Proteins for Oreochromis niloticus

Transcription

1 The Isreli Journl of Aquulture - Bmidgeh 62(3), 2010, The IJA ppers now exlusively s peerreviewed on-line Open Aess journl t 15 N: n Inditor of the Assimiltion of Dietry Proteins for Oreohromis nilotius Hnno Slwski 1 *, Klus-Peter Götz 2, Crsten Shulz 1,3 1 Gesellshft für Mrine Aqukultur mh, Hfentörn 3, Büsum, Germny 2 Deprtment of Crop Siene Tropis nd Sutropis, Humoldt-Universität zu Berlin, Alreht-Ther-Weg 5, Berlin, Germny 3 Deprtment of Mrine Aquulture, Christin-Alrehts-Universität zu Kiel, Olshusenstr. 40, Kiel, Germny (Reeived , Aepted ) Key words: dietry mrkers, stle isotope, 15 N mmoni, nitrogen ssimiltion Astrt The potentil of enrihed stle isotopes ( 15 N) for protein digestion nd ssimiltion studies in juvenile fish ws evluted. Fishmel nd mggot mel were enrihed with 15 N, inorported into two diets (rude protein 30.2%). Four groups of ten Oreohromis nilotius (47.5±8.3 g) were fed n experimentl diet t the sme strting time. After 15 min, 2 h, 4 h, nd 6 h, five fish were seleted, the stomh nd gut were extrted, nd the liver, kidney, gills, nd musulus lterlis mgnus were smpled. Conentrtions of 15 N in the stomh 2-6 h fter feeding indite tht fishmel pssed through the stomh fster thn mggot mel. Differenes in digestive trt pssge etween protein soures were proly due to the hrd digestile hitin present in the mggot mel. In the liver, kidney, gills, nd musulus lterlis mgnus, 15 N in fishmel nd mggot mel nitrogen ws ssimilted t similr speeds. Results indite tht 15 N n provide omprehensive informtion out protein digestion nd ssimiltion in fish nutrition studies. * Corresponding uthor. E-mil: hnno.slwski@vti.und.de

2 Use of 15 N s dietry mrker 147 Introdution Fishmel prodution hs remined stle from the lte 1980s t pproximtely million tons per nnum (FAO, 2007). Limittion is use of serious onern for the long-term vilility of fishmel s protein soure in fish diets nd the growth of quulture prodution (Benedito-Plos et l., 2007). Sustitutes for fishmel must e evluted for their nutritionl vlue, s well s digestiility nd ssimiltion of the min nutrients to lrify their potentil s fish feed ingredients (Glenross et l., 2007). Digestiility of individul dietry ingredients is ommonly determined diretly or indiretly y the mount of nutrients ingested nd lter exreted. Diret methods mesure the mounts of feed onsumed y fish nd the resulting exret in n quti metolism hmer (Smith, 1971). Indiret methods re sed on nondigestile mrkers, suh s hromi oxide, yttrium oxide, rude fier, or other (Wetherup nd MCrken, 1998; Middleton et l., 2001; Vndenerg nd De L Noüe, 2001; Opstvedt et l., 2003; Toppe et l., 2006). It is ssumed tht the rtio of the mrker in the feed nd fees remins onstnt throughout gut pssge nd tht it will e found in the olleted fees. The differene etween the feed nd fel onentrtions of the mrker nd the nutrient mkes lultion of nutrient digestiility possile (Cho et l., 1982). Common diret nd indiret methods provide eptle pproximtion of rel nutrient digestiility. But oth methods gin no diret informtion out temporry nutrient sorption nd ssimiltion in