This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
Author's personal copy
Animal Reproduction Science 119 (2010) 169–171
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Sex-preselected buffalo (Bubalus bubalis) calves derived from artificial
insemination with sexed sperm
Yangqing Lua, Ming Zhanga, Shengsheng Lua, Dianxin Xub,
Weihong Huangb, Bing Menga, Huiyan Xua, Kehuan Lua,∗
aAnimal Reproduction Institute, Guangxi Key Laboratory of Subtropical Bio-resource Conservation and Utilization,
Guangxi University, Nanning 530004, PR China
bGuangxi Livestock and Poultry Breeding Station, Nanning 530001, PR China
a r t i c l ei n f o
Received 8 September 2009
Received in revised form 5 December 2009
Accepted 8 January 2010
Available online 14 January 2010
a b s t r a c t
Flow cytometry sorting of X- and Y-chromosome bearing sperm has been emerging as a
objective of this study was to evaluate the efficiency of AI by using the sexed sperm to pro-
duce sex-preselected calves in buffalo species. A total of 43 buffalo cows were inseminated
with X-sorted sperm, 30 of which were confirmed pregnant 3mo following AI. In terms of
conception rate, significant difference was observed between AI with sexed sperm derived
from different bulls (P<0.05), but not between sexed and non-sexed sperm (P>0.05), nor
between heifers and parous buffalo cows (P>0.05). A total of 29 sex-preselected calves, 24
females and 5 males, developed to term and were viable on delivery. Results of this study
indicate the feasibility of the application of the sexing technology to accelerate the genetic
improvement in swamp buffalo.
© 2010 Elsevier B.V. All rights reserved.
Buffalo is an important domestic animal in agricul-
ture and animal production industry in many Asian and
provinces in southern China (FAO, 2003). As the consump-
tion of high quality dairy product increase, there is a need
to expand the genetic merit of river type buffalo, whose
milk production is much higher than that of swamp buf-
falo. So far it has been demonstrated that the application
of sexed bovine sperm using AI is effective in altering the
sex ratio and rapidly expanding dairy herds carrying high
genetic value animals (Johnson, 2000; Garner, 2006). Flow
∗Corresponding author. Tel.: +86 771 3235724; fax: +86 771 3238064.
E-mail addresses: email@example.com (Y. Lu), firstname.lastname@example.org,
email@example.com (K. Lu).
subsequent use in IVF has been previously reported (Lu et
al., 2006; Lu et al., 2007), which validated the feasibility
of this sex-preselecting technology in buffalo species. The
practical application of sexed sperm in buffalo breeding
would be of great interest both in biological and economic
Over the last several decades, AI has been shown
to be one of the most successful breeding strategies to
quickly disseminate genes from the best available males
for improvement of production traits. Previous reports
revealed that, following Ovsynch protocol and AI with
sexed sperm into the utero-tubal junction, a concep-
tion rate of 42.8% was observed in Mediterranean Italian
buffaloes (Presicce et al., 2005). However, buffalo in house-
holds of China and many Asian countries are usually raised
in very small herds (2–5 buffaloes per family) and the
ficial insemination following spontaneous estrous should
be a more viable way to rapidly disseminate the sexed buf-
falo sperm at present. In this study, semen was collected
0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
Author's personal copy
Y. Lu et al. / Animal Reproduction Science 119 (2010) 169–171
from river type buffalo (Nili-Ravi and Murrah) and sorted
for X-sperm enriched sample. This was inseminated by AI
into the deep uterine horn of spontaneous estrous buffalo.
Conception rate was recorded to determine the efficiency
of the use of this technology for buffalo.
2. Materials and methods
One Murrah and one Nili-Ravi bull of proven fertil-
ity were used in semen collection by artificial vagina and
unsexed sperm used in the experiment was collected from
10 bulls of proven fertility. All bulls were raised in Guangxi
Livestock and Poultry Breeding Station, China. The swamp
type buffalo cows and F1 crossbreeds of river type and
swamp type used for AI were raised in households in Ling-
shan county, Guangxi, China.
2.2. Sperm sorting
Only ejaculates with >75% morphologically normal
sperm and >65% progressive motility were used. Sperm
sexing procedure was similar to the report of Lu et al.
(2007). Briefly, sperm were stained in 40?g/mL Hoechst
sorted into X-chromosome bearing sperm enriched popu-
lations by a modified BD FACSVantage SE flow cytometer
sure, with a 197mM Tris Base sheath fluid. Sperm were
sorted into 50-mL tubes containing 2mL 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. The purity of
sexed sperm was determined by flow-cytometric reanaly-
sis following re-staining and sonication.
2.3. Deep uterine horn AI
Each 0.25mL straw used in this study contained 2×106
sexed sperm, while the conventional semen 0.25mL straw
contained 20×106non-sexed sperm. A total of 43 buffalo
cows were inseminated with sexed sperm, of which 29
were swamp type and 14 were F1 crossbreed. A total of
2325 buffalo cows, of which over 85% were swamp type
and the rest were F1 crossbreed, were inseminated with
non-sexed sperm. The animals used in this study were pri-
marily raised on pasture, exhibited spontaneous estrous
and were inseminated by the same technician during the
year of 2006.
