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Effects of equine chorionic gonadotropin and type of ovulatory

stimulus in a timed-AI protocol on reproductive responses

in dairy cows

A.H. Souza a, S. Viechnieski b, F.A. Lima c, F.F. Silva d, R. Araujo a,G.A. Bo e, M.C. Wiltbank f, P.S. Baruselli a,*

a Department of Animal Reproduction, FMVZ-USP, Brazilb Iguacu Farm-StarMilk, Cascavel, PR, Brazil

c Veterinary Policlinic-Pioneiros, Carambeı, PR, Brazild Department of Informatics, UFV, Brazil

e Institute of Animal Reproduction-Cordoba, IRAC, Cordoba, Argentinaf Department of Dairy Science, UW-Madison, Madison, WI, USA

Received 1 July 2008; received in revised form 1 December 2008; accepted 20 December 2008

Abstract

The objectives were to evaluate the effects of equine chorionic gonadotropin (eCG) supplementation (with or without eCG) and

type of ovulatory stimulus (GnRH or ECP) on ovarian follicular dynamics, luteal function, and pregnancies per AI (P/AI) in

Holstein cows receiving timed artificial insemination (TAI). On Day 0, 742 cows in a total of 782 breedings, received 2 mg of

estradiol benzoate (EB) and one intravaginal progesterone (P4) insert (CIDR). On Day 8, the CIDR was removed, and all cows were

given PGF2a and assigned to one of four treatments in a 2 � 2 factorial arrangement: (1) CG: GnRH 48 h later; (2) CE: ECP; (3)

EG: eCG + GnRH 48 h later; (4) EE: eCG + ECP. There were significant interactions for eCG � ovulatory stimulus and

eCG � BCS. Cows in the CG group were less likely (28.9% vs. 33.8%; P < 0.05) to become pregnant compared with those

in the EG group (odds ratio [OR] = 0.28). There were no differences in P/AI between CE and EE cows (30.9% vs. 29.1%;

OR = 0.85; P = 0.56), respectively. Thinner cows not receiving eCG had lower P/AI than thinner cows receiving eCG (15.2% vs.

38.0%; OR = 0.20; P < 0.01). Treatment with eCG tended to increase serum progestesterone concentrations during the diestrus

following synchronized ovulation (P < 0.10). However, the treatment used to induce ovulation did not affect CL volume or serum

progesterone concentrations. In conclusion, both ECP and GnRH yielded comparable P/AI. However, eCG treatment at CIDR

removal increased pregnancy rate in cows induced to ovulate with GnRH and in cows with lower BCS.

# 2009 Elsevier Inc. All rights reserved.

Keywords: eCG; Estrogen; Progesterone; Timed AI; Dairy cow

www.theriojournal.com

Available online at www.sciencedirect.com

Theriogenology xxx (2009) xxx–xxx

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* Corresponding author at: Departamento de Reproducao Animal –

VRA, Av: Prof. Dr. Orlando Marques de Paiva, no. 87 – Cidade

Universitaria, CEP: 05508-000, Sao Paulo – SP, Brazil.

Tel.: +55 11 3091 7674; fax: +55 11 3091 7412.

E-mail address: barusell@usp.br (P.S. Baruselli).

0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2008.12.025

1. Introduction

Timed artificial insemination (TAI) protocols, to

inseminate without the need for detection of estrus, have

been developed to increase service rates in cows [1–3].

Generally, these protocols use GnRH and PGF2a

(Ovsynch protocol [1]) to precisely synchronize the

e chorionic gonadotropin and type of ovulatory stimulus in a

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time of AI. In addition, short-acting estrogens in

conjunction with progestagen treatments have been

used to synchronize the emergence of a new follicular

wave during TAI protocols [3] and to avoid the

production of persistent follicles during progesterone-

based protocols [4]. Although service rates are

generally improved during TAI procedures, fertility

following synchronized ovulation is sub-optimal [5].

One potential problem with TAI protocols in

lactating dairy cows is the decrease in circulating

estradiol concentrations prior to TAI [6]. This reduction

in circulating estradiol is partly caused by the high rate

of steroid metabolism in dairy cows, due to increased

hepatic blood flow, in association with the elevated feed

intake of high-producing cows [7]. In addition, TAI

protocols induce premature ovulation of the follicle in

order to synchronize ovulation in all cows; this can

further reduce peak circulating estradiol concentrations

[6,8]. Previous studies have reported some benefits of

supplementing estrogen during the final stages of TAI

protocols [6,9–11]. Thus, supplementation with estro-

gens near TAI may be a rational approach to improve

fertility in TAI protocols for lactating cows.

A further consequence of prematurely inducing

ovulation during TAI protocols is that the diameter of

the ovulatory follicle is somewhat reduced [8,12,13].

Vasconcelos et al. [12] reported a close relationship

between diameter of the ovulatory follicle and CL

volume as well as circulating progesterone (P4)

concentrations. Thus, premature ovulation following

TAI procedures might induce ovulation of smaller

follicles, formation of a smaller CL, and with

concurrent high liver metabolism of ovarian steroids,

decrease circulating P4 during the luteal phase of high-

producing cows. Several studies have indicated that low

circulating P4 may suppress early embryonic develop-

ment [14,15]. Treatment with eCG prior to ovulation

has increased circulating P4 during the subsequent

luteal phase [16]. Studies in beef cattle in which P4-

based TAI protocols were supplemented with eCG

concurrent with removal of the P4 insert provided

evidence that eCG increased the percentage of cows that

ovulated to the TAI protocol, increased circulating P4

following TAI, and increased fertility to TAI [17].

Accordingly, strategies to increase circulating P4 after

ovulation could improve embryo survival for TAI

protocols.

We chose estradiol cypionate (ECP), a long-acting

conjugated estradiol, as a source of estrogen to be given

concurrent with P4 insert removal, to increase

circulating estrogen concentrations during the peri-

ovulatory period and minimize animal handling. In

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addition, cows were treated with or without eCG at

CIDR removal, to increase subsequent luteal function.

The main hypotheses of this study were: (1) eCG

treatment will increase CL volume, circulating P4 and

P/AI; (2) ECP will increase P/AI; and (3) combining

eCG with ECP will produce the greatest increase in P/

AI following TAI. Therefore, this experiment was

designed to test whether eCG treatment at CIDR

removal (with or without eCG) and type of stimulus

(ECP or GnRH) used to induce ovulation, can increase

fertility in lactating cows following a TAI protocol.

