222 IN VITRO FERTILIZATION OF OOCYTES OBTAINED THROUGH TRANSVAGINAL OOCYTE RETRIEVAL FROM CYCLIC MURRAH BUFFALOES (BUBALUS BUBALIS)

Abstract

Very limited information is available in the literature on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of oocytes collected through the transvaginal oocyte retrieval (TVOR) technique in buffaloes. Therefore, the present study was undertaken to examine the post IVF cleavage rates and embryonic development up to the blastocyst stage in the Murrah breed of buffalo subjected to TVOR. The five cyclic Murrah buffaloes were synchronized for estrus by a single prostaglandin injection. The animals were subjected to TVOR once weekly for seven weeks. TVOR was performed using an ultrasound machine with a transvaginal convex transducer (5 MHz), a needle guide, a single-lumen 19-gauge 60-cm-long needle, and a vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm), and large (e10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes with e5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. IVM, IVF and IVC were carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). Briefly, the oocytes were cultured for 24 h in a CO₂ incubator (5% CO₂ in air) at 38.5°C for in vitro maturation. Frozen-thawed semen was used in Bracket and Olyphant medium for capacitation and fertilization. The in vitro-fertilized and cleaved embryos were cultured further for 9 days in modified synthetic oviductal fluid. The small follicles constituted a major proportion (60%) of the total observed follicles, although a substantial proportion of medium (19%) and large (21%) follicles were also present. A total of 76 oocytes were recovered by aspiration of 110 follicles, with an overall recovery rate of 70% (range 67-74%). Of these, 45 (59%) were of grades A and B, and 31 (41%) were of grades C and D. The mean number of total follicles and the oocytes recovered per session did not differ significantly among individual donors. Out of the 37 oocytes subjected to IVM and IVF, 19 (51%) cleaved at Day 2 post-insemination. A total of four embryos (11%) developed into morulae/blastocysts. This study demonstrates the use of TVOR as a mean of obtaining oocytes, their fertilization, and further embryo development in the Murrah breed of buffalo.