我们感谢 Cellerino, 泰隆 Genade, 安妮深色, 夏普, 米奇鲍威尔, 西蒙妮 Keil, Yumi 金, 帕特里克史密斯, 凯 Mathar 和 Valenzano 实验室的所有成员在最大的普朗克生物研究所为促进不同方面目前鳉鱼畜牧业的协议。

鱼在28°c 被上升在水循环系统 (参见水参量), 与 10-20% 每日水处置。三不同的坦克大小推荐: 0.8 升, 2.8 升和9.5 升。每罐接收恒定的水流2毫升/秒。
1. 试剂制备 (不含材料)
注: 非洲绿松石鳉鱼 (Nothobranchiusfurzeri) 可从已建立的实验室库存中提供。每年鳉鱼干燥的胚胎可以通过邮寄方式运送。在 8-30 °c 温度范围内运送胚胎是至关重要的。
<ol><li>在系统水中溶解1克/升腐植酸, 制备腐植酸 (孵化) 溶液。蒸压釜和贮存在4摄氏度, 长达10周。</li>
	<li>为制备 HUFA 卤虫浓缩, 每天在盐水虾溶液浓度为500µL/升的盐水虾孵化中加入 HUFA 富集。</li>
	<li>在以前的蒸压系统水中溶解100µL/升的亚甲基蓝溶液, 制备亚甲基蓝液。由于亚甲基蓝是光敏的, 保持溶液在黑暗的瓶子, 或覆盖与箔。存储在 RT。</li>
	<li>准备椰子纤维作为胚胎孵化的固体基质。或者, 使用过滤纸 (见1.5 节)。<ol>
<li>Presoak 椰子纤维用蒸馏水。蒸压釜和贮存在4摄氏度, 长达5周。</li>
		<li>在胚胎移植当天, 用湿润椰子纤维制备培养皿。</li>
		<li>填充一个90毫米直径的培养皿与椰子纤维下的油烟罩和旁边的火焰, 以减少污染的酵母和细菌。</li>
		<li>椰子纤维紧凑, 高度为1厘米, 无菌组织。把大部分的水分从椰子纤维上按在盘子上的纸巾上, 让纸张吸收多余的水。在火焰上加热一个金属勺子, 然后按下整个椰子纤维表面。这可以防止椰子纤维的真菌/细菌污染。</li>
	</ol>
</li>
	<li>准备过滤纸作为胚胎孵化的固体基质。<ol>
<li>在胚胎移植当天, 放置3层过滤纸盘, 适合90毫米培养皿。加入5毫升腐植酸溶液以保持湿度。</li>
	</ol>
</li>
</ol>2. 育种
<ol><li>
鳉鱼绿松石菌的选育 注意: 根据这项协议, 性成熟度达到4周后孵化和生育高峰之间 7-9 周。重要的是要注意, 生育力取决于喂养频率和食物质量;因此, 建议至少每天喂养两个饲养池, 以提高胚胎产量 (见5.6 节)。<ol>
<li>设置一个9.5 升繁殖罐。填充系统水并添加一条雄性和两只雌鱼。</li>
		<li>由于雄性非洲绿松石鳉鱼在交配期间表现出优势, 这可能会导致对雌性的骚扰, 所以选择一个比雌性体型稍小一点的雄性来减少交配压力, 增加生殖产量。成立了5周大的男性, 6/7 周大的女性。</li>
		<li>填充一个塑料容器 (10 x 10 x 5 厘米), 蒸压砂达到最终深度为 2-3 厘米, 并将砂箱放在繁殖槽的中心。</li>
		<li>让绿松石鳉鱼连续繁殖, 每周收获一次胚胎孵化。 注: 使用砂基板对集中式过滤系统构成挑战, 今后应采用替代方法替代。可能的替代方案可能是使用斑马鱼养殖坦克。</li>
	</ol>
</li>
	<li>
转基因育种 注: 用于注射的胚胎需要在单细胞阶段同步, 这需要在受精后立即收集。<ol>
<li>为了获得用于注射和生成转基因品系的一细胞级胚胎, 建立了一只雄性和两条雌鱼的繁殖槽 (与2.1 相同)。</li>
		<li>在胚胎采集前两天, 将雄性单独放在一个水箱中, 并保持雄性与成年雌性的视觉接触。</li>
		<li>在收集日, 将雄性和一个沙盒添加到繁殖罐中, 让它们产卵2小时。</li>
	</ol>
</li>
</ol>3. 胚胎饲养
<ol><li>
胚胎采集 注: 胚胎采集是通过从砂盒中筛选和收获胚胎进行的。在正常情况下, 每个砂箱应包含从30到200胚胎。<ol>
<li>在收集日, 从饲养箱中取出砂盒。将砂箱清空至过滤器 (0.9 毫米应变大小), 并用系统水冲洗。这可以通过一个大的坦克来收集沙子为热处理。</li>
		<li>将过滤器部分浸入系统水中, 轻轻旋转, 让胚胎聚集在中心。</li>
