在保健和本协议描述的代表性结果产生鱼的使用是完全按照规定的机构动物护理和使用委员会在波士顿儿童医院，协议＃14-05-2673R的指导方针进行。

1.轮虫文化<ol><li> 文化系统使用的是100升培养容器基本组件 <ol><li>收集所有组件的轮虫培养设置。轮虫培养装置由一个培养容器（CV）发展壮大的轮虫;类似的船只，以保持喂料轮虫（喂料培养容器，FCV）;圆底孵化缸（订阅水库，FR），用于存储所述藻类饲料混合物（AFM）的;气源（AS），以充气的简历，燃料电池汽车和FR;蠕动泵计量定时器（PMT）来控制藻类交付饲料进入CV和燃料电池汽车;和牙线微粒过滤器（FPF），坐落在简历里面。 注 :耗材和组件的完整列表中的材料清单提供。 </li></ol></li><li> 组态 <ol><li>提升支架上或桌子上的简历和燃料电池汽车，这样的文化可以通过排水管装修成一个集合续轻松收获ainer（ 图1）。使用灵活的送风管到AS连接到每个培养容器的长度刚性管。确保管足够长以将空气输送到CV或燃料电池汽车的底部。 </li><li>使用小容量的空气线到AS连接到一个长度刚性管的延伸到包含AFM对FR的底部。安装一个阀门到每个空气管路调节风量。帧中继连接到与饲料输送管的PMT和运行从PMT的管导入钻入CV / FCV的一侧，在顶部附近的孔。 图1。 </li></ol></li><li> 启动 <ol><li>培养容器填充到90％的可用容量与反渗透水（RO）的。如果RO不可用，使用清洁，脱氯市政供水;然而，应该执行一个生物安全风险评估，以确保没有潜在的致病微生物存在于水源。注:这种分析，可以执行任何资质的水质检测实验室。 </li><li>剂量的培养容器水与水族箱盐达到15克/升的盐度。置的空气流进入容器，使得它保持一个"滚动沸腾"，然后慢慢盐的测定量添加到培养容器，直到它完全被曝气溶解。继续曝气水为&gt; 1小时，以确保其完全氧化。 </li><li>使藻类进料混合物。向3升的干净，脱氯鲜（0ppm的）水加入100克碳酸氢钠和100克的氨中和剂（钠hydroxymethylsulfonate）。这最后的试剂提供中和从自来水或漂白残基的任何残余的氯从消毒的培养设备的额外的好处。以确保这些化合物完全溶解，这一点至关重要。然后加入1升藻浓缩液（生物干重〜15％）。添加进料混合物，以FR和储存在4℃。 </li><li>加5的首发文化年 - 1000万元<eM&gt;含充气15克/ L矿化度水褶皱臂尾轮虫轮虫的简历。如果轮虫已装运或储存过程中被冷却，它们应该是逐渐（在30分钟期间或更长的时间）驯化的水在培养容器中的温度（25 - 27℃）。 </li><li>打开PMT，并开始抽藻送入轮虫培养容器。使用PMT的定时器功能，设置藻类饲料混合物的排出流量，使得〜1.6 ml的藻类饲料混合物的每百万轮虫在培养递送，每天。分布在一小部分的喂食定期超过24小时的周期过程;越频繁喂食，效果更佳。 </li><li>通过手动开启PMT为在设定时间内（例如，1分钟）校 ​​准泵的输出率，并收集它此间隔成量筒或烧杯中泵藻类。例如，如果该PMT剂量5 ml的藻类中1分钟，然后将剂量率将是5 ml的藻类/分钟。 </li><li>乘以本轮虫的数以百万，1.6 ml的计算所需的日常饲养率。例如，对于在100万轮虫人口尺寸的轮虫培养需要〜160 ml的饲料，每天（100×1.6毫升）的。 </li><li>设置的PMT，在整个24小时内的剂量以规则间隔的总日饲料需求。例如，递送的160毫升的总日饲料量可以以能够部分被递送一次，每3小时，在24小时内使用的PMT组为5毫升/分钟4分钟，每日8次的计量泵速率（5毫升/分钟×4分钟= 20毫升×8喂食= 160毫升）。 </li><li>让文化成长，直到它生成所需的人口，一般为48 - 72小时，收获前。在24小时后启动，添加牙线粒子滤波器到培养容器中，并开始正常的维护。 </li></ol></li><li> 保养 注:培养运行在一个连续的基础上，并要求routin而理想地应在同一时间的每一天进行，按照以下顺序ë维修。 <ol><li>燃料电池汽车填充到90％的有效量的清洁，脱氯淡水，给药10克/升的水族馆的盐。确保水充分混合，并且所有的盐的完全溶解。置的空气流进入容器，使得它保持一个"滚动沸腾"。测量盐度用折射并确保盐度为10克/升。要达到这个目标，不要超过它，这一点至关重要。 </li><li>样品中的CV轮虫:确保培养充分混合，然后收集3个样本的2 - 3各自使用一个移液管或autopipettor毫升，从培养的不同部分。结合这些样品中的管或小瓶方便的大小（例如，10ml）中的。 </li><li>转移1 - 2毫升组合样本的上一个培养皿，以便它可以在解剖显微镜下观察。