British Journal of Nutrition

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1 British Journl of Nutrition (2010), 104, q The Authors 2010 doi: /s Dietry histidine supplementtion prevents trt development in dult Atlnti slmon, Slmo slr L., in sewter R. Wgø 1 *, C. Tröße 1, W. Koppe 2, R. Fontnills 2 nd O. Brek 3 1 Ntionl Institute of Nutrition nd Sefood Reserh (NIFES), PO Box 2029, Nordnes, N-5817 Bergen, Norwy 2 Skretting Aquulture Reserh Center, ARC, Stvnger, Norwy 3 Mrine Hrvest Norwy, Bergen, Norwy (Reeived 7 Jnury 2010 Revised 24 My 2010 Aepted 25 My 2010 First pulished online 9 August 2010) British Journl of Nutrition The im of the present study ws to investigte the trt preventive effet of dietry histidine regimes in dult Atlnti slmon (Slmo slr L.) in sewter, oth through mnipulting the dietry histidine level nd feeding period. Men ody weight of individully tgged Atlnti slmon t the strt of the experiment ws 1662 (SD 333) g. Low prevlene of mild trts were reorded in the eginning of June. Three fishmel nd fish oil-sed extruded diets (rude protein: 375 g/kg nd ft: 342 g/kg), differing only in histidine ontent (low (L): 9 3, medium (M): 12 8 nd high (H): 17 2 g histidine/kg diets), were fed to duplite net pens in sewter. The experimentl period ws divided into three sesons (June July; July Septemer; Septemer Otoer), eh strting nd ending with individul trt exmintion, ssessment of somti dt, nd smpling of lens nd musle tissues for nlysis of histidine nd histidine derivtives. In July nd Septemer, prt of the popultion fed L- nd H-histidine feeds were trnsferred (rossed over) to respetive series of replite net pens fed L-, M- nd H-histidine diets (i.e. eleven experimentl feeding groups t tril onlusion). The fish douled their ody weight from June to Otoer, with no systemti effets on weight gin of dietry histidine feeding regimes. Development of severe trts ws oserved etween July nd Septemer. The trt severity ws diretly relted to the dietry histidine level fed during the first nd seond periods. Feeding histidine-supplemented diets (M or H) in the first period from June to July mitigted lter trt outreks. The sttus of seleted free imidzoles in musle nd lens tissues refleted the dietry histidine feeding regimes, reltive to oth feed onentrtion nd feeding durtion. The study shows the risk for trt development for dult Atlnti slmon, 1 yer fter the trnsfer of slmon smolts from freshwter to sewter, whih to mjor extent n e prevented y histidine supplementtion just efore nd during the erly phse of trt development. Atlnti slmon: Ctrt: Fish welfre: Histidine: N-etyl-histidine: Amino ids The eye disorder trt hs een reltively ommon oserved symptom in the fish frming history, relted to environmentl, geneti, infetious, toxiologil nd nutritionl ftors (1,2). Ctrts in Atlnti slmon smolts hve een oserved in Europen quulture for the lst 20 yers, nd n epidemiologil survey performed in 1998 in sewter frms long the Norwegin ost found trt prevlene s high s 82 % (3). The losses for the quulture industry vry nd n e onsiderly high in more severe sesons (4). Ctrts hve ltely lso een oserved in slmon growers (.1 5 kg) in their seond yer in sewter. Besides eing n importnt ethil welfre issue for the fish frming industry, trt development in the finl phse of slmon prodution my lso represent eonomil losses from redued growth, redued feed effiienies, seondry infetious diseses nd qulity down grding t slughter (4). The mino id histidine hs een identified s key preventtive ftor in the development of trt in Atlnti slmon (Slmo slr L.) smolts (5 7). The type of trt tht ourred in the nineties t high inidenes nd severities in frmed slmon smolts ws proly used y the omission of lood mel from ommeril slmon diets (8). Blood mel ws nned s feed ingredient s mesure to prevent the spred of ovine spongiform enephlopthy (9). Blood mel is espeilly rih in histidine (pproximtely 50 g histidine/ kg in spry-dried produts) (10). Brek et l. (6) showed tht feed ould e supplemented with histidine either in the form of lood mel, high histidine fishmel or s pure histidine dditive, nd tht ll forms prevented trt development in slmon smolts eqully well. The ext mehnism for the trt mitigtion y elevted dietry histidine level is not ompletely understood. It hs eome ler tht histidine nd relted ompounds (imidzoles) over mny importnt iohemil roles in the slmon tissues esides its funtion s essentil mino id in protein synthesis, suh s omponents relted to lens osmoregultion (11,12), musle ph uffering (13,14), tissue ntioxidtion (15,16) nd detoxifition of ytotoxi-retive ronyl speies (17,18). The smoltifition phse in slmonids hs een onsidered s risk period for trt development (7,8). Musle histidine Arevitions: H, high histidine diet; L, low histidine diet; M, medium histidine diet; NAH, N-etyl histidine. * Corresponding uthor: Professor R. Wgø, fx þ , emil rw@nifes.no

