Theriogenology 85 (2016) 792–799

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Theriogenology
journal homepage: www.theriojournal.com

Different doses of equine chorionic gonadotropin on

ovarian follicular growth and pregnancy rate of suckled Bos
taurus beef cows subjected to timed artificial insemination
protocol
G.A. Pessoa a, A.P. Martini a, G.W. Carloto b, M.C.C. Rodrigues c, I. Claro Júnior d,
P.S. Baruselli e, C.C. Brauner c, M.I.B. Rubin a, M.N. Corrêa c, F.G. Leivas b,
M.F. Sá Filho a, e, *
a
Embryolab - Laboratório de Embriologia Animal, Departamento de Clínica de Grandes Animais-DCGA, Centro de Ciências Rurais –
CCR, Universidade Federal Santa Maria (UFSM), Santa Maria RS, Brazil
b
Laboratório de Biotecnologia da Reprodução (Biotech), Universidade Federal do Pampa, Uruguaiana, Rio Grande do Sul, Brazil
c
Núcleo de Pesquisa, Ensino e Extensão em Pecuária (NUPEEC), UFPel, Pelotas, Rio Grande do Sul, Brazil
d
Zoetis Indústria de Produtos Veterinários Ltda, São Paulo, São Paulo, Brazil
e
Departamento de Reprodução Animal (VRA), FMVZ/USP, São Paulo, São Paulo, Brazil

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

Article history: This study evaluated the effect of different doses of eCG (control, 300 or 400 IU) administered
Received 17 June 2015 at progesterone (P4) device removal in suckled Bos taurus beef cows undergoing a timed
Received in revised form 10 September 2015 artificial insemination (TAI) protocol. A total of 966 cows received a P4 insert and 2.0 mg
Accepted 14 September 2015
intramuscular estradiol benzoate at the onset of the synchronization. After 9 days, P4 insert
was removed, and 12.5 mg of dinoprost tromethamine and 1 mg of estradiol cypionate were
Keywords:
administered, followed by TAI 48 hours later. Then, the cows received one of three treatments
Bovine
as follows: control (n ¼ 323), 300 (n ¼ 326), or 400 IU of eCG (n ¼ 317). A subset (n ¼ 435) of
eCG
Fixed-time artificial insemination cows in anestrus had their ovaries evaluated using ultrasound at the time of P4 removal and at
Dominant follicle TAI. Data were analyzed by orthogonal contrasts (C): C1 (eCG effect) and C2 (eCG dose effect).
Reproduction Estrous occurrence (control ¼ 53.7%, 300 IU ¼ 70.6%, and 400 IU ¼ 77.0%) and pregnancy per
artificial insemination (control ¼ 29.7%, 300 IU ¼ 44.8%, and 400 IU ¼ 47.6%) were improved
by eCG treatment (C1; P ¼ 0.0004 and P < 0.0001, respectively). Furthermore, the
cows receiving eCG presented larger follicles at TAI (control ¼ 13.5  0.3 mm, 300
IU ¼ 14.0  0.2 mm, and 400 IU ¼ 15.1  0.3 mm; P < 0.0001; C1). However, there was no effect
of eCG dose on any response variables studied (C2; P > 0.15). In conclusion, the eCG treatment
administered at the time of P4 removal increased the occurrence of estrus, the larger follicles
at TAI, and pregnancy per artificial insemination of suckled B taurus beef cows. Despite the
greater occurrence of estrus in noncyclic cows receiving 400 IU of eCG, both eCG doses
(300 and 400 IU) were equally efficient to improve pregnancy to artificial insemination.
Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction (BS) are critical for herd profitability. As a result, repro-

ductive programs, such as synchronization of ovulation for
In pasture-based cow–calf production systems, high timed artificial insemination (TAI), have been increasingly
pregnancy rates at the beginning of the breeding season incorporated in cow–calf operations focusing on enhancing
pregnancy rates at the beginning of the BS [1–5]. Among
* Corresponding author. Tel./fax: þ55 5532208501. the available hormonal therapies that allow the use of
E-mail address: [email protected] (M.F. Sá Filho). TAI, the estradiol (E2)-plus-progesterone (P4)–based

