纤细背孔吸虫的卵黄细胞发育和虫卵超微结构观察

应用透射电镜和扫描电镜观察纤细背孔吸虫（Notocotylus attenuatus）的卵黄细胞发育和虫卵超微结构。结果显示，纤维背孔吸虫卵黄细胞的发育过程分为3个时期：I期为未发育的卵黄细胞期，细胞外形不规则，核大；Ⅱ期为发育中的卵黄细胞期，细胞增大，胞质内出现大量粗面内质网，卵黄颗粒明显增加；Ⅲ期为成熟卵黄细胞期，胞质内可见单个类脂滴并充满卵黄球，卵黄球内含卵黄颗粒9～37个（X^-=20.45,n=20）。虫卵两端具有卵丝，一端具有卵盖，卵盖与卵壳交接处有2条环状隆起，卵内含有胚细胞和脂滴。胚细胞的胞质内含有核二样的细胞质体和线粒体。     

奶牛性控IVF胚胎移植效果的初探

于2004年9月至2005年4月,在内蒙古大正美联奶牛工程有限公司移植的591头奶牛性控IVF胚胎的资料分析:细管常规冷冻法的冷冻胚胎移植效果高于(P<0.05)微滴玻璃化;移植双胚和单胚、发情后第6和7天的受体母牛的移植效果不显著(P>0.05);A级胚胎的移植效果高于B级,差异显著(P<0.05).     

胚胎一分为二移植产犊成功

河北省奶牛胚胎移植技术研究中心,在总结奶牛鲜胚移植,奶牛胚移黄牛等多项技术成果基础上,为探讨扩大胚胎来源技术途径和降低胚胎移植成本的先进方法,以胚胎质量和黄体状况为研究重点,并进行了系统、综合、配套技术的科学探索,取得明显效果。在胚胎冷冻上,突破了胚胎-7℃     

提高奶牛胚胎移植妊娠率的措施

随着奶牛价格的大幅降低,奶牛胚胎移植技术推广应用受到了大大限制,要想推广技术必须提高奶牛胚胎移植妊娠率,降低胚胎移植犊牛生产成本。1胚胎方面除了选择优秀供体奶牛外,还要选择品质优良种公牛,精液活力高,一般冻精活力在0.6以上,精子含量在1000万个/m L。一般鲜胚A、B、C级胚胎受胎率分别为64%、45%、33%;冷冻胚胎A级受胎率为45%,B级受胎率为40.02%,C级受胎率为12.19%。所以一般冻胚移植A、B级胚胎,C级胚胎直接进行鲜移植,不进行冷冻。由于冷冻对胚胎有一定的危害,当条件允许时,尽量采用鲜胚进行移植。2受体方面受体牛选择要按照严…     

成都奶牛胚胎移植中心的现状和展望

成都市目前有1万多头奶牛。为提高奶牛单产,从70年代开始采用冷冻精液技术,80年代广泛应用奶牛胚胎移植技术。成都市从事奶牛胚胎移是从1985年开始的。在中国科学院遗传所专家的指导下,熟练、准确地掌握了从同步发情、超数排卵、冲胚、检胚、分割到移植等复杂的技术,已获得胚胎侈植牛只近70头,鲜胚移植成功率在50％,冻胚     

体外生产的牛胚胎超微结构的研究

利用两种常规体外培养系统研究了体外培养及不同培养条件对牛体外受精胚胎早期发育各时期超微结构的影响。结果显示：体外发育的牛胚胎在发育的各个时期（从原核期到囊胚）胞质中均含有丰富的脂滴，胚胎中特别是在桑椹胚及囊胚阶段可见到一些异常细胞。两系统下发育的胚胎中脂滴形态不同，从桑椹胚阶段开始，胚胎线粒体的发育在M199-BOEC-FCS培养系统中明显滞后于其在SOFaa-BSA培养系统中的对照。     

体外生产的牛胚胎超微结构研究

利用两种常规体外培养系统研究了体外培养及不同培养条件对牛体外受精胚胎早期发育各时期超微结构的影响。结果显示:体外发育的牛胚胎在发育的各个时期(从原核期到囊胚)胞质中均含有丰富的脂滴,胚胎中特别是在桑椹胚及囊胚阶段可见到一些异常细胞。两系统下发育的胚胎中脂滴形态不同,从桑椹胚阶段开始,胚胎线粒体的发育在M199 BOEC FCS培养系统中明显滞后于其在SOFaa BSA培养系统中的对照。     

玻璃化冷冻对猪卵母细胞超微结构的影响

本研究以GV期和MII期卵母细胞作为试验材料,通过透射电镜方法观察卵母细胞冷冻前后的超微结构变化,结果发现,透射电镜下观察卵母细胞发现,GV期卵母细胞与透明带连接紧密,微绒毛伸入透明带中,皮质区分布大量的线粒体。脂滴分为两种,一种为灰色脂滴,一种为深色脂滴。MII期卵母细胞排出第一极体,质膜下分布大量的皮质颗粒。微绒毛缩短成矮柱状,线粒体常聚集存在,卵周隙出现。冻后GV期卵母细胞微绒毛消失,线粒体肿胀,线粒体内有囊泡样结构,包围脂滴的内质网不完整,胞质内溶解为絮状,未见高尔基体。冻后MII期卵母细胞透明带损伤、微绒毛、细胞膜损伤甚至消失、极少量皮质颗粒分布于皮质区。脂滴部分溶解,相互连接,脂滴周围伴随大量肿胀呈圆形的线粒体,嵴不明显,内呈囊泡样,未见到高尔基复合体结构。     

影响猪卵母细胞玻璃化冷冻保存效果的因素

对卵母细胞进行冷冻保存,可以使卵源不再受到时间和空间限制,并能为体外受精、核移植和转基因等胚胎工程技术的研究提供充足的原材料.猪卵母细胞中富含脂滴,且对低温十分敏感,因此,猪卵母细胞冷冻保存的研究进展相对较慢.玻璃化冷冻是有效的卵母细胞冷冻方法之一.综述了卵母细胞发育阶段、冷冻栽体和冷冻保护剂等因素对猪卵母细胞玻璃化冷冻保存效果的影响.     

牛胚胎性别鉴定与取样胚胎移植应用技术的研究

本研究应用ＰＣＲ技术扩增牛ＳＲＹ序列进行奶牛胚胎性别鉴定。经１０９枚鲜、冻胚的移植，获鲜胚移值妊娠率５８．６％（３４／５８），常规冷冻胚胎移植妊娠率４４．４％（１２／２７），一步细管冷冻解冻胚胎移值妊娠率１６．７％（４／２４）。犊牛性别验证与ＳＲＹ鉴定结果均相符合。实验中对胚胎发育时期的划分，胚胎质量评定和胚龄的确定，胚龄与受体发情时间在移植中的关系，胚胎的切割取样，取样胚胎的冷冻进行了研究。     

Electron microscopic studies of the morphogenesis of duck enteritis virus

The morphogenesis of duck enteritis virus (DEV) and distribution in vivo were observed by electron microscopy after ducks were infected experimentally with DEV virulent strain. The investigation showed that a few typical herpesvirus virions and nucleocapsids were first observed in the spleen, thymus, and bursa of Fabricius (BF), and many nucleocapsids, mature viruses, and viral inclusion bodies could be found in the nucleus and cytoplasm of infected liver, small intestine, spleen, thymus, and BF when the ducks died. Nucleocapsids assembled both in nucleus and cytoplasm and could be divided into four different types according to their structures. Typical herpesvirus, light particles (L-particles), and virions without tegument could be observed at the same time. With the replication, assembly, and maturation of the viruses, intracytoplasmic and intranuclear inclusion bodies, electron-density particles, microtubules, hollow tubes, and coated electron-density bodies were observed in infected cells.     

Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi)

The object of this study was to examine the erythrocytes, leukocytes and thrombocytes of the giant lizard of El Hierro (Gallotia simonyi) by light and electron (TEM) microscopy, and cytochemical staining. Smears were prepared from blood from the ventral coccygeal vein of 10 healthy adult lizards (five males and five females) from the Giant Lizard of El Hierro Reproduction and Research Centre, Canary Islands, Spain. The cytochemical stains used were: benzidine peroxidase (BP), chloroacetate esterase (CAE), alpha-naphthyl acetate esterase (ANAE), acid phosphatase (AP), periodic acid-Schiff (PAS), toluidine blue (TB) and May-Grünwald-Giemsa (MGG). Electron microscopy was also performed on all samples. Heterophils had granules that were heterogeneous in both size and electron density, and stained with BP, PAS and ANAE. Eosinophil granules were homogeneously electron-dense and stained for AP, CAE and ANAE. Basophils had both highly and moderately electron-dense granules, and stained with TB and ANAE. Azurophil granules were of low electron-density and stained for AP, CAE and ANAE. Azurophil cytoplasm was vacuolated on TEM. The cytoplasm of lymphocytes contained many ribosomes and was positive for AP. Monocytes had a large nucleus and a vacuolated cytoplasm but did not stain by any of the cytochemical methods used. Thrombocytes had a relatively large nucleus but little cytoplasm; they did not stain cytochemically. The blood cells of the giant lizards of El Hierro differ from those of other members of the Order Squamata both morphologically and cytochemically. The variation in cytochemical responses in the blood of reptiles makes it necessary to study species individually if meaningful clinical decisions are to be made.     

Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach

In this study, the fluorescent lipid dye Nile Red, was used to demonstrate that the lipid content of immature bovine oocytes is correlated with the morphological appearance of the ooplasm. Oocytes with a uniform dark cytoplasm contained significantly more intracellular lipids in lipid droplets compared with oocytes with a granulated or pale cytoplasm (p < 0.05). Furthermore, this lipid-analysing technique was applied for the first time on single bovine in vitro embryos, showing a significant increase of the lipid content in lipid droplets after culture in the presence of serum (p < 0.05).     

Immunoferritin and immune electron microscopic study of bovine herpesvirus strain DN-599.

