The Effects of Rate-restricted Feeding Regimes in Cycles on Digestive Enzymes of Gilthead Sea-bream, Sparus aurata

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1 Turkish Journl of Fisheries nd Aquti Sienes 8: (2008) The Effets of Rte-restrited Feeding Regimes in Cyles on Digestive Enzymes of Gilthed Se-rem, Sprus urt Orhn Tufn Eroldoğn 1, *, Cüneyt Suzer 2, Oğuz Tşozn 1, Surhn Tkoğlu 1 1 Çukurov University, Fulty of Fisheries, Deprtment of Aquulture, 01330, Adn, Turkey. 2 Ege University, Fulty of Fisheries, Deprtment of Aquulture, 35440, İskele-Url, İzmir, Turkey. * Corresponding Author: Tel.: (Ext ); Fx: ; E-mil: mtufn@u.edu.tr Astrt Reeived 16 July 2007 Aepted 03 Jnury 2008 In order to understnd enzymti ltertion during the restrited-to-fed feeding shedules in yles, the modultion of key enzyme tivities in digestion ws studied in different prt of gstrointestinl trk of juvenile gilthed se rem (Sprus urt). Juveniles (6.4 g) were stoked into 12 tnks t density of 16 fish per tnk. Four different feeding shedules were tested on triplite groups of juvenile fish: (1) fish were fed to pprent stition twie dy throughout 48 dys, (2) strvtion for 1 dy nd then re-fed for 2 dys (S1), (3) 50% stition for 2 dys nd then re-fed to pprent stition for 2 dys (R2), (4) 50% stition for 6 dys nd then re-fed to pprent stition for nother 6 dys (R6). The tivity of totl protese, mylse nd lipse in the ontrol group ws higher thn those S1 nd R6 in whole gstrointestinl trk. The restrited feeding in R2 enhned the tivities of lipse, mylse, nd, to some extent, totl protese in fish, with the mostly pronouned effet exhiited in the pylori e. Generlly, totl protese nd lipse tivity of fish in R2 exhiited no signifint differene ompred to the ontrol, exept mylse tivity in mid-intestine. Thus, euse of preserved digestive enzyme tivities, some food restrition (R2) yles my e reommended s routine proedure in ommeril prodution of juvenile gilthed se rem. Key words: Sprus urt, restrited feeding, totl protese, lipse, mylse. Introdution The osts of formulted feed nd lour ssoited with feeding re the mjor omponents of the expenditure of rnivore fish prodution (Love, 1970; Joling, 1994; De Silv nd Anderson 1998). Optimizing the feeding strtegy is prime onsidertion in intensive rnivorous fish prodution mngement due to high protein ontent in their diet. Protein (s fish mel) is n expensive ingredient in rnivorous mrine fish diet. Thus, reserh in quulture nutrition is eing direted towrds the improvement of feed nd protein utiliztion (Gómez- Requeni et l., 2004; Eroldoğn et l., 2004; Eroldoğn et l., 2006; 2006). Theoretilly the mximum feed nd protein utiliztion of fish ours t feeding rte ove the mintenne feeding level ut elow the mximum or stition feeding level (Eroldoğn et l., 2004; Rowlnd et l., 2005; Eroldoğn et l., 2008). Feed restrition shedules up to 25-50% of stition without growth suppression re suggested to provide mny dvntges suh s esy feed mngement, high feed utiliztion nd low wter pollution (Pirhonen nd Forsmn 1998; Einen et l., 1999). The reltionship with growth rte relted with feed utiliztion my e the result of speifi digestive enzymes pity (i.e. totl protese, α-mylse, nd lipse) whih ould e used y hnges in protein metolism nd ppetite mehnism. In this sense, study of digestive enzymes is n essentil step towrds understnding the mehnism of digestion nd how orgnisms dpt to hnges in the nutritionl environment (Sunde et l., 2004). Anlysis of digestive enzymes tivities is n esy nd relile methodology tht n e used s inditor of digestive proesses nd nutritionl ondition of fish. Chnges in digestive enzyme tivity in response to periods of fsting my indite the most ritil nutrient nd energy reserves, nd those metolized or onserved in the fe of inresing food deprivtion or restrition (Hrms et