Aquaculture 358–359 (2012) 79–84

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Oxidative status and gut morphology of white sea bream, Diplodus sargus fed solublenon-starch polysaccharide supplemented diets

P. Enes a,⁎, A. Pérez-Jiménez a,b, H. Peres a, A. Couto a,c, P. Pousão-Ferreira d, A. Oliva-Teles a,c

a CIMAR/CIIMAR- Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas 289, 4050‐123 Porto, Portugalb Departamento Zoología, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, 18071 Granada, Spainc Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, Edifício FC4, 4169‐007 Porto, Portugald IPIMAR/CIMSul, Av. 5 de Outubro s/n, 8700‐305 Olhão, Portugal

⁎ Corresponding author. Tel.: +351 22 040 2736; faxE-mail address: enes@portugalmail.com (P. Enes).

0044-8486/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.aquaculture.2012.06.020

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 March 2012Received in revised form 14 June 2012Accepted 18 June 2012Available online 26 June 2012

Keywords:Guar gumHistologyIntestineOxidative statusWelfareWhite sea bream

The present work aims to study the effect of a soluble non-starch polysaccharide (NSP), guar gum on whitesea bream intestinal health through oxidative status and distal intestine morphology evaluation. A controldiet was formulated to contain 40% crude protein, 14% crude lipids and 35% pregelatinized maize starch.Three other diets were formulated to include the same levels of all ingredients as control diet except forguar gum, which was included at 4% (diet GG4), 8% (diet GG8) or 12% (diet GG12). Compared to the control,lower catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activities and higher SOD,glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities were observed inthe whole intestine of fish fed with diets GG4 and GG12, respectively. Thiobarbituric acid-reacting substances(TBARS) levels were unaffected by diet composition. No morphological alterations in the distal intestine werefound among groups. Overall, data on key enzymes of the antioxidant enzymatic defense mechanismsuggested that diet-induced reactive oxygen species (ROS) generation is dependent on guar gum inclusionlevels and low dietary inclusion of soluble NSP (up to 4%) may have a gut oxidation stress prevention effect.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Non-starch polysaccharides (NSPs) include a wide variety of mole-cules that can be classified according to their chemical–physical proper-ties (Sinha et al., 2011). According to the classification proposed byBailey (1973), NSPs can be divided into three main groups: cellulose,non-cellulosic polymers and pectic polysaccharides. In aquafeeds NSPsare present as components of plant ingredients, as non-nutritive fillers,binders or pellet and faeces stabilizers. Among non-cellulosic polymers,guar gumhas been used in animal nutrition for different purposes. Guargum is a water-soluble NSP polysaccharide isolated from Cyamopsistetragonolobus seeds. Chemically it is a galactomannan composed of achain of mannose units with β-(1→4) linkages to which galactoseunits are attached by α-(1→6) linkages (Chawla and Patil, 2010). Infish nutrition, guar gum plays an important role as a binder to reducewastage from fish feeding (Amirkolaie et al., 2005; Combs andBurrows, 1958). Additionally, many studies were already conducted toknow the effects of NSPs in general and galactomannans such as guargum, in the nutrient digestibility due to its effects over digesta viscosity,alterations of gut morphology and physiology or its interaction with di-etary nutrient metabolism due to decrease of gastric emptying rate

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(Ikegami et al., 1990; Leenhouwers et al., 2006; Sinha et al., 2011;Storebakken, 1985; Storebakken and Austreng, 1987; Yamamoto andAkiyama, 1995).