检查文化素质（游泳的行为轮虫，存在脱落的鸡蛋，污染原生动物）。 </li><li>通过加入100微升50％Lugols碘溶液到样品固定剩余组合的样品中的轮虫。在几秒钟内加入Lugols后，观察轮虫停止游泳。现在，很容易算轮虫。 注:乙醇，稀漂白剂，或醋可以代替Lugols的使用。醋（2滴/ 10毫升）中有被优点无危险，没有失去强度中存储作为漂白和碘溶液可以，并没有使轮虫合同，所以纤毛的电晕和"脚"保持伸出并且动物看起来更自然。 </li><li>确保样品充分混合（固定轮虫会沉淀迅速），然后迅速采取约2ml的子样本在一个塑料吸管和分配1 ml的成塞奇威克-拉夫特计数滑动件（20×50 1毫米正方形）（图2 ）。使用解剖或复合式显微镜，算上完整轮虫和Total号码附着到这些轮虫蛋（图2）。计数一样多的滑动面积的必要来算〜100轮虫。计算每毫升轮虫的数量，并在电子表格或日志记录此。 </li><li>收获〜于CV轮虫的体积的30％:除去空气供应和牙线过滤，慢慢在CV的底部打开阀并允许水流入一个浮游生物集电极与一个53微米网状筛。收集过滤成桶或漏极后流出收集器的底部的水。使用温和到中等流量，以免损坏轮虫。不要让轮虫干在屏幕上。 </li><li>出于一致性的原因，建议建立与燃料电池汽车的每一天，标准号轮虫。因此，基于已知的CV轮虫密度和收获量，调整燃料电池汽车总体积达到一致的最终密度（例如，1500轮虫/毫升）。添加收获烤肉FERS到FCV:（ - 24 g / L的10），轻轻地从采集窗口使用的是洗瓶子装满干净的盐水转移轮虫。反转屏幕在燃料电池汽车，并用食盐水的温柔流洗轮虫到燃料电池汽车。启动PMT提供饲料（〜每天每百万轮虫1.57毫升）的燃料电池汽车。 </li><li>磨砂CV的整个内部用干净，柔软的尼龙刷或擦洗垫。 </li><li>使加入盐的适量的清洁RO水测定量在5加仑桶取代的水的体积丢失收获15克/升的水一新的组合。的盐添加到水在水桶和剧烈搅拌，直至其完全溶解，然后添加到CV。 </li><li>在水槽采用高压喷淋，冲洗绒毛过滤器，直到它是免费的碎片，然后将其返回到CV。 </li><li>调整藻类通过改变每个给药事件的持续时间递送到CV的进料速率，根据轮虫/毫升每日计数。使用在步骤1.3.8提供的计算，上述确定进料待递送的适当的量。 </li><li>约24小时后，重复上述过程。开始通过收获残留在燃料电池汽车（即没有所需的前一天）的轮虫中相同的方式，如上所述（步骤1.4.2 - 10年1月4日）。集中起来在2升的新鲜，洁净的脱氯水（5克/升的盐）。这些可以存储在4℃下作为备用电源，或用于饲料的鱼后期阶段，超出了在该协议描述。 注意:此协议允许高达2 - 3天减压培养维护（与正常的自动喂养），因为在CV轮虫可以容忍收成遗漏无严重的后果。 </li></ol></li></ol> 2.混养<ol><li> 建立 <ol><li>通过茶型过滤器浇催生的胚胎，然后用无菌FIS轻轻漂洗收集从产卵事件斑马鱼胚胎h进水（或适当地调节溶液的其它任何未污染源;例如，胚胎培养基，E3等）从一个洗涤瓶入培养皿中。 </li><li>孵育胚胎在25 - 28℃下在培养皿中在40密度 - 每皿50胚胎5天。 </li><li>开始第5天受精后，或当大于90％的孵幼虫的正在积极游动在水柱混养相。 </li></ol></li><li> 接种 <ol><li>添加500毫升轮虫培养直接从FCV到3.5升育苗池;列入轮虫养殖水体藻类提供饲料用于维护轮虫混养过程中的营养成分。 </li><li>从一个培养皿中的幼虫轻轻倒入幼儿园坦克。确保没有幼虫留在菜。 </li><li>从再循环系统或专用水源加入500毫升干净，空调鱼水到容器达到1升的终体积和最终的小号alinity 5克/升。 注意:因为如果盐度是&gt; 7克/升和轮虫生存将受到负面影响，斑马鱼幼体生存将受到负面影响这盐度是至关重要的，如果盐度&lt;2克/升。 </li></ol></li><li> 混养阶段 注:混养相应持续长达4天接种后（共5天，对应于天5-9受精后）。 <ol><li>在此期间观察混养罐至少每天一次，以确保轮虫和鱼存在和生长。确保轮虫在整个水柱可见。确保了fishs也是可见的水柱内，轮虫中游泳。 </li><li>开始正常的水流量通过罐。放置一个屏幕或挡板在泄油口，以确保幼虫不会被清理出的坦克。 注:在此阶段结束时，鱼会大到足以消耗较大的猎物如卤虫125微米 - 在75的尺寸范围内的无节幼体或饲料加工物品。 注意:一代表混养罐内的轮虫种群动态通过取样测定/如步骤1.4.2描述计数从以相同的方式将罐轮虫 - 1.4.5。这是从混养阶段开始，直到完成完成每天一次。 </li></ol></li></ol>

