Anesthetic induction and recovery of Hippocampus reidi exposed to the essential oil of Lippia alba

The aim of this study was to identify the times of anesthetic induction and recovery in slender seahorses (Hippocampus reidi) that were exposed to the essential oil of Lippia alba (EO), as well as the efficacy of EO as a stress-reducing agent in the transport of this species. Slender seahorses were placed in 1-L aquaria containing different concentrations of EO (0, 10, 20, 50, 150, 300 and 450 μL L), and after induction, fish were transferred to aquaria that were free of anesthetic to evaluate their recovery time. In an additional experiment, slender seahorses were transported in plastic bags with 15 μL L of EO for 4 or 24 h. The increased concentration of EO proportionally decreased the time required for the induction of anesthesia. EO treatment (15 μL L) inhibited the increase in blood glucose levels that was provoked by transportation for 4 or 24 h. Transportation for 24 h also decreased the number of lymphocytes and increased the neutrophil count, and these effects were avoided with the addition of EO to the water. These results demonstrate that EO was effective as an anesthetic at concentrations of 10-20 μL L for slight sedation and transport and at 150 μL L for deep anesthesia in the slender seahorse.


Introduction
The slender seahorse, Hippocampus reidi, is one of the most exported Brazilian marine ornamental fish species (Monteiro-Neto et al., 2003).This species is also collected for folk medicine, souvenirs and religious purposes (Rosa et al., 2002(Rosa et al., , 2005)).The seahorses produced in Brazil are usually exported to the United States.Mortality generally occurs either during transport or within weeks of arrival at the destination as a delayed physiological stress response to collection, handling and transport.Stress-related damage is most likely to be highest between collection and export, as the conditions for holding and transport are poorest at this level (Koldewey et al., 2005).Severe or chronic stress is often associated with poor performance and has long been suspected to cause immunosuppression in cultured fish (Pickering, 1998).Stress may induce the release of epinephrine and norepinephrine by chromaffin tissue in response to stimulation of the sympathetic nervous system, which might increase plasma glucose levels (Gomes et al., 2006), neoglucogenesis, the deposition of glycogen in the liver, immunosuppression and leukocyte counts (Ross & Ross, 2008).Similarly, Ellsaesser & Clem (1987) showed that both the number and immunological competence of circulating lymphocytes in the blood of channel catfish (Ictalurus punctatus) were reduced following chronic stress.
Anesthetics are known to be effective at reducing or minimizing stress in fish.Certain anesthetics have been used in the transport of fish, such as benzocaine in Menidia estor (Ross et al., 2007) and amylobarbitone, barbital sodium, chloral hydrate, MS222, quinaldine, tertiary amyl alcohol and urethane in Indian carp (Das & Goswami, 2003).The essential oil of Lippia alba (Mill.)N. E. Brown (EO) is effective as an anesthetic for silver catfish (Rhamdia quelen) (Cunha et al., 2010a), but no studies on EO have been performed in other species or on its effects on fish during transport.Therefore, the aim of this study was to identify the anesthetic induction and recovery times in slender seahorses that were exposed to EO, as well as the efficacy of EO as a stress-reducing agent in the transport of this species.This is also the first study dealing with seahorse transport.

Animals
Juvenile slender seahorses were acquired from a local producer in the city of Serra -ES, Brazil.They were transported to the laboratory and kept in continuously aerated 100-L aquaria with controlled temperature (22.3ºC), salinity 27.5 ± 0.2 and dissolved oxygen levels 5.5 ± 0.5 mg L -1 .A voucher specimen was registered in the ichthyologic collection at the Universidade Federal do Espírito Santo (CI-UFES 1027).

Plant material
Lippia alba (Mill.)N.E.Brown was cultivated at the Universidade Federal de Santa Maria (UFSM) campus, Santa Maria, RS, Brazil.Samples of the aerial portions of the plant were collected in October, 2008.The plant material was identified by Dr. Gilberto Dolejal Zanetti from the Departamento de Farmácia Industrial, UFSM.A voucher specimen (SMDB No. 10050) was deposited in the herbarium of the Departamento de Biologia, UFSM.

Essential oil extraction
The essential oil used was obtained from fresh leaves of the plant by hydro-distillation using a Clevenger type apparatus for 2 h (European Pharmacopoeia, 2007).Essential oil samples were stored at -20 ºC in amber glass bottles.

Anesthesia induction and recovery
Slender seahorse juveniles (2.5 ± 0.5 g and 10.0 ± 1.0 cm) that had been fasted for 24 hours were transferred to aquaria containing 1 L of water and EO at concentrations of 10, 20, 50, 150, 300 and 450 μL L -1 (equivalent to 8, 16, 40, 120, 240 and 360 mg L -1 , respectively, because the density of this EO is approximately 0.80), with the EO first being diluted in ethanol (1:10).Control experiments were performed using aquaria that contained only ethanol at a concentration equivalent to the dilution used for the 450 μL L -1 EO treatment.To evaluate the time required for anesthesia induction, 10 seahorses, each of which were placed in individual aquaria, were used for each concentration tested, and each juvenile was used only once, according to the method of Schoettger & Julin (1967).The maximum observation time was 45 min, except in the seahorses exposed to the lower concentrations of EO (10 and 20 μL L -1 ), which were observed for a period of 6 h to determine the concentration to be used for transport.After induction, the juveniles were transferred to anesthetic-free aquaria to measure the anesthesia recovery time.

