Water temperature and dietary histidine affect cataract formation in Atlantic salmon (Salmo salar L.) diploid and triploid yearling smolt

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1 Journl of Fish Diseses 2017, 40, oi: /jf Wter temperture n ietry histiine ffet trt formtion in Atlnti slmon (Slmo slr L.) iploi n triploi yerling smolt F Smrus 1,2, P G Fjelll 1, S C Remø 3, E M Hevrøy 4, T O Nilsen 2, A Thorsen 5, T J Hnsen 1 n R Wgø 3 1 Institute of Mrine Reserh (IMR), Mtrel, Norwy 2 Deprtment of Biology, University of Bergen, Bergen, Norwy 3 NIFES, Ntionl Institute of Nutrition n Sefoo Reserh, Bergen, Norwy 4 Crgill Innovtion Center, Dirl, Norwy 5 Institute of Mrine Reserh (IMR), Bergen, Norwy Journl of Fish Diseses This is n open ess rtile uner the terms of the Cretive Commons Attriution Liense, whih permits use, istriution n reproution in ny meium, provie the originl work is properly ite. Astrt The im of the present stuy ws to investigte trt evelopment in iploi (2N) n triploi (3N) Atlnti slmon smolts n post-smolts t two wter tempertures (10 n 16 C) given iets with ifferent histiine supplementtion (LH, 10.4 n HH, 13.1 g kg 1 ) efore n fter sewter trnsfer. In freshwter, severe trt outrek ws reore in oth ploiies rere t 16 C. The trt sore ws signifintly higher in triplois ompre to iplois, n the severity ws lower in oth ploiies fe the HH iet. The trt evelopment t 10 C ws minor. Low gill N +, K + -ATPse tivity in fish rere t 16 C efore sewter trnsfer ws followe y osmoregultory stress with elevte plsm eletrolyte onentrtions n high mortlity in se wter. Both iplois n triplois rere t 10 C evelope trts uring the sewter perio, with higher severities in triplois thn iplois n reue severity in the fish fe the HH iet. The finings of this stuy emonstrte the importne of environmentl onitions in the husnry of Atlnti slmon, n prtiulrly triplois, with regr to smoltifition n juste iets to mitigte trt evelopment in fresh n se wter. Corresponene F Smrus, Institute of Mrine Reserh (IMR), Mtre 5, 5984 Mtrel, Norwy (e-mil: florin.smrus@imr.no) 1195 Keywors: Atlnti slmon, trt, histiine, smoltifition, temperture, triploi. Introution The use of sterile frme slmon is urrently eing teste s ountermesure to protet the genetis of wil slmon, s it hs emonstrte tht espe frme slmon hyriize with wil onspeifis (Glover et l. 2012). Triploiiztion is the inution of sterility n urrently the only lterntive to proue sterile slmon with regr to ommeril emns (Piferrer et l. 2009). However, the option of triploi Atlnti slmon in the inustry hs een restrine ue to welfre issues (reviewe y Frser et l. 2012; Benfey 2015) n impee growth n higher mortlity ompre to iplois t high wter tempertures (Glreth et l. 1994; MGehy, Benfey & Frirs 1995; O Flynn et l. 1997). Triploi slmon iffer from iplois y ontining fewer ut lrger ells in most orgns tht, epenent on ell shpe, n result in reue ellulr surfe to volume rtio tht represents the sis of physiologil ifferenes (Benfey 1999). Differenes in gut morphology etween iploi n triploi slmon suggest ifferenes in utiliztion n metolism etween ploiies (Peruzzi, Hgen & Joling 2015), n lso ifferenes in the suseptiility to trts (Wll & Rihrs 1992; Oppel,

2 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses Trnger & Hnsen 2003; Lelerq et l. 2011; Tylor et l. 2013). Ctrt is efine s opity of the lens n is ommon in eye pthology in fish (Hrgis 1991). Cuses of trtogenesis in slmonis re multiftoril, ut often relte to environmentl onitions (Bjerks et l. 1996; Bjerks et l. 2003). In prtiulr, slmonis s visul feeers n e signifintly impte y severe trt formtion in terms of reue fee intke n growth (Brek & Sveier 2001), seonry iseses n lso mortlity (Hrgis 1991). Ctrts re ffilite with ethil n welfre issues in slmon quulture, ut n lso result in eonomi losses (Menzies et l. 2002). In prtiulr, wter temperture ppers to hve mjor influene on trt formtion in Atlnti slmon (Bjerks & Bjørnest 1999; Bjerks et l. 2001; Wgø et