Animal Reproduction Science 129 (2011) 132–139

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Induction of ovarian follicular wave emergence and ovulation in

progestin-based timed artificial insemination protocols for Bos indicus
cattle
M.F. Sá Filho a,∗ , J.M. Baldrighi a , J.N.S. Sales a , G.A. Crepaldi a , J.B.P. Carvalho b , G.A. Bó c ,
P.S. Baruselli a,∗
a
Departament of Animal Reproduction, FMVZ-USP, CEP 05508-050, São Paulo, SP, Brazil
b
APTA, Pólo Vale do Paraíba, CEP 12400-970, Pindamonhangaba, SP, Brazil
c
Instituto de Reproducción Animal Cordoba (IRAC), Cordoba Paraje Poco del Tigre, General Paz, 5147 Córdoba, Argentina

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

Article history: The present study aimed to evaluate the efficacy of different inducers of new follicular wave
Received 3 July 2010 emergence (FWE) and ovulation in fixed-time artificial insemination (FTAI) synchronization
Received in revised form 6 December 2011 protocols using norgestomet ear implants (NORG) in Bos indicus cattle. In Experiment 1, the
Accepted 12 December 2011
synchronization of FWE was evaluated when two different estradiol esters in different doses
Available online 17 December 2011
[2 mg estradiol benzoate (EB), 2.5 mg EV or 5 mg estradiol valerate (EV)] were administered
with NORG implant insertion in B. indicus cattle (estrous cyclic heifers and cows with suck-
Keywords:
ling calves; n = 10 per treatment). After estradiol treatment, ovarian ultrasonic exams were
GnRH
performed once daily to detect the interval between treatment and FWE. There were signif-
Estradiol
Nelore icant treatment-by-animal category interaction (P = 0.05) on the interval from the estradiol
Follicular wave emergence treatment to FWE. An earlier (P < 0.0001) and less variable (P = 0.02) interval from estradiol
FTAI treatment to FWE was observed in heifers treated with EB (2.5 ± 0.2; mean ± SE) than in
those treated with 2.5 mg EV (4.2 ± 0.3) or 5 mg EV (6.1 ± 0.6). Cows treated with 5 mg EV
(4.0 ± 0.5) had longer (P = 0.05) interval than cows receiving EB (2.5 ± 0.2), however, there
was an intermediate interval in those cows treated with 2.5 mg EV (3.1 ± 0.4). In Experi-
ment 2, the number of uses of the NORG implant (new; n = 305 or previously used once;
n = 314) and three different ovulation induction hormones [0.5 mg estradiol cypionate (EC)
at implant removal (n = 205), 1 mg EB given 24 h after implant removal (n = 219), or 100 ␮g
gonadorelin (GnRH) given at FTAI (n = 195)] were evaluated in Nelore heifers (2 × 3 factorial
design). Similar pregnancy per AI (P/AI; 30 days after FTAI; P > 0.05) were achieved using
each of the three ovulation induction hormones (EB = 40.6%; EC = 48.3%, or GnRH = 48.7%)
and with a new (47.2%) or once-used NORG implant (44.3%). In Experiment 3, the effect
of different ovulation induction hormones for FTAI [1 mg EC at NORG implant removal
(n = 228), 10 ␮g buserelin acetate at FTAI (GnRH; n = 212) or both treatments (EC + GnRH;
n = 215)] on P/AI was evaluated in suckled beef cows treated with a once-used NORG implant
and EB to synchronize the FWE. Similar P/AI (P = 0.71) were obtained using GnRH (50.9%),
EC (51.8%) or both treatments (54.9%) as ovulation induction hormones. Therefore, both
doses of EV (2.5 or 5.0 mg) with NORG implant delayed and increased the variation of the
day of new FWE compared with EB in B. indicus cattle. These effects were more pronounced

© 2011 Elsevier B.V. All rights reserved.

∗ Corresponding authors. Tel.: +55 0113091 7674.

E-mail addresses: manoelsa@usp.br (M.F. Sá Filho), barusell@usp.br
(P.S. Baruselli).

