Theriogenology 65 (2006) 89101

www.journals.elsevierhealth.com/periodicals/the

The timing of ovulation and insemination

schedules in superstimulated cattle
Gabriel A. Bo a,*, Pietro S. Baruselli b, Pablo M. Chesta a,
Claudiney M. Martins b
a
Instituto de Reproduccion Animal Cordoba (IRAC), J.L. de Cabrera 106,
X5000GVD Cordoba, Argentina
b
Departamento de Reproducao Animal, FMVZ-USP, Sao Paulo 05508-000, Brazil

Abstract

The development of treatments that control follicular wave dynamics during the bovine estrous
cycle has resulted in interesting possibilities for the precise control of follicular wave emergence and
the time of ovulation. For superstimulation, follicular wave emergence can be controlled by
ultrasound-guided follicle ablation with FSH treatments initiated 1 or 2 d later, or injection of
estradiol combined with progesterone at the time of insertion of a progestogen releasing device and
FSH treatments beginning 4 d later. These are the most widely used protocols for superstimulation of
donor cows because they offer the convenience of being able to initiate treatments quickly and at a
self-appointed time, without reducing the number of transferable embryos. However, these protocols
still require precise estrus detection of donors following superstimulation in order to conduct AI at the
most appropriate time. Recent studies have been designed to develop superstimulation protocols that
involve fixed-time AI of donors, without regard to estrus detection. Results presented herein indicate
that delaying the removal of a progestogen releasing device, combined with the administration of
GnRH or porcine LH (pLH) 12 or 24 h later results in predictable, synchronous ovulations, permitting
fixed-time AI without reducing the numbers or quality of embryos. These protocols facilitate the
application of on-farm embryo transfer programs because they are practical, easy to administer by
farm personnel, and more importantly, they eliminate the need for detecting estrus.
# 2005 Elsevier Inc. All rights reserved.

Keywords: Bovine; Follicular development; Superovulation; Ovulation; Fixed-time AI

* Corresponding author. Tel.: +54 351 710669; fax: +54 351 710559.
E-mail address: gabrielbo@iracbiogen.com.ar (G.A. Bo).

0093-691X/$ see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2005.10.008
90 G.A. Bo et al. / Theriogenology 65 (2006) 89101

1. Introduction

Although embryo transfer techniques are widely used around the world, with more than
500,000 bovine embryos being transferred each year, variability in response to the
superstimulatory treatments remains an important limitation [1]. The better understanding
of ovarian function, acquired in recent years through the use of ultrasonography, has
provided possibilities for controlling follicular development and ovulation. Recent
protocols, designed to control follicular wave emergence and ovulation, permit the
initiation of superstimulatory treatments at a self-appointed time. However, these
treatments still require precise estrus detection of donors so that AI is conducted at the most
appropriate time. The intention of this manuscript is to review these protocols, present
information about the timing of ovulation in superstimulated donors and propose
modifications, developed recently, that will permit fixed-time AI of donors, thereby,
avoiding the necessity of estrus detection.

2. Manipulation of the follicular wave for superstimulation

The conventional protocol for 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
812 d after estrus (reviewed in [2]). However, none of these early studies specifically
evaluated follicular status of animals when gonadotropin treatments were initiated.
Monitoring follicular development ultrasonically in cattle was, in most cases, in the early
stages of development and not available in many laboratories.
Through information generated by ultrasonography, it is now known that 812 d after
estrus (equivalent to days 711 after ovulation) is the approximate time of emergence of the
second follicular wave in cows exhibiting two- or three-wave cycles [3], and a cohort of
growing follicles should be available at that time. However, it has been shown that
superovulatory response was higher when gonadotropin treatments were initiated at the
precise time of follicular wave emergence rather than later [4,5]. An alternative to ensure
that treatments are initiated at the beginning of a follicular wave in normally cycling cattle
is to use treatments that mechanically or hormonally control the timing of follicular wave
emergence.
One approach to controlling the time of follicular wave emergence involves transvaginal
ultrasound-guided ablation of all follicles 5 mm [6] or the two largest follicles present in
the ovaries [7], at random stages of the estrous cycle. Follicular ablation is followed by an
FSH surge and follicular wave emergence 12 d later; gonadotropin treatments are
initiated at that time. Others have used ultrasound-guided follicle ablation of all follicles
[8] or just the dominant follicle [9,10] 2 d prior to superstimulation during mid-diestrus,
with improved superovulatory responses in most cases [810] and a higher number of ova/
embryos, but a comparable number of transferable embryos to when cows were
superstimulated 713 d after estrus [11].
Follicular wave emergence has also been synchronized in estrus synchronization
protocols through the use of GnRH or pLH. However, the reported asynchrony in follicular
 G.A. Bo et al. / Theriogenology 65 (2006) 89101 91

