The document summarizes 4 experiments evaluating strategies to improve pregnancy rates in Bos indicus cows undergoing fixed-time artificial insemination (TAI) using protocols involving progesterone, estradiol, and progesterone treatments. In Experiment 1, temporary weaning for 48 hours or treatment with 400IU of eCG both improved pregnancy rates compared to controls, but 300IU of eCG was not more effective. In Experiment 2, FSH treatment was less effective than temporary weaning or 400IU of eCG at improving pregnancy rates. Experiment 3 found that adding 400IU of eCG to temporary weaning did not further improve pregnancy rates. Experiment 4 analyzed over 64,000 cow records and found pregnancy rate was affected by various farm,

1. Available online at www.sciencedirect.com
Theriogenology 72 (2009) 210–218
www.theriojournal.com
Fixed-time artificial insemination with estradiol and progesterone
for Bos indicus cows II: Strategies and factors affecting fertility
O.G. Sa Filho a, M. Meneghetti a, R.F.G. Peres a, G.C. Lamb b, J.L.M. Vasconcelos a,*
´
a
¸˜ ´
Departamento de Producao Animal, Faculdade de Medicina Veterinaria e Zootecnia-UNESP, Botucatu SP 18618-000, Brazil
b
North Florida Research and Education Center, University of Florida, Marianna 32446, Florida, USA
Received 29 September 2008; received in revised form 5 February 2009; accepted 8 February 2009
Abstract
In Experiments 1, 2, and 3, we evaluated the effects of temporary weaning (TW), equine chorionic gonadotropin (eCG), and
follicle-stimulating hormone (FSH) treatments on results of a fixed-time artificial insemination (TAI) protocol in postpartum Bos
indicus cows. In Experiment 1, treatment with 400 IU eCG or with TW for 48 h consistently improved pregnancy rates (PRs) at TAI,
but, in Experiment 2, FSH treatment was less effective than eCG or TW. In Experiment 3, the inclusion of eCG treatment in cows
subjected to TW did not improve PRs. We concluded that TW or 400 IU eCG should be included in the TAI protocol in postpartum
Bos indicus cows to enhance fertility. In Experiment 4, we used records from heifers and cows treated with the proposed protocol
during the 2006–2007 (n = 27,195) and 2007–2008 (n = 36,838) breeding seasons from multiple locations in Brazil to evaluate
factors potentially affecting PRs. Overall PR at TAI was 49.6% (31,786 of 64,033). Pregnancy rate differed (P < 0.01) among farm
within location (results ranging between 26.8% and 68.0%; P < 0.01), cow group within farm, by breed (Bos indicus, 48.3%
[26,123 of 54,145]; Bos taurus, 61.7% [3652 of 5922]; and crossbred Bos indicus  Bos taurus, 50.7% [2011 of 3966]), category
(nulliparous, 39.6% [2095 of 5290]; suckled primiparous, 45.2% [3924 of 8677]; suckled multiparous, 51.8% [24,245 of 46,767];
and nonsuckled multiparous, 46.1% [1522 of 3299]), body condition score at TAI ( 2.5, 43.0% [3409 of 7923]; 3.0, 49.6% [18,958
of 38,229]; and !3.5, 52.7% [9419 of 17,881]). Days postpartum at beginning of protocol did not affect PR (30 to 60 d, 47.6% [4228
of 8881]; 61 to 90 d, 51.7% [16,325 to 31,572]; and 91 to 150 d, 50.8% [7616 to 14,991]; P > 0.1). Pregnancy rate was also
consistently affected (P < 0.01) by sire (results ranging from 7.2% to 77.3%) and artificial insemination technician (results ranging
from 15.1% to 81.8%).
# 2009 Elsevier Inc. All rights reserved.
Keywords: Bos indicus; Estradiol; Fixed-time artificial insemination; Pregnancy rate; Progesterone
1. Introduction containing 1.9 g of progesterone (CIDR) plus 2.0 mg im
estradiol benzoate (EB) on Day 0; 12.5 mg im dinoprost
In a previous study [1] we proposed a protocol that tromethamine (PGF2a) on Day 7; CIDR withdrawal
allowed fixed-time artificial insemination (TAI) in plus 0.5 mg im estradiol cypionate (ECP) plus
anestrous and cycling postpartum Bos indicus cows, temporary weaning on Day 9; TAI (48 h after CIDR
which consisted of insertion of an intravaginal device withdrawal) plus reuniting of calves with their dams on
Day 11. Although satisfactory fertility had been
achieved, more studies are necessary to improve results
and provide better knowledge of factors potentially
* Corresponding author. Tel.: +55 14 3811 7185;
fax: +55 14 3811 7180. affecting pregnancy rates in cows submitted to that
E-mail address: vasconcelos@fca.unesp.br (J.L.M. Vasconcelos). protocol.