The condition defined as non-infectious sub-acute enteritis wasoriginally described in the distal intestine of Atlantic salmon, Salmosalar (Baeverfjord and Krogdahl, 1996; van den Ingh et al., 1991,1996) and later in the distal intestine of other fish species (Bureau etal., 1998; Heikkinen et al., 2006; Olsen et al., 2007; Romarheim et al.,2008; Uran et al., 2008; Yamamoto et al., 2008) in response to anti-nutritional factors present in soybean meal, but no unique causingagent was yet identified. Regarding NSPs effects on gut morphology,several studies have been conducted mainly in monogastric farm ani-mals and observed results include increased size and length of digestiveorgans, reduced villi length, increased depth of intestinal crypts, dehy-dration related to impaired water absorption and increased rate of in-testinal cells turnover (Sinha et al., 2011). In fish, studies are scarce. InAfrican catfish, Clarias gariepinus the intestinal somatic index tendedto increase with dietary guar gum supplementation denoting an in-creased rate of cell proliferation, cell size and protein synthesis(Leenhouwers et al., 2006). In contrast, in Atlantic salmon fed dietswith either cellulose, native or extruded NSPs, alone or in combinationshowed no effects on gut morphology (Kraugerud et al., 2007).

An increasing number of studies aimed to study the possibleimmunostimulating effect of NSPs in fish. Several mechanisms could

Table 1Ingredient composition and proximate analysis of the experimental diets.

Diets

Control GG4 GG8 GG12

Ingredients (% dry weight basis)Fish meala 46.9 44.9 43.0 41.3Soluble fish protein concentrateb 5.7 5.4 5.2 5.0Cod liver oil 8.8 8.4 8.0 7.7Pregelatinized maize starchc 35.2 33.7 32.3 31.0Guar gumd – 4.3 8.3 12.0Vitamin premixe 0.6 0.5 0.5 0.5Mineral premixf 1.1 1.1 1.0 1.0Choline chloride 0.6 0.5 0.5 0.5Binderg 1.1 1.1 1.0 1.0

Proximate analysis (% dry weight basis)Dry matter 92.4 93.6 94.4 94.9Crude protein 41.2 39.7 38.1 36.4Crude fat 13.6 12.9 12.2 11.7Ash 10.5 10.1 9.9 9.7Starch 34.2 32.0 32.5 30.5Gross energy (kJ g−1 DM) 21.2 20.7 14.7 14.8

DM, dry matter; CP, crude protein; GL, gross lipid.a Pesquera Diamante, SteamDried LT. Austral Group, S.A. Peru (CP: 75% DM; GL: 8% DM).b Sopropèche G, France (CP: 79.4% DM; GL: 19.7% DM).c C-Gel Instant— 12016, Cerestar, Mechelen, Belgium.d Labsolve. Lisboa, Portugal.e Vitamins (mg kg−1 diet): retinol, 18 000 (IU kg−1 diet); cholecalciferol, 2 000 (IU kg−1

diet); alpha tocopherol, 35;menadion sodium bis., 10; thiamin, 15; riboflavin, 25; Ca panto-thenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin,1.5; ascorbyl monophosphate, 50; inositol, 400.

f Minerals (mg kg−1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate,200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganeseoxide, 26; sodiumselenite, 0.66; zinc oxide, 37.5; potassiumchloride, 1.15 (g kg−1 diet); so-dium chloride, 0.44 (g kg−1 diet).

g Aquacube. Agil, England.

80 P. Enes et al. / Aquaculture 358–359 (2012) 79–84

be involved in this beneficial effect. Thus, some NSPs includinggalactomannan have the ability to adhere to the same receptors thatare used by pathogenic microbes, compromising their capacity of colo-nisation (Kumar et al., 2005; Raa, 2000; Sinha et al., 2011). On the otherhand, NSPsmay enhance phagocytesmediated non-specific cellular de-fencemechanisms (Ali et al., 1996; Kumar et al., 2005). Under these cir-cumstances, an increase of reactive oxygen species (ROS) to kill sometypes of bacteria occurs. Under acute or uncontrolled circumstancesthis may compromise cellular integrity and overall animal health andwelfare.

