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Theriogenology 71 (2009) 1120–1126

www.theriojournal.com

The effect of addition of equine chorionic gonadotropin to a

progesterone-based estrous synchronization protocol in buffaloes
(Bubalus bubalis) under tropical conditions
K. Murugavel a, D. Antoine a, M.S. Raju a, F. López-Gatius b,*
a
Department of Animal Reproduction, Gynaecology and Obstetrics, Rajiv Gandhi College of Veterinary and Animal Sciences,
Kurumbapet, Pondicherry 605009, India
b
Department of Animal Production, University of Lleida, Lleida, Spain
Received 21 June 2008; received in revised form 10 December 2008; accepted 10 December 2008

Abstract
Poor estrus expression and anestrus decrease the reproductive efficiency of buffaloes. The objective of this study was to
determine whether the addition of equine chorionic gonadotropin (eCG) to an estrous synchronization protocol and timed
insemination could improve ovulation and pregnancy rates of anestrous buffalo cows under tropical conditions. The study
population comprised 65 lactating Murrah buffalo cows which were assigned to CIDR (n = 33) or CIDR + eCG (n = 32) treatment
groups. Cows in the CIDR group were fitted for 8 d with a controlled intravaginal drug release (CIDR) device containing 1.38 g
progesterone, received GnRH (10 mg i.m.) on D 0, PGF2a (750 mg i.m.) on D 7, and GnRH (10 mg i.m.) on D 9; whereas cows in the
CIDR + eCG group received the same treatment plus eCG (500 IU, i.m.) at the time of PGF2a treatment. All cows were inseminated
16–20 h after the second GnRH treatment. Blood samples were obtained 10 d before the start of synchronization treatment (Day –
10) and at the onset of treatment (Day 0). Cows with plasma progesterone concentrations <1 ng/mL recorded in both samples
(Low–Low levels of P4) were classified as non-cyclic cows. Similarly, when either one or both of the sample pair contained
concentrations of serum progesterone 1 ng/mL (High–High, Low–High, or High–Low levels of P4), the buffaloes were classified
as cyclic cows. Ovulation rate, defined as the number of buffaloes with at least one corpus luteum 10 days after insemination, was
significantly higher (P = 0.018) in the CIDR + eCG (84.4%) cows than in the CIDR cows (57.6%). Pregnancy rate was numerically
lower in CIDR (27.3%) than CIDR + eCG (40.6%) cows, though differences were not significant (P = 0.25). Pregnancy rates for
CIDR + eCG cows were similar to that of cows inseminated after natural estrus (40.9%; 29/71). In the non-cyclic animals, higher
ovulation rates (P = 0.026) were recorded for the CIDR + eCG (81%) than for the CIDR cows (47.4%). Our results indicate that the
addition of eCG to a progesterone-based estrous synchronization regimen substantially improves the ovulation rate in non-cyclic
buffaloes. When this treatment is followed by timed AI, pregnancy rates achieved in anestrous buffaloes, whether cyclic and non-
cyclic, may approach the rates observed in cows inseminated at natural estrus.
# 2009 Elsevier Inc. All rights reserved.

Keywords: eGG; Progesterone-based estrous synchronization; Anestrus; Pregnancy; Buffalo cows

1. Introduction

About 97% of the world’s buffaloes are reared by

* Corresponding author. Tel.: +34 973702500. small holders in South and Southeast Asia, most
E-mail address: flopez@prodan.udl.cat (F. López-Gatius). enterprises having fewer than 10 animals [1]. Due to