l. 2010) n espeilly in triplois (Tylor et l. 2015). Further, the trnsition from fresh to se wter uring prr-smolt trnsformtion (smoltifition) ssoite with osmoregultory stress ppers to e n importnt stge for trt outreks (Iwt et l. 1987; Bjerks et l. 2003; Brek & Sveier 2001; Brek et l. 2005; Brek et l. 2005; Remø et l. 2014). The onsequene of osmoti imlne in the lens is the formtion of louiness tht susequently reues vision (Rhoes et l. 2010; Tr oe et l. 2010). In ontrst to trt tht pper erlier in fresh or lter uring the sewter stge, the osmoti inue trts re onsiere reversile (Hrgis 1991). Aleit speifi risk perios hve een ientifie, trt formtion in Atlnti slmon hs een oserve uring freshwter (Bjerks et l. 1996) s well s in the first (Brek et l. 2005) n seon (Wgø et l. 2010) yer t se, respetively. Nutritionl imlnes ttriute to trt formtion in slmon frming hve een extensively stuie with the onlusion tht the mino i histiine is key omponent for the prevention of trts in slmon (Brek et l. 2003; Bjerks & Sveier 2004; Bjerks, Brek & Wgø 2006; Tr oe et l. 2010; Wgø et l. 2010; Remø et l. 2014). However, the histiine requirement for growth (8 g kg 1, Sott 1998; NRC 2011) oes not meet the requirement for minimizing trts in ommeril Atlnti slmon iets, ove ll not in triploi slmon (Tylor et l. 2015). The unerlying mehnism for trt mitigtion is suggeste to e through the histiine metolite N-etyl histiine (NAH), 1196 whih hs lso een foun in the hert n rin; however, in the slmon lens, NAH funtions s n osmolyte (Rhoes et l. 2010) n my lso funtion s n intrellulr ntioxint (Remø et l. 2011). Lens NAH onentrtions orrelte positively with the ietry histiine onentrtion n negtively with trt severity (trt sore) in Atlnti slmon (Brek et l. 2005; Tr oe et l. 2010; Wgø et l. 2010; Remø et l. 2014). The ipeptie nserine is the mjor histiine ompoun (imizole) foun in white musle tissues in slmon n epens on the ietry histiine level (Remø et l. 2014). The roles of nserine re relte to musle uffering n urst swimming pity in se wter, n the musle onentrtion inreses uring smoltifition n therefter (Ogt, Konno & Silverstein 1998; Brek et l. 2005). Nonetheless, more reserh is neee to evelop est prtie rering protools for frming triploi Atlnti slmon in orer to ensure welfre n performne. The present feeing stuy exmine whether low or high levels of ietry histiine supplementtion ove the requirement for growth mitigte the effet of trtogenesis in iploi n triploi Atlnti slmon smolt efore n uring smoltifition t moerte n high tempertures. Mterils n methos The experiment ws onute t the Institute of Mrine Reserh (IMR), Mtre, Norwy, n pprove y the Norwegin Animl Reserh Authority, performe oring to previling niml welfre regultions (FOTS i 5283). Fish stok n rering onitions Diploi n triploi eye eggs were quire from AquGen (AquGen AS, Postoks 1240, Sluppen, 7462 Tronheim). The eggs were inute t 6 C, n pek hthing took ple on 11 Jnury First feeing strte on 5 Mrh 2013 t 10.5 C. On 7 Mrh 2013, the temperture ws rise to 13 C. The fish were rere in hete wter until 18 June 2013, when the temperture ws hnge to mient (12 C). The photoperio ws LD24:0 from first feeing to 1 Otoer 2013, when it ws hnge to simulte nturl photoperio. The fish were vinte (Norvx â, MINOVA 6 vet. Intervet Interntionl B.V.) on 25 Novemer 2013 n iniviully tgge

3 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses (Trovn trnsponer ID-100A Mirotrnsponer, Trovn â BTS Sninvi) on 5 Ferury Ploiy verifition Ploiy ws etermine se on re loo ell imeter (Benfey, Sutterlin & Thompson 1984; Peruzzi et l. 2005; Opst et l. 2013). On 17 Septemer 2013, 120 loo smers of eh ploiy th were tken. After ir rying, the loo smers were photogrphe t resolution of pixels lm 1 using Lei DMRE mirosope (type ) t 409 mgnifition n Sion mer (CFW- 1312C). Three pitures were tken from eh loo smer. From the pitures (verge 286), loo ells from eh fish were utomtilly size mesure using ImgeJ ( n slightly moifie version of the OjetJ ( nwi.uv.nl//ojetj/) projet Elliptil ooytes ( ytes/ooytes.htm). On verge, the iploi fish h mjor xis loo ell imeter of (SD), while the orresponing vlue for the triploi fish ws There ws no overlp in men re loo ell size etween fish from the iploi n triploi group n therefore the inution of triploiy ws onsiere to e 100% suessful. Experimentl esign On 5 Mrh 2014, 938 iploi n 925 triploi PIT-tgge Atlnti slmon (1+) presmolt were rnomly istriute into 32 qurti tnks (455 L) with 16 tnks ploiy 1 n fish tnk 1. The fish were rere uner simulte nturl light onitions from tnk stoking to experimentl termintion n t mient temperture (5.3 C) uring the limtion perio until the experimentl strt on 27 Mrh. Constnt freshwter flow ws supplie t 100% DO. The fish were fe in exess etween the hours of 08:00 n 18:00. Previous n uring the limtion perio, ommeril iet (Nutr Olympi 3 mm, Skretting AS; 9.0 g histiine kg 1 ) ws provie oring to the mnufturers tles n y utomti feeers (ARVO-TEC T Drum 2000, Arvote). Feeing n photoperio were ontrolle y ustomize omputer softwre (SD Mtre, Normti AS). The experimentl perio lste from 27 Mrh until 24 June n 12 August 2014 for the 16 C n10 C groups, 1197 respetively. Boy weight, fork length n onition ftor t the experimentl strt were g 0.9; 22.7 m 0.05; n g 0.9; 23.6 m 0.05 n (men SEM; n = 938; 925) for iplois n triplois, respetively. Therefter the fish were supplie with one of two experimentl iets, iffering in the mount of supplemente histiine. A low histiine iet (LH) ontining 10.4 g (espite terme low histiine iet, the histiine onentrtion is ove stnr ommeril iets) n high histiine (HH) iet with 13.1 g histiine kg 1 (4.5 mm) formulte n mnufture y Crgill Aqu Nutrition, Florø, Norwy. Fee ingreients, fee omposition n mino i profile re shown in Tle 1. On 31 Tle 1 Ingreients, proximte nutritionl omposition n mino i omposition of two experimentl iets (4.5 mm) vrying in the level of histiine: ommeril (pre): 9.0, low (LH): 10.4; high (HH): 13.1 g kg 1 Experimentl iet Ingreients (g kg 1 ) Fish mel Euro Soy protein onentrte Fish oil Rpesee oil Whet gluten Tpio Pe protein onentrte MCP Vitmin n minerl premix 3 3 Miro nutrients 10 5 Histiine 0 4 Proximte omposition (g kg 1 ) Protein Lipi Strh Totl phosphorous Amino i omposition (mg g 1 ) Hyp His Tu Ser Arg Gly Asp Glu Thr Al Pro Lys Tyr Met Vl Ile Leu Phe Σ mino is LH HH

4 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Mrh 2014, the wter temperture of ll tnks ws elevte y 1 C y 1 until eight iploi n eight triploi tnks rehe 10 C (4 April 2014), the remining 8 iploi n 8 triploi tnks were further elevte until 16 C (10 April 2014; Fig. 1). Both ploiies (2N, 3N), experimentl iets (LH, HH) n tempertures (10, 16 C) were use in full rossover esign, resulting in eight experimentl groups (2N10LH; 2N10HH; 3N10LH; 3N10HH; 2N16LH; 2N16HH; 3N16LH; n 3N16HH) with quruplite tnk replites. On 14 My 2014, the wter supply ws swithe from freshwter to se wter (heneforth referre s sewter trnsfer) with 35 g L 1 slinity n mintine DO (80 100%). The temperture ws not hnge uring sewter trnsfer. Due to high mortlity in the 16 C groups fter sewter trnsfer, the wter temperture ws lowere from 16 to 14 C from 17 My until 5 June 2014 (Fig. 1). Smpling proeures Three smplings were onute in the FW phse (7 weeks, 3 weeks, 2 ys prior to hnge to SW) n four in the SW phse (3 ys, 3 weeks, 6 weeks, 13 weeks fter hnge to SW; Fig. 1). The fish were strve pproximtely 40 h prior to eh smpling. As there ws no tretment of temperture n iet t the experimentl strt, only one fish per tnk (n = 32; 16 fish ploiy 1 ) ws smple, s well s one whole fish for ny unforeseen nlyses. Length, weight n PIT tg ientity were reore from ll fish. Therefter three fish per tnk n smpling point (n = 12 tretment group 1 smpling 1 ) were smple n three itionl whole fish tnk 1 were remove fter 3 ys (12 My) n fter 6 weeks (24 June) in SW. The fish were kille y n overose th of nesthetis (Finquel â ) followe y single low to