		<li>用10毫升的巴斯德吸管收集胚胎。</li>
		<li>将胚胎移植到90毫米培养皿中, 在40毫升的系统水中。</li>
		<li>在光显微镜下检查培养皿中的胚胎, 除去那些呈现破裂的绒毛膜和损伤的迹象。</li>
		<li>直接进行胚胎漂白。 注意: 每次鱼菌株都要使用一个过滤器, 以防止潜在的交叉应变胚胎污染。</li>
	</ol>
</li>
	<li>
胚漂白 注意: 胚胎漂白可防止鱼缸中的微生物污染孵化介质。<ol>
<li>漂白前, 使用一次性的巴斯德吸管从含有采集胚胎的培养皿中取出系统水。</li>
		<li>为防止不必要的真菌和细菌生长, 将50毫升新制备的 H2O2 (在蒸压系统水中1% 伏/v) 添加到所收集的胚胎中。</li>
		<li>在50毫升溶液中, 在90毫米培养皿中以低速速度摇动胚胎5分钟。</li>
		<li>将 H2O2解决方案与一次性的巴斯德吸管和洗涤胚胎三次为5分钟与50毫升亚甲基蓝溶液。移除亚甲基蓝溶液。</li>
		<li>在蒸压系统水中添加50毫升 H2O2 (1% 伏/v) 到胚胎并摇动5分钟。</li>
		<li>删除 H2O2解决方案, 并用50毫升亚甲基蓝溶液清洗三次5分钟。</li>
		<li>孵化28°c 的胚胎, 以增加同步胚胎发育, 最高密度为100胚每90毫米培养皿在40毫升亚甲基蓝溶液。 注意: 不要在漂白溶液中延长胚孵化。这可能会对鸡蛋绒毛膜造成损害, 并增加胚胎死亡率。胚胎漂白可能会导致鸡蛋绒毛膜的物理化学变化, 可能导致改变绒毛膜生理和孵化成功。</li>
	</ol>
</li>
	<li>
亚甲蓝胚孵化 注: 在亚甲蓝溶液中的液体孵化可以防止寄生虫生长, 并能检测死胚和未受精的卵。<ol>
<li>检查孵化的胚胎, 从培养皿中除去任何死胚 (由亚甲蓝染蓝), 以防止真菌和细菌污染影响活健康胚胎的生存。</li>
		<li>除去旧的亚甲基蓝溶液, 用新鲜溶液代替。</li>
		<li>将 Petri 盘返回到28°c 孵化器 (图 1A)。在 7-10 天内, 确保已发育的胚胎显示可见的黑色眼睛。将这些胚胎转移到椰子纤维或过滤纸固体基质介质 (图 1B)。</li>
		<li>保留未发育的亚甲蓝胚胎, 每日监测, 一旦黑眼睛发育, 就转移到固体基质培养基中。</li>
		<li>重复步骤 3.3. 1-3. 3.3 每天直到胚胎有肉眼可见的黑眼睛。 注意: 胚胎对亚甲基蓝的持续暴露可能会导致成年鱼类生理的长期变化。</li>
	</ol>
</li>
	<li>
胚胎移植到过滤纸上 注: 绿松石鳉鱼胚可在干燥基质上发育, 综述自然条件。此外, 干胚孵化使研究人员能够同步胚胎和孵化他们在同一天。<ol>
<li>随着发育的胚胎在 7-10 天内会有肉眼可见的黑眼睛, 使用一次性的巴斯德吸管或细弯镊子将胚胎从亚甲基蓝溶液转移到先前制备的滤纸板上。</li>
		<li>将胚胎与镊子分开5毫米, 每90毫米板可达100个胚胎 (图 1B)。</li>
		<li>用 parafilm 封住培养皿。</li>
		<li>孵化胚胎在28°c 2-3 周, 直到他们已经完全开发了金色的虹膜, 并准备孵化 (图 1C)。 注意: 不要延长孵化胚胎的时间超过2周, 因为它们的生存能力将大大减少。</li>
	</ol>
</li>
	<li>
椰子纤维的胚胎移植 注: 蒸压、无菌椰子纤维 (或有机泥炭苔藓) 可用作固体基质孵化的有效替代培养基。<ol>
<li>使用一次性的巴斯德吸管或细弯曲镊子将胚胎从亚甲基蓝溶液转移到现成的椰子纤维板上。</li>
		<li>将胚胎分布在5毫米之外, 每90毫米板最多100个胚胎 (图 1B)。</li>
		<li>用 parafilm 封住培养皿。</li>
		<li>孵化胚胎在28°c 2-3 周, 直到他们已经完全开发了金色虹膜 (例如在图 1C)。 注: 对于长期贮存 (最多一年), 在3天后从亚甲基蓝溶液到17摄氏度的固基板上转移胚胎。孵化胚胎直到它们发育成黑色的眼睛。</li>
	</ol>
</li>
</ol>4. 孵化绿松石鳉鱼
注: 绿松石鳉鱼胚胎可以成功孵化在腐植酸溶液14中.
