Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods.

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl–pyrrolidone and 0.4% trehalose and they were warmed in a 0.3 M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P < 0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P < 0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified–warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.