Theriogenology 81 (2014) 38–48

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Theriogenology
journal homepage: www.theriojournal.com

40th Anniversary Special Issue

Historical perspectives and recent research on superovulation in cattle

Gabriel A. Bó a, b, *, Reuben J. Mapletoft c
a
Instituto de Reproducción Animal Córdoba (IRAC), Córdoba, Argentina
b
Instituto A.P. de Ciencias Básicas y Aplicadas, Medicina Veterinaria, Universidad Nacional de Villa María, Córdoba, Argentina
c
Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Superovulation protocols have evolved greatly over the past 40 to 50 years. The devel-
Received 31 July 2013 opment of commercial pituitary extracts and prostaglandins in the 1970s, and partially
Received in revised form 16 September 2013 purified pituitary extracts and progesterone-releasing devices in the 1980s and 1990s have
Accepted 17 September 2013
provided for the development of many of the protocols that we use today. Furthermore,
the knowledge of follicular wave dynamics through the use of real-time ultrasonography
Keywords:
and the development of the means by which follicular wave emergence can be controlled
Superovulation
have provided new practical approaches. Although some embryo transfer practitioners still
FSH
Follicle wave initiate superstimulatory treatments during mid-cycle in donor cows, the elective control
GnRH of follicular wave emergence and ovulation has had a great effect on the application of on-
Estradiol farm embryo transfer, especially when large groups of donors need to be superstimulated
at the same time. The most common treatment for the synchronization of follicular wave
emergence for many years has been estradiol and progestins. In countries where estradiol
cannot be used, practitioners have turned to alternative treatments for the synchronization
of follicle wave emergence, such as mechanical follicle ablation or the administration of
GnRH to induce ovulation. An approach that has shown promise is to initiate FSH treat-
ments at the time of the emergence of the new follicular wave after GnRH-induced
ovulation of an induced persistent follicle. Alternatively, it has been suggested recently
that it might be possible to ignore follicular wave status, and by extending the treatment
protocol, induce small antral follicles to grow and superovulate. Recently, the mixing of
FSH with sustained release polymers or the development of long-acting recombinant FSH
products have permitted superstimulation with a single or alternatively, two gonadotropin
treatments 48 hours apart, reducing the need for animal handling during super-
stimulation. Although the number of transferable embryos per donor cow superstimulated
has not increased, the protocols that are used today have increased the numbers of
transferable embryos recovered per unit time and have facilitated the application of on-
farm embryo transfer programs. They are practical, easy to administer by farm
personnel, and more importantly, they eliminate the need for detecting estrus.
Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction number of transferable embryos with a high probability of

The objective of treatments to induce superovulation in
embryo transfer programs is to obtain the maximum
* Corresponding author. Tel.: þ54 (0) 3543 477214; fax: þ54 (0) 3543
477214.
E-mail address: gabrielbo@iracbiogen.com.ar (G.A. Bó).
producing pregnancies. However, wide ranges in super-
ovulatory response and embryo yield in several different
species have been reported [1]. In fact, Gordon [2] stated in
1975 that “although superovulation as a method of
obtaining a supply of eggs has taken many forms over the
years; it still leaves much to be desired. In fact, it must still
be regarded as a major problem blocking progress in

0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2013.09.020
 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
exploiting egg transfer in increasing numbers of progeny
from genetically superior cattle”. Although considerable
recent progress has been made in the study of ovarian
physiology, manipulation of ovarian function and gonado-
tropin biochemistry, factors inherent to the donor animal
which affect superovulatory response are only partially
understood. Thus, a high degree of unpredictability in
superovulatory response still exists more than 35 years
later, creating problems which affect the efficiency and
profitability of commercial embryo transfer.
The earliest reports on superovulation in cattle and
sheep came from Wisconsin, in the United States (cited in
[2]). Basically, gonadotropins were administered several
days in advance of the anticipated normal estrus in an
effort to induce multiple ovulations. This was often fol-
lowed by an injection of human chorionic gonadotropin
(hCG) approximately 5 days later (or at the onset of estrus)
to induce ovulation. Although studies on the use of anterior
pituitary preparations from sheep, horse, cattle, and pigs
were reported (cited in [2]), the use of equine chorionic
gonadotropin (eCG or PMSG) became the standard
approach. Betteridge [3] indicated that in the early days of
bovine embryo transfer there was general consensus that
eCG had to be administered during the transition from the
luteal to the follicular phase, at approximately Day 16 of the
estrous cycle, in animals undergoing natural luteolysis. In
that regard, Hafez et al. [4] developed a protocol that was
widely used in the cow; 2000 to 3000 IU of eCG was
administered on Day 16 of the cycle, followed by 10 mg of
estradiol-17b on Day 19 and 20 and 2000 IU of hCG on Day
21. It was intended that the estradiol would ensure luteal
regression, but a more important consequence was that
most donors showed estrus [2]. This protocol became the
standard superovulation protocol used at that time.
The introduction of commercial preparations of PGF2a in
the 1970s resulted in its use in superovulation protocols.
Elsden et al. [5] reported that donors that received PGF2a 48
hours after the administration of eCG appeared to have
higher superovulation rates than those that simply
received eCG on Day 16 of the cycle. The use of PGF2a also
meant that it was possible to initiate gonadotropin treat-
ments at other times during the estrous cycle and most
practitioners began treating with gonadotropins during
midcycle. Indeed, Phillippo and Rowson [6] reported a
higher superovulatory response when eCG was adminis-
tered between Days 8 and 12 than when it was adminis-
tered at other times during the estrous cycle. This has
subsequently been demonstrated to be at the approximate
time of emergence of the second follicular wave [7].