0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2011.12.005
 M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139 133

in B. indicus heifers than cows. Synchronization protocols for FTAI with either a new or
once-used NORG implant with EB at insertion to induce a new FWE and either the use of
EB, EC or GnRH as ovulation induction hormones may be successful in B. indicus heifers.
Also, when a once-used NORG implant was used, either the administration of EC, GnRH or
both as ovulation inducers resulted in similar P/AI in suckled B. indicus cows, showing no
additive effect of the combination of both ovulation induction hormones.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction of the LH peak and ovulation in suckled B. indicus beef cows,

Fixed time artificial insemination (FTAI) programs are
widely used due to the ability of these programs to increase
the number of females inseminated without the neces-
sity of estrus detection. A variety of protocols have been
developed to design specific treatments for different ani-
mal categories and to minimize time and labor, yielding
satisfactory pregnancy outcomes.
Estradiol 17␤-estradiol (17␤-E2) or one of its esters
have been successfully used to synchronize follicu-
lar wave emergence (FWE) in cattle (reviewed by Bó
et al., 2002). The association between estradiol (E2) and
progestin/progesterone (P4) in cattle induces follicular
regression and subsequent synchronous FWE (Bó et al.,
1995, 1996; Caccia and Bó, 1998). In Bos taurus cattle, estra-
diol valerate (EV) another ester of E2 with longer half life
than estradiol benzoate (EB) or 17␤-E2 has been reported
to result in a longer FSH suppression and follicular wave
emergence (Bó et al., 1993; Martinez et al., 2005). How-
ever, treatment with 5 mg of EV resulted in a longer and
less synchronous FWE than treatment with a dose of 2 mg
of EV (Mapletoft et al., 2004), suggesting that the interval
from treatment to FWE is dependent on both the ester of
E2 and the dose administered.
In spite of those differences, both E2 esters (EV or EB)
have been successfully applied in FTAI synchronization
protocols for suckled B. taurus (Odde, 1990; Geary et al.,
1998) and suckled Bos indicus beef cows (Sá Filho et al.,
2010a,b; Meneghetti et al., 2009). However, limited reports
have studied the use of these different E2 esters on induc-
tion of new FWE in B. indicus heifers in progestin-based
FTAI synchronization protocols.
After luteolysis, commercial FTAI synchronization pro-
tocols use inducers of ovulation to achieve synchronized
ovulation. GnRH-based protocols have been successfully
used in synchronization protocols for FTAI in beef and dairy
cattle (Pursley et al., 1997a,b; Geary et al., 2001). Addition-
ally, in the E2 plus progestin/P4 protocols, EB have been
successfully used for induction the ovulation (Macmillan
and Peterson, 1993; Macmillan et al., 1997). Estradiol cypi-
onate (EC) is another ester of E2 with a low water solubility
that delays its release from the site of injection. EC has been
routinely used in P4-based FTAI protocols to synchronize
time of ovulation in suckled B. indicus cows (Sá Filho et al.,
2009; Meneghetti et al., 2009; Peres et al., 2009).
However, previous studies demonstrated that the treat-
ment with EC at intravaginal P4 device removal is less
effectively to synchronize the time of the preovulatory LH
surge (Souza et al., 2009) and ovulation (Martins et al.,
2005) than the use of GnRH or EB as ovulatory stimulus.
Nonetheless, in spite of the increased variability in timing
satisfactory pregnancy per AI (∼50%) have been described
following the use of EC as inducer of ovulation (Penteado
et al., 2006; Sá Filho et al., 2009, 2011).
Previous study demonstrated that GnRH treatment at
the moment of AI in a progestin-based FTAI protocol in B.
indicus suckled cows prevented the occurrence of delayed
ovulations, improving synchronization of ovulations and
pregnancy per FTAI (P/AI) (Sá Filho et al., 2010a). There-
fore, GnRH treatment at the moment of FTAI in those cows
receiving EC at NORG implant removal could improve the
synchronization of ovulation time preventing the occur-
rence of delayed ovulations, improving the P/AI following
progestin-based FTAI protocol in suckled B. indicus cows.
Thus, the present series of studies were designed to
compare EB and different doses of EV to synchronize FEW