<ol><li>使用细弯曲镊子, 仔细转移 50-100 开发的胚胎进入孵化盒填充的腐植酸溶液在4摄氏度。腐植酸溶液由系统水中的1克/升腐植酸组成。蒸压釜和贮存在4摄氏度, 长达10周。要确保所有的胚胎都完全浸入。腐植酸溶液必须浅, 不超过2厘米。 注: 腐植酸溶液的低温改善孵化和完全浸泡的胚胎在解决方案允许同步孵化。</li>
	<li>将孵化箱放入28°c 孵化孵化器。用盖子盖住孵化箱。要提供足够的曝气, 用油管与供气连接孵化箱。 注: 在孵化过程中没有足够的曝气导致高率的鱼苗无法填满气体膀胱 ("肚皮滑块" 表型, 见5.1 节中的说明)</li>
	<li>从孵化后的次日起, 在孵化箱中保持足够的水质, 每天加蒸压水系统的比例为 1:1, 保持最后深度2厘米。</li>
	<li>将孵化胚胎转移回固体基质, 并在一周后尝试孵化。 注: 孵化后, 绿松石鳉鱼很容易吸收和食用活食物。为最佳生长, 饲料鱼苗每天两次与过剩新鲜孵化盐水虾 (卤咸藻)。充分饱足的标志是橙色的腹部后, 每喂养 10-15 分钟。每天用巴斯德吸管抽出多余的、吃剩的和分解过的盐水虾。</li>
</ol>5. 饲养幼鱼和成年鱼类
<ol><li>孵化后五天, 将幼仔移至水循环系统。使用一次性塑料吸管 (或塑料勺), 小心地转移每 0.8 L 坦克配备400µm 油炸屏幕 (图 2) 的五青少年。 注: 有可能部分少年鳉鱼不会填满气膀胱, 导致典型的 "腹部滑块" 表型, 其特征是鱼没有达到适当的浮力, 迫使他们连续游泳, 造成严重畸形成年鱼。这些鱼不能用于生存化验或有效育种, 需要被审查。</li>
	<li>每日两次喂养幼鱼, 新鲜孵化的盐水虾超过14天后孵化。每天从每罐底部抽出碎片。</li>
	<li>在14天的年龄, 转移幼鱼到2.8 升坦克配备了850µm 油炸屏幕。从这一点开始, 标签每个坦克与鱼的 ID, 表明舱口日期, 应变信息, 鱼类性别和鱼类识别号码 (图 3)。为生存化验, 单独地房子每条鱼在单坦克从这一点开始。</li>
	<li>在接下来的7天里, 每条鱼用2毫升的盐水虾每天喂两次幼仔。在这个阶段鱼可以补充与 1-3 活的血液蠕虫 (如果血液蠕虫幼虫是太大为鱼, 砍他们入更小的片断用剃刀刀片)。为防止水质恶化, 每周抽出两次吃剩的食物和额外的废物。</li>
	<li>从孵化3周后, 从罐体背面取出油炸筛, 每天两次喂鱼2毫升盐水虾和0.5 毫升的血蠕虫。在这个阶段, 青少年应该达到1厘米的身体大小, 应该能够摄取全尺寸的血蠕虫。</li>
	<li>在4周的年龄, 每条鱼每天两次喂养2毫升的盐水虾和1毫升的血蠕虫。雌性可以被共同安置在密度高达3女性每 2.8 L 坦克。</li>
	<li>在这个阶段确保鱼达到完全性成熟。检查是否存在大背, 肛门和尾鳍的迹象, 男性和圆形腹部满卵的雌性。 注: 在单个贮罐中饲养成年鱼以进行生存队列研究可能对鱼类的行为和健康产生负面影响。然而, 群体住房的生存队列研究增加了重要的混杂因素, 由于建立社会优势和男性领土, 导致严格的社会等级制度。</li>
</ol>6. 喂养
注: 实验室绿松石鳉鱼可喂养婴儿盐水虾 (卤虫藻幼体) 和血蠕虫 (摇幼虫) 的组合.绿松石鳉鱼鱼苗只喂养婴儿盐水虾。幼鱼和成年鱼类每天喂两次盐水虾和血蠕虫 (图 2)。理想情况下, 鱼可以每天喂食多次, 超过本议定书所示的2喂养。
<ol><li>
盐水虾养殖
	<ol>
<li>将10升反渗透 (RO) 水和350克红海盐添加到盐水虾孵化中, 用曝气管曝气溶解。</li>
		<li>用5毫升高不饱和脂肪酸 (HUFA) 来丰富培养。</li>
		<li>在孵化液中加入20克盐水虾囊肿。检查盐水虾囊肿不漂浮在水面上, 并确保适当的氧合和循环的文化。 注: 每日整除数的干盐水虾囊肿可储存在4摄氏度。</li>
		<li>第二天下午, 供应的文化与另一整除5毫升的 HUFA。</li>
	</ol>
</li>
	<li>
收获孵化的盐水虾 注: 从起动培养到36小时后, 盐水虾就可以收割 (二龄期)。<ol>
<li>收集 5 L 的文化在一个容器中使用水龙头底部的孵化器, 让坐10分钟。</li>