l., 1991; Johnston et l., 2004). There re few studies foused on the effets of strvtion on digestive pity in fish (Mommsen et l., 2003; Krogdhl nd Bkke-MKellep, 2005). Some pprohes were tken in some old wter speies i.e. Atlnti od, Gdus morhu, (Bélnger et l., 2002); Jpnese flounder, Prlihthys oliveus, (Bolsin et l., 2006); nd Atlnti slmon, Slmo slr (Krogdhl nd Bkke-MKellep, 2005). Cyli restrited feeding regimes in reltion to niml digestive enzyme tivity hve not een studied in ny of mrine fish speies so fr. Although the tivity of the min digestive enzymes in gilthed se rem hs een reently ssessed (Alrón et l., 2001; Munill-Morán nd Sorido-Rey, 1996), little ttention hs een pid to the hnges in digestive enzyme tivity of different setions of the gstrointestinl trk of this speies when restrited feeding shedules were exposed in yles. Suh informtion is interesting for ulturists nd feed mnufturers striving to improve the feed utiliztion. Centrl Fisheries Reserh Institute (CFRI) Trzon, Turkey nd Jpn Interntionl Coopertion Ageny (JICA)

2 50 O.T. Eroldoğn et l. / Turk. J. Fish. Aqut. Si. 8: (2008) Therefore, the ojetives of the urrent study were, (i) to exmine the effets of the restrited feeding regimes on digestive enzyme tivity, (ii) to ompre the gstrointestinl setions so s to ssess if there re ny regionl differenes in intestinl digestive pities y mesuring tivities of totl protese, mylse nd lipse; (iii) to ssess possile digestion of rohydrte rther thn proteins in juvenile gilthed se rem under restrited feeding ondition. Mterils nd Methods Fish, Experimentl Design nd Feeding Gilthed se rem (Sprus urt) juveniles weighing 6.35±0.05 g (men±sd) were otined from lol ommeril frm nd trnsported to n indoor system where they were held in two 1000-L fierglss tnks for period of three weeks (limtion period) prior to the strt of the experiment. Feed (3 mm in dimeter) ws in the form of sinking extruded pellets mnuftured for gilthed se rem y Çmlı Feed Ltd., Turkey. Proximte omposition of the feed ws 45% rude protein, 12% rude ft, 12% sh nd 12% moisture. The fish were rndomly distriuted mong 12 irulr plsti tnks (155-L), giving 16 fish per tnk. Eh tnk ws ontinuously supplied with flowthrough sewter (40 ppt) filtered y 80 µm snd filter t flow rte of pproximtely 2 L min -1. Throughout the 48-dy experimentl period, the rering wter in eh tnk ws permnently sturted with oxygen y supplying ir ontinuously through ir-stones from n ir-lower. The dissolved oxygen nd ph were mg L -1 nd , respetively. Averge wter temperture rnged from 23.5 C to 24.5 C. The photoperiod ws held t 12 drk: 12 light during the experiment. The tretments implemented in the present study were: (A) Control: fish were fed to pprent stition twie dy throughout the experimentl period, (B) S1: strvtion for 1 dy nd then re-fed for 2 dys to pprent stition level (16 yles throughout 48 dys), (C) R2: 50% restrited feeding for 2 dys nd then re-fed for 2 dys to pprent stition (12 yles throughout 48 dys), (D) R6: 50% restrited feeding for 6 dys nd then re-fed for 6 dys to pprent stition (4 yles throughout 48 dys). Feeding rtio (% ody weight/dy) ws lulted s follow: 100 x [(dry food fed per dy) / ((finl weight + initil weight) / 2) / numer of dy)]. Throughout the experiment, exept the stition feeding periods, the restrited groups lwys reeived 50% of the mount of feed onsumed y the ontrol group. For exmple, if the lulted feeding rte for the ontrol ws lulted s 5% ody weight/dy, the mount of feed given to the groups during the next restrited feeding period ws djusted s 2.5% ody weight/dy. Aording to feeding shedules, diet ws given twie dy (etween 09:00-10:00 nd 18:00-19:00) nd ws hieved y presenting smll quntity of feed every few minutes until the fish esed to show interest. Enzymti Assys At the end of the experiment, fish