and EB, EC or GnRH to synchronize ovulation in B. indicus
cattle treated with NORG implant and FTAI. The hypotheses
tested were as follows: treatment with EV induces a longer
interval from E2 treatment to FWE than EB (Experiment
1); similar P/AI are achieved with the use of three differ-
ent protocols to induce ovulation, GnRH at the time of AI,
EB 24 h after removing NORG implant, or EC at the time
of NORG implant removal (Experiment 2); and similar P/AI
are achieved using GnRH or EC to induce ovulation with
an positive effect of the combination of EC and GnRH to
induce ovulation (Experiment 3) in B. indicus cattle treated
with NORG implants and FTAI.
2. Materials and methods
2.1. Animals and management
2.1.1. Experiment 1
Experiment 1 was conducted at an Institutional Center
named APTA, Pólo Regional do Vale do Paraiba, Pindamon-
hangaba, in the São Paulo state, southeastern Brazil. Cattle
were maintained on a Brachiaria brizantha pasture with free
access to mineralized salt and water. A total of 30 Nelore
cows with suckling calves, ranging from 70 to 90 days post-
partum [63% (19/30) were estrous cyclic] and 29 estrous
cyclic Nelore heifers (22–28 months of age) were used.
Cattle were examined by transrectal ultrasonography to
determine the presence of corpora lutea (CL) 10 days before
(Day −10) and on the first day of the experiment (Day 0).
2.1.2. Experiment 2
Experiment 2 was performed on three commercial
farms located in southern and central eastern Brazil. Cattle
were maintained on a Brachiaria decumbens pasture with
free access to mineralized-salt and water. A total of 619
cycling Nelore heifers (Farm A = 87, Farm B = 421 and Farm
134 M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139
C = 111) were selected according to the presence of a CL on
the first day of the synchronization protocol (Day 0). Their
body condition score (BCS) was evaluated on Day 0 using a
1 to 5 scale (1 = emaciated, 5 = obese; Ayres et al., 2009).
2.1.3. Experiment 3
Experiment 3 was performed on four commercial farms
located in southern and central eastern Brazil. A total of
655 suckled B. indicus beef cows [Farm A = 97; Farm B = 148;
Farm C = 144; Farm D = 266 (two cohorts of cows 121 and
145)] ranging 30–60 days postpartum were maintained on
a B. brizantha pasture with free access to mineralized-salt
and water. Cows had their BCS evaluated on Day 0, using a
1–5 scale (1 = emaciated, 5 = obese; Ayres et al., 2009).
2.2. Experimental design
2.2.1. Experiment 1
At random stages of the estrous cycle, designated as Day
0, heifers and cows received a subcutaneous ear implant
containing 3 mg norgestomet (NORG, Crestar® , Intervet,
Boxmeer, Netherlands) and were randomly assigned to
receive one of three treatments. Ten cows and 10 heifers
were treated with 2.0 mg EB intramuscularly (i.m.) (Estro-
gin, Farmavet® , Sao Paulo, Brazil). Ten cows and 10 heifers
were treated with 2.5 mg EV i.m. ((1/2)EV) plus 1.5 mg of
norgestomet (1 mL of the Crestar injectable portion, Inter-
vet), and 10 cows and nine heifers received 5.0 mg EV i.m.
plus 3.0 mg of norgestomet (total recommended dose; i.e.
2 mL of the Crestar injectable portion, Intervet).
2.2.2. Experiment 2
At random stages of the estrous cycle, heifers were
submitted to a protocol for synchronization of ovulation
time for FTAI. On the first day of the synchronization pro-
tocol (i.e. Day 0), the BCS was recorded, 2 mg EB were
administered i.m. and heifers were randomly assigned into
one of two experimental groups according to the num-
ber of uses of the norgestomet (NORG) ear implant: new
or previously used for 9 days (once-used). By the time of
implant removal (8 days after insertion), heifers received
0.15 mg d-cloprostenol (Preloban® , Intervet,) and 300 IU
eCG (Folligon® , Intervet) i.m. Thereafter, heifers from both
groups were equally re-assigned to receive one of three
treatments: EB, EC or GnRH (factorial 2 × 3). Heifers in
the EB group received 1 mg EB i.m. 24 h after NORG ear
implant removal (Day 9), heifers in the EC group received
0.5 mg EC (ECP® , Pfizer Animal Health, São Paulo, Brazil)
i.m. at NORG removal (Day 8), and heifers in the GnRH
group received 100 ␮g gonadorelin (Fertagil® , Intervet) at
FTAI (Day 10). All heifers were inseminated 52–58 h after
NORG ear implant removal. One AI technician performed
all inseminations. Six different sires were used, depending
