ESSENTIAL OIL OF Aloysia triphylla IS EFFECTIVE IN NILE TILAPIA TRANSPORT

The study evaluated the use of the essential oil of Aloysia triphylla (EOAT) as stress reducer in transporting Nile tilapia, Oreochromis niloticus. Juveniles were transported for 8 h in plastic bags by car with water containing 0 (control), 20 or 30 μL L-1 EOAT (three replicates). We evaluated water quality, net ion fluxes (Cl−, Na+ and K+) and, plasma parameters (cortisol, glucose, lactate and paraoxonase) and ventilatory rate (VR) of Nile tilapia. There was no mortality in juveniles undergoing transport. Nile tilapia transported with 20 or 30 μL L-1 EOAT presented lower pH than control group and with 30 μL L-1 EOAT presented lower water conductivity, alkalinity and Cl−, Na+ and K+ net ion fluxes compared to other treatments. The EOAT did not alter plasma cortisol and paraoxonase levels, but reduced VR, and 30 μL L-1 EOAT reduced plasma glucose levels compared to the control group after transport. The use of 20 μL L-1 EOAT increased plasma lactate levels compared to other treatments. The use of 30 μL L-1 EOAT for sedation in 8 h transport of Nile tilapia is recommended because it improved water quality and reduced ionic losses, plasma glucose levels and VR.


INTRODUCTION
The production of fish depends on various factors, of which transportation is one of the most important.During transportation, water quality can change (e.g.decrease in oxygen levels, pH changes and increase in ammonia levels) and hence affect the well-being and survival of fish (COLT et al., 2011;SENA et al., 2016;BOSISIO et al., 2017).The use of sedative substances in fish transport can reduce physical activity and prevent physical injury and reduce metabolism without total loss of equilibrium (BECKER et al., 2012;ZEPPENFELD et al., 2014;SENA et al., 2016).
Fish react to stress with a primary neuroendocrine response, represented by a rapid release of catecholamines (adrenaline and noradrenaline) and by the activation of the hypothalamic-pituitary-interrenal (HPI) axis.The increase of these hormones may induce a wide range of secondary responses that can be observed through disturbance of metabolic processes (WENDELAAR-BONGA, 1997), like plasma lactate, glucose and cortisol levels and/or ion loss in freshwater fish.Evaluation of ionic flow, plasma chemistry and hormones, especially cortisol concentrations in plasma, is broadly used as stress response indicators in fish (BARTON and IWAMA, 1991), since such parameters may reflect alterations of physiological homeostasis, demonstrating its application as biomarkers in the evaluation of stress effects, as well as paraoxonase, an enzyme that acts to protect the fish against oxidative stress (SENA et al., 2016).A sedative must have a stress-reducing capacity, blocking the HPI axis, and render the fish unable to respond to additional stressors (BOLASINA, 2006).In addition, sedation can contribute to the maintenance of water quality by reducing oxygen consumption and ammonia excretion and contribute to the fish culture.
Essential oils derived from different plant species have been used for sedation and transporting fish (BECKER et al., 2012;LIMMA-NETTO et al., 2016;HOHLENWERGER et al., 2017).The essential oil of Aloysia triphylla (L'Hér.)Britton (EOAT) contributes to maintaining water quality (PARODI et al. 2014) and improving physiological conditions of silver catfish (Rhamdia quelen) after transport (ZEPPENFELD et al., 2014).EOAT has also proved to be efficient for sedation, anesthesia and stress reducer in Nile tilapia (TEIXEIRA et al., 2017) and for transporting albino and grey strains of silver catfish (PARODI et al., 2014) and post larvae of white shrimp (Litopenaeus vannamei) (PARODI et al., 2012).Although EOAT has not been evaluated for the transport of Nile tilapia in a closed system such as a plastic bag, clove oil is not recommended due to increased osmoregulatory disorder and mortality (SIMÕES et al., 2011), although it did not influence glucose levels (OLIVEIRA et al., 2009).These data suggest the necessity for studies with essential oils which can be recommended for the transport of Nile tilapia species.
The Nile tilapia (Oreochromis niloticus) adjusts easily to different environments and is one of the most important species of freshwater fish for global aquaculture, primarily because of its known hardiness and good adjustment to captivity conditions (EL-SAYED, 2002).However, there are no studies about EOAT in Nile tilapia transport.Therefore, the purpose of this investigation was to verify the efficacy of EOAT for use during the transport of Nile tilapia.So, we performed experimental trials to compare the influence of EOAT on survival, water quality, net ion fluxes (Cl − , Na + and K + ), biochemical determinations (plasma cortisol, glucose, lactate and paraoxonase) and ventilatory rate (VR) of Nile tilapia.

