1. Animal Reproduction Science 124 (2011) 12–18
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Fixed-time AI protocols replacing eCG with a single dose of FSH were
less effective in stimulating follicular growth, ovulation, and fertility in
suckled-anestrus Nelore beef cows
J.N.S. Sales a , G.A. Crepaldi a , R.W. Girotto b , A.H. Souza a , P.S. Baruselli a,∗
a
Departamento de Reproducão Animal, FMVZ-USP, Rua Prof. Orlando Marques de Paiva 87, CEP: 05508-270, São Paulo, SP, Brazil
¸
b
RG Genética Avancada, Rua 1, Sala 03, n◦ 481, CEP: 78635-000, Água Boa, MT, Brazil
¸
a r t i c l e i n f o a b s t r a c t
Article history: The aim of the present study was to evaluate the effects of a single treatment with FSH
Received 8 June 2010 on diameter of the largest follicle and on conception rates of suckled Bos indicus beef cows
Received in revised form 19 January 2011
submitted to timed artificial insemination (TAI). Four hundred fifty-six suckled anestrous
Accepted 7 February 2011
Nelore beef cows at 30–60 days postpartum were assigned to treatments. At the first day
Available online 13 February 2011
of the estrous synchronization protocol (Day 0), all cows received a progesterone-releasing
intravaginal device plus 2 mg of estradiol benzoate. On Day 8, cows were assigned to blocks
Keywords:
according to the diameter of the largest follicle and then allocated to one of three treat-
FSH
Bos indicus ment groups (Control, FSH, or eCG) within each block. Simultaneously to progesterone
Ovulation device withdrawal on Day 8, cows in the eCG treatment group (n = 150) received 300 IU of
Follicular growth eCG and cows in FSH treatment group (n = 153) received 10 mg of FSH, and Control cows
Estrous synchronization (n = 153) did not receive any additional treatment. Additional treatments with 150 ␮g of
cloprostenol and 1 mg of estradiol cypionate (EC) were also administered concurrently to
progesterone device removal in all cows on Day 8. Two days later (D10), TAI and ovarian
ultrasonic examinations to evaluate follicle size were performed in all cows. On Day 12,
a subset of cows (n = 389) were submitted a second ultrasonic exam to confirm ovulation.
Final follicular growth (mm/day) was less (P = 0.006) in both Control (0.95 ± 0.11) and in
FSH-treated cows (0.90 ± 0.10) than in eCG-treated cows (1.40 ± 0.13). Interestingly, there
was a treatment-by-BCS interaction in ovulation results (P = 0.03), in which, eCG treatment
increased percentage of cows having ovulations with a lesser BCS. Similarly, there was a
treatment-by-BCS interaction for conception (P = 0.04), where the eCG treatment increased
fertility in cows with a lesser BCS. In conclusion, FSH failed to stimulate final follicular
growth, ovulation, and conception rate in sucked-anestrous beef cows submitted to TAI as
effectively as eCG. However, physiological effects of eCG seem to be more evident in cows
with a lesser BCS.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
The majority of cattle in tropical and subtropical
areas are of zebu (Bos indicus) origin. This is prob-
ably determined by several physiological advantages
∗ Corresponding author. Tel.: +55 11 3091 7674; fax: +55 11 3091 7920.
encountered in zebu breeds over Bos Taurus includ-
E-mail addresses: barusell@usp.br, znlogan@yahoo.com.br
ing greater resistance to environmental challenges (high
(P.S. Baruselli). temperature and humidity) and greater adaptation to
0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2011.02.007

2. J.N.S. Sales et al. / Animal Reproduction Science 124 (2011) 12–18 13
tropical grazing systems (based on seasonal grasses). March 2009. Multiparous suckled Nelore (B. indicus)
Regardless of these evolutional advantages in tropical cows (n = 533), at 30–60 days postpartum and aver-
climates, B. indicus cows have longer postpartum ane- age body condition score (BCS) of 2.81 ± 0.35 (1–5
strous periods, which together with other behavioral point scale, Ayres et al., 2009) were used. All cows
traits, leads to a lesser expression of estrus and poor were kept on pasture (Brachiaria brizantha), supple-
detection of estrus results (reviewed by Baruselli et al., mented with mineral salt and ad libitum access to
2004). water.
