Isolation of somatic cell derived from ear tissue of collared peccary (Pecari tajacu linnaeus, 1758) submitted to different vitrification techniques

The cryopreservation of tissue samples from wildlife species, especially collared peccary (Pecari tajacu), is an interesting step in obtainment and conservation of somatic cells for use in nuclear transfer (cloning). However, tissue vitrification protocols need to be optimized to ensure maximum preservation of the viable characteristics of cells. Therefore, the aim of this study was to compare by in vitro culture (IVC) two ear tissue cryopreservation techniques [conventional vitrification directly, CVD, as Silvestre et al. (2004, Theriogenology 49, 221–229) or solid-surface vitrification, SSV, according to Carvalho et al. (2011, Theriogenology 76, 933–941)]. Thus, ear fragments (9.0 mm3 ) of eight collared peccaries (4 males and 4 females) with 7-12 months old, from Center Multiplication of Wild Animals (CEMAS/UFERSA), were collected and cryopreserved in solution containing Dulbecco Modified Eagles Medium (DMEM) plus 3.0 M dimethylsulfoxide, 3.0 M ethylene glycol, 0.25 M sucrose and 10% fetal bovine serum. After two weeks, fragments warmed and not cryopreserved (control) were cultured in vitro and assessed for cell morphology, adhesion, early subconfluence (70% of the plate covered by cells) and cell viability by trypan blue (in %). All data from eight animals were analyzed by ANOVA followed by the appropriate post-hoc test. After collection of tissue samples, a total of 96 fragments were distributed for the three experimental groups (CVD: 32; SSV: 32 and control: 32 fragments). Of these, only one fragment of control group did not promote adherence after the IVC. All fragments adhered in all groups were able to cell confluence, with cell proliferation from days 7°, 5° and 3° for the CVD, SSV and control groups, respectively. Differences in initiation of proliferation were observed between the cryopreserved and control groups (P 0.05). No difference was observed for the viability of growing cells (CVD: 89% vs. SSV: 86% vs. control: 91%, P>0.05). Additionally, whereas the control group reached as maximum viability, cells derived from the CVD and SSV groups showed a viability of 98% and 95%, respectively. In conclusion, both vitrification techniques may be used for the cryopreservation of somatic tissue of collared peccary, allowing the isolation of viable cells for the cloning and genetic conservation of this species.