Investigating the effect of anaesthesia on the metabolism of zebrafish (D. rerio)

Abstract

In recent years, zebrafish (D. rerio) have emerged as valuable research models, being widely used as models for aquaculture research, but also to model human metabolic diseases. The ethical guidelines stipulate that anaesthetics need to be applied before sampling of zebrafish. The effect of the anaesthetics on the metabolic profiles of zebrafish and the implications that this may have for zebrafish as research models, have mostly been overlooked. The primary hypothesis of this study was that anaesthetics will have an effect on zebrafish metabolism. The possibility for new hypotheses to be generated were not eliminated, since untargeted metabolomics are more hypothesis-generating than hypothesis-testing techniques. In this study, multi-platform metabolomics were used to investigate the metabolic alterations in zebrafish caused by three commonly used anaesthetics namely tricaine methanesulfonate (MS-222), eugenol and 2- phenoxyethanol (2-PE). The metabolomic analyses indicated that anaesthesia caused minimal metabolic alterations, with the concentration of only a small number of metabolites altered. It is hypothesized that these concentration changes are caused by ATP depletion and a stress response, which both lead to the upregulation of protein and lipid catabolism. Glycolysis and tricarboxylic acid cycle (TCA) intermediates remained relatively unaffected, but there are some indications that gluconeogenesis may be slightly upregulated. These effects are, however, limited by the short induction times of the anaesthetics. It was also determined that eugenol exhibited significantly less metabolic perturbations than the other anaesthetics with only three significantly altered metabolites, making it the preferred anaesthetic for metabolomic studies in zebrafish. General pathways impacted during anaesthesia include but are not limited to ẞ-oxidation, gluconeogenesis, urea cycle and amino acid transamination. In conclusion, anaesthetics used during sampling are not expected to have a significant effect on the suitability of zebrafish as research models. Further investigations are needed to confirm the hypotheses generated in this study.