Production of the first viable ovum pick–up and in vitro embryo produced (OPU–IVEP) buffalo calf in India
Authors: Shiv Prasad, Beerendra Singh, Sumit Singhal, FA Khan, JK Prasad, HP Gupta
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This report puts on record production of the first viable calf following ovum pick-up in vitro embryo production (OPU-IVEP) technique in buffalo (Bubalus bubalis) in India. A total of 313 transvaginal OPU sessions were performed in six elite Murrah buffaloes and 1 171 follicles were aspirated. From these follicles, a total of 729 immature oocytes were recovered. The recovered oocytes were graded as A (59, 8.1%), B (111, 15.2%), C (293, 40.2%) and D (262, 35.9%) following standard procedures. Maturable grade oocytes (A, B, and C; 473, 68.9%) were subjected to in vitro maturation in Medium 199 supplemented with 10% FBS, 0.5 μg/mL FSH, 10 IU/mL LH, 1 μ g/mL estradiol-17 β, 20 ng/mL EGF and 50 μM cysteamine in CO2 incubator at 39 °C temperature, 5% CO2 and high humidity. The matured oocytes (427 out of 473; 90.3%) were co-incubated with 1×106 /mL buffalo sperms in modified synthetic oviductal fluid (mSOF) containing 10 ng/mL heparin for 22 hrs for in vitro fertilization. After fertilization the presumptive zygotes were stripped off of remaining cumulus cells and cultured in mSOF for 7 days to study embryonic development. A total of 278 (65.1%) oocytes cleaved after 24-36 hrs of fertilization. Out of the cleaved oocytes, 249 (89.6%), 216 (77.7%), 167 (60.1%), 139 (50.0%) and 73 (26.3%) developed up to 4-cell, 8-cell, 16-cell, morula, and blastocyst stages, respectively. A total of 29 embryos were transferred into 27 synchronized recipient buffaloes. Two of these buffaloes were found pregnant at 90 days. One of them aborted at 4.5 months of gestation (male fetus) and the other gave birth to a live male calf weighing 25 kg. Currently, the calf is normal and has attained the age of 4.5 years.