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Theriogenology 72 (2009) 179189

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Fixed-time artificial insemination with estradiol and progesterone

for Bos indicus cows I: Basis for development of protocols
M. Meneghetti a, O.G. Sa Filho 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, FL 32446, USA
Received 29 September 2008; received in revised form 5 February 2009; accepted 8 February 2009

Abstract
Five experiments were conducted on commercial farms in Brazil aiming to develop a fixed-time artificial insemination (TAI)
protocol that achieved pregnancy rates between 40% and 55% in Bos indicus cows. These studies resulted in the development of the
following protocol: insertion of an intravaginal device containing 1.9 g of progesterone (CIDR) plus 2.0 mg im estradiol benzoate
on Day 0; 12.5 mg im dinoprost tromethamine on Day 7 in cycling cows or on Day 9 in anestrous cows; CIDR withdrawal plus
0.5 mg im estradiol cypionate plus temporary calf removal on Day 9; TAI (48 h after CIDR withdrawal) plus reuniting of calves
with their dams on Day 11. Reduced dose of prostaglandin F2a (PGF2a; 12.5 mg im dinoprost tromethamine) effectively caused
luteolysis. In cycling cows, fertility was greater when the treatment with PGF2a was administered on Day 7 than on Day 9, but in
anestrous cows, no effects of time of the PGF2a treatment were found. Estradiol cypionate effectively replaced estradiol benzoate or
gonadotropin-releasing hormone as the ovulatory stimulus, reducing labor and cost. In this protocol, CIDR inserts were successfully
used four times (9 d each use) with no detrimental effects on fertility.
# 2009 Elsevier Inc. All rights reserved.

Keywords: Bos indicus; Estradiol; Fixed-time artificial insemination; Pregnancy rate; Progesterone

1. Introduction reduced estrus detection and pregnancy rates and

In tropical climates, most beef cattle herds rear Bos
indicus cattle due to their greater capacity to adapt to
environmental challenges of climate and disease.
Despite this adaptation, Bos indicus cattle have longer
periods of postpartum anestrus [1], which contributes to
poorer reproductive efficiency of zebu cows when
compared with that of Bos taurus. In addition, length of
estrous behavior in Bos indicus cattle is shorter than that
of European breeds [2]. These factors together lead to
* Corresponding author. Tel.: +55 14 3811 7185;
fax: +55 14 3811 7180.
E-mail address: vasconcelos@fca.unesp.br (J.L.M. Vasconcelos).
increased calving-conception intervals in tropical beef
herds.
Several treatments have been developed recently
allowing induction of ovulation in anestrous cows and
artificial insemination without the need for detection of
estrus. In this series of studies, our goals were to develop
a fixed-time artificial insemination (TAI) protocol for
Bos indicus cows. The basis for the protocol was to (1)
synchronize follicle wave emergence with progesterone
plus estradiol; (2) increase dominant follicle develop-
ment with an intravaginal progesterone insert plus
temporary weaning; (3) prevent premature luteolysis
after a synchronized ovulation by treating anestrous
cows before the synchronized ovulation with proges-
terone; (4) reduce the cost of the protocol by decreasing

0093-691X/$ see front matter # 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2009.02.010
180 M. Meneghetti et al. / Theriogenology 72 (2009) 179189
the dosage of prostaglandin F2a (PGF2a), replacing
gonadotropin-releasing hormone (GnRH) with estradiol
as an ovulatory stimuli, and using the same insert as
many as 4 times.
2. Materials and methods
2.1. Experiment 1
The goal of this experiment was to evaluate the effect
of decreasing the PGF2a dose on the incidence of corpus
luteum (CL) regression, ovulation, conception, and
pregnancy rates of suckled cycling Nelore cows subjected
to synchronization of ovulation using a protocol that
utilized progesterone, estradiol benzoate, and temporary
weaning. The hypothesis was that reducing the dose of
PGF2a by 50% would not alter the rates of CL regression,
ovulation, conception, and pregnancy.
Suckled multiparous Nelore cows (n = 932; 40 to
110 d postpartum) with body condition score (BCS) 3.0
to 4.0 on a 1 to 5 scale [3] were evaluated by ultrasound
(Aloka SSD-500 with a 7.5 MHz linear-array transrec-
tal transducer; Tokyo, Japan) to determine presence of
CL on Day 0. In this and in the following studies, BCS
was evaluated always on Day 0 by the same
veterinarian. Cows with evidence of luteal tissue
(n = 370) received a synchronization of ovulation
protocol and were maintained on a Brachiaria
decumbens pasture with ad libitum access to water
and a mineral supplement during the experimental
period. On Day 0, all cows received an intravaginal
insert containing 1.9 g progesterone (CIDR; Controlled
Intravaginal Drug Release, Pfizer Animal Health, Sao
Paulo, SP, Brazil) and 2.0 mg im estradiol benzoate
(EB; 2.0 mL of Estrogin; Farmavet, Sao Paulo, SP,
Brazil). On Day 9, CIDRs were removed, and calves
were temporally weaned (Fig. 1). In this and the
following experiments, during temporary weaning time
(48 h), calves were held in pens out of sight from their
dams and had ad libitum access to water. On Day 7,
cows were evaluated by ultrasound, and those having
Fig. 1. Schematic diagram of the basic synchronization 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; TW
(48 h), temporary weaning for 48 h.
