Superovulation and embryo transfer in Bos indicus cattle

Theriogenology 65 (2006) 77–88
www.journals.elsevierhealth.com/periodicals/the
Superovulation and embryo transfer
in Bos indicus cattle
Pietro S. Baruselli a,*, Manoel F. de Sá Filho a,
Claudiney M. Martins a, Luiz F. Nasser a,
Marcelo F.G. Nogueira b, Ciro M. Barros b, Gabriel A. Bó c
a
Departament of Animal Reproduction, FMVZ-USP, Rua Prof. Orlando Marques de Paiva,
87, CEP 05508-000, Sao Paulo-SP, Brazil
b
Departamento de Farmacologı́a, Instituto de Biociências, UNESP, Botucatu-SP, Brazil
c
Instituto de Reproducción Animal Córdoba, Universidad Católica de Córdoba, Córdoba, Argentina
Abstract
Compared to Bos taurus breeds, Bos indicus breeds of cattle present several differences in
reproductive physiology. Follicular diameter at deviation and at the time of ovulatory capability are
smaller in B. indicus breeds. Furthermore, B. indicus breeds have a greater sensitivity to gonadotropins, a shorter duration of estrus, and more often express estrus during the night. These differences
must be considered when setting up embryo transfer programs for B. indicus cattle. In recent studies,
we evaluated follicular dynamics and superovulatory responses in B. indicus donors with the
objective of implementing fixed-time AI protocols in superstimulated donors. Protocols using
estradiol and progesterone/progestrogen releasing devices to control follicular wave emergence
were as efficacious as in B. taurus cattle, allowing the initiation of superstimulatory treatments (with
lower dosages of FSH than in B. taurus donors) at a self-appointed time. Furthermore, results
presented herein indicate that delaying the removal of progesterone/progestogen-releasing devices,
combined with the administration of GnRH or pLH 12 h after the last FSH injection, results in
synchronous ovulations, permitting the application of fixed-time AI of donors without the necessity
of estrus detection and without compromising the results.
# 2005 Elsevier Inc. All rights reserved.
Keywords: Superstimulation; Embryo transfer; Bos indicus; Fixed-time AI; Ultrasonography
* Corresponding author.
E-mail address: [email protected] (P.S. Baruselli).
0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2005.10.006
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1. Introduction
Bovine embryo transfer has been applied widely around the world. This technology
increases the number of offspring obtained from donors with high genetic value and is used to
disseminate desirable genetics around the world. In Brazil and in other tropical countries,
there has been an increasing demand to multiply the genetics of valuable Bos indicus females.
However, there are important differences in the physiology and the reproductive behavior
between B. indicus and Bos taurus cattle that can affect the efficiency of superstimulation
programs. Traditional superstimulation protocols have some limitations: (1) necessity of
handling and detecting estrus to establish the ‘‘base heat’’, (2) inability to start
superstimulatory treatments at the optimal time of follicular development, (3) necessity
to detect estrus to determine time of AI, (4) high variability in embryo production per donor,
and (5) 20–30% of unresponsive donors that do not produce embryos.
2. Factors that influence superovulatory response
Variability in superovulatory responses after gonadotropin treatments continues to be
the greatest problem for commercial embryo transfer [1–3]. Individual variation in
superovulatory response has also been observed in Nelore cattle using a ‘‘cross-over’’
experimental design [4]. Numbers of CL, ova/embryos and embryos suitable for freezing
varied significantly among donors. A recent study involving high producing Holstein cows
in a ‘‘cross-over’’ experimental design in a tropical environment also reported significant
individual variation in the number of follicles >8 mm in diameter at the time of estrus and
in the number of CL at the time of ova/embryo collection [5].
In the conventional protocol for superstimulation, gonadotropin treatments are initiated
during mid-cycle (8–12 d post-ovulation). This approach presents difficulties because it
requires estrus detection prior to initiation of gonadotropin treatments, and because there is
a great individual variation in the day of emergence of the second follicular wave. These
difficulties can adversely affect superovulatory responses [3].
Several studies have demonstrated the importance of initiating gonadotropin treatments
at the time of follicular wave emergence. The absence of a dominant follicle at the
beginning of treatment increased the efficacy of the superstimulatory treatments [3,6].
Nasser et al. [7] obtained a higher superstimulatory response when gonadotropin
treatments were initiated on the day of follicle wave emergence than when treatments were
initiated 1 or 2 d later. Therefore, alternatives to control follicular wave emergence at
random stages of the estrus cycle, without necessity to detect estrus to establish a ‘‘base
heat’’, would facilitate management of B. indicus donors, and possibly increase the
efficiency of embryo transfer programs in cattle of Zebu breeding.
3. Control of follicular dynamics for superstimulation
Mechanical (follicle ablation) [8] or pharmacological (GnRH) [9], LH, hCG or estradiol
plus progesterone (P4) [10,11] methods of controlling follicular wave emergence have
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
79
been developed in the last 10 year. Treatment with GnRH resulted in ovulation of the
dominant follicle and emergence of a new follicular wave 1–2 d later [12]. In B.
taurus B. indicus heifers, the administration GnRH at random stages of the estrus cycle
induced ovulation in only 45.7% (16/35) of heifers [13]. Low pregnancy rates following
administration of GnRH have been observed in B. indicus cattle kept on tropical regions
[14]. The results of these studies raise doubts about the efficacy of GnRH to synchronize
wave emergence in B. indicus cattle.
