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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생물학

使用软件和冷却装置进行鱼类精子评估

Published: July 28, 2018
doi:
10.3791/56823
,
1PROiSER R+D S.L., 2Department of Cellular Biology, Functional Biology and Physical Anthropology,University of Valencia

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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…

Representative Results

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?…

Discussion

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,…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Tags

Keywords: Fish SpermSperm QualityCASA TechnologyMotility AnalysisCooling DevicesArtificial FertilizationEuropean EelSperm Sample CollectionSperm DilutionSperm ActivationCooler BlockCASA Software Configuration
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Caldeira, C., Soler, C. Fish Sperm Assessment Using Software and Cooling Devices. J. Vis. Exp. (137), e56823, doi:10.3791/56823 (2018).
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