Besides phagocytosis processes, free radical and ROS are continuouslygenerated as products of cells oxidative metabolism in all aerobic organ-isms, includingfish (Winston andDiGiulio, 1991). In this sense, the nutri-ent metabolism alterations induced by NSPs in fish nutrition, like thoseobserved in Nile tilapia, Oreochromis niloticus and African catfish, inwhich intestinal volatile fatty acid (VFA) production was increased byNSPs (Amirkolaie et al., 2006; Leenhouwers et al., 2007), may also inducea modification in normal cellular oxidative balance. To minimize ROS re-lated cell damage, fish as other aerobic organisms possess an antioxidantdefence scavenging systembased on non-enzymatic (vitamins E, C, carot-enoids, amino acids, glutathione, etc.) as well as enzymatic mechanisms(Halliwell and Gutteridge, 2007; Martínez-Álvarez et al., 2005). Key en-zymeswith antioxidant activity are superoxide dismutase (SOD), catalase(CAT) and glutathione peroxidase (GPX). Additionally, other enzymessuch as glutathione reductase (GR) and glucose-6-phosphate dehydroge-nase (G6PD) also play an important role in the maintenance of anadequate reduced/oxidized glutathione ratio. Under homeostasis, antiox-idant defences prevent the uncontrolled generation of ROS by the cells.However, when ROS generation rate exceeds that of removal oxidativestress occurs (Sies, 1985; Tocher et al., 2002). The oxidative status is con-ditioned by multiple biotic and abiotic factors, including phylogeneticposition (Rudneva, 1997), age (Díaz et al., 2010; Rudneva, 1999), environ-mental conditions (Grim et al., 2010; Lushchak, 2011; Martínez-Álvarezet al., 2002; Pérez-Jiménez et al., 2012a) and nutritional condition (Bayiret al., 2011; Morales et al., 2004; Olsvik et al., 2011; Pérez-Jiménez et al.,2009, 2012b). In this sense, nutrition plays an important role in animalwelfare by maintaining this fragile oxidative balance, either by supplyingnutrients that enhance the antioxidant system or avoiding those thatwould induce an increase of ROS production by different physiologicalpathways. Most studies in this area were conducted to evaluate the po-tential effect of micronutrients such as vitamins and minerals (Bell et al.,1987; Hidalgo et al., 2002; Kiron et al., 2004; Stephan et al., 1995;Tocher et al., 2002) whereas macronutrient role in oxidative status hasbeen less researched (Álvarez et al., 1998; López Bote et al., 2001;Pérez-Jiménez et al., 2009; Rueda-Jasso et al., 2004). Unfortunately, toour best knowledge, no studies regarding the effect of NSPs on fish oxida-tive statuswere yet performed. In pigs it was however shown that dietaryfiber at an inclusion level of 10% resulted in higher number of epithelialcells exhibiting DNA fragmentation in jejunum and ileum compared tothe control group thus denoting programmed cell death (Jin et al.,1994). In contrast, in rats it was shown that dietary fibers can protectenterocytes from oxidative damages (Kawaguchi et al., 1999; Kohen etal., 1993).

White sea bream, Diplodus sargus is a Mediterranean sparid withpotential for aquaculture diversification due to its high marketvalue, easy adaptation to captivity and production technology similarto that of other sparids (Abellán and Basurco, 1999). Moreover, it is anomnivorous species which tolerates relatively high levels of dietarycarbohydrates (Sá et al., 2008) and thus is well positioned for usingplant feedstuffs in order to reduce feed costs and environmental im-pacts. However, no study using dietary NSPs supplementation hasbeen yet performed in this species.

Taking into account the scarce studies performed with dietaryNSPs inclusion in fish nutrition, the aim of the present study was toevaluate the potential effects of soluble NSP - guar gum - on intestinalmorphology and oxidative status of white sea bream juveniles.

2. Materials and methods

2.1. Experimental diets

A control diet was formulated to contain 40% crude protein, 14%crude lipids and 35% pregelatinized maize starch. Three other dietswere formulated to include the same levels of all ingredients as controldiet except for the soluble guar gum, which was included at 4% (dietGG4), 8% (diet GG8) or 12% (diet GG12). No adjustment to diet compo-sition was otherwise made. All dietary ingredients were finely ground,well mixed and dry pelleted in a laboratory pellet mill (CPM) througha 3 mm die. Diets were then air-dried in an oven at 40 °C for 24 h andstored in plastic containers until used. Ingredients and proximate com-position of the experimental diets are presented in Table 1.