the he. Length (to the nerest 1 mm), weight (to the nerest 1 g) n the PIT tg ientity were reore efore loo ws rwn from the ul vessel using heprinize neeles n syringes. White musle smples were tken, n susequently lenses refully issete. Gills (7 weeks, 2 ys efore s well s 3 weeks fter hnge to SW) were issete n ll smples immeitely frozen in liqui nitrogen efore store t 80 C until further nlysis. At the experimentl strt (27 Mrh) n 2 ys efore hnge to SW (12 My) s well s 6 weeks fter hnge to SW (24 June) n t termintion (12 August), ll fish were mesure for length, weight n PIT tg ientity. Aitionlly, 200 iploi n 200 triploi fish were inspete for trt y using slit lmp mirosope (HEINE â HSL 150 hn-hel slit lmp, HEINE Optotehnik) t the experimentl strt, therefter of 10 fish tnk 1 (40 fish group 1 ) for the sme tes. Ctrts were gre s trt sore oring to its severity on sle from 0 to 4 for eh eye n 0 8 for eh fish, respetively (Wll & Bjerks 1999). For lens n white musle nlyses, smples were poole oring to the following tretments in the initil smpling (1 smple tnk 1 ; 4 poole Temperture ( C) Dte Journl of Fish Diseses Figure 1 Experimentl esign to test the effet of wter temperture (10 vs. 16 C) n ietry histiine supplementtion (LH: 10.4 vs. HH: 13.1 g kg 1 ) on the performne of iploi n triploi Atlnti slmon in fresh n se wter. Soli rrows inite smpling tes, n lk ots ove rrows inite smpling tes when ll PIT-tgge fish were itionlly mesure for weight n length. A single she rrow inites the trnsfer to se wter. The wter temperture ws temporrily reue in groups rere t 16 C ue to high mortlity fter sewter trnsfer. 1198

5 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses smples ploiy 1 ) n therefter within eh tnk (3 smples tnk 1 ; 4 poole smples replite 1 ). Bloo n plsm Bloo ws rwn from the ul vein n eh smple entrifuge for 1 min t g t 6 C (Eppenorf, 5415R) llowing plsm to seprte. Plsm hlorie ws etermine with AVL 9180 Eletrolyte Anlyzer (Rohe Dignostis) using n ion-seletive eletroe. Plsm osmollity ws etermine y freeze point etermintion (Fiske miro-osmometer Moel 210). Amino i n histiine imizole etermintion Amino nlyser ninhyrin etetion (Amershm Phrmi Bioteh) ws use to nlyse fee. Musle (free si) n mino i (totl free) onentrtions were nlyse oring to Brek et l. (2005). Lens histiine n NAH onentrtions were nlyse y reverse phse HPLC (Wters Corportion) oring to moifie metho (Brek 2004), originlly esrie y O Dow et l. (1990). Gill N +,K + -ATPse tivity Gill N +, K + -ATPse (NKA) tivity ws etermine y the metho of MCormik (1993). Briefly, this kineti ssy utilizes the hyrolysis of ATP, whih is enzymtilly ouple to the onversion of NADH to NAD + y pyruvte kinse n lti ehyrogense with or without the ition of ouin, speifi inhiitor of NKA. Reings were one t 340 nm for 10 min t 25 C n enzyme tivity is expresse s lmol ADP * mg protein 1 * h 1. Clultions n sttistil nlysis The speifi growth rte (% growth y 1 ) ws lulte s SGR = (e q 1) 100 (Houe & Shekter 1981) where q = [ln(w 2 ) ln(w 1 )]/ (t 2 t 1 ) (Bgenl & Tesh 1978), W 2 n W 1 re oy weight t times t 1 n t 2, respetively. Fulton s onition ftor ws lulte s f = 100 * weight [g]/fork length 3 [m]. The t were sttistilly nlyse using Sttisti 11 with signifine level of 5% (P < 0.05) If not ifferently stte, the results re presente s men SEM. Prmeters were nlyse y neste ANOVA esigns onsiering temperture, ploiy n iet s fixe ftors n nesting these in tnk replites s rnom ftor. Signifint neste ANOVAs were followe y ftoril ANOVA esigns with temperture, ploiy n iet s inepenent vriles to etet possile intertions etween the vriles. Signifint ftoril ANOVAs were followe y Stuent Newmn Keul (SNK) post ho tests to etet possile ifferenes etween the iniviul groups. A one-wy ANOVA with ploiy s inepenent vrile ws performe with t from the initil smpling point (27 Mrh) when the vriles temperture n iet were not present. Dt were rsine-trnsforme prior to sttistil nlysis when neessry. Results Growth At the strt of the