Você está na página 1de 8

Animal Reproduction Science 129 (2011) 132–139

Contents lists available at SciVerse ScienceDirect

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
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,∗
Departament of Animal Reproduction, FMVZ-USP, CEP 05508-050, São Paulo, SP, Brazil
APTA, Pólo Vale do Paraíba, CEP 12400-970, Pindamonhangaba, SP, Brazil
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-
ling calves; n = 10 per treatment). After estradiol treatment, ovarian ultrasonic exams were
performed once daily to detect the interval between treatment and FWE. There were signif-
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.
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,

satisfactory pregnancy per AI (∼50%) have been described
Fixed time artificial insemination (FTAI) programs are following the use of EC as inducer of ovulation (Penteado
widely used due to the ability of these programs to increase et al., 2006; Sá Filho et al., 2009, 2011).
the number of females inseminated without the neces- Previous study demonstrated that GnRH treatment at
sity of estrus detection. A variety of protocols have been the moment of AI in a progestin-based FTAI protocol in B.
developed to design specific treatments for different ani- indicus suckled cows prevented the occurrence of delayed
mal categories and to minimize time and labor, yielding ovulations, improving synchronization of ovulations and
satisfactory pregnancy outcomes. pregnancy per FTAI (P/AI) (Sá Filho et al., 2010a). There-
Estradiol 17␤-estradiol (17␤-E2) or one of its esters fore, GnRH treatment at the moment of FTAI in those cows
have been successfully used to synchronize follicu- receiving EC at NORG implant removal could improve the
lar wave emergence (FWE) in cattle (reviewed by Bó synchronization of ovulation time preventing the occur-
et al., 2002). The association between estradiol (E2) and rence of delayed ovulations, improving the P/AI following
progestin/progesterone (P4) in cattle induces follicular progestin-based FTAI protocol in suckled B. indicus cows.
regression and subsequent synchronous FWE (Bó et al., Thus, the present series of studies were designed to
1995, 1996; Caccia and Bó, 1998). In Bos taurus cattle, estra- compare EB and different doses of EV to synchronize FEW
diol valerate (EV) another ester of E2 with longer half life and EB, EC or GnRH to synchronize ovulation in B. indicus
than estradiol benzoate (EB) or 17␤-E2 has been reported cattle treated with NORG implant and FTAI. The hypotheses
to result in a longer FSH suppression and follicular wave tested were as follows: treatment with EV induces a longer
emergence (Bó et al., 1993; Martinez et al., 2005). How- interval from E2 treatment to FWE than EB (Experiment
ever, treatment with 5 mg of EV resulted in a longer and 1); similar P/AI are achieved with the use of three differ-
less synchronous FWE than treatment with a dose of 2 mg ent protocols to induce ovulation, GnRH at the time of AI,
of EV (Mapletoft et al., 2004), suggesting that the interval EB 24 h after removing NORG implant, or EC at the time
from treatment to FWE is dependent on both the ester of of NORG implant removal (Experiment 2); and similar P/AI
E2 and the dose administered. are achieved using GnRH or EC to induce ovulation with
In spite of those differences, both E2 esters (EV or EB) an positive effect of the combination of EC and GnRH to
have been successfully applied in FTAI synchronization induce ovulation (Experiment 3) in B. indicus cattle treated
protocols for suckled B. taurus (Odde, 1990; Geary et al., with NORG implants and FTAI.
1998) and suckled Bos indicus beef cows (Sá Filho et al.,
2010a,b; Meneghetti et al., 2009). However, limited reports 2. Materials and methods
have studied the use of these different E2 esters on induc-
tion of new FWE in B. indicus heifers in progestin-based 2.1. Animals and management
FTAI synchronization protocols.
After luteolysis, commercial FTAI synchronization pro- 2.1.1. Experiment 1
tocols use inducers of ovulation to achieve synchronized Experiment 1 was conducted at an Institutional Center
ovulation. GnRH-based protocols have been successfully named APTA, Pólo Regional do Vale do Paraiba, Pindamon-
used in synchronization protocols for FTAI in beef and dairy hangaba, in the São Paulo state, southeastern Brazil. Cattle
cattle (Pursley et al., 1997a,b; Geary et al., 2001). Addition- were maintained on a Brachiaria brizantha pasture with free
ally, in the E2 plus progestin/P4 protocols, EB have been access to mineralized salt and water. A total of 30 Nelore
successfully used for induction the ovulation (Macmillan cows with suckling calves, ranging from 70 to 90 days post-
and Peterson, 1993; Macmillan et al., 1997). Estradiol cypi- partum [63% (19/30) were estrous cyclic] and 29 estrous
onate (EC) is another ester of E2 with a low water solubility cyclic Nelore heifers (22–28 months of age) were used.
that delays its release from the site of injection. EC has been Cattle were examined by transrectal ultrasonography to
routinely used in P4-based FTAI protocols to synchronize determine the presence of corpora lutea (CL) 10 days before
time of ovulation in suckled B. indicus cows (Sá Filho et al., (Day −10) and on the first day of the experiment (Day 0).
2009; Meneghetti et al., 2009; Peres et al., 2009).
However, previous studies demonstrated that the treat- 2.1.2. Experiment 2
ment with EC at intravaginal P4 device removal is less Experiment 2 was performed on three commercial
effectively to synchronize the time of the preovulatory LH farms located in southern and central eastern Brazil. Cattle
surge (Souza et al., 2009) and ovulation (Martins et al., were maintained on a Brachiaria decumbens pasture with
2005) than the use of GnRH or EB as ovulatory stimulus. free access to mineralized-salt and water. A total of 619
Nonetheless, in spite of the increased variability in timing 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 previously used for 9 days. On Day 8, the NORG ear implant
the first day of the synchronization protocol (Day 0). Their was removed and cows received 300 IU eCG and 0.15 mg
body condition score (BCS) was evaluated on Day 0 using a d-cloprostenol. Then, the cows were allocated into one of
1 to 5 scale (1 = emaciated, 5 = obese; Ayres et al., 2009). three treatments, taking into account BCS and the sire used
for FTAI. In Group EC, cows were treated with 1 mg EC
2.1.3. Experiment 3 on Day 8. In Group GnRH, cows were treated with 10 ␮g
Experiment 3 was performed on four commercial farms buserelin acetate (SincroForte® , Ourofino Animal Health,
located in southern and central eastern Brazil. A total of Ribeirao Preto, Brazil) at FTAI. In Group EC + GnRH, cows
655 suckled B. indicus beef cows [Farm A = 97; Farm B = 148; received both treatments. The FTAI was performed 48–50 h
Farm C = 144; Farm D = 266 (two cohorts of cows 121 and after the NORG implant removal.
145)] ranging 30–60 days postpartum were maintained on One AI technician performed all inseminations. Seven
a B. brizantha pasture with free access to mineralized-salt different sires were used for the insemination depending
and water. Cows had their BCS evaluated on Day 0, using a on the farm location (Farm A = one sire; Farm B = two sires;
1–5 scale (1 = emaciated, 5 = obese; Ayres et al., 2009). Farm C = two sires; Farm D = three sires). All sires used had
previously resulted in satisfactory pregnancy outcomes in
2.2. Experimental design FTAI programs.

