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 unfroze
n controls, (ii) frozen once and cultured, (iii) subjected to two cons
ecutive freeze-thaw cycles, (iv) frozen and thawed, and then cultured
for 18-30 h before being frozen a second time, and (v) frozen three ti
mes in succession without being cultured. To assess their functional s
urvival after freezing and thawing, all embryos were cultured in vitro
to the hatched blastocyst stage in Whitten's medium. In one experimen
t, hatched embryos that developed after one, two or three cycles of fr
eezing and thawing were stained with Hoechst 33342 to determine their
mean cell number. More embryos of the culture control group and the on
ce-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 am
ong 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 d
amage to their zonae pellucidae. Furthermore, frozen mouse embryos had
fewer cells per embryo at the time of hatching than the unfrozen embr
yos. Nevertheless, these results demonstrate that mouse embryos can su
rvive even three successive freeze-thaw cycles yet still be capable of
in-vitro development.