The adoption of artificial insemination (AI) using pro-
tocols combining progesterone and estrogens to improve 2.2. Experimental design
AI-submission rates normally produces consistent con-
ception results (∼50%) in beef herds. However, in some Postpartum anestrous cows received a progesterone-
physiological circumstances such as limited nutrition releasing intravaginal device (Sincrogest® , Ouro Fino,
(either insufficient and/or inadequate quality of feed), early Brazil) plus 2 mg of estradiol benzoate i.m. (Estrogin® , Far-
postpartum, deficient body condition scores, and when mavet, Brazil) on Day 0. On Day 8, 500 ␮g of cloprostenol
used in primiparous cows, timed AI protocols tend to i.m. (Sincrocio® , Ouro Fino, Brazil) and 1 mg of estradiol
produce inconsistent results (reviewed by Baruselli et al., cypionate i.m. (ECP® , Pfizer, Brazil) were administered
2008). In these situations, most of the available scientific concurrently to progesterone device removal. Simultane-
literature indicates that equine chorionic gonadotropin ously to these hormones given at progesterone device
(eCG) should be used at the time of progesterone-device removal (Day 8), cows were assigned to blocks accord-
removal. Treatment with eCG normally improves ovulation ing to the diameter of the largest follicle and allocated
and conception rates in beef cattle (Baruselli et al., 2004; to one of three treatment groups (Control, FSH, or eCG)
Bó et al., 2003; Macmillan and Peterson, 1993; Peres et al., within each block. Cows in the eCG-treated Group received
2009). 300 IU of eCG i.m. (Folligon® , Intervet, Netherland; n = 150),
Alternatively, FSH is another gonadotropin that can cows in the FSH-treated group received 10 mg of FSH
potentially be used to enhance follicular growth. The i.m. (Folltropin® , Bioniche, Canada; n = 153), and Control
underlying cellular mechanism by which FSH induces fol- group (n = 153) received no additional treatment. In all
licular development is mediated through greater numbers experimental groups, insemination was performed 48 h
of FSH receptors in granulosa cells (Fortune et al., 2001). after progesterone device removal. Frozen semen straws
Normally, porcine FSH is used in cattle undergoing super- from single ejaculates of each of the two sires were
ovulatory treatments in B. indicus (reviewed by Baruselli homogenously distributed among experimental groups.
et al., 2006) and in Bos taurus (reviewed by Bó et al., 2006; All inseminations were performed by the same tech-
Sales et al., 2008). More recently, FSH has been used in asso- nician who had no knowledge of the treatment group
ciation with timed AI protocols in non-superovulated cows of the cows at the time of insemination. Cows were
with inconsistent results. For example, Santos et al. (2007a) not exposed to bulls either before or during the exper-
observed that 10–20 mg of FSH can be used to increase fer- imental period. The experimental design is depicted in
tility to timed AI in B. indicus cows. Furthermore, Santos Fig. 1.
et al. (2007b) have shown that 10 mg of FSH supplemen-
tation at time of progesterone device removal had similar 2.3. Ultrasonic examinations
effects than eCG on percentage of cows having ovulations.
In contrast, Sá Filho et al. (2009) and Lima et al. (2010) found At the beginning of the estrous synchronization pro-
that FSH supplementation at progesterone device removal cedures (Day 0) ultrasonic examinations were performed
had lesser conception results following timed AI than cows (US; Chison 600 VET, 5 MHz linear transducer, China) to
receiving eCG and/or calf weaning management. Collec- evaluate ovarian status (presence of CL and follicles ≥8 mm
tively, this somewhat inconclusive scientific evidence of or <8 mm, Baruselli et al., 2004). At the first day of the
the potential benefits of FSH in timed AI procedures are estrous synchronization protocol, cows were also classified
herein used as a rationale for further investigations. according to their ovarian status in: 1 = largest follicle (LF)
Thus, the objective of the present study was to evaluate ≥8 mm and 2 = LF <8 mm in diameter. On Day 8, all cows
the effect of FSH supplementation in timed AI procedures were submitted to another US to measure the diameter of
in anestrus suckled B. indicus beef cows. The hypothesis the LF and then were reclassified to the following four cat-
was that FSH would produce similar effects to the pre- egories: (a) LF <8 mm, (b) LF between 8 mm and 10 mm, (c)
established positive results observed with the use of the LF between 10 mm and 12 mm, and (d) LF >12 mm. Then,
eCG, enhancing the final follicular growth, ovulation, and cows were distributed randomly among the experimental
conception rates in suckled B. indicus beef cows submitted groups (FSH-treated Group, eCG-treated Group or Control
to TAI. Group) these four categories of follicular diameter. Cows
with no visible CL structures on Day 0 and Day 8 were
2. Materials and methods considered in anestrous and then enrolled in the trial. Fur-
ther ultrasonic examinations were performed on Days 10
2.1. Animal management and 12 to measure the diameter of the LF and ovulation,
respectively. The final follicular growth rate was calcu-
The experiment was conducted in a commercial farm lated based on the difference in LF diameters between Days
located in northwestern Brazil from January through 8 and 10. Percentage of cows having ovulations (n = 389)

3. 14 J.N.S. Sales et al. / Animal Reproduction Science 124 (2011) 12–18
Fig. 1. Experimental design (Ø – diameter; US – ultrasonic examinations; BCS – body condition score; P4 – progesterone; EB – estradiol benzoate; EC –
estradiol cypionate).