were strved for one dy nd three fish from eh tnk were srified using slpel to sever the spine. Smpling of the digestive trks ws done within the first mel (09:00-10:00). Fish were disseted on glss utting ord kept on ie. The digestive trk nd ssoited orgns were immeditely removed from the rss fter the fish were killed. The intestines were freed from the other orgns nd ll visile ft removed. The digestive system ws then divided into the following setions: stomh (ST), pylori intestine with ee (PC) nd mid-intestine without distl hmer (IN). Three 0.5 m 2 piees from eh region were ut, lotted nd pooled for four groups of three fish eh efore homogenizing in n queous suspension (5 volumes v/w of ie-old distilled wter). Extrks utilized for enzyme ssys were otined fter homogeniztion of smples (~35 mg ml 1 ) in old 50 mm Tris HCl uffer, ph 8.0, followed y entrifugtion (13,500 g; 30 min t 4 C). The superntnts were trnsferred to mrked test tues immeditely nd stored t -20 C freezer until they were nlyzed the following dy. The totl protese tivity ws ssessed using the sein-hydrolysis method of Wlter (1984) s desried y Furné et l. (2005). The uffers used were KCl HCl 0.1 M (ph 1.5), glyine HCl 0.2 M (ph 3.0), itrte 0.1 M-phosphte 0.2 M (phs 4.0 nd 7.0), Tris HCl 0.1 M (ph 8.5 nd 9.0) nd glyine NOH 0.1 M (ph 10.0). We hose none-speifi tehnique due to the ft this method enles the quntifition of different photolyti tivities s funtion on ph: the tivity of pepsin (idi ph), hymotrypsin nd trypsin tivity (neutrl or slightly si ph) nd other enzymes suh s roxypeptidses, elstses, nd ollgenses (si ph) (Furné et l., 2005; De Almeid et l., 2006). The α-mylse (E.C ) tivity ws determined y strh-hydrolysis method, ording to Royt nd Wheln (1968). Mltose ws used s stndrd nd the tivity unit α-mylse ws defined s the qulity of enzyme tht produed one mmol of mltose ml -1 min -1 t ph 7.5 nd 25 C. Asorne ws determined t 600 nm. This method ws previously used to evlute mylse tivity in gilthed se rem (Moyno et l., 1996), rinow trout nd Adriti sturgeon (Furné et l., 2005). The lipse (E.C ) tivity ws ssyed y

3 O.T. Eroldoğn et l. / Turk. J. Fish. Aqut. Si. 8: (2008) 51 the evlution of the degrdtion of triylglyerols, diylglyerols, nd monoylglyerols to free ftty ids, following the method of Bier (1955) s desried in Furné et l. (2005). The enzyme tivities mesured in the intestine re given s speifi tivities in homogentes lulted s follows: speifi tivity= enzyme tivity (U ml -1 ) / protein (mg ml -1 ). Protein ws ssyed ording to Brdford proedure (1976). Sttistil nlysis of dt ws performed with the J.M.P. version sttistil softwre (SAS Institute, 1996). Followed y Tukey Krmer HSD test, men results per tnk were sujeted to one-wy nlyses of vrine (ANOVA) with feeding shedules s the independent vrile. The level of signifine ws hosen t P<0.05, nd the results re presented s groups mens (n=3). Results Throughout the experiment, survivl rnged from 98% to 100%. Over the ourse of 48 dys, verge weight gins (g) per tnk (±S.D.) were 37.8±0.10, 26.9±0.75, 28.7±0.61 nd 27.3±0.70 g in ontrol, S1, R2 nd R6, respetively. A signifint differene in tivities is tht ll three digestive enzymes were found mong the tested groups wheres there ws no tnk vrition within the replition of the tnks. Results of totl protese tivity mesured in the stomh (ST), pylori (PC) nd intestine (IN) showed different distriution of this enzyme long the limentry trk (Figure 1). However, the tivity of this enzyme in ontrol nd R2 ws signifintly higher thn tht of S1 nd R6 throughout the digestive trk (Figure 1). Amylse tivity ws the most importnt tivity identified long the gstrointestinl trk, eing minly oserved in the PC nd the IN extrks. As shown in Figure 2, mylse ws deteted throughout the gstrointestinl trk (ST, PC nd IN), ut highest mylse tivity ws