luteal tissue (n = 250) were randomly assigned to
receive either 12.5 mg im (half-dose; n = 124) or
25.0 mg im (full-dose; n = 126) dinoprost trometha-
mine (Lutalyse; Pfizer Animal Health). Cows with no
luteal tissue visible by ultrasound on Day 7 were
excluded from the analyses. On Day 10, cows received
1.0 mg im EB, and TAI was performed 30 to 36 h later
(Day 11). Immediately after TAI, calves were returned
to their dams. Diameter of the largest follicle was
determined on Day 11 by averaging the length and
width measurements from ovarian ultrasonography. The
rates of visual CL regression, ovulation, conception,
and pregnancy were evaluated by ultrasound examina-
tions. Corpus luteum regression was considered in cows
that did not have a visible CL on Day 13 or had a
residual luteal tissue but ovulated in response to EB
treatment. Ovulation was considered when cows had
presence of a follicle with diameter 8.5 mm at 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, and
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 investigate the
effects of three different protocols using estradiol
cypionate (ECP), EB, or gonadorelin (GnRH) as
ovulatory stimuli on ovulation, conception, and
pregnancy rates of suckled Nelore cows subjected to
a synchronization of ovulation protocol that used
progesterone, EB, PGF2a, and temporary weaning.
The hypothesis was that the three ovulatory stimuli
would have similar efficiency.
Suckled Nelore cows (n = 945; 45 to 80 d
postpartum; BCS 3.0 to 4.0), maintained on a
Brachiaria decumbens pasture with ad libitum access
to water and a mineral supplement, were treated with
the synchronization of ovulation protocol described in
Fig. 1 plus 12.5 mg im dinoprost tromethamine on Day
7. On Day 9, cows were assigned randomly to receive
one of the following treatments: (1) cows received
0.5 mg im estradiol cypionate (0.25 mL of E.C.P.; Pfizer
Animal Health) immediately after CIDR withdrawal
(ECP; n = 315); (2) cows received 1.0 mg im estradiol
benzoate 24 h (Day 10) after CIDR withdrawal (EB;
n = 318); (3) cows received 100 mg im gonadorelin
(1.0 mL of Fertagyl; Intervet, Sao Paulo, SP, Brazil)
48 h (Day 11) after CIDR withdrawal (GnRH; n = 312).
On Day 11 (48 h after CIDR withdrawal), all cows were
 M. Meneghetti et al. / Theriogenology 72 (2009) 179189
inseminated and calves reunited with their dams
immediately after insemination. Cycling status of cows
was evaluated by two ultrasound examinations on Days
0 and 7. Cows with evidence of having luteal tissue
during at least one of the examinations (n = 150) were
considered cyclic. Follicular diameter on Day 11,
ovulation, conception, 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 administering a CIDR that had been previously used
once or twice (9 d each use) on the ovulation, conception,
and pregnancy rates of suckled Nelore cows subjected to
a synchronization of ovulation protocol that used
progesterone, EB, PGF2a, ECP, and temporary weaning.
The hypothesis was that a CIDR that had been used
previously once or twice (9 d each use) could be used with
the same efficiency as that of new CIDR inserts.
Suckled Nelore cows (n = 1487; 30 to 90 d
postpartum; BCS 3.0 to 4.0), maintained on a
Brachiaria decumbens pasture with ad libitum access
to water and mineral supplement, were treated with the
synchronization of ovulation protocol described in
Fig. 1 plus 12.5 mg im dinoprost tromethamine on Day
7 plus 0.5 mg im ECP on Day 9. On Day 11 (48 h after
CIDR withdrawal), all cows were inseminated and
calves reunited with their dams immediately after
insemination. Treatment groups were as follows: (1)
CIDR inserts used in this group had never been
previously used (CIDRnew; n = 541); (2) CIDR inserts
from this group had been used previously for 9 d
(CIDR2nd-use; n = 532); and (3) CIDR inserts from this
group had been used previously twice, 9 d each use
(CIDR3rd-use; n = 414). Cycling status of cows was
evaluated as described in Experiment 2. A total of 236
cows were considered cyclic. Follicular diameter on
Day 11, ovulation, conception, and pregnancy rates
were determined as described for Experiment 1.
The used inserts were, after each previous use,
washed with water and dipped in iodophor with 2.6%
iodine (Biocid; Pfizer Animal Health) diluted with
water (1:100) for 15 min. The inserts were then rinsed
with water, air dried, and placed in plastic bags at room
temperature until treatments were performed. This same
procedure was applied in Experiment 4.
