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Theriogenology 69 (2008) 822–826

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In vitro embryo production in buffalo (Bubalus bubalis)

using sexed sperm and oocytes from ovum pick up
X.W. Liang b,1, Y.Q. Lu a,1, M.T. Chen b, X.F. Zhang b, S.S. Lu a,
M. Zhang a, C.Y. Pang b, F.X. Huang b, K.H. Lu a,*
a
Animal Reproduction Institute, Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization,
Guangxi University, Nanning 530004, China
b
Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Science, Nanning 530001, China
Received 6 April 2007; accepted 8 November 2007

Abstract
The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected
bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly)
ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF,
using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were
Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from
abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P = 0.357) and blastocyst development rate (16.0 vs. 23.9%,
P = 0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%,
P = 0.978) or blastocyst development (15.3 vs. 19.1%, P = 0.291) after IVF using OPU-derived oocytes. Of the embryos produced
in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients
established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%)
transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed
buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing
technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for
further research and wider field application of these technologies in buffalo.
# 2008 Published by Elsevier Inc.

Keywords: Buffalo; Sexed sperm; In vitro embryo production; OPU; IVF

1. Introduction in DNA content is the most reliable and repeatable

To date, flow-cytometric sexing of X- and Y-
chromosome bearing sperm based on the difference
* Corresponding author. Tel.: +86 771 3235724;
fax: +86 771 3238064.
E-mail address: khlu@gxu.edu.cn (K.H. Lu).
1
These authors contributed equally to this work.
method to produce sex-preselected animals. Since the
first report by Johnson et al. [1] in rabbits, flow-
cytometric sexing technology has been shown to be
efficacious in several species [2–5]. The difference of
the DNA content between X- and Y-chromosome
bearing buffalo sperm (approximately 3.6%) is the basis
of the flow-cytometric sperm sexing [6].
Pregnancies have been reported following AI of
sexed sperm in Mediterranean Italian buffaloes [7].

0093-691X/$ – see front matter # 2008 Published by Elsevier Inc.

doi:10.1016/j.theriogenology.2007.11.021
 X.W. Liang et al. / Theriogenology 69 (2008) 822–826
Nevertheless, since sperm sexing is slow and requires
expensive equipment, this technology is most likely
to be used when the additional costs are justified by
improved production and/or efficiency [8]. One
option for using sexed sperm is in vitro production
(IVP) of embryos. In that regard, approximately 10–
15 oocytes (from abattoir-derived ovaries) could be
fertilized with 40,000–80,000 sexed buffalo sperm in
vitro [5]; the blastocyst development rate (approxi-
mately 20%) was not significantly different from that
achieved with unsexed sperm [5]. Furthermore,
transfer of presumed X-embryos into a recipient
resulted in the birth of female twins [5], providing
proof of concept. However, abbatoir-derived ovaries
are typically collected from culled buffaloes, with
unknown (but typically low) genetic merit. In
contrast, ovum pick up (OPU) technology could be
used to recover large quantity of meiotically
competent oocytes from antral follicles in live
animals of proven genetic merit, thereby greatly
increasing the value of IVEP programs [9,10]. The
objective of the present study was to explore the use
of sexed sperm and OPU-derived oocytes in an IVP
system to produce sex-preselected bubaline embryos.
2. Materials and methods
2.1. Sperm preparation
Semen samples were collected from three Murrah
and Nili-Ravi buffaloes (2–6-year-old and of proven
fertility) at the Livestock and Poultry Breeding
Station of Guangxi, China. Only ejaculates with
>75% morphologically normal sperm and >65%
progressive motility were used. Sperm sexing was
similar to that described by Johnson and Welch [11].
Briefly, sperm were stained in 40 mg/mL Hoechst
33342, incubated at 35 8C for 45 min, filtered, and
then sorted into X- or Y-chromosome bearing sperm
enriched populations by a modified BD FACSVantage
SE flow cytometer (Becton, Dickinson & Company,
San Jose, CA, USA), operating at 40 psi pressure,
with a 197 mM Tris Base sheath fluid. Sperm were
sorted into 50-mL tubes containing 2 mL of Tris
extender supplemented with 20% (v/v) egg-yolk.
After collection, sexed sperm were frozen in Tris
extender supplemented with 20% (v/v) egg-yolk and
6% (v/v) glycerol [12]. The purity of sexed sperm was
determined by flow-cytometric reanalysis following
re-staining and sonication [13]. The unsexed sperm
used in the following experiments was collected from
the same buffaloes and similarly cryopreserved.