2 Histidine nd trt in Atlnti slmon 1461 British Journl of Nutrition onentrtions reorded during smoltifition in wild slmon demonstrted hnges in the pttern of imidzoles t or fter the trnsfer from freshwter to sewter. This ws ssumed to reflet the introdution of neroi urst swimming in sewter with inresed need for musle ph uffering (19,20). The mjor histidine ompound in slmonid white musle is the dipeptide nserine, whih ours t high onentrtion fter smoltifition (7,20). The iosynthesis of nserine (-lnyl-3-methyl-histidine) inludes methyltion of the preursor rnosine (-lnyl-l-histidine), whih ours t muh lower onentrtions in the musle tissue. The trt preventtive tion of histidine in slmon smolts seems, however, to e medited through the derivtive N-etyl-histidine (NAH) in the lens (11,12). Lens NAH our in high onentrtion rnges s sme s nserine in white musle, nd the onentrtion depends on the dietry histidine levels efore nd during smoltifition. Low lens NAH onentrtion orrelted with the development of severe trt fter trnsferring to sewter (7). From the results otined in the susequent feeding experiments, Brek et l. (12) suggested rpid synthesis nd turnover of NAH in the lens to ope with osmoti hllenges, supporting proposed model of this histidine ompound s n importnt lens osmolyte (11,21). Histidine defiieny hs not een regrded s n re of onern, eyond the oserved redutions in growth nd feed effiieny in young Atlnti slmon fed histidine elow the estlished minimum requirement of 7 g/kg (22). The underlying experiments did not, however, report on free imidzoles in tissue s mrkers for histidine sttus. Feeding experiments with Atlnti slmon smolts fed diets with norml (11 7 g/kg) nd high (17 6 g/kg) levels of histidine showed tht oth musle nserine nd lens NAH onentrtions depended diretly on dietry histidine inlusion level, despite the ft tht these dietry histidine levels fr exeeded the estlished requirement for slmonids (7). The requirement of histidine for dult Atlnti slmon growers under prtil onditions is not known, ut it is normlly suggested to e lower thn for smolts, in line with generl lower reltive protein requirement in older fish. The min hypothesis of the present experiment ws tht dietry histidine exerts trt preventive effet in Atlnti slmon growers depending on oth the dietry onentrtion nd feeding period. A feeding study with Atlnti slmon during their seond yer in sewter ws performed t n experimentl se frming site in Southern Norwy with previous history of trt development. The experimentl design inluded three dietry histidine levels. Two hnges in diet during the experimentl period (rossover design) were inluded to identify ritil periods of histidine feeding. The experiment ws supported y ssessment of somti dt, eye inspetion using slit lmp iomirosope nd nlyses of free mino ids in musle nd lens tissues. Mterils nd methods Adult Atlnti slmon (Slmo slr L.) were limtised for 2 weeks in two sewter net pens t Lerng Reserh Sttion (Skretting ARC, Roglnd, Norwy), efore eing divided rndomly into six experimentl net pens (5 m 2 ). Men ody weight of the 1834 individully pssive integrted trnsponder-tgged (Eletroni I, In., Dlls, TX, USA) slmon t the strt of the experiment ws 1662 (SD 333) g. Three fishmel nd fish oil-sed extruded high-energy diets (protein: 375 g/kg nd ft: 342 g/kg), differing only in histidine ontent (low (L) 9 3, medium (M) 12 8 nd high (H) 17 2 g histidine/kg diets), were fed to duplite net pens y the use of feeding utomts nd hnd feeding ording to in-house tles onsidering iomss nd wter temperture. The three experimentl diets were produed y Skretting Aquulture Reserh Center for the whole feeding period in one prodution using ommerilly ville feed ingredients (Tle 1). Vitmins nd minerls were supplemented s ommeril premix fulfilling the requirements for slmonids given y the Norwegin Reserh Counil (10). Proximte omposition (Tle 1) ws determined y n in-house ner-ir spetrosopy nlysis. The dt otined from ner-ir spetrosopy methods were lirted nd heked y redited Tle 1. Ingredients, proximte omposition nd mino id profiles (g/kg) of the extruded experimentl diets, vrying only in histidine (low (L): 9 3, medium (M): 12 8 nd high (H): 17 2 g histidine/kg diet) Experimentl histidine diets L M H Ingredients LT Fish mel* Whet Soy Hi Pro Fish oil Vit/Min premixk Histidine HClk Proximte omposition Moisture Protein Ft Ash DE (MJ/kg)** Amino id omposition Arg His Ile Leu Lys Met Cys Phe Tyr Thr Trp NA NA NA Vl Al Asp Glu Gly Pro Ser Sum of mino ids DE, digestile energy; NA, not nlysed. * LT Skndinvi, Chr Holtermnn AS, Norwy. Sttkorn, Oslo, Norwy. Denof, Fredrikstd, Norwy. Nordsildmel, Bergen, Norwy. k Vitmin nd minerl premix; Skretting, Stvnger, Norwy (fulfilling reommendtions for slmonids given y Norwegin Reserh Counil (10) ). { Kyow Hkko, Tokyo, Jpn. ** Clulted DE (protein: 18 kj/g; lipid: 33 5 kj/g; rohydrtes: 12 5 kj/g).

