123 BOVINE OOCYTE VITRIFICATION USING THE CRYOTOP METHOD: EFFECT OF CUMULUS CELLS AND VITRIFICATION PROTOCOL ON SURVIVAL AND SUBSEQUENT DEVELOPMENT

Abstract

The ability to successfully cryopreserve mammalian oocytes has numerous practical and economic ethical benefits that may positively affect animal breeding programs and assisted conception in humans. However, oocyte survival and development following cryopreservation remain poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) oocytes, (2) to compare empirical and theoretical vitrification protocol, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. Bovine oocytes were collected from the ovaries of slaughtered cross-bred beef heifers. In Experiment 1, cumulus-enclosed and partially denuded GV and MII oocytes were vitrified in 15% EG + 15% DMSO + 0.5 M sucrose in 2 steps. In Experiment 2, GV oocytes were vitrified as above or using theoretical modelling based on permeability and osmotic tolerance characteristics (Wang et al. Reprod. Fertil. Dev. 21 141) in 30% EG +11.4% trehalose in 3 steps or 40% EG + 11.4% trehalose in 4 steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA, USA) 1% X-1000, 1% Z-1000, or both in 3 steps. The survival, cleavage, and blastocyst rate of cumulus-enclosed oocytes was significantly higher (ANOVA) than those of partially denuded oocytes when vitrified at GV (93.8% v. 81.3%, 65.8% v. 47.3%, 11.3% v. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% v. 91.8%, 35.2% v. 36.8%, 5.0% v. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher development competence than those vitrified at MII (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage, and blastocyst rates among 3 protocols (86.0% v. 92.8% v. 91.2%, 44.8% v. 54.4% v. 45.6%, 5.0% v. 5.4% v. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than nonvitrified control oocytes. In Experiment 3, the presence of ice-blockers did not improve rate or blastocyst development (P < 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes. Theoretical analysis of permeability characteristics and tolerance limits alone may not be sufficient to improve vitrification protocols.