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Human Reproduction, Vol. 10, No. 2, pp. 396-402, 1995
© 1995 European Society of Human Reproduction and Embryology

research-article

Fertilization and early embryology: Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow-cooled mouse pronuclear and 4-cell embryos

J.M. Shaw1,2, C. Ward3 and A.O. Trounson1

1Centre for Early Human Development, Institute for Reproduction and Development, Monash University 268 Clayton Road, Clayton, Victoria 3168, Australia 3C/- Prof. N.Hallam, Department of Ecology and Evolutionary Ecology, Monash Medical Centre, Monash University 268 Clayton Road, Clayton, Victoria 3168, Australia

Correspondence: 2To whom correspondence should be addressed

Mouse pronuclear and 4-cell embryos were cryopreserved by slow cooling to –33°C in 1.5 M 1, 2-propanediol or 1.5 M ethylene glycol, with or without 0.1 M sucrose. Straws were thawed by immersion into a 37°C water bath, immediately after their removal from liquid nitrogen (protocol 1), or after being held in air for 15 (protocol 2) or 30 s (protocol 3). Others were held in air until the ice melted (protocol 4). Embryos which formed blastocysts that hatched and attached to the Petri dish in vitro (plated) were considered viable. The thawing protocol did not significantly influence the viability of embryos frozen in propanediol with 0.1 M sucrose (52–72% of pronuclear and 69–97% of 4-cell embryos plated). In the other solutions tested, propanediol without sucrose and ethylene glycol with/without sucrose, only protocol 2 resulted in uniformly high development of both pronuclear (45–65% plating) and 4-cell embryos (70–97% plating). Thawing protocol 4 significantly reduced development, in particular for embryos frozen in ethylene glycol (0% 1-cell; 0–25% 4-cell plating). The difference between thawing protocols 2 and 4 was reduced by continuing slow cooling of ethylene glycol solutions to lower temperatures (–41°C). Adding antifreeze proteins type I or III did not improve survival or development. Thus, although mouse pronuclear and 4-cell embryos can be frozen-thawed in either ethylene glycol or propanediol without significant loss of viability, an appropriate thawing protocol is essential for embryos frozen in ethylene glycol or propanediol-sucrose.

Key words: antifreeze protein/cryoprotectant/embryo/glycols/warming rate


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