Twenty-four-hour post-anaesthetic behavioural effects of menthol and thymol compared to conventional anaesthetics in zebrafish.

The search for welfare-improving anaesthetic alternatives to MS-222 has highlighted monoterpenes. However, their long-term behavioural consequences remain unclear. This study evaluated the 24-h post-anaesthetic behavioural effects of menthol and thymol compared to MS-222 and eugenol in adult zebrafish. Fish were randomly assigned to six groups. Individual fish (n = 10/group) were maintained for 10 min in MS-222 (150 mg/L), eugenol (80 mg/L), menthol (50 mg/L) and thymol (25 mg/L) or a control (water and ethanol (EtOH) 0.1%) after Stage 3 anaesthesia was reached. Twenty-four hours post-exposure, fish were tested in comprehensive behavioural approach, assessing exploratory and locomotor activity, anxiety-related responses, aggression, shoaling, social preference, and learning and memory, complemented by principal components analysis and hierarchical clustering to characterise treatment-specific behavioural phenotypes. The results revealed compound-specific behavioural profiles 24 h after anaesthesia. MS-222 and eugenol were primarily associated with alterations in anxiety-related and exploratory endpoints, whereas menthol and thymol were mainly associated with consistent modulations of social-related behaviours. Behavioural phenomics further indicated clustering between MS-222 and menthol, while thymol displayed a distinct behavioural profile and eugenol clustered closer to control groups. Overall, the findings highlight that anaesthetic agents are associated with distinct and lasting behavioural profiles in fish, underscoring the importance of considering post-anaesthetic behavioural outcomes when selecting anaesthesia protocols. Further studies are warranted to refine the application of monoterpene-based anaesthetics across different experimental and applied contexts.