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. 2018 Jun:208:29-37.
doi: 10.1016/j.cbpc.2017.11.004. Epub 2017 Nov 21.

The validation of a sensitive, non-toxic in vivo metabolic assay applicable across zebrafish life stages

Ross M Reid  1 , Andrea L D'Aquila  2 , Peggy R Biga  3
Affiliations

The validation of a sensitive, non-toxic in vivo metabolic assay applicable across zebrafish life stages

Ross M Reid et al. Comp Biochem Physiol C Toxicol Pharmacol. 2018 Jun.

Abstract

Energy expenditure and metabolism, is a well-studied field as it is linked to many diseases as dysregulation of metabolism is associated with cancer, neurodegeneration, and aging. Classical methods of studying metabolism in vivo are well established, but most are tedious and expensive, thus, finding methods of accurately measuring metabolism in living organisms that is quick and non-invasive is of strong interest. In this work, we validate the use of resazurin; a compound that is conformationally changed into fluorescent resorufin upon metabolic reduction by NADH2, as a metabolic assay for adult zebrafish. This assay is based on the principle that increases in resorufin fluorescence intensity (FI) conveys relative changes in metabolic output of the organisms. We demonstrate the effectiveness of resazurin in measuring metabolic changes in zebrafish larvae and adults in relation to number of pooled fish, as well as temperature alteration. Moreover, we provide details on the appropriate and optimized diluents and concentrations of resazurin. Further, by using a novel sample collection technique, we can increase the temporal possibilities that were previously limited, as well as show that samples can be stored and measured at a later time point with no decrease in accuracy. Thus, the validation of this assay in adult zebrafish may increase the versatility and complexity of the types of experiments that can be performed and have many practical applications in the field.

Keywords: Energy expenditure; Metabolic assay; Metabolism; NADH(2); Zebrafish.