Essential oil of Aloysia triphylla
Leaves of A. triphylla cultivated at Frederico Westphalen, RS, Brazil, were collected in winter 2013.A voucher specimen was registered in the herbarium of the Department of Biology of the Universidade Federal de Santa Maria (SMDB no. 11169).Oil extraction from fresh leaves was performed by hydrodistillation and was stored in amber glass bottles (-4°C).The chemical constituents were verified by TEIXEIRA et al. (2017), where geranial (28.97%) and neral were the main constituents (16.12%).

Animals
Sex-reversed male Nile tilapia juveniles of Gift lineage (92.66 ± 28.76 g and 17.33 ± 1.66 cm) were purchased from Bebedouro Fish Farm, Petrolina, PE, Brazil.The fish were housed for 14 days in continuously aerated 300-L masonry tanks with a semi-static system.The fish were fed twice a day at a ratio of 5.0% body mass with a commercial diet (320 g kg -1 crude protein; 3500 kcal digestible energy, Purina Nutripeixe SI).Individuals were fasted for a period of 24 h prior to the experiments.
We performed two experiments separately.Initially, we evaluated the effects of EOAT on ionic fluxes, water quality and biochemical responses in plasma of fish during transport (N=12 before transport + N=36 after transport for each treatment).Subsequently, we performed a second experiment to evaluate the effect of EOAT on ventilatory rate of fish (N=24).The experiments were approved by the Ethical Committee of the Biology Institute of the Universidade Federal da Bahia under registration no.19-2014.

Evaluation of stress during transport
The transport began at 8 a.m. from the Bebedouro Fish Farm, Petrolina, PE towards Universidade Federal da Bahia, Salvador, BA, Brazil (a distance of 534 km) and the Nile tilapia juveniles were transported in a car for 8 h, mean time of transport of juveniles in conditions of long distances.The fish were placed in 30-L plastic bags, closed with elastic straps, containing 10-L of water and the rest completed with oxygen.Fish were transported at a loading density of 12 fish per plastic bag (density of 9.26 g L -1 ).Fish were divided into three treatments (three replicates each): 0 (control), 20 and 30 μL L -1 EOAT, both first diluted in ethanol (1: 10).Previous studies demonstrated that ethanol does not induce sedation or anesthesia at concentrations up to 4500 μL L -1 in Nile tilapia (HOHLENWERGER et al., 2016;TEIXEIRA et al., 2017).
Another 12 fish were not subject to transport and designated 'before transport'.They remained in water free of EOAT throughout the trial.The concentrations of EOAT were chosen according to the range recommended for sedation of Nile tilapia by TEIXEIRA et al. (2017).Additionally, a pilot study was performed which showed that the fish reached only light sedation during 8 h of exposure at these concentrations.After transportation, fish were placed in 250-L tanks and mortality was assessed after 96 h.Pesca 2018, 44(1): 17-24. DOI: 10.20950/1678-2305.2018.263Water quality parameters and net ion fluxes Physical and chemical parameters of water were measured before and after transportation (in triplicate).Dissolved oxygen and temperature were measured with an oxygen meter (Politerm POL 60), pH with a pH meter (Hanna Combo HI 98130), electrical conductivity with a portable meter (Phtek CD310) and alkalinity, hardness and nitrite were measured with a commercial kit (Alfatecnoquímica, Florianópolis, Brazil).Total ammonia was measured by a colorimetric method and reading in a spectrophotometer (photoLab ® S12) and un-ionized ammonia levels were calculated according to COLT (2002).The levels of Na + and K + were determined by spectrophotometry (Espectro Blue, model FME) and the levels of Cl − were determined by ion chromatography (Dionex ® , ICS 1000).
The net ion fluxes were calculated based on the following equation: , where [ion 1 ] and [ion 2 ] are the ion concentrations in the water before and at the end of the experimental period, respectively, V is the water volume (L), M is the mass of the fish (kg) and t is the duration of the exposure (h) (BALDISSEROTTO et al., 2008).

