In-vitro development of refrozen mouse embryos

To evaluate the effects of sequential, repetitive freezing on their in-vitro development, mouse embryos at the eight- to 16-cell stage weresubjected to one of five treatments. They were (i) cultured as unfrozencontrols, (ii) frozen once and cultured, (iii) subjected to two consecutivefreeze-thaw cycles, (iv) frozen and thawed, and then cultured for 18-30 hbefore being frozen a second time, and (v) frozen three times in successionwithout being cultured. To assess their functional survival after freezingand thawing, all embryos were cultured in vitro to the hatched blastocyststage in Whitten's medium. In one experiment, hatched embryos thatdeveloped after one, two or three cycles of freezing and thawing werestained with Hoechst 33342 to determine their mean cell number. Moreembryos of the culture control group and the once-frozen group developedinto hatching blastocysts than those of the refrozen groups. There was nodifference in the second post-thaw rate of in-vitro development for embryosrefrozen with the culture-refreeze or direct-refreeze procedure.Furthermore, there was no difference among in-vitro development rates forembryos frozen two or three times. However, among those embryos subjectedto repeated cycles of freezing and thawing that did not survive, there wasa considerable amount of damage to their zonae pellucidae. Furthermore,frozen mouse embryos had fewer cells per embryo at the time of hatchingthan the unfrozen embryos. Nevertheless, these results demonstrate thatmouse embryos can survive even three successive freeze-thaw cycles yetstill be capable of in-vitro development.