In B. indicus cattle, an elective treatment of induction of follicular wave emergence is
the association between estradiol and P4. The efficacy of this association has been
demonstrated in several studies in B. taurus cattle [10,11,15–17]. We have also studied the
effects of estradiol and P4 treatment for the synchronization of follicular wave emergence
in B. indicus, B. indicus B. taurus crosses, and B. taurus heifers that were kept under the
same tropical conditions [18]. There were no differences in the mean interval from
treatment to wave emergence between B. indicus, B. taurus or B. taurus B. indicus
heifers. However, B. indicus heifers recruited more follicles into the wave than B. taurus
heifers, suggesting the probability of a higher superstimulatory response following
gonadotropin treatments in B. indicus cattle (Table 1).
Different esters of estradiol, including estradiol benzoate (EB), estradiol valerate (EV),
and estradiol cypionate (ECP) are commercially available in South America. All have been
reported to induce follicle regression when administered in the presence of elevated plasma
P4 concentrations [11]. Both EV and ECP have a long half-life, resulting in delayed and
more variable intervals to follicular wave emergence [11,19,20] than the shorter-acting
estradiol-17b [11] or EB [21]. We have recently evaluated the effect of administration of
2 mg EB at the time of insertion of a 3 mg norgestomet ear implant (Crestar; Intervet, Sao
Paulo, Brazil) or an intravaginal insert (CIDR; Pfizer Animal Health, Sao Paulo, Brazil) in
cycling B. indicus heifers [22]. The interval from treatment to follicular wave emergence
was not difference between heifers treated with Crestar ear implants (2.9 0.1 d) and
those treated with CIDR inserts (3.1 0.1 d). In a more recent experiment [23], we
evaluated the effect of EB and two different doses of EV on the time and synchrony of
follicular wave emergence in B. indicus cows and heifers receiving Crestar ear implants.
There were significant effects of treatment (Table 2), but no treatment-by-class interaction
on the mean interval to, and synchrony of, wave emergence.
We have recently evaluated the effect of administering 50 mg of P4 intramuscularly
along with EB at the time of insertion of a DIB vaginal device (1 g of progesterone; Syntex,
Buenos Aires, Argentina) on the interval to, and synchrony of, follicular wave emergence
Table 1
Mean (S.E.M.) interval from treatment to follicular wave emergence and number of follicles recruited per wave
in Bos indicus, B. indicus Bos taurus, and B. taurus heifers treated with 2 mg estradiol benzoate and CIDR insert
Heifers
N
Interval to follicular
wave emergence (d)
Number of follicles
recruited per wave
B. indicus
B. indicus B. taurus
B. taurus
23
25
22
3.1 0.1
3.3 0.1
3.2 0.1
33.4 3.2 a
29.6 2.5 ab
25.4 2.5 b
Adapted from [18]. Means within the same column with different superscripts (a, b) differ (P < 0.05).
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Table 2
Mean (S.E.M.) interval (d), and range, from treatment to follicular wave emergence in B. indicus cows and
heifers treated with 2 mg estradiol benzoate (EB), 2.5 mg EV plus 1.5 mg norgestomet (1/2 EV + Nor), or 5 mg of
estradiol valerate plus 3 mg norgestomet (EV + Nor) at the time of insertion of a Crestar ear implant
Class
EB
1/2 EV + Nor
EV + Nor
N treated animals
Heifers
10
2.5 0.2 a
2–3x
10
2.5 0.2 a
2–4x
10
4.2 0.3 b
3–6xy
10
3.1 0.4 ab
2–6xy
9
6.1 0.6c
3–8y
10
4.0 0.5b
2–6y
N treated animals
Cows
P-value
<0.001
<0.05
<0.05
<0.05
Adapted from [23].
in B. indicus cattle [24]. Treatment with P4 plus EB at the time of DIB insertion resulted in
a delayed and more synchronous wave emergence (4.2 0.0 d) than treatment with EB
alone at DIB insertion (2.8 0.2 d). These results are in agreement with those previously
reported in B. taurus cattle [25]. In yet another experiment [26], we examined the effect of
different doses of EB (2 or 3 mg), time of EB administration and the addition of a P4
injection in 50 Nelore cows treated with DIB devices (n = 10/group). Treatment with EB
plus P4 on the day of DIB insertion resulted in more synchronous emergence of a new
follicular wave (4.0 0.0 d) than 2 mg EB given alone at DIB insertion (3.6 0.2 d) or
1 d later (4.3 0.2 d), or 3 mg EB given alone on the day of DIB insertion (4.0 0.2 d) or
1 d later (4.2 0.3 d). In conclusion, treatment with EB plus injectable P4 at the time of
insertion of a progesterone releasing intravaginal device induced the most synchronous
follicular wave emergence in B. indicus cattle.