Biochemical determinations
At the end of transportation, blood samples (1 mL) were collected from 12 fish per plastic bag with heparinized syringes, transferred to micro tubes (2 mL) and centrifuged at 3000 rpm for 15 min (at 6°C) to obtain the plasma.Another 12 fish were also used to evaluate plasma parameters before transport.The plasma samples were stored under constant refrigeration (-20 °C) and were sent to the Laboratory of Clinical Biochemistry of the Universidade Federal da Bahia to measure cortisol, glucose, lactate and paraoxonase levels.
A cortisol S kit was used for the determination of cortisol in the plasma aliquots in mini VIDAS ® equipment, using the enzyme-linked fluorescent assay technique.The measurement values of the VIDAS ® cortisol S kit ranged from 2 to 650 ng mL -1 .The analytical detection limit was 2 ng mL -1 .The observed values of total precision, dependent on serum concentration, ranged from 7.42 to 12.98% (coefficient of variance).
The plasma glucose levels were determined enzymatically by glucose oxidase/glucose peroxidase in BT 3000 apparatus (Wiener Lab, Rosario, Argentina).Lactate analysis was performed using the same equipment.The paraoxonase activity was determined by measuring p-nitrophenol reaction product according to the method described by SENTI et al. (2003), where paraoxonase activity = factor × Δ abs/min.The evaluation of paraoxonase activity has already been evaluated in previous studies with essential oil of Lippia alba in fish transport (HOHLENWERGER et al., 2016;SENA et al., 2016).

Ventilatory rate
To test the possible sedative effect of EOAT, the second experiment about VR was quantified by visually counting 20 successive opercular/buccal movements, measuring the elapsed time with a chronometer (HOHLENWERGER et al., 2017).
Eight fish per treatment (one individual for an 8-L aquarium) that were not submitted to transport were exposed to the same EOAT concentrations used in transport treatments.The evaluation times of VR were: 0, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h.After the VR experiment, fish were placed in 250-L tanks and mortality was assessed after 96 h and, each juvenile was used only once.

Statistical analyses
All data are expressed as the mean ± SEM.All data were subjected to Levene's test to verify the homogeneity of the variances.The data normality was assessed by Shapiro-Wilk test.Because the data exhibited homogeneous variances, comparisons between different treatments were made using two-way ANOVA (time X EOAT concentration) for VR and one-way ANOVA for biochemical determinations, net ion fluxes and water quality parameters post-transport followed by Tukey post-hoc tests.We used t-test for comparison between the "before transport" and "control" groups.Significance was set at a critical level of 95% (P<0.05).

Survival and water quality parameters
No mortality or injury in the Nile tilapia juveniles was observed immediately after or 96 h after transport.
The dissolved oxygen and temperature after transport were significantly lower and higher, respectively, than before transport (P<0.05).After transport, pH was significantly lower with the use of 20 or 30 μL L -1 EOAT compared with the control group (P<0.05).The electrical conductivity and alkalinity after transport was significantly higher than before transport and electrical conductivity and alkalinity of the fish transported with 30 µL L -1 EOAT was significantly lower than control group or 20 μL L -1 EOAT (P<0.05).
The total ammonia and un-ionized ammonia were significantly higher after transport than before transport (P<0.05).After transport, total ammonia was significantly higher in the 20 μL L -1 EOAT group compared with the control group (P<0.05).Hardness and nitrite levels were not significantly affected by transport and treatments (Table 1).

