Name: handling and treatment of male european eels (anguilla anguilla) for hormonal maturation and sperm cryopreservation
Uploaded: 2018-01-28T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation at JoVE.com

这份出版物是由欧洲联盟的2020玛丽·斯卡洛多斯卡·居里-居里赠款协议 642893 (打动), 成本办公室 (成本行动法 1205, AQUAGAMETE) 和卓越研究中心资助的研究和创新方案-1476-4/2016/FEKUT。

本议定书所述的与欧洲鳗鲡有关的所有程序均由大学 Politècnica de València 的动物实验伦理学委员会批准, 遵循西班牙的法律和法规, 控制实验并活动物的程序。
1. 鱼类保养
<ol><li>把欧洲鳗鲡带到一个研究设施, 并把它们放在一个200升水族馆与循环系统。使用恒温器和冷却机保持恒定的20° c 温度。</li>
	<li>保持在黑暗的条件下的鱼, 以避免压力22 , 并在实验中没有食物。</li>
	<li>在头3天内将鱼放在淡水中。然后每隔一天更换1/3 的水, 再加满海水, 直到达到37.0 ±0.3 克/升的盐度。</li>
</ol>2. 荷尔蒙治疗
注: 荷尔蒙治疗包括每周注射人绒毛膜促性腺激素 (hCG) 在整个持续时间 (九周) 的实验。
<ol><li>通过稀释生理盐水 (0.9% 氯化钠) 中的激素, 在1µL 的浓度下提前准备 hCG 激素。 注意: 在-18 ° c 的时候, 荷尔蒙可以在这个浓度下被稀释一周以上。</li>
	<li>为了麻醉鱼, 准备一个40升的柔性桶与5升的系统水。<ol>
<li>在250毫升瓶, 稀释300毫克的因在100毫升的70% 乙醇。</li>
		<li>将稀释后的因倒入桶中 (最终浓度 0.36 mM) 并适当混合。这是最后的因浓度为60毫克/升。</li>
		<li>将鱼单独放入水中, 因, 等待几秒钟, 直到因生效, 鱼被适当地麻醉。 注意: 要确认鱼是被麻醉的, 把鱼放在仰卧位, 检查它是否保持在那个位置。</li>
	</ol>
</li>
	<li>为了管理荷尔蒙, 在1.5 毫升的塑料管中, 称鱼和准备 hCG 激素, 剂量为1.5 的鱼/克。<ol>
<li>用塑料管中的 hCG 激素填充1毫升注射器。</li>
		<li>将麻醉鱼置于仰卧位, 并在辅助或注射器中, 在腹腔内仔细注射激素。</li>
	</ol>
</li>
	<li>把鱼放回水族馆, 并监测它直到完全恢复。 注意: 荷尔蒙管理必须在整个实验中每周进行一次。通常情况下, 欧洲鳗鲡在激素治疗6-7 周后开始 spermating。</li>
</ol>3. 精子取样
<ol><li>要获得最好的质量样本, 在荷尔蒙管理后提取精子 24 h6,23。</li>
	<li>麻醉在步骤2.2 中描述的因的鱼。</li>
	<li>将麻醉鱼放在仰卧位, 用蒸馏水清洗生殖器区域, 用纸擦干, 避免在生殖器区出现粪便污染, 或通过与水族馆的海水接触, 防止意外精子活化。</li>
	<li>把手指放在鱼的两侧, 按摩小心地从胸鳍侧向生殖器区域施压, 迫使精子出来。<ol>
<li>重复按摩直到没有更多的精子从生殖器口出来。</li>
	</ol>
</li>
	<li>使用真空泵将精子收集到15毫升塑料管中。</li>
	<li>稀释被提炼的精子1:10 在修改的 Tanaka´s 扩展器解答24 (137 毫米氯化钠, 76.2 毫米 NaHCO3, 在蒸馏水) 在 4 c。 注: 扩能剂溶液中的精子应保持在 4 c。</li>
</ol>4. 精子质量评价
<ol><li>准备人工海水13 (以 mM 为单位: 氯化钠 354.7, 氯化镁2 52.4, CaCl2 9.9, Na2如此4 28.2, 氯化钾 9.4, 在蒸馏水中) 与2% 牛血清白蛋白 (BSA) (w: v), 调整 pH 值到 8.2, 渗透到 1100 mOsm/千克, 并保持它在 4 c, 以避免细菌生长。</li>
	<li>打开计算机辅助精子分析软件 (CASA), 并选择鱼精子模块。<ol>
<li>单击属性打开软件的系统设置。在那里, 设置捕获选项每秒60图像。选择负位相光学, Makler 室和10X 尺度。</li>
		<li>然后在分析值上, 选择Fish作为种类和粒子区域大于2µm2和小于20µm2。</li>
		<li>在速度参数上, 当单元格在10到45µm/秒之间移动时设置为慢速, 当在45和100µm/秒之间移动时设置为中, 并在移动速度超过100µm/秒时设置为快速。 注: 速度低于10µm/秒的精子被认为是 immotile。</li>
