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Essential oil of Aloysia citriodora Paláu and citral: sedative and anesthetic efficacy and safety in Rhamdia quelen and Ctenopharyngodon idella

Published:October 29, 2021DOI:https://doi.org/10.1016/j.vaa.2021.10.004

      Abstract

      Objective

      To verify the efficacy of citral in inducing sedation and anesthesia in silver catfish (Rhamdia quelen) and grass carp (Ctenopharyngodon idella) and to assess the safety of essential oil (EO) of Aloysia citriodora and citral in inducing and maintaining anesthesia in silver catfish.

      Study design

      Clinical study, randomized, parallel, multi-arm with control group in target species.

      Animals

      A total of 96 juvenile and 72 adult silver catfish and 80 juvenile grass carp were used.

      Methods

      Silver catfish and grass carp were exposed to different concentrations of citral, 15–675 and 15–600 μL L–1, respectively, during the maximum period of 30 minutes to verify sedation and anesthesia induction and recovery times. In addition, for anesthetic induction, silver catfish were exposed to the EO of A. citriodora and citral at 225 μL L–1 for 3.5 minutes. Then, fish were transferred to an anesthesia maintenance solution at 50 μL L–1 for 10 minutes to assess hematologic and biochemical variables at 60 minutes, 2 and 6 days after treatment.

      Results

      Citral only induced sedation from 15, 25 and 40 μLL–1 in both species. Anesthesia without mortality was induced in silver catfish at 50–600 μL L–1 and grass carp at 75–450 μL L–1. At 675 and 600 μL L–1, mortality was recorded in silver catfish and grass carp, respectively. The EO of A. citriodora and citral were safe in inducing and maintaining anesthesia in silver catfish, with mean corpuscular hemoglobin concentration being the only variable that varied in relation to time and treatments.

      Conclusions and clinical relevance

      Citral was effective in inducing sedation and anesthesia in both species. In addition, A. citriodora EO and citral were safe in inducing and maintaining anesthesia in silver catfish. Both agents are promising substances for the development of new drugs for fish.