2. Superovulation and commercial embryo transfer
The commercial embryo transfer industry in North
America developed in the early 1970s with the introduction
of continental breeds of cattle which were then in short
supply. Superovulation and embryo transfer offered the
means by which their numbers could be increased rapidly.
Although eCG was used initially (reviewed in [2,3]), intense
research resulted in very rapid changes in practitioner’s
approaches to superovulation. In 1978, Elsden et al. [8]
reported a greater superovulatory response after
39
treatment with a crude pituitary extract containing FSH
plus 20% LH compared with the use of eCG. Monniaux et al.
[9] also observed that ovulation rate and the percentage of
cows with more than three transferable embryos tended to
be greater after the use of a pituitary extract containing FSH
rather than eCG. However, others found no differences
between pituitary extracts containing FSH and eCG [10,11].
Endocrine studies have revealed that eCG-treated animals
more frequently had abnormal profiles of LH and proges-
terone [12,13] which was associated with reductions in
ovulation and fertilization rates [14] compared with FSH-
treated cows.
The main reason that most practitioners switched from
eCG to FSH was related to problems associated with the
prolonged stimulation of the ovaries after a single injection
of eCG. The half-life of eCG has been shown to be 40 hours
in the cow and eCG persists for up to 10 days in the bovine
circulation [15]. The long half-life of eCG causes continued
ovarian stimulation, unovulated follicles, abnormal endo-
crine profiles, and reduced embryo quality [13,16–18]. The
problems associated with the half-life of eCG were largely
overcome by the intravenous injection of antibodies to eCG
at the time of the first insemination, 12 to 18 hours after the
onset of estrus [19,20]. Unfortunately, the pharmaceutical
company that developed a commercial monoclonal anti-
body to eCG (Neutra-PMSG; Intervet, The Netherlands)
discontinued the product in the 1990s. Another hormone
preparation used in human fertility clinics, human meno-
pausal gonadotropin (hmG), has also resulted in a super-
ovulatory response that was comparable with pituitary
extracts [21]; however, it has not been used extensively by
practitioners because of its high cost.
Most cows nowadays are superstimulated using pitui-
tary extracts containing FSH. However, there is also a large
amout of LH in crude pituitary extracts, and there is
considerable variability in FSH and LH content of all crude
gonadotropin preparations [22]. Radioreceptor assays and
in vitro bioassays have revealed variability in the FSH and
LH activity of eCG, not only among pregnant mares, but also
within the same mare at different times during gestation
[15]. The effects of the FSH/LH ratio of eCG on the super-
ovulatory response has been examined and there was
a positive correlation between the ratio of FSH/LH activity
and superovulatory response. Lower ratios of FSH/LH
activity resulted in a reduced ovulatory response in im-
mature rats and LH added to eCG reduced the super-
ovulatory response in cattle [15,22].
Purified pituitary extracts with low LH contamination
have been reported to improve the superovulatory
response in cattle. Chupin et al. [23] superstimulated three
groups of dairy cows with an equivalent amount of 450 mg
pure FSH and varying amounts of LH and showed that the
mean ovulation rate and the number of recovered and
transferable embryos increased as the dose of LH
decreased. It has been suggested that high levels of LH
during superstimulation cause premature activation of the
oocyte [16]. Although several experiments with an LH-
reduced pituitary extract [24] using several different total
doses ranging from 100 to 900 mg of NIH-FSH-P1 revealed
no evidence of detrimental effects of dose on embryo
quality [25,26], doubling the dose of crude pituitary
40 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
extracts containing both FSH and LH resulted in signifi-
cantly reduced percentages of fertilized ova and transfer-
able embryos [25]. Collectively, the data support the
hypothesis that the detrimental effects of high doses of
pituitary gonadotropins on ova/embryo quality is because
of an excess of LH.
Although it is generally believed that some LH is
required for successful superovulation, endogenous LH
levels might be adequate to support the final follicle
growth. Looney et al. [27] reported that recombinantly-
produced bovine FSH induced high superovulatory re-
sponses without the addition of exogenous LH. These data
suggest that LH is not needed in superstimulatory prepa-
rations and that embryo quality might be superior with use
of pure FSH. Further, these results and more recent results
using another recombinant bovine FSH [28] indicate that
the future lies in the use of recombinantly-derived
gonadotropins.
2.1. Current superstimulation treatment protocols in cattle
The biological half-life of FSH in the cow has been
estimated to be 5 hours or less [29] so it must be admin-
istered twice daily to successfully induce superovulation
[9,30]. The usual regimen has been twice daily intramus-
cular treatments with FSH for 4 or 5 days, with a total dose
of 28 to 50 mg (Armour) of a crude pituitary extract or 400
mg NIH-FSH-P1 of a partially-purified pituitary extract
(Folltropin-V; Bioniche Animal Health Inc., Belleville,
Ontario, Canada). Forty-eight or 72 hours after initiation of
treatment, PGF2a is administered to induce luteolysis.
Estrus occurs in 36 to 48 hours, with ovulations beginning
24 to 36 hours later [31,32].
Many practitioners prefer decreasing FSH dose sched-
ules and others use constant-dose schedules. Many treat
with PGF2a on the third day of the treatment protocol, and
others prefer to treat with PGF2a on the fourth day, and
many do not treat with FSH on the day after the adminis-
tration of PGF2a. Although there would appear to be no
experimental data supporting one approach over the other,
recent experiments have indicated that ovulation rate can
be improved in at least some donors if FSH treatments are
administered over 6 or 7 days [33,34]. Regardless, most
superstimulation protocols have been successful in
inducing superovulation under most circumstances
(reviewed in [32]). Still other practitioners incorporate a
progestin insert into the protocol which ensures that do-
nors do not come into estrus early, especially if it is not
possible to confirm the presence of a CL before initiating
FSH treatments. In all cases, inseminations are normally
done 12 and 24 hours after the onset of estrus [31,35].
