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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。

The experiment results were as following:All cytoplast recipients derived from oocytes of rabbit, bovine and goat can support early development of cloned embryo, the fusion rate of reconstructed embryos of goat-bovine and goat-rabbit were 59.91% and 74.10%, their cleaved rate were 57.48% and 72.57%, blastocyst rate were 9.59% and18.06% respectively, and the fusion of reconstructed embryos of goat-rabbit and goat-goat were 60.01% and 74.10%, their cleaved rate were 56.68% and 72.57%, blastocyst rate were 11.76% and18.06% respectively.

试验获得了如下结果:1。以山羊耳成纤维细胞为供体,以牛、羊、兔卵母细胞做为胞质受体构建重构胚,并经体外培养,发现不同的胞质受体均能支持重构胚的早期发育,但受体胞质对重构卵的重构胚早期发育速度和卵裂率均有显著影响。

At the small growth stage, three kinds of the sex steroid receptors usually were localized in the cytoplasm or nuclear membrane of oogonia and early primary oocyte, and localized in the cytoplasm and nucleoplasm of oocytes at the large growth stage and mature stage.

在小生长期,3种受体通常定位在卵原细胞和早期初级卵母细胞的胞质或核膜;在大生长期和成熟期,则定位在胞质和核质。

Methods: Lecithin and the sucrose ester-phospholipids liposome were prepared using the ethyl ether injection method by mixing sucrose ester with lecithin, sphingomyelin and cholesterol according to different lipids ratio.

按不同比例将乙酸异丁酸蔗糖酯与卵磷脂、鞘磷脂和胆固醇混合,溶于乙醚中,然后注入pH7.2的PBS缓冲液中,制成卵磷脂脂质体及蔗糖酯-卵磷脂脂质体。

Recombinant plasmid pSVH 7 DNA of avian influenza virus H7 subtype heamagglutinin gene was encapsulated with DC-chol/DOPE liposomes and PC/chol/SA liposomes separately. Two-week old SPF chickens were intramuscularly inoculated with 50 μ g/0.2ml of the liposome entrapped PSVH 7 DNA. Four-weeks later, each chicken was challenged with 0.1ml 〓 AIV . One week after the challenge, the secretion of the cloacas was collected and transfected to chicken embryos to isolate the virus. The virus was isolated from 6/6 of the control group, 1/6 of the naked DNA group, 1/6 of the PC/chol/SA entrapped DNA group and 0/6 of the DC-chol/DOPE liposome entrapped group. The HI antibody titers (log2) of the four groups were 6. 83±0.98, 7. 0±1. 26, 7. 83±1. 17 and 8. 00±0.89 respectively 1-week after challenge, and 8. 5±0.55, 8. 17±0.82, 8. 68±0.45 and 9. 33±0.54 respectively 2-week after challenge. The results showed that inoculation of liposome entrapped DNA significantly enhanced resistance to virosis in animals.

将含禽流感病毒H7亚型血凝素基因的重组质粒pSVH7用DC-chol阳离子脂质体和胆固醇/卵磷脂/十八胺脂质体包裹,免疫2周龄SPF鸡,4周后用同型禽流感病毒进行人工感染,1周后采集泄殖腔分泌物分离病毒,结果未免疫组6/6分离到病毒,裸质粒DNA免疫组1/6分离到病毒胆固醇/卵磷脂/十八胺脂质体包裹DNA免疫组1/6分离到病毒,DC-chol脂质体DNA免疫组没有分离到病毒(0/6):人工感染后1周各组的HI抗体(Log2)分别为6.38±0.98,7.00±1.26,7.83±1.17,8.00±0.89,2周后为8.50±0.55,8.67±0.82,8.68±0.45,9.33±0.52,脂质体包裹组在同期均高于未免疫组和裸DNA免疫组,表明脂质体包裹质粒DNA免疫动物后,能增加动物对病毒感染的抵抗力和反应能力。

The new GV preparation method we developed allowed the best visualization of chromatin distribution after Hoechst or CMA3 staining. Our results showed that GV chromatin configurations are different among different species, and thus, while chromatin condensed into a ring around the nucleolus in porcine and bovine oocytes, no such a ring was observed in caprine oocytes; Porcine oocytes were synchronized at GV1, goat oocytes at GV3n and bovine oocytes at GVf configurations before GVBD occurred, indicating that these configurations are progressive towards final maturation rather than atresia; Changes of chromatin configurations were associated with gene activity of transcription; GV chromatin configurations were affected by the environmental changes such as serum starvation and elevated temperature.

应用该技术对猪、山羊和牛不同大小、不同健康程度卵泡及体内和体外成熟过程中卵母细胞染色质构型的变化发现:1)不同动物卵母细胞染色质构型不同;猪和牛卵母细胞染色质围绕核仁凝集形成环,但山羊卵则没有此环。2)卵母细胞在GVBD之前都同步在某一染色质构型;猪同步在GV1,山羊GV3n,牛GVf,说明这些染色质构型代表着进行性变化。3)染色质构型的变化反映基因转录活动的变化。4)染色质构型受环境变化的影响。

This paper reviews the ooplasmic transfer in summary, the relation between ooplasm and fertilization and embryo development, the hereditary behavior of heterogenous mitochondria DNA and it's examination after ooplasmic transfer.

