应用流式细胞技术鉴定桑树倍性的样品选择

利用流式细胞仪对桑树不同叶位的叶片、同一叶位的不同部位、叶芽和种子胚根等进行倍性测定,筛选合适的桑树倍性鉴定材料。结果表明,未完全展开幼叶的检测效果优于第1叶位叶和第2叶位叶,第1叶位叶的检测效果优于第2叶位叶;同一叶位幼叶叶基部分的检测效果优于叶尾部分;叶芽的检测效果最好,而且制备样品的杂质较少,是用于流式细胞术研究的最佳材料。胚根也可以作为流式细胞仪检测的材料,这为更早检测桑树的倍性提供了候选材料。研究结果为选取不同时间段的不同材料进行桑树倍性检测提供了依据,也为其他植物倍性鉴定取材提供了参考。     

Antigens of Goat Spermatozoa that Recognize Homologous Zona Pellucida

Goat sperm surface proteins obtained from purified plasma membrane (PPM) vesicles (purity of membrane checked by marker enzymes and transmission electron microscopy) were size fractionated on an fast protein liquid chromatography (FPLC) gel filtration column. All the seven surface proteins (129, 100, 46, 28, 27, 18 and 10 kDa) obtained were further fractionated and purified on high-efficiency gel filtration (GFC-HPLC) as well as ion exchange (DEAE-HPLC) columns. Antibodies were generated against the PPM and the protein fractions. Such resolved and purified surface antigens were tested by Dot Blot Immunoassay and homologous in vitro sperm-zona binding assays. It was revealed that the binding of goat spermatozoa to homologous zona pellucida was inhibited by antisera raised against the five lower molecular weight surface antigens. Further, the components of FPLC-AIII (46 kDa; A represents antigenic protein) and IV (28 kDa) were most promising as the antibodies against these fractions inhibited sperm binding to zona pellucida even at a dilution of 1 : 1000 as tested by the sperm-zona binding assays.     

Effects of Seminal Fluid Fractions on Plasma and Acrosome Membrane Integrity and Mitochondrial Membrane Potential Determined by Flow Cytometry in Chilled Canine Spermatozoa

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third [prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm‐rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5°C for 72 h in egg yolk‐TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT‐SF) or third (EYT‐PF) ejaculated fractions. After cold storage, groups EYT‐SF and EYT‐PF showed significantly higher percentages of sperm cells with an intact acrosome [68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1% respectively]. Only in EYT‐SF was PS translocation significantly reduced compared to EYT‐PF and EYT [3.9 ± 0.4%, 10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT‐SF and EYT‐PF compared to EYT [36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in ΔΨm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.     

流式细胞术在霉菌毒素研究中的应用

世界范围内受霉菌毒素污染的农作物越来越多,这不仅造成巨大的经济损失,也给人和动物健康带来严重危害.近年来,由于流式细胞术能够对混合物中的单个细胞进行快速准确的分析,而逐步成为研究霉菌毒素细胞毒性作用的一种合理可行的方法.作者主要介绍了流式细胞术在检测霉菌毒素及其细胞毒性作用研究中的应用,以期为进一步探究霉菌毒素的毒性作用机制提供参考.     

流式细胞术及其在动物病理学研究中的应用

本文简单介绍了流式细胞仪和流式细胞术的主要功能，并结合作者的实际工作，讨论了流式细胞术在动物分子病理学研究中的应用，包括检测免疫细胞的分化抗原、细胞凋亡、自然杀伤细胞活性、吞噬机能等。     

山羊源伪狂犬病病毒的分离与鉴定

为确诊一例发病山羊是否感染伪狂犬病病毒,采集发病羊体的肺脏和脑组织进行伪狂犬病病毒gE基因PCR检测,并将病料接种至PK-15细胞分离病毒,以及进行小鼠感染试验和gD基因分析。结果表明,PCR检测结果 PRV阳性,病料接种PK-15细胞24h后,细胞开始出现细胞病变;将病毒感染小鼠,36h后小鼠出现局部奇痒、死亡;gD基因序列分析发现,分离毒株与GenBank中的PRV gD基因序列同源性均在98%以上,氨基酸同源性在99%以上;在分离毒株gD基因的808bp~837bp位置上存在缺失与变异、高变重复区。本研究成功分离获得一株羊源伪狂犬病毒,为云南省羊伪狂犬病防控和基础研究提供资料。     

