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Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen)

Published:February 22, 2017DOI:https://doi.org/10.1111/vaa.12386

      Abstract

      Objectives

      To document the time for anesthesia induction and recovery using different concentrations of essential oils (EOs) of Cymbopogon flexuosus and Aloysia triphylla in silver catfish (Rhamdia quelen), and to determine whether the mechanism of action of either EO involves the benzodiazepine (BDZ) site of the GABAA receptor.

      Study design

      Experimental study.

      Animals

      A total of 144 silver catfish, length 7.5 ± 1.1 cm, weighing 3.95 ± 0.85 g.

      Methods

      Essential oils were evaluated at concentrations of 25, 150 and 300 μL L−1, and also ethanol alone (seven groups, n = 6 per group). Induction of sedation or anesthesia and recovery were assessed. In a further six groups (n = 6 per group), fish were exposed to both EOs (25, 150 or 300 μL L−1) with diazepam 150 μm, and also diazepam (10 μm) alone. Flumazenil (5 or 10 μm) was added to the recovery water of fish exposed to diazepam (150 μm) or both EOs (150 and 300 μL L−1) (total of 10 groups = 60 fish).

      Results

      Both EOs induced anesthesia at concentrations of 150 and 300 μL L−1, and sedation at 25 μL L−1. There was no significant difference between EOs for reaching deep anesthesia; there was a significantly longer recovery time for the EO of C. flexuosus. The addition of diazepam (150 μm) resulted in faster induction of anesthesia with both EOs, with no significant change in recovery times. Flumazenil (10 μm) reversed the diazepam-induced anesthesia, but not the anesthesia induced by EOs.

      Conclusions and clinical relevance

      The EO of C. flexuosus induced effective sedation (25 μL L−1) and anesthesia (150 and 300 μL L−1) without short-term mortality. The modulation of the BDZ site of the GABAA receptor in the anesthetic action mechanism of both EOs was not demonstrated.

