Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes

This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (approximately 53% vs approximately 68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (approximately 47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (approximately 20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (approximately 15%) than did the non-delipated oocytes, whether centrifuged or not (approximately 4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, approximately 39% were activated and male pronucleus formation was observed in approximately 40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.     

肉种鸽繁殖性能低下的原因与对策

在进行肉鸽强制配对过程中，由于饲养人员缺乏基本的雌雄鉴别方法，将两只雄鸽配在一起，配对后长时间不见产蛋。这种情况在种鸽初次配对时出现较多。种鸽配对后要做好产蛋记录，一般配对后7～10天就会产蛋，如超过1月仍未见产蛋，要进行重新鉴定性别，重新配对。肉鸽性成熟后雌雄鉴别并不困难，主要通过性行为表现和耻骨特征来进行区分。雄鸽进入发情期后发情明显，追逐雌鸽，发出“咕咕”的叫声，并向雌鸽频频点头示爱。触摸耻骨厚硬，两耻骨间距窄或闭合，耻骨到胸骨末端距离窄，表明腹部容积很小。雌鸽发情不明显，受到雄鸽追逐，无叫声，到处躲藏，耻骨薄软，两耻骨间距宽，     

生物法处理养殖污水中氨氮的研究进展

综述近几年生物方法处理养殖污水中氨氮的研究进展及实际应用,并对大规模处理养殖污水方面提出了一些思考和展望,期望在今后的研究工作中能够探索出高效率、低成本、无二次污染的生物方法处理养殖污水中的氨氮.     

过瘤胃氨基酸在奶牛生产中的应用

本文综述了过瘤胃氨基酸的概念、稳定性、保护效果的测定方法及过瘤胃氨基酸对奶牛生产性能的影响和影响过瘤胃氨基酸效果的因素。     

结缕草种子打破休眠过程呼吸途径的研究

采用呼吸抑制剂测定了结缕草休眠与打破休眠种子在发芽过程中的各个呼吸代谢途径－糖酵解呼吸途径（ＥＭＰ）、三羧酸循环呼吸途径（ＴＣＡ）、磷酸戊糖呼吸途径（ＰＰＰ）的呼吸速率及其占总呼吸速率的比例，结果表明，休眠种子的总呼吸速率显著低于打破休眠种子的总呼吸速率，造成这种差异的原因是磷酸戊糖途径及三羧酸循环途径的呼吸速率低，这是休眠结缕草种子不能发芽的原因之一。     

影响猪精子介导基因转移效果的因素研究

为优化猪精子载体法技术参数,利用荧光定量PCR、荧光显微镜检测和精液常规检测方法,分析不同DNA转染剂、不同孵育温度和不同形态DNA对猪精子转染外源DNA的影响。结果表明,PEI和TransFast转染剂能极显著提高猪精子转染效果,且PEI优于TransFast,而NanoFect转染剂包裹DNA不能转染精子。随着转染温度的升高,精子的活力和活率均明显下降,而转染率和内化外源DNA量基本保持稳定,而吸附外源DNA量呈下降趋势。环状DNA和线性化DNA在与精子共孵育后,两者的精子活力、活率、转染率和内化外源DNA量差异均不显著,精子吸附线性化DNA量极显著高于环状DNA量。综合分析表明,外源DNA形态对猪精子转染效果无显著影响;PEI和TransFast能够显著提高猪精子转染效果;共孵育温度以17℃为最佳,本结果为开展精子载体法制备转基因猪研究提供了基础试验依据。     

鸭瘟病毒dUTPase基因克隆、原核表达及在病毒感染宿主亚细胞中的定位分析

根据笔者实验室新发现的鸭瘟病毒（DPV）dUTPase基因（GenBank登录号DQ486149）序列设计1对引物,PCR扩增后将产物亚克隆至pET32a（＋）原核表达载体,获得表达质粒pET32a-DU。然后将其转化至E．coliBL21（DE3）进行IPTG诱导表达。SDS-PAGE检测显示诱导表达蛋白约66．8ku,与预期表达蛋白大小一致。薄层扫描分析表明重组蛋白占菌体总蛋白的36．2％,主要以包涵体的形式存在。重组蛋白纯化后制备兔抗血清,间接EUSA效价为1：409600。通过免疫荧光技术进行DPVdUTPase亚细胞定位检测,结果显示最早可在感染后4h的胞质中检测到特异性荧光,12h大量点状绿色荧光聚集于胞核;24h后胞核内点状荧光减弱,而胞质点状荧光增强;48h胞核和胞质荧光均显著减弱。本研究为DPVdUTPase基因功能和DPV致病机理的研究提供了重要数据。     

猪伪狂犬病毒和猪细小病毒双重PCR方法的建立及初步应用

目的本研究旨在建立一种适用于临床样品和动物源性生物制品中猪伪狂犬病毒和猪细小病毒同时检测的双重PCR技术。方法针对猪伪狂犬病毒(PRV)的gE基因和猪细小病毒(PPV)的VP2基因的保守区域分别设计引物。结果经条件优化后,所建立的双重PCR方法能特异性地检测出样品中的PRV(581bp)和PPV(202bp)。结论本方法具有良好的特异性、敏感性和稳定性,适用于临床样品中对PRV和PPV的同时检测,也可用于猪源性生物制品的检测。     

应用Excel软件分析敌敌畏农药对两广二号家蚕的安全间隔期

应用Excel软件中的曲线方程分析判断敌敌畏农药对两广二号家蚕的安全间隔期。结果表明：敌敌畏1000倍液的安全间隔期是6d多,其数学模型为幂函数曲线方：y=88．0653x-1834本方法克服以往家蚕的农药安全期无数学模型可循或使用的BASIC方法陈旧繁杂的缺点,也避免英文统计软件SPSS难以在农村普及的不足,是一种计...     

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser⁸³→Arg⁸³ or Ser⁸³→Asn⁸³. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.