To determine the time of insemination, overt signs of
heat were observed, followed by transrectal palpation to
check for the presence of the dominant follicle. Semen
was deposited into the deep uterine horn ipsilateral to the
Pregnancy status was diagnosed 3mo after insemination.
Statistical analysis of the conception rate was done by Chi-
square test using SPSS 11.0 (SPSS, Inc., Chicago, IL, USA)
3. Results and discussion
3.1. Conception rates in AI with sexed and non-sexed
Flow cytometry sorting of X- and Y-sperm followed by
insemination for production of sex-preselected offspring
has been successful in various animals (Cattle: (Seidel et
al., 1999a); Sheep: (Cran et al., 1997; Hollinshead et al.,
2002); Horse: (Buchanan et al., 2000); Pig: (Johnson, 1991;
Grossfeld et al., 2005)). However, the low dose of sexed
sperm using in AI, combined with the negative effect of
ception rate. In cattle, the conception rate of AI using sexed
sperm, with one tenth the sperm number of non-sexed
sperm, is around 70–80% of those achieved by non-sexed
sperm (Garner, 2006). Some reports reveal no significant
difference in AI with sexed and non-sexed sperm (Bodmer
et al., 2005; Sun et al., 2007). In the current study, the con-
and 66.5% (1545/2325) for non-sexed sperm, not statisti-
temperature for breeding was 10–23◦C (Gwazdauskas et
al., 1981) and conception rate decreased significantly dur-
ing the summer because of heat stress (Shearer and Beede,
1990). In southern China, estrous cycles in buffalo cows are
largely observed between August and January, which rep-
resents a mild climate in this region (Li, 2007). In this study
inseminations of sexed sperm were carried out between
AI were from records taken through out the whole year.
The optimal breeding season for sexed sperm could be the
primary contribution resulting in a comparable conception
rate with the non-sexed sperm in buffalo.
3.2. Conception rate in AI with sexed sperm derived from
It has long been acknowledged that the fertility of
sperm from different bulls could vary (Collins et al., 1962;
Davidson and Farver, 1980). Such variation has also been
observed in AI (Seidel et al., 1999b) and IVF (Zhang et al.,
2003) by using sexed Holstein sperm. Our finding for AI
with sexed sperm from different buffalo bulls was similar
to the previous reports in Holsteins. The conception rate of
sexed sperm derived from NL388 was 55.0% (11/22), sig-
nificantly lower than that of 83.6% (19/23) from ML445
(P<0.05). This variation in fertility could be attributed to
the difference in morphology and genetics of sperm and
their ability to withstand the sexing procedures (Dai et
al., 2009) and therefore, in terms of economic efficiency,
the selection of bulls for sperm sexing would be of great
3.3. Conception rate in AI with sexed sperm in heifer and
parous buffalo cows
Studies on cattle have shown that the conception rate
in AI with sexed sperm in heifer was 66.1%, higher than
Author's personal copy Download full-text
Y. Lu et al. / Animal Reproduction Science 119 (2010) 169–171
45.2% in parous cows (Sun et al., 2007). However, in this
study, conception rate following AI with sexed sperm in
heifers is 77.8% (7/9), slightly higher than that of 67.6%
(23/34) in parous buffalo cows, but not statistically differ-
ent (P=0.699). The different results in buffalo and Holstein
cows may be due to the difference in the way of animal
maintenance. The buffaloes used in the present study were
swamp type and F1 crossbreed, mainly used for beef pro-
duction and draught, which suffered little from milking
stress. Moreover, these animals were mainly pastured and
had more exercise than those milking cows, which were
and thus results in a high fertility in the parous cows. We
found in this study that the conception rate of the primi-
(7/9) in heifers, which indicated a similar uterus condition
and fertility in these animals.
3.4. Sex-preselected buffalo calves born following AI with
A total of 30 pregnancies were confirmed 3mo after
insemination, of which 1 aborted and 29, 24 females and 5
males, developed to term and gave birth to healthy calves
in 2007. The sexed sperm used in AI was X-sorted, gated
around 90% purity. However, 82.8% (24/29) of the calves
delivered were female, lower than usually observed in AI
The lower efficiency in sex alteration could be possibly due
to the smaller DNA difference between X- and Y-sperm in
buffalo (Lu et al., 2006) than those in cattle (Garner, 2006),
of buffalo sperm during sorting and thus a lower purity.
sorted into X-sperm enriched population and inseminated
into spontaneously estrous buffaloes, resulting in 69.7%
conception rate and 82.8% sexing accuracy. This represents
an efficient alteration of the sex ratio by using flow cytom-
etry sexing and AI technologies in this species. However,
the data of the present study is limited and a more exten-
sive study should be carried out to allow optimization of
the procedure before it is commercially applied.