wave emergence (from 3 d before to 5 d after treatment) [12], and following

superstimulation, the lower number of ova/embryos collected than when the follicular
wave was synchronized with estradiol or follicular ablation [13] suggests that this approach
may not be optimal for the synchronization of follicular wave emergence prior to
superstimulation.
The most common approach to the synchronization of follicular wave emergence for
superstimulation in donor cows has been treatment with estradiol-17b (E-17b) and
progesterone (P4) by intramuscular (im) injection at the time of progesterone/progestogen
device insertion [14]. Data from experiments and commercial embryo transfer programs
[2,15] have shown that superovulatory response of donors treated with E-17b and P4 at
unknown stages of the estrous cycle followed by FSH, beginning 4 d later, was comparable
to, or better than that of donors superstimulated beginning 812 d after observed estrus.
Estradiol-17b is not readily available for commercial use in many countries. Therefore,
we investigated the possibility of using other commercially available estrogen esters such
as estradiol benzoate (EB) or estradiol valerate (EV) to induce follicular wave emergence.
Treatment with 2.5 mg EB and 50 mg P4 at the time of CIDR (1.9 g of progesterone; Pfizer
Animal Health, Buenos Aires, Argentina) insertion resulted in synchronous emergence of a
new follicular wave 34 d later [16]. Superstimulatory treatments initiated 4 d after that
treatment resulted in superovulatory responses comparable to those initiated 4 d after
treatment with 5 mg E-17b and 50 mg P4 [17] or 2.5 mg E-17b and 50 mg P4 [18], or those
initiated 812 d after estrus [19]. Treatment with 5 mg EV and 3 mg norgestomet resulted
in less synchronous follicular wave emergence and a lower superovulatory response than
5 mg E-17b and 100 mg P4 in cows with two norgestomet ear implants [20]. However,
dosages of 1.0 or 2.0 mg EV in CIDR-treated cows resulted in follicular wave emergence in
34 d, with little variability [20]. These results suggest that lower dosages of estradiol
esters may be useful in the synchronization of follicular wave emergence for
superstimulation. However, experimental and field studies are needed to confirm these
observations.
Collectively, these studies demonstrated that exogenous control of follicular 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. It also
eliminates the need for detection of estrus (to determine the base heat) and eliminates
waiting 812 d to initiate superstimulatory treatments. However, this approach still
requires effective estrus detection for the AI of donors following the superstimulatory
treatments.

3. Time of ovulation and fixed-time AI in beef donors

Estrus detection is one of most difficult tasks to perform in any breeding program. The
timing of estrus, the endogenous LH surge and ovulation are especially variable among
superstimulated donor cattle [21,22], and a significant inverse relationship has been
reported between ovulation rate and the interval from administration of PGF to the LH
surge in donors [23]. In a preliminary study [24], we evaluated the interval between PGF
administration and ovulation in cows treated with DIB vaginal devices (1 g of
92 G.A. Bo et al. / Theriogenology 65 (2006) 89101

progesterone; Syntex, Buenos Aires, Argentina), 2.5 mg EB and 50 mg P4 on day 0 and