2.2.1. Experiment 1 2.3. Ultrasonographic examinations

At random stages of the estrous cycle, designated as Day
0, heifers and cows received a subcutaneous ear implant 2.3.1. Experiment 1
containing 3 mg norgestomet (NORG, Crestar® , Intervet, Transrectal ultrasonography of both ovaries was per-
Boxmeer, Netherlands) and were randomly assigned to formed on Day −10 and Day 0 (NORG implant insertion).
receive one of three treatments. Ten cows and 10 heifers Then, ovaries were assessed once daily to detect emergence
were treated with 2.0 mg EB intramuscularly (i.m.) (Estro- of new follicular wave and to confirm follicular domi-
gin, Farmavet® , Sao Paulo, Brazil). Ten cows and 10 heifers nance. The dominant follicle was identified as the newest
were treated with 2.5 mg EV i.m. ((1/2)EV) plus 1.5 mg of growing follicle reaching a diameter larger than 8 mm.
norgestomet (1 mL of the Crestar injectable portion, Inter- The day of FWE was retrospectively defined as the day
vet), and 10 cows and nine heifers received 5.0 mg EV i.m. when the dominant follicle was first detected at a diame-
plus 3.0 mg of norgestomet (total recommended dose; i.e. ter of 3 mm. A real-time ultrasonic scanner equipped with
2 mL of the Crestar injectable portion, Intervet). a 5.0 MHz linear transducer was used (Aloka 500-V, Coro-
metrics Medical Systems, Wallingford, CT, USA).
2.2.2. Experiment 2
At random stages of the estrous cycle, heifers were
2.3.2. Experiment 2
submitted to a protocol for synchronization of ovulation
At FTAI, all heifers had their ovaries scanned by ultra-
time for FTAI. On the first day of the synchronization pro-
sonography to identify and measure the largest follicle
tocol (i.e. Day 0), the BCS was recorded, 2 mg EB were
(LF). The LF at FTAI was classified based on their diame-
administered i.m. and heifers were randomly assigned into
ter (<3.6 mm, 3.6–8.0 mm, 8.1–12.5 mm or >12.5 mm), as
one of two experimental groups according to the num-
previous described by Sá Filho et al. (2010b). All heifers
ber of uses of the norgestomet (NORG) ear implant: new
were examined for pregnancy diagnosis by transrectal
or previously used for 9 days (once-used). By the time of
ultrasonography 30 days after FTAI. The detection of an
implant removal (8 days after insertion), heifers received
embryonic vesicle with a viable embryo (presence of heart-
0.15 mg d-cloprostenol (Preloban® , Intervet,) and 300 IU
beat) was used as an indicator of pregnancy. The P/AI was
eCG (Folligon® , Intervet) i.m. Thereafter, heifers from both
calculated as the proportion of heifers pregnant 30 days
groups were equally re-assigned to receive one of three
after FTAI divided by the number of heifers inseminated.
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 2.3.3. Experiment 3
0.5 mg EC (ECP® , Pfizer Animal Health, São Paulo, Brazil) All cows were examined for pregnancy diagnosis by
i.m. at NORG removal (Day 8), and heifers in the GnRH transrectal ultrasonography 55 days after FTAI. Similar to
group received 100 ␮g gonadorelin (Fertagil® , Intervet) at Experiment 2, the visualization of the fetus and its heart-
FTAI (Day 10). All heifers were inseminated 52–58 h after beat was used as an indicator of pregnancy.
NORG ear implant removal. One AI technician performed
all inseminations. Six different sires were used, depending 2.4. Statistical analysis
on farm location (Farm 1 = one sire; Farm 2 = three sires;
Farm 3 = two sires). All sires used in the present study had 2.4.1. Experiment 1
previously resulted in satisfactory pregnancy outcomes (i.e. Statistical analysis was conducted using the SAS Sys-
around 50% of P/AI) in FTAI programs. tem for Windows (SAS Institute Inc., Cary, NC, USA, 2003).
The explanatory variables included in the statistical model
2.2.3. Experiment 3 were the type of E2 ester (EB, (1/2)EV and EV), animal
At random stages of the estrous cycle (Day 0), suck- category (cows and heifers) and the interaction between
led cows received 2 mg EB i.m. and a NORG ear implant treatments and animal category. The dependent variable
M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139 135