fish. Enrihed stle isotopes s dietry mrkers ould provide insight into the ptterns of pssge of dietry proteins in the digestive trt nd their ssimiltion in tissues of fish. Enrihed stle isotopes hve een used in nutrition studies on fish lrve (see review y Coneição et l., 2007) nd shrimps (Preston et l., 1996). We used 15 N leled fishmel nd mggot mel in feeding experiment on juvenile Oreohromis nilotius, pying speil interest on the potentil of 15 N to show temporry nitrogen ssimiltion in seleted fish tissues. By this, we wish to provide n insight into the ssimiltion of nitrogen from dietry proteins y tilpi. Mterils nd Methods 15 N enrihment of protein soures. Leled dietry protein soures were not otinle, therefore, fishmel nd mggot mel were enrihed with 15 N. In the se of fishmel, 75 rps (Cyprinus rpio L.; 22.3 ± 1.5 g) were kept in 140-l fish tnk ontining 15 N leled wter (100 mg 15 N exess/l wter) from mmonium hloride ( 15 NH 4 Cl, 95 tom%). The fish were given proteinfree feed for 12 dys nd then were killed. Complete fish odies were freezedried nd ground to pss through 1-mm sreen (Bergner et l., 1993). In the se of mggot mel, sustrte with high 15 N onentrtion ws provided to mture egg-lying flies. For this, 4000 mg 15 N exess (mmonium hloride, 95 tom%), 3.0 kg pig liver, nd 2.0 kg semolin were homogenized in utter. A PVC ox (0.8 x 0.6 x 0.3 m) ws filled with the sustrte nd the ox ws exposed to the sun. The ox ws overed with lk lid to

3 148 Slwski et l. support wrmth. Slits in the sides of the ox provided ess for lueottle fly speies (Clliphoride) to the egg-lying sustrte. When enough developing eggs were oserved, the entrne slits were losed. Lrve strted feeding on the sustrte immeditely fter hthing. Outgrown mggots were olleted y hnd d fter hthing nd killed with hot wter. They were wshed thrie with old wter, lened from sustrte remins, dried for 48 h t 60 C in n oven, rushed with mortr nd pestle, nd stored in refrigertor t 4 C efore use in feed prodution. The protein soures were nlyzed for nutrient omposition nd 15 N onentrtions were determined (Tle 1). Tle 1. Nutritionl omposition of fishmel nd mggot mel. Fishmel Mggot mel Dry mtter (DM, %) Crude protein (% DM) Crude ft (% DM) Ash (% DM) Atom% 15 N exess mg 15 N exess/g DM Preprtion of experimentl diets. Two diets (30.2% protein) were enrihed with 15 N sed on fishmel nd mggot mel (Tle 2). Further, two unleled diets identil to the experimentl diets were formulted for enzymti hitution in fish digestive trt efore feeding with the leled diets. Dry diet omponents were mixed with fish oil nd wter (50 C) ws dded to otin liquid mixture. The mixture ws poured on flt pltes in thin lyers nd put in n oven t 30 C for drying. After 48 h, the dry mixture ws srthed off the pltes with knife, produing feed flkes. Experimentl diets were stored in refrigertor t 2 C until use. Tle 2. Ingredients nd nutritionl omposition of experimentl diets. Ingredient (g/kg orgni mtter) Fishmel Mggot mel Fishmel Mggot mel Pe seed mel 0 0 Fish oil Vitmin/minerl mix* Potto strh Whet gluten Nutritionl omposition Dry mtter (DM, %) Crude protein (%DM) Crude ft (%DM) Ash (%DM) mg 15 N exess/g DM * Spezilfutter Neuruppin -VM BM 55/13 Nr (per 1 kg dry feed): vitmin A IE, vitmin D IE, vitmin E 160 mg, vitmin K3 6.4 mg, vitmin B1 12 mg, vitmin B2 16 mg, vitmin B6 12 mg,vitmin B g, niotini id 120 mg, iotin 800 g, foli id 4.8 mg, pntotheni id 40 mg, inositol 240 mg, vitmin C 160 mg, ntioxidnts (BHT) 120 mg, iron 100 mg, zin 24 mg, mngnese 16 mg, olt 0.8 mg, iodine 1.6 mg, selenium 0.08 mg

