Field-applicable skin tissue cryopreservation protocols for mammalian genetic banking: Evidence from Neotropical cervids

Cryopreserved genetic resources from threatened species face substantial logistical challenges, particularly in field settings where laboratory infrastructure is limited. This study evaluated three simplified cryopreservation protocols for wildlife biobanking using threatened Neotropical cervids as experimental models. Ultra-rapid freezing (UF) with cryoprotectants (CPA) using short (s) and long (l) exposure times (30 s and 5 min, corresponding to sCPA-UF and lCPA-UF, respectively), and CPA-free UF (CPAf-UF), were compared with no cryopreservation (fresh tissue, FT). In sCPA-UF, tissues were frozen immersed in the freezing medium, whereas in lCPA-UF, excess medium was removed before ultrarapid freezing. Post-thaw assessment included cell viability analysis, histomorphological evaluation, DNA damage quantification, mitotic index determination, and functional characterization through cell culture, migration, and proliferation assays. All cryopreservation methods yielded viable and proliferating fibroblasts. lCPA-UF demonstrated the highest cell viability (often comparable to FT), while CPAf-UF and sCPA-UF protocols achieved significantly lower but functionally adequate viability (>50%). Histomorphological analysis revealed minor, non-significant structural alterations across all cryopreserved groups, with slightly increased apoptotic nuclei in CPAf-UF and sCPA-UF treatments. Despite reduced proliferation rates compared to lCPA-UF, all protocols maintained cellular adhesion, migration capacity, and proliferative potential. Mitotic indices were largely preserved. This study demonstrates that, although cryoprotectant-based ultrarapid freezing provides superior cell survival, cryoprotectant-free ultrarapid freezing represents a functionally viable alternative for wildlife tissue preservation, offering practical logistical advantages for field applications.