Bighorn Sheep Cloned Embryos Produced by ISCNT via HMC from Domestic Sheep Oocytes Treated with Resveratrol During IVM

The bighorn sheep is listed on the IUCN Red List of Threatened Species. By interspecies somatic cell nuclear transfer (ISCNT), other endangered species are cloned using somatic cells as nuclear donors, fusing them with enucleated oocytes from heterologous domestic species. On the other hand, resveratrol added during in vitro maturation (IVM) of domestic sheep oocytes favors the development of embryos produced in vitro. The aim of this study was to treat O. aries oocytes with resveratrol during IVM using them as cytoplasts in ISCNT via handmade cloning (HMC), evaluating its effect on the in vitro development of Mexican bighorn sheep (O. c. mexicana) cloned embryos. Post-mortem skin fibroblasts from an adult male specimen from the Chapultepec Zoo were frozen for 8 years, thawed, and reseeded for 8 cell passages. For IVM, O. aries oocytes were treated with 0, 0.5, or 1.0 µM resveratrol. Matured oocytes were manually enucleated, and triplets (O. aries cytoplast-O. c. mexicana karyoplast-O. aries cytoplast) were formed and electrically fused. The reconstructed embryos were chemically activated and cultured until they developed into blastocysts. For IVM, no differences were found between treatments, yet at 0.5 µM, resveratrol significantly increased (p<0.05) the blastocyst rate and decreased the fragmentation rate.