Evaluation of Different Vitrification Methods and Cryoprotectants on Viability of In Vitro Matured Buffalo Oocytes

The developmental competence of buffalo oocytes is greatly influenced by the vitrification process and the cryoprotectants used during cryopreservation. The present study was undertaken to evaluate the effect of different cryoprotectants on the developmental potential of vitrified buffalo oocytes in terms of cleavage, and blastocyst formation. A total of 201 (18.3±0.8/session) buffalo ovaries were collected from the slaughterhouse in 11 sessions, and 447 (40.6±3.5/session) cumulus-oocyte complexes (COCs) were recovered from 663 aspirated follicles. Following in vitro maturation (IVM), 397 (36.1±3.5/session; 87.83%) oocytes attained maturity and were allocated to four experimental groups: Group I (fresh, non-vitrified control; n=198), Group II (Vitrogen vitrification; n=64), Group III (DMSO + EG vitrification; n=74), and Group IV (DMSO + EG + Sucrose vitrification; n=61). After warming and in vitro fertilisation (IVF), a total of 318 presumptive zygotes were cultured in vitro (IVC). Fresh oocytes exhibited superior developmental competence compared with vitrified oocytes. The cleavage rate was highest in the fresh control group, with 130 out of 198 presumptive zygotes (11.8±1.8; 63.23%) undergoing cleavage, whereas vitrified groups (Groups II, III, and IV) showed markedly lower cleavage rates of 11.61% (0.7±0.3), 11.97% (0.6±0.2), and 11.61% (0.5±0.2), respectively. Similarly, blastocyst formation was highest (n=15 BLs) in the fresh control group (8.09%; 1.4±0.3), in comparison to Groups II, III, and IV, 2.44% (0.2±0.1), 0.91% (0.09±0.8), and 1.52% (0.1±0.08), respectively. Although minor variations were observed among the results of different vitrification protocols, all vitrified groups demonstrated substantially reduced embryonic development compared with fresh oocytes.