Antioxidant enzymes, hematology and histology of spleen in Nile tilapia fed supplemented diet with natural extracts challenged with Aeromonas hydrophila

Fish and Shellfish Immunology 79 (2018) 175–180

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Antioxidant enzymes, hematology and histology of spleen in Nile tilapia fed supplemented diet with natural extracts challenged with Aeromonas hydrophila

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Geovana Dottaa, Jaqueline Inês Alves de Andradea, Patrícia Garciaa, Gabriel Fernandes Alves Jesusa, José Luiz Pedreira Mouriñoa, Jacó Joaquim Mattosb, Afonso Celso Dias Bainyb, Maurício Laterça Martinsa,∗ a

AQUOS-Aquatic Organisms Health Laboratory, Aquaculture Department, Federal University of Santa Catarina (UFSC), Rod. Admar Gonzaga 1346, 88040-900 Florianópolis, SC, Brazil Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, Biochemistry Department, CCB, UFSC, SC, Brazil

b

A R T I C LE I N FO

A B S T R A C T

Keywords: Oreochromis Biochemistry Hematology Immunology Bacterium

This study investigated the effects of dietary supplementation with the extrats of propolis and Aloe barbadensis (aloe) on the antioxydant enzime activity, hematology and histology of the spleen of Nile tilapia challenged with Aeromonas hydrophila. Seventy two juvenile Nile tilapia were divided in four treatments and three replicates and fed extract mixture for 15 days: fish fed supplemented diet with 1% of the mixture of extracts of propolis and aloe (1:1) injected with phosphate-buffered saline (PBS); fish fed suplemented diet with 1% of the mixture of extracts of propolis and aloe (1:1) injected with the A. hydrophila, fish fed supplemented diet with the mixture of propolis extracts and aloe, injected with PBS and injected with A. hydrophila. The influence of the supplementation of propolis and Aloe extracts on the immunomodulation in tilapias was observed by the evaluation of the survival of the animals after challenge with A. hydrophila. Non-supplemented fish had a 44.5% survival rate and those supplemented with 1% of the mixture of extracts showed 55.6% survival 7 days after challenge. The supplemented animals also showed a significant increase in the number of lymphocytes in the evaluation of the blood parameters and, consequently, in the histopathological evaluation, presented greater presence of centers of melanomacrophages. In addition, the activity of the antioxidant enzymes glutathione reductase (GR) in the spleen presented a significant difference in fish supplemented with 1% of the extracts mixture, being superior in the animals injected with PBS when compared to those challenged with A. hydrophila.

1. Introduction Bacterial diseases are among the most important causes of economic losses in fish farming [1], Aeromonas spp., Pseudomonas fluorescens, Vibrio anguillarum, Flavobacterium columnare, Edwardsiella tarda, Streptococcus spp. and Enterococcus sp. are the main cause of mortality [2]. Excessive use of antibacterial drugs in fish farming to combat pathogens is responsible for changes such as immunosuppression, nephrotoxicity, low growth, development of resistant bacterial strains, environmental problems such as effluents from fish farming and bioaccumulation in fish [3]. According to Yonar et al. [4], these products may interact with lymphoid tissues and may alter immune system functions and balance, leading to undesirable effects such as immunosuppression, uncontrolled cell proliferation, and other changes that may unbalance the body's ∗

Corresponding author. E-mail address: [email protected] (M.L. Martins).

https://doi.org/10.1016/j.fsi.2018.05.024 Received 1 February 2018; Received in revised form 4 May 2018; Accepted 11 May 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved.

defense mechanisms against pathogens. Some substances have been used to stimulate the immune system, acting through different mechanisms seeking for the improvement of the defense response. Substances of plant origin have been studied for the purpose of immunostimulating fish against possible bacterial infections [5–7]. The extracts of propolis and Aloe barbadensis Miller have shown good results as immunostimulants [7–9]. The administration of immunostimulatory substances prior to infection can improve the fish defense system and protect against infections [10]. Biochemical, metabolic and hematological characteristics can be used to evaluate the influence of these products on the immune system. Studies with experimental bacterial infection in fish are usually carried out [11,12], but few demonstrate the hematological and enzymatic changes that the infection can cause, especially in fish supplemented with natural extracts of propolis and aloe.

