Tissue Preservation, Life care and Biobanking (B2B & Networking)

Cryopreservation is an important tool for conservation of biological materials. This study reports effects of vitrification on the morphology of ovine immature cumulus oocytes complexes, and mRNA content to observe the expression of key apoptotic genes, BAD, BAK, BAX, BID, BOK, BCL2A1, BCL2, MCL1, P53, and GAPDH on immature oocytes. For morphological evaluation using Zoom stereo-microscope, 853 oocytes were divided into 3 groups for vitrification using 40% Glycerol or 40% DMSO or 20% Glycerol + 20% DMSO. After thawing, a total of 819 oocytes were recovered, resulting in a loss of 34 oocytes during handling. The morphological examination of the oocytes revealed that the percentage of oocytes recovered in a morphologically normal state was minimum 84.2±2.3% for glycerol 40% and maximum 94.09±1.1% for 20% Glycerol + 20% DMSO. The proportion of oocytes recovered in a morphologically normal state was significantly lower (P<0.01) for 40% Glycerol (84.2±2.3%) compared to that for these groups. For scanning electron, microscopic studies 451 oocytes were used in this experiment for vitrification using a combination of 20% Glycerol + 20% DMSO and percentage of oocytes recovered in a morphologically normal state was 95.7±2.07 recorded. The real-time PCR results for the comparative expression of the apoptotic gene using mRNA abundance showed the down-regulation BAD, BCL2A1 and MCL1. The expression of BCL2, BAX and BOK are similar in both the groups. However, BID and BAK were up-regulated. In addition, P53 was also downregulated proves vitrification safe for the preservation of immature oocytes in ovine.