2.4. Experiment 4
The goal of this experiment was to evaluate the
effects of administering a CIDR that had been
181
previously used 3 times (9 d each use) on the ovulation,
conception, and pregnancy rates of suckled Nelore cows
subjected to a synchronization of ovulation protocol
that used progesterone, EB, PGF2a, ECP, and temporary
weaning. The hypothesis was that a CIDR that had been
used previously 3 times (9 d each use) could be used
with the same efficiency as that of CIDR inserts used
less than 3 times.
Suckled multiparous Nelore cows (n = 578; 30 to 60
d postpartum; BCS 2.5 to 3.5), maintained on a
Brachiaria humidicola pasture with ad libitum access to
water and mineral supplement, were treated with the
synchronization of ovulation protocol described in Fig 1
plus 12.5 mg im dinoprost tromethamine plus 0.5 mg
im ECP on Day 9. On Day 11 (48 h after CIDR
withdrawal), all cows were inseminated and calves
reunited with their dams immediately after insemina-
tion. Treatment groups were as follows: (1) CIDR
inserts used in this group had never been previously
used (CIDRnew; n = 69); (2) CIDR inserts from this
group had been used previously once for 9 d (CIDR2nd-
use; n = 180); (3) CIDR inserts from this group had
been used previously twice, 9 d each use (CIDR3rd-use;
n = 224); (4) CIDR inserts from this group had been
used previously 3 times, 9 d each use (CIDR4th-use;
n = 105).
On Day 9, cows were submitted to ovarian
ultrasonography. In a subgroup of 60 cows (15 from
each treatment group) with absence of CL, blood
samples were collected from the coccygeal vein into
Vacutainer tubes (Becton Dickinson Co., Franklin
Lakes, NJ, USA) for progesterone concentration
analysis. Blood was allowed to clot at 4 8C for 24 h
and centrifuged at 1500  g for 15 min at 25 8C. Serum
was removed and frozen at 20 8C until assays were
performed. Serum concentrations of progesterone were
determined using a solid-phase radioimmunoassay kit
containing antibody-coated tubes and 125I-labeled
progesterone (Coat-a-count; Diagnostic Products Cor-
poration, Los Angeles, CA, USA) according to the
manufacturers instructions. The assay sensitivity was
0.01 ng/mL, and the intra-assay CV was 4.25%.
Follicular diameter on Day 11, ovulation, conception,
and pregnancy rates were determined as described for
Experiment 1.
2.5. Experiment 5
The goal of this experiment was to evaluate the effects
of time of PGF2a treatment (48 h before vs. immediately
after CIDR withdrawal) on pregnancy rates of suckled
Nelore cows subjected to a synchronization of ovulation
182 M. Meneghetti et al. / Theriogenology 72 (2009) 179189
protocol that used progesterone, EB, PGF2a, ECP, and
temporary weaning. The hypothesis was that the
PGF2a treatment given 2 d before CIDR withdrawal in
cows having a CL at this time of the protocol may
improve pregnancy rate, whereas in anestrous cows there
would be no effect of time of PGF2a treatment on
pregnancy rate.
Suckled multiparous Nelore cows (n = 1332; 30 to
90 d postpartum; BCS 2.5 to 3.5), maintained on a
Brachiaria brizantha pasture with ad libitum access to
water and mineral supplement, were treated with the
synchronization of ovulation protocol described in
Fig. 1 plus 0.5 mg im ECP on Day 9. On Day 7, cows
were submitted to ovarian ultrasonography to determine
presence (n = 326) or absence (n = 1006) of luteal
tissue. Within each luteal status group, cows were
randomly assigned to receive 12.5 mg im dinoprost
tromethamine either on Day 7 (PGF2ad7) or on Day 9
(PGF2ad9) of the protocol (With CLPGF2ad7,
n = 171; With CLPGF2ad9, n = 155; Without CL
PGF2ad7, n = 603; Without CLPGF2ad9, n = 403). On
Day 11 (48 h after CIDR withdrawal), all cows were
inseminated and calves reunited with their dams
immediately after insemination. Blood samples were
collected on Day 9 from a random subgroup of 76 cows
with presence of CL (38 from each time of PGF2a
treatment) for progesterone concentration analysis as
described for Experiment 4. The assay sensitivity was
0.01 ng/mL, and the intra-assay CV was 1.3%.
2.6. Statistical analyses
Binomial variables such as CL regression, ovula-
tion, 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, and cyclicity
were used in the model as classes. Explanatory
variables considered covariates, such as follicle
diameter on Day 11 and serum concentration of
progesterone on Day 9, were previously subjected to
univariate analysis and when found to be significant
were included in the multivariate models. For
conception and pregnancy data, original models also
included the effects of sire and artificial insemination
(AI) technician. For all logistic regression models, all
two-way interactions were tested. In the final logistic
regression model, variables were removed by a
backward elimination (according to the criterion of
Wald) when P > 0.2. When a 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)].
