Repeated Blood Collection for Blood Tests in Adult Zebrafish

Summary

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Abstract

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel “repeated” blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be ≤0.4% of body weight every week or ≤1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Introduction

Zebrafish are gaining increasing popularity as a valuable model of human diseases because their organs and genetics are similar to those of humans^1,2. In the field of developmental biology, many studies have demonstrated that zebrafish and human show marked similarity in hematopoiesis³, hemostasis^4,5, and myelopoiesis⁶. Adult zebrafish are also used for studying immunological⁷, neurodegenerative⁸ and obesity-related diseases⁹ because this model organism shares common pathways with those disrupted in human diseases. For obesity and obesity-related diseases (diabetes, hepatic steatosis and nona....

Protocol

All animal procedures were approved by the Ethics Committee of Mie University, and were performed according to Japanese animal welfare regulation ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan) and complied with international guidelines.

1. Preparation of the Needle  

NOTE: All experiments were performed under anesthesia, and all efforts were made to minimize suffering. For euthanasia, fish were immersed in an ice–water bath (5 parts ice/1 part water at ≤4 °C) for ≥20 min.

1. Prepare the glass microcapillary needles by pulling a 1.0-mm....

Results

This blood collection method causes minimal injury to zebrafish (a <1 mm puncture; Figure 1J) and yields a very low mortality rate of 2.3%. We examined the maximum volume of blood that could be collected from a single fish and evaluated the relationship to its body weight (Figure 3). We found that the maximum blood volume collected was linearly correlated with body weight (R = 0.813). The largest volume of blood collected from an individual fish (body weight = 1.071 g) was 25 µl, and .......

Discussion

We present here a detailed protocol for serially obtaining blood from adult zebrafish. This method is straightforward to carry out and we use it in lab on a daily basis. This blood collection method is based on inserting a glass capillary needle into the zebrafish’s dorsal aorta. During this procedure, it is critical to be careful to not ablate the spine because it is the criterion for searching for the dorsal aorta. Reducing the spine injury will improve the survival rate. Although this technique is simple and eas.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass capillaries with filament	Narishige	GD-1	1.0 mm outer diameter.
Needle puller	Narishige	PC-10	To produce the needles
Heparin	Wako Pure Chemical Industries	081-00136	For heparinization
Aspirator tube assembly	Drummond	2-040-000	For blood collection
Bulb dispenser	Drummond	1-000-9000	For blood collection
2-phenoxyethanol	Wako Pure Chemical Industries	163-12075	For anesthetizing the fish
DRI-CHEM3500V	Fujifilm	-	For hemoglobin measurement
DRI-CHEM Slides	Fujifilm	Hb-WII	For hemoglobin measurement
Glutest Neo Super	Sanwa Kagaku Kenkyusho	-	For bood glucose measurement
Wako L-type TG kit	Wako Pure Chemical Industries	464-44201	For TG measurement
Wako L-type CHO kit	Wako Pure Chemical Industries	460-44301	For total cholesterol measurement
Parafilm M	Alcan Packaging	PM996	To expel the blood on