通过卵胞质转移研究概况、卵胞质与受精和胚胎发育、异种线粒体DNA遗传方式和卵胞质转移遗传物质的检测4个方面对卵胞质转移技术进行讨论。

The results showed that immunopositive substance existed in the ovary and testis of different stages. At the early developmental stage of ovary, the cytoplasm and nucleolar membrane of oogonia and early primary oocyte showed immunopositive reaction with strong or medium intensity to 17 b -estradiol, testosterone and progesterone antibodies.

在卵巢发育早期,卵原细胞和卵母细胞的胞质和核仁膜对雌二醇、睾酮和孕酮抗体显示强或中等强度免疫阳性反应;在大生长期和成熟期,卵母细胞胞质和核仁膜对睾酮和孕酮抗体的免疫阳性反应明显减弱,而对雌二醇则显著增强。

Stage I of both male and female stone flounder appears only once in all its life, and the germ cells are comprised by spermatogonia or oogonia. The gonad from Mar to Aug keeps at stage II. The gonad index of testis at this stage is 0.037%, and the amount of spermatogonia is increased quickly. There is some linear germ plasm in the cytoplasm of spermatogina. In ovary at this stage it is mostly composed by oocyte of phase 2 which the character is the appearance of yolk nucleus, and no zona radiate in membrane. The mean GI of ovary is 1.95%. From Sep to Oct gonad is at stage III which testis is composed by lots of spermatogina and few spermatocytes, and the mean GI of testis at this stage is 0.086%. In ovary the ooctyes at phase 3 are in dominate position, the yolk nucleus disappear. And the GI of this stage is 3.35%. Both testis and ovary are at stage IV in Nov. hi testis the germ cells are in spermiogenesis, and the mean GI is 0.93%. hi ovary the oocytes are mostly at phase 4, which are filled in the cytoplasm with vitellin granule, and the zona radiate in membrane begins to formation. Nucleus moves to one side of the oocyte gradually. The mean GI of ovary at this stage is 9.37%.

在每年的3月-8月期间性腺处于Ⅱ期,此期精巢中精原细胞明显增多,胞质局部可见有线状的生殖质存在,平均成熟系数为0.037%;卵巢中以2时相卵母细胞为主,可见细胞质中出现强嗜碱性的卵黄核,细胞外由一层滤泡细胞包围,但尚无放射带,平均成熟系数为1.95%。9月-10月期间性腺处于Ⅲ期,此期精巢中仍有大量精原细胞,同时可见部分精母细胞,平均成熟系数为0.086%;卵巢中以3时石鲜孟加限加$玩印面n洲匆s性腺发生、分化及发育的周年变化相卵母细胞为主,细胞质中的卵黄核己消失,平均成熟系数为3.35%。11月性腺处于IV期,此期精巢内精细胞正处于不同的形成过程中,平均成熟系数为0.93%;卵巢中以4时相卵母细胞为主,胞质中充满染成桔红色的卵黄颗粒。

In the present study, we collected cumulus cells oocyte complex from ovaries of two different strain mice. The cumulusenclosed oocytes were cultured for 6 h in MEM supplemented with growth factor and FSH. The meiotic maturation of these oocytes has progressed to pro-metaphse Ⅰ stage and the condensed chromosomes are visible under DIC microscope, metaphase Ⅰ spindle even can be detected under Polscope. The metaphase Ⅰ spindles of oocytes were exchanged under such microscopes. After electric stimuli, 91. 6% and 91. 6% karyoplasts-cytoplasm pairs were fused respectively. The resulting oocytes were cultured further in MEM and over 80% of oocytes released the first polar body. 79% and 77% of oocytes formed two pronuclei after in vitro fertilization and the embryos were cultured in KSOM supplemented with amino acids. Over 60% of embryos developed to blastocyst stage.

在本研究中我们在取得两种不同品系小鼠的卵丘卵母细胞复合体后,先将卵丘卵母细胞复合体置于含有多种生长因子和激素的MEM培养液中培养6小时,此时卵母细胞已进入第一次减数分裂的前中期,并且在DIC倒置显微镜下可以看到浓缩的染色体,用Polscope可以发现明显的纺锤体,借助这种显微镜通过显微操作将两种不同品系小鼠来源的卵母细胞的MI纺锤体进行互换,经过三次直流电脉冲作用后,分别有91.6%的胞质—MI核质体对融合,经过进一步的培养后,超过80%的重组卵母细胞排出第一极体,体外受精后分别有79%和77%的重组卵形成双原核,受精后的胚胎在KSOM胚胎培养液中体外培养4天后,超过60%的胚胎发育至囊胚。

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