0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2015.09.057
 G.A. Pessoa et al. / Theriogenology 85 (2016) 792–799 793

synchronization protocols have been successfully used to synchronized with a GnRH-plus-PGF2a–based TAI proto-
control follicular dynamics and luteal life span to syn- col. Therefore, because of the relative high cost of the eCG
chronize ovulation, enabling the TAI [3,6,7]. Currently, treatment in relation to the cost of the entire TAI protocol,
these P4-plus-E2–based synchronization protocols are finding the minimal effective dose of eCG for suckled B
widely used and represent the main commercial hormonal taurus beef cows could help reduce costs of TAI programs
treatment for TAI of postpartum beef cows in South and deserves further investigation.
America [1–5]. Thus, the aims of the present study were twofold: (1) to
Postpartum anestrus beef cows have insufficient pul- evaluate the impact of eCG treatment administrated at P4
satile release of LH to support the final stages of ovarian removal and (2) to verify the effect of decreasing the eCG
follicular development and ovulation [8], limiting the effi- dose from 400 to 300 IU on ovarian follicular growth and
ciency of TAI programs [5,7,9]. Exogenous P4 increases LH pregnancy results in suckled B taurus beef cows. In addition,
pulse frequency during and after treatment, inducing the present study was designed to evaluate the impact of
ovulation [10]. However, despite progestin-induced post- occurrence of estrus and the ovarian follicle size at TAI on the
partum cyclicity, there are some situations with high inci- likelihood of pregnancy of synchronized cows. Our hypoth-
dence of anestrus, typically observed in grazing suckled eses were that, in suckled B taurus beef cows submitted to a
beef herds, so that the efficiency of such treatment could be P4-plus-E2–based TAI protocol, (1) eCG increases the rate of
compromised [1,11]. Thus, gonadotropins, such as the eCG, final dominant follicle growth, the occurrence of estrus, and
have been included in the synchronization protocols for TAI P/AI and (2) the reduction of the eCG dose from 400 to 300 IU
aiming to improve ovulation and pregnancy rates [3]. decreases its positive effect on studied variables.
The eCG glycoprotein is a long half-life molecule pro-
duced by the cells of the endometrial cups in pregnant mares 2. Materials and methods
[12]. It has both FSH- and LH-like activity owing to its ability
to bind to both FSH and LH receptors, located at the granulosa 2.1. Herd and animal management
and theca cells [13], stimulating ovarian follicular growth and
ovulation in cattle [12]. The administration of eCG at P4 This experiment was conducted during the 2014/2015
removal is an efficient alternative to increase ovulatory spring–summer BS at five commercial beef farms located in
response and pregnancy rates after TAI programs in Bos three distinguished regions of Rio Grande do Sul State,
indicus [4,9,14,15] and Bos taurus [16–18] suckled beef cows. Brazil. A total of 966 suckled B taurus beef cows were
The traditional dose of eCG used for both B indicus and B enrolled in the study. On the first day of the synchronization
taurus suckled beef cows is 400 IU [9,16–19]. However, protocol, the body condition score (BCS) was 2.77  0.02 on
recent reports indicate that, at least for suckled B indicus the basis of a 1- to 5-point scale (1 ¼ emaciated, 5 ¼ obese)
beef cows, the dose of 400 IU of eCG could be reduced to [24]. The cows were maintained in a native pasture, with
300 IU without compromising pregnancy results [4]. In free access to water and mineralized salt. All animal
addition, other studies reported the satisfactory efficiency handling procedures in this trial followed general guidelines
of the administration of 300 IU of eCG on the improvement for animal welfare. Information regarding the cow breed (B
of the final dominant follicle growth, ovulatory and preg- taurus or crossbreed), parity (primiparous or multiparous),
nancy outcomes of suckled B indicus beef cows [14,15]. BCS at the beginning of the TAI protocol, and breeding group
However, B indicus cows have subtle differences in their within each farm location is shown in Table 1.
reproductive physiology, such as greater sensitivity to
exogenous gonadotropins, compared to B taurus breeds 2.2. Reproductive management and experimental design
[20–22]. Marquezini et al. [23] failed to find any improve-
ment on pregnancy per artificial insemination (P/AI) after Suckled beef cows, between 40 and 70 days postpartum,
administering 200 IU of eCG in B taurus suckled beef cows were blocked by parity and BCS, and within each block,

Table 1
Descriptive analyses according to farm location.

