Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution

Hredzák, R. and Ostró, A. and Ždilová, Viera and Maraček, I. and Kačmárik, J. (2006) Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution. Acta Veterinaria Hungarica, 54 (1). pp. 117-125. ISSN 0236-6290

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Abstract

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196°C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitroculturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitrowithout freezing in a culturing medium in an environment consisting of 95% air and 5% CO₂. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitroculturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.

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