Anesthesia of Epinephelus marginatus with essential oil of Aloysia polystachya : an approach on blood parameters

This study investigated the anesthetic potential of the essential oil (EO) of Aloysia polystachya in juveniles of dusky grouper (Epinephelus marginatus). Fish were exposed to different concentrations of EO of A. polystachya to evaluate time of induction and recovery from anesthesia. In the second experiment, fish were divided into four groups: control, ethanol and 50 or 300 μL L EO of A. polystachya, and each group was submitted to induction for 3.5 min and recovery for 5 or 10 min. The blood gases and glucose levels showed alterations as a function of the recovery times, but Na and K levels did not show any alteration. In conclusion, the EO from leaves of A. polystachya is an effective anesthetic for dusky grouper, because anesthesia was reached within the recommended time at EO concentrations of 300 and 400 μL L. However, most evaluated blood parameters showed compensatory responses due to EO exposure.

The EO used in the present study was obtained from leaves of A. polystachya (Griseb.)Moldenke (Verbenaceae), an aromatic native plant widely distributed in subtropical regions of South America, mainly in Paraguay and North Argentina, and popularly known as "burrito", "poleo de Castilla" or "poleo riojano".This plant is referred to as a sedative (Del Vitto and Petenatti 1997) and is also used against gastrointestinal pain in folk medicine (Filipoy 1994).Studies with mice and rats indicated that the hydro-ethanolic extract from the aerial parts of A. polystachya has anxiolytic and antidepressant-like effects (Mora et al. 2005, Hellión-Ibarrola et al. 2006, 2008).
The dusky grouper, Epinephelus marginatus (Serranidae) has a wide distribution, occurring along the Mediterranean Sea and in the Indian Ocean to the southeast of the African continent (Fennessy 2006).On the west coast of the Atlantic Ocean, the dusky grouper occurs from Rio de Janeiro to the New Gulf region in Argentinean Patagonia (Figueiredo andMenezes 1980, Irigoyen et al. 2005).Since E. marginatus is a target species for aquaculture (Cunha et al. 2013, Cavalli 2014, Sanches et al. 2014), the present study investigated the anesthetic potential of the EO of A. polystachya in dusky grouper juveniles.Some blood parameters were also analyzed, aiming to evaluate possible side effects of this EO.

PLANT MATERIAL AND ESSENTIAL OIL EXTRACTION
Aloysia polystachya (Griseb.)Moldenke (Verbenaceae) was cultivated in the medicinal plant garden of "Nature's Pharmacy", Municipality of Jardinópolis, SP, Brazil.The leaves were harvested in September 2012 at 10 am and dried in an oven with forced air circulation at a temperature of 45 °C for 48 h.The voucher specimen (UPMU No. 1213) was identified by Dr. Rossi from the Institute of Botany of São Paulo, and a voucher was deposited in the Herbarium of Medicinal Plants at the University of Ribeirão Preto, SP, Brazil.The EO was extracted from dried leaves by hydrodistillation using a Clevenger-type apparatus according to the European Pharmacopoeia (2007).

ESSENTIAL OIL ANALYSIS
The EO samples were analyzed by GC-MS with an Agilent 6890A gas chromatograph equipped with a 5973C mass selective detector using a nonpolar HP5-MS fused silica capillary column (5% phenyl, 95% methylsiloxane, 30 m x 0.25 mm i.d.x 0.25 µm film thickness) and electron ionization mode at 70 eV.Helium was used as carrier gas at a flow rate of 1.0 mL min −1 ; the injector and detector temperatures were set at 250 and 280 °C, respectively.Oven temperature was kept at 40 °C for 4 min and then gradually raised to 320 °C at 4 °C min −1 .Injections were performed in split inlet mode (ratio 1:100).Kovats retention indices were calculated using a homologous series of C7-C31 n-alkanes injected under the same conditions.The EO constituents were identified by comparison of the mass spectra and Kovats retention indices with literature data and with the National Institute of Standards and Technology (NIST) Mass Spectral Library (NIST 2008, Adams 2009).FID analysis was performed in an equivalent column and using the same oven parameters as described for GC-MS.Both injection and detection temperatures were set at 300 °C and the split inlet mode ratio was 1:50.The percentage of EO compounds was calculated by under peak area integration.