4. Fixed-time AI in superstimulated donors
Although the control of follicular development allows for the elective initiation of
gonadotropin treatments, the time of AI continues to be dependent on estrus detection.
Unfortunately, estrus detection in B. indicus cattle is difficult, variable and subject to errors.
Estrus is shorter than that in B. taurus cattle and there is a greater tendency for B. indicus
cattle to show estrus during the night [27]. Therefore, several studies have been conducted
to investigate the pharmacological control of the time of ovulation in superstimulated B.
indicus cattle, with the objective of developing a fixed-time AI protocol.
A superstimulation protocol (named P-36) has been developed in Brazil [28]. In this
protocol the progesterone/progestogen-releasing device is removed 36 h after PGF
treatment (thus, P-36) and ovulation is induced with pLH (Lutropin-V; Bioniche Animal
Health, Belleville, Ont., Canada), administered 12 h after progesterone/progestogenreleasing device removal (i.e., 48 h after PGF administration). Since ovulation occurs
between 24 and 36 h after pLH treatment [28], fixed-time AI is done 12 and 24 h after pLH
injection, avoiding the inconvenience of estrus detection. Recently, the effectiveness of the
P-36 protocol was tested in a commercial herd of Nelore cattle. In 136 superstimulations
and embryo collections performed with the P-36 protocol (CIDR or DIB and 3 mg EB on
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
81
Table 3
Mean (S.E.M.) number of ova and embryos and transferable embryos in Nelore cows superstimulated 5 d after
synchronization of follicular wave emergence with 3 mg estradiol benzoate and a progesterone-releasing
intravaginal device (CIDR or DIB)
Progesterone device
Number of treated cows
Total ova/embryos
Transferable embryos
Dose of Plh
CIDR
DIB
25.0 mg
12.5 mg
53
12.7 1.1
8.7 0.9
83
13.6 1.0
9.8 0.9
43
13.3 1.3
9.8 1.1
93
13.2 0.9
9.2 0.8
The progesterone-releasing intravaginal device was removed 36 h after PGF treatment (P-36 protocol) and cows
were treated with pLH (25.0 or 12.5 mg) 12 h later. Fixed-time AI was done 12 and 24 h after pLH treatment.
Adapted from [29]. Means did not differ.
Day 0 and superstimulation initiated on Day 5), the number of ova/embryos and
transferable embryos and pregnancy rates following nonsurgical transfer of fresh embryos
were 13.3 0.8, 9.4 0.6 and 43.5% (528/1213), respectively [29]. Additionally, the type
of progesterone-releasing intravaginal device (DIB versus CIDR) and dose of pLH
(12.5 mg versus 25.0 mg) did not influence embryo yield significantly (Table 3). These
results were comparable those reported in studies in which Nelore cows were inseminated
12 and 24 after onset of behavioral estrus [28–31].
Another experiment was designed to evaluate the effect of the time of removal of the
progesterone-releasing device in superstimulated B. indicus cattle that were fixed-time
inseminated [32]. Ten Nelore cows were placed at random into one of two groups in a
‘‘cross-over’’ experimental design and were superstimulated twice 35 d apart with
Folltropin-V (Bioniche Animal Health). The cows received a CIDR and were treated with
2.5 mg EB plus 50 mg of P4 at random stages of the estrus cycle (Day 0). Superstimulation
treatments (133 mg NIH-FSH-P1 of Folltropin-V) divided into eight decreasing doses 12 h
apart were initiated on Day 4. Cows received PGF on the morning of Day 6. CIDR were
removed in the morning (P-24) or afternoon (P-36) of Day 7. Cows also received 25 mg
pLH (Lutropin-V) in the morning of Day 8 (12 h after the last Folltropin-V treatment or
48 h after PGF) and were fixed-time inseminated 12 and 24 h later. No differences in
superovulatory responses or ova/embryo quality were observed between Groups P-24 and
P-36 (Table 4). These results demonstrate that the progesterone-releasing device can be
Table 4
Mean (S.E.M.) number of ova/embryo, fertilized ova and transferable embryos in superstimulated Nelore donors
treated with progesterone releasing devices (CIDR) for 7 (P-24) or 7.5 d (P-36), pLH on Day 8 a.m. and AI 12 and
24 h later
Number of treated cows
Total ova/embryos
Fertilized ova
Transferable embryos
Adapted from [31]. Means did not differ (P > 0.05).
P-24 protocol
P-36 protocol
10
21.2 4.8
16.0 3.9
9.3 2.4
10
17.7 3.7
15.5 3.8
10.3 1.9
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P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
removed either 24 h (P-24) or 36 h (P-36) after PGF treatment without affecting
superovulatory response or ova/embryo quality following for fixed-time AI in superstimulated B. indicus donors.