2.2. Animals and experimental conditions

This experiment was directed by trained scientists (followingFELASA category C recommendations) and was conducted accordingto European Union Directive 2010/63/EU on the protection of animalsfor scientific purposes. The growth trial was performed at the MarineZoology Station, Porto University (Portugal) in a thermoregulated rec-irculating water system equipped with a battery of fiberglass tanks(100 L capacity) supplied with continuous flow of filtered seawater(6.0 L min−1), temperature regulated to 22.0±1.0 °C, salinity of37.0±1.0 g L−1 and dissolved oxygen at 95% saturation. White seabream (D. sargus) juveniles were obtained from IPIMAR/CRIPSul,Olhão, Portugal, and after transportation to the experimental facilitiesthey were submitted to a quarantine period of 15 days. Thereafter, 12groups of 10 fish with an initial mean body weight of 55 g wereestablished and each diet randomly assigned to triplicate groups. Thetrial lasted 63 days and during this period fish were fed by hand twicea day, 6 days a week on a pair-feeding scheme as follows: the group re-ceiving the control diet was fed to apparent visual satiation, while the

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other groups received 105% (diet GG4), 109% (diet GG8) and 114% (dietGG12) of the mean ration given to the control group at each daily meal.With this pair-feeding scheme it was assured that every group was fedthe same amount of all nutrients except dietary guar gum. Utmost carewas taken to assure that all food supplied was consumed.

2.3. Sampling

Fish in each tankwere bulk-weighed at the beginning and at the endof the trial, after 1 day of food deprivation. After the final weighing andin order to minimize manipulation stress, the fish continued to be fedfor 3 more days, after which 6 fish from each tank were randomly sam-pled 4 h after the morning meal and then sacrificed with a sharp blowon the head. The whole intestine was excised from 3 fish, immediatelyfrozen in liquid nitrogen and then stored at−80 °C until measurementof enzymatic activity and lipid peroxidation. The remaining 3 fish weredissected and a sample (ca. 5×5 mm) from the distal intestine, distin-guished from themid intestine by an enlarged diameter and darkermu-cosa, was collected for histological analysis. The samples were collectedand immediately fixed in phosphate buffered formalin (4%; pH 7.4) for24 h and transferred to alcohol at 70% until histological processing.

2.4. Analytical methods

2.4.1. Proximate analysisChemical composition of the diets was analysed using the following

procedures: drymatter after drying at 105 °C until constantweight; ashby incineration in a muffle furnace at 450 °C for 16 h; protein content(N×6.25) according to the Kjeldahl method, using Kjeltec digestorand distillation units (Tecator Systems, Höganäs, Sweden; model 1015and 1026, respectively); lipid by petroleum ether extraction in a SoxTecextraction system (Tecator Systems, Höganäs, Sweden; extraction unitmodel 1043 and service unit model 1046); gross energy by direct com-bustion in an adiabatic bomb calorimeter (PARR Instruments, Moline,IL, USA; PARR model 1261) and starch according to Beutler (1984).

2.5. Enzyme activity

Intestine samples were homogenized in 4 volumes of ice-cold 100‐mM Tris–HCl buffer containing 0.1‐mM EDTA and 0.1% (v/v) TritonX-100, pH 7.8. The procedure was performed on ice. Homogenateswere centrifuged at 30,000g for 30 min at 4 °C and the resultant super-natants were kept in aliquots and stored at −80 °C until use. All en-zyme assays were carried out at 25 °C and the changes in absorbancewere monitored to determine the enzyme activity using a microplatereader (ELx808; Bio-Tek Instruments). The optimal substrate and pro-tein concentrations for measurement of maximal activity of each en-zyme were established in preliminary assays. The molar extinctioncoefficients used for H2O2 and NADPH were 0.039 and 6.22 cm/mM,respectively.

Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured bythe ferricyto-chrome cmethodusing xanthine/xanthine oxidase as sourceof superoxide radicals and the reaction monitored at 550 nm (McCordand Fridovich, 1969). The reaction mixture consisted of 50‐mMpotassium phosphate buffer (pH 7.8), 0.1‐mM EDTA, 0.1‐mM xanthine,0.012‐mM cytochrome c and 0.025‐IU/ml xanthine oxidase. Activity isreported in units of SOD/mg of protein, with one activity unit defined asthe amount of enzyme necessary to produce 50% inhibition of the fer-ricytochrome c reduction rate.

Catalase (CAT, EC 1.11.1.6) activity was determined by measuringthe decrease in H2O2 concentration at 240 nm according to Aebi(1984). The reaction mixture contained 50‐mM potassium phosphatebuffer (pH 7.0) and 10‐mMH2O2 freshly added.

Glutathione peroxidase (GPX, EC 1.11.1.9) activity was measuredfollowing the method of Flohé and Günzler (1984). The oxidized gluta-thione (GSSG) generated byGPXwas reduced by glutathione reductase,

and NADPH oxidation monitored at 340 nm. The reaction mixtureconsisted of 50‐mM potassium phosphate buffer (pH 7.1), 1‐mMEDTA, 3.9‐mM reduced glutathione, 3.9‐mM sodium azide, 1‐IU/mlGR, 0.2‐mMNADPH and 0.05‐mMH2O2.

Glutathione reductase (GR, EC 1.6.4.2) activity was assayed as de-scribed by Morales et al. (2004), measuring the oxidation of NADPH at340 nm. The reaction mixture consisted of 0.1‐M sodium phosphatebuffer (pH 7.5), 1‐mM EDTA, 0.63‐mMNADPH and 0.16‐mM GSSG.

Glucose 6-phosphate dehydrogenase activity (G6PD, EC 1.1.1.49)was measured as described by Morales et al. (1990), using a reactionmixture containing 50‐mM imidazole–HCl buffer (pH 7.4), 5‐mMMgCl2, 2‐mM NADP and 1‐mM glucose-6-phosphate.

Soluble protein concentration was determined by the method ofBradford (1976), using bovine serum albumin as standard.

Except for SOD, for which the units of expression were indicatedabove, enzyme activity is expressed as units (CAT) or milliunits(G6PD, GPX and GR) permg of soluble protein, with one unit of enzymeactivity defined as the amount of enzyme required to transform 1 μmolof substrate/min under the aforementioned assay conditions.

2.6. Lipid peroxidation

Concentration of thiobarbituric acid-reacting substances (TBARS)was determined according to Buege and Aust (1978). An aliquot of ho-mogenate supernatant (100 ml) was mixed with 500 ml of a solutioncontaining 15% (w/v) TCA, 0.375% (w/v) thiobarbituric acid, 80% (v/v)HCl 0.25 M and 0.01% (w/v) butylated hydroxytoluene. The mixturewas heated to 100 °C for 15 min and after cooling to room temperaturecentrifuged at 1500g for 10 min. Absorbance in the supernatant wasmeasured at 535 nm and compared with a blank. Concentration wasexpressed as nmol malondialdehyde/g of tissue, calculated from a cali-bration curve.

2.7. Histological processing and morphological evaluation

Fixed samples of the distal intestine were routinely dehydrated inethanol, equilibrated in xylene and embedded in paraffin according tostandard histological techniques, sections cut and mounted in glassslides. Sections were then stained with hematoxylin and eosin. Blindedevaluation of the histological samples was performed using a light mi-croscope. Considering the following criteria; (1) widening and shorten-ing of the intestinal folds, (2) loss of the supranuclear vacuolization inthe absorptive cells (enterocytes) in the intestinal epithelium, (3) wid-ening of the lamina propria within the intestinal folds, (4) infiltration ofa mixed leucocyte population in the lamina propria and submucosa(Krogdahl et al., 2003). Images were acquired with software AxioVison Rel. 4.8 (Carl Zeiss, Jena, Germany).

