle at the time superstimulatory treatments are initiated may be expected to depress superovulatory response, and the presence of actively growing follicles in the range of 3 - 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 [19]. However, the presence of more than six or seven follicles 3 - 6 mm in diameter 8 - 10 days after ovulation, in the presence of a large follicle, provides strong evidence for the beginning of a new follicle wave [74].

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 even pre-antral follicles, so that a larger, more uniformly responsive group is available when gonadotrophin treatments are initiated [16]. Alternatively, it is possible to mimic the effects of the dominant follicle and suppress the development of all antral follicles: gonadotrophin treatments initiated at selected times after the termination of follicle suppressing treatments, could be expected to catch a developing cohort of responsive follicles as they begin to grow [13,15].

Hormone Profiles in Superovulated Cows Low progesterone concentrations at the time of initiation of gonadotrophin treatment have been shown to be related to a reduced superovulatory response indicating the importance of a functional CL [54]. Progesterone increased within 24 h after treatment with pregnant mare serum/equine chronic gonadotrophin (eCG) or a crude pituitary extract suggesting a luteotrophic action of these gonadotrophins [6]. This did not occur with more highly purified pituitary extracts. Normally, the decline in progesterone is rapid after treatment with PGF [8,37,70]. Levels of progesterone dropped to less than 1 ng/ml of serum within 10 - 32 h. Ovulation was shown to occur approximately 72 h after PGF treatment in superstimulated cows

Figure 2. Two pairs of superstimulated 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 likely is 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. - To view this image in full size go to the IVIS website at www.ivis.org . -

[16]. At the onset of estrus, progesterone concentrations were lower in superstimulated cows that yielded good quality embryos than in cows that yielded unfertilized ova [6]. A high level of progesterone at the time of estrus may affect LH release and sperm transport and capacitation. After ovulation, the increase in serum progesterone concentration occurred earlier and the slope of the curve was steeper as the number of CL increased in superovulated cattle.

Although embryo transfer techniques are widely used around the world, variability in response to the hormonal treatments remains one of the limitations to the widespread application and success of this technology [32,39,48]. With a better understanding of ovarian function has come a greater capability of controlling it. Recent protocols, designed to control both luteal and follicular function, permit the initiation of superstimulatory treatments at a self-appointed time and provide the possibilities for superstimulation of cows that have abnormal ovarian function.

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 superstimulatory treatments were initiated 8 - 12 days after estrus [reviewed in 54]. However, none of these early studies evaluated follicular status at the time that gonadotrophin treatments were initiated.

Through information generated by ultrasonography, it is now known that 8 - 12 days after estrus (equivalent to Days 7 - 11 after ovulation) would be the approximate time of emergence of the second follicular wave in 2- or 3-wave cycles and a cohort of growing follicles would be present. However, the day of emergence of the second follicular wave differs among individuals and is 1 or 2 days later in two- versus three-wave cycles. In this regard, it has been clearly shown that superovulatory response was higher when superstimulatory treatments were initiated at the time of wave emergence; as little as 1 day asynchrony significantly reduced the superovulatory response compared to initiating treatments on the day of wave emergence [59].

Based on duration of the developmental phases of the dominant follicle in two- and three-wave interovulatory intervals, approximately 20% (4 or 5 days) of the estrous cycle is available for initiating treatment at the time of follicular wave emergence. Therefore, 80% of the estrous cycle is not conducive to an optimal superovulatory response. The necessity of waiting until mid-cycle to initiate superstimulatory treatments implies monitoring estrus and an obligatory delay. 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.

Follicle Ablation One approach to the synchronization of follicle wave emergence involves transvaginal ultrasound-guided follicle ablation of all follicles 5 mm, regardless of stage of the estrous cycle [9,28]. This removes the suppressive effects of follicle products (estradiol and inhibin) on FSH release; as a results FSH surges and new follicular wave emerges 1 day later. Superstimulatory treatments are then administered, beginning 1 day after ablation, and PGF is administered 48 or 72 h later [10]. The timing of estrus was more synchronous when a progestin device was inserted for the period of superstimulation and 2 injections of PGF were administered on the day of progestin removal. Combined over 2 experiments, there was no difference in the superovulatory response between the ablated and non-ablated control groups. Transvaginal ultrasound-guided follicle ablation of all follicles [10] or just the dominant follicle [19,72] during mid-diestrus, followed in 2 days by superstimulation, also resulted in a higher superovulatory response than cows in which 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 than cows superstimulated 7 - 13 days after estrus [40]. In a more recent study, ablation of the 2 largest follicles at random stages of the cycle was as efficacious as ablating all follicles 5 mm in synchronizing follicular wave emergence for superstimulation [7]. However, it is always advised that a progestin device be used during superstimulation.