0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2008.12.012
 Author's personal copy
K. Murugavel et al. / Theriogenology 71 (2009) 1120–1126
the tropical and subtropical conditions of these areas
buffalo cows show; a marked ovarian inactivity,
suboptimal functioning of the hypothalamus–pituitary–
gonadal axis [2–5] associated with low peaks of FSH and
LH [6,7], and variable plasma progesterone levels in the
luteal phase [8,9]. Therefore, it is difficult to successfully
implement estrous synchronization (ES) and artificial
insemination (AI) protocols in buffalo cows.
Reproductive similarities between bubaline (Buba-
lus bubalis) and bovine (Bos taurus and Bos indicus)
reproduction allow reproductive management technol-
ogies developed in cattle to be applied to buffalo [10].
However, many ES protocols used in dairy cows show
variable responses in buffalo cows [11]. One reason for
this discrepancy between bovine and buffalo could be
that buffalo cows have a higher degree of variability in
the interval from the preovulatory LH surge to
ovulation [12] than bovine cows in both naturally
and hormonally induced ovulations [11]. A timed
insemination protocol (Ovsynch) has been developed
in dairy cows to precisely control the time of ovulation
[13,14]. In the Ovsynch program, 100 mg of GnRH are
given at random during the estrous cycle, followed by
25 mg of PGF2a on D 7 and a second dose of 100 mg
GnRH 36–48 h later. Artificial insemination is per-
formed 16–20 h after the second GnRH treatment.
Notwithstanding, the Ovsynch protocol was found to
be effective in buffaloes, particularly cyclic buffalo
cows, for both double [15–17] and single fixed-time AI
[18–21]. However, anestrous dairy cows [22–25] and
acyclic buffaloes [19,21] respond poorly to the
Ovsynch protocol yielding poor conception/pregnancy
rates, compared to cyclic cows. Pregnancy rates in non-
cyclic/anestrous cows [25–29] and buffaloes [19],
however, seem to be improved by adding exogenous
progesterone with an intravaginal progesterone device
to the Ovsynch protocol. Previous experiments on
anestrous buffaloes have shown that a progesterone-
based estrus synchronization protocol, with equine
chorionic gonadotropin (eCG) given at the time of
progesterone withdrawal, improved the ovulation rate
[30–32]. However, due to the unpredictability of
the timing of ovulation, at least two inseminations
[15,32,33] or AI at observed estrus [6,30] were required
to obtain acceptable pregnancy rates. Furthermore, few
studies have been designed to evaluate ES protocols
applied in buffaloes under field conditions in a tropical
climate. Most ES protocols have been tested in well-
managed animals reared under farm conditions [1,11]
and environmental and nutrition factors have a major
impact on the conception rate and response to estrus
synchronization treatment [34].
1121
In order to promote ovulation [30–32], we hypothe-
sized that the addition of eCG to a progesterone-based
ES protocol may improve ovulation and pregnancy rates
of anestrous buffaloes in response to fixed-time AI. The
objective of our study was to evaluate the effect of
addition of eCG to a progesterone-based protocol on
anestrous cyclic and acyclic Murrah buffalo cows under
conditions of village herd management and a tropical
climate.
2. Materials and methods
2.1. Animals
The study was conducted on multiple small buffalo
herds in the Pondicherry area, southern India (latitude
11.598N, longitude 79.508E) over the period June 2004–
April 2005. The study population was comprised of 65
postpartum lactating multiparous Murrah buffalo cows
(B. bubalis) with more than 120 d in milk. None of the
cows used in this experiment were observed in estrus
from calving to 10 d before the start of treatment and
were considered to be anestrus. Cows were kept in
stalls, suckled during 30–40 s and then milked by hand
twice daily, fed with mixed rations and allowed to graze
