JOURNAL OF THEWORLD AQUACULTURE SOCIETY

Vol. 45, No. 3June, 2014

doi: 10.1111/jwas.12120

Effects of Dietary Supplementation of Barodon on GrowthPerformance, Innate Immunity and Disease Resistance of JuvenileOlive Flounder, Paralichthys olivaceus, Against Streptococcus iniae

Chang-Hoon Shin

Department of Animal Life system, Kangwon National University, Chuncheon 200-701,South Korea

Hien Thi Dieu Bui, Samad Rahimnejad, Ji-Hoon Cha

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, South Korea

Byung-Woo Yoo, Bo-Kyeun Lee and Hyung-Jin Ahn

Cargill Agri Purina Inc, Seongnam, South Korea

Soo-Il Choi and Yun-Jeong Choi

Barodon – S.F. Corp, Ansung, South Korea

Yong-Ho Park

College of Veterinary Medicine, Seoul National University, Seoul, South Korea

Jeong-Dae Kim

Department of Animal Life system, Kangwon National University, Chuncheon 200-701,South Korea

Kyeong-Jun Lee1

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, South Korea

AbstractThis study was conducted to evaluate the effects of dietary supplementation of Barodon, an anionic

alkali mineral complex, on growth, feed utilization, humoral innate immunity and disease resistance ofolive flounder. A basal experimental diet was used as a control and supplemented with 0.1, 0.2, 0.3, 0.4,or 0.5% Barodon. Triplicate groups of fish (26.4± 0.2 g) were fed one of the diets to apparent satiationtwice daily for 10 wk. The growth performance was enhanced (P< 0.05) linearly and quadraticallyin fish fed diets containing Barodon compared with that in fish fed the control. Feed utilization wassignificantly improved by Barodon supplementation. Serum lysozyme and antiprotease activities wereincreased quadratically in Barodon fed groups. Also, significantly higher superoxide dismutase activitywas found in Barodon-fed fish. Dietary supplementation of 0.1–0.3% Barodon resulted in significantenhancement of fish disease resistance against Streptococcus iniae. The findings in this study indicatethat dietary supplementation of Barodon can enhance growth, feed utilization, innate immunity, anddisease resistance of olive flounder and that the optimum level seems to be 0.1% in diets.

Disease outbreaks cause significant economiclosses in aquaculture production. Traditionally,antibiotics have been used for treatment of

1 Corresponding author.

the infectious bacterial diseases in aquacul-ture. Nevertheless, there have been concernsregarding the serious side effects of antibioticssuch as the development of antibiotic resistantstrains of bacteria and the presence of antibiotic

© Copyright by the World Aquaculture Society 2014

258

BARODON IMPROVES INNATE IMMUNITY IN OLIVE FLOUNDER 259

residues in aquaculture products (Nakanishiet al. 2002; Sahu et al. 2007). Vaccine treat-ment is considered as one of the most effectiveways in controlling fish diseases. However, thepractical application of fish vaccines in fishfarming faces several restrictions includinghigh handling costs, treatment stresses, andpathogen specificity (Siwicki et al. 1994; Ardóet al. 2008). Therefore, during the last twodecades immunostimulants have emerged as apromising approach for disease management inaquaculture (Anderson 1992; Sakai 1999; Misraet al. 2006). Immunostimulants can improve theoverall resistance of fish to infectious diseasescaused by various pathogens by enhancing theinnate immunity (Sakai 1999). Fish encounterhigh concentration of various pathogenic agentsin ambient water, therefore, enhancement ofthe nonspecific immunity seems to be one ofthe most effective therapies in prevention offish disease (Ellis 2001). Immunostimulantsinclude a wide variety of synthetic chemicals,polysaccharides, bacterial components, animalor plant extracts, and nutritional factors (Raa1996; Sakai 1999; Ringø et al. 2012; Liu et al.2013).