		<li>10分钟后, 从5升容器的顶部除去盐水虾壳 (棕色), 并通过网格过滤孵化的盐水虾 (橙色)。注意排除在容器底部发现的沉淀物, 因为它含有非孵化蛋和死盐水虾。</li>
		<li>用 RO 水将孵化后的盐水虾冲洗成2升的容器, 让它坐10分钟。</li>
		<li>10分钟后, 再用滤网过滤盐水虾, 并在2升的 RO 水中采集。</li>
		<li>重复前三步骤, 直到盐水虾解决方案没有孵化囊肿和盐水虾壳。</li>
		<li>将盐水虾从2升容器中转移到挤瓶中进行喂养。 注: 养殖盐水虾是相当健壮和可靠的。但是, 为了避免盐水虾在孵化失败的情况下短缺, 可以使用较小的 (500 毫升) 备份 hatchers。</li>
	</ol>
</li>
	<li>
设置备份孵化器
	<ol>
<li>用曝气法将17.5 克的红海盐溶解在500毫升的 RO 水中。</li>
		<li>以500µL 的 HUFA 丰富文化。</li>
		<li>加入2克盐水虾囊肿。</li>
		<li>在 18-24 h 以后, 再供应文化以500µL HUFA。 注: 盐水虾准备在24小时后收割。</li>
	</ol>
</li>
	<li>
活血蠕虫的制备
	<ol>
<li>在喂食之前, 用 RO 水过滤适量的血虫。 注: 活血蠕虫可以储存在4°c 7-10 天。</li>
		<li>用少量的 RO 水将血蠕虫冲洗成塑料容器。</li>
		<li>用一个塑料巴斯德吸管 (狭窄的尖端去除), 拿起血液蠕虫混合物喂养。 注: 用来自联合国控制源的活食物喂养实验室鳉鱼菌落增加了来自寄生虫和潜在致病微生物群落的外部污染物的风险。今后应开发一种特殊的不育鱼饲料。</li>
	</ol>
</li>
</ol>7. 鳉鱼实验室菌株基因分型
注意: 为了区分绿松石鳉鱼菌株, 以及确定每个菌株中的性别, 可以使用特定的基因 (微卫星) 标记 9 ( 表1)。
<ol><li>
采样
	<ol>
<li>把鱼牢牢地放在湿海绵顶部的网中。</li>
		<li>拭子 2-3 鳞片从鱼体从笼盖到尾翅使用棉签。</li>
		<li>在含氢氧化钠溶液的1毫升管 (200 µL 0.5 摩尔/L 氢氧化钠, 1% β巯基乙醇, 0.5% 聚乙烯醇吡咯烷酮) 中, 选取从拭子中提取的鳞片并转移 2-3 鳞片。</li>
		<li>在十五年代旋转 PCR 管以确保鳞片完全浸入氢氧化钠溶液中。</li>
	</ol>
</li>
	<li>
基因组 DNA 分离
	<ol>
<li>在95摄氏度孵化样品20分钟。</li>
		<li>冷却在 RT, 中和样品以1/10 容量 1 M 三 HCl, pH 8.0。</li>
		<li>以全速离心样品5分钟。</li>
	</ol>
</li>
</ol>8. 水参数
注: 在目标物种的整个生命周期内, 其预期用途为成人分型的生物体的畜牧业需要高度稳定的畜牧业条件。因此, 培养水生生物, 如绿松石鳉鱼, 必须严格控制水的参数。水循环, 再加四步水过滤, 确保了可靠的基础, 以达到对水参数的控制, 提供所有的水箱, 随着时间的推移相同的水条件。建议将系统水从反渗透 (RO) 水中重新使用, 并加入商业海水盐和碳酸氢钠。
<ol><li>水循环系统方案: 第一, 从鱼缸中流出的废水通过固体微粒金属过滤器来捕获所有未被吃掉的食物碎片和更大的微粒。金属过滤器每周冲洗三次;其次, 在第一次机械过滤后, 水被输送到大污水坑中, 然后注入到生物滤池中, 细菌将氨转化为亚硝酸盐和硝酸盐;第三, 从生物滤池中, 水被泵送到 25-µm 过滤套管, 它可以捕获更细的颗粒。最后, 水流经紫外线 (紫外线) 灯, 从细菌和病毒中消毒水。经过这四步, 过滤后的水返回到鱼缸。</li>
	<li>为了减少微生物在水箱中的生长, 防止硝酸盐积聚, 降低总盐度, 每天处理 10-20% 的系统水。</li>
	<li>保持水温恒定在28摄氏度, 水 pH 常数在7.0 到7.5 范围内。</li>
	<li>虽然鳉鱼容忍广泛的盐度, 以避免 oodinosis, 保持电导率范围内 650-710 微西门子。一年长达12小时的光/暗循环确保了群体的健康和生产力。 注: 鳉鱼可耐受1500微西门子的水电导率。</li>
</ol>