3. Ovarian follicular waves and superovulation
The conventional protocol of initiating ovarian super-
stimulation during midcycle was originally based on anec-
dotal and experimental information in which a greater
superovulatory response was reported when super-
stimulatory treatments were initiated 8 to 12 days after
estrus (reviewed in [36]). However, none of these early
studies evaluated the specific follicular status of the animals
when superstimulation treatments were initiated. This was
mainly because monitoring follicular development ultra-
sonically in cattle was, in most cases, in the early stages of
development and not available in many laboratories.
Through information generated using ultrasonography,
it is now known that 8 to 12 days after estrus (equivalent to
7 to 11 days after ovulation) would be the approximate
time of emergence of the second follicular wave [7], and a
cohort of growing follicles would be present around that
time. However, the day of emergence of the second follic-
ular wave has been shown to differ between two-wave
cycles and three-wave cycles (1 or 2 days earlier in three-
wave cycles), and between individual animals [7]. In this
regard, it has been shown clearly that superovulatory
response was optimized when superstimulatory treat-
ments were initiated at the time of follicle wave emergence
[1,37,38]. Initiating gonadotropin treatments as little as 1
day before or after wave emergence significantly reduced
the superovulatory response compared with initiating
treatments on the day of wave emergence [1,37,38].
Based on duration of the developmental phases of the
dominant follicle in two-wave and three-wave inter-
ovulatory intervals, the probability at any given time that
the dominant follicle is not functionally dominant (late-
static or regressing phases) is approximately 30% (6 of 20
days) for two-wave animals and 35% (8 of 23 days) for
three-wave animals. Thus, only approximately 20% (4 or 5
days) of the estrous cycle is available for initiating treat-
ment 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
midcycle to initiate superstimulatory treatment also im-
plies monitoring estrus and an obligatory delay. Therefore,
most treatment protocols used today involve the initiation
of superstimulatory treatments subsequent to the exoge-
nous control of follicular wave emergence [32].
4. Manipulation of follicle wave emergence
4.1. Estradiol and progestin
The ability to electively induce follicular wave emer-
gence permits initiation of superstimulation without re-
gard to the stage of the estrous cycle and eliminates the
need for estrus detection and waiting 8 to 12 days to initiate
gonadotropin treatments [35]. In the 1990s, we reported on
the use of progestins and estradiol to induce synchronous
emergence of a new follicular wave (reviewed in [36]); this
approach to the induction of superovulation in the cow has
been reviewed extensively [32,35,43]. It has been used by
practitioners around the world and has recently been
incorporated into protocols that permit fixed-time AI (FTAI)
of donors [31,44].
The most common protocol to synchronize the emer-
gence of a new follicular wave for superstimulation involves
the administration of 2.5–5 mg estradiol-17b or 2–2.5 mg
estradiol benzoate plus 100 or 50 mg progesterone using
intramuscular injection at the time of insertion of an intra-
vaginal progestin device (reviewed in [31,35,36,43,44]). The
estradiol treatment suppresses FSH release and induces
follicle atresia. When estradiol has been metabolized, FSH
 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
surges and a new follicular wave emerges, on average 4 days
after treatment [36]. Gonadotropin treatments are initiated
at that time, i.e., 4 days after treatment. Therefore, this
treatment allows for a new cohort of 3- to 5-mm follicles,
to grow at the same time. Although the number of trans-
ferable embryos has not always been greater than when
cows were superstimulated between 8 to 12 days after
estrus, the fertilization rate in donor cows superstimulated
after estradiol and progestin treatments was significantly
greater than in the control cows in a least two well
controlled studies [36,39]. Furthermore, heifers super-
stimulated 4 days after the insertion of a progestin device
without the administration of estradiol also had a lower
percentage of fertilized ova and transferable embryos than
those treated with estradiol at the time of insertion of the
progestin device. These results suggest that the adminis-
tration of exogenous FSH might not only induce growth of
healthy follicles but also those that have already undergone
atresia. Although the superovulatory response might not
have been affected, poor ova/embryo quality might have
resulted from the ovulation of incompetent oocytes. In
contrast, estradiol and progestin treatments resulted in the
development of a new follicular wave and consequently a
more uniform group of viable follicles with competent oo-
cytes at the time of gonadotropin treatment. It has been
shown that subordinate follicles can be saved from atresia
by dominant follicle ablation [40] or FSH treatments at the
time of dominant follicle selection [41]. Furthermore,
Goulding et al. [42] have shown that supestimulatory
treatment early in the luteal phase in heifers with a pro-
gestin device resulted in poor embryo quality which was
apparently because of the stimulation of subordinate folli-
cles that were already undergoing atresia, with oocytes that
were undergoing degeneration. Therefore, estradiol and
progestin treatments not only eliminated the need for estrus
synchronization and detection before superstimulation, but
also tended to improve ova/embryo quality.
