An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution

Summary.This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1·5, 3·0 or 4·5 m-DMSO. Rapidly cooled straws containing 4·5 m-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4·5 m-DMSO was not influenced by straw handling as 91–92% formed blastocysts in vitro, 77–78% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing <4.5 m-DMSO ice formation occurred throughout (1·5 m-DMSO), or in parts (3·0 m-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3·0 or 1·5 m-DMSO solutions was reduced both in vitroand in vivoand structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3·0 m-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4·5 m-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitroand in vivo.Keywords:dimethyl sulphoxide; rapid freezing; ice formation; handling; chromosome rearrangements; mouse; embryos