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Reproduction, Fertility and Development, 2009, 21, 749756

Ejaculate characteristics, short-term semen storage

and successful artificial insemination following
synchronisation of oestrus in the Indian
blackbuck antelope (Antilope cervicapra)
Sadanand D. SontakkeA , Manoj S. PatilA , Govindhaswamy UmapathyA ,
K. Ramachandra RaoA and Sisinthy ShivajiA,B
A Laboratory

for Conservation of Endangered Species (LaCONES), Centre for Cellular and

Molecular Biology Annexe-I (CSIR), Attapur Ring Road, Hyderabad 500 048, India.
B Corresponding author. Email: shivas@ccmb.res.in

Abstract. The blackbuck (Antilope cervicapra) is a small (2030 kg) Indian antelope that is listed on Schedule I of
the Indian Wildlife Protection Act, 1972. Studies were undertaken to develop assisted reproductive technologies, such
as synchronisation of oestrus and non-surgical AI, to support the conservation and genetic management of this Indian
antelope. Semen characteristics, testosterone levels and the feasibility of short-term cold storage of semen were investigated. Furthermore, different oestrous synchronisation protocols (norgestomet implants and prostaglandin injections)
were evaluated for successful AI, defined as the birth of live young. Norgestomet ear implants and i.m. administration
of pregnant mares serum gonadotropin (PMSG) resulted in successful pregnancies in two of five inseminated females,
but both had twin pregnancies that were delivered prematurely. In contrast, two injections of prostaglandin 11 days apart
were effective in synchronising oestrus in the blackbuck. Transcervical AI in oestrous-synchronised animals 72 and 96 h
after the second prostaglandin injection resulted in successful pregnancies in four of six inseminated females (67%) and
resulted in the delivery of three live fawns. These studies demonstrate the potential application of AI technology for the
conservation of endangered ungulates. To our knowledge, this is the first report regarding the synchronisation of oestrus
and successful non-surgical AI in blackbuck.
Additional keywords: assisted reproduction, gamete biology, norgestomet, prostaglandin, ungulate.

Introduction
The blackbuck (Antilope cervicapra) is a small (2030 kg) Indian
antelope that lives primarily on the grassy plains throughout India. The International Union for Conservation of Nature
(IUCN) 2008 Red List of Threatened Species (IUCN 2008) lists
blackbuck as near threatened. However, in India, the blackbuck
has been listed on Schedule I (meaning it is endangered) of the
Indian Wildlife Protection Act, 1972, because of a serious threat
to its survival in the wild due to hunting for meat and trade, loss
of habitat and inbreeding. Therefore, there is an urgent need to
conserve this native species using both in situ and ex situ conservation approaches. It has been clearly demonstrated recently
that ex situ methods involving reproductive biotechnologies,
such as semen preservation (Howard et al. 2002; Shivaji et al.
2003), AI (Howard et al. 2003; Pukazhenthi and Wildt 2004;
Sontakke 2007) and embryo transfer (Pope et al. 1997), augment the genetic management of endangered species, especially
when the surviving population consists of only a few individuals
(Wildt et al. 1997; Holt and Pickard 1999). Some of these studies have documented successful AI in various antelope species
(e.g. Spekes gazelle, addax, blackbuck, suni antelope, gaur,
CSIRO 2009

