A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability

Abstract

Mouse morulae were exposed to solutions containing 30-50% of permeable agents (ethylene glycol, glycerol, propylene glycol) in modified phosphate-buffered saline (PB1 medium) at 20.degree. C for 20 min. A high percentage of them developed to expanded blastocysts in culture, after exposure to 30% and 40% ethylene glycol (98 and 84%, respectively), or 30% glycerol (88%). Ethylene glycol and glycerol were diluted to 30 and 40% with PB1 medium or with PB1 containing 30% Ficoll or 30% Ficoll+0.5 M-sucrose, immersed in liquid nitrogen in straws and warmed in 20.degree. C water. Solutions containing 40% of a permeable agent with Ficoll did not crystallize during cooling or warming. Mouse morulae were exposed to 40% ethylene glycol in PB1 medium containing 30% Ficoll (EF) or PB1 medium+30% Ficoll+0.5 M-sucrose (EFS) for 5-20 min at 20.degree. C. EFS solution was non-toxic to the embryos during 5 min of exposure. When embryos, equilibrated in EFS solution for 2 or 5 min at 20.degree. C, were vitrified at -196.degree. C and were warmed rapidly, nearly all embryos developed in culture (97-98%), and 51% developed to live young at term after transfer. This method, which results in virtually no decrease in embryonic viability, may be of practical use for embryo preservation.