Regions of state Farms Breeding No. of Multiparous, % (n) Crossbreeds, % (n)a Mean BCS Cyclicity, % (n) Estrus, % (n)b P/AI, % (n)c
groups cows (1–5 scale)
Southwest A 1 57 35.1 (20) 47.4 (27) 2.61  0.02 17.5 (10) 63.2 (36) 29.8 (17)
B 2 146 47.3 (69) 100 (146) 3.10  0.07 14.0 (6) 60.3 (88) 37.7 (55)
C 3 145 72.4 (105) 100 (145) 2.92  0.03 52.4 (76) 33.1 (48)
Center-west D 4 132 100 (132) 22.7 (30) 2.61  0.03 20.5 (27) 75.8 (100) 52.3 (69)
5 91 100 (91) 22 (20) 2.69  0.03 18.7 (17) 62.6 (57) 55.0 (50)
6 122 100 (122) 18.9 (23) 2.82  0.03 24.6 (30) 60.7 (74) 41.8 (51)
7 82 100 (82) 0 (0) 2.83  0.03 31.7 (26) 70.7 (58) 53.7 (44)
Southeast E 8 66 78.8 (52) 100 (66) 2.92  0.04 28.8 (19) 77.3 (51) 24.4 (16)
9 71 67.6 (48) 98.6 (70) 3.10  0.04 9.9 (7) 81.7 (58) 32.4 (23)
10 54 79.6 (43) 100 (54) 2.94  0.04 18.5 (10) 90.7 (49) 37.0 (20)
Overall 966 79.1 (764) 60.1 (581) 2.82  0.01 21.2 (152) 67.0 (647) 40.7 (393)

Abbreviations: AI, artificial insemination; BCS, body condition score.

a
Proportion of crossbred cows.
b
Occurrence of estrus between progesterone insert removal and timed artificial insemination (TAI).
c
Pregnancy per AI (P/AI) 30 days after TAI.
794 G.A. Pessoa et al. / Theriogenology 85 (2016) 792–799