3 1462 R. Wgø et l. British Journl of Nutrition onventionl Assoition of Offiil Anlytil Chemists methods (1995). Feed mino id profiles (Tle 1) were nlysed t Skretting ARC ording to n Europen Union stndrd method (23). The experimentl period from June to Otoer ws divided into three sesons ((June 6 July 14 (6 weeks), July 17 Septemer 6 (13 weeks) nd Septemer 7 Otoer 12 (18 weeks)), eh strting nd ending with eye exmintion for trt (slit lmp iomirosopy), ssessment of somti dt (ody weight, length nd ondition ftor), smpling of lens nd musle tissues for nlysis of histidine nd histidine derivtives nd gene expression nlyses. Approximtely, 200 individuls were lloted to eh net pen elonging to L nd H histidine groups, while the replites of M histidine groups were housed eh with eighty fish. Within respetive replites, pproximtely thirty fish fed L-histidine feed were trnsferred (rossed over) to M (group only reeiving fish) nd H histidine dietry groups t the smplings in July nd Septemer. Similrly, thirty fish fed high-histidine feed were trnsferred (rossed over) to M nd L-histidine dietry groups t the smplings in July nd Septemer; ending up with seven dietry groups in the seond period (LL, LM, LH, MM, HL, HM nd HH) nd eleven experimentl groups in the third period (LLL, HLL, HHL, LLM, LMM, MMM, HMM, HHM, LLH, LHH nd HHH) of pproximtely similr size, ut vrying in histidine level nd feeding period. Fig. 1 detils the experimentl design with movement of fish. Sine the M dietry groups reeived fish only during the two rossovers, they were housed with fewer fish t the strt; mening tht ll the net pens would hve pproximtely equl iomss in the ourse of the tril. The fish were exposed to nturl light regime in sewter (men slinity of 30 g/l). Wter temperture (4 m depth) rose from 12 to 18 58C from June to July, nd delined slowly to 168C in the end of Septemer nd further delined to 14 48C L M H June 6 Strt n 6 July 14 Smpling 1 Septemer 6 Smpling 2 Otoer 12 Smpling 3 n 6 n 6 n 6 Fig. 1. Detils of the experimentl design run in duplite net pens in sewter. Experimentl feeds with low (L: 9 3 g/kg), medium (M: 12 8 g/kg) nd high (H: 17 2 g/kg) histidine were fed during three periods, eh ending with ssessment of iologil somti performne dt (thirty individuls per feed replite), trt exmintion (thirty per feed replite) nd tissue smpling (six per feed replite). At smplings in July nd Septemer, individully mrked fish from L nd H were trnsferred to respetive replites of L, M nd H ording to rossover design, ending up with omintions of seven (LL, LM, LH, MM, HL, HM nd HH) nd eleven (LLL, LLM, LLH, LMM, LHH, MMM, HLL, HMM, HHL, HHM nd HHH) dietry groups. t the end of Otoer. Wter oxygen onentrtion mesured t regulr intervls ws never elow 8 2 mg/l. The Lerng Reserh Sttion is pproved, nd the tril ws onduted ording to the guidelines of the Norwegin Stte Commission for Lortory Animls. Ctrt exmintion When estlishing the six experimentl groups, sixty fish from the two limtistion net pens were nesthetised (metin ws dded to the smpling try ording to n in-house protool), weighed, mesured nd exmined for trt using slit lmp iomirosope t 15 mgnifition. The fish were inspeted nd sored individully for hnges (0 4 per eye; i.e. 0 8 for oth the eyes per fish) ording to the previously desried proedure (24). Fish with visul physil eye dmges were exluded from the experiment. At lter exmintions (July 15, Septemer 5 nd Otoer 12), thirty fish per replite (n 60 per dietry tretment) from ll the experimentl groups were exmined ording to the sme proedure. The exmined fish, exept six tken for smpling, were returned to their respetive net pens. Smplings Initilly (June 6), nd t July 15, Septemer 5 nd Otoer 12, musle nd lens tissues of six fish were smpled per replite (n 12 per dietry group) for nlysis of histidine ompounds nd gene expression profiling (25). The fish were fsted for 1 d efore smpling. Individul fish were nesthetised, inspeted for trt, mesured for weight nd length (n 30 per replite), nd smpled for lens nd musle tissues (n 6 per replite). Skin-free epxil white musle tissue ws smpled from ehind the dorsl fin nd ws frozen diretly on dry ie. Lenses were refully disseted, gently rolled on len filter pper to remove liquid nd musle tthments, immeditely frozen on liquid nitrogen/dry ie, nd stored t 2808C until nlysis. Anlysis of free mino ids Individul musle smples of six fish were nlysed for free mino ids, while individul lenses were nlysed for histidine nd NAH. Free mino ids were nlysed using Biohrom 20 Plus Amino Aid Anlyser (Amershm, Cmridge, UK), ording to stndrdised proedure from the mnufturer (Biohrom AAAFAQ08), with post-olumn ninhydrin derivtistion nd olorimetri detetion t 570 nd 440 nm. NAH nd free histidine in the lens were nlysed y reverse phse HPLC nd UV detetion t 210 nm (26), with modifitions ording to Brek et l. (7). Sttistil nlyses The dt were nlysed y the CSS:Sttisti for Windowse Sttistil Softwre (version 9.0; Sttsoft, In., Tuls, OK, USA, ). Differenes etween groups were ssessed y nested ANOVA (net pen individuls nested in dietry groups) nd Tukey s honestly signifint differene post ho test t level of P, All vlues re given s men vlues with their stndrd errors if not stted otherwise.

