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Theriogenology 72 (2009) 210218

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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 20062007 (n = 27,195) and 20072008 (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

In a previous study [1] we proposed a protocol that
allowed fixed-time artificial insemination (TAI) in
anestrous and cycling postpartum Bos indicus cows,
which consisted of insertion of an intravaginal device
* Corresponding author. Tel.: +55 14 3811 7185;
fax: +55 14 3811 7180.
E-mail address: vasconcelos@fca.unesp.br (J.L.M. Vasconcelos).
estradiol benzoate (EB) on Day 0; 12.5 mg im dinoprost
tromethamine (PGF2a) on Day 7; CIDR withdrawal
plus 0.5 mg im estradiol cypionate (ECP) plus
temporary weaning on Day 9; TAI (48 h after CIDR
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
affecting pregnancy rates in cows submitted to that
protocol.

0093-691X/$ see front matter # 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2009.02.008
 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218
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
follicle development; (2) evaluate if temporary weaning
could be excluded from or replaced by eCG or FSH
treatments in the protocol; (3) evaluate whether eCG
treatment would improve fertility of cows submitted to
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
locations in Brazil.
2. Materials and methods
2.1. Experiment 1
The goal of this experiment was to evaluate the
effects of temporary weaning for 48 h or treatment with
two doses of eCG (300 IU or 400 IU; 1.5 or 2.0 mL of
Folligon [Intervet, Sao Paulo, SP, Brazil]) on the
ovulation, conception, and pregnancy rates of suckled
Nelore cows submitted to a synchronization of
ovulation protocol based on progesterone, EB, PGF2a,
and ECP. The hypothesis was that inclusion of
temporary weaning, treatment with 300 IU eCG, or
treatment with 400 IU eCG in the protocol would
improve ovulation and pregnancy rates of cows without
altering conception rates.
Suckled multiparous Nelore cows (n = 891; 40 to
90 d postpartum; body condition score (BCS) at Day 0
between 2.5 and 3.5 on a 1 to 5 scale [2]), maintained
in Brachiaria decumbens pasture with ad libitum
access to water and mineral supplement, were treated
with the following basic synchronization of ovulation
protocol (Fig. 1): insertion of an intravaginal insert
containing 1.9 g progesterone (CIDR; Controlled
Intravaginal Drug Release, Pfizer Animal Health,
Sao Paulo, SP, Brazil) plus 2.0 mg im EB (2.0 mL of
Estrogin; Farmavet, Sao Paulo, SP, Brazil) on Day 0;
and 12.5 mg im PGF2a (2.5 mL of Lutalyse; Pfizer
Animal Health) on Day 7. On Day 9, all cows had
CIDR inserts removed and received 0.5 mg im ECP
(0.25 mL of E.C.P.; Pfizer Animal Health). Immedi-
ately after CIDR withdrawal, cows were assigned
randomly to receive one of the following treatments:
(1) cows received no additional treatment (Control;
n = 221); (2) cows received 300 IU im eCG (1.5 mL of
Folligon) immediately after CIDR withdrawal
211
Fig. 1. Schematic diagram of the basic synchronization of ovulation
protocol. EB (2.0 mg), 2.0 mg im estradiol benzoate; CIDR (9 d),
treatment with an intravaginal insert containing 1.9 g progesterone
during 9 d; PGF2a (12.5 mg), 12.5 mg im dinoprost tromethamine;
ECP (0.5 mg), 0.5 mg im estradiol cypionate; TAI, fixed-time artificial
insemination.
(eCG300; n = 208); (3) cows received 400 IU im
eCG (2.0 mL of Folligon) immediately after CIDR
withdrawal (eCG400; n = 223); (4) cows had their
calves temporarily weaned for 48 h, beginning
immediately after CIDR withdrawal (TW; n = 239).
In this and the following experiments, during
temporary weaning time, calves were held in pens
out of sight from their dams, with ad libitum access to
water. On Day 11 (48 h after CIDR withdrawal), all
cows received TAI. Calves submitted to temporary
weaning were reunited with their dams immediately
after insemination. Cycling status of cows was
previously evaluated by two ultrasound examinations
(Aloka SSD-500 with a 7.5 MHz linear-array trans-
rectal transducer; Tokyo, Japan) on Days 0 and 7.
Cows with evidence of luteal tissue during at least one
of the examinations (n = 291) were considered cyclic.
