Obtaining a culture of somatic cells using tissue material from the ear of dead sheep/snow sheep hybrid

Abstract

Production and cryopreservation of somatic cells (SCs) from valuable and endangered animals allows a preservation of genetic diversity and ensuring their future reproduction. The aim of present work was to isolate SCs from the ear of unique hybrid sheep (Ovis aries) and snow sheep (Ovis nivicola borealis) post-mortem. In this purpose, enzymatic and mechanical methods of tissue preparation were compared.Materials and Methods. Ears from deceased animal were brought to the laboratory 12 hours after the death in a pasture, and biological material was thoroughly washed under running water. The hairs were removed from the part of the ear shell by the blade. Skin fragments were treated with 70% ethyl alcohol, washed three times in a saline solution with antibiotics and ground up to small pieces. The ear pieces were washed several times in phosphate buffer saline and divided into two parts. One part of the explants started in vitro culture without enzymatic treatment (group 1), whereas another part was pre-treated with a 0.25% trypsin/EDTA solution. After trypsinization, either tissue fragments (group 2), or cell complexes separated from cell suspension fraction (group 3) were taken for in vitro culture for 9 days. Monitoring of cell colony formation and growth was carried out daily. Results. In the group 3, cell colonies were formed on the second day of in vitro culture. In groups 1 and 2, cell growth was observed from tissue fragments after five days regardless of the treatment. On the 9th day, all the groups produced the primary cultures, represented by two types of SCs. In general, single cell complexes from the group 3 formed cell growth zones more quickly than tissue explants from the groups 1 and 2, however, final cultures of SCs and their morphological aspects were no different between the groups. Conclusion. Methodological protocols were proposed and successfully used to obtain in vitro cultures of SCs from the ear of dead sheep/snow sheep hybrid animal, 12 hours post-mortem that may allow further storage of valuable genetic material.