on farm location (Farm 1 = one sire; Farm 2 = three sires;
Farm 3 = two sires). All sires used in the present study had
previously resulted in satisfactory pregnancy outcomes (i.e.
around 50% of P/AI) in FTAI programs.
2.2.3. Experiment 3
At random stages of the estrous cycle (Day 0), suck-
led cows received 2 mg EB i.m. and a NORG ear implant
previously used for 9 days. On Day 8, the NORG ear implant
was removed and cows received 300 IU eCG and 0.15 mg
d-cloprostenol. Then, the cows were allocated into one of
three treatments, taking into account BCS and the sire used
for FTAI. In Group EC, cows were treated with 1 mg EC
on Day 8. In Group GnRH, cows were treated with 10 ␮g
buserelin acetate (SincroForte® , Ourofino Animal Health,
Ribeirao Preto, Brazil) at FTAI. In Group EC + GnRH, cows
received both treatments. The FTAI was performed 48–50 h
after the NORG implant removal.
One AI technician performed all inseminations. Seven
different sires were used for the insemination depending
on the farm location (Farm A = one sire; Farm B = two sires;
Farm C = two sires; Farm D = three sires). All sires used had
previously resulted in satisfactory pregnancy outcomes in
FTAI programs.
2.3. Ultrasonographic examinations
2.3.1. Experiment 1
Transrectal ultrasonography of both ovaries was per-
formed on Day −10 and Day 0 (NORG implant insertion).
Then, ovaries were assessed once daily to detect emergence
of new follicular wave and to confirm follicular domi-
nance. The dominant follicle was identified as the newest
growing follicle reaching a diameter larger than 8 mm.
The day of FWE was retrospectively defined as the day
when the dominant follicle was first detected at a diame-
ter of 3 mm. A real-time ultrasonic scanner equipped with
a 5.0 MHz linear transducer was used (Aloka 500-V, Coro-
metrics Medical Systems, Wallingford, CT, USA).
2.3.2. Experiment 2
At FTAI, all heifers had their ovaries scanned by ultra-
sonography to identify and measure the largest follicle
(LF). The LF at FTAI was classified based on their diame-
ter (<3.6 mm, 3.6–8.0 mm, 8.1–12.5 mm or >12.5 mm), as
previous described by Sá Filho et al. (2010b). All heifers
were examined for pregnancy diagnosis by transrectal
ultrasonography 30 days after FTAI. The detection of an
embryonic vesicle with a viable embryo (presence of heart-
beat) was used as an indicator of pregnancy. The P/AI was
calculated as the proportion of heifers pregnant 30 days
after FTAI divided by the number of heifers inseminated.
2.3.3. Experiment 3
All cows were examined for pregnancy diagnosis by
transrectal ultrasonography 55 days after FTAI. Similar to
Experiment 2, the visualization of the fetus and its heart-
beat was used as an indicator of pregnancy.
2.4. Statistical analysis
2.4.1. Experiment 1
Statistical analysis was conducted using the SAS Sys-
tem for Windows (SAS Institute Inc., Cary, NC, USA, 2003).
The explanatory variables included in the statistical model
were the type of E2 ester (EB, (1/2)EV and EV), animal
category (cows and heifers) and the interaction between
treatments and animal category. The dependent variable
 M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139
Table 1
Effect of different estradiol esters on the induction of a new follicular wave emergence (FWE) in Nelore Bos indicus cattle treated with norgestomet ear
implant (Experiment 1).
Treatments1
Heifers Cows
EB (1/2)EV EV EB
Number of animals 10 10 9 10
FWE (days)2 2.5 ± 0.2a 4.2 ± 0.3b,x 6.1 ± 0.6c,x 2.5 ± 0.2a
Range 2–3 3–6 3–8 2–4
Mean ± SE values of FWE within same animal category (heifers or cow) with different superscript letters (a = / b=/ c) differ significantly (P < 0.05).
Mean ± SE values of FWE within same estradiol ester (EB, (1/2)EV or EV) with different superscript letters (x =
1
Heifers and cows received 3 mg norgestomet ear implant and three different i.m. treatments at implant insertion: BE (2 mg estradiol benzoate), (1/2)EV
(2.5 mg EV plus 1.5 mg norgestomet) or EV (5 mg EV plus 3 mg norgestomet).
2
FWE = interval, in days, from estradiol injection to new follicular wave emergence.
(interval from E2 treatment to FWE) was tested accord-
ing to their homogeneity and normality of variances using
Guide Data Analysis from SAS and subjected to log10 trans-
formation. As the Levene’s test revealed heterogeneity of
variances, the median test was used to compare intervals