When effect of a class with more than two levels
was detected, Bonferroni test was used to compare
means. In Experiment 1, for analysis of CL regression
rate, the final model included effects of treatment and
BCS at Day 0; for analysis of ovulation, conception,
and pregnancy rates, the final models included the
effects of treatment, BCS at Day 0, and follicle
diameter on Day 11. In Experiments 2 and 3, for the
analysis of ovulation, conception, and pregnancy
rates, the final models included the effects of
treatment, BCS at Day 0, cyclicity, parity, and
follicular diameter on Day 11. In Experiment 4, for
the analysis of ovulation, conception, and pregnancy
rates, the final models included the effects of
treatment and follicular diameter on Day 11. In
Experiment 5, for the analysis of pregnancy rate, the
final model included the effects of treatment,
presence of corpus luteum on Day 7, BCS at Day
0, and the interaction between treatment and presence
of corpus luteum on Day 7. A further analysis was
performed to evaluate the effect of serum progester-
one concentration on Day 9 on probability of
pregnancy.
Continuous data, such as serum concentration of
progesterone on Day 9 and follicle diameter on Day 11,
were analyzed using PROC GLM of the SAS program.
A previous test for normality of residues (Shapiro-Wilk
test) and 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 and BCS at Day 0; in Experiments 2
and 3, the final models included the effects of treatment,
BCS at Day 0, parity, and cyclicity. In Experiment 4, for
the analysis of serum progesterone concentration on
Day 9 and follicle diameter on Day 11, the final models
included the effect of treatment. In Experiment 5, for
the analysis of serum progesterone concentration on
Day 9, the final model included the effects of treatment,
presence of corpus luteum on Day 7, BCS at Day 0, and
the interaction between treatment and presence of
corpus luteum on Day 7. When effects of a class with
more than two levels were detected, t-test was used for
mean separation.
For all experiments, differences were considered
significant when P < 0.05, whereas tendencies were
considered when 0.1 < P  0.05.
 M. Meneghetti et al. / Theriogenology 72 (2009) 179189 183

3. Results Table 1
Ovulation conception, and pregnancy rates of suckled Bos indicus
cows treated in Experiments 1 and 2.
3.1. Experiment 1
Item Proportion, n (%)
Diameter of the largest follicle measured at the TAI Ovulation ratea Conception rateb Pregnancy ratec
did not differ between treatments (Half-dose, 12.30 Experiment 1
 0.37 mm; Full-dose, 11.96  0.35 mm; P > 0.1) or Treatmentd
BCS (3.0, 12.06  0.74 mm; 3.5, 12.06  0.40 mm; 4.0, Half-dose 110/124 (88.7) 76/110 (69.1) 76/124 (61.3)
12.27  0.26 mm; P > 0.1). Follicle diameter positively Full-dose 119/126 (94.4) 81/119 (68.1) 81/126 (64.3)
affected ovulation rate (P < 0.05; Fig. 2) but had no Body condition scoree
3.0 27/30 (90.0) 18/27 (66.7) 18/30 (60.0)
effects on conception and pregnancy rates (P > 0.1). The 3.5 68/72 (94.4) 47/68 (69.1) 47/72 (65.3)
rate of structural CL regression was not affected by 4.0 134/148 (90.5) 92/134 (68.7) 92/148 (62.2)
treatment and BCS (96.0%; P > 0.1). Treatments and
Experiment 2
BCS did not affect ovulation, conception, or pregnancy Treatmentf
rates (P > 0.1; Table 1). ECP 282/315 (89.5) 160/282 (56.7) 160/315 (50.8)
EB 289/318 (90.9) 165/289 (57.1) 165/318 (51.9)
3.2. Experiment 2 GnRH 284/312 (91.0) 169/284 (59.5) 169/312 (54.2)
Body condition scoree
3.0 329/369 (89.2) 176/329 (53.5) 176/369 (47.7)
Diameter of the largest follicle measured at the TAI 3.5 288/316 (91.1) 170/288 (59.0) 170/316 (53.8)
did not differ among treatment groups (ECP, 12.12 4.0 238/260 (91.5) 148/238 (62.2) 148/260 (56.9)
 0.13 mm; EB, 12.02  0.13 mm; GnRH, 12.31  Cycling statusg
0.13 mm; P > 0.1) or parity (Primiparous, 12.38  Anestrous 718/795 (90.3) 406/718 (56.6) 406/795 (51.1)
Cycling 137/150 (91.3) 88/137 (64.2) 88/150 (58.7)
0.11 mm; Multiparous, 12.31  0.14 mm; P > 0.1) but
Parity
was affected by BCS (3.0, 11.80  0.14 mm; 3.5, Primiparous 197/214 (92.1) 114/197 (57.9) 114/214 (53.3)
12.25  0.13 mm; 4.0, 12.41  0.12 mm; P < 0.05) Multiparous 658/731 (90.0) 380/658 (57.8) 380/731 (52.0)
and cyclicity (Anestrous, 12.36  0.07 mm; Cycling, a
Percentage of cows ovulating after PGF2a compared with all cows
11.94  0.17 mm; P < 0.05). Follicle diameter posi- treated.
tively affected ovulation (P < 0.01; Fig. 2) and b
Percentage of cows pregnant to TAI compared with cows that
pregnancy rate (P < 0.01; Fig. 3) but had no effects on ovulated.
c
conception rate (P > 0.1). Treatments, BCS, cyclicity, Percentage of cows pregnant to TAI compared with all cows
treated.
and parity did not affect ovulation, conception, or d
Cows treated with 12.5 mg (Half-dose) or 25 mg (Full-dose)
pregnancy rates (P > 0.1; Table 1). dinoprost tromethamine on Day 7 of a TAI protocol.
e
Body condition scores assessed on a 1 to 5 scale [3].
f
3.3. Experiment 3 Cows were treated with estradiol cypionate (ECP) on Day 9,
estradiol benzoate (EB) on Day 10, or gonadotropin-releasing hor-
mone (GnRH) on Day 11 relative to initiation of a TAI protocol.
