Vitrification of Saiga (Saiga Tatarica) Skin Fibroblasts: Effect of Cryoprotectant Composition and Ultra-Rapid Cooling at −205 °c

Background. The saiga antelope (Saiga tatarica) remains exposed to recurrent mass mortality, and a bank of viable somatic cells can insure its genetic diversity. Vitrification of saiga cells has not been studied. Aim. To establish fibroblast lines from neonatal saiga calves and to compare three vitrification solutions under two cooling regimens. Methods. Ear skin explants from 50 calves (25 males, 25 females) aged up to two weeks from the Betpak-Dala region were cultured in DMEM. Cells were loaded stepwise with 20% dimethyl sulfoxide (DMSO), 20% ethylene glycol (EG) or 20% DMSO + 20% EG, each with 0.5 M sucrose, and cooled in 0.25 mL straws either in liquid nitrogen (−196 °C) or in subcooled nitrogen at −205 °C (super-cooling ultra-rapid vitrification, SCURV). Viability was assessed by trypan blue exclusion (n = 50 lines per group). Results. Cell outgrowth began on days 6–10 of culture. Two-way ANOVA showed effects of both the cooling regimen (p < 0.001; η² = 0.19) and the solution (p < 0.001; η² = 0.04), without interaction. SCURV increased viability by 6.1 percentage points (95% CI 4.7–7.5), and the DMSO + EG mixture by 3.1 points relative to either single agent (p = 0.001); DMSO and EG did not differ. The highest viability, 53.9 ± 5.9%, was obtained with DMSO + EG under SCURV. Conclusions. The DMSO + EG mixture combined with ultra-rapid cooling at −205 °C was the most effective vitrification protocol. Samples of all 50 calves were added to the saiga somatic cell cryobank.