Biochemical parameters of silver catfish (Rhamdia quelen) after transport with eugenol or essential oil of Lippia alba added to the water / Parâmetros bioquímicos do jundiá (Rhamdia quelen) após transporte com eugenol ou óleo essencial de Lippia alba adicionado à água

Abstract The transport of live fish is a routine practice in aquaculture and constitutes a considerable source of stress to the animals. The addition of anesthetic to the water used for fish transport can prevent or mitigate the deleterious effects of transport stress. This study investigated the effects of the addition of eugenol (EUG) (1.5 or 3.0 µL L-1) and essential oil of Lippia alba (EOL) (10 or 20 µL L-1) on metabolic parameters (glycogen, lactate and total protein levels) in liver and muscle, acetylcholinesterase activity (AChE) in muscle and brain, and the levels of protein carbonyl (PC), thiobarbituric acid reactive substances (TBARS) and nonprotein thiol groups (NPSH) and activity of glutathione-S-transferase in the liver of silver catfish (Rhamdia quelen; Quoy and Gaimard, 1824) transported for four hours in plastic bags (loading density of 169.2 g L-1). The addition of various concentrations of EUG (1.5 or 3.0 µL L-1) and EOL (10 or 20 µL L-1) to the transport water is advisable for the transportation of silver catfish, since both concentrations of these substances increased the levels of NPSH antioxidant and decreased the TBARS levels in the liver. In addition, the lower liver levels of glycogen and lactate in these groups and lower AChE activity in the brain (EOL 10 or 20 µL L-1) compared to the control group indicate that the energetic metabolism and neurotransmission were lower after administration of anesthetics, contributing to the maintenance of homeostasis and sedation status.

Resumo O transporte de peixes vivos é uma prática rotineira na aquicultura e constitui uma fonte considerável de estresse para os animais. A adição de anestésicos na água utilizada para o transporte de peixes pode prevenir ou mitigar os efeitos nocivos do estresse de transporte. Este estudo investigou os efeitos da adição de eugenol (EUG) (1,5 ou 3,0 mL L-1) e óleo essencial de Lippia alba (OEL) (10 ou 20 mL L-1) em parâmetros metabólicos (níveis de glicogênio, lactato e proteína total) no fígado e músculos, a atividade da acetilcolinesterase (AChE) no músculo e no cérebro, e os níveis de proteína carbonil (PC), ácido tiobarbitúrico (TBARS) e grupos tióis não proteicos (NPSH) e atividade da glutationa-S-transferase no fígado de jundiás (Rhamdia quelen; Quoy e Gaimard, 1824) transportados por quatro horas em sacos plásticos (densidade de carga de 169,2 g L-1). A adição de várias concentrações de EUG (1,5 ou 3,0 mL L-1) e OEL (10 ou 20 mL L-1) na água de transporte é aconselhável para o transporte do jundiá, uma vez que ambas concentrações destas substâncias aumentaram os níveis do antioxidante NPSH e diminuíram os níveis de TBARS no fígado. Além disso, os níveis mais baixos de glicogénio e lactato no fígado destes grupos e menor atividade de AChE no cérebro (EOL 10 ou 20 mL L-1) em comparação com o grupo controle indicam que o metabolismo energético e neurotransmissão foram menores após a administração dos anestésicos, contribuindo para a manutenção do estado de homeostase e sedação.

Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (Rhamdia quelen): evaluation of acetylcholinesterase and GABAergic activity

This study evaluated the anesthetic potential of thymol and carvacrol, and their influence on acetylcholinesterase (AChE) activity in the muscle and brain of silver catfish (Rhamdia quelen). The AChE activity of S-(+)-linalool was also evaluated. We subsequently assessed the effects of thymol and S-(+)-linalool on the GABAergic system. Fish were exposed to thymol and carvacrol (25, 50, 75, and 100 mg/L) to evaluate time for anesthesia and recovery. Both compounds induced sedation at 25 mg/L and anesthesia with 50-100 mg/L. However, fish exposed to carvacrol presented strong muscle contractions and mortality. AChE activity was increased in the brain of fish at 50 mg/L carvacrol and 100 mg/L thymol, and decreased in the muscle at 100 mg/L carvacrol. S-(+)-linalool did not alter AChE activity. Anesthesia with thymol was reversed by exposure to picrotoxin (GABAA antagonist), similar to the positive control propofol, but was not reversed by flumazenil (antagonist of benzodiazepine binding site), as observed for the positive control diazepam. Picrotoxin did not reverse the effect of S-(+)-linalool. Thymol exposure at 50 mg/L is more suitable than carvacrol for anesthesia in silver catfish, because this concentration did not cause any mortality or interference with AChE activity. Thymol interacted with GABAA receptors, but not with the GABAA/benzodiazepine site. In contrast, S-(+)-linalool did not act in GABAA receptors in silver catfish.

Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.

Effects of anesthesia with the essential oil of Ocimum gratissimum L. in parameters of fish stress / Efeitos da anestesia com o óleo essencial de Ocimum gratissimum L. em parâmetros de estresse de peixes

RESUMO: The effects of anesthesia with the essential oil of Ocimum gratissimum (EOO) in parameters of stress after handling were investigated in silver catfish (Rhamdia quelen). EOO was obtained from the aerial parts by hydrodistillation. Juveniles were anesthetized with 70 or 300 mg L-1 EOO and submitted to air exposure for 1 minute. The fishes were sampled immediately or transferred to anesthetic-free aquaria until sampling. In the first experiment, juveniles had their blood collected at 0, 1, 4, and 8 h after handling to assay plasma cortisol and blood glucose levels. The unanesthetized animals were restrained manually for blood collection. In the second experiment, water samples of the recovery aquaria were collected to evaluate net ion fluxes at 0 - 4 h and 4 - 8 h. Water and ethanol controls were also performed under the same conditions. The results showed that the cortisol levels did not differ among the treatments. Hyperglycemia was verified in fish exposed to 70 and 300 mg L-1 EOO at 1 h and 4 h after handling. After 8 h, cortisol and glucose concentrations were lower or similar than those from immediately after handling for all treatments. EOO anesthesia prevented Na+ efflux observed in the control groups in both flux periods. There were net Cl- and K+ effluxes at 0 - 4 h and influxes at 4 - 8 h after handling in most treatments, and these fluxes did not differ among the treatments. The results suggest that EOO did not impair stress recovery and did not act as an additional handling stressor in silver catfish.

RESUMO: Os efeitos da anestesia com o óleo essencial de Ocimum gratissimum (EOO) em parâmetros de estresse após manuseio foram investigados em jundiás (Rhamdia quelen). EOO foi obtido a partir das partes aéreas por hidrodestilação. Os juvenis foram anestesiados com 70 ou 300 mg L-1 de EOO e expostos ao ar por 1 minuto. Os peixes foram amostrados imediatamente ou transferidos para aquários sem anestésico até amostragem. No primeiro experimento, os juvenis tiveram seu sangue coletado em 0, 1, 4, e 8 h após manuseio para avaliar os níveis de cortisol e glicemia. Os animais não anestesiados foram contidos manualmente para coleta sanguínea. No segundo experimento, amostras de água foram coletadas do aquário de recuperação dos animais para avaliação do fluxo iônico entre 0 - 4 h e 4 - 8 h. Grupos controles em água e etanol também foram realizados sobre as mesmas condições. Os resultados demonstraram que os níveis de cortisol não diferiram entre os tratamentos. Hiperglicemia foi detectada em peixes expostos a 70 e 300 mg L-1 de EOO em 1 h e 4 h após o manuseio. Após 8 h, os teores de cortisol e glicose foram menores ou similares aqueles imediatamente após o manuseio para todos os tratamentos. A anestesia com EOO preveniu o efluxo de Na+ observado para os grupos controle em ambos os períodos avaliados. Ocorreram efluxos de Cl- and K+ entre 0 - 4 h e influxos entre 4 - 8 h após o manuseio para a maioria dos tratamentos, e estes eventos não diferiram entre os tratamentos. Os resultados sugerem que o EOO não prejudica a recuperação do animal frente ao evento estressor ou atua como estressor adicional ao manuseio em jundiás.

Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich.) Briq. and their isolated components in silver catfish (Rhamdia quelen)

This study evaluated the sedative and anesthetic effects of the essential oils (EO) of Hyptis mutabilis (Rich.) Briq. and their isolated components on silver catfish (Rhamdia quelen). Quantitative chemical differences between the EOs obtained from leaves and inflorescences were verified, and a new chemotype rich in globulol was described. Although there were no significant differences in the time of induction for sedation and anesthesia between the EOs, only the leaf EO at 344 mg/L anesthetized all fish without side effects. Fractionation of the leaf EO was carried out by column chromatography. The isolated compounds [(+)-1-terpinen-4-ol and (-)-globulol] showed different activity from that detected for the leaf EO in proportional concentrations and similar sedation to a eugenol control at 10 mg/L. However, fish exposed to 1-terpinen-4-ol (3 and 10 mg/L) did not remain sedated for 30 min. Anesthesia was obtained with 83-190 mg/L globulol, but animals showed loss of mucus during induction and mortality at these concentrations. Synergism of the depressor effects was detected with the association of globulol and benzodiazepine (BDZ), compared with either drug alone. Fish exposed to BDZ or globulol+BDZ association showed faster recovery from anesthesia in water containing flumazenil, but the same did not occur with globulol. In conclusion, the use of globulol in aquaculture procedures should be considered only at sedative concentrations of 10 and 20 mg/L, and its mechanism of action seems not to involve the GABAA-BDZ system.

Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N.E. Brown essential oil

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABA A receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABA A receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish.

Ion fluxes and hematological parameters of two teleosts from the Rio Negro, Amazon, exposed to hypoxia / Fluxos iônicos e parâmetros hematológicos em duas espécies de teleósteos do Rio Negro, Amazônia, expostos à hipoxia

The aim of this study was to describe the effect of hypoxia on whole body ion fluxes and hematological parameters in two Amazonian teleosts: Serrasalmus eigenmanni and Metynnis hypsauchen. The increase of Na+ and Cl- effluxes on M. hypsauchen exposed to hypoxia may be related to an increase of gill ventilation and effective respiratory surface area, to avoid a reduction in the oxygen uptake, and/or with the decrease of pHe, that could inhibit Na+ and Cl- transporters and, therefore, reduce influx of these ions. Effluxes of Na+ and Cl- were lower in hypoxia than in normoxia for S. eigenmanni, possibly because in hypoxia this species would reduce gill ventilation and oxygen uptake, which would lead to a decrease of gill ion efflux and, consequently, reducing ion loss. The increase on hematocrit (Ht) during hypoxia in M. hypsauchen probably was caused by an increase of the red blood cell volume (MCV). For S. eigenmanni the increase on glucose possibly results from the usage of glucose reserve mobilization. Metynnis hypsauchen showed to be more sensitive to hypoxia than Serrasalmus eigenmanni, since the first presented more significant alterations on these osmoregulatory and hematological parameters. Nevertheless, the alterations observed for both species are strategies adopted by fishes to preserve oxygen supply to metabolizing tissues during exposure to hypoxia.

O objetivo deste trabalho foi descrever o efeito da hipoxia no fluxo iônico corporal e nos parâmetros hematológicos em duas espécies de teleósteos da Amazônia: Serrasalmus eigenmanni e Metynnis hypsauchen. O aumento dos efluxos de Na+ e Cl- em M. hypsauchen expostos à hipoxia pode estar relacionado ao aumento da ventilação branquial e da eficiência da área da superfície respiratória, a fim de evitar redução na captação de oxigênio; e/ou com a diminuição do pHe, que pode inibir os transportadores de Na+ e Cl- e, então, reduzir o influxo destes íons. Os efluxos de Na+ e Cl- foram menores em hipoxia do que em normoxia para a espécie S. eigenmanni, possivelmente porque esta espécie em hipoxia poderia reduzir a ventilação branquial e a captação de oxigênio, a qual levaria a uma diminuição do efluxo branquial de íons e, conseqüentemente, à redução da perda de íons. O aumento do hematócrito (Ht) durante hipoxia em M. hypsauchen provavelmente foi causado pelo aumento do volume das células vermelhas do sangue (MCV). Para a espécie S. eigenmanni, o aumento da glicose possivelmente foi resultado do uso da mobilização da reserva de glicose. A espécie Metynnis hypsauchen mostrou ser mais sensível à hipoxia do que a espécie Serrasalmus eigenmanni, uma vez que a primeira espécie apresentou mais alterações significativas em seus parâmetros osmorregulatórios e hematológicos. Contudo, as alterações observadas em ambas as espécies são estratégias adotadas pelos peixes a fim de preservar o suprimento de oxigênio para metabolização nos tecidos durante exposição à hipoxia.