Barodon is an anionic alkali mineral complexsolution. The main compositions of Barodoninclude silicon, sodium, and potassium ions inan alkaline solution (pH 13.5). Barodon has beenpatented as a nonspecific immunostimulator inthe USA, Korea, and South Africa (Choi et al.2002a; Choi et al. 2002b; Choi et al. 2004a,2004b). Immunomodulatory activity of Barodonhas been evaluated in vivo in terrestrial animals,such as swine (Yoo et al. 2001, 2002) and equine(Koo et al. 2006). However, there is limitedinformation on Barodon in fish.

Olive flounder, Paralichthys olivaceus, hasbeen the most important marine aquaculturespecies in Korea. Its production increasedsharply from 48,073 m.t. in 2001 to 91,125 m.t.in 2006 (Kim et al. 2007). However, lossescaused by infectious bacterial diseases havebeen a main constraint to its culture. Strep-tococcosis caused by Streptococcus iniaeis one the most frequently occurred dis-eases in flounder farms with high mortalities(Pham et al. 2006). Therefore, this study was

undertaken to examine supplemental effectsof Barodon on growth, feed utilization, innateimmunity, and disease resistance of oliveflounder against S. iniae.

Materials and Methods

Experimental Diets

A basal experimental diet (47% crude pro-tein, 21.8 MJ/kg gross energy) was prepared andregarded as a control and supplemented with 0.1,0.2, 0.3, 0.4, or 0.5% Barodon (designated as,Control, 1×, 2×, 3×, 4×, and 5×, respectively).Composition of Barodon used in this study isshown in Table 1. Formulation and proximatecomposition of the experimental diets are shownin Table 2. All dry materials were thoroughlymixed with squid liver oil and double distilledwater containing Barodon solution. The mixturewas extruded through a meat grinder (SMC2,Kuposlice, Busan, Korea) at 3 mm in diameter.Diets were dried with electric fans at room tem-perature and stored at −20 C until used.

Experimental Fish and Feeding Trial

Juvenile olive flounder were transported froma local fish farm in Jeju Island to Marine andEnvironmental Research Institute, Jeju NationalUniversity (Jeju, Korea). The fish were fed thecontrol diet for 2 wk to be acclimated to theexperimental diets, conditions, and facilities.At the end of the acclimation period, the fish(initial mean body weight, 26.4± 0.2 g) wererandomly distributed into 18 polyvinyl circulartanks of 200 L capacity at a density of 45 fish pertank. The tanks were supplied with sand-filtered

Table 1. Composition of major ingredients for Barodon.1

Ingredient Amount

Na2SiO3 30 gK2CO3 15 gNa2CO3 0.45 gC12H22O11 q.s.AgNO3 q.s.NaCl q.s.Na2S2O3 q.s.

q.s.= quantum satis.1Composition is based on g per 1000 mL H2O.

260 SHIN ET AL.

Table 2. Formulation and proximate composition of thesix experimental diets (% dry matter).

Diets

Control 1× 2× 3× 4× 5×

IngredientsWhite fish

meal50.0 50.0 50.0 50.0 50.0 50.0

Soybean meal 6.0 6.0 6.0 6.0 6.0 6.0Corn gluten

meal6.0 6.0 6.0 6.0 6.0 6.0

Wheat flour 24.0 24.0 24.0 24.0 24.0 24.0Mineral mixa 1.0 1.0 1.0 1.0 1.0 1.0Vitamin mixb 1.0 1.0 1.0 1.0 1.0 1.0Squid liver oil 11.0 11.0 11.0 11.0 11.0 11.0Carboxymethyl

cellulose1.0 1.0 1.0 1.0 1.0 1.0

Barodonc 0 0.1 0.2 0.3 0.4 0.5Chemical composition (% dry mater)

Moisture 10.7 10.8 10.8 10.9 10.8 10.9Protein 47.1 46.5 47.1 47.1 46.1 46.9Lipid 15.2 15.2 15.4 15.5 15.2 15.0Ash 15.2 15.2 15.4 15.5 15.2 15.0Gross energy,

