该项目从成本协会 (粮食和农业成本行动 FA1205: AQUAGAMETE, 以及欧洲联盟2020《玛丽玛丽·斯卡洛多斯卡·居里-居里项目》下的研究和创新方案中获得了资金 (GA 642893)。我们要感谢 PROiSER 的科学团队, 特别是本德雷利伯纳乌的学生, 他积极参与了这个项目的录像记录。

涉及动物问题的程序已获得批准 (2015/变现/豌豆/00064) 的一般方向的农业生产和牲畜在大学 Politècnica de València。
1. 在圈养的欧洲鳗鱼中收集精子
注: 使用欧洲鳗鱼雄性在水箱中保持海水和循环系统在恒温 (20 摄氏度)。通过每周腹腔注射 (人绒毛膜促性腺激素 (hCG) 来治疗激素; 1.5 的每克鱼体重)。在淡水中逐渐开始使用3天的鱼, 然后用海水替换 (水箱中总水的 1/3) 每2天, 直到达到37.0 克/毫升的盐度。
<ol><li>麻醉24小时后, 通过激素注射获得更好的精子质量样品。<ol>
<li>预先准备麻醉: 添加300毫克的苯佐卡因到100毫升70% 乙醇。混合好, 并存储在4摄氏度。</li>
		<li>在5升的系统水的柔性桶中稀释苯佐卡因, 以获得60毫克/升的最终浓度, 并适当混合。</li>
		<li>用系统水和苯佐卡因把鱼转到水桶里。等几分钟, 直到鱼平静下来。</li>
	</ol>
</li>
	<li>用清水清除生殖器区, 用吸水纸干燥, 以避免粪便、尿液或海水污染精子样本。</li>
	<li>在腹部使用温和的压力, 并将精子样本收集到15毫升的塑料管用真空泵。精子体积可达6-7 毫升, 取决于雄性。</li>
	<li>将精子样本保持在4摄氏度, 至少1小时, 直到进行动力分析。</li>
</ol>2. 冷冻和稀释精子样本
<ol><li>预先准备稀释解决方案。 注: 精子样本应稀释在特定于每个物种的扩展剂解决方案。<ol>
<li>制备鳗鱼精子样品的非活化剂培养基 (P1 培养基)。添加0.42 克碳酸氢钠, 1.828 克氯化钠, 0.127 克氯化镁, 0.56 克氯化钾和0.037 克氯化钙到250毫升蒸馏水。混合好溶化。存储在4摄氏度。</li>
	</ol>
</li>
	<li>将冷却器块设置为4摄氏度, 以保持样品在恒温下的温度。等到温度稳定下来。</li>
	<li>使用特定于每个物种的稀释溶液稀释精子样本。 注: 稀释率为特定物种, 必须定义为规范该协议。首先, 根据在运动前分析中获得的浓度来估计精子浓度, 如步骤3和4所述。通过这种分析, 也可以根据运动的百分比选择最佳精子样本。在此之后, 研究员可以开始收集数据的实验使用最好的样本与正确的浓度。<ol>
<li>稀释鳗鱼精子样品在 P1 培养基中的比例为1:50。为制备500µL 的鳗鱼稀释精子, 添加50µL 的新鲜精子样本和450µL P1。混合好。</li>
	</ol>
</li>
</ol>3. 评估精子运动参数
<ol><li>
建立软件的运动模块
	<ol>
<li>将冷却器的温度设定在4摄氏度, 等到它稳定下来。</li>
		<li>打开软件并选择运动模块。如有必要, 请创建用户名和密码。</li>
		<li>在开始精子分析之前, 选择属性并选择所需的参数。<ol>
<li>点击物种 |鱼。</li>
			<li>根据每个物种的最佳技术条件, 选择每秒帧数和图像数量。将两个选项设置为每秒120个图像。</li>
			<li>选择负对比度。这是必须的精确重建精子轨迹16。</li>
			<li>选择相应的计数室和摄像机的刻度。为实验中使用的放大透镜校准摄像机。将 SpermTrack10 设置为计数室和10X 刻度。 注: 一般而言, 在计数室和10X 放大透镜的深度10µm 是推荐的条件, 因为它们提供了更好的聚焦所有精子和捕获最高数量的细胞。然而, 建议对每个物种的运动分析的最佳条件进行前期研究。</li>
			<li>调整每个物种的粒子面积和连通性。设置最小粒子面积在2µm2和连通性在7µm。 注意: 对于鱼来说, 粒子面积的最小值介于2-5 µm 之间, 连通性取决于精子速度和帧速率。</li>
			<li>保存设置。</li>
		</ol>
</li>
		<li>选择捕获。</li>
	</ol>
</li>
	<li>
用软件分析欧洲鳗鱼精子
	<ol>
<li>将0.946 克商业盐添加到25毫升蒸馏水中, 用 2% BSA 制备活化剂溶液 (人工海水)。</li>
		<li>采取500毫升活化剂溶液 (人工海水) 和地方在冷却器块。 注意: 一般来说, 鱼精子是由渗透休克事件激活的, 虽然对某些物种来说, 离子浓度也很重要。对于淡水物种, 活化剂介质应具有低渗渗透, 而对于海洋物种则应为高渗介质。</li>
		<li>将计数室置于冷却阶段, 等到温度稳定到4摄氏度。</li>
		<li>混合活化剂和稀释精子激活精子, 并启动运动视频记录后5-10 秒激活。<ol>
<li>取4µL 活化剂溶液并放置在计数室中。</li>
			<li>通过摇动离心3次, 轻轻地融汇稀释的精子, 以避免精子细胞的损伤。</li>
			<li>服用0.5 µL 稀释精子, 与活化剂混合, 并迅速将盖子放在计数室中。 注意: 此步骤不应超过5秒, 以尽快开始分析。</li>
			<li>关注精子细胞并找到最佳视觉区域, 这是由少量的精子 (150 到200细胞) 定义的, 以避免细胞间的拦截 (补充图 1A)。选择捕获视频以获取精子轨迹, 并根据速度进行分离。</li>
			<li>选择 "捕获" 以获取3到7个字段, 这是在结果中获取较低可变性的最佳间隔, 并继续进行。记录视频直到120s 后激活, 以避免在计数室的封面上凝结。</li>
			<li>选择 "退出" 可拥有所有字段的一般视图。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 获取动力数据
<ol><li>选择域 |部分 |保存并选择一个文件名。单击 "保存"。 注意: 电子表格提供了精子轨迹的部分数据、图表和图像的平均值。</li>
	<li>选择常规数据 |文件名 |保存。 注: 数据为各个领域的个体精子提供了动力学值。</li>
</ol>