Diameter of the largest follicle was determined on day
of TAI by averaging the length and width measure-
ments from ovarian transrectal ultrasonography. The
rates of ovulation, conception, and pregnancy were
evaluated by ultrasound examinations. Ovulation was
considered when cows had presence of a follicle with
diameter 8.5 mm on Day 11 and absence of this
follicle 48 h later (Day 13). Pregnancy diagnosis was
performed 28 d after TAI. Conception rate was
calculated by dividing the number of pregnant cows
by number of cows that ovulated. Pregnancy rate was
calculated by dividing the number of pregnant cows
by the number of treated cows.
2.2. Experiment 2
The goal of this experiment was to evaluate the
effects of replacing temporary weaning or eCG with
FSH on the diameter of ovulatory follicle and the
pregnancy rates of suckled Nelore cows submitted to a
synchronization of ovulation protocol based on pro-
gesterone, EB, PGF2a, and ECP. The hypothesis was
212 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218
that FSH could replace temporary weaning or eCG
treatment in the protocol.
Suckled Nelore cows (n = 713; 30 to 90 d
postpartum; BCS at Day 0 between 2.25 and 2.75),
maintained in Brachiaria brizantha pasture with ad
libitum access to water and mineral supplement, were
treated with the same basic synchronization of
ovulation protocol described for Experiment 1. On
Day 9, cows were assigned randomly to receive one
of the following treatments: (1) cows had their
calves temporarily weaned for 48 h, beginning
immediately after CIDR withdrawal (TW; n = 198);
(2) cows received 400 IU im eCG immediately after
CIDR withdrawal (eCG400; n = 187); (3) cows
received 10 mg im FSH (0.5 mL of Folltropin-V;
Bioniche Animal Health, Belleville, ON, Canada;
FSH:LH proportion >80.0%) immediately after
CIDR withdrawal (Folltropin; n = 216); (4) cows
received 25 IU im FSH (0.5 mL of Pluset; Hertape-
Calier, Sao Paulo, SP, Brazil; FSH:LH proportion
about 50.0%) immediately after CIDR withdrawal
(Pluset; n = 112). On Day 11 (48 h after CIDR
withdrawal), all cows received TAI. Calves submitted
to temporary weaning were reunited with their dams
immediately after insemination. Follicle diameter and
pregnancy rates were determined as described for
Experiment 1.
2.3. Experiment 3
The goal of this experiment was to evaluate the
effects of two doses of eCG (200 IU or 400 IU) on the
ovulation, conception, and pregnancy rates of suckled
Nelore cows submitted to a synchronization of
ovulation protocol based on progesterone, EB, PGF2a,
ECP, and temporary weaning. The hypothesis was that
treatment with either 200 or 400 IU eCG would improve
reproductive performance in cows treated with a
synchronization of ovulation protocol that involved
temporary weaning from the progesterone insert with-
drawal until the time of insemination.
Suckled Nelore cows (n = 887; 40 to 110 d
postpartum; BCS at Day 0 between 2.5 and 3.5),
maintained in Brachiaria decumbens pasture with ad
libitum access to water and mineral supplement, were
treated with the same basic synchronization of
ovulation protocol described in Experiment 1. On
Day 9, all cows were submitted to temporary weaning
and assigned randomly to receive one of the following
treatments: (1) cows received no additional treatment
(TW; n = 297); (2) cows received 200 IU im eCG
immediately after CIDR withdrawal (TW + eCG200;
n = 299); (3) cows received 400 IU im eCG immedi-
ately after CIDR withdrawal (TW + eCG400; n = 291).
On Day 11, 48 h after CIDR withdrawal, all cows were
inseminated and calves reunited with their dams
immediately after insemination. Cycling status of cows
was previously evaluated by two ultrasound examina-
tions on Days 0 and 7. Cows with evidence of luteal
tissue during at least one of the examinations (n = 142)
were considered cyclic. Follicular diameter, ovulation,
conception, and pregnancy rates were determined as
described for Experiment 1.
