Effect of warming rate on mouse embryos frozen and thawed in glycerol

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Abstract
Summary. Mouse embryos (8-cell) fully equilibrated in 1·5 M-glycerol were cooled slowly (0·5°C/min) to temperatures between − 7·5 and − 80°C before rapid cooling and storage in liquid nitrogen (−196°C). Some embryos survived rapid warming (~500°C/min) irrespective of the temperature at which slow cooling was terminated. However, the highest levels of survival of rapidly warmed embryos were observed when slow cooling was terminated between − 25 and − 80°C (74–86%). In contrast, high survival (75–86%) was obtained after slow warming ( ~ 2°C/min) only when slow cooling was continued to − 55°C or below before transfer into liquid N2. Injury to embryos cooled slowly to − 30°C and then rapidly to −196°C occurred only when slow warming (~2°C/min) was continued to − 60°C or above. Parallel cryomicroscopical observations indicated that embryos became dehydrated during slow cooling to − 30°C and did not freeze intracellularly during subsequent rapid cooling ( ~ 250°C/min) to − 150°C. During slow warming (2°C/min), however, intracellular ice appeared at a temperature between −70 and − 65°C and melted when warming was continued to − 30°C. Intracellular freezing was not observed during rapid warming (250°C/min) or during slow warming when slow cooling had been continued to − 65°C. These results indicate that glycerol provides superior or equal protection when compared to dimethyl sulphoxide against the deleterious effects of freezing and thawing.

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