0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2009.02.008

2. O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218 211
In this series of studies, our goals were to evaluate
additional strategies to improve results and to elucidate
factors that may affect the results of this protocol. The
basis for these studies was to (1) increase dominant
follicle development with temporary weaning, equine
chorionic gonadotropin (eCG), or follicle-stimulating
hormone (FSH) supplementation at final stages of
Fig. 1. Schematic diagram of the basic synchronization of ovulation
follicle development; (2) evaluate if temporary weaning protocol. EB (2.0 mg), 2.0 mg im estradiol benzoate; CIDR (9 d),
could be excluded from or replaced by eCG or FSH treatment with an intravaginal insert containing 1.9 g progesterone
treatments in the protocol; (3) evaluate whether eCG during 9 d; PGF2a (12.5 mg), 12.5 mg im dinoprost tromethamine;
treatment would improve fertility of cows submitted to ECP (0.5 mg), 0.5 mg im estradiol cypionate; TAI, fixed-time artificial
insemination.
temporary weaning; and (4) evaluate factors that may
impact the TAI pregnancy rate using data from 64,033
cows treated with the proposed protocol from multiple (eCG300; n = 208); (3) cows received 400 IU im
locations in Brazil. eCG (2.0 mL of Folligon) immediately after CIDR
withdrawal (eCG400; n = 223); (4) cows had their
2. Materials and methods calves temporarily weaned for 48 h, beginning
immediately after CIDR withdrawal (TW; n = 239).
2.1. Experiment 1 In this and the following experiments, during
temporary weaning time, calves were held in pens
The goal of this experiment was to evaluate the out of sight from their dams, with ad libitum access to
effects of temporary weaning for 48 h or treatment with water. On Day 11 (48 h after CIDR withdrawal), all
two doses of eCG (300 IU or 400 IU; 1.5 or 2.0 mL of cows received TAI. Calves submitted to temporary
˜
Folligon [Intervet, Sao Paulo, SP, Brazil]) on the weaning were reunited with their dams immediately
ovulation, conception, and pregnancy rates of suckled after insemination. Cycling status of cows was
Nelore cows submitted to a synchronization of previously evaluated by two ultrasound examinations
ovulation protocol based on progesterone, EB, PGF2a, (Aloka SSD-500 with a 7.5 MHz linear-array trans-
and ECP. The hypothesis was that inclusion of rectal transducer; Tokyo, Japan) on Days 0 and 7.
temporary weaning, treatment with 300 IU eCG, or Cows with evidence of luteal tissue during at least one
treatment with 400 IU eCG in the protocol would of the examinations (n = 291) were considered cyclic.
improve ovulation and pregnancy rates of cows without Diameter of the largest follicle was determined on day
altering conception rates. of TAI by averaging the length and width measure-
Suckled multiparous Nelore cows (n = 891; 40 to ments from ovarian transrectal ultrasonography. The
90 d postpartum; body condition score (BCS) at Day 0 rates of ovulation, conception, and pregnancy were
between 2.5 and 3.5 on a 1 to 5 scale [2]), maintained evaluated by ultrasound examinations. Ovulation was
in Brachiaria decumbens pasture with ad libitum considered when cows had presence of a follicle with
access to water and mineral supplement, were treated diameter !8.5 mm on Day 11 and absence of this
with the following basic synchronization of ovulation follicle 48 h later (Day 13). Pregnancy diagnosis was
protocol (Fig. 1): insertion of an intravaginal insert performed 28 d after TAI. Conception rate was
containing 1.9 g progesterone (CIDR; Controlled calculated by dividing the number of pregnant cows
Intravaginal Drug Release, Pfizer Animal Health, by number of cows that ovulated. Pregnancy rate was
˜
Sao Paulo, SP, Brazil) plus 2.0 mg im EB (2.0 mL of calculated by dividing the number of pregnant cows
˜
Estrogin; Farmavet, Sao Paulo, SP, Brazil) on Day 0; by the number of treated cows.