from E2 treatment to FWE using the two-way ANOVA of
PROC GLM. The Tukey test was used to determine differ-
ences between groups.
2.4.2. Experiment 2
The P/AI was analyzed using the procedure GLIMMIX
of SAS, with heifers treated as a random effect. Addition-
ally, for the final logistic regression model, variables were
removed by backward elimination, based on the Wald
statistics criterion, when P > 0.20. The variables included
in the final model were the effects of farm, BCS, LF at FTAI
and treatments (number of uses of the NORG ear implant
and ovulation inductions hormones).
The diameter of the LF at FTAI was analyzed using
PROC MIXED to detect the effect of farm, treatment, BCS
and their interactions. The variables included in the final
models were the effects of farm, BCS and treatments
(number of uses of the NORG ear implant and ovula-
tion induction hormones). The relationships between the
diameter of the LF at FTAI and the probability of preg-
nancy for all heifers were determined. Logistic regression
curves were created using the coefficients provided by
the interactive data analysis from SAS and the formula
y = exp(˛ × x + b)/[1 + exp(˛ × x + b)].
2.4.3. Experiment 3
The P/AI was analyzed using the procedure GLIMMIX
of SAS, with cows treated as a random effect. Also, for the
final logistic regression model, variables were removed by
backward elimination based on the Wald statistics crite-
rion when P > 0.20. The variables included in the final model
were the effects of farm, BCS and treatments.
3. Results
3.1. Experiment 1
There were significant effects of treatment (P < 0.0001),
animal category (P = 0.002), and treatment-by-animal cat-
egory interaction (P = 0.05). Emergence of a new follicular
wave was detected in all animals after treatments with EB,
135
P-Values
Animal Estradiol Interaction
category esters
(1/2)EV EV
10 10 – – –
3.1 ± 0.4ab,y 4.0 ± 0.5b,y 0.002 <0.0001 0.05
2–6 2–6 – – –
/ y) differ significantly (P < 0.05).
EV, or (1/2)EV (Table 1). However, an earlier (P < 0.0001)
and less variable (P = 0.02) interval from E2 treatment to
FWE (mean ± SE) was observed in heifers treated with EB
(2.5 ± 0.2; 2–3 days) than in those treated with (1/2)EV
(4.2 ± 0.3; 3–6 days) or EV (6.1 ± 0.6; 3–8 days). Cows
treated with EV (4.0 ± 0.5; 2–6 days) had similar intervals
from treatment to FWE than cows treated with (1/2)EV
(3.1 ± 0.4; 2–6 days), but this interval was longer (P = 0.05)
than that in cows receiving EB (2.5 ± 0.2; 2–4 days). The
effect of EB was similar in heifers and cows (P = 0.92),
however treatments with (1/2)EV (P = 0.02) or EV (P = 0.02)
resulted in a greater delay on the FWE in heifers than in
cows.
3.2. Experiment 2
The BCS at the first day of the estrous synchroniza-
tion protocol was 3.0 ± 0.03. No interactions were observed
between the different ovulation induction hormones eval-
uated (EB, EC, or GnRH) and the number of uses of NORG
implant (new or once-used) on the diameter of the LF
at FTAI (P = 0.89) and P/AI (P = 0.55). A larger diameter of
the LF (P = 0.00004) was observed in GnRH-treated heifers
(9.4 ± 0.03) than in EC-treated heifers (8.1 ± 0.03) and EB-
treated heifers (8.3 ± 0.03). Also, heifers treated with new
NORG implant (9.2 ± 0.02) had larger diameters of the
LF (P = 0.01) than heifers treated with a once-used NORG
(7.9 ± 0.02). Results are shown in Table 2.
On the first day of the synchronization protocol, an
effect of BCS was observed on the diameter of the LF
at FTAI and P/AI. Heifers with a greater BCS (≥3.25)
showed increased (P = 0.03) LF diameters (9.3 ± 0.03 mm)
and greater (P = 0.03) P/AI (54.1%; 93/172) than heifers with
intermediate (3.00) BSC (8.3 ± 0.03 mm and 43.1%; 84/195)
or lesser (≤ 2.75) BCS (8.2 ± 0.03 mm and 41.5%; 105/253).
Heifers with larger LF at FTAI had the greatest rates of
pregnancy 30 days after FTAI (P = 0.0001; Fig. 1). More-
over, follicular diameter was categorized, and heifers with
follicles ranging from 8.1 to 12.5 mm in diameter had
similar P/AI (52.6%; 142/270) as those with LF > 12.5 mm
(57.4%; 62/108), but both had greater P/AI (P = 0.001) than
heifers with follicles ranging from 3.6 to 8.0 mm in diam-
eter (24.3%; 17/70) and those with LF < 3.6 mm (34.3%;
58/169). The P/AI also differed with the farm location
[Farm A = 55.2% (48/87), Farm B = 41.1% (173/421) and Farm
136 M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139