4 Histidine nd trt in Atlnti slmon 1463 British Journl of Nutrition Results Growth nd mortlity There were no systemti differenes in somti dt relted to dietry histidine regimes t ny smplings (Tles 2 nd 3). In the first period, the fish grew from 1662 g (men vlues; n fish from two replites) to 2251 (SEM 62), 2135 (SEM 61) nd 2200 (SEM 59) g in the L, M nd H histidine groups, respetively (nested ANOVA, P. 0 05). Similrly, no differenes in ondition ftor were oserved, rnging from 1 17 to 1 18; neither were ny differene in growth nd somti dt oserved in the seond period, with men ody weight rnging etween 2361 nd 2652 g. At the onlusion of the tril, fish ontinuously fed on the high-histidine diet (HHH) were signifintly hevier thn fish fed ontinuously on the M-histidine diet (MMM) nd the rossover diet (HMM) (nested ANOVA, P, 0 05). There were, however, no differenes in the length (rnge of mens from 60 7 to 63 1 m) nd ondition ftor (rnge of mens from 1 18 to 1 26) etween the groups. Exluding the sexully mture individuls reorded t the smplings (18 5 % in Septemer nd 28 % in Otoer) improved the men individul speifi growth rte ( %/d) in the dietry groups, ut did not hnge the growth results reltive to dietry histidine regimes. Ctrt ssessment At the strt of the experiment, minor hnges in one eye (sore 1) ws oserved in sixteen fish, two fish showed sore 2, nd one individul ws totlly lind (sore 8), ending with trt prevlene of 30 % nd men sore of 0 4 of sixty-three inspeted fish. A similr overll outome ws oserved in July, with pproximtely 30 % trts in the fish popultion. All the groups showed very mild trt hnges, with no differene in inidene nd men trt sores (#0 7; Fig. 2). From July to Septemer, mjor outrek of trts ws reorded, with men trt sores refleting the previous 6 dietry histidine feeding regimes. Fish groups fed diet tht ws not supplemented with histidine (L) from June to July developed signifintly more trt (men sore of 4 4 (SEM 0 2)) thn fish fed M- or H-histidine diets, oth with sores, 1 5 (P, 0 05). The trt sores were lso influened y the feeding levels of histidine from July to Septemer, where fish rossed over from L to H histidine diet (LH) showed signifintly lower trt sore (2 9 (SEM 0 2)) thn the L histidine group (LL), while fish rossed over from L to M histidine diet (LM) showed n intermedite higher sore (3 7 (SEM 0 2)). No further development in trt ws oserved from Septemer to Otoer in ny group, ending with pproximtely similr trt severities s oserved in Septemer. All the groups fed diets supplemented with histidine during the first feeding periods (H, M nd respetive rossover diets) showed low ut modertely inresed trt sores from the strt (0 4) to the end (#2 0; Fig. 2). Considering ll individully exmined fish t the end of the experiment, no differene in trt sores ws oserved etween sexully mture fish nd immture fish. Free mino id omposition in musle Free histidine in white musle tissue refleted systemtilly the feed histidine regimes, oth in dietry onentrtion nd feeding time (Fig. 3). In July, efore the trt outrek, there were ler differenes etween the groups fed the L (0 13 mmol/g)-, M (0 70 mmol/g)- nd H (1 86 mmol/g)-histidine diets (P, 0 05), while in Septemer fter the outrek, musle histidine levels were loser in onentrtion with signifintly higher onentrtions in fish ontinuously fed H-histidine diet (HH) thn in fish ontinuously fed the L-histidine diet (LL). Fish rossed over to the H (LH)- nd L-histidine diets (HL) showed intermedite musle histidine levels. In Otoer, fter seond rossover, lrger nd systemti vritions were oserved mong the eleven dietry groups; lrgely dividing into three onentrtion levels nd refleting the dietry histidine levels during the finl period (Fig. 3). White musle nserine onentrtion onstituted etween 45 nd 50 % of the free mino ids nd ws less vrile Ctrt sore , June July Septemer Otoer Musle histidine (µmol/g) ,,,, July Septemer Otoer d,d, Fig. 2. Development of trts (men trt sore of oth eyes; 0 8) in Atlnti slmon fed the experimentl histidine diets: low (L), medium (M) nd high (H) (see Fig. 1) during three periods from June to Otoer (men of thirty exmined fish per experimentl duplite).,, Men vlues with unlike letters were signifintly different (P, 0 05; nested ANOVA nd Tukey s honestly signifint differene post ho test)., LLL;, LLM;, LLH;, LMM;, LHH;, MMM;, HLL;, HMM;, HHL;, HHM;, HHH. Fig. 3. Development of white musle histidine in Atlnti slmon fed the experimentl histidine diets: low (L), medium (M) nd high (H) during three periods from June to Otoer. Dt re expressed s mens with their stndrd errors (six per experimentl duplite; see Fig. 1). d Men vlues with unlike letters were signifintly different (P, 0 05; nested ANOVA nd Tukey s honestly signifint differene post ho test)., LLL;, LLM;, LLH;, LMM;, LHH;, MMM;, HAA;, HMM;, HHL;, HHM;, HHH.

5 1464 R. Wgø et l. Tle 2. Somti dt from duplite groups of dult Atlnti slmon fed three levels of dietry His for 5 weeks efore mssive outrek of trt (Men vlues with their stndrd errors) Somti dt Ctrt sore Weight (g) Length (m) CF (unit) Left eye (0 4) Right eye (0 4) Totl (0 8) Feed ode* Men SEM Men SEM Men SEM n Men SEM Men SEM Men SEM L M H ANOVA NS NS NS NS NS NS L, low; M, medium; H, high. * The single pitl letter odes indite the use of diets L, M or H in the period from June to July. Initil vlues: weight, 1662 g (n 1834). The dt were evluted y nested ANOVA nd Tukey s honestly signifint differene post ho test (NS: P.0 05). British Journl of Nutrition (6 8 % differene) mong the dietry histidine groups (Tle 4; Fig. 4). Musle nserine ws signifintly higher in fish fed M (17 0 mmol/g)- nd H (16 7 mmol/g)-histidine diets, thn in fish fed the unsupplemented histidine diet (15 7 mmol/g). Musle nserine inresed modertely from July to Septemer in ll the groups, while for fish rossed over from L to H histidine diets (LH), this inrese ws signifint. Moderte delines in musle nserine were pprent from Septemer to Otoer, mostly in fish ontinuously fed the L-histidine diet (LLL; Fig. 4). Musle rnosine ws signifintly higher in the M thn in the L histidine group t the first smpling in July (Tle 4), nd delined in ll the groups in Septemer (Tle 5) exept in fish rossed over from L to M histidine diet (LM), however, with no signifint differenes etween the groups. Finlly, rnosine