Stepwise vitrification of in vitro produced buffalo Blastocysts

The 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 &#956;l 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 cryoprotectants, 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 2448 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.