CRYOPRESERVATION OF IMMATURE BUFFALO OOCYTES BY VITRIFICATION

Abstract

Cryopreservation of collected oocytes from slaughtered animals of high genetic value, for production of embryos may provide an opportunity to replenish the valuable germplasm lost. The aim of this study was to cryopreserve the immature buffalo oocytes by vitrification. Experiments were conducted to study the effect of using different ethylene glycol concentrations (10, 20 and 40%) in the equilibration solution, equilibration periods (3, 5 and 10 min) and vitrification solutions (ethylene glycol with 0.5 M sucrose, 0.3 M trehalose and 20% dimethyl sulfoxide) with a non vitrified group served as control on morphological survival, in vitro maturation and embryo development of vitrified-warmed immature buffalo oocytes. The selected cumulus oocyte complexes with compacted cumulus cells and evenly granulated ooplasm were vitrified. The present results revealed that, equilibration solution and equilibration time significantly (P < 0.01) decreased the proportion of morphologically normal oocytes. Using sucrose in the vitrification solution, an equilibration time of 5 min and an equilibration solution with 20% ethylene glycol (EG) yielded the highest proportion of morphologically normal oocytes (93.73%) and the lowest proportion of morphologically abnormal oocytes (6.11%). Moreover, vitrification of buffalo oocytes with different vitrification solutions, sucrose, trehalose or DMSO, significantly (P < 0.5) decreased the in vitro maturation rate (47.27, 26.92 and 42.03%, respectively), in vitro fertilization rate (30.36, 13.21 and 23.08%, respectively), cleavage rate (23.91, 6.67 and 21.43%, respectively) and morula development (4.35, 0.00 and 2.38%, respectively) compared to the control treatment (78.67, 58.73, 44.64 and 19.64%, respectively). The current results also showed that, all the oocytes that vitrified in different vitrification solutions failed to develop to the blastocyst stage compared to the control treatment (10.71%). Therefore, from the current results we can conclude that immature buffalo oocytes could be frozen by vitrification technique. Acceptable vitrification protocol of immature buffalo oocytes was observed when immature buffalo oocytes were vitrified using 20% EG in the equilibration solution, an equilibration time of 5 min, and a vitrification solution containing 20% EG and 0.5 M sucrose. However, further studies are needed to improve the in vitro embryo development of the vitrified buffalo oocytes.