<ol>
	<li>Valenzano, D. R., Aboobaker, A., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-canonical+aging+model+systems+and+why+we+need+them.">Non-canonical aging model systems and why we need them.</a> EMBO J. 36 (8), 959-963 (2017).</li><li>Harel, I., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Turquoise+Killifish:+A+Model+for+Exploring+Vertebrate+Aging+and+Diseases+in+the+Fast+Lane.">The African Turquoise Killifish: A Model for Exploring Vertebrate Aging and Diseases in the Fast Lane.</a> Cold Spring Harb Symp Quant Biol. 80, 275-279 (2015).</li><li>Smith, P., 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=Regulation+of+life+span+by+the+gut+microbiota+in+the+short-lived+African+turquoise+killifish.">Regulation of life span by the gut microbiota in the short-lived African turquoise killifish.</a> Elife. 6, (2017).</li><li>Cellerino, A., Valenzano, D. R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+bush+to+the+bench:+the+annual+Nothobranchius+fishes+as+a+new+model+system+in+biology.">From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology.</a> Biol Rev Camb Philos Soc. 91 (2), 511-533 (2016).</li><li>Kim, Y., Nam, H. G., Valenzano, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+short-lived+African+turquoise+killifish:+an+emerging+experimental+model+for+ageing.">The short-lived African turquoise killifish: an emerging experimental model for ageing.</a> Dis Model Mech. 9 (2), 115-129 (2016).</li><li>Blazek, R., 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=Repeated+intraspecific+divergence+in+life+span+and+aging+of+African+annual+fishes+along+an+aridity+gradient.">Repeated intraspecific divergence in life span and aging of African annual fishes along an aridity gradient.</a> Evolution. 71 (2), 386-402 (2017).</li><li>Terzibasi, 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=Large+differences+in+aging+phenotype+between+strains+of+the+short-lived+annual+fish+Nothobranchius+furzeri.">Large differences in aging phenotype between strains of the short-lived annual fish Nothobranchius furzeri.</a> PLoS One. 3 (12), e3866 (2008).</li><li>Kirschner, J., 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=Mapping+of+quantitative+trait+loci+controlling+lifespan+in+the+short-lived+fish+Nothobranchius+furzeri--a+new+vertebrate+model+for+age+research.">Mapping of quantitative trait loci controlling lifespan in the short-lived fish Nothobranchius furzeri--a new vertebrate model for age research.</a> Aging Cell. 11 (2), 252-261 (2012).</li><li>Valenzano, D. R., 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=Mapping+loci+associated+with+tail+color+and+sex+determination+in+the+short-lived+fish+Nothobranchius+furzeri.">Mapping loci associated with tail color and sex determination in the short-lived fish Nothobranchius furzeri.</a> Genetics. 183 (4), 1385-1395 (2009).</li><li>Reichwald, K., 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=Insights+into+Sex+Chromosome+Evolution+and+Aging+from+the+Genome+of+a+Short-Lived+Fish.">Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.</a> Cell. 163 (6), 1527-1538 (2015).</li><li>Valenzano, D. R., 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=The+African+Turquoise+Killifish+Genome+Provides+Insights+into+Evolution+and+Genetic+Architecture+of+Lifespan.">The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.</a> Cell. 163 (6), 1539-1554 (2015).</li><li>Harel, I., 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=A+platform+for+rapid+exploration+of+aging+and+diseases+in+a+naturally+short-lived+vertebrate.">A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate.</a> Cell. 160 (5), 1013-1026 (2015).</li><li>Valenzano, D. R., Sharp, S., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-Mediated+Transgenesis+in+the+Short-Lived+African+Killifish+Nothobranchius+furzeri,+a+Vertebrate+Model+for+Aging.">Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging.</a> G3. 1 (7), 531-538 (2011).</li><li>Harel, I., Valenzano, D. R., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+engineering+approaches+for+the+short-lived+African+turquoise+killifish.">Efficient genome engineering approaches for the short-lived African turquoise killifish.</a> Nat Protoc. 11 (10), 2010-2028 (2016).</li><li>Polacik, M., Blazek, R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+breeding+of+the+short-lived+annual+killifish+Nothobranchius+furzeri.">Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri.</a> Nat Protoc. 11 (8), 1396-1413 (2016).</li><li>Avdesh, A., 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. (69), e4196 (2012).</li></ol>