GnRH Another method of synchronizing follicular wave emergence for superstimulation could involve the use of GnRH or porcine LH (pLH) to induce ovulation of a dominant follicle followed by emergence of a new follicle wave 2 days later [49,56,64,82]. However, the administration of GnRH or pLH does not always induce ovulation, and if ovulation does not occur, follicle wave emergence will not be synchronized [56]. 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 [56]. In a study involving 3 different experiments [22], GnRH or pLH treatments consistently resulted in a lower number of embryos than when follicular wave emergence was synchronized with other methods. Therefore, we do not recommend using GnRH or pLH to synchronize follicular wave emergence prior to superstimulation.

Estradiol and Progesterone Traditionally, estradiol has been administered near the beginning of progestin treatment to induce luteolysis and allow for

shortened progestin treatment periods [13,60,86]. However, we have shown that the benefit of estradiol in shortened progestin treatment protocols may also be associated with the fact that it causes follicular regression [13,14]. The mechanism involves suppression of FSH and possibly LH. The initial suppression of FSH and LH 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-17 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 [13,14]. Estradiol-17 is normally injected with 50 to 100 mg of progesterone at the same time as placement of a progestin device [13,14,17,55]. The progesterone prevents an estrogen-induced preovulatory-like LH surge in those animals that do not have a functional CL, and appears to cause regression of LH-dependent follicles.

Therefore, our preferred approach for synchronization of follicular wave emergence prior to superstimulation is an injection of 5 mg estradiol-17 + 100 mg progesterone at the time of insertion of progestin releasing device, with FSH beginning 4 days later [13,14]. Data from experimental [14] and commercial [15] superovulation programs have shown that the superovulatory response of donors given estradiol-17 and progesterone at unknown stages of the estrous cycle was comparable to that of donors superstimulated 8 - 12 days after observed estrus (Table 1).

Many practitioners are now utilizing estradiol-17 and progesterone along with one of the many progestin-releasing devices that are now available to synchronize follicle wave emergence for superstimulation of donors [reviewed in 54]. On Day 0 (random stages of the estrous cycle; try to avoid the last 2 or 3 days of the cycle), donor cows receive a progestin device and an injection of 5 mg of estradiol-17 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 - 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 - 12 days to initiate gonadotrophin treatments is eliminated.

This is a fairly robust protocol; for example, it is possible to use 2.5 mg estradiol-17 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 h 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; we have shown that it is not necessary to administer FSH on the day after PGF is given [16,54].

Unfortunately, estradiol-17 is not readily available for commercial use in many countries. 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 CIDR insertion, resulted in synchronous emergence of a new follicular wave 3 - 4 days later [15]. 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-17 + 50 mg progesterone or 2.5 mg estradiol-17 + 50 mg progesterone or those initiated 8 - 12 d after estrus [15]. 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-17 + 100 mg progesterone [21]. However, a dose of 1.0 or 2.0 mg estradiol valerate has been shown recently to result in follicular wave emergence in 3.2

Table 1. Superovulatory response in beef and dairy cattle superstimulated 8 - 12 days after estrus or 4 days after treatment with estradiol-17, progesterone and a progestin releasing device (P4 + E-17; adapted from

[15].

Beef cattle Dairy cattle

Treatment No. of donors Total ova/embryos

Transferable embryos

No. of donors Total ova/embryos

Transferable embryos

8 - 12 days after estrus

1073 12.8 0.3 6.6 0.2 254 8.9 0.4 5.1 0.3

P4 + E-17

307 12.1 0.9 6.3 0.6 187 10.3 0.5 6.0 0.4

Means did not differ (P>0.2).

and 3.4 days, respectively, with little variability, which should be suitable for superstimulation [reviewed in 54].

Traditionally, donor cows have been subjected to embryo collection at 2-month intervals. However, the elective synchronization of follicular wave emergence has resulted in cows being superstimulated successfully every 25 - 30 days, without regard to expression of estrus [16]. 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 this approach can be used to superstimulate cows every 30 days, without the need for estrus detection and without compromising results.