for 3–4 h per day, and had access to water throughout
each day. Over the year, air temperature ranged from 24
to 32 8C. Body condition scores were assigned at the
start of treatment by the same veterinarian using a five-
point scale: 1 = thin to 5 = obese [18]. Only buffaloes
with body condition scores between score 2 and 3.5
were included in this study. Efforts were made to reduce
the variation in the general health status of the animals,
so that failure to ovulate or conceive could be attributed
to factors other than the clinical condition of the
buffaloes during the study. Animals with diseases
detected during the course of the study, such as digestive
disorders, abnormal genital discharges and abnormal-
ities of the reproductive tract detectable by palpation per
rectum, were withdrawn from the trial.
2.2. Treatment and AI
At the start of the experiment sixty-seven buffalo
cows were assigned randomly to one of two treatments
9.7  3.53 after calving to two progesterone-based ES
protocols with (CIDR + eCG) or without (CIDR) the
addition of equine chorionic gonadotropin (eCG;
Fig. 1). Buffaloes in the CIDR group (n = 33) were
treated with GnRH (Buserelin 10 mg i.m.; Receptal Vet,
Intervet International, GmbH, Germany) and received a
controlled internal drug release device (CIDR) contain-
 Author's personal copy
1122 K. Murugavel et al. / Theriogenology 71 (2009) 1120–1126
Fig. 1. Diagram showing the protocols for estrous synchronization used in the two treatment groups.
ing 1.38 g progesterone (Ezibred, Livestock Improve-
ment, Hamilton, NewZealand) on D 0 (a.m.). On D 7
(p.m.), buffaloes received a luteolyic dose of PGF2a
(Tioprost 750 mg i.m.; Ilerin, Intervet International,
GmbH, Germany). The CIDR’s were removed on D 8
(a.m.). A second dose of GnRH (10 mg i.m.) was
administered 48 h after the PGF2a treatment. Buffaloes
in the CIDR + eCG group (n = 34) received the same
treatment except that they were also given a dose of
equine chorionic gonadotropin (eCG) (Serum Gonado-
trophin 500 IU, i.m.; Folligon, Intervet International
B.V., Boxmeer, Holland) at the time of PGF2a treatment
(Fig. 1).
During the treatment period cows were prevented
from wallowing, to reduce the occurrence of vaginal
infection due to the presence of the CIDR device. Two
CIDR + eCG cows lost the CIDR device and were
excluded from the study. Thus, the final numbers of
buffaloes in the CIDR and CIDR + eCG groups were 33
and 32, respectively.
Estrous signs following the treatment were not
registered and all the animals were inseminated 16–20 h
after the second GnRH dose using frozen/thawed semen
from a single Murrah buffalo bull of proven fertility. All
inseminations were performed by the same veterinarian.
2.3. Ovulation and pregnancy diagnosis
Ovulation rate was determined by rectal palpation of
ovarian structures for the presence or absence of corpora
lutea on each ovary 10-d after timed-AI. Pregnancy rate
was determined by rectal palpation of ovarian structures
and uterine contents 45 d after AI.
Ovulation rate was defined as the number of
buffaloes with at least one corpus luteum 10 d after
insemination as a percentage of the total number of
animals in each group. Pregnancy rate was defined as
the number of pregnant buffaloes after AI as a
percentage of the total number of buffaloes in each
group. The pregnancy rate of buffaloes in the same
village inseminated following natural estrus during the
study period was used as reference.
2.4. Determination of cycling status
Blood samples were collected from the jugular vein
of each animal into heparinized vacuum tubes 10-d
before and at the start of the ES period. These blood
samples were centrifuged (10 min, 1600  g) within
2 h after collection, and plasma stored at 20 8C until
assayed. Plasma progesterone concentrations were
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K. Murugavel et al. / Theriogenology 71 (2009) 1120–1126 1123