2. Materials and methods

2.1. Cows and management

Lactating Holstein cows were used in this experiment

(ovarian follicular dynamics, n = 96 cows; field fertility

test, n = 742 cows in a total of 782 breedings) having

average (mean � S.E.M.) body condition score [18] of

2.88 � 0.01 (CG = 2.89 � 0.02; CE = 2.89 � 0.02;

EG = 2.87 � 0.02; and EE = 2.86 � 0.02), milk produc-

tion (daily average from Day 0 to Day 8) of

36.2 � 0.4 kg/d (CG = 35.9 � 0.6 kg/d; CE = 36.8 �0.6 kg/d; EG = 35.4 � 0.6 kg/d; and EE = 36.6 �0.6 kg/d), and average days in milk of 151.6 � 3.5

(CG = 151.1 � 6.7; CE = 153.7 � 7.0; EG = 145.5 �6.8; and EE = 156.2 � 7.8). The study was replicated

in 22 free-stall dairy herds in southeastern Brazil. Healthy

postpartum cows with >50 d in milk and cows not

pregnant from the last AI before enrollment in the study

were assigned to hormonal treatments. The experimental

period began in December 2005 and ended in September

2006. On all farms, cows were milked twice daily and fed

a TMR that consisted of corn silage and alfalfa silage as

forage, with a corn-soybean meal-based concentrate. The

TMR was balanced to meet or exceed minimum

nutritional requirements for lactating dairy cows [19].

All animal procedures were approved by the Animal Care

Committee of the Faculty of Veterinary Medicine and

Husbandry, University of Sao Paulo, Sao Paulo, SP,

Brazil.

2.2. Hormones and experimental design

All cows received 2 mg of estradiol benzoate (EB;

Estrogin; Farmavet Produtos Veterinarios, SP, Brazil)

and one intravaginal progesterone insert (CIDR; 1.9 g

of P4; Pfizer Animal Health, SP, Brazil) on Day 0. At

CIDR removal (Day 8), all cows received 25 mg of

PGF2a (Lutalyse Sterile Solution; Pfizer Animal

Health). Simultaneously (Day 8), cows were randomly

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Fig. 1. Experimental design. US = ovarian ultrasound examination and BS = blood sample. *Ultrasound evaluation performed every 12 h for 96 h

after CIDR removal or until ovulation was detected. **Blood samples were collected every 4 h from 30 to 58 h after CIDR removal. ***Blood

Q2 samples and ovarian ultrasound examinations were performed every 2–3 d for 21 d after synchronized ovulation. Pregnancy diagnosis was

performed by ultrasound 33 d after timed AI.

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assigned within each farm to four treatments (Fig. 1), in

a 2 � 2 factorial design, as follows: (1) Group CG:

GnRH (100 mg Fertagyl; Intervet Schering-Plough, SP,

Brazil) 48 h after CIDR removal (Day 10); (2) Group

CE: ECP (1 mg ECP; Pfizer Animal Health) at CIDR

removal (Day 8); (3) Group EG: eCG (400 IU; Folligon;

Intervet Schering-Plough) on Day 8 + GnRH (100 mg)

on Day 10; (4) Group EE: eCG (400 IU) + ECP (1 mg)

on Day 8.

2.3. Follicular dynamics

In a subset of the cows (Subset A; n = 45), transrectal

ovarian ultrasound examinations were performed every

24 h from Day 0 (CIDR insertion) to Day 8 (CIDR

removal). Wave emergence was defined retrospectively

when the dominant follicle was 4–5 mm in diameter, as

described previously [20]. After CIDR removal, ovarian

ultrasound examinations were performed every 12 h in

two subsets of cows (Subset A, n = 45 and Subset

B = 51; total = 96 cows) from CIDR removal to

Please cite this article in press as: Souza AH, et al. Effects of equin

timed-AI protocol on reproductive responses in dairy cows. Therio

disappearance of the ovulatory follicle, or 96 h after

CIDR withdrawal, whichever occurred first. In another

subset of cows (Subset B; LH analysis, n = 40) blood

samples were collected every 4 h from 30 to 58 h after

CIDR removal to evaluate the time of the LH peak.

Cows in Subsets A and B were not inseminated.

Subsequently, ovulating cows in Subset A were

evaluated every 2 or 3 d after synchronized ovulation

for 21 d. The total volume of the CL formed after TAI in

ovulating cows was calculated with the following

formula: V = (4/3)pR3; R was defined as R = (Da/

2 + Db/2)/2, where Da and Db are the perpendicular

diameters of the CL. If the CL had a cavity, it was

calculated with the same formula and deleted from the

total volume of the CL. An ultrasound scanner with

linear-array transducer (6.0/8.0 MHz) was used to

perform all measurements of ovarian structures (Falco

100, Pie Medical Equipment B.V., Maastricht, Hol-

land). On the day of CIDR removal (Day 8), all cows in

Subsets A and B (n = 96) were fitted with a Kamar (heat

mount patch) and visually checked for signs of estrus

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(defined as standing estrus, with an activated Kamar)

every 4 h for 48 h after CIDR removal. Day 0 of the

estrous cycle subsequent to hormonal treatments was

defined as the day of the synchronized ovulation.

2.4. Field fertility trial

In the fertility study, 742 cows (in a total of 782

breedings) randomly received the same hormonal

treatments as a 2 � 2 factorial design (Fig. 1). The

term ‘‘cow’’ and ‘‘breeding’’ is used interchangeably

throughout the manuscript to define our experimental

unit ‘‘breeding’’. Semen from 30 sires was used and

homogenously distributed among treatments and farms.

Timed AI was performed 58 h after CIDR removal and

pregnancy diagnosis was performed by ultrasonography

33 d later. Pregnancy was confirmed based on the

echodensity of the fluid in the uterine horns and the

presence of a heartbeat in the embryo.

2.5. Hormone assays

Blood samples were collected by puncture of

coccygeal vessels, immediately refrigerated, centrifuged

(3000 � g for 20 min), and serum samples were stored at

�20 8C until assayed for P4 concentrations. Circulating

P4 was evaluated from unextracted sera using an

antibody-coated-tube RIA kit (Diagnostic Products

Corporation, Los Angeles, CA, USA). The intra- and

inter-assay CVs were 4.3% and 4.6%, respectively.