Day 0 - Insert CIDR and inject 5 mg estradiol-17 + 100 mg progesterone Day 4 - 80 mg Folltropin-V bid by deep intramuscular injection Day 5 - 60 mg Folltropin-V bid by deep intramuscular injection Day 6 AM - 40 mg Folltropin-V by deep intramuscular injection; PGF Day 6 PM - 40 mg Folltropin-V by deep intramuscular injection; remove CIDR Day 7 - 20 mg Folltropin-V bid by deep intramuscular injection Day 8 PM - AI Day 9 AM - AI Day 15 - Embryo collection, freezing and/or transfer; PGF Day 30 Insert CIDR and inject 5 mg estradiol-17 + 100 mg progesterone

Collectively, these studies demonstrate that exogenous control of follicle wave emergence offers the advantage of initiating superstimulatory treatments at a time that is optimal for follicle recruitment, regardless of the stage of the estrous cycle. 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 - 12 days to initiate gonadotrophin treatments. Synchronization of follicular wave emergence by follicle ablation or estradiol + progesterone treatments has resulted in comparable superovulatory responses. Furthermore, the estradiol + progesterone plus progestin approach to superstimulation makes it possible to superstimulate cows that are not cycling or have abnormal ovarian function [reviewed in 54].

Gonadotrophins and Superovulation Three different types of gonadotrophins have been used to induce superovulation in the cow: gonadotrophins from extracts of domestic animal pituitaries (FSH), equine chorionic gonadotrophin (eCG) and human menopausal gonadotrophin (hMG) [6,54]. PGF has been used for the induction of luteolysis in a superstimulatory regimen, to allow for precise timing of onset of estrus and of ovulation. As the biological half-life of pituitary FSH in the cow has been estimated to be 5 h or less, it must be injected intramuscularly twice daily to induce superovulation. The usual regimen is 4 or 5 days of twice daily treatments of FSH in decreasing doses. Forty-eight or 72 h after initiation of treatment, PGF is injected to induce luteolysis. Estrus and preovulatory LH release occurs within 36 - 48 h, with subsequent ovulation 24 - 36 h later. Purified pituitary extracts (LH removed) are available in most countries today. One product, Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) is a porcine pituitary extract with approximately 84% of the LH content removed [6,54]. It is available in bottles containing the equivalence of 400 mg NIH-FSH-P1. 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 [6].

Equine chorionic gonadotrophin is a complex glycoprotein with both FSH and LH activity and it has been shown to have a half-life of approximately 40 h in the cow, persisting for up to 10 days in the animal's circulation; thus it is normally injected once followed by PGF 48 h later [6,54]. The long half-life of eCG also causes protracted ovarian stimulation, non-ovulating follicles, abnormal endocrine profiles and reduced embryo quality. These problems have been largely overcome by the intravenous injection of antibodies to eCG at the time of the first insemination, 12 - 18 h after the onset of estrus. However, antibodies to eCG are not commercially available, and so eCG is seldom used in cattle. Recommended doses of eCG range from 1500 to 3000 IU/animal with 2500 IU by intramuscular injection commonly chosen. Human menopausal gonadotrophin, although used in rare individual cases, has not gained favor in bovine embryo transfer because of cost and no greater efficacy [6].

Individual animal variability has been an over-riding factor in all superovulation studies. Breed may also be a factor to be considered. In one study, Holstein cows required a higher proportion of FSH whereas Charolais cows required a higher proportion of LH for maximal superovulation [6,54]. It has also been reported that the purified pituitary extracts were more efficacious than crude pituitary extracts when used under conditions of heat-stress, whereas there was no difference during more moderate environmental temperatures [54]. 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 in winter months. Winter temperatures may be stressful to Bos indicus cattle. It would appear that stress is the problem and that under stressful conditions, purified pituitary extracts should be used [54].

Most recently, we have investigated the use of a single bolus injection of a purified porcine pituitary extract (Folltropin-V) for the superstimulation of donor cows [6,54]. A single subcutaneous injection of Folltropin-V at a dose equivalent to 400 mg NIH-FSH-P1 resulted in a superovulatory response equivalent to that of a twice daily intramuscular treatment regime over 4 days. However, a more consistently high superovulatory response occurred when the subcutaneous injection was made behind the shoulder as opposed to in the neck region. Whether cows responded to a neck injection seemed to depend on body condition. In fact, anything that increased absorption of Folltropin-V (e.g., intramuscular injection or injection in the neck region of lean cows) resulted in a reduced superovulatory response and a reduced number of cows expressing estrus after a single subcutaneous injection. In large part, we have overcome the problem in lean cows by dividing the total dose of Folltropin-V given subcutaneously behind the shoulder; 75% is given at the start of treatment and 25% is given at the time of PGF injection.