they were randomly assigned into one of three treatments by visualization of an embryo with heartbeat, and preg-
according to the dose of eCG (control untreated [n ¼ 323], nancy rate was defined as the number of pregnant cows
300 IU [n ¼ 326], or 400 IU [n ¼ 317]) administered at P4 divided by the number of cows submitted to TAI in each
insert removal of the synchronization protocol (Fig. 1). The treatment.
synchronization protocol consisted of the insertion of an
controlled internal drug releasing device (CIDR, 1.9 g of P4; 2.4. Detection of estrus
Zoetis, Campinas, São Paulo, Brazil) and 2 mg of intra-
muscular (IM) estradiol benzoate (Gonadiol; Zoetis). On The cows had their tailheads marked with chalk
Day 9, the P4 device was removed, and 12.5 mg of dinoprost (Raidl-Maxi; RAIDEX GmbH, Dettingen/Erms, Germany) at
tromethamine IM (Lutalyse; Zoetis) plus 1 mg of estradiol the removal of the P4 insert (Day 9). The occurrence of
cypionate IM (Zoetis) were administered to all animals. The estrus was evaluated at TAI and was determined on the
cows were timed artificially inseminated 48 hours later. basis of the removal of the tailhead mark. Estrus was
Semen from 15 different sires within each farm location deemed to have occurred in cattle without a tailhead
was collected during TAI. All batches from each bull were chalk mark at TAI.
analyzed. Only batches with sperm quality features
exceeding the minimum requirements of the Brazilian 2.5. Statistical analysis
College of Animal Reproduction, namely 35% progressive
sperm motility and a maximum of 20% total abnormal Statistical analysis was performed using the GLIMMIX
sperm cells [25] after thawing, were used. procedure of SAS (SAS 9.3; 2003; SAS Institute Inc., Cary,
NC, USA) for Windows. Continuous variables were analyzed
by ANOVA using the GLIMMIX procedure fitted to normal
2.3. Ultrasound examination
distribution. Categorical data were analyzed by logistic
regression with models fitted to binary distribution. The
At the insertion (Day 0) and at removal (Day 9) of the P4
Kenward–Roger method was used to calculate the domi-
insert, transrectal ultrasound examinations (Mindray DP
nator degrees of freedom to approximate the F tests in the
220 with 5-MHz linear array transducer, Shenzhen, China)
mixed models.
were performed in a subset of cows (n ¼ 718) to evaluate
The occurrence of estrus and P/AI were analyzed using
the presence of a CL. The cows without a visible CL on Days
the GLIMMIX procedure of SAS. The variables that were
0 and 9 were considered in anestrus and were enrolled in
initially included in the models were the treatments
the ovarian response evaluation. A subset of anestrus cows
(control, 300 IU, or 400 IU), farm location (1–5), cow breed
(n ¼ 435) were also submitted to additional ultrasound
(B taurus or crossbreed) and BCS at the first day of the syn-
examinations: at P4 insert removal (Day 9) and at Day 11
chronization protocol (categorized as 2.75 or >2.75), and
(immediately before the TAI) to measure the diameter of
their interactions. For the analysis of P/AI, the bull sire within
the largest follicle (LF). The final follicular daily growth rate
each breeding group in each farm location was included as a
was calculated on the basis of the difference in LF diameters
random effect. For the final logistic regression model, vari-
between Days 9 and 11 divided by two.
ables were removed through backward elimination on the
Pregnancy diagnosis was performed by transrectal
basis of the Wald statistics criterion when P > 0.20.
ultrasound 30 days after TAI. Pregnancy was characterized
In the subset of cows (n ¼ 435) subjected to ovarian
response evaluation by ultrasound, continuous variables
Control (n = 323 suckled cows) (i.e., diameter of the LF at P4 insert removal, diameter of the
12.5mg dinoprost
LF at TAI, and LF daily growth rate) were analyzed by
2mg EB 1mg E.C.P US
TAI
ANOVA using the GLIMMIX procedure of SAS. The cows
P4 device
within each breeding group were considered as a random
48 h
effect. Response variables were tested according to their
300 IU eCG (n = 326 suckled cows)
homogeneity and normality of variances using Guide Data
300 IU
12.5mg dinoprost
Analysis from SAS and transformed if necessary to reach
2mg EB 1mg E.C.P US the normality. In addition, a second analysis (n ¼ 718)
TAI
regarding the interaction between cyclic status (presence
P4 device 48 h
of CL at P4 insert insertion and/or removal) and treatment
on occurrence of estrus and on P/AI were performed.
400 IU eCG (n = 317 suckled cows)
400 IU The data were analyzed by orthogonal contrast to
12.5mg dinoprost
2mg EB 1mg E.C.P US
determine significant differences among the groups. The
TAI contrasts established were: C1 (eCG effect): control versus
P4 device
48 h 300 þ 400 IU of eCG and C2 (effect of eCG dose): 300-
versus 400-IU eCG.
Day 0 9 11 41 In addition, the effect of the diameter of the LF on Day 11
on probability of estrus occurrence and P/AI was analyzed.
Fig. 1. Experimental design. EB ¼ 2 mg of estradiol benzoate; ECP ¼ 1 mg of The model included the fixed effects of treatment, cow
estradiol cypionate; dinoprost ¼ 12.5 mg of dinoprost tromethamine;
TAI ¼ timed artificial insemination performed 48 hours after removal of the
breed (B taurus or crossbreed), LF diameter at Day 11 as
progesterone (P4) insert; US ¼ ultrasound examinations (pregnancy linear or quadratic term and the random effect of the
diagnosis). breeding group within each farm location. Logistic
 G.A. Pessoa et al. / Theriogenology 85 (2016) 792–799 795

Table 2
Effect of dose of eCG on reproductive responses after the synchronization of estrus and ovulation in Bos taurus suckled beef cows.

Item Treatmentsa P values (contrasts)b

Control 300 IU 400 IU eCG treatment eCG dose

No. of animals 323 326 317 d d
BCS (1–5 scale)c 2.8  0.02 2.8  0.02 2.8  0.02 0.88 0.88
Ovarian follicle responses (subset of 435 cows)
No. of animals 146 153 136 d d
LF diameter at P4 insert removal, mmd 11.0  0.2 10.7  0.2 11.0  0.2 0.32 0.38
Growth rate of the LF, mm/day 1.2  0.1 1.6  0.1 2.1  0.1 <0.0001 0.33
LF diameter at TAI, mm 13.5  0.3 14.0  0.2 15.1  0.3 0.04 0.19
Estrus and pregnancy responses (all cows)
Occurrence of estrus, %e 53.7 70.6 77.0 0.0004 0.58
P/AI, % 29.7 44.8 47.6 <0.0001 0.55