MJ/kgd21.8 21.8 21.8 21.9 21.7 21.7

aMineral premix (g/kg mixture): l-ascorbic acid, 121.2;dl-αtocopheryl acetate, 18.8; thiamin hydrochloride, 2.7;riboflavin, 9.1; pyridoxine hydrochloride, 1.8; niacin, 36.4;Ca-d-pantothenate, 12.7; myo-inositol, 181.8; d-biotin, 0.27;folic acid, 0.68; p-aminobezoic acid, 18.2; menadione, 1.8;retinyl acetate, 0.73; cholecalficerol, 0.003; cyanocobalamin,0.003.

bVitamin premix (g/kg mixture): MgSO4.7H2O, 80.0;NaH2PO4.2H2O, 370.0; KCl, 130.0; Ferric citrate,40.0; ZnSO4.7H2O, 20.0; Ca-lactate, 356.5; CuCl2,0.2; AlCl3. 6H2O, 0.15; Na2Se2O3, 0.01; MnSO4.H2O, 2.0;CoCl2.6H2O, 1.0

cBarodon was provided by Barodon – SF Corp, Ansung,Gyounggi, 456-880, Korea.

dGross energy of experimental diets was calculatedaccording to gross energy values 5.64 kcal/g crude protein,4.11 kcal/g carbohydrate, and 9.44 kcal/g crude fat, respec-tively (NRC 1993).

seawater at a flow rate of 3 L/min and aeration tomaintain enough dissolved oxygen. A photope-riod of 12-h light and 12-h dark was used. Theaverage water temperature during the feedingtrial was 22.0± 1.8 C. Triplicate groups of fishwere fed one of the experimental diets to appar-ent satiation (twice a day, 0800 and 1800 h) for10 wk. Uneaten feed was collected 30 min afterfeeding and reweighed to determine feed intake.The growth of fish was measured every 3 wk. In

order to minimize stress on the fish, feeding wasstopped 24 h prior to weighing.

Sample Collection

At the end of the feeding trial, all the fish ineach tank were bulk-weighed and counted forcalculation of growth parameters and survival.Three fish per tank (nine fish per dietary treat-ment) were randomly captured and anesthetizedwith 2-phenoxyethanol solution (200 mg/L) andblood samples were taken from the caudal veinwith heparinized syringes for determination ofhematocrit, hemoglobin, and respiratory burstactivity. Then plasma samples were separated bycentrifugation at 5000 g for 10 min and stored at−70 C for determination of total immunoglobu-lin (Ig) level. Another set of blood samples wastaken from the caudal vein of three fish from eachtank using nonheparinized syringes and allowedto clot at room temperature for 30 min. Thenthe serum was separated by centrifugation for10 min at 5000 g and stored at−70 C for the anal-ysis of lysozyme, superoxide dismutase (SOD),antiprotease, myeloperoxidase (MPO), andglutathione peroxidase (GPX) activities.

Analytical Methods

Hematocrit was determined by the micro-hematocrit technique (Brown 1980) andhemoglobin was determined by using an auto-mated blood analyzer (Slim, Seac Inc, Florence,Italy).

Analysis of moisture and ash contents ofdiets was performed by the standard procedures(AOAC 1995). Crude protein was measured byusing automatic Kjeltec Analyzer Unit 2300(FossTecator, Höganäs, Sweden) and crude lipidwas determined using the Soxhlet method withextraction in diethyl ether (Soxhlet ExtractionSystem C-SH6, Seoul, Korea).