5. Optimum time for induction of ovulation for fixed-time AI in superstimulated
B. indicus donors
Currently, we are investigating the most appropriate time for induction of ovulation
for fixed-time AI in superstimulated Nelore (B. indicus) and Holstein (B. taurus)
donors. Physiological differences between Nelore and Holstein cows, especially the
diameter of the dominant follicle at the time of deviation, have been reported. The
diameter of the dominant follicle at the time of deviation has been reported to be
6.0 mm [33] or 6.3 mm [34] in Nelore and 8.5 mm in Holstein [35] cattle. A study was
designed to determine the diameter at which the dominant follicle acquired the capacity
to ovulate in response to treatment with pLH in Nelore heifers [36]. The onset of
ovulatory capacity of the dominant follicle was found to be between 7 and 8.4 mm
(30%; 3/9), and there was a linear increase in response from 8.5 to 10 mm (80.0%; 8/
10). In heifers with follicles >10 mm, 90% ovulated in response to pLH treatment.
These results differed from those reported in Holstein cows where ovulatory capacity
was found to begin at 10 mm [37].
In a recent study, we evaluated the effect of delaying the induction of ovulation in
superstimulated Nelore donors by 12 h (i.e., 12 or 24 h after the last Folltropin-V
injection) [38]. The objective of this experiment was to improve ovulation rates by
increasing follicle diameter (and ovulatory capacity) at the time of pLH treatment. Twenty
Nelore cows were placed at random into one of two experimental groups and
superstimulated twice utilizing a ‘‘cross-over’’ experimental design. All cows received a
DIB plus 2.5 mg EB and 50 mg P4 on Day 0, and on Day 4, were superstimulated with a
total of 100 mg Folltropin-V in a twice-daily decreasing dose schedule over 4 d (Days 4–
7). On Day 6, PGF was given, and in the morning of Day 7, DIB were removed (P-24).
Cows were subdivided to receive 25 mg pLH 12 or 24 h after the last Folltropin-V
injection (Day 8 a.m. and p.m.) and were fixed-time inseminated 12 and 24 h later with the
same batch of frozen-thawed semen. Timing of ovulations was determined by twice daily
ultrasonography and on Day 15, ova/embryos were collected and classified according to
the IETS criteria. Delaying treatment with pLH from 12 to 24 h after the last Folltropin-V
injection increased (P < 0.01) the number of degenerated embryos, and decreased
(P < 0.005) the number transferable and freezable embryos (Table 5). The decreased
embryo quality following the delay in induction of ovulation may be explained by the
increased (P < 0.05) interval between first to last ovulation and the tendency (P = 0.08)
for increased variability in ovulation times after treatment at 24 h. These results indicate
that the optimum time for treatment with pLH for fixed-time AI in superstimulated Nelore
donors is 12 h after the last Folltropin-V treatment (i.e. the morning of Day 8 in the
EB + P4/P-24 superstimulation protocol).
The results obtained in Nelore donors differ from those observed in Holstein donor
cows. In high producing Holstein cows, pLH treatment 24 h after the last Folltropin-V
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
83
Table 5
Mean (S.E.M.) number of ova/embryo, fertilized ova and transferable embryos in superstimulated Nelore donors
treated with progesterone releasing devices (DIB) for 7 d (P-24), pLH 12 (Day 8) or 24 h (Day 8.5) after the last
Folltropin-V injection and AI 12 and 24 h later
Number of treated cows
Number of follicles >8 mm at pLH injection
Number of CL at embryo collection (Day 15)
Total ova/embryos collected
Unfertilized ova
Degenerate embryos
Transferable embryos (Grades 1, 2 and 3)
Freezable embryos (Grades 1 and 2)
Interval—first to the last ovulation (h)
Interval—last FSH injection to ovulation (h)
Interval-pLH injection to ovulation (h)
pLH 12 h
(Day 8)
pLH 24 h
(Day 8.5)
20
12.5 1.5
9.1 1.4
7.5 1.0
0.7 0.3
0.6 0.3
6.2 1.0
5.9 0.9
15.0 2.1
42.3 0.6
30.3 0.6
20
9.4 1.0
6.0 0.7
5.1 0.5
0.6 0.2
1.5 0.3
3.1 0.5
2.5 0.5
21.0 1.7
56.8 1.1
32.8 1.1
P-value
Treatment
Donor
0.11
0.08
0.05
0.63
0.008
0.004
0.002
0.02
0.0001
0.03
0.001
0.01
0.006
0.51
0.05
0.02
0.04
0.04
0.13
0.13
Adapted from [38].
injection resulted in an improved superovulatory response and higher numbers of
transferable embryos [5,38,39]. Rodrigues et al. [39] also observed a reduction in the
number of unovulated follicles (>10 mm) at ova/embryo collection and a numerically
higher number of transferable embryos when GnRH was administered 24 h after the last
FSH injection, compared to GnRH administered at 12 h after the last FSH treatment.
Martins et al. [5], utilizing a ‘‘cross-over’’ experimental design and the same batch of
semen, also observed an increase in the number of transferable embryos when pLH was
administered 24 h after the last FSH treatment in Holstein cows rather than 12 h after the
last FSH. Based on these results, it was concluded that 24 h after the last FSH injection
(60 h after treatment with PGF) was the optimum time for induction of ovulation with pLH
for fixed-time AI in superstimulated high-producing Holstein cows, whereas a 12 h interval
from the last treatment with FSH (48 h after PGF treatment) was more appropriate for
Nelore cows.