4.2. Follicle ablation
An alternative for the synchronization of follicular wave
emergence is to eliminate the suppressive effect of the
dominant follicle using ultrasound-guided follicle ablation
[45,46]. Initial studies involved the ablation of all follicles
5 mm [47], but we subsequently showed that it was
necessary to ablate only the two largest follicles [48] to
ensure that the dominant follicle was removed. Super-
stimulatory treatments are then initiated 1 to 2 days later,
at the time of emergence of a new follicular wave. Although
follicle ablation has been shown to be highly effective
(reviewed in [31]), ultrasound equipment and trained
personnel are required, making it most appropriate for
embryo production centers, where the equipment and
personnel are present, and donors are maintained. Follicle
ablation is difficult to use in the field, especially when do-
nors are some distance from the embryo production center.
4.3. GnRH
It has been shown that after GnRH-induced ovulation, a
new follicular wave will emerge approximately 1.5–2 days
41
after treatment. However, follicular wave emergence oc-
curs only when GnRH induces ovulation [49], and recent
studies have shown that the administration of GnRH at
random stages of the estrous cycle results in ovulation in
44% to 54% of dairy cows [50,51], 56% of beef heifers [49],
and 60% of beef cows [52]. Therefore, the interval from
GnRH treatment to wave emergence might be too incon-
sistent for superstimulation. Indeed, Deyo et al. [53] re-
ported unsatisfactory embryo production after
synchronization of follicular wave emergence with GnRH.
However, recent results from commercial embryo transfer
practitioners [54] and a research report involving 411 dairy
donors [55] have revealed more promising results. Basi-
cally, a progestin device is inserted at random stages of the
estrous cycle and GnRH is administered 2 or 3 days later
with superstimulation treatments beginning 1.5 to 2.5 days
later. Unfortunately, no studies using ultrasonography have
been performed to evaluate the efficiency of GnRH given 2
or 3 days after insertion of a progestin device to induce
ovulation and synchronize a follicle wave; ovulatory
response (and synchronization of follicle wave emergence)
might have been improved because the presence of the
progestin device has prevented ovulation of mature folli-
cles thus increasing the efficacy of GnRH.
4.3.1. Improving the ovulatory response to GnRH
An alternative for the synchronization of follicle wave
emergence for superstimulation is to synchronize ovula-
tion and then initiate FSH treatments at the time of emer-
gence of the first follicular wave [1,38,56]. Nasser et al. [57]
induced synchronous ovulation in Nelore (Bos indicus) do-
nors, with an estradiol-based protocol designed for FTAI.
Superstimulatory treatments were then initiated at the
expected time of ovulation (and emergence of the first
follicular wave). Superovulatory response did not differ
from a contemporary group superstimulated 4 days after
treatment with estradiol. However, the number of trans-
ferable embryos was reduced in cows superstimulated
during the first follicular wave unless the protocol was
accompanied by the use of a progestin device. Similar re-
sults were obtained by Rivera et al. [58] who inserted two
progesterone releasing devices (CIDR-B; Zoetis Animal
Health, Florham Park, NJ) devices in Holstein cows super-
stimulated during the first follicular wave.
Most FTAI protocols using GnRH to synchronize follicle
wave emergence use a form of presynchronization to im-
prove the ovulatory response to the first injection of GnRH
[59]. Bó et al. [60] recently reported on a series of experi-
ments with the overall objective of developing a protocol for
superstimulation after ovulation induced synchronously by
the administration of GnRH. This approach was based on a
previous study in which ovulatory response was increased by
causing a persistent follicle to develop with the administra-
tion of PGF2a and the insertion of a progestin device 7 to 10
days before the administration of GnRH [61]. In that study,
ovulation occurred in a very high percentage of treated ani-
mals and follicle wave emergence occurred 1 to 2 days after
the administration of GnRH, indicating that this approach
could be used in groups of randomly cycling donors.
The recommended superstimulation protocol consists
of the administration of PGF2a at the time of insertion of a
42 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
progestin device. Seven days later (with the progestin de-
vice still in place), GnRH is administered to induce ovula-
tion of the persistent follicle and synchronization of follicle
wave emergence; FSH treatments are initiated 36 hours
after the administration of GnRH [62]. The remainder of the
protocol is as described previously for estradiol-
synchronized follicle wave emergence. Although this pro-
tocol was designed for 4 days of FSH treatments, a 5-day
superstimulation protocol can be accomplished by simply
delaying the removal of the progestin device by 1 day.
Overall, in this series of experiments, more than 95% of
animals ovulated to the first GnRH administration and
superovulatory response and ova/embryo numbers and
quality were similar to that obtained when estradiol was
used to synchronize follicular wave emergence [60].
4.4. Subordinate follicle breakthrough
During a normal follicular wave, subordinate follicles
regress because of decreasing concentrations of circulating
FSH, caused by the secretions (estradiol and inhibin) of the
cohort and especially of the dominant follicle [63]. Small
follicles require FSH to continue their growth, and evidence
suggests that follicles as small as 1 mm in diameter will
commence growth under the influence of FSH (reviewed in
[64]). We hypothesized that all that is required to recruit
follicles for superstimulation is to cause small antral folli-
cles to grow to a diameter of 3 or 4 mm at which time the
regular 4- or 5-day superstimulatory treatment protocol
could be initiated. Assuming a growth rate of 1 to 2 mm per
day, it should take 2 to 3 days for a 1-mm follicle to reach 3
or 4 mm, i.e., add 2 to 3 days to the superstimulation
treatment protocol. As the exogenous FSH replaces
endogenous levels that are depressed by the secretory
products of the dominant follicle, we hypothesized that the
presence of a dominant follicle would not have any effect
on superovulatory response.
Beef donors were superstimulated successfully using
this approach at random stages of the estrous cycle and
without regard for the presence of a dominant follicle [33].
Small doses of FSH were administered twice daily for 2 days
and then the regular FSH treatment protocol was begun
with no increase in the total amount of FSH administered.