      Keywords

      Introduction

      The Aloysia citriodora Paláu plant, with synonyms Aloysia triphylla (L'Hér.) Britton, Lippia citriodora Kunth, Lippia triphylla (L'Hér.) Kuntze, Verbena triphylla L'Hérit. and Verbena citriodora Cav., popularly known in Brazil as cidró, in South American Spanish-speaking countries as cedrón, and in English as lemon verbena, among other names, is a shrub of the Verbenaceae family which has leaves and flowers rich in essential oil (EO). The plant has been cultivated and used worldwide as a spice and in folk medicine as a sedative, among other uses (
      • Bahramsoltani R.
      • Rostamiasrabadi P.
      • Shahpiri Z.
      • et al.
      Aloysia citrodora Paláu (Lemon verbena): A review of phytochemistry and pharmacology.
      ).
      The benefits of sedative and anesthetic procedures in aquaculture have been proven (
      • Zahl I.H.
      • Samuelsen O.
      • Kiessling A.
      Anaesthesia of farmed fish: implications for welfare.
      ). Initially, the focus was on studying several synthetic drugs such as tricaine methanesulfonate (MS-222), benzocaine, quinaldine, 2-phenoxyethanol and etomidate (
      • Schoettger R.A.
      • Julin A.M.
      Efficacy of MS-222 as an anesthetic on four salmonids.
      ;
      • Gilderhus P.A.
      • Marking L.L.
      Comparative efficacy of 16 anesthetic chemicals on rainbow trout.
      ;
      • Kazuń K.
      • Siwicki A.K.
      Propiscin: a safe new anaesthetic for fish.
      ). Currently, the study of substances of natural origin, such as the EOs of plants and substances isolated from these EOs, has been a promising alternative for the development of new drugs for use in fish (
      • da Cunha M.A.
      • Barros F.M.C.
      • Garcia L.O.
      • et al.
      Essential oil of Lippia alba: a new anesthetic for 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).
      ;
      • Benovit S.C.
      • Silva L.L.
      • Salbego J.
      • et al.
      Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima in silver catfish Rhamdia quelen.
      ;
      • Teixeira R.R.
      • de Souza R.C.
      • Sena A.C.
      • et al.
      Essential oil of Aloysia triphylla in Nile tilapia: anaesthesia, stress parameters and sensory evaluation of fillets.
      ;
      • Hoseini S.M.
      • Mirghaed A.T.
      • Yousefi M.
      Application of herbal anaesthetics in aquaculture.
      ;
      • Aydına B.
      • Barbas L.A.L.
      Sedative and anesthetic properties of essential oils and their active compounds in fish: a review.
      ).
      The anesthetic effect results from the concentration and the exposure time, and sedation is a preliminary state of anesthesia on a continuum (
      • Ross L.G.
      • Ross B.
      The features of anaesthetic agents.
      ). The EO of A. citriodora has proven sedative and anesthetic efficacy in fish. Several studies place the product as promising in developing new drugs for the mentioned uses in fish (
      • 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’Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status.
      ;
      • 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.
      ,
      • Parodi T.V.
      • Gressler L.T.
      • Silva L.L.
      • et al.
      Chemical composition of the essential oil of Aloysia triphylla under seasonal influence and its anaesthetic activity in fish.
      ;
      • Zeppenfeld C.C.
      • Toni C.
      • Becker A.G.
      • et al.
      Physiological and biochemical responses of silver catfish, Rhamdia quelen, after transport in water with essential oil of Aloysia triphylla (L’Herit) Britton.
      ;
      • dos Santos A.C.
      • Bandeira Junior G.
      • Zago D.C.
      • et al.
      Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen).
      ;
      • Teixeira R.R.
      • de Souza R.C.
      • Sena A.C.
      • et al.
      Essential oil of Aloysia triphylla in Nile tilapia: anaesthesia, stress parameters and sensory evaluation of fillets.
      ). The major component verified for the A. citriodora EO in these studies has been a terpenoid, the citral (a mixture of α-citral or geranial and β-citral or neral). However, the component has not been definitively linked to the related effects.
      The silver catfish (Rhamdia quelen) is widely used in studies on sedation and anesthesia with an emphasis on EOs or isolated components of EOs (
      • da Cunha M.A.
      • Barros F.M.C.
      • Garcia L.O.
      • et al.
      Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen.
      ;
      • 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’Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status.
      ;
      • Heldwein C.G.
      • Silva L.L.
      • Gai E.Z.
      • et al.
      S-(+)-linalool from Lippia alba: sedative and anesthetic for 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.
      ;
      • Zeppenfeld C.C.
      • Toni C.
      • Becker A.G.
      • et al.
      Physiological and biochemical responses of silver catfish, Rhamdia quelen, after transport in water with essential oil of Aloysia triphylla (L’Herit) Britton.
      ;
      • Benovit S.C.
      • Silva L.L.
      • Salbego J.
      • et al.
      Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima in silver catfish Rhamdia quelen.
      ;
      • dos Santos A.C.
      • Bandeira Junior G.
      • Zago D.C.
      • et al.
      Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen).
      ). It is native to South America, suitable for fish farming for human consumption (
      • Gomes L.C.
      • Golombieski J.I.
      • Gomes A.R.C.
      • Baldisserotto B.
      Biologia do jundiá Rhamdia quelen (Teleostei, Pimelodidae).
      ). The grass carp (Ctenopharyngodon idella) is the most raised fish species for consumption worldwide (
      FAO
      The state of world fisheries and aquaculture: meeting the sustainable development goals.
      ). There is a lack of data on sedation and anesthesia procedures for this species, with only one study reporting the effectiveness of etomidate (
      • Kazuń K.
      • Siwicki A.K.
      Propiscin: a safe new anaesthetic for fish.
      ) and another one regarding the effect of the EO of Ocimum micranthum (
      • Zeppenfeld C.C.
      • Brasil M.T.B.
      • Cavalcante G.
      • et al.
      Anesthetic induction of juveniles of Rhamdia quelen and Ctenopharyngodon idella with Ocimum micranthum essential oil.
      ).
      The growing concern about the presence of toxic residues from veterinary drugs in the environment and the meat of fish intended for consumption suggests that natural substances or phytochemicals isolated from these products may be good alternatives to the synthetic substances for the development of new drugs that are less toxic to consumers and the environment (
      • Aydına B.
      • Barbas L.A.L.
      Sedative and anesthetic properties of essential oils and their active compounds in fish: a review.
      ). Therefore, this study aimed to verify the effectiveness of citral as a sedative and anesthetic agent in silver catfish and grass carp exposed to different concentrations of citral using verification of elapsed times for anesthetic induction and recovery. The study also aimed to verify the safety of A. citriodora EO and citral in inducing and maintaining anesthesia in silver catfish from the verification of mortality data and the determination of several hematologic and biochemical variables after procedures. The hypotheses assumed were that citral would effectively induce sedation and anesthesia in silver catfish and grass carp and that the EO and the isolate will be safe for inducing and maintaining anesthesia in silver catfish.

      Materials and methods

      Study design

      This was a clinical study, randomized, parallel, multi-arm with control group in target species (
      VICH
      Good clinical practice VICH GL9: guidance for industry.
      ;
      • Juszczak E.
      • Altman D.G.
      • Hopewell S.
      • Schulz K.
      Reporting of multi-arm parallel-group randomized trials: extension of the CONSORT 2010 statement.
      ). The Animal Use Ethics Committee of Universidade Federal de Santa Maria (UFSM), Brazil (no. 074/2014), approved this study. The procedures were performed in accordance with the principles of reduction, refinement and replacement of animal experimentation.

      Plant material, essential oil of Aloysia citriodora and citral

      Aloysia citriodora Paláu was cultivated in the campus of the UFSM located in the city of Frederico Westphalen (RS, Brazil). The agronomist Renato Aquino Záchia identified the species. A voucher was deposited in the herbarium of the Department of Biology of UFSM (SMDB 16.111). 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 sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae).
      in the Industrial Pharmacy sector of UFSM. The constituents were identified by mass spectra and Kovats retention index comparison with data from the Mass Spectral Library of the National Institute of Standards and Technology (
      NIST
      Mass spectral library and search/analysis programs.
      ) and from the literature (
      • Adams R.P.
      Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy.
      ). The EO density, determined by weighing, was 0.9. Citral natural ≥ 96% from Sigma-Aldrich (MO, USA) was used.