and 12.5 mg im PGF2a (2.5 mL of Lutalyse; Pfizer
Animal Health) on Day 7. On Day 9, all cows had 2.2. Experiment 2
CIDR inserts removed and received 0.5 mg im ECP
(0.25 mL of E.C.P.; Pfizer Animal Health). Immedi- The goal of this experiment was to evaluate the
ately after CIDR withdrawal, cows were assigned effects of replacing temporary weaning or eCG with
randomly to receive one of the following treatments: FSH on the diameter of ovulatory follicle and the
(1) cows received no additional treatment (Control; pregnancy rates of suckled Nelore cows submitted to a
n = 221); (2) cows received 300 IU im eCG (1.5 mL of synchronization of ovulation protocol based on pro-
Folligon) immediately after CIDR withdrawal gesterone, EB, PGF2a, and ECP. The hypothesis was

3. 212 O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218
that FSH could replace temporary weaning or eCG n = 299); (3) cows received 400 IU im eCG immedi-
treatment in the protocol. ately after CIDR withdrawal (TW + eCG400; n = 291).
Suckled Nelore cows (n = 713; 30 to 90 d On Day 11, 48 h after CIDR withdrawal, all cows were
postpartum; BCS at Day 0 between 2.25 and 2.75), inseminated and calves reunited with their dams
maintained in Brachiaria brizantha pasture with ad immediately after insemination. Cycling status of cows
libitum access to water and mineral supplement, were was previously evaluated by two ultrasound examina-
treated with the same basic synchronization of tions on Days 0 and 7. Cows with evidence of luteal
ovulation protocol described for Experiment 1. On tissue during at least one of the examinations (n = 142)
Day 9, cows were assigned randomly to receive one were considered cyclic. Follicular diameter, ovulation,
of the following treatments: (1) cows had their conception, and pregnancy rates were determined as
calves temporarily weaned for 48 h, beginning described for Experiment 1.
immediately after CIDR withdrawal (TW; n = 198);
(2) cows received 400 IU im eCG immediately after 2.4. Experiment 4
CIDR withdrawal (eCG400; n = 187); (3) cows
received 10 mg im FSH (0.5 mL of Folltropin-V; The objective was to retrospectively assess factors
Bioniche Animal Health, Belleville, ON, Canada; affecting pregnancy rate of heifers and cows treated
FSH:LH proportion >80.0%) immediately after with a synchronization of ovulation protocol based
CIDR withdrawal (Folltropin; n = 216); (4) cows on progesterone, EB, PGF2a, and ECP. Lactating
received 25 IU im FSH (0.5 mL of Pluset; Hertape- and nonlactating beef cows and heifers from various
˜
Calier, Sao Paulo, SP, Brazil; FSH:LH proportion locations in Brazil during the 2006–2007 (n = 27,195)
about 50.0%) immediately after CIDR withdrawal and 2007–2008 (n = 36,838) breeding seasons were
(Pluset; n = 112). On Day 11 (48 h after CIDR submitted to the same basic synchronization of
withdrawal), all cows received TAI. Calves submitted ovulation protocol described in Experiment 1. In
to temporary weaning were reunited with their dams suckled cows, a 48 h temporary weaning was performed
immediately after insemination. Follicle diameter and between CIDR withdrawal and TAI. BCS was evaluated
pregnancy rates were determined as described for always on Day 0 on a 1 to 5 scale [2]. In both breeding
Experiment 1. seasons, the cattle were from the same 71 farms in the
following Brazilian states: Goias (n = 23), Mato Grosso
2.3. Experiment 3 (n = 20), Mato Grosso do Sul (n = 9), Minas Gerais
(n = 9), Para (n = 6), Parana (n = 2), and Rondonia
The goal of this experiment was to evaluate the (n = 2). Cows were managed according to local
effects of two doses of eCG (200 IU or 400 IU) on the procedures, and in all places nutritional management
ovulation, conception, and pregnancy rates of suckled was based only on grass pastures with water and
Nelore cows submitted to a synchronization of appropriate ad libitum access to a mineral supplement.
ovulation protocol based on progesterone, EB, PGF2a, Each breeding season lasted from October until March
ECP, and temporary weaning. The hypothesis was that of the following year but varied among farms. Only data
treatment with either 200 or 400 IU eCG would improve recorded from cows with no recorded reproductive
reproductive performance in cows treated with a abnormalities were used for analysis. Pregnancy was
synchronization of ovulation protocol that involved diagnosed by transrectal ultrasonography at 28 to 35 d
temporary weaning from the progesterone insert with- after TAI.
drawal until the time of insemination.