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 P-Values

Heifers Cows Animal Estradiol Interaction

category esters

EB (1/2)EV EV EB (1/2)EV EV

Number of animals 10 10 9 10 10 10 – – –
FWE (days)2 2.5 ± 0.2a 4.2 ± 0.3b,x 6.1 ± 0.6c,x 2.5 ± 0.2a 3.1 ± 0.4ab,y 4.0 ± 0.5b,y 0.002 <0.0001 0.05
Range 2–3 3–6 3–8 2–4 2–6 2–6 – – –

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 =
/ y) differ significantly (P < 0.05).
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).
FWE = interval, in days, from estradiol injection to new follicular wave emergence.

(interval from E2 treatment to FWE) was tested accord- EV, or (1/2)EV (Table 1). However, an earlier (P < 0.0001)
ing to their homogeneity and normality of variances using and less variable (P = 0.02) interval from E2 treatment to
Guide Data Analysis from SAS and subjected to log10 trans- FWE (mean ± SE) was observed in heifers treated with EB
formation. As the Levene’s test revealed heterogeneity of (2.5 ± 0.2; 2–3 days) than in those treated with (1/2)EV
variances, the median test was used to compare intervals (4.2 ± 0.3; 3–6 days) or EV (6.1 ± 0.6; 3–8 days). Cows
from E2 treatment to FWE using the two-way ANOVA of treated with EV (4.0 ± 0.5; 2–6 days) had similar intervals
PROC GLM. The Tukey test was used to determine differ- from treatment to FWE than cows treated with (1/2)EV
ences between groups. (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
2.4.2. Experiment 2 effect of EB was similar in heifers and cows (P = 0.92),
The P/AI was analyzed using the procedure GLIMMIX however treatments with (1/2)EV (P = 0.02) or EV (P = 0.02)
of SAS, with heifers treated as a random effect. Addition- resulted in a greater delay on the FWE in heifers than in
ally, for the final logistic regression model, variables were cows.
removed by backward elimination, based on the Wald
statistics criterion, when P > 0.20. The variables included 3.2. Experiment 2
in the final model were the effects of farm, BCS, LF at FTAI
and treatments (number of uses of the NORG ear implant The BCS at the first day of the estrous synchroniza-
and ovulation inductions hormones). tion protocol was 3.0 ± 0.03. No interactions were observed
The diameter of the LF at FTAI was analyzed using between the different ovulation induction hormones eval-
PROC MIXED to detect the effect of farm, treatment, BCS uated (EB, EC, or GnRH) and the number of uses of NORG
and their interactions. The variables included in the final implant (new or once-used) on the diameter of the LF
models were the effects of farm, BCS and treatments at FTAI (P = 0.89) and P/AI (P = 0.55). A larger diameter of
(number of uses of the NORG ear implant and ovula- the LF (P = 0.00004) was observed in GnRH-treated heifers
tion induction hormones). The relationships between the (9.4 ± 0.03) than in EC-treated heifers (8.1 ± 0.03) and EB-
diameter of the LF at FTAI and the probability of preg- treated heifers (8.3 ± 0.03). Also, heifers treated with new
nancy for all heifers were determined. Logistic regression NORG implant (9.2 ± 0.02) had larger diameters of the
curves were created using the coefficients provided by LF (P = 0.01) than heifers treated with a once-used NORG
the interactive data analysis from SAS and the formula (7.9 ± 0.02). Results are shown in Table 2.
y = exp(˛ × x + b)/[1 + exp(˛ × x + b)]. On the first day of the synchronization protocol, an
effect of BCS was observed on the diameter of the LF
2.4.3. Experiment 3
at FTAI and P/AI. Heifers with a greater BCS (≥3.25)
The P/AI was analyzed using the procedure GLIMMIX
showed increased (P = 0.03) LF diameters (9.3 ± 0.03 mm)
of SAS, with cows treated as a random effect. Also, for the
and greater (P = 0.03) P/AI (54.1%; 93/172) than heifers with
final logistic regression model, variables were removed by
intermediate (3.00) BSC (8.3 ± 0.03 mm and 43.1%; 84/195)
backward elimination based on the Wald statistics crite-
or lesser (≤ 2.75) BCS (8.2 ± 0.03 mm and 41.5%; 105/253).
rion when P > 0.20. The variables included in the final model
Heifers with larger LF at FTAI had the greatest rates of
were the effects of farm, BCS and treatments.
pregnancy 30 days after FTAI (P = 0.0001; Fig. 1). More-
3. Results over, follicular diameter was categorized, and heifers with
follicles ranging from 8.1 to 12.5 mm in diameter had
3.1. Experiment 1 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
There were significant effects of treatment (P < 0.0001), heifers with follicles ranging from 3.6 to 8.0 mm in diam-
animal category (P = 0.002), and treatment-by-animal cat- eter (24.3%; 17/70) and those with LF < 3.6 mm (34.3%;
egory interaction (P = 0.05). Emergence of a new follicular 58/169). The P/AI also differed with the farm location
wave was detected in all animals after treatments with EB, [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