Effects of the number of uses of a norgestomet ear implant (new and once-used) and the administration of different ovulatory inducers on the largest follicle diameter at FTAI and P/AI in Nelore (Bos indicus)

Heifers received a new or once-used (previously used for 9 days) norgestomet ear implant and 2 mg of estradiol benzoate at implant insertion on the first day of the protocol for synchronization of ovulation
time. The implant was removed 8 days later, and the heifers received three different treatments: EC (0.5 mg of estradiol cipionate at implant removal), EB (1 mg of estradiol benzoate 24 h after implant removal)

Implant = effect of the number of uses of norgestoment ear implant (New vs. Once-used); Ovulation inducer = effect of different ovulatory inducer (EB vs. EC vs. GnRH); Interactions = interaction between
100

Interaction

0.89

0.55
80

Probability of Pregnanvcy
Ovulation 60

0.00004
inducer

40

0.47
–

20
P-Valuesb

Implant

0
0 5 10 15 20 25
0.14
0.01

Largest Follicle Diameter at FTAI (mm)

–

Fig. 1. Probability of pregnancy 30 days after fixed time artifi-

cial insemination (FTAI) in Bos indicus heifers (n = 617), according
to the diameter of the largest follicle (LF) at FTAI [probability of
8.9 ± 0.5

pregnancy at 30 days = exp(−1.1788 + 0.1171 × diameter of the LF at

FTAI/1 + exp(−1.1788 + 0.1171 × diameter of the LF at FTAI); P < 0.0001)].
GnRH

50.0
100

C = 54.1% (60/111); P = 0.008], but there was no farm by

treatment interaction (P = 0.64).
7.3 ± 0.5

3.3. Experiment 3
45.9
EC

98

The average BCS on the first day of the FTAI synchroniza-

tion protocol was 2.5 ± 0.03 and no interactions between
treatments and BCS or farm (P > 0.05) were detected.
However, P/AI was influenced by farm [Farm A = 45.3%
7.6 ± 0.5
Once-used

(44/97); Farm B = 43.3% (64/148); Farm C = 67.4% (97/144);

Farm D = 52.3% (139/266); P = 0.0002]. Similar P/AI were
37.9
116

achieved among cows treated with different protocols

EB

to induce ovulation [Group EC = 51.8% (118/228), Group

GnRH = 50.9% (108/212) and Group EC + GnRH = 54.9%
(118/215); (P = 0.71)].
9.9 ± 0.4

4. Discussion
GnRH

47.4
95

In the present study, the mean interval to, and variability

of FWE varied with the type and dose of ester of E2 that was
administered at the start of treatment with a NORG implant
in B. indicus cattle. Treatment with EV (5 or 2.5 mg) resulted
8.6 ± 0.4

in a longer and more variable interval from treatment to

50.5

FEW than EB treatment, confirming our hypothesis. Also,

107
EC

B. indicus heifers had a longer interval from EV treatment

or GnRH (100 ␮g of gonadorelin at fixed time AI).

to FWE than B. indicus cows.

Treatmentsa

9.0 ± 0.5

The E2 and progestin/P4 combination has been the most

successful hormone therapy used to synchronize FWE in
42.7
New

103

cattle (reviewed by Bó et al. (2002)). In a series of stud-

EB

ies (reviewed by Bó et al. (2002, 2005)), E2 treatment was

Implant and Ovulation inducer.

found to suppress the growing phase of the dominant fol-

licle and suppression was more profound when given in
heifers (Experiment 2).

combination with P4. The mechanism responsible for E2-

Number of animals
Largest Follicle at

induced suppression of follicle growth appears to involve

Pregnancy per AI

suppression of FSH (Bó et al., 1994, 1995; O’Rourke et al.,

FTAI (mm)