Suckled Nelore cows (n = 887; 40 to 110 d 2.5. Statistical analyses
postpartum; BCS at Day 0 between 2.5 and 3.5),
maintained in Brachiaria decumbens pasture with ad Binomial variables such as ovulation, conception,
libitum access to water and mineral supplement, were and pregnancy rates were analyzed using PROC
treated with the same basic synchronization of LOGISTIC of the SAS program (SAS Institute Inc.,
ovulation protocol described in Experiment 1. On Cary, NC, USA). Explanatory variables such as
Day 9, all cows were submitted to temporary weaning treatment, BCS at Day 0, parity, categorized number
and assigned randomly to receive one of the following of days postpartum, cyclicity, breeding season, location,
treatments: (1) cows received no additional treatment and farm were used in the model as classes. Follicle
(TW; n = 297); (2) cows received 200 IU im eCG diameter at Day 11, considered a covariate, was
immediately after CIDR withdrawal (TW + eCG200; previously submitted to univariate analysis and

4. O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218 213
included in the multivariate models if found to be
significant. For conception and pregnancy data, original
models also included the effects of sire and artificial
insemination (AI) technician. For all logistic regres-
sion models, all two-way interactions were tested. In
the final logistic regression model, variables were
removed by a backward elimination (according to the
Wald’s criterion) when P > 0.2. When significant
effect of a covariate x on a dependent variable y was
detected, logistic regression curves were created
using the coefficients provided by the software and
the formula y = EXP(a  x + b)/[1 + EXP(a  x + b)]. Fig. 2. Effect of follicle diameter (mm) at TAI on the probability of
When effect of a class with more than two levels was ovulation in postpartum Nelore cows (P < 0.01).
detected, Bonferroni test was used to compare
means. In Experiment 1, for analysis of ovulation, For all experiments, differences were considered
conception, and pregnancy rates, the final models significant when P < 0.05, whereas tendencies were
included the effects of treatment, BCS at Day 0, considered when 0.1 < P 0.05.
cyclicity, and follicle diameter at Day 11. In
Experiment 2, for analysis of pregnancy rate, the 3. Results
final model included the effects of treatment, BCS at
Day 0, parity, and follicle diameter at Day 11. In 3.1. Experiment 1
Experiment 3, for the analysis of ovulation, concep-
tion, and pregnancy rates, the final models included Diameter of the largest follicle measured at TAI did
the effects of treatment, BCS, cyclicity, parity, and not differ among treatment groups (Control, 10.18
follicle diameter at Day 11. In Experiment 4, for the Æ 0.13 mm; eCG300, 10.13 Æ 0.14 mm; eCG400,
analysis of pregnancy rate, the final model included 10.37 Æ 0.13 mm; TW, 10.28 Æ 0.13 mm; P > 0.1) or
the effects of breeding season (2006–2007 and 2007– BCS at Day 0 (2.5, 10.30 Æ 0.21 mm; 3.0, 10.34 Æ
2008), breed of cow (Bos indicus, Bos taurus, or 0.14 mm; 3.5, 10.38 Æ 0.11 mm; P < 0.1) but was
crossbred Bos indicus  Bos taurus), category (nulli- affected by cyclicity (Anestrous, 10.49 Æ 0.08 mm;
parous, suckled primiparous, suckled multiparous, or Cycling, 10.09 Æ 0.13 mm; P < 0.01). Follicle dia-
nonsuckled multiparous), BCS at TAI ( 2.5, 3.0, or meter positively affected ovulation (P < 0.01; Fig. 2)
!3.5), days postpartum at initiation of the protocol and pregnancy rate (P < 0.01; Fig. 3) but had no effects
(30 to 60 d, 61 to 90 d, or 91 to 150 d), sire, AI on conception rate (P > 0.1). Treatments and BCS did
technician, location, farm within location, and cow not affect ovulation and conception rates (P > 0.1;
group within farm. Table 1). Pregnancy rate was affected by treatment and
Follicle diameter on Day 11 data were analyzed BCS (P < 0.05; Table 1). Cyclicity did not affect
using PROC GLM of the SAS program. A previous test ovulation, conception, or pregnancy rates (P > 0.1;
for normality of residues (Shapiro-Wilk test) and Table 1).