Circulating concentrations of LH were determined by

RIA, as previously described [21]. The intra- and inter-

assay CVs for LH were2.3% and 3.6%, respectively. The

LH peak was defined as an increase in LH< 2SD above

the overall within-cow mean of LH concentrations [22].

2.6. Statistical analyses

The binomial distribution was assumed for the

categorical response variables, such as expression of

estrus, ovulation rate, double ovulation rate, and P/AI.

The variable P/AI was analyzed by using procedure

GLIMMIX of SAS [23], with cow treated as a random

effect. Variables initially considered for inclusion in the

models were eCG (with or without eCG), type of

ovulatory stimulus (GnRH or ECP), farm within season

(warmer or cooler months), parity (categorized as 1 or

�2), days in milk (continuous variable), BCS (categor-

ized as <2.75 or �2.75), milk production (continuous

variable), sire, CL presence at CIDR insertion, and

interactions. The variable farm was nested within

season in the model, because not all farms had cows

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treated in both seasons. The final logistic regression

model removed variables by backward elimination,

based on the Wald statistics criterion when P > 0.20.

Variables that were included in the final model for

analysis of P/AI were: eCG (with or without eCG), type

of ovulatory stimulus (GnRH or ECP), BCS, interaction

eCG � type of ovulatory stimulus, interaction

eCG � BCS, interaction among eCG � type of ovula-

tory stimulus � BCS, and farm within season. Some

variables such as expression of estrus, number of LH

peaks detected, ovulation rate, and double ovulation

rate, were analyzed with the GENMOD procedure of

SAS. The final model for these dependent variables

accounted for effects of eCG, ovulatory stimulus, and

interaction between eCG and ovulatory stimulus.

Comparisons of CL volume and circulating P4 during

the cycle following synchronized ovulation were

performed using the MIXED procedure of SAS [24].

Square root transformation was used for CL volume and

log transformation was used for circulating progester-

one to attain normality. The model included the effects

of eCG, ovulatory stimulus, BCS, time, interaction

between eCG � time, interaction between ovulatory

stimulus � time, interaction between BCS � time,

interaction among eCG � ovulatory stimulus � time,

milk production, number of ovulations, and cow, which

was treated as a random effect and was the subject for

the repeated measures. Variables such as time to

ovulation, follicular wave emergence, dominant follicle

size 48 h after CIDR removal, time to LH peak, LH

peak amplitude, and area under the curve of LH release

(calculated by the trapezoid method), were analyzed

using the GLM procedure of SAS, and the final model

accounted for the effects of eCG, ovulatory stimulus,

and interaction between eCG � ovulatory stimulus.

Correlations between milk production and day of

follicular wave emergence after CIDR insertion were

evaluated with the CANCORR procedure of SAS. The

correlation model considered effects of days in milk,

parity and BCS. Levene’s test was used to compare

variances in the time of LH peak and ovulation after

CIDR removal. Probabilities of P < 0.05 were con-

sidered significant, whereas probabilities between 0.05

and 0.10 were considered tendencies.

3. Results

3.1. Follicular dynamics and hormonal responses

following CIDR + EB

Combining a CIDR with 2 mg of EB was effective in

synchronizing follicular wave emergence, with 84.4%

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Fig. 2. Day of the follicular wave emergence (mean � S.E.M.) after estradiol benzoate (EB) treatment and CIDR insert, according to milk

production class (kg/d). Means without a common superscript (a and b) differed (P < 0.05).

Fig. 3. Distribution of the time of ovulation (h) after CIDR removal,

according to each treatment.

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CIDR + EB treatment (Fig. 2). Out of 45 cows, we did

not detect emergence in five cows and two of them had

late emergence. Thus, lack of regression of the

dominant follicle (n = 3), lack of ovarian structures

(n = 2), and late follicular wave emergence (>5 d

following EB + CIDR insert; n = 2) were the major

reasons for failures in synchronization of follicular

wave emergence after EB plus CIDR insert. All cows

not presenting regression of the dominant follicle had

above average milk production (37.5, 45.6 and 49.4 kg/

d). However, time of follicular wave emergence

(mean � S.E.M.) was similar among treatments

(P > 0.10), occurring an average of 3.9 � 0.2 d after

CIDR insertion. Level of milk production and time to

follicular emergence tended to be negatively correlated

(r = �0.36; P = 0.08). Analysis of the time of wave

emergence by level of milk production also indicated a

link between these variables (Fig. 2). Cows with lower

milk production (<25 kg/d) had the longest interval to

wave emergence (4.6 � 0.3 d), whereas, cows with the

highest milk production (>35 kg/d) had the earliest

wave emergence (3.5 � 0.2 d).

3.2. Hormonal and ovulatory responses

An LH surge was detected in 82.5% (33/40) of cows

that had intensive blood samples taken for analysis of

this value. Overall, time of the LH peak averaged 43.6 h

after CIDR removal (Fig. 3). There was no difference

among the four treatments in percentage of cows that

had an LH surge detected after treatments. There were

no significant effects of eCG, ovulatory stimulus, or

interactions between eCG treatment and type of

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ovulatory stimulus on any of the LH parameters

evaluated (Table 1). The LH peak amplitude averaged

14.0 ng/mL and the area under the LH peak averaged

27.2 ng2, with no effects of eCG, ovulatory stimulus,

and interaction between eCG and ovulatory stimulus

(Table 1). However, there was a greater (P < 0.05)

variability in time to LH peak for cows treated with ECP

than cows treated with GnRH (S.E.M. = 2.6 vs. 1.8).

A total of 83.3% (80/96) of cows had ovulation in

response to hormonal treatments and there was no effect

of eCG, ovulatory stimulus, and interaction between

eCG and ovulatory stimulus for the percentage of cows

that ovulated following treatment (Table 2). The time to

ovulation in the cows intensively analyzed for peak LH

concentrations (Table 1) averaged 69.1 h, or 25.5 h after

detection of the LH peak. A similar time to ovulation

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Table 1

Characteristics (mean � S.E.M.) of LH peak and time of ovulation after CIDR removal (cows in Subset B).