<ol><li>Kime, D. E., et al. <a target="_blank" href="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/pii/S0166445X96008065">Use of computer-assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals.</a> Aquatic Toxicology. 36, 223-237 (1996).</li>
<li>Kime, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+sperm+analysis+%28CASA%29+as+a+tool+for+monitoring+sperm+quality+in+fish%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+-+Part+C%3A+Toxicology+%26+Pharmacology%22%5BJournal%5D)">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology. 130, 425-433 (2001).</li>
<li>Bobe, J., Labbé, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Egg+and+sperm+quality+in+fish%5BTitle%5D)+AND+%22General+and+Comparative+Endocrinology%22%5BJournal%5D)">Egg and sperm quality in fish.</a> General and Comparative Endocrinology. 165, 535-548 (2010).</li>
<li>Rurangwa, E., Kime, D. E., Ollevier, F., Nash, J. P. <a target="_blank" href="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/abs/pii/S0044848603008330">The measurement of sperm motility and factors affecting sperm quality in cultured fish.</a> Aquaculture. 234, 1-28 (2004).</li>
<li>Bekkevold, D., Hansen, M. M., Loeschcke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Male+reproductive+competition+in+spawning+aggregations+of+cod+%28Gadus+morhua%2C+L.%29%5BTitle%5D)+AND+%22Molecular+Ecology%22%5BJournal%5D)">Male reproductive competition in spawning aggregations of cod (Gadus morhua, L.).</a> Molecular Ecology. 11, 91-102 (2002).</li>
<li>Chong, A. P., Walters, C. A., Weinrieb, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neglected+laboratory+test%3A+the+semen+analysis%5BTitle%5D)+AND+%22Journal+of+Andrology%22%5BJournal%5D)">The neglected laboratory test: the semen analysis.</a> Journal of Andrology. 4, 280-282 (1983).</li>
<li>Overstreet, J. W., Katz, D. F., Hanson, F. W., Fonesca, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laboratory+tests+for+human+male+reproductive+risk+assessment%5BTitle%5D)+AND+%22Teratogenesis%2C+Carcinogenesis%2C+and+Mutagenesis%22%5BJournal%5D)">Laboratory tests for human male reproductive risk assessment.</a> Teratogenesis, Carcinogenesis, and Mutagenesis. 4, 67-82 (1984).</li>
<li>Gallego, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardization+of+European+eel+%28Anguilla+anguilla%29+sperm+motility+evaluation+by+CASA+software%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software.</a> Theriogenology. 79, 1034-1040 (2013).</li>
<li>Fauvel, C., Suquet, M., Cosson, J. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1439-0426.2010.01529.x">Evaluation of fish sperm quality.</a> Journal of Applied Ichthyology. 26, 636-643 (2010).</li>
<li>Mortimer, S. T., Schoëvaërt, D., Swan, M. A., Mortimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+observations+of+flagellar+motility+of+capacitating+human+spermatozoa%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Quantitative observations of flagellar motility of capacitating human spermatozoa.</a> Human Reproduction. 12, 1006-1012 (1997).</li>
<li>Bompart, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CASA-Mot+technology%3A+How+results+are+affected+by+the+frame+rate+and+counting+chamber%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">CASA-Mot technology: How results are affected by the frame rate and counting chamber.</a> Reproduction, Fertility and Development. , (2018).</li>
<li>Vladić, T., Järvi, T. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1095-8649.1997.tb01632.x">Sperm motility and fertilization time span in Atlantic salmon and brown trout - the effect of water temperature.</a> Journal of Fish Biology. 50, 1088-1093 (1997).</li>
<li>Rurangwa, E., Volckaert, F. A. M., Huyskens, G., Kime, D. E., Ollevier, F. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11245263">Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardizes fertilisation in African catfish (Clarias gariepinus).</a> Theriogenology. 55, 751-769 (2001).</li>
<li>Cosson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marine+fish+spermatozoa%3A+racing+ephemeral+swimmers%5BTitle%5D)+AND+%22Reproduction%22%5BJournal%5D)">Marine fish spermatozoa: racing ephemeral swimmers.</a> Reproduction. 136, 277-294 (2008).</li>
<li>Castellini, C., Dal Bosco, A., Ruggeri, S., Collodel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+the+best+frame+rate+for+evaluation+of+sperm+motility+in+different+species+by+computer-assisted+sperm+analysis%3F%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">What is the best frame rate for evaluation of sperm motility in different species by computer-assisted sperm analysis?</a> Fertility and Sterility. 96, 24-27 (2011).</li>
<li>Soler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+holographic+solution+for+sperm+motility+analysis+in+boar+samples.+Effect+of+counting+chamber+depth%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">A holographic solution for sperm motility analysis in boar samples. Effect of counting chamber depth.</a> Reproduction, Fertility and Development. , (2018).</li>
