41 DEVELOPMENTAL POTENTIAL OF BUFFALO OOCYTES VITRIFIED AT THE GERMINAL VESICLE STAGE: EFFECTS OF DIFFERENT CRYOPROTECTANT COMBINATIONS AND CRYODEVICES

Abstract

The cryopreservation of immature oocytes would generate a readily available, nonseasonal source of female gametes for both research and reproduction. In domestic animals, the most promising results in the field of oocyte cryopreservation have been reported in cattle, and a few experiments have been conducted on buffalo. The aim of the present study was to compare the effects of different cryoprotectant combinations and different cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle stage. Cumulus-oocyte complexes obtained at slaughter from mature buffalos were vitrified by using either straw or open pulled-straw or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol or 20% EG + 20% dimethylsulfoxide (DMSO). Following vitrification and warming, viable oocytes were matured in vitro for 22 h. Matured oocytes were either evaluated for nuclear maturation or fertilized and cultured in vitro for 7 days. Recovery rate was significantly higher (P < 0.05) in the oocytes vitrified by straw in 20% EG + 20% glycerol (92.6%) as compared with the other groups. Percentages of viable oocytes were significantly higher (P < 0.05) in the oocytes vitrified in 20% EG + 20% DMSO using SSV (95.7%) than those in the other groups (from 80 to 88.0%). Among the vitrified groups, the highest maturation rate was achieved in SSV with 20% EG + 20% DMSO group (56.7%). This value was comparable with those in the control group (62.1%). After IVF and embryo culture, the highest cleavage and blastocyst rates were obtained in SSV with 20% EG + 20% DMSO group (35.7 and 21.4%, respectively), and these values were nearly similar to those in the control group (38.7 and 25.8%, respectively). Vitrification of oocytes by straw or open pulled-straw resulted in significantly lower (P < 0.05) blastocyst rates (2.6 and 11.5%) as compared with the control. In conclusion, buffalo oocytes vitrified at the germinal vesicle stage can be matured, fertilized, and developed in vitro and produce high frequencies of blastocyst embryos. Solid surface vitrification may be superior to straw and open pulled-straw in vitrification of immature buffalo oocytes because this technique results in higher survival and embryo development rates.