牛磺酸、谷酰胺、改良CR1和改良CR2培养液对牛受精卵体外发育的影响

本研究探讨了牛磺酸、谷酰胺、改良CR1 (CR1 无机盐成分 10% 胎牛血清) 和改良CR2 培养液(改良CR1 液 1 m m ol/L谷酰胺和绵羊输卵管分泌液浓度的多种氨基酸)对牛卵子体外受精后的分裂率、囊胚率和囊胚细胞数的影响。结果表明: 牛磺酸和谷酰胺对牛卵子受精后体外发育的影响依赖于胚胎培养液种类; 利用改良CR1 和改良CR2 培养液可成功生产可移植胚胎, 但它们所生产的可移植胚胎的效率无显著差异, 利用改良CR1 培养液生产可移植胚胎的成本更低一些。     

Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

利用IVM/IVF技术生产试管犊牛的研究

本研究利用卵母细胞体外成熟(IVM)、体外受精(IVF)技术,体外生产牛胚胎,以获得试管牛.结果表明:卵母细胞体外培养22～24h后,其成熟率为85％;体外受精率为83％.体外受精卵分别在颗粒细胞单层(GCM)和输卵管细胞单层(OCM)上培养,其胚胎最后产量(以授精时卵母细胞的数目为基数)分别为19％和29％,差异极显著(P<0.01).若体外受精卵先在GCM中培养72h后,再将已发生卵裂的(>4细胞)胚胎移到OCM中培养,其胚胎最后产量为35％.由此表明,早期胚胎在其发育过程中至少存在着3个发育相期,即1～8细胞、8～16细胞和16细胞～囊胚3个阶段,各阶段需要不同的培养环境.IVM/IVF胚胎移植到受体黄牛体内后,其足月时的妊娠率为15％.第一头试管犊牛已于1993年4月18日凌晨产出.     

克服昆明白小鼠胚胎发育阻滞的初步研究

为克服昆明白小鼠胚胎的2—细胞阻滞并使受精卵发育到囊胚期,本研究设两个实验组和一个对照组。第1实验组所用培养液为Brinster氏化学合成培养液与输卵管上皮和输卵管液混合的协同培养液;第2实验组用改良Brinster氏化学合成培养液;对照组用经典的Brinster培养液。培养是在5％CO_2和95％空气,37℃的CO_2箱内进行。3次体外培养试验结果是:第1实验组小鼠胚胎从受精卵发育到囊胚期平均发育率为43.7±7.76％;第2实验组的平均发育率为17.1±5.06％;对照组的平均发育率是15.3±3.06％。统计学分析表明,添加输卵管上皮和输卵管液的Brinster培养液对克服昆明白小鼠胚胎2—细胞阻滞具有一定作用。     

葡萄双受精过程的形态学观察

观察了葡萄双受精过程中雌雄性核的融合。结果表明:花粉管通过珠孔穿入退化助细胞,释放出二精子;精子核接近并贴附在卵核和次生核上,二者核膜融合,精核沉入卵核及次生核,染色质松解,并与雌性核染色质融合,出现雄性核仁,两性核仁融合,形成合子及初生胚乳核。卵核完成受精较次生核晚。受精属有丝分裂前型。精核进入卵核前看到了它的核仁。少数胚囊具有两套卵器。     

黄花菜胚囊受精前后的超微结构观察

 应用透射电镜技术研究黄花菜（Hemerocallis citrine Baroni）助细胞、卵细胞、中央细胞、 合子与初生胚乳核的超微结构特征。结果如下：助细胞有高度发达的丝状器;一个助细胞在花粉管进入 后退化;宿存助细胞在合子期退化。受精前,卵细胞、助细胞合点端无壁,细胞膜完整;受精后,合子 被完整的细胞壁包围。受精前超微结构特征显示卵细胞代谢活动低,合子期的代谢活性被激活;中央细 胞、初生胚乳核代谢活性较高。卵细胞质中的精核由双层核膜包被,染色质高度凝聚,核孔数少。助细 胞、卵细胞、中央细胞缺乏中央大液泡是黄花菜成熟胚囊的显著特征。     