Practical Considerations Donor cows must be a minimum of 50 days post-partum, cycling normally, and on an increasing plane of nutrition with no specific nutritional deficiencies. Some recommend trace mineral supplementation before superstimulation, and although apparently 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.

Superstimulatory treatments are normally initiated on Days 8, 9 or 10 of the estrous cycle. The length of a cow's cycle may provide a clue as to the most appropriate time to start superstimulatory treatments. A cow with a 21 - 23 day cycle should be started on Day 9, whereas, a cow with an 18 - 20 day cycle should be started on Day 10. 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-17 or estradiol benzoate and progesterone can be used to synchronize follicular wave emergence. Superstimulatory treatments are usually initiated in 4 days and the progesterone releasing device is removed 60 - 84 h later (12 - 24 h after PGF treatment). This is a reliable means by which superstimulatory treatments can be initiated at predetermined times without consideration of the stage of the cycle.

PGF may be administered on either the third or fourth day of FSH treatment; i.e., 48 or 72 h after initiating treatment and a double dose of PGF is often divided over two FSH treatments. Cows are normally in estrus 36 - 48 h after PGF and are inseminated with a single straw of high fertility semen at 12 and 24 h after the onset of estrus or 60 and 72 h 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 gonadotrophins in subsequent treatment protocols. 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.

Several gonadotrophin preparations are available for use. Within optimal dose ranges, they all work well. An antibody to eCG given at the time of first insemination results in high ovulation rates and a low incidence of unovulated follicles. Evidence indicates that gonadotrophins must be administered by deep intramuscular injection [54]. 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 superovulation. Regardless of whether a single subcutaneous injection 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.

Embryo Recovery In the early days of commercial bovine embryo transfer, embryos were collected surgically from the cow around Day 4 after estrus [11,12]. Three methods of non-surgical embryo recovery were described in 1976 [23,25,67]. Non-surgical techniques are preferred as they are not damaging to the reproductive tract, are repeatable and can be performed on the farm [51,52]. Briefly, the donor cow is placed in a squeeze chute and the rectum is evacuated of feces and air. The number of CL is estimated at this time or just prior to collection. The perineal region and vulvar labia are thoroughly washed and dried, and the tail is tied out of the way. Collection 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 collection rates 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 [51,52]. 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. 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. Many groups still use the Foley catheter as it is inexpensive and readily available. However, the

rubber is soft and the catheter is difficult to thread into the uterine horn. Furthermore, the distance from the cuff to the catheter tip is short. The two-way Rusch catheter is preferred by many. 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.

Basically, there are two methods of embryo collection [52]: the continuous or interrupted flow, closed-circuit system and the interrupted-syringe technique. However, any combination 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 bacteria or chemicals. 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. Again, embryo transfer suppliers now provide disposable equipment for closed-circuit systems.

Flushing medium is prepared before preparation of the cow. Dulbecco's phosphate-buffered saline (PBS) in 500 to 1000 ml bottles is usually refrigerated ready for use. In addition, quantities of heat-inactivated fetal calf serum (FCS), and of an antibiotic/antimycotic solution are kept frozen so that a single quantity of each is required for each bottle of PBS. Collection medium will then contain 1 to 2 % FCS and the appropriate concentration of antibiotics (normally, 100 IU of penicillin, 100 g of streptomycin and 25 g of fungizone per ml). The holding medium, containing 10% serum, 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) [35]. Similarly, if syringes are used in the flushing procedure, it is recommended that those with rubber plungers be washed and heat sterilized before use. Culture medium is normally passed through a disposable 0.22 m Millipore filter prior to use, but the first 4 - 5 ml should be discarded as it may also affect embryo survival. Nowadays, ready-made embryo collection and holding media are commercially available; they are ready for use. 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 have become available making it unnecessary to worry about refrigeration.

Temperature does not seem to be critical to embryo survival. Room temperature seems satisfactory, provided chills and drafts are avoided. Similarly, sterility is not possible but every attempt should be made to be as clean as possible. Embryo collection must be a clean technique but cannot be sterile. Sterilization with chemicals is as likely to kill embryos as bacterial contaminants. Thorough washing of embryos with sterile medium has been shown to remove all infectious agents. As a routine, embryos should be passed through 10 washes of fresh medium prior to transfer or freezing.

