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

CARINE O. FOGLIARINI QUELEN I. GARLET THAYLISE V. PARODI ALEXSSANDRO G. BECKER LUCIANO O. GARCIA BERTA M. HEINZMANN ANA MARIA S. PEREIRA BERNARDO BALDISSEROTTO About the authors

ABSTRACT

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−1 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−1. However, most evaluated blood parameters showed compensatory responses due to EO exposure.

Key words:
anesthetic efficacy; blood gases; glucose; hemoglobin; plasma ion levels

INTRODUCTION

Several procedures of fish culture such as handling, blood sampling, transporting and vaccination often generate a stress response in the animals (Kiessling et al. 2009KIESSLING A, JOHANSSON D, ZAH LIH and SAMUELSEN OB. 2009. Pharmacokinetics, plasma cortisol and effectiveness of benzocaine, MS-222 and isoeugenol measured in individual dorsal aorta-cannulated Atlantic salmon (Salmo salar) following bath administration. Aquaculture 86: 301-308. , Zahl et al. 2012ZAHL IH, SAMUELSEN O and KIESSLING A. 2012. Anaesthesia of farmed fish: implications for welfare. Fish Physiol Biochem 38: 201-218. ). Firstly, activation of the hypothalamic-pituitary-interrenal axis occurs, with subsequent release of catecholamines and cortisol. As a consequence, glucose and lactate increase and osmoregulatory disturbances occur (Zahl et al. 2012). Anesthetics obtained from plants, such as the essential oil (EO) of Lippia alba (Cunha et al. 2010CUNHA MA, BARROS FMC, GARCIA LO, VEECK APL, HEINZMANN BM, LORO VL, EMANUELLI T and BALDISSEROTTO B. 2010. Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen. Aquaculture 306: 403-406. , Azambuja et al. 2011AZAMBUJA CR, MATTIAZZI J, RIFFEL APK, FINAMOR IA, GARCIA LO, HELDWEIN CG, HEINZMANN BM, BALDISSEROTTO B, PAVANATO MA and LLESUY SF. 2011. Effect of the essential oil of Lippia alba on oxidative stress parameters in silver catfish (Rhamdia quelen) subjected to transport. Aquaculture 319: 156-161. , Becker et al. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM and BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796. , Heldwein et al. 2012HELDWEIN CG, SILVA LL, RECKZIEGEL P, BARROS FM, BALDISSEROTTO B, MALLMANN CA, SCHMIDT D, CARON BO and HEINZMANN BM. 2012. Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill) N E Brown essential oil. Braz J Med Biol Res 45: 436-443., Salbego et al. 2014SALBEGO J ET AL. 2014. The essential oil from Lippia alba induces biochemical stress in the silver catfish (Rhamdia quelen) after transportation. Neotrop Ichthyol 12: 811-818.), Ocimum gratissimum (Silva et al. 2012SILVA LL, PARODI TV, RECKZIEGEL P, GARCIA VO, BURGER ME, BALDISSEROTTO B, MALLMANN CA, PEREIRA AMS and HEINZMANN BM. 2012. Essential oil of Ocimum gratissimum L. anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen. Aquaculture 350: 91-97. ), Hesperozygis ringens (Silva et al. 2013SILVA LL, SILVA DT, GARLET QI, CUNHA MA, MALLMANN CA, BALDISSEROTTO B, LONGHI SJ, PEREIRA AMS and HEINZMANN BM. 2013. Anesthetic activity of Brazilian native plants in silver catfish (Rhamdia quelen). Neotrop Ichthyol 11: 443-451. , Toni et al. 2014TONI C, BECKER AG, SIMÕES LN, PINHEIRO CG, SILVA LL, HEINZMANN BM, CARON BO and BALDISSEROTTO B. 2014. Fish anesthesia: effects of the essential oils of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiol Biochem 40: 701-714. ) and Aloysia triphylla (Gressler et al. 2014GRESSLER LT ET AL. 2014. Silver catfish Rhamdia quelen immersion anesthesia with essential oil of Aloysia triphylla (L'Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status. Aquac Res 45: 1061-1072. , Parodi et al. 2014PARODI TV, CUNHA MA, BECKER AG, ZEPPENFELD CC, MARTINS DI, KOAKOSKI G, BARCELLOS LJG, HEINZMANN BM and BALDISSEROTTO B. 2014. 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. Fish Physiol Biochem 40: 323-334. , Zeppenfeld et al. 2014ZEPPENFELD CC ET AL. 2014. Physiological and biochemical responses of silver catfish, Rhamdia quelen, after transport in water with essential oil of Aloysia triphylla (L'Herit) Britton. Aquaculture 418-419: 101-107.) showed efficacy and safety for use in aquaculture procedures.

