Journal of Applied Biological Sciences 9 (2): 37-42, 2015 ISSN: , E-ISSN: ,

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1 Journl of Applied Biologicl Sciences 9 (2): 37-42, 15 ISSN: 17-11, E-ISSN: , The Effects of Blnced Diets with Soy Ben Extrct or Met nd Bone mel on Muscle nd Liver Tissue Protein nd Glycogen Levels of the Nile Tilpi (Oreochromis niloticus L.) Infected with Virio nguillrum Ferl ÖZKAN YILMAZ 1* Kenn ENGIN 2 Arzu ÖZLÜER HUNT 2 1 Mersin University, Fculty of Fisheries, Deprtment of Bsic Sciences, Mersin, Turkey 2 Mersin University, Fculty of Fisheries, Deprtment of Aquculture, Mersin, Turkey * Corresponding uthor Received: My 14, 15 E-mil: ferl1111@hotmil.com Accepted: June 25, 15 Astrct This study investigted the effects of lnced diets with solvent extrcted soy en nd met nd one mel on the levels of protein nd glycogen in muscle nd liver tissues of the Nile tilpi (Oreochromis niloticus L.) infected with Virio nguillrum. Compred to the control diet (fishmel only), diets with solvent extrcted soy en (SBM) nd met nd one mel (MBM) significntly lowered (P<.5) the protein levels in liver tissues. However, glycogen levels in muscle nd liver tissues were lso lower in fish fed with SBM nd MBM diets thn fish fed with control diet. When fish infected or not infected with V. nguillrum compred within the sme diet groups, the liver protein levels were mesured significntly higher (P<.5) on dy1, dy3 nd dy5 of following injection. Glycogen levels oth in muscle nd liver tissues were lso found to e significntly higher (P<.5) in infected fish compred to control fish during the experiment. Considering the level of increse of metolic reserves in muscle nd liver tissues, it might e concluded from this experiment tht the Nile tilpi hs utilized SBM diet etter thn MBM diet under stress. Further reserch concentrting on higher mounts of solvent extrcted soy en nd met nd one mel, different environmentl conditions nd doses of infection with V. nguillrum nd immune response prmeters including physiologicl nd cellulr stress responses is needed in order to identify the effects of dietry plnt nd niml protein sources on overll moiliztion of metolic reserves in the Nile tilpi infected with V. nguillrum. Key Words: Oreochromis niloticus, Virio nguillrum, protein, glycogen, soy en, met nd one mel INTRODUCTION Sustinle quculture is only possile using costeffective qu feeds since the feed cost comprises of up to 6% of opertionl costs in qu frms. Fish mel is one of the most importnt nd widely used protein source in qu feeds nd its production levels vry tremendously from one yer to nother resulting lmost equl vriility in its per unit price. Therefore, incresing mount of reserch is eing conducted to identify cheper nd undnt lterntive plnt nd niml protein sources tht could e used prtilly or totlly in qu feeds [1, 2]. Since supplying the ody with minimum required nutrients tht re well lnced to ech other is extremely importnt for helthy growth [3], lterntive proteins should e investigted first for their qulity nd nutritionl vlue for fish species [4]. Hving higher crude protein level, mino cid profile similr to the fish mel nd higher nutrient digestiility increses the chnce of lterntive proteins eing used for fish mel replcement in qu feeds. Previous studies showed tht there ws liner reltionship etween the protein source in diets nd the resistnce ginst stress depending on these protein sources eing plnt or niml origin [5, 6]. Most of the infectious diseses occur minly fter exposing to su-optiml environmentl conditions nd the severity of the infection is even worse when eting foods with lower nutritionl qulity. Nutrition could e n importnt tool for the prevention nd cure of mny diseses [7] nd this is minly vlid for frmed nimls. In this respect, formulting lnced diets ccording to the nutritionl requirements of fish species is the most importnt spects of fish nutrition nd feeding under cptivity [8]. Dietry protein qulity nd its mino cid profile nd digestiility vlue re importnt defining fctors of nutritionl vlue of fish feeds. Proteins nd other nitrogenous compounds nd enzymes re lso importnt elements in cell structure nd cellulr functions of hormones. Protein synthesis, for exmple, is strongly ffected y the qulity of dietry protein sources. Fish mel is considered one of the est protein source for fish feeding due minly to its excellent protein qulity judged y the well lnced mino cid profile for fish growth. Reserch is vigorously eing conducted to investigte the effects of fish mel replcement y lterntive plnt nd niml protein sources in fish feeds on growth performnce nd generl well eing including orgn histology. Solvent extrcted soy en nd met nd one mel ws lso tested for some importnt frmed quculture species [9]. It is reported tht feed contining either plnt or niml protein sources cn ffect the prevlence of infections in fish differently []. Neji nd de l Noue [] demonstrted tht the sensitivity in feeding ehviors ws higher mong infected fish fed with minly plnt proteins thn fish fed with niml proteins. Glycogen is the form of glucose stored in muscle nd liver of living orgnisms. Although muscle tissue spend glycogen only for its need, liver glycogen is the precursor for plsm glucose [11]. In cse of need, glycogen stored in oth muscle nd liver tissues is ctolized through process clled glycogenolysis nd the level depletes. In

