P-27: Conservation Cloning of Esfahan Mouflon

Background: Among wide range of bio-conservational strategies envisaged, recent accomplishments in the field of somatic cell nuclear transfer (SCNT) holds considerable promise due to its unique potential to decelerate or prevent rapid loss of animal genetic resources, and even to revive extinct species. This study was undertaken to investigate whether domestic sheep in vitro matured and enucleated cytoplast can support reprogramming and in vitro development of cryo-banked somatic cell nuclei of Ovis orientalis isphahanica (Esfahan Mouflon), a vulnerable species classified by IUCN, into cloned blastocysts and whether these embryos can implant in the uterus of domestic sheep and develop to viable cloned offspring. Materials and Methods: Cryo-banked fibroblast cell lines of one male mouflon from a genome resource bank and a domestic sheep were prepared and used for karyotyping analysis. Prior to SCNT, fibroblast donor cells were serum starved for 5 days. Using zona-free SCNT technique, in vitro matured and enucleated domestic sheep oocytes were reconstructed with nuclei donor cells of mouflon and domestic sheep and their competence for in vitro development to the cloned blastocyst was compared. The cloned mouflon blastocysts were then transplanted in the uterus of the synchronized domestic sheep. Results: Karyotyping analysis confirmed that cryo-banked somatic cells of Esfahan mouflon had the correct number of diploid chromosomes (2n=54). Evaluation of 907 activated reconstructs [Esfahan mouflon (n=667), domestic sheep (n=240)] revealed no significant difference in overall blastocyst development (7.6 ± 0.5% vs. 9.3 ± 0.5%, respectively). After transfer of 12 cloned Esfahan mouflon blastocysts into 5 domestic sheep recipients, 2 (40.0%) pregnancies were established and both (100%) sustained until cesarean section at days 147 and 150 of pregnancy, which both culminated in the live birth of cloned Esfahan mouflon lambs. Conclusion: The newborns did not survive and died soon after birth. Karyotype and genetic analyses confirmed that both clones 1) had correct diploid chromosome number (2n=54), and 2) were genetically identical to each other and to their original cell donor. This study highlights the importance of conservation cloning using closely related abundant alternates.