Rhamdia quelen ( Quoy & Gaimard , 1824 ) , submitted to a stressful condition : effect of dietary addition of the essential oil of Lippia alba on metabolism , osmoregulation and endocrinology

The aim of this study was to evaluate the effect of the essential oil of Lippia alba (EOLA) as a feed additive on ionoregulatory and metabolic parameters and pituitary hormones expression in silver catfish, Rhamdia quelen, submitted to a stressful condition (stocking density of 10.6 kg m-3 and limited space). Fish were fed with different concentrations of EOLA (0.0 – control, 0.25 and 0.50 mL kg food-1) for 20 days. Metabolic parameters were not affected by the diet, with the exception of alanine aminotransferase, which was higher in the liver of fish fed 0.50 mL EOLA kg food-1. Plasma ions and activity of H+-ATPase did not change, but fish fed 0.25 mL EOLA kg food-1 presented higher Na+/K+-ATPase activity. Somatolactin expression in the pituitary was higher in the fish fed 0.25 mL EOLA kg food -1, but the expression of growth hormone and prolactin did not change. Therefore, dietary EOLA does not exert a protective effect in R. quelen submitted to a stressful situation because it did not alter most measured parameters. The use of 0.25 mL EOLA kg food-1 seems to be more suitable than 0.50 mL EOLA kg food-1 since the latter may be related to liver damage.


Introduction
Addition of herbal extracts in fish feed is increasingly seen as a safe and practical alternative to synthetic pharmaceuticals.Some studies showed that dietary addition of plants has several advantages.The addition of 0.5% of Massa medicata, Crataegi fructus, Artemisia capillaries or Cnidium officinale to the diet led to better use of lipids in stress and recovery of Pagrus major (Ji et al., 2009).The use of the oregano essential oil (Origanum heracleoticum) as a supplement in the food improved growth, antioxidant status and resistance against Aeromonas hydrophila in channel catfish (Ictalurus punctatus) (Zheng et al., 2009) and the extract of Allium sativum in the diet also promoted growth and reduced mortality rate of Nile tilapia (Oreochromis niloticus) challenged with Aeromonas hydrophila (Shalaby et al., 2006).
Rhamdia quelen can be found from Argentina to southern Mexico (Perdices et al., 2002), and is the most raised native species in South Brazil (Baldisserotto, 2009).A recent study demonstrated that feeding R. quelen with different levels of dietary EOLA for 60 days does not affect growth, but alters some metabolic parameters and improves the antioxidant status (Saccol et al., 2013).However, it is not known whether the effect of EOLA on anesthesia and antioxidant status may induce any protective effect in R. quelen exposed to non-optimal culture situations, as at a stocking density of 10.6 kg m -3 , which is considered stressful for this species when fish are maintained in a limited space (tanks or cages around up to 300-400 L) (Barcellos et al., 2004;Menezes et al., 2015).Consequently, the aim of this study was to evaluate metabolic, osmoregulatory and endocrinological parameters of R. quelen fed with a diet containing different levels of EOLA and submitted to a stressful situation.

Material and Methods
Essential oil.The plant L. alba was cultivated in the Centro de Educação Superior do Norte (CESNORS-UFSM) -Frederico Westphalen Campus.A voucher specimen (SMDB 10050) was deposited in the herbarium of the Department of Biology (UFSM).Botanical identification was made by Gilberto Dolejal Zanetti (Department of Industrial Pharmacy, UFSM).The EOLA was obtained from fresh leaves of L. alba by hydrodistillation in a Clevenger apparatus for 2 h (Council of Europe (COE), 2007) and stored at -20 o C until use.The composition of the EOLA was the same as that described by Saccol et al. (2013): the major components of the EO were linalool (55.26%), 1,8-cineole (7.85%),γ-muurolene (4.63%), β-caryophyllene (3.15%) and E-carveol (2.79%).
Experimental design.The experiment was conducted through January and February 2013 (summer), natural photoperiod (15 h light, 9 h darkness) in a continuously aerated recirculation system at the Laboratory of Fish Physiology, Universidade Federal de Santa Maria (UFSM).Adult R. quelen (132.74 ± 10.24 g, 24.05 ± 0.55 cm, voucher number UFRGS 19612, Ichthyology Laboratory, Universidade Federal do Rio Grande do Sul) of both sexes were obtained from the Fish Culture sector (UFSM) and placed in 60-L tanks (4 fish/tank) at a stocking density of 10.6 kg m -3 .
The animals were fed to satiation once a day (15:00) with a diet (34% crude protein) formulated according to Lazzari et al. (2007) (Table 1).Three concentrations of the EOLA in the diet (0-control, 0.25 and 0.50 mL kg food -1 ) were added to the ingredients together with canola oil.The EOLA concentrations were the lowest concentrations that improved antioxidant status in R. quelen (Saccol et al., 2013).The amount of feed offered and the unconsumed remains were weighed to determine feed intake.The fish (n=10/dietary EOLA concentration) were fed with the control diet for one week prior to the experiment for acclimation to the system and to the diet, and then for additional 20 days with the treatment diets.Uneaten food and feces were siphoned 30 minutes after feeding, and the water removed in this process was replaced with water under the same conditions and proportions found in the system.