		<li>选择渐进值作为80% 的直线度 (STR) 并保存属性。</li>
		<li>单击捕获字段(将打开带有照相机的实时图像的窗口)。 注: 计算机辅助精子分析系统由显微镜、摄像机和图像分析软件组成。</li>
	</ol>
</li>
	<li>在计数室, 投入4µL 人工海水和增加0.5 µL 的精子溶液 (精子稀释剂溶液)。<ol>
<li>将图像与显微镜聚焦在10X 放大的负相位。十五年代激活后, 单击计算机辅助精子分析软件中的从视频捕获。</li>
		<li>分析每一个样品一份, 并选择样本的运动高于70% 的冷冻保存。 注意: 这是非常重要的选择, 只有高质量的精子, 以成功的这一冷冻保存协议。</li>
	</ol>
</li>
</ol>5. 精子冷冻法
<ol><li>预先准备液氮 (大约2.5 升) 在 34 cm x 34 cm x 30 cm 和 5 cm 厚实的发泡胶盒。 注: 在任何时候保持4-5 厘米高的液氮水平。<ol>
<li>建立一个浮动结构, 预冻结精子。使用2块聚苯乙烯 (20 厘米 x 5 厘米), 用2塑料管将其绑定, 并将结构放置在液氮上。这一结构需要漂浮在液氮上的大约高度3厘米的表面 (图 1)。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg"> 图 1.用于液氮预冻结的浮动结构示意图.该结构由两片低密度发泡胶 20 cm x 4 cm x 5 cm 连接与塑料管 14 cm。秸秆被放置在塑料管在3厘米以上的液氮。<a href="//cloudflare.jove.com/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>将精子、延长剂溶液和甲醇在1.5 毫升塑料管中掺入 1:8: 1, 并轻轻搅拌, 以制备冷冻液。</li>
	<li>在吸管的帮助下, 将冷冻液的480µL 加入到500µL 的吸管中, 并在必要时用模拟粘土将其结束。</li>
	<li>把稻草放在漂浮装置上, 在3厘米高的液体氮气上3分钟。然后, 将稻草放入液氮中。</li>
	<li>10-15 分钟后, 用长钳将吸管转入液氮贮罐, 随时将其浸入液氮中。在这里, 精子可以无限期地储存。</li>
</ol>6. 解冻方法
<ol><li>准备一个带有液氮的泡沫塑料盒, 如步骤5.1 所述, 并准备一个水浴使用 3 L 烧杯与自来水在 40 c。</li>
	<li>用长钳将储罐中的冷冻精子从贮槽中转移到带有液氮的发泡胶盒中。<ol>
<li>把每根稻草放入水浴在 40 c 十三年代。</li>
		<li>将精子倒入1.5 毫升塑料管中, 用剪刀把吸管的闭合端切下来。</li>
	</ol>
</li>
	<li>使用计算机辅助精子分析系统分析精子运动, 如步骤4.1 所述。</li>
	<li>保留100µL 的精子, 用流式细胞仪分析精子的生存能力。</li>
</ol>7. 流式细胞仪
<ol><li>使用含有碘碘化物 (PI) 的荧光试剂盒, 它是一种红色的荧光化合物, 它可以染色死细胞的细胞核, 以及一种膜状 permeant 核荧光化合物, 即绿色会使活细胞的细胞核染色。<ol>
<li>准备绿色荧光染色液, 稀释它从股票溶液 (1 毫米) 1:10 在 Tanaka´s 介质的工作解决方案, 100 µM。</li>
		<li>不要稀释 PI 溶液。股票的解决方案是在2.4 毫米。</li>
	</ol>
</li>
	<li>对于每个样本, 采取50µL 新鲜或解冻精子和添加0.5 µL 绿色荧光染色工作溶液 (最终浓度1µM) 和2µL PI 溶液 (最终浓度100µM)。</li>
	<li>在黑暗中孵育含有染料 (PI 和绿色荧光染色) 的样品5分钟。<ol>
<li>稀释500µL Tanaka´s 扩剂溶液中的样品, 并与流式细胞仪进行分析。</li>
	</ol>
</li>
	<li>打开流式细胞仪, 并创建一个新的协议, 其中包含至少2地块: SS 日志 vs FS 日志和 FL1 vs FL3。 注意: 绿色荧光染色和碘碘化物可以用可见光波长的光来激发。当与 DNA 结合时, 这些染料的最大荧光量分别为 516 nm 和 617 nm。<ol>
<li>调整不同激光器的电压: SS = 199;FS = 199;FL1= 377;FL3= 372</li>
		<li>建立符合最大事件的购置设置 = 5000 或十五年代 (样品的最终浓度应该是大约100万的细胞/毫升)。使用低流读取该示例。</li>
		<li>选择阅读模式单管固定位置模式。</li>
		<li>把样品放在旋转木马的正确编号</li>
		<li>阅读示例 (按 F9) 并将收集到的数据保存到 excel 文件 (按 F7) 以进行进一步分析。</li>
	</ol>
</li>
</ol>