2.8. Statistical analysis

Results are presented as means and standard errors. Data were ana-lyzed by one-way ANOVA after testing the homogeneity of varianceswith the Levene test. When F values were significant (Pb0.05), meanswere compared using Duncan multiple range test. All statistical analy-ses were carried out using the SPSS version 18.0 for Windows softwarepackage (SPSS, Inc.).

3. Results

3.1. Growth performance, feed efficiency and feed intake

Fish accepted the experimental diets well and during the trial mor-tality ranged from 3 to 7% and was not statistically different amonggroups (Table 2). Final body weight, weight gain, feed intake and feedefficiency were all similar among groups. As expected, guar gum intake

82 P. Enes et al. / Aquaculture 358–359 (2012) 79–84

significantly increased with the increase of dietary guar gum inclusionlevel (Table 2).

3.2. Enzyme activities and lipid peroxidation

Specific activities of antioxidant enzymes, G6PD and TBARS levels inthe intestine are shown in Table 3. SOD, CAT and GR activities werelower in fish fed diet GG4 than in the control while SOD, GPX andG6PD activities were higher in fish fed the diet GG12 than in all othergroups. TBARS levels were not affected by diet composition, althougha trend to increase with dietary guar gum levels was noticed.

3.3. Distal intestine morphology

No differences were noticed among groups on the intestinal mor-phology, height or density of the intestinal villous (Fig. 1). The epitheli-um consisted of a single layer of prismatic cells with apical microvilli—enterocytes — and goblet cells. Enterocytes showed basal nuclei andthere were no differences in number or size of vacuoles between thefish fed the control and the guar gum supplemented diets. Epithelial in-tegrity was similar among groups, with no signs of hypertrophy or hy-perplasia of the goblet cells and minimal number of intraepithelialleucocytes being noticed. The lamina propria was thin, without in-creased cellularity and the submucosa showed similar width in allgroups. The muscular layer also showed no differences among groupsin the thickness and morphological aspect of its circular and longitudi-nal layers or on the very thin connective tissue separating the twomus-cular layers.

4. Discussion

The majority of studies considering the effects of NSPs on gut mor-phology were performed in farm monogastric animals and reportedintestinal pathologies related to dietary NSP. An increased rate of intesti-nal cells turnover characterized by decreased villous length and in-creased crypt depth associated with increased rate of cell proliferationand evidence of programmed cell death was reported by several authorsin pigs fed high dietary fibre food (see review by Sinha et al., 2011). Al-though Sinha et al. (2011) considered that similar effects might as welloccur in fish, present data showed an absence of morphological alter-ation in the distal intestine of white sea bream fed guar gum. Similarly,in Atlantic salmon no gut morphology alterations were noticed in fishfed a diet including 10% cellulose or purified soy-NSP (Kraugerud et al.,2007).

Antioxidant defence mechanisms are initiated by SOD which dis-mutases superoxide anion (O2

−) to oxygen and hydrogen peroxide

Table 2Growth performance and feed utilization efficiency of white sea bream fed the exper-imental diets.

Diets

Control GG4 GG8 GG12

Final body weight (g) 99.7±2.9 103.5±2.2 100.3±1.2 96.2±1.4Weight gain(g kg/ABM†/day)

8.0±0.2 8.4±0.1 8.3±0.2 7.8±0.2

Feed intake(g kg/ABM†/day)

14.5±1.1 15.3±0.5 16.0±0.2 16.8±0.1

Guar gum intake(g kg/ABM†/day)

– 0.61±0.03a 1.28±0.02b 2.01±0.02c

Feed efficiency1 0.56±0.04 0.55±0.02 0.52±0.00 0.46±0.01Mortality 6.7±11.5 6.7±11.5 3.3±5.8 0.0±0.0

Values are means±S.E. (n=3).For each parameter, letters in the same column indicate differences (Pb0.05) due todiet composition.