Ion fluxes and hematological parameters of two teleosts from the Rio Negro, Amazon, exposed to hypoxia

The aim of this study was to describe the effect of hypoxia on whole body ion fluxes and hematological parameters in two Amazonian teleosts: Serrasalmus eigenmanni and Metynnis hypsauchen. The increase of Na+ and Cl- effluxes on M. hypsauchen exposed to hypoxia may be related to an increase of gill ventilation and effective respiratory surface area, to avoid a reduction in the oxygen uptake, and/or with the decrease of pHe, that could inhibit Na+ and Cl- transporters and, therefore, reduce influx of these ions. Effluxes of Na+ and Cl- were lower in hypoxia than in normoxia for S. eigenmanni, possibly because in hypoxia this species would reduce gill ventilation and oxygen uptake, which would lead to a decrease of gill ion efflux and, consequently, reducing ion loss. The increase on hematocrit (Ht) during hypoxia in M. hypsauchen probably was caused by an increase of the red blood cell volume (MCV). For S. eigenmanni the increase on glucose possibly results from the usage of glucose reserve mobilization. Metynnis hypsauchen showed to be more sensitive to hypoxia than Serrasalmus eigenmanni, since the first presented more significant alterations on these osmoregulatory and hematological parameters. Nevertheless, the alterations observed for both species are strategies adopted by fishes to preserve oxygen supply to metabolizing tissues during exposure to hypoxia.

O objetivo deste trabalho foi descrever o efeito da hipoxia no fluxo iônico corporal e nos parâmetros hematológicos em duas espécies de teleósteos da Amazônia: Serrasalmus eigenmanni e Metynnis hypsauchen. O aumento dos efluxos de Na+ e Cl- em M. hypsauchen expostos à hipoxia pode estar relacionado ao aumento da ventilação branquial e da eficiência da área da superfície respiratória, a fim de evitar redução na captação de oxigênio; e/ou com a diminuição do pHe, que pode inibir os transportadores de Na+ e Cl- e, então, reduzir o influxo destes íons. Os efluxos de Na+ e Cl- foram menores em hipoxia do que em normoxia para a espécie S. eigenmanni, possivelmente porque esta espécie em hipoxia poderia reduzir a ventilação branquial e a captação de oxigênio, a qual levaria a uma diminuição do efluxo branquial de íons e, conseqüentemente, à redução da perda de íons. O aumento do hematócrito (Ht) durante hipoxia em M. hypsauchen provavelmente foi causado pelo aumento do volume das células vermelhas do sangue (MCV). Para a espécie S. eigenmanni, o aumento da glicose possivelmente foi resultado do uso da mobilização da reserva de glicose. A espécie Metynnis hypsauchen mostrou ser mais sensível à hipoxia do que a espécie Serrasalmus eigenmanni, uma vez que a primeira espécie apresentou mais alterações significativas em seus parâmetros osmorregulatórios e hematológicos. Contudo, as alterações observadas em ambas as espécies são estratégias adotadas pelos peixes a fim de preservar o suprimento de oxigênio para metabolização nos tecidos durante exposição à hipoxia.

Ion fluxes of Metynnis hypsauchen, a teleost from the Rio Negro, Amazon, exposed to an increase of temperature

The aim of this study was to investigate the effect on an increase of temperature on the net ion fluxes on Metynnis hypsauchen, a teleost species from the Rio Negro. Fish were collected in the Anavilhanas archipelago, Rio Negro, Amazon. After 24 h adaptation fish were placed in individual chambers served with a steady flow of recirculated water. Na+ and Cl- fluxes were determined at 26 and 33ºC. After 18 h in the chambers, fish presented an influx of Na+ and Cl-, and the temperature raise to 33ºC led to an efflux of both ions, which remained even after 6 h in this temperature. Six hours were not enough to promote a significant reduction of net ion effluxes, but certainly the fluxes would be in net balance after a longer period of time, since this species can be exposed to this temperature in its natural environment

Effect of Plantago australis leaves on different gastric ulcer models

The anti-ulcerogenic effect of the crude ethanolic extract (CEE) of Plantago australis leaves was tested against ethanol-, indomethacin-, and cold restrain-induced stress ulcers. The CEE (500 and 1000 mg/kg) reduced the lesion index (LI) and the ulcer index in ethanol-induced ulcers, and the dose of 1000 mg/kg increased the amount of mucous. The highest dose of the CEE reduced the LI of cold restraint-induced stress ulcers when compared to the control group. The indomethacin-induced ulcers were not affected by this extract.

Ion fluxes of Metynnis hypsauchen, a teleost from the Rio Negro, Amazon, exposed to an increase of temperature

The aim of this study was to investigate the effect on an increase of temperature on the net ion fluxes on Metynnis hypsauchen, a teleost species from the Rio Negro. Fish were collected in the Anavilhanas archipelago, Rio Negro, Amazon. After 24 h adaptation fish were placed in individual chambers served with a steady flow of recirculated water. Na+ and Cl- fluxes were determined at 26 and 33ºC. After 18 h in the chambers, fish presented an influx of Na+ and Cl-, and the temperature raise to 33ºC led to an efflux of both ions, which remained even after 6 h in this temperature. Six hours were not enough to promote a significant reduction of net ion effluxes, but certainly the fluxes would be in net balance after a longer period of time, since this species can be exposed to this temperature in its natural environment.