Abbreviations: P4, progesterone; P/AI, pregnancy per artificial insemination; TAI, timed artificial insemination.
a
Cows received a P4 insert and 2 mg of intramuscular estradiol benzoate on the first day of the protocol for synchronization. The insert was removed
9 days after, and cows were received 1 mg of estradiol cypionate and eCG treatment with 0, 300, or 400 IU.
b
The orthogonal contrasts established were (1) eCG effect: control versus 300 þ 400 IU of eCG and (2) effect of eCG dose: 300- versus 400-IU eCG.
c
Body condition score evaluated at the onset of the synchronization protocol.
d
LF ¼ largest follicle diameter.
e
Occurrence of estrus ¼ percentage of cows that displayed estrus between P4 insert removal and the TAI.

regression curves were created using the coefficients pro- TAI that combined the highest sensitivity and specificity for
vided by Interactive Data Analysis from SAS and the for- P/AI was 15.0 mm. The area under the curve was 66.1 (95%
mula, y ¼ exp (˛X þ b)/[1 þ exp(˛X þ b)]. Also, receiver confidence interval was 0.661–0.780; P < 0.001). The dis-
operating characteristic (ROC) analysis using MedCalc tribution of cows according to the LF diameter on the day of
version 9.5.1.0 (MedCalc software, Mariakerke, Belgium) TAI was 40.9% (15.0 mm) and 59.1% (>15.0 mm), defined
was performed to determine the critical LF diameter at TAI according to the ROC curve. The cows presenting LF greater
to optimize the P/AI on the basis of sensitivity and than 15.0 mm at TAI presented greater occurrence of estrus
specificity. and greater P/AI (Table 3). Linear relationships were
An additional retrospective analysis, regarding the observed between the LF diameter at TAI and the proba-
impact of occurrence of estrus between the P4 insert bility of occurrence of estrus (P < 0.0001) and P/AI
removal and TAI, was performed. Information from indi- (P < 0.0001). There were no quadratic relationships be-
vidual cows was compiled into a single data set for statis- tween the LF diameter at TAI and occurrence of estrus
tical analysis. Data were analyzed by logistic regression (P ¼ 0.19) or P/AI (P ¼ 0.30). Thus, as the diameter of the LF
using the GLIMMIX procedure. at the TAI increased, the occurrence of estrus and the P/AI
Means (standard error) and percentages were used to on Day 30 after TAI also increased (Fig. 3).
describe all the response variables. Differences with
P  0.10 were considered statistically significant. 100 Treatment * Status (P = 0.06)
a Control
a
a 87.5 300 IU eCG
85.4
3. Results 80.2 400 IU eCG
b
80
72.6
3.1. Effect of treatment on ovarian response, occurrence of c Treatment * Status (P = 0.05)

estrus, and P/AI 59.9

60 a 55.4 54.2
b 52.1
49.7
48.2 a
%

Similar BCS at Day 0 (P ¼ 0.88) was observed among the 41.6

treatment groups (Table 2). Similar diameter of the LF at P4 40
b
insert removal (P > 0.05) was found; however, eCG-treated 25.8
cows presented greater follicular growth and larger diam-
20
eter of the LF at TAI (Table 2). The cows treated with eCG
exhibited a greater occurrence of estrus and P/AI (Table 2). 182 197 187 56 48 48 182 197 187 56 48 48

No differences between eCG doses were observed on any 0

response variables. In the subset of cows that had their Non-cyclic Cyclic Non-cyclic Cyclic

cyclic status evaluated, an interaction between cyclic status Occurrence of estrus P/AI
and treatment was observed on estrus occurrence
(P ¼ 0.06) and P/AI (P ¼ 0.05; Fig. 2). Fig. 2. Occurrence of estrus and pregnancy per artificial insemination (P/AI)
in suckled Bos taurus beef cows (subset of cows; n ¼ 718) according to
treatment with different doses of eCG (control, 300 IU, or 400 IU) and the
3.2. Impact of ovarian follicular diameter at TAI and the estrus cyclic status evaluated by ultrasound (presence of CL at insertion and/or
occurrence on ovarian and pregnancy response removal of P4 insert). There was an interaction (P ¼ 0.06) between treat-
ment and cyclic status on occurrence of estrus. Significant interaction
(P ¼ 0.05) between treatment and cyclic status on P/AI was found. Bars
The average diameter of the LF at TAI was 14.2  0.2 mm. without a common letter differ. Numbers in boxes indicate the number of
The ROC analysis indicated that the critical LF diameter at cows.
796 G.A. Pessoa et al. / Theriogenology 85 (2016) 792–799