As an alternative, the 2 days of FSH pretreatment could be
replaced with a single injection of eCG. In one study, the
administration of 500 IU of eCG 2 days before initiating FSH
treatments tended to increase the superovulatory response
in beef donors [65] and in another study, pretreatment
with 400 IU of eCG resulted in increased embryo produc-
tion in beef donors that had previously produced unsatis-
factory numbers of embryos after superstimulation
without eCG (cited in [33]). It was hypothesized that the
eCG recruited additional follicles into the wave before the
initiation of FSH treatments.
4.5. Exogenously induced follicular waves
Recently, we have investigated the effect of lengthening
the superstimulatory treatment protocol from the tradi-
tional 4-day protocol to 7 days to recruit more follicles into
the wave and provide the time required for them to acquire
the capacity to ovulate [34]. Lengthening the FSH treatment
protocol to 7 days, without increasing the total amount of
FSH administered, increased the number of ovulations and
the synchrony of ovulations, and tended to increase the
mean numbers of total ova/embryos, fertilized ova, and
transferable embryos. In other words, the lengthened
superstimulatory treatment protocol resulted in more fol-
licles reaching an ovulatory size and acquiring the capacity
to ovulate with an increased number of ovulations, and
with no decrease in oocyte/embryo quality. In a more
recent study, the 7-day FSH treatment protocol resulted in
2.5 times more transferable embryos (morulae and blas-
tocysts) per animal after ultrasound-guided oocyte collec-
tion and in vitro embryo production than the regular 4-day
FSH treatment protocol (5.6 and 2.5 embryos per oocyte
donor, respectively; P ¼ 0.04) [66]. It was concluded that
prolonged FSH treatment protocols might be an effective
strategy to recruit small follicles into the follicular cohort
available for superstimulation, and provide the additional
time needed for these follicles to reach an ovulatory size
and acquire the capacity to ovulate. In addition, these re-
sults suggest that traditional 4-day superstimulatory
treatment protocols might not provide adequate time for
all follicles within the cohort to acquire the capacity to
ovulate. Obviously this requires further study.
4.6. Superstimulation of donor cattle with abnormal ovarian
function
Cows with abnormal ovarian function are difficult to
superstimulate because they usually do not have a func-
tional CL, or they come into estrus at unpredictable times.
In addition, it is almost impossible to predict the stage of
follicle development in these cows. Progestin inserts and
elective synchronization of follicle wave emergence have
been used in superstimulation protocols for donor cattle
with abnormal ovarian function. It was the need to super-
stimulate cows with abnormal ovarian function that led to
the use of estradiol before the administration of FSH. Em-
bryo production did not differ between 190 problem cows
superstimulated 7 days after receiving a norgestomet ear
implant and an injection of norgestomet and estradiol
valerate (EV) and 260 control cows superstimulated be-
tween Days 8 and 12 of the estrous cycle [67]. Although this
protocol was developed to provide an artificial CL using the
norgestomet implant, it was subsequently shown that EV
played a role by synchronizing emergence of a new follicle
wave [1]. Benefits probably accrued from the use of the
implant (artificial CL) and the EV (induction of a follicle
wave). Follicle ablation might also be used to synchronize
follicular wave emergence in problem cows that had
received a progestin device. Although the use of progestin
devices by themselves have not been critically evaluated in
problem cows, the insertion of a progestin device and the
initiation of superstimulatory treatments 3 to 5 days later
might be efficacious.
In cows with a history of low superovulatory responses
because of anovulation, the progestin/estradiol treatment
has been followed with the administration of porcine
LH (pLH; Lutropin-V, Bioniche Animal Health) at the time of
first AI to induce ovulation [68]. Administration of 25 mg
 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
pLH in problem cows increased the number of transferable
embryos (4.1  1.2) compared with the previous super-
stimulation without pLH (1.0  0.4; P < 0.03). However, pLH
did not improve embryo production in normal cows [68].
4.7. Follicle numbers and superovulatory response
Monniaux et al. [9] suggested that the number of folli-
cles present in the ovary at the time of gonadotropin
treatments affects superovulatory response in cattle. She
identified two classes of cows that responded poorly to
superstimulatory treatments. The first group had 50 to 200
growing follicles (0.7 mm in diameter) per ovary,
compared with 600 or more follicles in the ovaries of cows
that responded well. The second class of cows that did not
respond had a large number of follicles but many of them
were undergoing atresia at the time that gonadotropin
treatments were initiated. Cows in this later group might
have responded well to superstimulation if the gonado-
tropin treatments had been initiated at the time of follicle
wave emergence. However, the former group of animals
might not have had a large enough follicle population;
consequently, this condition might not be subject to
amelioration.
These early observations were confirmed recently in
studies in which the number of follicles in ovarian follicular
waves in cattle were reported to be highly variable among
animals, but very highly repeatable within individuals
[69,70]. For example, peak number of follicles during
different follicular waves of an estrous cycle have been
reported to be consistently as low as 8 in some cattle, but as
high as 56 in others [69]. In addition, it was shown that
cows with relatively high numbers of follicles had higher
serum inhibin-A concentrations, but lower serum FSH and
similar estradiol concentrations during the first follicular
wave compared with cows with low numbers of follicles
[69,70]. Singh et al. [71] have reported differences in
superstimulatory response between cows that had more
than 30 three- to 5-mm follicles compared with those with
fewer than 30 follicles. Similarly, Ireland et al. [70] also
reported that beef heifers with a low number of follicles
(15 follicles 3 mm in diameter) at the time of wave
emergence also had lower superovulatory response than
those with high number of follicles (i.e., 25 follicles) at
wave emergence. Therefore, it might be possible to select
donor cows based on the number of follicles present at the
time of follicle wave emergence detected using ultraso-
nography. It is also noteworthy that the tremendous vari-
ation in follicle numbers between animals might be the
reason that current treatment protocols have failed to in-
crease the number of transferable embryos per super-
stimulation treatment. Although synchronizing follicle
wave emergence might be critical in cows with medium to
low numbers of follicles (i.e., cows with less than 15 and
between 15 and 25 follicles per wave), the effect might not
be as apparent in cows with more than 25 follicles entering
the follicle wave and might be confounded by our inability
to recover all the embryos present in the uterus after
superovulation.
