Name: fluorimetric techniques for the assessment of sperm membranes
Uploaded: 2018-11-28T13:00:00
Description: Watch this Scientific Journal Video about Fluorimetric Techniques for the Assessment of Sperm Membranes at JoVE.com

作者感谢 "sion" 以色列公司的人工授精和育种 (以色列 Hafetz-Haim) 的帮助与合作, 并感谢 li na 女士 (imv technologies, l ' aigle, 法国) 在仪器设置和培训方面提供的协助。

所有实验都是根据1994年以色列动物福利准则进行的。牛精子由以色列商业公司提供, 用于人工授精和繁殖。本研究对11只公牛的射精进行了评估。
1. 精子样品制备
请注意:该程序是基于罗斯实验室的协议1,3。
<ol><li>在室温下, 在15毫升管中获得大约1-6 毫升的公牛精液。</li>
	<li>在每1毫升精液中, 加?...

<ol>
	<li>Komsky-Elbaz, A., Roth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+herbicide+atrazine+and+its+metabolite+DACT+on+bovine+sperm+quality.">Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality.</a> Reprod Toxicol. 67, 15-25 (2016).</li><li>G&#252;rler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. 86 (2), 562-571 (2016).</li><li>Komsky-Elbaz, A., Saktsier, M., Roth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxin+B1+impairs+sperm+quality+and+fertilization+competence.">Aflatoxin B1 impairs sperm quality and fertilization competence.</a> Toxicology. 393, 42-50 (2018).</li><li>Beltr&#225;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ion+Channels+in+the+Sperm+Acrosome+Reaction.">Role of Ion Channels in the Sperm Acrosome Reaction.</a> Adv Anat Embryol Cell Biol. 220, 35-69 (2016).</li><li>Breitbart, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+sperm+capacitation+and+acrosome+reaction.">Signaling pathways in sperm capacitation and acrosome reaction.</a> Cell Mol Biol (Noisy-le-grand). 49 (3), 321-327 (2003).</li><li>Almadaly, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+factors+affecting+the+results+of+staining+frozen-thawed+fertile+and+subfertile+Japanese+Black+bull+spermatozoa+for+acrosomal+status.">Methodological factors affecting the results of staining frozen-thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status.</a> Anim Reprod Sci. 136 (1-2), 23-32 (2012).</li><li>Jankovicov&#225;, J., Simon, M., Antal&#237;kov&#225;, J., Horovsk&#225;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrosomal+and+viability+status+of+bovine+spermatozoa+evaluated+by+two+staining+methods.">Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods.</a> Vet Hung. 56 (1), 133-138 (2008).</li><li>Lybaert, P., Danguy, A., Leleux, F., Meuris, S., Lebrun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodology+for+the+detection+and+quantification+of+the+acrosome+reaction+in+mouse+spermatozoa.">Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.</a> Histol Histopathol. 24 (8), 999-1007 (2009).</li><li>Whitfield, C. H., Parkinson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+fertility+of+bovine+semen+and+in+vitro+induction+of+acrosome+reactions+by+heparin.">Relationship between fertility of bovine semen and in vitro induction of acrosome reactions by heparin.</a> Theriogenology. 38 (1), 11-20 (1992).</li><li>Celeghini, E. C. C., de Arruda, R. P., de Andrade, A. F. C., Nascimento, J., Raphael, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Techniques+for+Bovine+Sperm+Simultaneous+Fluorimetric+Assessment+of+Plasma,+Acrosomal+and+Mitochondrial+Membranes.">Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes.</a> Reprod Domest Anim. 42 (5), 479-488 (2007).</li><li>Ramalho-Santos, J., Varum, S., Amaral, S., Mota, P. C., Sousa, A. P., Amaral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+in+reproduction:+from+gonads+and+gametes+to+embryos+and+embryonic+stem+cells.">Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.</a> Hum Reprod. 15 (5), 553-572 (2009).</li><li>Eddy, E. M., O&#8217;Brien, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The spermatozoon. , (1994).</li><li>Gallon, F., Marchetti, C., Jouy, N., Marchetti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functionality+of+mitochondria+differentiates+human+spermatozoa+with+high+and+low+fertilizing+capability.">The functionality of mitochondria differentiates human spermatozoa with high and low fertilizing capability.</a> Fertil Steril. 86 (5), 1526-1530 (2006).</li><li>Espinoza, J. a., Paasch, U., Villegas, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+disruption+pattern+in+human+sperm.">Mitochondrial membrane potential disruption pattern in human sperm.</a> Hum Reprod. 24 (9), 2079-2085 (2009).</li><li>H&#252;ttemann, M., Lee, I., Pecinova, A., Pecina, P., Przyklenk, K., Doan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+oxidative+phosphorylation,+the+mitochondrial+membrane+potential,+and+their+role+in+human+disease.">Regulation of oxidative phosphorylation, the mitochondrial membrane potential, and their role in human disease.</a> J Bioenerg Biomembr. 40 (5), 445-456 (2008).</li><li>Sellem, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+combinations+of+in+vitro+quality+assessments+to+predict+fertility+of+bovine+semen.">Use of combinations of in vitro quality assessments to predict fertility of bovine semen.</a> Theriogenology. 84 (9), 1447-1454 (2015).</li><li>Odhiambo, J. F., Sutovsky, M., DeJarnette, J. M., Marshall, C., Sutovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+ubiquitin-PNA+based+sperm+quality+assay+for+semen+evaluation+by+a+conventional+flow+cytometer+and+a+dedicated+platform+for+flow+cytometric+semen+analysis.">Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.</a> Theriogenology. 76 (6), 1168-1176 (2011).</li><li>Barrier Battut, I., Kempfer, A., Becker, J., Lebailly, L., Camugli, S., Chevrier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+fertility+prediction+model+for+stallion+semen,+including+flow+cytometry.">Development of a new fertility prediction model for stallion semen, including flow cytometry.</a> Theriogenology. 86 (4), 1111-1131 (2016).</li></ol>

