CLC对冷休克和冻融猪精子活力及线粒体膜电位的影响

来自3头公猪的混合精液(n=5)用于本次试验。试验用β-环糊精胆固醇包合物(CLC)作为冷冻保护剂,分对照组和0.75,1.5,3.0g/L CLC等3个试验组,检测冷休克及冻融精子的活力及线粒体膜电位。结果显示:用CASA分析,添加1.5g/L CLC的试验组精子活力显著好于其他试验组(P<0.05);利用JC-1对精子进行荧光染色分析,在4,17,-196℃下添加1.5g/L CLC的试验组精子线粒体膜电位显著高于对照组(P<0.05)。结果表明:在稀释液中添加CLC能提高冻融后精子的活力和减少线粒体的损伤。     

国产精子计数板在长白公猪精液检测上的应用研究

文章以精子计数板为研究对象,应用SPSS 19.0软件的单因素方差分析法对81份精液检测数据进行分析,以探讨国产的精子计数板代替进口的精子计数板的可行性。试验结果显示,进口产品组精子计数板测得的随机3个视野平均精子数、精子密度、精子活力、正常精子比例分别为566.06个、249.56×10^6/ml、92.65%、71.52%;国产产品组的对应值测得为577.02个、252.68×10^6/ml、91.32%、72.65%。两产品的同一指标相比均差异不显著。结果表明:国产的精子计数板替代进口的精子计数板是可行的。     

沙冬青生物碱对MDCC-MSB1细胞增殖及细胞线粒体膜电位的影响

采用体外细胞培养技术、噻唑蓝(MTT)还原法、流式细胞技术检测梯度浓度沙冬青总生物碱对体外培养鸡马立克氏病肿瘤HDCC-MSB1细胞株的增殖抑制及对线粒体膜电位的影响。结果显示,沙冬青生物碱可显著抑制HDCC-MSB1细胞增殖,且这种抑制具有剂量和时间效应关系。经沙冬青生物碱作用的HDCC-MSB1细胞培养样本中有明显的DNA低含量颗粒(亚G1期峰),细胞周期各时相分布发生改变,细胞增殖在G1被阻滞。细胞线粒体膜电位(△Ψm)明显下降,也呈现出剂量和时间效应关系。结果表明,沙冬青生物碱对HDCC-MSB1细胞增殖有显著抑制效应,且抑制依赖于诱导HDCC-MSB1细胞线粒体膜电位(△Ψm)的下降,并进一步诱导了该细胞的凋亡。     

精子质量分析仪在猪精液检查中的应用

应用精子质量分析仪 (SQA)对猪精子的活力、密度和正常形态进行了检测并与常规分析作了比较。通过检测鲜精 87个样本活力为 0 .794 2± 0 .0 74 59,染色死活计数法活力为 0 .80 1 8± 0 .0 1 0 1 3两者差异不显著 (P >0 .0 5)。目测活力为 0 .82 37± 0 .1 640差异显著 (P <0 .0 5)。SQA检测密度 (N =2 7)平均为1 .788亿 /ml± 0 .31 94亿 /ml,计数板测定为 1 .898亿 /ml± 0 .51 2 7亿 /ml,精子密度仪测定为 1 .976亿 /ml± 0 .674 5亿 /ml。三种检测密度的方法差异均不显著。SQA检测精子正常形态率为 51 .81 2 5%± 4 .61 72 % ,抹片染色检查为 87.562 5%± 4 .6485% ,差异巨大 (P <0 .0 0 1 )。此两种方法呈现良好线性相关 ,相关系数r为 0 .82 56,r非常显著 (P <0 .0 1 ) ;得回归方程 ^y =4 4.4 9+0 .831 3x     

手持式猪精子密度仪在牛精子密度测定中的应用

为建立牛精子密度实用测定方法,实现密度仪一机多用,降低购买成本,以多头牛精液为材料,用手持式猪精子密度仪和血细胞计数板为检测工具,0.9％ NaCl为检测液,建立牛精液吸光度-精子密度标准曲线(函数),并进行验证.结果 显示:以0.9％ NaCl为检测液时,牛精液吸光度-精子密度呈三次函数关系,回归曲线方程为Y=0.4...     

