3. Vitrification of Bovine Blastocysts Obtained by In Vitro Culture of Oocytes Matured and Fertilized In Vitro(Papers presented at the 38th Annual Meeting)

Abstract

Three experiments were conducted to assess the viability of bovine blastocysts by vitrification procedures which were obtained by in vitro fertilization of oocytes matured in vitro. In experiment 1, the optimal concentrations of glycerol and 1, 2-propanediol as the basic medium (modified 199 supplemented with 20% calf serum) for producing vitrification were determined. When both glycerol and 1, 2-propanediol were present in the solution (> 45% v/v in total), vitrification of the solution was achieved. In experiment 2, blastocysts were equilibrated to the vitrification solution in step wise manners from one to 16 steps using exposure to gradually concentrated solutions. Then blastocysts were vitrified by plunging the straws containing them into liquid nitrogen, thawed and cultured. Higher survial rates were obtained for blastocysts equilibrated in 4, 8 and 16 steps (79%, 82% and 87%, respectively) than for those in 1 step (0%) and 2 steps(10%). Inexperiment 3, freeze-replica observations were carried out on blastocysts vitrified by the 16 step method. The freeze-replicas showed an absence of ice crystals throughout the cytoplasm, cavity of blastocyst and extracellular areas. In addition, little ultrastructure change in the plasma membrane of the blastocysts was observed. It was suggested that successful cryopreservation of blastocysts using the 16 step equilibration was established not only by the complete vitrification of whole specimens but also by minimization of the plasma membrane ultrastructural changes.