Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

日本大耳白兔TLR3部分cDNA克隆及mRNA在组织中的分布

采用RT—PCR技术从日本大耳白兔的脾脏组织克隆出兔的Toll样受体基因3（命名为。TLR3）,并对其mRNA在兔的17种组织中的分布情况进行了检测。结果显示,RTLR3核苷酸序列与GenBank中登载的穴兔（Oryctolagus cuniculus）TLR3序列（登录号：NM_001082219）相似性为100％;兔的胸腺、脾脏、睾丸等14种组织中检测出TLR3 mRNA的转录产物,而在骨骼、胃和皮肤中未能发现。推导的氨基酸序列同源性比对表明,兔TLR3与人、野猪、牛、猫、褐鼠等动物的同源性最高,为80％-85％;与原鸡同源性次之,为61％;与草鱼、绿河豚等同源性在45％～48％之间;系统进化树分析表明,兔TLR3与马的亲缘关系最接近,与猪、人／绵羊、牛、猩猩／猫的亲缘关系依次渐远,与鼠的亲缘关系最远。可见,TLR3在不同种属动物及不同组织中所起的作用具有生物多样性。     

睾丸支持细胞的功能、分离纯化及鉴定研究进展

支持细胞对维持精子形成过程中的微环境起决定作用,它可以通过分泌功能、细胞间连接形成的血睾屏障功能以及吞噬功能等来促进精子的形成过程,其发育异常会导致不同程度的雄性生殖缺陷。基于支持细胞在雄性动物生殖过程中的作用,体外培养高纯度支持细胞可成为研究睾丸两大核心功能-精子发生和性激素分泌功能相关调节机制重要的细胞模型。此外,体外培养睾丸支持细胞也可作为生殖毒理学等新兴热点领域的细胞模型,为评估和研究环境因素对雄性生殖的影响提供便利。因此,作者系统地归纳、总结了目前关于动物支持细胞生物功能的研究及常用的体外分离纯化、培养及鉴定方法,以期为利用动物支持细胞开展雄性生殖领域的研究提供参考。     

改良背主动脉注射法对鸡胚发育与转基因表达效率的影响

背主动脉注射是生产转基因鸡的经典方法,该方法不需换壳培养,但壳内注射技术难度大,并且无法实时观察后期胚胎发育情况.本实验对背主动脉注射法进行了改进,将增强型绿色荧光蛋白(EGFP)腺相关病毒(Adeno-associated Virus,AAV)壳外注射至150枚HH 14～16期(Hamburger-Hamilton...     

抑制瘤胃甲烷排放的酵母菌的筛选及培养条件优化

减少瘤胃甲烷排放措施的研究已成为当前的研究热点之一。本研究以酒、面包和畜禽专用酿酒酵母产品为原材料,从中分离到三株酵母菌,将其制成冻干粉,通过体外产气试验,筛选出了一株抑制瘤胃甲烷排放效果最为显著的酵母菌株。然后以该酵母菌株作为研究对象,进行分子生物学鉴定;采用分光光度计比浊法测定该酵母菌株的生长曲线,确定菌体生长规律;并以此为依据,设计单因素试验考察该酵母菌株最适宜的生长条件。结果表明,分离得到的酵母菌株均为酿酒酵母saccharomyces cerevisiae;抑制瘤胃体外甲烷排放效果最显著的1株酵母菌分离自面包中;该酿酒酵母菌株的对数生长期为8～24h,其最佳培养条件为:生长温度30℃,pH 5.5,培养基中葡萄糖的浓度4.0%。     

高效液相色谱法检测动物脂肪组织中辛硫磷残留量的研究

建立了高效液相色谱法检测动物脂肪组织中辛硫磷残留量的方法.均质后的动物脂肪组织在微波液化后,经乙腈提取,正己烷脱脂,旋转蒸发浓缩后净化,残余物用80％乙腈-水溶解,供高效液相色谱定量(紫外检测器)检测.该方法在浓度0.05 ～2 μg/mL之间呈线性相关,相关系数(R2)为0.9999;在空白脂肪组织样品中添加20(定量限)、200、400、800 μg/kg的辛硫磷,回收率为80％～110％,相对标准偏差(RSD)小于10％.该方法简单快速,灵敏度高,重现性好,适合动物组织中辛硫磷残留量的测定.     

