Blood Collection for Biochemical Analysis in Adult Zebrafish

Summary

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Abstract

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies¹.

There are relatively few publications related to adult zebrafish physiology of organs and systems², which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking³. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues⁴ who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta⁵. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year⁶. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish⁷. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.⁸. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started.

Recently, Gupta et al.⁹ described how to dissect adult zebrafish organs, Kinkle et al.¹⁰ described how to perform intraperitoneal injections, and Pugach et al.¹¹ described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.

The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

Protocol

1. Protocol Text

1. Before collecting zebrafish blood, it is necessary to prepare anesthetizing water. Pour ~200 ml of aquarium water into a container with a 500-ml capacity. Add ~200 g of ice chips. The temperature should be about 4 °C. As the ice chips melt, it will be necessary to add more ice chips to maintain a constant temperature near 4 °C.
2. When the anesthetizing water is ready, prepare the materials needed for blood collection. Put a low-retention tip on a P20 pipettor and leave the pipettor where it can be easily accessed. Do not allow the pipette tip to contact any sources of contamination.
3. Cover a Petri d....

Discussion

This paper presents a simple technique that allows further blood and serum analysis in zebrafish experiments. This technique has the potential to contribute to future zebrafish hematologic studies requiring blood parameter data. It should also allow for greater applications of the zebrafish as an experimental model.

This technique does not require special skills or implementation of a precise technique. Moreover, it enables up double the amount of blood to be collected relative to other techni.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Low retention tips	Applied Biosystems	022493020
Eppendorf Centrifuge 5415D	Eppendorf	Discontinued