Based on the results of the previous study in Nelore cows, we have recently
performed an experiment to test the hypothesis that it is possible to use a single fixedtime insemination in B. indicus donors without adversely affecting fertilization rates or
ova/embryo quality. Ten Nelore cows were subdivided into two experimental groups
utilizing a ‘‘cross-over’’ experimental design. Treatments were similar to those in the
previous study except that DIB were removed in the afternoon of Day 7 (P-36 protocol).
All cows received pLH 12 h after the last Folltropin-V injection (Day 8 a.m.; 12 h after
DIB removal or 48 h after PGF treatment) and cows were fixed-time inseminated with
the same batch of frozen-thawed semen twice, 12 and 24 h after pLH, or only once, 16 h
after pLH treatment. There was no difference in superovulatory response or ova/embryo
quality between one or two fixed-time inseminations (Table 6). Results indicate that
it is possible to perform a single fixed-time AI 16 h after pLH in superstimulated
B. indicus donors using the P-36 treatment protocol without compromising ova/embryo
production.
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Table 6
Effect of number of fixed-time inseminations (1 vs. 2 FTAI) on embryo production in Nelore donors superstimulated with Folltropin-V and induced to ovulate with Lutropin-V 12 h after DIB removal and last FSH
injection (mean S.E.M.)
Number of follicles >8 mm at pLH
Number of CL at ova/embryo collection
Total ova/embryos collected
Unfertilized ova
Degenerated embryos
Transferable embryos (Grades 1, 2 and 3)
Freezable embryos (Grades 1 and 2)
Interval from the first to the last ovulation (h)
1 FTAI (n = 10)
2 FTAI (n = 10)
P-value
16.2 1.4
10.5 1.3
8.2 0.9
0.6 0.2
3.3 0.9
4.3 0.7
2.9 0.6
32.4 1.8
14.8 1.2
9.2 0.7
7.2 0.8
0.8 0.2
2.2 0.3
4.2 0.6
2.8 0.4
33.6 1.6
0.20
0.23
0.35
0.57
0.36
0.91
0.89
0.61
6. Use of different dosages of FSH for superstimulation of B. indicus cattle
B. indicus breeds have been shown to have a reduced capacity for LH secretion and a
greater sensitivity to exogenous gonadotropins than B. taurus cattle [40]. Baruselli et al. [4]
evaluated superovulatory response in 23 Nelore cows to three different doses of FolltropinV (100, 133 or 200 mg) in a ‘‘cross-over’’ experimental design. All cows received 2.5 mg
EB and 50 mg P4 at the time of CIDR insertion (Day 0) and superstimulatory treatments
(twice daily im injections over 4 d) were initiated on Day 4. PGF was given in the morning
and afternoon of Day 6, CIDR were removed in the afternoon of Day 7 and pLH was
administered in the morning of Day 8. All cows were fixed-time AI, utilizing the same
batch of frozen-thawed semen, 12 and 24 h after pLH and ova/embryos were collected on
Day 15. There were no significant differences in any of the parameters evaluated (Table 7).
Results indicate that it is possible to reduce the dose of Folltropin-V to 100 mg in a fixedtime AI, superstimulatory protocol in Nelore cattle without compromising superovulatory
response and ova/embryo quality.
7. Superstimulatory protocols for in vitro embryo production
Ovarian superstimulation with gonadotropins has been reported to increase the number
of oocytes retrieved by ultrasound-guided oocyte aspiration as compared to nonstimulated
B. taurus cattle [41–44]. However, forcing a population of follicles to continue in the
growth phase by administering FSH may not provide an ideal follicular environment for
cumulus-oocyte complexes (COC) to acquire developmental competence [45]. There is
evidence in B. taurus cattle that a ‘‘coasting’’ period between hormonal stimulation and
slaughterhouse ovary collection [46] or from ovary collection to oocyte aspiration [47] will
affect the in vitro developmental potential of COC. In both situations, follicles in phases of
pseudodominance or early atresia provided COC with an ideal environment in which to
acquire developmental competence [45].
Blondin et al. [45] achieved a surprisingly high blastocyst rate (80%), following
superstimulation of Holstein heifers with FSH and delaying COC retrieval for 48 h i.e., a
stage of early atresia was induced in these follicles by depriving them of FSH for the 48 h
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
85
Table 7
Means (S.E.M.) superovulatory response and ova/embryo production in Nelore cows utilizing three different
doses of Folltropin-V
Total dose of Folltropin-V
Number of treated cows
Number of CL
Total ova/embryos
Transferable embryos (Grades 1, 2 and 3)
Freezable embryos (Grades 1 and 2)
100 mg
133 mg
200 mg
23
13.0 7.2
10.0 7.8
7.7 7.4
6.4 6.5
23
12.1 7.0
9.9 7.0
5.6 4.1
4.4 3.2
23
14.9 11.6
10.6 8.6
6.5 7.7
5.7 7.0
Adapted from [4]. Means did not differ.