superstimulated with 400 mg NIH-FSH-P1 of Folltropin-V (Bioniche Animal Health,
Belleville, ON, Canada), starting on day 4. Cows were randomly allocated to receive PGF
in the morning and afternoon of day 6 or 7 and DIB were removed with the last PGF
injection. All cows were examined by twice daily ultrasonography to determine the time
and distribution of ovulations. Ovulations occurred between 60 and 108 h after the first
PGF treatment and there were no differences in the mean interval from PGF treatment to
ovulation in cows in which DIB were removed on day 6 (87.5  3.4 h) or day 7
(82.3  3.5 h). However, when cows were evaluated based on their superovulatory
response, cows with more than four CL ovulated earlier (79.6  1.8 h; P = 0.023) than
those with less than four CL (90.2  3.7 h). Obviously, variability based on superovulatory
response, makes estrus detection critically important to ensure that AI is conducted at the
most appropriate time to maximize fertilization rate and embryo quality.
The control of the LH surge in superstimulatory treatment protocols has been studied.
The main strategy has been to postpone the LH surge in relation to PGF treatment, thereby,
allowing more follicles to develop and acquire the capacity to ovulate. Rieger et al. [25]
used a GnRH antagonist to prevent the LH surge in superstimulated cattle; ovulation was
induced with hCG 4872 h after the administration of PGF. Although the number of
transferable embryos did not differ between cows treated at 72 h (6.2  2.0) and those in
the control group (no hCG treatment; 5.6  5.7), both were numerically higher than those
treated with hCG 48 h after PGF (4.0  4.2). Other studies have involved postponing the
removal of a norgestomet ear-implant [26] or a CIDR insert [27,28] with the administration
of GnRH or pLH at the time of implant or CIDR removal. However, these treatments
appeared to have an adverse effect on embryo quality. The authors speculated that poor
embryo quality was due to adverse effects of progestogens on the oviduct microenviron-
ment and early embryonic development [29].
DOcchio et al. [22] used a Deslorelin implant to block LH release in superstimulated
Brahman heifers; ovulation was induced with 25 mg im of pLH (Lutropin-V; Bioniche
Animal Health) at 48, 60 or 72 h after PGF administration. There was a nonsignificant
increase in the number transferable embryos when pLH was given 60 h after PGF. This
would correspond to approximately 12 h after the expected time of LH release in
superstimulated cattle, and may have permitted less mature follicles to acquire sufficient
development to ovulate. However, there was no significant difference in the number of
transferable embryos in a follow up study in which heifers treated with Deslorelin implants
and pLH 60 h after PGF, followed by a single AI 12 h later were compared to control
heifers that were AI at the onset of estrus, 12 and 24 h later [30]. Unfortunately, Deslorelin
implants for cattle are not commercially available in most countries.
Recent studies, mainly performed in Brazil, have been directed toward the development
of a superstimulation protocol that allows for fixed-time AI in Nelore cattle in which
follicular wave emergence was synchronized with EB on day 0 and superstimulatory
treatments were initiated on day 4 [28,31,32]. In all cases, PGF was given in the morning
and afternoon of day 6 and progesterone/progestogen devices were removed at varying
times after PGF, but before induction of ovulation with pLH 48 h after the first PGF (i.e.,
the morning of day 8). Treatments were named depending on the time from the first PGF
treatment to removal of the progesterone/progestogen-releasing device. Therefore, when
 G.A. Bo et al. / Theriogenology 65 (2006) 89101 93

