Zebrafish 24 hpf-embryos as a model organism for measuring oxygen consumption rate (OCR)

The alterations in mitochondrial function are involved in various pathological conditions and hence the evaluation of such damage is crucial to determine the mitotoxic potential of different chemicals. Due to their 71% genetic similarity to humans, the similar mitochondrial functions, and metabolic processes of zebrafish (Danio rerio) have been used in analyses for this purpose. The aim of this study was to establish a standardized technique to assess mitochondrial dysfunction by analyzing oxygen consumption rate (OCR) in zebrafish embryos 24 h post-fertilization using the OROBOROS O2k Oxygraph. The technique involved the use of mitochondrial modulators and the OROBOROS O2k Oxygraph to directly assess mitochondrial and electron transport chain complex activity during embryonic development. Embryos were treated with a respiratory medium supplemented with malate, succinate, and pyruvate, and with digitonin to permeabilize the chorion and membranes for mitochondrial analysis. OCR measurements were performed in the presence of specific mitochondrial modulators: oligomycin, FCCP, rotenone and antimycin A. Optimized evaluation was achieved using 20 embryos per assay. Therefore, through the development of a protocol for synchronization analysis of OCR in zebrafish embryos, several parameters related to the effectiveness of the oxidative phosphorylation process could be rapidly determined. Since zebrafish are particularly useful to study mitochondrial dysfunction in toxicants, this protocol describes the procedure to quantitate the effect of toxicants on mitochondrial activity which turns out to be valuable to understanding the mechanism of xenobiotic toxicity.