Embryo Transfer And Lipids Analysis In The Production Of Omega-3 Fatty Acid Desaturase(sFat-1)-transgenic Pigs By Somatic Cell Nuclear Transfer

Polyunsaturated fatty acids (PUFAs) have been broadly investigated and have been paid much attention to by the public. PUFAs mainly includes two groups,ω-3 PUFAs andω-6PUFAs,ω-6 PUFAs is not crucial for human beings. Contrarily, their excess will bring many healthy problems. However the source ofω-6 PUFAs is abundant but that ofω-3 PUFAs is very limited, which lead to the level ofω-3 PUFAs in mammals is relatively too low compared withω-6 PUFAs, the ratio ofω-6/ω-3 PUFAs as high as 16 or more. Therefore, the only thing need to solve is to increase the level ofω-3 PUFAs content in human's rational utilizing of fatty acids, and how to obtain abundant source ofω-3 PUFAs is a focus in the world.The technique classical transfection of somatic cells in combination with somatic cell nuclear transfer (SCNT) provides a more efficient approach to produce transgenic animals,and how to obtain abundant source ofω-3 PUFAs is a focus in the world. This research to improve somatic cell cloning of transgenic pig production efficiency, to study pig somatic cell cloning and embryo transfer in the process of the best transport temperature, time and the best time for embryo transfer and rich inω-3 fatty acid desaturase gene was cloned pig microsatellite identification and detection of fatty acids.This study compared the different transport temperature and time on development of parthenogenetic embryo in order to establish the best cloned embryos Pig transport temperature and time. The experiments included that: (1) The effects of different temperatures (37℃, 38℃, 39℃) on parthenogenetic embryo cleavage and blastocyst rate; (2) The effects of different times (2 h, 3 h, 4 h) on parthenogenetic embryo cleavage and blastocyst rate . The results were as follows: (1) There were statistical differences (P<0.05) of the parthenogenetic cleavage rate (90.0±4.3% VS. 72.6±6.9%,80.6±5.5%) and blastocyst rate (33.8±7.4%VS18.9±1.5%,26.0±2.2%) between development of parthenogenetic embryo in 39℃and37℃.38℃;(2)In3h,the parthenogenetic cleavage rate (81.6±6.1 %) and blastocyst rate (31.2±3.6%); in 2h,4h, he parthenogenetic cleavage rate (64.9±9.6%,49.2±2.4%) and blastocyst rate (18.2±6.7%,19.7±2.9%).In this study, we evaluated the effect of different ovulation status on pregnancy rates, in order to determine the optimal stage of the estrus cycle to achieve the highest pregnancy rates in surrogate recipient gilts receiving SCNT embryos. Porcine somatic cell cloned embryos on 2d in vitro were transplanted into the 1-2d estrous recipient gilts. Recipient gilts were divided into two categories according to follicular development and ovulation status: just prior to ovulation & Ovulating (group1); per-ovulation & after ovulation (group2) . The results were as follows: (1) Group 1 the pregnancy rate on 30 d of six recipient gilts was 100%; and Group 2 the pregnancy rate on 30d of ten recipient gilts was 40%,Significantly different between Group 1 and Group 2 (P<0.05); (2) For the full-term pregnancy rate of recipient gilts, significant difference was observed between Group1 and Group2 (100% vs. 20%, P<0.05).Then we useω-3 fatty acids desaturase genes to produce transgenic pig,at last 21 piglet born, 15 survived. Analysis by PCR and southern blot, 13 of them are transgenic. The tissue of the transgenic pig muscle produced 0.968% of DHA( averagely) and 7.523% of totalω-3 PUFAs, all of which far exceed the former studies ofω-3 desaturase transgenic anmals.These results suggest: (1) Significantly different between3h and 2h,4h. These results suggest that at 39℃, 3 h parthenogenetic embryos to carry out the transport, to obtain a higher cleavage rate, blastocyst rate;(2) that development stage of porcine somatic cloning embryos on 2d in vitro with recipient gilt's womb environment of ovulation status of just prior to ovulation & ovulating is the best in the same period. It may achieve the best efficiency of embryo transfer; (3)Our research produced transgene large-scale livestock yield a longer chain, more valuable DHA by sFat-1 gene cloned embryos by somatic cell nuclear transfer.