# Production of Cloned Bighorn Sheep Embryos Using ISCNT via HMC from Domestic Sheep Oocytes Treated with Resveratrol During IVM

## TL;DR

Scientists cloned embryos of endangered bighorn sheep using domestic sheep oocytes treated with resveratrol, improving embryo development rates.

## Contribution

This study demonstrates that resveratrol treatment during oocyte maturation enhances cloned bighorn sheep embryo development using post mortem fibroblasts.

## Key findings

- Resveratrol-treated oocytes led to higher blastocyst rates in cloned bighorn sheep embryos.
- Fragmentation rates were significantly reduced in embryos from resveratrol-treated oocytes.
- Post mortem fibroblasts cryopreserved for 8 years were successfully used for cloning.

## Abstract

The bighorn sheep is listed on the IUCN Red List of Threatened Species. The purpose of this study was to clone bighorn sheep (O. c. mexicana) embryos using, as cytoplasts, oocytes from domestic sheep (O. aries) treated with resveratrol, an antioxidant with beneficial effects on the development of in vitro-produced embryos, and, as karyoplasts, fibroblasts from a post mortem adult male bighorn sheep from the Chapultepec Zoo, which had been frozen for 8 years. The bighorn sheep embryo clones were reconstructed using handmade cloning (HMC), and their development rate was evaluated. A higher blastocyst rate and a lower fragmentation rate were observed in cloned bighorn sheep embryos produced from sheep oocytes treated with 0.5 µM resveratrol. It was concluded that resveratrol improves the blastocyst rate and reduces fragmentation in cloned post mortem bighorn sheep embryos produced using ISCNT via HMC.

The aim of this study was to treat sheep oocytes with resveratrol during in vitro maturation (IVM) and use them as cytoplasts in ISCNT via handmade cloning (HMC), evaluating the effect of resveratrol on the in vitro development of cloned Mexican bighorn sheep embryos. Post mortem skin fibroblasts from an adult male specimen were frozen for 8 years, thawed, and reseeded for eight cell passages. For IVM, Ovis aries oocytes were treated with 0, 0.5, or 1.0 µM resveratrol. Matured oocytes were manually enucleated, and triplets (O. aries cytoplast–Ovis canadensis mexicana karyoplast–O. aries cytoplast) were formed and electrically fused. The reconstructed embryos were chemically activated and cultured for in vitro development (IVD). The IVM rate was 81.8 ± 10.4% for CG, 81.9 ± 6.7% for EG1, and 76.3 ± 7.7% for EG2, with no significant differences between groups. For IVD, EG1 showed a statistically significant increase (p < 0.05) in the blastocyst rate (31 ± 12.0%) and a statistically significant decrease in the fragmented embryo rate (25 ± 10.4) when compared with the other groups. It was concluded that better rates of cloned bighorn sheep blastocysts could be obtained in ISCNT via HMC when fusing O. aries oocytes supplemented with resveratrol during IVM with post mortem adult male O. c. mexicana fibroblasts that had been cryopreserved for 8 years.