所有的作者都宣称没有竞争的金融和非金融利益。

我们描述了一项关于绿松石鳉鱼实验室培养的协议, 包括胚胎采集, 孵化, 以及成年鱼类的住房, 饲养和饲养。我们的议定书专门针对从事成年鱼类研究的实验室, 特别是关于衰老和寿命的实验研究。绿松石鳉鱼可以在标准的斑马鱼设施上饲养;然而, 鳉鱼畜牧业的重要方面不同于标准的斑马鱼护理 16.这些调整包括从盐水虾只饮食的早期过渡到饮食补充富含蛋白质的血液蠕虫, 以及胚胎孵化的具体步骤, 包括液体和干燥孵化阶段。
协议中的关键步骤包括在 8-30 °c 温度范围内运送胚胎。在繁殖的情况下, 生育力取决于摄食频率和食物质量;因此, 我们建议至少每天喂养两个饲养池, 以提高胚胎产量 (见5.6 节)。在胚胎漂白过程中, 不要在漂白溶液中延长胚孵化。这可能会对鸡蛋绒毛膜造成损害, 增加胚胎死亡率。当用亚甲蓝孵化胚胎时, 不要延长准备孵化胚胎的潜伏期超过2周, 因为它们的生存能力将大大减少。为孵化绿松石鳉鱼, 腐植酸溶液的低温改善孵化和完全浸泡的胚胎在解决方案允许同步孵化。没有足够的曝气在孵化期间导致高率的鱼苗无法填满气体膀胱 ("肚皮滑块" 表型, 见5.1 节附注)。
育种议定书的限制包括使用对集中过滤系统构成挑战的砂基板, 今后应改用替代方法。可能的替代方案可能是使用斑马鱼养殖坦克。胚胎漂白可能会导致鸡蛋绒毛膜的物理化学变化, 可能导致改变绒毛膜生理和孵化成功。胚胎持续暴露于亚甲基蓝可能会引起成年鱼类生理的长期变化。在单独的坦克中饲养成年鱼进行生存队列研究可能会对鱼类的行为和健康产生负面影响。然而, 群体住房的生存队列研究增加了重要的混杂因素, 由于建立社会优势和男性领土, 导致严格的社会等级制度。因此, 我们认为, 分离雄性鱼类进行生存研究是一个合理的妥协。喂养实验室鳉鱼菌落和来自非控制源的活食物增加了来自寄生虫和潜在致病微生物群落的外部污染物的风险。今后应开发一种特殊的不育鱼饲料。
今后对该议定书的改进将集中在受控的非生活饮食, 这仍然导致 3-4 周内完成性成熟。总之, 我们的协议为绿色鳉鱼实验室培养到广泛的科学界提供了方便。

