Theriogenology 68 (2007) 450–453

www.theriojournal.com

Advanced reproductive technology in the water buffalo

M. Drost
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136, USA

Abstract
Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in
buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of
recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic.
Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The
average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably
improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield
more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between
buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein
heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine
oocytes and subsequent development to the blastocyst stage have been reported.
# 2007 Elsevier Inc. All rights reserved.

Keywords: Reproductive technology; Embryo transfer; Superovulation; Water buffalo; Bubalus bubalis

1. Introduction livestock. They are prerequisites to success with

advanced procedures such as embryo transfer, transfer
Reproductive technology in water buffalo (Bubalus of frozen embryos and in vitro-derived embryos,
bubalis) has not developed to the extent it has in cattle including cloned embryos. Embryo transfer is a
(Bos taurus, Bos indicus). Although AI is practiced factorial process that consists of a series of carefully
commercially, it is hampered by difficulties in estrus integrated sequential steps including donor selection,
detection. More advanced procedures such as embryo donor treatment, recipient selection, insemination of the
transfer and IVF remain in the experimental stages due donor, embryo recovery, embryo handling and evalua-
to poor yield of embryos. There are some preliminary tion, embryo transfer, and recipient care.
successes with the esoteric approaches of intergeneric Although bovine embryo transfer technology has
embryo transfer and nucleus transfer. This condensed been successfully applied in the water buffalo, progress
review will touch on the current status of the above has been slow and results have been modest to poor.
approaches. The first successful transfer in buffalo was performed
in the United States of America in 1983 [1]. Subsequent
2. Embryo transfer
successful transfers have been reported in Bulgaria [2],
India [3] and Thailand [4]. Live buffalo calves have
Estrus detection, AI, and estrous cycle control are
also been born after the transfer of in vivo-derived
basic methods in assisted reproductive technology in
embryos frozen by conventional freezing methods
[5]. Generally the numbers of animals involved have
E-mail address: drost@ufl.edu. been small.

0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2007.04.013
 M. Drost / Theriogenology 68 (2007) 450–453 451

It is of interest to look at the differences between the of this is that on Day 5 the corpus luteum has not yet
two genera, Bubalus and Bos, which contribute to lower reached full development and is both small and
success rates in buffalo. First among the inherent relatively soft, hence difficult to identify by palpation
differences is the lower level of fertility in buffalo. The per rectum. The latter is in addition to the fact that the
latter is largely due to lack of selection of breeding corpus luteum of the buffalo is already smaller per se
stock. Problems of delayed puberty and prolonged and more deeply embedded (hence more difficult to
calving intervals can be reduced with adequate palpate) than that of the cow. This difficulty extends to
nutrition, parasite control, and disease management the proper evaluation of synchronized recipients on Day
as has been demonstrated in the excellent (large) milk 5, in order for the embryo to be transferred to the horn
buffalo herds in Italy and Bulgaria. Seasonality of the ipsilateral to the corpus luteum. The use of ultrasono-
breeding season may further restrict the success rate of graphy has become a valuable, if not indispensable,
embryo recovery. adjunct.

3. Superovulation 4. In vitro embryo production

Superovulation is even less predictable in buffalo The birth of a live calf was reported as a result of the
than in cattle [1,3,5–9]; an optimal treatment regimen transfer of fresh in vitro-derived buffalo embryos to
with FSH remains elusive. The buffalo ovary has a buffalo recipients in 1991 [17]. Subsequently, in 2004,
smaller population of recruitable follicles at any given six live calves were born after the transfer of 95 in vitro-
time than the ovary of the cow, at birth an average of derived vitrified buffalo embryos to 55 recipients (1–3
12,000 primary follicles in the buffalo [10] versus an per recipient) [18]. Although numbers were small, no
average of 133,000 in the cow [11]. signs of abnormal offspring syndrome calves were
Estrus detection in buffaloes is problematic because observed.
overt signs are few. All available techniques should be That IVF could considerably increase embryo
employed, including frequent observation, use of a non- production (relative to the inconsistent response to
entry teaser animal [12,13] transrectal palpation, and superovulation) has led to an increasing interest in
ultrasonography. Accurate determination of the onset of large-scale embryo production and the opportunity to
estrus (estrus = Day 0) is critical for precise determina- enhance the maternal contribution to genetic improve-
tion of the stage of development of the embryo, as the ment. Oocytes recovered from abbatoir-derived ovaries
onset of estrus is used as a reference point for the age of generally have little genetic value. The mean number of
the embryo. oocytes collected per ovary and used for IVF was
The Ovsynch protocol effectively synchronized approximately 0.43–0.70 in India, and 2.4–3.3 in Italy
ovulation in Murrah buffaloes and resulted in concep- [19]. This number increased to 2.25 when oocytes were
tion rates (to two fixed-time inseminations) that were collected by transvaginal ultrasound guided puncture,
comparable to those achieved with a single insemina- often referred to as Ovum Pick Up (OPU) [20,21].
tion after detection of estrus [14]. This study indicates These differences may depend on genetic, nutritional,
the opportunity for the use of Ovsynch, with confirma- environmental, and stressful conditions. However, this
tion of ovulation by transrectal palpation or ultrasono- variation substantially affects the potential of IVF. The
graphy, to synchronized embryo recipients. buffalo ovary is smaller than that of cattle [22]. With the
Embryo development is more rapid in buffalo than in OPU method, the recovery rate by aspiration of 2–8 mm
cattle [15,16]. The zona pellucida serves as a follicles is much lower, from 0.7 [23] to 2.4 per ovary
distinguishing feature in the identification of embryos [24], compared with cattle in which 8–12 good quality
in the effluent recovery medium. After hatching, when oocytes are obtained on average from abbatoir-derived
this characteristic landmark has been shed, the ovaries [25]. The technique of OPU is superior to
blastocyst is difficult to recognize, particularly because superovulation because it can yield more transferable
the blastocele is frequently collapsed. Hatching gen- embryos per donor on a monthly basis (2.0 versus 0.6,
erally occurs 6.5–7.0 days after the beginning of estrus respectively). Therefore this technology has the greater
in buffalo compared to 8.5–10.0 days in cattle. potential to improve to improve the genetic potential of
Consequently, nonsurgical recovery of buffalo embryos the water buffalo via the maternal lineage [26].
must be done between 5 and 6 days after the onset of Overall, high oocyte maturation, fertilization, and
estrus (versus 7 days in the bovine) when they are in the cleavage rates, but low rates of blastocyst yield, and
morula and blastocyst stages. The negative implication calving after the transfer of in vitro produced buffalo
452 M. Drost / Theriogenology 68 (2007) 450–453

embryos have been obtained. The efficiency of IVF in When bubaline adult fibroblasts were used as donor
buffalo is much lower than that in cattle. Several cells, there were no differences in the cleavage rates
problems need to be resolved before IVF technology (66.2% versus 64.0%) between bovine and bubaline
can be used commercially in producing buffalo recipient oocytes, but more embryos derived from
breeding stock [27]. bovine cytoplasts developed to blastocysts than from
bubaline cytoplasts (13.3% versus 3.0%). When bovine
5. Intergeneric embryo transfer adult fibroblasts were used as donor nuclei, both
cleavage rate (45.3%) and blastocyst yield (4.5%) of NT
The feasibility of reciprocal embryo transfer embryos derived from bubaline cytoplasts were lower
between water buffalo and domestic cattle has been than those of NT embryos derived from bovine
investigated [28]. Eight mature water buffalo females cytoplasts (65.5% and 11.9%). The authors concluded
were superovulated and hand-bred naturally to a male that embryos constructed by intergeneric NT of adult
water buffalo. Sixteen buffalo embryos were recovered fibroblasts between buffalo and cattle developed to
nonsurgically with 13 flushes 5.5, 6.0, 7.0, and 7.5 days blastocysts, but bovine cytoplasts may facilitate
after they were first bred by the bull. Three morulas embryonic development more effectively than bubaline
were recovered on Day 5.5, six blastocysts on Day 6.0, cytoplasts, irrespective of donor cell genus [37].
one hatched blastocyst on Day 7, and six hatched Additional success has been reported with the inter-
blastocysts on Day 7.5. Compared to cattle embryos, the generic nucleus transfer of Bubalus bubalis fetal and
diameter of the embryo was generally the same, and the adult somatic cell nuclei into Bos indicus cytoplasts and
color, particularly that of the inner cell mass, darker. subsequent development to the blastocyst stage [38].
The stage of development was 24–36 h in advance of
that of bovine embryos collected on corresponding days 6. Conclusions
[29]. No pregnancies resulted after transfer of 13
buffalo embryos to synchronized Holstein heifers. Despite the inherent infertility of the water buffalo
Intergeneric embryo transfers have been reported (Bubalus bubalis) slow progress has been made in the
between sheep and goats [30–32] but have otherwise application of reproductive technology in this genus.
largely been limited to descriptions of attempts in The domestic cow (Bos taurus) serves as a good model,
rodents [33,34]. Selection of a recipient that is capable but idiosyncrasies of estrous behavior, ovarian char-
of hybridizing with the donor has been suggested [35] acteristics, and embryo development in the water
which would maximize the chances for an interspecies buffalo must be acknowledged. Although AI is
embryo transfer. There are no reports of hybridization practiced commercially, embryo transfer, in vitro
(by natural service or AI) between Bubalus bubalis and embryo production, and nucleus transfer remain in
Bos taurus, whose respective chromosome numbers are the realm of experimentation. A recent report from
2n = 48 (swamp buffalo) or 2n = 50 (river buffalo) and China affirmed that even sexed semen by flow
2n = 60 in cattle. cytometric sorting of X- and Y-bearing sperm of
The feasibility of producing hybrid embryos of buffalo is feasible [39].
domestic cattle (Bos taurus) and water buffalo (Bubalus
bubalis) was investigated by exposing cattle oocytes to Illustrations
buffalo sperm, and buffalo oocytes to cattle sperm [36].
The cleavage rate in buffalo oocytes exposed to cattle Go to the Visual Guide to Theriogenology (http://
sperm was low (40.8%), with only 8.8% of these hybrid drostproject.vetmed.ufl.edu), select the Bubaline Guide
embryos reaching the blastocyst stage. Cattle oocytes and/or the Bovine Guide, then select Reproductive
exposed to buffalo sperm showed 86.3% cleavage, Technology.
whereas 25.9% of these hybrid embryos attained the
blastocyst stage [36]. References
A different approach to establish intergeneric
pregnancies was explored by using adult fibroblasts [1] Drost M, Wright Jr JM, Cripe WS, Richter AR. Embryo transfer
as donor cells in nucleus transfer (NT) between in water buffalo (Bubalus bubalis). Theriogenology 1983;20:
buffaloes and cattle. Bubaline and bovine oocytes, 579–84.
[2] Drost M, Vlahov K, Alexiev A, Cripe WS, Karaivanov CH,
matured in vitro for 22 h, were enucleated by Leonards AP, et al. Successful nonsurgical embryo transfer in
micromanipulation. Subsequently, an ear fibroblast buffaloes (Bubalus bubalis) in Bulgaria. Theriogenology
was injected into the enucleated oocyte (cytoplast). 1988;30:659–68.
 M. Drost / Theriogenology 68 (2007) 450–453 453

