Thymol and menthol as anaesthetics for short transportation of zebrafish larva

Transportation-induced stress is a major concern in aquaculture, often causing physiological and behavioural disruptions in fish. To alleviate these effects, anaesthesia is commonly used, but the most widely used anaesthetics, MS-222 and eugenol, have been linked to adverse effects, and alternatives are lacking. This study evaluated the anaesthetic efficacy of thymol and menthol, in comparison with MS-222 and eugenol, under stress conditions induced by a simulated 4-h transportation of 96 hpf zebrafish larvae. Based on LC₅₀ values, the transport simulation was conducted with 200 mg L^-1 MS-222, 80 mg L^-1 eugenol, 15 mg L^-1 thymol, and 50 mg L^-1 menthol. Parameters assessed included heart rate, biochemical markers, stress responses, metabolic rate, protein expression related to oxidative stress and cell death, and potential inflammation. No changes in cortisol levels were observed possibly reflecting negative feedback of the HPI axis. However, signs of stress were evident through increased metabolic rate and reduced ATPase activity. The use of anaesthetics reduced glucose spikes and lowered heart rate and ATPase activity suggesting a shift towards a hypometabolic state. In terms of oxidative stress, GST activity returned to baseline, but GSH levels were further reduced by anaesthetics. Anaesthetics increased SOD levels and decreased CAT activity, with menthol causing a significant rise in Nrf2. Despite changes in oxidative stress markers, no alterations in cellular damage or inflammation markers were found. While further studies are needed, thymol and menthol did not significantly disrupt physiological balance, supporting their potential use in aquaculture.