Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions

Experiments were conducted to find optimal conditions for obtaining high survival of expanded mouse blastocysts after vitrification. The vitrification solutions used were designated EFS20, EFS30 and EFS40, and contained 20%, 30% and 40% ethylene glycol, respectively, diluted in PB1 medium containing 30% Ficoll plus 0.5 mol sucrose l-1. In the toxicity test of the solutions and each cryoprotectant, ethylene glycol was found to be toxic to embryos. For vitrification, expanded blastocysts were exposed to the vitrification solutions at 10, 20 or 25 degrees C for various periods; they were then cooled rapidly in liquid nitrogen, after which they were warmed rapidly. When the embryos were directly exposed to EFS40 at 20 degrees C for 2 min before vitrification, 66% of them re-expanded during 48 h of post-warming culture. The re-expansion rates decreased when exposure time was shortened (0.5 min), when exposure temperature was lowered (10 degrees C), or when embryos were vitrified in EFS20 and EFS30, although these conditions should be less toxic. When embryos had been pretreated in a dilute (10-20%) ethylene glycol solution for 5 min, followed by short exposure (0.5 min) to EFS40 at 20 degrees C, post-vitrification survival rate increased to 83-84%; furthermore, the rate reached 94% when the temperature was increased to 25 degrees C. Expanded blastocysts cryopreserved by this two-step method developed into live young as well as control embryos after transfer.(ABSTRACT TRUNCATED AT 250 WORDS)