| The pig is a ideal animal mode with broad application prospect in transgenic bioreactordomain, so we needs to establish a set of effective in vitro reproductive technology fortransgenic pigs production to provide the massive high quality mature oocytes and theembryos for transgenic pig production.. This paper mainly focus on establish of earlyactivation system for pocine somatic cell clone and in vitro embryo production, and we alsohave researched oocyte mature system, orphaned female activation system in vitro,fertilization system in vitro and the freezing method.The result indicated that: the percentage trend of all levels of ovaries each month:denpending on the weather hotter, the first Level of ovaries gradually decrease, and itachieved the least in June and July; the proportions of the second levels ovaries was steady;the proportions of the third levels of ovaries was opposited to the weather; the averageEGGS every ovary was at least from June september, insteadly, the ovarian quality andaverage EGGS were quite well in april, may, september and october with cool weather.The maturation rate impact of oocytes, having hormone or not, was not significant(P＜0.01)in the half mature culture system of Oocytes; during cultivaing the oocytes, maturation ratewas the best when the cell concentration was 100/400μl; the NCSU-23+10% FBS was thebest of matural cultivation effection of different matural system; meantime, theNCSU-23+BSA was the best for cultivation effection of oocytes development, afteroocytes activation.During electricity activation using the CRY-3 cellactivation meter, the pulse voltageshould be below 200V, the pulse time interval might be 20μs, 40μs, 80μs, the pulsefrequency should be below 3 times, 120v/20μs per time, 150v/20μs two time was the bestactivations effect group. When using electricitical activation and chemical Activationtogether, the electricity+CB+γ-BLI was the best; compared with activation effection ofγ-BLI different concentration, we found that 600μM was the best.Along with hatching time and sperm density increasing, frozen sperm IVF andpolyspermia rate increased. The effection of zygote altogether 6h was the best. When we taking IVF using sperm vitality 0.1 after defrost, the sperm density 10~7 was suitable. Thecombined effect of two kind of different ovocytes mature culture medium and two kind ofdifferent ovocytes before fertilization removing the oviferous showed that the removingcumulus partly was better than removing completely, and the IVF development effectionwas the best after removing cumulus partly NCSU-23+FBS. The fertilization effection offresh semen was better than frozen semen, the difference of every indicators was notsignificant except 8- cell rate(78.01±1.53%vs68.09±1.08%)(P＞0.05).The result of frozen MⅡoocytes using three methods indcated that the self-restraintspear method was better than OPS, and the effection of frozen altogether with self-restraintspear method and centrifugal was the best; the frozen effection was not ideal using fluidspear for 8- cell ovocyte. The cleavage rate of ovocyte IVF of MⅡovocyte frozen withOPS method and activation method was higher remarkably than fluid spearmethod(P＜0.01), the cleavage rate of 2-cell after electrical activation was higher than IVF.We recommend the matural cultivation system of pig in the experiment thatNCSU-23+10%FBS of continuous culture 48h, cell density 100 per 400ul, anddevelopment culture system NCSU-23+BSA; activated by CRY-3 was 150v/20us two timeand 600μMγ-BLI; when IVF with frozen semen, the sperm density was 10~7, incubator timewas 6h, and removed cumulus cells partly when matural treatment; when frozen ofMⅡoocytes, the method was fluid spear and centrifuge method, the 2-cell cleavage rate ofactived after frozen was higher than IVF, the effection of season for Oocytes quality andaverage ovulation was significant(P＜0.05).
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