OSMOTIC AND CRYOPROTECTIVE EFFECTS OF A MIXTURE OF DMSO AND ETHYLENE-GLYCOL ON RABBIT MORULAE

Comparisons were made of the osmotic and cryoprotective effects on rab bit embryos preserved by vitrification with 2 solutions and by convent ional freezing. Embryos obtained from rabbits killed 70 to 72 h after mating were used in the study (n = 948). Initially, toxicity of the 3 cryoprotectants was studied in fresh (unfrozen) embryos (n = 135). Sub sequently, embryos placed in ethylene glycol (EG, 40% v/v; n = 88) and ethylene glycol with dimethyl sulfoxide (EG+DMSO, 20% v/v each, respe ctively; n = 344) were loaded into straws and plunged directly into li quid nitrogen. Embryos placed in 1.5 M DMSO and 20% heat inactivated r abbit serum were subjected to conventional freezing in a programmable freezer (control group, n = 363). The osmotic effect was estimated by measuring the changes in the embryonic and interzonal volume (crossect ional area) and in the thickness of the mucin coat (n 18). Cryoprotect ive effectivity was determined by development to the blastocyst stage in vitro, or birth of normal pups after transfer into synchronized rec ipients. Osmotic effects of cryoprotective solutions on embryonic and interzonal volume and mucin coat thickness were variable and overall n ot significant. Survival rate of cryopreserved embryos in vitro and de velopment to blastocysts, was worst in the EG-treated embryos. Surviva l rate at birth was higher in vitrified vs frozen embryos. We conclude that rabbit morulae can be vitrified successfully in EG+DMSO medium.