      Animals

      Juveniles and adults of silver catfish and juveniles of grass carp (males and females) that were apparently healthy, with no external signs of lesions or body imbalances, were purchased from a local fish farm and transported to the Fish Physiology Laboratory at UFSM. The juveniles were transported in 60 L plastic bags and adults in plastic barrels with 200 L of water.
      The adult fish were placed in a 1000 L tank to proceed with the random allocation, whereas the juveniles were divided by species into two 500 L tanks. From these tanks, the fish were randomly captured and transferred one by one, in a determined cyclic sequence, to 250 L tanks. The fish were randomly allocated to the 250 L tanks from a slight movement of the capture net, initially through the bottom of the 500 and 1000 L tanks, where the animals were forced to swim in a block through the most superficial part of the water, being captured by same net when passing in the central superficial part of the tank. All tanks mentioned in this process were circular in shape.
      The number of 250 L tanks or fish per tank varied according to the experiment to be performed. A 5 day acclimation period was observed before performing the experiments. The fish remained under continuous aeration, biological and mechanical filtration, and the photoperiod was 12/12 hours.
      The water quality parameters were checked daily: dissolved oxygen at 8.67 ± 0.55 mg L–1, pH of 7.4 ± 0.2 and temperature of 22.5 ± 2.0 °C. To maintain total ammonia and/or nitrite concentrations approximately 0.9 ± 0.7 and 0.06 ± 0.02 mg L–1, respectively, partial water exchanges were sometimes required. An oxygen meter (Model Y5512; YSI Inc., OH, USA), pH meter (DMPH-2; Digimed Analytical Instrumentation, SP, Brazil), mercury thermometers and commercial kits for total ammonia and nitrite (NO2) (Amônia Tóxica and Nitrito NO2; LabcomTest, Camboriú, SC, Brazil) were used to measure the water parameters.

      Sedation and anesthesia

      Sedation and anesthesia were assessed employing a protocol adapted from
      • 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.
      . Stages I and II correspond to a progressive state of sedation of the animals. Stage I describes a decrease in swimming activity and/or environmental responsiveness. In stage II, the fish stays at the bottom of the aquarium or exhibits partial loss of balance and/or erratic swimming. Stage III is the anesthesia stage, with no movement and no response to external stimuli (light pressure with a glass rod on the caudal peduncle of the fish or biometric procedures for measuring length and weight). Stage IV is defined as respiratory failure and death.

      Efficacy verification procedure

      A total of 96 silver catfish juveniles (8.0 ± 1.7 cm, 4.5 ± 1.3 g, n = 8 per treatment) and 80 grass carp juveniles (8.4 ± 1.8 cm, 10 ± 2.5 g, n = 8 per treatment) were allocated in 16 tanks with 250 L (eight tanks for each species) and exposed to different concentrations of citral: 15, 25, 40, 50, 75, 150, 225, 300, 450, 600 and 675 μL L–1 for silver catfish and 15, 25, 40, 75, 150, 225, 300, 450 and 600 μL L–1 for grass carp. Citral was previously dissolved (95% ethanol, 1:10). For control, two ethanol groups (n = 8 per treatment) were performed at a concentration equivalent to the dilution of 675 and 600 μL L–1 of citral for silver catfish and grass carp, 6075 and 5400 μL L–1 ethanol, respectively.
      Sedation and anesthesia induction and recovery times were used in the development of concentration-response curves. The fish were exposed one by one to different concentrations of citral or ethanol only in 1 L aquaria under continuous aeration. Lack of sensitivity to pressure on the caudal peduncle with a glass rod was the stimulus used to confirm stage III and determined the passage of fish from the anesthetic solution to the recovery solution. The induction time to different stages of anesthesia was evaluated for up to 30 minutes.
      After recovery, the fish were measured with a ruler and weighed on an AD 10K scale (Marte Científica, MG, Brazil). Finally, the fish were placed in 30 L tanks (fish of the same species submitted as the same treatments in the same 30 L tank; 22 tanks, 12 for silver catfish and 10 for grass carp), in conditions similar to the original ones. Each fish was used only once. Short-term mortality was assessed for 2 days after the procedure.
      In this study, the definition of anesthetic efficacy for rapid anesthesia was induction and recovery times of < 3 and 5 minutes, respectively (
      • Marking L.L.
      • Meyer F.P.
      Are better anesthetics needed in fisheries?.
      ). The ideal citral concentration for rapid anesthesia in silver catfish and grass carp was the minimum concentration that induced the highest level of the observed effect with the shortest recovery time.