Probability of Pregnanvcy
Ovulation 60






0 5 10 15 20 25

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)].


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


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

(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

achieved among cows treated with different protocols


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


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


FEW than EB treatment, confirming our hypothesis. Also,


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.


9.0 ± 0.5

The E2 and progestin/P4 combination has been the most

successful hormone therapy used to synchronize FWE in


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


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


interval post-treatment regardless of the phase of follicle

M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139 137

development at which treatment was initiated. The use suckled B. indicus cows treated with an intravaginal P4
of 17␤-E2 in progestin-implanted B. taurus cattle was fol- device, the use of GnRH to synchronize ovulation reduced
lowed consistently by a new FWE, on average, 4.3 ± 0.2 days the risk of occurrence of estrus and the P/AI when com-
later (Bó et al., 1995). The administration of 5 or 2.5 mg of pared with either the use of EC or the combination of EC
17␤-E2 (Bó et al., 2002) or 2.5 mg of EB (Caccia and Bó, and GnRH (Sá Filho et al., 2011). It seems that EC could
1998) or EV (Colazo et al., 2005) in progestin-implanted be particularly important for suckled B. indicus cows under
cattle at random stages of the estrous cycle was followed tropical pasture conditions (i.e. greater incidence of post-
by a new FWE approximately 4 days later. partum anestrous) or cows with low BCS by increasing the
In the present study, although inclusion of treatment E2 concentration during the proestrus phase (Sellars et al.,
with an ester of E2 at the initiation of a progestin-based 2006; Hillegass et al., 2008; Souza et al., 2009) enhancing
treatment resulted in the emergence of a new follicular the occurrence of estrus and the odds of pregnancy follow-
wave, the administration of EV at either dosage used in ing FTAI (Sá Filho et al., 2011). Differences in P/AI observed
this study (2.5 or 5.0 mg) resulted in a longer and more following induction of ovulation with GnRH or EC in some
variable interval from treatment to emergence of a new studies but not in the present study may be attributable to
follicular wave than the administration of EB. O’Rourke differences in treatments with progestins between studies
et al. (2000) observed that the interval from E2 treatment to (Kojima et al., 1992; Rathbone et al., 2001). In the current
FWE seemed to depend on FSH resurgence, which has been study use of NORG implant may have resulted in a less sup-
reported to occur after E2 concentrations decreased below pressive effect on the frequency of LH release, resulting in
a threshold level. EV has a long circulating half-life, and its an increase in growth and synthesis of E2 by the domi-
prolonged suppressive effect on FSH and ovarian follicu- nant follicle compared to studies that treated cows with an
lar growth (Bó et al., 1993; Martinez et al., 2005) could be intravaginal P4 releasing insert. As a result in the current
responsible for the variability and length of interval from study critical threshold values for maturity of preovulatory
treatment to FWE observed in the current study. Hence, follicles were achieved in cows in each of the treatment
E2 preparation and dose can affect the timing of FWE in B. groups whereas in other studies differences in the maturity
indicus cattle. of preovulatory follicles may have differed at the time when
The present data also demonstrate that the adminis- treatments were applied resulting in differences in the size
tration of the EV in B. indicus heifers induced a prolonged and/or maturity of ovulatory follicles. Conversely, the asso-
interval from treatment to FWE compared with lactating B. ciation between GnRH and EC treatment did not improve
indicus cows. Furthermore, different than cows, half dose of the P/AI, probably due to a satisfactory synchronization of
EV (2.5 mg) still presented a longer interval from treatment ovulation by the use of EC in these cows. Therefore, it is
to FEW than EB in B. indicus heifers. These differences could possible that circulating E2 concentration were sufficient
be associated with differences in steroid metabolisms in to influence pregnancy rates in cows treated with GnRH
the two animal categories (Sartori et al., 2004; Wolfenson at FTAI and the addition effect of injectable EC at NORG
et al., 2004). Although it has not been specifically studied, implant removal was not evident.
it is conceivable that, similar to what occurs in Holsteins, Similarly to previous studies, the follicular diameter
suckled B. indicus cows could have a faster clearance rate of at the moment of FTAI influenced the odds of pregnancy
E2 than heifers. Therefore, that could influence the time at (Perry et al., 2007; Meneghetti et al., 2009; Sá Filho et al.,