动物组织碱水解处理技术的研究进展

碱水解是近年来发展起来的一种动物组织处理技术,通过NaOH或KOH等碱性物质在高温高压条件下催化动物组织水解为无害的固体残渣和废液并实现组织的灭菌和分解,具有彻底灭活病原微生物、不产生有害气体、操作简单、费用低廉等优点。本文从原理、应用及研究进展等方面,综述了碱水解处理技术的发展与现状。比较分析了碱水解相对于传统组织处理方法的优势,介绍了组织处理机的类型、存在的问题和未来发展趋势。     

牦牛StAR基因克隆及其生物信息学与组织表达特性的研究

旨在克隆获得牦牛StAR基因编码序列（CDS）并进行生物信息学分析,探究其mRNA组织表达特性。本研究以屠宰场采集的成年母牦牛心、肝、脾、肺、肾、卵巢、输卵管、子宫组织（n=5）,不同年龄（胎牛、1岁、2岁）牦牛的卵巢（n=3）,不同发情周期（卵泡期、黄体期）的牦牛卵巢（n=3）,黄体期黄牛的卵巢（n=3）及实验室冻存的牦牛颗粒细胞为研究材料。以牦牛黄体期卵巢cDNA为模板,用逆转录PCR克隆StAR基因,并使用MEGA7.0和ExPASy-ProtParam等软件分析其生物信息学特性;采用实时荧光定量PCR技术分析牦牛StAR基因组织表达特性。结果发现,StAR基因CDS区长858 bp,编码285个氨基酸,StAR蛋白总体带正电荷,属于碱性亲水稳定蛋白,无跨膜结构及信号肽,主要存在于细胞质和线粒体; StAR基因具有较高的保守性,符合物种进化规律。牦牛StAR基因在卵巢表达水平最高（P<0.01）,且2岁时卵巢表达水平极显著高于胎牛和1岁龄（P<0.01）,黄体期卵巢表达水平极显著高于卵泡期（P<0.01）;黄体期黄牛卵巢中StAR基因的表达量极显著高于牦牛（P<0.01）;在颗粒细胞的体外培养过程中StAR基因表达量逐渐上升,在培养24 h时达到高峰（P<0.01）,随后显著降低。综上所述,StAR基因序列较为保守,在牦牛卵巢组织中表达最高,且表达水平随年龄与卵巢周期而变化,提示StAR基因可能参与牦牛卵巢及黄体功能相关的繁殖调控。     

Effect of follistatin on pre‐implantational development of pig parthenogenetic embryos

The present study was designed to explore effects of follistatin (FST) on pre‐implantational development of parthenogenetically activated embryos (PAEs) in pigs. First, we investigated the FST messenger RNA expression level and dynamic FST protein expression patterns in porcine oocytes and PAEs. Then, PAEs were placed in embryo culture medium supplemented with 10 ng/mL of FST‐288, FST‐300, and FST‐315. Next, PAEs were cultured with 0, 1, 10 and 100 ng/mL of FST‐315 protein throughout the in vitro culture (IVC) duration. Further, 10 ng/mL of FST‐300 was added from the start of IVC in which PAEs were treated for 30, 48 and 60 h. The results showed that 1 ng/mL FST‐315 could significantly increase the total cell numbers of blastocyst and trophectoderm cell number in PAEs. Exogenous FST‐300 supplementation could significantly promote the early cleavage divisions and improve the blastocyst formation rate of porcine embryos. FST‐300 appeared to affect early embryonic development before activation of the embryonic genome. In all, the study confirmed for the first time that FST plays a role in promoting early embryonic development in pigs, which differed with different FST subtypes. FST‐300 could facilitate the initial cleavage time and improve the blastocyst formation rate, and FST‐315 could improve the blastocyst quality.     

Mouse embryo co‐culture with autologous cumulus cells and fetal development post‐embryo transfer

This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague‐Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co‐cultured with autologous cumulus cells. We also experimented with co‐culture in 100 µL drops. Drop co‐culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co‐cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post‐transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co‐culture in Cook medium with cumulus cells compared to in vivo‐derived blastocysts. The advantage of the co‐culture system is in generating more blastocysts available for transfer.