| Although many types of somatic cloning animal successfully have been produced, the efficiency of nuclear transfer is still low. The research in nuclear transfer fields mainly focus on molecular mechanism in nuclear reprogramming, and especially all kinds of small chemical compounds are researched in improving the efficiency. Many kinds of iPS cells are successfully induced after using iPS technique as the other kind of nuclear reprogramming methods, but it is a problem that the chimera to prove the totipotency of iPSc similar to embryonic stem cells is only successfully applied in mouse. Therefore, it is very important to research in producing porcine chimera, which will be a model for human disease and xenotransplantation in future. This study is to research in the effects of two kinds of small chemical compounds (ITS and Vitamin C) on the development of porcine embryos in vitro and analyze the factors of affecting to producing porcine chimera using porcine iPSc, and the experimental restuls shown as following.1. The effects of insulin–tranferrin–selenium (ITS) on nuclear and cytoplasmic maturation of porcine oocytes and subsequent embryonic development were researched(1) The rate of oocyte in vitro maturation (IVM) in experimental group treated with hormones for 42 h was significantly increased than that in control group without hormones treatment (47.8% vs. 11.7%, respectively, P<0.05). When reduced the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormones treatment (44.8%), the rate of oocyte IVM was still higher than that of control group (P<0.05).(2) To improve porcine oocyte nuclear maturation, 1% ITS was added in medium supplemented with hormones. The rate of nuclear maturation in ITS treated group was significantly higher than ITS untreated group (78.6% vs 54.4%, respectively, P<0.05).(3) ITS treatment was also significantly reduced the ratio of oocytes with type I and type III CGs distributions, and significantly increased the ratio of oocytes with type II CGs distribution (85.3%). The results indicated that synchronization rates of nuclear and ooplasmic maturation reached to 67.04% (78.56% x 85.33%).In conclusion, the group of modified Tissue Culture Medium 199 (mM199) + 10 ng/ml EGF + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulate hormone (FSH) + 1%ITS is suitable for culturing porcine oocytes in vitro.2. The effects of Vc on development of porcine embryos were researched in vitroIn order to clear out the effects of Vc (0, 2.5, 5, 10, 20, 40μg/ml; group A, B, C, D, E, F) adding in PZM-3 were choosed to culture parthenogenetic presumptive diploid zygotes produced by electroactivation after oocytes maturation in vitro.(1)the ROS level in embryos significantly decreased in the Vc adding groups compared to the group A (the control group ),and the group E is significantly decreased ROS level in embryos compared to other groups with adding Vc(Group B, C, D, F).(2)The Rate of blastocyst in group E (27.74%) is significantly higher than in other groups (P<0.05) but there was not significant differences in the rate of embryo cleavage in different groups(P>0.05), and the total nuclei number in blastocyst of Group E(62.33) is significantly higher than Group A and B(34.67 Vs 46.67, P<0.05), but there was not significant difference in Group C,D,E and F(52.67, 58, 62.33 and 56.67; P>0.05).(3)The relative expression of Bcl-xl to Bax gene in Group E is significantly higher than other groups (P<0.05), and it was almost 4 folds to the control.(4) It is showed that TUNEL index in adding Vc groups (Group C and E: 12.21% and 11.94%) significantly decreased compared to nonsupplemented Group (Conrol: 23.17%, P<0.05) through analyzing TUNEL results (Group A, C, E).(5) The expression of Nanog gene in Group E and F is significantly higher than other groups (P<0.05) but there is not significant difference in this two groups (P>0.05) and Group E and F was almost 5 folds to the control.These data thus suggest that Vc , which 20μg/ml in PZM-3 was the best suitable dosage, improves the development rate and quality of porcine parthenotes by preventing oxidative damage in the process of porcine embryo development in vitro.3. The effects of different superovulation procotols on ovary reaction of Baimei gilts were researchedThe objective of this study was to compare the superovulation effects between equine chorionic gonadotropin (eCG , Group A) and FSH (Group B-D) in Chinese Bamei gilts.(1) The results show that though eCG could produce more corpus lutea (CL, 14.3) than the control (CL, 9.2), the FSH treatments were significantly increased the number of CL than that of eCG treatment.(2) Within the different FSH protocols, the numbers of CL were significantly more in Group B (CL, 77.8) and C (CL, 66.8) than that in Group D (CL, 42.7), however, ovarian cysts were observed in Group B and C, but not in Group D. These data suggest that Group D (280IU FSH) is a suitable protocol to facilitate the development of ovarian follicles and may obtain more useful embryos per gilt through embryos recovery.The optimal FSH protocol of superovulation in Bamei gilts is: D13/100 IU, D14/80 IU, D15/60 IU, D16/40 IU plus PG 0.2 mg, D17/hCG 1000 IU4. Research on creating chimera embryos using porcine iPS cells as donor cellsThe research of producing gilts of iPSc chimera is very important because it has been a model for human disease and xenotransplantation in future. The objective of this study is to choose suitable iPSc for chimera embryo transfer through analyzing the development of reconstructed embryos using iPSc as donor cells through nuclear transfer and chimera embros in vitro before transferring embryos into gilts, and research in time and methods of obtaining the porcine embryos in vivo.(1)The nuclear transfer embryos cannot develop to blastocyte stage using iPS-3 as donor cells and the iPS-3 cell cannot involve in the development of chimera embryos through using blasocytes from in vitro and vivo, the ratio of blastocyte in reconstructed embryos development usig iPS-1 and iPS-3(6.98%, 8.07%) as donor cells is significantly lower than the control(13.16%,P<0.05).(2) The ratio of blastocystes in recovering embryos between 4.5 to 5 days(37.71%) is significantly higher than that below 4.5days after first mating(4.56%, P<0.05),and there is not significant difference in the rate of recovering porcine embryo between surgical and in vitro uterus methods(83.33% vs 85.92%, P>0.05).Therefore, the system of choosing suitable iPSc for donor cells and obtaining many porcine embryos with good quality is basically built for the next porcine iPSc chimera research. |
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