823
Unless stated otherwise, all chemicals used were
purchased from Sigma–Aldrich Co. (St. Louis, MO,
USA).
2.2. Ultrasound-guided transvaginal ovum pick up
Twenty healthy, cycling Murrah and Nili-Ravi
buffaloes (5–12 years of age and 580–720 kg body
weight) were used. They were maintained under the
general feeding and management conditions at the farm
of Guangxi Buffalo Research Institute, China. Oocyte
retrieval was conducted as described [14] twice weekly
during August, September and October 2006. Briefly,
buffalo were restrained in a squeeze chute, and oocyte
retrieval was performed with an ultrasound scanner
(HS2000B, Fujihira Co., Japan) equipped with a
5.0 MHz Aloka sector scanner and a 60 cm long sterile
dorsal needle. A constant vacuum pressure (40 mmHg)
was used to aspirate fluid into a sterile 50-mL Falcon
tube.
The flushing medium was DPBS (GibcoTM, Invitro-
gen Co., Grand Island, NY, USA), supplemented with
0.3% new born cow serum (Sijiqing Co., Hangzhou,
China). After aspiration, the flushing medium was
filtered through an embryo filter, and the embryos were
washed two or three times with the flushing medium. A
zoom stereomicroscope was used to assess, retrieve, and
grade oocytes (Grades A–D, according to their
morphological appearance [14]). Only Grades A or B
oocytes were used in subsequent maturation culture and
IVF procedures.
2.3. In vitro fertilization
Oocytes collected by OPU or aspirated from follicles
2–6 mm in diameter (from abbatoir-derived ovaries)
were washed in TCM199 (GibcoTM, Invitrogen Co.,
Grand Island, NY, USA) supplemented with 0.3% BSA
and once more in maturation medium. Oocytes were
matured in vitro in TCM199 supplemented with 5%
estrous cow serum and 10 mg/mL FSH (Institute of
Zoology, Chinese Academy of Sciences, Beijing,
China), cultured at 38.5 8C and 5% CO2 for 22–24 h
[5]. Following oocyte maturation, the cumulus cells
surrounding the COCs were stripped off by repeated
pipetting.
Buffalo semen (sexed and unsexed) was thawed in
38 8C water bath for 20 s, followed by a centrifugation
(700  g for 25 min) in Percoll gradient (90:45%) to
remove dead sperm. The live sperm pellet was washed in
fertilization medium by centrifugation at 400  g for
5 min. Ten to fifteen oocytes were added to a drop of
824 X.W. Liang et al. / Theriogenology 69 (2008) 822–826
40 mL modified Tyrode’s medium supplemented with
0.6% BSA, 2.0 mM caffeine, and 20 mg/mL heparin. The
final concentration of sexed or unsexed sperm added into
the fertilization medium was 1–2  106 sperm/mL.
Eight to 10 h after insemination, presumptive zygotes
were transferred to culture dishes with granulosa cell
monolayers. The embryo culture medium was TCM199
supplemented with 10% newborn cow serum [5].
Cleavage of the oocyte was evaluated on Day 2
(insemination = Day 0); on Days 6–8, blastocyst devel-
opment was evaluated under a stereomicroscope.
Embryo cryopreservation was done with an automated
embryo freezer (Freeze Control CL8000; Cryologic,
Mulgrave, Victoria, Australia), using TCM199 supple-
mented with 1.8 M ethylene glycol and 0.2 M sucrose, as
described [15].
2.4. Embryo transfer
The recipients used in this study were Murrah
(n = 75) and Nili-Ravi (n = 67) buffaloes in sponta-
neous estrus. Blastocysts at Days 6–8 of development
were transferred into recipients 4–6 days after visual
detection of estrus. To determine the side of ovulation,
transrectal palpation was done to determine the side of
the preovulatory follicle, and was also done at the time
of embryo transfer to verify the presence of a CL. Each
recipient received one fresh or frozen-thawed embryo
transferred nonsurgically to the uterine horn ipsilateral
to the CL. Sixty days after transfer, pregnancy status
was determined by ultrasonography.
2.5. Statistical analysis
Rates of cleavage and blastocyst formation were arc
sine transformed and the transformed data were
analyzed using one-way ANOVA; significant differ-
ences were located using Duncan’s Multiple Range test.
Pregnancy rates following embryo transfer were
evaluated by Chi-square. All the statistical analyses
were done with the SPSS 12.0 statistical package for
windows (SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Oocytes recovered by OPU
Twenty buffalo cows were used in the OPU program.