由于短寿命和快速生命周期, 绿松石鳉鱼正在迅速成长为一个有前途的新的模式生物在生物学 1, 2, 3.这个物种的特点是一个独特的生命周期的硬骨鱼类, 包括胚胎滞育, 快速性成熟, 和延长后生殖生命阶段4, 5.最近的工作有助于阐明这一物种的生物学在圈养和野生6,7。绿松石鳉鱼生活在津巴布韦和莫桑比克的非洲大草原雨季期间形成的季节性淡水体中。在旱季, 由于不缺水, 胚胎在干燥的泥浆中存活, 这是一种叫做滞育的抗应力生命期。
该物种的遗传图谱已生成8,9, 最近他们的基因组已经测序和组装10,11。一些近交的实验室鱼类菌株已经开发, 转基因和基因组编辑通过 CRISPR/Cas9 已成为在这个物种,事实上促进绿松石鳉鱼作为一个竞争性实验室脊椎动物模型生物12,13,14。
虽然已经为这个物种发布了一个实验室协议15, 但在本议定书中, 我们制定了一份全面的实验实验室指南, 专门针对调查衰老和生存的研究。本议定书使已经熟悉斑马鱼和鳉畜牧业的研究人员通过最小数量的关键调整来精通绿松石鳉鱼畜牧业。同时, 该议定书还为研究人员提供了在渔业方面没有经验的重要工具, 以提高蓬勃发展的绿松石鳉鱼殖民地。

<table><tbody><tr><td>Probe calibration buffer solution pH=7.0</td><td>Roth</td><td>A518.1</td><td>1L buffer solution pH=7.0 to calibrate water system pH-electrode</td></tr><tr><td>Probe calibration buffer solution pH=4.0</td><td>Roth</td><td>P712.1</td><td>1L buffer solution pH=4.0 to calibrate water system pH-electrode</td></tr><tr><td>Conductivity standard</td><td>VWR</td><td>83607.260</td><td>500 mL Conductivity standard 1,413 uS/cm to calibrate water system conductivity-electrode</td></tr><tr><td>Easy Strips Test 6in1</td><td>JBL</td><td>2533900</td><td>Test strips for determination chlorine values of system water</td></tr><tr><td>Ammonia Test</td><td>JBL</td><td>2536500</td><td>Test to determine ammonia content of system water</td></tr><tr><td>Red Sea Salt</td><td>Red Sea</td><td></td><td>22 kg bucket</td></tr><tr><td>Sodium hydrogen carbonate</td><td>VWR</td><td>27780.360</td><td></td></tr><tr><td>Humic acid</td><td>Sigma- Aldrich</td><td>53680-50G</td><td></td></tr><tr><td>New HUFA Artemia enrichment</td><td>ZM Systems UK</td><td></td><td>75g bottle</td></tr><tr><td>Methylene blue</td><td>Roth</td><td>AE64.1</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma- Aldrich</td><td>31642-1L</td><td>30% (w/w)</td></tr><tr><td>Coconut fiber</td><td>Dragon</td><td>ZCS010</td><td></td></tr><tr><td>Whatman paper</td><td>GE healthcare</td><td>3030-690</td><td></td></tr><tr><td>Ethanol pure</td><td>VWR</td><td>20821.467</td><td>100%</td></tr><tr><td>Silica sand</td><td>local pet shop</td><td></td><td></td></tr><tr><td>Artemia Eggs Premium Grade</td><td>Sanders</td><td></td><td></td></tr><tr><td>Bloodworm</td><td>local distributor</td><td></td><td>Poseidon Aquakultur Germany</td></tr><tr><td>dNTPs solution mix</td><td>Biolabs</td><td>N04472</td><td>10mM</td></tr><tr><td>Taq DNA polymerase</td><td>Invitrogen</td><td>18038-042</td><td>5U/uL</td></tr><tr><td>PCR 10x Buffer</td><td>Invitrogen</td><td>18038-042</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Invitrogen</td><td>18038-042</td><td>50mM</td></tr><tr><td>NaOH</td><td>Sigma- Aldrich</td><td>S8045-500g</td><td>50mM</td></tr><tr><td>Tris-HCl, pH=8.0 solution</td><td>Sigma- Aldrich</td><td>T2694-1L</td><td>1M</td></tr><tr><td>HCl 37%</td><td>Sigma- Aldrich</td><td>H1758-500mL</td><td></td></tr><tr><td>Fish tanks</td><td>Aquaneering</td><td></td><td>volume: 0.8L, 2.8L, 9.5L; equipped with baffles, fry mesh and lids</td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>89032-100</td><td>model 5000</td></tr><tr><td>Microbiological incubator</td><td>Thermo Scientific, Heratherm</td><td>50125882</td><td>model IMC18; for storage embryos in the liquid phase, set to t=27-28&amp;deg;C</td></tr><tr><td>Cooling Incubator</td><td>Binder</td><td>9020-0209</td><td>model KT115; for storage embryos in the solid phase, set to t=27-28&amp;deg;C</td></tr><tr><td>Hatching incubator</td><td>Thermo Scientific, Heratherm</td><td>51028114</td><td>model OGS180; for embryos hatching, set to t=27-28&amp;deg;C</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td></td><td>model M80</td></tr><tr><td>Breeding sand/hatching boxes</td><td>Roth</td><td>1598.1</td><td>1000mL</td></tr><tr><td>Petri dish</td><td>Sarstedt</td><td>82.1473</td><td>92x16mm</td></tr><tr><td>50 mL Conical tube</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>15 mL Conical tube</td><td>Sarstedt</td><td>62.554.002</td><td></td></tr><tr><td>Disposable Plastic Pasteur pipette</td><td>Roth</td><td>EA71.1</td><td>2mL; For fish feeding with bloodworms, or embryos selection cut off the tip to open 3-4mm diameter</td></tr><tr><td>Serological pipette</td><td>Sarstedt</td><td>86.1689.001</td><td>50mL</td></tr><tr><td>Syringe</td><td>Henke Sass Wolf</td><td>4100-000V0</td><td>10mL</td></tr><tr><td>Metal strainer</td><td></td><td></td><td>fineness &amp;lt;1mm; for embryos collection</td></tr><tr><td>Tweezers</td><td>Dumont</td><td>0508-5/45-PO</td><td>type5/45; for embryos transfer</td></tr><tr><td>25 L Brine shrimp hatcher</td><td>Aquaneering</td><td>ZHBS25</td><td>main hatcher</td></tr><tr><td>500 mL Brine shrimp hatcher</td><td>JBL</td><td>6106100</td><td>model Artemio 1; backup hatcher</td></tr><tr><td>Narrow-mesh fish nets</td><td>JBL</td><td></td><td></td></tr><tr><td>Sand beaker</td><td>VWR</td><td>BURK7102-5000</td><td>5000mL</td></tr><tr><td>Brine shrimp separation beaker</td><td>VWR</td><td>BURK7102-2000</td><td>2000mL</td></tr><tr><td>Plastic zipper bag</td><td>Roth</td><td>P279.2</td><td>for dead fish storage</td></tr><tr><td>Pipetboy</td><td>Integra</td><td>155000</td><td>model Pipetboy acu2</td></tr><tr><td>Parafilm</td><td>P-Lab</td><td>P701605</td><td></td></tr><tr><td>Air tubing</td><td>www.zajac.de</td><td>AQ380</td><td>4-6 mm diameter</td></tr><tr><td>1 L Glass bottle</td><td>VWR</td><td>215-1595</td><td></td></tr><tr><td>2 L Glass bottle</td><td>VWR</td><td>215-1596</td><td></td></tr><tr><td>500 mL Squeeze bottle</td><td>Roth</td><td>K665.1</td><td>for fish feeding with brine shrimp</td></tr><tr><td>120-&amp;#956;m brine shrimp strainer</td><td>Florida Aqua Farms</td><td>BB-PC2</td><td>for brine shrimp/bloodworm collection</td></tr><tr><td>Finish filter socks</td><td>Aquaneering</td><td>MFVB025C</td><td>25-&amp;#956;m</td></tr><tr><td>Central filtration fish housing system</td><td></td><td></td><td>Aquaneering, Techniplast, Aquatic Habitats, Aqua Schwarz</td></tr></tbody></table>