      Safety verification procedure

      The anesthetic safety of A. citriodora EO and citral was defined as not inducing short-term mortality, plus the absence of significant changes in hematologic and biochemical variables determined after induction and maintenance of anesthesia in silver catfish. To perform a longer procedure, the concentration chosen for the induction of anesthesia by citral was that immediately below, in relation to the concentration established as ideal for rapid induction and recovery times. In addition, an anesthetic maintenance solution was developed from the lowest concentration of citral capable of inducing anesthesia within the maximum proposed period of 30 minutes. The maintenance period of 10 minutes was chosen to reflect the handling period most required by fish workers, according to
      • Marking L.L.
      • Meyer F.P.
      Are better anesthetics needed in fisheries?.
      . The safety evaluation for citral was repeated for the EO of A. citriodora, and for two control groups, water and ethanol.
      A total of 72 adult silver catfish (31.0 ± 3.1 cm, 138.7 ± 34.5 g) were allocated into four groups (four 250 L tanks; n = 18 per treatment). Fish were exposed to different treatments: water control group (WCG), ethanol control group (ECG), citral group (CG) and Aloysia group (AG). The fish were individually placed in 10 L aquaria and exposed to anesthetic solutions (CG or AG at 225 μL L–1) or other treatments (WCG or ECG) for 3.5 minutes to induce anesthesia (
      • 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.
      ). The solution for group ECG contained ethanol equivalent to that used in groups CG and AG for 225 μL L–1 concentration (2025 μL L–1). Then, the fish were transferred to 10 L aquaria containing maintenance solutions for groups CG and AG at 50 μL L–1, water for group WCG and ethanol concentration of 450 μL L–1 for group ECG for 10 minutes.
      Sensitivity to external stimuli was verified with a glass rod when the fish were passed from induction solutions to anesthetic maintenance solutions. Biometric measurements were obtained at the end of the maintenance period. Fish were placed in aquaria with untreated water for recovery; recovery time was not recorded. After recovery, all fish were allocated to the other four 250 L tanks. Short-term mortality was assessed for 8 days after the procedure.

      Blood collection

      Access to the caudal vessels was made with the fish in lateral recumbency on a moistened towel on a table, in the lateral line of fish, cranially to the caudal peduncle in the region ventral to the most caudal third of the adipose fin. Blood samples were collected from six fish from each group at three time points: 60 minutes after recovery from anesthesia (T1), 2 days after recovery (T2) and 6 days after recovery (T3). The fish were captured and briefly removed from the water for collection of 1 mL of blood for hematologic and plasma measurements. The percutaneous puncture was made using 25 gauge, 0.7 mm needles and heparinized 3 mL syringes for 0.5 mL of blood. A second venipuncture was made more cranially in the fish lateral line for collection of 0.5 mL blood into syringes without heparin to obtain serum. After blood collection, the animals were placed in other 250 L tanks in conditions similar to the original ones.

      Hematologic variables

      Determinations of hematologic variables were according to
      • Ranzani-Paiva M.J.T.
      • Pádua S.B.
      • Tavares-Dias M.
      • Egami M.I.
      Métodos para Análise Hematológica em Peixes.
      . The hematocrit (Hct) was measured after micro-centrifugation at 13,680 g for 5 minutes. Erythrocyte (RBC) count was performed in a Neubauer chamber after dilution 1:200 in Natt and Herrick solution. Hemoglobin (Hb) was determined by the cyanomethemoglobin method after centrifuging the mixture (2318 g for 10 minutes) to remove free cores of RBC. White blood cell count and leukocyte differential were counted in blood smears by counting the total leukocytes and differential in a proportion of 2000 RBC. Blood smears were stained with rapid hematology stain Diff-Quik (Panótico rápido; Laborclin, PR, Brazil).
      Total plasma protein (TPP) was determined by refractometry. The mean corpuscular volume and the mean corpuscular hemoglobin concentration (MCHC) were calculated through the formula (Hct/RBC) × 10 and (Hb/Hct) × 100, respectively.

      Biochemical variables

      Biochemical variables were determined through commercial kits and a BIO-200 semiautomatic spectrophotometer (Bioplus, SP, Brazil). The variables were alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyl transferase (GGT), urea (URE), creatinine (CRE), uric acid (UA), glucose (GLU), cholesterol (CHO), triglycerides (TRI), lactate (LAC), total protein (TP), albumin (ALB) (respective commercial kit references: K049, K048, K021, K080, K056, K067, K139, K082, K083, K117, K084, K031, and K040; Bioclin, MG, Brazil), chloride (Cl) and sodium (Na+) (respective commercial kit references: 04950-2 and 573351; In Vitro Diagnóstica, MG, Brazil).
      Serum and plasma were separated by centrifugation at 2318 g for 10 minutes. Alanine aminotransferase, AST, GGT, URE, CRE, UA, GLU, CHO and LAC were determined in plasma. Alkaline phosphatase, TRI, TP, ALB, Cl, and Na+ were determined in serum. Globulins were determined by the difference between TP and ALB.

      Statistical analysis

      All data are expressed as mean ± standard error (SE). Data related to evaluating the sedative and anesthetic efficacy of citral and safety of A. citriodora EO and citral were submitted to Levene’s test with conversions of the data to logarithms when necessary, followed by the application of one-way analysis of variance and Tukey’s test. All statistical analyses were performed using Statistica Version 7.0 (StatSoft Inc., OK, USA). The minimum level of significance was p < 0.05.

