囊胚

By statistic analysis, there were significant differences with the average cell number of each morulae, early blastula and blastula between in vivo and in vitro, there were no differences with the hatching of blastula between in vivo and in vitro.

经统计分析体内桑椹胚、早期囊胚、囊胚的每一胚胎平均细胞数与体外桑椹胚、早期囊胚、囊胚的每一胚胎平均细胞数有显著差异；扩张囊胚的体内，体外每一胚胎平均细胞数没有显著差异。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明：利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程，在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样，呈现高度甲基化状态；在细胞核开始融合的时候，甲基化水平急速下降，在细胞核完全融合的时候甲基化水平达到最低点；随着胚胎继续分裂，胚胎甲基化水平逐渐增加，在桑葚胚期甲基化水平最高；但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别，这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cyto-plasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methy-lation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribu-tion of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明：利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程，在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样，呈现高度甲基化状态；在细胞核开始融合的时候，甲基化水平急速下降，在细胞核完全融合的时候甲基化水平达到最低点；随着胚胎继续分裂，胚胎甲基化水平逐渐增加，在桑葚胚期甲基化水平最高；但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别，这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastcysts fellowed by fusion of cytoplasm and nucleus and cleavage in vitro.After fusion of cytoplasm, the DNA methylation levels of the fused embryos was very high as well as two-cell diploid embryos in vivo.Then the embryos was rapiddly demethylated when the nucleus begin to fuse, resulting the lowest DNA methylation levels when the nucleus fused completely.After that, the DNA methylation levels of fused embryos were gradually increased until the blastocysts stage.However, whereas an asymmetric distribution of DNA methylation was established in an vivo-derived blastocysts with a higher methylation level in the inner cell mass than in the trophectoderm, in most vitro-derived tetraploid blastocysts, we can not detect the asymmetric distribution.

结果表明：利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程，在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样，呈现高度甲基化状态；在细胞核开始融合的时候，甲基化水平急速下降，在细胞核完全融合的时候甲基化水平达到最低点；随着胚胎继续分裂，胚胎甲基化水平逐渐增加，在囊胚期甲基化水平最高；但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别，这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

The results showed that 1 there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 μg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 μg/mL) for 1 h, and the rate of cytoplast protusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2 There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 μg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 μg/mL colchicine for 0.5 h. 3 A maturation time of 18–23 h did not affect the rates of cytoplast protusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4 The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21–23 h.

结果表明： 1 卵母细胞在0.4 mg/mL的秋水仙素溶液中分别孵育0.5 h和1 h，胞质突起率和去核率没有显著的差异，突起率可高达85.4%，去核率达到100%； 2 0.2 mg/mL或0.4 mg/mL秋水仙素溶液将卵母细胞处理0.5 h，对去核效果没有显著影响； 3 对于体外成熟18~23 h的卵母细胞，随着成熟时间的延长，盲吸法的去核率降低，但没有影响秋水仙素诱导胞质突起的比率和去核率； 4 两种去核方法对重构胚的发育没有产生显著影响，但成熟21~23 h卵母细胞重构胚囊胚的发育率显著高于成熟18~20 h卵母细胞重构胚囊胚的发育率。

With the prolonging of treatment with 6-DMAP,the developmental ability of parthenogenetic embryos decreased.

随着6-DMAP处理时间加长，孤雌胚的发育能力降低。6-DMAP处理1小时的桑葚胚／囊胚的发育率(72％)远远高于6-DMAP处理6小时的桑葚胚／囊胚的发育率(28％)。

Then we compared cleavage rate and blastocyst rate among different groups. After that we evaluated the quality of blastocysts by using ICM/TE (Inner cell mass/Trophectoderm cells) ratio via staining with propidium iodide and Hoechest333258, and analyzed the level of genome methylation and histone acetylation by immunofluorescence.

比较各组受精卵的卵裂率和囊胚发育率；并应用碘化丙啶和Hoechest333258对囊胚进行染色，利用ICM/TE值评价各组胚胎体外发育的质量；同时将囊胚进行免疫荧光染色，观察其基因组甲基化和组蛋白乙酰化的水平。

This experiment object was 1-cell embryo of KUNMING mouse , ROS was added in different developmental phases through basal medium with CZB medium, cultured to morulae, early blastula, blastula and the hatching of blastula, observed the change of cell number and cleavage index of embryo.

