High Throughput Danio Rerio Energy Expenditure Assay

Summary

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH₂ sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Abstract

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH₂ production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH₂ production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Introduction

The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 - 4 months) and surpasses even the mouse in ease of genetic manipulation ^1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens ³.

To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, in....

Protocol

Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee

1. Zebrafish Breeding and Embryo Maintenance

1. Prepare E3 embryo medium⁸. To Make 60x stock solution, dissolve 34.8 g NaCl, 1.6 g KCl, 5.8g CaCl₂^●2H₂O, and 9.78 g MgCl₂^●6H₂O in 1.95 L ddiH₂O. Adjust pH to 7.2 using NaOH and HCl Adjust volume to 2 L using ddiH₂O.
2. To prepare 1x E3 embryo medium, dilute 16.5 ml 60x stock in 1 L ddiH20. Ad....

Results

Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P<0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are p.......

Discussion

Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AlamarBlue	Fisher Scientific	10-230-103	Manufactured by Thermo Scientific
Fine Tip DisposableTransfer Pipette	Fisher Scientific	13-711-26	Manufactured by Thermo Scientific
Fisherbrand Graduated Disposable Transfer Pipette	Fisher Scientific	13-771-9AM	Manufactured by Thermo Scientific
Black sided clear bottom 96-well plates	Fisher Scientific	50-320-785	Manufactured by E&K Scientific Products Inc.