Embryo Handling Embryos are located under 10 X magnification with a stereoscopic dissecting microscope after filtering the collection medium through a filter with pores that are approximately 50 - 70 m in diameter [52]. Although embryos are usually transferred as soon as possible after collection, it is possible to maintain embryos for several hours at room temperature in holding medium. It is also possible to cool bovine embryos in holding medium and to maintain them in the refrigerator for 2 - 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. As indicated earlier, embryo collection holding and freezing media that are free of animal products have recently become available, avoiding the need for refrigeration and increasing biosecurity.

Embryo Evaluation Evaluation of bovine embryos must be done at 50 to 100 X magnification, with the embryo in a small culture dish. 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. The International Embryo Transfer Society (IETS) considers the export of poor and fair quality embryos to be improper [50].

Figure 3. Evaluation of bovine embryos must be done at 50 to 100 X magnification, with embryos in small culture dish. A and B are IETS quality code 1 morulae while C is an IETS quality code 1 early blastocyst [50]. - To view this image in full size go to the IVIS website at www.ivis.org . -

Classification Embryos are classified and evaluated by morphological examination at 50 to 100 X magnification according to the Manual of the International Embryo Transfer Society [50]. 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 until 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 even 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 collapsed and should not contain debris on its surface. Embryos of good and excellent quality 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 for the synchrony of the recipient.

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, 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 highly 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.

Hatched Blastocyst: Embryos recovered at this developmental stage can be undergoing the process of hatching or may have completely shed the zona pellucida. Hatched blastocysts may be spherical with a well defined blastocoele or may be collapsed. Identification of embryos at this stage can be difficult unless it re-expands.

Quality Evaluation 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

Figure 4. A bovine embryo with about 16 cells, as it would appear in the uterus of a cow about 4 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). - To view this image in full size go to the IVIS website at www.ivis.org . -

Figure 5. 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. - To view this image in full size go to the IVIS website at www.ivis.org . -

Figure 6. 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). - To view this image in full size go to the IVIS website at www.ivis.org . -

Figure 7. 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 pelucida. (Courtesy of Dr. John K. Thibodeaux). - To view this image in full size go to the IVIS website at www.ivis.org . -

vesicles but an apparently viable embryo mass. These are generally not of transferable quality.

Recommended Quality Code [50] 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 should be relatively minor and at least 85% of the cellular material should be 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 should be 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 should be 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 [50] 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 are 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 hatching blastocysts within the same flush. 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, at the developmental stages of compact morula to blastocyst yield the highest pregnancy rates, even after freezing. Fair and poor quality embryos yield poor pregnancy rates after freezing and should be transferred fresh. It is advisable to select the stage of the embryo for the synchrony of the recipient. It would also seem that fair and poor quality embryos are most likely to survive transfer if they are placed in the most synchronous recipients.

Embryo Transfer Transfer of embryos in the cow will result in a high pregnancy rate providing the preceding estrus in the donor and recipient occurred within 24 h of each other [33]. Alternately, recipients must be synchronous with the stage of development of embryos that had been previously frozen. Recipients can be made available by maintaining a large herd to obtain natural heats 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 [11,12]. Surgical transfers were first done by way of a midline incision, which necessitated the use of general anesthesia and rather elaborate facilities. During the mid to late 1970's, this gave way to a standing flank approach, an approach that was quicker and because of lesser requirements in facilities made "on farm" embryo transfer possible. More recently, the use of non-surgical embryo transfers has increased the utilization of embryo transfer because of even less elaborate requirements [68,88].

Non-surgical embryo transfer techniques utilized today involve the use of an artificial insemination pipette and more recently, specialized embryo transfer pipettes. After confirming 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 the straw is loaded in the embryo transfer pipette. Care must be taken to insure 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 transfer so as to prevent trauma to the endometrium during passage. The embryo is deposit slowly and firmly while slightly withdrawing the tip of the transfer pipette. 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 and 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.

In summary, with existing technology, an average of 8 to 10 ova/embryos will be collected from each superstimulated donor cow and 5 to 6 embryos will be transferred, resulting in 3 to 4 pregnancies. It must be emphasized that very few donor cows are average. Pregnancy rates are generally around 60% with fresh embryos and range from 50% to 60% with frozen embryos. One can anticipate a death loss of 10% from pregnancy diagnosis until the calf is six months old. It is worthy of note that this is not different from that of the normal cattle population and that embryo transfer procedures have been shown to result in no increase in calf abnormalities.