Soybean meal 30
Meat and bone meal 35

Sample collection.
After being fasted for 24 h, fish were anesthetized with 50 mg L -1 eugenol and blood was collected by caudal puncture with heparinized 5 mL syringes.The samples were centrifuged at 1000 xg for 5 min and the plasma was stored at -20 o C until analyses.The fish were then euthanized and the pituitary gland, gills, liver and muscle were excised and stored in a -80° C freezer.Cortisol.Plasma cortisol was determined in duplicates using an enzyme-linked immunosorbent assay (ELISA) kit (Diagnostics Biochem Canada Inc., Canada).Absorbance was determined in spectrophotometer at 450 nm, and the inter-and intra-assay variation coefficients were 5.15±0.53%and 4.13±0.67%respectively.The specificity of the test was evaluated by comparing the standard curve and serial dilutions of the plasma samples.The curve obtained using serial dilutions of R. quelen plasma ran parallel to the curve constructed with human standards provided by the kit.

RNA
Biochemical measurements.The protein content in liver and muscle was measured according to Lowry et al. (1951) using bovine serum albumin as standard.Plasma glucose and lactate were measured with Labtest kits (Lagoa Santa, MG, Brazil).Glycogen and glucose in the liver and muscle were determined according to Dubois (1956).Lactate in the muscle was determined as in Harrower & Brown (1972) and total lipids were determined in the liver and muscle by the method of Bligh & Dyer (1959).Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma and liver were determined colorimetrically according to Reitman & Frankel (1957).
Ionoregulatory measurements.Plasma Cl -levels were determined according to Zall et al. (1956) and Na + and K + with a flame spectrophotometer B262 (Micronal, São Paulo, Brazil).Standard solutions were made with analytical reagent grade (Vetec Merck) dissolved in deionized water, and standard curves for each ion were tested at five different concentrations.The activities of Na + /K + -ATPase and H + -ATPase were assessed in the gills as described by Gibbs & Somero (1989).
Statistical Analyses.A Levene test was conducted to evaluate the homogeneity of variances.The data were compared using one-way analysis of variance (ANOVA) followed by the Tukey's test.The data regarding GH were not homocedastic and were compared using the Kruskal-Wallis test followed by the multiple comparison of mean ranks for all groups.All analyzes were performed with the software Statistica 7.0 (Stat Soft, Tulsa, OK).The minimum level of significance was P <0.05.The results were expressed as the mean ± standard error of the mean (SEM).
Dietary essential oil for stressed Rhamdia quelen 710
Diet (mL EO kg food  Ions and enzyme activities.Fish fed 0.25 mL EOLA kg food -1 presented significantly higher Na + /K + -ATPase activity compared to the other treatments, but plasma Na + , K + and Cl -levels, as well as H + -ATPase activity, were not significantly affected by the treatments (Fig. 3).
Different letters indicate significant differences (P < 0.05) between treatments.

Hormones expression. Pituitary GH and PRL expressions
did not change significantly between groups, but SL expression was higher in the group treated with 0.25 mL EOLA kg food -1 compared to the others (Fig. 4).