Table 1
Effects of the two treatment regimes on ovulation and pregnancy rates in anestrous buffaloes.
Group CIDRa (n = 33) CIDR + eCGb (n = 32) P-value
c
Ovulation rate (%) 57.6 (19/33) 84.4 (27/32) 0.018
Pregnancy rate (%) d 27.3 (9/33) 40.6 (13/32) 0.25
a
Cows were fitted for 8 d with a controlled intravaginal drug release (CIDR) device containing 1.38 g progesterone, received GnRH (10 mg i.m.)
on D 0, PGF2a (750 mg i.m.) on D 7, and GnRH (10 mg i.m.) on D 9, and inseminated 16–20 h after the second GnRH treatment.
b
Cows received the same treatment as for the CIDR cows plus 500 IU eCG i.m. along with PGF2a.
c
The number of buffaloes with at least one corpus luteum 10 d after insemination.
d
The number of pregnant buffaloes 45 d after insemination.

determined using solid-phase RIA kits (Immunotech, suffer subestrus or silent estrus, since these animals
Beckman Coulter, France). The sensitivity of the assay showed no estrous signs from the time of calving until
was 0.05 ng/mL progesterone. Plasma samples showing the outset of treatment.
hormone concentrations below this value were assigned Overall ovulation and pregnancy rates and ovulation
the sensitivity value. Intra- and inter-assay coefficients and pregnancy rates within cycling status were evaluated
of variation of the P4-RIA technique were 5.8% and using Chi-square analyses. P-values less than 0.05 were
9.0%, respectively. The intra-assay variation was considered significant and values are expressed as the
assessed as the coefficient of variation of 10 replicate mean  standard deviation (S.D.).
determinations in the same assay. The inter-assay
variation was assessed as the coefficient of variation of 3. Results
18 duplicate determinations in different assays.
Over the study period, 71 buffaloes showing natural
2.5. Statistical analyses estrus were inseminated and 29 (40.9%) of them
resulted in pregnancy.
Plasma concentrations of progesterone 1 ng/mL Mean lactation numbers and intervals from parturi-
were considered indicative of the presence of an active tion to AI/treatment were 2.6  1.29 lactations (1–7
corpus luteum [35]. Accordingly, buffaloes with plasma lactations) and 2.4  1.13 lactations (1–6 lactations);
progesterone concentrations <1 ng/mL recorded in two and 8.9  2.82 months (5–21 months) and 10.5  4
samples taken on Days 10 and 0 (Low–Low levels of months (4–22 months) for CIDR and CIDR + eCG
P4) were classified as anestrous or non-cyclic cows. cows, respectively.
Similarly, when either one or both of the sample pair Ovulation rates were significantly higher (P = 0.018)
contained concentrations of serum progesterone 1 ng/ in the CIDR + eCG (84.4%;) than in the CIDR cows
mL (High–High, Low–High, or High–Low levels of (57.6%) (Table 1). Pregnancy rates did not differ
P4), the buffaloes were classified as subestrous or cyclic (P = 0.25) but numerically lower for CIDR (27.3%)
cows [23]. Thus, cyclic buffaloes were considered to cows than CIDR + eCG (40.6%) cows.
Table 2
Effects of the two treatment regimes on ovulation and pregnancy rates in anestrous buffaloes classified as cyclic or non-cyclic at the onset of
treatment.
Total (n = 65) CIDRa (n = 33) CIDR + eCGb (n = 32) P-value
Ovulation rate (%)
Non-cyclicc 65 (26/40) 47.4 (9/19) 81 (17/21) 0.026
Cyclicd 80 (20/25) 71.4 (10/14) 90.9 (10/11) 0.23
Pregnancy rate (%)
Non-cyclicc 30 (12/40) 21.1 (4/19) 38.1 (8/21) 0.24
Cyclicd 40 (10/25) 35.7 (5/14) 45.5 (5/11) 0.62
a
Cows were fitted for 8 d with a controlled intravaginal drug release (CIDR) device containing 1.38 g progesterone, received GnRH (10 mg i.m.)
on D 0, PGF2a (750 mg i.m.) on D 7, and GnRH (10 mg i.m.) on D 9, and inseminated 16–20 h after the second GnRH treatment.
b
Cows received the same treatment as for the CIDR cows plus 500 IU eCG i.m. along with PGF2a.
c
Cows with plasma progesterone concentrations <1 ng/mL recorded in both samples 10 d before and at the onset of treatment (Low–Low levels of P4).
d
Cows with either one or both of the sample pair (10 d before and at the onset of treatment) contained concentrations of serum progesterone
1 ng/mL (High–High, Low–High, or High–Low levels of P4).
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1124 K. Murugavel et al. / Theriogenology 71 (2009) 1120–1126
Percentages of non-cyclic animals at the outset of
treatment were 57.6% (n = 19) and 65.6% (n = 21)
for the CIDR and CIDR + eCG cows, respectively. In
non-cyclic buffaloes, the ovulation rate was higher
(P = 0.026) for the CIDR + eCG (81%) than CIDR
