Phenotypic and physiological responses of Zebrafish (Danio rerio) to three commonly used anaesthetics: clove oil, 2-Phenoxyethanol (2-PE), and MS-222.

Abstract

The use of chemical anaesthesia in fish reduces the risk of mechanical injuries or stress in any husbandry practices or handling procedures in aquaculture, research, transport, surgery, or other veterinary practices. However, the type of the anaesthetic agent needed, the dosage required to induce a desired level of anaesthesia, and the physiological effect incurred can differ between species of fish. Since zebrafish are the most held lab fish and widely used in the aquarium trade, it is essential to determine protocols to anaesthetise zebrafish to reduce the risk of injury and stress due to their high demand and importance in these fields. It is also key to understand the physiological consequence of the anaesthetics for the benefit of studies centred around the use of physiological endpoints. This study focussed on assessing the efficacy and the metabolomic consequence of the three most used anaesthetics for fish: clove oil, ethyl 3-aminobenzoate methane sulphonic acid (MS-222), and 2-phenoxyethanol (2- PE). The study exposed adult zebrafish to various concentrations of the three anaesthetic agents and recorded the anaesthetics induction times and made observations of the phenotypic responses to each concentration. The time it took to recover from each exposure was recorded along with the phenotypic changes that occurred. The exposures were repeated using only dosages that induced the fish to stage IV (surgical) anaesthesia in 60-90 s and samples were taken for metabolomics analyses. Clove oil at 60 mg/L and 100 mg/L was the recommended anaesthetic for fast induction of surgical anaesthesia. For maintaining fish in surgical anaesthesia for an extended duration without artificial respiration, 2-PE at 500 mg/L was most effective, and for fish transport, 2-PE at 100 mg/L was suitable. Induction time was faster at higher concentrations across anaesthetics. Anaesthesia with clove oil yielded the shortest induction time at effective concentrations but required longer for full recovery at lower doses. However, recovery was comparably rapid at higher concentrations. Fish anaesthetised with MS-222 recovered quickly, as did those with 2-PE. The metabolic consequence of clove oil anaesthesia was less widespread and severe than that of 2-PE and MS-222-anaesthised zebrafish, while MS-222 affected a wider range of metabolic pathways and metabolites in comparison to 2-PE and clove oil. While all three anaesthetics elicited a stress-related metabolic response, 2-PE and clove oil did not induce a response indicative of acute or life-threatening physiological disruption. These findings serve as a basis for selecting suitable anaesthetics and dosages for zebrafish, striking a balance between physiological safety and procedural requirements to promote both animal welfare and the integrity of scientific findings.