Table 3 Table 4
Largest follicle (LF) diameter at timed artificial insemination (TAI) on Impact of estrus occurrence between progesterone (P4) insert removal
occurrence of estrus and pregnancy per artificial insemination (P/AI) of and timed artificial insemination (TAI) on ovarian follicular growth (subset
suckled noncyclic Bos taurus cows. of 435 noncyclic cows) and pregnancy per artificial insemination (P/AI; all
966 cows) of suckled Bos taurus beef cows subjected to TAI.
Item LF diameter at TAI P value
Item Occurrence of estrus P value
15 mm >15 mm
No. of animals 178 257 d No estrus Estrus
LF diameter at TAI, mm 12.5  0.2 17.0  0.2 <0.001 No. of animals 319 647 d
Occurrence of estrus, % 63.0 84.8 <0.001 LF diameter at P4 insert 9.8  0.1 11.3  0.1 <0.0001
P/AI, % 22.6 53.9 <0.001 removal, mma
Growth rate of the LF, mm/daya 13.0  0.2 14.7  0.2 <0.0001
LF diameter at TAI, mma 1.6  0.1 1.7  0.1 0.03
P/AI, % 24.8 48.5 <0.001
Estrus was detected in 67% of the cows at TAI. The cows
Abbreviation: LF, largest follicle.
that expressed estrus had greater (P < 0.001) LF diameter at a
Only evaluated in the subset of 435 suckled noncyclic beef cows.
P4 insert removal and TAI and also increased P/AI than the
cows that did not express estrus (Table 4).
rates when administered in suckled zebu beef cows
4. Discussion [4,9,11,15,19]. However, eCG treatment in suckled beef cows
subjected to TAI has been more effective to increase
This study demonstrates the positive effect of the follicular development and pregnancy rates in herds
administration of eCG at P4 removal on ovarian follicular exhibiting high prevalence of anestrus cows or in cows
growth and pregnancy rate of suckled B taurus beef cows presenting a low BCS at the onset of the synchronization
subjected to an E2-plus-P4–based TAI protocol. Cows program [4,9,11,15]. This particular effect may be explained
treated with eCG presented greater dominant follicle by the fact that the incidence of anestrus due to insufficient
growth from P4 insert removal to TAI and consequently LH pulses is a common problem in suckled beef cows under
increasing LF at TAI. Also, eCG-treated cows displayed more grazing conditions [8,27]. Thus, the positive effects of eCG
estrus and had greater P/AI. Nonetheless, the improvement found in the present study could be explained by the high
on pregnancy restricted to anestrus cows demonstrates incidence of anestrus (w79%) and relatively low BCSs at the
that this synchronization of the ovulation protocol may onset of the synchronization protocol (w2.8; 1–5 scale).
become an important tool to increase fertility, especially in No differences were found after decreasing the dose of
suckled anestrus beef cows. Furthermore, no differences eCG from 400 to 300 IU on ovarian follicle growth, estrus
were found on ovarian follicular dynamics or P/AI with occurrence rate, and P/AI when all cows were analyzed.
decreasing the dose of eCG from 400 to 300 IU. In addition, Interestingly, despite similar P/AI, noncyclic cows treated
regardless of the treatment, the increased diameter of the with 300 IU of eCG presented lower occurrence of estrus
LF at TAI was positively associated with the increased after the synchronization protocol than cows treated with
occurrence of estrus and P/AI. 400 IU. Previous studies clearly reported that synchronized
The eCG treatment increased final follicular develop- cows displaying estrus before TAI exhibited larger domi-
ment, follicular diameter at TAI, and ovulation rate in B nant follicles, greater E2 concentration during the
indicus cows submitted to TAI [4,9,14,15], and consequently, proestrus, greater luteal function on the subsequent estrus
cows with a larger dominant follicle at TAI had an increased cycle, and greater P/AI than cows that did not display estrus
probability of ovulation after the synchronization protocol [28–32]. In the present study, despite the lower
[15,26]. Moreover, eCG treatment also increased pregnancy estrus occurrence, noncyclic cows treated with 300 IU