homogeneity of variances (F-max test) indicated that
variables fulfilled the assumptions for analysis of
variance. For the least squares analysis of variance
models, all two-way interactions were tested, and
variables having P > 0.2 were removed from the final
models. For the analysis of follicle diameter on Day 11,
in Experiment 1, the final model included the effects of
treatment, BCS at Day 0, and cyclicity; in Experiment 2,
the final model included the effects of treatment, BCS at
Day 0, and parity; in Experiment 3, the final model
included the effects of treatment, BCS at Day 0, parity,
and cyclicity. When effects of a class with more than
two levels were detected, t-test was used for mean Fig. 3. Effect of follicle diameter (mm) at TAI on the probability of
separation. pregnancy in postpartum Nelore cows (P < 0.01).

5. 214 O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218
Table 1
Ovulation, conception, and pregnancy rates of suckled Bos indicus cows treated in Experiments 1 and 2.
Item Proportion, n (%)
Ovulation rate a Conception rateb Pregnancy ratec
Experiment 1
Treatmentd
Control 181/221 (81.9) 92/181 (50.8) 92/221 (41.6)x
eCG300 168/208 (80.8) 97/168 (57.7) 97/208 (46.6)xy
eCG400 187/223 (83.9) 121/187 (64.7) 121/223 (54.3)y
TW 207/239 (86.6) 123/207 (59.4) 123/239 (51.5)y
Body condition scoree
2.5 199/247 (80.6) 105/199 (52.8) 105/247 (42.5)x
3.0 341/407 (83.8) 202/341 (59.2) 202/407 (49.6)xy
3.5 203/237 (85.7) 126/203 (62.1) 126/237 (53.2)y
Cycling statusf
Anestrous 498/600 (83.0) 282/498 (56.6) 282/600 (47.0)
Cycling 245/291 (84.2) 151/245 (61.6) 151/291 (51.9)
Experiment 2
Treatmentg
TW – – 83/198 (41.9)w
eCG400 – – 81/187 (43.3)w
Folltropin – – 74/216 (34.3)z
Pluset – – 36/112 (32.1)z
Body condition scoree
2.25 – – 126/325 (38.8)
2.5 – – 65/195 (33.3)
2.75 – – 83/193 (43.0)
Parity
Primiparous – – 24/80 (30.0)w
Multiparous – – 250/633 (39.5)z
x,y
Means with different letters differ (P < 0.05; Bonferroni test). w,zMeans with different letters tend to differ (P < 0.1; Bonferroni test).
a
Percentage of cows ovulating after PGF2a compared with all cows treated.
b
Percentage of cows pregnant to TAI compared with cows that ovulated.
c
Percentage of cows pregnant to TAI compared with all cows treated.
d
Cows received no treatment (Control), 300 IU eCG (eCG300), 400 IU eCG (eCG400), or temporary weaning (48 h; TW) on Day 9 relative to
initiation of an ovulation synchronization protocol.
e
Body condition scores assessed on a 1 to 5 scale.
f
Cycling status of cows was previously evaluated by two ultrasound examinations on Days 0 and 7. Cows with evidence of having luteal tissue
during at least one of the examinations were considered cyclic.
g
Cows received temporary weaning (48 h; TW), 400 IU eCG (eCG400), 10 mg Folltropin (Folltropin), or 25 IU Pluset (Pluset) on Day 9 relative to
initiation of a TAI protocol.
