Improvement of vitrification of in vitro produced buffalo embryos with special reference to sex ratio following vitrification.

Cryopreservation and sexing of embryos are integrated into commercial embryo transfer technologies. To improve the effectiveness of vitrification of in vitro produced buffalo embryos, two experiments were conducted. The first evaluated the effect of exposure time (2 and 3 min) and developmental stage (morula and blastocysts) on the viability and development of vitrified buffalo embryos. Morphologically normal embryos and survival rates (re-expansion) significantly increased when vitrified morulae were exposed for 2 min compared to 3 min (P<0.001). On the other hand, morphologically normal and survival rates of blastocysts significantly increased when exposed for 3 min compared to 2 min (P<0.001). However, there were no significant differences between the two developmental stages (morulae and blastocystes) in the percentages of morphologically normal embryos and re-expansion rates after a 24 h culture. The second experiment aimed to evaluate the effect of viability on the sex ratio of buffalo embryos after vitrification and whether male and female embryos survived vitrification differently. A total number of 61 blastocysts were vitrified for 3 min with the same cryoprotectant as experiment 1. Higher percentages of males were recorded for live as compared to dead embryos; however, this difference was not significant. In conclusion, the post-thaw survival and development of in vitro produced morulae and blastocysts were found to be affected by exposure time rather than developmental stage. Survivability had no significant effect on the sex ratio of vitrified blastocysts; nevertheless, the number of surviving males was higher than dead male embryos.