A B
100 100
Probability of estrus occurrence, %

Probability of pregnancy per AI, %

90
80
80

60
70

60
40

50
20
40

30 0
4 6 8 10 12 14 16 18 20 22 24 26 4 6 8 10 12 14 16 18 20 22 24 26
Largest follicle at TAI, mm Largest follicle at TAI, mm

Fig. 3. Probability of occurrence of estrus (panel A) and pregnancy per artificial insemination (P/AI; panel B) on Day 30 after timed artificial insemination (TAI) in
suckled noncyclic Bos taurus cows (n ¼ 435) according to the diameter of the largest follicle (LF) at TAI, regardless of the eCG treatment (all treatment groups).
Probability of occurrence of estrus {exp[1.5503 þ 0.1802  LF at TAI/1 þ exp(1.5503 þ 0.1802  LF at TAI); P < 0.0001]} and probability of P/AI {exp
[3.4719 þ 0.1985  LF at TAI/1 þ exp(3.4719 þ 0.1985  LF at TAI); P < 0.0001]}.
 G.A. Pessoa et al. / Theriogenology 85 (2016) 792–799 797

of eCG presented similar P/AI to cows treated with 400 IU of E2 on the hypothalamus [42], and E2 concentration is
eCG. These contrasting findings on the impact on P/AI could directly related to the diameter of the ovulatory follicle
be explained by other important underlying physiological [30]. Also, a positive relationship has been observed be-
factors such as severity of postpartum anestrus, metabolic tween the concentration of E2 in circulation and the
status of the cow, or even changes in BCS during the duration and/or intensity of estrus [43,44]. Therefore,
postpartum period [10,33,34]. Similarly, it was observed greater estrus occurrence in cows receiving eCG at P4 insert
that the 400-IU eCG dose had a positive influence on estrus removal, compared to nontreated cows, could be related to
occurrence even on cyclic cows. Altogether, decreasing eCG the greater E2 concentration and greater number of cows
dose from 400 to 300 IU in E2-plus-P4–based synchroni- reaching the threshold of estrus occurrence.
zation protocols did not appear to have negative effects on Previous exposure to P4 before estrus, after greater
P/AI results. concentrations of E2 during the proestrus phase and,
In addition, earlier reports indicate that, at least for finally, a greater concentration of P4 during the estrous
suckled B indicus beef cows, the traditional dose of 400 IU cycle subsequent to the artificial insemination, is important
of eCG could be reduced to 300 IU without compromising to the establishment of pregnancy [45,46]. Proestrus E2
pregnancy outcomes [4]. However, this reduction was concentration plays an important role in sperm transport
supported by greater sensitivity of B indicus to exogenous and viability, in modulation of the uterus for the subse-
gonadotropins compared to B taurus breeds [20–22]. In a quent luteal phase [47,48], and in pregnancy establishment
recent study [23], the authors failed to find improvement success [36]. In addition, gene expression of bovine endo-
on the P/AI after the use of 200 IU of eCG in B taurus suckled metrium changes according to the phase of the estrous
cows synchronized with a GnRH-plus-PGF2a–based TAI cycle, and it is closely controlled by circulating concentra-
protocol. These later results might indicate that the effect of tions of E2 and P4 and the ratio between these steroid re-
decreasing the eCG dose may vary according to the type of ceptors in target tissues [47,49]. In suckled beef cows, high
the synchronization protocol used. Further research is concentrations of E2 during the proestrus induce changes
required to confirm this hypothesis. in the uterus throughout the subsequent estrous cycle
In this study, cows having an LF diameter at TAI pre- regarding messenger RNA expression for E2 and P4 re-
sented greater occurrence of estrus and greater P/AI on Day ceptors (ERS1 and RMP, respectively), oxytocin receptors,
30 after TAI. Optimization of follicle size and health has and a variety of proteins such as cyclooxygenase-2 and beta
been an important objective in current reproductive pro- subunit inhibin serpin-14 [50]. These changes are reduced
grams, especially in TAI [35]. In accordance with this when females have lower concentrations of E2 during the
statement, females presenting larger ovulatory follicles proestrus [47,50]. Furthermore, beef heifers exposed to a
exhibited a greater ovulation rate, improved endogenous longer exposure of E2 during the proestrus presented al-
E2 production, larger CL diameter, greater concentration of terations in the pattern of receptor expression and other
P4 in the subsequent estrous cycle, and greater P/AI in beef proteins associated with the uterine receptivity to preg-
cattle [14,29,36]. Nevertheless, when beef cattle are syn- nancy [51]. Therefore, the positive effect of the eCG treat-
chronized with GnRH-plus-PGF2a–based TAI protocols or ment on the occurrence of estrus and on pregnancy rate
when lactating dairy cows are synchronized with E2-plus- could be related to the greater E2 concentration during
P4- or GnRH-plus-PGF2a–based protocols, there is proestrus, determined by greater final follicular growth,
frequently a point where follicle size reaches a maximum larger dominant follicle at TAI, resulting in a greater E2
beyond which fertility declines [30,37,38]. In these partic- priming during the synchronized proestrus.
ular situations, cows that fail have a new follicular wave In conclusion, eCG treatment administered at P4
emergence after the onset of the synchronization protocol removal increases the occurrence of estrus, the diameter of
may produce larger ovulatory follicles; however, a longer the LF at TAI, and P/AI in suckled B taurus beef cows. Both
period of dominance can also lead to an aged oocyte eCG doses (300 and 400 IU) were equally efficient to
resulting in drastic reductions in fertility [39–41]. There- improve pregnancy results, although the 400 IU eCG dose
fore, the results from the present study suggest that the induced greater estrus occurrence in noncyclic cows. In
E2-plus-P4 treatment used at the beginning of the addition, the increased diameter of the LF at TAI was
synchronization protocol was efficient to induce a new positively associated with the increased occurrence of
follicular wave emergence and, consequently, promoting estrus and P/AI, and these results were independent from
the formation of a larger follicle bearing a healthy oocyte. the treatment group. As expected, suckled cows that dis-
As a result, we observed a linear positive association played estrus had a larger follicle at TAI and greater P/AI in
between the development of the LF diameter and the P/AI comparison to cows that did not display estrus.
in suckled B taurus beef cows.
Synchronized females displaying estrus before TAI Acknowledgments
exhibited a larger dominant follicle and greater P/AI when
compared to females that did not display estrus [28–31]. In Zoetis Animal Health, Brazil, donated all hormonal
agreement with these studies, the present results showed products applied in this trial. The authors would like to
that suckled B taurus cows receiving eCG treatment thank Normelio Alves and Mauro Meneghetti from Zoetis
regardless of the dose, in E2-plus P4–based TAI protocols, for the support in this research. They also thank the Agro-
had a greater proportion of cows displaying estrus, allow- pecuária Odair Gonzáles, Fazenda Dois Angicos e Estância
ing greater pregnancy rate after TAI than non-eCG–treated Nova Aurora (Uruguaiana, Rio Grande do Sul, Brazil),
cows. Estrus behavior is modulated by the direct action of Cabanha da Aguada (Pinheiro Machado, Rio Grande do Sul,
798 G.A. Pessoa et al. / Theriogenology 85 (2016) 792–799

