1Hospital de Clínicas de Porto Alegre, Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental, 2Universidade Federal do Rio Grande do Sul, UFRGS. Porto Alegre, RS, Brasil
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Pedroso, G. L., Hammes, T. O., Escobar, T. D. C., Fracasso, L. B., Forgiarini, L. F., da Silveira, T. R. Blood Collection for Biochemical Analysis in Adult Zebrafish. J. Vis. Exp. (63), e3865, doi:10.3791/3865 (2012).
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.
1. Protocol Text
2. Representative Results
It was possible to collect 5 to 20 μl of whole blood from each fish what represents even 4 times more blood than previously described techniques (Table 2). Biochemical analysis of total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides were performed after blood collection using this technique. Two groups of both sexes fish were fasted for 24 hours before blood collection to avoid food intake interference. The analyses were done with small-scaled colorimetric tests (Labtest Diagnóstica S.A., Brazil) For total cholesterol and triglyceride analyses, 3 μl of serum were used. For LDL-cholesterol and HDL-cholesterol analysis, 4 μl and 10 μl of serum were used, respectively. These analyses were performed on pooled samples of 10 zebrafish per sample.
Serum lipidic levels were compared between fish that accessed their own eggs and those that, in a bottom covered aquarium, did not have access to their own eggs for an experimental duration of 2 weeks. Serum analysis showed that the serum levels of total cholesterol (with eggs 362 ± 42 mg/dL and without eggs 357 ± 13 mg/dL), HDL-cholesterol (with eggs 91.22 ± 1.79 mg/dL and without eggs 72.14 ± 2.89 mg/dL), and LDL-cholesterol (with eggs 55.68 ± 10.88 mg/dL and without eggs 44.18 ± 9.84 mg/dL) did not differ significantly between the groups. However, triglyceride levels were significantly lower in the experimental group (without eggs 292 ± 64 mg/dL) than in the control group (with eggs 457 ± 25 mg/dL; P = 0.03).
|With access to eggs||Without access to eggs|
|Total Cholesterol (mg/dL)||362.82 ± 73.11||357.69 ± 23.08|
|LDL – Cholesterol (mg/dL)||55.69 ± 18.84||44.19 ± 17.05|
|HDL – Cholesterol (mg/dL)||91.23 ± 3.11||72.14 ± 5.01|
|Triglycerides (mg/dL)||457.64 ± 43.78*||292.36 ± 111.28|
Table 1. Cholesterol and triglycerides seric levels for both studied groups (with access to eggs and without access to eggs) expressed in mean ± standard deviation.
*Statistically significant (P=0.03). Student t test.
|Authors||Place of incision||Harvest method||Anesthesia||Amount of collected blood|
|Jagadeeswaran et. al., 1999 Murtha et al., 2003||Micro dissection posterior to dorsal fin||Micropipette||Not mentioned
MS222 3% in cold water
|1 a 5 μl
5 a 10 μl
|Eames et al., 2010||Decapitation||Micro capillaries tube||MS222 0,02%
28 °C water
|5 a 10 μl|
|Present Study||Incision between anal fin and caudal fin||Micropipette and low retention tips||Water and ice chips||5 a 20 μl|
Table 2. Comparison between the previously described blood collection techniques and the one described at the present study.
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 techniques, thereby allowing for the use of fewer fish to obtain the needed amount of biological material. The technique has one critical step, which is that the blood samples be handled carefully as zebrafish blood can incur hemolysis very easily. The time delay between blood collection and centrifugation must be strictly limited. A 10-minute limit should prevent hemolysis. The speed and duration of centrifugation (0,5 g for 10 minutes) should also be strictly followed.
Other blood collection techniques were attempted before this technique was developed. However, the number of animals used was large and very small amounts of blood were collected from each fish. This new technique allowed the use of fewer animals, was demonstrated to be feasible with low skill level practitioners, and gave better results than other techniques in terms of the amount of blood collected from each fish.
No conflicts of interest declared.
FIPE/HCPA - Fundo de Incentivo a Pesquisa e Eventos
|Low retention tips||Applied Biosystems||022493020|
|Eppendorf Centrifuge 5415D||Eppendorf||Discontinued|