Most recently, it has been reported that follicle numbers
were associated with circulating concentrations of anti-
43
Müllerian hormone (AMH) [72,73]. Anti-Müllerian hor-
mone is a glycoprotein belonging to the transforming
growth factor-b family that is produced by the granulosa
cells of all growing follicles and is greatest in healthy small
antral follicles, which contribute most significantly to AMH
endocrine levels [73]. In adult cows and goats, AMH levels
were highly variable between individuals, but characteristic
of each animal over a period of several months [73].
Therefore, it might be possible to evaluate the ovarian
reserve of gonadotropin-responsive follicles through
detection of AMH concentrations in the circulation. Rico
et al. [74] have shown a correlation between AMH produc-
tion and superovulatory response in Holstein donors and
have suggested values that could be used to differentiate
between cows producing more or less than 10 transferable
embryos. Obviously more research is needed in this area.
5. Additional treatments and superovulatory
response
The need for FSH and LH at different times during
superstimulation has been debated. Basic studies on
follicular development have shown that FSH is required for
follicle recruitment and growth [63], until the dominant
follicle reaches 8.5 mm in diameter in Bos taurus breeds of
cattle [75] and 6.2 mm in Bos indicus cattle [76]. After se-
lection, dominant follicles acquire LH receptors and
become LH-dependent (reviewed in [77]). Therefore, folli-
cles of superstimulated cattle might benefit from the in-
clusion of LH near the end of the treatment protocol. Equine
chorionic gonadotropin is a gonadotropin with FSH and LH
activity [15] and could provide a constant stimulus to the
LH receptors of the growing follicles near the end of a
conventional FSH superstimulation treatment protocol.
Barros et al. [78] conducted an experiment in which
Nelore cows were superstimulated with FSH over 3 days;
the last two FSH injections (on the fourth day) were
replaced with two injections of 200 IU eCG. Treatment with
eCG significantly increased the number of ova/embryos and
increased the number of transferable embryos over that in
control animals. Although Sartori et al. [79] and Davis et al.
[80] found no beneficial effect, eCG treatment resulted in
more transferable embryos in Red Sindhi [81], Brangus
[82], Brahman, Senepol, and Angus [83] donors. In most of
the studies showing a positive effect of eCG treatment,
donors in the control group produced average to below
average numbers of transferable embryos, whereas in the
study by Davis et al. [80], donors in the control group
produced above average numbers of transferable embryos,
suggesting that this treatment might be especially useful
when embryo production is depressed. More recently,
Barros et al. [84] reported a similar improvement in embryo
production when 1 mg pLH was added to each of the last
two Folltropin-V administrations in Angus donors.
6. Improved convenience
6.1. Repeated superstimulation
Traditionally, donor cows have been subjected to
embryo collection at approximately 60-day intervals.
44 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48

However, more recent information suggests that cows can
be superstimulated as often as every 30 days [85]. In one
study, a luteolytic dose of PGF2a given at the time of ova/
embryo collection resulted in luteal regression by 3.6  0.8
days and 7 of 8 (88%) donors showed estrus by 7 days after
treatment [86]. In a more recent study in which a second
injection of PGF2a was administered 1 day after ova/embryo
collection, luteal regression was hastened by approximately
1 day (4.1  0.6 days vs. 5.3  2.2 days; P ¼ 0.03), but no
differences were detected in the interval to estrus and
ovulation [87]. The proportion of cows ovulating within 19
days after collection was 87% and the interval to ovulation
was 10.2  0.4 days. However, the interval from PGF2a
administration to ovulation was related to the number of CL
at ova/embryo collection (4.0  0.5 days in cows with 1 to 2
CL, 7.6  1.5 days in cows with 3 to 4 CL, and 10.4  0.9, 11.7
 0.6, and 10.8  0.8 in cows with 5 to 10, 11 to 15, and >15
CL, respectively) [88]. As indicated earlier (Section 4.3.1),
when the cow reovulates, follicular wave patterns are re-
established. Therefore, the cow could be superstimulated
the day after expression of estrus (first follicular wave;
using a progestin device), 8 to 12 days later, or after syn-
chronization of follicle wave emergence. In this way, donor
cows can be superstimulated in groups at approximately
30- to 35-day intervals. In a recent analysis from com-
mercial data from our laboratory, no differences were
detected in embryo production when cows were super-
stimulated at intervals ranging from 28 to 30 days to more
than 90 days (Table 1).
developing follicles to “catch-up”, followed by induction of
ovulation with GnRH or pLH which prevented late ovula-
tions. In this protocol, follicular wave emergence was syn-
chronized with estradiol and a progestin device on Day
0 and FSH treatments were initiated on Day 4. On Day 6,
PGF2a was administered in the morning and evening and
the progestin device was removed in the morning of Day 7
(24 hours after the first administration of PGF2a). On the
morning of Day 8 (24 hours after the removal of the pro-
gestin device), GnRH or pLH was administered and FTAI
was done 12 and 24 hours later. Delaying the removal of the
progestin device to the morning of Day 7 resulted in a
greater number of ova/embryos and fertilized ova than
removal on the evening of Day 6 (Table 2) [90]. From a
practical perspective, FTAI of donors has been shown to be
useful in eliminating the need for estrus detection for busy
embryo transfer practitioners [91].