Diameter of the largest follicle measured at the TAI g
Cycling status of cows was previously evaluated by two ultrasound
did not differ among treatment groups (CIDRnew, examinations on Days 0 and 7. Cows with evidence of having luteal
12.08  0.14 mm; CIDR2nd-use, 12.23  0.11 mm; tissue during at least one of the examinations were considered cyclic.

CIDR3rd-use, 12.21  0.12 mm; P > 0.1) or parity

Fig. 2. Effect of follicle diameter (mm) at TAI on the probability of
ovulation in postpartum Nelore cows (P < 0.01).
(Primiparous, 12.37  0.15 mm; Multiparous, 12.27
 0.10 mm; P > 0.1) but was affected by BCS
(3.0, 11.88  0.14 mm; 3.5, 12.30  0.13 mm; 4.0,
12.50  0.13 mm; P < 0.05) and cyclicity (Anestrous,
12.46  0.09 mm; Cycling, 11.98  0.17 mm; P <
0.05). Follicle 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, cyclicity, and parity did not affect
ovulation, conception, or pregnancy rates (P > 0.1;
Table 2).
184 M. Meneghetti et al. / Theriogenology 72 (2009) 179189

Table 2
Ovulation, conception, and pregnancy rates of suckled Bos indicus
cows treated in Experiments 3 and 4.
Item Proportion, n (%)
Ovulation ratea Conception rateb Pregnancy ratec
Experiment 3
Treatmentd
CIDRnew 496/541 (91.7) 278/496 (56.1) 278/541 (51.4)
CIDR2nd-use 482/532 (90.6) 286/482 (59.3) 286/532 (53.8)
CIDR3rd-use 373/414 (90.1) 208/373 (55.8) 208/414 (50.2)
Body condition scoree
3.0 525/580 (90.5) 279/525 (53.1) 279/580 (48.1)
Fig. 3. Effect of follicle diameter (mm) at TAI on the probability of 3.5 452/498 (90.8) 267/452 (59.1) 267/498 (53.6)
pregnancy in postpartum Nelore cows (P < 0.01). 4.0 374/409 (91.4) 226/374 (60.4) 226/409 (55.3)
Cycling statusf
3.4. Experiment 4 Anestrous 1136/1251 (90.8) 642/1136 (56.5) 642/1251 (51.3)
Cycling 215/236 (91.1) 130/215 (60.5) 130/236 (55.1)
Parity
Diameter of the largest follicle measured at the TAI Multiparous 1042/1151 (90.5) 597/1042 (57.3) 597/1151 (51.9)
did not differ among treatment groups (CIDRnew, Primiparous 309/336 (92.0) 178/309 (57.6) 178/336 (53.0)
10.99  0.22 mm; CIDR2nd-use, 11.29  0.14 mm; Experiment 4
CIDR3rd-use, 11.49  0.12 mm; CIDR4th-use, 11.55 Treatmentg
 0.18 mm; P > 0.1). Follicle diameter positively CIDRnew 55/69 (79.7) 30/55 (54.5) 30/69 (43.5)
affected ovulation (P < 0.01; Fig. 2) and pregnancy rate CIDR2nd-use 144/180 (80.0) 88/144 (61.1) 88/180 (48.9)
CIDR3rd-use 188/224 (83.9) 113/188 (60.1) 113/224 (50.5)
(P < 0.01; Fig. 3) but had no effects on conception rate
CIDR4th-use 90/105 (85.7) 52/90 (57.8) 52/105 (49.5)
(P > 0.1). Treatments did not affect ovulation, concep-
a
tion, or pregnancy rates (P > 0.1; Table 2) but affected Percentage of cows ovulating after PGF2a compared with all cows
treated.
serum concentrations of progesterone on Day 9 b
Percentage of cows pregnant to TAI compared with cows that
of the protocol (P < 0.05; Fig. 4). Only one cow in ovulated.
CIDR4th-use treatment had serum concentration of c
Percentage of cows pregnant to TAI compared with all cows
progesterone < 1.0 ng/mL on Day 9 (0.56 ng/mL). treated.
d
Cows received a not previously used CIDR (CIDRnew), a CIDR
used previously once for 9 d (CIDR2nd-use), or a CIDR used pre-
3.5. Experiment 5
viously twice, 9 d each use (CIDR3rd-use) at initiation of a TAI
protocol.