Transport
Slender seahorses (2.3 ± 0.8 g and 9.8 ± 1.1 cm) were placed in plastic bags (one seahorse per bag) containing 0.5 L of water with 15 μL L -1 of EO that had previously been diluted in ethanol (1:10) (n= 10 for treatment) and transported for 4 or 24h.This concentration was used because there was no significant difference observed between the 10 and 20 μL L -1 treatments with respect to the anesthetic stage reached.The control group was subjected to the same procedures but no anesthetic was added to the water.Bags were then inflated with oxygen, tied with rubber strings and packed in plastic boxes, as described by Gomes et al. (2006).Water samples were collected before the plastic bags were closed and after transport for determination of dissolved oxygen, temperature and salinity with an oxygen meter YSI (model Y5512 Yellow Springs, USA).Blood samples (n= 10 for each treatment) were collected at the end of the transport period and from a group that was not subjected to transportation.An aliquot of the blood was used for glucose determination with a digital Accu-Check™ apparatus, and another aliquot was smeared on clean slides (two per fish), which were dried at room temperature for 24 h, and then fixed in 100% methanol for 10 min.Subsequently, they were stained with 4% Giemsa solution for 10 min, air-dried, and then prepared for counting lymphocytes, eosinophils, neutrophils, thrombocytes, monocytes, and basophiles; one hundred random leukocytes cells had been counted for each individual with the aid of a Leica Galen III optical microscope, as described by Tavares-Dias et al. (2000).

Statistical analyses
To verify the homogeneity of variances, all data were submitted to Levene's test.As the data were homoscedastic, they were analyzed using two-way ANOVA and Tukey tests.STATISTICA (version 5.1) was used for these analyses, and significance was set at a level of 95% (P < 0.05).

Results
An increasing concentration of EO proportionally decreased the time required for anesthesia induction.Slender seahorses that were exposed to low concentrations (10 and 20 μL L -1 ) of the EO for 6 h maintained a uniform state of sedation, i.e., they remained at stage 2 (Table 1).No mortality resulted from anesthesia induction within the range tested.The application of 4500 μL L -1 ethanol alone (concentration equivalent to the dilution used for the 450 μL L -1 EO treatment) did not produce an anesthetic effect.No significant difference was found in the recovery time at the different concentrations of the EO tested.
There was no significant difference in the dissolved oxygen levels (16.60±4.20 mg L -1 ), salinity (27.6±0.2) or temperature (22.8±0.6 o C) after transportation, and no mortality was observed in any of the treatments.The lymphocyte counts of the control slender seahorses transported for 24 h presented significantly lower values than before transport.However, the neutrophil counts increased significantly after the transport compared to before transport.Slender seahorses transported in water with EO did not shown any significant change in these parameters at the end of transport.No significant change was observed in eosinophil, thrombocyte, monocyte and basophil counts (Table 2).
The blood glucose levels of control slender seahorses increased significantly after 4 and 24 h of transportation compared to before transport.Fish transported under conditions of 15 μL L -1 EO did not exhibit any significant change in their blood glucose levels compared to before transport (Fig. 1).

Anesthesia induction and recovery
The maximum allowable time for the induction of deep anesthesia (stage 4) in fish is 10 min (Roubach et al., 2005;Ross & Ross, 2008).The lowest concentration of the EO used here that was capable of inducing deep anesthesia in the slender seahorse was 50 μL L -1 , but 150 -300 μL L -1 was required to obtain rapid (approximately 3 -4 min) deep anesthesia (Table 1).As slender seahorses anesthetized with these two concentrations did not show any significant difference in the recovery times, the lower concentration (150 μL L -1 ) is suggested as the optimal concentration for deep anesthesia induction.In the silver catfish, the lowest concentration of EO of L. alba that is able to induce deep anesthesia is 100 μL L -1 , while rapid anesthesia is reached with concentrations of 300-500 μL L -1 , and recovery time is 350-450 s (Cunha et al., 2010a).Apparently, the slender seahorse is more easily anesthetized by EO of L. alba than the silver catfish, but the time of recovery was similar in both species.Recovery time is usually faster at lower concentrations of anesthetic, and it becomes more prolonged as the concentration increases (Ross & Ross, 2008).However, the different concentrations of EO tested in the slender seahorse did not affect recovery time.
No other studies besides Cunha et al. (2010a) have been reported on anaesthetizing fish using EO of L. alba, but menthol at concentrations of 100-200 mg L -1 can provoke deep anesthesia in the tambaqui, Colossoma macropomum, after 1-2 min, and this results in a recovery time of 5-12 min (Façanha & Gomes, 2005), similar to what was found in the present study for slender seahorses.Deep anesthesia can be achieved in the tropical reef fishes Sergeant Major Abudefduf saxatilis, Cocoa damselfish Stegastes variabilis, Maria-nagô Pareques acuminatus, Doctorfish Acanthurus chirurgus, Budião-batata Sparisoma axillare, Schoolmaster snapper Lutjanus apodus, and Frillfin goby Bathygobius soporator with 20 mg L -1 clove oil, with associated induction and recovery times of less than 3 and 5 min, respectively (Cunha & Rosa, 2006).Silver catfish can reach deep anesthesia with 20 mg L - 1 eugenol (the main compound of clove oil), but for rapid anesthesia is necessary to use 50 mg L -1 (Cunha et al., 2010b).The effect of clove oil varies according to the species, but usually 20-50 mg L -1 induced stage 4 of anesthesia within 120-360s (Cho & Heath 2000;Keene et al., 1998;Sladky et al., 2001;Iversen et al., 2003).Therefore a lower concentration of clove oil or eugenol than EO of L. alba is necessary to induce deep anesthesia in the teleosts species studied so far.