† Average body weight: (initial body weight+final body weight)/2.1 FE: (wet weight gain/dry feed intake).

(H2O2) (Halliwell and Gutteridge, 2007). In the present study, SOD activ-ity in the intestine increased as dietary guar gum increased, which sug-gests a rise in O2

− generation related to soluble NSP levels. It is knownthat guar gum tends to increase digesta viscosity and thus delay gastricemptying rate in Nile tilapia, African catfish and rainbow troutOncorhynchus mykiss (Amirkolaie et al., 2005; Leenhouwers et al.,2006; Storebakken, 1985). This delay of digesta passage through the gutmay stimulate NSPs fermentation by intestine microbiota thus leadingto an increase of VFA production. As in mammals, these VFA mighthave beenmobilized for energy production, leading to a rise of O2

− levelsand thus an increase of SOD activity (Choct and Kocher, 2000). Accord-ingly, feeding Nile tilapia and African catfish with diets including NSPslead to intestinal VFA production, with acetic acid as the main VFA pro-duced followed by very low levels of propionic and butyric acids(Amirkolaie et al., 2006; Leenhouwers et al., 2007). Another possibilityfor the suggested rise of O2

− levels would be an increase in intestinal mu-cosal cell turnover and consequently need of energy supply for the pro-cess in fish fed increasing levels of NSPs, as observed in pigs fed highdietaryfiber (Jin et al., 1994). However, if such increased cell turnover oc-curred in the present study it was not associated with modifications ondistal intestine morphology, although the occurrence of morphologicalmodifications in other intestinal portions cannot be disregarded.

The increase of SOD activity results in increasedH2O2 levels and thusa rise in CAT and/or GPX activities are expected to happen, as these en-zymes are involved in the detoxification of H2O2 to molecular oxygenand water (Halliwell and Gutteridge, 2007). In this study, only GPX ac-tivity was higher in fish fed diet GG12 than in the control. Overall, GPXactivitywas alsomuchmore important than that of CAT suggesting thatit was the main responsible for detoxification of H2O2.

An absence of good correlation between SOD and CAT activities wasalso reported in other studies in fish (Cooper et al., 2002; García-Medinaet al., 2010; Pascual et al., 2003; Pérez-Jiménez et al., 2009, 2012b). Con-trary to GPX, which is induced by low H2O2 levels, CAT is known to beactive preferentially when H2O2 production is high (Halliwell andGutteridge, 2007). Therefore, it is possible that H2O2 produced in thepresent study was not high enough to induce high CAT response. Be-sides being involved in the breakdown of H2O2, GPX also reduces organ-ic peroxides (ROOH) to the corresponding stable alcohols (ROH). Thus,if VFA production increased due to NSPs microbial fermentation, an in-crease in organic peroxides level is also expected to occur as result oflipid catabolism also contributing for the increment of GPX activity inthe GG12 group.

Glutathione reductase plays a crucial role in the modulation of GPXactivity as it catalyses the NADPH-dependent regeneration of reducedglutathione (GSH) from the oxidized form (GSSG) generated by GPX(Halliwell and Gutteridge, 2007). Thus, higher GR activity was to beexpected in fish fed diet GG12, in an attempt to maintain the GSSG:GSH ratio. Although not statistically significant, a tendency for GR activ-ity increase with dietary guar gum was noticed in this study.

Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme ofthe pentose phosphate pathway and is involved in NADPH regenera-tion,which plays an important role in the protection of cells fromoxida-tive stress, as it is a coenzyme of GR and it is also essential for CATactivity (Kirkman et al., 1999; Leopold and Loscalzo, 2000; Pandolfi etal., 1995; Scott et al., 1991). A correspondence between G6PD and GRactivities is thus expected to occur and that was indeed observed inthe present study.