experiment, triploi fish were signifintly longer n hevier, ut oth triploi groups going to e hel 16 C h lower onition ftor thn ll other groups (SNK, P < 0.05). The evelopment in length, weight n the onition ftor uring the experiment is presente in Tle 2. Wheres length growth ws similr etween ploiies t 16 C, in freshwter, iplois h higher weight growth (SGR: 0.89 n 0.94% for LH n HH, respetively) thn the remining groups whih h similr SGRs etween 0.70% n 0.78% (ftoril ANOVA, temperture 9 ploiy, P < 0.001; Tle 3). Higher length growth (mm y 1 ) in triplois t 10 C resulte in signifint intertion etween temperture n ploiy in freshwter (ftoril ANOVA, P < 0.001). Men weight n length of groups t 16 C in se wter (Tle 2) re mske ue to high mortlity (Tle 4) n the SGR n growth in mm 1 (Tle 3) give therefore etter representtion of performne uring this perio. During the first 6 weeks in se wter, groups t 10 C grew (SGR n mm 1 ) similrly to eh other, ut signifintly etter (SNK, P < 0.05) ompre to ll 16 C groups (neste ANOVA, P < 0.001). Within groups t 16 C, iplois mintine their weight, wheres triploi h

6 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses 1200 Tle 2 Development of length (L), weight (W) n onition ftor (CF) (mensem) in iploi (2N) n triploi (3N) Atlnti slmon t 10 n 16 C, fe two experimentl iets iffering in the level of histiine (LH: 10.4; HH: 13.1 g kg 1 ). The experimentl iets reple ommeril iet on n the wter temperture ws then elevte from mient (5.3 C) to either 10 or 16 C (1 C y 1 ). The wter inflow ws hnge from freshwter to se wter on Groups t 16 C were terminte on 24.06, while 10 C groups were terminte on Groups t 16 C were terminte erly on Exept for (one-wy ANOVA), n sterisk inites sttistil P-vlues from neste ANOVA esign. Smpling L W CF L W CF L W CF L W CF Group 2N10LH N10HH N10LH N10HH N16LH N16HH N16LH N16HH Effet P-vlue Temperture* >0.001 >0.001 > >0.001 >0.001 Ploiy* >0.001 >0.001 >0.001 >0.001 >0.001 >0.001 > >0.001 > >0.001 Diet* T 9 P >0.001 > T 9 D P 9 D T 9 P 9 D Supersripts enote signifint ifferenes (SNK, P < 0.05) etween the groups for eh prmeter t eh perio. P-vlues in ol inite signifint effet of the respetive vrile or intertion.

7 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Tle 3 Speifi growth rte n length growth (men SEM) of iploi (2N) n triploi (3N) Atlnti slmon rere t 10 n 16 C n fe two experimentl iets iffering in the level of histiine (LH: 10.4; HH: 13.1 g kg 1 ). The experimentl iets reple ommeril iet on n the wter temperture ws then elevte from mient (5.3 C) to either 10 or 16 C (1 C y 1 ). The wter inflow ws hnge from freshwter to se wter on Groups t 16 C were terminte on 24.06, while 10 C groups were terminte on Asterisk inites sttistil P-vlues from neste ANOVA esign Group SGR mm 1 SGR mm 1 SGR mm 1 2N10LH N10HH N10LH N10HH N16LH N16HH N16LH N16HH Effet P-vlue Temperture* <0.001 <0.001 <0.001 <0.001 Ploiy* < < Diet* T 9 P <0.001 < T 9 D P 9 D T 9 P 9 D Supersripts enote signifint ifferenes (SNK, P < 0.05) etween the groups t eh perio. P-vlues in ol inite signifint effet of the respetive vrile or intertion. Journl of Fish Diseses negtive growth rtes (Tle 3). At oth tempertures, speifi growth rtes in iplois were higher thn in triploi groups. The onition ftor erese in ll groups, ut less in iplois thn triplois, less t 10 C thn 16 C n less in fish fe the high histiine iet (neste ANOVA, P < 0.05; Tle 2). From week seven in se wter until termintion (24 June to 12 August), ll groups t 10 C h stle SGRs (>1% 1 ). Among groups fe the high histiine iet, iplois h signifintly higher SGR ompre to triplois, wheres the respetive triploi group grew fster in length (Tle 3) resulting in lower onition ftor (SNK, P < 0.05; Tle 2). The onition ftor t termintion ws signifintly higher in iplois thn triplois n lso higher in oth ploiies fe the high histiine iet (neste ANOVA, P < 0.05; SNK, P < 0.05). Mortlity There ws no mortlity (Tle 4) in the perio etween tnk stoking n 2 ys