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Fish Biol. 60 (1), 139-146 (2002).</li><li>Gallego, V., 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=Subpopulation+pattern+of+eel+spermatozoa+is+affected+by+post-activation+time,+hormonal+treatment+and+the+thermal+regimen.">Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.</a> Reprod. Fertil. Dev. 27 (3), 529-543 (2015).</li><li>Asturiano, J. F., Cabrita, E., Horv&#225;th, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress,+challenges+and+perspectives+on+fish+gamete+cryopreservation:+A+mini-review.">Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review.</a> Gen. Comp. Endocrinol. , (2016).</li><li>Kime, D. 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=Computer-assisted+sperm+analysis+(CASA)+as+a+tool+for+monitoring+sperm+quality+in+fish.">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comp. Biochem. Physiol. C Toxicol. Pharmacol. 130 (4), 425-433 (2001).</li><li>Rurangwa, E., Kime, D. E., Ollevier, F., Nash, J. 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+measurement+of+sperm+motility+and+factors+affecting+sperm+quality+in+cultured+fish.">The measurement of sperm motility and factors affecting sperm quality in cultured fish.</a> Aquaculture. 234 (1-4), 1-28 (2004).</li><li>Verstegen, J., Iguer-Ouada, M., Onclin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+assisted+semen+analyzers+in+andrology+research+and+veterinary+practice.">Computer assisted semen analyzers in andrology research and veterinary practice.</a> Theriogenology. 57 (1), 149-179 (2002).</li><li>Magnotti, 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=Cryopreservation+and+vitrification+of+fish+semen:+a+review+with+special+emphasis+on+marine+species.">Cryopreservation and vitrification of fish semen: a review with special emphasis on marine species.</a> Rev. Aquacult. , (2016).</li><li>Dziewulska, K., Rzemieniecki, A., Czerniawski, R., Domaga&#322;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-thawed+motility+and+fertility+from+Atlantic+salmon+(Salmo+salar+L.)+sperm+frozen+with+four+cryodiluents+in+straws+or+pellets.">Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets.</a> Theriogenology. 76 (2), 300-311 (2011).</li><li>Babiak, I., Bolla, S., Ottesen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitable+methods+for+cryopreservation+of+semen+from+Atlantic+halibut,+Hippoglossus+hippoglossus+L.">Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.</a> Aquac. Int. 16 (6), 561-572 (2008).</li><li>M&#252;ller, T., 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=Japanese+eel+(Anguilla+japonica+Temminck+&amp;+Schlegel,+1846)+propagation+using+cryopreserved+sperm+samples.">Japanese eel (Anguilla japonica Temminck &amp; Schlegel, 1846) propagation using cryopreserved sperm samples.</a> J. Appl. Ichthyol. 33 (3), 550-552 (2017).</li></ol>

