38 ROCKY MOUNTAIN BIGHORN SHEEP (OVIS CANADENSIS CANADENSIS) EMBRYOS PRODUCED USING SOMATIC CELL NUCLEAR TRANSFER

Abstract

The present objective was to determine if interspecies somatic cell nuclear transfer (SCNT) could be a viable option for producing cloned Rocky Mountain bighorn sheep (RMBS) embryos, a model for other endangered ovine species or sheep of high genetic value. A primary question was whether viable cells from a harvested 5.5-year-old male RMBS (Ovis canadensis canadensis) could be cultured 4 days postmortem for SCNT embryo production. Domestic sheep ovaries from an abattoir were placed in TCM199 holding medium containing Hanks’ salts, kept at ambient temperature, and transported to the laboratory in less than 3 h. Oocytes were collected using 2 methods, aspirating visible follicles (G1) or slicing ovary tissues (G2), and matured in TCM199-based medium. At 20 h postmaturation, oocytes were stripped of cumulus cells and those with an extruded polar body underwent SCNT using ViaGen's protocol. Tissue samples were collected from the RMBS, placed in plastic bags, and shipped to the laboratory for cell culture; that is, tissue disinfection and mincing, placement in cell culture flasks with cell culture medium and into an incubator at 38.5°C in a humidified atmosphere of 5% CO₂. At confluency, cells having fibroblast-like cell morphology were cryopreserved using DMSO-containing medium and stored at –196°C. For SCNT, 4-day postmortem donor cells were thawed and cultured for at least 3 days. Matured oocytes were enucleated using a micromanipulation system and Hoechst 33342 to ensure nucleus removal. A donor cell was inserted into an enucleated cytoplast and fusion pulses were applied to reconstruct embryos. Cloned embryos were cultured in modified SOF in 5% CO₂, 5% O₂, and 90% N₂ for 6 days before shipment in a portable incubator at 38.5°C in SOF for analysis on Day 7. Pearson's χ² test and Fisher's exact test were used to determine statistical differences (α = 0.05). The 60 ovaries yielded 324 oocytes (5.4 oocytes/ovary) and 217 matured (67%) of which 204 were enucleated, 156 fused (77%), and 102 cleaved (65%), producing 25 blastocysts and morulae (25/156 = 16%). G1 resulted in 62 cleaved embryos of 91 fused, whereas G2 resulted in 40 cleaved embryos of 65 fused (χ² = 0.728; P = 0.393). Using the IETS stage and grading system, 3 blastocysts (one 5–2, one 6–1, one 6–2) and 22 morulae (4–1) appeared viable, but the rest had degenerated. Specifically, G1 produced 3 blastocysts and 8 stage 4–1 embryos versus 14 stage 4–1 embryos in G2 (based on fused or cleaved embryos: χ² = 2.516, P = 0.113 and χ² = 3.914, P = 0.048, respectively). Fibroblast-like cell cultures were obtained from 4- and 5-day postmortem tissue samples; however, only Day 4 cultured cells were used for RMBS SCNT embryo production. Although G1 produced more blastocysts than G2, the difference was not statistically significant (P > 0.05).