Net ion fluxes
The use of EOAT in the transport water led to a significant decrease in Cl − and Na + (20 and 30 μL L -1 EOAT) and K + net fluxes (30 μL L -1 EOAT) compared to the control group (P<0.05).Additionally, the net Cl − efflux was significantly lower in the 30 μL L -1 EOAT group than in the 20 μL L -1 EOAT group (P<0.05)(Figure 1).

Biochemical determinations
Nile tilapia juveniles of the control group had significantly higher plasma cortisol, glucose and, paraoxonase levels than fish not subjected to transport (P<0.05) (Figure 2A, B and C).Nitrite (mg L −1 N-NO 2 ) 0.015±0.000.025±0.00a 0.025±0.00a 0.025±0.00a Different letters indicate significant difference between treatments post transport.Asterisk denote differences between the "before transport" and "control" groups (P<0.05).The plasma glucose levels of fish transported with 30 μL L -1 EOAT were significantly lower than fish of the control group or transported with 20 μL L -1 EOAT (P<0.05)(Figure 2B).The plasma paraoxonase levels were not significantly affected by transport or treatments (Figure 2C).Plasma lactate levels of fish transported with 20 μL L -1 EOAT were significantly higher than fish of the control group or transported with 30 μL L -1 EOAT (P<0.05)(Figure 2D).

Ventilatory rate
After 0.5 h, fish sedated with EOAT (20 and 30 μL L -1 ) presented lower VR than those from the control group up to the end of the experiment (8 h) (P<0.05).The VR was significantly lower in the 20 and 30 μL L -1 EOAT groups after 0.5 h (except at time 8 h) compared with time zero (P<0.05).In general, fish from the control group presented higher VR after 0.5 h compared to time zero (P<0.05)(Figure 3).

Water quality parameters
Dissolved oxygen in water levels are normally reduced during transport due to fish respiration (COLT et al., 2011).The values found in this study were above the minimum dissolved oxygen level for Nile tilapia culture (2.9 mg L -1 at 32.7 °C) (SODERBERG, 2006).The water temperature before transport was lower due to the environmental conditions of this experiment, with lower temperatures typical of early morning hours.Water temperatures throughout the experiment remained within the thermal comfort range for Nile tilapia (24-33°C) (BARAS et al., 2000).
The electrical conductivity of water is closely related to the dissolved ions (WENDELAAR BONGA, 1997) and the increase in conductivity values after transport was possibly caused by ion net fluxes by the fish.The lower electrical conductivity values observed in the water of Nile tilapia transported with 30 μL L -1 EOAT were probably related to the lower net ion net fluxes observed in this treatment.Water pH remained within tolerance values for Nile tilapia (EL-SHERIF and EL-FEKY, 2009).The highest pH value of the water in the control group was probably due primarily to un-ionized ammonia excretion (BALDISSEROTTO et al., 2008).
Similar to our data, increases in alkalinity levels after transport were reported in previous studies with silver catfish (BECKER et al., 2012;PARODI et al., 2014).These authors reported that the increase in alkalinity and hardness was probably due to regurgitation of commercial food (which contained calcitic limestone) by the fish.Food debris in the transport plastic bags was not observed in the present study because there was a 24 h fasting period prior to transport, as recommended for Nile tilapia (OLIVEIRA et al., 2009).The alkalinity increase in the control group after transport was probably due to the increase in the pH of the water and the higher efflux of Na + and K + , which were associated with the carbonates and bicarbonates present in the water (BOYD et al., 1992).Moreover, the absence of regurgitation observed in the present study may explain the maintenance of levels of water hardness after 8 h of transport.
Total ammonia levels were higher after transport than before transport, as expected (BECKER et al., 2012;PARODI et al., 2014;ZEPPENFELD et al., 2014).However, in the present study the lower pH in the 20 and 30 μL L -1 EOAT treatments compared to the control group contributed to the lack of significant difference in un-ionized ammonia levels.Un-ionized ammonia levels remained well below lethal levels for Nile tilapia (above 7400 µg L -1 ) (BENLI and KÖKSAL, 2005) suggesting that Nile tilapia could be transported for a longer period without mortality due to ammonia toxicity levels under the conditions used in this experiment.

