Controlled-rate slow freezing as a promising strategy for juvenile feline testicular tissue preservation

Testicular tissue cryopreservation represents a promising fertility preservation strategy for prepubertal males lacking mature spermatozoa, as it allows the storage of germ-cell populations within their native seminiferous niche, potentially maintaining their spatial relationships with supporting somatic cells, which contribute to the microenvironment required for germ-cell survival and development. However, no standardized protocol is currently available for domestic cats. This study compared vitrification and controlled-rate slow freezing of testicular fragments from 17 juvenile cats, both immediately after warming/thawing and after 48 h of in vitro culture. Fresh fixed and fresh cultured fragments served as controls. Tissue preservation was evaluated by histomorphometry, histomorphology, and immunohistochemistry for MCM-7, PGP9.5, SYCP3, and activated caspase-3. Immediately after warming or thawing, both procedures maintained tubular, fibrotic, and necrotic areas comparable to fresh tissue, indicating preservation of the overall tissue compartment distribution. However, slow freezing better preserved the proportion of normal tubules and MCM-7-, PGP9.5-positive cell densities, whereas vitrification reduced these parameters. By contrast, SYCP3-positive cell density and activated caspase-3 immunoreactivity, remained comparable to fresh tissue after both cryopreservation procedures. Culture after cryopreservation revealed additional structural and cellular injuries in both groups, but vitrified tissue showed the greatest impairment, with the lowest tubular area, greatest cell detachment and pyknosis, and lowest proliferating and spermatogonial cell densities. Accordingly, the better preservation of slow-frozen tissue during subsequent culture indicates greater short-term resilience to cryopreservation-induced stress. Post-cryopreservation culture also proved useful for revealing structural and cellular alterations that were not fully apparent immediately after thawing or warming. Together, these findings support controlled-rate slow freezing as a promising strategy for feline testicular tissue banking and future in vitro applications.