On average, 4.6 oocytes were retrieved per buffalo per
session, of which 70.9% were either Grades A or B.
Overall, 1064 oocytes recovered by OPU were matured
and inseminated.
3.2. Embryo development from oocytes derived
from OPU and abattoir ovaries after IVF with sexed
sperm
Oocytes retrieved from OPU were matured simulta-
neously with those collected from abattoir-derived
ovaries on the same day; oocytes from both sources
were fertilized with frozen-thawed sexed sperm on the
same date. The mean (S.D.) cleavage rates of oocytes
derived from OPU and abattoir ovaries were 57.6  12.9
and 50.4  6.4%, respectively (P = 0.357), and the
proportions of oocytes that developed into blastocysts
were 16.0  7.8 and 23.9  11.4% (P = 0.237).
3.3. Embryo development from OPU oocytes after
IVF with sexed and unsexed sperm
Overall, 909 oocytes recovered from OPU were
randomly divided into two groups and fertilized with
sexed or unsexed sperm. For these two sperm sources,
cleavage rates were 50.5  13.0 and 50.9  18.9%
(P = 0.978), whereas blastocyst development rates were
15.3  7.4 and 19.1  10.3% (P = 0.291).
3.4. Embryo transfer
A total of 142 embryos produced by sexed and
unsexed sperm were transferred (Table 1). There was no
difference in pregnancy rates between sexed versus
unsexed fresh embryos (26.5 vs. 26.9%, P = 0.969), nor
between sexed versus unsexed frozen-thawed embryos
(11.6 vs. 15.4%, P = 0.618). However, there was a
decrease in pregnancy rate following transfer of frozen
embryos in comparison to fresh embryos; pregnancy
rates for fresh and frozen sexed embryos were 26.5 and
11.6% (P = 0.094), respectively, whereas pregnancy
rates of fresh and frozen unsexed embryos were 26.9
and 15.4% (P = 0.255), respectively. When the data of
sexed and unsexed embryo were combined, there was a
difference (P = 0.047) in pregnancy rates between
fresh (16/60, 26.7%, and frozen embryos (11/82,
13.4%). As of 30 November 2007, 11 buffalo calves had
been born (10 females and one male) and three aborted
(two females and one male) following transfer of
female embryos produced by X-sorted sperm. Among
embryos produced by unsexed sperm and following
their transfer into suitable recipients, 10 calves were
born (six females and four males) and three abortions
(all males) occurred. The proportion of abortions from
pregnancies established by sexed embryos (3/14,
21.4%) was similar to that of unsexed embryo (3/13,
23.1%; P = 0.918).
 X.W. Liang et al. / Theriogenology 69 (2008) 822–826
Table 1
Pregnancies in buffalo following transfer of fresh or frozen-thawed embryos produced by in vitro fertilization of OPU-derived oocytes with sexed or
unsexed frozen-thawed sperm
Sperm/embryo No. of embryos transferred
Sexed/fresh 34
Sexed/frozen 43
Unsexed/fresh 26
Unsexed/frozen 39
4. Discussion
The average number of oocytes recovered per
buffalo per session (4.6) in this study, seemed lower
than a previous report (5.4, [16]), but higher than two
other reports (2.7 and 2.0, [17,18]). Overall, 70.9% of
the oocytes were Grades A or B oocytes, which seemed
higher than previously reported [16–18]. Apparent
differences between studies may have been due to
technical and biological factors. In that regard, the
quantity and quality of the oocytes recovered by OPU in
cattle were attributed to needle geometry, vacuum
pressure, donor animal, hormonal prestimulation,
timing and frequency of OPU, and the experience of
the OPU operators [19].
In the present study, embryo development was not
significantly different between oocytes from OPU
versus abattoir-derived ovaries after IVF with sexed
sperm. It was previously speculated that OPU could
synchronize ovarian follicular development, reduce
the number of atretic follicles, and result in a better
quality oocyte and a higher blastocyst yield after IVF
than oocytes collected from abattoir-derived ovaries
[9]. However, in this study, the proportion of Grade
A oocytes recovered by OPU and from abattoir-
derived ovaries were 20 and 70%, in contrast to the
previous study [9], in which more than 80% of
oocytes from both OPU and abattoir-derived ovaries
were Grade A.