Studies in high-producing Holstein cows (Bos taurus) in
Brazil have indicated that it is preferable to allow an
additional 12 hours before removing the progestin device
(i.e., Day 7 evening; P-36) followed by GnRH or pLH 24
hours later, i.e., Day 8 evening [92]. Baruselli et al. [44]
confirmed that it was preferable to remove the progestin
device on the evening of Day 7 (P-36), followed by GnRH 12
hours later (i.e., morning of Day 8) in Bos indicus breeds.
Although donors are typically inseminated twice, 12 and 24
hours after administration of pLH or GnRH [31], Baruselli
et al. [44] were successful using a single insemination of
high quality semen 16 hours after pLH.

6.2. Fixed-time AI of donors

6.3. Reducing the numbers of gonadotropin treatments
Barros and Nogueira [89] have developed a super-
Because the half-life of pituitary FSH is short in the
stimulatory protocol for Bos indicus cattle that they refer to
cow [29], traditional superstimulatory treatment protocols
as the P-36 protocol. In this protocol, the progestin device
consist of twice daily intramuscular injections over 4 or 5
that is inserted before the initiation of superstimulatory
days [35]. This requires frequent attention by farm
treatments is left in place for 36 hours after PGF2a admin-
personnel and increases the possibility of failures because
istration and ovulation is induced by the administration of
of noncompliance. In addition, twice-daily treatments
pLH 12 hours after withdrawal of the progestin device (i.e.,
might cause undue stress in donors with a subsequent
48 hours after PGF2a administration). Because ovulation
decreased superovulatory response and/or altered
occurs between 24 and 36 hours after pLH administration
[31], FTAI is scheduled 12 and 24 hours later, eliminating
the need for estrus detection. Table 2
In a series of experiments in which the timings of ovu- Mean ( SEM) number of ova/embryos, fertilized ova, and transferable
lations were monitored ultrasonically, Bó et al. [31] devel- embryos in superstimulated Red Angus and Red Brangus donors treated
with a progestin device for 6.5 or 7 days, with or without GnRH treatment
oped a protocol for FTAI in Bos taurus donors without
on the morning of Day 8, and AI on Days 8.5 and 9.
the need for estrus detection and without compromising
results. Basically, the time of progestin device removal Time of GnRH N Total Fertilized Grade 1
progestin device (Day 8) ova/ ova and 2
was delayed to prevent early ovulations and allow late
removal embryo embryos
Day 6.5 (P-12) No 26 8.1  1.4 6.4  1.2 4.2  0.7
Table 1 Day 6.5 (P-12) Yes 27 9.9  1.4 7.1  1.2 5.7  1.0
Interval (days) between ova/embryo collections and embryo production in Day 7 (P-24) No 24 11.3  1.8 8.4  1.3 5.9  1.1
beef donors. Day 7 (P-24) Yes 30 12.9  1.8 9.1  1.3 5.8  0.9
Main effects
Interval, N Total ova/ Fertilized Transferable Freezable No 50 9.7  1.2 7.4  0.9 5.0  0.7
days embryos ova embryos embryos Yes 57 11.4  1.2 8.1  0.9 5.7  0.7
28 to 30 360 11.1  0.5 8.1  0.4 6.5  0.3 4.8  0.3 Day 6.5 (P-12) 53 9.0  1.1a 6.7  0.9c 5.0  0.6
31 to 45 244 9.8  0.4 7.0  0.3 6.1  0.3 4.1  0.2 Day 7 (P-24) 54 12.1  1.3b 8.8  0.9d 5.8  0.7
46 to 60 132 10.9  0.5 7.6  0.4 6.2  0.3 4.6  0.3
Abbreviations: P12, progestin device removal 12 hours after the first
61 to 90 325 9.2  0.7 6.1  0.5 5.1  0.5 3.1  0.3
PGF2a; P24, progestin device removal 24 hours after the first PGF2a.
91 to 180 134 9.5  0.8 6.8  0.6 6.1  0.3 4.0  0.4 a,b
Means differ (P < 0.05).
c,d
Means did not differ (P > 0.15). Data from Angel and Bó (unpublished). Means tended to differ (P < 0.09). Adapted from [90].
 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
preovulatory LH surge [93]. Simplified protocols might be
expected to reduce donor handling and improve response,
particularly in less tractable animals.
A single subcutaneous administration of FSH has been
shown to induce a superovulatory response equivalent to
the traditional twice-daily treatment protocols in beef
cattle in high body condition (i.e., body condition score of
>3 out of 5) [94], but results were not repeatable in Hol-
stein cows, which had less subcutaneous adipose tissue.
However, superovulatory responses were improved in
Holstein cows when the single injection was split into two;
75% of the FSH dose was administered subcutaneously on
the first day of treatment and the remaining 25% was
administered 48 hours later, when PGF2a is normally
administered [95].
An alternative to induce a consistent superovulatory
response with a single injection of FSH is to combine the
pituitary extract with agents that cause the hormone to be
released slowly over several days. In a series of experiments
in which FSH diluted in a 2% hyaluronan solution was
administered as a single intramuscular injection (to avoid
the effects of body condition), a similar number of ova/
embryos was produced as in the traditional, twice-daily
FSH protocol [96]. However, 2% hyluronan was viscous
and difficult to mix with FSH, especially in the field.
