The Complete og opdateret "hjuldyr polykultur Method" til opdræt First Feeding Zebrafisk
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
Zebrafisken (Danio rerio) er en fremtrædende forsøgsdyr anvendt i et stigende antal videnskabelige discipliner, herunder, men ikke begrænset til udviklingsmæssige genetik, toksikologi, adfærd, akvakultur, regenerativ biologi og modellering af mange menneskelige lidelser 1 – 5. Selvom arten er relativt let at vedligeholde i laboratoriet, er der en række ledelsesmæssige udfordringer forbundet med deres kultur 6. Den mest fremtrædende af disse er larveopdræt, især når fiskene først begynder at fodre efter gas blære inflation 7. Under normale, kontrollerede forhold, denne udviklingsmæssige begivenhed indtræffer på ~ 5 dage efter befrugtning (DPF), med de følgende 3 – 5 dages vækst er særligt kritiske 7. Den centrale tekniske vanskeligheder i løbet af denne fase er i tilstrækkelig grad at opfylde de ernæringsmæssige krav til første fodring larver – foder elementer skal være af passende størrelse, Digestible, attraktiv og tilgængelig på en næsten kontinuerlig basis, uden at skabe overdreven affald i dyrkning tanke. Historisk dette er opnået typisk ved at levere en lang række små mængder foder til fisk i tanke, sammen med rutinemæssig vandudskiftning 8,9. Mens disse metoder er til en vis grad en succes, er de ineffektive, kræver høje arbejdskraft input, og returnere eneste variabel og begrænsede vækstrater og overlevelse 10.
I naturen, zebrafisk larver formentlig foder på rigelige små zooplankton til stede i vandsøjlen 11. Af denne grund, larviculture protokoller, der inkorporerer live feeds såsom Paramecium, hjuldyr, Artemia og er typisk mest effektive 7. I 2010, Bedste og medforfattere viste, at det var muligt at dyrke larvernes zebrafisk i statisk, brakvand sammen med saltvand hjuldyr for de første 5 dage af eksogen fodring 12. Denne tilgang, som udnytteres den naturlige høje produktivitet hjuldyr kulturer til at give rigelig, meget nærende bytte uden at forurene vandet, udbytter meget høje larvernes vækst og overlevelse med lav arbejdskraft 12,13. I de senere år har et stigende antal laboratorier rundt om i verden vedtaget variationer af denne protokol, og mange er nu dyrkning hjuldyr i en kontinuerlig måde at støtte planteskole systemer 14.
I løbet af de sidste mange år, har metoder til både hjuldyr / zebrafisk polykultur og hjuldyr produktion blevet forfinet og forbedret til at blive mere standardiseret og let skalerbar. Denne artikel indeholder trin-for-trin instruktioner til 1) kontinuerlig og robust hjuldyr produktion og 2) etablering af hjuldyr / zebrafisk polykultur system, der anvendes til at understøtte robust vækst af fisk i de første 5 dage af eksogen fodring.
Protocol
Representative Results
Discussion
En vellykket gennemførelse af hjuldyr polykultur metode til fodring tidlige larve zebrafisk kræver effektive protokoller for to opgaver: etablering og vedligeholdelse af et kontinuerligt hjuldyr kultur system til at fodre fisk, og dyrkning første fodring zebrafisk larver sammen med hjuldyr i samme tank.
Opsætningen i en sammenhængende saltvands hjuldyr produktionssystem for zebrafisk laboratorier først beskrevet af Lawrence og medforfattere 14 er blevet ændret og udvidet i …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Pleje og brug af fisk genereret for repræsentative resultater, der er beskrevet i denne protokol blev udført i fuld overensstemmelse med de retningslinjer, der er fastsat af Institutional Animal Care og brug Udvalg på Boston Børnehospital, protokol # 14-05-2673R.
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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