精子受精的可能性取决于反映其质量的多种因素。高浓度的精子和高比例的高度运动精子可能被认为是高质量的精液。然而, 这种评价没有考虑到其他细胞和功能参数。使用 "台式" 微毛细管流细胞仪可以很容易地适应评估各种精子结构使用荧光探针, 如前面的其他17和演示 (技术 #3)。例如, 精子顶体完整性对于成功的自然受精的发生是非常重要的, 因此, 准确的评估顶体状态是必要...

精子膜的完整性和功能是表明精子活力和受精潜力的几个因素。质膜作为细胞内和细胞外隔间之间的屏障, 从而保持细胞渗透平衡1。任何导致对质膜完整性的损害的压力都可能损害体内平衡, 降低生存能力和受精能力, 并增加细胞死亡。例如, 冷冻保存降低精子的生存能力, 由于其质膜的损害, 由于温度变化和渗透应力2。我们以前报告说, 将公牛精子暴露在低浓度的食源性污染物中, 如农药阿特拉津、其主要代谢物二胺氯水嗪或霉菌毒素黄曲霉毒素 b1, 降低精子的生存能力1,3.这是通过用 dapi 与 pi 组合标记双链 dna 来确定的, pi 与受损质膜的细胞 dna 结合在一起。
伴随反应 (ar) 包括外顶膜和覆盖质膜的融合, 导致顶体酶4,5的释放。这些都是重要的事件, 为氧化锆渗透和进一步合并的精子与卵母细胞6。因此, 对顶体膜完整性的评价是评价精液质量和男性生育力的一个有用参数, 7,8,9。几种荧光技术适用于对丙烯酸的完整性验证, fitc-pna 或 fitc-psc8,10。在我们以前的研究中, 利用 fitc-sa 染色1,3 的模式, 我们提供了准确的定义, (i) 完整的顶体, (ii) 受损的尖锐湿疣膜和 (iii) 反应的顶体。在本报告中, 我们使用精子专用流式细胞仪评估顶体状态, 并将结果与荧光显微镜的结果进行比较。
线粒体是参与 atp 合成、活性氧生产、钙信号转导和凋亡等方面的多功能细胞器。生理功能障碍, 包括男性和女性不育, 与线粒体功能改变11。精子线粒体排列在中段, 在精子活力12中发挥着至关重要的作用。人们普遍认为, 高线粒体膜电位 (m) 与正常运动和高施肥能力有关。相反, 低 m 与活性氧物种水平升高和受精率降低14有关.尽管如此, 各种环境化合物, 例如内分泌干扰物, 可以诱导细胞压力, 并导致短暂增加的 m, 超极化1,3, 自由基的生产增加, 并最终,细胞凋亡15。荧光探针 5, 5 ', 6, 6 '-四氯-1, 1 ', 3, 3 '-四乙基苯并咪唑基碘化物 (jc-1), 使检查例如, 食源性毒素对精子的影响 m1,3。
标准的精原图, 基于生理和形态学参数, 是不够好预测精液质量。需要更准确的方法来确保精子质量。在这里, 我们提供了两种可行的方法来确定精子质量的基础上, 精子膜的评估: 同时四染色与特定的荧光探针和荧光显微镜, 在我们的研究1,3中描述和先进的精子专用流式细胞仪, 最近在我们的实验室使用, 并已被其他人使用 16,17,18。