3.2. Experiment 2 3.3. Experiment 3
Diameter of the largest follicle measured at TAI did not Diameter of the largest follicle measured at TAI
differ among treatment groups (TW, 13.49 Æ 0.19 mm; did not differ among treatment groups (TW,
eCG400, 13.40 Æ 0.19 mm; Folltropin, 13.45 Æ 0.17 12.67 Æ 0.15 mm; TW + eCG200, 12.66 Æ 0.15 mm;
mm; Pluset, 13.32 Æ 0.27 mm; P > 0.1) or BCS at TW + eCG400, 12.56 Æ 0.12 mm; P > 0.1), parity
Day 0 (2.25, 13.58 Æ 0.15 mm; 2.5, 13.31 Æ 0.19 mm; (Primiparous, 12.72 Æ 0.17 mm; Multiparous, 12.54
2.75, 13.32 Æ 0.18 mm; P > 0.1) but was affected by Æ 0.09 mm; P > 0.1), or BCS (2.5, 12.64 Æ 0.15 mm;
parity (Primiparous, 12.25 Æ 0.32 mm; Multiparous, 3.0, 12.63 Æ 0.14 mm; 3.5, 12.62 Æ 0.17 mm; P > 0.1)
13.51 Æ 0.11 mm; P < 0.01). Follicle diameter posi- but was affected by cyclicity (Anestrous, 12.67 Æ
tively affected pregnancy rate (P < 0.01; Fig. 3). 0.09 mm; Cycling, 12.19 Æ 0.20 mm; P < 0.05). Fol-
Pregnancy rate tended to be affected by treatment and licle diameter positively affected ovulation (P < 0.01;
parity (P < 0.1) but was not affected by BCS (P > 0.1; Fig. 2) and pregnancy rate (P < 0.01; Fig. 3) but had no
Table 1). effects on conception rate (P > 0.1). Treatments, BCS,

6. O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218 215
cyclicity, and parity did not affect ovulation, concep- from 50.1% to 60.0% at 27 farms; and from 60.1% to
tion, or pregnancy rates (P > 0.1; Table 2). 68.0% at 6 farms. Pregnancy rate was affected
(P < 0.01) by breed (Bos indicus, 48.3% [26,123 of
3.4. Experiment 4 54,145]; Bos taurus, 61.7% [3652 of 5922]; and
crossbred Bos indicus  Bos taurus, 50.7% [2011 of
Pregnancy rate did not differ between breeding 3966]), category (nulliparous, 39.6% [2095 of 5290];
seasons, and no interactions between breeding season suckled primiparous, 45.2% [3924 of 8677]; suckled
and other explanatory variables were found. Grouping multiparous, 51.8% [24,245 of 46,767]; and nonsuckled
data from 2006–2007 and 2007–2008 breeding seasons, multiparous, 46.1% [1522 of 3299]), BCS at TAI ( 2.5,
overall pregnancy rate at TAI was 49.6% (31,786 of 43.0% [3409 of 7923]; 3.0, 49.6% [18,958 of 38,229];
64,033). Pregnancy rate did not differ among location and !3.5, 52.7% [9419 of 17,881]). Days postpartum at
(P > 0.1) but varied among farm within location beginning of protocol did not affect pregnancy rate (30
(results ranging between 26.8% and 68.0%; to 60 d, 47.6% [4228 of 8881]; 61 to 90 d, 51.7%
P < 0.01) and cow group within farms. During the [16,325 of 31,572]; and 91 to 150 d, 50.8% [7616 of
2006–2007 breeding season, pregnancy rate ranged 14,991]; P > 0.1). Pregnancy rate was also consistently
from 29.5% to 40.0% at 10 farms; from 40.1% to 50.0% affected (P < 0.01) by sire (results ranging from 7.2%
at 26 farms; from 50.1% to 60.0% at 30 farms; and from to 77.3%) and AI technician (results ranging from
60.1% to 65.0% at 5 farms. During the 2007–2008 15.1% to 81.8%).