<li>Elliot, F. I., Sherman, J. K., Elliot, E. J., Sullivan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+photo+method+of+measuring+sperm+motility%5BTitle%5D)+AND+%22Journal+of+Animal+Science%22%5BJournal%5D)">A photo method of measuring sperm motility.</a> Journal of Animal Science. 37, 310(1973).</li>
<li>Katz, D. F., Dott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+of+measuring+swimming+speed+of+spermatozoa%5BTitle%5D)+AND+%22Journal+of+Reproduction+and+Fertility%22%5BJournal%5D)">Methods of measuring swimming speed of spermatozoa.</a> Journal of Reproduction and Fertility. 45, 263-272 (1975).</li>
<li>Liu, Y. T., Warme, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computerized+evaluation+of+sperm+cell+motility%5BTitle%5D)+AND+%22Computers+and+Biomedical+Research%22%5BJournal%5D)">Computerized evaluation of sperm cell motility.</a> Computers and Biomedical Research. 10, 127-138 (1977).</li>
<li>Jecht, E. W., Russo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+system+for+the+quantitative+analysis+of+human+sperm+motility%5BTitle%5D)+AND+%22Andrologia%22%5BJournal%5D)">A system for the quantitative analysis of human sperm motility.</a> Andrologia. 5, 215-221 (1973).</li>
<li>Holt, W. V., Palomo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+a+continuous+real-time+computerized+semen+analysis+system+for+ram+sperm+motility+assessment%2C+and+evaluation+of+four+methods+of+semen+preparation%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">Optimization of a continuous real-time computerized semen analysis system for ram sperm motility assessment, and evaluation of four methods of semen preparation.</a> Reproduction, Fertility and Development. 8, 219-230 (1996).</li>
<li>Stephens, D. T., Hickman, R., Hoskins, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Description%2C+validation%2C+and+performance+characteristics+of+a+new+computer-automated+sperm+motility+analysis+system%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Description, validation, and performance characteristics of a new computer-automated sperm motility analysis system.</a> Biology of Reproduction. 38, 577-586 (1988).</li>
<li>Mortimer, D., Goel, N., Shu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+CellSoft+automated+semen+analysis+system+in+a+routine+laboratory+setting%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Evaluation of the CellSoft automated semen analysis system in a routine laboratory setting.</a> Fertility and Sterility. 50, 960-968 (1988).</li>
<li>Mortimer, S. T., Swan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinematics+of+capacitating+human+spermatozoa+analysed+at+60+Hz%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Kinematics of capacitating human spermatozoa analysed at 60 Hz.</a> Human Reproduction. 10, 873-879 (1995).</li>
<li>Holt, W. V., O'Brien, J., Abaigar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+and+interpretation+of+computer-assisted+sperm+analyses+and+sperm+sorting+methods+in+assisted+breeding+and+comparative+research%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">Applications and interpretation of computer-assisted sperm analyses and sperm sorting methods in assisted breeding and comparative research.</a> Reproduction, Fertility and Development. 19, 709-718 (2007).</li>
<li>Gill, H. Y., Van Arsdalen, K., Hypolote, J., Levin, R., Ruzich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+study+of+two+computerized+semen+motility+analyzers%5BTitle%5D)+AND+%22Andrologia%22%5BJournal%5D)">Comparative study of two computerized semen motility analyzers.</a> Andrologia. 20, 433-440 (1988).</li>
<li>Jasko, D. J., Lein, D. H., Foote, R. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/2254179">A comparison of two computer-assisted semen analysis instruments for the evaluation of sperm motion characteristics in the stallion.</a> Journal of Andrology. 11, 453-459 (1990).</li>
<li>Vantman, D., Koukoulis, G., Dennison, L., Zinaman, M., Sherins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+semen+analysis%3A+Evaluation+of+method+and+assessment+of+the+influence+of+sperm+concentration+on+linear+velocity+determination%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Computer-assisted semen analysis: Evaluation of method and assessment of the influence of sperm concentration on linear velocity determination.</a> Fertility and Sterility. 49, 510-515 (1988).</li>
<li>Kime, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+sperm+analysis+%28CASA%29+as+a+tool+for+monitoring+sperm+quality+in+fish%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+-+Part+C%3A+Toxicology+%26+Pharmacology%22%5BJournal%5D)">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology. 130, 425-433 (2001).</li>
<li>Scherr, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidics+and+numerical+simulation+as+methods+for+standardization+of+zebrafish+sperm+cell+activation%5BTitle%5D)+AND+%22Biomedical+Microdevices%22%5BJournal%5D)">Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation.</a> Biomedical Microdevices. 17, 65-75 (2015).</li>