Intracellular antigens of strain DN-599 bovine herpesvirus were detected in the cytoplasm and the nucleus of infected bovine embryonic kidney cells by the indirect immunoferritin (IF) technique. Specific tagging was observed in viral envelope and capsids. Aggregates of viral particles heavily coated with antibody were seen by immune electron microscopy (IEM).     

Studies on the replication of a bovine parvovirus

Optimal replication of a bovine parvovirus type 1 was found to occur when parasynchronous bovine embryonic lung cells were infected during the S phase of the cell cycle, just prior to maximum DNA synthesis. Viral antigen was first detected in the cytoplasm by immunofluorescence at 8 h post-infection, reaching a maximum at this location by 16 h and then disappearing. In the nucleus, antigen was first detected at 12 h, concurrent with early inclusion body formation and first detection of intracellular virus production. Intranuclear antigen then increased rapidly to a maximum at 20 h, as the inclusions progressively matured, large amounts of virus were produced within the cell, with some release to the environment. From 24 h, the nuclear inclusions became increasingly shrunken and basophilic as virus migrated to the cytoplasm and was progressively released to the exterior concurrent with cell degeneration and fragmentation. The majority of virus remained cell associated, even at 28 h post-inoculation. Two morphological types of early and late stage intranuclear inclusions were produced by the virus, these appearing to be a distinct feature of bovine strains. In other aspects, the replication of bovine parvovirus appeared similar to that of other members of the genus.     

牛病毒性腹泻病毒侵染宿主细胞的电镜观察

用电镜观察了牛病毒性腹泻病毒Oregon C24株在侵染新生犊牛睾丸细胞中的形态发生。成熟的病毒颗粒呈直径约为50nm的球形颗粒，内含直径约为30nm的核芯。病毒侵染宿主细胞后，在胞质内复制，通过糙面内质网膜出芽成熟，病毒可通过侵染细胞外排或在细胞死亡后含有病毒颗粒的空泡破溃而释放到胞外。     

Bovine herpesvirus-5 (DN599) antigens in cells derived from bovine ocular squamous cell carcinoma.

Bovine herpesvirus 5 antigens were detected by indirect immunofluorescence in the cytoplasm of tumor cells derived from bovine ocular squamous cell carcinomas. The number of tumor cells expressing bovine herpesvirus 5 antigens was increased following exposure to ultraviolet light or maintenance in pH 8.4 media. Infectious virus could not be detected in homogenates of tumors or by explantation of tumors. However, medium removed from the ocular tumor explants induced cytoplasmic vacuolization, inclusions and syncytia when inoculated onto bovine fetal spleen monolayers. Acridine orange staining revealed these cytoplasmic inclusions to contain double-stranded deoxyribonucleic acid. These results provide evidence that bovine herpesvirus 5 may be associated with bovine ocular squamous cell carcinoma.     

Heterospecific Nuclear-transferred Embryos Derived from Equine Fibroblast Cells and Enucleated Bovine Oocytes

This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin with 2 mm/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The results showed that the first completed cleavage of xenonuclear transfer equine embryos occurred between 30 and 48 h following activation. 52% of the injected oocytes were successfully fused, 72% of the fused eggs underwent the first egg cleavage and 17% of the heterospecific nuclear-transferred zygotes developed to 4- or 8-cell embryo stages. This study demonstrated that the reconstructed embryos have undergone the first embryonic division and the reprogramming of equine fibroblast nuclei can be initiated in bovine-enucleated oocytes.     

亮甲酚蓝染色法筛选卵母细胞对牛体细胞克隆胚体外发育的影响

为探索用亮甲酚蓝染色法筛选卵母细胞是否有利于牛体细胞克隆胚的体外发育,将采集的卵母细胞分成3组:对照组,采集后的卵母细胞直接放入成熟液培养,不用亮甲酚蓝处理;控制对照组,卵母细胞于PBS 中放置90 min后放入成熟液中培养;试验组,将采集的卵母细胞在含有26 μmol/L亮甲酚蓝的PBS中放置90 min.然后将处理过的卵母细胞根据着色情况分为两组,即BCB+(胞质呈蓝色,成熟卵母细胞)和BCB-(胞质未着色,生长期卵母细胞).各组卵母细胞24 h体外成熟培养,统计各组卵母细胞成熟率.分别用成熟后的各组卵母细胞用于细胞核移植,统计体细胞核移植的卵裂数、囊胚数和囊胚细胞数.结果表明,BCB+组卵母细胞的成熟率明显高于BCB-组卵母细胞的成熟率(74.0%,53.4%),差异显著(P<0.05).BCB+组的囊胚率(45.0%)与对照组、控制对照组和BCB-组的囊胚率(34.9%,36.4%和5.2%)相比差异显著(P<0.05).另外BCB+组的囊胚细胞总数(114.5±7.5)较其他组差异显著(P<0.05).表明通过亮甲酚蓝染色法筛选出成熟的高质量牛卵母细胞用于体细胞核移植,可以获得更高的克隆囊胚率.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.