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 1997DEL VITTO LA and PETENATTI EM. 1997. Recursos herbolarios de San Luis (República Argentina) Primeira parte: plantas nativas. Multequina 6: 49-66. ) and is also used against gastrointestinal pain in folk medicine (Filipoy 1994FILIPOY A. 1994. Medicinal plants of the Pilaga of Central Chaco. J Ethnopharmacol 44: 181-193. ). 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. 2005MORA S, DÍAZ-VÉLIZ G, MILLÁN R, LUNGENSTRASS H, QUIRÓS S, COTO-MORALES T and HELLIÓN-IBARROLA MC. 2005. Anxiolytic and antidepressant-like effects of the hydroalcoholic extract from Aloysia polystachya in rats. Pharmacol Biochem Behav 82: 373-378. , 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 2006FENNESSY YST. 2006. Reproductive biology and growth of the yellowbelly rockcod Epinephelus marginatus (Serranidae) from South-East Africa. Afr J Mar Sci 28: 1-11. ). 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 and Menezes 1980FIGUEIREDO JL and MENEZES NA. 1980. Manual de peixes marinhos do sudeste do Brasil III Teleostei (2) São Paulo, Museu de Zoologia, Universidade de São Paulo, 90 p., Irigoyen et al. 2005IRIGOYEN AJ, GALVAN DE and VENERUS LA. 2005. Occurrence of dusky grouper Epinephelus marginatus (Lowe, 1834) in gulfs of northern Patagonia, Argentina. J Fish Biol 67: 1741-1745. ). Since E. marginatus is a target species for aquaculture (Cunha et al. 2013CUNHA ME, RÉ P, QUENTAL-FERREIRA H, GAVAIA PJ and POUSÃO-FERREIRA P. 2013. Larval and juvenile development of dusky grouper Epinephelus marginatus reared in mesocosms. J Fish Biol 83: 448-465., Cavalli 2014CAVALLI RO. 2014. Maricultura. In: Castello JP. Introdução às ciências do mar, Pelotas: Editora Textos, p. 418-455., Sanches et al. 2014SANCHES ES, SILVA FC, LEITE JR, SILVA PKA, KERBER CE and SANTOS PA. 2014. A incorporação de óleo de peixe na dieta pode melhorar o desempenho da garoupa-verdadeira Epinephelus marginatus? Bol Inst Pesca 40: 147-155.), 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.

MATERIALS AND METHODS

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)EUROPEAN PHARMACOPOEIA. 2007. European directorate for the quality of medicines, 6th ed., Strassbourg..

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 non-polar 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 2009ADAMS RP. 2009. Identification of essential oil components by gas chromatography/mass spectrometry, 4th ed., Illinois: Allured Publishing Corporation, 804 p.). 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 (1952BENDSCHNEIDER K and ROBINSON RJ. 1952. A new spectrophometric method for the determination of nitrite in sea water. J Mar Res 11: 87-96. ) and alkalinity (149.75 ± 0.67 mg CaCO3 L−1) by the method of Baumgarten et al. (1996BAUMGARTEN MGZ, ROCHA JMB and NIENCHESKY LF. 1996. Manual de análises em oceanografia química, Rio Grande: FURG, 172 p. ). 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. (2014PARODI TV, CUNHA MA, BECKER AG, ZEPPENFELD CC, MARTINS DI, KOAKOSKI G, BARCELLOS LJG, HEINZMANN BM and BALDISSEROTTO B. 2014. 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. Fish Physiol Biochem 40: 323-334. ) 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 (2003SMALL BC. 2003. Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus. Aquaculture 218: 177-185. ): 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 (Ca2+), bicarbonate (HCO3 −), pH, gases (PvO2, PvCO2), 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 fish species (Cooke et al. 2008COOKE SJ ET AL. 2008. Effects of different capture techniques on the physiological condition of bonefish Albula vulpes evaluated using field diagnostic tools. J Fish Biol 73: 1351-1375., Kristensen et al. 2010KRISTENSEN T, ROSSELAND BO KIESSLING A, DJORDEVIC B and MASSABAU JC. 2010. Lack of arterial PO2 downregulation in Atlantic salmon (Salmo salar L.) during long-term normoxia and hyperoxia. Fish Physiol Biochem 36: 1087-1095. , Paust et al. 2011PAUST LO, FOSS A and IMSLAND AK. 2011. Effects of chronic and periodic exposure to ammonia on growth, food conversion efficiency and blood physiology in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 315: 400-406. ).

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.

RESULTS

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%).

TABLE I
Chemical composition of the essential oil from dried leaves of Aloysia polystachya.

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.

TABLE II
Time (in seconds) required for induction and recovery from anesthesia using the essential oil of Aloysia polystachy a leaves (EO) in Epinephelus marginatus.

BLOOD PARAMETERS

Blood pH was not significantly affected by treatments. The PvO2 and PvCO2 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 PvO2 values than control fish. The PvO2 values were also higher in fish exposed to 50 µL L−1 EO than in those exposed to ethanol. The PvO2 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 PvCO2 values than the control group. In addition, fish recovered for 5 min presented significantly higher PvCO2 values than the control and ethanol groups, but after 10 min recovery these values were significantly lower than in the ethanol group. The HCO3 concentration decreased in all groups at both recovery times when compared to exposure, but was not affected by treatments (Table III).

TABLE III
Blood parameters of Epinephelus marginatus evaluated after exposure (3.5 min) and recovery times from anesthesia induction by essential oil of Aloysia polystachya.

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).

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).

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 Ca2+ levels in the control group were observed after 10 min recovery. Additionally, lower Ca2+ 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).

Figure 2
Plasma Na+ (a), K+ (b) and Ca2+ (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).

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. 2010GONZÁLEZ JOW, GUTIERREZ MM, MURRAY AP and FERRERO AA. 2010. Biological activity of essential oils from Aloysia polystachya and Aloysia citriodora (Verbenaceae) against the soybean pest Nezara viridula (Hemiptera: Pentatomidae). Nat Prod Commun 5: 301-306. ) and limonene in this EO (Cabanillas et al. 2003CABANILLAS CM, LOPEZ ML, DANIELE G and ZYGADLO JA. 2003. Essential oil composition of Aloysia polystachya (Griseb) Moldenke under rust disease. Flavour Frag J 18: 446-448.). 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. 2004DUSCHATZKY CB, MARTINEZ AN, ALMEIDA NV and BONIVARDO SL. 2004. Nematicidal activity of the essential oils of several Argentina plants against the root-knot nematode. J Essent Oil Res 16: 626-628. ).

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 1985MARKING LL and MEYER FP. 1985. Are better fish anesthetics needed in fisheries? Fisheries 10: 2-5., Gilderhus and Marking 1987GILDERHUS PA and MARKING LL. 1987. Comparative efficacy of 16 anesthetic chemicals on rainbow trout. N Am J Fish Manage 7: 288-292. , Keene et al. 1998KEENE JL, NOAKES DLG, MOCCIA RD and SOTO CG. 1998. The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum). Aquac Res 29: 89-101. , Park et al. 2009PARK IS, PARK MO, HUR JW, KIM DS, CHANG YJ, KIM YJ, PARK JY and JOHNSON SC. 2009. Anesthetic effects of lidocaine-hydrochloride on water parameters in simulated transport experiment of juvenile winter flounder, Pleuronectes americanus. Aquaculture 294: 76-79. ). Parodi et al. (2014PARODI TV, CUNHA MA, BECKER AG, ZEPPENFELD CC, MARTINS DI, KOAKOSKI G, BARCELLOS LJG, HEINZMANN BM and BALDISSEROTTO B. 2014. 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. Fish Physiol Biochem 40: 323-334. ) 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 2014ROOHI Z and IMANPOOR MR. 2014. Effects of spearmint (/-carvon) oil and methyl salicylate oil emulsion on anesthesia of common carp (Cyprinus carpio L., 1758). Aquac Res Dev 5: 1-5. ) and Atlantic salmon (Salmo salar) at 257 µL L−1 (Danner et al. 2011DANNER GR, MUTO KW, ZIEBA AM, STILLMAN CM, SEGGIO JA and AHMAD ST. 2011. Spearmint (l-carvone) oil and wintergreen (methyl salicylate) oil emulsion is an effective immersion anesthetic of fishes. J Fish Wildl Manage 2: 146-155.), but M. spicata EO alone induced anesthesia in less than 3 min only at 5000 µL L−1 (Roohi and Imanpoor 2015ROOHI Z and IMANPOOR MR . 2015. The efficacy of the oils of spearmint and methyl salicylate as new anesthetics and their effect on glucose levels in common carp (Cyprinus carpio L., 1758) juveniles. Aquaculture 437: 327-332. ). Interestingly, increased EO concentration promoted recovery time decrease. Other EOs produced higher recovery times as the EO concentration increased (Cunha et al. 2010CUNHA MA, BARROS FMC, GARCIA LO, VEECK APL, HEINZMANN BM, LORO VL, EMANUELLI T and BALDISSEROTTO B. 2010. Essential oil of Lippia alba: a new anesthetic for silver catfish, Rhamdia quelen. Aquaculture 306: 403-406. , Heldwein et al. 2012HELDWEIN CG, SILVA LL, RECKZIEGEL P, BARROS FM, BALDISSEROTTO B, MALLMANN CA, SCHMIDT D, CARON BO and HEINZMANN BM. 2012. Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill) N E Brown essential oil. Braz J Med Biol Res 45: 436-443., Silva et al. 2012SILVA LL, PARODI TV, RECKZIEGEL P, GARCIA VO, BURGER ME, BALDISSEROTTO B, MALLMANN CA, PEREIRA AMS and HEINZMANN BM. 2012. Essential oil of Ocimum gratissimum L. anesthetic effects, mechanism of action and tolerance in silver catfish, Rhamdia quelen. Aquaculture 350: 91-97. , 2013SILVA LL, SILVA DT, GARLET QI, CUNHA MA, MALLMANN CA, BALDISSEROTTO B, LONGHI SJ, PEREIRA AMS and HEINZMANN BM. 2013. Anesthetic activity of Brazilian native plants in silver catfish (Rhamdia quelen). Neotrop Ichthyol 11: 443-451. , Parodi et al. 2014PARODI TV, CUNHA MA, BECKER AG, ZEPPENFELD CC, MARTINS DI, KOAKOSKI G, BARCELLOS LJG, HEINZMANN BM and BALDISSEROTTO B. 2014. 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. Fish Physiol Biochem 40: 323-334. ). 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 2014RAUT JS and KARUPPAYIL SM. 2014. A status review on the medicinal properties of essential oils. Ind Crop Prod 62: 250-264. ).