Estrus Detection 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 [38]. 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.

The primary sign of estrus is a cow standing firm when mounted. Secondary signs of estrus include mounting other cows, mucus discharge, swollen vulva, hyperactivity, and bellowing. It is recommended that >80% of inseminations be based on standing estrus.

The two principal causes of estrus-detection problems are missed estrus and estrus detection errors. Indicators of missed estrus include prolonged intervals from calving to breeding, prolonged intervals between breedings, >10 to 15% non-pregnant at pregnancy examination, and

Estrus Synchronization for Embryo Transfer Acceptable pregnancy rates in 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 [33]. 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 [37] and in superovulated donors in 36 to 48 hours [15,16,54]. 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 [33], probably because of improved estrus detection. It must be remembered that exogenous steroid hormones will induce estrus and even ovulation in post-partum cows and prepubertal heifers [55]. Therefore, post-partum 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 from a herd 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 in the cow (described earlier), 2) pharmacological agents and their effects on the cow's 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 recipients will be described.

Prostaglandin PGF has become the most commonly used treatment for estrus synchronization in cattle [26,41,60,70]. 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 [37]. This variation 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 doses has been used because it represents the mid-point of the estrous cycle and theoretically, all cows 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 [26], 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 also 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).

Progestins Various progestins (progesterone-like compounds) have been utilized for estrus synchronization. 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 [55,66]. These effects are transitory, and fertility at the following estrus is normal.

Progesterone alters ovarian function in cattle; it suppresses estrus and prevents 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 [2,4]. 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 more than 14 days) result in synchronous estrus upon withdrawal, but fertility is low [66]. 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

Figure 10. Kamar Heatmount Detectors are valuable heat detection aids that assist in identifying cows that are in heat. 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 heat versus false mounting activity. Standing estrous behavior in cattle is the most reliable sign that a cow is ready to be inseminated. - To view this image in full size go to the IVIS website at www.ivis.org . -

Figure 11. The heat mount 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. - To view this image in full size go to the IVIS website at www.ivis.org . -

Figure 12. Heat detection using tail-chalk. Freshly applied chalk is positioned on the tail head. When cows are mounted, the chalk is removed, with more and longer mounts resulting in the removal of more chalk. (Courtesy of Dr. Glen Selk). - To view this image in full size go to the IVIS website at www.ivis.org . -

high LH pulse frequency and development of "persistent" follicles, which contain aged oocytes [55,66]. Ovulation of an aged oocyte results in poor fertility.

Melengestrol acetate (MGA) is the progestin that has been most commonly used for estrus synchronization in cattle [55]. The advantages of MGA include low cost (few cents per day), oral administration (usually mixed in grain) and extremely low toxicity. Because feeding of MGA will lead to the development of persistent follicles, animals are normally not bred at the first estrus following withdrawal from the feed. One regimen is to feed 0.5 mg MGA/head/day for 14 days, followed by treatment with PGF 17 days after cessation of MGA. This regimen has been reported to give well-synchronized estrus with good fertility. It appears to be more effective in cattle in moderate compared to fair or poor body condition. Furthermore, separating suckling calves from their dams for 48 hours, starting 2 days after the last feeding of MGA, increased the percentage of 2 and 3 yr old dams conceiving early in the breeding season compared to a similar regimen without calf removal or untreated controls.

The progesterone-impregnated CIDR-B (controlled internal drug release; Pfizer) intravaginal device has recently been approved in Canada and USA for synchronization of estrus in cattle [55]. 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 after device removal. Because of the short treatment period (7 days), the problem of persistent follicles is reduced. 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 [55].

Fixed-time Embryo Transfer Embryo transfer techniques can be further refined to completely eliminate the need of estrus detection in either donors or recipients [16,17]. The use of progestin devices and the synchronization of follicular wave emergence were described earlier as a means of initiating superstimulatory treatment without regard to the stage of the donor's estrous cycle. It is also possible to eliminate the need for estrus detection of recipients by taking advantage of protocols that have been developed for fixed-time AI in cattle [55]. Basically, two approaches are used: the so-called Ovsynch or Cosynch protocols utilizing GnRH [64,86] or pLH [56], with or without a progestin-releasing device [57], or estradiol to synchronize follicle wave emergence and ovulation in progestin-treated animals [17,55].