were determined by the absence of the LF on Day 12 probability of P < 0.05 and tendencies were assumed when
previously detected on Day 10. Pregnancy exams were con- P < 0.10.
ducted by transrectal ultrasonic examination 30 days after
AI.
3. Results
2.4. Statistical analysis 3.1. Ovarian follicular dynamics
Statistical analyses were performed using Statisti- Cows with a CL at first (Day 0; n = 77; which represented
cal Analysis System for Windows (SAS, 2001). Models 14.5% of the cows) and second (Day 8; n = 7; which rep-
included ovary status on Day 0 (follicle <8 mm or resented 1.3% of the cows) ultrasonic examinations were
≥8 mm), treatment (Control, eCG, FSH), BCS (<2.75 or not enrolled in the trial. Thus, 456 cows (or 84.2% of the
≥2.75) and interactions. The effect of the diameter of cows) that did not have a CL were available for further anal-
the LF on Day 8 (x) on probability of ovulation (y) was yses. Out of 456 cows without a CL, 33.1% had an LF < 8 mm
detected. Logistic regression curves were created using (n = 151), and 66.9% had follicles ≥8 mm (n = 305) at the
the coefficients provided by Interactive Data Analysis from beginning of the estrous synchronization procedures. After
SAS and the formula y = exp (˛ × X + b)/[1 + exp (˛ × X + b)]. the randomized-block distribution of the cows between
Percentage of cows having ovulations and pregnancy groups, all experimental groups had similar follicular diam-
rates were analyzed by logistic regression with back- eters of LF (P = 0.94).
ward elimination (probability of 0.20) with the GLIMMIX Data in Table 1 indicate the average sizes for LF found
procedure of SAS. Normality of the residues was ver- on Days 8 and 10, as well as the follicular growth rate
ified in the follicle diameter data from Days 8 and in all groups. There was no interaction among treatment,
10, and analyzed by the UNIVARIATE procedure (trans- ovary status on Day 0 and BCS on the size of the LF
formed when necessary) and subjected to Bartlett’s test measured on the day of the timed AI. In addition, follic-
to assess homogeneity of variances. The GLM proce- ular growth rates between Days 8 and 10 differed among
dure with Tukey adjustment was then used to determine groups (P = 0.006), with eCG-treated cows having the great-
significant differences among groups. Non-parametric est follicle growth (1.40 mm/day) compared to cows in
data were analyzed using the NPAR1WAY procedure Control group (0.95 mm/day), or in the FSH-treated group
(Wilcoxon). Significant differences were indicated by a (0.90 mm/day).

4. J.N.S. Sales et al. / Animal Reproduction Science 124 (2011) 12–18 15
Fig. 2. Probability of ovulation in cows (n = 456) in non-gonadotropin sup- Fig. 3. Pregnancy per AI by treatment (Control, eCG, and FSH) in suckled-
plemented Control (solid line), treated with FSH (dotted line) and eCG anestrus Nelore beef cows having ovulations following the protocol for
(dashed line) according to the diameter of the largest follicle at proges- synchronization of ovulation for TAI; a,b Bars with different superscripts
terone device removal (Day 8). are different (P = 0.04).
