Effect of Vitrification by Straw and Cryotop on DNA Integrity Using Comet Assay with Reference To Brilliant Cresyl Blue Exposure in Buffalo Oocytes

To improve the effectiveness of vitrification procedure of buffalo oocytes, two experiments were conducted. The first experiment evaluated the effect of cryodevices on DNA integrity of vitrified, matured buffalo oocytes. The in vitro matured oocytes were divided into two groups, the first was vitrified using conventional French straws, while the other was vitrified using Cryotops. DNA fragmentation level was evaluated by means of comet assay. There was a significant increase in the incidence of DNA damage after vitrification with both methods compared to control. The percentages of DNA damage of vitrified thawed buffalo oocytes were significantly higher in straws than Cryotops. The second experiment evaluated the effectiveness of application of the brilliant cresyl blue (BCB) stain on the selection of immature buffalo oocytes quality. Oocytes were classified according to their cytoplasmic coloration: blue coloration (BCB+) and colorless (BCB-). Both types were matured then vitrified and compared with non stained mature oocytes vitrified by straw methods. Significantly higher DNA damage was noted in both BCB+ (P 0.05) and BCB- (P 0.001) oocytes, compared to non-stained vitrified oocytes. The percentages of damage were significantly higher (P 0.05) in BCB- than BCB+ oocytes. In conclusion, DNA is a target of cryoinjury in vitrified buffalo oocytes and the damage decreased when using Cryotop than straw. Immature oocytes subjected to brilliant cresyl blue staining show a tendency towards DNA damage.