scimitar-horned oryx, Mohor gazelle, eland, banteng, Barbery

sheep, gerenuk and Spanish ibex) that resulted in pregnancies
(for a review, see Morrow et al. 2009). Sporadic live births have
also been reported for a few species, such as Spekes gazelle,
addax, blackbuck, gaur, scimitar-horned oryx, eland, banteng,
Barbery sheep and Spanish ibex. These earlier studies were the
impetus for the present study regarding AI in blackbucks.
An essential prerequisite for the application of assisted reproductive techniques in any threatened species is a sound knowledge of the reproductive physiology with respect to seasonality,
oestrous cycle, gestational length, reproductive anatomy, etc.
However, very little is known about the reproductive physiology
of the blackbuck. What is known is that the blackbuck breeds
throughout the year. The mean length of oestrous cycle has been
determined to be 16.9 0.6 days on the basis of behavioural
oestrus by Holt et al. (1988), which contrasts with a mean cycle
length of 14.4 0.5 days that we determined in a recent study
using ultrasonographic monitoring of follicular dynamics (S. D.
Sontakke, M. S. Patil, S. Shivaji, unpubl. data). The mean length
of gestation in the blackbuck is 183 days (Holt et al. 1988) and it
usually gives birth to only one young. Necropsies performed on
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Reproduction, Fertility and Development

captive blackbucks indicate that the ovaries of the female blackbuck are oval and symmetrical, the cervix has three annular rings,
there is a duplex uterus with two symmetrical large uterine horns
(67 cm in length) that bifurcate after the cervical annular rings
and the vagina has a thick muscular wall with longitudinal folds
(S. D. Sontakke, G. Umapathy, S. Shivaji, unpubl. data).
To our knowledge, there has been only one comprehensive scientific study performed on the blackbuck in the UK
(Holt et al. 1988) that provides limited data on semen volume, sperm concentration and semen preservation. In addition,
these authors also achieved live births following AI of animals
in natural oestrus. However, there was no attempt to achieve
synchronisation of oestrus in the blackbuck before AI.
It is well known that synchronisation of oestrus improves the
chances of success of AI, which depends on consistent induction of oestrus and ovulation combined with an ability to place
viable semen in the female reproductive tract at the optimum
time. Furthermore, the ability to tightly regulate ovulation allows
insemination of females at a predetermined time, thereby maximising labour efficiency and avoiding handling and anaesthetic
stress to females (Morrow et al. 2000). With this in mind, the
primary aim of the present study was to develop a reliable
protocol for the synchronisation of oestrus and a non-surgical
(transcervical) AI technique to achieve live births in endangered
blackbucks. In addition, we determined ejaculate characteristics
and testosterone levels for the blackbuck, as well as the feasibility
of the short-term cold storage of semen.
Materials and methods
Study area and animals
The present study was conducted between June 2005 and
December 2007 at the Nehru Zoological Park, Hyderabad, India
(78.3 E, 17.2 N). Twelve adult healthy male and 10 female
blackbucks (mean weight 23.3 1.9 and 22.2 2.3 kg, respectively) obtained from the Hyderabad, Tirupati and Vishakhapatnam zoos were maintained at the Nehru Zoological Park,
Hyderabad. Throughout the experimental period, stags were
housed individually in indoor cubicles (3 3 2 m) in the night,
but were set free in an adjoining paddock (12 11 m) during the day. Females were reared together in four adjoining
indoor pens (2.5 2.5 2 m each) at night and were set free
in another paddock (13 12 m) adjacent to the stags during the
day. Animals were exposed to a natural photoperiod and were
fed once in the morning with commercial cattle feed (Poshak
Feeds, Hyderabad, India) supplemented with mineral mixture
(Agrimin; GlaxoSmithKline India, Mumbai, India). Green fodder was also provided twice a day and animals had free access
to clean drinking water throughout the day. Prior to the study,
the blackbucks were allowed at least 4 months to acclimatise to
their new environment and enclosures. The Central Zoo Authority, Ministry of Environment and Forests, Government of India,
gave permission for the study.
Anaesthesia and semen collection
Food and water were withdrawn for 24 h before blackbucks were
anaesthetised. For semen collection, males were anaesthetised
with a combination of ketamine hydrochloride (2 mg kg1 , i.m.;

