The Techonoly Research On The Elite Boars Produced By Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) can be used as a technology to produce outstanding boars in a short time. However, there're still many problems on cloning efficiency and breeding the cloned pigs until now. Timing of the first zygotic cleavage is related to the developmental potential of mammalian embryos. The present study documented this phenomenon in porcine parthenogenetic (PA) and somatic cell nuclear transfer embryos. An important marker has been found about the high-quality SCNT embryos in the early development stage, which optimized the cloning conditions and improved the cloning efficiency; There're some problems about the health of newborn cloned piglets:physically weak, lower survive rate and so on. For these problems, we need to enhance the breeding and management, establish an effective system for reproducing boar quickly. The results were as fallows:Experiment 1:According to the performance measure results of boars in Beijing, three adult England Yorkshire boars were selected. Three ear fibroblast cell lines were established by using explant-seeding method. Cells still have a higher genetic stability at passage 10 in vitro.Experiment 2:Early embryo development is regulated by maternal substance. In order to explore the relationship between timing of the first zygotic cleavage and porcine PA embryos, In vitro matured pig oocytes were activated parthenogenetically. The developmental competence of PA embryos undergoing the first zygotic cleavage at different times (early-cleaving,20-24 h; mid-cleaving,24-36 h; late-cleaving,36-48 h and unselected controls,0-48 h) was evaluated by the proportions developing to blastocysts and expanded blastocysts and their total cell number. For PA embryos, the proportion of early-cleaving embryos that developed to blastocysts was significantly higher than for mid-cleaving, late-cleaving and controls (P< 0.05; 54.2±3.9% vs. 19.7±1.4%,5.5±0.6% and 18.9±2.3%, respectively); the same pattern was noted for formation of expanded blastocysts.Experiment 3:In this study, the relationship between timing of the first zygotic cleavage and the developmental potential of porcine SCNT embryos had been evaluated. The proportion of early-cleaving embryos developing to blastocysts was not significantly higher than for mid-cleaving embryos (32.6±3.9% vs.24.6±2.8%), but the late-cleaving embryos had poor developmental competence to blastocysts (4.0±0.4%). Development to expanded blastocysts was significantly higher for early-cleaving SCNT embryos than for mid-, late-cleaving and unselected control embryos (P< 0.05; 19.0±3.9% vs.6.0±4.8%,0.8±0.2,7.4±0.8, respectively).Total cell number in blastocysts declined across the three cleavage-timed groups.Experiment 4:Embryo transfer is an important part which determine the SCNT embryos development status in vivo. Developmental potential of SCNT cloned embryos was assessed in vivo after transferring early-cleaving (<24 h) (Group 1) and unselected (time of cleavage up to 48 h not determined) embryos (Group2) into recipient gilts. In Group 1, early-cleaving embryos (718) were transferred into 6 recipients,28 piglets (1 stillborn) were born. The largest litter size of cloned pigs were 11 piglets. In Group 2, unselected embryos (2719) were transferred into 12 recipients, 25 piglets (including 8 stillborns and 3 mummified fetuses) were delivered. Litter size and cloning efficiency (piglets born/transferred embryos) in recipients of early-cleaving embryos were significantly higher than those in recipients of unselected embryos (P< 0.05; 4.7 vs.2.1 piglets; 3.9% vs.0.9%). The data demonstrated that early-cleaving SCNT embryos had better developmental potential than did later-cleaving embryos.These results indicated that somatic cell nuclear transfer produced boars effectively; Timing of the first zygotic cleavage is an important marker of the developmental potential of cloned embryos in pigs; Transferring early-cleaving embryos improves overall cloning efficiency and provides an important technical aid for the industrialization of cloning technology and breeding of transgenic animals.