2 F. Özkn Yılmz et l / JABS, 9 (2): 37-42, ddition, excretion of ctecholmine, stress indictor, could induce drmtic chnge in the level of stored glycogen [12]. The im of this study ws to investigte the effects of lnced diets with solvent extrcted soy en or met nd one mel on the moiliztion of protein nd glycogen levels in muscle nd liver tissues of Nile tilpi (Oreochromis niloticus L.) on dys 1, 3, 5, 7, nd 14 following infection with Virio nguillrum. MATERIAL AND METHODS Fish nd the Mintennce The Nile tilpi used in this experiment were supplied y Fish Culture Unit t Fculty of Fisheries nd Aquculture, Mersin University. Prior to experimenttion ll the fish ws kept in L squre tnks which hd centrl dringe system nd fed with commercil trout pellets (Çmlı Fish Feed Compny, İzmir, Crude Protein:42%, 2-mm dry pellets). Experimenttion ws conducted using 9 glss quri mesuring H L W cm. Two weeks efore the experimenttion, fish (42.4 ± 2.3 g verge initil wet weight) rndomly selected from the stock tnks were housed in quri following individul weighing nd cclimtized to experimentl diets. The experimentl system ws constructed in two sections. The fish not infected with V. nguillrum were investigted in the first 15-dy period nd designted s controls for the infected fish in the second experiment. Fish were fed t 4% nd 3% BW/d in two equl mels from 9: to : h nd 17: to 18: h during the 2-week cclimtion period to experimentl diets nd in the ctul trils respectively. Fish were prevented from escping using perforted plstic sheeting on top of ech qurium. Aertion to experimentl system ws supplied y centrl ertion system using ir stones. The dissolved oxygen level in ech qurium ws not llowed to fll elow 5.5 mg/l during the experiments. Wter temperture ws kept t 26±2 ºC in the system using heters with thermosttic control. Uneten feed ws collected from ech qurium following feeding during the experiments. Diet Formultion, Preprtion nd Chemicl Anlysis Two experimentl diets were formulted to replce % of fish mel crude protein with SBM (solventextrcted soyen mel, Toros Feed Compny, Adn) nd MBM (met nd one mel, Toros Feed Compny, Adn). Diets were formulted to contin similr crude protein nd gross energy on dry mtter sis. Ingredients nd chemicl compositions of the diets re reported in Tle 1. The mount of minerl nd vitmin mixes incorported into diets ws decided in ccordnce with the dietry requirements of Nile tilpi (Tle 1). L-lysine ws dded to SBM nd MBM diets to ring the essentil mino cid profiles in line with the control diet [13]. For further description of the diets see Engin nd Özkn [14]. Diet smples were nlysed for crude protein (Kjeldhl, selenium ctlyst; %N 6.25). Gross energy in diets ws lso nlysed using om clorimeter (IKA WERKE, C Auto om, sic model, Stufen, Germny, clirted with enzoic cid). Crude ft nlysis in diets ws ccording to the soxhelet method where s dry mtter (g per kg DM) nd sh nlysis in diets were ccording to stndrd methods [15]. Tle 1. Formultion nd chemicl composition of the experimentl diets. DIETS Ingredients (g/kg diet) CONT SBM MBM Fish Mel L-lysine Soyen mel Met nd Bone mel Fish oil Dextrose Bentonite Croxymethylcellulose... Minerl mix * Vitmin mix ** St-C *** Cr 2O 3... Chemicl composition Dry mtter (DM g/kg diet) 937.5± ± ±7.5 Crude protein (g/kg DM) 7.6± ± ±8.7 Crude ft (g.kg DM) 128.4± ± ±1.8 NFE+crude fier **** (g/kg DM) Ash (g/kg DM) Gross Energy (MJ/kg 16.8± ± ±.8 DM) * Minerl mixture (mg/kg food): Mn;, Fe; 437.5, Zn; 625., Cu; 62.5, I; 25., Co; 5., Se; ** Vitmin mixture (mg/kg food): Vitmin A; 114. IU, Vitmin D 3; 14.2 IU, Vitmin E; 11., Vitmin K 3; 57., Vitmin B 1; 142.5, Vitmin B 2; 199.5, Vitmin B 6; 142.5, Vitmin B 12;.342, Biotin; 2.85, Folic cid; 57., Nicine; 1254., Pntothenic cid; 285., Vitmin C; 1425., Inositol; 11., Antioxidnts; Vitmin mixture ws supplied y Hoffmn L Roche, Istnul, Turkey. ***St-C (L- Ascoryl-2-polyphosphte) **** NFE=Nitrogen-free extrctives. Clculted s the reminder of crude protein+crude ft+sh The Experimentl Infection Procedure of V.nguillrum to the Fish V. nguillrum ws injected to juvenile Nile tilpi intrperitonelly. A 24-h fresh culture of cteri ws used in