S. D. Sontakke et al.

Ketamil; Troy Laboratories, Smithfield, NSW, Australia) and

xylazine hydrochloride (0.25 mg kg1 , i.m.; Ilium; Troy Laboratories) using a dart and blow-pipe (Sontakke et al. 2009a).
After a surgical level of anaesthesia was attained, physiological assessments of heart rate, respiratory rate, rectal temperature
and oxygen haemoglobin saturation (Sp o2 ) were made (Sontakke
et al. 2007, 2009a, 2009b). Semen was collected by electroejaculation, as described elsewhere (Shivaji et al. 1998, 2003;
Jayaprakash et al. 2001). Briefly, an electroejaculator (90240 V
AC; 2.5 A; Bioelectric, Washington, DC, USA) attached to a
Teflon-coated rectal probe (2.5 cm diameter) with three longitudinal electrodes and powered by a 1 kVA sine wave uninterrupted
power supply was used to deliver incremental series of a total of
40 stimuli (24 V in pulses lasting for 35 s each, with each series
separated by 5 min). Semen was collected into a prewarmed
50-mL plastic tube (Tarson, Calcutta, India). Immediately after
completion of these procedures, anaesthesia was reversed by
injection of 2 mg kg1 , i.v., tolazoline hydrochloride (SigmaAldrich, St Louis, MO, USA; Sontakke et al. 2009a). In total,
95 ejaculates were collected from 12 stags over a period of
2 years. A minimum of 1015 days was allowed between
collections in a single stag animal.
Semen evaluation
Immediately after collection, semen was maintained at 37 C in a
portable incubator. Volume was determined with a micropipette,
whereas pH was determined using colour indicator strips. Sperm
concentration was evaluated using a computer aided sperm
analyser (CASA; HTM-IVOS, Version 10; Hamilton Thorne
Research, Danvers, MA, USA; Sontakke et al. 2004). The percentage of motile and progressively motile spermatozoa was
also estimated using CASA, with slides mounted on a stage
warmer set at 37 C. For studies of sperm morphology, 5 L neat
semen was fixed in 100 L of 0.5% gluteraldehyde, smeared on a
glass slide and 200 spermatozoa were assessed by phase contrast
microscopy (400).
The percentage of spermatozoa with an intact plasma membrane (i.e. plasma membrane integrity; PMI) was determined
using a hypo-osmotic swelling test (HOST), as developed by
Jeyendran et al. (1984). Briefly, 10 L semen was added to 90 L
hypo-osmotic swelling (HOS) solution containing 0.7% sodium
citrate and 1.3% fructose (100 mOsmol L1 ) and incubated for
30 min at 37 C. An aliquot was then placed on a glass slide and
analysed under a phase contrast microscope (400). The number of spermatozoa with a coiled tail (indicating an intact plasma
membrane) or an uncoiled tail (damaged plasma membrane)
was recorded. To determine sperm acrosomal status, smears
were stained with fluorescein isothiocyanate-conjugated Arachis
hypogaea (peanut) agglutinin (FITC-PNA; Sigma-Aldrich), as
described by Roth et al. (1998), and the acrosomal status of
100 spermatozoa per sample was evaluated using fluorescent
microscopy (400; Axioplan-2; Carl Zeiss, Jena, Germany).
Spermatozoa were considered acrosome intact if the acrosome
stained bright green, the acrosomal membrane was intact and the
acrosomal cap was over the head region of the spermatozoon.
Damaged acrosomes or lost acrosomes were either unstained or
stained only faintly green at the equatorial region.