inresed during the lst feeding period in ll the groups exept those fed the L-histidine diet during the finl Tle 3. Somti dt from duplite groups of dult Atlnti slmon fed three levels of dietry His levels for 5 months* (Men vlues with their stndrd errors) Weight (g) Length (m) Condition ftor Feed ode Men SEM Men SEM Men SEM n LLL 2893, LLM 2850, LLH 2903, LMM 2842, LHH 2931, MMM HLL 3083, HMM HHL 2935, HHM 2996, HHH ANOVA P,0 05 NS NS L, low; M, medium; H, high., Men vlues within olumn with unlike supersript letters were signifintly different (P, 0 05). * The experimentl period ws divided into three sesons (the three pitl letter ode indites diets L, M nd H used in June July, July Septemer nd Septemer Otoer, respetively). Three-letter odes indite the use of diets L, M or H in the first, seond nd third periods. The dt were evluted y nested ANOVA nd Tukey s honestly signifint differene post ho test (NS: P.0 05). Tle 4. Free mino ids in white musle tissue (mmol/g) of dult Atlnti slmon fed low (L; 9 3 g/kg), medium (M; 12 8 g/kg) nd high (H; 17 2 g/kg) levels of histidine smpled in July (week 6) efore serious outrek of trt* Diets Amino id L M H Pooled SEM Essentil Thr Vl Met Ile , 0 00 Leu , 0 01 Phe 0 12, Lys His Arg Trp ND ND ND Non-essentil Ser , 0 03 Glu , Gln Pro Gly Al , Tyr Asp 0 13, Hydroxyproline Nitrogenous ompounds -Alnine Methyl-histidine Crnosine , 0 04 Anserine Turine O-phospho-ethnolmine Ure L--Amino-N-utyri id Ethnolmine Ammoni , Ornithine SFree mino ids ND, elow the detetion limit.,, Men vlues within row with unlike supersript letters were signifintly different (P, 0 05). * Six smples per duplite; evluted y nested ANOVA nd Tukey s honestly signifint differene post ho test t level of P,0 05. Single-letter odes indite the use of diets L, M or H in the first feeding period. Pooled SEM,

6 Histidine nd trt in Atlnti slmon 1465 British Journl of Nutrition Anserine (µmol/g) July Septemer Otoer,,, Fig. 4. Development of white musle nserine in Atlnti slmon fed the experimentl histidine diets: low (L), medium (M) nd high (H) during three periods from June to Otoer. Dt re expressed s mens with their stndrd errors (six per experimentl duplite; see Fig. 1).,, Men vlues with unlike letters were signifintly different (P,0 05; nested ANOVA nd Tukey s honestly signifint differene post ho test)., LLL;, LLM;, LLH;, LMM;, LHH;, MMM;, HLL;,HMM;, HHL;, HHM;, HHH. period (, 0 2 mmol/g). Musle rnosine ws four times higher in fish rossed over from L to H histidine diet fter the seond period (LLH; P, 0 05). After 6 weeks of feeding, severl free mino ids in white musle tissue, inluding some essentil mino ids (threonine, isoleuine, leuine nd lysine), were signifintly higher in slmon fed the L-histidine diet ompred with fish fed histidine-supplemented diets (M nd H; Tle 4). The sum of free mino ids ws, however, the sme in ll the dietry groups. After 13 weeks nd following the trt outrek, there were minor differenes mong the other free mino ids in musle thn the imidzoles, with non-systemti differenes in O-phospho-ethnolmine, sprti id, glutmine, lnine, tyrosine nd mmoni (P, 0 05) mong the groups (Tle 5). Agin, totl mino id onentrtion ws equl in the smplings, etween 34 nd 35 mmol/g. At the end of the tril, profile similr to previous free mino id profile in musle ws oserved (dt not shown), with signifint higher onentrtion of seleted mino ids (hydroxyproline, proline, threonine, glutmte, tyrosine, phenyllnine, ornithine, 1-methyl histidine) in slmon fed Tle 5. Free mino ids in white musle tissue (mmol/g) of dult Atlnti slmon fed low (L; 9 3 g/kg), medium (M; 12 8 g/kg) nd high (H; 17 2 g/kg) levels of His in two periods nd smpled in Septemer fter serious outrek of trt* Amino id LL LM LH MM HL HM HH Pooled SEM Essentil Thr Vl Met Ile Leu Phe Lys His ,, 1 13, 0 93,, 0 55, 0 77,, Arg Trp ND ND ND ND ND ND ND Non-essentil Ser Glu Gln 0 34, 0 36, 0 54, 0 47,, , 0 03 Pro Gly Al 1 94, , 2 02, 2 13, Tyr 0 09, , 0 08, 0 09, 0 06, 0 00 Asp 0 14, 0 16, , Hydroxyproline Nitrogenous ompounds -Alnine Methyl-histidine 0 13 e 0 12, 0 10,, ,, 0 11, 0 12,d,e 0 00 Crnosine Anserine 16 5,, , 17 2,, 17 2, ,,d 0 1 Turine O-phospho-ethnolmine 0 05, , 0 05, , 0 00 Ure L--Amino-N-utyri id Ethnolmine Ammoni 4 94,d , 4 46, 4 80, 4 66,,d 0 05 Ornithine SFree mino ids Diets ND, elow the detetion limit. e Men vlues within row with unlike supersript letters were signifintly different (P, 0 05). * Six smples per duplite; evluted y nested ANOVA nd Tukey s honestly signifint differene post ho test t level of P,0 05. Two-letter odes indite the use of diets L, M or H in the first nd seond feeding periods. Pooled SEM,

7 1466 R. Wgø et l Histidine (µmol/g) d,d,,d,,,, Lens NAH (µmol/g) British Journl of Nutrition 0 0 July Septemer Otoer Fig. 5. Development of lens histidine in Atlnti slmon fed the experimentl histidine diets: low (L), medium (M) nd high (H) during three periods from June to Otoer. Dt re expressed s mens with their stndrd errors (six per experimentl duplite; see Fig. 1).,,,d Men vlues with unlike letters were signifintly different (P,0 05; nested ANOVA nd Tukey s honestly signifint differene post ho test)., LLL;, LLM;, LLH;, LMM;, LHH;, MMM;, HLL;, HMM;, HHL;, HHM;, HHH. the L-histidine diet during the two lst feeding periods (HLL nd LLL groups) ompred with the other groups. Totl mino id onentrtion ws equl within the smpling, ut somewht lower thn tht oserved previously (31 33 mmol/g). Lens mino id omposition As in the white musle, lens histidine nd NAH refleted dietry histidine onentrtions stritly systemtilly reltive to oth onentrtion nd feeding period (Figs. 5 nd 6). Lrge differenes in the onentrtion of NAH etween the feeding groups ws oserved in July efore the trt outrek, with signifint differenes (P, 0 05) etween