| In vitro maturation of porcine oocytes, parthenogenetic activation and in vitro culture of embryos are considered as main items of embryos engineering such as somatic cloned pig. To provide effective technology parameter for production of cloned pigs, the current research was designed to study the effects of several media on maturation of porcine oocytes in vitro and different activation parametes or combined activation on parthenogenetic Activation. Meantime, I examined mostly the effects of several embryo cultural mediums and epidermal growth factor (EGF) or bisac fibroblast growth factor (bFGF) on the development of porcine parthenogenetic embryos in vitro. The results were as follows:Porcine cumulus-oocyte complexes(Cocs) were cultured in medium NCSU-23 with serum and without serum, maturation rates(73.31±3.03% vs 68.83±4.60%) were no significantly different(P>0.05). And porcine oocytes were cultured in PZM,NCSU-23 and TCM-199, maturation rates of NCSU-23 and TCM-199 (72.43±3.84% vs 67.91±5.11%)were no significantly different(P>0.05), but they were significantly higher than PZM's(56.09±2.12%)(P<0.05).Matured oocytes were stimulated electrically separately with different pulse strengths and pulse times ,one pulse, and were treated for 3-4h by CB(7.5μg/ml)after post-activation, the rates of cleavage (64.04±1.72%)and blastocysts formation(24.64±1.79%) were significantly higher than others after electrical stimulation at 1.20 KV/cm, 30μsec once , while the death rate was hoisting with increasing of pulses of strength and time.The studies were undertaken to determine the different effects of electrical activation combined with CB,CHX,6-DMAP,CB+CHX,CB+6-DMAP,CHX+6-DMAP,CB+CHX+6-DMAP on the parthenogenetic embryonic development in vitro. the rates of cleavage(80.50±4.52%) and blastocysts formation (32.22±1.92%)were significantly higher than others after electrical stimulation by 6-DMAP(P<0.05).And the rate of combination activation was significantly higher than the control(P<0.05).Porcine parthenogenetic embryos were cultured in NCSU-23,PZM-3 and BECM-3.The rates of cleavage (69.38±3.99%,69.87±3.16%)and blastocysts formation(39.91±2.17%,34.37±5.11%) and the total cell number in blastocysts (48.38±7.87,38.00±8.57)of NCSU23 and PZM-3 were higher than the BECM-3'(57.05±4.16,24.63±4.58,15.33±4.51)(P<0.05) ,And total cell number in blastocysts of PZM-3 was higher than the NCSU23'(P<0.05). The addition of EGF or bFGF to the culture medium significantly enhanced the rates of cleavage and blastocysts formation(36.20±5.37%,44.25±5.83% vs19.01±1.72%) of 2-4-cell stage porcine parthenogenetic embryos (P<0.05);Additionally, bFGF increased the numbers of blastocysts and the rates of blastocysts formation.Results: Under the condition of this experiment, as proputertal gilt sow, this optimized the activation condition of porcine in vitro matured oocytes parthenogenetic activation and culture scheme of in vitro culture of parthenogenetic embryos. the oocytes are activated better with electrical activation combined with 6-DMAP for 3h, and the parthenogenetic embryos cultured in PZM-3 have higher rates of blastocysts formation and the total cell number in blastocysts. Parthenogenetic embryos cultured in NCSU-23 with 10ng/ml bFGF the 2-4-cell stage have higher rates of blastocysts formation and the total cell number in blastocysts, Which will provide scientific foundation for the study and improvement of porcine embryos engineering technology... |
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