O objetivo deste trabalho foi verificar o efeito do aumento de temperatura nos fluxos iônicos de Metynnis hypsauchen, uma espécie de teleósteo do Rio Negro, Amazonas. Os peixes foram coletados no arquipélago de Anavilhanas, Rio Negro, Amazonas. Após adaptação de 24 h, os peixes foram colocados em câmaras individuais com fluxo constante de água recirculada. Os fluxos de Na+ e Cl- foram determinados a 26 e 33ºC. Após 18 h nas câmaras, os peixes apresentaram influxo de Na+ e Cl-, e o aumento da temperatura para 33ºC provocou efluxo de ambos os íons, o qual se manteve mesmo depois de 6 h nessa temperatura. Seis horas não foram suficientes para promover redução significativa dos efluxos iônicos, mas certamente esses fluxos atingiriam equilíbrio após um período mais prolongado, uma vez que essa espécie é regularmente exposta a essa temperatura em seu ambiente natural.

Changes in the electrophysiological parameters of the posterior intestine of Anguilla anguilla (Pisces) induced by oxytocin, urotensin II and aldosterone

In view of the importance of the intestine in the osmoregulation of freshwater fishes, we determined the effects of oxytocin, urotensin II (UII), and aldosterone added to the serosal side of the isolated posterior intestine of the freshwater-adapted teleost Anguilla anguilla on electrophysiological parameters. Oxytocin decreased the short-ciruit current (SCC) and transepithelial potential difference (TPD) at a centrations of 1 and 10 mU/ml (to 50 per cent and 42 per cent of control values, respectively), but did not alter these parameters at a concentration of 0.1 mU/ml. UII reduced SCC and TPD at concentrations of 10 nM, 50nM and 100 nM (to 85 per cent of control values), but increased these parameters at the concentration of 500 nM (to 115 per cent of control values). Aldosterone did not alter SCC or TPD at the concentrations tested (10 nM and 100 nM). Oxytocin may open Na+ channels in the apical membrane, allowing the flow of Na+ to the serosa, reduced SCC and TPD. Should this hypothesis be correct, oxytocin would be important for freshwater adaptation, since it would increase absorption. The reduction of SCC and TPD in the posterior intestine A. anguilla induced by UII is evidence that this neurohormone is also important for freshwater adaptation in teleosts. Aldosterone did not show this effect probaly due to the lack of receptors in this organ.

Effect of HCO3 and pH on ion transport in the posterior intestine of the freshwater-adapted teleost Anguilla anguilla

The intestinal mucosa may be exposed to acidic or alcaline solutions due to liberation of digestive secretions. In several situations blood pH may also change. Consequently, the effects of HCO-3, CO2, and pH variation of medium on the ion transport acrross the posterior intestine of the eel (Anguilla anguilla) adapted to freshwater were studied in terms of fractional values of short-circuit current (SCC), transepithelial potential difference (TPD) and conductance (G). Immature eels weighing 100-200 g were used. The control physiological solution contained: 118.5 mM NaCl, 25.0 mM NaHCO3, 3.0 mM CaCl2.2H2O, 4.7 mM KCl, 1.0 mM MgCl2.6H2O, 5.0 mMD-glucose, 10.0 mM D-mannitol, pH 7.80, and was gassed with 98 percent O2-2 percent CO2. Control values(N = 21) were: SCC = 51.90 + or - 51.90 + or - 2.8 µA.cm-2, TPD = 2.33 + or - 0.1 mV, G = 22.43 + or - 0.6 mS.cm-2. At constant pH, the reduction of HCO-3 concentration to 50 percent and 10 percent did not alter the values of SCC and TPD, but G increased with HCO-3 reduction to 10 percent. In the absence of HCO-3, SCC, TPD and G (slightly) decreased, but 1.5 mM HCO-3 still maintained the ion transport within control values, at constant pH. Comparing PH values from 6.65 to 8.61, higher values of SCC and TPD were observed at PH 7.45, but were little affected below and above this pH. There was a significant correlation between pH and SCC and TPD values; from the regression equations (1) SCC was zero at pHs below 6.62 and above 8.78 and (2) TPD was zero below 6.50 and above 8.71