| In this paper , we took 44 female New Zealand Rabbits as object, mainly researched in vitro maturation,fertilization of oocyte on ovarian surface and in vitro culture of zygote,our purposes were optimizing the system of in vitro maturation,fertilization of rabbit oocyte and in vitro culture of zygote,results were as follows.1. Cumulus and oocyte complex (COCs) on the surface of ovaries were gotten by the method of cutting with Blade or stab-extruding with needle,the number of COCs gotten by the method of stab-extruding with needle(23.63 per rabbit )were significant larger than by the method of cutting (16.50 per rabbit )(P <0.05).2. Supplemented with diffirent concentration of FSH+LH in the maturation medium,then COCs were IVM under the condition of 38.5°C,5 % CO2 and saturated humidity,the time of IVM was 30 h,the results showed that the rate of maturation by supplementing 1 U/mL FSH+2 U/mL LH was significent higher than 10 U/mL FSH+20 U/mL LH,and more significent higher than contrast group(TCM199+10 %FCS).The rate of maturation were respectively 46. 62 %,26.83 %,13. 71 %.3. This experiment compared the rate of maturation of oocytes by the time of 24 h,30 h,36 h or 40 h,the results indicated that culture time of 30 h or 36 h were better than 24 h or 40 h(P <0.05).The rate of maturation were 21.92 %,46.62 %,52.77 %, 30.71 % respectively.4. Basic maturation medium was TCM199+FCS+FSH+LH+E2+pyruvic sodium,added epidermal growth factor (EGF)50 ng/mL,EGF 100ng/mL,stem cell factor (SCF)10 ng/mL,SCF 20 ng/mL,leukemia inhibitory factor(LIF)10 ng/mL,LIF 20 ng/mL,then counted rate of first polar body emission by the time of 30 h .Results manifested the first polar body emission rate in group of Adding 50 ng / mL and 100 ng / mL EGF were significantly higher(P <0.05),first polar body emission rate of contrast group,50 ng / mL group and 100 ng / mL was respectively 49.04 %,61.39 % and 65.79 %,SCF could not enhance the first polar body emission rate,first polar body emission rate was respectively 54.17 %,50.00 % and 45.83 %,LIF obviously restrained the maturation of oocyte,first polar body emission rate was respectively 54.19 %,12.50 % and 4.17 %.5. Test compared three methods of sperm handlling on sperm recovery rate and cleavage rate , they were swimming-up , direct washing and density gradient centrfugation,results showed that the method of density gradient centrfugation was a ideal method(sperm recovery rate of the three ways were 7.8 %,93.9 % and 86.9 %,cleavage rate after the fertilization were 0 %,39.05 % and 54.29 %).6. Cleavage rate of in vitro fertilization and cleavage rate of intracytoplasmic sperm injection was compared,result was that the cleavage rate of former(63.97 %)was more significant higher than the later(25.65 %)(P <0. 01).7. The experiment compared embryo developmental potential capacity between TCM199+10 %FCS group and DMEM+10 %FCS group,the result indicated that they have no significant difference(P >0. 05).8. Supplemented with vitmin C(100μmol/L)and mercaptoethanol (50μmol/L)in the zygote culture medium,zygote of 2-cell (from in vitro fertilization)were cultured under the condition of 38.5℃,5% CO2 and saturated humidity,then statisticed the rate of 4-cell and of 8-cell or later,the results were that this three groups have no significat difference,Whether the rate of 4-cell zygote or rate of 8-cell zygote or later. |
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