There was interaction between presence of CL at e
Body condition scores assessed on a 1 to 5 scale [3].
f
Day 7 and treatment on serum progesterone concentra- Cycling status of cows was previously evaluated by two ultrasound
tion at Day 9 (P < 0.05). Treatment with PGF2a on Day examinations on Days 0 and 7 of a TAI protocol. Cows with evidence
of having luteal tissue during at least one of the examinations were
7 decreased serum progesterone concentration at Day 9
considered cyclic.
in cows with presence of CL (PGF2ad7, 4.44  0.58 ng/ g
Cows received a not previously used CIDR (CIDRnew), a CIDR
mL vs. PGF2ad9, 6.03  0.62; P < 0.05) but not in used previously once for 9 d (CIDR2nd-use), a CIDR used previously
cows without CL (4.32  0.66 ng/mL). Serum con- twice, 9 d each use (CIDR3rd-use), or a CIDR used previously 3 times,
centrations of progesterone at Day 9 were not affected 9 d each use (CIDR4th-use) at initiation of a TAI protocol.
by BCS (P > 0.1). Pregnancy rate was negatively
affected by serum concentration of progesterone on Day long length of postpartum acyclicity. Because of this
9 (P < 0.05; Fig. 5) and positively affected by BCS characteristic, treatments that induce cyclicity and
(P < 0.05; Table 3). Treatment with PGF2a on Day 7 of allow TAI have large positive impacts in beef herds.
the protocol improved pregnancy rate in cows with With this series of studies, we proposed a synchroniza-
presence of CL (P < 0.05) but had no effects on cows tion of ovulation protocol that used EB plus progester-
with absence of CL (P > 0.1; Table 3). one for synchronization of new follicular wave
recruitment and provided satisfactory rates of ovulation
4. Discussion (90%) and pregnancy (50%) in anestrous and
cycling cows. Confirming our previous expectations,
The most important challenge in suckled beef cows, 12.5 mg im treatment with dinoprost tromethamine did
mainly Bos indicus cattle in tropical climates, is the not reduce ovulation and pregnancy rates, 0.5 mg im
 M. Meneghetti et al. / Theriogenology 72 (2009) 179189
Fig. 4. Serum concentration of progesterone (P4) on Day 9 (least
square means  SEM) of a TAI protocol in anestrous Nelore cows that
received a not previously used CIDR (CIDRnew), a CIDR used
previously once for 9 d (CIDR2nd-use), a CIDR used previously
twice, 9 d each use (CIDR3rd-use), or a CIDR used previously 3
times, 9 d each use (CIDR4th-use) at initiation of a TAI protocol
(Experiment 4; P < 0.05).
treatment with ECP could be satisfactorily used as an
ovulatory stimulus on Day 9, and CIDR inserts could be
used as many as 4 times with no detrimental effects on
fertility of postpartum Nelore cows. Also, pregnancy
rate in cows with the presence of a CL on Day 7 was
found to be greater with PGF2a treatment on Day 7 than
on Day 9.
Synchronization of recruitment of a new follicular
wave is the first step of a TAI protocol. Pharmacolo-
gically, this can be performed by inducing ovulation of
the dominant follicle or by induction of follicular
atresia. In protocols using GnRH to synchronize
follicular wave emergence (e.g., Ovsynch protocol),
induction of ovulation in a high percentage of treated
cows at the start of treatment is critical for obtaining
satisfactory results, as shown previously in dairy cattle
[4]. The follicular dynamics in anestrous cows is
characterized by several nonovulatory waves of follicle
Fig. 5. Relationship between serum concentration of progesterone on
Day 9 of a TAI protocol and the probability of pregnancy in cycling
Nelore cows (Experiment 5; P < 0.05).
185
Table 3
Pregnancy rates of suckled Bos indicus cows treated in Experiment 5.
Item Pregnancy ratea
Proportion, n (%)
b c
CL presence Treatment
With CLPGF2ad7 86/171 (50.3)x
With CLPGF2ad9 56/155 (36.1)y
Without CLPGF2ad7 285/603 (47.3)x
Without CLPGF2ad9 195/403 (48.4)x
Body condition scored
2.5 204/507 (40.2)x
3.0 154/344 (44.8)x
3.5 264/481 (54.9)y
a
Percentage of cows pregnant to TAI compared with all cows
treated.
b
Presence of CL was evaluated by ultrasound examination on Day 7
of a TAI protocol.
c
Cows treated with 12.5 mg dinoprost tromethamine on Day 7 (48 h
before CIDR withdrawal; PGF2ad7) or on Day 9 (immediately after
CIDR withdrawal; PGF2ad9) of a TAI protocol.
d
Body condition scores assessed on a 1 to 5 scale [3].
x ,y
Means with different letters differ (P < 0.05; Bonferroni test).
growth, in a manner that follicle deviation occurs but
does not reach final stages of development, due to
insufficient frequency of luteinizing hormone (LH)
pulses [5]. Therefore, the probability of ovulation to a
GnRH treatment is lower in anestrous than in cycling
cows [6,7]. Furthermore, anestrous cows that do not
ovulate to the first ovulatory stimulus but ovulate to the
second are likely to have premature luteolysis because
ovulation occurs with no preexposure to progesterone
[79]. Although exogenous progesterone can be
provided between first and second ovulatory stimuli
to avoid premature luteolysis, cows not ovulating to the
first ovulatory stimulus do not have synchronized
follicular wave, reducing the probability of success of
the protocol [4,7].