fter sewter trnsfer (15 My). On 16 My, fter 3 ys in se wter, groups t 16 C experiene signifint mortlity n triplois more thn iplois. No mortlity ws reore in ny ploiy t 10 C (ftoril ANOVA, temperture 9 ploiy, 1201 P < 0.001). Mortlity ontinue in the 16 C tretments etween the thir n fifth week in se wter, however without n effet of ploiy (Tle 4). Smoltifition n osmoregultion Gill NKA enzyme tivity ws reore 3 weeks (23 April) n 2 ys (12 My) efore, s well s 3 weeks fter (3 June) sewter trnsfer. On 23 April, fish t 10 C h generlly higher gill NKA tivity thn fish t 16 C (neste ANOVA, P < 0.05; Fig. 2+). Shortly efore sewter trnsfer, the 10 C groups expresse nerly threefol the mount of gill tivity ompre to their respetive groups t 16 C (neste ANOVA, P < ) n still twofol the mount fter 3 weeks in SW (neste ANOVA, P < ; Fig. 2+). Initil levels of plsm osmollity (mosm; Fig. 2+) n hlorie (Cl, Fig. 2e+f) were similr etween ploiies n evelope in omprle pttern throughout the experiment. Two ys efore sewter trnsfer, the 16 C groups h higher levels of plsm mosm (neste ANOVA, P = 0.022). After 3 ys in se wter, osmollity n hlorie onentrtions in fish t 16 C peke high ompre to fish of the

8 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses 1202 Tle 4 Mortlity (%SE) evelopment etween smpling points of iploi (2N) n triploi (3N) Atlnti slmon t 10 n 16 C, fe two experimentl iets iffering in the level of histiine (LH: 10.4; HH: 13.1 g kg 1 ). The experimentl iets reple ommeril iet on n the wter temperture ws then elevte from mient (5.3 C) to either 10 or 16 C (1 C y 1 ). The wter inflow ws hnge from freshwter to se wter on Groups t 16 C were terminte erly on Unlike lower se, supersripts enote signifint ifferenes (SNK, P < 0.05) etween the groups t eh smpling point; ol P-vlues for temperture (T), ploiy (P), iet (D) n intertions inite signifint effets (ftoril ANOVA, P < 0.05) Group P-vlue 2N10LH 2N10HH 3N10LH 3N10HH 2N16LH 2N16HH 3N16LH 3N16HH T P D T 9 P T 9 D P 9 D T 9 P 9 D Perio <0.001 < < < Supersripts enote signifint ifferenes (SNK, P < 0.05) etween the groups t eh perio. P-vlues in ol inite signifint effet of the respetive vrile or intertion. respetive 10 C groups (neste ANOVA, P < ) n higher osmollity levels in triplois t 16 C use signifint intertion etween temperture n ploiy (ftoril ANOVA, P = 0.035). A signifint effet of temperture remine on oth prmeters fter 6 weeks in se wter (neste ANOVA, P < 0.05). There were no ifferenes reore etween ploiy n iet within the 10 C groups fter thirteen weeks in se wter. From winter solstie (21 Deemer) until the y efore the temperture ws grully inrese (26 Mrh), the men wter temperture ws C (min 4.3 C; mx 6.4 C; egreeys 523) n until sewter trnsfer (14 My), the 16 C groups were rere t 220 egree-ys more thn the 10 C groups (986 vs 1206 egree-ys). Ctrt evelopment The oservtion of trts t the experimentl strt, with previously fe ommeril iets n uner mient wter temperture, ws low n low gre, ut iffere sttistilly etween ploiies (men sore 2N: ; 3N: ; n = 200 ploiy 1 ; Fig. 3, +). The prevlene ws 3% in the iploi n 11.5% in the triploi popultion n the men sore of ffete iniviuls ws 1.17 n 1.52 in iplois n triplois, respetively. In most ffete fish in oth ploiies, the lotion of trts ws t the nterior ortex. At the en of the freshwter perio, groups t 16 C evelope signifintly higher trt sores (from (2N16LH) to (3N16LH)) ompre to ll groups t 10 C (neste ANOVA, P < 0.001). Within groups t 16 C, trts evelope systemtilly more in triplois (men sore > 3) ompre to iplois (men sore < 3) n lso signifintly more in groups fe the low histiine iet (ftoril ANOVA, temperture 9 ploiy, temperture 9 iet, P < 0.001; SNK, P < 0.05). Groups t 10 C evelope minor trts n only 3N10LH ( ) h signifintly higher (SNK, P < 0.05) trt sore thn 2N10HH ( ). The lenses of fish fe the low histiine iet t 16 C preominntly revele jellylike or louy strutures in the nterior ortex, whih were not oserve mong fish rere t 10 C.

