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 compounds administered in water baths cause toxic lesion to zebrafish gills before the intestinal effects that may be under study occur.⁶

Gavage is a standard method used in other laboratory animal species to administer precise quantities of a product with a known concentration for study in biomedical and pharmaceutical research. Only recently have methods for gavaging zebrafish been described in the literature. One technique described is a two-person method using a 24-G catheter sheath attached to a 2-20 μl pipette to deliver 5 μl of solution.⁷ The method described had a mortality rate of 8.7% and 39% on the first and second trial, respectively; most of the mortality was attributed to gravid females. The second gavage technique described used blunt-tipped gavage syringes to administer 5 μl of solution to medakas (Oryzias latipes).⁸ Information on mortality was not provided and the exact process of performing the gavage was not described. According to the Canadian Council on Animal Care and the Laboratory Zebrafish, up to 1% of the fish's body weight can be administered via gavage.^9,10 Our goal was to develop a repeatable, safe and efficient method of delivering precise volumes of compounds orally to adult zebrafish of both sexes and in all stages of reproduction. This procedure would be applicable for any study in which accurate oral dosing of a compound is required.

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 one fish for each new group being used to get the best estimate of the length needed.
2. Push the cut clear implantation tubing onto the ½-inch 22-G needle until it is secure and there is no risk of detaching. If a ½ 22-C needle is not available, cut a longer needle to approximately that length.
3. When complete, the catheter implantation tubing should extend about 1 cm from the tip of the needle, or shorter if the fish are smaller.
4. Attach the 22-G needle hub to a 1-cc luerlok syringe.

3. Preparing for Gavage

1. Cut a groove into a sponge with a scalpel blade.
2. Soak the sponge in system water free from MS-222.
3. Place the moistened sponge on a flat surface.
4. Draw up the appropriate 5 μl solution for gavage into the 22-G catheter tubing and a 1-cc syringe.

4. Anesthesia and Gavage

1. Fast the fish for at least 24 hr before the procedure.
2. Place the fish in the MS-222 solution until it loses its righting reflex, and does not respond to a tail fin pinch, but maintains opercular movement. This typically takes about 2-3 min.
3. Remove the fish from the anesthetic solution and place it into the groove in the sponge with the head slightly protruding from the sponge, but the gills covered by the sponge.
4. Move the sponge into a vertical position.
5. Open the zebrafish's mouth using the 22-G catheter tubing.
6. Gently insert the tubing until the tip is past the gills (approximately 1 cm or the length of the tubing). The implantation tubing should not need to be forced. Resistance suggests the tube may be hitting the gill arch or heart.
7. If there is resistance, gently withdraw, reposition and try again.
8. Inject the material slowly.
9. While injecting, make sure that the solution does not exit via the gills or the mouth.
10. Remove the fish from the sponge and place into the recovery tank.
11. Recovery typically occurs in less than 1 min and is indicated by the fish swimming upright and maintaining equilibrium.
12. Monitor fish for regurgitation as shown by visualizing the fish actively expelling material from its mouth, or no opercular movement.
13. Fish can be returned to their regular tank once they have recovered.

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