THE EFFECT OF GENTLE PRE-EQUILIBRATION ON SURVIVAL AND DEVELOPMENT RATES OF BOVINE IN VITRO MATURED OOCYTES VITRIFIED IN DROPLETS

Vitrification of bovine oocytes performed using traditional straw systems, have not given satisfactory results. Some alternative methods (Biol. Reprod. 54:1059-1069, 1996; Theriogenology 49:176, 1998 ) were described recently with relatively much higher survival and subsequent fertilization and development rates of vitrified oocytes. We report here a simple and efficient method for vitrification of bovine oocytes. Abattoir derived, in vitro matured and denuded oocytes were subjected to equilibration in 2 ml of 0, 1, 2, 3, 4, 5 or 6% ethylene glycol (EG) solution in TCM 199 medium supplemented with 20% FCS at 32-35°C. After 12-15 min of equilibration, groups of 5 to 8 oocytes were transferred within 30 sec through 3 changes of VS14 vitrification solution (5.5 M E G and 1.0 M sucrose, J. Reprod. Fertil. 98:459-465, 1993) and dropped directly into LN2 in droplets containing about 6 ~1 of vitrification medium. Droplets were collected to cryotubes using fine forceps and kept in LN2 1 to 4 h. Thawing was performed by means of direct immersing and stirring of each droplet in 2 ml of warm TCM 199/20% FCS medium. After washing in fresh medium, oocytes were subjected to IVF (Day 0) and IVC procedures (Theriogenology 47:357, 1997) and cultured up to Day 10. Data representing 3 or 4 replicates were analysed using Chisquare test. The cleavage, blastocyst and hatched blastocyst rates are presented in Table 1. The highest cleavage and blastocyst rates were obtained after preequilibration of oocytes