本试验以昆明小鼠1-细胞期胚胎为实验对象，以CZB培养液为基础液在不同发育阶段添加外源性ROS，培养至桑椹胚、早期囊胚、囊胚、扩张囊胚阶段，分别观察胚胎的胚细胞数、分裂指数的变化。

In the experiment, the FSH+LH combinations were adopted to superovulate KM mice to ascertain the proper phase to gain morulae and blastocysts, three ways were adopted to split the morulae and blastocysts, demi-embryos were cultured in D-PBS, mTCM199, CZB, mM16 added with FCS or MS of different concentration, and transplantation of the blastocysts derived from demi-embryos was performed.

本试验以昆明白小鼠为实验动物，试采用FSH和LH进行超排处理，确定了该处理程序下获取桑椹胚、囊胚的恰当时间，比较了三种方法对桑椹胚和囊胚分割的效果，采用了添加不同浓度FCS、MS的4种培养基对分割胚进行体外培养，并进行了半胚移植。

Our study includes four aspects. In the first aspect we study several important conditions of porcine oocytes maturation in vitro and oocytes cleavage after parthenogenetic activation and found mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15% PFF+0.57mMcysteine is a good culture condition .When the Cocs are cultured in it ,the maturation rate and oocytes cleavage rate are higher than those of foreign covered. Our result are (86.7±3.35)% and (86.3±4.16)% and the highest report of foreign is(85.7±4.1)%.In the second aspect we study the effect of different chemical activations on development of porcine parthennogenetic embryo and found two best activation method. The first one is that putting the maturation MII oocytes in the 20μmol/L ionomycin for 30 minutes and then putting them in the NCSU-23 condition containing 5μg/mICB and 5mM/L6-DMAP for 3.5 hours, the oocytes cleavage rate and morulae/blastocysts development rate are (76.7±7.6)% and (37.1±6.4)%.The second one is that putting the maturation MII oocytes in the 200μM/L Thimerosal for 20 minutes and then putting them in the NCSU-23 condition containing 8mM DTT for 30 minutes

本研究分为4个部分，第一部分对影响猪卵母细胞体外成熟和孤雌激活后胚胎分裂的几个重要条件进行了比较研究，确立了一种较好的培养方法：与颗粒细胞共培养，找到了一种适合猪卵母细胞体外成熟的培养基：mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15%PFF+0.57mM半胱氨酸，成熟率和分裂率分别为(86.7±3.35)%和(86.3±4.16)%，国外报道的最高成熟率为(85.7±4.1)%；第二部分对猪卵母细胞孤雌激活的化学方法进行了研究，确立了化学激活猪卵母细胞的两种最佳方法：1将成熟的去卵丘颗粒细胞的MII期卵母细胞用20μmol/Lionomycin作用30min，再将卵母细胞培养于含5μg/mlCB和5mM/L 6-DMAP(6-二甲基氨基嘌呤)的NCSU-23培养液中，卵裂率和桑囊胚发育率达到(76.7±7.6)%和(37.1±6.4)%2将成熟的去卵丘颗粒细胞的MII期卵母细胞在200μM/L的Thimerosal中处理20min，再与8mM的DTT共孵育30min，卵裂率和桑/囊胚形成率为(81.0±2.8)%和(39.6±2.7)%；第三部分对孤雌激活胚胎的培养条件进行了研究，确立了一种最佳的胚胎培养条件：在SOF简单培养基中添加颗粒细胞进行前3天的培养，然后转入添加胎牛血清的NCSU—23培养基并和输卵管上皮细胞进行后期的培养，其桑椹胚和囊胚的发育率为(59.5±3.2)%；第四部分研究了IGF-I

But we don't care about Battlegrounds.

但我们并不在乎沙场中的显露。

Ah! don't mention it, the butcher's shop is a horror.

啊！不用提了。提到肉，真是糟透了。