Item No eCG eCG P-value

GnRH ECP GnRH ECP eCG OSa eCG � OS

LH peaks detectedb 9/10 8/10 9/10 7/10 0.80 0.23 0.73

Time of LH peakc,d (h) 45.5 � 2.7 42.0 � 4.1 42.4 � 2.4 44.3 � 3.4 0.84 0.81 0.40

LH peak amplitude (ng) 12.1 � 1.3 12.9 � 2.9 15.0 � 2.5 16.2 � 3.8 0.27 0.72 0.94

Area under LH curvee (ng2) 24.2 � 1.6 27.2 � 4.3 30.1 � 2.8 27.4 � 3.4 0.28 0.96 0.37

Time of ovulation (h) 69.0 � 3.4 71.3 � 4.3 67.5 � 2.4 69.0 � 3.9 0.49 0.34 0.78

a Ovulatory stimulus (GnRH or ECP).b Only 40 cows out of 51 were available for LH analysis.c Blood samples were collected every 4 h from 30 to 58 h after CIDR removal.d LH peak was defined as an increase in LH > 2SD above the overall within-cow mean of LH concentrations.e Area under LH curve was calculated with trapezoid method.

Table 2

Proportion of cows showing estrus within 48 h after CIDR removal, ovulation rate, diameter of the dominant follicle at 48 h after CIDR removal

(mean � S.E.M.), time of ovulation after CIDR removal (mean � S.E.M.), percentage of double ovulations, and P/AI 33 d after timed AI.

Item No eCG eCG P-value

GnRH ECP GnRH ECP eCG OSa eCG � OS

Estrusb �48 h (%, n/n) 8.7 (2/23) 20.8 (5/24) 12.5 (3/24) 32.0 (8/25) 0.36 0.04 0.89

Ovulation ratec (%, n/n) 82.6 (19/23) 87.5 (21/24) 79.2 (19/24) 84.0 (21/25) 0.61 0.60 0.81

Diameter of DFd (mm) 13.1 � 0.6 14.3 � 0.5 14.7 � 0.6 13.6 � 0.4 0.40 0.93 0.11

Time of ovulatione (h) 74.8 � 2.2 75.1 � 2.7 72.9 � 2.1 70.2 � 2.9 0.17 0.63 0.70

Double ovulationf (%, n/n) 21.7 (5/23) 20.8 (5/24) 12.5 (3/24) 12.0 (3/25) 0.48 0.36 0.85

Pregnancy per AI d 33 (%) 28.9 (56/194)b 30.9 (60/194)ab 33.8 (67/198)a 29.1 (57/196)ab <0.01 0.46 0.03

Within a row, proportions without a common letter (a and b) differed (P < 0.05).a Ovulatory stimulus (GnRH or ECP).b Detected with Kamar1 heat mount detectors starting at CIDR removal for 48 h.c Ovulation rate = percentage of cows that ovulated at least one follicle within 96 h after CIDR removal.d DF = largest dominant follicle in single ovulated cows, 48 h after CIDR removal.e The ovaries of each cow were visualized every 12 h starting at CIDR removal to disappearance of the ovulatory follicle or 96 h after CIDR

withdrawal, whichever happened first; and time of ovulation was defined as the time of disappearance of dominant follicle minus 6 h.f Double ovulation was confirmed positive if a given cow had two dominant follicles at 48 h after CIDR removal and two CL structures in the same

ovarian site 10 d after CIDR removal.

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(73.2 h) was calculated for the entire group of cows with

intensive ultrasound evaluations for time of ovulation.

Also, there were no significant effects of the main

treatments or interaction between eCG supplementa-

tion � ovulatory treatment in time to ovulation

(Tables 1 and 2).

The percentage of cows with a detected standing

estrus prior to 48 h after CIDR removal was fairly low

(18.8%; 18/96) and there was no effect of eCG and

interaction eCG � ovulatory stimulus. In contrast, there

was a significant effect of ECP on percentage of cows

with an early estrus, with 26.5% (13/49) of cows

receiving ECP having early estrus compared to only

10.6% (5/47) of cows not receiving ECP. Cows detected

in estrus <48 h after CIDR removal also ovulated

earlier (P < 0.05) and had greater variation among cows

(P < 0.05) compared to cows without premature estrus

Please cite this article in press as: Souza AH, et al. Effects of equin

timed-AI protocol on reproductive responses in dairy cows. Therio

(estrus �48 h = 63.3 � 2.1 h vs. not detected in estrus

until 48 h = 75.7 � 1.2 h).

The size of the ovulatory follicle at 48 h after CIDR

removal averaged 13.9 mm. In addition, there were no

significant effects of eCG, ovulatory drug and interac-

tion of eCG � ovulatory on diameter of the ovulatory

follicle. A total of 16 cows had double ovulation (16/

96 = 16.7% of all cows analyzed; 16/80 = 20% of

ovulating cows) and there were no significant differ-

ences between treatments or interactions on double

ovulation (Table 2).

3.3. Size of CL and circulating P4 after TAI

protocol

There were no significant effects of eCG, ovulatory

stimulus or interactions in CL volume or progesterone

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025

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Fig. 4. Main effect (mean � S.E.M.) of eCG on CL volume (A) and on serum progesterone concentrations (B) during diestrus following

synchronized ovulation in lactating Holstein cows (n = 37; 8 of 45 cows did not ovulate and were not further evaluated). Data were normalized to the

day of ovulation (Day 0). Means without a common superscript (a and b) differed (P < 0.05). Means without a common superscript (A and B)

differed (P < 0.10).

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Rconcentrations during diestrus following synchronized

ovulations. Similarly, milk production did not sig-

nificantly affect either CL volume or circulating P4. In

contrast, number of ovulations affected both CL volume

and circulating progesterone. The interaction of eCG by

day tended to be significant for CL volume (Fig. 4A).

Also, eCG tended to increase circulating P4 concentra-

tions throughout the diestrus that followed synchro-

nized ovulation. However, the major increase in

circulating P4 seemed to occur only at 12 (P < 0.05)

and 14 d (P < 0.10) after ovulation (Fig. 4B).

Cows were classified into two BCS classes (<2.75 or

�2.75) based on previous studies [6,25,26]. In these

studies, cows with BCS < 2.75 had lower fertility

compared with cows with greater BCS. Body condition

score of the cows tended to affect CL volume, but had

no influence on circulating P4 concentrations following

synchronized ovulations. Thus, the overall volume of

Please cite this article in press as: Souza AH, et al. Effects of equin

timed-AI protocol on reproductive responses in dairy cows. Therio

the CL tended to be smaller in cows with lower BCS.

Furthermore, there were no significant interactions

between eCG treatment and BCS, or ovulatory

treatment and BCS.