The oxidative radical production by phago-cytes during respiratory burst was measuredthrough the nitro-blue-tetrazolium (NBT)assay described by Anderson and Siwicki(1995). Briefly, blood and NBT (0.2%) (Sigma,St. Louis, MO, USA) were mixed in equalproportion (1:1) and incubated for 30 minat room temperature. Then 50 μL was taken

BARODON IMPROVES INNATE IMMUNITY IN OLIVE FLOUNDER 261

out and dispensed into glass tubes; 1 mL ofdimethylformamide (Sigma) was added andcentrifuged at 2000 g for 5 min. Finally, theoptical density of supernatant was measured at540 nm using a spectrophotometer (DU®730,Beckman Coulter Inc, Indianapolis, IN, USA).Dimethylformamide was used as the blank.

A turbidometric assay was used to determineserum lysozyme activity using the methoddescribed by Hultmark (1980) with slightmodifications. Briefly, Micrococcus lysodeik-ticus (0.75 mg/mL) was suspended in sodiumphosphate buffer (pH 6.4, 0.1 M), 200 μL ofsuspension was placed in each well of a 96-wellplate, and 20 μL of serum was added. The reduc-tion in absorbance of samples was determinedat 570 nm in a microplate reader (UVM 340,Biochrom, Cambridge, UK) after incubationin room temperature for 0 and 30 min. Henegg white lysozyme (Sigma) was used for thestandard curve. Values are expressed as μg/mL.

Serum MPO activity was measured accord-ing to Quade and Roth (1997). Briefly, 20 μLof serum was diluted with Hanks balancedsalt solution (HBSS) without Ca2+ or Mg2+

(Sigma) in 96-well plates. Then, 35 μL of3,3′,5,5′-tetramethylbenzidine hydrochloride(TMB, 20 mM) (Sigma) and H2O2 (5 mM)were added. The color change reaction wasstopped after 2 min by adding 35 μL of4 M sulfuric acid. Finally, the optical den-sity was read at 450 nm in the microplatereader.

SOD activity was measured by the percent-age reaction inhibition rate of enzyme with watersoluble tetrazolium dye (WST) substrate andxanthine oxidase using a SOD assay kit (Sigma,19160) according to the manufacturer’s instruc-tions. Each endpoint assay was monitored byabsorbance at 450 nm (the absorbance wave-length for the colored product of WST reactionwith superoxide) after 20 min of reaction time at37 C. The percent inhibition was normalized bymg protein and presented as SOD activity units.

Serum antiprotease activity was measuredaccording to the method described by Ellis(1990) with slight modifications (Magnadóttiret al. 1999). Briefly, 20 μL of serum was incu-bated with 20 μL of standard trypsin solution

(Type II-S, from porcine pancreas, 5 mg/mL,Sigma) for 10 min at 22 C. Then, 200 μL ofphosphate buffer (0.1 M, pH 7.0) and 250 μLazocasein (2%) (Sigma) were added and incu-bated for 1 h at 22 C. Five hundred microlitersof trichloro acetic acid (10%) were added andfurther incubated for 30 min at 22 C. The mix-ture was centrifuged at 6000 g for 5 min and100 μL of the supernatant was transferred tothe wells of a 96-well flat bottomed microplatecontaining 100 μL of NaOH (1 N). Opticaldensity was read at 430 nm. For a 100% pos-itive control, buffer replaced the serum, whilefor the negative control buffer replaced bothserum and trypsin. The trypsin inhibition per-centage was calculated using the followingequation:

Trypsin inhibition (%) =(A1 –A2∕A1

)× 100

where A1 = control trypsin activity (withoutserum); A2 = activity of trypsin remained afterserum addition.

The level of plasma total Ig was determinedaccording to the method described by Siwickiand Anderson (1993). Briefly, plasma total pro-tein content was measured using a micro pro-tein determination method (C-690; Sigma), andthe Ig was precipitated from the plasma with12% solution of polyethylene glycol (Sigma).The total Ig was determined from the differ-ence in the total plasma protein before and afterprecipitation of the plasma Ig.