period. Additionally, LH was administered 6 h before oocyte aspiration in an attempt to
render the COC more competent. Recently, the same protocol was evaluated against
other protocols used for oocyte aspiration and in vitro embryo production (IVP) in
Nelore cattle [48]. Eighteen Nelore cows were randomly placed into the following three
groups: Group 1: oocyte aspiration was performed without superstimulation; Group 2:
superstimulation was induced before oocyte aspiration; Group 3: superstimulation and
FSH deprivation were induced before oocyte aspiration. Three oocyte aspiration
sessions were performed on each cow in a ‘‘cross-over’’ design so that all cows were
subjected to all three-treatment protocols. At random stages of the estrus cycle, all
follicles 6 mm were aspirated to induce a new follicular wave 2 d later (Day 0). In
Group 1, oocyte aspiration was performed on Day 2. In Group 2, cows were
superstimulated with twice daily injections of 30 mg Folltropin-V for 3 d (starting on
Day 0), 12.5 mg pLH 6 h after the last FSH treatment, and oocyte aspiration 6 h later
(12 h after the last FSH treatment). In Group 3, cows received the same treatment as in
Group 2, except that pLH was administered 36 h after the last treatment with FSH and
oocyte aspiration was performed 6 h later. In this group, follicles were deprived of
exogenous FSH for 48 h before oocyte aspiration. Regular in vitro production
procedures were used to mature and fertilize COC and culture presumptive zygotes.
Results indicate that ovarian superstimulation associated with deprivation of FSH prior
to oocyte aspiration did not increase in vitro embryo production in Nelore cattle
(Table 8). On the contrary, the highest rate of hatched blastocysts was observed in
oocytes from nonstimulated cows (Group 1).
Table 8
Number of oocytes collected, cleavage, blastocyst and hatched blastocyst rates in Nelore cows treated with three
different protocols prior to oocyte aspiration: Group 1 (control), Group 2 (superstimulation and oocyte aspiration),
and Group 3 (superstimulation, FSH deprivation and oocyte aspiration)
Group 1
Group 2
Group 3
Number of oocytes
Cleavage (%)
Blastocysts (%)
Hatched blastocysts (%)
185
139
159
77.8
75.5
63.5
42.7
31.7
33.3
30.3a
11.5b
15.7b
Adapted from [48]. Percentages within the same column with different superscripts (a, b) differ (P < 0.01).
86
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
Fig. 1. Treatment protocol for superstimulation, induction of ovulation, and fixed-time AI in Nelore embryo
donors. Treatment consists of insertion of progesterone releasing intravaginal device and administration of
estradiol benzoate (EB) and progesterone (P4) im on Day 0. Superstimulatory treatments are initiated on Day 4,
with FSH given twice daily over 4 d. Donors receive PGF treatment in the a.m. and p.m. of Day 6 and progesterone
devices are removed with the last FSH, in the p.m. of Day 7. Donors also receive pLH in the a.m. of Day 8 and are
inseminated without estrus detection 12 and 24 h later, or once on Day 8 (16 h after pLH). Ova/embryos are
collected nonsurgically on Day 15.
8. Conclusion
The results of the studies presented herein demonstrate that it is possible to synchronize
follicular wave emergence and initiate superstimulatory treatments at random stages of the
estrus cycle in B. indicus cattle. Moreover, treatment with pLH 12 h after the last FSH
treatment synchronized ovulations, allowing for the application of fixed-time AI protocols
in superstimulated B. indicus cattle. Finally, the number of inseminations was also reduced
to only one (16 h after pLH treatment) without adversely affecting ova/embryo quality
(Fig. 1).
References
[1] Barros CM, Nogueira MFG. Superovulação em zebuı́nos de corte. In: Proceedings of the first simpósio
internacional de reprodução animal aplicada Londrina; 2004. p. 212–22.
[2] Nogueira MFG, Barros BJP, Teixeira AB, Trinca LA, D’Occhio MJ, Barros CM. Embryo recovery and
pregnancy rates after the delay of ovulation and fixed-time insemination in superstimulated beef cows.
Theriogenology 2002;57:1625–34.
[3] Mapletoft RJ, Steward KB, Adams GP. Recent advances in the superovulation in cattle. Reprod Nutr Dev
2002;42:601–11.
[4] Baruselli PS, Marques MO, Reis EL, Nasser LFT, Silva RCP, Menegatti JA, et al. Adequação da dose de FSH
(Folltropin-V) em protocolos de superovulação de vacas nelore (Bos taurus indicus) com inseminação
artificial em tempo fixo. Acta Sci Vet 2003;31(Suppl 1):244–5.
[5] Martins CM, Castricini ESC, Reis EL, Torres-Júnior JRS, Gimenes LU, Sá Filho MF, et al. Produção
embrionária de vacas holandesas a diferentes protocolos de superovulação com inseminação artificial em
tempo fixo. Acta Sci Vet 2005;3(Suppl 1):286 (abstract).
[6] Adams GP. Control of ovarian follicular wave dynamics in cattle: implications for synchronization and
superstimulation. Theriogenology 1994;54:19–24.
[7] Nasser L, Adams GP, Bó GA, Mapletoft RJ. Ovarian superstimulatory response relative to follicular wave
emergence in heifers. Theriogenology 1993;40:713–24.
