Study On Black Pig Of Putian By Somatic Cell Nuclear Transfer

Cloned pigs by somatic cell have great potential as models for human disease and as human xenotransplantation,which becomes a hot research field.In this study, the ear fibroblast cells of Putian Pig were used as donor cells and the porcine oocytes were used as recipient cells,the porcine oocytes were used as recipient cells in order to establish an efficient system of porcine somatic cell nuclear transfer.The research optimized culture conditions of porcine oocytes maturation in vitro,studied parthennogenetic activation of mature porcine oocytes and the effect of micro-electrode fusion method on the fusion rate of reconstructed embryos.Then the open elongated straw (OPS) was applicated in vitrificating the reconstructed embryos.The results showed that:1.Ear skin fibroblast cell of 3~5d Putian Pigs were cultured using explant-seeding method with a lot of stabilizated donorcells lines received by descending,purificating and freezing.2.The effects of several factors such as the culture methods,the addition of paraffin oil,combinations of exogenous hormones,ITS adding on the oocytes in vitro maturation(IVM) were studied and analized in the test.Results showed that:(1) The maturation rate of porcine oocyte in groove dish were higher than four hole plate and 30mm plastic dish,but the difference was not significant (P>0.05).(2) The addition of paraffin oil can significantly increase (P<0.05) the Cumulus Expansion and maturation rate of oocyte in vitro.(3)The maturation rate of hormone PMSG+HCG+FSH was significantly higher than that of Grade FSH+LH and Grade HMG(80.1%vs68.3%vs53.3%),the differences between each other (P<0.05) were significant.(4) As a results, adding 1% ITS to oocyte maturation medium did not improve the maturation rate,but significantly increased the cleavage rate and blastocyst rate of parthenogenesis embryos(63.3%vs55.1%,18.7%vs12.1%, P<0.05).In conclusion, adding 1%ITS to the medium of porcine oocyte maturation can significantly improve the development of oocyte parthenogenetic embryos power in vitro.3. The experiment evaluated the effects of ionomycin concentration and treatment time and combined with 6-DMAP on porcine oocyte parthenogenetic activation of cells in order to optimize and improve the oocytes'parthenogenetic activation.Results showed that, the cleavage/blastocyst rate was the highest(66.7%,13.6%)for 5μmol/L ionomycin treating 4 minites,the effect of different electrical activation parameters on parthenogenetic activation were also compared,numerous tests showed that the parthenogenetic activation parameters 120v/mm,60μs,activation of 1DC was the best by studying the effect of different culture medium on parthenogenetic embryos.The results showed that there was no significant difference in cleavage rate (55.2%,56.1%)between NCSU-23 and PZM-3 as for the culture of parthenogenetic embryos.But for the blastocyst rate,PZM-3 was significantly higher than NCSU-23 (13.8%vs7.84%,P<0.05).4. Microelectrode fusion protocol was used in this study so as to improve the electrofusion efficiency of pig reconstructed oocytes, microelectrode fusion (84.8%vs64.4%,P<0.05) has been significiently increased compared to traditional chamber fusion.Repeated parthenogenetic activation experiments confirmed that 16v,20μs,1DC was suitable to improve cleavage rate and blastocyst rate,but there was no statistical difference (P>0.05)between cleavage rate and blastocyst rate.5. Cloned embryo morula and blastocyst were vitrificated using open-elongated straws (OPS),the results were 72.3% normal embryos were obtained after thawing.