4 Use of 15 N s dietry mrker 149 Nutritionl nlysis of experimentl diets. Experimentl diets were nlyzed for nutritionl omposition nd sh ontent. Eh nlysis ws rried out in duplite. Dry mtter ws determined y freeze-drying for onstnt weight. Crude protein (N x 6.25) ws determined ording to the Kjeldhl method (Kjelte System; Tetor, Hogns, Sweden). Crude ft ws nlysed y petroleum ether extrtion in Soxhlet pprtus (Soxte System HT; Tetor). Ash ws determined y urning in muffle furne t 550 C for 10 h. Experimentl design. The feeding tril ws onduted in the experimentl filities of the Institute of Animl Siene, Humoldt-Universität zu Berlin, Germny. Ten tilpi (47.5±8.3 g) were stoked in eh of eight 240-l experimentl tnks, orgnized in two reirultion systems. Eh system omprised four tnks nd filtrtion unit with sedimenttion hmer nd trikling filter for nitrifition. Wter qulity ws similr in oth systems: men temperture ws 24.2±0.29 C in system A nd 24.6±0.25 C in system B, men ph ws 8.00±0.08 in system A nd 7.82±0.15 in system B, O 2 ontent ws 5.6±0.23 mg/l in system A nd 5.4±0.28 mg/l in system B, nd ondutivity ws 816±5.7 μs/m in system A nd 828±5.2 μs/m in system B. To ensure enzymti hitution prior to the tul experiment, fish were fed similr unleled diets seven dys efore the feeding tril. The dily feed mount ws 3% of fish ody weight given in two portions. On the morning of dy 8, fish were fed 15 N leled diets t 1.5% of their ody weight. They reeived the diets egerly nd me to rest few minutes fter feeding, swimming lmly in the tnks. One fish group of ten individuls ws tken out of tnks fifteen minutes, 2, 4, nd 6 hours fter initil feeding. Fish were srified nd lid in iy wter. The dominl vity ws opened nd the gut nd stomh, with its ontents, were extrted. Both orgns were mnully lened from ft remins, pled in plsti gs, nd immeditely ooled in liquid nitrogen. The sme ws done with smples of liver, kidney, gills, nd musulus lterlis mgnus. Tissue smples were stored in refrigertor t -18 C until nlysis. Determintion of 15 N in tissues. The dominl vity ws opened nd, s fr s detetle, sex ws determined. Only mle fish with low developed gonds were further investigted. Aout five of the ten fish were mle. Smples of gut nd stomh (n = 5) were nlyzed for 15 N enrihment y emission spetrometry (Isonitromt 5200, F. Sttron, Fürstenwlde, Germny). The enrihment of liver, kidney, gills, nd musulus lterlis mgnus (n = 5) ws mesured y mss spetrometry using Finnign Delt C MS (ThermoFinnign, Bremen, Germny) oupled with Conflo II Interfe on Crlo Er 1108 (Crlo Er, Milno, Itly). Anlyses were rried out in triplite. Sttistis. The dt were nlyzed using sttistil softwre from SPSS, version 14.0 (ANOVA) oupled with Tukey s test (p<0.05).

