Background: Many years have been devoted to preserve fertility, but the effect of cryopreservation on gene functionality in primary, secondary and tertiary follicular stages; is still unclear. The present study was designed to assess the effect of vitrification on the histological structures and expression of follicular cells related genes. Methods: The buffalo ovarian cortical tissues were vitrified in two-steps. The sliced cortical tissues were incubated with VS1 (8.5% DMSO and 8.5% EG) for 10 min and sequentially incubated and stored in liquid nitrogen in VS2 (16.5% DMSO, 16.5% EG and 0.1M Sucrose). Morphological differences were assessed by hematoxylin and eosin staining, which indicated similar structures in both groups. Further, functionality of these follicles were evaluated by the relative gene expression of folliculogenesis-related genes; FOXO3, NLRP5, WNT4, SF1, VEGFA and HAS2. No significant difference was observed between the vitrified groups as compared to control. Moreover, toxicity in follicular cells during vitrification was rectified by MTT assay which also showed no significant difference. Conclusion: Present study can be considered as the key work that helps in filling the gaps regarding the growth of follicles after cryopreservation in buffalo species, as this imparts nonsignificant injury on follicular functionality and development.
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