A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate.

Survival of mouse 2-cell embryos was evaluated after exposure to 1.38, 2.75 or 5.5 M single cryoprotectants [dimethylsulphoxide (DMSO), acetamide (Ac) and propylene glycol (PG)], components frequently utilized as a vitrification solution, for 0.5, 1, 2 and 10 minutes at room temperature prior to vitrification. More than 80 % of the treated embryos developed to normal blastocysts in culture, after exposure to 1.38-2.75 M of each reagent for 0.5 minutes, although Ac tended to provide with have a deleterious effect on their survival. When the embryos were vitrified with solutions containing DP (2.75 M DMSO and 2.75 M PG) plus 0, 0.5 and 1.0 M Ac after a 0.5-minute exposure, their in vitro survival rates to the blastocysts were 44, 41 and 37%, respectively, showing no significant difference among them (x(2)=0.1-0.6, P>0.05). This indicates that the presence of Ac is not always needed for vitrifying mouse 2-cell embryos. Embryos, that had been vitrified with DP solution supplemented with 1.0 M sucrose (DPS) after a 0.5- minute exposure, exhibited significantly higher in vitro survival rate (82%) than those vitrified with DP (44%) (x(2)=23.4, P<0.001). Similar high survival rate (81%) was obtained when they were vitrified with DP plus 0.16 M raffinose (DPR) (x(2)=28.3, P<0.001). In vivo survival of embryos vitrified with DPS or DPR after a 0.5-minute exposure was both 49%, and there was no significant difference comparing to the unvitrified control group (60%). This method is rapid, efficient and reliable, and thus may be of practical use for cryopreserving mouse 2-cell embryos.