We acknowledge the staff at Animal Husbandry and
Veterinarian Service Station in Lingshan County for
assistance in carrying out the AI. This research was
jointly supported by National High-tech R & D Pro-
gram (2008AA101004), National Science and Technology
Supporting Program (No. 2006BAD04A18), Guangxi Sci-
ence and Technology R&D Program (No. 0630006-5B,
Guangxi University Key Program (No. 2005ZD05).
Bodmer, M., Janett, F., Hassig, M., den Daas, N., Reichert, P., Thun, R., 2005.
Fertility in heifers and cows after low dose insemination with sex-
sorted and non-sorted sperm under field conditions. Theriogenology
Buchanan, B.R., Seidel Jr., G.E., McCue, P.M., Schenk, J.L., Herickhoff,
L.A., Squires, E.L., 2000. Insemination of mares with low numbers
of either unsexed or sexed spermatozoa. Theriogenology 53, 1333–
Collins, W.E., Inskeep, E.K., Tyler, W.J., Casida, L.E., 1962. Variation
in conception rates of Guernsey cattle. J. Dairy Sci. 45, 1234–
Cran, D.G., McKelvey, W.A.C., King, M.E., Dolman, D.F., McEvoy, T.G.,
Broadbent, P.J., Robinson, J.J., 1997. Production of lambs by low
dose intrauterine insemination with flow cytometrically sorted and
unsorted sperm. Theriogenology 47, 267.
Dai, L., Zhao, Z., Zhao, R., Xiao, S., Jiang, H., Yue, X., Li, X., Gao, Y., Liu,
J., Zhang, J., 2009. Effects of novel single nucleotide polymorphisms
of the FSH beta-subunit gene on semen quality and fertility in bulls.
Anim. Reprod. Sci. 114, 14–22.
Davidson, J.N., Farver, T.B., 1980. Conception rates of Holstein bulls for
artificial insemination on a California dairy. J. Dairy Sci. 63, 621–626.
FAO, 2003. www.fao.org/dad.it.
Garner, D.L., 2006. Flow cytometric sexing of mammalian sperm. Theri-
ogenology 65, 943–957.
Grossfeld, R., Klinc, P., Sieg, B., Rath, D., 2005. Production of piglets with
sexed semen employing a non-surgical insemination technique. The-
riogenology 63, 2269–2277.
Gwazdauskas, F.C., Lineweaver, J.A., Vinson, W.E., 1981. Rates of con-
ception by artificial insemination of dairy cattle. J. Dairy Sci. 64,
Hollinshead, F.K., O’Brien, J.K., Maxwell, W.M., Evans, G., 2002. Production
of lambs of predetermined sex after the insemination of ewes with
low numbers of frozen-thawed sorted X- or Y-chromosome-bearing
spermatozoa. Reprod. Fertil. Dev. 14, 503–508.
Johnson, L.A., 1991. Sex preselection in swine: altered sex ratios in off-
sperm. Reprod. Domestic Anim. 26, 309–314.
the state-of-the-art. Anim. Reprod. Sci. 60–61, 93–107.
Li, Z.Q., 2007. Study on the seasonal estrus and artificial insemination
in water buffalo. Guangxi J. Anim. Husbandry Vet. Med. 23, 116–
Lu, Y.Q., Liang, X.W., Zhang, M., Wang, W.L., Kitiyanant, Y., Lu, S.S., Meng,
B., Lu, K.H., 2007. Birth of twins after in vitro fertilization with flow-
cytometric sorted buffalo (Bubalus bubalis) sperm. Anim. Reprod. Sci.
Lu, Y.Q., Zhang, M., Meng, B., Lu, S.S., Wei, Y.M., Lu, K.H., 2006. Identifica-
Anim. Reprod. Sci. 95, 158–164.
Presicce, G.A., Verberckmoes, S., Senatore, E.M., Klinc, P., Rath, D., 2005.
First established pregnancies in Mediterranean Italian buffaloes
utero tubal junction. Reprod. Domestic Anim. 40, 73–75.
Seidel Jr., G.E., Schenk, J.L., Herickhoff, L.A., Doyle, S.P., Brink, Z., Green,
R.D., Cran, D.G., 1999a. Insemination of heifers with sexed sperm.
Theriogenology 52, 1407–1420.
Seidel, G.E.J., Cran, D.G., Herickhoff, L.A., Schenk, J.L., Doyle, S.P., Green,
R.D., 1999b. Insemination of heifers with sexed frozen or sexed liquid
semen. Theriogenology 51, 400.
on production, reproduction, and health of dairy cattle. Agri-Practice
Sun, S.C., Shang, R.Z., Sun, G.Q., Li, D.H., Zhu, W.S., Li, W.J., Zhao, Z.J., 2007.
Effect of artificial insemination with sexing semen on pregnanct rate
and female calf rare of dairy cattle. Heilongjiang J. Anim. Reprod. 15,
Zhang, M., Lu, K.H., Seidel, G.E., 2003. Development of bovine embryos
after in vitro fertilization of oocytes with flow cytometrically sorted,
stained and unsorted sperm from different bulls. Theriogenology 60,