GnRH Gonadotrophin releasing hormone became available in the 1970's as a treatment for follicular cysts [24]. Treatment of a cow with a growing dominant follicle with GnRH induces ovulation [49,82] with emergence of a new follicular wave approximately 2 days later [55,56]. 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 [82,88]. An ovulation synchronization scheme utilizing GnRH for fixed-time AI called "Ovsynch" was developed by Pursley et al., [64]. The first injection of GnRH is followed 7 days later with an injection of PGF followed in 48 hours by a second injection of GnRH; fixed-time AI is performed 0 to 24 hours later. The Ovsynch protocol has been more efficacious in lactating dairy cows than in heifers. 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 [64]. In addition, 19% of heifers returned to estrus before the injection of PGF making fixed-time AI impossible [88]. 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 [56]. However, the addition of a CIDR to a 7-day GnRH-based protocol improved pregnancy rates after fixed-time AI in heifers [57], and improved pregnancy rates in non-cycling, lactating dairy cows [55].

Ovsynch protocols have been used to synchronize ovulation in recipients prior to embryo transfer without estrus detection [8]. In two studies, the overall pregnancy rate (recipients pregnant over recipients treated) was higher following treatment with the Ovsynch protocol than treatment with only PGF, because it was not dependent on estrus detection. In another study, pregnancy rates did not differ between recipients treated with an Ovsynch protocol or an Ovsynch protocol plus a

Figure 13. 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 of the growing dominant follicle(s). - To view this image in full size go to the IVIS website at www.ivis.org . -

Figure 14. Various progestin-releasing devices used to control the estrous cycle in cattle available around the world. (Courtesy of Dr. Gabriel Bo). - To view this image in full size go to the IVIS website at www.ivis.org . -

progestin for 7 days or recipients transferred 6 to 8 days after detected estrus, but more recipients were selected for embryo transfer in the Ovsynch groups. In a field trial involving 1637 recipients treated with an Ovsynch protocol plus progestin for 7 days, without estrus detection, an overall pregnancy rate of 59.9% was achieved.

Estradiol and Progesterone As indicated earlier, treatment with progestins and estradiol has been used for several years to synchronize estrus, but it was not until recent discoveries of the effects of estradiol on follicular development that the reason for these effects was understood [13]. 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-17 emergence of a new follicular wave occurred 3 to 5 days later, regardless of the stage of follicular growth at the time of treatment [13]. For estrus synchronization, estradiol need not be injected with progesterone, but a progestin device must be in place [17,55]. 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 later [55]. Pregnancy rates to fixed-time AI have been high with this protocol.

A series of experiments evaluated estradiol/progestin protocols to synchronize recipients without estrus detection [17]. Treatments evaluated were similar to those used of fixed-time AI (progestin device insertion and an injection of 2 mg estradiol benzoate on Day 0, PGF at device removal (Days 7 or 8) and 1 mg estradiol benzoate 24 h later). Pregnancy rates to fixed-time embryo transfer (8 d after the second estradiol benzoate) were comparable to that of recipients synchronized with 2 doses of PGF 14 d apart and embryo transfer 7 d after an observed estrus. Clearly, acceptable pregnancy rates can be achieved following protocols, which synchronize ovulation, without the necessity of estrus detection [17].

Resynchronization of Non-pregnant Recipients Progestin devices have also been used to resynchronize cattle that previously received an embryo [17,55]. Animals receive a new or used device at the time of transfer, or on Days 12 or 13 after estrus. When devices are removed on Day 21, 50 to 60% of the non-pregnant animals were detected in estrus on Days 22 - 25 [17]. Cows not detected in estrus were presumed pregnant, whereas those in estrus can be examined by ultrasonography 7 d later and if found not to be pregnant can be reused for embryo transfer. In any case, embryo transfer programs can be designed, utilizing 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 2 and sometimes 3 opportunities to become pregnant.

Management Factors 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 been shown to have reduced conception rates and may also have reduced pregnancy rates after receiving a viable embryo by embryo transfer [17]. In a field study, recipients were condition scored at the time of embryo transfer on a scale of 1 (thin) to 5 (fat). Pregnancy rates were significantly higher in recipients scoring 3 and 4 than in those scoring 1, 2 or 5 [52]. 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.

Embryo Freezing Basic Principles The freezing of a living cell constitutes a complex physiochemical 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 [43,61].

Normally, the medium that contains the embryos 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 2C below its freezing point (-4 to -7C) by seeding the medium with an ice crystal [61]. 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, the solute concentration rises and the embryo responds osmotically by losing water into the extracellular unfrozen medium.

