Title: Stepwise vitrification of in vitro produced buffalo Blastocysts
Abstract: he purpose of this study was to evaluate the
use of a stepwise vitrification as a method for
cryopreservation of in vitro-produced (IVP)
buffalo blastocysts and to compare the results
with post-thaw survival rate of buffalo
blastocysts frozen by stepwise vitrification with
those frozen by conventional vitrification (one
step method).
Selected IVP blastocysts were exposed to a
vitrification solution consisting of 40% ethylene
glycol (EG) plus 0.3 M trehalose and 20%
polyvinyl pyrrolidone (PVP) for 1 min and loaded
in 0.25 ml plastic mini straws containing 100 μl of
10% sucrose. The loaded cryostraws were
cryopreserved by either the stepwise vitrification
or one step vitrification and stored in liquid nitrogen
for one month.
After thawing and removal of cryopro-
tectants, embryos exhibiting intact zona pellucida
and uniform blastomeres were considered
suitable for in vitro culture. Of the embryos
cryopreserved by stepwise and one step
vitrification, 100 and 60%, respectively, recovered
embryos post-thawing. Similarly 95.4 and 71.1%
of embryos cryopreserved by stepwise and one
step vitrification were exhibiting good embryos
post-thawing.
Post-thaw blastocysts were serially washed
in tissue culture medium 199 (TCM-199) for 5 min
in both cases. They were then cultured in TCM-199
supplemented with 10% fetal calf serum for 24-
48 h. Development to hatched blastocyst stage
was considered the initial indicator of success of
cryopreservation of embryos. The rates of
blastocyst re-expansion and hatching of stepwise
vitrified blastocysts (66 and 55%, respectively)
were significantly higher (p<0.01) than the
corresponding values with one step method (40%
and 20%, respectively) and it was nearly similar
to that of the control group (68% and 58%,
respectively). This is the first report on stepwise
vitrification of buffalo embryos. Present results
suggest that stepwise vitrification supports better
in vitro survival of frozen thawed buffalo embryos.