黄花棘豆生物碱胚胎发育毒性的体外实验研究

应用大鼠全胚胎培养（ＷＥＣ）方法，观察了黄化棘豆主要有毒成分－生物碱对体外胚胎生长发育、形态分化的影响，首先用醇类溶剂提取法从黄花棘豆中提取生物碱，薄板层析鉴定于少有４种生物碱成分。在选定０．１％ＤＭＳＯ为该生物碱溶剂的基础上，分别在含有２５、５０、１００和２００μｇ／ｍｌ生物碱的大鼠血清中培养９．５ｄ胚胎４８ｈ，结果在５０μｇ／ｍｌ以上可影响胚胎生长发育和器官分化，颅臂长、体节数、蛋白质含量及１     

猪核移植重组胚胎的发育能力

以ＭｃＧｒａｔｈ－Ｓｏｌｔｅｒ（１９８３）提供的去（注）核方法，将猪胚胎的单个卵裂球细胞注入去核的次级成熟卵母细胞，借助电融合的方法（ＡＣ；５Ｖ，１．５ＭＨｚ，１２－１５ｓ；ＤＣ：２ｋＶ／ｃｍ，４０μｓ。电激活／融合液：０．３ｍｏｌ／Ｌ甘露醇＋－．１ｍｍｏｌ／ＬＭｇＳＯ４＋０．１ｍｍｏｌＣａｃＬ２＋７．５ｍｇ／ｍｌＣＣＢ）融入受体胞质构成重级胚，体外培养（培养液为改衣ＴＣＭ－１９９；培养条件为５％     

牛体外受精胚胎细胞核移植的实验研究

将采自肉联厂屠宰车间的牛卵巢中的卵母细胞置于ＴＣＭ－１９９＋１０％ＥＣＳ（０ｄ）＋ＦＳＨ（１万ＩＵ／Ｌ）＋ＬＨ（５ｍｇ／Ｌ）中培养成熟，用含０．３％透明质酸酶的消化液将卵母细胞周围的卵丘细胞去掉，并以７．５ｍｇ／Ｌ的ＣＢ液处理后，用微吸管吸去透明带内的第一极体及极体下面的部分卵母细胞质，然后将经体外受精并发育至８～１６细胞期胚胎的卵裂球注入去核卵母细胞的卵周隙中，再将移核胚置于电融合槽内进行融合（电场强度为１２００Ｖ／ｃｍ，持续时间为８０μｓ，１次脉冲刺激）。将融合的胚胎移入生长有单层贴壁颗粒细胞的发育培养液（ＴＣＭ－１９９＋１０％牛血清）中培养，观察移核胚的融合率及发育率，部分去核卵母细胞经染色后观察去核率。结果表明：（１）移核胚的融合率为８６．９％（５３／６１）；（２）发育至２～８细胞期的胚胎占培养的移核胚胎总数的２１．３％（１０／４７）；（３）卵母细胞的去核率为６８．２％（４５／６６）。     

红三叶组织培养及再生植株

本文采用ＭＳ，Ｂ５和ＰＣ一Ｌ２作为基本培养基，在不同激素组合条件下对红三叶（Ｔｒｉｆｏｌｉｕｍｐｒａｔｅｎｓｅ）子叶及下胚轴进行组织培养。在ＭＳ，Ｂ５和ＰＣ一Ｌ２附加２ｍｇ／Ｌ２，４－Ｄ和０．５ｍｇ／ＬＢＡ的培养基上，子叶及下胚轴愈伤组织诱导率均在８９％以上。愈伤组织继代在Ｂ５无机盐十三倍ＭＳ有机成分＋３％蔗糖＋０．５ｍｇ／Ｌ２，４－Ｄ＋０．５ｍｇ／Ｌ６ＢＡ０．２ｍｇ／ＬＮＡＡ，＋１０ｍｇ／ＬＶｃ的培养基上生长良好。愈伤组织在Ｂ５附加０．１ｍｇ／Ｌ６ＢＡ＋０．２ｍｇ／ＬＩＢＡ的培养基上再生植株。下胚轴在ＭＳ附加２ｍｇ／Ｌ６ＢＡ＋１ｍｇ／ＬＮＡＡ的培养基上直接分化形成丛生苗，转入无激素ＮＳ培养基上再生根。实验表明ＶＥ，Ｖｃ，ＰＶＰ和活性碳对愈伤组织褐化有抑制作用，１０ｍｇ／ＬＶｃ抗褐化作用最好。     

牛Y染色体特异DNA序列的体外扩增

本试验建立了用聚合酶链反应（Ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）体外扩增牛Ｙ染色体特异ＤＮＡ序列的方法。在牛、山羊和绵羊Ｙ染色体特异ＤＮＡ同源序列高度保守区设计一对引物，两条引物均为２０ＮＴ，对公牛Ｙ染色体特异的３０７ｂｐ序列进行体外扩增。从屠宰的成年公牛、母牛肝提取ＤＮＡ，用作ＰＣＲ扩增的模板。ＰＣＲ反应总体积为５０μＬ，各成分含量为：２５ｍｍｏ１／ＬＴｒｉｓ－ＨＣＩ（ｐＨ８．２），２５ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４、２ｍｍｏｌ／ＬＭｇＣｌ２、０．１ｍｇ／ｍＬｇｅｌａｔｉｎ、５％Ｆｏｒｍａｍｉｄｅ、ｌμｇ模板、引物各为２５ｐｍｏｌ、ｄＮＴＰ各为２００μｍｏｌ／Ｌ、ＤＮＡ聚合酶２ＩＵ。反应在微机控制ＰＣＲ仪上进行，采用９２．５℃变性，５５℃退火，７２℃延伸，共３５个循环。扩增产物在２％琼脂糖凝胶上电泳，用ｐＧＥＭ－７ｚｆ（＋）Ｈａｅ　Ⅲ作分子量标准。电泳结果，公牛个体样品显示清晰可见的特异ＤＮＡ扩增带，而母牛个体样品无扩增片段出现。扩增产物用ＰｓｔⅠ酶解后出现３条区带，与预期结果一致。     

首乌蜜精营养素分析研究

定量分析首乌蜜精所含的氨基酸、粗蛋白、还原糖、蔗糖，Ｖｃ、ＶＢ１、ＶＢ２、ＶＥ、ＶＤ、ＶＡ、灰份和矿物质。结果表明，首乌蜜精含有１６种氨基酸，其总含量为２９８．１９ｍｇ／１００ｍ１；粗蛋白含量为３６６．００ｍｇ／１００ｍ１；还原糖、蔗糖、灰份含量为５５．９７％、８．５９％、０．２２％；Ｖｃ、ＶＢ１ＶＢ２、ＶＥ、ＶＤ、ＶＡ含量（每１００ｍｌ）分别为０．０６ｍｇ、１７．４６ｍｇ、０．２１ｍｇ、２．３５ｍｇ、４１．０６μｇ、１８，６２μｇ；含有民民Ｚｎ、Ｃｏ、Ｆｅ、Ｍｎ、Ｍｇ、Ｎａ、Ａｌ、Ｃａ、Ｃｕ、Ｓｒ’Ｔｉ、Ｋ１４种矿物质，其总含量为１１４７．７５ｍｇ／Ｌ，不含对人体有害的矿物质。首乌蜜精所含的各种营养素是其多种食疗保健作用的物质基础。     

超排山羊卵巢卵母细胞体外成熟和体外受精的研究

以ＴＣＭ１９９＋１０ｍｍｏｌ／ＬＨＥＰＥＳ＋青霉素（６ｍｇ／Ｌ）＋链霉素（５ｍｇ／Ｌ）为基础培养液（ＢＭ），再分别加入不同成分，配成５种卵母细胞成熟液：（Ａ）ＢＭ＋１０％ＥＧＳ；（Ｂ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ（２．５ｍｇ／Ｌ）；（Ｃ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２（１ｍｇ／Ｌ）；（Ｄ）ＢＭ＋１０％ＦＣＳ＋ＨＣＧ＋Ｅ２；（Ｅ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２＋颗粒细胞（１．５×１０６～３．０×１０６个／ｍＬ）。在３８．５℃、５％ＣＯ２下培养２６ｈ，排卵后第１天卵巢卵母细胞体外成熟率分别为６７．６７％（２０／３０），６０．４２％（２９／４８），８３．６７％（４１／４９）和８０．８２％（５９／７３），排卵后第５天卵巢卵母细胞，在培液Ｄ中体外成熟率为７１．４３％（４５／６３）。排卵后第１天的９８枚卵巢卵母细胞，体外成熟体外受精卵裂率为２１．４３％，２１枚２细胞胚移植５头受体获２头羔羊。研究表明，超排山羊卵巢卵母细胞经体外成熟可获得大量廉价的成熟卵母细胞，并可通过体外受精获得试管山羊     

传染性支气管炎病毒中国地方分离株RFLP基因分型的研究

为初步确定我国不同地区流行的传染性支气管炎病毒（ＩＢＶ）的基因分型，对分离自国内８个不同地域疫区的ＩＢＶＱＤ、ＧＺ、ＺＺ、ＴＪ、ＤＬ、ＹＣ、ＪＳ１和ＪＳ２及参考株Ｍ４１、Ｈ５２和Ｔ的Ｓ１基因ＲＴ－ＰＣＲ扩增ｃＤＮＡ进行ＨａｅⅢ的ＲＦＬＰ分析。结果，ＱＤ与ＭＤ４１、Ｈ５２同属Ｍａｓｓａｃｈｕｓｓｅｔｅ基因型，ＧＺ、ＺＺ、ＹＣ与Ｔ的基因型相同，ＤＬ、ＪＳ１和ＪＳ２则表现为各自独立的基因型，而ＴＪ则为ＤＬ和Ｔ２种基因型毒株的混合感染，表明我国的广大地域内存在着Ｍａｓｓ基因型、Ｔ基因型和可能的变异株ＩＢＶ的流行。     

用PCR鉴别牛胚胎性别的研究

本试验建立了用聚合酶链反体外扩增牛Ｙ染色体特异ＤＮＡ序列鉴别牛胚胎性别的方法。选择３头成年健康西门塔尔母牛为供体，在其自然发性后第９～１４ｄ，用ＦＳＨ超排处理，人工授精，发情后的第７ｄ（发情日为０ｄ），用非手术法采集胚胎，共获得１１枚Ａ．Ｂ级胚胎。在立体显微镜下分别对胚胎进行分割，各获得２个半胚。一个法胚在２０μＬ含１０ｍｍｏｌ／Ｌ Ｔｒｉｓ－ＨＣＬ（ｐＨ８．３）、５０ｍｍｏｌ／Ｌ ＫＣｌ，２ｍｏ     

抗氧化微营养素对缓解蛋鸡热应激的研究

在热应激条件下,向蛋鸡日粮中添加不同水平的Ｖｃ、Ｓｅ、Ｃｕ、Ｚｎ四种抗氧化微营养素。结果表明,抗氧化微营养素（Ｖｃ１０００ｍｇ／ｋｇ,Ｓｅ０．３ｍｇ／ｋｇ,Ｃｕ４０ｍｇ／ｋｇ,Ｚｎ８５ｍｇ／ｋｇ）能有效缓解热应激,提高产蛋率和饲料报酬,同时改善蛋壳品质。     

Investigation of direct toxic and teratogenic effects of anticoagulants on rat embryonic development using in vitro culture method and genotoxicity assay

Heparin and low molecular weight heparins (LMWHs) are used to reduce the incidence of venous thromboembolism in pregnancy. Although, these agents have been shown to be safe when used during pregnancy, the studies about direct toxic and teratogenic effects of these drugs on embryonic development are limited. In this study, the effects of heparin and LMWHs on rat embryonic development were investigated by using in vitro embryo culture and micronucleus (MN) assay methods. Rat embryos were cultured in vitro in the presence of different concentrations of heparin (5-40 IU/ml), dalteparin (2.5-20 IU/ml), enoxaparin (25-100 microg/ml) and nadroparin (1-4 IU/ml). Effects of anticoagulants on embryonic developmental parameters were compared and embryos were evaluated for the presence of any malformations. After culturing the embryos, classic MN assay was performed. Anticoagulants significantly decreased all growth and developmental parameters dose-dependently. Dalteparin and enoxaparin were found to cause more developmental toxicity than heparin and nadroparin. Along with haematoma in general, heparin and nadroparin caused maxillary deformity, situs inversus and oedema most frequently, while neural tube defects were observed with dalteparin and enoxaparin. All agents also significantly induced MN formation in rat embryonic blood cells. These results indicate the possible genotoxic effects of anticoagulant agents on the developing rat embryo when applied directly.     

Teratogenicity of edoferon kappa A, a molecule derived from salicylate, in cultured rat embryos: differences from salicylate and interaction with free oxygen radical scavenging enzymes

The effect of edoferon kappa A (E-KA), a non-specific immunomodulatory and anti-neoplastic chemical substance derived from the methyl form of salicylate (acetyl salicylic acid; ASA), on mammalian embryos was studied and compared to the effects of ASA. Rat embryos were cultured in vitro from 9.5 days of gestation for 48 h. E-KA (0.1-12.8 mg/ml) and ASA (0.1-0.6 mg/ml) were added to the whole rat serum. To investigate the interaction of these molecules with antioxidant agents, the lowest effective concentrations of E-KA (0.6 mg/ml) and ASA (0.3 mg/ml) for all parameters were added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 mumol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. E-KA and ASA decreased growth and development in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology (haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud). There were no statistically significant differences between the control and embryos grown in the presence of 0.1-0.4 mg/ml E-KA, although the effects of ASA started at a concentration of 0.2 mg/ml. Embryos showed significant growth retardation in all scoring criteria and severe malformations when 0.5-3.2 mg/ml E-KA and 0.3-0.6 mg/ml ASA were added. When SOD was added, there was a significant decrease in the incidence of malformations and growth and developmental parameters were increased but this decrease never reached the control level. We concluded that E-KA has direct toxic effects on the developing embryo but at much higher concentrations than ASA, and the teratogenic effects of these molecules might be related to free oxygen radicals.     

铁对牛卵母细胞体外成熟和体外受精的影响

本试验研究铁对体外生产牛胚胎的影响。从屠宰场收集牛卵巢,抽取卵巢表面的卵母细胞,采用体外成熟和体外受精的方法,研究不同浓度的铁(0.45mg/L,0.81mg/L,1.96mg/L,2.78mg/L)对牛卵母细胞体外成熟和体外受精的影响。结果如下:当卵母细胞在体外培养22h时,不同浓度的铁之间对卵母细胞体外成熟影响较小,差异不显著(p>0.05),且与对照组之间差异不显著(p>0.05);在体外受精的研究中,铁浓度为1.96mg/L和2.78mg/L的受精卵培养液可以明显提高8细胞胚胎发育率,差异显著(p<0.05);铁浓度为1.96mg/L的受精卵培养液可以明显提高囊胚发育率,囊胚发育率为31.6%,与其他浓度的铁相比,差异显著(p<0.05)。本试验结果说明:在卵母细胞体外成熟阶段,培养液中的铁对卵母细胞体外成熟没有影响,但对受精后早期胚胎的发育有促进作用,1.96mg/L是比较合适的早期胚胎培养液中铁的添加量。     

两种抗生素的效价比及其对甘农3号紫花苜蓿愈伤组织生长发育的影响

两种抗生素羧苄青霉素和头孢霉素对农杆菌LBA4404的抑菌效果及其对甘农3号紫花苜蓿愈伤组织的生长、体细胞胚分化的影响进行了研究。结果表明,羧苄青霉素的抑菌效果较好,适宜浓度为200~300 mg/L;在浓度200~400 mg/L的羧苄青霉素可促进芽分化;头孢霉素对甘农3号下胚轴离体培养的毒性大,浓度为600 mg/L时完全抑制芽分化。因此,羧苄青霉素为适宜抑菌剂,愈伤诱导阶段的适宜浓度为300 mg/L,体胚形成及芽诱导分化阶段的适宜浓度为200 mg/L。     

Effect of Anti-Basic Fibroblast Growth Factor (Anti-bFGF) on In Vitro Embryonic Development in Rat

In this study, we aimed at the in vitro effects of anti-fibroblast growth factor-2 (anti-FGF-2 or anti-bFGF) on embryo culture in rats. In vitro effects of anti-bFGF on total embryonic development were investigated in 40 rat embryos (which were divided into four groups) (obtained from five pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), and in WRS+ 2.5, 5, and 10 μg/ml anti-bFGF. After 48 h of culturing, the embryos from each group were harvested to be analysed morphologically according to a morphological scoring system and biochemically to obtain the embryo protein content. The morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in WRS+ anti-bFGF had significant embryonic retardation. Mean morphological scores for the embryos grown in WRS, in the presence of 2.5, 5 and 10 μg anti-FGF-2 were 61.4 ± 1.64, 46.3 ± 8.42, 27 ± 2.58 and13.6 ± 0.96 respectively. These results suggest that bFGF is very important for normal embryonic development and rat anti-bFGF neutralizes bFGF effect.     