ANIMALS AND WATER CONDITIONS
Dusky grouper (82.0 ± 2.3 g; 16.7 ± 0.1 cm) juveniles obtained from a fish culture in Rio Grande, southern, Brazil, were maintained for one week in 250 L continuously aerated tanks to acclimate to laboratory conditions.The animals were fed once a day with commercial feed and kept fasted for a period of 24 h prior to the experiments that were conducted in accordance with the Ethical Committee and the Animal Welfare Committee of UFSM (process number 074/2014).The water parameters were measured as follows: dissolved oxygen (6.20 ± 0.11 mg L −1 ) and temperature (26.29 ± 0.12 °C) with a YSI oxygen meter (model DO 200A), pH (7.2 ± 0.1) with a pH meter (Hanna Instruments, Woonsocket, RI, USA; model HI 8424), total ammonia nitrogen (0.25 ± 0.06 mg N L −1 ) measured by the salicylate method (UNESCO 1983), nitrite (0.08 ± 0.03 mg L −1 ) determined as described by Bendschneider and Robinson (1952) and alkalinity (149.75 ± 0.67 mg CaCO 3 L −1 ) by the method of Baumgarten et al. (1996).In addition, salinity was maintained throughout the experiment at 29 ppt.

EXPERIMENT 1: ANESTHESIA INDUCTION AND RECOVERY
The water conditions for this experiment were similar to those reported for acclimation.Juveniles were transferred with a net to a 10 L aquarium with the EO from the leaves of A. polystachya at 50, 75, 100, 200, 300 or 400 µL L −1 , firstly diluted in ethanol (1:10).Moreover, the possible anesthetic effect of ethanol was tested with the highest concentration used to dilute the EO.The EO concentrations were chosen based on the study of Parodi et al. (2014) with the EO of A. triphylla.To evaluate the time required for anesthesia induction, six (n = 6) juveniles were individually tested using aquaria at the respective concentration.Each animal was used only once and the anesthesia stages were determined according to Small (2003): Stage 1: sedation -decreased reactivity to external stimuli; Stage 2: partial loss of equilibrium and erratic swimming; Stage 3: total loss of equilibrium and cessation of locomotion.The maximum observation time was 30 min.After the induction of anesthesia, juveniles were transferred to anesthetic-free aquaria to measure the recovery time.Animals were considered to have recovered when they demonstrated normal swimming and reaction to external stimuli.
EXPERIMENT 2: BLOOD ANALYSIS OF ANESTHETIZED AND RECOVERED FISH Animals were divided into the following groups (n = 6 per treatment and time of collection): control (without anesthetic), ethanol, 50 or 300 µL L −1 A. polystachya leaf EO.The collection times for each group were: exposure (3.5 min) and recovery times (5 or 10 min).Each fish was sampled only once.Recovery was performed in anesthetic-free aquaria.After exposure or recovery times, blood was collected from the caudal vein of each fish by heparinized 1 mL syringes and immediately analyzed using an i-STAT portable clinical analyzer with CG8+ cartridge (Abbott Laboratories, Chicago, IL, USA).The parameters measured were: sodium (Na + ), potassium (K + ), calcium (Ca 2+ ), bicarbonate (HCO 3 − ), pH, gases (PvO 2 , PvCO 2 ), glucose, hemoglobin (Hb) and hematocrit (Hct).The clinical analyzer temperature was corrected to the water temperature according to the manufacturer's specifications.The efficacy of i-STAT measurements has been proved for several CARINE O. FOGLIARINI et al.

STATISTICAL ANALYSIS
All data are expressed as mean ± SEM.The homogeneity of variances between treatments was calculated with Levene's test.As the data exhibited homogeneous variances, comparisons between different groups and times were made using two-way ANOVA and Tukey's test.Analyses were performed using Statistica ver.7.0 software (StatSoft, Tulsa, OK, USA) with the minimum significance level set at P < 0.05.

CHEMICAL COMPOSITION
A total of 19 compounds were identified in the EO obtained from the dried leaves of A. polystachya (Table I).The main constituents found in this EO were carvone (58.76%) and α-limonene (33.68%).

INDUCTION AND RECOVERY TIMES FROM ANESTHESIA
As expected, by increasing EO concentration there was a proportional decrease in the time required for sedation and anesthesia induction, but not for recovery.Fish exposed up to 75 µL L −1 A. polystachya EO reached sedation (Stage 1), but no evidence indicated a possible deep anesthesia (Stage 3) during the evaluation time (maximum 30 min).Concentrations above 100 µL L −1 EO were able to induce sedation and anesthesia.Recovery time was significantly faster at 200 and 300 µL L −1 EO than at 100 µL L -1 , and the highest concentration tested (400 µL L −1 EO) presented the fastest recovery time (Table II).Mortality was not observed throughout the anesthesia induction procedure.Ethanol added to the water did not produce any anesthetic effect.
BLOOD PARAMETERS Blood pH was not significantly affected by treatments.The PvO 2 and PvCO 2 values increased and decreased, respectively, in fish placed in the simulated recovery, as well as in those recovering from ethanol exposure for 5 min compared to those exposed to ethanol.Groupers exposed to both EO concentrations presented higher PvO 2 values than control fish.The PvO 2 values were also higher in fish exposed to 50 µL L −1 EO than in those exposed to ethanol.The PvO 2 values of fish recovered from 50 µL L −1 EO exposure were significantly lower than in exposed fish, but in those recovered from 300 µL L −1 EO exposure, these values were higher than in exposed fish and in the recovered control and ethanol groups.Groupers exposed to 300 µL L −1 showed significantly higher PvCO 2 values than the control group.In addition, fish recovered for 5 min presented significantly higher PvCO 2 values than the control and ethanol groups, but after 10 min recovery these values were significantly lower than in the ethanol group.The HCO 3 − concentration decreased in all groups at both recovery times when compared to exposure, but was not affected by treatments (Table III).
The levels of glucose, hemoglobin and hematocrit were not significantly different between exposure times in the control group (Fig. 1).Glucose levels also did not differ between times in the ethanol group.The groups treated with 50 or 300 µL L −1 EO showed higher glucose levels after 10 min recovery compared to other times.Fish exposed to 50 µL L −1 EO had increased glucose levels after 10 min recovery compared to the control and ethanol groups, but those exposed to 300 µL L −1 EO had decreased levels after 5 min recovery compared to the ethanol group (Fig. 1a).
Hemoglobin and hematocrit levels in the ethanol and 50 µL L −1 EO groups were lower after 5 min recovery compared to the other exposure times.Groupers exposed to 50 µL L −1 EO showed lower hemoglobin and hematocrit levels than the ethanol group and lower hematocrit than the control group.After 10 min recovery, fish that were exposed to 300 µL L −1 EO presented lower hematocrit and hemoglobin levels than exposed fish.Fish exposed to 300 µL L −1 EO showed higher hemoglobin and hematocrit levels after 5 min recovery compared to the fish that recovered from ethanol exposure for the same amount of time (Fig. 1b, c).Na + and K + levels did not differ between groups or exposure times (Fig. 2a, b).The lowest Ca 2+ levels in the control group were observed after 10 min recovery.
Additionally, lower Ca 2+ levels were observed in animals anesthetized with 50 µL L −1 EO after 10 min recovery compared to the ethanol group at the same time (Fig. 2c).

DISCUSSION
The main constituents found in the EO obtained from leaves of A. polystachya were the monoterpenes carvone (58.76%) and α-limonene (33.68%).Other studies also demonstrated the presence of carvone (González et al. 2010) and limonene in   Small (2003), where sedation corresponds to Stage 1 and anesthesia corresponds to Stage 3. The equations fitted above represent a relationship between the times of anesthesia and concentrations of EO, where y = time to reach the stage and x = concentration of EO of A. polystachya (in µL L −1 ).this EO (Cabanillas et al. 2003).High contents of α-thujone and β-thujone (which were not found in the present study) were previously detected in this EO (Cabanillas et al. 2003, Duschatzky et al. 2004).

TABLE III Blood parameters of
The present study demonstrated that A. polystachya EO has an anesthetic effect on grouper juveniles.This EO induced sedation at all concentrations tested and anesthetized animals within 3 and 1.5 min (300 and 400 µL L −1 , respectively).Recovery time for both concentrations was about 8 min and 5 min, respectively, and no mortality was observed as a result of anesthesia induction.These findings are in accordance with literature criteria (Marking and Meyer 1985, Gilderhus and Marking 1987, Keene et al. 1998, Park et al. 2009).Parodi et al. (2014) tested the anesthetic effect of the EO obtained from another species of Aloysia, namely A. triphylla, in concentrations ranging between 20 and 800 µL L −1 on two strains (albino and gray) of silver catfish (Rhamdia quelen), and 200 µL L −1 EO was the best concentration to induce anesthesia in the albino strain, while for the gray strain it was 400 µL L −1 EO.An emulsified mixture composed of Mentha spicata EO and methyl salicylate oil (containing 28.4% L-carvone) anesthetized common carp (Cyprinus carpio) within the recommended time at 395 µL L −1 (Roohi and Imanpoor 2014) and Atlantic salmon (Salmo salar) at 257 µL L −1 (Danner et al. 2011), but M. spicata EO alone induced anesthesia in less than 3 min only at 5000 µL L −1 (Roohi and Imanpoor 2015).Interestingly, increased EO concentration promoted recovery time decrease.Other EOs produced higher recovery times as the EO concentration increased (Cunha et al. 2010, Heldwein et al. 2012, Silva et al. 2012, 2013, Parodi et al. 2014).Since EOs are complex mixtures of compounds, a particular biological activity such as induction and recovery times from anesthesia depends on the specific chemical characteristics of each EO, including the qualitative composition and the proportions of each component in the oil (Raut and Karuppayil 2014).
The sedative and anesthetic activity of the EO of A. polystachya can be explained mainly by the combined action of its major components, the monoterpenoid-derived compounds carvone and limonene, which account for 92.44% of its total chemical composition.Carvone has a central nervous system (CNS) depressant effect detected in different pre-clinical studies in mice (Sousa et al. 2007), while limonene acts as an agonist for adenosine A2A receptors, and consequently can induce sedative effects (Park et al. 2011).Limonene also inhibited stimulant-induced behavioral changes in mice and rats, by regulating dopamine levels and 5-HT receptor function (Yun 2014).However, it is likely that a contribution to the activity detected in groupers is made by the minor components, such as limonene epoxide, for which an anxiolytic-like effect was detected in mice.The CNS effects of this compound were reversed by flumazenil, indicating a GABAergic mechanism of action (Almeida et al. 2012).
The blood pH values of the groupers, regardless of exposure time or group, were similar to or slightly lower than those reported for red pacu (Piaractus brachypomus) exposed to MS-222 and eugenol at 50, 100 and 200 mg L −1 (Sladky et al. 2001), yellow perch (Perca flavescens), walleye pike (Sander vitreus) and common carp anesthetized with buffered MS-222 (150 mg L −1 ) (Hanley et al. 2010); silver catfish transported for 4 h in plastic bags with eugenol (1.5 or 3.0 µL L −1 ) and L. alba EO (10 or 20 µL L −1 ) added to the water (Becker et al. 2012).The pH values observed in the blood of groupers were not affected by A. polystachya EO exposure, in accordance with the lack of effect of eugenol and L. alba EO on this parameter in silver catfish (Becker et al. 2012).
The PvO 2 , PvCO 2 and HCO 3 − values were within the range reported by other studies (Sladky et al. 2001, Souza et al. 2001, Hanley et al. 2010, Becker et al. 2012) In the present study, the handling of simulated exposure did not induce a significant increase in blood glucose levels, but groupers anesthetized with 50 µL L −1 A. polystachya EO after 10 min recovery showed increased levels compared to those after exposure.Repetitive blood sampling increased blood glucose levels in rainbow trout (Oncorhynchus mykiss) and anesthesia with clove oil or MS-222 did not change this pattern (Wagner et al. 2003).
Hematocrit values found in this study (26-31%) in fish exposed to control conditions were similar to those reported by Becker et al. (2012) (26-33%) andCarneiro et al. (2009) (27-30%) in silver catfish.The lower hematocrit and hemoglobin values in recovered ethanol and 50 µL L −1 EO (5 min) and 300 µL L −1 EO (10 min) groups compared to exposed fish indicate possible hemodilution or decreased red cell number.Contrary to our results, anesthesia with clove oil or MS-222 reduced hematocrit decrease produced by repetitive sampling in rainbow trout (Wagner et al. 2003).
Ionoregulatory homeostasis is important to ensure proper cell function (Hwang et al. 2011).Moreover, stress due to usual aquaculture procedures is known to affect fish ionoregulation (Ashley et al. 2007).Addition of some sedatives to water can reduce ion loss in fish (Becker et al. 2012).Ionoregulation in groupers was not affected by anesthesia with A. polystachya EO, because the only change was lower Ca 2+ in fish after 10 min recovery from 50 µL L −1 EO exposure compared to those recovered from ethanol exposure.Anesthesia with H. ringens and L. alba EOs (150, 300 or 450 µL L −1 ) and recovery did not considerably affect plasma Na + and Cl − levels either (Toni et al. 2014).Blood Na + also did not change, but K + increased in red pacu anesthetized with MS-222 and eugenol (Sladky et al. 2001).