which concentrations of E2 in circulation fail to maintain 2010b). However, when the treatments (Experiment 2)
suppression of the pre-emergence increase in FSH and thus were considered, despite the larger diameters of the LF
the timing of a new FWE (Burke et al., 2003). The greater at FTAI in heifers treated with a new NORG implant or
suppressive effects of the EV on follicle development in with GnRH in the current study when compared to those
B. indicus heifers need to be considered for the devel- treated with EB or EC or with a once-used NORG implant,
opment of new synchronization protocols and requires the larger follicular diameter had no effect on improving
further study. P/AI. It is important to state that the overall P/AI rate of
In the current study, similar P/AI were achieved with 34.3% observed in heifers with follicles of ≤3.5 mm in diam-
the use of three different protocols to induce ovulation eter might be overestimated, as it is possible that some of
using GnRH at the time of FTAI, EB 24 h after removing these heifers ovulated before the FTAI. Previous study with
NORG, or EC at the time of NORG implant removal, con- suckled B. indicus cows reinforces this possibility once the
firming the hypothesis for the present study. Similar effects majority of the females (79.4%) with a small follicle at FTAI
were observed with these three different treatments to ovulated before the day of FTAI (Sá Filho et al., 2010b).
induce ovulation using either with a new or once-used The follicular size at FTAI and the occurrence of pre-
NORG impalnt. Also similar P/AI using EB or EC (at implant mature ovulation could be influenced by several variables
removal or 24 h later, respectively) in the protocol to syn- such as the age of preovulatory follicle (Sanchez et al.,
chronize time of ovulations in B. taurus beef heifers (Colazo 1995; Austin et al., 1999), the LH profiles during the pro-
et al., 2003) and in B. indicus suckled beef cows (Penteado gestin/P4 treatment (Adams et al., 1992; Savio et al., 1993;
et al., 2006) were observed. Carvalho et al., 2008; Cerri et al., 2011) and the type and
Likewise, in suckled B. indicus cows, progestin-based the moment of ovulatory stimulus administration prior to
FTAI synchronization protocols using EC or GnRH to induce FTAI (Colazo et al., 2003; Souza et al., 2009). In spite of
ovulation presented similar P/AI. Also, there was no addi- the satisfactory predictability of the moment of ovulation
tive effect of the combination of EC and GnRH on the P/AI, provided by the use of EB to synchronize the ovulation
rejecting our hypothesis. Conversely, in a recent study with 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 Pennacchi Agropastoril (Cascavel, PR, Brazil), Fazenda
follicle at the time of the ovulatory stimulus treatment Primeiro de Maio (Sonora, MS, Brazil) and Fazenda Jacare-
(Neves, 2010). Neves (2010) reported that cows experi- una (Espigão do Leste, MT, Brazil) for allowing the use of
encing premature ovulation (i.e. ovulation occurring from their animals and facilities for this study. This research
48 to 59 h after P4 device removal) had larger ovulatory was supported by Intervet/Schering-Plough, São Paulo,
follicle (14.0 ± 2.2 mm) than cows with delayed ovula- SP, Brazil and FIRMASA-Technologies, Campo Grande, MS,
tion (11.4 ± 2.2 mm; 73–96 h after P4 device removal) and Brazil.
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 References
lower circulating P4, such as when using a previously used
NORG implant, could have a larger follicle size at the time Adams, G.P., Matteri, R.L., Ginther, O.J., 1992. Effect of progesterone on
of the implant removal, resulting in a greater occurrence of ovarian follicles, emergence of follicular waves and circulating follicle-
stimulating hormone in heifers. J. Reprod. Fertil. 95, 627–640.
earlier ovulations. Austin, E.J., Mihm, M., Ryan, M.P., Williams, D.H., Roche, J.F., 1999. Effect
In addition, the larger follicle diameter at FTAI of the of duration of dominance of the ovulatory follicle on onset of estrus
heifers treated GnRH could be due to the moment that and fertility in heifers. J. Anim. Sci. 77, 2219–2226.
Ayres, H., Ferreira, R.M., Torres-Júnior, J.R.S., Demétrio, C.G.B., de Lima,
the follicle was measured relatively to the timing of the C.G., Baruselli, P.S., 2009. Validation of body condition score as a pre-
expected moment of the LH surge. The LH surge after the dictor of subcutaneous fat in Nelore (Bos indicus) cows. Livestock Sci.
GnRH administration occurs in approximately 2 h (i.e. 2 h 123, 175–179.
Bó, G.A., Adams, G.P., Nasser, L.F., Pierson, R.A., Mapletoft, R.J., 1993. Effect
after the FTAI; Chenault et al., 1990) when compared to
of estradiol valerate on ovarian follicles, emergence of follicular waves
the 48 h after EC administration (i.e. approximately 7 h and circulating gonadotropins in heifers. Theriogenology 40, 225–239.
before the FTAI; Sales et al., 2008) and the 20 h following Bó, G.A., Adams, G.P., Pierson, R.A., Tribulo, H.E., Caccia, M., Mapletoft,
