In Vitro Viability of Split Rabbit Embryos before and after Vitrification

Of 125 embryos (category I and II) recovered from 18 does, 25 were maintained intact as control, 25 bisected using a simplified splitting protocol, 25 frozen thawed according to a vitrification method, 25 bisected-vitrified, and 25 vitrified-bisected. The survivability of intact embryos and demiembryos were evaluated after 24 hours of in vitro culture by development ability, morphological assessment, and cell count with double staining method. The development rate after in vitro culture of intact control embryos (96%) was higher (P<0.001) than vitrified embryos (36%). The development rate of demiembryos was higher (74%) than bisected-vitrified embryos (34%), and vitrified-bisected embryos (10%) as well (P<0.001). A higher number of cells and percentage of surviving cells was observed in control embryos (91%), followed by fresh demiembrios (84%), vitrified embryos (74%) (P<0.01), bisected-vitrified demiembryos (67%), and vitrified-bisected demiembryos (61%). Embryos and demiembryos originated from category I embryos showed higher development rate than those obtained from category II. The reduced survivability for the vitrified-bisected embryos might be due to the combined effects of loss of cohesion and reduction of cells as a result of the bisection and to the crioprotectant toxicity during hydratation. In conclusion, bisection and vitrification can be used together without micromanipulation and freezing complex systems, but the embryo quality is the main factor in the success of these procedures.