Cryopreservation of Saiga (Saiga Tatarica) Somatic Cells: Effect of Non-Equilibrium and Equilibrium Freezing on Fibroblast Viability

Background. The saiga antelope (Saiga tatarica) has recovered numerically in Kazakhstan, but its populations remain exposed to mass mortality events that can remove a large share of a population within weeks. Viable somatic cell banking can insure genetic diversity, yet no cryopreservation protocol had been reported for saiga cells. Aim. To establish primary fibroblast cultures from saiga ear skin and to compare post-thaw survival after passive and controlled-rate slow freezing. Methods. Ear skin biopsies were collected from 50 adult saigas of the Betpak-Dala and Ustyurt populations. Fibroblasts obtained by explant culture were frozen at passage 2 either passively in cryovials with 10% dimethyl sulfoxide (DMSO; ≈ −1 °C/min to −70 °C) or in 0.5 mL straws with 1.5 M DMSO or 1.5 M ethylene glycol (EG) after 2 h equilibration at 5 °C, using a programmable freezer. Viability was assessed by trypan blue exclusion. Results. Cell outgrowth began on days 6–10 of culture. Passive freezing reduced viability from 97.6% to 44.5% (p < 0.001), with high inter-individual variability (9–77%; CV 30%). Controlled-rate freezing yielded 52.8 ± 1.1% viable cells with DMSO and 62.1 ± 1.0% with EG versus 85.8 ± 0.8% in fresh controls (p < 0.001); EG exceeded DMSO by 9.3 percentage points and gave 27% more viable cells per straw. Both controlled-rate treatments outperformed passive freezing and showed 2.9–3.5-fold lower variance. Conclusions. Controlled-rate slow freezing with 1.5 M EG was the most effective protocol and was adopted for the first somatic cell cryobank of the saiga in Kazakhstan.