R.J., 1994. Follicular wave dynamics after estradiol-17␤ treatment of
EB administration (i.e. approximately 7 h before the FTAI;
heifers with or without a progestogen implant. Theriogenology 41,
Lammoglia et al., 1998; Sales et al., 2008). Therefore, heifers 1555–1569.
treated with GnRH at FTAI could present a larger period of Bó, G.A., Adams, G.P., Pierson, R.A., Mapletoft, R.J., 1995. Exogenous control
the preovulatory follicle growth than heifers treated with of follicular wave emergence in cattle. Theriogenology 43, 31–40.
Bó, G.A., Caccia, M., Martínez, M.F., Mapletoft, R.J., 1996. Follicle wave
EC at or EB 24 h after the NORG implant removal. emergence after treatment with estradiol benzoate and CIDR vagi-
The data reported herein demonstrate that a greater nal devices in beef cattle. In: Proc. Int. Congr. Anim. Reprod., vol. 22,
BCS resulted in greater conception rates after AI, indicating Sydney, Australia (abstract).
Bó, G.A., Baruselli, P.S., Moreno, D., Cutaia, L., Caccia, M., Tribulo, R., Tribulo,
that nutritional status is critical for FTAI synchronization H., Mapletoft, R.J., 2002. The control of follicular wave development
protocol responses in cyclic B. indicus heifers. The farm for self-appointed embryo transfer programs in cattle. Theriogenology
location also accounted for some variation in reproduc- 57, 53–72.
Bó, G.A., Adams, G.P., Caccia, M., Martínez, M.F., Pierson, R.A., Mapletoft,
tive performance. This is an extremely important aspect R.J., 2005. Ovarian follicular wave emergence after treatment with
that compromises the commercial application of FTAI in B. progestogen and estradiol in cattle. Anim. Reprod. Sci. 39, 193–204.
indicus cattle. The effect of farm location can be related to Burke, C.R., Macmillan, K.L., Boland, M.P., 1996. Oestradiol potentiates a
prolonged progesterone-induced suppression of LH release in ovariec-
BCS, sexual maturity (heifers), estrous cyclic status (suck-
tomised cows. Anim. Reprod. Sci. 45, 13–28.
led cows), sire used and type of feeding. All of these factors Burke, C.R., Mussard, M.L., Gasser, C.L., Grum, D.E., Day, M.L., 2003.
usually differ among farms (Sá Filho et al., 2009), and can Estradiol benzoate delays new follicular wave emergence in a dose-
dependent manner after ablation of the dominant ovarian follicle in
potentially influence the pregnancy outcomes of a repro-
cattle. Theriogenology 60, 647–658.
ductive program. Caccia, M., Bó, G.A., 1998. Follicle wave emergence following treatment of
In summary, the administration of EV (2.5 or 5.0 mg) CIDR-implanted beef cows with estradiol benzoate and progesterone.
with a NORG implant delayed the day of FWE in comparison Theriogenology 49, 34 (abstract).
Carvalho, J.B., Carvalho, N.A., Reis, E.L., Nichi, M., Souza, A.H., Baruselli,
to treatment with 2.0 mg of EB in B. indicus cattle. Further- P.S., 2008. Effect of early luteolysis in progesterone-based timed AI
more, the effects of EV on FWE seem to be more pronounced protocols in Bos indicus, Bos indicus × Bos taurus, and Bos taurus heifers.
in B. indicus heifers than in B. indicus cows. Synchroniza- Theriogenology 69, 167–175.
Cerri, R.L., Chebel, R.C., Rivera, F., Narciso, C.D., Oliveira, R.A., Amstalden,
tion protocols for FTAI with either new or once-used NORG M., Baez-Sandoval, G.M., Oliveira, L.J., Thatcher, W.W., Santos, J.E.,
implant and EB, EC or GnRH as ovulation induction hor- 2011. Concentration of progesterone during the development of the
mones may be successfully used in B. indicus heifers. Finally, ovulatory follicle. II. Ovarian and uterine responses. J. Dairy Sci. 94,
the administration of either EC, GnRH or both as ovulation Chenault, J.R., Kratzer, D.D., Rzepkowski, R.A., Goodwin, M.C., 1990. LH and
induction hormones resulted in similar P/AI in suckled B. FSH response of Holstein heifers to fertirelin acetate, gonadorelin and
indicus cows, showing no additive effect of the combination buserelin. Theriogenology 34, 81–98.
Colazo, M.G., Kastelic, J.P., Mapletoft, R.J., 2003. Effects of estradiol cyp-
of both EC and GnRH on P/AI.
ionate (ECP) on ovarian follicular dynamics, synchrony of ovulation,
and fertility in CIDR-based, fixed-time AI programs in beef heifers.
Acknowledgements Theriogenology 60, 855–865.
Colazo, M.G., Martínez, M.F., Small, J.A., Kastelic, J.P., Burnley, C.A., Ward,
D.R., Mapletoft, R.J., 2005. Effect of estradiol valerate on ovarian folli-
The authors are grateful to Dr. Henderson Ayers and cle dynamics and superovulatory response in progestin-treated cattle.
Dr. Roberta Ferreira (University of São Paulo, Brazil) for Theriogenology 63, 1454–1468.
their help with manuscript preparation and statistical anal- Geary, T.W., Whittier, J.C., Downing, E.R., LeFever, D.G., Silcox, R.W., Hol-
land, M.D., Nett, T.M., Niswender, G.D., 1998. Pregnancy rates of
yses. The authors also thank the staff of APTA-Polo Regional postpartum beef cows that were synchronized using Syncro-Mate-B
Vale do Paraiba (Pindamonhangaba, São Paulo, Brazil), or the Ovsynch protocol. J. Anim. Sci. 76, 1523–1527.
M.F. Sá Filho et al. / Animal Reproduction Science 129 (2011) 132–139 139

