Gavaging Adult Zebrafish

Summary

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Abstract

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 μl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

Introduction

Current methods for administering compounds orally to zebrafish include administering compounds top-coated onto feed, mixed into gelatin diets¹, bioencapsulated into brine shrimp², mixed with lipid encapsulated diets³, wax spray beads⁴ and via gluten-based diets.⁵ Limitations of these methods of oral administration include high leaching rates and incomplete or unpredictable compound consumption by individual fish. These variables are problematic because in infectious disease studies, knowing the infective dose administered to the fish may be critical for study success. Also, previous work has shown that certain....

Protocol

1. Preparing the Anesthetic Solution

1. Prepare a solution of 150 mg/L of MS-222 in system water either from a stock solution or from the powdered form.
2. Verify the pH using a pH meter.
3. Buffer with sodium bicarbonate as needed until the pH is between 7-7.5.
4. Prepare a tank of system water free from MS-222 for the fish to recover in. The recovery time is typically less than 1 min.

2. Preparing the Gavage Apparatus

1. Cut the clear 22-G implantation tubing to a little more than approximate length from the mouth to the intestinal bulb of the fish. Initially, we suggest anesthetizing....

Results

Both sexes of fish, including gravid females were successfully gavaged (Figure 1). A successful procedure takes less than one minute with no liquid seen exiting the gills or the mouth. The tubing enters easily, without force, with no blood seen upon catheter removal. The procedure is rapid, requiring approximately 10 min to gavage 3 to 4 fish, with an average of 30-45 sec per fish.

Zebrafish should not be gavaged with more than 5 μl of any solution. Gavage with 10 μl sol.......

Discussion

Significance

This technique is an improvement and provides several advantages over the two previously described techniques for gavaging adult zebrafish. First, the use of a sponge to restrain the fish allows one person to perform the procedure, as opposed to a previously described technique requiring two people.⁷ Second, catheter implantation tubing is more flexible than IV catheters, which can help minimize secondary trauma due to perforation similarly to using blunt-tipped syringes.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
A sponge
1-cc luer lock syringe	Becton Dickinson Co
22-G Micro-Renathane Implantation tubing (cut)	Braintree Scientific, Inc.
1 22-G needle (cut, if needed)	Becton Dickinson Co
pH meter	Hanna probe
1 scalpel blade
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Reagent/Material
MS-222 (Finquel)	Argent Laboratories