外源一氧化氮对镉胁迫下黑麦草生长和抗氧化酶活性的影响

利用一氧化氮(NO)供体硝普钠(sodium nitroprusside,SNP)0.1 mmol/L、Cd2 200 mg/L和SNP 0.1mmol/L Cd2 200 mg/L分别处理黑麦草幼苗.结果表明,与对照比较,SNP 0.1 mmol/L处理可增加黑麦草生物量、叶绿素含量、根系活力,降低丙二醛(malondialdehyde,MEA)含量、超氧阴离子(O2-·)产生速率和质膜透性,增强抗氧化酶活性;与对照比较,200 mg/L的Cd2 处理降低了黑麦草生物量、叶绿素含量、根系活力,使MDA含量和O2-·产生速率增加、质膜透性增大,抗氧化酶活性下降,植株体内积累Cd2 ;与Cd2 200 mg/L处理比较,SNP 0.1 mmol/L cd2 200 mg/L处理可提高黑麦草的生物量、叶绿素含量、根系活力,降低MDA含量和O2-·产生速率、质膜透性,而抗氧化酶活性增强,植株体内Cd2 积累量也明显下降.因此,外源NO对Cd胁迫下黑麦草生长有一定的缓解作用,可以增强黑麦草对Cd毒害的抗性.     

甘氨酸对延边黄牛体细胞克隆重组胚发育的影响

为了提高延边黄牛体细胞克隆重组胚的发育率,研究了在体外培养液中添加不同浓度的甘氨酸对重组胚发育的影响。分别在体外培养液中添加0、7、14、21mmol/L的甘氨酸,结果表明,在体外培养液中添加甘氨酸能够显著提高延边黄牛体细胞克隆重组胚的发育率,且以7mmol/L浓度的甘氨酸囊胚的发育率最高。     

金属螯合剂对延边黄牛体细胞核移植重组胚发育的影响

由于在核移植试验中,所用的水和化学物质都不可避免的会被一些金属离子轻微污染,造成胚胎内抗氧化物和过氧化物之间难以保持平衡,从而导致胚胎发育率降低,本试验以此为出发点,探讨了在延边黄牛体细胞核移植重组胚早期培养液中添加乙二胺四乙酸钠（EDTA-Na）和柠檬酸钠（sodium citrate）2种金属螯合剂类抗氧化剂,对其后续发育的影响,以期筛选出最佳的体外培养条件。结果表明：适合延边黄牛体细胞核移植重组胚后期发育的EDTA-Na和柠檬酸钠的最佳浓度分别为50μmol/L和0.6mmol/L。     

香根草叶片水提液对重金属胁迫下两种草坪草种子萌发的影响

为改善重金属胁迫下草坪草种子的萌发,采用室内水分发芽床试验,研究香根草叶片水提液对单一不同浓度重金属(锌、铜、镉)胁迫下高羊茅和多年生黑麦草种子萌发和幼苗生长的影响.结果表明,香根草叶片水提液对高羊茅和多年生黑麦草种子萌发无显著化感作用,但香根草叶片水提液相较对照(蒸馏水)显著提高了Cd2+(200mg/L)胁迫下多年...     

酸、铝胁迫对苜蓿种子发芽和幼苗生长的影响

本试验采用水培法,以金皇后品种的紫花苜蓿为材料,探讨了酸(pH 3.0,4.5,6.0,7.5)、Al³⁺(0,50,100,200 mg/L)胁迫处理对苜蓿种子发芽和幼苗生长的影响。结果表明,随着酸度的增加对苜蓿种子发芽和幼苗生长的抑制作用增强,微酸(pH 6.0)环境下几乎没有抑制作用,甚至有促进作用。溶液铝含量在0,50 mg/L时,苜蓿生长正常,发芽率、发芽势、发芽指数、茎长、根长等均明显高于其他处理(P<0.05);当铝含量在100~200 mg/L时,苜蓿种子发芽和幼苗生长受到严重抑制,发芽率、发芽势、发芽指数、茎长、根长等均显著低于(P<0.05)其他处理。酸、铝共同胁迫对苜蓿种子发芽和幼苗生长的影响大于单一胁迫对它的影响,当pH<4.5、Al³⁺浓度达到50 mg/L以上时,即可对苜蓿芽苗的生长产生明显的抑制作用。