作者声明他们没有竞争的金融利益。

该协议描述了欧洲鳗鲡成熟、处理和精子冷冻保存的完整过程。这里描述的畜牧业条件是最佳的快速成熟和生产高质量精子在这个种类6,7,25。这一冷冻保存协议的成功及其在解冻后的潜在用途很大程度上取决于新鲜精子的质量26。因此, 选择高质量的精子是非常重要的。请注意, 主观的精子质量评估取决于对样...

在过去25年中, 欧洲鳗鲡 (安圭拉) 到达欧洲海岸的数量稳步下降, 有 90% 1,2,3。有几个因素解释这一急剧下降, 包括污染, 感染, 过度捕捞和栖息地破坏。所有这一切都对这一物种产生了深远的影响, 导致将欧洲鳗鲡列入国际自然保护联盟 (IUCN) 名单, 作为严重濒危的4。因此, 在圈养的繁殖技术和规程的发展是必要的。
欧洲鳗鲡在圈养中的成熟是通过荷尔蒙治疗来实现的5,6,7但是在两性的配子生产上很难同步8。虽然新的雄激素植入物的发展已经显示出加速卵子在鳗鱼9,10, 雌性的最终成熟时间仍然是高度可变的, 难以控制11。因此, 短期储存精子12,13,14和冷冻保存技术是复制管理所必需的, 使配子同步不必要的8。
欧洲鳗鲡精子的超低温保存自 2003年15,16开始开发。一些研究人员设计成功的协议使用二甲基亚砜 (亚砜) 或甲醇作为冷冻16,17,18,19,20。尽管两种协议都已成功使用, 但用亚砜保存的解冻精子所获得的细胞活力低于甲醇20,21。此外, 鳗鱼精子被激活与亚甲基亚砜接触, 需要更繁琐的精子操作19, 因此, 甲醇是一个更适合剂欧洲鳗鲡精子比亚砜。
在这里, 欧洲鳗鲡最佳处理和荷尔蒙治疗的协议将在下面描述。此外, 最好的欧洲鳗鱼精子冷冻保存协议, 使用甲醇作为剂和一个协议的精子质量的评估也将描述。

<table><tbody><tr><td>AQUACEN BENZOCA&amp;Iacute;NA +D2A2:D24</td><td>AQUACEN</td><td>3394ESP</td><td>Benzocaine</td></tr><tr><td>NaCl</td><td>Avantor&amp;nbsp;</td><td>3624-19</td><td></td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>305501</td><td>Syringe 1 mL</td></tr><tr><td>FC500</td><td>Beckman Coulter</td><td></td><td>Flow cytometer</td></tr><tr><td>Air pump 100</td><td>EHEIM</td><td>4011708370032</td><td>Modified for suction</td></tr><tr><td>Eppendorf 1.5</td><td>Eppendorf</td><td>175508</td><td>Plastic tubes 1.5 mL</td></tr><tr><td>Falcon tubes</td><td>Falcon</td><td>175747</td><td>Plastic tubes 15 mL</td></tr><tr><td>Flexible bucket</td><td>Fiel</td><td>1040101</td><td>40 L, Black color.</td></tr><tr><td>Ethanol</td><td>Guinama SL</td><td>Mg96270</td><td></td></tr><tr><td>Cyopreservation straws&amp;nbsp;</td><td>IMV Technologies</td><td>14550</td><td>500 &amp;micro;L straws</td></tr><tr><td>Live/Dead Sperm Viability Kit</td><td>Invitrogen</td><td>L7011</td><td>Cytometer Kit</td></tr><tr><td>Modelling clay</td><td>JOVI</td><td>Art 30</td><td>4 different colors</td></tr><tr><td>Precision scale PCB</td><td>KERN &amp;amp; Sohn GmbH</td><td>PCB35002</td><td>Digital scale</td></tr><tr><td>Saline solution Vitulia 0.9%</td><td>Laboratorios Ern, S.A.</td><td>C.N. 999790.8</td><td>Saline solution</td></tr><tr><td>Forceps</td><td>Levantina de Lab. SL</td><td>3710025</td><td>30 cm metal forceps</td></tr><tr><td>Scissors</td><td>Levantina de Lab. SL</td><td>3700014</td><td>Scissors 14cm</td></tr><tr><td>Ovitrelle (r-hCG)</td><td>Merck SL</td><td>EMEA/H/C/000320</td><td>Human chorionic gonadotropin</td></tr><tr><td>Nikon Eclipse 80i</td><td>Nikon</td><td></td><td>Microscope</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>2112211210</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>3257091611</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>1416381210</td><td></td></tr><tr><td>782M Camera</td><td>Proiser</td><td>782M</td><td>Camera for CASA</td></tr><tr><td>ISAS v1</td><td>Proiser</td><td>ISAS v1</td><td>CASA software</td></tr><tr><td>SpermTrack-10</td><td>Proiser</td><td>SpermTrack-10</td><td>Countig chamber for CASA</td></tr><tr><td>Beaker Pyrex 3L</td><td>PYREX</td><td>2110668</td><td>Glass beaker 3L</td></tr><tr><td>Flask Pyrex 250 GL-45</td><td>PYREX</td><td>21801365</td><td>Glass flask 250 mL</td></tr><tr><td>KCl</td><td>Scharlab SL</td><td>PO02000500</td><td></td></tr><tr><td>Methanol</td><td>Scharlab SL</td><td>ME03061000</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Scharlab SL</td><td>MA00370500</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>05482</td><td>Bovine serum albumina</td></tr><tr><td>Styrofoam box</td><td></td><td></td><td>34x34x30 cm and 5cm thick</td></tr></tbody></table>