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2.4. Hematological analysis

Nile tilapia (Oreochromis niloticus) fed with diet containing 1% propolis-ethanolic-extract, presented increase in the specific growth rate, food efficiency, hematocrit percentage, monocyte number, lysozyme activity, bactericidal activity of the serum and protection against A. hydrophila, when compared to those fed unsuppelemented diet or with diet containing crude propolis [13]. Similarly, studies have shown that Nile tilapia (GIFT variety) fed diet containing different concentrations of Aloe vera (0.5% 1%, 2%, and 4%) presented enhanced growth, food conversion and hemato-biochemical parameters, but Aloe had no significant effect on the survival of fish when compared to control [14]. The present study evaluated the effects of supplemented diet with propolis and aloe on the activity of antioxidant enzymes, hemato-immunological parameters and histology of the spleen in Nile tilapia after challenge with A. hydrophila.

After the feeding period, nine animals from each treatment were anesthetized with eugenol Vetec® (100 mg L−1) for collection of blood by puncture of the caudal vessel using a syringe containing 10% EDTA solution. The collected blood was separated into aliquots for different analyzes: percentage of phagocytosis, erythrocyte counts in Neubauer's chamber and confection of duplicate blood extensions later stained with May-Grunwald/Giemsa by the Rosenfeld method [16], later used for differential counting of leukocytes and total count of the number of thrombocytes and leukocytes [17]. The hematocrit rate was performed according to [18] and, after reading, the capillary was broken slightly above the white blood cell range and the plasma was transferred to the total protein refractometer for total plasma protein (PPT) according to [19].

2. Material and methods

2.5. Collection and preparation of spleen samples

2.1. Experimental conditions

After blood collection, the animals were euthanized by deepening the anesthetic state (Ethics Committee on the Use of Animals: no 23080.009240/CEUA/UFSC) for the collection of spleen samples individually packed in cryogenic tubes rapidly frozen in nitrogen liquid, stored in a freezer −80 °C. For biochemical assays, the samples were homogenized in 20 mM HEPES buffer, pH 7.4 and centrifuged at 20,000 g for 30 min (4 °C). The supernatant was used for the determination of the enzymatic activities [22]. For the histological analysis, fragments of the spleen were fixed in 10% buffered formalin solution and submitted to routine histological procedures of dehydration, diaphanization and paraffin inclusion. Duplicate cuts, 4 μm thick were stained with Harris hematoxylin and 1% aqueous eosin [20]. Based on the quantification of the melanomacrophage centers (CMM), the histopathological analysis was done by stereology using the Weibel graticule coupled to the optical microscope and the fraction of the organ volume calculated according to formula [21].

Nile tilapia juveniles (n = 72; 57.3 ± 11.2 g weight and 17.6 ± 4.2 cm of total length) from the same spawning, were stocked in polyethylene water tanks with 100 L capacity, equipped with biological filter, heater and constant aeration, maintained in the following conditions: average temperature of 24.0 ± 2.8 °C, pH 6.51 ± 0.43 (Alfakit®, AT-350), total ammonia 0.90 ± 0.33 mg L−1 (Alfakit®, colorimetric method) and dissolved oxygen 6.0 ± 0.7 mg L−1 (Hanna®, HI 9146). Seven days after acclimatization, the experiment was started with the supplemented diets for a period of 15 days. For this, the animals were randomly distributed in tanks, with four treatments in triplicate: fish fed supplemented diet with 1% of the mixture of extracts of propolis and aloe (1:1) injected with phosphate-buffered saline (PBS); fish fed suplemented diet with 1% of the mixture of extracts of propolis and aloe (1:1) injected with the A. hydrophila, fish fed supplemented diet with the mixture of propolis extracts and aloe, injected with PBS and injected with A. hydrophila.

2.6. Biochemical analyzes

2.2. Preparation of experimental diet

The quantification of total protein levels was determined by the Bradford method [22] using bovine serum albumin as standard, in order to normalize the enzymatic activity. Samples homogenized in buffer solution were used to determine the biochemical parameters. The enzymatic assays were performed in a final volume of 500 μL, with the exception of catalase activity, whose final volume was 1 mL. It was also used between 5 and 100 μL of the sample, depending on the parameter to be analyzed.

Commercial diet Nicoluzzi® 4 mm 36% crude protein was used throughout the experimental period. The extracts of propolis and aloe were prepared at the Laboratory of Morphogenesis and Plant Biochemistry, Department of Plant Science, UFSC; being the propolis used in this experiment was from apiaries of the state of Santa Catarina, from Eucalyptus grandis shoots and from Araucaria angustifolia resin. For each experimental unit used, the biomass of fish was calculated to determine the amount of feed at equivalent to 3% of live weight. A dilution of the extracts in 50% alcohol was prepared to form the 1:1 mixture in concentration 1%, included by spraying the feed pellets.

2.6.1. Glutathione reductase (GR) The activity of glutathione reductase was based on the method described by Carlberg and Mannervik [23]. By reducing the glutathione disulfide substrate (GSSG), GR oxidizes NADPH, which can be monitored by decreasing absorbance at the wavelength of 340nm. Thus, the rate of NADPH consumption expresses the activity of this enzyme.

2.3. Challenge with Aeromonas hydrophila After 15 days of feeding, the fish were challenged with A. hydrophila. The strain prepared in the Marine Shrimp Laboratory, Department of Aquaculture, UFSC, was incubated in test tubes with brain culture infusion (BHI) liquid culture medium at 30 °C for 24 h, after which the contents were centrifuged for 15 min at 10000 g and the precipitate was resuspended in 10 mL of sterile physiological solution. For the bacterial count present in the inoculum, five serial 1:10 dilutions were performed, seeded on plates containing tryptone soya agar (TSA) culture medium. After checking the concentration of the bacteria in the inoculum, the dilution was performed in order to reach the ideal concentration for the challenge. The experimental infection was performed by injecting 100 μL of A. hydrofila per fish, at a concentration of 5 × 106 CFU mL−1 [15].

2.6.2. Glutathione-S-transferase (GST) The conjugation of glutationa (GSH) to the chlorodinitrobenzene (CDNB) substrate catalyzed by GST yields a compound that can be detected at 340nm (ε = 9600 M−1 cm−1). The enzymatic activity is proportional to the rate of production of the conjugated compound [24]. From this activity is taken the basal reaction obtained by reading the reaction between the GSH of the assay and the CDNB, without the presence of the sample. The 5 min enzyme assay was performed in 100 mM potassium phosphate buffer (KPi), 1 mM EDTA, pH 7.0, containing 1 mM GSH. As the starting substrate 1 mM of CDNB was used. The basal absorbance was discounted from reading the test reaction in the absence of the sample. 176

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Table 1 Haematological parameters of Nile tilapia fed supplemented diet with propolis and Aloe barbadensis at concentrations 1% and not supplemented, challenged with Aeromonas hydrophila. Lowercase letters indicate significant difference between the injection of PBS and Aeromonas for each concentration of the extract and uppercase letters indicate significant difference between the concentrations of each extract (p < 0.05) by the Tukey test. PPT: total plasma protein. Haematological parameters

Treatments Supplemented 1% Injection PBS

Hematocrit (%) PPT (g.dL−1) Erythrocytes (x 106.μL−1) Leukocytes (x 103.μL−1) Thrombocytes (x 103.μl−1) Lymphocytes (x 103.μl−1) Neutrophils (x 103.μl−1) Monocytes (x 103.μl−1)

aA

25 ± 0.6 5.7 ± 0.5aA 2.32 ± 0.74aA 49.73 ± 16.47aAB 27.78 ± 25.73aA 0.43 ± 0.14aAB 0.04 ± 0.01aA 0.02 ± 0.01aA

Not Supplemented Injection Aeromonas aA

23 ± 0.7 5.5 ± 0.4aA 1.81 ± 0.50aA 61.94 ± 36.17bB 27.01 ± 11.07aA 0.57 ± 0.37aB 0.03 ± 0.02aA 0.01 ± 0.01aA

Injection PBS aA

28 ± 0.5 5.7 ± 0.6aA 2.22 ± 0.43aA 56.03 ± 16.30bB 35.49 ± 12.05aA 0.53 ± 0.13aB 0.03 ± 0.02aA 0.02 ± 0.02aA

Injection Aeromonas 22 ± 0.7aA 5.7 ± 0.5aA 1.72 ± 0.59aA 47.41 ± 27.41aAB 22.77 ± 15.06aA 0.42 ± 0.25aAB 0.03 ± 0.02aA 0.01 ± 0.00aA

2.7. Survival

2.6.3. Glucose 6-phosphate dehydrogenase (G6PDH) In the presence of glucose-6-phosphate, NADPH is formed from nicotinamide adenine dinucleotide phosphate (NADP +) and thus the increase in absorbance is measured at 340 nm (ε = 6220 M−1 cm−1) [25]. It is deduced from the reaction the basal activity obtained by the formation of NADPH by reading the enzymatic assay without the presence of the substrate. The 5 min enzyme assay is performed in 50 mM TRIS/HCL buffer, pH 7.4, containing 0.127 mM NADP + and 3 mM magnesium chloride (MgCl2). As the starting substrate, 1.5 mM glucose6-phosphate (G6P) is used. The basal absorbance was discounted from reading the assay reaction in the absence of G6P.

