DESIGN OF VITRIFICATION SOLUTIONS FOR THE CRYOPRESERVATION OF EMBRYOS

A series of experiments was performed to determine the concentrations at which ten cryoprotectants singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous when warmed by plunging into a water bath at 25 degrees C. From these tests eight solutions (VS) were selected for testing of toxicity to mouse morulae in vitro. One of these (VSI) was modified as a further five VS of which one (VS11) was tested for toxicity to all stages of mouse embryos and to sheep compacted morulae. The concentrations at which the cryoprotectants vitrified on cooling were: butylene glycol, 3.0 mol l(-1); propylene glycol, 4.0 mol l(-1); dimethyl sulfoxide (DMSO) and glycerol 5.0 mol l(-1); ethylene glycol, 6.5 mol l(-1) None of these, at the highest concentration tested, remained vitreous during warming. Methanol and the high molecular weight polymers, dextran, Ficoll, polyethylene glycol and polyvinylpyrrolidone, did not vitrify at the concentrations tested. Toxicity studies showed the order of increasing toxicity to be ethylene glycol, methanol, DMSO, glycerol, propylene glycol and butylene glycol. Of the mixtures composed of two cryoprotectants, those containing ethylene glycol and glycerol were the least toxic at vitrifying concentrations. VS11 (6.0 mol ethylene glycol l(-1) and 1.8 mol glycerol l(-1)) was well tolerated by mouse morulae, less well by eight- and one-cell embryos and poorly by two-cell embryos. Dilution of the VS11 from mouse embryos by exposure to 1.0 mol sucrose l(-1) for to min did not enhance their survival. VS14 (5.5 mol ethylene glycol l(-1) and 1.0 mol sucrose l(-1)) was a good vitrifying mixture and was non-toxic to mouse embryos when they were exposed for up to 30 min. The survival of sheep compacted morulae in vitro was not affected by exposure to VS11 for up to 20 min when dilution of the VS11 was done by exposure to 1.0 mol sucrose l(-1) for 10 min. Without sucrose dilution, exposure to VS11 for 10 min was detrimental to embryo survival.