3.4. Comparison of pregnancies per AI (P/AI)

A total of 782 breedings were available for analysis

of treatment effects on P/AI. There was a significant

interaction between eCG treatment and type of

ovulatory stimulus, and between eCG treatment and

BCS on P/AI (Table 2). Cows receiving CG were less

likely to become pregnant compared with EG cows

(odds ratio [OR] = 0.28; 95% CI = 0.12, 0.65; P < 0.05;

Table 2). There were no differences in P/AI between CE

and EE (OR = 0.85; 95% CI = 0.49, 1.48; P = 0.56).

Cows with lower BCS not receiving eCG were less

likely to become pregnant compared with cows with

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025

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Fig. 5. Pregnancy rate per AI for cows receiving or not eCG and with

BCS < 2.75 or �2.75. Columns without a common superscript dif-

fered (P < 0.05). *Body condition score [18] data were not available

for 11 cows.

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Rlower BCS that received eCG (OR = 0.20; 95%

CI = 0.06, 0.69; P < 0.01). In contrast, the same trend

was not verified in cows with higher BCS (OR = 1.18;

95% CI = 0.75, 1.88; P = 0.35). Thus, there was a strong

positive effect of eCG in cows with low BCS, but not in

cows with higher BCS (Fig. 5). In addition, there was no

significant interaction between type of ovulatory

stimulus and BCS on P/AI.

Body condition score and farm within season also

affected P/AI (P < 0.05). Thinner cows (<2.75) were

less likely to become pregnant compared with cows

with higher BCS (�2.75) at breeding (OR = 0.55; 95%

CI = 0.33, 0.92; P = 0.02). There was no significant

(P > 0.10) effect of parity, days in milk, CL presence at

CIDR insertion, sire, and milk production on P/AI.

4. Discussion

Treatment with eCG did not increase size of the

dominant follicle 48 h after CIDR removal. These

results were in agreement with preliminary studies

performed in beef [17,25] and dairy cattle [26]. For

instance, Veneranda et al. [26], working with Holstein

cows given either 400 IU of eCG or no eCG, reported

that diameter of the ovulatory follicle was similar in

cows that did or did not receive eCG. However, in other

studies, eCG treatment at the end of progesterone

treatment increased the diameter of the ovulatory

follicle in anestrous beef cattle [27,28]. It is widely

known that anestrous beef cows have low blood LH

concentrations [29]. In addition, the incidence of

anestrous due to insufficient LH pulses is less of a

Please cite this article in press as: Souza AH, et al. Effects of equin

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problem in dairy cows compared with beef cows [29].

Thus, differences between these studies in the effect of

eCG on follicle size may relate to differences in blood

LH concentrations between dairy and beef cattle, or

even between similar breeds of cows in different

environmental situations. Nevertheless, in this study

using high-producing dairy cattle, 400 IU of eCG had

no effect on diameter of the ovulatory follicle.

Based on most evidence, we inferred that eCG

treatment at the time of P4 insert removal is highly

recommended in beef herds that have a high percentage

of cows that are not cycling [17,30]. Baruselli et al. [17]

found that the improvement in fertility after eCG

supplementation was due to an increase in percentage of

cows that ovulated after the protocols, as well as

increases in circulating P4 following tTAI, perhaps

minimizing early embryonic loss. In the current trial,

eCG did not increase the percentage of cows that

ovulated to the TAI protocol. However, addition of eCG

at CIDR withdrawal increased circulating P4 during the

subsequent cycle, particularly at Days 12 and 14, a

critical time in embryo development. Treatments to

increase P4 during the luteal phase increased embryo

development [14,15], which could enhance fertility in

dairy cattle [31]. It was somewhat surprising that there

was an increase in circulating P4 at Day 12 without a

detectable increase in ovulatory follicle size or CL

volume. The physiological mechanisms that lead to

enhanced luteal P4 production following eCG treatment

were not identified in the present study; however, they

could involve increased proportion, size, or function of

large luteal cells in the CL. Large luteal cells are

responsible for nearly 80% of luteal P4 production

(reviewed by Ref. [32]). Furthermore, increases in

circulating P4 have been related to increases in large

luteal cells during the CL lifespan [33], making changes

in large luteal cells a strong candidate for the changes in

P4 production following eCG.

As expected, ECP increased the percentage of cows

detected in estrus within 48 h after CIDR removal. Also

as might be expected, cows with premature signs of

estrus also ovulated earlier and less synchronously

compared with cows without premature estrus. This

information has practical importance, since cows in

premature estrus would be less likely to conceive to TAI

58 h after CIDR removal. Thus, if ECP is used at CIDR

removal, pregnancy rates may be improved results if

cows are monitored for estrus, and those with premature

estrus are bred at a more appropriate time than at the

later TAI. Another option would be to use ECP

supplementation at 24 h rather than at CIDR removal. In

spite of premature estrus, several reports have found

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025

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positive effects on fertility by supplementing estrogen

in the pro-estrus period [6,34]. However, other studies

[35–37], including the present results, did not detect an

overall positive effect of estrogen on conception rates.

The timing, dose, or type of estrogen, as well as breed

and BCS of cow and experimental environment, may all

have contributed to differences between studies in the

effects of estrogen supplementation.

Cows with lower BCS had lower P/AI, tended to have

reduced CL volume, but had no significant difference in

circulating P4 during diestrus after synchronized ovula-

tion. Santos et al. [31], reported that lactating cows with

lower BCS (�2.75) tended to have lower circulating P4 in

diestrus compared to cows with higher BCS. Data from

Santos et al. [31] might indicate that cows with lower

BCS have either greater steroid metabolism and/or lower

luteal P4 production. It has been shown that dairy cows

with lower BCS have greater feed intake than higher BCS

cows [38], potentially increasing rates of steroid

metabolism in the liver [7] and decreasing circulating

P4. In addition, Gansworthy and Topps [38] found that

cows with lower BCS produced more milk than fatter

cows. However, in the current trial, milk production was

similar between cows with lower and higher BCS (lower

BCS = 33.0 � 2.6 kg/d vs. higher BCS = 35.6 � 1.0 kg/

d; P > 0.10). Regardless, in the current trial, there was a

significant interaction between eCG and BCS on P/AI.

Perhaps eCG treatment either improved ovulation rate or

CL development, particularly in cows with lower BCS,

and this may have lead to increased fertility in these cows.

Due to a limited number of cows with lower BCS during

the follicular dynamics evaluation, we were unable to

detect a significant interaction between eCG treatment

and BCS on ovulation results. Thus, other eCG-induced

mechanisms underlying the enhancement of fertility in

low BCS cows, such as improved ovulation rate,

follicular or oocyte function or increased circulating

estradiol, cannot be excluded.