breeding season, pregnancy rate ranged from 26.8% to
40.0% at 11 farms; from 40.1% to 50.0% at 27 farms; 4. Discussion
Table 2 In beef cows, suckling and negative energy balance
Ovulation, conception, and pregnancy rates of suckled Bos indicus are the two most important factors increasing the length
cows treated in Experiment 3. of anestrus due to their negative effect on frequency of
Item Proportion, n (%) LH pulses and follicular development [3]. Because the
size of the dominant follicle at the end of protocols is an
Ovulation ratea Conception rate b Pregnancy ratec
important factor affecting ovulation and pregnancy
d
Treatment rates [1,4,5], associations of estrus/ovulation synchro-
TW 264/297 (88.9) 158/264 (59.8) 158/297 (53.2)
nization protocols and treatments that increase fre-
TW + eCG200 278/299 (93.0) 155/278 (55.8) 155/299 (51.8)
TW + eCG400 261/291 (89.7) 152/261 (58.2) 152/291 (52.2) quency of LH pulses or directly stimulate follicle
development are likely to improve pregnancy rates. In
Body condition scoree
2.5 276/310 (89.0) 147/276 (53.3) 147/310 (47.4)
this series of studies, we observed that pregnancy rates
3.0 260/285 (91.2) 153/260 (58.9) 153/285 (53.7) were improved with strategies that provided gonado-
3.5 268/292 (91.8) 165/268 (61.6) 165/292 (56.5) tropic support during proestrus after a TAI protocol,
Cycling statusf such as temporary weaning or intramuscular treatment
Anestrous 673/745 (90.3) 381/673 (56.6) 381/745 (51.1) with 400 IU eCG. We also found that BCS and parity
Cycling 130/142 (91.6) 84/130 (64.6) 84/142 (59.2) were reliable factors to predict pregnancy rate in cows
Parity submitted to the TAI protocol and that sire and AI
Primiparous 186/202 (92.1) 107/186 (57.5) 107/202 (53.0) technician were critical for the success of TAI
Multiparous 618/685 (90.2) 358/618 (57.9) 358/685 (52.3) programs.
a
Percentage of cows ovulating after PGF2a compared with all cows Results from Experiment 1 confirmed those of
treated. previous studies in which temporary calf removal
b
Percentage of cows pregnant to TAI compared with cows that improved pregnancy rate of postpartum cows submitted
ovulated. to hormonal treatments for induction of ovulation [4,6].
c
Percentage of cows pregnant to TAI compared with all cows
treated. Short-term temporary weaning increased gonadotropin-
d
Cows received temporary weaning (48 h; TW), temporary wean- releasing hormone (GnRH) and LH pulse frequency, so
ing plus 200 IU eCG (TW + eCG200), or temporary weaning plus 400 that pulse frequency was similar to those in proestrus [7]
IU eCG (TW + eCG400) on Day 9 relative to initiation of a TAI and increased ovulation rate in response to GnRH and
protocol. EB treatments [4]. A dominant follicle that is exposed to
e
Body condition scores assessed on a 1 to 5 scale.
f
Cycling status of cows was previously evaluated by two ultrasound
higher frequency of LH pulses may have a more
examinations on Days 0 and 7. Cows with evidence of having luteal desirable final maturation [8] and improved preovula-
tissue during at least one of the examinations were considered cyclic. tory estradiol production, which is important to activate

7. 216 O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218
responsiveness of the pituitary to GnRH stimulus [9], treatment, in disagreement to a previous report in which
preovulatory LH secretion [10], and to provide an treatments with 10 or 20 mg Folltropin-V were similar
adequate oviductal/uterine environment for embryo to 400 IU eCG in anestrous Bos indicus cattle submitted
development [11,12]. The frequency of LH pulses also to TAI [24].
positively affects follicular growth after divergence, Some important differences among physiologic
which improves likelihoods of estrus and ovulation responses to the treatments may explain results from
[1,4,5,13]. Cows submitted to temporary weaning had Experiment 2. Temporary weaning for 48 h improves
greater secretion of LH after exogenous GnRH LH pulse frequency during the period when calves were
treatment [14], and this may be another factor positively separated from their dams [7]; eCG has both FSH- and
affecting ovulation rate and luteinization. Temporary LH-like effects and a long half-life, remaining active in
weaning is a low-cost intervention that in combination the blood for as long as 5 to 7 d after administration
with the proposed protocol provided an opportunity to [18,25]. Folltropin-V and Pluset are reported to contain
improve fertility in postpartum Bos indicus cows. 87% [26] and 50% FSH, respectively, and their
Similar to temporary weaning, intramuscular treat- metabolism seems to be relatively rapid as, after
ment with 400 IU eCG improved pregnancy rates in intramuscular administration, the half-life and the
postpartum cows, in agreement with previous studies disappearance of porcine FSH were estimated at 5 h
[15,16]. The length of the half-life of eCG is relatively and 10 to 12 h, respectively [27]. These differences in
long [17], and it has the capacity to bind to both LH and half-life between eCG and FSH are well reported in
FSH receptors [18], which may stimulate theca and superovulatory protocol studies, in which a single
granulosa cells of the dominant follicle [19] and also intramuscular dose of eCG is sufficient to stimulate
progesterone secretion by the early corpus luteum [15]. growth of multiple follicles [28,29], whereas to
Thus, eCG treatment potentially improves follicular superstimulate a cow with FSH, it was necessary to
development and provides a more adequate endocrine use multiple intramuscular administrations [30], a
environment during proestrus (greater circulating single intramuscular dose with slow-release vehicles
estradiol concentrations) and diestrus (greater circulat- [31], or a single subcutaneous dose [32]. Thus, the poor
ing progesterone concentrations), which is favorable for effect of FSH treatment found in Experiment 2 may be
fertility. Interestingly, in Experiment 3, treatments with due to an inadequate dosage used in this study and/or a
either 200 or 400 IU eCG did not improve conception or short half-life of FSH, which may not remain for a
pregnancy rates in cows that were temporarily weaned. sufficient time in circulation to stimulate follicle
This indicates that temporary weaning likely stimulated development.
