Optimization Of The Protocols In Sheep Somatic Cell Nuclear Transfer And The Application Of The Cloned Sheep

Somatic cell nuclear transfer(SCNT)enables the donor cells acquire totipotency after being injected into the enucleated oocytes cytoplasm.By this way,cloned animals inherit genotype and excellent characters from dornors.It is an important technique in rapid breeding of superior herds.Nevertheless,a series of problems such as low pregnancy,high abortion,abnormal fetal development and high birth-death,which restrict the applicaton of SCNT in production.In this study,we systematically modified some steps of SCNT in sheep and established a more efffective sheep SCNT system for production in firm.The ecological adaptability of cloned Dorper to free-grazing in harsh enviornment was also studied.1.Modification of sheep nuclear transfer proceduresThis study focused on modification of sheep somatic cell cloning procedures.The results showed that: 1)Two-step oocyte maturation: Oocytes were cultured in vitro for a period to remove the cumulus cells and then cultivated till maturation.Oocytes matured in two step have significantly higher development in morula /blastocyst compared with one-step(P<0.05).2)Blind extracting the nuclear in minimally invasive manner:At IVM 18 h,approximately 80% of oocytes entered into AI/TI stage and the first polar bodies(Pb1)were still tigntly connected to the oocyte nuclei by the spindles.And then 6 hrs later,Pb1 was still adjacent to the oocyte nuclear.Enucleation could reach 96% by extracting the Pb1 only with a little cytoplasm(about less than 1/30 of the volume).3)Different activation methods had effect on the cleavage of reconstructed embryo.The cleavage by Iono + CHX and Iono + 6-DMAP was 80.1% and 71.0%(P<0.05)respectively,but there was no significant difference in morula /blastocyst(P>0.05).4)Increasing the activation frenqency could improve the development of embryo.Twice treatment of fused embryos with ionomycin showed higher cleavage(87.6% vs 79.1%,P<0.05)and morula /blastocyst development(30.8% vs 22.5%,P<0.05)than once.This demonstrated that embryo development can be improved significantly by our optimized system.2.Research on massvie sheep production in firm by somatic cell nuclear transferThis research based on the firm to produce cloned purebred Dorper sheep and hybrids we called Dorper-Mongolian sheep massively.The efficiency of cloning and pregnancy in production was evaluated.Moreover,the reproductive performance of cloned sheep together with growth status and hematological valules and serum biochemical values of Dorper and Dorper-Mongolian sheeps were aslo investigated respectively.The results showed that all 4 cell lines could be used as the donor cells,they had no difference on cleavage and morula /blastocyst development(P>0.05).Because of the relatively low efficentcy of morula/blastocyst of embryos,we generally choose 1-cell or 2-cell to carry out embryo transplantation in scale production.The state of receptors has great effects on the pregnancy.The pregnancy of receptors and also the birth rate under barn feeding conditions were higher than under grazing(P<0.05).We totally got 61 lived cloned Dorper sheeps.The body weight of the cloned sheep was significantly higher than the controls in the first three months after birth(P<0.05).With their growth,the difference in the body weight and body size(body length,body height and chest circumference)between the cloned sheep and the control group gradually decreased and reached to the similar sizes.The hematological valules(19/20)and serum biochemical values(18/18)of the cloned sheep were almost entirely within the standard range of clinically relevant range.When the cloned rams grew to adult,their semen qualities were examined.The results showed that the quantity and motility of the cloned Dorper semen were similar with controls as the growth performencce.By artificial insemination,we got 1213 purebreed Dorpers.To the end of 2014,total of more than 68,000 Dorper-Mongolian F1 hybrids crossed by the purebreed Dorper rams with local Mongolia ewes,which have good quality in meat and leather and have shown a good economic and social performence.3.Research of ecological adaptability of cloned sheep under grazingSince the birth of the first cloned sheep,somatic cell nuclear transfer technology has been successfully used to clone a variety of mammals.Cloned livestock have no apparent health risks,and the quality and safety of the cloned animal products are similar to non-cloned animals.The social behavior and environmental adaptability of postnatal cloned animals,especially when used for grassland farm production purposes,is unknown.In the present study,the cloned Dorper sheep equipped with GPS location devices were free-grazed in a harsh natural environment similar to conditions commonly experienced by Mongolian sheep.The main findings of this research were as follows.1)Under free-grazing conditions,the cloned sheep showed excellent climatic and ecological adaptability.In extreme temperature conditions ranging from-30 to 40 °C,the cloned sheep maintained acceptable body condition and behaved as other sheep.2)The cloned sheep quickly adapted from a herd feeding strategy to the harsh environment and quickly exhibited a grazing regimen as other free-grazing sheep.3)The cloned sheep exhibited free-grazing patterns and social behavior as other sheep.4)The cloned sheep in the harsh environment thrived and produced healthy lambs.Overall,the cloned Dorper sheep exhibited excellent ecological adaptation,which is an important consideration for breeding meat sheep by cloning.The Dorper sheep readily adapted to the free-grazing conditions on the Mongolian plateau grassland,which attests to their ability to withstand harsh environmental conditions.