Oestrous synchronisation and AI in blackbuck

Short-term cold storage of semen

To determine the feasibility of the short-term storage of semen
at 5 C, semen samples were diluted at ambient temperature
to a concentration of approximately 5075 106 spermatozoa
mL1 with either Tes-n-tris buffer (TEST; Abaigar et al. 2001)
or TEST supplemented with 0.09 m trehalose (TEST-trehalose).
Both buffers contained 5% (v/v) egg yolk to avoid cold shock
because preliminary experiments had shown that buffers without egg yolk resulted in poor sperm motility (<20%) following
cold storage. Aliquots of 1 mL were then placed in 1.8-mL
polypropylene cryotubes (Nunc; Nalge, Rochester, NY, USA)
and transferred to a cryocontainer before being stored in a refrigerator (at approximately 5 C) for 7 days. Semen assessments,
such as sperm motility, PMI and acrosomal integrity, were performed at 0 h and after every 24 h for up to 7 days of cold
storage. All assessments were performed after the aliquots had
been rewarmed to 37 C. Experiments were repeated five times.
Testosterone levels
Blood samples were collected only once from all stags into heparinised tubes (Akuret; Eastern Medikit, Gurgaon, India) by
jugular venipuncture just before electroejaculation. The blood
plasma was separated by centrifugation (600g for 10 min) and
stored at 20 C until assayed for testosterone. Free testosterone concentrations were measured, in duplicate, with a commercially available ELISA kit for human serum (Diagnostic
Biochem Canada, London, ON, Canada) in accordance with the
manufacturers instructions. The sensitivity of the ELISA was
0.17 pg mL1 , with intra- and interassay CVs of 4.717.0% and
5.312.4%, respectively, as reported by the manufacturer.
Comparing oestrous synchronisation protocols
A series of experiments was conducted to optimise the oestrousinduction protocol and the time ofAI using norgestomet implants
and prostaglandin (PG) F2 in five groups of animals. Animals
in Group I (n = 5) were treated with the commercially available
Crestar kit (Intervet, Boxmeer, The Netherlands) under anaesthesia to synchronise oestrus, as reported earlier for addax, sika
deer, mouflon sheep and axis deer (Densmore et al. 1987; Willard
et al. 1996; Ptak et al. 2002; Umapathy et al. 2007). The Crestar
treatment consisted of a norgestomet ear implant and i.m. injections of norgestomet (3 mg) and oestradiol valerate (5 mg) at
the time of implantation. On Day 12, the implant was removed
from anaesthetised blackbuck and 100 IU pregnant mares serum
gonadotropin (PMSG; Folligon; Intervet) was injected intramuscularly. In Group II (n = 5), oestrus was induced using the
same protocol as for Group I, but PMSG was not administered.
Fixed-time transcervical AI with freshly collected semen was
performed 48 and 56 h after implant withdrawal (Umapathy et al.
2007).
Animals in Groups III, IV and V were given two intramuscular injections of 10 mg PGF2 (Lutalyse; Pfizer, Mumbai,
India) 11 days apart. Females in Group III (n = 4) were monitored for behavioural oestrus using a teaser male and were
inseminated once on the day of behavioural oestrus (approximately 70 3 h after the second PGF2 injection) using freshly
collected semen. In Group IV, female blackbuck (n = 6) were

