Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation.

Abstract

While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.