      Keywords

      Introduction

      Aquaculture practices such as biometry, the collection of blood and other materials for analysis, hormonal implants and transportation, often stress fish, affecting their subsequent performance and resulting in disorders contributing to disease and mortality (
      • Barton B.A.
      Stress.
      ). Synthetic drugs, such as tricaine methanesulfonate (MS-222), quinaldine, etomidate, metomidate, benzocaine, barbiturates and propofol, have been used in various aquatic species to minimize the stress induced by these procedures (
      • Amend D.F.
      • Goven B.A.
      • Elliot D.G.
      Etomidate: effective dosages for a new fish anesthetic.
      ,
      • Mattson N.S.
      • Ripple T.H.
      Metomidate: a better anesthetic for cod (Gadusmorhua) in comparison with benzocaine, MS-222, chlorobutanol, and phenoxy-ethanol.
      ,
      • Gomes L.C.
      • Chippari-Gomes A.R.
      • Lopes N.P.
      • et al.
      Efficacy of benzocaine as an anesthetic in juvenile tambaqui Colossoma macropomum.
      ,
      • Ross L.G.
      • Ross B.
      Anaesthetic and Sedative Techniques for Aquatic Animals.
      ,
      • Gressler L.T.
      • Parodi T.V.
      • Riffel P.K.
      • et al.
      Immersion anaesthesia with tricaine methanesulphonate or propofol on different sizes and strains of silver catfish Rhamdia quelen.
      ).
      Studies investigating alternatives to these synthetic substances have identified sedative and anesthetic effects of plant essential oils (EOs) or compounds isolated from EOs, often with similar activity (
      • Palic D.
      • Herolt D.M.
      • Andreasen C.B.
      • et al.
      Anesthetic efficacy of tricaine methanesulfonate, metomidate and eugenol: effects on plasma cortisol concentration and neutrophil function in fathead minnows (Pimephalespromelas Rafinesque, 1820).
      ,
      • Gonçalves A.F.N.
      • Santos E.C.C.
      • Fernandes J.B.K.
      • et al.
      Mentol e eugenol como substitutos da benzocaína na indução anestésica de juvenis de pacu. [Menthol and eugenol as benzocaine substitutes in anesthetic induction of pacu juveniles] Acta.
      ,
      • Gressler L.T.
      • Riffel A.P.K.
      • Parodi T.V.
      • et al.
      Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Herit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status.
      ).
      The plant Aloysia triphylla (Verbenaceae) is an aromatic shrub with an EO that is primarily a mixture of α-citral (geranial) and β-citral (neral). Anesthetic activities of this EO were reported in white shrimp (Litopenaeus vannamei) (
      • Parodi T.V.
      • Cunha M.A.
      • Heldwein C.G.
      • et al.
      The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and subadults of Litopenaeus vannamei (Crustacea, Penaeidae).
      ) and silver catfish (Rhamdia quelen) (
      • Gressler L.T.
      • Parodi T.V.
      • Riffel P.K.
      • et al.
      Immersion anaesthesia with tricaine methanesulphonate or propofol on different sizes and strains of silver catfish Rhamdia quelen.
      ,
      • Parodi T.V.
      • Cunha M.A.
      • Becker A.G.
      • et al.
      Anesthetic activity of the essential oil of Aloysia triphylla and effectiveness in reducing stress during transport of albino and gray strains of silver catfish, Rhamdia quelen.
      ). The plant Cymbopogon flexuosus (Poaceae) is an aromatic grass, and also has an EO with citral as the predominant component (
      • Taskinen J.
      • Mathela D.K.
      • Mathela C.S.
      Composition of the essential oil of Cymbopogon flexuosus.
      ).
      Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the central nervous system. Modulation of the GABAergic system through the benzodiazepine (BDZ) site of the GABAA as an anesthetic mechanism of action of EOs of Lippia alba and Ocimum gratissimum has recently been suggested in silver catfish (
      • Heldwein C.G.
      • Silva L.L.
      • Reckziegel P.
      • et al.
      Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N. E. Brown essential oil.
      ,
      • Silva L.L.
      • Parodi T.V.
      • Reckziegel P.
      • et al.
      Essential oil of Ocimumgratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen.
      ). Expressions of this system have been identified in several species of vertebrates, from fish to mammals, with GABAergic functional evidence (immunocytochemistry) being described in the brain of zebrafish, Danio rerio (
      • Kim Y.J.
      • Nam R.H.
      • Yoo Y.M.
      • Lee C.J.
      Identification and functional evidence of GABAergic neurons in parts of the brain of adult zebrafish (Danio rerio).
      ,
      • Delgado L.
      • Schmachtenberg O.
      Immunohistochemical localization of GABA, GAD65, and the receptor subunits GABAAalpha1 and GABAB1 in the zebrafish cerebellum.
      ).
      Benzodiazepines are positive modulators of the BDZ site of the GABAA receptors, resulting in sedative, hypnotic, muscle relaxant and anxiolytic effects in mammals (
      • Foster A.C.
      • Kemp J.A.
      Glutamate- and GABA-based CNS therapeutics.
      ). Flumazenil (RO 15-1788) is an imidazo-benzodiazepine that antagonizes the central effects of benzodiazepines by competition for the BDZ site (
      • Darragh A.
      • Lambe R.
      • Kenny M.
      • et al.
      RO 15-1788 antagonises the central effects of diazepam in man without altering diazepam biovailability.
      ).
      As both EOs have the same main compound (citral), it is possible that the EO of C. flexuosus induces anesthesia and that the mechanism of action of both EOs are related to the BDZ site of the GABAA. This study aimed to evaluate the anesthetic efficacy of the EO of C. flexuosus in silver catfish and whether this EO induces anesthesia, and to compare the time for anesthesia induction and recovery for different concentrations with the EO of A. triphylla. The study also aimed to evaluate any potentiation offered by the inclusion of diazepam and to determine the involvement of the EOs in the BDZ site of the GABAA by observing responses to treatment with flumazenil. The hypotheses of this study were that the EO of C. flexuosus would induce anesthesia and that flumazenil would confirm that the action of both EOs is through the BDZ site of the GABAA in silver catfish.

      Material and methods

      Plant material

      The A. triphylla was cultivated and identified according to
      • Parodi T.V.
      • Cunha M.A.
      • Heldwein C.G.
      • et al.
      The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and subadults of Litopenaeus vannamei (Crustacea, Penaeidae).
      . The C. flexuosus was cultivated in the city of Tiradentes do Sul, Rio Grande do Sul state (RS), Brazil. The species was identified by agronomist engineer Luiz Volnei of the Regional University of Northeastern Rio Grande do Sul (UNIJUI), and a voucher specimen was deposited in the Herbarium of Forest Sciences Department (HDCF no. 6748) at the Federal University of Santa Maria (UFSM).