Animals belonging to the control groups and those challenged with an injection of 5 × 106 UFC mL−1 from A. hydrophila were used for the survival test. After challenge with A. hydrophila, mortality was observed for 7 days and survival calculated according to the formula:

% Relative of survivors =

Number of surviving fishes Number of infected fish

× 100

2.8. Statistical analysis The homocedasticity of the data was verified through the Bartlett test. The data were submitted to analysis of variance (ANOVA) followed by Tukey's test for comparison of means. Data transformations were applied when necessary. For all tests, the software Statistica 10.0 was used and a significance level of 5% was considered.

2.6.4. Catalase (CAT) The high rate of reaction of this enzyme, associated to a low “affinity”, allows the determination of its activity with high concentrations of H2O2 (10 mM). The activity was determined by the rate of H2O2 consumption in the first minute of the reaction, 240 nm (ε = 40 M−1cm−1) [26]. The disappearance of hydrogen peroxide without the presence of the sample is also discounted. The enzyme assay of 40 s is performed in 50 mM potassium phosphate (KPi) buffer, 0.5 mM EDTA, pH 7.0, containing 0.012% Triton X-100. As the starting substrate, 10 mM H2O2 is used. The basal absorbance is discounted from the reading of the assay reaction in the absence of the sample.

3. Results 3.1. Hematological parameters There was a significant difference (p < 0.05) in the values for the total number of leukocytes, which were lower in fish fed with 1% of dietary extracts, both for PBS and for those challenged with A. hydrophila. This result was reflected in the lymphocyte values during the differential count. The percentage of hematocrit; total plasma protein levels; number of erythrocytes; thrombocytes; neutrophils and monocytes showed no significant difference between treatments (Table 1). ƞmol∗ mg−1∗ mol−1

2.6.5. Glutathione peroxidase (GPx) It is indirectly accompanied by the disappearance of NADPH. The enzyme when using GSH to degrade an organic peroxide, such as t-butyl peroxide (t-BOOH) or cumene, generates oxidized glutathione (GSSG) which, in turn, is reduced by glutathione reductase, added to the reaction medium, with the consumption of NADPH (ε = 6220 M−1cm−1). This consumption of NADPH is accompanied by a spectrophotometer in 340nm, similar to the determination of GR [27]. From this rate of consumption, the basal NADPH consumption, obtained by reading the enzymatic assay without the presence of the substrate (peroxide), is discounted. The 5 min enzyme assay is performed in 50 mM potassium phosphate buffer (KPi), 0.5 mM EDTA, pH 7.0, containing 0.2 mM NADPH, 1 mM GSH and 0.2 μM L −1 GR purified from yeast. It takes between 5 and 10 min incubation with the reagents (except primer substrate) for enzyme activation. A total of 1 mM CUOOH was used as the starting substrate.

μmol∗ mg−1∗ mol−1 3.2. Biochemical analyzes The activity of the antioxidant enzymes glutathione reductase (GR) and catalase (CAT) in the spleen presented a significant difference in the group fed with 1% of the extracts mixture, being superior in the animals injected with PBS and without significant difference in those challenged with A. hydrophila, which maintained the tendency of the other group under study. Glutathione S-transferase (GST), glucose 6phosphate dehydrogenase (G6PDH), glutathione peroxidase (GPx) and superoxide dismutase (SOD) showed no significant difference between treatments (Fig. 1).

2.6.6. Superoxide dismutase (SOD) The activity of SOD was determined according to the methodology described by Mccord and Fridovich [28], which is based on the inhibition of the reduction of cytochrome c by O2-, produced by the xanthine/xanthine oxidase system. This inhibition is measured by increasing the absorbance at 550 nm, at 25 °C.