鼠尾藻有性繁殖和幼孢子体发育的形态学观察

野外跟踪观测烟台芦洋湾地区鼠尾藻的成熟繁殖状况,采集临近成熟的鼠尾藻至实验室培养,跟踪观测其卵子的排放、卵子和受精卵的发育、假根的生长以及幼孢子体的前期发育。试验结果表明,本地区鼠尾藻繁殖期集中在6—8月,鼠尾藻的卵子发育属8核1卵型,卵子受精后约20 h开始脱离生殖托,受精卵先连续进行2次的横分裂,而后再由一端的1个细胞发育成假根,另外2个发育成体细胞。幼孢子体在约2 mm时开始形成分枝突起。     

几种天然饵料对清洁虾亲虾繁殖的影响

采用6种天然饵料喂养清洁虾亲虾,分析其对亲虾性腺发育、繁殖性能以及受精卵质量的影响。性腺发育试验发现,投喂6种不同饵料亲虾性腺发育周期差异显著(P<0.01),最长的为鱼肉组(12.82±1.91)d,最短的为卤虫无节幼体组(11.46±1.76)d;卤虫无节幼体与桡足类组性腺指数、干物质含量及总蛋白质含量随着性腺发育显著增加(P<0.01),增加均在2倍左右。繁殖性能与受精卵质量结果表明,鱼肉组(3 357±621)(粒/g)亲虾相对怀卵量最大,卤虫无节幼体组最小(1 829±213)(粒/g);卤虫无节幼体[(0.110 9±0.016 3)mm3]组亲虾受精卵体积最大,乌贼组最小[(0.096 2±0.014 0)mm3];鱼肉组[(37.33±1.75)μg]的受精卵干重最大,虾肉组最小[(31.00±1.26)μg];桡足类组蛋白质绝对含量[(367.17±25.68)mg/g]在干物质中所占比例(72.34%)最高。此外,6个饵料组中所有亲虾在各抱卵周期内抱卵率与抱卵间期无显著差异。     

Reproductive Deterioration in Buckwheat (Fagopyrum esculentum) under Summer Conditions

To resolve the problem of low and unstable yield in buckwheat under unsuitable environmental conditions, five varieties were grown and compared in summer and autumn. Vegetative and reproductive growth characteristics were investigated, and embryo development was observed with Nomarski's differential interference contrast (DIG) microscopy. In summer, under high temperatures and longer day-length, autumn-type varieties showed faster vegetative growth and later reproductive development as compared to summer-types. In the autumn, with generally good climatic condition for buckwheat growth in southern part of Japan, the embryo sacs and zygotes of both types developed normally. At 3 days after pollination (DAP), the embryos reached the globular stage. In summer, however, mostly abnormal embryo sacs were observed 3 DAP, leading to incomplete development, failure of fertilization or abortion of embryo. The abnormal embryo sacs were classified into 6 types. Abnormality of either type occurred in 91—100 % of the tetraploids, which is higher than in the diploids (50—77 %). The present study, conducted to clarify the factors causing low seed set in buckwheat under summer conditions, provides detailed information for further breeding of environment-tolerant buckwheat. Di-ploid genotypes which are less sensitive to unfavourable conditions and, thus, show more efficient fertilization in summer, should be considered for use as breeding material in buckwheat.     

三角帆蚌怀卵期外侧瓣鳃结构初步研究

对怀卵期三角帆蚌的外侧瓣鳃分别从水平、横、纵等三个方向作组织切片,利用光镜观察其组织形态结构。结果显示,在怀卵期外侧瓣鳃增至(2 876.77±123.58)μm,两鳃小瓣之间被鳃间隔分成许多鳃小腔,其宽度为(474.06±32.01)μm(纵切),(410.74±70.69)μm(水平切)。瓣间隔的厚度为(244.92±17.23)μm(纵切),(288.80±35.16)μm(水平切)。从横切面观察,瓣间隔又由横向隔板组成瓣间隔孔,隔板宽度为(309.2±50.56)μm,两隔板间的孔宽度为(365.22±37.74)μm。鳃小腔通过瓣间隔孔而互相连通,从而形成鳃腔育儿囊。同时还观察到:在胚胎发育初期,受精卵被粘液状物质包裹,随着孵化的进行包裹物逐渐消失。