2.4. Experiment 4
The objective was to retrospectively assess factors
affecting pregnancy rate of heifers and cows treated
with a synchronization of ovulation protocol based
on progesterone, EB, PGF2a, and ECP. Lactating
and nonlactating beef cows and heifers from various
locations in Brazil during the 20062007 (n = 27,195)
and 20072008 (n = 36,838) breeding seasons were
submitted to the same basic synchronization of
ovulation protocol described in Experiment 1. In
suckled cows, a 48 h temporary weaning was performed
between CIDR withdrawal and TAI. BCS was evaluated
always on Day 0 on a 1 to 5 scale [2]. In both breeding
seasons, the cattle were from the same 71 farms in the
following Brazilian states: Goias (n = 23), Mato Grosso
(n = 20), Mato Grosso do Sul (n = 9), Minas Gerais
(n = 9), Para (n = 6), Parana (n = 2), and Rondonia
(n = 2). Cows were managed according to local
procedures, and in all places nutritional management
was based only on grass pastures with water and
appropriate ad libitum access to a mineral supplement.
Each breeding season lasted from October until March
of the following year but varied among farms. Only data
recorded from cows with no recorded reproductive
abnormalities were used for analysis. Pregnancy was
diagnosed by transrectal ultrasonography at 28 to 35 d
after TAI.
2.5. Statistical analyses
Binomial variables such as ovulation, conception,
and pregnancy rates were analyzed using PROC
LOGISTIC of the SAS program (SAS Institute Inc.,
Cary, NC, USA). Explanatory variables such as
treatment, BCS at Day 0, parity, categorized number
of days postpartum, cyclicity, breeding season, location,
and farm were used in the model as classes. Follicle
diameter at Day 11, considered a covariate, was
previously submitted to univariate analysis and
 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218 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
Walds 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  0.13 mm; eCG300, 10.13  0.14 mm; eCG400,
analysis of pregnancy rate, the final model included 10.37  0.13 mm; TW, 10.28  0.13 mm; P > 0.1) or
the effects of breeding season (20062007 and 2007 BCS at Day 0 (2.5, 10.30  0.21 mm; 3.0, 10.34 
2008), breed of cow (Bos indicus, Bos taurus, or 0.14 mm; 3.5, 10.38  0.11 mm; P < 0.1) but was
crossbred Bos indicus  Bos taurus), category (nulli- affected by cyclicity (Anestrous, 10.49  0.08 mm;
parous, suckled primiparous, suckled multiparous, or Cycling, 10.09  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).
214 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218

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
differ among treatment groups (TW, 13.49  0.19 mm;
eCG400, 13.40  0.19 mm; Folltropin, 13.45  0.17
mm; Pluset, 13.32  0.27 mm; P > 0.1) or BCS at
Day 0 (2.25, 13.58  0.15 mm; 2.5, 13.31  0.19 mm;
2.75, 13.32  0.18 mm; P > 0.1) but was affected by
parity (Primiparous, 12.25  0.32 mm; Multiparous,
13.51  0.11 mm; P < 0.01). Follicle diameter posi-
tively affected pregnancy rate (P < 0.01; Fig. 3).
Pregnancy rate tended to be affected by treatment and
parity (P < 0.1) but was not affected by BCS (P > 0.1;
Table 1).
Diameter of the largest follicle measured at TAI
did not differ among treatment groups (TW,
12.67  0.15 mm; TW + eCG200, 12.66  0.15 mm;
TW + eCG400, 12.56  0.12 mm; P > 0.1), parity
(Primiparous, 12.72  0.17 mm; Multiparous, 12.54
 0.09 mm; P > 0.1), or BCS (2.5, 12.64  0.15 mm;
3.0, 12.63  0.14 mm; 3.5, 12.62  0.17 mm; P > 0.1)
but was affected by cyclicity (Anestrous, 12.67 
0.09 mm; Cycling, 12.19  0.20 mm; P < 0.05). Fol-
licle diameter positively affected ovulation (P < 0.01;
Fig. 2) and pregnancy rate (P < 0.01; Fig. 3) but had no
effects on conception rate (P > 0.1). Treatments, BCS,
 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218 215

cyclicity, and parity did not affect ovulation, concep-
tion, or pregnancy rates (P > 0.1; Table 2).
