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
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

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

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.
Figure 1. Bovine ovarian follicular wave dynamics during a 2-wave estrous cycle. The top panel
shows the relationship between progesterone secretion and luteinizing hormone (LH) release. The
shaded area represents progesterone secretion in ng/ml in serum shown on the left axis. The dashed
line represents typical serum concentrations of LH in ng/ml on the right axis. 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 in serum shown on the right axis. The solid line represent
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 (in mm on the left axis) of follicles within follicle
waves 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 10 days later. Approximately 3 days after
ovulation, selection of the dominant follicle occurs and subordinate follicles undergo atresia. The
dominant follicle of the first wave undergoes regression beginning approximately 9 days after
ovulation, while the dominant follicle of second wave ovulates (OV) approximately 20 days after the
previous ovulation. (Modified from Adams [1]). - To view this image in full size go to the IVIS
website at www.ivis.org . 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 <1 ng/ml and synchronous growth
and ovulation of a viable follicle [1,2]. It follows, therefore, that PGF is effective only when a fully developed CL is
present (Days 7 - 18 of the cycle) and that withdrawal of exogenous progesterone is only effective if either natural or
induced regression of the CL has occurred. If properly implemented, within the physiological constraints of their
mechanism of action, current techniques for estrus synchronization are highly successful. However, variation in ovarian
follicular wave dynamics makes it difficult to control the time of estrus and ovulation precisely.

Superovulation
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. 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 cow [48]. 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, embryo recovery from 987
dairy cows yielded slightly fewer ova/embryos, on the average, but there was similar variability in response [32]. 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 superstimulatory treatments such as gonadotrophin
preparation, batch of gonadotrophin, duration of treatment, timing of treatment with respect to the estrous cycle, total dose
of gonadotrophin and the use of additional hormones in the superstimulation protocol [6,13,27,45,54,71]. Additional,
perhaps more important sources of variability are factors inherent to the animal and its environment. These factors may
include nutritional status, reproductive history, age, season, breed, ovarian status at the time of treatment and the effects of
repeated superstimulation. While considerable recent progress has been made in the field of bovine reproductive

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].
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 . -

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

[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 ultrasoundguided 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 progestinimplanted 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 LHdependent 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).

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 + E17

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).

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

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, nonovulating 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 fullydisposable 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 onecell 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.
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 frozenthawed 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 . -

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

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.
Figure 8.The primary and most definitive sign of estrus in the cow is standing firm when mounted. To view this image in full size go to the IVIS website at www.ivis.org . -

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% nonpregnant at pregnancy examination, and <50% of potential estrous periods detected. Several factors can contribute to
missed estrus. Often the observer does not spend adequate time observing the cattle for estrus or tries to combine estrus
detection with other activities (e.g., feeding). 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 breeding and interbreeding intervals <17 d or >25 d.
In some studies, up to 20% of cattle had 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 inducing estrus, allocating adequate time for observation, using estrus detection aids, and predicting the next estrus.
Figure 9. Estrus detection is the most important factor affecting widespread use of AI and an
impediment to the successful use of embryo transfer. (Courtesy of Dr. Gabriel Bo). - To view this
image in full size go to the IVIS website at www.ivis.org . -

Estrus detection aids include heat-mount detectors, tail-head chalk or paint, pedometers, androgen-treated marker animals
and electronic estrus detection systems [38]. These methods should be utilized in addition to, and not as a substitute for,
visual observation of estrous 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 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 . -

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 midcycle (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 dosedependent 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

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].
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 . -

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 fixedtime 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

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 "onestep 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 freezing embryos can be identified.
Finally, standard procedures for labeling embryo freezing containers are also recommended 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 IETS [50].

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