There was no significant effect of sexed versus
unsexed sperm on either cleavage or blastocyst rates. In
a previous study [5], both of these rates were lower after
IVF with sexed versus unsexed sperm. Furthermore, in
other reports in cattle [20,21], sexed sperm had lower
rates of fertilization and embryo development, espe-
cially blastocyst formation. These apparent differences
may have been due to oocyte collection or maturation,
and the IVF protocol used. Furthermore, although
cleavage rates were nearly identical, rates of blastocyst
formation were nearly four percentage points lower
825
No. of pregnancies (%) No. of live births No. of abortions
Male Female Male Female
9 (26.5%) 1 6 1 1
5 (11.6%) 0 4 0 1
7 (26.9%) 2 3 2 0
6 (15.4%) 2 3 1 0
with sexed sperm; perhaps with a larger number of OPU
oocytes, the difference in blastocyst development would
be significant.
In the present study, there was no significant
difference in pregnancy rates between sexed and
unsexed fresh embryos, or sexed and unsexed frozen-
thawed embryos following transfer to the buffalo cows.
Furthermore, the abortion rate for sexed and unsexed
embryo was also similar. These findings were consistent
with a report on sheep embryos produced in vivo by
sexed ram sperm, in which the rates of pregnancy,
embryo survival, and lambing were similar following
transfer of sexed and unsexed embryos [22]. Therefore,
the sexing procedures did not impair the ability of
embryos to develop to term.
Pregnancy rates were reduced following transfer of
sexed or unsexed frozen-thawed embryos, compared to
fresh embryos, in this study; this was attributed to
sensitivity to cryopreservation. Techakumphu et al. [23]
reported a 35.7% (5/14) pregnancy rate and a 14.3% (2/
14) calving rate using fresh embryos, but only a 5.9%
(1/17) pregnancy rate and no development to term from
frozen-thawed embryos. Furthermore, Hufana-Duran
et al. [24] reported a 16.4% pregnancy rate following
transfer of vitro-derived vitrified warmed embryos to
recipient buffalos. The low success could have also been
associated with the in vitro embryo production
procedures, considering that buffalo embryos produced
in vivo and cryopreserved by slow-freezing resulted in
28.2% (11/39) pregnancy rate [25].
A major factor limiting IVP of bubaline embryos is
the relative lack of oocytes with superior genetic merit.
Under field conditions, using oocytes collected from
abattoir-derived ovaries of culled buffalo without
knowledge of pedigree or quality of germplasm would
limit the use of IVP, especially with sexed sperm.
However, using OPU for serial collection of oocytes
from genetically superior donors, and IVF with sexed
semen (from genetically superior sires) would be a very
effective utilization of IVP. In the present study, OPU,
826 X.W. Liang et al. / Theriogenology 69 (2008) 822–826
sperm sexing technology, IVP, and embryo transfer,
were used to produce sex-preselected buffalo calves.
This study provided proof of concept for further
research and wider field application of these technol-
ogies in buffalo.
Acknowledgements
We acknowledge the staff at Livestock and Poultry
Breeding Station of Guangxi for assistance in collection
of buffalo semen. This research was jointly supported by
National Science and Technology Supporting Program
(no. 2006BAD04A18), Guangxi Key Technology R&D
Programs (nos. 0537001-1, 0447001, 0630006-5B and
07109006) and Guangxi University Key Program (no.
2005ZD05).
References
[1] Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits:
live births from X and Y sperm separated by DNA and cell
sorting. Biol Reprod 1989;41:199–203.
[2] Johnson LA. Sexing mammalian sperm for production of off-
spring: the state-of-the-art. Anim Reprod Sci 2000;60–61:93–
107.
[3] Seidel Jr GE, Garner DL. Current status of sexing mammalian
spermatozoa. Reproduction 2002;124:733–43.
[4] Maxwell WM, Evans G, Hollinshead FK, Bathgate R, De Graaf
SP, Eriksson BM, et al. Integration of sperm sexing technology
into the ART toolbox. Anim Reprod Sci 2004;82–83:
79–95.
[5] Lu YQ, Liang XW, Zhang M, Wang WL, Kitiyanant Y, Lu SS,
et al. Birth of twins after in vitro fertilization with flow-cyto-
metric sorted buffalo (Bubalus bubalis) sperm. Anim Reprod Sci
2007;100:192–6.
[6] Lu YQ, Zhang M, Meng B, Lu SS, Wei YM, Lu KH. Identifica-
tion of X- and Y-chromosome bearing buffalo (Bubalus bubalis)
sperm. Anim Reprod Sci 2006;95:158–64.