ll the three groups (Fig. 6). All the dietry groups hd inresed lens NAH from July to Septemer, exept fish rossed over from H to L histidine diet (HL). The lens NAH inreses were most signifint in fish with low NAH sttus in July, nd depended on dietry histidine onentrtion in this seond feeding period. NAH (µmol/g) , e d,e,d,e,d July Septemer Otoer, Fig. 6. Development of lens N-etyl-histidine (NAH) in Atlnti slmon fed the experimentl histidine diets: low (L), medium (M) nd high (H) during three periods from June to Otoer. Dt re represented s mens with their stndrd errors (six per experimentl duplite; see Fig. 1).,,,d,e Men vlues with unlike letters were signifintly different (P,0 05; nested ANOVA nd Tukey s honestly signifint differene post ho test)., LLL;, LLM;, LLH;, LMM;, LHH;, MMM;, HLL;, HMM;, HHL;, HHM;, HHH Individul trt sore (0 8) Fig. 7. Correltion etween individul lens N-etyl-histidine (NAH) nd trt sores in dult Atlnti slmon fed experimentl diets differing in histidine from June to Septemer nd exmined fter the seond period with serious outrek of trt (n 60). y ¼ x x þ ; R In August, the lens NAH sttus orrelted negtively with severity of trt given s trt sore (Fig. 7). At the end of the tril, lens NAH showed 3-fold differene etween the groups, depending on dietry histidine feeding regimes (Fig. 6). The H-histidine diet oviously supported sturtion level of lens NAH t mmol/g. Conentrtions of most free mino ids in the lens were signifintly higher in the group fed L histidine ompred with the group fed M nd H histidine efore the trt outrek (Tle 6). In sme mnner s the sum of free mino ids, onentrtion of seleted free mino ids like hydroxyproline, glutmte, lnine, isoleuine, leuine, tyrosine, phenyllnine nd 1-methyl histidine grdully deresed in fish fed L-, M- nd H-histidine diets. Adding NAH to the other lens mino ids gve, however, totl mino id level of 24 3 mmol/g in L nd H histidine groups, nd somewht lower level in lenses from the M histidine group (20 1 mmol/g). In Septemer, fter the outrek of trt, similr elevted onentrtion of mjor mino ids were oserved in the L histidine group (LL) ompred with the groups fed histidine-supplemented diets in either of the two periods (Tle 7). Agin, the totl mino id, inluding NAH, in lens ws similr mong the groups within the rnge of mmol/g. At the onlusion of the tril in Otoer, free mino ids like glutmte, glyine, phenyllnine, turine nd 1-methyl histidine in lens were still elevted in slmon fed ontinuously on the L-histidine diet (LLL), espeilly ompred with fish fed the H-histidine diet (HHH) (P, 0 05; dt not shown). Disussion The present study demonstrtes for the first time tht dult Atlnti slmon hve prtil requirement for dietry histidine levels ove present reommendtions y Norwegin Reserh Counil (10) to prevent trt development during their seond yer in sewter. Low prevlene nd severity of trts were reorded t the strt of the experiment. A mssive outrek of trts ws reorded in Septemer, proly initited y period of inresed wter temperture in July. Modertely elevted nd flututing tempertures hve een demonstrted to represent risk ftors for trt development, oth experimentlly nd in field (27,28), proly

8 Histidine nd trt in Atlnti slmon 1467 British Journl of Nutrition Tle 6. Free mino ids in lens tissue of dult Atlnti slmon fed low (L; 9 3 g/kg), medium (M; 12 8 g/kg) nd high (M; 17 2 g/kg) levels of His smpled in July (week 6) efore serious outrek of trt* Diets Amino id L M H Pooled SEM Essentil Thr Vl Met Ile Leu Phe Lys His Arg ND ND ND Trp Non-essentil Ser Glu Gln Pro Gly Al Tyr Asp Hydroxyproline , Cys Asn Nitrogenous ompounds -Alnine Methyl-histidine , Turine O-phospho-ethnolmine Ure , 0 05 L--Amino-N-utyri id Ammoni Phospho-serine Srosine Amino dipi id Cystthionine Cystthionine SFree mino ids NAH SFree mino ids inluding NAH ND, elow the detetion limit; NAH, N-etyl-histidine.,, Men vlues within row with unlike supersript letters were signifintly different (P, 0 05). * Six smples per duplite; evluted y nested ANOVA nd Tukey s honestly signifint differene post ho test t level of P,0 05. Single-letter odes indite the use of diets L, M or H in the first feeding period. Pooled SEM, relted to inresed growth nd metolism. The trt severity mong the dietry groups ws systemtilly relted to the dietry histidine level nd histidine feeding period. All the groups of slmon fed the unsupplemented diet with 9 g histidine/kg during the first period developed severe trt, while histidine-supplemented diets $ 13 g histidine/kg, s in the M- nd H-histidine diets prevented trt. However, fish rossed over from low to oth histidine-supplemented diets (LM nd LH groups) developed intermedite severities of trt, suggesting tht feeding histidine-supplemented diet from onset July ws too lte for mximum trt preventive effet. On the other hnd, fish rossed over from the H-histidine diet in the first period to L-histidine diet in the tul period with trt outrek (HL group) were fully proteted. In summry, oth dietry histidine supplementtion nd histidine feeding period were ritil for trt prevention. Sine the present histidine supplementtion levels ($13 g histidine/kg) prevented trt in dult Atlnti slmon fter reltively short period of feeding, this represents n effetive ounter mesure in periods with inresed risk for trt, like rises in wter temperture during the summer seson. In previous study with Atlnti slmon smolts, dietry histidine levels of 12 nd 18 g/kg were ompred, nd showed tht the former ws not le to prevent trt during 26-week feeding period (7). This ws first indition tht histidine requirement to support growth nd helth in Atlnti slmon prr nd smolts under prtil onditions my e higher thn the given minimum requirement of 7 g/ kg (10). Slmonids undergo smoltifition to prepre for