      Drugs, extraction and analysis of EOs

      Flumazenil (Flumazenil injectable solution 0.1 mg mL−1; Cristália Produtos Químicos e Farmacêuticos Ltda, SP, Brazil) and diazepam (Compaz injectable solution 5 mg mL−1; Cristália Produtos Químicos e Farmacêuticos Ltda) were obtained from local trade sources (LDT Produtos Farmaceuticos e Hospitalares Ltda, RS, Brazil). The EO of A. triphylla was obtained from fresh leaves and the extraction and chromatographic analysis of the constituents of EOs were performed as described by
      • Parodi T.V.
      • Cunha M.A.
      • Heldwein C.G.
      • et al.
      The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and subadults of Litopenaeus vannamei (Crustacea, Penaeidae).
      . The EO of C. flexuosus was obtained from fresh leaves by hydrodistillation for 2 hours, according to the European Pharmacopoeia (
      • European Directorate for the Quality of Medicines
      European Pharmacopoeia.
      ), and stored in amber bottles at –4 °C. The gas chromatography-mass spectrometry (GC-MS) analysis was performed on an Agilent 7890A gas chromatograph coupled to a 5975C mass spectrometer with a nonpolar HP5-MS fused silica capillary column (5% phenyl – 95% methylsiloxane, 30 m × 0.25 mm internal diameter × 0.25 μm film thickness) and electron ionization MS of 70 eV (Agilent Technologies Brasil Ltda, SP, Brazil). The operating conditions were as follows: carrier gas, He, at a flow rate of 1 mL minute—1; split inlet, 1:100; injector and detector temperatures, 250 °C; temperature program, 40 °C for 4 minutes and 40–320 °C at 4 °C minute−1. The constituents of the EO were identified by comparison of the mass spectra and Kovats retention index with the literature and a mass spectral data bank (
      • Adams R.P.
      Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy.
      ,
      • NIST/EPA/NIH
      NIST/EPA/NIH Mass Spectral Library and Search/Analysis Programs.
      ). The quantification of the components was performed using Agilent 7890A with a flame ionization detector (FID) for HP5 column (30 m × 0.25 mm internal diameter × 0.25 μm film thickness) in accordance with the parameters mentioned earlier (carrier gas, He, at a flow rate of 1 mL minute−1; the split less mode; injector and detector temperature, 300 °C; temperature program, 40 °C for 4 minutes and 40–320 °C at 4 °C minute−1). Samples of EO were injected in triplicate. The relative percentage of the EO compounds was estimated by integrating the areas under the peaks obtained from FID chromatograms.

      Animals and culture conditions

      Juveniles of silver catfish, of both sexes (144 fish; 7.5 ± 1.1 cm, 3.95 ± 0.85 g) were acquired from a local producer (Bela Vista Fish Culture, RS, Brazil). They were kept for 10 days in a continuously aerated recirculation system with 30 L aquaria, at a stocking density of 4.94 ± 1.2 g L−1, and with the following parameters: temperature, 23.0 ± 1 °C; dissolved oxygen, 8.87 ± 0.51 mg L−1; pH 7.40 ± 0.11; and total ammonia, 0.99 ± 0.22 mg L−1. Fish were fed daily with commercial feed 28.0% crude protein and 3500 kcal kg−1. The aquaria were siphoned 30 minutes after feeding to remove food debris and other impurities. Measurements of dissolved oxygen and temperature, pH and total ammonia were performed daily with the aid of an oxygen meter YSI (Model Y5512; YSI Inc., OH, USA), a DMPH-2 pH meter (Digimed Analytical Instrumentation, MS, Brazil), and as described in
      • Eaton A.D.
      • Clesceri L.S.
      • Rice E.W.
      • Greenberg A.E.
      Standard Methods for the Examination of Water & Wastewater.
      , respectively. Fish with no visual abnormalities in the mucosa, skin, body volume and/or feeding habits and behavior were used in the study.
      The methodology used in this study was approved by the Institutional Animal Care and Use Committee of the Federal University of Santa Maria (no. 5935290715). The number of animals used in each experiment was the lowest possible to meet the institution's policy of reduction, refinement and substitution of experimental animals.