流式细胞术在细胞凋亡研究中的应用

流式细胞术做为一种高新生物技术,在动物医学的各个领域,包括细胞生物学、病毒学、肿瘤学、免疫学和病理学中都得到广泛应用,为细胞凋亡研究提供了有效的技术手段。该技术具有简便、快速、多参数分析等优点,可针对细胞在凋亡时产生的一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases 激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质 Ca2 浓度升高,DNA片段化及含量变化等特点,进行定性、定量测试分析,从而实现对细胞凋亡的准确测定。     

Flow Cytometric Assessment of Acrosomal Status and Viability of Dog Spermatozoa

Contents
A procedure was attempted to simultaneously evaluate viability and acrosomal integrity of dog spermatozoa by flow cytometry and the dual staining technique using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and propidium iodide (PI). Three ejaculates were obtained from three dogs; each of which was divided into five aliquots and increasing concentrations (0–288 μmol/l) of lysophosphatidylcholine (LPC) were added to each one to artificially induce the acrosome reaction in different proportions of spermatozoa. Data obtained by flow cytometric analysis of each sample were compared with those obtained by microscopic evaluation under epifluorescence illumination and by light microscopy evaluation of smears stained with Spermac^® staining. Regression analysis was used to compare the flow cytometric assay with the epifluorescence and light microscopic techniques, and the results indicated that flow cytometry was highly correlated with the Spermac^® staining whereas the correlation with the epifluorescence microscopy was lower. In comparison with the Spermac^® staining, the results from this study validate flow cytometry as a precise method for evaluating the acrosomal integrity of canine spermatozoa.     

山羊不同输精方式和精子数目的受胎效果比较

影响山羊人工授精效果的因素主要是人工输精方式和输入的有效精子数目。山羊人工输精方式有子宫颈口法和子宫内法,目前主要采用子宫颈口法。这种方法虽然简单方便,但是精液消耗量大,受胎率低,复配率高。随着腹腔镜的应用,子宫内输精已经得到了普遍的推广和应用。试验通过比较山羊不同人工输精方式和输入的有效精子数目,进一步探讨了子宫内输精的应用条件,为该技术的推广利用奠定基础。1材料与方法1.1材料1.1.1试验山羊和分组选择健壮的2～4岁经产、空怀、发情母羊共123只,随机分为4组,试验处理见表1。1.1.2主要试剂卡那霉素、BSA、HEPES…     

猪精子质量的流式细胞检测分析

本试验将流式细胞术应用于猪精子分析,同时检测精子功能的多个特性,以保证精子具备多种特性且功能完整,顺利完成授精过程。分别采用SYBR-14/PI（Propidium Iodide）、YO-PRO-1/PI、PNA/PI及Mito Tracker/YO-PRO-1 4种染色方法对荣昌猪精子的质膜完整性、细胞凋亡、顶体完整性及线粒体功能进行了检测和分析;以SYBR-14⁺/PI^-表示质膜完整的活精子,YO-PRO-1^-/PI^-表示活精子,PNA^-/PI^-表示未发生顶体反应的活精子,Mito Tracker⁺/YO-PRO-1^-表示线粒体活性高的活精子,以上述检测指标的综合结果判定精子功能的完整性。本试验通过流式细胞术对荣昌猪精子进行质量检测和分析,结果表明,该方法可为临床诊断种猪不孕症提供理论依据,对有效治疗不孕症有重要意义。     

Detection and Analysis of the Pig Sperm Quality by Flow Cytometry

The flow cytometry was used in this trail to determine the pig sperm quality,in order to ensure the sperm had complete functions during the fertilization process.Four stain methods of SYBR-14/PI (Propidium Iodide),YO-PRO-1/PI,PNA/PI,Mito tracker/YO-PRO-1 were used to detect the plasma membrane integrity,apoptosis,top body ntegrity and mitochondrial function of Rongchang pig sperm.SYBR-14⁺/PI^- expressed live sperm with complete plasma membrane,YO-PRO-1^-/PI^- expressed live sperm,PNA^-/PI^- expressed live sperm had no top body reaction,and Mito Tracker⁺/YO-PRO-1^-expressed live sperm with high mitochondrial activity.The functional integrity of the tested sperm was evaluated according to the above comprehensive results.Through the study of Rongchang pig sperm quality detection by flow cytometry,it suggested that the method could provide theoretical basis for clinical diagnosis,and was very important for swine infertility treatments.     