GPX activity was assayed using a kit (Bio-vision, Inc., Milpitas, CA, USA). In this assay,cumene hydroperoxide was used as a peroxidesubstrate (ROOH), and glutathione reductase(GSSG-R) and NADPH (b-nicotinamide ade-nine denucleotide phosphate, reduced) wereincluded in the reaction mixture. The changein 340 nm due to NADPH oxidation was moni-tored for GPX activity. Briefly, 50 μL of serumwas added to 40 μL of the reaction mixtureand incubated for 15 min and then 10 μL ofcumene hydroperoxide was added and OD1read at 340 nm. After 5 min of incubation,OD2 was read in 340 nm by the microplatereader. Activity of GPX was calculated asnmol/min/mL.

262 SHIN ET AL.

Table 3. Growth performance and feed utilization of olive flounder fed the six experimental diets for 10 wk.

Diets P-value

Control 1× 2× 3× 4× 5× SEM Linear Quadratic

IBW 26.4 26.2 26.4 26.3 26.5 26.3 0.3 0.973 0.983FBW (g) 125 145 142 143 145 145 8 0.011 0.005Survival (%) 99.3 91.9 93.3 100.0 87.4 93.3 8.5 0.161 0.386FIa (g/fish) 121 128 121 125 117 129 12 0.890 0.870FCRb 1.23 1.09 1.05 1.08 0.99 1.08 0.10 0.020 0.004PERc 1.94 2.21 2.29 2.21 2.48 2.21 0.21 0.034 0.013

FBW=final body weight; FCR= feed conversion ratio; FI= feed intake; IBW= initial body weight.aFeed intake= dry feed consumed (g)/fish.bFeed conversion ratio= dry feed fed/wet weight gain.cProtein efficiency ratio=wet weight gain/total protein given.

Bacterial Challenge

At the end of the feeding trial, 15 fish pertank (45 fish per treatment) were randomlycaptured and intraperitoneally injected with0.1 ml of S. iniae (ATCC 29178) suspension(1× 109 CFU/mL). S. iniae was provided bythe Marine Applied Microbes and AquaticOrganism Disease Control Laboratory at theDepartment of Aquatic Biomedical Sciences,Jeju National University. Injected fish weredistributed into 18 plastic tanks of 60 L capacityand their behavior and mortality were monitoredfor 34 days.

Statistical Analysis

All diets were assigned by a completely ran-domized design. Data on fish growth and innateimmune parameters were analyzed using linearand quadratic orthogonal polynomial contrastsin SPSS version 19 (SPSS Inc., Chicago, IL,USA). The results of fish survival after challengewith S. iniae were analyzed by Kaplan–Meiersurvival test. Statistical significance was deter-mined at 5% (P≤ 0.05). Percentage data werearcsine transformed before analysis.

Results

Dietary supplementation of Barodon resultedin linear (P= 0.011) and quadratic (P= 0.005)increase of growth performance (Table 3). Also,feed utilization was significantly affected byBarodon; leading to lower feed conversion ratioand higher protein efficiency ratio (P< 0.05).

However, survival rate and feed intake were notsignificantly different among dietary treatments(P> 0.05).

The hematological parameters includinghematocrit and hemoglobin values were notsignificantly affected by dietary treatments(P> 0.05) (Table 4). The results showedquadratic increase of lysozyme (P= 0.028) andantiprotease (P= 0.005) activities by Barodonsupplementation (Table 4). Also, SOD activitywas significantly increased (linear, P= 0.005;quadratic, P= 0.0004) in Barodon-treated fish.However, NBT, MPO, and GPX activities andplasma Ig level were not significantly influencedby dietary treatments.

The groups of fish offered 1×, 2×, and 3×diets exhibited significantly higher disease resis-tance against S. iniae challenge (93.3, 97.8, and95.6% survival, respectively) compared with fishfed the control diet (64.4% survival) (Fig. 1).However, significant decreases in survival wereobserved at higher supplementation levels than0.3% Barodon and no significant differenceswere found among the groups fed 4×, 5×, andcontrol diets.

Discussion

The correlation between immunostimulationand growth performance has been reportedby several studies. Growth-promoting activityseems to be an additional effect of immunos-timulants (Sakai 1999). Cha et al. (2013)found that Bacillus subtilis supplementation indiets enhanced growth performance and feed

BARODON IMPROVES INNATE IMMUNITY IN OLIVE FLOUNDER 263

Table 4. Hematological and non-specific immune parameters of olive flounder fed the six experimental diets for 10 wk.

Diets P-value

Control 1× 2× 3× 4× 5× SEM Linear Quadratic

Hematocrit (%) 28.8 30.6 33.6 32.3 32.1 33.8 4.7 0.076 0.158Hemoglobin (g/dL) 3.97 4.37 4.14 4.11 3.92 4.10 0.59 0.709 0.844NBT (absorbance) 1.01 1.03 1.02 1.07 1.09 1.06 0.08 0.074 0.194MPO (absorbance) 2.04 2.33 2.36 2.34 2.29 2.28 0.37 0.323 0.373Lysozyme (μg/mL) 34.1 61.2 60.5 47.7 49.0 48.1 5.8 0.678 0.028Ig (mg/mL) 15.3 21.4 18.9 19.8 19.6 20.0 3.9 0.076 0.053Antiprotease (% inhibition) 21.0 30.8 34.6 29.7 30.6 27.8 5.4 0.254 0.005SOD (% inhibition) 57.7 68.1 70.7 68.4 70.0 69.8 3.9 0.005 0.0004GPX (nmol/min/mL) 75.0 102.6 81.9 73.4 89.4 77.6 13.1 0.593 0.684

Ig= total immunoglobulin; NBT= nitro blue tetrazolium activity; MPO=myeloperoxidase activity; SOD= superoxidedismutase; GPX= glutathione peroxidase activity.

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9

Surv

ival

(%

)

Days after infection

CON 1X 2X 3X 4X 5X

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Figure 1. Survival rate of olive flounder after Streptococcus iniae challenge.

utilization of olive flounder. Such effects werealso found in rainbow trout, Oncorhynchusmykiss, fed ginger containing diets (Nya andAustin 2009) and in orange-spotted grouper,Epinephelus coioides, fed garlic-supplementeddiets (Guo et al. 2012). In this study, growthperformance and feed utilization of olive floun-der were significantly improved with Barodonsupplementation. Similarly, Yoo et al. (2002)reported improvement of daily gain and feedutilization in porcine fed Barodon. The exactmechanism through which Barodon influencesgrowth performance is still unclear. However,Jurkic et al. (2013) suggested that sodium sili-cate, the main mineral ingredient, is responsible

for growth promoting effect of Barodon anddeclared that it decomposes quantitatively intobioavailable ortho-silicic acid in the acidicgastric juice and is absorbed in the body. Also,Carlisle (1969, 1970) reported that silicon,which plays critical roles in growth and skeletaldevelopment, is localized in active growth areasin bones of animals. From another point of view,Barodon provides a good source of mineralsthat are involved in maintenance of skeletalformation and colloidal system, acid–base equi-librium and are used for biologically importantcompounds like the hormones and enzymes(Watanabe et al. 1997).

264 SHIN ET AL.

Although, hemoglobin and hematocrit reveallittle information about the immunostimulatoryeffect of dietary treatment (Siwicki et al. 1994),they play important roles in indicating the phys-iological stress response as well as the generalhealth condition in fish. Hemoglobin and hemat-ocrit values in this study were not significantlydifferent among the experimental groups andassumed to be within a normal range for oliveflounder (Pham et al. 2007; Lim and Lee 2008;Song et al. 2012).