      Results

      Chemical characterization of Aloysia citriodora essential oil

      The main constituents of A. citriodora EO were citral (α-citral or geranial 17.71% and β-citral or neral 14.85%), trans-caryophyllene oxide (9.91%) and limonene (L-limonene 8.31% and D-limonene 7.71%). A total of 34 phytochemicals were detected (Table 1).
      Table 1Chemical composition of Aloysia citriodora Paláu essential oil determined by chromatographic analysis. KIC, Calculated Kovats Index; KIL, Literature Kovats Index [
      NIST
      Mass spectral library and search/analysis programs.
      (Mass spectral library);
      • Adams R.P.
      Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy.
      ]; NI, nonidentified compound; RT, retention time
      PeakRTCompoundKICKILReference%
      117.50L-Limonene10311028Nist8.31
      217.59D-Limonene10321035Nist7.71
      317.68E-3-Octen-2-one10341036Nist3.34
      422.66S-Linalool10911100Nist0.47
      522.72R-Linalool10981105Nist0.43
      623.30Limonene oxide, cis-11401132Nist0.93
      723.40Limonene oxide, trans-11431139Nist0.33
      823.48NI1175_Nist/Adams0.25
      923.66NI1176_Nist/Adams0.32
      1023.74NI1196_Nist/Adams0.19
      1124.27NI1207_Nist/Adams0.15
      1224.32NI1210_Nist/Adams0.18
      1327.15NI1238_Nist/Adams0.30
      1427.64β-Citral (Neral)12421244Nist14.85
      1527.75Nerol12461254Nist2.49
      1628.73Geraniol12501258Nist2.91
      1728.82α-Citral (Geranial)12511250Nist17.71
      1829.01NI1423_Nist/Adams0.40
      1930.43Nerol acetate13391342Nist0.81
      2030.75NI1342_Nist/Adams0.33
      2130.86NI1350_Nist/Adams0.60
      2231.29Geraniol acetate13571352Nist3.67
      2332.24NI1379_Nist/Adams1.82
      2432.77NI1388_Nist/Adams1.20
      2533.54α-Curcumene14641473Nist5.10
      2633.68NI1469_Nist/Adams1.84
      2734.53Nerolic acid15081499Nist2.00
      2834.65Guaiene15131504Nist0.47
      2936.81(+)-Nerolidol15441535Nist0.80
      3037.96Caryophylene oxide-cis15731576Nist2.71
      3138.07Caryophylene oxide-trans15771576Nist9.91
      3238.24NI1582_Nist/Adams0.39
      3338.43(-)-Spathulenol15841578Nist4.53
      3440.09tau.-Cadinol16191625Nist2.56
      Total identified94.24

      Efficacy of citral

      Citral added to water induced sedation (stages I and II) without inducing anesthesia (stage III) within 30 minutes at concentrations of 15, 25 and 40 μL L–1 in silver catfish and grass carp. At concentrations of 15 and 25 μL L–1, both species reached stage I only, with significantly longer onset time in the carp at 15 μL L–1 (p < 0.05). At a concentration of 40 μL L–1, both species reached stage II, with carp reaching stage I significantly earlier. Anesthetic recovery times were not recorded for concentrations that did not induce stage III within 30 minutes (Fig. 1).
      Figure 1
      Figure 1Time required for sedation induction by citral in silver catfish (Rhamdia quelen, RQ) and grass carp (Ctenopharyngodon idella, CI) (n = 8 per treatment) 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.
      . Data are presented as mean ± standard error. Stage I sedation, black columns; Stage II sedation, grey columns. ∗Significant difference between the species at the same citral concentrations (p < 0.05). a–cDifferent superscript low case letters indicate a significant difference between the concentrations of the same species (p < 0.05).
      The anesthetic effect of citral demonstrated a concentration-dependent pattern in silver catfish and grass carp, with faster induction and slower recovery following the increase in the concentrations used (Fig. 2a & b). Anesthesia without mortality was induced in silver catfish at concentrations of 50 to 600 μL L–1 and in carp at 75 to 450 μL L–1.
      Figure 2
      Figure 2Time required for induction and recovery from anesthesia by citral in (a) silver catfish (Rhamdia quelen, RQ) and (b) grass carp (Ctenopharyngodon idella, CI) (n = 8 per treatment) 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.
      . Data are presented as mean ± standard error. Stage I, first stage of sedation, black columns; Stage II, second stage of sedation, light grey columns; Stage III, general anesthesia, dark grey columns; Recovery, recovery from anesthesia, white columns. a–eDifferent superscript low case letters indicate significant differences between the citral concentrations within the same species (p < 0.05).
      The most suitable concentrations for rapid induction and recovery from anesthesia were the concentrations of 300 μL L–1 for silver catfish and grass carp. For the silver catfish, stage III was induced in < 3 minutes (149 ± 11 seconds) with recovery time approximately 5 minutes (287 ± 33 seconds), mean ± SE. For grass carp, stage III developed in < 3 minutes (112 ± 11 seconds) with a recovery time > 10 minutes (722 ± 74 seconds).
      The onset of stage I and transition from stage I to III at citral concentrations of 150 and 300 μL L–1, respectively, were faster in grass carp than in silver catfish (Fig. 3). The recovery times were significantly longer in grass carp at concentrations of 150–300 μL L–1 (Fig. 3). The fish showed some hyperactivity on initial contact with citral solutions, which did not occur in the control groups with ethanol. No undesirable side effects were observed during induction and recovery from the sedative and anesthetic procedures performed. Ethanol alone did not induce sedation or anesthesia in silver catfish or grass carp.
      Figure 3
      Figure 3Time required for induction and recovery from anesthesia by citral in silver catfish (Rhamdia quelen, RQ) and grass carp (Ctenopharyngodon idella, CI) (n = 8 per treatment) 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.
      . Data are presented as mean ± standard error. Stage I, first stage of sedation, black columns; Stage II, second stage of sedation, light grey columns; Stage III, general anesthesia, dark grey columns; Recovery, recovery from anesthesia, white columns. ∗Significant difference between species at the same citral concentration (p < 0.05).
      After exposure to citral concentrations of 675 and 600 μL L–1, two (25%) silver catfish and one (12.5%) grass carp, respectively, died. The registered deaths were verified by respiratory arrest, with the fish already allocated for anesthetic recovery in untreated water. There was no short-term mortality in fish treated with the other concentrations during the 2 days after anesthesia.