CONCLUSIONS
The EO from the leaves of A. polystachya was an effective sedative and anesthetic for dusky grouper juveniles in concentrations similar to those found for other EOs in other species.Some of the tested blood parameters showed compensatory responses due to EO exposure, but most changes returned to control values after 10 min of recovery.

Figure 1
Figure 1 -Glucose (a), hemoglobin (b) and hematocrit (c) levels in Epinephelus marginatus after exposure to essential oil (EO) of Aloysia polystachya added to the water (n = 6).Values are represented as mean ± SEM.Different letters indicate significant differences between times in the same treatment (P < 0.05).* indicates significant difference from the control group at the same time and # indicates significant difference from the ethanol group at the same time (P < 0.05).

Figure 2 -
Figure 2 -Plasma Na + (a), K + (b) and Ca 2+ (c) levels in Epinephelus marginatus after exposure to essential oil (EO) of Aloysia polystachya added to the water (n = 6 animals per group).Values are represented as mean ± SEM.Different letters indicate significant differences between times in the same treatment (P < 0.05).* indicates significant difference from the control group at the same time and # indicates significant difference from the ethanol group at the same time (P < 0.05).

TABLE I Chemical composition of the essential oil from dried leaves of Aloysia polystachya.
RT = retention time (in min); RI experimental = calculated Kovats retention index; RI literature = reference Kovats retention index; N = NIST Mass Spectral Library (2008) and A = Adams (2009).

TABLE II Time (in seconds) required for induction and recovery from anesthesia using the essential oil of Aloysia polystachya leaves (EO) in Epinephelus marginatus.
Values are represented as mean ± SEM (n = 6).Different letters indicate significant differences between essential oil concentrations in the recovery stage (P < 0.05).Stages of anesthesia induction are according to . The handling (simulated recovery) of control groupers increased PvO 2 and decreased PvCO 2 and HCO 3 − values, probably due to hyperventilation.Exposure to 50 µL L −1 A. polystachya EO (and 300 µL L −1 compared to control fish) increased PvO 2 , as was also observed by Hanley et al. (2010) in perch, walleye and common carp anesthetized with MS-222.PvCO 2 only increased in groupers exposed to 50 µL L −1 A. polystachya EO, as observed by Sladky et al. (2001) in red pacu exposed to MS-222 and eugenol.However, as in groupers exposed to 50 µL L −1 A. polystachya EO, Hanley et al. (2010) reported that PvCO 2 did not change in perch, walleye or common carp anesthetized with MS-222.