the experimentl infection procedure. Following the 24-h incution period, cteri were centrifuged for min. t 3 rpm. The superntnt ws then re-centrifuged nd wshed twice with Phosphte Buffered Sline (PBS) solution. After eing diluted with PBS, this cteril suspension ws prepred in four different doses [16]. The dilution consisting cteril numers s 1 6 cfu (colony forming units) ws chosen nd injected into fish using plstic syringes (1cc per fish). Muscle nd Liver Tissue Smpling nd Determintion of Protein nd Glycogen Levels Muscle nd liver tissue smples were collected from rndomly selected fish in ech qurium of ech tretment on dys 1, 3, 5, 7, nd 14 (d1, d3, d5, d7, nd d14) y killing fish with sudden low to the hed throughout the experiments. Following killing muscle nd liver tissues were quickly excised from smple fish, weighed nd frozen until nlysis. Before determintion of protein levels, tissue

3 Protein (mg/g) Protein (mg/g) Protein (mg/g) Protein (mg/g) F. Özkn Yılmz et l / JABS, 9 (2): 37-42, smples were weighed nd homogenized in.3 M sucrose solution t 24. rpm (Ultr-Turrx T-25 Homogenizer, Germny) for 5 min. then centrifuged t 3 rpm for min. Totl protein concentrtion in homogentes were ccording to Lowry method [17]. Tissue smples nlysed for glycogen reserves were first put into centrifuge tues for protein nd lipid extrctions nd then dded with 3 ml KOH solution ( %) nd plced in hot wter th for mins. Following hot wter th tretment, smples were dded with.5 ml concentrted N 2 SO 4 nd 3 ml 95% ethyl lcohol nd oiled for 15 min. on hot plte. Afterwrds smples were centrifuged t 3 rpm for min. nd superntnt were discrded. Suspension in tues were then dissolved in 2 ml distilled wter nd dded with 2.5 ml 95% ethyl-lcohol nd further centrifuged t 3 rpm for min. nd superntnt were discrded. Suspension free from protein nd lipid were then diluted to ml nd mde redy for the glycogen nlysis [17]. Glycogen concentrtions in smples were ccording to Antron method [18]. MUSCLE (A) Time (Dys) MUSCLE CONT (B) SBM MBM Sttisticl Anlysis All dt were sujected to one-wy ANOVA using SPSS. nd reported s men ± stndrd error throughout the text. When significnt tretment effect ws oserved, Tukey-Krmer HSD test ws used to compre mens. Significnce ws ccepted t the proility of.5 or less. Student s t-test ws lso used to identify the significnt differences etween non-infected nd infected groups for diets nd individul smpling dys. RESULTS There ws no mortlity throughout the experiment. The effects of lnced diets with solvent extrcted soy en or met nd one mel on muscle tissue protein levels in the infected nd non-infected fish re given in Figure 1. There were no significnt differences (P>.5) in muscle protein levels of fish fed with CONT, SBM nd MBM diets (Figure 1). However, the protein levels of fish fed with MBM diet ws significntly lower (P<.5) fter the d7 compred to the other tretments (Figure 1). The liver protein levels of the infected nd non-infected fish fed with CONT, SBM nd MBM diets re given in Figure 2. The liver protein levels of fish fed with SBM nd MBM diets were significntly lower (P<.5) when compred to tht of fish fed with CONT diets. The mgnitude of decrements were even significntly higher (P<.5) on d7, d nd d14 in fish fed with MBM thn tht of fish fed with SBM diet (Figure 2). The sme decrements in liver protein levels were lso oserved in the infected fish fed with SBM nd MBM diets compred to tht of fish in the non-infected group (P<.5) (Figure 2). Muscle glycogen levels of infected nd non-infected fish fed with lnced diets contining SBM nd MBM re given in Figure 3. There were no significnt differences etween muscle glycogen levels of fish fed with CONT nd SBM diets during the first 15-dy of the experimenttion (non-infected fish or control group). However fish fed with MBM diet showed significntly decresed (p<.5) muscle glycogen levels during this period (Figure 3). Fish experimentlly infected with V. nguillrum, however, were demonstrted to hve significntly lower (P<.5) muscle glycogen levels when fed with SBM nd MBM diets compred to tht of fish fed with CONT (Fish mel only diet) diet Time (Dys) CONT SBM MBM Figure 1. Muscle tissue protein levels of O. niloticus (A) noninfected nd (B) infected with V. nguillrum when fed CONT, SBM nd MBM diets. Ech vlue is the men±se (n=3). Different letters indicte significnt difference (P<.5) etween mens recorded for the sme dy in ech group LIVER (A) c Time (Dys) LIVER (B) c c CONT SBM MBM Time (Dys) CONT SBM MBM Figure 2. Liver tissue protein levels of O. niloticus (A) noninfected nd (B) infected with V. nguillrum when fed CONT, SBM nd MBM diets. Ech vlue is the men±se (n=3). Different letters indicte significnt difference (P<.5) etween mens recorded for the sme dy in ech group.