Net ion fluxes
The transport of Nile tilapia caused net ion (Cl − , Na + and K + ) net fluxes, as expected, because the stress of transport can cause an increased release of catecholamines in fish, with a resulting increase in blood flow and gill permeability.In freshwater fish this implies ion loss to the water, especially Cl − and Na + (URBINATI et al., 2004).
Previous studies also found that EOAT (30-50 μL L -1 ) reduced net ion (Na + , Cl − and K + ) losses in silver catfish transported for 5-6 h (PARODI et al., 2014;ZEPPENFELD et al., 2014).On the other hand, for the transport of Nile tilapia (6 to 24 h), clove oil (9 and 18 mg L -1 ) is not recommended because it increases net ion fluxes and mortality (SIMÕES et al., 2011).We hypothesized that the lower ion losses with the use of EOAT in Nile tilapia transport were due to lower catecholamine release during the transport process and, consequently, there was lower gill blood flow.However, further studies should be conducted for a more accurate determination of the role of EOAT on ionic uptake in fish.

Biochemical determinations
A stressor can result in an endocrine response, such as a release of cortisol.Transport by car generates a change in the water flow of the plastic bags and exposes the fish to ambient temperature and insolation (SENA et al., 2016).This is consistent with our findings to lower plasma cortisol levels before transport.Additionally, in this study, Nile tilapia transported with EOAT did not exhibit an increase in plasma cortisol levels compared with the control group.Similarly, HOHLENWERGER et al. (2017) found no change in plasma cortisol levels in Nile tilapia transported with essential oil of L. alba (10-20 μL L -1 ).Previous studies of fish transport have demonstrated that the addition of EOAT (30-50 μL L -1 ) had conflicting results in silver catfish: plasma cortisol levels did not change or decreased in the grey strain (PARODI et al., 2014;ZEPPENFELD et al., 2014), but increased in the albino strain compared to fish transported without EOAT (PARODI et al., 2014).
Physiological stress responses include plasma hormones, metabolic processes and ionic balance (BARTON and IWAMA, 1991).The elevation of plasma glucose levels in fish after transport is due to the action of catecholamines and/or corticosteroids, which stimulate muscle and liver glycogenolysis (BARTON and IWAMA, 1991) as an adaptive response for supplying energy during transport and to maintain homeostasis (SENA et al., 2016).Our results showed that 30 μL L -1 EOAT was effective in reducing plasma glucose levels, reinforcing the hypothesis of lower catecholamine release.According to TAKAHASHI et al. (2006) plasma glucose can give better answers than cortisol in assessing the physiological condition due to stress caused by transport.In this study, EOAT presented sedative effect in Nile tilapia transport and reduced the energy demand.
Additionally, stress can result in a rapid increase in the plasma lactate levels, because lactate is a final product of anaerobic glycolysis for cellular energy supplies (URBINATI et al., 2004).We found that 30 μL L -1 EOAT, did not cause elevation of plasma lactate levels, which could indicate that sedation with EOAT at this concentration did not require the use of an anaerobic metabolic pathway as an important energy source during the transport (HOHLENWERGER et al., 2016) as verified for Nile tilapias under handling (500 μL L -1 ) or transport (10 or 20 μL L -1 ) conditions with use of essential oil of L. alba (HOHLENWERGER et al., 2017).Another hypothesis is that lactate has been produced and immediately consumed in the muscle (GLADDEN, 2000).In this sense, the lactate increase was already verified in Nile tilapia juveniles immediately after handling with 300 μL L -1 EOAT, but not for 1 or 4 hours after handling (TEIXEIRA et al., 2017).So, further studies should be conducted for a clarification about the lactate response in the transport of Nile tilapia with the use of EOAT.Additionally, the increased plasma lactate levels observed in fish exposed to 20 μL L -1 EOAT could be related to increased total ammonia levels seen in this treatment.According COPATTI et al. (2015), the increase in NH 3 concentrations triggers the use of an anaerobic route with the use of lactate as substrate for gluconeogenesis.
Plasma paraoxonase is an important indicator of protection against oxidative stress, preventing damage in the immune response and changes in total plasma proteins (KARATAS and KOCAMAN, 2014).In this study, plasma paraoxonase and cortisol levels indicate that exposure to EOAT did not alter the protection mechanism for stress in Nile tilapia after 8 h of transport, unlike what occurred in the control group, where the activation of antioxidant defences may be an adaptive response which involves physiological adjustments caused by stress o transport.However, few studies have investigated paraoxonase in fish, and its relation to the use of anaesthetics or sedatives in fish should be further better investigated (SENA et al., 2016).