[8] Bergfelt DR, Bó GP, Mapletoft RJ, Adams GP. Superovulatory response following ablation-induced
follicular wave emergence at random stages of the oestrous cycle in cattle. Anim Reprod Sci 1997;
49:1–12.
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
87
[9] Kohram H, Twagiramungu H, Bousquet D, Durocher J, Guilbault LA. Ovarian superstimulation after
follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle.
Theriogenology 1998;49:1175–86.
[10] Bó GA, Adams GP, Nasser LF, Pierson RA, Mapletoft RJ. Effect of estradiol valerate on ovarian follicles,
emergence of follicular waves and circulating gonadotropins in heifers. Theriogenology 1993;40:225–39.
[11] Bó GA, Adams GP, Caccia M, Martinez M, Pierson RA, Mapletoft RJ. Ovarian follicular wave emergence
after treatment with progestogen and estradiol in cattle. Anim Reprod Sci 1995;39:193–204.
[12] Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF2a and GnRH.
Theriogenology 1995;44:915–23.
[13] Baruselli OS, Marques MO, Carvalho NAT, Valentim R, Berber RCA, Carvalho Filho AF, et al. Aumento da
taxa de prenhez em receptoras de embrião bovino pela utilização do protocolo ‘‘Ovsynch’’ com inovulação
em tempo fixo. Arq Fac Vet UFRGS 2000;28:216 (abstract).
[14] Fernandes P, Teixeira AB, Crocci AJ, Barros CM. Timed artificial insemination in beef cattle using GnRH
agonist, PGF2a and estradiol benzoate (EB). Theriogenology 2001;55:1521–32.
[15] Bó GA, Adams GP, Pierson RA, Trı́bulo HE, Caccia M, Mapletoft RJ. Follicular wave dynamics after
estradiol-17b treatment of heifers with or without a progestogen implant. Theriogenology 1994;41:1555–69.
[16] Bó GA, Pierson RA, Mapletoft RJ. The effect of estradiol valerate on follicular dynamics and superovulatory
response in cows with Syncro-Mate-B implants. Theriogenology 1991;36:169–83.
[17] Martinez MF, Adams GP, Kastelic JP, Bergfelt DR, Mapletoft RJ. Induction of follicular wave emergence for
estrus synchronization and artificial insemination in heifers. Theriogenology 2000;54:757–69.
[18] Carvalho JBP. Sincronização da ovulação com dispositivo intravaginal de progesterona (CIDR) em novilhas
B. indicus, B. indicus B. taurus e B. taurus. PhD Thesis, São Paulo: Universidade de São Paulo; 2004.
[19] Colazo MG, Kastelic JP, Mapletoft RJ. Effects of estradiol cypionate (ECP) on ovarian follicular dynamics,
synchrony of ovulation, and fertility in CIDR-based, fixed-time AI programs in beef heifers. Theriogenology
2003;60:855–65.
[20] Colazo MG, Martinez MF, Small JA, Kastelic JP, Burnley CA, Ward DR, et al. Effect of estradiol valerate on
ovarian follicle dynamics and superovulatory response in progestin-treated cattle. Theriogenology 2005;
63:1454–68.
[21] Bo GA, Baruselli PS, Moreno D, Cutaia L, Caccia M, Trı́bulo R, et al. The control of follicular wave
development for self-appointed embryo transfer programs in cattle. Theriogenology 2002;57:53–72.
[22] Sá Filho MF, Gimenes LU, Ayres H, Carvalho NAT, Carvalho JB, Baruselli PS. Dinâmica folicular de
novilhas Bos indicus tratadas com implante auricular de norgestomet ou com dispositivo intravaginal de
progesterona. Acta Sci Vet 2005;33(Suppl 1):266 (abstract).
[23] Sá Filho MF, Reis EL, Ayres H, Peres AAC, Carvalho, CAB, Carvalho JB, Araújo, CASC, Baruselli PS.
Effect of oestradiol valerate or benzoate on induction of a new follicular wave emergence in Bos indicus
cows and heifers treated with norgestomet implant. Reprod Fertil Dev 2006; (abstract), in press.
[24] Martins CM, Castricini ESC, Sá Filho MF, Gimenes LU, Baruselli PS. Dinâmica folicular em novilhas e
vacas nelore (Bos indicus) tratadas com dispositivo intravaginal de progesterona novo ou reutilizado
associado ou não à progesterona injetável. Acta Sci Vet 2005;33(Suppl 1):227 (abstract).
[25] Moreno D, Cutaia L, Villata ML, Ortisi F, Bó GA. Follicular wave emergence in beef cows treated with
progesterone releasing devices, estradiol benzoate and progesterone. Theriogenology 2001;55:408
(abstract).
[26] Sá Filho MF, Gimenes LU, Torres-Júnior JRS, Carvalho NAT, Kramer MPS, De Faria MH, et al. Emergência
folicular conforme a dose e o momento da aplicação de benzoato de estradiol e da sua associação com
progesterona injetável em vacas Nelore (Bos indicus) tratadas com dispositivo intravaginal de progesterona.
In: Proceedings of the anais VI simpósio internacional de reproducción animal, CD-ROM; 2005.