      Safety of Aloysia citriodora essential oil and citral

      The MCHC of group WCG was significantly higher at time points T2 and T3 than at T1 (p < 0.05). This variable was also significantly higher in fish in groups CG and AG at T1 than fish in group WCG but not different from group ECG (Table 2). There were no significant differences among groups at different time points in TPP (Table 2), leukogram (Table 3) and biochemical variables (Tables S1 & S2).
      Table 2Erythrogram and total plasma protein (TPP) of silver catfish (Rhamdia quelen) after exposure to Aloysia citriodora essential oil (group AG) or citral (group CG) at 225 μL L–1 for 3.5 minutes then 50 μL L–1 for 10 minutes, or equivalent concentrations of ethanol (group ECG) or water (group WCG). Blood was collected from six fish in each group on the same day after recovery from anesthesia (T1), 2 days after anesthesia (T2) and 6 days after anesthesia (T3). Data are presented as mean ± standard error. Hb, hemoglobin; Hct, hematocrit; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cells.
      GroupTime pointHct (%)RBC (× 106 μL–1)Hb (g dL–1)MCV (fL)MCHC (%)TPP (g dL–1)
      WCGT130 ± 41.54 ± 0.175.09 ± 0.88190.6 ± 5.116.7 ± 1.64.9 ± 0.3
      T227 ± 21.42 ± 0.105.90 ± 0.57188.5 ± 9.322.1 ± 1.05.2 ± 0.2
      T328 ± 11.52 ± 0.075.93 ± 0.20185.6 ± 6.021.2 ± 0.44.7 ± 0.2
      ECGT131 ± 21.68 ± 0.086.13 ± 0.45187.5 ± 15.319.6 ± 0.34.9 ± 0.3
      T226 ± 21.62 ± 0.115.74 ± 0.48163.4 ± 0.721.6 ± 0.34.6 ± 0.2
      T329 ± 21.59 ± 0.116.98 ± 0.56183.1 ± 3.523.9 ± 0.75.2 ± 0.4
      CGT128 ± 11.53 ± 0.105.97 ± 0.35188.8 ± 11.421.0 ± 1.0∗5.1 ± 0.3
      T230 ± 31.65 ± 0.116.41 ± 0.74182.0 ± 6.621.0 ± 0.54.9 ± 0.1
      T323 ± 21.36 ± 0.144.85 ± 0.34169.1 ± 7.121.6 ± 1.44.1 ± 0.4
      AGT126 ± 21.40 ± 0.035.34 ± 0.33183.0 ± 9.820.8 ± 1.0∗4.7 ± 0.3
      T227 ± 21.36 ± 0.075.77 ± 0.45195.1 ± 6.121.6 ± 0.84.6 ± 0.2
      T329 ± 11.65 ± 0.065.84 ± 0.26174.5 ± 4.920.3 ± 0.24.8 ± 0.1
      ∗Significantly different from group WCG at the same time point (p < 0.05). Significantly different from T1 in the same group (p < 0.05)
      Table 3Leukogram of silver catfish (Rhamdia quelen) after exposure to Aloysia citriodora essential oil (group AG) or citral (group CG) at 225 μL L–1 for 3.5 minutes then 50 μL L–1 for 10 minutes, or equivalent concentrations of ethanol (group ECG) or water (group WCG). Blood was collected from six fish in each group on the same day after recovery from anesthesia (T1), 2 days after anesthesia (T2) and 6 days after anesthesia (T3). Data are presented as mean ± standard error. There was no significant difference from group WCG at the same time point and from T1 in the same group (p < 0.05)
      GroupTime pointTotal leukocytes (μL–1)Neutrophils (μL–1)Monocytes (μL–1)Lymphocytes (μL–1)
      WCGT128020 ± 276020538 ± 17571049 ± 2676433 ± 1188
      T221575 ± 262412017 ± 1833531 ± 1029027 ± 1774
      T325400 ± 221214687 ± 25561054 ± 1899659 ± 777
      ECGT127400 ± 116720648 ± 1696538 ± 956214 ± 681
      T223650 ± 189813582 ± 2495721 ± 1939348 ± 1008
      T322325 ± 432611563 ± 34691102 ± 5189660 ± 2008
      CGT125050 ± 409016082 ± 3834743 ± 2888191 ± 1978
      T230260 ± 353020064 ± 2850903 ± 2439293 ± 1177
      T327100 ± 429520790 ± 4540794 ± 3185515 ± 742
      AGT124920 ± 494319060 ± 4256745 ± 1495116 ± 1196
      T220967 ± 182413996 ± 1965789 ± 1306182 ± 546
      T321533 ± 313014340 ± 3427990 ± 1986203 ± 597
      There was no short-term mortality during the observation period, 8 days after anesthesia.