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS [3-N-morphilino propanesulfonic acid]</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sigma</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Neubauer Germany</td>
			<td></td>
			<td>hemocytometer</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>PI (propidium iodide )</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>FITC-PSA (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin )</td>
			<td>Sigma</td>
			<td>L0770</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>JC-1 (5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide)</td>
			<td>ENZOBiochem, New York, NY, USA</td>
			<td>ENZ52304</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>Annexin V conjugated to FITC</td>
			<td>MACS, Miltenyi Biotec</td>
			<td>130-093-060</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer 20X stock solution</td>
			<td>MACS, Miltenyi Biotec</td>
			<td>130-092-820</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>Nikon Eclipse, TE-2000-u</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>Nis Elements</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Nikon DXM1200F</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Guava EasyCyte Plus</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td></td>
			<td>microcapillary sperm flow cytometer</td>
		</tr>
		<tr>
			<td>CytoSoft</td>
			<td>Guava Technologies Inc., Hayward, CA, USA; distributed by IMV Technologies</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Buffered solution for cytometry</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>023862</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>Viability and concentration kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>024708</td>
			<td>kit for viability assessment</td>
		</tr>
		<tr>
			<td>Mitochondrial activity kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>024864</td>
			<td>kit for mitochondrial activity assessment</td>
		</tr>
		<tr>
			<td>Viability &#38; acrosome integrity kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>025293</td>
			<td>kit for acrosome integrity assessment</td>
		</tr>
		<tr>
			<td>JMP-13</td>
			<td>SAS Institute Inc., 2004, ary, NC, USA</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Bovine sperm</td>
			<td>&#34;SION&#34;, Israeli company for artificial insemination and dreeding, Hafetz-Haim, Israel</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorimetric techniques for the assessment of sperm membranes

描述精子质量的标准精子图主要基于生理和视觉参数, 如射精体积和浓度、运动和渐进运动、精子形态和活力。然而, 这些评估都不足以预测精液质量。鉴于维持精子的活力和受精潜力取决于细胞膜的完整性和细胞内的功能, 对这些参数的评估可能有助于更好地预测精子受精能力。在这里, 我们描述了三种可行的方法来评估精子质量使用特定的荧光探针结合荧光显微镜或流式细胞仪分析。使用 4 ', 6-二胺-2-苯丙二醇 (dapi) 和碘化物 (pi) 和顺丁烯 (pi) 评估了评估的质膜完整性, 使用荧光素异硫氰酸酯结合化皮素凝集素 (fitc-sa) 和线粒体膜完整性使用 5, 5 ', 6, 6 '-四氯-1, 1 ', 3, 3 '-四乙基苯并咪唑基卡宾氰酸酯 (jc-1)。还介绍了这些方法的组合。例如, 使用环素 v 与 pi 荧光结合, 可以评估细胞凋亡和计算凋亡精子的比例 (凋亡指数)。我们认为, 这些方法, 这是基于检查精子膜, 是非常有用的精子质量的评估。

图 1显示了使用 pi、dapi、fitc-psa 和 jc-1 同时对精子膜 (血浆、顶体和线粒体) 进行荧光评估的情况。使用四个荧光探针同时染色来评估精子膜, 例如, 可以评估精子在每一类中的比例--活的与死的;高与低 m;完好无损vs受损的精子--同时为每个精子。
图 2显示了使用荧...

Watch this Scientific Journal Video about Fluorimetric Techniques for the Assessment of Sperm Membranes at JoVE.com

Fluorimetric Techniques for the Assessment of Sperm Membranes

在这里, 我们提出的方法来评估精子细胞膜的完整性, 一个细胞特征与精子受精能力。我们描述了精子膜荧光测量评估的三种技术: 与特定荧光探针同时染色, 荧光显微镜, 和先进的精子专用流式细胞仪。还介绍了合并这些方法的实例。