Although more dilute preparations of hyaluronan were less
efficacious as a single administration, their use was
improved by splitting them into two injections 48 hours
apart as was done with single subcutaneous injection of
FSH in Holstein cows [97]. The split intramuscular treat-
ment protocol consists of diluting the FSH lyophilized
powder with 10 mL of a 1% or 0.5% hyaluronan solution and
the administration of two-thirds of the total dosage of FSH
on the first day, followed by administration of the
Fig. 1. Recommended estradiol-based superstimulatory treatments for: beef Bos taurus, Bos indicus, and lactating Holstein (i.e., dairy Bos taurus) donors. D, Day;
E2, 2.5–5 mg estradiol-17b or 2–2.5 mg estradiol benzoate; P4, 50 or 100 mg progesterone; PGF, PGF2a.
45
remaining one-third 48 hours later, when PGF2a is normally
administered. When compared with the twice-daily treat-
ment protocol (control cows) in 29 beef cows super-
stimulated three times in a crossover design, the numbers
of transferable embryos obtained using the split-injection
protocol (1.0% hyaluronan: 5.0  0.9; 0.5% hyaluronan: 6.1
 1.3) were not different from control cows (4.0  0.8) [97].
Data were interpreted to suggest that splitting the FSH dose
in either concentration of hyaluronan into two intra-
muscular injections 48 hours apart would result in a
superovulatory response comparable with the traditional
twice-daily intramuscular injection protocol in beef cattle.
Furthermore, the less concentrated solutions of hyaluronan
were not difficult to mix with FSH, even in field conditions.
A recent report derived from commercial embryo transfer
data has now confirmed these results in beef cattle in North
America [98].
7. Concluding remarks
A great deal of effort has been expended over the years
in improving superstimulation treatment protocols. Treat-
ments have evolved from depending on natural luteolysis
to complete control of follicular development and ovula-
tion. These advances have made superovulation treatments
more “user friendly” and have helped the widespread
application of the embryo transfer technology around the
world. Superstimulation of a synchronized cohort of folli-
cles can be accomplished by initiating treatments 4 days
after estradiol and progesterone (Fig. 1), 2 days after follicle
ablation, or 1.5 to 2 days after GnRH-induced ovulation
(Fig. 2). Hormonal treatments are usually preferred by most
practitioners because of practicality and convenience.
Furthermore, progestin devices are used in almost all
46 G.A. Bó, R.J. Mapletoft / Theriogenology 81 (2014) 38–48
Fig. 2. Recommended GnRH superstimulatory treatment for Bos taurus donors. D, Day; PGF, PGF2a.
protocols to maintain progesterone concentrations greater
than 1 ng/mL during superstimulation and to control the
time of ovulation. Although these approaches have been
shown to be effective across cattle breeds, the recom-
mended FSH dosages vary significantly from very low in Bos
indicus breeds to very high in lactating Holstein donors,
with intermediate dosages in beef Bos taurus breeds.
Furthermore, FTAI of donors can be performed successfully
but the optimum interval from progestin device removal to
GnRH treatment differ between breeds. The following
alternatives are suggested: (1) for Bos taurus beef donors:
remove progestin devices with the penultimate FSH
administration, give GnRH or pLH 24 hours later, and AI 12
and 24 hours after GnRH administration; (2) for Bos indicus
donors: remove the progestin devices with the last FSH
administration, give GnRH or pLH 24 hours later, and AI 12
and 24 hours after GnRH administration; and (3) for Bos
taurus lactating dairy donors: remove progestin devices
with the last FSH administration, give GnRH or pLH 24
hours later, and AI 12 and 24 hours after GnRH
administration.
Although the major advances might have been hindered
by the tremendous individual variation in superovulatory
response, recent studies have confirmed that the major
source of variability is the follicular population in the
ovaries of the donor animal. Although this recent knowl-
edge has not resulted in more embryos from any given
donor, it has provided us with the tools by which selected
donors have a greater chance of responding satisfactorily to
the superstimulatory treatments and might provide the
opportunity to titrate FSH dosages more accurately.
Furthermore, it has provided us the opportunity to examine
different approaches with donors that have previously
responded unsatisfactorily to conventional treatments. The
addition of eCG 2 days before starting FSH treatment,
adding eCG at the end of the superstimulatory treatment,
and/or extending the superstimulation treatment with FSH
to 6 or 7 days might prove to be very useful in increasing
the number of transferable embryos in these poor
responders.
New advances in molecular biology might also direct us
to other factors influencing follicle wave dynamics, and the
identification of genes that play important roles in normal
follicle function might lead us to ways by which we can in-
fluence gene expression or their products to increase follicle
recruitment and decrease or postpone follicle atresia.
Finally, it is expected that recombinant gonadotropins will
soon provide another alternative for the superstimulation of
cattle. Gonadotropin products currently available for use in
cattle are all produced by extracting and purifying the
desired hormones from pituitary tissue, blood, or urine.
Considering the potential risk of pathogen transmission
arising from the use of animal-derived products, it is fore-
seeable that regulatory authorities will eventually require
that these products be serum- and animal-derived product-
free, thereby making recombinant production the most
likely source. Recombinant gonadotropins can also be made
to have an extended action, facilitating a form of single
administration. These hormones are already available for
use in human fertility clinics; thus, it is reasonable to expect
that in the not too distant future, recombinant hormones
with prolonged action will be licensed for use in veterinary
medicine.
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