5 150 Slwski et l. Results 15 N onentrtions in fishmel, mggot mel, nd diets. The protein soures were suessfully enrihed with 15 N t ut t different onentrtions depending on protein soure, protein ontent, leling proedure, nd lel uptke. As result, the diets ontined different 15 N onentrtions. 15 N onentrtions in the digestive trt. Due to different orgn weights etween individuls nd the smll size of the fish, it ws unfesile to ompre urte quntittive stomh nd gut onentrtions. Thus, investigtion of stomh nd gut 15 N ontents were sed on reltive 15 N onentrtions. In fish fed the fishmel diet, onentrtions in the fish stomh plus its ontent signifintly differed 2-6 h fter ingestion from the initil onentrtion t 15 min fter ingestion (Fig. 1). Conentrtions in the fish gut plus its ontent 15 min fter feeding styed stle. There were no signifint differenes etween smples in fish fed the mggot mel diet. 15 N ontent (mg/g) 0.16 A: Fishmel diet 0.16 B: Mggot mel diet Stomh Gut min 2 h 4 h 6 h Stomh Gut 15 min 2 h 4 h 6 h Fig. 1. Conentrtion of 15 N (mg 15 N/g) in the stomh nd gut nd their ontent of fish fed the fishmel nd mggot mel diets 15 min, 2, 4, nd 6 h fter feeding (mens±stndrd error, n = 5). Different supersripts indite signifint differenes of 15 N exess etween smpling times of stomh or gut (Tukey s test, p<0.05). 15 N exess in liver, kidney, gills, nd musulus lterlis mgnus. There were differenes etween protein soures in protein nitrogen ssimiltion s seen y temporry 15 N enrihment (tom% 15 N exess) in smpled tissues. In the fishmel diet, the 15 N exess in liver, kidney, nd gills of fish signifintly differed 4 h fter feed intke from the initil sttus; in the musulus lterlis mgnus, the 15 N enrihment signifintly differed 2-6 h fter feed intke from the initil 15 N enrihment t 15 min fter feed ingestion (Fig. 2). In fish fed the mggot mel diet, the 15 N enrihment signifintly differed in the liver, kidney, gills, nd musulus lterlis mgnus of fish 2 h fter diet intke. Exept for the musulus lterlis mgnus, the 15 N enrihment further inresed signifintly 4 h fter diet intke. The 15 N exess ontinued to inrese in the gills nd musulus lterlis mgnus 6 h fter ingestion.

6 Use of 15 N s dietry mrker 151 Atom% 15 N exess Atom% 15 N exess A: Liver MM FM MM FM B: Kidney min 2 h 4 h 6 h 15 min 2 h 4 h 6 h C: Gills D: M. lt. mgnus d min 2 h 4 h 6 h 15 min 2 h 4 h 6 h MM FM MM FM Fig N enrihment (tom% 15 N exess) in smples of gills, kidney, liver, nd musulus lterlis mgnus of fish (n = 5) 15 min, 2, 4, nd 6 h fter feeding fishmel nd mggot mel diets. For eh diet, different supersripts indite signifint differenes of 15 N exess etween smpling times (Tukey s test, p<0.05). Disussion Enrihed stle nitrogen isotopes hve een used s trers in nutrition studies on shrimps (Preston et l., 1996) nd fish lrve (see review y Coneição et l., 2007). The 15 N trer is diet element tht n give insight on digestive trt pssge ptterns of dietry nutrients nd their ssimiltion in fish tissues. This is n dvntge over non-nutritive mrkers suh s hromi oxide nd others tht n influene the digestion proess itself or show different pssge ptterns long the gstro intestinl trt with respet to the digest (Hnley, 1987; Vndenerg nd De L Noüe, 2001; Glenross et l., 2005) without providing dt on nutrient ssimiltion. Our experiment provides n insight into temporry pssge ptterns of protein from fishmel nd mggot mel long the gstro intestinl trt of juvenile Nile tilpi fed ompound diet. We oserved fster stomh pssge of 15 N from fishmel s ompred to mggot mel. The differenes etween fishmel nd mggot mel in stomh evution time ould e due