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. 2007SOUSA DP, NÓBREGA FFF and ALMEIDA RN. 2007. Influence of the chirality of (R)-(-)- and (S)-(+)-carvone in the central nervous system: a comparative study. Chirality 19: 264-268. ), while limonene acts as an agonist for adenosine A2A receptors, and consequently can induce sedative effects (Park et al. 2011PARK HM, LEE JH, YAOYAO J, JUN HJ and LEE SJ. 2011. Limonene, a natural cyclic terpene, is an agonistic ligand for adenosine A2A receptors. Biochem Biophys Res Commun 404: 345-348. ). Limonene also inhibited stimulant-induced behavioral changes in mice and rats, by regulating dopamine levels and 5-HT receptor function (Yun 2014 YUN J. 2014. Limonene inhibits methamphetamine-induced locomotor activity via regulation of 5-HT neuronal function and dopamine release. Phytomedicine 21: 883-887. ). 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. 2012ALMEIDA AAC, COSTA JP, CARVALHO RBF, SOUSA DP and FREITAS RM. 2012. Evaluation of acute toxicity of a natural compound (+)-limonene epoxide and its anxiolytic-like action. Brain Res 1448: 56-62. ).

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. 2001SLADKY KK, SWANSON CR, STOSKOPF MK, LOOMIS MR and LEWBART GA. 2001. Comparative efficacy of tricaine methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus brachypomus). Am J Vet Res 62: 337-342.), yellow perch (Perca flavescens), walleye pike (Sander vitreus) and common carp anesthetized with buffered MS-222 (150 mg L−1) (Hanley et al. 2010HANLEY CS, CLYDE VL, WALLACE RS, PAUL-MURPHY J, PATTERSON TA, KEULER NS and SLADKY KK. 2010. Effects of anesthesia and surgery on serial blood gas values and lactate concentrations in yellow perch (Perca flavescens), walleye pike (Sander vitreus), and koi (Cyprinus carpio). JAVMA - J Am Vet Med A 236: 1104-1108. ); 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. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM and BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796. ). 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. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM and BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796. ).