[27] Bo GA, Baruselli PS, Martinez MF. Pattern and manipulation of follicular development in Bos indicus cattle.
Anim Reprod Sci 2003;78:307–26.
[28] Nogueira MFG, Barros CM. Timing of ovulation in Nelore cows superstimulated with P36 protocol. Acta Sci
Vet 2003;31:509 (abstract).
[29] Nogueira MFG, Fragnito PS, Trinca LA, Barros CM. Effectiveness of the P36 superstimulatory protocol,
associated with fixed-time AI, on embryo production in a commercial herd of beef cattle. Theriogenology
2005; submitted for publication.
88
P.S. Baruselli et al. / Theriogenology 65 (2006) 77–88
[30] Barros CM, Nogueira MFG. Embryo transfer in Bos indicus cattle. Theriogenology 2001;56:1483–96.
[31] Nogueira MFG, Barros BJP, Teixeira AB, Trinca LA, D’Occhio MJ, Barros CM. Embryo recovery and
pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows.
Theriogenology 2002;57:1625–34.
[32] Zanenga CA, Marques MO, Santos ICC, Valentin R, Baruselli PS. Comparação entre dois protocolos de
superovulação com inseminação artificial em tempo fixo em vacas Nelore (Bos taurus indicus). Acta Sci Vet
2003;31(Suppl 1):626–7.
[33] Sartorelli ES, Carvalho LM, Bergfelt DR, Ginther OJ, Barros CM. Morphological characterization of follicle
deviation in Nelore (Bos indicus) heifers and cows. Theriogenology 2005;63:2382–94.
[34] Gimenes LU, Sá Filho MF, Madure EH, Trinca LA, Barros CM, Baruselli PS. Estudo ultrasonográfico da
divergência folicular em novilhas Bos indicus. Acta Sci Vet 2005;33(Suppl 1):210 (abstract).
[35] Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant follicle in cattle. Biol
Reprod 1996;55:1187–94.
[36] Gimenes LU, Carvalho NAT, Sá Filho MF, Santiago LL, Carvalho JBP, Mapletoft RJ, et al. Capacidade
ovulatória em novilhas Bos indicus. Acta Sci Vet 2005;33:209 (abstract).
[37] Sartori R, Fricke PM, Ferreira JCP, Ginther OJ, Wiltbank MC. Follicular deviation and acquisition of
ovulatory capacity in bovine follicles. Biol Reprod 2001;65:1403–9.
[38] Baruselli PS, Sá Filho MF, Martins CM, Reis EL, Nasser LF, Bó G. Novos avanços nos tratamentos de
superovulação em doadoras de embriões bovino. In: Proceedings of the anais VI simpósio internacional de
reproducción animal, CD-ROM; 2005.
[39] Rodrigues CA, Mancilha RF, Reis EL, Ayres H, Gimenes LU, Sá Filho MF, et al. Efeito do número de
implantes de norgestomet e do momento da administração do indutor de ovulação em vacas holandesas
superovuladas. Acta Sci Vet 2005;33(Suppl 1):229 (abstract).
[40] Randel RD. Seasonal effects on female reproductive functions in the bovine (Indian breeds). Theriogenology
1984;21:170–85.
[41] Goodhand KL, Staines ME, Hutchinson JSM, Broadbent PJ. In vivo oocyte recovery and in vitro embryo
production from bovine oocyte donors treated with progestogen, estradiol and FSH. Anim Reprod Sci
2000;63:145–58.
[42] Pieterse MC, Vos PLAM, Kruip THAM, Wurth YA, Van Bebede THH, Willemse AA, et al. Repeated
transvaginal ultrasound-guided ovum pick-up in eCG-treated cows. Theriogenology 1992;37:273 (abstract).
[43] Looney CR, Lindsey BR, Gonseth CL, Johnson DL. Commercial aspects of oocyte retrieval and in vitro
fertilization (IVF) for embryo production in problem cows. Theriogenology 1994;41:67–72.
[44] Stubbings RB, Walton JS. Effect of ultrasonically guided follicle aspiration on estrous cycle and follicular
dynamics in Holstein cows. Theriogenology 1995;43:705–12.
[45] Blondin P, Bousquet D, Twagiramungu H, Barnes F, Sirard MA. Manipulation of follicular development to
produce developmentally competent bovine oocytes. Biol Reprod 2002;66:38–43.
[46] Goodhand KL, Watt RG, Staines ME, Hutchinson JSM, Broadbent PJ. In vivo oocyte recovery and in vitro
embryo production from bovine oocyte donors aspirated at different frequencies or following FSH treatment.
Theriogenology 1999;51:951–61.
[47] Blondin P, Guilbault LA, Sirard MA. The time interval between FSH-P administration and slaughter can
influence the developmental competence of beef heifer oocytes. Theriogenology 1997;48:803–13.
[48] Barros CM, Gonzaga Ferreira MM, Potiens JR, Eberhardt BG, Melo DS, Monteiro FM. Influence of
superstimulation and hormonal deprivation protocols on in vitro production of Nelore embryos (Bos taurus
indicus). Reprod Fertil Dev 2006; (abstract), in press.