Discussion
High plasma cortisol levels were observed in the control group, demonstrating that the stocking density and the limited space of the 60 L tanks of the present experiment was stressful for R. quelen as previously indicated by Barcellos et al. (2004) and Menezes et al. (2015).According to Barcellos et al. (2001) plasma cortisol values for non-stressed adult R. quelen maintained in 100 m 3 ponds with stocking density of 0.8 kg m 3 are 15-30 ng mL -1 .Immersion anesthesia with EOLA prevented plasma cortisol increase in R. quelen subjected to handling (Cunha et al., 2010), but dietary EOLA did not change the levels of such hormone, indicating that it did not have an effect upon stress parameters when administered through this route.Lactate, glycogen, glucose, protein and lipids are biochemical parameters commonly used to assess the metabolic state of fish tissues (Pretto et al., 2014).Apparently, dietary addition of both EOLA concentrations for 20 days did not change Rhamdia quelen metabolism since these parameters were not affected.R. quelen fed with dietary EOLA for 60 days also did not show significant alteration in most of these biochemical parameters in the plasma, but the liver and muscle glycogen increased and hepatic glucose decreased in R. quelen fed 0.50 mL EOLA kg food -1 (Saccol et al., 2013).Therefore, probably dietary EOLA only change R. quelen metabolism after a long-term treatment.
The enzymes AST and ALT are mainly used as biomarkers to assess liver damage, although they are also found in organs such as skeletal muscle, heart, pancreas and kidneys (Gharaei & Ghaffari, 2010).The increased ALT activity in the liver of silver catfish fed with the highest dietary EOLA concentration suggests that there was hepatocyte damage.However, the lack of alteration in plasma ALT and in plasma and liver AST indicates that the observed increase in hepatic ALT did not cause any serious damage.In agreement with this hypothesis, R. quelen fed with up to 2.0 mL EOLA kg food -1 for 60 days only decreased the glucose levels in the liver, resulting in an increase in the glycogen and lactate reserves in the liver (Saccol et al., 2013).
The addition of EOLA to the water of transport reduced the net Na + , K + and Cl -losses in R. quelen (Becker et al., 2012), and immersion anesthesia with this oil increased gill Na + /K + -ATPase and H + /ATPase activities in this species (Toni et al., 2014), indicating an osmoregulatory effect.However, the only osmoregulatory effect of dietary EOLA observed in the present experiment was the increase in gill Na + /K + -ATPase activity in fish fed with 0.25 mL EOLA kg food -1 .The expression of SL also increased in R. quelen fed with that concentration of EOLA, indicating that both effects may be related.Previous studies verified a negative relationship between expression of SL and gill Na + /K + -ATPase activity in chum salmon (Oncorhynchus keta) migrating from the ocean to the rivers (Onuma et al., 2010) and in Atlantic salmon (Salmo salar) sampled in the later weeks of smoltification compared to those at the beginning (O' Keeffe et al., 2008).However, both studies compared fish with different reproductive stages, which may have altered expression of SL.The Na + /K + -ATPase plays a major role in fish osmoregulation (Duarte et al., 2013), while SL seems to be involved with acid-base regulation in rainbow trout (Oncorhynchus mykiss) (Kakizawa et al., 1996) and correction of plasma osmotic balance in Mozambique tilapia (Oreochromis mossambicus) exposed to acidic freshwater (Furukawa et al., 2010).The absence of significant difference in gill Na + /K + -ATPase activity and SL expression in the fish fed with 0.50 mL EOLA kg food -1 may be because at such concentration the effect was faster and the osmotic and/or acid-base equilibrium was fully reestablished.
Maintenance of GH expression is in accordance with the findings by Saccol et al. (2013), which demonstrated that dietary EOLA did not change R. quelen growth.Prolactin is considered one of the most important hormones related to freshwater adaptation, and it is essential for ion uptake as well as reduction in ion and water permeability of osmoregulatory surfaces (Sakamoto & McCormick, 2006).Its unaltered expression in R. quelen fed with dietary EOLA is probably related to the unchanged plasma ion levels.
In conclusion, dietary EOLA does not have a protective effect in R. quelen submitted to a stressful situation because it did not alter most measured parameters.The use of 0.25 mL EOLA kg food -1 may be more suitable than 0.50 mL EOLA kg food -1 since the latter increased ALT, indicating a possible liver damage.
Baldisserotto et al. (2014)lyzed through qRT-PCR, using the StepOnePlus™ RT-PCR system (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems).The sequences used to design all the primers were according toBaldisserotto et al. (2014)using the Primer Express software 3.3 (Applied Biosystems).The results were normalized to the expression of the constitutive gene β-actin according toBaldisserotto et al. (2014).The calculation of relative expression was performed as recommended byPfaffl (2001).
Delaware, USA; Abs 260/280 nm ratio) spectrophotometer.Ratios above 1.7 were considered pure and samples below this threshold were discarded.Total RNA (1µg) was treated with DNase (Invitrogen) at 37 °C for 5 min to digest any contaminating DNA.The reverse transcriptase reaction was performed with iScript cDNA synthesis kit (Bio-Rad) in a final volume of 20 µL.Pituitary expression of GH, PRL and SL mRNA.

Table 2 .
Biochemical parameters in liver and muscle of Rhamdia quelen fed with diets containing different concentrations of the essential oil (EO) of Lippia alba.