Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro

Abstract

Two experiments were conducted to assess the viability of bovine blastocysts obtained by in vitro fertilization of oocytes matured in vitro (IVM-IVF) and cryo-preserved by vitrification. In Expt 1, the optimal concentrations of glycerol and 1,2-propanediol in the basic medium (modified TCM 199) for cooling and warming without formation of ice crystals were determined by plunging the solution into liquid nitrogen and then warming it in a water bath at 15°C; when both glycerol and 1,2-propanediol were present in the solution (>45% v/v), vitrification of the medium was observed. In Expt 2, IVM-IVF blastocysts were equilibrated to the mixture of glycerol and 1,2-propanediol (0% to 45%) at 15°C in a stepwise manner as follows: (i) in one step, for 18 min to the final vitrification solution; (ii) in two steps, for 8 min in the first step and 10 min in the second step; (iii) in four steps, for 4 min in the first three steps and 6 min in the last step; (iv) in eight steps, for 2 min in each step, but 4 min in the last step; and (v) in 16 steps, for 1 min in each step, but 3 min in the last step. After removal of cryoprotectants, the blastocysts were cultured for 24h in vitro. The survival rates for the embryos equilibrated in 1, 2, 4, 8 and 16 step(s) were 56, 89, 100, 100 and 100%, respectively. The blastocysts equilibrated in 1, 2, 4, 8 and 16 steps were vitrified by plunging the straws containing them into liquid N2, thawed and cultured in vitro. Higher survival rates were obtained for blastocysts equilibrated in 4, 8 and 16 steps (79, 82 and 87%, respectively) than for those in one (0%) or two (10%) steps. Ten blastocysts that survived after vitrification were transferred to ten recipients and six of these became pregnant. These results indicate that vitrification can be used for cryo-preservation of blastocysts obtained by in vitro culture of IVM-IVF bovine follicular oocytes.