Reproduction, Fertility and Development

751

inseminated at 72 and 96 h (n = 3 at each time-point) without

monitoring behavioural oestrus. Finally, two females in Group V
were inseminated only once 96 h after the second injection of
PGF2 .
Transcervical AI and confirmation of pregnancy
Prior to AI, female blackbuck were anaesthetised with ketamine
xylazine, as described above for males (Sontakke et al. 2009a).
Each anaesthetised female was placed on a stretcher in the ventral
recumbent position, with hindquarters elevated. A 5-mm diameter, 30 fibre optic telescope (Karl Storz, Tuttlingen, Germany)
attached to a halogen cold light fountain source (Karl Storz) was
inserted into the vagina after lubrication with sterile spermicidefree jelly (Petro-Gel; Hy-Glass, Hyderabad, India) to visualise
the cervical folds. No attempts were made to grasp the cervical
os with forceps. Once the cervical os was located, an insemination catheter (30 cm long, 16 gauge; Labotect, Gottingen,
Germany) loaded with 0.5 mL freshly collected semen (containing approximately 75150 106 spermatozoa) was deposited
deep into the cervix and manipulated gently through the cervical folds. Immediately after completion of the insemination
procedure, anaesthesia was reversed by intravenous injection of
tolazoline hydrochloride (Sontakke et al. 2009a).
Real-time, -mode ultrasonography, equipped with a 5-MHz
transrectal curved-array transducer (240 Parus Vet; Pie Medical, Maastricht, The Netherlands), was used to detect pregnancy
5563 days after insemination in anaesthetised female
blackbuck.
Statistical analyses
All statistical analyses were performed using SPSS 11.1 for
Windows (SPSS, Chicago, IL, USA). Data are presented as the
mean s.d. and P 0.05 was considered significant.A repeatedmeasures analysis of variance (ANOVA; General Linear Model)
was used to determine the effects of cold storage over time. The
MannWhitney U-test was used to compare the efficacy of the
different buffers during the cold storage of spermatozoa.
Results
Ejaculate characteristics
In total, 95 ejaculates were collected from 12 stags over a period
of 2 years. Semen could be collected consistently from all anaesthetised males (100%) by electroejaculation and every attempt
yielded good-quality ejaculates. Usually five to seven electrical stimuli of 23 V were sufficient to induce ejaculation in
the males without considerable extrusion of the penis from the
prepuce. The first fraction of the ejaculate that was normally
obtained after six to eight stimuli was lemony yellow in colour,
turbid, viscous and had a higher concentration of spermatozoa
(450500 106 spermatozoa mL1 ). Although the second fraction was also lemony yellow in colour, it was less viscous and
less concentrated (125160 106 spermatozoa mL1 ). Table 1
lists the characteristics of the ejaculate from the 12 stags collected over the 2 years; for the sake of clarity, data for both
fractions were pooled. There was no seasonal variation observed
for semen characteristics, except for a low sperm count between
April and May (data not shown). Ejaculates contained 1035% of

Reproduction, Fertility and Development

S. D. Sontakke et al.

5.3 1.4 (29.3)

7.1 0.4 (68.5)
332 9 (310340)
224 60 (100350)
73.7 9.2 (4590)
80.3 0.9 (6090)
77.5 7.6 (5891)
82.7 6.0 (6592)
81.0 4.8 (6590)
19.1 5.2 (1035)
30.8 13.6 (2.2150)

structurally abnormal spermatozoa and spermatozoa with cytoplasmic droplets and coiled tails were predominant (data not
shown). The mean blood plasma testosterone concentration of
blackbucks was 30.8 13.6 pg mL1 . Serial blood sampling for
testosterone concentrations over the period of the study was not
performed.
Short-term cold storage of semen
The results indicate that both TEST and TEST-trehalose were
suitable for the short-term cold storage of blackbuck spermatozoa at 5 C for up to 7 days (Fig. 1ac). All sperm characteristics
(e.g. motility, PMI and acrosomal integrity) decreased significantly (P < 0.001 for all, repeated-measures ANOVA) with
time in both buffers. However, comparison of these parameters between the two buffers indicated that spermatozoa stored
in TEST-trehalose had significantly higher motility, PMI and
acrosomal integrity over time than those stored in TEST alone
(U = 353, P < 0.001 for motility; U = 330, P < 0.001 for PMI;
U = 616, P < 0.05 for acrosomal integrity). After 7 days storage in TEST-trehalose buffer, sperm viability was maintained at
>50% compared with only 25% for spermatozoa stored in TEST
buffer (Fig. 1). Furthermore, following cold storage, a significant reduction (P < 0.01) in sperm motility and PMI was noted
for spermatozoa stored in TEST compared with TEST-trehalose
buffer after 2 days storage (Fig. 1a, b). Levels of acrosomal damage between the buffers did not differ significantly up to Day 4
of cold storage; however, significantly (P < 0.05) higher levels
of acrosomal damage were observed for spermatozoa stored in
TEST compared with TEST-trehalose buffer after 4 days storage
(Fig. 1c).
Oestrous synchronisation and successful AI
Table 2 gives an overview of the comparative effectiveness of the
oestrous-induction protocols, conception rate, gestation length
and live births following AI in blackbuck. Two of the five females
inseminated (40%) in Group I (implant + PMSG) conceived, as
detected by ultrasonography on Day 55. However, both females
had twin pregnancies and live young were delivered prematurely

100

Motile spermatozoa (%)

Semen volume (mL)

Semen pH
Semen osmolarity (mOsmol L1 )
Sperm concentration (106 mL1 )
Sperm motility (%)
Sperm forward progressive motility (%)
Plasma membrane integrity (%)
Acrosome-intact spermatozoa (%)
Morphologically normal spermatozoa (%)
Morphologically abnormal spermatozoa (%)
Blood plasma testosterone (pg mL1 )

(a)

80
60

40
20
0

(b)
100
Intact plasma membrane (%)

Table 1. Ejaculate characteristics and testosterone concentrations in

captive blackbuck (Antilope cervicapra)
In total, 95 ejaculates were collected from 12 adult stags over a period of
2 years. Values are the mean s.d., with the range given in parentheses

80
60

40
20
0

(c)
100
Intact acrosomes (%)

752

80
60
40
20
0
0

Storage time (days)

Fig. 1. Effect of cold storage on (a) motility, (b) plasma membrane
integrity and (c) acrosomal integrity of blackbuck spermatozoa stored in
Tes-n-tris buffer (TEST; ) and TEST buffer supplemented with 0.09 m
trehalose (
).

(1525 days before term). In contrast, none of the females in

Group II (implant only) conceived.
In Group III, behavioural oestrus (Flehman reaction, mounting attempts by the teaser male and clear cervical mucus at the
time of AI) was detected in all four females 70 3 h after the
second PGF2 injection. However, none of these females conceived following a single insemination at 70 3 h after PGF2

Reproduction, Fertility and Development

753

B Behavioural

A Time

1/2 (50%)
PGF2 on Days 1 and 12
Group V

96C

PGF2 on Days 1 and 12

Group IV

after implant withdrawal.

oestrus (e.g. Flehman reaction and mounting attempts) was detected with a teaser male, which was allowed to run with the female after the second PGF2 injection.
CTime after the second prostaglandin injection.
DTwo pregnancies that were detected by ultrasonography on Day 63 were not carried to term.
E Crestar treatment consisted of a norgestomet ear implant and i.m. injection of norgestomet and oestradiol valerate.

23 August 2007
14 September 2008
5 October 2008
1
1
1
Female
Male
Female
180
177
177

0/3 (0%)

On the day of behavioural oestrusB

(i.e. 70 3)C
72 and 96C
PGF2 on Days 1 and 12

4/6D (67%)

2/5 (40%)
0/5 (0%)
48 and 56A
48 and 56A
Implant plus PMSG on Day 12
Implant but no PMSG

Crestar
Group I
Group II
PGF2
Group III

0
0

No. live births

Sex of fawn
Gestation length (days)
Conception rate
Insemination time (h)
Protocol
treatmentE

Table 2. Comparative efficacy of oestrus-synchronisation protocols and successful artificial inseminations in the blackbuck
PGF2 , prostaglandin 2; PMSG, pregnant mares serum gonadotropin

Date of parturition

Oestrous synchronisation and AI in blackbuck

Fig. 2. Ultrasonographic image of a 58-day-old blackbuck fetus. The pregnant animal delivered the first live fawn (named Blacky) after 180 days
gestation.