      Anesthesia induction and recovery

      Anesthesia was evaluated according to
      • Small B.C.
      Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus.
      , which involves four stages: stage I, sedation and decreased responsiveness to external stimuli; stage II, partial loss of balance and erratic swimming; stage III, no movement and no response to stimuli; stage IV, respiratory failure and death. Juveniles were fasted for 24 hours before the experiments and then placed in continuously aerated 1 L aquaria. The times for anesthesia induction and recovery of the fish after exposure to EOs of A. triphylla and C. flexuosus at concentrations of 25, 150 or 300 μL L−1 were measured and compared (Table 1). The fish were chosen randomly and used once. Each juvenile was assessed individually and it was considered anesthetized when it reached stage III. The maximum time of observation was 30 minutes. After this, juveniles were removed from the anesthetic bath, placed in an aquarium with water only to assess the anesthetic recovery, and monitored until complete recovery. Juveniles were considered recovered when normal swimming and reaction to external stimuli were observed. The EOs were previously diluted 1:10 in 95% ethanol, and therefore a group exposed to ethanol at the highest concentration used in this study was included (2700 μL L−1 ethanol).
      Table 1Sequence of experimental groups for 144 silver catfish (six fish per group). The maximum observation period was 30 minutes after exposure to essential oil (EO), ethanol or diazepam (D), where indicated. Fish were recovered in water, with flumazenil (F) if indicated, and observed until the fish were fully recovered
      Experimental sequenceGroups
      EO of Aloysia triphyllaEO of Cymbopogon flexuosusControls
      I. Effects of EOs
      25 μL L−125 μL L−1Ethanol
      150 μL L−1150 μL L−1
      300 μL L−1300 μL L−1
      II. Effects of EOs and diazepam
      25 μL L−1 + D 150 μm25 μL L−1 + D 150 μmD 150 μm
      150 μL L−1 + D 150 μm150 μL L−1 + D 150 μmD 150 μm + F 5 μm
      300 μL L−1 + D 150 μm300 μL L−1 + D 150 μmD 150 μm + F 10 μm
      III. Effects of flumazenil on recovery from EO
      150 μL L−1 + F 5 μm150 μL L−1 + F 5 μm
      150 μL L−1 + F 10 μm150 μL L−1 + F 10 μm
      300 μL L−1 + F 5 μm300 μL L−1 + F 5 μm
      300 μL L−1 + F 10 μm300 μL L−1 + F 10 μm

      Effects of diazepam or flumazenil

      To determine whether the effects of EOs were potentiated by diazepam, fish were exposed to the EOs of A. triphylla or C. flexuosus (25, 150 or 300 μL L−1) with diazepam 150 μm (Table 1). Anesthetic induction and recovery times were compared between the groups with and without diazepam. The effects of diazepam (150 μm) alone, and of administration of flumazenil (5 or 10 μm) in recovery were also studied.
      The impact of flumazenil (5 or 10 μm) on the recovery time from EO-induced anesthesia (150 or 300 μL L−1) was studied in an additional 48 fish (Table 1). The results were compared with the results from fish that were not administered flumazenil.

      Statistical analysis

      All data were submitted to a Levene test to check the homogeneity of variances. As data were homoscedastic, one-way anova followed by Tukey test was performed. The minimum significance was 95% and the data are presented as means ± standard error (Version 9, STATISTICA; StatSoft Inc., TX, USA).

      Results

      EO composition

      The major components of the EO of A. triphylla were α-citral (29.41%), β-citral (20.78%) and limonene (11.90%). The major components of the EO of C. flexuosus were α-citral (48.90%), β-citral (37.47%) and heptene-one <6-methyl-5> (2.67%).

      EO anesthesia

      The EOs of A. triphylla and C. flexuosus induced stage III anesthesia at 150 and 300 μL L−1, and induced up to stage II at 25 μL L−1, within 30 minutes. Time to anesthesia induction followed a negative concentration-dependent pattern, but the recovery time demonstrated a positive concentration-response relationship. As 25 μL L−1 of both EOs did not induce stage III, recovery times were not registered.
      The EO of C. flexuosus induced stages I and II of anesthesia, at the three concentrations tested, significantly more quickly than the EO of A. triphylla, but the recovery times of the EO of C. flexuosus were significantly longer (p < 0.05) (Fig. 1). Ethanol alone failed to induce sedation or anesthesia.
      Figure thumbnail gr1
      Figure 1Time required for induction and recovery from anesthesia by essential oil (EO) of Aloysia triphylla (At) and EO of Cymbopogon flexuosus (Cf) (six fish per group). Data are means ± standard error. Different letters in the columns indicate a significant difference between the concentrations of the same EO. *Significant difference between the different EOs at the same concentrations (p < 0.05).

      Anesthetic effects of EOs with diazepam

      The addition of diazepam to the EOs induced stage I of anesthesia significantly more quickly. The association of diazepam with 25 μL L−1 of EO of A. triphylla significantly decreased the time to reach stage II. Diazepam also made it possible to induce stage III anesthesia with 25 μL L−1 of both EOs, but there was no significant difference in the anesthetic recovery times (Figs 2a & b) (p < 0.05).
      Figure thumbnail gr2
      Figure 2Time required for anesthesia induction and recovery after exposure to essential oil (EO) of Aloysia triphylla (At) (a) and EO of Cymbopogon flexuosus (Cf) (b) alone and in combination with 150 μm diazepam (D) (six fish per group). Data are means ± standard error. *Significantly different from the EO without diazepam at the same concentration (p < 0.05).

      Effect of flumazenil

      Flumazenil (10 μm) reversed the diazepam-induced anesthesia, but not the anesthesia induced by EOs at concentrations of 150 and 300 μL L−1. No anesthetic procedure in the tested groups was reversed by 5 μm of flumazenil (Fig. 3) (p < 0.05). There was no short-term mortality in any of the experimental periods.
      Figure thumbnail gr3
      Figure 3Time required for recovery from anesthesia in water or water and flumazenil after exposure to diazepam (D) at 150 μm, essential oil (EO) of Aloysia triphylla (At) at 150 or 300 μL L−1 and EO of Cymbopogon flexuosus (Cf) at 150 or 300 μL L−1 (six fish per group). Data are means ± standard error. *Significantly different from recovery in water (p < 0.05).

      Discussion

      Both EOs induced sedation (25 μL L−1) and anesthesia (150 and 300 μL L−1) at different concentrations. Short-term mortality during induction or recovery was not observed, similar to what is reported by
      • Cunha M.A.
      • Barros F.M.C.
      • Garcia L.O.
      • et al.
      Essential oil of Lippia alba: a new anesthetic for silver catfish.
      for EO of L. alba,
      • Silva L.L.
      • Parodi T.V.
      • Reckziegel P.
      • et al.
      Essential oil of Ocimumgratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen.
      for EO of O. gratissimum and
      • Gressler L.T.
      • Riffel A.P.K.
      • Parodi T.V.
      • et al.
      Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Herit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status.
      and
      • Parodi T.V.
      • Cunha M.A.
      • Becker A.G.
      • et al.
      Anesthetic activity of the essential oil of Aloysia triphylla and effectiveness in reducing stress during transport of albino and gray strains of silver catfish, Rhamdia quelen.
      for EO of A. triphylla in relation to mortality.
      Several authors referred to 3 and 5–10 minutes as the maximum periods for anesthesia induction and recovery, respectively (
      • Gilderhus P.A.
      • Marking L.L.
      Comparative efficacy of 16 anesthetic chemicals on rainbow trout.
      ,
      • Small B.C.
      Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus.
      ,
      • Ross L.G.
      • Ross B.
      Anaesthetic and Sedative Techniques for Aquatic Animals.
      ). The concentration of 300 μL L−1 of both EOs considered here was within these parameters.
      Both EOs showed a negative concentration-response relationship for anesthesia induction, but recovery times were directly proportional to concentration. Similar patterns for the time of induction and recovery of anesthesia were described for the 100–800 μL L−1 concentration range for EO of A. triphylla (
      • Parodi T.V.
      • Cunha M.A.
      • Becker A.G.
      • et al.
      Anesthetic activity of the essential oil of Aloysia triphylla and effectiveness in reducing stress during transport of albino and gray strains of silver catfish, Rhamdia quelen.
      ).
      • Cunha M.A.
      • Barros F.M.C.
      • Garcia L.O.
      • et al.
      Essential oil of Lippia alba: a new anesthetic for silver catfish.
      also described a negative relationship for the time of anesthesia induction for EO of L. alba at concentrations of 100–500 μL L−1, but recovery times did not follow any relationship.
      Further studies are needed to establish the sedative and anesthetic concentrations of both EOs for different fish species and strains, since significantly different concentrations of the EO of A. triphylla were necessary to reach sedation, anesthesia and recovery in grey or albino silver catfish (
      • Parodi T.V.
      • Cunha M.A.
      • Becker A.G.
      • et al.
      Anesthetic activity of the essential oil of Aloysia triphylla and effectiveness in reducing stress during transport of albino and gray strains of silver catfish, Rhamdia quelen.
      ).
      The mechanisms of action of several psychoactive herbs remain undefined, even after their therapeutic effects have been well established. Additional studies are needed to understand the role of each substance isolated from both EOs studied and whether there are cooperative or antagonistic interactions. The different anesthesia induction and recovery times following exposure to the EO of C. flexuosus as compared with the EO of A. triphylla are probably a result of the different percentages of citral and limonene in the EOs. It has been reported that citral and limonene induced sedation and muscle relaxation in mice (
      • Vale T.G.
      • Furtado E.C.
      • Santos J.G.
      • Viana G.S.
      Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (Mill.) n.e. Brown.
      ) and citral inhibited neural excitability in rats (
      • Sousa D.G.
      • Sousa S.D.G.
      • Silva R.E.R.
      • et al.
      Essential oil of Lippia alba and its main constituent citral block the excitability of rat sciatic nerves.
      ).
      Although further studies are needed to understand the effects of combining diazepam with the EOs of A. triphylla and C. flexuosus, in the present study these combinations resulted in significant reductions in anesthesia induction time, mainly at the lowest concentration (25 μL L−1). Diazepam also accelerated anesthesia induction time when combined with the EOs of O. gratissimum (
      • Silva L.L.
      • Parodi T.V.
      • Reckziegel P.
      • et al.
      Essential oil of Ocimumgratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen.
      ) and L. alba (
      • Heldwein C.G.
      • Silva L.L.
      • Reckziegel P.
      • et al.
      Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N. E. Brown essential oil.
      ). Furthermore, similar to reports by
      • Silva L.L.
      • Parodi T.V.
      • Reckziegel P.
      • et al.
      Essential oil of Ocimumgratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen.
      and
      • Heldwein C.G.
      • Silva L.L.
      • Reckziegel P.
      • et al.
      Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N. E. Brown essential oil.
      , the addition of diazepam to EOs resulted in a deeper stage of anesthesia at the lowest concentration.
      Flumazenil reversed the anesthetic effects caused by diazepam, but did not change the anesthetic recovery times from EOs of A. triphylla and C. flexuosus. Therefore, the BDZ site of the GABAA is not involved in the EOs' mechanism of action, unlike what was found for the EOs of L. alba (
      • Heldwein C.G.
      • Silva L.L.
      • Reckziegel P.
      • et al.
      Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N. E. Brown essential oil.
      ) and O. gratissimum (
      • Silva L.L.
      • Parodi T.V.
      • Reckziegel P.
      • et al.
      Essential oil of Ocimumgratissimum L.: anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen.
      ). Some anesthetic compounds isolated from EOs, such as linalool (L. alba) and (—) globulol (H. mutabilis), also did not have their anesthetic effects reversed by flumazenil (
      • Silva L.L.
      • Garlet Q.I.
      • Benovit S.C.
      • et al.
      Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich) Briq. and their isolated components in silver catfish (Rhamdia quelen).
      ,
      • Heldwein C.G.
      • Silva L.L.
      • Gai E.Z.
      • et al.
      S-(+)-linalool from Lippia alba: sedative and anesthetic for silver catfish (Rhamdia quelen).
      ).
      These results confirm the hypothesis that EO of C. flexuosus induces anesthesia, but does not confirm the hypothesis that the mechanisms of action of both EOs involve the BDZ site of the GABAA in silver catfish.

      Conclusion

      The EO of C. flexuosus induced effective sedation (25 μL L−1) and anesthesia (150 and 300 μL L−1) without short-term mortality in silver catfish. In comparison with the EO of A. triphylla, the EO of C. flexuosus induced the initial stages of anesthesia (I and II) more rapidly, but also resulted in significantly longer anesthetic recovery times. The combination of diazepam with both EOs significantly decreased the time required to reach anesthesia, without changing recovery times. Flumazenil did not reduce EO recovery times, and therefore modulation of the BDZ site of GABAA was not confirmed as a mechanism of anesthetic action of EOs of A. triphylla and C. flexuosus in silver catfish.

      Authors' contributions

      ACS: study design, data acquisition and interpretation, preparation of the manuscript. GBJ: data acquisition, ethical project and plant voucher routing. DCZ: preparation of the manuscript. CCZ: data interpretation, statistical analysis. DTS and BMH: distillation and determination of essential oils. BB: study design. MAC: study design, ethical project routing. All authors were involved in critical revision and approval of the final manuscript.

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