7 152 Slwski et l. to nti nutritionl ftors present in mggot mel. Aminopolyshride hitin (1,4β-N-etyl gluosmine) is the most prominent nti nutritionl ftor in mggot mel. Anti nutritionl ftors n dely trnsit time of ingested food from the stomh to the intestine of quti orgnisms (Storekken, 1985; Shiu et l., 1988; González-Peñ et l., 2002). Chitin dereses the stomh evution rte in rinow trout s hitin prtiles n e lrger thn the pylorus in the fish stomh, even fter whole orgni sustnes re digested (Kionk nd Windell, 1972). Smll hitin prtiles pssed the fish stomh t similr speed s the orgni sustnes. It n e ssumed tht the hitin present in mggot mel used the longer stomh evution time of mggot mel in ontrst to fishmel. Differenes in 15 N enrihment in smpled tissues mke temporry protein nitrogen ssimiltion in fish ody visile. In generl, most sored mino ids proeed to the portl venous lood nd re trnsported to the liver. After demintion of mino ids in the liver the resulting mmoni is trnsported to the gills nd kidney for exretion while mino ids enter fish musles (Smutná et l., 2002). The 15 N ssimiltion ws similr in tissues of fish fed fishmel nd mggot mel diets, similr to hydrolyses, digestion, nd sorption of fishmel nd mggot mel proteins. This ws seen in the similr ssimiltion ptterns of 15 N from fishmel nd mggot mel throughout the period. However, when ompring the protein soures (fishmel nd mggot mel) for 15 N enrihment in tissues, the different 15 N enrihment of the diets must e onsidered (1.51 tom% 15 N exess for fishmel nd 0.86 for mggot mel), i.e., the mggot mel diet ontined reltively less 15 N thn the fishmel diet. Therefore, the effiieny of nitrogen sorption nd ssimiltion in fish tissues seems reltively higher for mggot mel thn for fishmel. The totl 15 N onentrtions in the fish gstro intestinl trt indite tht digestion nd sorption of fishmel nd mggot mel ws not omplete until 6 h fter diet ingestion. Studies indite tht juvenile Nile tilpi n effetively use mggot mel s soure of protein (Ajni et l., 2004; Ogunji et l., 2007). However, when low qulity fishmel ws sustituted with mggot mel, deresed digestiility of dietry protein for tilpi ws oserved (Fshin-Bomt nd Blogun, 1997). This finding my e relted to mggot mel hitin. Beside our ssumption tht hitin slowed stomh pssge of 15 N from mggot mel, hitin ould lso depress protein digestiility in fish gut. Chitin is not digested y three tilpi speies (Buddington, 1980). It seems possile tht, fter whole gut pssge, reltive nitrogen sorption from mggot mel might e lower thn oserved. It seems likely tht n oservtion period inluding nutrient pssge long the whole gstro intestinl trt will provide different results for nitrogen sorption nd ssimiltion from fishmel nd mggot mel. In onlusion, the use of 15 N s dietry mrker of ompound diet provided insight into the temporry pssge, sorption, nd ssimiltion ptterns of different dietry proteins in the gstro intestinl trt nd orgn tissues of juvenile Nile tilpi. Enrihment of proteins with 15 N n help mximize protein nd mino id ssimiltion from quulture diets. Our limited oservtion period did not permit omplete ssessment of protein

8 Use of 15 N s dietry mrker 153 digestion plus nitrogen sorption nd ssimiltion from fishmel nd mggot mel. This will e the im of further investigtions. Aknowledgements We thnk Mrs. Susnne Moryson (HU zu Berlin, Germny) for her skilled nlytil ssistne, nd Dr. K. Dittert (CAU zu Kiel, Germny) for providing dt of mss spetrometri nlysis. Referenes Ajni E.K., Nwnn L.C. nd B.O. Mus, Replement of fishmel with mggot mel in the diets of Nile tilpi, Oreohromis nilotius. World Aqu., 35: Benedito-Plos L., Ser-Vil A., Clduh-Giner J.A., Kushik S. nd J. Pérez-Sánhez, Comined replement of fish mel nd oil in prtil diets for fst growing juveniles of gilthed se rem (Sprus urt L.). Aquulture, 267: Bergner H., Götz K.P., Simon