devices were removed in the morning of day 7, the treatment was called P24, and when
devices were removed in the afternoon of day 7, the treatment was called P36. All donors
were AI 12 and 24 h after pLH treatment on day 8. No differences in the number of
transferable embryos were detected between the P24 and the P36 treatments [33], or
between the P36 treatment and a control group in which cows were AI 12 and 24 h after the
onset of estrus [34].
Based on these results, we designed a series of experiments to evaluate the effect of the
time of removal of a progesterone/progestogen releasing device and GnRH or pLH
treatment on the distribution of ovulations and embryo production in beef donors
superstimulated with Folltropin-V. In the first experiment, Red Angus and Red Brangus
donors (53 cows and 42 heifers), with a body condition score of 2.53.5 (15 scale), were
blocked by parity and randomly assigned to one of four treatment groups in a 2  2
factorial design. On day 0, all donors received a DIB device and an injection of 2.5 mg E-
17b and 50 mg P4 (Lab Rio de Janeiro, Santa Fe, Argentina). Superstimulatory treatments
were initiated on day 4 with a total dose of 320 mg (Angus cows), 200 mg (Angus heifers),
260 mg (Brangus cows) or 180 mg (Brangus heifers) of Folltropin-V in twice daily
injections over 4 d. All cows received PGF (150 mg D-(+)-cloprostenol, Ciclase; Syntex) in
the morning and afternoon of day 6 and were divided randomly to have DIB removed at the
time of the second PGF treatment (day 6.5 or P12) or 12 h later (day 7 or P24). On day 8
a.m. (48 h after the first PGF), donors were further subdivided to receive GnRH (0.050 mg
Lecirelina, Gonasyn; Syntex) or no further treatment, and all animals were fixed-time AI
60 and 72 h after the first PGF (day 6 a.m.). On day 15, ova/embryos were collected non-
surgically and classified following IETS recommendations.
A subgroup of 37 cows and 17 heifers representing all treatment groups were randomly
selected and examined every 8 h for a period of 120 h by ultrasonography, to determine the
time and distribution of ovulations, commencing at the time of DIB removal [35]. Hour 0
was designated as the time of the first PGF injection. All follicles >10 mm at each
examination were recorded and the time of each ovulation was determined to be the time
when a previously identified follicle was no longer observed. For each cow, a mean time of
ovulation was calculated, and the resulting mean value was then considered as the time of
ovulation for this animal in the statistical analysis. Data were first analyzed by Bartletts
test to evaluate homogeneity of variances. If variances did not differ, data were analyzed by
ANOVA, but if variances differed, data were analyzed using Friedman two-way non-
parametric ANOVA. There was a significant breed effect on the number of ovulations
(Angus: 14.0  1.0; Brangus: 22.0  1.8; P < 0.001), but not on the time of ovulation
(Angus: 81.0  1.2 h; Brangus: 77.6  1.3 h; P > 0.13). Furthermore, no significant
differences were detected between cows and heifers (P > 0.13) for the parameters
evaluated. There was no effect (P > 0.22) of treatment on the number of ovulations
(Table 1). However, there were differences among groups in the distribution of ovulations
(P < 0.01). When DIB were removed on day 7 (P24) and GnRH was administered on day
8, the variability in the time of ovulation was less than in the other three treatment groups
(Table 1). Individual ovulations (irrespective of individual donors) occurred from 56 to
104 h after PGF in the day 6.5control (no GnRH) group; 6496 h in the day 6.5GnRH
group; 7296 h in the day 7control group; 6496 h in the day 7GnRH group.
Furthermore, delaying the removal of the DIB resulted in a delay in the onset of ovulation;
94 G.A. Bo et al. / Theriogenology 65 (2006) 89101

Table 1
Mean (S.E.M.) numbers of and intervals to (in hours after the first PGF) ovulation in superstimulated Red Angus
and Red Brangus donors treated with progesterone releasing devices (DIB) for 6.5 or 7 d, with or without GnRH
treatment on day 8 (a.m.)
Time of DIB removal GnRH (day 8) N Ovulations Time of ovulation (h)
Mean Mean Variance
Day 6.5 (P12) No 13 15.2  2.2 79.4  2.0 50.2 b
Day 6.5 (P12) Yes 14 16.6  1.6 77.1  2.0 53.8 b
Day 7 (P24) No 13 18.1  2.3 83.6  2.0 53.0 b
Day 7 (P24) Yes 14 17.9  2.4 79.1  0.7 7.6 a

Main effects
No 26 16.6  1.6 81.5  1.4 d 53.9
Yes 28 17.3  1.4 78.1  1.1 c 30.6
Day 6.5 (P12) 27 15.9  1.3 78.2  1.4 51.6
Day 7 (P24) 27 18.0  1.6 81.2  1.1 33.5
Means or variances in the same column with different letters differ: a and b: P < 0.01; c and d: P < 0.05.

when DIB were removed on day 6.5, 11/27 (40.7%) of the donors began to ovulate before
72 h after PGF, but when DIB were removed on day 7, 1/27 (3.7%) began to ovulate before
72 h (P < 0.001). The mean numbers of ova/embryos and transferable embryos were only
numerically (not significantly) higher in donors treated with GnRH than those that were not
and when DIB were removed on day 7 than on day 6.5 (Table 2), suggesting that in addition
to the time of ovulation, many other factors are involved in the production of high quality
embryos. However, removing the DIB on day 7 a.m. and giving GnRH on day 8 a.m.
resulted in synchronous ovulations.
Based on these results, a second experiment was designed to determine if synchrony of
ovulation and the number of transferable embryos can be increased by delaying
progesterone/progestogen device removal another 12 h (day 7.5 or P36) and giving GnRH
12 or 24 h later. In this experiment, Red Angus and Red Brangus donors (46 cows and 36
heifers), with body condition scores between 3 and 4, were blocked by parity and randomly

Table 2
Mean (S.E.M.) number of ova/embryos, fertilized ova and transferable embryos in superstimulated Red Angus
and Red Brangus donors treated with progesterone releasing devices (DIB) for 6.5 or 7 d, with or without GnRH
treatment on day 8 (a.m.) and AI on days 8.5 and 9
Time of DIB removal GnRH (day 8) N Total ova/embryo Fertilized ova Grade 1 and 2 embryos
Day 6.5 (P12) No 23 8.8  1.6 6.6  1.3 4.2  0.8
Day 6.5 (P12) Yes 23 10.4  1.8 7.0  1.4 5.4  1.1
Day 7 (P24) No 22 10.8  1.8 8.2  1.4 5.6  1.2
Day 7 (P24) Yes 27 13.3  2.1 9.2  1.4 5.4  0.9
Main effects No 45 9.8  1.2 7.4  1.0 4.9  0.7
Yes 50 12.0  1.4 8.2  1.0 5.4  0.7
Day 6.5 (P12) 46 9.6  1.2 6.8  0.9 4.8  0.7
Day 7 (P24) 49 12.2  1.4 8.7  1.0 5.5  0.7
Means did not differ (P > 0.1).
 G.A. Bo et al. / Theriogenology 65 (2006) 89101 95

Table 3
Mean (S.E.M.) numbers of and interval to (in hours after the first PGF) ovulation in superstimulated Red Angus
donors treated with progesterone releasing devices (DIB) for 7 or 7.5 d and GnRH on day 8 or 8.5
Time of DIB removal GnRH N Ovulations Interval to ovulation (h)
Mean Mean Variance
Day 7 (P24) Day 8 11 10.0  1.3 81.1  0.6 a 4.4 a
Day 7.5 (P36) Day 8 13 11.7  1.9 81.7  0.6 a 5.0 a
Day 7.5 (P36) Day 8.5 13 12.1  2.0 91.1  1.3 b 19.6 b
Means or variances in the same column with different letters differ: a and b: P < 0.05.

assigned to one of three treatment groups. On day 0, all donors received a DIB plus 2.5 mg
E-17b and 50 mg P4. Superstimulatory treatments were initiated on day 4 with the same
dosages of Folltropin-V used in the first experiment. All donors received PGF treatment in
the a.m. and p.m. of day 6 and were randomly divided to have DIB removed in the a.m. of
day 7 or 12 h later (day 7.5). The group in which DIB were removed on day 7 a.m. received
GnRH in the a.m. of day 8, whereas, the group in which DIB were removed on day 7.5 was
divided to receive GnRH in the a.m. of day 8 or 12 h later (day 8.5). All donors were fixed-
time AI 12 and 24 h after GnRH treatment. Ova/embryos were collected non-surgically on
day 15 in the morning, in donors which received GnRH in the morning of day 8 and 12 h
later in those that received GnRH on day 8.5. In this experiment, a subgroup of Red Angus
donors (19 cows and 7 heifers) representing all treatment groups were randomly selected
and examined every 8 h by ultrasonograhy to determine timing of ovulations. Data analysis
was performed as in the first experiment. Although ovulations were more synchronous in
the two groups that received GnRH in the morning of day 8 (DIB removal on day 7 or 7.5;
Table 3), ova/embryo numbers did not differ among groups (Table 4).
Results obtained so far can be interpreted to suggest that all treatments result in
comparable superovulatory responses and embryo quality. Therefore, all of these
treatments can be used in a superstimulation scheme in beef cattle involving fixed-time AI.
Delaying the removal of the progesterone/progestogen device from day 6.5 to 7 or 7.5 had
the largest effect in preventing early ovulations. Although the administration of GnRH
seemed to result in more synchronous ovulations, it did not affect the number or quality of
ova/embryos in these studies. In a recent study in which all cows had a CIDR insert
removed in the morning of day 7, the number of transferable embryos did not differ
between those that were AI 12 and 24 h after the onset of estrus (detected by Heat-Watch)

Table 4
Mean (S.E.M.) number of ova/embryo, fertilized ova and transferable embryos in superstimulated Red Angus
and Red Brangus donors treated with progesterone releasing devices (DIB) for 7 or 7.5 d, GnRH on day 8 or 8.5
and AI 12 and 24 h later
Time of DIB GnRH N Total Fertilized Grade 1 and
removal (d) ova/embryo ova 2 embryos
Day 7 (P24) Day 8 33 9.1  1.4 6.4  1.2 4.2  1.0
Day 7.5 (P36) Day 8 36 9.4  1.3 5.9  0.8 4.3  0.7
Day 7.5 (P36) Day 8.5 36 8.4  1.2 6.1  1.0 4.5  0.8
Means did not differ (P > 0.1).
96 G.A. Bo et al. / Theriogenology 65 (2006) 89101

and those that were fixed-time AI 12 and 24 h after the administration of 12.5 mg im of
pLH (Lutropin-V, Bioniche) in the morning of day 8 [36]. Collectively, results suggest that
either GnRH or pLH can be used to synchronize ovulation for fixed-time AI in
superstimulated donors.

4. Superstimulation and fixed-time AI in high-producing Holstein cows

Recently, the protocols for fixed-time AI in superstimulated donors have been evaluated
in high-producing Holstein cows in Brazil (i.e., 40 kg of milk per day) [37]. In the first
experiment, 40 Holstein cows were treated with one or two Crestar implants (Intervet, Sao
Paulo, Brazil) plus 3 mg EB and 100 mg P4 on day 0; FSH treatments were initiated on day
4 and PGF was given in the a.m. and p.m. of day 6. Crestar implants were removed in the
p.m. of day 7 (P36) and GnRH was given either in the a.m. (day 8) or p.m. (day 8.5) of day 8
with AI 12 and 24 h later. No differences in superovulatory response and the number of
transferable embryos were detected between one or two Crestars (Table 5). However,
treatment with GnRH 24 h after Crestar removal (day 8.5) resulted in fewer (P < 0.05)
unovulated follicles (i.e., follicles >10 mm at the time of ova/embryo collection) than
when GnRH was given 12 h after Crestar removal (day 8). Although this was not reflected
in a significant increase in the number of transferable embryos, numbers favored treatment
with GnRH 24 h after Crestar removal.
A follow up study was designed to determine the effects of time of removal of a
progesterone/progestogen device and the time of pLH treatment on the distribution of
ovulations and embryo production in high-producing Holstein cows [38]. Twelve Holstein
cows were superstimulated four times each in a cross-over experimental design, in which
all cows were treated with the four treatments in four replicates and all treatment groups
were represented in each replicate. All donors received a DIB on day 1 and 3 mg EB on
day 0. Superstimulatory treatments were initiated on day 4, with a total dose of 200 mg

Table 5
Mean (S.E.M.) numbers of ovulations (CL) and unovulated follicles (>10 mm), ova/embryos, fertilized ova and
transferable embryos in superstimulated Holstein cows treated with one or two Crestars implants for 7.5 d, GnRH
12 or 24 h after Crestar removal and AI 12 and 24 h later
Number of GnRH N CL Follicles Total Grade 1 and
implants >10 mm ova/embryos 2 embryos
One Crestar Day 8 (12 h) 10 10.9  3.6 2.8  0.6 bc 8.0  4.0 1.1  0.6
Day 8.5 (24 h) 10 10.1  2.4 2.3  0.5 b 8.9  2.5 1.9  0.9
Two Crestars Day 8 (12 h) 10 9.4  2.4 4.2  0.8 c 8.6  2.6 2.1  0.9
Day 8.5 (24 h) 10 9.7  2.9 1.1  0.3 a 8.4  3.2 3.8  1.9
Main effects
One Crestar 20 10.5  2.1 2.6  0.4 8.4  2.3 1.5  0.5
Two Crestars 20 9.6  1.8 2.7  0.5 8.5  2.0 3.0  1.0
Day 8 (12 h) 20 10.2  2.1 3.5  0.5 a 8.3  2.3 1.6  0.6
Day 8.5 (24 h) 20 9.9  1.8 1.7  0.3 b 8.6  2.0 2.9  1.0
Means in the same column with different letters differ: a, b and c: P < 0.05.
 G.A. Bo et al. / Theriogenology 65 (2006) 89101 97

Table 6
Mean (S.E.M.) numbers of and intervals to (in hours after the first PGF) ovulation in superstimulated Holstein
cows treated with progesterone releasing devices (DIB) for 7 or 7.5 d, pLH on day 8 or 8.5 and AI 12 and 24 h later
Day of DIB removal pLH N Ovulations Interval to ovulation (h)
Mean Mean Variance
Day 7 (P24) Day 8 11 6.2  1.4 79.5  2.0 42.8
Day 7 (P24) Day 8.5 10 8.6  1.9 82.7  2.9 85.7
Day 7.5 (P36) Day 8 11 5.9  1.1 77.8  2.3 56.9
Day 7.5 (P36) Day 8.5 11 9.2  1.8 84.9  1.5 24.9
Main effects Day 8 22 6.1  0.9 c 78.6  1.5 a 48.2
Day 8.5 21 8.9  1.3 d 83.9  1.6 b 52.3
Day 7 (P24) 21 7.3  1.2 81.0  1.7 62.7
Day 7.5 (P36) 22 7.6  1.1 81.3  1.5 52.3
Means in the same column with different letters differ: a and b: P < 0.03; or tended to differ: c and d: P < 0.08.

Folltropin-V administered twice daily over 4 d. All cows received PGF treatment in the
a.m. and p.m. of day 6 and were randomly assigned to have DIB removed in the a.m. of day
7 (P24) or 12 h later (day 7.5; P36). In the morning of day 8, donors were further subdivided
to receive 25 mg pLH (Lutropin-V, Bioniche Animal Health) at that time or 12 h later (day
8.5). All cows were fixed-time AI 12 and 24 h after pLH treatment. On day 15, ova/
embryos were collected nonsurgically and classified following IETS recommendations.
All cows were also examined every 12 h by ultrasonography to determine the time and
distribution of ovulations.
Average time of ovulation and the distribution of mean ovulation times are depicted in
Table 6 and superovulatory responses and ova/embryo production are depicted in Table 7.
There was no effect of the time of DIB removal on any of the end points evaluated.
However, pLH treatment 60 h after PGF (day 8.5) resulted in a delayed mean time of
ovulation (P < 0.03) and a tendency for a higher number of follicles ovulating (P < 0.08)
during the examination period (Table 6). The mean interval from the first to the last
ovulation was also shorter (P < 0.05) in cows treated with pLH on day 8.5 (10.9  2.0 h)

Table 7
Mean (S.E.M.) number of ova/embryos, fertilized ova and transferable embryos in superstimulated Holstein
cows treated with progesterone releasing devices (DIB) for 7 or 7.5 d, pLH on day 8 or 8.5 and AI 12 and 24 h later
Day of DIB removal pLH N Total ova/embryo Fertilized ova Grade 1 and 2 embryos
Day 7 (P24) Day 8 12 4.0  1.2 3.4  0.8 2.3  0.8
Day 7 (P24) Day 8.5 12 4.8  1.3 4.5  1.1 3.5  1.1
Day 7.5 (P36) Day 8 12 3.6  0.9 3.2  0.7 1.9  0.7
Day 7.5 (P36) Day 8.5 12 6.5  1.5 6.4  1.4 5.9  1.3
Main effects Day 8 24 3.8  0.7 3.3  0.5 2.1  0.5 a
Day 8.5 24 5.7  1.0 5.4  0.9 4.7  0.9 b
Day 7 (P24) 24 4.4  0.9 3.9  0.7 2.9  0.7
Day 7.5 (P36) 24 5.0  0.9 4.7  0.9 3.8  0.8
Means in the same column with different letters differ: a and b: P < 0.01.
98 G.A. Bo et al. / Theriogenology 65 (2006) 89101

