Optimization of the Procedures for the Vitrification of Expanded Mouse Blastocysts in Glycerol-Based Solutions.

Abstract

Experiments were conducted to find optimal conditions for obtaining high survival of expanded mouse blastocysts after vitrification in glycerol-based solutions. The solutions were GFS20, GFS30, GFS40 and GFS50, which contained 20%, 30%, 40% and 50% glycerol, respectively, diluted in PB1 medium containing 30% Ficoll + 0.5 M sucrose. The toxicity tests of the solutions and glycerol showed that longer exposure to a higher concentration of glycerol is more injurious to the embryos. For vitrification, expanded blastocysts were exposed to the GFS solution at 20 or 25 C for various periods; they were then vitrified in liquid nitrogen, and were warmed rapidly. When the embryos were directly exposed to GFS40 for 2 min at 25 C, 68% of them re-expanded during 48 h of post-warming culture. The re-expansion rates decreased, when exposure time was shortened or extended, when the exposure temperature was lowered (20 C), or when embryos were vitrified in GFS20, GFS30, and GFS50. When embryos had been pretreated in a dilute (10-20%) ethylene glycol solution for 5 min, followed by short exposure (0.5 min) to GFS40 at 25 C, the post-vitrifica-tion survival rate increased to 82-87%; furthermore, the rate reached 92% when GFS40 was re-placed by GFS50. Expanded blastocysts cryopreserved in GFS50 had the ability to develop into live young. The findings showed that glycerol-based solutions are as effective as ethylene glycol-based solutions, although the optimal solution needs a higher concentration of glycerol, probably because glycerol permeates the embryos more slowly than ethylene glycol.