2000) and LH (Burke et al., 1996). The combination of E2 and

NORG was subsequently used to determine if suppression
Table 2

of follicle growth would induce a new FWE at a consistent

(%)
a

interval post-treatment regardless of the phase of follicle

 M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139
development at which treatment was initiated. The use
of 17␤-E2 in progestin-implanted B. taurus cattle was fol-
lowed consistently by a new FWE, on average, 4.3 ± 0.2 days
later (Bó et al., 1995). The administration of 5 or 2.5 mg of
17␤-E2 (Bó et al., 2002) or 2.5 mg of EB (Caccia and Bó,
1998) or EV (Colazo et al., 2005) in progestin-implanted
cattle at random stages of the estrous cycle was followed
by a new FWE approximately 4 days later.
In the present study, although inclusion of treatment
with an ester of E2 at the initiation of a progestin-based
treatment resulted in the emergence of a new follicular
wave, the administration of EV at either dosage used in
this study (2.5 or 5.0 mg) resulted in a longer and more
variable interval from treatment to emergence of a new
follicular wave than the administration of EB. O’Rourke
et al. (2000) observed that the interval from E2 treatment to
FWE seemed to depend on FSH resurgence, which has been
reported to occur after E2 concentrations decreased below
a threshold level. EV has a long circulating half-life, and its
prolonged suppressive effect on FSH and ovarian follicu-
lar growth (Bó et al., 1993; Martinez et al., 2005) could be
responsible for the variability and length of interval from
treatment to FWE observed in the current study. Hence,
E2 preparation and dose can affect the timing of FWE in B.
indicus cattle.
The present data also demonstrate that the adminis-
tration of the EV in B. indicus heifers induced a prolonged
interval from treatment to FWE compared with lactating B.
indicus cows. Furthermore, different than cows, half dose of
EV (2.5 mg) still presented a longer interval from treatment
to FEW than EB in B. indicus heifers. These differences could
be associated with differences in steroid metabolisms in
the two animal categories (Sartori et al., 2004; Wolfenson
et al., 2004). Although it has not been specifically studied,
it is conceivable that, similar to what occurs in Holsteins,
suckled B. indicus cows could have a faster clearance rate of
E2 than heifers. Therefore, that could influence the time at
which concentrations of E2 in circulation fail to maintain
suppression of the pre-emergence increase in FSH and thus
the timing of a new FWE (Burke et al., 2003). The greater
suppressive effects of the EV on follicle development in
B. indicus heifers need to be considered for the devel-
opment of new synchronization protocols and requires
further study.
In the current study, similar P/AI were achieved with
the use of three different protocols to induce ovulation
using GnRH at the time of FTAI, EB 24 h after removing
NORG, or EC at the time of NORG implant removal, con-
firming the hypothesis for the present study. Similar effects
were observed with these three different treatments to
induce ovulation using either with a new or once-used
NORG impalnt. Also similar P/AI using EB or EC (at implant
removal or 24 h later, respectively) in the protocol to syn-
chronize time of ovulations in B. taurus beef heifers (Colazo
et al., 2003) and in B. indicus suckled beef cows (Penteado
et al., 2006) were observed.
Likewise, in suckled B. indicus cows, progestin-based
FTAI synchronization protocols using EC or GnRH to induce
ovulation presented similar P/AI. Also, there was no addi-
tive effect of the combination of EC and GnRH on the P/AI,
rejecting our hypothesis. Conversely, in a recent study with
137
suckled B. indicus cows treated with an intravaginal P4
device, the use of GnRH to synchronize ovulation reduced
the risk of occurrence of estrus and the P/AI when com-
pared with either the use of EC or the combination of EC
and GnRH (Sá Filho et al., 2011). It seems that EC could
be particularly important for suckled B. indicus cows under
tropical pasture conditions (i.e. greater incidence of post-
partum anestrous) or cows with low BCS by increasing the
E2 concentration during the proestrus phase (Sellars et al.,
2006; Hillegass et al., 2008; Souza et al., 2009) enhancing
the occurrence of estrus and the odds of pregnancy follow-
ing FTAI (Sá Filho et al., 2011). Differences in P/AI observed
following induction of ovulation with GnRH or EC in some
studies but not in the present study may be attributable to
differences in treatments with progestins between studies
(Kojima et al., 1992; Rathbone et al., 2001). In the current
study use of NORG implant may have resulted in a less sup-
pressive effect on the frequency of LH release, resulting in
an increase in growth and synthesis of E2 by the domi-
nant follicle compared to studies that treated cows with an
intravaginal P4 releasing insert. As a result in the current
study critical threshold values for maturity of preovulatory
follicles were achieved in cows in each of the treatment
groups whereas in other studies differences in the maturity
of preovulatory follicles may have differed at the time when
treatments were applied resulting in differences in the size
and/or maturity of ovulatory follicles. Conversely, the asso-
ciation between GnRH and EC treatment did not improve
the P/AI, probably due to a satisfactory synchronization of
ovulation by the use of EC in these cows. Therefore, it is
