Approaching Standardization of Bovine Ovarian Cortex Cryopreservation: Impact of Cryopreservation Protocols and Tissue Size on Preantral Follicle Population

Cryopreservation of bovine ovarian cortical tissue offers a promising strategy for preserving female fertility and genetic resources, yet outcomes remain variable and influenced by both protocol and tissue size. This study investigated how slow freezing-thawing (SFT) and two vitrification-warming procedures (VW1 and VW2) affect preantral follicle morphology and granulosa cell proliferation in bovine ovarian cortex fragments of two dimensions (1 × 10 × 5 mm and 1 × 10 × 10 mm). Tissue from six cows was processed for histological evaluation and Ki67 immunostaining. Small fragments subjected to SFT showed no significant reduction in the proportion of morphologically normal follicles compared with fresh controls, representing the best overall preservation. In contrast, vitrification decreased morphological integrity, with VW2 performing better than VW1 in both fragment sizes. Small SFT pieces contained more morphologically normal follicles than large ones. Granulosa cell proliferation capacity was largely maintained across cryopreservation protocols, increasing with follicular stage; a size-related difference only appeared on VW2, where small fragments displayed higher Ki67 positivity. These findings underscore the relevance of jointly evaluating cryopreservation protocol and fragment size to optimize bovine ovarian tissue preservation, strengthening the evidence supporting SFT of small fragments as a robust option for safeguarding cortical integrity and improving tissue-based fertility preservation strategies.