Testicular cryopreservation of prepubertal and adult Morada Nova rams: Slow freezing versus vitrification

Cryopreservation of testis is an alternative for preserving the reproductive potential of prepubertal animals that do not yet produce spermatozoa or endangered adult animals that die unexpectedly. This study aimed to evaluate slow freezing (SFR) and vitrification (VIT) for testicular tissue cryopreservation in prepubertal and adult Morada Nova rams. Small testicular fragments (3 ×3 ×1 mm) were maintained fresh (CTR) or cryopreserved using Mr. Frosty™ for SFR or the ovarian tissue cryosystem for VIT. Fragments were evaluated for histomorphology, ultrastructure, protein (β-catenin, activated caspase-3), and gene expression (OCT4, β-CATENIN, BAX, BCL2), and proteomic profiles. Morphologically, SFR better preserved seminiferous tubule integrity than VIT, especially in prepubertal animals. Ultrastructural analysis confirmed superior cell preservation with SFR. Immunofluorescence revealed higher β-catenin expression in SFR compared to VIT (p < 0.05), and increased caspase-3 activation post-VIT in adults (p < 0.05). OCT4 gene expression was upregulated after cryopreservation (p < 0.05), suggesting a stress response. Proteomic analysis detected 288 proteins, with distinct expression patterns in prepubertal tissue. PGK2 and EEF1A1 were downregulated, while HNRNPA2B1 was upregulated in cryopreserved groups, especially in VIT, suggesting metabolic suppression and RNA regulation responses. In contrast, adult tissues showed no significant proteomic changes across groups. These results support SFR as the preferable method, particularly for prepubertal testes, due to better structural preservation and more adaptive molecular responses to cryoinjury.