Fish fed diet GG4 showed lower SOD, CAT andGR activity levels thanthe control group. Thus, in contrast towhat was observed for fish fed ondiet GG12, these results suggest that diet GG4 was responsible for a de-crease in O2

– generation. Such a decrease can be related with a decreasein fat digestion and absorption and thus on lipid catabolism. In fact, inNile tilapia and rainbow trout a decrease in fat digestibility was ob-served with guar gum dietary inclusion levels up to 8% due to an in-crease of digesta viscosity (Amirkolaie et al., 2005; Storebakken,1985). A reduction in fat digestibility was also reported in Atlantic

Fig. 1. Histomorphological appearance of the distal intestine of white sea bream fed the experimental diets: (A) Control; (B) GG4; (C) GG8; (D) GG12. (H–E staining; original mag-nification ×100).

Table 3Specific activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD) and ox-idative damage biomarker for lipids in the whole intestine of white sea bream fed the experimental diets.

Diets SOD CAT GPX GR G6PD Lipidperoxidation

Control 243.4 ±18.7b 58.9 ±5.6b 184.2 ±11.1a 40.8 ±2.7b 14.4 ±1.1a 65.3 ±7.5GG4 182.3 ±6.6a 41.5 ±3.3a 159.7 ±11.2 a 31.2 ±2.2 a 10.6 ±1.1 a 50.9 ±5.9GG8 214.6 ±20.5ab 63.7 ±6.6b 160.7 ±11.9a 38.0 ±3.7ab 15.4 ±1.4a 60.1 ±6.6GG12 294.3 ±22.8c 63.0 ±3.6b 220.8 ±13.2b 45.0 ±3.5b 32.3 ±2.7b 72.4 ±9.3

Values are means±S.E. (n=9). CAT and SOD are expressed as U mg protein−1. GPX, GR and G6PD are expressed as mU mg protein−1. Lipid peroxidation is expressed as nmolmalondialdehyde (MDA) g tissue−1. For each parameter, letters in the same column indicate differences (Pb0.05) due to diet composition.

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salmon fed a soybeanmeal diet with 4% of soluble NSPs (Øverland et al.,2009). Such an effectwas relatedwith theNSP content of the diet whichreduces fat absorption by disturbing micelle formation in the gastroin-testinal tract. It is also possible that dietary guar gum at an inclusionlevel of 4%may act as an antioxidant per se. Accordingly, in vitro studiesperformed with rat jejune mucosa showed that pectin, also a solubleNSP efficiently scavenged peroxy (ROO), O2

− and hydroxyl (OH) radi-cals, thus preventing oxidative stress (Kohen et al., 1993). Moreover,pectin, alginate and cellulose were able to protect small intestinal mu-cosa of rat from oxidative damage, by depressing lipid catabolism(Kawaguchi et al., 1999).

Malonaldehyde (MDA) concentration is one of themostwidely usedindices for assessing oxidative stress in animals as thismetabolite is oneof the most abundant end product of lipid peroxidation (Moore andRoberts, 1998). In the present studyMDA concentrationwas unaffectedby dietary guar gum level, suggesting that diet-induced ROS generationwas effectively counteracted by the fish antioxidant enzymatic system.

In conclusion, data on key enzymes of the antioxidant enzymatic de-fensemechanism suggested that diet-inducedROS generation is depen-dent on guar gum inclusion levels and that low dietary inclusion ofsoluble NSP (up to 4%) might have a gut oxidation stress prevention ef-fect. Overall, the inclusion of plant feedstuffs with soluble NSPs up to12% can be used in with sea bream diets without compromising intesti-nal health.

Acknowledgments

The present work was supported by Fundação para a Ciência e aTecnologia (pluriannual funding) through the PIDDAC Program fundsfrom the Portuguese government. The first author was supported bya grant (BPD/39688/2007) from Fundação para a Ciência e aTecnologia, Portugal. The second author was supported by a grant(BPD/64684/2009) from Fundação para a Ciência e a Tecnologia,Portugal. A. Couto was supported by a grant (BD/47495/2008) fromFundação para a Ciência e a Tecnologia, Portugal. We would like toexpress our thanks to Mr. P. Correia for the technical assistance dur-ing the trial.

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