<li>Mortimer, S. T., Swan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+image+sampling+frequency+on+established+and+smoothing-independent+kinematic+values+of+capacitating+human+spermatozoa%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Effect of image sampling frequency on established and smoothing-independent kinematic values of capacitating human spermatozoa.</a> Human Reproduction. 14, 997-1004 (1999).</li>
<li>Hoogewijs, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+counting+chamber+type+on+CASA+outcomes+of+equine+semen+analysis%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Influence of counting chamber type on CASA outcomes of equine semen analysis.</a> Equine Veterinary Journal. 44, 542-549 (2012).</li>
<li>Soler, C., et al. <a target="_blank" href="https://www.researchgate.net/publication/289987642_Effect_of_counting_chamber_on_seminal_parameters_analyzing_with_the_ISASv1_R">Effect of counting chamber on seminal parameters, analyzing with the ISASv1®.</a> Revista Internacional de Andrología. 10, 132-138 (2012).</li>
<li>Didion, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+semen+analysis+and+its+utility+for+profiling+boar+semen+samples%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Computer-assisted semen analysis and its utility for profiling boar semen samples.</a> Theriogenology. 70, 1374-1376 (2008).</li>
<li>David, G., Serres, C., Jouannet, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/2384341">Kinematics of human spermatozoa.</a> Gamete Research. 4, 83-95 (1981).</li>
<li>Björndahl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+normal+semen+quality%3F+On+the+use+and+abuse+of+reference+limits+for+the+interpretation+of+semen+results%5BTitle%5D)+AND+%22Human+Fertility+%28Cambridge%29%22%5BJournal%5D)">What is normal semen quality? On the use and abuse of reference limits for the interpretation of semen results.</a> Human Fertility (Cambridge). 14, 179-186 (2011).</li>
<li>Verstegen, J., Iguer-ouada, M., Onclin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+semen+analyzers+in+andrology+research+and+veterinary+practice%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Computer-assisted semen analyzers in andrology research and veterinary practice.</a> Theriogenology. 57, 149-179 (2002).</li>
<li>Alavia, S. M. H., Cosson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sperm+motility+in+fishes.+I.+Effects+of+temperature+and+pH%3A+a+review%5BTitle%5D)+AND+%22Cell+Biology+International%22%5BJournal%5D)">Sperm motility in fishes. I. Effects of temperature and pH: a review.</a> Cell Biology International. 29, 101-110 (2005).</li>
<li>Islam, M. S., Akhter, T. <a target="_blank" href="http://article.sapub.org/10.5923.j.als.20110101.03.html">Tale of Fish Sperm and Factors Affecting Sperm Motility: A Review.</a> Advancements in Life Sciences. 1, 11-19 (2011).</li>
<li>Dadras, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+common+carp+Cyprinus+carpio+sperm+motility+and+lipid+composition+using+different+in+vitro+temperatures%5BTitle%5D)+AND+%22Anim.+Reprod.+Sci%22%5BJournal%5D)">Analysis of common carp Cyprinus carpio sperm motility and lipid composition using different in vitro temperatures.</a> Anim. Reprod. Sci. 180, 37-43 (2017).</li>
<li>Soler, C., García, A., Contell, J., Segervall, J., Sancho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinematics+and+subpopulations%27+structure+definition+of+blue+fox+%28Alopex+lagopus%29+sperm+motility+using+the+ISASV1+CASA+system%5BTitle%5D)+AND+%22Reproduction+in+Domestic+Animals%22%5BJournal%5D)">Kinematics and subpopulations' structure definition of blue fox (Alopex lagopus) sperm motility using the ISASV1 CASA system.</a> Reproduction in Domestic Animals. 49, 560-567 (2014).</li>
<li>Vásquez, F., Soler, C., Camps, P., Valverde, A., GarcíaMolina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spermiogram+and+sperm+head+morphometry+assessed+by+multivariate+cluster+analysis+results+during+adolescence+%2812-18+years%29+and+the+effect+of+varicocele%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Spermiogram and sperm head morphometry assessed by multivariate cluster analysis results during adolescence (12-18 years) and the effect of varicocele.</a> Asian Journal of Andrology. 18, 824-830 (2016).</li>
<li>Soler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dog+sperm+head+morphometry%3A+its+diversity+and+evolution%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Dog sperm head morphometry: its diversity and evolution.</a> Asian Journal of Andrology. 19, 149-153 (2017).</li>
<li>Valverde, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphometry+and+subpopulation+structure+of+Holstein+bull+spermatozoa%3A+variations+in+ejaculates+and+cryopreservation+straws%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Morphometry and subpopulation structure of Holstein bull spermatozoa: variations in ejaculates and cryopreservation straws.</a> Asian Journal of Andrology. 18, 851-857 (2016).</li>
</ol>

