Cryopreservation and transplantation of ovarian cortical tissue are novel techniques to preserve fertility in young patients undergoing gonadotoxic treatments that may affect fertility. Vitrification has demonstrated growing success in restoring ovarian function and achieving pregnancy post-grafting. It helps maintain communication between follicles and interstitial tissue, which is essential for follicular growth. This study compares two ovarian cortex dissection techniques (strips and layers) using an ovine animal model. The results indicate that manipulation of the ovarian cortex affects primordial follicle activation and stromal tissue during vitrification, potentially compromising oocyte viability and reproductive potential. Additionally, the distribution of primordial follicles in ovarian tissue varies, influencing transplantation efficiency. Both dissection methods increase follicle activation, suggesting that mechanical manipulation impacts outcomes. These findings underscore the need to optimize tissue processing to enhance fertility preservation, particularly by understanding the roles of the stromal and extracellular matrix (ECM) in maintaining follicle dormancy and viability post-vitrification and reimplantation.