3.2. Percentage of cows having ovulations
4. Discussion
Overall, ovulation response was 71.6% (96/134) in cows
Our main hypothesis was that FSH would produce sim-
in the Control group, 71.9% (95/132) in cows receiving FSH,
ilar effects to the pre-established positive results observed
and 88.6% (109/123) for cows receiving eCG. Interestingly,
with the use of the eCG, enhancing the final follicular
there was a treatment-by-BCS interaction (P = 0.02), and a
growth, ovulation, and ultimately conception rates in suck-
tendency for a treatment-by-follicle size on Day 0 (P = 0.08)
led B. indicus beef cows submitted to TAI. Thus, based on
interaction. As a result, there was a greater percentage
the results of both follicular dynamics and field concep-
of cows with BCS < 2.75 (Table 2) receiving eCG that had
tion rates the primary hypothesis in the present study is
ovulations than Controls and FSH-treated cows. The admin-
refuted. However, eCG produced consistent results on final
istration of eCG also increased the probability of ovulation
follicular stimulation, which was consistent with the field
at the end of the protocol when only small- or medium-
conception results achieved in the field trial. The apparent
sized LF (4–8 mm in diameter) were observed in the ovaries
beneficial results of eCG described herein are in agreement
at progesterone device removal (Fig. 2).
with other recent scientific literature. In addition, because
most published literature describes that gonadotropins are
3.3. Conception rate beneficial mostly in cows undergoing anestrus (Baruselli
et al., 2004), a focus of the present study was to use only
There was a treatment-by-BCS interaction (P = 0.04) on cows without CL at the beginning of the trial, so as to be
conception rate. Consequently, eCG treatment increased able to evaluate differing gonadotropins in a more targeted
conception rate in cows with a lesser BCS (<2.75) as com- population of cows. This allowed us to test and clearly mea-
pared with cows with a greater BCS (P = 0.0001; Table 2). In sure any possible positive effects of FSH that have been
contrast, there was no interaction between treatment and previously described.
follicle size on Day 0 (P = 0.51). In the analysis of the results Therefore, our main finding is that FSH supplementation
considering only cows having ovulations, treatment with failed to enhance follicular growth, ovulation, and con-
eCG improved conception results compared to FSH-treated ception results in beef cows receiving progesterone-based
cows (P = 0.04; Fig. 3). In addition, when considering only timed AI protocols. These results are in agreement with a
cows having ovulations, BCS did not seem to have any effect preliminary finding (Sá Filho et al., 2009), in which treat-
on conception (P = 0.43). ment with FSH did not increase conception rates in cows
Table 1
Effects of treatment (FSH, eCG, and Control) at progesterone device removal on diameter of the largest follicle in suckled-anestrous Nelore beef cows
submitted to timing artificial insemination (TAI).
Controlc FSH eCG P-value
Number of experimental units 153 153 150
Ø Largest follicle (mm)
At device removal (Day 8) 11.0 ± 0.3 11.0 ± 0.3 11.1 ± 0.2 0.94
At TAI (Day 10) 12.9 ± 0.3b 12.8 ± 0.3b 13.9 ± 0.2a 0.006
Follicular growth from Days 8 to 10 (mm/day) 0.95 ± 0.1b 0.90 ± 0.1b 1.40 ± 0.1a 0.006
a,b
Ø – diameter; Means within a row with different superscripts are different (P < 0.05).
c
No further treatment.

5. 16 J.N.S. Sales et al. / Animal Reproduction Science 124 (2011) 12–18
Effects of treatment (FSH, eCG, and Control) at progesterone device removal according to body condition score (BCS) class on percentage of cows having ovulations and pregnancy per AI in suckled-anestrus
exposed to TAI procedures. Lima et al. (2010) found sim-
ilar results, where cows treated with FSH (22.7%, 22/97)
Treat × BCS
had lesser conception rates compared to eCG-treated cows
(41.5%, 95/229; P < 0.05), but unfortunately this trial was
0.02
0.01
lacking a negative control as compared to the design in
the present study to independently measure FSH effects in
association with timed AI in beef cattle. In contrast, Santos
et al. (2007a,b) and Martins et al. (2010) described compa-
0.25
0.08
BCS
rable results of FSH to eCG. These inconsistent results could
be due the proportion of anestrous cows in each group, or
even due to differences in days post-partum at breeding. It
P-value
is widely known that FSH is a potent gonadotropin regu-
0.008
0.001
Treat
larly used to superovulate cattle in multiple ovulation and
embryo transfer (MOET) regimens. In addition, although
follicles are more dependent on LH after the process of devi-
ation (Ginther et al., 1996), previous research (Hampton
84.8 (67/79)
60.0 (57/95)
et al., 2004; Xu et al., 1995) has indicated presence of active
FSH receptors even in dominant follicles (>10 mm). Thus,
based on these scientific evidence, in addition to the fact
eCG
that only anestrous cows were used in the present study,
the working model indicated FSH would serve as a good
alternative to eCG in synchronization protocols for beef
cattle.