4 F. Özkn Yılmz et l / JABS, 9 (2): 37-42, 15 Figure 3. Muscle tissue glycogen levels of O. niloticus (A) noninfected nd (B) infected with V. nguillrum when fed CONT, SBM nd MBM diets. Ech vlue is the men±se (n=3). Different letters indicte significnt difference (P<.5) etween mens recorded for the sme dy in ech group. Liver glycogen levels of infected nd non-infected fish fed with lnced diets consisting SBM nd MBM re given in Figure 4. Liver glycogen levels were found to e significntly lower (P<.5) in fish fed with SBM nd MBM diets compred to tht of fish fed with CONT diet. The decrements were lso more evident eginning d5 onwrds during the first 15-dy of the experimenttion (Figure 4). Fish infected with V. nguillrum, however, hd significntly lower liver glycogen levels (P<.5) throughout the second 15-dy of the experimenttion when fed with MBM diet compred to tht of non-infected fish in the first 15-dy of the experimenttion (Figure 4). DISCUSSION Protein synthesis is iochemicl process through which ll the necessry mino cids re sufficiently supplied nd linked in correct wy. Therefore, protein sources tht re deficient of certin essentil mino cids could disrupt the protein synthesis in living orgnisms. In ddition, fish immune system hs previously een shown tht it is directly ffected y the type of dietry protein source whether it is plnt or niml origin [19]. Furthermore, it ws demonstrted tht tissue protein levels could sustntilly e ffected y simply the ltertion in protein synthesis nd the hzrds tht infections might cuse on tissues [7]. Figure 4. Liver tissue glycogen levels of O. niloticus (A) noninfected nd (B) infected with V. nguillrum when fed CONT, SBM nd MBM diets. Ech vlue is the men±se (n=3). Different letters indicte significnt difference (P<.5) etween mens recorded for the sme dy in ech group. This study showed tht SBM diet hd decresing effect on the liver protein levels of the Nile tilpi throughout the study indicting proly the ltertion in protein synthesis due to nti-nutritionl fctors tht soy en mel hs. Soy en is considered one of the most suitle plnt protein lterntives for fish mel in qu feeds due to its resonly high crude protein content nd s nerly eqully lnced essentil mino cid profile s fish mel [1]. However, the deficiency of some sulfur contining mino cids nd protese inhiitor s n ntinutritionl fctor in soy en mel could minly prevent its use in higher mounts in qu feeds. It ws previously demonstrted tht supplementtion of sulfur deficient mino cids to feeds nd mechnicl tretment of soy en mel hve oth incresed its use in tilpi feeds []. Feeding trout with diets contining plnt proteins hs sustntilly ltered the liver protein levels compred to tht of fish fed with fish mel only diets [21]. They lso concluded tht this ltertion ws minly due to the limittion in protein use or the vilility of essentil mino cids cused y nti-nutritionl fctors present in plnt proteins [21]. Liver is the min orgn where proteins nd other nitrogenous compounds re synthesized. Any disruptions to this process could not only severely ffect the protein utiliztion efficiency ut lso the cpcity of orgnisms to fight disese infections in vertertes. Previously it hs een reported tht net protein utiliztion of tilpi (Oreochromis niloticus x O. ureus) sustntilly ffected y inclusion of soy en mel into their diets [22].