Brazil), and Fazenda Nova EsperançadAgropecuária Posto benzoate and equine chorionic gonadotropin (eCG). Annual Meeting
International Embryo Transfer Society. Auckland, Nueva Zelanda:
Branco (Júlio de Castilhos, Rio Grande do Sul, Brazil) for
Theriogenology; 2003. p. 216.
allowing the use of their animals and facilities for this [18] Small JA, Colazo MG, Kastelic JP, Mapletoft RJ. Effects of pro-
study. They would like to thank Professor Daniela S. Brum gesterone presynchronization and eCG on pregnancy rates to
from Universidade Federal do Pampa for the analysis of the GnRH-based, timed-AI in beef cattle. Theriogenology 2009;71:
698–706.
semen used in the trial. Also, they are grateful to Alex H. [19] Penteado L, Ayres H, Madureira EH, Reis EL, Baruselli PS. Efeito do
Souza from Ceva Santé Animale and Julia G. Soares and eCG e do desmame temporário na taxa de prenhez de vacas
MSc. Bruno Freitas from Universidade de São Paulo (USP) Nelore lactantes inseminadas em tempo fixo. XVIII Reunião Anual
da Sociedade Brasileira de Transferência de Embriões (SBTE).
for the English editing. Barra Bonita, São Paulo, Brazil. Acta Veterinariae Scientiae 2004;
32:223.
[20] Baruselli PS, Sá Filho MF, Martins CM, Nasser LF, Nogueira MFG,
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