ABSTRACT
To evaluate the effects of sequential, repetitive freezing on their in-vitro development, mouse embryos at the eight- to 16-cell stage were subjected to one of five treatments. They were (i) cultured as unfrozen controls, (ii) frozen once and cultured, (iii) subjected to two consecutive freeze-thaw cycles, (iv) frozen and thawed, and then cultured for 18-30 h before being frozen a second time, and (v) frozen three times in succession without being cultured. To assess their functional survival after freezing and thawing, all embryos were cultured in vitro to the hatched blastocyst stage in Whitten's medium. In one experiment, hatched embryos that developed after one, two or three cycles of freezing and thawing were stained with Hoechst 33342 to determine their mean cell number. More embryos of the culture control group and the once-frozen group developed into hatching blastocysts than those of the refrozen groups. There was no difference in the second post-thaw rate of in-vitro development for embryos refrozen with the culture-refreeze or direct-refreeze procedure. Furthermore, there was no difference among in-vitro development rates for embryos frozen two or three times. However, among those embryos subjected to repeated cycles of freezing and thawing that did not survive, there was a considerable amount of damage to their zonae pellucidae. Furthermore, frozen mouse embryos had fewer cells per embryo at the time of hatching than the unfrozen embryos. Nevertheless, these results demonstrate that mouse embryos can survive even three successive freeze-thaw cycles yet still be capable of in-vitro development.