精子膜评价的荧光技术

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

这些程序的目标是使用特定的荧光探针与荧光显微镜或流式细胞学分析相结合来评估精子膜的完整性。这些技术可能有助于回答生殖领域的关键问题，如预测受精潜力和精子样本的质量。这些技术的主要优点是，它们能够精确评估已知与精子受精潜力关联的多个精子膜。这些技术的影响延伸到射精质量的诊断，因为它能够更准确地评估精子血浆和杂技膜的完整性以及线粒体膜电位。要开始这个程序，在室温下在15毫升管中获取大约1到6毫升的公牛精液。将六毫升 NKM 缓冲液预热至 37 摄氏度，每毫升精液。在 600 次 g 下离心 8 分钟。重复离心一次或两次，直到上清液清晰。在此之后，立即丢弃大部分上经剂，在颗粒上方留下大约一厘米的上经液。小心地将管子以30度角倾斜在培养箱中，等待20至30分钟，让精子游起来。使用微管，小心地去除剩余的一毫升上那液，现在含有动量精子，并将其转移到一个新的1.5毫升管。将精子保持在37摄氏度，直到准备好使用。首先，准备文本协议中概述的所有所需库存解决方案。接下来，将每毫升浓度为2500万精子的133微升的精子转移到新的1.5毫升管中。加入 17 微升的 DAPI 工作溶液，在 37 摄氏度下孵育 10 分钟。在1000次g下离心5分钟。丢弃上流液，在颗粒中加入100微升的NKM缓冲液。接下来，添加 50 微升 FITC-PSA、JC-1 的 2 微升和 3 微升的 PI。在37摄氏度下孵育10分钟。在1000次g下离心5分钟。丢弃上流液，将颗粒重新在 40 微升 NKM 缓冲液中。然后将样品的 10 微升转移到玻璃幻灯片中。涂抹样品并添加盖玻片。在此之后，立即开始使用荧光显微镜对样品进行可视化，该视像为 40 倍，并且使用文本协议中概述的三重滤镜，使用数码相机为每个滤镜捕获单独的图像。使用相机软件中的合并选项合并从过滤器接收的三个图像，以 JPEG 格式保存新文件。使用油漆工具打开合并的图像，并使用画笔选项标记已计数的精子。接下来，根据每个探针发出的荧光对精子进行分类，确保每张幻灯片至少评估200个精子。所有单元格应从 DAPI 显示为蓝色。计算显示为紫色的死细胞，以评估生存能力。然后使用荧光染色模式来评估杂技状态。最后，通过区分具有高线粒体膜电位的精子粒体膜电位来评估线粒体膜电位，该精子具有红色染色的中片，具有低线粒体膜电位，具有绿色染色的中层。分别计算红色和绿色中间件并计算其比率。要开始等离子膜完整性评估，请从可行性和浓度套件中选择所需的井数。将水井转移到工作基座上，用灵活的盖子盖住它们，以保护其免受光线的光。在每个井中加入199微升缓冲溶液，用于细胞测量。然后以每毫升5700万个细胞的浓度加入一微升的同质精液，通过移液进行均质化。用盖子盖住盘子，在37摄氏度下孵育10分钟。在此之后，使用可行性设置在流量环流仪中运行样本。要开始评估线粒体膜电位，请从线粒体活性试剂盒中拿出所需的井数。将它们转移到工作基地。在每个井中加入10微升绝对乙醇，并使用移液器重新暂停井内的粉末。每井添加 190 微升 PBS，通过移液均质。在每一井中加入0.75微升的同质精液，每毫升浓度为5700万细胞，通过移液使其均质。用盖子盖住盘子，在37摄氏度下孵育30分钟。在此之后，使用线粒体活性设置通过流式细胞仪运行样本。要开始评估杂技膜的完整性，请从可行性和杂技完整性套件中拿出所需的孔数。将水井转移到工作基座上，用灵活的盖子盖住它们，以保护其免受光线的光。在每个井中加入200微升缓冲溶液，用于细胞测量。然后以每毫升5700万个细胞的浓度加入0.7微升的同质精液，通过移液使均质化。用盖子盖住盘子，在37摄氏度下孵育45分钟。在此之后，使用洞察设置在流式测速仪中运行样本。在这项研究中，用不同的技术评估精液质量，其中一种技术使用荧光探针评估精子膜。可以评估精子样本质量在膜完整性方面的差异。例如，与公牛一号射精相比，牛7号射精的死细胞比例相对较低，具有伪反应杂技体的精子比例较低，线粒体膜电位较高。然后对样本进行可行性和线粒体活性评估。这些代表性示例的直方图代表未被污染的精子和碎片以及封闭的精子。这些直方图也代表活细胞和死细胞以及精子与极化和去极化线粒体膜的分布。然后用即用的试剂盒进行评估，然后用流动的细胞学读取，将该区域划分为三个标记区域，这些区域表示可忽略不计的低荧光细胞，具有完好无损的杂技体、低荧光细胞，其中残留的杂技部分，以及具有被破坏的杂技细胞的极致。在尝试这些程序时，重要的是要确保正确准备初始精子样本。这些技术可以提供精子质量评估的见解，并可以应用于各种生物体，如牛，家禽和人类。两种技术之间的比较，四重染色和流式细胞学表明，在生存能力、线粒体膜电位和杂技完整性方面没有显著差异，表明这两种技术产生了匹配的结果。

Research

JoVE Journal

Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

心肌细胞DNA的合成和倍性的同时评价：一个方法来帮助心肌细胞再生和营业额的量化

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...