完整的和更新的“轮虫混养法”饲养第一移送斑马鱼
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Rotifer Culture Infrastructure | |||
| 100 Liter Culture Vessel | Aquaneering | Custom | Polycarbonate culture vessel, conical bottomed, with drain valve |
| 5 Gallon Culture Bucket Kit | Reed Mariculture | CCS Starter Kit | Small volume culture vessel for small facilities |
| Rigid Clear Tubing 1/2" O.D., 36” | Pentair Aquatic Ecosystems | 16025 | Rigid clear tubing for air delivery |
| Mesh tube | Pentair Aquatic Ecosystems | RT444X | Mesh tube support for floss filter |
| Rotifer Floss | Reed Mariculture | Rotifer floss 12” x 42” | Particulate waste trap |
| Peristaltic Metering Timer Pump, 5 GPD | Grainger | 38M003 | Metering pump with timer for dosing feed to rotifers |
| Peristaltic Metering Timer Pump, 1-100 mL/h (for smaller-scale culture) | Coral Vue | SKU: IC-LQD-DSR | Metering pump with timer for dosing feed to rotifers |
| Silicone Tubing | Cole Parmer | Tubing for algae delivery to rotifer vessel | |
| Rigid Clear Tubing " O.D.,36” | Pentair Aquatic Ecosystems | 16025 | Rigid clear tubing for air delivery to algae paste |
| Rigid Clear Tubing O.D., 36” | Pentair Aquatic Ecosystems | 16025 | Rigid clear tubing for algae delivery |
| Rotifers | |||
| Live Rotifers Brachionus plicatilis Type L | Reed Mariculture | Type L 5 million | Rotifer stock culture for system startup |
| Rotifer Feed | |||
| Sodium hydroxymethylsulfonate | Reed Mariculture | ClorAm-X® 1lb tub | Ammonia reducer for algae feed mix |
| Sodium Bicarbonate | Fisher Scientific | S25533B | pH buffer for algae feed mix |
| Microalgae concentrate | Reed Mariculture | Rotigrow Plus® 1 liter bag | Nutritionally optimized rotifer feed |
| Water Preparation | |||
| Reef Crystals Reef Salt | That Fish Place | 198210 | Salt for making culture water (NOTE: this item is an example only; any contaminant free salt formulations may be used). |
| Refractometer | Pentair Aquatic Ecosystems | SR6 | measuring salinity |
| Rotifer Culture Equipment | |||
| Plankton Collectors 12" Dia, 53 microns | Pentair Aquatic Ecosystems | BBPC20 | Mesh screen for collecting rotifers |
| Scrub Pads | Pentair Aquatic Ecosystems | SCR-58 | Scrub pad for cleaning inside of culturing vessels |
| Scrub Brush | |||
| Bucket | Grainger Supply | 43Y530 | Graduated bucket for mixing culture water |
| Hatching Jar | Pentair Aquatic Ecosystems | J30 | Storage of algae feed mix |
| Lugol’s Solution, Dilute | Fisher Scientific | S99481 | Agent used to immobilize live rotifers for counting |
| Sedgewick-Rafter plankton counting slide with grid | Pentair Aquatic Eco-Systems | M415 | Counting rotifers |
| Miscelleneous | |||
| Tea Strainer | Kitchenworks | 971972 | Used for collecting zebrafish embryos after spawning |
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