In intensive aquaculture, practical procedures as weighing, measuring, spawning or transport are frequent, which can induce stress in fish and influence their welfare. To minimize these negative effects which can have significant economic impacts, the use of anaesthetics is increasingly recommended. Tricaine methanesulphonate (MS-222) is a synthetic anaesthetic often used in aquaculture and research, however some studies support that this compound may induces further changes on different parameters of the fish welfare. In this sense, it is necessary to find compounds that have anaesthetic properties and are harmless to fish, the environment and the consumer with the purpose of enable safe and sustainable use. Thymol and menthol are natural antioxidant compounds with proven anaesthetic properties in different species. The zebrafish is a teleost fish which has been used as a model fish for aquaculture research. Its larval stages are highly suitable for addressing welfare mechanisms by anaesthetics as most receptors are found after 72 hours post-fertilization (hpf). Therefore, the objective of this work was to evaluate the anaesthetic profile of menthol and thymol and biochemical changes in anaesthetized and recovered animals.
Material and methods
Zebrafish larvae (72 hpf) were anaesthetized with different concentrations of thymol (12.5 – 400 mg L-1) and menthol (25 – 600 mg L-1) prepared in E3 medium (negative control). MS-222 at 200 mg L-1 was used as a positive control group. The time it took to lose the ability to swim and the response to a touch stimulus on the tail was registered. Then, the animals were placed to recover in E3 medium and the time until the animals returned to swimming normally noted. After finding the concentrations best suited for aquaculture procedures (induction time be less than 180s and of recovery less than 300s), a new set of animals was anesthetized and collected while others were allowed to recover to evaluate biochemical changes. After 24 and 48h of recovery, the animal’s heartbeat was evaluated.
The induction and recovery time of MS-222 was 35.60 (17.00 – 60.45), 86.40 (60.90 – 159.60) seconds, respectively. The natural compounds tested had an induction time very similar with that of MS-222: for thymol 200 mg L-1 it was 33.92 (30.00 – 48.83) seconds and for menthol 400 mg L-1 it was 21.00 (17.00 – 60.45) seconds. However, the recovery time was higher in all concentrations tested in comparison to MS-222. In the heartbeat evaluation, all animals had a normal heartbeat after 48 hours of recovery. At the biochemical level, larvae under the effect of thymol showed an increase in SOD activity and a decrease in LDH and ATPase activity. After the recovery, there was an increase in SOD activity and a decrease in GSH and GSSG levels and ATPase activity. In the case of menthol, there was an increase in SOD, GST and GPx activities and a decrease in GSH, GSSG, LPO and LDH after induction. After the recovery, a decrease in LPO level and ATPase activity and an increase in ROS, AChE, CarE and GST activities were observed.
Discussion and conclusion
Natural compounds tested showed a good anaesthetic profile similar to MS-222 with a low induction while increased recovery times. No mortality rates were obtained, and no alterations in heartbeat rate were observed. Regarding the biochemical changes observed, the induction of SOD and other antioxidant defences activity by thymol or menthol during and after anaesthesia could be an adaptive response to the stress which neutralizes the impact of ROS generated induced, as described in other studies. LPO reduction with menthol anaesthesia may indicate that the compound used prevents the oxidation process of the lipids present in cell membranes. The decrease in LDH activity in relation to control might represent a reduction of cellular anaerobic processes and a possible reduction of oxidative damage. In general, the antioxidant improvement of defenses seems to be happening for both thymol and menthol.
Though further studies are needed to understand the concentration-time relationship and the physiological changes at these development stages of Danio rerio as the literature is scarse, the use of these natural compounds to avoid stress in teleost fish seems to be a very interesting way and with great benefit for both animals and the environment.
Menthol; Thymol; Anaesthetic profile; Anaesthesia; Zebrafish
The authors wanted to thank the FCT - Foundation for Science and Technology, I.P., under the PhD grant (SFRH/BD/144904/2019), R. Vieira and Projects IDB/04033/2020 and POCI-01-0145- FEDER-029542 (PTDC/CVT-CVT/29542/2017).