Human Reproduction, Vol 12, 310-316, Copyright © 1997 by Oxford University Press
NJ Vitale, MW Myers, RS Denniston, SP Leibo and RA Godke
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.
ARTICLES
In-vitro development of refrozen mouse embryos
Department of Animal Science, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803, USA.
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