group (47.4%). The CIDR + eCG group appeared a
higher, albeit not significant (P = 0.24), pregnancy rate
(38.1%) than the CIDR Group (21.1%). Ovulation and
pregnancy rates for the cyclic cows failed to differ
(P > 0.2) between treatments (Table 2).
4. Discussion
Suboptimal functioning of the hypothalamus–pitui-
tary–gonadal (HPG) axis and low FSH and LH peaks
have been related by several authors to anestrus in the
buffalo cow under tropical and subtropical conditions [2–
5,7]. In another study, eCG subsequent to 13 d of
progesterone treatment increased plasma concentrations
of LH and FSH and enhanced the LH peak during estrus
in non-cyclic buffalo heifers [6]. Thus, administration of
eCG probably helps complete the recovery of HPG axis
function already stimulated by the progesterone treat-
ment. Our results are in partial agreement with these
findings in that the authors reported that their GnRH–
progesterone-based ES protocol (lacking eCG) failed to
sufficiently trigger the HPG axis in non-cyclic buffaloes.
This could explain the higher (P = 0.026) ovulation rate
observed in our CIDR + eCG cows (81%) compared to
the CIDR cows (47.4%) in non-cyclic buffaloes. As a
result, although not significantly, the pregnancy rate
including both cyclic and non-cyclic anestrous animals
appeared lower in the CIDR (27.3%) than that in the
CIDR + eCG (40.6%) group; the latter rate being similar
to that noted in the cows inseminated following a natural
estrus used as reference (40.9%). This result needs to be
confirmed in further studies.
We were unable to find a treatment effect in cyclic
anestrous buffaloes. In effect, the pregnancy rate for all
treated cyclic anestrous buffaloes (40%) was comparable
to that shown by buffaloes inseminated at natural estrus
(40.9%), and to the rate of 40% previously reported for
buffaloes inseminated at overt estrus under similar
tropical–subtropical conditions [36,37]. However,
although not significant, there was a clear tendency in
the CIDR cows for a higher pregnancy rate in cyclic
(35.7%) versus non-cyclic (21.1%) animals. The same
tendency was observed in a previous study performed on
Mediterranean buffaloes subjected to a similar proges-
terone-based protocol [19]. In this latter study, pregnancy
rates were 51.5% and 30% for cyclic and non-cyclic
buffaloes, respectively, which are higher than the rates
recorded here. This difference in response to similar
treatment may be attributed to differences in breed,
management and environmental conditions. As men-
tioned above, the present study was undertaken in a
tropical area in which high temperatures throughout the
year may impair ovarian function.
Based on plasma progesterone concentrations deter-
mined on Days 10 and 0 of treatment, we stratified our
data according to whether the anestrous animals were
cyclic or non-cyclic at the start of treatment (Table 2).
Thus, 25 (38.5%) of the 65 anestrous buffaloes showed
ovarian activity but no estrus signs. This observation
confirms the findings of previous studies in which silent
ovulation or low intensity estrous behavior can occur in
as many as 30–40% buffalo cows [38,39].
The hypothesis that the addition of eCG to a
progesterone-based protocol might improve the ovula-
tion and pregnancy rates of anestrous buffaloes in
response to fixed-time AI was supported by our findings.
The treatment improved substantially the ovulation
rate in non-cyclic buffaloes, whereas pregnancy rates
whether cyclic and non-cyclic, albeit not significant,
were numerically higher for the CIDR + eCG cows. In
conclusion, our results indicate that the addition of eCG
to a progesterone-based ES protocol substantially
improves the ovulation rate in non-cyclic buffaloes.
They also reveal that similar ovulation and pregnancy
rates to timed AI as those observed in cows inseminated
at natural estrus may be achieved in anestrous buffaloes,
including cyclic and non-cyclic animals.
Acknowledgments
The authors thank Ana Burton for assistance with the
English translation. This research was supported by
grants from the Department of Science, Technology and
Environment, Government of Pondicherry, Pondi-
cherry, India (G.O. Rt. No. 14/Envt./2004). We also
thank the National Institute of Animal nutrition and
Physiology, Bangalore, India for help with the hormone
assays. The authors gratefully acknowledge the Dean of
the College of Veterinary and Animal Sciences,
Pondicherry, India, Rajiv Gandhi, for allowing us to
undertake the lab work at the college.
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