Lactating cows with BCS < 2.75 had lower fertility

compared with cows with greater BCS [6,39,40].

Interestingly, in the current trial, cows with lower

BCS not receiving eCG were less likely to become

pregnant than cows with lower BCS that received eCG.

Veneranda et al. [26] reported that lactating cows

supplemented with eCG had greater fertility than

control cows. However, in a second study done by this

author [29], no fertility differences were detected in

cows treated with eCG compared with controls.

Nevertheless, when all three experiments were taken

together [41], pregnancy rates were higher (P < 0.01) in

eCG-treated cows than in non-eCG-treated cows. Other

studies suggested that eCG increased conception

Please cite this article in press as: Souza AH, et al. Effects of equin

timed-AI protocol on reproductive responses in dairy cows. Therio

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outcomes in cows not cycling before TAI treatments

[42]. In the current trial, any improvements in P/AI

following the use of eCG seemed to be limited to cows

with lower BCS, but not to cyclicity before TAI.

Regardless, future studies using eCG supplementation

in cattle should compare treatments with different eCG

doses, and also take into account the possibility of anti-

eCG antibody after repeated treatments with high doses

of eCG [43].

There was no significant effect of ovulatory stimulus

or interaction between type of ovulatory stimulus and

BCS on P/AI. These results were not in agreement with

recent reports that described beneficial effects of

supplementing with a short-acting estrogen [6] in

protocols for TAI in lactating cows. A more recent

report [37], also using lactating cows, using the same

long-acting estrogen used in the current trial (ECP) did

not report improvements in fertility in animals with low

BCS treated for TAI. Thus, based on this previous study

and our present results, we inferred that improvements

in fertility with ECP supplementation in TAI protocols

were somewhat inconsistent. Because more cows

showed premature estrus in ECP group, improvements

in fertility might be possible with additional estrus

detection and breeding of cows expressing premature

estrus, or by delaying ECP treatment to avoid premature

estruses. However, this topic warrants further research.

The time of LH peak after CIDR removal was not

influenced by eCG, ovulatory treatment or their

interaction, and occurred on average 43.6 � 1.5 h after

CIDR removal. Consequently, the mean time of

ovulation was also similar among treatments. Colazo

et al. [8], using beef heifers, reported that long-lasting

estrogens, e.g. ECP, can be effectively used concurrent

with removal of the P4 insert, to synchronize ovulation

in TAI protocols. In the present study using high-

producing cows, it seems likely that ECP, also given at

CIDR removal, decreased the synchrony of the LH peak

and ovulation, compared to cows treated with GnRH

48 h later. In addition, more frequent sampling for the

LH peak (every 4 h) allowed detection of the greater

variation in this variable; whereas, greater numbers of

cows were required to statistically detect differences in

variation for time to ovulation, due to less frequent

evaluations (every 12 h) to detect ovulation following

CIDR removal. Together, data presented in the current

report clearly detected differences in variation in the

time of ovulation in cows treated with ECP versus

GnRH; there were some indications that time to

ovulation was more variable in ECP-treated cows.

Milk production might also contribute to variation in

the timing of the LH peak and ovulation in ECP-treated

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025

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cows, due to greater estradiol metabolism in cows with

greater milk production. Thus, greater variation in the

time of ovulation in ECP-treated cows might be

expected to compromise fertility due to inconsistent

and, in some cases, inadequate intervals from AI to

ovulation [44,45]. Nonetheless, in spite of the increased

variability in timing of the LH peak and ovulation in

ECP-treated cows, we did not detect any differences in

fertility between groups that had ovulation synchro-

nized with ECP or GnRH. Perhaps, the increased

variability in time of ovulation in cows treated with ECP

was not sufficient to compromise fertility. Alternatively,

an increase in circulating estradiol concentrations in

ECP-treated cows may have provided some enhance-

ment in fertility, as previously observed [6,10,11] that

overcame any negative effects of increased variability in

time of ovulation. Additionally, in GnRH-treated cows,

it is also likely that the ideal (�16 h) interval from

GnRH treatment to insemination used in the current trial

(�10 h) was not attained as suggested by previous

research [45], which could also have lowered overall

conception rate results in GnRH-treated cows. Thus,

more experiments comparing these two types of

ovulatory stimulus, given at different times in relation

to device removal and TAI, are needed.

The reasons for the significant interaction between

eCG and type of ovulatory stimulus on P/AI are unclear.

However, differences in time to ovulation following

CIDR removal between cows treated with ECP or

GnRH might provide some rationale for this interaction.

Accordingly, it is likely that EG cows underwent a

better combination of synchronization of ovulation

produced by the GnRH treatment associated with

enhancements in CL function produced by the eCG

treatment. Thus, in cows treated only with GnRH, CL

function could have been compromised. Alternatively,

ECP groups had greater variation in time to ovulation.

Thus, cows ovulating prematurely were bred somewhat

late and eCG was not able to improve fertility, due to

inadequate insemination-to-ovulation intervals. Con-

versely, in cows with delayed ovulations, perhaps the

overexposure of the oocyte to the LH-like actions of

eCG hastened oocyte maturation, causing the ovulation

of an aged oocyte. Another physiological explanation

for the significant eCG by type of ovulatory stimulus

interactions was potential increases to more optimal

blood estradiol concentrations near the time of AI in EG

but not CG cows, and/or excessive estrogen concentra-

tions near AI in EE but not CE cows.

An interesting finding in the current trial, not

prospectively hypothesized, was that level of milk

production seemed to affect the time of follicular

Please cite this article in press as: Souza AH, et al. Effects of equin

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emergence after EB and CIDR insertion. Burke et al.

[46], studying the effects of four doses of EB (0, 1, 2, or

4 mg) with P4 in a P4-based TAI protocol, reported that

greater doses of estradiol delayed follicular emergence.

Because follicular emergence only occurred after

circulating estradiol concentrations returned to basal,

these researchers indicated that the time of follicular

emergence seemed to depend on the rate of estradiol

clearance from the blood. In this regard, Sangsritavong

et al. [7] found that rate of steroid metabolism by the liver

was much higher in lactating dairy cows compared to

non-lactating cows. It is possible that in the current study

higher producing cows had greater estradiol clearance

that could lead to an earlier return of circulated estradiol

to basal concentrations after an EB treatment, with

subsequent earlier emergence of the next follicular wave.