3.3. Histopathological analysis Fifteen days after feeding, no significant differences were observed 177

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Fig. 2. Percentage of survival of nile tilapia fed with feed supplemented with a mixture of propolis and Aloe barbadensis extracts (0% and 1%) after challenge with Aeromonas hydrophila. Lower case letters indicate difference between concentrations (p < 0.05) by Tukey's test.

in the analysis of histopathological sections of spleen in relation to the number and total area of melanomacrophage centers. However, fish fed supplemented diet with 1% of the mixture of the extracts showed on average 20.52 melanomacrophagous centers and an area of 0.0952 mm2, being superior to the group not supplemented, which presented 15.37 and 0.0711 mm2 (Table 2). 3.4. Survival There were no statistically significant difference between treatments. Non-supplemented fish had a 44.5% survival rate and those supplemented with 1% of the mixture of extracts showed 55.6% survival 7 days after challenge (Fig. 2). 4. Discussion The cells involved in the immune response have regulatory function, and may promote or suppress immune responses [29]. This regulatory mechanism ensures that responses are appropriate both qualitatively and quantitatively [30], showing that the immune system itself is regulated by the release of immunomodulating substances [31,32]. Knowledge of these processes and the discovery of immunomodulators has made it possible to manipulate the immune system in favor of a healthy state for the animal organism [33]. In this study, supplementation with propolis and aloe extracts in the diet of tilapia did not cause alterations in hematological parameters, but alterations were observed in the number of leukocytes, which mainly reflected the circulating lymphocytes of fish, being superior in those after challenge and supplemented with 1% of extracts. Lymphocytes act as immunocompetent cells [34] and in defense against infections by protozoa, helminths [35] and other pathogens [36]. Several studies have reported the presence of T-lymphocytes in several species of fish [37,38]. These cells are responsible for the activation and maintenance of the immune response, mediating both cellular and humoral immune responses [39] [40]. evaluated the immunomodulation in tilapia experimentally infected with A. hydrophila and as in this study, they observed that the animals supplemented with β-glucans and Sacharomyces cerevisiae showed a greater number of lymphocytes than those not supplemented. The increase in lymphocyte numbers usually occurs when the fish defense system is activated. Increased in the number of lymphocytes and leukocytes 24 h after inoculation with Enterococcus sp. in Nile tilapia was also found [41] and others [42] have reported an increase in the percentage of lymphocytes in tilapia fed 1% of propolis extract for 28 days after challenge with A. hydrophila, similar to the one found in the present study. Lymphocytes are specialized cells of the immune system that

Fig. 1. Antioxidant enzymes in the spleen of nile tilapia. GR (glutathione reductase), GST (glutathione S-transferase), G6PDH (glucose 6-phosphate dehydrogenase), CAT (catalase), GPx (glutathione peroxidase), SOD (superoxide dismutase). Lowercase letters indicate a significant difference between the concentrations (0% and 1%) of the blend of propolis and Aloe barbadensis extracts. Upper case letters indicate difference between injections of PBS and fish challenged with Aeromonas hydrophila (p < 0.05) by the Tukey test.

Table 2 Histological analysis with morphometry and melanomacrophage centers counting in spleen of Nile tilapia fed supplemented diet with propolis and Aloe barbadensis at concentrations 1%. CMM

Amount (nο) Area (mm2)

Treatments Supplemented 1%

Not Supplemented

20.52 ± 4.28a 0.0952 ± 0.0383a

15.37 ± 8.99a 0.0711 ± 0.0257a

Lower case letters indicate a significant difference between the analyzed groups (p < 0.05) by the Tukey test. CMM: Melanomacrotic centers, VTB: total spleen volume.