Innate immunity acts as the first line ofdefense against invading bacteria and playsan instructive role in the acquired immuneresponse (Magnadóttir 2006). Fish dependsmore heavily on nonspecific defense mechanism(Anderson 1992) because limited factors do notallow fish to develop complex physiologicalpathways for the development of the adaptiveimmune response (Siwicki et al. 1994). Theinnate immunity of fish is triggered with immu-nunostimulants (Sakai 1999). There are twoapproaches to elucidate the immunomodulatoryeffects of immunostimulants, in vitro by esti-mating the cellular and humoral parameters ofthe innate immune system and in vivo by chal-lenging against pathogenic bacteria (Sakai 1999;Murray et al. 2003). In this study, the immunos-timulatory effect of Barodon was investigatedby both analyses of humoral parameters of thenonspecific immune system and challenge testagainst S. iniae.

Immunostimulatory effect of Barodon hasbeen demonstrated in terrestrial animals. Immu-noenhancing properties of Barodon in pigshas been described by the proliferation andactivation of porcine immune cells, particularlyCD4+CD8+ double-positive T lymphocytes inperipheral blood and in the secondary lymphoidorgan (Yoo et al. 2001, 2002). Additionally,Barodon has been identified as an immunos-timulant in horses and shown to enhancedisease resistance against Streptococcus equiand Staphylococcus aureus which are causativeagents of horse respiratory disease and highantibiotic resistance (Koo et al. 2006). Barodonwas shown to have an adjuvant effect on hogcholera vaccine efficiency by increasing theantibody titer and immune cell proportions

(Park et al. 2000). Although previous studies didnot elucidate the exact mechanism of Barodon,these authors assumed that minerals are the maincomponents of Barodon that play essential rolesin the biological mechanism in the body includ-ing immune responses. Silica, the main mineralingredient present in Barodon solution, might beresponsible for these immunoenhancing effects(Koo et al. 2006; Jurkic et al. 2013). Subchronicand short-term exposure to silica activated andenhanced the lung phagocytes and respiratorydefense mechanism by increasing neutrophils,natural killer (NK) cells, T lymphocytes, andmacrophage activation in rats (Antonini et al.2000a, 2000b). Additionally, Kumar (1989)found that injected mice with silica increasedT and B lymphocytes and the number of cellsexpressing CD4 and CD8 antigens.

It is well known that lysozyme activity isan important humoral indicator of nonspecificimmunity in fish. Lysozyme protects the hostby lysing the 1, 4-beta-linkages in the peptido-glycan layer found in bacteria cell walls espe-cially gram-positive bacteria, and thus can stim-ulate the complement system and phagocytesas an opsonin (Ellis 1999). In this study thelysozyme activity was significantly increased inthe fish fed Barodon containing diets comparedwith the control group. This result is in agree-ment with enhancement of fish serum lysozymeactivity after administration of other immunos-timulants such as yeast glucan (Engstad et al.1992), organic chromium (Gatta et al. 2001),the Indian medicinal plant, Solanum trilobatum,(Divyagnaneswari et al. 2007), and fermentedgarlic powder (Kim et al. 2010).

Once penetrated in the body of fish, bacterianeed to digest the host protein as the source ofamino acids for their intrusion to the body cellsand growth. On the other hand, antiproteasesin fish plasma, principally α1-antiprotease,α2-antiplasmin, and α2-macroglobulin, playimportant roles in restricting the ability of bacte-ria to invade and grow in vivo (Ellis 2001). In thisstudy, significantly higher antiprotease activitywas found in Barodon-treated fish comparedwith the control. Similar effects on antiproteaseactivity were previously reported in rainbowtrout fed with garlic (Nya and Austin 2009),

BARODON IMPROVES INNATE IMMUNITY IN OLIVE FLOUNDER 265

Indian major carp, Catla catla, with herbalextract (Rao and Chakrabarti 2005), and rain-bow trout with bovine lactoferrin (Rahimnejadet al. 2012).

Regarding NBT and MPO activities, although,no significant differences were found, numeri-cally higher MPO levels were detected in Bar-odon fed groups. Cha et al. (2013) also foundno significant difference in MPO activities whenolive flounder were fed with diets supplementedwith Bacillus spp., but did find significant dif-ference in NBT activity. In contrast, Song et al.(2012) reported that dietary supplementation ofinosine monophosphate significantly increasedMPO activity of olive flounder but did not revealany effect on NBT activity.

Total immunoglobulins play important roles ininnate and acquired immunity response (Mag-nadóttir 2006) and can be enhanced by severalimmunostimulants (Sakai 1999). Therefore,total Ig is a good indicator for the action ofthe immunostimulants (Siwicki et al. 1994). Inthis study, higher total Ig levels were obtainedin Barodon-fed fish, although the differenceswere not significant. This finding is similar tothose of the studies by Kumar et al. (2013) forgoldfish fed with an Azadirachtin containingdiet and Siwicki et al. (1994) for rainbow troutfed with different kinds of immunostimulantsincluding Macrogard® (KS Biotec-Mackzymal,Tromso, Norway), Candida utilis, Saccha-romyces cerevisiae, Evetsel® (Polfa, Krakow,Poland), Chitosan® (Sea Fisheries Institute,Gdynia, Poland), and FinnStim® (FinnsugarBioproducts, Helsinki, Finland).

Reactive oxygen species (ROS) are chemicallyreactive molecules containing oxygen, includ-ing oxygen ions and peroxides. The overpro-duction of ROS may result in damage to cellmembranes (Liu et al. 2007). Cells can defendthemselves against ROS damage with radicalscavenging enzymes such as SOD and GPX.The SOD enzyme catalyzes the dismutation ofsuperoxide into oxygen and hydrogen peroxide,while GPX scavenges hydrogen peroxide andlipid hydroperoxides (Halliwell and Gutteridge1996). Thus, SOD and GPX enzymes play essen-tial roles in controlling and rapid elimination ofROS which is detrimental to the proper function

and health of aquatic organisms. In this study,SOD activities in Barodon-fed groups weresignificantly higher than in the control group,and GPX activity was enhanced numerically.The evidences in this study show that Barodoncan act as an antioxidant in fish.

Streptococcus iniae, a gram-positive bac-terium, is one of the causative agents ofstreptococcosis in many species of cultured fish(Park et al. 2009). In Korea, streptococcosis hasbeen reported to occur frequently in olive floun-der farms causing mass economic losses duringthe summer season. Infected fish show hemor-rhagic septicemia clinical signs accompaniedwith hemorrhagic septicemia, exophthalmia,and meningitis (Nho et al. 2009). According toSakai (1999), immunostimulants can enhanceresistance of fish to several bacterial pathogensincluding Streptococcus spp. The correlationbetween the immune response and diseaseresistance against S. iniae was well studied infishes. Li et al. (2004) demonstrated that dietaryoligonucleotides positively affect the immuneresponse and disease resistance of juvenilehybrid striped bass, Morone chrysops×Moronesaxatilis, to S. iniae infection. Similarly, Guoet al. (2012) concluded that inclusion of 1.3%garlic in diets for orange-spotted grouper, E.coioides, can enhance resistance to S. iniae. Inthis study, after 10 wk of the feeding trial, allfish groups were challenged with S. iniae. Theselected dose of bacteria in this challenge testwas higher than that reported by Song et al.(2012) and Cha et al. (2013). The result of thechallenge test revealed that incorporation of Bar-odon in the diet of olive flounder can remarkablyenhance disease resistance. Enhancement of fishresistance against bacterial diseases can be dueto the improvement in the above mentionednonspecific immunity of Barodon fed groups.

In conclusion, the findings in this study indi-cate that inclusion of Barodon in diets can pos-itively affect growth performance, feed utiliza-tion, innate immunity, and disease resistance ofolive flounder. The suggested supplemental levelof Barodon in the diet of fish seems to be 0.1%.

266 SHIN ET AL.

Acknowledgments

This study was supported by Cargill AgriPurina, Inc. and Barodon – S.F. Corp. Salarieswere partly supported by the National ResearchFoundation of Korea (NRF) grant funded bythe Korea government (MEST) (grant number2011-0015925).

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