Viability of vitrified mouse embryos using various cryoprotectant mixtures

Abstract

Mouse morulae and blastocysts were cryopreserved by vitrification using six types of solutions. Each solution was composed of two types of cryoprotectants, glycerol (GL) + ethylene glycol (EG), GL + propylene glycol (PG), GL + dimethyl sulfoxide (DMSO), EG + PG, EG + DMSO, and PG + DMSO at an each cryoprotectant concentration of 25% v/v. Embryos were exposed to each type of vitrification solutions, which had been diluted 50% in PBS, for 5 minutes at room temperature, then for another 5 minutes at 4 °C. The embryos were loaded into straws containing vitrification solution at 4°C and plunged into liquid nitrogen within 30 seconds. After warming in water at 0°C and following one-step dilution of the cryoprotectant in 0.5 M sucrose+PBS, the embryos were cultured in vitro. The survival rates of morulae were 51, 16, 78, 44, 79 and 50%, respectively, for the six solutions. The survival rates of the morulae using GL + DMSO and EG + DMSO were significantly higher than those of the other solutions (P<0.01). The survival rates of blastocysts were 72, 29, 55, 46, 79 and 46%, respectively, for the six solutions. The survival rates using GL + EG and EG + DMSO were significantly higher than those of the other solutions (P<0.05). A high in vitro survival rate was obtained when both morulae and blastocysts were vitrified using EG + DMSO. Therefore, embryos vitrified in EG + DMSO were transferred to recipient mice, and their development into live fetuses was investigated. The in vivo development rates of morulae and blastocysts were 34 and 49%, respectively. These values were not significantly different from those of fresh control embryos.