In this series of studies, we used protocols based on
synchronization of follicular wave emergence with
progesterone plus estradiol, because this treatment was
more efficient in postpartum cows under tropical
climates than those based on synchronization of
follicular wave emergence with GnRH [10]. Although
there is a range (3 to 6 d) in the time that follicular wave
begins [11,12], this treatment resulted in satisfactory
results in both anestrous and cycling beef cows [13].
Treatment with progesterone plus estradiol efficiently
suppressed FSH and follicular growth, synchronizing
follicular wave emergence in bovine females regardless
of the stage of follicular development at the time of
treatment [14]. Furthermore, these protocols provide
exogenous progesterone necessary to prevent premature
186 M. Meneghetti et al. / Theriogenology 72 (2009) 179189
luteolysis after first postpartum ovulation. One concern
regarding previously used CIDR inserts is whether it
provides sufficient progesterone to prevent premature
luteolysis in anestrous cows after first ovulation. The
facts that conception rates were similar between cows
receiving new versus used CIDRs, in both Experiments
3 and 4, and that serum progesterone concentrations
were >1.0 ng/mL in cows without CL treated with a
CIDR previously used 3 times indicated that progester-
one provided by a CIDR used as many as 4 times (9 d
each use) was sufficient to prevent premature luteolysis
in anestrous cows induced to ovulate. However, limited
literature is available comparing treatments with inserts
previously used 2 or 3 times. Cerri et al. [15] reported
that treatment with a 7-d used CIDR (containing 1.38 g
progesterone) was as efficacious as a new CIDR at
increasing induction of cyclicity in anestrous high-
producing dairy cows. In contrast with our findings,
Colazo et al. [16] observed that Bos taurus beef cows
treated with twice-used CIDRs had lower pregnancy
rate than did those treated with new or once-used
inserts. A possible explanation for this difference may
be due to physiologic differences between genetic
groups. Carvalho et al. [17], comparing Bos indicus, Bos
taurus, and crossbred heifers, verified that treatment
with CIDR in Bos indicus heifers resulted in higher
circulating progesterone concentrations and lower
follicular growth and ovulation rates than those in
the other breeds. In fact, we found in Experiment. 5 a
negative linear effect of serum concentration of
progesterone on Day 9 on pregnancy rate to TAI,
which may have been due to lower frequency of LH
pulses and, consequently, lower rates of dominant
follicle growth and ovulation in cows with higher
progesterone in the last days of treatment with CIDR
[1821]. Also, in Experiments 2 and 3, it was
surprising that anestrous cows had greater follicular
diameter at Day 11 than did cycling cows. It is
possible that an optimal range of circulating proges-
terone concentration during the protocol exists, such
that high progesterone concentrations cause negative
effects on follicle development and fertility [19]. In
cycling cows treated with protocols with EB plus
progesterone, because there are two sources of
progesterone (CIDR and CL), this concern may be
more critical, and previously used inserts that release
less progesterone could reduce these negative impacts
on reproduction [18,19]. The lack of effect of number
of CIDR uses on follicular diameter at TAI and on
ovulation rate, found in Experiments 3 and 4, indicates
that the number of uses of the inserts did not alter
follicular development in postpartum cows, probably
due to the high incidence of animals without CL and
use of PGF2a, on Day 7.
The second step of a synchronization of ovulation
protocol is a decrease in circulating concentrations of
progesterone for the beginning of proestrus and
development of the ovulatory follicle. The interval
between beginning of proestrus and ovulation may
affect LH secretion [20,21], follicular development and
steroidogenic capacity [22,23], and uterine environment
[23,24]. Considering the 3- to 4-d interval between EB
plus progesterone treatment and recruitment of the new
follicular wave, it is expected that deviation will occur
around 7 d after the beginning of the protocol. In cycling
cows, administration of PGF2a treatment on Day 7
ensures a high rate of luteolysis [25] and causes a
decrease in circulating concentrations of progesterone
when the follicle becomes dependent on LH for
development [26]. This may be why, in Experiment
5, cows with CL receiving PGF2a treatment on Day 7
had greater pregnancy rate than that of those receiving
PGF2a treatment on Day 9, and the lack of effect of
treatment in cows without CL may be attributed to the
fact that the only role of PGF2a treatment in the protocol
is to induce luteolysis, which is not necessary in
noncycling cows. Regarding dosages of PGF2a, pre-
vious studies demonstrated that reduced doses of PGF2a
administered via an intravulvo-submucosal route were
as effective at inducing luteolysis as doses recom-
mended by the manufacturers that were administered
intramuscularly [6,27]. In Experiment 1, the lack of
effect of treatment on follicle diameter at TAI and rates
of visual CL regression and ovulation indicate that a
50% reduction in the recommended dose of dinoprost
tromethamine when administered intramuscularly was
as effective in causing luteolysis in Nelore cows, with a
CL older than 7 d, as the standard recommended dose.