injection. At the same time, when females were inseminated

72 and 96 h after the second PGF2 injection (Group IV), four
of the six females inseminated (67%) conceived, as evidenced
by ultrasonography 5863 days after AI (Fig. 2). Unfortunately,
only two females carried to term and delivered live fawns without assistance after 177 and 180 days gestation. Furthermore, in
Group V, one of two females (50%) that was inseminated only
once at 96 h conceived and delivered a live female fawn after
177 days gestation.
Discussion
The most widely applied assisted reproductive technology in
wildlife conservation is AI (Wildt et al. 1997; Sontakke 2007).
However, the reported success of AI in antelopes is limited. Sporadic live births after AI have been reported in Spekes gazelle
(Boever et al. 1980), addax (Densmore et al. 1987), blackbuck
(Holt et al. 1988), gaur (Junior et al. 1990), scimitar-horned
oryx (Garland et al. 1992; Morrow et al. 2000), Barbery sheep
(Johnston et al. 2000), eland (Bartels et al. 2001), banteng (Johnston et al. 2002) and Spanish ibex (Santiago-Moreno et al.
2006). Previously, Holt et al. (1988) produced six live blackbuck
offspring by AI in animals in natural oestrus. However, synchronising oestrus and ovulation before insemination has not been
studied in this species.
In the present study, all males (100%) responded consistently
to electroejaculation under surgical anaesthesia with ketamine
xylazine, yielding quality ejaculates without any contamination
by urine in any of the attempts. In the present study, the ejaculate volume was comparatively higher (5.3 v. 1.9 mL) and the
sperm concentration was lower (224 v. 338 106 mL1 ) than
reported earlier in the blackbuck (Holt et al. 1988), as well
as in other equivalent-sized ungulates, such as Mohor gazelles
(0.93.0 mL and 6461160 106 mL1 ; Holt et al. 1996;

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Reproduction, Fertility and Development

Cassinello et al. 1998), Dorcas gazelles (0.5 mL and

922 106 mL1 ; Cassinello et al. 1998), Cuvieri gazelles
(0.7 mL and 483 106 mL1 ; Cassinello et al. 1998) and
gerenuk (1.75 mL and 387 106 mL1 ; Penfold et al. 2005).
Undoubtedly, semen cryopreservation is a valuable tool in
wildlife conservation because it facilitates the dispersion of
the existing gene pool without the risks associated with the
transportation of valuable sires between zoos. However, the
maintenance of cryopreserved semen is costly and, in addition,
freezethawing can cause cellular and functional damage to the
spermatozoa, leading to reduced rates of conception. If semen is
to be used within a short period of time (e.g. within 4896 h), a
preferred alternative could be the short-term storage of semen at
5 C in the refrigerator. Such semen could be easily shipped from
one zoo to another in a cold container within 4872 h of collection by road or air (Abaigar et al. 2001). This technique has been
used successfully for wild species such as the Mohor gazelle
(Abaigar et al. 2001) and eland (Bissett and Bernard 2005).
In the present study, our short-term preservation of blackbuck
semen was very effective using TEST buffer, as reported earlier
for Mohor gazelle (Abaigar et al. 2001), and yielded comparable
recovery (50%) of spermatozoa after 72 h storage as reported
for Mohor gazelle. However, whenTEST was supplemented with
trehalose (0.09 m), all sperm characteristics (motility, PMI and
acrosome intactness) improved significantly compared with storage in TEST buffer alone. Trehalose, a disaccharide, has been
shown to have a cryoprotective action and its supplementation in
sperm diluents protects spermatozoa against freezing damage in
many species (Aboagla and Terada 2003; Berlinguer et al. 2007).
In eland, the motility of epididymal spermatozoa was maintained
only up to 96 h of cold storage (Bissett and Bernard 2005). To
our knowledge, the cold storage of semen for up to 7 days is
probably the longest period reported, with a higher efficiency
in the present study compared with earlier reports on the cold
storage of semen of either domestic or wildlife species (Bissett
and Bernard 2005; Soler et al. 2005). However, the fertilising
efficiency of the cold-stored spermatozoa needs to be addressed
and such studies are currently underway.
Successful application of AI technology to wildlife conservation depends on the development of a successful oestroussynchronisation protocol and determination of the exact timing
of insemination to obtain live births (Morrow et al. 2000).
However, the optimal time of insemination following oestrous
synchronisation varies between species (Garde et al. 2006). Progesterone implants (Crestar or other controlled internal drug
release (CIDR) devices), with or without PMSG supplementation, have been used effectively to synchronise oestrus in
domestic and wild animals (Densmore et al. 1987; Willard
et al. 1996; Cardwell et al. 1998; Ptak et al. 2002; Umapathy et al. 2007). Although our treatment of blackbucks with an
implant + PMSG were successful in inducing oestrus, as well
as conception in two of five females (40%), both pregnancies
were twin pregnancies, as detected by ultrasonography. Blackbucks are monotochous animals and give birth to a single fawn,
but it is likely that multiple ovulations may have resulted into
twin pregnancies leading to premature delivery of fetuses in
both females. Thus, the use of PMSG is not recommended in
the blackbuck, as is the case for fallow deer, which also exhibit