<ol>
	<li>Ribas, L., Piferrer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+(Danio+rerio)+as+a+model+organism,+with+emphasis+on+applications+for+finfish+aquaculture+research.">The zebrafish (Danio rerio) as a model organism, with emphasis on applications for finfish aquaculture research.</a> Reviews in Aquaculture. 6, 209-240 (2014).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+tissue+regenerative+capacity+and+mechanisms+in+animals.">Advances in understanding tissue regenerative capacity and mechanisms in animals.</a> Nature reviews. Genetics. 11, 710-722 (2010).</li><li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends in genetics TIG. 29, 611-620 (2013).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+modeling+human+disease+in+zebrafish.">Hooked! modeling human disease in zebrafish.</a> Journal of Clinical Investigation. 122, 2337-2343 (2012).</li><li>Selderslaghs, I. W. T., Blust, R., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+study+of+the+zebrafish+assay+as+an+alternative+method+to+screen+for+developmental+toxicity+and+embryotoxicity+using+a+training+set+of+27+compounds.">Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds.</a> Reproductive Toxicology. 33 (2), 142-154 (2012).</li><li>Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+husbandry+of+zebrafish+(Danio+rerio):+A+review.">The husbandry of zebrafish (Danio rerio): A review.</a> Aquaculture. 269, 1-20 (2007).</li><li>Harper, C., Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Zebrafish (Laboratory Animal Pocket Reference). , (2010).</li><li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish, A Practical Approach. , (2002).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish. , (2007).</li><li>Carvalho, P., Arau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+zebrafish+(Danio+rerio)+larvae+without+live+food:+evaluation+of+a+commercial,+a+practical+and+a+purified+starter+diet+on+larval+performance.">Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance.</a> Aquaculture Research. 37, 1107-1111 (2006).</li><li>Spence, R., Gerlach, G., Lawrence, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+ecology+of+the+zebrafish,+Danio+rerio.">The behaviour and ecology of the zebrafish, Danio rerio.</a> Biol Rev Camb Philos Soc. 83 (1), 13-34 (2008).</li><li>Best, J., Adatto, I., Cockington, J., James, A., Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+rearing+first-feeding+larval+zebrafish:+polyculture+with+Type+L+saltwater+rotifers+(Brachionus+plicatilis).">A novel method for rearing first-feeding larval zebrafish: polyculture with Type L saltwater rotifers (Brachionus plicatilis).</a> Zebrafish. 7 (3), 289-295 (2010).</li><li>Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+zebrafish+husbandry+and+management.">Advances in zebrafish husbandry and management.</a> Methods in Cell Biology. 104, 429-451 (2011).</li><li>Lawrence, C., Sanders, E., Henry, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+saltwater+rotifers+(Brachionus+plicatilis)+for+rearing+larval+zebrafish.">Methods for culturing saltwater rotifers (Brachionus plicatilis) for rearing larval zebrafish.</a> Zebrafish. 9, 140-146 (2012).</li><li>Tucker, C. S., Hargreaves, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology and Culture of Channel Catfish. 34, 634-657 (2004).</li></ol>