found in PC, regrdless of tretment (Figure 2). Tukey Krmer HSD test showed tht mylse tivity in ST ws signifintly lower in R6 ompred to the ontrol, S1 nd R2. Speifi tivity of this enzyme in IN ws higher in ontrol ompre to ll other feeding regimes (Figure 2). As expeted, there ws no signifint differene in lipse tivity in ST for ll tested groups (Figure 3). On the other hnd, it ws ler tht tivity of lipse in PC (14-fold) nd IN (5-fold) ws higher thn in ST, irrespetive of tretment. Lipse tivity in PC of the ontrol, S1 nd R2 ws signifintly higher thn tht in R6. Speifi tivity of this enzyme in IN ws higher in ontrol nd R2 ompre to S1 nd R6 (Figure 3). Disussion The inresing feed intke nd rpid weight gin in fish during the reovery period re often ompnied y improved food onversion (Russell nd Wootton, 1992; Joling, 1994). However, in 1.2 1, , ,0 Control S1 R2 R6 Speifi Ativity (U/mg protein) 0.9 0, , , , , , , ,2 Stomh Pylori Intestine Digestive Trt Setion Figure 1. Ativity of totl protese mesured in the different setions of the digestive trt of gilthed se rem sujeted to four feeding regimes during 48 dys of rering. Vlues re mens ± S.D. (n=3, eh n onsist of mesurements of triplite nlysis).

4 52 O.T. Eroldoğn et l. / Turk. J. Fish. Aqut. Si. 8: (2008) Control S1 Speifi Ativity (U/mg protein) R2 R6 3 2 Stomh Pylori Intestine Digestive Trt Setion Figure 2. Ativity of mylse mesured in the different setions of the digestive trt of gilthed se rem sujeted to four feeding regimes during 48 dys of rering. Vlues re mens ± S.D. (n=3, eh n onsist of mesurements of triplite nlysis). Speifi Ativity (U/mg protein) 2.5 2, , , , , ,0 Control S1 R2 R6 Stomh Pylori Intestine Digestive Trt Setion Figure 3. Ativity of lipse mesured in the different setions of the digestive trt of gilthed se rem sujeted to four feeding regimes during 48 dys of rering. Vlues re mens ± S.D. (n=3, eh n onsist of mesurements of triplite nlysis). some ses, fish fed restrited rtio hve een reported to show improved feed effiieny without inresing feed intke (Russell nd Wootton, 1992; Wng et l., 2000; Eroldoğn et l., 2004). This is thought to e due to high digestive enzyme tivities indued y restrited feeding regimes s lso suggested y Brrington (1957) nd Joling (1994). Thus it would e of interest to study orreltion etween restrited feeding rtion nd digestive enzymes in juvenile gilthed se rem. As ommonly known protein digestion is omplex proess in fish nd ourred not only in stomh ut lso other prts of digestive systems suh s pylori e nd intestine. Hene, idi protese, pepsin, nd peptidses re minly ppointed in protein digestion in digestive system. Within this ontext, in the present study, the idi totl protese tivity in ST ws found to e higher ompred to tht in PC nd IN. Regrding tretments, restrited feeding in R2 nd ontrol showed higher totl protese tivity thn in other tested groups, suggesting tht restrited feeding inreses the totl protese tivity in digestive trk whih is lso in greement with results otined s in other rnivorous fish speies i.e. Atlnti od (Bélnger et l., 2002) nd Asin se ss (Hrpz et l., 2005). Furthermore, the signifint higher tivity of totl protese in whole prt of the digestive trk in R2 ompred with its tivity in S1 nd R6 my e

