High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Summary

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Abstract

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Introduction

The mechanisms by which the intestine regulates dietary lipid processing, the liver controls complex lipid synthesis and lipoprotein metabolism, and how these organs work with the central nervous system to control food intake are incompletely understood. It is of biomedical interest to elucidate this biology in light of the current epidemics of obesity, cardiovascular disease, diabetes, and non-alcoholic fatty liver disease. Studies in cell culture and mice have provided the majority of our understanding of the mechanistic relationships between dietary lipids and disease, and zebrafish (Danio rerio) are emerging as an ideal model to complement this work.

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Protocol

These protocols have been approved by the Carnegie Institution for Science Institutional Animal Care and Use Committee (protocol no. 139).

1. Animal Preparation

1. Maintain adults and larvae at 28 °C on a 14 hr:10 hr light:dark cycle. Feed adults twice daily with shell free Artemia (decapsulated, non-hatching, starting at 14 dpf) and commercial micropellets.
2. These protocols are optimized for the use of 6-7 dpf larvae collected by natural spawning of the AB background. Protocols can be modified for larvae of other ages and backgrounds. Do not provide exogenous food prior to 6 dpf.
3. Anesthetize larvae with Tricaine in emb....

Results

When fed on a rocker at 29-31 °C, the majority of healthy larvae (≥95%) will eat within 1 hr. Upon consuming the egg yolk emulsion, the larval intestine darkens in color. Very dark intestines can be observed at 2 hr (Figure 1). If larvae are unfed or fail to feed, the intestine remains clear. Larvae fed egg white exhibit a distended intestinal lumen that does not darken in color.

Discussion

The techniques described here allow researchers to treat larval zebrafish with a lipid-rich feed, visualize dietary lipid processing in live larvae, and quantify larval food intake. To ensure success, special attention should be given to several critical steps. Commercial chicken eggs vary; to minimize potential variability we perform all assays on organic eggs from cage-free chickens that have not been enriched for omega-3 fatty acids. Lower feeding rates may be observed in fish younger than 6 dpf with remaining endogen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tricaine (ethyl 3-aminobenzoate methanesulofnate salt)	Sigma-Aldrich	A5040-25G	Anesthesia for larval zebrafish
Chicken eggs	N/A	N/A	Organic, cage-free eggs, not enriched for omege-3 fatty acids
Ultrasonic processor 3000 sonicator	Misonix, Inc.	S-3000	To make egg yolk liposomes
Sonabox acoustic enclosure	Misonix, Inc.	432B	To make egg yolk liposomes
1/8” tapered microtip	Misonix, Inc.	419	To make egg yolk liposomes
Amber vials (4 ml, glass)	National Scientific	13-425	Lipid storage; includes vials, open-top caps, and cap septa
Incu-Shaker Mini	Benchmark	1222U12	Incubated shaker for feeds
BODIPY FL C16	Thermo Fisher Scientific	D3821	Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid)
BODIPY FL C12	Thermo Fisher Scientific	D3822	Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)
BODIPY FL C5	Thermo Fisher Scientific	D3834	Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoic Acid)
BODIPY FL C5	Thermo Fisher Scientific	D2183	Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid)
TopFluor cholesterol	Avanti Polar Lipids Inc.	810255	Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol
Fatty acid-free BSA	Sigma-Aldrich	A0281-1G	For TopFluor cholesterol solubilization
Methyl cellulose	Sigma-Aldrich	M0387	Mounting media for live larval imaging; 75 x 25 x 1 mm
Low melt agarose	Thermo Fisher Scientific	BP165-25	Mounting media for live larval imaging; 22 x 30
VWR microscope slides	VWR	16004-422	Mounting larvae for live imaging
Coverslips	Cover Glass	12-544A	Mounting larvae for live imaging
Super glue	Loctite	LOC01-30379	Mounting larvae for live imaging
FluoroDish (glass bottom dish)	World Precision Instruments, Inc.	FD35-100	Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness
Confocal microscope	Leica Microsytems	SP-2, SP-5	Microscope for high magnification live imaging
Stereoscope	Nikon	SM21500	Microscope for low magnification live imaging
Glass culture tubes	Kimble	73500-13100	Lipid extraction; (13 x 100 mm; 13 ml)
Savant SpeedVac Plus	ThermoQuest	SC210A	Lipid extraction
Channeled TLC plates	Whatman Scientific	WC4855-821	Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued
Channeled TLC plates	Analtech, Inc.	66911	Food intake assay; Direct replacement for Whatman Scientific TLC plates
Typhoon 9410 Variable Mode Imager	GE Healthcare	9410	Fluorescent plate reader for food intake assay
ImageQuant software	GE Healthcare	29000605	Analysis of food intake assay
5 3/4’ Wide bore, borosilicate disposable pasteur pipets	Kimble	63A53WT	Transfering larvae