29 THE NOVEL CRYOPROTECTIVE AGENT CARBOXYLATED ε-POLY-L-LYSINE IS EFFECTIVE FOR VITRIFICATION OF PRE-IMPLANTATION MOUSE EMBRYOS AT THE DIFFERENT STAGES

Abstract

Embryo cryopreservation is one of the important technologies in various research and clinical areas. Very recently, carboxylated ε-poly-L-lysine (COOH-PLL) has been developed as a new cryoprotective regent, and we previously demonstrated that combined treatment of COOH-PLL with ethylene glycol (EG), which is a commonly used cryoprotective regent, is effective for vitrification of unfertilized mouse oocytes and pronuclear embryos. In this study, we examined different concentration of COOH-PLL and tried to vitrify the embryos at the different stages beyond the 2-cell stage using the same protocol. Embryos at the pronuclear, 2-cell, 4-cell, 8-cell, morulae, and blastocyst stages were treated with 7.5% (vol/vol) EG and 7.5% (vol/vol) COOH-PLL for 3 min and then vitrified with 15% EG (vol/vol) and 15% (vol/vol) COOH-PLL (E15P15) for 1 min by the Cryotop. The embryos except for pronuclear embryos showed low rates of survival, blastocyst, and hatched blastocysts. To improve the survival and developmental ability of the vitrified embryos, blastocysts were vitrified with different concentrations of COOH-PLL. At the results, the survival and developmental rates of vitrified blastocyst in E20P10 group were higher (98.0 ± 2.5% and 93.3 ± 4.0%) than those of the E30, E25P5, and E15P15 groups (P < 0.05). When 2-cell, 4-cell, and 8-cell embryos or morulae were vitrified with E20P10, survival, blastocyst, and hatched blastocyst rates were dramatically improved (more than 86.0%). Taken together, vitrification with 20% COOH-PLL and 10% EG is available for vitrification of mouse pre-implantation embryos at the different stages.