9 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Gill N +, K + -ATPse tivity (µmol ADP* mg protein 1 * h 1 ) () 2N10LH 2N10HH 3N10LH 3N10HH () 2N16LH 2N16HH 3N16LH 3N16HH 0 Osmollity (mosm kg 1 ) () 2N10LH 2N10HH 3N10LH 3N10HH () 2N16LH 2N16HH 3N16LH 3N16HH Chlorie (µmol L 1 ) (e) 2N10LH 2N10HH 3N10LH 3N10HH (f) 2N16LH 2N16HH 3N16LH 3N16HH 120 Dte 120 Dte Figure 2 Development of gill N +,K + -ATPse tivity (, ), plsm osmollity (, ) n plsm hlorie levels (e, f) (men SEM) in iploi (2N) n triploi (3N) Atlnti slmon smolts n post-smolts (sewter hnge ) t 10 n 16 C, fe two iets iffering in the level of histiine (LH: 10.4 g kg 1 ; HH: 13.1 g kg 1 ). The experimentl iets were reple y ommeril iet on n the wter temperture ws then elevte from mient (5.3 C) to 10 n 16 C (1 C y 1 ), respetively. Different supersripts inite signifint ifferenes (SNK, P < 0.05) etween the groups, ut within eh prmeter (, ;, ; e, f), t eh smpling point. Journl of Fish Diseses During the first 6 weeks in se wter, iplois n triplois tht fe the low histiine iet t 16 C evelope trts further (men sore > 5), wheres the onspeifi groups tht fe the high histiine iet hlte trt formtion (2N) or only slightly inrese (3N) (ftoril ANOVA, ploiy 9 iet, P < 0.05). Among groups t 10 C, trts inrese signifintly more in 3N10LH ompre to the remining groups. Triplois fe the HH iet n iploi fe 1203 the LH iet evelope similr trt severity, n only the trt sore in iplois fe the high histiine iet i not inrese (ftoril ANOVA, temperture 9 iet, P < 0.05). The nterior ortex ws mostly ffete in fish t 10 C, wheres jelly n louy strutures previle in fish t 16 C, often in form of ring or str ppernes. At termintion, trts were systemtilly higher in triplois thn iplois (neste ANOVA, P < 0.001) n groups fe the

10 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Ctrt sore (0-8) 6.0 () 6.0 () N10LH 2N10HH 3N10LH 3N10HH 2.0 e ef ef f 1.0 e N16LH 2N16HH 3N16LH 3N16HH Lens histiiine (µmol g 1 ) () 2N10LH 2N10HH 3N10LH 3N10HH () 2N16LH 2N16HH 3N16LH 3N16HH Lens NAH (µmol g 1 ) (e) 2N10LH 2N10HH 3N10LH 3N10HH Dte (f) 2N16LH 2N16HH 3N16LH 3N16HH Dte Figure 3 Development of trt (, ) (sum sore 0-8; men SEM), lens histiine (, ) n lens N-etyl-histiine (e, f) (men SEM; n = 4 poole smples) onentrtions in iploi (2N) n triploi (3N) Atlnti slmon smolts n post-smolts (sewter hnge 14.05) t 10 n 16 C, fe two iets iffering in the level of histiine (LH: 10.4 g kg 1 ; HH: 13.1 g kg 1 ). On 30.03, the experimentl iets reple ommeril iet n the wter temperture ws then elevte from mient (5.3 C) to 10 n 16 C (1 C y 1 ), respetively. Different supersripts inite signifint ifferenes (SNK, P < 0.05) etween the groups, ut within eh prmeter (, ;, ; e, f), t eh smpling point. Journl of Fish Diseses high histiine iet h lower trt sores ompre to groups fe the low histiine iet (neste ANOVA, P < 0.001). Distint jelly strutures, often str shpe, were foun in oth ploiies fe the low histiine iet, wheres groups fe the respetive high histiine iet were moertely ffete in the nterior or showe trt formtion with singulr or multiple spots. In the lst 7 weeks of the experiment, oth ploiies fe the low histiine iet signifintly inrese in trt sore, wheres the groups fe the high histiine 1204 iet hlte trt formtion (neste ANOVA, ploiy, iet, P < 0.001). Thus, iploi slmon fe the high histiine iet n rere t low temperture showe minor trt evelopment throughout the experiment. Lens histiine n NAH Lens histiine levels were similr etween ploiies t the experimentl strt (2N: vs. 3N: lmol g 1 ; Fig. 3+). At the