5 O.T. Eroldoğn et l. / Turk. J. Fish. Aqut. Si. 8: (2008) 53 the results of n inresed effort of fish to digest proteins in order to mximize protein utiliztion in R2 groups. Low mylse tivity in rnivorous fish is the generl ssumption (Hidlgo et l., 1999; Krogdhl et l., 2005). As rnivorous speies, gilthed se rem hs low mylse tivities in the liver nd intestine ompred to rp (Cyprinus rpio) nd gold fish (Crssius rssius) (Hidlgo et l., 1999). However, under stress ondition (i.e. strvtion, restrited feeding) fish n hnge rohydrte metolism. Sngio-Alvrellos et l. (2005) found n inresing pity to export gluose whih is moilized from liver glyogen stores in gilthed se rem exposed to food deprivtion for 2 weeks, inditing possile rohydrte metolism. Indeed, mylse is stimulted y glyolyti hins, glyogen, nd strh in fish lrve nd juveniles (Péres et l., 1998; Krogdhl et l., 2005). Thus, expeting n inrese mylse tivity in digestive trk is not surprising in fish sujeted to strvtion. In the present study, mylse tivity ws the min tivity identified long the digestive trk, eing minly oserved in the PC nd IN. Interestingly, tivity of this enzyme in ST of se rem ws deteted in the present study. This is onsistent with the findings of Alrón et l. (2001) who lso oserved mylse of tivity in stomh of 50-g gilthed se rem. In ft, it should e diffiult to explin the presene nd tivity of these enzymes, whih hve n optimum ph neutrl to lkline, onsidering the id environment existing in the stomh. However, tivity of mylse in ST in the reent study ws onsidered n rteft resulting from ontmintion of stomh extrts with pnreti tissue during mnipultion for dissetion. On the other hnd, in the present study, the rtio of mylse: protese tivity in ontrol, S1, R2 nd R6 ws 12.6, 16.7, 13.0, 17.0 in PC, respetively (unpresented dt). Hidlgo et l. (1999) postulted tht the high mylse: protese tivity rtio in gilthed se rem nd eel ould possily e due to the digestion of rohydrte rther thn proteins. It would pper tht similr phenomenon my hve ourred in our tested fish, lthough it ws not possile to nlyze this in detil s only single point in time ws smpled. It is ommonly known tht lipse showed reltively higher tivity t n lkline region etween ph (Iijim et l., 1998) nd tivity of this enzyme ws found in extrts of the pnres, pylori ee nd upper intestine (De Silv nd Anderson, 1998). Similrly, present findings in our study supported tht lipse tivity in PC nd IN were reltively higher thn those in ST where lipse tivity ws lmost non-existent. Most fish speies rely on ody lipid nd protein stores during period of food deprivtion nd diverging results re otined with respet to the importne of glyogen s n energy reserve (Ojveer et l., 1996; Hemre et l., 2002). In generl, inrese in lipse tivity is inditive of lipid use in fish. However, in the present study, there ws no signifint differene in lipse tivity in the tested groups, suggesting tht lipid is tively tolised in ontrol, S1 nd R2, with the exeption of fish in R6. These dt lso suggest tht the durtion of food restrition (R2) nd strvtion (S1) in yles ws too short to use potentil derese in lipse tivity nd tolised lipid ws used for energy during short-term restrited rtion nd strvtion. In onlusion, the signifint inrese in totl protese, lipse nd mylse tivity in digestive trk in fish fed with restrited feeding regime (R2) suggests tht protein utiliztion is ourring nd tht protein is n importnt energy soure under onditions of food deprivtion. Tking the oserved enzymti tivity into ount, some food restrition yles my e reommended s routine proedure in ommeril prodution of gilthed se rem to inrese feed effiieny due to inresing digestive enzyme tivity in gstrointestinl trk. 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Biohem., 72: De Almeid, L.C., Lundstedt, L.M. nd Mores, G Digestive enzyme responses of tmqui (Colossom mropomum) fed on different levels of protein nd lipid. Aquult. Nutr., 10: De Silv, S.S. nd Anderson, T.A Fish Nutrition in Aquulture. Chpmn nd Hll, London, 319 pp. Einen, O., Mørkøre, T., Rørå, A.M.B. nd Thomssen, M.S Feed rtion prior to slughter- potentil tool for mnging produt qulity of Atlnti slmon (Slmo slr). Aquulture, 178: Eroldoğn, O.T., Kumlu, M. nd Aktş, M Optimum feeding rte for Europen se ss Dientrrhus lrx rered in sewter nd freshwter. Aquulture, 231(1-4):

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