Cryopreservation of zona-hatched mouse blastocysts

Experiments were conducted to determine the conditions for successful and efficient cryopreservation of hatched mouse blastocysts, using simple vitrification procedures. Hatched blastocysts were obtained by culture of morulae in vitro. Vitrification solutions used were EFS40 and GFS40, which were 40% (v/v) ethylene glycol and 40% (v/v) glycerol, respectively, diluted in PB1 medium containing 30% Ficoll (w/v) and 0.5 mol sucrose l-1. In the one-step method, embryos were directly exposed to the vitrification solutions at 25 degrees C for 0.5 or 2 min; in the two-step method, embryos were equilibrated with a dilute (10-20%, v/v) ethylene glycol or glycerol solution for 5-10 min, before a 0.5 min exposure to EFS40 or GFS40, respectively. They were then vitrified in liquid nitrogen. When the embryos were vitrified in EFS40, the post-warming survival rates, assessed by the re-expansion of the blastocoel during 16 h of culture, were higher in embryos that had hatched from the zona earlier (120-132 h after hCG) than in those hatched later (142-150 h after hCG); however, the highest survival rate was only 65%, which was obtained by a one-step method. When embryos were vitrified in GFS40, a high survival rate (89-94%) was obtained especially by the two-step methods. Vitrified blastocysts developed into live young just as well as did fresh blastocysts; survival was highest after transfer to recipients on day 3 or day 4 of pseudopregnancy. These findings show that hatched mouse blastocysts can be successfully cryopreserved by a simple vitrification method, and that a glycerol-based vitrification solution is more suitable than the corresponding ethylene glycol-based solution for the vitrification, probably because slower permeation of glycerol avoids toxic injury.