A comparative analysis of vitrification and two slow freezing methods for gonocyte-containing neonatal calf testicular tissue and subsequent in vitro culture

The cryopreservation of neonatal testicular tissue containing gonocytes is crucial for preserving genetic diversity, advancing research, and developing reproductive technologies. In this study, we investigated three cryopreservation techniques, slow freezing (in which the rate of freezing was controlled or uncontrolled) and vitrification, using neonatal bovine testicular tissues containing gonocytes, followed by in vitro culture to evaluate cell functionality. Vitrification resulted in a significantly lower proportion (19.15 ± 1.82%) of seminiferous tubules with > 70% attachment to the basement membrane in comparison to both the controlled slow freezing group (47.89 ± 10.98%) and the uncontrolled slow freezing group (39.05 ± 4.15%) (<i>P</i> < 0.05). No significant differences were observed in the proportion of seminiferous tubules containing PGP9.5-positive germ cells when compared between the three methods. Comparable densities of germ cells per unit area were observed in the controlled/uncontrolled slow freezing groups and the vitrification group (7.89 ± 1.83, 7.75 ± 1.75, and 7.92 ± 1.23/104 µm², respectively). In addition, the proportions of Sertoli cells (vimentin-positive) and proliferating cells (Ki67-positive) were similar across the three cryopreservation methods. There were no significant differences in cell membrane integrity and the expression of selected genes when compared between the three cryopreservation groups. Compared to fresh tissue, the uncontrolled slow freezing groups exhibited significantly higher levels of apoptosis (<i>P</i> < 0.05); there was no significant change in the controlled slow freezing and vitrification group. Notably, all in vitro cultures of testicular cells, from both fresh and freeze/thawed tissues, displayed the formation of germ cell colonies. Our data demonstrate that vitrification effectively preserves neonatal bovine testicular tissues containing gonocytes, safeguarding cell membrane integrity, promoting proliferation, and protecting against apoptosis. Collectively, these findings propose vitrification as a promising alternative cryopreservation method for immature testicular tissue (ITT) in clinical applications.