| In this study, we carried porcine oocytes in vitro maturation, parthenogenetic activation, in vitro fertilization, fibroblast cell culture, somatic cell nuclear transfer and embryo culture techniques out testing and analysis, and thus to establish and improve the pig in vitro embryo production and somatic cell nuclear transfer (SCNT) system for further study of transgenic pigs to provide technical support:This study included the following main elements: (1) The effects of different hormones combination, different concentrations of FSH and LH and amino acid combination supplemented in the IVM basic medium were studied to oocytes IVM. (2) The effect of IVM oocytes in different maturation systems were parthenogenetic activation, and their subsequent developmental potential were studied; the effect of different activation voltage and electrical pulse duration on porcine oocyte parthenogenetic activation efficiency and the ability of embryonic development; Application of CHX on porcine oocytes parthenogenetic activation efficiency and the ability of embryonic development. (3) The effects of IVF medium, fertilization time, floating time of sperm, sperm donor, granular cells and other conditions on fertilization and embryo development (4) Established new-born piglets ear fibroblast cells line and its biological characteristics. (5) The effect of different tissue-derived cells, reagents in parthenogenesis, or amino acid culture conditions on capacity of somatic cell nuclear transfer embryonic development. The major findings are as follows:Test 1 The factors affecting in vitro maturation of oocytes(1) The oocytes were IVM 22h in medium containing hormones than mature in non-hormone medium to 44h, The hormones plaied a great role on porcine oocytes maturation, compared different hormones combination role on porcine oocytes IVM, we found that, the medium with any hormones had the lowest rate of oocytes nuclear maturation and only33.8±2.2%; PMSG+hCG group had the highest maturation rate, up to85.9±2.1%, than the FSH+LH group and the E2 group;(2) During IVM, FSH had greater impact on porcine oocyte nuclear maturation rate, and its effect is related with its concentration; LH in low concentration had little effect on porcine oocytes IVM, when the concentration reached 10IU/mL, LH had positive effect on the mature; FSH and LH had a synergistic effect with each other, when supplemented 10IU/mL FSH and 20IU LH/mL in IVM medium, the porcine oocyte maturation rate reached 80.9±2.2%.(3) It would promote porcine oocytes proportion of nuclear maturation in IVM when amino acids were added to medium. To add non-essential amino acids in IVM medium had the largest percentage of nuclear maturity up to 83.0±2.8%, the group of without amino acids percentage of nuclear maturity was only 74.9±2.4%, the group of essential amino acids was 78.6±1.5%, while adding two kinds of amino acids wae 79.9±1.1%, slightly lower than the non-essential amino acid group.Test 2 The study of parthenogenetic activation Condition(1) The oocytes from mediums with different hormone had markedly different developmental ability after parthenogenetic activation. The non-hormone group had the lowest cleavage rate and of blastocyst only 24.4±4.3% and 0% after parthenogenetic activation, and the PMSG+hCG group had the highest rate of cleavage rate and blastocyst were 85.2±2.4% and 55.3±1.3%;(2) There were some directly relationship among the efficiency of porcine oocytes parthenogenetic activation and the electric field strength and pulse duration. Among them, the electric field strength had the most direct impact, from 1.4 kV/cm to 2.0kV/cm, there were an increase in the activation efficiency, when the electric field strength higher than 2.0kV/cm, the activation efficiency were decreased. The best conditions for parthenogenetic activation was 2.0kV/cm, 30 ms, 1DC, and the cleavage rate was 90.3±1.4%, and blastocyst rate was 46.7±1.9%.(3) The CHX could raise the parthenogenetic activation efficiency to IVM oocytes, which effect depend on the time and the mode of action. After electrical activation, the oocytes were activated in CHX for 8h, the cleavage rate and blastocyst rate were 87.1±1.6% and 42.9±1.1%. In addition, pretreated with CHX would also increase the efficiency of activation, in which pretreatment 15 minutes had the best results.Test 3 The factors of influence on the results of in vitro fertilization of pig oocytes(1) The effect of porcine oocytes in vitro fertilization affected by many factors, this study, we found that under the conditions of sperm floating 60min, with a small amount of granular cells, in mTBM medium, and semen and sperm were incubated 6h, got the best effect on fertilization, cleavage rate was 77.6±2.3%, and blastocyst rate was 25.7±1.6%.(2) Different individual semen had significant different efficiency for IVF.Test 4 The factors of affecting the efficiency of somatic cell nuclear transfer embryo development(1) Different activation agents had markedly different effect on the activation of reconstructed embryos, CHX had the most competent with 67.4±0.6% cleavage rate and 19.6±2.1%blastocyst rate; 6-DMAP followed; CB was the worst among the three groups.(2) Different tissue-derived cells had a great impact for the ability of the reconstructed embryos development, among them the newborn piglets ear fibroblast cells as nuclear donor had the best embryos developmental ability, and the newborn piglets tail fibroblasts were the poor, and the cumulus granulosa cells were between them.(3) The non-essential amino acids could improve the rate of nuclear transfer embryo development, but essential amino acids led to a decline in capacity of embryonic development, while adding two types of amino acids, the embryo development rate is less than only non-essential amino acids group, but higher than essential amino acids group..
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