217 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF MATURED BOVINE OOCYTES AFTER VITRIFICATIONAND ON SUBSEQUENT EMBRYO DEVELOPMENT

Abstract

Prolonged exposure of oocytes to cryoprotectants causes cell injury, whereas a short exposure time results in insufficient permeation because of ice formation. This study was designed to determine the optimal pre-equilibration time and its effect on the survival rate of matured bovine oocytes after warming and on subsequent embryo development. Bovine COC were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL^–1 of FSH at 38.5°C in 5% CO₂ in air. Then, the COC were partially denuded. The oocytes were pre-equilibrated in 100 μL of vitrification solution 1 (VS1) containing 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS in TCM-199 for 0, 1, 3, and 5 min. Then, the oocytes were moved through 100-μL drops of vitrification solution 2 (VS2) containing 30% EG, 30% DMSO, 0.5 m sucrose (Suc), and 20% CS in TCM-199 for 30 s, loaded into cryotops, and immersed into liquid nitrogen. Oocytes were warmed by plunging the cryotops into 1 m Suc in TCM-199 supplemented with 20% CS for 1 min, placed in 0.5 m Suc in TCM-199 supplemented with 20% CS for 3 min, and finally in TCM-199 supplemented with 20% CS alone for 5 min. Frozen–thawed semen from a single bull (5 × 10⁶ spermatozoa mL^–1) was used for fertilization. Zygotes were vortexed to remove the cumulus cells 18 h after fertilization and cultured in CR1aa for 9 days. Data were analyzed by the chi-square test. Results are presented in Table 1. There were no differences in the survival rates of the control and vitrified oocytes. The cleavage rate of controls at both 24 and 48 h was greater (P < 0.01) than that of vitrified oocytes. Among pre-equilibration times, the cleavage rate of 0, 1, and 3 min pre-equilibrations at both 24 and 48 h was greater than with the pre-equilibration time of 5 min (P < 0.01). With respect to blastocyst development, the control oocytes showed greater development rates than the vitrified oocytes (P < 0.01), whereas the development rates were lower with the pre-equilibration time of 5 min (P < 0.01) than with the other pre-equilibration times. In conclusion, the results indicated that matured bovine oocytes could survive after vitrification and subsequently develop into blastocysts after IVF. However, the pre-equilibration time before vitrification affects cleavage and blastocyst development; a longer exposure time resulted in lower blastocyst development.

Table 1. Effect of pre-equilibration time on matured bovine oocytes survival and on subsequent embryo development