Geary, T.W., Salverson, R.R., Whittier, J.C., 2001. Synchronization of ovu- Pursley, J.R., Kosorok, M.R., Wiltbank, M.C., 1997a. Reproductive manage-
lation using GnRH or hCG with the CO-Synch protocol in suckled beef ment of lactating dairy cows using synchronization of ovulation. J.
cows. J. Anim. Sci. 79, 2536–2541. Dairy Sci. 80, 301–306.
Hillegass, J., Lima, F.S., Sá Filho, M.F., Santos, J.E., 2008. Effect of time of Pursley, J.R., Wiltbank, M.C., Stevenson, J.S., Ottobre, J.S., Garverick, H.A.,
artificial insemination and supplemental estradiol on reproduction of Anderson, L.L., 1997b. Pregnancy rates of cows and heifers insemi-
lactating dairy cows. J. Dairy Sci. 91, 4226–4237. nated at a synchronized ovulation or synchronized estrus. J. Dairy Sci.
Kojima, N., Stumpf, T.T., Cupp, A.S., Werth, L.A., Roberson, M.S., Wolfe, 80, 295–300.
M.W., Kittok, R.J., Kinder, J.E., 1992. Exogenous progesterone and pro- Rathbone, M.J., Kinder, J.E., Fike, K., Kojima, F., Clopton, D., Ogle, C.R., Bunt,
gestins as used in estrous synchrony regimens do not mimic the C.R., 2001. Recent advances in bovine reproductive endocrinology and
corpus luteum in regulation of luteinizing hormone and 17 beta- physiology and their impact on drug delivery system design for the
estratadiol in circulations of cows. Biol. Reprod. 47, 1009–1017. control of the estrous cycle in cattle. Adv. Drug Deliv. Rev. 50, 277–320.
Lammoglia, M.A., Short, R.E., Bellows, S.E., Bellows, R.A., MacNeil, M.D., Sá Filho, O.J., Meneghetti, M., Peres, R., Lamb, G., Vasconcelos, J.L.M., 2009.
Hafs, H.D., 1998. Induced and synchronized estrus in cattle: dose titra- Fixed-time artificial insemination with estradiol and progesterone for
tion of estradiol benzoate in peripubertal heifers and postpartum cows Bos indicus cows. II. Strategies and factors affecting fertility. Theri-
after treatment with an intravaginal progesterone-releasing insert ogenology 72, 210–218.
and prostaglandin F2alpha. J. Anim. Sci. 76, 1662–1670. Sá Filho, M.F., Ayres, H., Ferreira, R.M., Marques, M.O., Reis, E.L., Silva,
Macmillan, K.L., Peterson, A.J., 1993. A new intravaginal progesterone R.C., Rodrigues, C.A., Madureira, E.H., Bó, G.A., Baruselli, P.S., 2010a.
releasing device for cattle (CIDR-B) for oestrus synchronisation, Equine chorionic gonadotropin and gonadotropin-releasing hormone
increasing pregnancy rates and the treatment of post-partum enhance fertility in a norgestomet-based, timed artificial insemina-
anoestrus. Anim. Reprod. Sci. 33, 1–25. tion protocol in suckled Nelore (Bos indicus) cows. Theriogenology 73,
Macmillan, K.L., Taufa, V.K., Day, A.M., 1997. Manipulating ovaries’ fol- 651–658.
licle wave patterns can partially synchronize returns to service and Sá Filho, M.F., Crespilho, A.M., Santos, J.E.P., Perry, G.A., Baruselli, P.S.,
increases the pregnancy rate to second insemination. Proc. N. Z. Soc. 2010b. Ovarian follicle diameter at timed insemination and estrus
Anim. Prod. 57, 23 (abstract). response influences the likelihood of ovulation and pregnancy after
Mapletoft, R.J., Colazo, M.G., Small, J.A., Ward, D.R., Kastelic, J.P., 2004. synchronization with progesterone or progestin-based protocols in
Effect of dose of estradiol valerate on ovarian follicular dynamics in suckled Bos indicus cows. Anim. Reprod. Sci. 120, 23–30.
CIDR-treated beef cows. Reprod. Fertil. Dev. 16, 130 (abstract). Sá Filho, M.F., Santos, J.E., Ferreira, R.M., Sales, J.N., Baruselli, P.S., 2011.
Martinez, M.F., Kastelic, J.P., Bó, G.A., Caccia, M., Mapletoft, R.J., 2005. Importance of estrus on pregnancy per insemination in suckled Bos
Effects of oestradiol and some of its esters on gonadotrophin release indicus cows submitted to estradiol/progesterone-based timed insem-
and ovarian follicular dynamics in CIDR-treated beef cattle. Anim. ination protocols. Theriogenology 76, 455–463.
Reprod. Sci. 86, 37–52. Sales, J.N.S., Carvalho, J.B.P., Crepaldi, G.A., Maio, J.R.G., Carvalho, C.A.B.,
Martins, C.M., Castricini, E.S.C., Sá Filho, M.F., Gimenes, L.U., Baruselli, P.S., Baruselli, P.S., 2008. Rate and timing of ovulation in Nelore cows
2005. Follicular dynamics of the Nelore cows treated with estradiol treated with estradiol cypionate or benzoate to induce ovulation on
cipionate or estradiol benzoate in timed artificial insemiantion proto- FTAI protocols. Reprod. Dom. Anim. 43, 181 (abstract).
cols. Acta Sci. Vet. 33, 285 (abstract). Sanchez, T., Wehrman, M.E., Kojima, F.N., Cupp, A.S., Bergfeld, E.G., Peters,
Meneghetti, M., Sá Filho, O.J., Peres, R., Lamb, G., Vasconcelos, J.L.M., 2009. K.E., Mariscal, V., Kittok, R.J., Kinder, J.E., 1995. Dosage of the syn-
Fixed-time artificial insemination with estradiol and progesterone for thetic progestin, norgestomet, influences luteinizing hormone pulse
Bos indicus cows. I. Basis for development of protocols. Theriogenology frequency and endogenous secretion of 17 beta-estradiol in heifers.
72, 179–189. Biol. Reprod. 52, 464–469.
Neves, K.A.L., 2010. Effect of interval between insemination and ovulation Sartori, R., Haughian, J.M., Shaver, R.D., Rosa, G.J.M., Wiltbank, M.C.,
in conception rates in Nelore cows timed AI with sex-sorted. Master 2004. Comparison of ovarian function and circulating steroids in
Diss. University of Sao Paulo, Brazil, p. 57. estrous cycles of holstein heifers and lactating cows. J. Dairy Sci. 87,
O’Rourke, M., Diskin, M.G., Sreenan, J.M., Roche, J.F., 2000. The effect of 905–920.
dose and route of oestradiol benzoate administration on plasma con- Savio, J.D., Thatcher, W.W., Morris, G.R., Entwistle, K., Drost, M., Mattiacci,
centrations of oestradiol and FSH in long-term ovariectomised heifers. M.R., 1993. Effects of induction of low plasma progesterone concen-
Anim. Reprod. Sci. 59, 1–12. tration with progesterone-releasing intravaginal device on follicular
Odde, K.G., 1990. A review of synchronization of estrus in postpartum turnover and fertility in cattle. J. Reprod. Fertil. 98, 77–84.
cattle. J. Anim. Sci. 68, 817–830. Sellars, C.B., Dalton, J.C., Manzo, R., Day, J., Ahmadzadeh, A., 2006. Time and
Penteado, L., Ayres, H., Torres-Júnior, J.R., Souza, A.H., Baruselli, P.S., incidence of ovulation and conception rates after incorporating estra-
2006. Conception rate of the lactating Nelore cows synchronized with diol cypionate into a timed artificial insemination protocol. J. Dairy
intravaginal progesterone device associated with estradiol benzoate Sci. 89, 620–626.
or estradiol cipionate. Acta Sci. Vet. 34, 401 (abstract). Souza, A.H., Viechnieski, S., Lima, F.A., Silva, F.F., Araújo, R., Bó, G.A.,
Peres, R.F., Claro Jr., I., Sá Filho, O.G., Nogueira, G.P., Vasconcelos, J.L., Wiltbank, M.C., Baruselli, P.S., 2009. Effects of equine chorionic
2009. Strategies to improve fertility in Bos indicus postpubertal heifers gonadotropin and type of ovulatory stimulus in a timed-AI protocol
and nonlactating cows submitted to fixed-time artificial insemination. on reproductive responses on dairy cows. Theriogenology 72, 10–21.
Theriogenology 72, 681–689. Wolfenson, D., Inbar, G., Roth, Z., Kaim, M., Bloch, A., Braw-Tal,
Perry, G.A., Smith, M.F., Roberts, A.J., MacNeil, M.D., Geary, T.W., 2007. R., 2004. Follicular dynamics and concentrations of steroids and
Relationship between size of ovulatory follicle and pregnancy success gonadotropins in lactating cows and nulliparous heifers. Theriogenol-
in beef heifers. J. Anim. Sci. 85, 684–689. ogy 62, 1042–1055.