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propolis. Catalase is present in all vertebrate tissues, with high erythrocyte activity [59]. Studies suggest that catalase is more effective in controlling oxidative stress when intracellular concentrations of hydrogen peroxide (H2O2) are high. H2O2 is a product of cellular metabolism in organisms exposed to atmospheric oxygen and is related to several pathologies related to oxidative stress [60]. Toxic to cells, H2O2 has to be rapidly converted into a chemical species that is innocuous. Some cells of the immune system produce H2O2 for use as an antibacterial agent. As catalases are located in the peroxisomes of most cells and are involved in the metabolism of fatty acids, changes in their activity may be difficult to interpret. Therefore, the catalytic activity in erythrocytes may be a more appropriate marker for oxidative exposure in fish [61]. Immunomodulatory substances may increase host defense mechanisms (immunostimulants) or decrease them (immunosuppressants) [62]. In this study, the influence of the supplementation of propolis and Aloe extracts on the immunomodulation in tilapias was confirmed by the evaluation of the survival of the animals after challenge with A. hydrophila. Active compounds of medicinal plants are considered promising as therapeutic resources [63] which could be used as treatment of immunosuppressive and immunodeficient states [64]. The supplemented animals also showed a significant increase in the number of lymphocytes in the evaluation of the blood parameters and, consequently, a greater presence of centers of melanomacrophages. In addition, in the supplemented animals, the increase in GR levels (important cellular antioxidant) reinforces the importance of the use of plants and alternative products as a source of immunomodulating substances. However, it still needs more studies, especially regarding the specific mechanisms of action of these agents in the animal organism.

capture and present microbial antigens and effector cells that can eliminate pathogens [43] and mature lymphocytes are produced in the lymphoid organs. In teleosts, hematopoiesis occurs mainly in the stroma of the spleen and in the interstitial tissue of kidney and, to a lesser extent, in the peri-portal areas of the liver, submucosa of the intestine and thymus [44], since fish are devoid of bone marrow and of lymph nodes [45]. On this view, the study of the spleen of the animals supplemented with the mixture of extracts in this work was extremely important. Analyzes such as antioxidant enzyme activity and histology characterizing melanomacrophage centers act as sensitive indicators of stressful conditions and/or toxicity in the aquatic environment. A concern in assessing the efficacy of natural substances in the treatment of aquatic animals is the possibility that they may lead to intoxication, allergy or intolerance rather than to animal health benefits. In fish, melanomacrophages can be found free or clustered, forming discrete centers in which leukocytes are also found, but in salmonids and cartilaginous fish they are not discreet, because they have a high proportion of pigments and are distributed by the tissues in which they occur [46]. The increase in melanomacrophage centers may be due to their involvement in detoxification processes [47] and with involvement in innate and adaptive immunity [48]. Following observations on cichlids, Fishelson [49] concluded that an abnormal increase in the number of melanomacrophage centers identifies environmental stress. Melanomacrophages of the spleen of Nile tilapia was studied after inclusion of the homeopathic nucleus Homeopatila 100® in the diet showing variation between 14.94 and 27.96 in CMM size [50], similar to that found in this study for the same species. The melanomacrophage centers have important physiological functions such as erythrocyte storage [51], catabolism of erythroid tissues [52] and the reuse of substances that can be reused in hematopoiesis [53]. Changes in melanomacrophage centers have been considered as reliable biomarkers [54] and sensitive biomarkers of immunotoxicology [55]. However, in this study there was no significant difference between supplemented and non-supplemented animals and could be indicative that the substances used did not impair the health of the animals. All living organisms have an intracellular environment of reductive nature, and there is a balance between oxidized and reduced forms of molecules such as nicotinamide adenine dinucleotide (NADH), a balance maintained by enzymes at the expense of metabolic energy. Disturbances in this redox balance can lead to the production of peroxides and free radicals that damage all cellular components, including proteins, lipids and DNA. To protect cells against damage from free radicals or Reactive Oxygen Species (ROS), organisms have efficient antioxidant defense systems [56,57]. These cellular systems can be enzymatic and non-enzymatic, and act in preventing the formation of ROS, inactivating the oxidative reactions or repairing the damage caused. Among the enzymatic antioxidant systems are the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST). In the present study, the activity of the GST, GPx and G6PDH enzymes in the spleen did not present a significant difference between treatments. In this study, the activity of the antioxidant enzymes glutathione reductase (GR) in the spleen presented a significant difference in fish supplemented with 1% of the extracts mixture, being superior in the animals injected with PBS when compared to those challenged with A. hydrophila. Kuang et al. [58] studying the activity of antioxidant enzymes in Cyprinus carpio spleen and kidney supplemented with methionine hydroxyl analogues (MHA) and challenged with A. hydrophila, observed that only GR activity of the spleen had a positive correlation with levels of supplemented substance in the diet, all other parameters presented negative correlation. The authors suggest that enzymatic activity may present great discrepancy between different organs of the animal organism. As in this assay [4], also reported increased CAT activity in the spleen of Oncorhynchus mykiss supplemented with

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