作者没有什么可透露的。

在这个协议中使用的精子分析软件已经被全世界的研究人员用于不同的物种, 包括鱼类。然而, 鱼有一些特定的特点, 可以影响精子评估。在活化的瞬间, 鱼精子表现出很高的速度, 在活化后迅速下降, 并导致短暂的运动时间。此外, 繁殖的温度是物种依赖性的, 在某些情况下, 可能是大约4°c2,4,8,12。因此, 有必要对提高鱼类精子分析系统的效率进行一些适应。动力参数的基本原理是对运动精子的连续图像进行采集和分析。大多数系统使用标准帧速率 (16、25、30、50或 60 fps), 因为硬件和软件的限制为17、18、19、20、21、22。然而, 一些研究表明, 较高的帧速率增加了一些速度参数, 如 VCL, STR, 光华15,23,24。这是特别重要的, 当需要评估精子的 hyperactivation 和发现精子的最大速度11,17,18,19,25. 在分析温度方面, 光学显微镜用的冷却板, 以及在恒温下冷藏样品的新技术也可以改善分析的时间。本研究为解决鱼类精子运动分析和温度敏感性两个主要问题提供了方法。尽管研究结果侧重于一些淡水物种, 但这种方法可用于海洋物种, 尽管应为每个物种调整活化培养基。
在市场上, 有一系列的产品, 甚至不同版本的同一产品。即使系统可能有相同的原则, 每一个都有不同的细节导致结果不兼容26,27,28。此外, 对鱼类精子质量的评价仍有方法和技术上的局限性。精子活化技术和活化后分析时间是影响精子质量评价29、30的两个重要因素。在精子样本活化和图像稳定之间的时间应该是大约5秒, 因为第一秒钟 (5-20 s 的时间间隔) 提供最有用的数据2,4。由于玻璃滑动的凝结问题, 精子分析的时间可能会受到限制。然而, 鱼类精子保持活跃, 运动不到两分钟2。在技术层面上, 有必要知道 "最优" 帧速率, 以获得详细信息, 准确地重建精子轨迹15。在较低帧速率下, 弹道的细节丢失, 特别是对于快速和非线性精子, 而在较高帧率, 信息成为冗余15,31。对某些鱼类来说, 帧速率 (高达 250 fps) 不足以找到某些鱼类的精子的最大速度, 如格雷林和鲑鱼。这种变异是特定物种的, 必须为每个物种定义, 以标准化协议, 并获得可靠的结果15,23,24。计数室的深度可能限制鱼的大鞭毛的运动, 精子不能达到最大速度11,32,33。此外, 当细胞相交时, 该软件可能对实际精子的检测有一定的限制。
对精子质量的经典评价是在对其浓度和运动百分率的估计的基础上进行的主观分析, 可以产生变异的结果。然而, 计算机系统提供了快速、准确和定量的运动参数测量。这一客观分析给出了大量的数据, 并减少了结果34、35、36、37的变异性。精子运动行为的反应取决于分析的温度, 不同物种38、39、40之间的变化。精子分析的最佳温度可以提供精子速度和运动期的持续时间, 类似于生殖38,40的自然条件。因此, 用冷却装置进行精子分析软件, 提高了鱼精子质量的评价。
精子质量分析结果可以提高精子亚群对鱼类精子特征和质量的认识, 这可能是41、42所有动物物种精子评估的未来。在实际水平上, 该技术可以作为高品质育种者的指标, 有助于获得更多的关于精子选择过程的信息, 人工授精的精量计算, 基于速度和累进的精子竞争, 以及生育潜能。所有这些知识可以应用到不同的研究领域, 包括水产养殖和保护项目, 这刺激了对鱼类精子储存和冷冻保存的兴趣在过去的几十年2,13。由于环境问题, 毒理学效应也得到了特别的关注, 并研究了基于精子运动特性的 phylogenetical 研究的发展43,44。
这项工作表明了如何优化的鱼精子运动分析的自动软件, 提高了结果。标准化的协议必须考虑到分析的温度和帧率, 这可能是不同的, 取决于物种。然而, 分析鱼类精子的运动有两个关键步骤, 研究者应该注意。由于粪便、尿液和水 (海水或淡水) 的污染, 精子收集是第一个关键步骤, 可以摧毁样本。另一个关键点是与精子活化的瞬间和录像的开始有关。这一步应尽快进行, 以收集数据时, 精子有剧烈的运动。