than in those treated with pLH on day 8 (17.5  2.3 h). Furthermore, pLH on day 8.5
resulted in a higher number of transferable embryos (P < 0.01; Table 7). In this study,
delaying the time of pLH treatment was beneficial, probably due to the development of
follicles that were more responsive to LH.
The results in Holstein cows differed from those in the Angus and Brangus donors, in
this manuscript, and those in Nelore (Bos indicus) cattle, in which the same protocols were
used [39]. In Nelore donors, delaying the time of pLH from day 8 to 8.5 resulted in a
numerically lower number of transferable embryos (6.0  1.5 versus 4.0  0.9,
respectively) and a tendency (P = 0.06) for a higher number of degenerate embryos
(0.7  0.4 versus 1.3  0.4, respectively).
Differences in results from the studies in Bos taurus and Bos indicus beef donors may be
related to differences in stage of follicle development at the time that ovulation was
induced. For example, the diameter at which dominant follicles diverge from subordinates
has been shown to occur earlier and at a smaller diameter in Bos indicus cattle (6.0
6.3 mm) [40,41] than in Bos taurus cattle of a dairy breed (8.5 mm) [42]. In addition,
follicles acquired ovulatory capacity at a larger diameter in Bos taurus dairy cows than in
Bos indicus cows [43,44]. Sartori et al. [43] reported that dairy cows ovulated after LH
administration when dominant follicles were 10 mm in diameter. Conversely, it has been
shown in Bos indicus cattle that follicles will ovulate at 78.4 mm [44]. Therefore, it is
conceivable that delaying the pLH treatment was beneficial in Holstein cows because it
allowed more follicles to achieve ovulatory capacity, while this was not necessary in
Nelore cows. Therefore, administration of pLH in the morning of day 8 induced ovulation
of the smaller follicles in the Nelore cows, whereas delaying pLH treatment until day 8.5
was necessary for the induction of ovulation of larger follicles in Holstein cows. It is also
possible that waiting either 12 or 24 h after progesterone/progestogen device removal
before GnRH treatment caused similar results in Angus and Brangus cattle. Although no
direct studies have been done to determine the diameter that the dominant follicle diverges
from subordinates in Angus or Brangus cattle, it is likely that the maximum diameter of the
dominant follicle is smaller in these breeds than in lactating Holsteins [45,46], but larger
than that in Nelore [47].

5. Summary and conclusions

Incorporation of protocols designed to control follicular wave dynamics, like those

discussed herein, offers the convenience of being able to initiate treatments quickly and at a
self-appointed time, without the necessity of estrus detection and without sacrificing
superovulatory results. Furthermore, evidence is mounting that these treatments can be
incorporated into protocols that involve fixed-time AI of donor cows, without sacrificing
ova/embryo quality. However, additional studies are required to confirm these conclusions
and recommendations. Nevertheless, results from the current studies suggest the following
alternatives for superstimulated beef cattle: (1) remove progesterone/progestogen devices
in the a.m. of day 7 and accurately detect estrus and AI 12 and 24 h after the onset of estrus;
(2) remove the progesterone/progestogen devices in the a.m. of day 7, give GnRH or pLH
in the a.m. of day 8 and AI 12 and 24 h later; or (3) remove the progesterone/progestogen
 G.A. Bo et al. / Theriogenology 65 (2006) 89101 99

devices in the p.m. of day 7, give GnRH or pLH in the p.m. of day 8 and AI 12 and 24 h
later. Based on the more synchronous ovulations and a higher number of transferable
embryos in these studies, this latter alternative is also preferable in high-producing
Holstein cows. Embryo collection should be done later with this alternative to avoid the
collection of early morulae, which are generally not preferred for embryo freezing [48].
These superstimulation treatment protocols facilitate the application of on-farm embryo
transfer programs because they are practical, easy to follow by farm personnel and, more
importantly, the need for detecting estrus or ovulation is eliminated.

Acknowledgements

Research was supported by grants from FAPESP (2002/06363-0) Brazil, and the
Agencia Cordoba Ciencia and Instituto de Reproduccion Animal Cordoba (IRAC),
Argentina. We also thank Bioniche Animal Health for Folltropin-V and Lutropin-V, and
Syntex for DIB and Gonasyn. Special thanks to our colleagues of IRAC and University of
Sao Paulo for technical assistance.

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