adequate secretion of LH in a manner that additional Several negative effects on fertility may occur in
gonadotropin support may not be required for final cows having small follicles at proestrus, such as lower
follicular development. ovulation rate [1,4,5,13,33], formation of smaller
As follicles grow and bypass the follicular diver- corpora lutea with lower capacity of progesterone
gence stage, their dependence on FSH and LH for production [33], lower growth and capacity of produc-
growth is decreased and increased, respectively. This tion of IFN-t of embryos [34], insufficient preovulatory
change in gonadotropin dependence is due to an estradiol secretion [5,33], alterations on oviductal and
increase in the abundance of mRNA expression and uterine environments [11,12,35], higher percentage of
number of LH receptors in granulosa cells of the cows experiencing premature luteolysis [33,36], and
dominant follicle [20,21]. Despite the fact that the increased likelihood of pregnancy losses [8]. Interest-
dominant follicle depends more on LH than on FSH for ingly, in Experiments 1 and 3, cycling cows had smaller
growth, the level of FSH receptor mRNA did not change follicles at time of AI than those of anestrous cows,
with stage of the first follicular wave in cattle [20], and which may be due to circulating concentrations of
the ability of granulosa cells to bind FSH did not vary progesterone in cycling cows. At high concentrations,
significantly with follicular size [22]. Furthermore, low progesterone decreases LH pulse frequency in a manner
concentrations of FSH at the time of divergence are still that dominant follicles have less gonadotropic support
required for continued growth of the largest follicle for growth [37]. Considering the mentioned negative
[23]. This indicates that FSH may exert some stimulus effects of ovulation of small follicles, it would be
on dominant follicles, which was the basis of the expected that poorer fertility results in cycling cows, but
hypothesis of Experiment 2. However, in Experiment 2, in Experiments 1 and 3 there was no effect of cyclicity
cows treated with FSH had lower pregnancy rates than on ovulation, conception, or pregnancy rates, which
that of cows that received temporary weaning or eCG may be due to the fact that ovulatory stimulus treatment

8. O.G.S. Filho et al. / Theriogenology 72 (2009) 210–218 217
was estradiol cypionate and that most of the cows in Differences in breed, BCS, category, sire, and AI
these experiments received treatments to improve technician were noted in this study and may account for
gonadotropic support during proestrus (i.e., TW and some of the variation in reproductive performance that
eCG). was observed between and within farms.
In Experiment 4, the lack of effect of days In conclusion, the proposed TAI protocol should be
postpartum on pregnancy rates of cows greater than associated with 48 h temporary weaning or intramuscular
30 d postpartum at the beginning of the synchronization treatment with 400 IU eCG to improve pregnancy rate.
protocol indicates that this is not the most reliable Because this protocol is efficient in synchronizing
variable on predicting fertility with the TAI protocol ovulation, factors like sire and AI technician are critically
used in this study. In contrast, category and BCS important variables that affect pregnancy rates. When
significantly affected pregnancy rates and appear to be using this protocol with adequate sire/semen quality and
reliable predictors of probability of pregnancy when the well-trained AI technicians, results can be predicted with
proposed TAI protocol is used, as we have previously satisfactory accuracy, considering BCS and parity of
reported [38]. Cows in negative energy balance are cows. The variation between farms and between cow
more sensitive to the negative feedback effect of groups within a similar farm may be due to the interaction
estradiol on gonadotropin release, and this may be the of the several factors affecting TAI results.
explanation of why cows with greater BCS had higher
pregnancy rates: cattle with greater BCS are likely to be
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