繁殖的功效取决于配子 (卵和精子) 的质量1,2。这是促成受精成功的主要因素, 允许3、4的存活后代的发育。对配子质量的方便评价是确定标本生育潜能的最佳工具。
混合精子的多男性是一个常见的做法, 生产许多水生商业物种4。然而, 男性之间的精子变异可能导致精子的竞争, 因此, 并非所有的男性都同样贡献的基因池5。从这个意义上说, 正确评估个别的射精/精子特征, 如运动, 是获得有关男性生育潜能的歧视性信息的根本。直接观察精子的运动可以产生不准确和主观的数据, 因为它需要时间和经验, 这导致缺乏一致性和不相容的结果6,7。然而, 有许多创新, 快速和定量的技术, 可以提供可靠的精子质量分析2,4。
通过计算机辅助精子分析, 提供准确的精子质量数据8。这项技术包括开发与相衬显微镜相关的软件, 它允许对精子运动进行评估。然而, 运动参数的限制因素是摄像机的帧速率。个体精子轨迹是基于精子头质心位置的连续帧的视频录音, 这是相关的鞭毛运动模式3,9,10, 11. 测量的主要动力学参数为直线速度 (VSL)、曲线速度 (VCL) 和平均路径速度 (VAP)。VSL 是精子所采取的起始点和终点之间的距离除以时间。VCL 是沿精子精确轨迹的真实速度。VAP 是沿着导出的轨迹平滑路径的速度。这些参数允许额外的动力学信息, 包括线性 (LIN), 直线度 (STR), 抖动 (WOB) 和跳动测量, 如振幅的横向头部运动 (ALH) 和节拍交叉频率 (光华)4,10。
精子分析系统最初用于哺乳类, 而系统的要求之一是在捐献者的体温下操作 (约37摄氏度)。该软件还可用于鱼类;但是, 有必要对精子分析结果的误差进行一些适应性的调整。在一些鱼类, 如鲑鱼和鳗鱼8,12, 施肥发生在低温 (约4°c)2,4。因此, 应开发冷却装置, 以避免不舒适的工作条件。此外 , 鱼精子在中 immotile , 需要渗透休克激活运动。对于淡水物种, 活化剂介质应具有低渗渗透, 而对于海洋物种则应为高渗介质。然而, 对于某些物种, 作为鲑鱼, 离子浓度也可能是重要的3,4,9。活化后, 鱼类精子的特征是运动速度的快速下降 (少于2分钟)13、14和高速, 对于确定最佳帧速率以获得可靠数据15至关重要。
本研究的目的是设计和应用用于鱼类精子样品的冷冻系统。此外, 该协议还定义了如何根据种类确定标准协议的最佳帧速率。使用这项议定书在鱼类精估的背景下打开新的门, 用欧洲鳗鱼作为模型。