[7] Presicce GA, Verberckmoes S, Senatore EM, Klinc P, Rath D.
First established pregnancies in Mediterranean Italian buffaloes
(Bubalus bubalis) following deposition of sexed spermatozoa
near the utero tubal junction. Reprod Domest Anim 2005;40:
73–5.
[8] Seidel Jr GE. Sexing mammalian sperm—intertwining of com-
merce, technology, and biology. Anim Reprod Sci 2003;79:
145–56.
[9] Neglia G, Gasparrini B, Caracciolo di Brienza V, Di Palo R,
Campanile G, Antonio Presicce G, et al. Bovine and buffalo in
vitro embryo production using oocytes derived from abattoir
ovaries or collected by transvaginal follicle aspiration. Therio-
genology 2003;59:1123–30.
[10] Gasparrini B. Advance on in vitro embryo production in water
buffalo. In: Proceedings of the 5th Asian Buffalo Congress;
2006. p. 15–21.
[11] Johnson LA, Welch GR. Sex preselection: high-speed flow
cytometric sorting of X and Y sperm for maximum efficiency.
Theriogenology 1999;52:1323–41.
[12] Schenk JL, Suh TK, Cran DG, Seidel Jr GE. Cryopreservation of
flow-sorted bovine spermatozoa. Theriogenology 1999;52:1375–
91.
[13] Welch GR, Johnson LA. Sex preselection: laboratory validation
of the sperm sex ratio of flow sorted X- and Y-sperm by sort
reanalysis for DNA. Theriogenology 1999;52:1343–52.
[14] Huang YJ, Presicce GA, Gasparrini B, Yang BZ, Liang XW,
Zhang XF, et al. In vitro fertilization of the oocytes derived from
ovum pick up in water buffalo (in Chinese). Chin Anim Husband
Vet Med 2004;31:22–4.
[15] Shi DS, Ling ZJ, Wei YM, Lu KH. Studies on cryopreservation
of bovine IVF embryos (in Chinese). J Guangxi Agric Univ
1998;17:305–11.
[16] Techakumphu M, Promdireg A, Na-Chiengmai A, Phutikanit N.
Repeated oocyte pick up in prepubertal swamp buffalo (Bubalus
bubalis) calves after FSH superstimulation. Theriogenology
2004;61:1705–11.
[17] Boni R, Roviello S, Zicarelli L. Repeated ovum pick-up in Italian
Mediterranean buffalo cows. Theriogenology 1996;46:899–909.
[18] Gupta V, Manik RS, Chauhan MS, Singla SK, Akshey YS, Palta
P. Repeated ultrasound-guided transvaginal oocyte retrieval from
cyclic Murrah buffaloes (Bubalus bubalis): oocyte recovery and
quality. Anim Reprod Sci 2006;91:89–96.
[19] van Wagtendonk-de Leeuw AM. Ovum pick up and in vitro
production in the bovine after use in several generations: a 2005
status. Theriogenology 2006;65:914–25.
[20] Merton JS, Haring RM, Stap J, Hoebe RA, Aten JA. Effect of
flow cytometrically sorted frozen/thawed semen on success rate
of in vitro bovine embryo production. Theriogenology
1997;47:295 [abstract].
[21] Lu KH, Cran DG, Seidel Jr GE. In vitro fertilization with flow-
cytometrically sorted bovine sperm. Theriogenology
1999;52:1393–405.
[22] de Graaf SP, Beilby K, O’Brien JK, Osborn D, Downing JA,
Maxwell WM, et al. Embryo production from superovulated
sheep inseminated with sex-sorted ram spermatozoa. Theriogen-
ology 2007;67:550–5.
[23] Techakumphu M, Sukavong Y, Yienvisavakul V, Buntaracha B,
Pharee S, Intaramongkol S, et al. The transfer of fresh and frozen
embryos in an elite swamp buffalo herd. J Vet Med Sci
2001;63:849–52.
[24] Hufana-Duran D, Pedro PB, Venturina HV, Hufana RD, Salazar
AL, Duran PG, et al. Post-warming hatching and birth of live
calves following transfer of in vitro-derived vitrified water buffalo
(Bubalus bubalis) embryos. Theriogenology 2004;61:1429–39.
[25] Kasiraj R, Misra AK, Mutha Rao M, Jaiswal RS, Rangareddi NS.
Successful culmination of pregnancy and live birth following the
transfer of frozen-thawed buffalo embryos. Theriogenology
1993;39:1187–92.