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 border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:151px;">AQUACEN BENZOCA&#205;NA +D2A2:D24</td>
			<td style="width:128px;">AQUACEN</td>
			<td style="width:119px;">3394ESP</td>
			<td style="width:141px;">Benzocaine</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td>Avantor&#160;</td>
			<td>3624-19</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringe</td>
			<td>BD Plastipak</td>
			<td>305501</td>
			<td>Syringe 1 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">FC500</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Air pump 100</td>
			<td>EHEIM</td>
			<td>4011708370032</td>
			<td>Modified for suction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Eppendorf 1.5</td>
			<td>Eppendorf</td>
			<td>175508</td>
			<td>Plastic tubes 1.5 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Falcon tubes</td>
			<td>Falcon</td>
			<td>175747</td>
			<td>Plastic tubes 15 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flexible bucket</td>
			<td>Fiel</td>
			<td>1040101</td>
			<td>40 L, Black color.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ethanol</td>
			<td>Guinama SL</td>
			<td>Mg96270</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyopreservation straws&#160;</td>
			<td>IMV Technologies</td>
			<td>14550</td>
			<td>500 &#181;L straws</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Live/Dead Sperm Viability Kit</td>
			<td>Invitrogen</td>
			<td>L7011</td>
			<td>Cytometer Kit</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Modelling clay</td>
			<td>JOVI</td>
			<td>Art 30</td>
			<td>4 different colors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Precision scale PCB</td>
			<td>KERN &#38; Sohn GmbH</td>
			<td>PCB35002</td>
			<td>Digital scale</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Saline solution Vitulia 0.9%</td>
			<td>Laboratorios Ern, S.A.</td>
			<td>C.N. 999790.8</td>
			<td>Saline solution</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Forceps</td>
			<td>Levantina de Lab. SL</td>
			<td>3710025</td>
			<td>30 cm metal forceps</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Scissors</td>
			<td>Levantina de Lab. SL</td>
			<td>3700014</td>
			<td>Scissors 14cm</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ovitrelle (r-hCG)</td>
			<td>Merck SL</td>
			<td>EMEA/H/C/000320</td>
			<td>Human chorionic gonadotropin</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Nikon Eclipse 80i</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CaCl2</td>
			<td>Panreac Quimica SAU</td>
			<td>2112211210</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Na2SO4</td>
			<td>Panreac Quimica SAU</td>
			<td>3257091611</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaHCO3</td>
			<td>Panreac Quimica SAU</td>
			<td>1416381210</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">782M Camera</td>
			<td>Proiser</td>
			<td>782M</td>
			<td>Camera for CASA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ISAS v1</td>
			<td>Proiser</td>
			<td>ISAS v1</td>
			<td>CASA software</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SpermTrack-10</td>
			<td>Proiser</td>
			<td>SpermTrack-10</td>
			<td>Countig chamber for CASA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beaker Pyrex 3L</td>
			<td>PYREX</td>
			<td>2110668</td>
			<td>Glass beaker 3L</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flask Pyrex 250 GL-45</td>
			<td>PYREX</td>
			<td>21801365</td>
			<td>Glass flask 250 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KCl</td>
			<td>Scharlab SL</td>
			<td>PO02000500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Methanol</td>
			<td>Scharlab SL</td>
			<td>ME03061000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MgCl2</td>
			<td>Scharlab SL</td>
			<td>MA00370500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>05482</td>
			<td>Bovine serum albumina</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">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 ...

Hi, my name is Juan Asturiano from the Polytechnic University of Valencia, and we are with some of our colleagues from the Szent Istv

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. Hi, my name is Juan Asturiano from the Polytechnic University of Valencia, and we are with some of our colleagues from the Szent Istv

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

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

修改MicroSecure玻璃化:一种安全，简便，高效的冷冻程序人胚泡

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

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, ...

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

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

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

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

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 ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

将成像与兴奋剂递送系统相结合以研究活体小鼠精子

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

作者聚焦:一种研究小鼠精子中钙信号传导和顶体胞吐作用的活细胞成像技术 

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

活小鼠精子顶体钙动力学和胞吐作用的实时成像

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...