migrtion from fresh wter to sewter, nd musle nd eye histidine metolism seems to e prt of these dpttions (7,20). Feeding studies with Atlnti slmon throughout the prr smolt trnsformtion indited tht the histidine requirements my vry ording to the physiologil sttus of the fish. After trnsferring to sewter nd when provided with suffiient dietry His support, Atlnti slmon seems to uild up tissue-speifi histidine ompounds, nserine nd NAH in musle nd lens, respetively, therey trpping histidine intrellulrly nd mking it unville for protein synthesis (7). These speifi histidine ompounds re elieved to hve importnt physiologil nd iohemil stilising funtions, relted to trt development. The underlying historil studies for estlishing the histidine requirement in slmonids (10) did not onsider these tissue imidzoles s mrkers for histidine sttus. With feed trget omposition to fulfil the requirements for optiml growth nd protein utilistion, mino id defiienies hve not een onsidered to e n re of onern with respet to fish helth (22). Somti growth seemed not to vry ording to the present dietry histidine regimes. All the diets hd levels well ove the minimum requirement for histidine, nd ll the dietry groups showed resonle growth during the experiment (speifi growth rte $ 0 40 %/d). Low temperture fishmel nd soyen protein onentrtes represent high protein qulities with highly digestile histidine (10). Likewise, the free supplemented histidine slt is highly digestile nd shows superior sorption (6,7,12). However, the present study shows tht the use of fishmel-sed diet without histidine supplementtion did not supply suffiient histidine to fulfil essentil funtions of histidine eyond protein synthesis. A signifint prt of the fish popultion (28 %) in ll net pens hd initited sexul mturtion during the tril. Exluding these individuls t the finl smpling generlly improved growth rtes, ut did not ffet the overll outome of growth mong the experimentl groups. Brek et l. (7) suggested growth-promoting effet of histidine in slmon smolts, whih ws rgued to e relted to diret effets on mino id metolism or y improved physiologil onditions of the tissue. A similr growth promotion ould not e onfirmed in dult slmon in the present study. The growth nd mronutrient deposition differ, however, etween smolts nd growers, whih my msk the potentil modest effets of histidine on protein synthesis (12).

9 1468 R. Wgø et l. Tle 7. Free mino ids in lens tissue of dult Atlnti slmon fed low (L; 9 3 g/kg), medium (M; 12 8 g/kg) nd high (M; 17 2 g/kg) levels of His smpled in Septemer (week 13) fter the outrek of trt* Amino id LL LM LH MM HL HM HH Pooled SEM Diets British Journl of Nutrition Essentil Thr , 0 24, 0 22, 0 24, 0 23, Vl Met Ile , 0 48, , 0 46, Leu Phe Lys His Arg Trp Non-essentil Ser Glu Gln Pro Gly Al , 0 45, , Tyr Asp Hydroxyproline 0 13, 0 12, , Cys , 0 06, 0 03, , 0 03, 0 01 Asp ,, 0 10, ,, 0 08,, 0 09,, 0 01 Nitrogenous ompounds -Alnine , Methyl-histidine , Turine , 0 82, , 0 66, 0 69, 0 04 O-phospho-ethnolmine Ure , 0 44, , 0 40, 0 02 L--Amino-N-utyri id , , Ammoni , , 0 06, 0 01 Phospho-serine , , Srosine , 0 44, 0 55, 0 41, 0 50, Amino dipi id Cystthionine Cystthionine Methyl-histidine SFree mino ids , NAH , 10 9, 8 9, SFree mino ids inluding NAH NAH, N-etyl-histidine.,, Men vlues within row with unlike supersript letters were signifintly different (P, 0 05). * n 6 smples per duplite; evluted y nested ANOVA nd Tukey s honestly signifint differene post ho test t level of P,0 05. Two-letter odes indite the use of diet L, M or H in the first nd seond feeding periods. Pooled SEM, White musle nserine, representing the mjor imidzole in slmon ody, ws ffeted signifintly y dietry histidine regimes ut within nrrow onentrtion rnge. Aording to the present high musle onentrtions nd minor hnges etween groups nd smplings, nserine synthesis seems to e prioritised in ll the groups, even in groups with ritil histidine supply nd sttus during the outrek of trt. Between Septemer nd Otoer, musle nserine generlly delined modertely, with onomitnt rise in preursors to nserine iosynthesis (rnosine nd -lnine) in most groups, exept those fed on L-histidine diet. This indites ompenstory up-regultion of nserine synthesis in the finl period, however, gin depending on ville free histidine s sustrte. While elevted musle nserine during prr smolt trnsformtion reflets need for uffering pity reltive to inresed neroi urst swimming tivity in sewter, nserine hs lso een suggested to e more importnt ntioxidnt in rinow trout growers thn juveniles (15). Mximl musle tissue onentrtion of nserine ws hieved y use of the M-histidine diet with 12 8 g histidine/kg. Musle rnosine, representing low onentrtion imidzole intermedite in nserine iosynthesis ws generlly higher in onentrtion efore thn fter the outrek of trt. In July, there seemed to e sturtion in nserine synthesis in oth fish fed M nd H histidine-supplemented diets, s evluted y signifintly lower -lnine nd rnosine nd higher nserine onentrtions in these two groups ompred with fish fed the L-histidine diet. At lter smplings, oth nserine preursors were lower in ll the groups. Low onentrtion levels of NAH in the lens hve een onsidered diret risk ftor for trt development (7). Dietry levels of 17 6 g histidine/kg mitigted trt