Thus, based on our results combined with this previous

information, we speculated that greater estrogen doses

might be required in cows with greater milk production in

order to consistently cause dominant follicle atresia and

eventually improve the synchrony of follicular emer-

gence in these TAI protocols.

In conclusion, improvements in fertility in response to

eCG were limited to cows with lower BCS. Estradiol

cypionate can be successfully used to induce ovulation in

P4-based TAI protocols for lactating cows. Nevertheless,

in this study, there was greater variation in time of

ovulation with ECP compared to GnRH. Although no

fertility differences were detected between these two

treatments to induce ovulation, perhaps better timing of

ECP treatment following CIDR removal might reduce

variation in time of ovulation and improve fertility. Thus,

additional estrus detection and breeding seem to be

required when ECP is used at CIDR removal. More

studies are necessary toexplain the underlyingphysiology

for eCG enhancement of fertility and its interactions with

BCS and type of ovulatory stimulus on the bovine CL.

Acknowledgments

The authors thank dairy producers from Parana, Rio

Grande do Sul, and Sao Paulo, Brazil and their staff, for

all the help and the use of their herds to conduct these on-

farm trials. We also thank the reviewers for their critical

inputs in data analysis. This research was supported by

FAPESP of Brazil (process number 05/59009-0).

References

[1] Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovula-

tion in dairy cows using PGF2 and GnRH. Theriogenology

1995;44:915–23.

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025

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729

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734

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736

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737

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744

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761

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763

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785

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787

788

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790

791

792

793

794

795

796

797

798

799

UN

CO

R

[2] Martinez MF, Adams GP, Kastelic JP, Bergfelt DR, Mapletoft

RJ. Induction of follicular wave emergence for estrus synchro-

nization and artificial insemination in heifers. Theriogenology

2000;54: 757–69.

[3] Bo GA, Adams GP, Caccia M, Martinez M, Pierson RA,

Mapletoft RJ. Ovarian follicular wave emergence after treatment

with progestagen and estradiol in cattle. Anim Reprod Sci

1995;39: 193–204.

[4] Kinder JE, Kojima FN, Bergfeld EG, Wehrman ME, Fike KE.

Progestin and estrogen regulation of pulsatile LH release and

development of persistent ovarian follicles in cattle. J Anim Sci

1996;74:1424–40.

[5] Santos JEP, Juchem SO, Cerri RLA, Galvao KN, Chebel RC,

Thatcher WW, et al. Effect of bST and reproductive management

on reproductive and lactational performance of Holstein dairy

cows. J Dairy Sci 2004;87:868–81.

[6] Souza AH, Gumen A, Silva EPB, Cunha AP, Guenther JN, Peto

CM, et al. Supplementation with estradiol-17b before the last

GnRH of the Ovsynch protocol in lactating dairy cows. J Dairy

Sci 2007;90:4623–34.

[7] Sangsritavong S, Combs DK, Sartori R, Armentano LE, Wilt-

bank MC. High feed intake increases liver blood flow and

metabolism of progesterone and estradiol-17b in dairy cattle.

J Dairy Sci 2002;85:2831–42.

[8] 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.

[9] Hanlon DW, Williamson NB, Wichtell JJ, Steffert IJ, Craigie

AL, Pfeiffer DU. The effect of estradiol benzoate administration

on estrus response and synchronized pregnancy rate in dairy

heifers after treatment with exogenous progesterone. Theriogen-

ology 1996;45:775–85.

[10] Cerri RL, Santos JE, Juchem SO, Galvao KN, Chebel RC. Timed

artificial insemination with estradiol cypionate or insemination

at estrus in high-producing dairy cows. J Dairy Sci 2004;87:

3704–15.

[11] Colazo MG, Kastelic JP, Whittaker PR, Gavaga QA, Wilde R,

Mapletoft RJ. Fertility in beef cattle given a new or previously

used CIDR insert and estradiol, with or without progesterone.

Anim Reprod Sci 2004;81:25–34.

[12] Vasconcelos JL, Silcox RW, Rosa GJ, Pursley JP, Wiltbank MC.

Synchronization rate, size of the ovulatory follicle, and preg-

nancy rate after synchronization of ovulation beginning on

different days of the estrous cycle in lactating dairy cows.

Theriogenology 1999;52:1067–78.

[13] Mussard ML, Burke CR, Behlke EJ, Gasser CL, Day ML.

Influence of premature induction of a luteinizing hormone surge

with gonadotropin-releasing hormone on ovulation, luteal func-

tion, and fertility in cattle. J Anim Sci 2007;85:937–43.

[14] Garrett GE, Geisert RD, Zavy MT, Morgan GL. Evidence for

maternal regulation of early conceptus growth and development

in beef cattle. J Reprod Fertil 1988;84:437–46.

[15] Mann GE, Fray MD, Lamming GE. Effects of time of proges-

terone supplementation on embryo development and interferon-t

production in the cow. Vet J 2006;171:500–3.

[16] Murphy BD, Martinuk SD. Equine chorionic gonadotropin.

Endocr Rev 1991;12:27–43.

[17] Baruselli PS, Reis EL, Marques MO, Nasser LF, Bo GA. The use

of hormonal treatments to improve reproductive performance of

anestrous beef cattle in tropical climates. Anim Reprod Sci

2004;82/83:479–86.

Please cite this article in press as: Souza AH, et al. Effects of equin

timed-AI protocol on reproductive responses in dairy cows. Therio

EC

TED

PR

OO

F

[18] Edmonson AJ, Lean IJ, Weaver LD, Farver T, Webster G. A body

condition scoring chart for Holstein dairy cows. J Dairy Sci

1989;72:68–78.

[19] NRC. Nutrient requirements of dairy cattle, 7th rev. ed.,

Washington, DC: National Academic Science; 2001.

[20] Ginther OJ, Knopf L, Kastelic JP. Temporal associations among

ovarian events in cattle during oestrous cycles with two and three

follicular waves. J Reprod Fertil 1989;87:223–30.

[21] Bolt DJ, Scott V, Kiracofe GH. Plasma LH and FSH after

estradiol, norgestomet and Gn-RH treatment in ovariectomized

beef heifers. Anim Reprod Sci 1990;23:263–71.

[22] Haughian JM, Ginther OJ, Kot K, Wiltbank MC. Relationships

between FSH patterns and follicular dynamics and the temporal

associations among hormones in natural and GnRH-induced

gonadotropin surges in heifers. Reproduction 2004;127:23–33.