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Figures

Figure 1
Figure 1. Larvae and Adult Zebrafish Assay Preparation and Collection Schedule
(A) Zebrafish larvae (5 dpf) were pipetted into 5 of the 6 wells of a 6-well plate containing 2 mL of the resazurin solution (0.02 mg/mL). (B) Adult zebrafish were individually placed in a 50-mL conical tube with 25 mL of the resazurin solution (0.02 mg/mL) and positioned at a 45° angle. (C) Timeline for adult and larvae sample collections of total resorufin at the designated time points (T0 – T48) with collections being at 3, 6, 12, 24, and 48 hours post initial T0 collection.
Figure 2
Figure 2. Optimization of resazurin concentration for sensitivity and non-toxicity
(A) Individual adult zebrafish were placed in conical tubes containing serial dilutions of resazurin stock solution to determine optimal resazurin concentration for sensitivity for in vivo studies at 23°C. (B) Resazurin optimization experiment was repeated with individual adult zebrafish at 16°C [n=3 (warm), n=3 (cold)]. (C) This dose is non-toxic to fish for at least 5 days [n=5].
Figure 3
Figure 3. Tank water is an appropriate control for the ‘blank’ wells in the assay
(A) Samples of deionized water (diH20), autoclaved diH20, tank water and autoclaved tank water were added to resazurin solution and measured over 48 hours. This assay demonstrated that diH20 and tank water have inherently different baseline fluorescence (P<0.0001). There was no significant difference between diH20 and autoclaved diH20, although there was a significant difference between tank water and autoclaved tank water at 48 hours (P=0.0003). (B) Results of the control blank assay compared to wells with larvae, which had significantly higher AFU than blank wells (P<0.0001). Statistical test: Two-way RM ANOVA, Sidak’s post-hoc test. [n=3 (blanks), n=4 (larvae)]
Figure 4
Figure 4. Increasing the number of pooled larvae zebrafish significantly increases the total resorufin fluorescence intensity (AFU) in a linear response
(A) 6-well plate wells that contained resazurin solution housed either 10, 20 or 40 zebrafish larvae (5 dpf). Significant increases in total resorufin fluorescence intensity were seen at 24 hr (P=0.033) between 10 and 40 larvae. After 48 hr, 40 larvae had significantly higher total resorufin fluorescent intensity when compared to 10 larvae (P=0.0015) and 20 larvae (P=0.02). Statistical test: Two-way RM ANOVA, Sidak’s post-hoc test. [n=4 (10 larvae), n=5 (20 larvae), n= 5 (40 larvae)]. (B) Larval response kinetics were analysed by measuring the relative change in AFU/g of 20 and 40 larvae compared to 10 larvae, and it had an R2 value of 0.9998.
Figure 5
Figure 5. Increasing the number of adult zebrafish significantly increases the total resorufin fluorescence intensity (AFU) in a linear response
(A)1, 2 or 3 adult zebrafish were added to resazurin solution in conical tubes and samples were collected over 24 hours. At 24 hours, the metabolic rates of the number of fish are all significantly different (P<0.0001), where the greater number of fish result in higher AFU. [Statistical test: Two-way RM ANOVA, Sidak’s post-hoc test. [n=5 (1 fish), n=4 (2 fish), n=4 (3 fish)]. (B) Adult response kinetics were analysed by measuring the relative change in AFU of 3 and 2 fish compared to 1 fish, and it had an R2 value of 0.9765.
Figure 6
Figure 6. Pooled larvae zebrafish had significantly lowered metabolism after exposure to cold temperature for 48 hours
Larvae zebrafish that were pooled in 6-well plate (10 larvae/well) contained in resazurin solution were exposed to either 23°C (warm) or 16°C (cold) temperature for 48 hours. Larvae had significantly (P<0.0001) lower metabolism at 48 hours as measured by total resorufin fluorescent intensity (AFU). Statistical test: Two-way RM ANOVA, Sidak’s post-hoc test. [n=3 (cold), n=4 (warm)]
Figure 7
Figure 7. Adult zebrafish had significantly lowered metabolism after exposure to cold temperature for 24 hours
Individual adult zebrafish contained in resazurin solution were exposed to either 23°C (warm) or 16°C (cold) temperature for 24 hours. At 12 hours, the animals had significantly lower metabolic rate (P=0.0002), which was maintained until 24 hours (P<0.0001), as measured by total resorufin fluorescent intensity (AFU). Statistical test: Two-way RM ANOVA, Sidak’s post-hoc test. [n=8 (cold), n=5 (warm)]
Figure 8
Figure 8. Fresh samples and frozen samples exhibit the same fluorescence intensity even after 3 weeks of storage
Samples were collected from individual adult zebrafish and assayed either immediately after collection or frozen (−20°C) and assayed 3 weeks later. This test shows no significant difference between samples at any time point. Statistical test: Two-way RM ANOVA, Sidak’s post-hoc test. [n=5 (fresh), n=5 (frozen)]
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References

    1. Argenton F, Zecchin E, Bortolussi M. Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo. Mech Dev. 1999;87:217–221. - PubMed
    1. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, Bedell JA, McPherson JD, Johnson SL. The syntenic relationship of the zebrafish and human genomes. Genome research. 2000;10(9):1351–1358. - PMC - PubMed
    1. De Fries R, Mitsuhashi M. Quantification of mitogen induced human lymphocyte proliferation: Comparison of alamarbluetm assay to 3h‐ thymidine incorporation assay. Journal of clinical laboratory analysis. 1995;9(2):89–95. - PubMed
    1. Del Pozo A, Sanchez-Ferez JA, Sanchez-Vazquez FJ. Circadian rhythms of self-feeding and locomotor activity in zebrafish (Danio rerio) Chronobiology international. 2011;28(1):39–47. - PubMed
    1. Finkel T. The metabolic regulation of aging. Nature medicine. 2015;21(12):1416. - PubMed

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