Transport
Sedation can be beneficial in fish transportation, especially in cases where long distances are to be covered, but there may also be advantages of sedating animals for short journeys.Stage two of anesthesia, or deep sedation, which was characterized by Schoettger and Julin (1967) as "partial loss of equilibrium, no reaction to external stimuli", is Table 1.Time required for induction and recovery from anesthesia using the essential oil of Lippia alba in slender seahorse juveniles.Stages according to Schoettger & Julin (1967).Maximum observation time was 45 min.The time to reach each stage in seconds (s) is shown.N = 10 for each concentration tested.No relationship between concentrations of the essential oil of L. alba and time of recovery was found.y = time to reach the stage, and x = concentration of the essential oil of L. alba (μL L -1 ).considered an ideal condition for transporting fish because fish sedated at this level exhibit reduced activity but are able to maintain partial equilibrium, swimming capacity, and avoid physical damage resulting from collision with plastic bags (Cooke et al., 2004).
The number of leukocytes present in an organism changes in situations of stress, depending on the studied species.Transportation of slender seahorses for 24 h decreased their lymphocyte and increased their neutrophil counts.No significant change occurred in slender seahorses transported with EO of L. alba, indicating that this EO is beneficial to reduce stress.A reduction in the concentration of lymphocytes and an increase in the number of neutrophils was also observed in the common carp, Cyprinus carpio, and the channel catfish, Ictalurus punctatus, after being subjected to the stress of capture or transport (Sopinska, 1984;Ellsaesser & Clem, 1986), as well as in Nile tilapia following the stress of capture (Martins et al., 2004), the European eel, Anguilla anguilla, under the stress of handling (Johansson-Sjöbeck et al., 1978) and the common dab, Limanda limanda, after being subjected to acute stress (Pulsford et al., 1994).An increase in the number of leukocytes was found in tambaqui (Tavares-Dias et al., 2001) and in the hybrid tambacu C. macropomum x Piaractus mesopotamicus (Martins et al., 2002) after being subjected to handling.The number of thrombocytes decreased in pacu, P. mesopotamicus (Martins et al., 2000), and did not change in tambacu (Martins et al., 2002) subjected to handling.
Stress is associated with cortisol release in the blood following the activation of the hypothalamic-pituitary-interrenal (HPI) axis.This hormone binds to receptors in leukocytes, leading to immunosuppression in most situations.One of the well-known effects of cortisol is the regulation of leukocyte migration in tissues.Stress increases the number of neutrophils (leukocytes involved in the inflammatory response) and reduces the counts of lymphocytes (leukocytes involved in the immune response) (Bauer et al., 2001).It is noteworthy that these changes are due to cortisol and norepinephrine, which induce leukocyte migration from blood to tissues and vice-versa (Pulsford et al., 1994).In conclusion, EO is effective in inducing slight sedation in the slender seahorse at concentrations of 10-20 µL L -1 and deep anesthesia at concentrations of 50-450 µL L -1 , and for rapid deep anesthesia, a concentration of 150 µL L -1 is recommended.Furthermore, adding 15 µL L -1 of EO to the water in which seahorses are transported inhibits the elevation of blood glucose and neutrophils and decrease in lymphocytes that occur without this anesthetic in the slender seahorse, and therefore, its use in the transport of this species is suggested because apparently reduces the stress of transport.

Fig. 1 .
Fig. 1.Blood glucose levels of seahorses transported in plastic bags (one seahorse per bag) for 4 or 24 h.N= 10.BT= Before transport; Control= only water; EO = essential oil of L. alba previously diluted in ethanol (1:10) 15 µL L -1 .* significantly different from before transport using two-way ANOVA and Tukey's test (P < 0.05).+ significantly different from control group at the same time of transport using twoway ANOVA and Tukey's test (P < 0.05).

Table 2 .
Hematological parameters (%) of slender seahorses transported in plastic bags with the essential oil of L. alba (15 µL L -1 ).N= 10 for each treatment tested.Different letters in the columns indicate a significant difference between treatments based on two-way ANOVA and Tukey's test (P < 0.05).