利用双向电泳技术分析山羊精子冷冻前后蛋白组的差异

建立稳定的山羊精子蛋白质双向电泳的技术平台,对精子上样量、蛋白质提取方法、二维电泳程序等相关技术都进行了优化,得到了相对清晰的冻精和鲜精的二维电泳图谱,并运用ImageMaster TM 2D Platinum软件进行了分析。结果表明：通过改进的热Trizol法提取山羊精子蛋白,当取精子3&#215;10^7、上样量为150μL时能取得较好的电泳图谱,鲜精的蛋白位点重复性较高的有650个&#177;10个,冻精的蛋白位点重复性较高的有600个&#177;10个。冷冻导致精子蛋白丢失约50个,而且冻精多表现为大分子量蛋白的丢失,大部分分布于60～100kb之间,其中60～80kb之间蛋白丢失约18个,80～100kb之间蛋白丢失近20个,12～60kb之间蛋白丢失近12个。实验初步探明了山羊精子融冻前后精子的蛋白变化规律,为进一步了解精子冷冻损伤的确切机理奠定了基础。     

流式细胞术鉴定黄花苜蓿染色体倍性水平

采用流式细胞术检测47份黄花苜蓿种质材料的染色体倍性水平,同时结合传统的根尖染色体制片法进行验证.结果表明,47份黄花苜蓿种质材料中45份为四倍体(2n=4x=32),核DNA含量为1.85±0.09 pg/2c;2份为二倍体(2n=2x=16),核DNA含量为1.016±0.021 pg/2c.研究结果为黄花苜蓿遗传育种、种质资源的合理利用及苜蓿属的系统进化研究提供了理论依据.     

流式细胞技术在哺乳动物性别控制中的研究进展

在动物性别控制的各种技术中,应用流式细胞仪分离的X、Y精子授精可较准确地控制动物后代的性别.文章简单阐述了流式细胞仪分离X、Y精子的原理及其在动物性别控制中的应用,并结合最新研究进展详细地探讨了这项技术在实际生产与应用中的注意事项,旨在为这项技术的研究与应用提供必要的参考与建议.     

琉式细胞技术在哺乳动物性别控制中的研究进展

在动物性别控制的各种技术中,应用流式细胞仪分离的X、Y精子授精可较准确地控制动物后代的性别.文章简单阐述了流式细胞仪分离X、Y精子的原理及其在动物性别控制中的应用,并结合最新研究进展详细地探讨了这项技术在实际生产与应用中的注意事项,旨在为这项技术的研究与应用提供必要的参考与建议.     

流式细胞仪在快速测定鲤春病毒血症病毒滴度中的应用

本研究建立了流式细胞仪快速检测鲤春病毒血症病毒（spring viraemia of carp virus,SVCV）滴度的方法。运用荧光激活细胞分选（fluorescence-activated cell sorting, FACS）技术检测SVCV A1株对草鱼性腺细胞系（GCO）的感染情况。用SVCV病毒单克隆抗体为一抗,FITC标记的羊抗鼠抗体为二抗,运用FACS来检测感染后不同时间点,以及不同病毒接种量的阳性细胞率。感染第3天时为最佳的病毒滴度测定时间点,测得SVCV的病毒滴度为8.31×105 FIU/mL,最低检测病毒滴度为（1000 FIU/mL）,与传统空斑试验（plaque assay,PA）相比,两种方法测得的结果基本一致。实验结果表明,FACS是一种简捷、高效、直接的检测SVCV滴度的方法,是一种新型的病毒滴度测定方法。     

内蒙古白绒山羊附睾精子冷冻效果的研究

本试验把不同温度下的样品分成3个处理组,通过检测精子与繁殖力有关的重要指标研究3个处理组的冷冻效果,结果显示, 在3个不同的环境下收集附睾,室温下(19 ℃)收集附睾精子(处理1组)的冷冻效果最好,解冻后活率为(55.0±3.4)%;4 ℃下收集的精子(处理2组)冷冻解冻后的活率为(35.0±2.5)%;37 ℃下收集的精子(处理3组)的冷冻效果较差,解冻后活率为(23.0±2.5)%。通过对处理组中冷冻和解冻后两个阶段的精子指标的测定,研究了精子活率与其他指标的关系,得出白绒山羊附睾精子能够成功的保存。