Fish sedation may help to secure fish health, welfare, preventing damages and negative side effects, when carrying out some farming procedures such as fish selection, transportation, vaccination and fish handling. To supply a slight sedation, or synthetic and natural (plant-based) sedative can be used. For aquaculture industry, the use of plant-based sedative has become a suitable alternative to the synthetic product, being more appreciated by the consumers and potentially claiming a reduced environmental impact (Aydın & Barbas, 2020). Lemon balm (Melissa officinalis) is well-known for its pharmacological effects. It contains different classes of phytochemicals, such as polyphenolic compounds which have many biological properties, including antioxidants, anti-anxiety, antidepressant and neuroprotective properties. Moreover, this plant affects the nervous system decreasing the symptoms of neurological disorders such as sleeping disorders, stress, anxiety, and irritability (Petrisor et al., 2022). Among mineral feed additives, MgCl2 is use as relaxant for research and commercial applications because it acts as a calcium channel blocker, preventing calcium ions (Ca2+) from entering the cells and blocking the release of acetylcholine (Azizan et al., 2021). Also, zebrafish (Danio rerio) has emerged as a model in neurobehavioral research; there are several studies that examined zebrafish behavior and their response to different drugs and sedatives products. The aim of the present study was investigating the potential toxicity and sedative effects of a commercial blend of Melissa Officinalis and soluble magnesium (AFI calm prototype®, Artic Feed Ingredient AS, Norway), on zebrafish larvae and adults.
Material and methods
The experimental procedures were approved by the Animal Welfare Board of the University of Pisa and the Italian Ministry of Health (B290E.N.0F7.). The study consisted of two trials, the first carried out on zebrafish larvae and using 5 different water concentration of AFI calm prototype ® (0-, 0.5-, 1.0-, 2.0-, and 4.0- ml L-1). Since the 2nd day post-fertilization (dpf) onwards, the larvae were exposed to the solution and on 5th dpf tested for locomotor behavior (distance moved, velocity, movement cumulative time) using DanioVision® (Noldus©, The Netherlands). Based on the observed results, the second trial was carried out on 260 adult zebrafish. Four treatments (CTRL, CTRL-R, T1 and T2) and three different AFI calm prototype ® water concentration (0-, 1.0- and 2.0- ml L-1) were used. On the 3rd, 6th, 9th day of treatment, fish behavior was tested using the Novel Tank Test (NTT). Two video cameras were used for recording fish behavior from the side and the upper view. This latter camera was connected to a pc and video elaborated by Ethovision XT 16® software (Noldus©, The Netherland). Moreover, after 3 days of suspension of the treatment, all the fish were tested once again for evaluating the recovery time. Finally, for groups CTRL, T1 and T2 the same individuals were tested at each time point, while in order to highlight possible inurement test effect, for CTRL-R group naïve fish were used. Each NTT lasted 4 minutes and each minute was analyzed both individually and as a whole period.
The test carried out on zebrafish larvae showed toxicity effect and reduced larvae survival rate only when 4.0 ml L-1 water concentration was used (44±32 %). Locomotor performances showed sedative effect already at 1.0 ml L-1, with a dose-dependent effect (Fig. 1a, 1b, 1c). Based on locomotor performances, no sedative effects were observed on adults on 3rd and 6th days of treatment. Effects were detected on 9th day of treatment at 2.0 ml L-1 concentration and on 4th bin (4th minute of test) fish of the treatment T2 showed higher exploration attitude than T1 in terms of distance moved (645.80±303.48 and 332.40±359.59 cm, respectively), movement cumulative duration (45.61±18.66 and 30.37±25.16 s, respectively) and velocity (11.29±5.27 and 5.79±6.25 cm s-1 respectively). Moreover, at 9th day of treatment, T2 showed shorter latency time to reach the top section of the water column and spent more time exploring this section of the tank than T1 (Fig. 2a, 2b). Also, T2 entered more time the upper section of the water column than T1 and CTRL (Fig. 2c). Furthermore, at 9th day of treatment, CTRL-R showed shorter latency time to enter, and spent more time exploring the upper section of the water column than CTRL and T1 (Fig. 2a, 2b); CTRL-R, also showed more entries to the upper section of the water column than T1 (Fig. 2c). Finally, at recovery CTRL-R showed a shorter latency time to enter the upper section of the water column than CTRL and T1 (37.74±64.90, 16.86±35.10 and 14.50±18.21 s, respectively).
The water exposure of zebrafish larvae to 1.0 ml L-1 and 2.0 ml L-1 of the tested herbal and Mg blend, showed no negative effects on larvae survival rate and induced lower locomotor performances. Similarly, adult zebrafish exposed for 9 days to 2.0 ml L-1 of the tested herbal and Mg blend, showed more capacity to cope with the stress supplied through the NTT test, demonstrated also by an increased environment exploration attitude. Repeatedly supplying the NTT stressor, induced in fish a certain grade of habituation. Hence, the observed results (CTRL-R group) support the hypothesis that the exposure to the herbal and Mg blend may reduce the stressor sensitiveness of fish through the sedation effect. These results open to interesting applications in the aquaculture sector. Despite that, it might be of great interest investigating about the possible administration of the sedative compounds through the inclusion in aquafeeds.
Aydın, B., & Barbas, L. A. L. (2020). Sedative and anesthetic properties of essential oils and their active compounds in fish: A review. In Aquaculture (Vol. 520). Elsevier B.V. https://doi.org/10.1016/j.aquaculture.2020.734999
Azizan, A., Alfaro, A. C., Young, T., & Venter, L. (2021). Beyond relaxed: magnesium chloride anaesthesia alters the circulatory metabolome of a marine mollusc (Perna canaliculus). Metabolomics, 17(8). https://doi.org/10.1007/s11306-021-01820-4
Petrisor, G., Motelica, L., Craciun, L. N., Oprea, O. C., Ficai, D., & Ficai, A. (2022). Melissa officinalis: Composition, Pharmacological Effects and Derived Release Systems—A Review. In International Journal of Molecular Sciences (Vol. 23, Issue 7). MDPI. https://doi.org/10.3390/ijms23073591