Rescue of Oocytes from Early Antral Follicles Isolated from Cryopreserved Buffalo Ovaries Using an in Situ Oocyte Cryopreservation Method: Competence to Undergo Maturation, Fertilization and Development in Vitro

This study was undertaken to determine whether fully grown oocytes isolated from the early antral follicles (2-8 mm size) of frozen-thawed buffalo ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved just after being collected during slaughter from the local abattoir using in situ oocyte (ISO) cryopreservation. ISO cryopreservation is a multistep procedure that involves aspiration of follicular fl uid and then perfusion of antral follicles (10-12 mm size) and diffusion of whole buffalo ovaries with cryoprotectant agent (CPA), rapid cooling, storage, thawing and, fi nally, dilution and removal of the CPA with return to physiological environment. Data analysis revealed the quality of follicular oocytes isolated from ISO cryo ovaries appeared similar to that from fresh ovaries, and the percentages of the morphologically normal immature oocytes from ISO cryo ovaries appeared also similar to those from fresh ovaries. There were no signifi cant differences in the maturation (80.1%), cleavage (46.9%) and buffalo embryo development (30.4%) produced by immature oocytes of ISO cryo ovaries in comparison to the three observations in fresh oocytes of fresh non-cryopreserved ovaries (88.4%, 56.5% and 38.1%, respectively, p<0.05). Therefore, fully grown oocytes in early antral follicles (2-8 mm size) survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro.