The Enhancement of Scripted in the Development of Cloned Sheep Embryos

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recent reports have demonstrated that scripted treatment after somatic cell nuclear transfer (NT) improves success rate of bovine embryo cloning. The objective of this study is to test the effect of scripted in the improvement of the developmental rate of cloned sheep embryos in vitro. Sheep ovaries (2113 ovary) and ear skin fibroblast were collected from the central slaughter house at Riyadh, Saudi Arabia. The fibroblasts (ear skin cell or cumulus cells) used as somatic donor. The total of (4206) oocytes with their cumulus cells were collected from the sheep ovary and only (3025) ova cultured in the in vitro maturation (IVM) media in a CO2 incubator (38.5C) for 22 hrs. For the cloning treatment the (IVM) oocytes (1495) were enucleated under the micromanipulator and the somatic cells were transferred to the enucleated mature oocytes (1015 ova). The transferred somatic cells were electrically fused with the enucleated mature oocytes (710 ova). The fused cells were activated with calcium ionophore and cycloheximide (494 oocytes). Subsequently, they were cultured in TCM199 medium with 0.5 or 0.0micro M concentrations of scripted treated (244 oocytes) and control (250) oocytes). The embryos were cultured inside an incubating chamber supplemented with air and (5 % CO2 +5 % O2+ 90 % N2) in the incubator at 38.5C for 6-7days. The results show that the development embryo of the cloned sheep embryos in vitro most of them were degenerate (301 ~ 61 %) and 46 ~ 25 % remain in one cell stage, while 17 embryo developed to two cell stage (3 Scripted, 14control), 24 embryo reached the four cell stage (5 Scrited, 19 control), 51 embryo (26 control, 25 scripted) blocked in eight cell stage, while 12 embryo remain at 16 cell stage (8 control, 4 scripted), and 34 embryo developed to morula stage (27 scripted, 7 control). Finally 9 embryo reached the blastula stage (two from control and 7 from scripted group. About 60 developed embryos were transferred to 20 sheep surgically. Finally three pregnant females confirmed by the ultrasound, with 4 new borne one is twins. However, when both control or treated the pre-implantation development of cloned embryos was compered we get twice development with the scripted treated compared to control. So the scripted enhance the development of cloned sheep embryos. Key words: In vitro maturation (IVM), somatic cell nuclear transfer, cloning, sheep, embryo, Scripted. *This research was granted by the National Plan for Science and Technology, grant no. (Bio-1019-2-10).