10 Histidine nd trt in Atlnti slmon 1469 British Journl of Nutrition formtion in slmon smolts in their first seson in sewter ompred with ontrol diet with 11 7 g histidine/kg, representing lens NAH onentrtions of. 10 nd, 2 mmol/g, respetively. In the present study, the lens NAH refleted the L-, M- nd H-histidine diets y showing men vlues of 1 1, 3 1 nd 10 8 mmol/g efore the outrek of trt. During the outrek of trt, the onentrtions inresed to 5 3, 10 9 nd 11 8 mmol/g, respetively, illustrting n tive uild-up of lens NAH in ll the groups; however, the onentrtions were not inresed suffiiently in the L histidine groups. This onfirms the ppliility of NAH s n inditor for trt suseptiility, in dult slmon. The intermedite histidine feed level (M), inresing lens NAH from 3 1 to 10 9 mmol/g from July to Septemer ws suffiient to prevent trt during the outrek. Similrly, fish rossed over from H to L histidine diet (HL), with lens NAH etween 9 nd 11 mmol/g in Septemer showed equl trt prevention. In ontrst, fish rossed over from low to histidinesupplemented diet (LM or LH) showed more severe trt development; despite lens NAH levels. 8 mmol/g t the time of smpling. Clerly, the tul sttus of NAH in these groups during the first nd seond feeding periods my hve hnged etween the smplings, nd for the ltter groups, this represented ritil low NAH onentrtion whih onsequently led to trt development. Sine the sttus of lens NAH ws not reorded extly t the outrek of trt, it is not possile to estimte ritil vlue of this inditor to prevent trt. Men trt sores did not inrese muh from Septemer to Otoer, suggesting tht the mjor outrek ws over in Septemer. For individul fish smpled in Septemer, negtive orreltion ws pprent etween lens NAH onentrtion nd trt sores (Fig. 7), s ws oserved previously in slmon smolts fter trnsferring to sewter (7). Generlly, low level of NAH in the lens seems to represent lower defene towrds flututions in oth externl nd ioti ftors, whih eventully P to trt hnges. The reltively wide onentrtion rnges t eh sore level indites tht the individul fish my e t different phses of trt development nd repir t the time of smpling. The mehnisms ehind the trt lleviting effet of histidine re not fully understood. Histidine nd its relted imidzoles hve een suggested to over mny iohemil roles in the slmon tissues esides eing n essentil mino id in protein synthesis, suh s lens osmoregultion (7). The former hs een given support y reent studies on slmon lens ex vivo ultures (29), where exposure of lenses to hypoosmoti medium used n efflux of NAH from the lens nd into the medium. This finding ws interpreted s survivl mehnism y the lens; to djust osmollity ording to the surrounding medium to prevent swelling nd eventully rupture of the lens psule. In the present study, free mino id profiles in musle nd lens were reorded oth efore nd fter the trt outrek to determine if ny other mino id ws influened y the histidine feeding regimes. These dt indite first tht the musle nd lens did not suffer from defiieny of ny other essentil mino ids (methionine, ystine, tryptophn) tht hve een relted to trt development (1). Seondly, the onentrtions of mny free mino ids were signifintly elevted in the L histidine groups ompred with the histidine-supplemented groups, most proly to equlise osmollity or the totl onentrtion of mino ids in defiieny of histidine for NAH synthesis. This ltter rgument is lso supporting the role of NAH in lens osmoregultion. Oviously, NAH synthesis represents trpping of high onentrtions of histidine for use s n effetive osmolyte, ompred with less effiient mixed ttery of other mino ids, mong others turine, glutmi id, glutmine, glyine, vline, methionine, leuine, tyrosine nd phenyllnine; ll with onentrtions.1 mmol/g in the L histidine group efore the trt outrek nd somewht less fter the outrek. This is summrised in the Tles 6 nd 7 s firly equl sum of totl mino ids (inluding NAH) in the lens nd supports our erlier finding in slmon smolts (7). In summry, the present study demonstrtes for the first time histidine-relted trt in dult Atlnti slmon in their seond yer in sewter. Low lens NAH onentrtions in the first two feeding periods oinided with inresed severity of trt formtion. Feeding diet with 12 8 g histidine/ kg throughout the experimentl periods prevented trt development. Alterntively, it lso seemed possile to prevent trt y pre-feeding H-histidine diet in risk periods. Even ltely initited trt ounter mesures seem to redue trt outreks, where diet supporting 17 2 g histidine/kg dded more protetion ompred with diet with 12 8 g histidine/kg. A risk ftor in the present study ws proly rpid rise in the se temperture during summer. The outome of the present histidine feeding study my e used s nutritionl guideline for trt prevention in dult slmonids in periods of inresed risks. In seprte ommunition, we imed to explore potentil moleulr mehnisms for the present histidinerelted trt, oth with respet to ute trtogenesis from July to Septemer nd suggested repir phse from Septemer to Otoer. Ctrt v. non-trt lenses from seleted groups were sreened for differentilly expressed genes using mirorry experiment (25). The expression levels of suset of genes oding for proteins involved in ntioxidtion, osmoregultion, rohydrte metolism nd poptosis were identified, ll representing the mehnisms involved in trtogenesis nd potentilly influened y histidine sttus. Aknowledgements The uthors wish to thnk reserh engineer Johnny Elvrum t Skretting ARC, Lerng Reserh sttion, nd engineer Anit Birkenes t Ntionl Institute of Nutrition nd Sefood Reserh, for their vlule tehnil ssistne t the rering site nd the lortory, respetively. This reserh ws supported y the Norwegin Reserh Counil (#168435/S40), Mrine Hrvest Norwy, Skretting ARC, Norwy, Skretting Irelnd nd Ntionl Institute of Nutrition nd Sefood Reserh. There re no onflits of interest tht the uthors should dislose. Eh o-uthor hs ssisted the first uthor in prepring the mnusript, lthough they prtiipted differently in designing the reserh (O. B., R. W., W. K. nd R. F.), mking the feeds (W. K. nd R. F.), onduting the experiment nd smpling (C. T., O. B., R. W., W. K. nd R. F.), nd performing hemil (C. T.) nd sttistil nlyses (C. T., O. B. nd R. W.).

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