S. D. Sontakke et al.

multiple ovulations, resulting in twin pregnancies that cannot be

carried to term (Asher et al. 1993). In subsequent experiments
in the present study, when females were treated with the implant
but without PMSG to avoid multiple ovulations, none of the animals became pregnant. The failure of conception in the absence
of PMSG is most likely due to the timing of insemination. In
sheep and red deer, oestrus and ovulation are delayed without
the use of PMSG (Asher et al. 1993; Cardwell et al. 1998). Therefore, for insemination to be successful in the absence of PMSG,
it should be performed after the onset of oestrus, which could
be confirmed by using a vasectomised male.
Treatment with PGF2 is the preferred method of oestrus
synchronisation in wild ungulates because the administration of
PGF2 is less invasive and requires no anaesthesia and handling.
Although PGF2 analogues have been used in antelopes for the
synchronisation of oestrus, so far only three gaurs, one Spekes
gazelle and one addax have been born using this methodology
(for a review, see Morrow et al. 2009). In the present study, two
injections of PGF2 , 11 days apart, were effective in inducing
oestrus in blackbuck, as judged by successful conception and in
the resulting live births. Of the six females inseminated twice
72 and 96 h after the last injection of PGF2 , four were pregnant
after 63 days (67%), but only two of delivered live fawns after 177
and 180 days gestation. The remaining two pregnancies did not
reach term, and although there was no evidence of spontaneous
abortion, pregnancy in these two females was confirmed by the
presence of uterine cotyledons. The embryonic mortality could
be due to stress exerted during the chemical restraint of females
to enable ultrasonographic confirmation of pregnancy. Similar
failures have been reported for the Mohor gazelle (Holt et al.
1996) and addax (Densmore et al. 1987).
Based on the results of the insemination trials in Groups III
and IV, as well as the ultrasonographic observations of follicular
development, it was anticipated that AI at 96 h after the second
PGF2 injection would induce conception. Thus, we attempted a
single insemination at 96 h to avoid repeated anaesthesia and
handling stress to the animals. One of the two inseminated
females (50%) became pregnant and gave birth to a live female
fawn after 177 days gestation. A fixed-time AI is always advantageous in wild ungulates because repeated anaesthesia and
handling exert severe stress on the animals, impacting on the
conception rate. Overall, the conception rate following oestrous
synchronisation with PGF2 in the present study was 63% (in
five of eight animals), with the live birth of three fawns.
In conclusion, non-surgical AI following PGF2 -induced
oestrous synchronisation in blackbuck antelope resulted in the
birth of three live and healthy offspring. Evaluation of the characteristics of the ejaculates and testosterone concentrations of
blackbuck, as well as the feasibility of the short-term cold storage of semen, indicates the potential application ofAI technology
for genetic breeding and conservation of critically endangered
ungulates.
Acknowledgements
This research was supported by grants from the Department of Biotechnology, Government of India, the Central Zoo Authority, Government of India,
and a supra institutional grant from CSIR, Government of India. The authors
thank the Chief Wildlife Warden of Andhra Pradesh and the Director, Nehru

Oestrous synchronisation and AI in blackbuck

Zoological Park, Hyderabad, for granting permission for the study to be performed. The authors thank Shri A. V. Joseph for his cooperation and interest
in this project and Dr S. D. Kholkute for his constant encouragement. The
authors acknowledge the technical assistance of Dr U. Lakshmikantan during
the latter part of the study.

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Manuscript received 10 December 2008, accepted 2 April 2009

http://www.publish.csiro.au/journals/rfd