Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage and temperatures

Abstract

Proper tissue preservation from a wide range of animals of different species is of paramount importance, as these tissue samples could be used to reintroduce lost genes back into the breeding pool by somatic cloning. We aim to study the temporal and thermal post-mortem limits, tested in rabbits and pigs, within which there will be guarantees of obtaining living skin cells in goat, sheep, and cattle. We also intend to study the effect of vitrification on the ability of ear skin cells, stored at different times and temperatures, to attach to the substratum and grow in vitro after warming. Ears were stored either at 4 °C for 12, 252, and 348 h post-mortem (hpm), or at room temperature (22–25 °C) for 60 and 96 hpm. In all cases, skin samples from these ears were sorted into two groups: one group was in vitro cultured immediately after storage, and the other group was vitrified after storage and further in vitro cultured. In goat and sheep, no differences in attachment (100%: goat; 90–100%: sheep) or subconfluence (75–100%: goat; 70–100%: sheep) rates were observed between experimental groups. However, in days of culture to reach subconfluence, significant differences between non-vitrified and vitrified groups were observed when ears were stored at 4 °C for 12 and 252 hpm. In cattle, with respect to attachment rate, vitrified samples from ears stored at 22–25 °C for 60 hpm were different from non-vitrified control group (60 vs. 100%, respectively; P < 0.05). Also, days of culture to reach subconfluence were analysed by a non-parametric Cox Survival Analysis. In general, results from ANOVA and Survival Analysis were similar, because the proportion of censored data was quite low (9%), so the bias when using ANOVA is not too high. In spite of all the above, the lowest survival rates (75%: goat; 70%: sheep; and 40%: cattle) were sufficiently high to enable collection of skin samples from the majority of dead animals and their cryopreservation.