      Discussion

      The effects induced by EOs of plants in animal organisms are often linked to the most abundant components present, in addition to being the product of interaction between components (
      • Heldwein C.G.
      • Silva L.L.
      • Gai E.Z.
      • et al.
      S-(+)-linalool from Lippia alba: sedative and anesthetic for silver catfish (Rhamdia quelen).
      ;
      • Benovit S.C.
      • Silva L.L.
      • Salbego J.
      • et al.
      Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima in silver catfish Rhamdia quelen.
      ;
      • Parodi T.V.
      • Gressler L.T.
      • Silva L.L.
      • et al.
      Chemical composition of the essential oil of Aloysia triphylla under seasonal influence and its anaesthetic activity in fish.
      ). According to
      • Heldwein C.G.
      • Silva L.L.
      • Gai E.Z.
      • et al.
      S-(+)-linalool from Lippia alba: sedative and anesthetic for silver catfish (Rhamdia quelen).
      , the S-(+)-Linalool isolated from the EO of Lippia alba is the most abundant component with 59.66% of the total. Although S-(+)-Linalool can induce anesthesia in silver catfish, it cannot induce the same level of effect verified for the EO in a crude state. Although several of the most abundant components in the EO of Aloysia gratissima have demonstrated sedative and anesthetic properties in silver catfish, only (+)-Spathulenol was effective in inducing sedation and anesthesia without the undesirable side effects of involuntary muscle contractions during induction and recovery (
      • Benovit S.C.
      • Silva L.L.
      • Salbego J.
      • et al.
      Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima in silver catfish Rhamdia quelen.
      ). Notably, only 2.71% of the EO was composed of (+)-Spathulenol.
      Although citral is the most abundant component in samples of the EOs of A. citriodora, Cymbopogon flexuosus and L. alba (citral chemotype) that were effective in inducing sedation and/or anesthesia in fish, the link between the monoterpene and the verified effects did not exist (
      • dos Santos A.C.
      • Bandeira Junior G.
      • Zago D.C.
      • et al.
      Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen).
      ;
      • Souza C.F.
      • Baldissera M.D.
      • Bianchini A.E.
      • et al.
      Citral and linalool chemotypes of Lippia alba essential oil as anesthetics for fish: a detailed physiological analysis of side effects during anesthetic recovery in silver catfish (Rhamdia quelen).
      ). Thus, the present study demonstrated for the first time the sedative and anesthetic efficacy of citral in fish.
      Studies based on tricaine methanesulfonate (MS 222) were responsible, in large part, for the definition of the pattern for rapid induction (in less than 3 minutes) with rapid recovery (in less than 5 minutes) proposed by
      • Marking L.L.
      • Meyer F.P.
      Are better anesthetics needed in fisheries?.
      . At the same time, these proposed time limits have guided several studies on the effectiveness of new sedative and anesthetic substances for fish (
      • Ross L.G.
      • Ross B.
      The features of anaesthetic agents.
      ;
      • Teixeira R.R.
      • de Souza R.C.
      • Sena A.C.
      • et al.
      Essential oil of Aloysia triphylla in Nile tilapia: anaesthesia, stress parameters and sensory evaluation of fillets.
      ;
      • Hoseini S.M.
      • Mirghaed A.T.
      • Yousefi M.
      Application of herbal anaesthetics in aquaculture.
      ). Rapid induction of anesthesia is ideal for handling fish for spawning, fin clipping, marking, measuring, some surgical operations such as vaccination and immobilizing specimens for various physiological investigations (
      • Schoettger R.A.
      • Julin A.M.
      Efficacy of MS-222 as an anesthetic on four salmonids.
      ;
      • Marking L.L.
      • Meyer F.P.
      Are better anesthetics needed in fisheries?.
      ;
      • Ross L.G.
      • Ross B.
      The features of anaesthetic agents.
      ).
      In the present study, the induction of anesthesia times achieved the goal of 3 minutes in silver catfish at 300 μL L–1 and in grass carp at 225 and 300 μL L–1; however, the anesthetic recovery times for grass carp exceeded 5 minutes for concentrations 150–300 μL L–1 (
      • Marking L.L.
      • Meyer F.P.
      Are better anesthetics needed in fisheries?.
      ). Despite the longer recovery time, deaths only occurred at much higher concentrations than those determined to be effective in silver catfish and grass carp.
      The toxicity of anesthetic agents in fish varies according to the characteristics of the active substances used, with the use of increasing concentrations and extended duration of exposure to the substances (
      • Ross L.G.
      • Ross B.
      The features of anaesthetic agents.
      ). The type of anesthesia should be varied according to the procedure to be performed and the skills or intention of the fish farm worker who performs the procedures with the animals. Long periods of handling animals may be necessary for carrying out procedures such as tagging, marking, surgical operations or even longer spawning procedures. Many workers need to keep fish anesthetized in the anesthetic solution to facilitate handling, and periods of anesthetic maintenance of 5–10 minutes are necessary for many procedures. In these situations, the pattern of rapid induction and recovery is not suited to the requirements of the procedure, as it is intended for performing equally rapid procedures (
      • Schoettger R.A.
      • Julin A.M.
      Efficacy of MS-222 as an anesthetic on four salmonids.
      ;
      • Marking L.L.
      • Meyer F.P.
      Are better anesthetics needed in fisheries?.
      ;
      • Ross L.G.
      • Ross B.
      The features of anaesthetic agents.
      ).
      Significantly lower values of MCHC were found in European catfish (Silurus glanis) anesthetized with clove oil (
      • Velísěk J.
      • Wlasow T.
      • Gomulka P.
      • et al.
      Effects of clove oil anaesthesia on European catfish (Silurus glanis L.).
      ) and common carp (Cyprinus carpio) anesthetized with 2-phenoxyethanol and etomidate (
      • Witeska M.
      • Dudyk J.
      • Jarkiewicz N.
      Haematological effects of 2-phenoxyethanol and etomidate in carp (Cyprinus carpio L.).
      ). By contrast,
      • Velísěk J.
      • Wlasow T.
      • Gomulka P.
      • et al.
      Effects of 2-phenoxyethanol anaesthesia on sheat-fish (Silurus glanis L.).
      and
      • Křišt’an J.
      • Stará A.
      • Turek J.
      • et al.
      Comparison of the effects of four anaesthetics on haematological and blood biochemical profiles in pikeperch (Sander lucioperca L.).
      identified increases in MCHC in pikeperch (Sander lucioperca) anesthetized with 2-phenoxyethanol and etomidate, and in European catfish anesthetized with 2-phenoxyethanol, respectively.
      Unlike mammals, adult fish RBCs maintain the bloodstream nucleus, and mild to moderate anisocytosis and polychromasia are normal in several species. The immature RBC is smaller, has a larger nucleus and less abundant cytoplasm when compared with more differentiated cells. These cells have active metabolism producing Hb in the blood (
      • Thrall M.A.
      Hematologia de peixes.
      ). MCHC can be reduced by anesthetic toxicity damage to RBC (
      • Witeska M.
      • Dudyk J.
      • Jarkiewicz N.
      Haematological effects of 2-phenoxyethanol and etomidate in carp (Cyprinus carpio L.).
      ), whereas
      • Thrall M.A.
      Hematologia de peixes.
      postulated that stress induces the release of catecholamines that cause hemoconcentration and the increased Hct results in a decrease in calculated MCHC. Considering that MCHC in fish is a result of the relationship between the removal of damaged or senile RBCs from circulation and replacement via hematopoietic tissue and bloodstream hematopoiesis, further studies to understand the Hb kinetics in fish are needed to explain the variability of MCHC observed in the present study and the studies mentioned previously.
      The present study is limited by the fact that several studies are needed for the development and authorization of a new veterinary drug. In addition to proof of safety and efficacy, it is necessary to verify the safety of the product for consumers and for the environment (
      VICH
      Good clinical practice VICH GL9: guidance for industry.
      ). Therefore, complementary studies on tissue pharmacokinetics and safe residue levels in the meat of animals destined for consumption, in addition to the environmental impact of A. citriodora EO and citral, are necessary for the development and authorization of a new sedative and anesthetic drug for use in fish.

      Conclusion

      Under the conditions of this study, citral effectively induced sedation and anesthesia in silver catfish and grass carp with a concentration-response relationship, with no mortality in silver catfish at concentrations of 50–600 μL L–1 and in carp at 75–450 μL L–1. Although further studies are required by authorization procedures for the introduction of new veterinary drugs in country markets, such as the presence of drug residues in food of animal origin or in the environment, the A. citriodora EO and citral are promising natural active substances for use in fish.

      Acknowledgements

      This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) , Finance Code 001, Brazil, and in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil . The authors also thank agronomist Renato Aquino Záchia for identifying the plant.

      Supporting Information

      The following are the Supporting information to this article:

      Authors’ contributions

      ACdosS: study design, data acquisition and interpretation (in vivo experiments and statistics), fish allocation, manuscript writing. AEB: data acquisition and interpretation (in vivo experiments), manuscript writing and critical revision. GBJ: data acquisition and interpretation (in vivo experiments), critical revision of manuscript. QIG and BMH: data acquisition and interpretation (EO chromatography), critical revision of manuscript. MTdeBB: data acquisition and interpretation (statistics), critical revision of manuscript. BB and MAdaC: study design, critical revision of manuscript. BOC: data acquisition (EO distillation), critical revision of manuscript. All authors read and approved the final version of the manuscript.

      Conflict of interest statement

      The authors declare no conflict of interest.

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