Ventilatory rate
VR is easily changed in cases of stressful stimuli (BARRETO and VOLPATO, 2004;BECKER et al., 2012).According to BARRETO and VOLPATO (2004), in the absence of stress, the opercular movements of Nile tilapia vary between 96 and 109 per min.In the present study, except for time zero, the concentrations of 20 and 30 μL L -1 EOAT reduced movements in relation to the control group over 8 h, and remained below 61 opercular movements per min.
In this sense, our data showed that EOAT reduced the VR of Nile tilapia after 0.5 h of exposure, however, as plasma cortisol levels were not affected by EOAT in the water of transport, probably this essential oil reduced catecholamine release and/or metabolism, which led to the lower VR observed (HOHLENWERGER et al., 2017).

CONCLUSIONS
The use of 30 μL L -1 EOAT in Nile tilapia indicate that water quality was maintained, ionic balance improved since it reduced the loss of ions (Na + , Cl − and K + ) and metabolic responses were prevented or not modified during transportation, and VR was reduced after 0.5 h and remained so over 8 h.So, 30 μL L -1 EOAT is safe and effective as a sedative for Nile tilapia transport.

Figure 1 .
Figure 1.Effect of the essential oil of Aloysia triphylla (EOAT) added to the water on net ion fluxes (Cl − , Na + and K + ) in Nile tilapia (n=3 plastic bags per treatment) transported for 8 h.Data are expressed as mean ± SEM.Different letters indicate significant difference between treatments (P<0.05).

Figure 2 .
Figure 2. Effect of the essential oil of Aloysia triphylla (EOAT) added to the water on plasma cortisol (A), glucose (B), paraoxonase (C) and lactate (D) levels in Nile tilapia (n=12 before transport + 36 fish per treatment after transport) after 8 h transport.Data are expressed as mean ± SEM.Different letters indicate significant difference between treatments (P<0.05).*Indicate significant difference between before transport and control group (P<0.05).

Figure 3 .
Figure 3.Effect of the essential oil of Aloysia triphylla (EOAT) added to the water on ventilatory rate in Nile tilapia (n=8 per treatment).Data are expressed as mean ± SEM.Different capital letters indicate significant differences between treatments at the same time.Different lowercase letters indicate significant differences between different times for the same treatment (P<0.05).

Table 1 .
Water parameters before and after transport (8 h) of Nile tilapia (n=3 plastic bags per treatment) with essential oil of Aloysia triphylla (EOAT) added to the water.Data are expressed as mean ± SEM.