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

## TL;DR

This study compares cryopreservation methods for bovine ovarian tissue, finding that slow freezing of small tissue fragments best preserves follicle quality and potential for future use.

## Contribution

The study identifies slow freezing of small tissue fragments as a superior method for preserving bovine ovarian tissue quality.

## Key findings

- Slow freezing preserved follicle morphology better than vitrification, especially in smaller tissue fragments.
- Vitrification performance varied, with one method significantly reducing morphological integrity.
- Granulosa cell proliferation was maintained across cryopreservation methods, indicating preserved developmental potential.

## Abstract

Cryopreservation of ovarian cortical tissue is a promising tool to preserve the reproductive potential of domestic animals and safeguard genetic resources. This tissue contains a large population of immature preantral follicles that could be used in future reproductive or biotechnological applications. However, the efficiency of current cryopreservation procedures varies widely and is influenced by both the protocol used and the physical characteristics of the tissue fragments. In this study, we compared slow freezing and two vitrification methods using bovine ovarian cortex fragments of two different sizes (small—1 × 10 × 5 mm—and large—1 × 10 × 10 mm—). Our aim was to assess how protocol and tissue dimension affect follicular morphology and granulosa cell proliferation, two indicators of tissue quality after cryopreservation. We found that slow freezing preserved follicle morphology more effectively than vitrification, particularly in smaller tissue fragments. Vitrification performance differed markedly between protocols, with one method substantially reducing morphological integrity. Proliferation activity was generally maintained across treatments, indicating that morphologically normal follicles retain developmental potential after cryopreservation. These results highlight the importance of considering fragment size together with protocol when optimizing bovine ovarian tissue cryopreservation, and support slow freezing of small pieces as an efficient approach for preserving bovine ovarian tissue.

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.