3.4. Experiment 4
Pregnancy rate did not differ between breeding
seasons, and no interactions between breeding season
and other explanatory variables were found. Grouping
data from 20062007 and 20072008 breeding seasons,
overall pregnancy rate at TAI was 49.6% (31,786 of
64,033). Pregnancy rate did not differ among location
(P > 0.1) but varied among farm within location
(results ranging between 26.8% and 68.0%;
P < 0.01) and cow group within farms. During the
20062007 breeding season, pregnancy rate ranged
from 29.5% to 40.0% at 10 farms; from 40.1% to 50.0%
at 26 farms; from 50.1% to 60.0% at 30 farms; and from
60.1% to 65.0% at 5 farms. During the 20072008
breeding season, pregnancy rate ranged from 26.8% to
40.0% at 11 farms; from 40.1% to 50.0% at 27 farms;
from 50.1% to 60.0% at 27 farms; and from 60.1% to
68.0% at 6 farms. Pregnancy rate was affected
(P < 0.01) 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]), BCS 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 pregnancy rate (30
to 60 d, 47.6% [4228 of 8881]; 61 to 90 d, 51.7%
[16,325 of 31,572]; and 91 to 150 d, 50.8% [7616 of
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 AI technician (results ranging from
15.1% to 81.8%).
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
216 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218
responsiveness of the pituitary to GnRH stimulus [9],
preovulatory LH secretion [10], and to provide an
adequate oviductal/uterine environment for embryo
development [11,12]. The frequency of LH pulses also
positively affects follicular growth after divergence,
which improves likelihoods of estrus and ovulation
[1,4,5,13]. Cows submitted to temporary weaning had
greater secretion of LH after exogenous GnRH
treatment [14], and this may be another factor positively
affecting ovulation rate and luteinization. Temporary
weaning is a low-cost intervention that in combination
with the proposed protocol provided an opportunity to
improve fertility in postpartum Bos indicus cows.
Similar to temporary weaning, intramuscular treat-
ment with 400 IU eCG improved pregnancy rates in
postpartum cows, in agreement with previous studies
[15,16]. The length of the half-life of eCG is relatively
long [17], and it has the capacity to bind to both LH and
FSH receptors [18], which may stimulate theca and
granulosa cells of the dominant follicle [19] and also
progesterone secretion by the early corpus luteum [15].
Thus, eCG treatment potentially improves follicular
development and provides a more adequate endocrine
environment during proestrus (greater circulating
estradiol concentrations) and diestrus (greater circulat-
ing progesterone concentrations), which is favorable for
fertility. Interestingly, in Experiment 3, treatments with
either 200 or 400 IU eCG did not improve conception or
pregnancy rates in cows that were temporarily weaned.
This indicates that temporary weaning likely stimulated
adequate secretion of LH in a manner that additional
gonadotropin support may not be required for final
follicular development.
As follicles grow and bypass the follicular diver-
gence stage, their dependence on FSH and LH for
growth is decreased and increased, respectively. This
change in gonadotropin dependence is due to an
increase in the abundance of mRNA expression and
number of LH receptors in granulosa cells of the
dominant follicle [20,21]. Despite the fact that the
dominant follicle depends more on LH than on FSH for
growth, the level of FSH receptor mRNA did not change
with stage of the first follicular wave in cattle [20], and
the ability of granulosa cells to bind FSH did not vary
significantly with follicular size [22]. Furthermore, low
concentrations of FSH at the time of divergence are still
required for continued growth of the largest follicle
[23]. This indicates that FSH may exert some stimulus
on dominant follicles, which was the basis of the
hypothesis of Experiment 2. However, in Experiment 2,
cows treated with FSH had lower pregnancy rates than
that of cows that received temporary weaning or eCG
treatment, in disagreement to a previous report in which
treatments with 10 or 20 mg Folltropin-V were similar
to 400 IU eCG in anestrous Bos indicus cattle submitted
to TAI [24].
Some important differences among physiologic
responses to the treatments may explain results from
Experiment 2. Temporary weaning for 48 h improves
LH pulse frequency during the period when calves were
separated from their dams [7]; eCG has both FSH- and
LH-like effects and a long half-life, remaining active in
the blood for as long as 5 to 7 d after administration
[18,25]. Folltropin-V and Pluset are reported to contain
87% [26] and 50% FSH, respectively, and their
metabolism seems to be relatively rapid as, after
intramuscular administration, the half-life and the
disappearance of porcine FSH were estimated at 5 h
and 10 to 12 h, respectively [27]. These differences in
half-life between eCG and FSH are well reported in
superovulatory protocol studies, in which a single
intramuscular dose of eCG is sufficient to stimulate
growth of multiple follicles [28,29], whereas to
superstimulate a cow with FSH, it was necessary to
use multiple intramuscular administrations [30], a
single intramuscular dose with slow-release vehicles
[31], or a single subcutaneous dose [32]. Thus, the poor
effect of FSH treatment found in Experiment 2 may be
due to an inadequate dosage used in this study and/or a
short half-life of FSH, which may not remain for a
sufficient time in circulation to stimulate follicle
development.