<table><tbody><tr><td>Human Chorionic Gonadotropin</td><td>Argent Chemical Laboratories</td><td>hCG</td><td>Hormone</td></tr><tr><td>Benzocaine</td><td>Merck</td><td>E1501 Sigma</td><td>Anesthesia</td></tr><tr><td>sodium bicarbonate</td><td>Merck</td><td>S5761 Sigma&amp;nbsp;</td><td>P1 medium</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1.06406 EMD Millipore</td><td>P1 medium</td></tr><tr><td>magnesium chloride</td><td>Merck</td><td>1374248 USP</td><td>P1 medium</td></tr><tr><td>potassium chloride</td><td>Merck</td><td>P3911-500G</td><td>P1 medium</td></tr><tr><td>calcium chloride</td><td>Merck</td><td>C7902-500G</td><td>P1 medium</td></tr><tr><td>commercial salt</td><td>Aqua Medic&amp;nbsp;</td><td>Meersalz</td><td>Activator solution&amp;nbsp;</td></tr><tr><td>BSA</td><td>Merck</td><td>05470 Sigma</td><td>Activator solution&amp;nbsp;</td></tr><tr><td>Falcon tubes 15 ml</td><td>Merck</td><td>T1943-1000EA</td><td></td></tr><tr><td>Falcon tubes support</td><td>Merck</td><td>R5651-5EA</td><td></td></tr><tr><td>Eppendorfs</td><td>Merck</td><td>T9661-1000EA</td><td></td></tr><tr><td>Micropipet 20 &amp;micro;l</td><td>Gilson</td><td>PIPETMAN&amp;reg; Classic</td><td></td></tr><tr><td>Micropipet 10 &amp;micro;l</td><td>Merck</td><td>Z683787-1EA</td><td></td></tr><tr><td>Tips for micropipets 20 &amp;micro;l</td><td>Merck</td><td>Z740030-1000EA</td><td></td></tr><tr><td>Tips for micropipets 10 &amp;micro;l</td><td>Merck</td><td>Z740028-2000EA</td><td></td></tr><tr><td>Spermtrack</td><td>PROiSER</td><td>Counting chamber</td><td></td></tr><tr><td>TruMorph</td><td>PROiSER</td><td>TruMorph</td><td></td></tr><tr><td>Microscope UB 200i Serie</td><td>PROiSER</td><td>Microscope</td><td></td></tr><tr><td>Cooler plate</td><td>PROiSER</td><td></td><td>Prototype</td></tr><tr><td>Cooler block</td><td>PROiSER</td><td></td><td>Prototype</td></tr><tr><td>ISAS v1</td><td>PROiSER</td><td>ISAS</td><td>Software</td></tr></tbody></table>

fish sperm assessment using software and cooling devices

对于配子质量评价, 有创新的, 快速的和定量的技术, 可以为水产养殖提供有用的数据。建立了精子分析的计算机系统来测量几个参数, 最常用的测量方法之一是精子运动。
最初, 这种计算机技术是为哺乳动物的物种设计的, 虽然它也可以用于鱼类精子分析。鱼类具有特定的功能, 可以影响精子评估, 如激活后的短期运动时间, 并在某些情况下, 适应较低的温度。因此, 有必要对软件和硬件组件进行修改, 使运动分析更有效地进行鱼精子分析。对于哺乳动物精子, 加热板用于维持精子的最佳温度。然而, 对于某些鱼类来说, 使用较低的温度来延长运动的持续时间是有利的, 因为精子保持活性不到2分钟。因此, 在分析时, 包括在光学显微镜上, 冷却装置是必要的, 在恒定温度下冷藏样品。该协议描述了使用精子分析软件和新的冷却装置对鱼类精子运动进行分析以优化结果。