Induction of ovulation is the third step of a TAI
protocol and can be performed with drugs that aim to
cause ovulation of potential preovulatory follicles.
Induction of ovulation can be achieved using hormones
such as human chorionic gonadotrophin or LH, which
act directly on follicles, or hormones that induce
preovulatory surge of LH such as GnRH or esters of
estradiol. Intervals between injection and ovulation,
however, vary for each product. The reason to compare
protocols with different timings of ovulatory stimuli
treatment in Experiment 2 was to reduce labor and
management with animals, as stress is an important
negative factor affecting LH secretion [28] and Bos
indicus are more temperamental than Bos taurus breeds
[29]. Furthermore, estradiol esters are less expensive
drugs than GnRH. With circulating concentrations of
 M. Meneghetti et al. / Theriogenology 72 (2009) 179189
progesterone <1.0 ng/mL, EB induced an LH surge and
ovulation 16 and 40 h after intramuscular treatment,
respectively [30]. Therefore, treatment with EB should
be given 30 to 36 h before TAI to increase the
probability of fertilization. When the ovulatory stimulus
was GnRH, ovulation occurred at 24 to 32 h after
intramuscular treatment [31]. Traditionally, a GnRH
treatment 48 h after induction of luteolysis and AI 16 h
after GnRH treatment is recommended, but other
studies have indicated that intramuscular GnRH
treatment can be done at TAI with acceptable fertility
[32]. Estradiol cypionate caused ovulation 55.4 h after
1 mg intramuscular treatment when administered to
cows after induction of luteolysis [33]. Studies
indicated that replacing the last GnRH treatment of
the Ovsynch protocol for an intramuscular ECP
treatment given 24 h after induction of luteolysis
(48 h before TAI) resulted in satisfactory pregnancy
rates [34]. However, Colazo et al. [35] evaluating
protocols with progesterone plus estradiol, reported that
ECP treatment could be given at the time of
progesterone release insert withdrawal and TAI 48 h
later with no negative effects on pregnancy rate. In
Experiment 2, there was no effect of ovulatory stimulus
on pregnancy rates, which concurs with previous studies
[6,34]. Because ECP is less expensive than GnRH
treatment and time of administration requires less labor
than does EB treatment, ECP treatment is indicated as
the ovulatory stimulus to be used in synchronization
protocols in Nelore cows.
In Experiments 2, 3, and 4, there were positive
effects of follicle diameter at TAI on ovulation and
pregnancy rates, in agreement with Vasconcelos et al.
[7] and Sa Filho et al. [9], which may be explained by
the improvement on ovulatory capacity as follicles grow
[36]. However, in the current studies, there were no
effects of follicle diameter at TAI on conception rates, in
contrast with a study in which conception rate was
lower when beef cows ovulated small follicles [37]. In
that study, ovulation was induced with GnRH likely
prior to peak estradiol concentrations in most cows, and,
interestingly, in cows exhibiting estrus behavior there
was no effect of follicle diameter on conception.
Therefore, the endocrine environment during proestrus
may be a more consistent predictor of fertility than is
size of ovulatory follicle, as also suggested by Mussard
et al. [23]. An inadequate endocrine environment during
proestrus (e.g., low frequency of LH pulses, smaller
ovulatory follicle, and/or low circulating estradiol
concentrations) may lead to alterations in uterine
environment [23,24] and higher incidence of premature
luteolysis in the subsequent estrous cycle [9,3840].
187
Vasconcelos et al. [8] demonstrated that temporary
weaning increased the proportion of cows exhibiting
estrus behavior even when dominant follicles were
small (7 mm), and Souza [41] observed, in lactating
dairy cattle, that supplementation with estradiol before
the second GnRH treatment of the Ovsynch protocol
improved conception rate in primiparous cows, cows
with lower BCS, and at first postpartum service. The
lack of effect of follicle diameter on conception rate
observed in the current study may be due to the use of
temporary weaning and estradiol (except in Experiment
2, in which 33.0% of cows received GnRH) at the end of
protocols, which could provide a better endocrine
environment during proestrus, improving conception
rate even in cows ovulating smaller follicles [35].
In conclusion, we obtained satisfactory pregnancy
rates (40% to 55%) in postpartum Bos indicus cattle
with the following protocol: insertion of a CIDR insert
plus 2.0 mg im EB on Day 0; CIDR withdrawal plus
12.5 mg im dinoprost tromethamine plus 0.5 mg im
ECP + temporary weaning on Day 9; TAI (48 h after
CIDR withdrawal) plus reuniting of calves with their
dams on Day 11. In cycling cows, anticipation of
dinoprost tromethamine treatment to Day 7 improved
pregnancy rates. Estradiol cypionate could replace EB
or GnRH as the ovulatory stimulus, reducing labor and
cost. With this protocol, the CIDR may be used as many
as 4 times with no negative effects on pregnancy rates.
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