Several negative effects on fertility may occur in
cows having small follicles at proestrus, such as lower
ovulation rate [1,4,5,13,33], formation of smaller
corpora lutea with lower capacity of progesterone
production [33], lower growth and capacity of produc-
tion of IFN-t of embryos [34], insufficient preovulatory
estradiol secretion [5,33], alterations on oviductal and
uterine environments [11,12,35], higher percentage of
cows experiencing premature luteolysis [33,36], and
increased likelihood of pregnancy losses [8]. Interest-
ingly, in Experiments 1 and 3, cycling cows had smaller
follicles at time of AI than those of anestrous cows,
which may be due to circulating concentrations of
progesterone in cycling cows. At high concentrations,
progesterone decreases LH pulse frequency in a manner
that dominant follicles have less gonadotropic support
for growth [37]. Considering the mentioned negative
effects of ovulation of small follicles, it would be
expected that poorer fertility results in cycling cows, but
in Experiments 1 and 3 there was no effect of cyclicity
on ovulation, conception, or pregnancy rates, which
may be due to the fact that ovulatory stimulus treatment
 O.G.S. Filho et al. / Theriogenology 72 (2009) 210218
was estradiol cypionate and that most of the cows in
these experiments received treatments to improve
gonadotropic support during proestrus (i.e., TW and
eCG).
In Experiment 4, the lack of effect of days
postpartum on pregnancy rates of cows greater than
30 d postpartum at the beginning of the synchronization
protocol indicates that this is not the most reliable
variable on predicting fertility with the TAI protocol
used in this study. In contrast, category and BCS
significantly affected pregnancy rates and appear to be
reliable predictors of probability of pregnancy when the
proposed TAI protocol is used, as we have previously
reported [38]. Cows in negative energy balance are
more sensitive to the negative feedback effect of
estradiol on gonadotropin release, and this may be the
explanation of why cows with greater BCS had higher
pregnancy rates: cattle with greater BCS are likely to be
in a better energy status, or experience a less severe
postpartum negative energy balance period, and,
therefore, their follicular development and probability
of ovulation may be greater [3,6,16].
In Experiment 4, fertility at TAI was found to be
different among categories. The difference between
suckled primiparous and multiparous cows is likely due
to differences in energy status in these categories of
cows. Primiparous beef cows in pasture-based systems
have prolonged periods of postpartum anestrus [39] due
to their greater nutrient requirements associated with
growth and lactation. The majority of heifers analyzed
in Experiment 4 were Bos indicus (97.4%), and the
poorer pregnancy rates found in this category are in
agreement with previous reports [40,41], which might
have been due to treatment of heifers that had not
reached puberty or to a poorer response of cycling
heifers to the synchronization of ovulation protocol
used [40,41]. Cycling Bos indicus heifers treated with
CIDR likely maintain higher circulating concentrations
of progesterone than did Bos taurus heifers [42,43], and
this increased concentration of progesterone during the
protocol might negatively affect follicle development
and fertility [8,40,41]. The negative effect of proges-
terone in cycling cattle receiving a CIDR may also
affect cows [41], as pregnancy rates in nonlactating
cows, which are more likely to be cycling, were lower
than in lactating multiparous cows.
The great range in pregnancy rate of cows according
to sire and AI technician indicated that these factors are
critical for the success of TAI programs. Obtaining a
50% pregnancy rate in Bos indicus cattle submitted to
TAI is a reachable benchmark [44], and the current
series of studies showed that this goal is achievable.
217
Differences in breed, BCS, category, sire, and AI
technician were noted in this study and may account for
some of the variation in reproductive performance that
was observed between and within farms.
In conclusion, the proposed TAI protocol should be
associated with 48 h temporary weaning or intramuscular
treatment with 400 IU eCG to improve pregnancy rate.
Because this protocol is efficient in synchronizing
ovulation, factors like sire and AI technician are critically
important variables that affect pregnancy rates. When
using this protocol with adequate sire/semen quality and
well-trained AI technicians, results can be predicted with
satisfactory accuracy, considering BCS and parity of
cows. The variation between farms and between cow
groups within a similar farm may be due to the interaction
of the several factors affecting TAI results.
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