精子运动时间效应分析
在欧洲鳗鱼的情况下, 静态精子的百分比从十五年代增加到120s 在激活以后 (从24.4% 到 40.7%), 并且移动的进步精子的百分比减少 (从36.9% 到 20.9%)(图 1A和1B)。根据速度, 精子细胞显示速度随着时间的推移 (图 1C和1D) 减少。快速速度的精子比例下降了约 23% (从49.4% 到 26.6%), 中等速度精子的比例增加了约7%。
帧速率和计数室的影响
不同的物种需要不同的帧速率来获得最佳结果。俄罗斯鲟鱼每秒需要150帧 (fps;图 2A);鲤鱼需要 100 fps (图 2B);对于格雷林, 200 fps 是不够的, 以获得最佳的动能结果 (图 2C)。这些结果不依赖于所测试的计数室的深度 (图 2A-2C)。
精子亚群结构
精子亚群的识别是基于统计分析的。第一步是确定主成分 (主成分分析;PCA) 从完整的运动数据集。然后, 利用 PCA 后获得的精子衍生指标进行聚类处理。
对于这项研究, 我们使用大西洋鲑鱼精子, 显示四不同的亚人口, 命名为: 慢非线性 (低林, str 和 VCL), 快速非线性 (低 STR, 高 VCL), 快速线性 (高林, str 和 VCL) 和最快的线性 (最高林, str, 和 VCL)。这些亚人口的分布随世代不同, 从野生动物到养殖的物种 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56823/56823fig1.jpg"> 图 1: 时间后活化对欧洲鳗鱼精子累进和运动的影响。A 和 B) 累进的百分比分别为15和 120s;和 C 和 D) 的运动百分比的速度随着时间的推移 (15 和120s 后激活, 分别).累进分为三组: 渐进性运动细胞 (白色;VCL 平均值: 114.5 18.2 和 101.4, 分别为15和120s 的34.5 µm/秒), 无渐进运动细胞 (灰色;VCL 平均值: 84.8 17.4 µm/秒为十五年代和 84.6 120s 16.5 µm/秒) 和静态精子 (黑色)。精子速度分为4组: 快速 (VCL 平均值: 119.3 到13.7 µm/秒为十五年代和 118.9 * 13.3 µm/秒为 120s; 白色), 中等 (VCL 平均值): 79.7 秒15.5 µm/秒十五年代和 79.6 @ 15.5 µm/秒 120s; 浅灰色), 慢 (VCL 平均值: 32.9 6.5 µm/秒为十五年代和 32.6 120s 6.6 µm/秒; 深灰色) 和静态 (黑色)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56823/56823fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56823/56823fig2.jpg"> 图 2: 三种淡水鱼类的帧率和计数室的影响:(a) 俄罗斯鲟鱼 (B) 鲤鱼和 (C) 格雷林。精子分析是在 50, 100, 150 和 200 fps, 使用两个不同深度的商业计数室 (10 和20µm)。黑色圆圈代表了每个物种的最佳帧速率。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56823/56823fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56823/56823fig3.jpg"> 图 3: 三代大西洋鲑鱼的精子亚群结构: 野生雄性 (F0), 以及在圈养 (F1 和 F2) 中产生的前两代.在聚类分析后, 观察到四个亚群: 慢非线性精子 (低林、STR 和 VCL; 簇 1; 黑色), 快速非线性精子 (下 STR, 高 VCL; 簇 2; 深灰色), 快速线性精子 (高林, STR 和 VCL; 簇 3;中间灰色) 和最快的线性精子 (最高林, STR 和 VCL; 4 组; 浅灰色)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56823/56823fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/56823/56823figsup1.jpg"> 补充图 1。不同的视觉区域从运动分析欧洲鳗鱼精子定义为 (A) 最佳和 (B) 最坏的细胞浓度.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56823/56823figsup1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fish Sperm Assessment Using Software and Cooling Devices at JoVE.com

Fish Sperm Assessment Using Software and Cooling Devices

本议定书描述了一种使用计算机辅助精子分析和冷却装置进行鱼类精子评估的程序。该软件对基于精子运动的鱼类精子质量进行了快速、准确和定量分析, 可作为水产养殖中提高繁殖成功率的有用工具。

使用软件和冷却装置进行鱼类精子评估

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

fish-sperm-assessment-using-software-and-cooling-devices

Nanyang Technological University

Research

JoVE Journal

Biology

8.7K 次观看.  PROiSER R+D S.L.. 本议定书描述了一种使用计算机辅助精子分析和冷却装置进行鱼类精子评估的程序。该软件对基于精子运动的鱼类精子质量进行了快速、准确和定量分析, 可作为水产养殖中提高繁殖成功率的有用工具。

视频: Fish Sperm Assessment Using Software and Cooling Devices

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

科研

遗传学

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

发育生物学

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

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

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

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

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 ...

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

筛选精子以快速分离斑马鱼的种系编辑

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

生物学

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书"在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

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, ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

硬骨模型日本青鳉鱼（Oryzias latipes）中的精子收集和计算机辅助精子分析

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...

U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of&#160;centrifugation on sperm DNA.&#160;The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 &#181;L fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 &#181;L fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 &#176;C with 6% CO2 to equilibrate. Subsequently, 3 &#181;L of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% &#177; 3.2%, whereas the DFI in the UHS group was 1.7% &#177; 1.1%. The difference between the two groups was statistically significant (P &#60; 0.05).

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

U 型水平游泳技术制备低 DNA 片段化指数的高质量精子

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and ...