M. nd B. Rennert, N-leling of fishes using 15 N isotopes in qurium wter nd the effet of different protein nutrient on the 15 N elimintion fter the leling period. Arh. Anim. Nutr., 45: Buddington R.K., Hydrolysis-resistnt orgni mtter s referene for mesurement of fish digestive effiieny. Trns. Am. Fish. So., 109: Cho C.Y., Slinger S.J. nd H.S. Byley, 1982: Bioenergetis of slmonid fishes; energy intke, expenditure nd produtivity. Comp. Biohem. Physiol. B, 73: Coneição L.E.C., Moris S. nd I. Rønnestd, Trers in fish lrve nutrition: review of methods nd pplitions. Aquulture, 267: Fshin-Bomt H.A. nd O. Blogun, The effet of prtil or totl replement of fish mel with mggot mel in the diet of tilpi (Oreohromis nilotius) fry. J. Prosp. Si., 1: FAO, The Stte of World Fisheries nd Aquulture Fish. Dept., Food nd Agriulture Orgniztion, Rome. 169 pp. Glenross B.D., Hwkins W.E., Evns D., MCfferty P., Dods K., Ms R. nd S. Sipss, Evlution of the digestile vlue of lupin nd soyen protein onentrtes nd isoltes when fed to rinow trout, Onorhynhus mykiss, using either stripping or settlement fel olletion methods. Aquulture, 245: Glenross B.D., Booth M. nd G.L. Alln, A feed is only s good s its ingredients review of ingredient evlution strtegies for quulture feeds. Aqu. Nutr., 13: González-Peñ M.C., Gomes S.Z. nd G.S. Moreir, Effets of dietry fier on growth nd gstri emptying time of the freshwter prwn

9 154 Slwski et l. Mrorhium rosenergii (De Mn, 1879). J. World Aqu. So., 33: Hnley F., The digestiility of foodstuffs nd the effets of feeding seletivity on digestiility determintions in tilpi, Oreohromis nilotius (L.). Aquulture, 66: Kionk B.C. nd J.T. Windell, Differentil movement of digestile nd indigestile food frtions in rinow trout, Slmo girdneri. Trns. Am. Fish. So., 101: Middleton T.F., Ferket P.R., Boyd L.C., Dniels H.V. nd M.L. Gllgher, An evlution of o-extruded poultry silge nd ulled jewel sweet pottoes s feed ingredient for hyrid tilpi (Oreohromis nilotius x O. mossmius). Aquulture, 198: Ogunji J.O., Nimptsh J., Wiegnd C. nd C. Shulz, Evlution of the influene of housefly mggot mel (mgmel) diets on tlse, glutthione S-trnsferse nd glyogen onentrtion in the liver of Oreohromis nilotius fingerling. Comp. Biohem. Physiol. A, 147: Opstvedt J., Aksnes A., Hope B. nd I.H. Pike, Effiieny of feed utiliztion in Atlnti slmon (Slmo slr L.) fed diets with inresing sustitution of fish mel with vegetle proteins. Aquulture, 221: Preston N.P., Smith D.M., Kellwy D.M. nd S.E. Bunn, The use of enrihed 15 N s n inditor of the ssimiltion of individul protein soures from ompound diets for juvenile Peneus monodon. Aquulture, 147: Shiu S.Y., Yu H.L., Hw S., Chen S.Y. nd S.I. Hsu, The influene of roxymethylellulose on growth, digestion, gstri emptying time nd ody omposition of tilpi. Aquulture, 70: Smith R.R., A method for determintion of digestiility nd metolizle energy of feedstuffs for finfish. Prog. Fish-Cult., 33: Smutná M., Vorlová L. nd Z. Svoodová, Pthoiohemistry of mmoni in the internl environment of fish (review). At vet. Brno, 71: Storekken T., Binders in fish feeds 1. Effet of lginte nd gur gum on growth, digestiility, feed intke nd pssge through the gstrointestinl trt of rinow trout. Aquulture, 47: Toppe J., Aksnes A., Hope B. nd S. Alrektsen, Inlusion of fish one nd r y-produts in diets for Atlnti od, Gdus morhu. Aquulture, 253: Vndenerg G.W. nd J. de l Noüe, Apprent digestiility omprison in rinow trout (Onorhynhus mykiss) ssessed using three methods of fees olletion nd three digestiility mrkers. Aqu. Nutr., 7: Wetherup R.N. nd K.J. MCrken, Comprison of estimtes of digestiility of two diets for rinow trout, Onorhynhus mykiss (Wlum), using two mrkers nd two methods of fees olletion. Aqu. Res., 29: