104 Birth of First Buffalo (Bubalus Bubals) Calf Following Embryo Splitting and Polymerase Chain Reaction Sexing at Blastocyst Stage

Abstract

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192–196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245–1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211–1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4).

Current Status of animal biotechnology and option for improvement of animal reproduction in Asia

“…Conception rates did not differ between adult buffaloes and buffalo heifers; however there was a difference between bulls (Tasripoo et al, 2014). Most studies in buffaloes have reported the use of sexed semen fur insemination after an estrus synchronization protocol (Zhang et al, 2011). A recent study, however, evaluated the insemination of 4521 swamp or crossbred buffaloes during natural estrus with X-sorted semen from river buffaloes and recorded a 48.5% pregnancy rate and 87.6% sex accuracy (Yang and Pang, 2010).…”

“…The in vivo procedures involve induction of multiple ovulation, breeding and non-surgical recovery of embryos which are then transferred to synchronized recipients Inherently poor reproduction and seasonality in buffalo warrants the use of advanced reproductive technologies in this species. Efforts at multiple ovulation and embryo transfer in buffaloes have resulted in the birth of calves in many countries by transfer of fresh (Zhang et al, 2011) or cryopreserved embryos, however, the efficiency in terms of super ovulatory response and recovery of transferrable embryos continue to be low. Attempts to improve the ovulatory responses in buffaloes have shown marginal improvements (Tasripoo et al, 2014).…”

“…Attempts to produce embryos from follicular oocytes by in vitro maturation, in vitro fertilization and in vitro culture were successful and resulted in the birth of river (Kandil et al, 2014) and crossbred (50:50 river: swamp) buffalo calves (Jain et al, 2011). Cryopreserved in vitro produced embryos also resulted in the birth of river and swamp buffalo calves, including twins (Zhang et al, 2011). Embryos have also been produced in vitro by fertilization of oocytes using density gradient prepared or sex selected sperm (Tasripoo et al, 2014).…”

“…Efficiency of oocyte cryopreservation requires further refinement before it can beused for routine application. The high lipid content of buffalo oocytes may influence the success of cryopreservation (Zhang et al, 2011).…”

“…Furthermore, fertility of sexed sperm is lower than unsexed controls (Kandil et al, 2014) leaving room for improvement. Advances have been made in two main areas: increasing the number of sperm sexed accurately per unit time, and making the process less damaging to the sperm by optimizing the pressure in the flow cytometer (Zhang et al, 2011). The possibility of choosing a male or female calf can be done by the use of X and Y -sorted sperm in artificial insemination or in vitro fertilization programs.…”