Cells are injured during freezing and thawing primarily by solution effects and intracellular ice formation [43,61]. The

latter is especially detrimental when relatively large amounts of large crystals form. To avoid intracellular freezing, embryos must be cooled at 1C/min or slower. However, very low 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 [61].

The required thawing rate depends on the freezing regimen used. When embryos are cooled slowly to temperatures between -27 and -40C and then rapidly to -196C (liquid nitrogen), thawing must be rapid, e.g., about 200C/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 -60C before transfer to liquid nitrogen, thawing is then normally done slowly at about 20C/min [43]. 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 -196C. The only reactions that occur at -196C 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 [61]. Glycerol has been most often used for the protection of embryos during freezing but more recently, more permeating cryoprotectants such as ethylene glycol, have been preferred because they can be used with "direct transfer" i.e., transfer into a recipient without prior removal of the cryoprotectant [44,85].

During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes resulting in swelling or contraction [61]. Consequently, if the addition or particularly the dilution is carried out inappropriately, the viability of cells can be affected. 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 was to dilute it by the "step-wise" addition of PBS or to pipette the embryos into decreasing concentrations of glycerol, e.g., 0.25 M steps (90). However, Leibo and Mazur [43] 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 the cryoprotectant leaves the embryo, it will shrink in response to the extracellular hypertonic dilution medium. It regains its normal volume 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 thawed and processed in sucrose prior to transfer, without microscopic evaluation, resulted in a 42.4% pregnancy rate [42]. More recently, this method has given way to "Direct Transfer" utilizing highly permeating cryoprotectants, such as ethylene glycol, which do not osmotically harm the embryo if not removed prior to transfer. Recent pregnancy results for "Direct Transfer" in Canada, with more than 19,000 embryos, were not different from those achieved with glycerol and cryoprotectant removal prior to transfer [44].

Freeze-Thaw Procedures The following protocol has been proven successful for the cryopreservation of Day 7 bovine embryos in PBS supplemented with 10 to 20% FCS and 1.0 to 1.5 M glycerol [51]. Embryos are pipetted into the freezing medium at room temperature (20C) and left for eight to 10 minutes to permit the glycerol to 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 that are then securely sealed. The samples can be immediately transferred into the freezing chamber at -6 or -7C 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 pre-cooled in liquid nitrogen (do not touch the column of media that contains the embryo(s). The samples are held at the seeding temperature for an additional 10 min to allow the crystallization of the medium to progress to equilibrium. Next, embryos are cooled at 0.3 to 0.8C/min to a temperature between -30 and -40C, at which time they are immersed into liquid nitrogen (-196C) and stored.

Thawing is carried out by placing the straw into a water-bath at a temperature between 20 and 35C. 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 35C water bath; the thaw rate should be around 200C/minute.

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) [51]. The embryos are

then transferred back into PBS culture medium, washed and evaluated prior to transfer.

Direct Transfer Recently, 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 [44]. 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 19,000 bovine embryos in Canada resulted in an overall pregnancy rate of 58%, which was not different to that achieved by regular cryoprotectant dilution techniques. Several AI Centers have now trained technicians to do nothing but embryo transfer. The transfer of frozen/thawed bovine embryos is now becoming very similar to the use of frozen/thawed semen in AI.

Vitrification The freezing of bovine embryos is now commonplace and pregnancy rates are only slightly less than that achieved with fresh embryos [44]. However, freezing and thawing procedures are time consuming and require the use of biological freezers and a microscope. These steps can be replaced by a relatively simple procedure called vitrification [65]. High concentrations of cryoprotectants are used and the embryo in its cryoprotectant solution is placed directly into liquid nitrogen. Because of the high concentration of cryoprotectants, ice crystals do not form; the frozen solution forms a glass. As ice crystal formation is one of the most damaging processes in freezing, vitrification has much to offer in the cryopreservation of oocytes and IVF embryos. However, its greatest advantage is its simplicity in application. Vitrification procedures are now widely used experimentally and it is only a matter of time before they find commercial application. Recently, a procedure for the direct transfer of vitrified bovine embryos with pregnancy rates that did not differ from that of traditional techniques was reported [84]. Clearly, vitrification of bovine embryos in commercial bovine embryo transfer is on the horizon.

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 application of embryo transfer technology. The International Embryo Transfer Society (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 [50]. The IETS also allocates embryo-freezing codes that must appear on all embryo containers and all documentation so that the organization