EC亨利是由芦苇海水养殖公司，该公司提供的轮虫，藻类的浓缩，和其他物资，以水产养殖和鱼类爱好者市场就业。

成功实施轮虫混养方法喂养早期幼虫的斑马鱼，需要有效的协议两个任务:第一喂养以及在同一水箱轮虫斑马鱼幼体连续轮虫培养体系的建立和维护喂鱼，和培养。 设置用于连续盐水轮虫生产系统首先由劳伦斯和共同作者14所述斑马鱼的实验室已被修改和增强的多种方法，使得它更加在应用健壮和普遍。新的协议包含改进都在设备和方法。 存储系?...

斑马鱼（ 斑马鱼 ）是一个卓越的实验动物在越来越多的科学学科的利用，包括但不限于发育遗传学，毒理学，行为，水产养殖，再生生物学，以及许多人类疾病1的造型- 5。虽然品种相对容易在实验室中保持，有许多与它们培养6相关联的管理的挑战。最突出的是苗种培育，特别是当鱼第一次开始喂气膀胱充气7其后。在正常，控制的条件下，此发育事件发生在〜5天后受精（DPF），但有以下3 -生长5天是特别关键的7。在这一阶段的中心技术难度是充分满足第一馈送幼虫的营养需求 - 饲料项目必须适当尺寸，diges1149，有吸引力，并在几乎连续的基础上提供，而不在培养罐产生过多的浪费。历史上，这已经达到一般通过提供大量的少量饲料鱼的坦克，以及常规的水交换8,9。虽然这些方法有一定程度的成功，它们是效率低下的，要求高劳动投入，并返回唯一的变量和生长和存活10的速率限制。 在自然界中，斑马鱼幼体想必饲料丰富的小礼物在水体中浮游动物11。出于这个原因，结合了活饲料如草履虫 ，轮虫和卤虫 larviculture协议通常是最有效的7。 2010年，百思及其合作者表明，它是可能的静态，苦咸水以及海水轮虫的第5天的外源性饲养12幼虫生长的斑马鱼。这种方法，它利用ES轮虫培养的天然高生产率，以提供充足的，高营养的猎物，不污染水，幼虫生长和存活的低劳动力投入12,13的收益率非常高的速度。近年来，越来越多的世界各地的实验室都采用这个协议的变化，现在很多都培养轮虫以连续的方式来支持育苗系统14。 在过去的几年中，为轮虫/斑马鱼混养和轮虫的生产方式进行了细化和改进，变得更加标准化和轻松扩展。本文提供了一步一步的说明1）连续和强大的轮虫生产及2）建立用于支持鱼类生长健壮的第5天的外源性摄食轮虫/斑马鱼混养系统。