5 F. Özkn Yılmz et l / JABS, 9 (2): 37-42, Fish fed with MBM diet hd decresed muscle nd liver protein levels compred to tht of fish fed with CONT nd SBM diets in this study. The levels were even lower in the infected group fed with MBM diet proly indicting the fster moiliztion of energetic reserves due to stress cused y the comined effect of dietry protein source nd V. nguillrum infection [19]. MBM is not primry protein source tht would e used for fish mel replcement in qu feeds due minly to its unlnced mino cid profile nd lower nutritionl qulity to qutic orgnisms [23, 24]. In ddition, high mount of crude sh in met nd one mel ws demonstrted to e the min reson of lower dry mtter nd protein digestiility in gilt hed se rem (Sprus urt) [4, 25], fresh wter shrimp (Mcrorchium nipponense) [24] nd the Nile tilpi [14]. The decrements oserved in muscle nd liver glycogen levels of fish fed with SBM nd MBM diets in this study might primrily e ttriuted to ltertions in the metolic pthwys due to the nti-nutritionl fctors present in soy en nd the reltively lower pprent nutrient digestiility of met nd one mel in the Nile tilpi [14]. The vrition etween lood glucose nd liver glycogen levels is directly ffected y metolic pthwys tht re minly determined y the oxidtive sttus nd conditions of orgnisms [11, 13, 26]. Amino cids re the primry sustrtes for gluconeogenesis in fish [27]. Von Der Decken nd Lied [28] demonstrted tht incresing mount of soy en protein concentrtes in lnced diets cused lower muscle protein nd glycogen levels in cod (Gdus morhu). They lso concluded tht lower energetic reserves found in muscle tissue were proly due to ltered metolic functions nd decresed enzyme ctivities s result of plnt protein inclusion in diets []. The liver protein levels significntly incresed in the infected fish compred to tht of fish in the non-infected group in ech dietry tretment during the study. Liver is n indictor orgn for ny dietry nd physiologicl ltertions occurring in the ody []. It lso plys n importnt role on the iosynthesis of immunogloulin in the immune system [7]. Therefore, higher liver protein levels in the infected group might e n outcome of incresed iosynthesis of immunogloulin ginst cteril infection in the Nile tilpi s defense mechnism []. The muscle nd liver glycogen levels of infected fish were found to e higher thn tht of non-infected fish fed with CONT, SBM nd MBM diets in this study indicting proly the higher rtes of gluconeogenesis nd incresed glycogen deposition s result of cortisol excretion triggered y stress due to cteril infection. Circultion levels of stress hormones re gretly ffected y the esily occurring stressful conditions in qu frming units [, 31]. Therefore glucose production is expected to increse sustntilly s result of increments in the excretion of stress hormones like cortisol nd ctecholmine in fish under cptivity. Glucose, the most importnt metolic sustrte for tissues, is ctolized from glycogen in liver in order to provide incresed energy needs due to stress in nimls [32]. These hormones specificlly ply roles in the moiliztion of energetic or metolic reserves nd increse the gluconeogenetic potentil of cells [33, 34]. Pthogen orgnisms re considered s nturl stressors nd cuses sustntil chnges in niml physiology [31]. It ws previously demonstrted tht plsm cortisol levels incresed in the rinow trout due to V. nguillrum infection [35]. Similrly, increments of plsm cortisol levels cused y disese infections were lso reported in severl other importnt quculture species [36, 37]. In conclusion, muscle protein levels did not chnge considerly with dietry tretments oth in the infected nd non-infected Nile tilpi in this study. However, muscle glycogen levels were found to e significntly lower in fish fed with MBM diet in ech experimentl group suggesting SBM is etter lterntive protein source in the Juvenile Nile tilpi. The lowest liver protein nd glycogen levels were otined on fish fed with MBM diets lso indicted tht SBM ws preferred choice of the two lterntive sources in terms of utiliztion nd moiliztion of energetic reserves in liver during stress mngement. Further reserch specificlly investigting the ctivities of key enzymes plying role in the moiliztion of energetic reserves is needed to drw cler conclusion out the utiliztion of lterntive proteins under stress. Acknowledgements The uthors wish to thnk Mersin University Fish Culture Unit for the supply of the Nile tilpi. Thnks re lso extended to Dr. Necl Türk from Bornov Veterinry Reserch Center for the supply of cteri. REFERENCES [1] El Syed A-FM Totl replcement of fishmel with niml protein sources in nile tilpi Oreochromis niloticus (L.) feeds. Aqucult. 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