a protocol for laboratory housing of turquoise killifish (nothobranchius furzeri)

开发用于实验目的的非模型实验室鱼类的畜牧业做法, 大大得益于建立参考鱼类模型系统, 如斑马鱼和鳉。近年来, 一条新兴的鱼类--绿松石鳉鱼 (Nothobranchiusfurzeri)--已被越来越多的生物老化和生态学领域的研究小组采用。由于 4-8 月的圈养寿命, 这一物种是在圈养中饲养的最短的脊椎动物, 允许科学界在短时间内对实验性干预进行试验, 从而导致老化率和预期寿命的变化。鉴于这一物种独特的生物学特性, 其特点是胚胎滞育、爆炸性性成熟、明显的形态学和行为性异形以及它们相对较短的成年寿命--临时畜牧业的做法迫在眉睫. 需求。该议定书报告了一系列关键的畜牧业措施, 允许最佳的绿松石鳉鱼实验室护理, 使科学界能够采用这个物种作为一个强大的实验动物模型。

绿松石鳉鱼适当的畜牧业导致中位存活率在 GRZ 菌株的 12-18 周之间 (如图4A)。中位存活率的变化取决于饮食、喂养频率和住房温度条件。恶劣的畜牧业导致了生存曲线的出现, 从而增加了早期死亡率和重复的, 突然下降的生存时间, 特点是几个拐点 (图 4B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57073/57073fig1.jpg"> 图 1: 具有各自孵化基质的代表性胚胎发育阶段。(A)新鲜收集的胚胎, 孵化在28摄氏度的孵化器中的亚甲基蓝溶液中。(B)胚胎准备转移到固体介质中, 无论是过滤纸还是椰子纤维。(C)胚胎准备孵化, 显示典型的金色虹膜。刻度条等于1毫米.<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig1large.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/57073/57073fig2.jpg"> 图 2: 绿松石鳉鱼孵化后发育阶段。<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig2large.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/57073/57073fig3.jpg"> 图 3: 从阶段3开始的鱼的代表性鱼 ID 标记.<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57073/57073fig4.jpg"> 图 4:70 雄性绿松石鳉鱼的代表性生存曲线。(A)实验室饲养的绿松石鳉鱼的典型生存曲线。(B)在最佳畜牧业条件 (黑色) 和不良畜牧业 (红色和蓝色) 下养殖的鱼类生存曲线的比较。虚线红色水平线表示50% 生存, 相交生存曲线在中值寿命 (在 x 轴上表示)。<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Id</td>
			<td colspan="2">前进底漆</td>
			<td colspan="2">反向底漆</td>
			<td>尺寸范围 (bp)</td>
		</tr>
<tr>
<td>* NfuSU0007</td>
			<td colspan="2">GGCTAAGCCTTGCTGACAGA</td>
			<td colspan="2">CAGGGAGCTGAAAACCTCAG</td>
			<td>166-214</td>
		</tr>
<tr>
<td>* NfuSU0010</td>
			<td colspan="2">CGCAGTCTGATCAAATCGTGT</td>
			<td colspan="2">TGTTTGAAGGTTCACATTCATTATC</td>
			<td>220-272</td>
		</tr>
<tr>
<td>NfuSU0016</td>
			<td colspan="2">CATGGCTAAACCGTGATGAA</td>
			<td colspan="2">GAAGGACGCCAGCTATGAAG</td>
			<td>209-240</td>
		</tr>
<tr>
<td>NfuSU0022</td>
			<td colspan="2">AACACAGCTCTCGTAAGGAGGTA</td>
			<td colspan="2">TTCAGACTTGTCTTACTACCATGTTT</td>
			<td>198-238</td>
		</tr>
<tr>
<td>NfuSU0027</td>
			<td colspan="2">TCCAGCTGAATCGGTAATGA</td>
			<td colspan="2">AAACTCGAGGGTGCAATCTG</td>
			<td>164-226</td>
		</tr>
<tr>
<td>NfuSU0049</td>
			<td colspan="2">CTGGACAAAGTGCCAATCAC</td>
			<td colspan="2">CTCCCACAGTCCCAAAACAT</td>
			<td>196-197</td>
		</tr>
<tr>
<td>NfuSU0050</td>
			<td colspan="2">CCAGAATGAACAATACTCAGATCAA</td>
			<td colspan="2">GCAGCTTAGTTTAATGATATCACAATG</td>
			<td>252-295</td>
		</tr>
<tr>
<td>NfuSU0060</td>
			<td colspan="2">CTAGCCACTCCCCTGGTTTA</td>
			<td colspan="2">CCGTCACGATGTGCTGATAC</td>
			<td>216-248</td>
		</tr>
<tr>
<td>NfuFLI0030</td>
			<td colspan="2">CAGAAGCTAAAGGCCAGACG</td>
			<td colspan="2">GGGAAACAATAGGGAACCAC</td>
			<td>174-205</td>
		</tr>
<tr>
<td>* NfuFLI0091</td>
			<td colspan="2">ACGCTGACTCTACCCAGTC</td>
			<td colspan="2">CTGCCTGCTACTGACAATG</td>
			<td>355-373</td>
		</tr>
<tr>
<td colspan="6">* 性别决定标记</td>
		</tr>
</tbody></table>表 1: 菌株鉴定的基因型引物。

Watch this Scientific Journal Video about A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri at JoVE.com

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

在集中式水过滤系统中, 可以将绿松石鳉鱼的实验室房屋扩大到室内, 并有效地提高数以千计的个体鱼类, 同时采用与标准斑马鱼设施相同的基础设施。在这里, 我们详细列出了一个标准化的程序, 允许有效的鳉鱼维护。

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

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Research

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Biology

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

16.8K 次观看.  Max Planck Institute for the Biology of Ageing. 在集中式水过滤系统中, 可以将绿松石鳉鱼的实验室房屋扩大到室内, 并有效地提高数以千计的个体鱼类, 同时采用与标准斑马鱼设施相同的基础设施。在这里, 我们详细列出了一个标准化的程序, 允许有效的鳉鱼维护。

视频: A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

科研

环境科学

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

用原位曝光法测定淡水珍珠贻贝 Bioindication 的流环境适宜性

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

提高墨西哥龙舌兰对幼虫后表型和全体免疫组织化学的分析

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

遗传学

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

墨西哥辣鱼的行为跟踪与神经吻合

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

行为学

A Standardized Protocol for Preference Testing to Assess Fish Welfare

Animal welfare assessment techniques try to take into consideration the specific needs and wants of the animal in question. Providing enrichment (the addition of physical objects or conspecifics in the housing environment) is often a way to give captive animals the opportunity to choose who or what they interact with and how they spend their time. A fundamental component of the aquatic environment that is often overlooked in captivity, however, is the ability for the animal to choose to engage in physical exercise. For many animals, including fish, exercise is an important aspect of their life history, and is known to have many health benefits, including positive changes in the brain and behavior. Here we present a method for assessing habitat preferences in captive animals. The protocol could easily be adapted to look at a variety of environmental factors (e.g., gravel versus sand as a substrate, plastic plants versus live plants, low flow versus high flow of water) in different aquatic species, or for use with terrestrial species. Statistical assessment of preference is carried out using Jacob&#39;s preference index, which ranks the habitats from -1 (avoidance) to +1 (most preferred). With this information, it can be determined what the animal wants from a welfare perspective, including their preferred location.

A fundamental aspect of assessing the welfare of animals in captivity is to ask whether the animals have what they want. Here, we present a protocol to determine housing preference in the zebrafish (Danio rerio) with respect to the presence/absence of environmental enrichment and access to flowing of water.

偏好测试评估鱼类福利的标准化协议

Animal welfare assessment techniques try to take into consideration the specific needs and wants of the animal in question. Providing enrichment (the ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

主机表型的考试<em&gt;食蚊鱼</em&gt;继抗生素治疗

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

免疫与感染

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

生物学

Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

一个经济实惠，易于构建斑马鱼设施建设

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Here we present a protocol to isolate nuclei from the brains of the short-lived vertebrate model Nothobranchius furzeri for downstream applications such as single-nucleus RNA sequencing or single-nucleus assay for transposase-accessible chromatin with sequencing (ATAC-seq).

从成年非洲绿松石鳉鱼的脑组织中温和分离细胞核，这是一种自然短暂的衰老研究模型

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we ...

神经科学

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

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