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifer Culture Infrastructure</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">100 Liter Culture Vessel</td>
			<td style="width:135px;">Aquaneering</td>
			<td style="width:119px;">Custom</td>
			<td style="width:264px;">Polycarbonate culture vessel, conical bottomed, with drain valve</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">5 Gallon Culture Bucket Kit</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">CCS Starter Kit</td>
			<td style="width:264px;">Small volume culture vessel for small facilities</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rigid Clear Tubing 1/2&#34; O.D., 36&#8221;</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">16025</td>
			<td style="width:264px;">Rigid clear tubing for air delivery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Mesh tube</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">RT444X</td>
			<td style="width:264px;">Mesh tube support for floss filter</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rotifer Floss</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">Rotifer floss 12&#8221; x 42&#8221;</td>
			<td style="width:264px;">Particulate waste trap</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Peristaltic Metering Timer Pump, 5 GPD</td>
			<td style="width:135px;">Grainger</td>
			<td style="width:119px;">38M003&#160;</td>
			<td style="width:264px;">Metering pump with timer for dosing feed to rotifers</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Peristaltic Metering Timer Pump, 1-100 mL/h (for smaller-scale culture)</td>
			<td style="width:135px;">Coral Vue</td>
			<td style="width:119px;">SKU: IC-LQD-DSR</td>
			<td style="width:264px;">Metering pump with timer for dosing feed to rotifers</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Silicone Tubing&#160;</td>
			<td style="width:135px;">Cole Parmer</td>
			<td style="width:119px;"></td>
			<td style="width:264px;">Tubing for algae delivery to rotifer vessel</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rigid Clear Tubing &#34; O.D.,36&#8221;</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">16025</td>
			<td style="width:264px;">Rigid clear tubing for air delivery to algae paste</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rigid Clear Tubing O.D., 36&#8221;</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">16025</td>
			<td style="width:264px;">Rigid clear tubing for algae delivery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifers</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Live Rotifers Brachionus plicatilis Type L</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">Type L 5 million</td>
			<td style="width:264px;">Rotifer stock culture for system startup</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifer Feed</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:305px;">Sodium hydroxymethylsulfonate</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">ClorAm-X&#174; 1lb tub</td>
			<td style="width:264px;">Ammonia reducer for algae feed mix</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Sodium Bicarbonate</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">S25533B</td>
			<td style="width:264px;">pH buffer for algae feed mix</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:305px;">Microalgae concentrate</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">Rotigrow Plus&#174; 1 liter bag</td>
			<td style="width:264px;">Nutritionally optimized rotifer feed</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Water Preparation</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:305px;">&#160;Reef Crystals Reef Salt</td>
			<td style="width:135px;">That Fish Place</td>
			<td style="width:119px;">198210</td>
			<td style="width:264px;">Salt for making culture water (NOTE: this item is an example only; any contaminant free salt formulations may be used).&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Refractometer</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">SR6</td>
			<td style="width:264px;">measuring salinity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifer Culture Equipment</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Plankton Collectors 12&#34; Dia, 53 microns</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">BBPC20</td>
			<td style="width:264px;">Mesh screen for collecting rotifers</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Scrub Pads</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">SCR-58</td>
			<td style="width:264px;">Scrub pad for cleaning inside of culturing vessels</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Scrub Brush</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Bucket</td>
			<td style="width:135px;">Grainger Supply&#160;</td>
			<td style="width:119px;">43Y530</td>
			<td style="width:264px;">Graduated bucket for mixing culture water</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Hatching Jar</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">J30</td>
			<td style="width:264px;">Storage of algae feed mix</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Lugol&#8217;s Solution, Dilute</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">S99481</td>
			<td style="width:264px;">Agent used to immobilize live rotifers for counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Sedgewick-Rafter plankton counting slide with grid&#160;</td>
			<td style="width:135px;">Pentair Aquatic Eco-Systems</td>
			<td style="width:119px;">M415</td>
			<td style="width:264px;">Counting rotifers</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Miscelleneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Tea Strainer</td>
			<td style="width:135px;">Kitchenworks</td>
			<td style="width:119px;">971972</td>
			<td style="width:264px;">Used for collecting zebrafish embryos after spawning</td>
		</tr>
	</tbody>
</table>

the complete and updated "rotifer polyculture method" for rearing first feeding zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

这里所描述的连续轮虫培养系统是动态的，这是正常的轮虫数量的波动在小范围内随着时间的推移，如果有变化，日常饲养和收获率。在水产养殖设施在波士顿儿童医院的活性培养物中的一个的轮虫的人口，保持在上述的方式，进行30 天 （图3）监控。在此期间，平均培养密度为932轮虫/毫升，最大为1330轮虫/ ml和至少510轮虫/毫升。这代表保持按照...

Watch this Scientific Journal Video about The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish at JoVE.com

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

完整的和更新的"轮虫混养法"饲养第一移送斑马鱼

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

the-complete-updated-rotifer-polyculture-method-for-rearing-first

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

15.3K 次观看.  Boston Children's Hospital. Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

视频: The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

完整的和更新的"轮虫混养法"饲养第一移送斑马鱼

摘要

探索更多视频

此视频中的章节