[3] Misra AK, Joshi BV, Agrawala PL, Kasiraj R, Sivaiah S, up and in vitro embryo production in Italian buffalo cows. In:
Rangareddi NS, et al. Multiple ovulation and embryo transfer Proceedings of the fifth world buffalo congress; 1997. p. 787–92.
in Indian buffalo. Theriogenology 1990;33:1131–41. [21] Galli C, Duci R, Crotti G, Lazarri G. Embryo production by
[4] Chantaraprateep P, Lhachit C, Techakumphu M, Kabayashi G, ovum pick up in water buffalo. Theriogenology 1998;49:400.
Virakul P, Kunavongkrit A, et al. Early embryonic development [22] Vittoria A. Anatomy of the female genital tract in the buffalo. In:
in Thai swamp buffalo (Bubalus bubalis). Theriogenology Proceedings of the third course on biotechnology of reproduction
1989;31:1131–8. in buffaloes, vol. 4, suppl; 1997. p. 15–20.
[5] Kasirai R, Misra AK, Mutha RM, Jaiswal RS, Rangareddi NS. [23] Totey SM, Singh G, Taneja M, Pawshe CH, Talwar GP. In vitro
Successful culmination of pregnancies of embryo transfer in maturation, fertilization, and development of follicular oocytes
buffalo. Buffalo J 1991;1:71–5. from buffalo (Bubalus bubalis). J Reprod Fertil 1992;95:597–
[6] Sharifudin W, Jainudeen MR. Superovulation and nonsurgical 607.
collection of ova in water buffalo (Bubalus bubalis). In: Pro- [24] Kumar A, Solanki VS, Jindal SK, Tripathi VN, Jain GC. Ooctye
ceedings of the Xth international congress of animal reproduc- retrieval and histological studies of follicular population in
tion AI; 1984. p. 240–2. buffalo ovaries. Anim Reprod Sci 1997;47:189–95.
[7] Parnpai R, Timsard V, Kamonpatana M, Pensin C, Sophon S, [25] Gordon I. Aspiration techniques: old and new. In: Laboratory
Jetana T, et al. Recovery of swamp buffalo embryos using Production of Cattle Embryos. Wallingford, UK: CAB Interna-
nonsurgical technique. Buffalo J 1985;1:77. tional; 1994. p. 71–2.
[8] Vlahov K, Karaivanov KH, Petrov M, Kacheva D, Alexiev A, [26] Gasparrini B. In vitro embryo production in buffalo: State of the
Polihronov DA. Studies on superovulation and embryo transfer Art. Theriogenology 2002;57:237–56.
in water buffaloes. In: Proceedings of the first world buffalo [27] Nandi S, Raghu HM, Ravindranatha BM, Chauhan MS. Produc-
congress, vol. III; 1985. p. 510–2. tion of buffalo (Bubalus bubalis) embryos in vitro: premises and
[9] Karaivanov C, Vlahov K, Petrov M, Kacheva D, Alexiev A. promises. Reprod Domest Anim 2002;37:65–74.
Superovulation in buffaloes with FSH. Vet Med Sci 1992;24:57 [28] Drost M, Wright JM, Elsden RP. Intergeneric embryo transfer
(in Bulgaria). between water buffalo and domestic cattle. Theriogenology
[10] Danell B. Oestrous behavior, ovarian morphology and cyclical 1986;25:13–23.
variation in follicular system and endocrine pattern in water [29] Drost M, Elsden RP. Blastocyst development in the water buffalo
buffalo heifers. PhD dissertation, Uppsala: Sveriges Landbruck- (Bubalus bubalis). Theriogenology 1985;23(1):191.
suniversitet; 1987. p. 54–94. [30] Warwick BL, Berry RO. Intergeneric and intraspecific embryo
[11] Erickson BH. Development and senescence of the postnatal transfers. J Heredity 1949;40:297–303.
bovine ovary. J Anim Sci 1966;25:800–5. [31] Hancock JL, McGovern PT. Transfer of goat  sheep hybrid
[12] Drost M, Cripe WS, Richter AR. Oestrus detection in buffaloes eggs to sheep and reciprocal transfer of eggs between sheep and
(Bubalus bubalis): use of an androgenized female. Buffalo J goats. Res Vet Sci 1968;9:411–5.
1985;1:159–61. [32] Bratanov K, Dikov D. Recondation entre les especes brebis et
[13] Vale WG, Sousa JS, Ribeiro HFL, Ohashi OM, Lau HD, Silva chevres et obtention d’hybrides interspecies. In: Proceedings of
AOA. Preparation of a ‘‘teaser’’ buffalo bull for use in artificial the fourth international congress of animal reproduction AI;
insemination programs. Buffalo J 1994;1:75–9. 1962. p. 744–9.
[14] Paul V, Prakash BS. Efficacy of the Ovsync protocol for syn- [33] Tarkowski AK. Interspecific transfers of eggs between rat and
chronization of ovulation and fixed-time insemination of Murrah mouse. J Embryol Exp Morph 1962;10:476–95.
buffaloes (Bubalus bubalis). Theriogenology 2005;1049–60. [34] Brines H, Beatty RA. Interspecific transfer of rodent eggs. J Exp
[15] Drost M, Elsden RP. Blastocyst development in the water buffalo Zool 1954;125:99–118.
(Bubalus bubalis). Theriogenology 1985;23:191. [35] Durrant B, Benirschke K. Embryo transfer in exotic animals.
[16] Karaivanov C, Vlahov K, Petrov M, Kacheva D, Stojanova M, Theriogenology 1981;15:77–83.
Alexiev A, et al. Studies on pre-implantation development of [36] Kocchar HP, Rao KB, Luciano AM, Totey SM, Gandolfi F,
buffalo embryos. Theriogenology 1987;28:747–53. Basrur PK, et al. In vitro production of cattle-water buffalo (Bos
[17] Madan ML, Singla SK, Jailkhavi S, Ambrose JD. In vitro taurus–Bubalus bubalis) hybrid embryos. Zygote 2002;10:155–
fertilization in buffalo and birth of first ever IVF buffalo calf. 62.
In: Proceedings of the third world buffalo congress, vol. 7; 1991. [37] Lu F, Shi D, Wei J, Yang S, Wei Y. Development of embryos
p. 10–7. reconstructed by interspecies nuclear transfer of adult fibroblasts
[18] Hufana-Duran D, Pedro PB, Venturina HV, Hufana RD, Salazar between buffalo (Bubalus bubalis) and cattle (Bos indicus).
AL, Duran PG, et al. Post-warming hatching and birth of live Theriogenology 2005;64:1309–19.
calves following transfer of in vitro-derived vitrified water [38] Saikhun J, Pavasuthipaisit K, Jaruansuwan M, Kityanant Y.
buffalo (Bubalus bubalis) embryos. Theriogenology Xenonuclear transplantation of buffalo (Bubalus bubalis) fetal
2004;1429–39. and adult somatic cell nuclei into bovine (Bos indicus) oocyte
[19] Boni R, Rovelli S, Gasparrini B, Langella M, Zicarelli L. In vitro cytoplasm and their subsequent development. Theriogenology
production of buffalo embryos in chemically defined medium. 2002;57:1829–37.
Buffalo J 1999;15:115–20. [39] Lu YQ, Zhang M, Meng B, Lu SS, Wei YM, Lu KH. Identifica-
[20] Boni R, Roviello S, Gasparrini B, Zicarelli L. Pregnancies tion of X- and Y-chromosome bearing buffalo (Bubalus bubalis)
established after transferring embryos yielded by ovum pick- sperm. Anim Reprod Sci 2006;95:158–64.