[23] SAS Institute. SAS User’s Guide: Statistics, Version 9. 1 for

Windows. Cary, NC: SAS Ins.; 2002–2003.

[24] Littell RC, Milliken GA, Stroup W, Wolfinger RD. SAS system

of mixed models. Cary, NC: Statistical Analysis System Insti-

tute; 1996.

[25] Small GA, Colazo MG, Kastelic JP, Mapletoft RJ. Effects of

progesterone presynchronization and eCG on pregnancy rates to

GnRH-based, timed-AI in beef cattle. Theriogenology 2009;71:

698–706.

[26] Veneranda G, Filippi L, Racca D, Romero G, Balla E, Cutaia L,

et al. Pregnancy rates in dairy cows treated with intravaginal

progesterone devices and different fixed-time AI protocols.

Reprod Fertil Dev 2006;18:118.

[27] Sa Filho MF, Reis EL, Viel Jr JO, Nichi M, Madureira EH,

Baruselli PS. Follicular dynamics in anestrous lactating Nelore

treated with ear implant, eCG and GnRH. Act Sci Vet 2004;32:

235.

[28] Marana D, Cutaia L, Peres L, Pincinato D, Borges LFK, Bo GA.

Ovulation and pregnancy rates in postpartum Bos indicus cows

treated with progesterone vaginal inserts and estradiol benzoate,

with or without eCG and temporary weaning. Reprod Fertil Dev

2006;18:116–7.

[29] Wiltbank MC, Gumen A, Sartori R. Physiological classification

of anovulatory conditions in cattle. Theriogenology 2002;57:21–

52.

[30] Bo GA, Baruselli PS, Martinez MF. Pattern and manipulation of

follicular development in Bos indicus cattle. Anim Reprod Sci

2003;78:307–26.

[31] Santos JE, Thatcher WW, Pool L, Overton MW. Effect of human

chorionic gonadotropin on luteal function and reproductive

performance of high-producing lactating Holstein dairy cows.

J Anim Sci 2001;79:2881–94.

[32] Diaz FJ, Anderson LE, Wu YL, Rabot A, Tsai SJ, Wiltbank MC.

Regulation of progesterone and prostaglandin F2a production in

the CL. Mol Cell Endocrinol 2002;191:65–80.

[33] Alila HW, Hansel W. Origin of different cell types in the bovine

corpus luteum as characterized by specific monoclonal antibo-

dies. Biol Reprod 1984;31:1015.

[34] Lopes AS, Butler ST, Gilbert RO, Butler WR. Relationship of

pre-ovulatory follicle size, estradiol concentrations and season to

pregnancy outcome in dairy cows. Anim Reprod Sci 2007;99:

34–43.

[35] Stevenson JS, Tiffany SM, Lucy MC. Use of estradiol cypionate

as a substitute for GnRH in protocols for synchronizing ovula-

tion in dairy cattle. J Dairy Sci 2004;87:3298–305.

[36] Sellars CB, Dalton JC, Manzo R, Day J, Ahmadzadeh A. Time

and incidence of ovulation and conception rates after incorpor-

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025

A.H. Souza et al. / Theriogenology xxx (2009) xxx–xxx12

+ Models

THE 10941 1–12

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801

802

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805

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810

811

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813

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818

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820

820

821

822

823

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827

828

829

830

831

832

833

834

835

836

837

838

839

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841841

ating estradiol cypionate into a timed artificial insemination

protocol. J Dairy Sci 2006;89:620–6.

[37] Hillegass J, Lima FS, Sa Filho MF, Santos JEP. Effect of time of

artificial insemination and supplemental estradiol on reproduc-

tion of lactating cows. J Dairy Sci 2008;91:4226–37.

[38] Gansworthy PC, Topps JH. The effect of body condition of dairy

cows at calving on their food intake and performance when given

complete diets. Anim Prod 1982;35:113–9.

[39] Moreira F, Risco C, Pires MF, Ambrose JD, Drost M, DeLorenzo

M, et al. Effect of body condition on reproductive efficiency of

lactating dairy cows receiving a timed insemination. Theriogen-

ology 2000;53:1305–19.

[40] Galvao KN, Santos JE, Juchem SO, Cerri RL, Coscioni AC,

Villasenor M. Effect of addition of a progesterone intravaginal

insert to a timed insemination protocol using estradiol cypionate

on ovulation rate, pregnancy rate, and late embryonic loss in

lactating dairy cows. J Anim Sci 2004;82:3508–17.

[41] Bo GA, Cutaia L, Souza AH, Baruselli PS. Systematic repro-

ductive management in dairy herds. In: New Zealand Veterinary

Association (NZVA) Conference. New Zealand, July 5–July 7:

Christchurch Convention Centre; 2007. p. 155–68.

UN

CO

RR

Please cite this article in press as: Souza AH, et al. Effects of equin

timed-AI protocol on reproductive responses in dairy cows. Therio

PR

OO

F

[42] Bryan MA, Emslie R, Heuer C. Comparative efficacy of an 8-day

Cue-Mate/estradiol benzoate program with or without inclusion

of equine chorionic gonadotropin in anestrus dairy cows. Reprod

Fertil Dev 2008;20:85.

[43] Drion PV, De Roover R, Houtain J, McNamara EM, Remy B,

Sulon J, et al. Increase of plasma eCG binding rate after

administration of repeated high dose of eCG to cows. Reprod

Nutr Dev 2001;41:207–15.

[44] Dransfield MBG, Nebel RL, Pearson RE, Warnick LD. Timing

of insemination for dairy cows identified in estrus by a radio-

telemetric estrus detection system. J Dairy Sci 1998;81:1874–

82.

[45] Pursley RJ, Silcox RW, Wiltbank MC. Effect of time of

artificial insemination on pregnancy rates, calving rates,

pregnancy loss, and gender ratio after synchronization of

ovulation in lactating dairy cows. J Dairy Sci 1998;81:

2139–44.

[46] Burke CR, Mussard ML, Gasser CL, Grum DE, Day ML.

Estradiol benzoate delays new follicular wave emergence in a

dose-dependent manner after ablation of the dominant ovarian

follicle in cattle. Theriogenology 2003;60:647–58.

EC

TED

e chorionic gonadotropin and type of ovulatory stimulus in a

genology (2009), doi:10.1016/j.theriogenology.2008.12.025