51.5 (53/103)
78.5 (73/93)
However, cows treated with FSH did not differ from
Control cows on any explanatory variables evaluated in the
present study. For instance, FSH produced similar follicu-
FSH
lar growth rates to Controls, and both were significantly
less than cows receiving eCG. Furthermore, greater follicle
48.5 (48/99)
78.0 (71/91)
BCS ≥2.75
growth rates observed in cows receiving eCG seem to be
Control
independent of the size of the LF on Day 8. This observation
could be explained by the longer half-life described for eCG
as well as its greater LH-like effects in early pharmacologi-
cal studies. In this regard, some studies have indicated that
FSH treatments can sustain active physiological concentra-
95.5a (42/44)
66.7a (34/51)
tions for only 16 h (Bodensteiner et al., 1996; Demoustier
et al., 1988), while eCG will maintain active concentrations
in bloodstream for up to 3 days (Murphy and Martinuk,
eCG
1991). This longer half-life observed in eCG could be one
of the underlying reasons why this compound was more
successful than FSH to stimulate follicular growth that
Means within a row with different superscripts are different (P < 0.05).
was observed in the current trial. Equivalent finding have
56.4b (22/39)
25.5b (12/47)
been described previously (Sá Filho et al., 2010a,b), where
Nelore beef cows submitted to timed artificial insemination (TAI).
heifers and cows treated with eCG had doubled follicu-
FSH
lar growth rates (eCG – 1.2 to 1.5 mm/day compared to
the Control – 0.5 to 0.6 mm/day). Another possible expla-
nation for this greater follicular stimulation promoted by
58.1b (25/43)
34.7b (17/49)
BCS <2.75
eCG could be related to its greater LH-like effects com-
Control
pared to purified porcine FSH. Early studies have indicated
a high affinity between eCG and ovarian cell receptors for
both FSH and LH (Licht et al., 1979; Murphy and Martinuk,
1991; Stewart and Allen, 1981). The role of LH on follic-
ular development and its close association with receptors
Percentage having ovulations
in granulosa cells have been well studied, and it appears
that LH will trigger a cascade of molecular reactions that
will produce catalytic enzymes responsible for steroid pro-
Pregnancy per AI (%)
duction and, thereby, final development and growth of
the dominant follicle (Carroll et al., 1992; Grummer and
Carroll, 1988). It is thought that eCG will induce a similar
cascade of reactions, because there is evidence that eCG
Table 2
increases estradiol production by increasing the amount
of mRNA for cytochrome P45017␣ (Soumano et al., 1996,
a,b

6. J.N.S. Sales et al. / Animal Reproduction Science 124 (2011) 12–18 17
1998). Thus, apparently eCG acts as a final gonadotropic percentage of cows having ovulations and conception rate
support leading to greater percentage of cows having ovu- in suckled-anestrous Nelore beef cows submitted to TAI.
lations (Macmillan and Peterson, 1993; Peres et al., 2009). Thus, administration of single dose of FSH at progesterone
This is confirmed by field results, which describe that eCG device withdrawal does not appear be a good alternative to
tends to be more effective in cows with compromised pro- eCG in progesterone-based estrous synchronization proto-
duction and/or with poor release of LH into bloodstream cols.
(i.e. at early postpartum stages or lesser BCS; Baruselli et al.,
2004; Sá Filho et al., 2010a,b). Thus, although FSH may be
Acknowledgements
a plausible alternative to eCG, its relative short-half life of
only 5 h appears to be a limiting factor when used only once
The authors thankful to DIMAFE Agropecuária (Jacare-
during TAI (Demoustier et al., 1988). Either multiple FSH
una Farm – São Felix do Araguaia, MT, Brazil) for allowing
treatments, which may not be very practical, or different
the use of their animals and facilities for this study. This
methods to maintain circulating FSH at greater concentra-
research was supported by Ourofino Agronegócio (Cravin-
tions might be necessary. A lesser purified FSH form (with
hos, SP, Brazil) and RG Genética Avancada (Água Boa, MT,
¸
more LH-like effects) may also be able to produce compara-
Brazil).
ble results to eCG in progesterone-based estrous synchrony
protocols for beef cattle.
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