Elsevier

Aquaculture

Volume 482, 1 January 2018, Pages 49-56
Aquaculture

Lippia alba and Aloysia triphylla essential oils are anxiolytic without inducing aversiveness in fish

https://doi.org/10.1016/j.aquaculture.2017.09.023Get rights and content

Highlights

  • Lippia alba and A. triphylla EOs do not induce aversive or attractive behavior in fish.

  • Lippia alba and A. triphylla EOs induced an anxiolytic effect.

  • Exposure to the EO of A. triphylla induced stress in both fish species.

  • Exposure to the EO of L. alba attenuated the stress response in zebrafish.

  • Preference tests, commonly performed with zebrafish, can also be applied in jundiá.

Abstract

Lippia alba and Aloysia triphylla essential oils (EOs) are known to contain anesthetic properties. The majority of conventional anesthetics applied to fish cause aversive behavior in these animals. Therefore, questions arise whether EOs from L. alba and A. triphylla are also aversive and if they alter anxioly-like behaviors. To answer these questions, jundiá (Rhamdia quelen) and zebrafish (Danio rerio) were studied with the preference test using 50% of the anesthetic concentration of L. alba (corresponding to 150 μL L 1) and A. triphylla (corresponding to 150 μL L 1) EOs and the novel tank test immediately after exposure. Cortisol analysis was also performed. We show that EOs from L. alba and A. triphylla have an anxiolytic effect on zebrafish and catfish without altering locomotor patterns or inducing aversive behavior. To the best of our knowledge, this was the first time that preference tests were applied for natural anesthetics from plant extracts.

Introduction

Essential oils (EOs) are plant-based products known to play an important role in animal health because they contain bioactive components (Bandeira et al., 2017). Thus, several EOs or their isolated compounds are applied, either via baths or incorporated into feed, for therapeutic purposes (Van Hai, 2015) or as anesthetics and to reduce stress (Cunha et al., 2010, Gressler et al., 2014, Parodi et al., 2014) in aquaculture.

The plant species Aloysia triphylla is a South American shrub that was introduced into Europe at the end of the 17th century and is used in the preparation of teas (Carnat et al., 1999). Parodi et al. (2014) reported that A. triphylla EO produces adequate anesthesia when applied to the catfish Rhamdia quelen. This EO provides moderate antibacterial activity against Aeromonas spp. strains, which is dependent on the conditions of oil extraction (Parodi et al., 2013). It also stimulates antioxidant activities, prevention of lipid peroxidation, and suppression of cortisol release (Gressler et al., 2014). The use of this EO in fish transport water is recommended because it decreases plasma cortisol levels, decreases ammonia excretion, and improves oxidative status and ionic regulation (Parodi et al., 2014, Zeppenfeld et al., 2014). When utilized as a sedative in transport water, this EO delays the rate of oxidation of lipids in frozen fish fillets, thus increasing shelf life (Daniel et al., 2016).

Lippia alba is a shrub widely distributed in Central and South America (Hennebelle et al., 2008). Previous studies have shown that the EO from this plant species displays an anesthetic activity in R. quelen and inhibits the increase of plasmatic cortisol levels caused by handling, without altering the odor or flavor of the fillet (Cunha et al., 2010). There is the involvement of the GABAergic system in the anesthetic effect of this EO in jundiá (Heldwein et al., 2012). This EO is also a good antioxidant for the short periods of hypoxia or hyperoxia that occur during transport of fish from fish farms to their destination, thus improving animal welfare as well as product quality (Azambuja et al., 2011). Antimicrobial efficacy of this EO against Aeromonas spp. has previously been described via in vitro and in vivo tests, with the infected and treated fish displaying survival > 90% after 10 days (Sutili et al., 2015).

Some of the main anesthetics presently used for fish handling, as the tricaine methanesulfonate (MS222®), generate an aversive effect, which causes stress to zebrafish (Readman et al., 2013). Being aware of the anesthetic properties of EOs from these two plant species, the following question arises: are these substances aversive or attractive to fish species? To answer this question, we performed a preference test that included jundiá (Rhamdia quelen) and zebrafish (Danio rerio), which are two distant species on the evolutionary scale since they belongs to different orders, Siluriformes (Gomes et al., 2000) and Cypriniformes (Meyer et al., 1993) respectively.

In addition to the preference test, we performed a novel tank test during the immediate post-exposure period and cortisol values were measured (whole body for zebrafish and plasma for jundiá). The left or right preference test was first applied by Korver and Sprague (1989) and has been adapted for testing different anesthetics (Readman et al., 2013), psychoactive drugs (Abreu et al., 2016a) and agrochemicals (Rosa et al., 2016), which have always utilized zebrafish as the research model. In the present study, we validated the possibility of using this preference test for the jundiá also to test the aversivity and/or attractiveness of A. triphilla and L. alba EOs. Since the search and use of natural plant based anesthetics are a present reality, our data on their aversivity or attractiveness may be valuable to consolidat of A. triphilla and L. alba as natural anesthetics.

Section snippets

Animals and environmental design

A total of 144 fish were selected for the experiments. Sevent two seven-month-old zebrafish adults (3.6 ± 0.4 cm, 0.58 ± 0.1 g, ~ 50/50 male/female ratio) of the wild-type short-fin strain were maintained under constant aeration in a natural photoperiod and fed three times a day (08:00, 13:30, and 17:00) with commercial flake feed (Alcon Basic®, MEP 200 Complex). The water temperature was maintained at 28 ± 2 °C (Lawrence, 2007), dissolved oxygen concentrations ranged from 4.5 to 6 mg L 1, pH values ranged

Preference tests

In the pre-flux period, both jundiá (Fig. 2A) and zebrafish (Fig. 4A) showed no preference between lanes within each group. As expected, during the influx period, both species spent more time in the lane containing neutral pH water than in the lane with pH 3 water (positive control), which revealed, as expected, clear aversive behavior. None of the other treatments caused the fish to change their lane preference (Fig. 2A). No changes were observed in locomotor parameters in jundiá (Fig. 2B),

Discussion

Here we show, for the first time, that L. alba and A. triphylla EOs do not induce aversive or attractive behavior in either jundiá or zebrafish, and we also show an anxiolytic effect from both EOs. However, exposure to the EO of A. triphylla induced stress in both fish species, whereas the EO of L. alba attenuated the stress response only in the zebrafish, which is a promising result. Finally, we revealed that preference tests, commonly utilized in research with zebrafish, can also be applied

Acknowledgements

This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil). B. Baldisserotto (301156/2012-3), L.J.G. Barcellos (301992/2014-2) and B.M. Heinzmann (306449/2015-3) are recipients of CNPq fellowship grants. The authors would like to thank Denis Rosemberg from the Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Santa Maria, for help with data analysis.

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