Influence of cryopreservation techniques and low concentrations of permeating cryoprotectants on the conservation of ear cartilage and skin derived from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758)

Abstract

Considering the importance of efficiently obtaining somatic resource banks as an ex-situ conservation strategy for wild mammals, we evaluated two techniques (slow freezing - SF and solid-surface vitrification - SSV) for the cryopreservation of ear cartilage and skin from six-banded armadillos. Additionally, we analyzed the effects of two combinations of intracellular cryoprotectants (3.0 M or 6.0 M ethylene glycol - EG and dimethyl sulfoxide - Me2SO) on SSV. Tissues not subjected to cryopreservation were used as controls. All samples were evaluated for morphological analysis and cell ability during culture. The thickness of the basal layer was similar to the control only for tissues derived from SF, while SSV ensured the preservation of the cartilage thickness. Moreover, fragments derived from SF and SSV, especially in the 3.0 M EG-Me2SO group, resulted in dermis thickness and total skin similar to the control. All cryopreserved techniques maintained normal patterns of the fibroblasts, epidermal cells, and melanocytes. While only SF-derived fragments maintained the number of degenerated chondrocytes similar to the control, no difference was observed between groups for normal chondrocytes and lacunae. Moreover, SSV maintained the collagen fibers percentage of the tissues even after warming. After culture, SF and SSV techniques were efficient for the recovery of the somatic cells in all parameters evaluated, except the day of subconfluence, which was greater for the SSV group with 6.0 M EG-Me2SO. In summary, SSV, especially with 3 M EG-Me2SO, was as efficient as SF in preserving ear skin and cartilage derived from six-banded armadillos.