handling and treatment of male european eels (anguilla anguilla) for hormonal maturation and sperm cryopreservation

在过去的几年中, 几个研究小组一直致力于开发和改进欧洲鳗鲡处理和成熟的新协议。到目前为止, 每周注射人类绒毛膜促性腺激素 (hCG) 已证明成熟男性经过短短5-6 周的治疗, 生产高品质的精子在几个星期。此外, 使用不同的扩增剂、冷冻、冷却和解冻时间的精子冷冻保存协议已经被描述为欧洲鳗鲡。在这里, 我们表明, Tanaka´s 扩展解决方案可以直接用于施肥或冷冻保存, 使不必要的使用不同类型的解决方案和稀释。此外, 甲醇作为剂的使用使得该协议易于使用, 因为甲醇具有低毒性和不激活精子。精子不需要在加入剂后立即保存, 并且在解冻后可以长时间使用。此外, 在解冻后精子活力仍很高, 但比新鲜精子低。这项工作的目的是为欧洲鳗鱼的处理, 成熟和精子冷冻保存提供最佳的协议。

本研究选取了18只有70% 或更高精子活力的鳗鱼精子。结果表明, 与新鲜精子 (表 1和图 2) 相比, 解冻后所有质量参数都有所降低。运动结果 (平均± S.E.M., n=18) 显示了更高的总运动和在新鲜的精子的进步运动比总运动和进步运动发现在后解冻精子样品。
在对快速精子细胞的分析中也发现了同样?...

Watch this Scientific Journal Video about Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation at JoVE.com

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

欧洲鳗鲡成熟和精子冷冻保存的协议在过去的几年里得到了改善。这篇文章描述了最佳的协议可利用人绒毛膜促性腺激素 (hCG) 诱导成熟和甲醇为剂。

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 ...

handling-treatment-male-european-eels-anguilla-anguilla-for-hormonal

Research

JoVE Journal

Developmental Biology

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

7.8K 次观看.  Universitat Politècnica de València. 欧洲鳗鲡成熟和精子冷冻保存的协议在过去的几年里得到了改善。这篇文章描述了最佳的协议可利用人绒毛膜促性腺激素 (hCG) 诱导成熟和甲醇为剂。

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

环境科学

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, ...

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

一种新的微声学标记在少年太平洋兰普瑞和美洲鳗鱼中的植入

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

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

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

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 ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

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 ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

阿斯蒂亚纳斯墨西哥的Gamete收集与体外受精

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

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 ...