possible that circulating E2 concentration were sufficient
to influence pregnancy rates in cows treated with GnRH
at FTAI and the addition effect of injectable EC at NORG
implant removal was not evident.
Similarly to previous studies, the follicular diameter
at the moment of FTAI influenced the odds of pregnancy
(Perry et al., 2007; Meneghetti et al., 2009; Sá Filho et al.,
2010b). However, when the treatments (Experiment 2)
were considered, despite the larger diameters of the LF
at FTAI in heifers treated with a new NORG implant or
with GnRH in the current study when compared to those
treated with EB or EC or with a once-used NORG implant,
the larger follicular diameter had no effect on improving
P/AI. It is important to state that the overall P/AI rate of
34.3% observed in heifers with follicles of ≤3.5 mm in diam-
eter might be overestimated, as it is possible that some of
these heifers ovulated before the FTAI. Previous study with
suckled B. indicus cows reinforces this possibility once the
majority of the females (79.4%) with a small follicle at FTAI
ovulated before the day of FTAI (Sá Filho et al., 2010b).
The follicular size at FTAI and the occurrence of pre-
mature ovulation could be influenced by several variables
such as the age of preovulatory follicle (Sanchez et al.,
1995; Austin et al., 1999), the LH profiles during the pro-
gestin/P4 treatment (Adams et al., 1992; Savio et al., 1993;
Carvalho et al., 2008; Cerri et al., 2011) and the type and
the moment of ovulatory stimulus administration prior to
FTAI (Colazo et al., 2003; Souza et al., 2009). In spite of
the satisfactory predictability of the moment of ovulation
provided by the use of EB to synchronize the ovulation
time (averaging 66–72 h after P4 device removal), the
138 M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139
timing of ovulation is influenced by the diameter of the
follicle at the time of the ovulatory stimulus treatment
(Neves, 2010). Neves (2010) reported that cows experi-
encing premature ovulation (i.e. ovulation occurring from
48 to 59 h after P4 device removal) had larger ovulatory
follicle (14.0 ± 2.2 mm) than cows with delayed ovula-
tion (11.4 ± 2.2 mm; 73–96 h after P4 device removal) and
that cows that ovulated at the expected time of ovulation
(60–72 h after P4 device removal) showed ovulatory fol-
licles of intermediate diameter (13.6 ± 2.1 mm). Thereby,
heifer subjected to estrus synchronization protocols under
lower circulating P4, such as when using a previously used
NORG implant, could have a larger follicle size at the time
of the implant removal, resulting in a greater occurrence of
earlier ovulations.
In addition, the larger follicle diameter at FTAI of the
heifers treated GnRH could be due to the moment that
the follicle was measured relatively to the timing of the
expected moment of the LH surge. The LH surge after the
GnRH administration occurs in approximately 2 h (i.e. 2 h
after the FTAI; Chenault et al., 1990) when compared to
the 48 h after EC administration (i.e. approximately 7 h
before the FTAI; Sales et al., 2008) and the 20 h following
EB administration (i.e. approximately 7 h before the FTAI;
Lammoglia et al., 1998; Sales et al., 2008). Therefore, heifers
treated with GnRH at FTAI could present a larger period of
the preovulatory follicle growth than heifers treated with
EC at or EB 24 h after the NORG implant removal.
The data reported herein demonstrate that a greater
BCS resulted in greater conception rates after AI, indicating
that nutritional status is critical for FTAI synchronization
protocol responses in cyclic B. indicus heifers. The farm
location also accounted for some variation in reproduc-
tive performance. This is an extremely important aspect
that compromises the commercial application of FTAI in B.
indicus cattle. The effect of farm location can be related to
BCS, sexual maturity (heifers), estrous cyclic status (suck-
led cows), sire used and type of feeding. All of these factors
usually differ among farms (Sá Filho et al., 2009), and can
potentially influence the pregnancy outcomes of a repro-
ductive program.
In summary, the administration of EV (2.5 or 5.0 mg)
with a NORG implant delayed the day of FWE in comparison
to treatment with 2.0 mg of EB in B. indicus cattle. Further-
more, the effects of EV on FWE seem to be more pronounced
in B. indicus heifers than in B. indicus cows. Synchroniza-
tion protocols for FTAI with either new or once-used NORG
implant and EB, EC or GnRH as ovulation induction hor-
mones may be successfully used in B. indicus heifers. Finally,
the administration of either EC, GnRH or both as ovulation
induction hormones resulted in similar P/AI in suckled B.
indicus cows, showing no additive effect of the combination
of both EC and GnRH on P/AI.
Acknowledgements
The authors are grateful to Dr. Henderson Ayers and
Dr. Roberta Ferreira (University of São Paulo, Brazil) for
their help with manuscript preparation and statistical anal-
yses. The authors also thank the staff of APTA-Polo Regional
Vale do Paraiba (Pindamonhangaba, São Paulo, Brazil),
Pennacchi Agropastoril (Cascavel, PR, Brazil), Fazenda
Primeiro de Maio (Sonora, MS, Brazil) and Fazenda Jacare-
una (Espigão do Leste, MT, Brazil) for allowing the use of
their animals and facilities for this study. This research
was supported by Intervet/Schering-Plough, São Paulo,
SP, Brazil and FIRMASA-Technologies, Campo Grande, MS,
Brazil.
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