The PvO2, PvCO2 and HCO3 values were within the range reported by other studies (Sladky et al. 2001SLADKY KK, SWANSON CR, STOSKOPF MK, LOOMIS MR and LEWBART GA. 2001. Comparative efficacy of tricaine methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus brachypomus). Am J Vet Res 62: 337-342., Souza et al. 2001SOUZA RHD, SONCINI R, GLASS ML, SANCHES JR and RANTIN FT. 2001. Ventilation, gill perfusion and blood gases in dourado, Salminus maxillosus Valenciennes (Teleostei, Characidae), exposed to graded hypoxia. J Comp Physiol B 171: 483-489. , Hanley et al. 2010HANLEY CS, CLYDE VL, WALLACE RS, PAUL-MURPHY J, PATTERSON TA, KEULER NS and SLADKY KK. 2010. Effects of anesthesia and surgery on serial blood gas values and lactate concentrations in yellow perch (Perca flavescens), walleye pike (Sander vitreus), and koi (Cyprinus carpio). JAVMA - J Am Vet Med A 236: 1104-1108. , Becker et al. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM and BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796. ). The handling (simulated recovery) of control groupers increased PvO2 and decreased PvCO2 and HCO3 values, probably due to hyperventilation. Exposure to 50 µL L−1 A. polystachya EO (and 300 µL L−1 compared to control fish) increased PvO2, as was also observed by Hanley et al. (2010)HANLEY CS, CLYDE VL, WALLACE RS, PAUL-MURPHY J, PATTERSON TA, KEULER NS and SLADKY KK. 2010. Effects of anesthesia and surgery on serial blood gas values and lactate concentrations in yellow perch (Perca flavescens), walleye pike (Sander vitreus), and koi (Cyprinus carpio). JAVMA - J Am Vet Med A 236: 1104-1108. in perch, walleye and common carp anesthetized with MS-222. PvCO2 only increased in groupers exposed to 50 µL L−1 A. polystachya EO, as observed by Sladky et al. (2001)SLADKY KK, SWANSON CR, STOSKOPF MK, LOOMIS MR and LEWBART GA. 2001. Comparative efficacy of tricaine methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus brachypomus). Am J Vet Res 62: 337-342. 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)HANLEY CS, CLYDE VL, WALLACE RS, PAUL-MURPHY J, PATTERSON TA, KEULER NS and SLADKY KK. 2010. Effects of anesthesia and surgery on serial blood gas values and lactate concentrations in yellow perch (Perca flavescens), walleye pike (Sander vitreus), and koi (Cyprinus carpio). JAVMA - J Am Vet Med A 236: 1104-1108. reported that PvCO2 did not change in perch, walleye or common carp anesthetized with MS-222.

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. 2003WAGNER GN, SINGER TD and MCKINLEY RS. 2003. The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss W). Aquac Res 34: 1139-1146.).

Hematocrit values found in this study (26-31%) in fish exposed to control conditions were similar to those reported by Becker et al. (2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM and BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796. ) (26-33%) and Carneiro et al. (2009CARNEIRO PCF, KAISELER PHS, SWAROFSKY EAC and BALDISSEROTTO B. 2009. Transport of jundiá Rhamdia quelen juveniles at different loading densities: water quality and blood parameters. Neotrop Ichthyol 7: 283-288. ) (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. 2003WAGNER GN, SINGER TD and MCKINLEY RS. 2003. The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss W). Aquac Res 34: 1139-1146.).

Ionoregulatory homeostasis is important to ensure proper cell function (Hwang et al. 2011HWANG PP, LEE TH and LIN LY. 2011. Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. Am J Physiol-Reg I 301: R28-R47.). Moreover, stress due to usual aquaculture procedures is known to affect fish ionoregulation (Ashley et al. 2007ASHLEY PJ. 2007. Fish welfare: current issues in aquaculture. App Anim Behav Sci 104: 199-235. ). Addition of some sedatives to water can reduce ion loss in fish (Becker et al. 2012BECKER AG, PARODI TV, HELDWEIN CG, ZEPPENFELD CC, HEINZMANN BM and BALDISSEROTTO B. 2012. Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba. Fish Physiol Biochem 38: 789-796. ). Ionoregulation in groupers was not affected by anesthesia with A. polystachya EO, because the only change was lower Ca2+ 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. 2014TONI C, BECKER AG, SIMÕES LN, PINHEIRO CG, SILVA LL, HEINZMANN BM, CARON BO and BALDISSEROTTO B. 2014. Fish anesthesia: effects of the essential oils of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiol Biochem 40: 701-714. ). Blood Na+ also did not change, but K+ increased in red pacu anesthetized with MS-222 and eugenol (Sladky et al. 2001SLADKY KK, SWANSON CR, STOSKOPF MK, LOOMIS MR and LEWBART GA. 2001. Comparative efficacy of tricaine methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus brachypomus). Am J Vet Res 62: 337-342.).

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.

ACKNOWLEDGMENTS

This study was supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/Programa de Apoio a Núcleos de Excelência (FAPERGS/PRONEX, process 10/0016-8), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 470964/2009-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES) for research fellowships and financial support.

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    Contribution to the centenary of the Brazilian Academy of Sciences

Publication Dates

  • Publication in this collection
    16 Apr 2017
  • Date of issue
    2017

History

  • Received
    21 July 2016
  • Accepted
    01 Sept 2016
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