褪黑素的抗氧化机制及其在哺乳动物精子冷冻保存中的应用研究进展

哺乳动物中褪黑素(melatonin)主要是由松果体分泌的一种内源性吲哚胺,含有5-甲氧基和N-乙酰基侧链两个关键官能团,以此决定其特异性和双亲性。近年来,褪黑素及其代谢产物被认为具有抗氧化、抗炎、抗凋亡等多种生物活性,在哺乳动物复杂生理调节过程中发挥重要作用。本文主要围绕褪黑素生物合成与代谢、抗氧化机制及其在哺乳动物精子冷冻保存中的作用效应这3个方面对近期的研究进展进行综述,为深入研究褪黑素在动物细胞体外保存中的应用提供参考依据。     

精子质量分析仪在猪常温精液检查中的应用

用精子质量分析仪(SQA)和常规分析法对350个猪的鲜精样本进行精子活力、密度和畸形率的检查。结果发现,2种方法检测出的精子活力、密度、畸形率差异均不显著(P>0.05),说明在对猪常温精液的检测时应用精子质量分析仪可以提高工作效率。     

免疫荧光技术在检测精子中PKA蛋白表达中的应用

为了探测精子中蛋白激酶A(PKA)的定位表达,通过饲养8只性成熟昆明雄性小鼠,49天后采集其精子制备精子悬液,涂片。用PKA多克隆抗体与精子进行免疫试验,阴性对照组用PBS取代,然后用FITC标记的羊抗兔二抗进行荧光标记。用荧光显微镜镜检,结果显示精子中PKA蛋白的表达部位主要为精子尾部的主段和中段。     

应用荧光RT-PCR技术检测高致病性猪蓝耳病病毒

猪繁殖与呼吸综合症（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）引起的一种猪病毒性传染病。该病主要引起妊娠母猪早产、晚期流产、死胎、弱胎以及木乃伊胎。仔猪和育肥猪呼吸困难,呈间质性肺炎。公猪性功能下降,精液质量和精子活力受到影响。该病是进出口猪必须检疫的一种重要的猪传染病。     

精子载体法在转基因动物中的应用与前景

作为动物雄性生殖细胞的精子可与卵子受精而形成受精卵。利用精子的这一特点,以其作为载体将外源基因导入动物体内而制作转基因动物。精子载体法以其简便、有效而引起人们的关注。本文对精子载体法的可行性、研究现状、机理及存在的问题等作一综述。     

Effect of CB1 Receptors on Boar Sperm Plasma Membrane

Veterinary Research Communications - Pistilli, M.G., Bernabò, N., Gloria, A., Mattioli, M. and Barboni, B., 2007. Effect of CB1 receptors on boar sperm plasma membrane. Veterinary Research...     

Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology 相似文献   

Interaction of Potential Porcine Sperm Ligands with the Oocyte Plasma Membrane

We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm–oocyte plasma membrane interaction. Abilities of these proteins to bind to the oocyte plasma membrane and to inhibit sperm–oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa proteins were electroeluted from one dimensional SDS polyacrylamide gels, dialysed and proteins biotinylated. Following incubation with zona‐free porcine oocytes, bound protein was visualized with 20 μg TRITC‐avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control protein. Addition of unlabelled sperm plasma membrane proteins to the biotinylated protein visibly reduced binding. Addition of each of these protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration‐dependent manner. Binding of these sperm plasma membrane proteins to the oocyte plasma membrane and inhibition of fertilization are consistent with these proteins being involved in sperm–oocyte plasma membrane interaction.     

分离和冷冻处理对牛性控精液线粒体膜电位的影响

采用JC-1染色结合流式细胞仪和激光共聚焦显微镜,分别对4组牛精液样品(鲜精、分离精液、普通冻精和分离冻精)的线粒体膜电位进行检测,研究分离和冷冻处理对牛精子线粒体膜电位的影响。结果表明:分离处理对A和B种公牛的精子线粒体膜电位影响不显著(P0.05),但对C种公牛影响显著(P0.05);而冷冻处理后,A、B和C种公牛的线粒体膜电位均显著变化(P0.05);且鲜精时线粒体膜电位无显著差异的A和B种公牛,分离后线粒体膜电位出现显著差异(P0.05)。当精液从采集到处理的间隔时间在24h以内时,分离处理后精子线粒体膜电位无显著性变化(P0.05),但同样时间间隔内冷冻处理后,精子线粒体膜电位显著变化(P0.05)。实验表明和分离处理相比,冷冻处理对精子线粒体膜电位的影响更为显著,且不同种公牛对分离和冷冻处理的耐受性不同。     

The Relationship of Mitochondrial Membrane Potential,Reactive Oxygen Species,Adenosine Triphosphate Content,Sperm Plasma Membrane Integrity,and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.     

不同获能条件对猪精子体外获能和体外受精的影响

探讨猪精子在mTBM和TALP中分别获能2、3、4、5h和6h对体外获能的影响,并研究最适获能时间的精子体外受精的效果。结果表明:随着获能时间的延长,猪精子顶体反应率逐渐升高、质膜完整性逐渐下降,获能6h时的顶体反应率显著高于其他时间组,分别为52.5%和50.8%(P<0.05);质膜完整性显著低于其他时间组,分别为30.5%和31.4%(P<0.05);可获能4h的超激活运动率显著高于其他时间组,分别为56.6%和54.5%。将鲜精与冻融的精子获能4h后与成熟的猪卵母细胞进行体外受精,mTBM处理组的卵裂率显著高于TALP,分别为42.7%与40.2%和35.9%与33.4%,但鲜精与冷冻精液组间差异不显著。     

Osteopontin in Seminal Plasma and Sperm Membrane of Dogs

The objective of this work was to describe the presence of osteopontin (OPN) in canine seminal plasma and sperm membranes. A pool of seminal plasma and sperm membrane extract from 30 dogs was used. Polyacrylamide electrophoresis gels were performed and the bands were transferred to nitrocellulose paper and Western blot was undertaken using an antibody anti-OPN. Two and 12 bands were marked in the seminal plasma (77.2 and 15.6 kDa) and sperm membrane extracts (70.6–26.6 kDa), respectively. However, from 12 marked bands in the sperm membrane extract, only three (46.4, 37.7 and 36.5 kDa) were strongly marked. We conclude that, seminal plasma and sperm membranes from dogs contain different isoforms of OPN; yet, further studies will be necessary to determine their function in this species.     

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.     

猪精子获能前后细胞亚组分蛋白分离及酪氨酸磷酸化蛋白的鉴定

本研究对猪精子获能前后细胞亚组分蛋白进行分离以及对酪氨酸磷酸化蛋白进行鉴定,旨在为哺乳动物精子受精生物学研究奠定理论基础。利用动物精子体外获能培养、细胞亚组分分离技术及蛋白免疫印迹的方法,分离猪精子细胞亚组分蛋白及酪氨酸磷酸化蛋白鉴定。结果表明,猪精子经过获能培养后各项活力指标均得到显著提高,且与精子蛋白发生酪氨酸磷酸化修饰密切相关;获能精子中126、108、79ku的高分子量蛋白磷酸化程度明显高于未获能精子;分子质量约为25、47、50ku的膜蛋白及47ku胞浆蛋白发生酪氨酸磷酸化,其中25、47ku的膜蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05);分子量约为23、37、42～50ku的核蛋白发生酪氨酸磷酸化,获能精子中23ku的核蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05)。结果提示,猪精子细胞不同亚组分中,发生酪氨酸磷酸化修饰的蛋白以膜蛋白及核蛋白为主,同时有少量的胞浆蛋白。     

"雅士勇"口服液对猪的射精量和精子畸形率的影响

以大白猪和长白猪为研究材料,对照组不饲喂"雅士勇"口服液,两试验组分别饲喂25 mL和30 mL"雅士勇"口服液,研究"雅士勇"口服液对猪的射精量和精子畸形率的影响。结果表明,"雅士勇"口服液对射精量无明显影响(P>0.05),但对种公猪的精子畸形率有显著影响,对照组的精子畸形率均极显著地高于两个试验组(P<0.01),而两试验组之间精子畸形率差异不显著(P>0.05)。