11 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses en of the freshwter perio, ll groups signifintly erese lens histiine onentrtions, lthough groups t 10 C n fe the high histiine iet less thn groups t 16 C n fe the low histiine iet (ftoril ANOVA, temperture 9 iet, P = 0.015; ploiy 9 iet, P = 0.012). Shortly fter sewter trnsfer, ontinues erese in lens histiine ws reore in the 10 C groups, wheres fish t 16 C stgnte when fe the low histiine iet n inrese when fe the high histiine iet. Within oth tempertures, fish fe the high histiine iet h higher onentrtions of lens histiine ompre to fish fe the respetive low histiine iet (neste ANOVA, P < 0.05). After 6 weeks in se wter, lens histiine onentrtions were similr etween ploiies of the sme iet t 10 C (HH > LH), wheres iplois n triplois t 16 C tht were fe the low histiine iet ontine more thn twie s muh lens histiine ompre to the high histiine iet groups (SNK, P < 0.05). Diploi lenses ontine signifintly more NAH t the first smpling (one-wy ANOVA, P < 0.05; 2N: vs. 3N: lmol g 1 ; Fig. 3e+f). Until the en of the freshwter perio, onentrtions signifintly erese in ll groups, ut less in groups fe the high histiine iet (neste ANOVA, P < 0.05). The effet of iet on lens NAH levels remine fter 2 ys in se wter n onentrtions erese slightly in fish t 10 C, wheres the 16 C groups moertely inrese when fe the high histiine iet. After 6 weeks in se wter, lens NAH onentrtions were similr etween ploiies of the sme iet t 16 C n t groups fe the low histiine iet t 10 C. Among groups fe the high histiine iet t 10 C, iploi lenses ontine signifintly higher levels of NAH thn triplois (neste ANOVA, iet, P < 0.05; ftoril ANOVA, ploiy 9 iet, P < 0.05). White musle mino i onentrtion In Mrh, the level of white musle totl free mino is ws higher in iplois thn triplois. Triplois h signifintly higher onentrtions of nserine, wheres iplois were signifintly elevte in the non-essentil mino is serine n glyine (Tle 5). The sum of white musle free mino is erese y nerly 25% shortly efore sewter 1205 trnsfer in ll experimentl groups ue to signifint reutions of essentil (methionine n histiine) n non-essentil mino is (serine, proline, glyine, lnine) n nitrogenous ompouns (-lnine, ure n nserine, in groups fe the low histiine iet). Before sewter trnsfer, oth ploiies fe the high histiine iet h signifintly higher levels of nserine n rnosine ompre to groups fe the low histiine iet (ftoril ANOVA, P < 0.001), whih ounterlne the sum of mino is with n inrese in non-essentil mino is (serine, glyine, lnine, hyroxyproline) n nitrogenous ompouns (ure, L-mino-N-utyri i). The iet llotion reflete overll the onentrtion of histiine in the white musle uring freshwter. Diploi slmon ontine more histiine thn triplois t the experimentl strt; however, the ifferene ws not sttistilly ifferent (one-wy ANOVA, P = 0.053). Prior to sewter trnsfer, ll groups erese in white musle histiine, prtiulrly groups fe the low histiine iet t 10 C (ftoril ANOVA, P < 0.05). The fstest growing group uring freshwter iplois fe the high histiine iet t 16 C erese signifintly less thn the remining groups n ws le to mintin most of the initil tissue histiine ontent (Tle 5). Disussion The present stuy emonstrte tht triploi Atlnti slmon yerlings evelope more severe trts t high tempertures n with insuffiient ietry histiine from 7 weeks prior to thirteen weeks fter sewter trnsfer. However, the iet with higher histiine level suessfully mitigte trt outreks in oth ploiies when rere t 10 C. Aitionlly, the stuy hs shown tht high temperture h negtive effet on smoltifition, survivl n sewter performne in iploi, n more so in triploi slmon. Growth n smoltifition In freshwter, oth iploi groups rere t 16 C grew fster thn the remining groups whih grew similrly. This is in orne with higher fee intke in iplois ompre to triploi Atlnti slmon t high (19 C) wter tempertures in se wter (Hnsen et l. 2015). The

12 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses Tle 5 Free mino i onentrtion in white musle tissue (poole men SEM; n = 4 smples) in iploi n triploi Atlnti slmon t the experimentl strt (27.03) t mient wter temperture (5.3 C) n t the en of the freshwter perio (12.05) in 10 C n 16 C, fe iets with low (LH: 10.4 g kg 1 ) or high histiine (HH: 13.4 g kg 1 ) ontent 1206 Diploi Triploi C 16 C 10 C 16 C Amino i (lmol g 1 ) LH HH LH HH LH HH LH HH Essentil Threonine * * * * Vline * Methionine * * * * * Isoleuine Leuine * * * Phenyllnine * * * * * * * * Lysine * * * * * * Histiine * * * * * * * Arginine * * * * * * Tryptophn n.. n.. n.. n.. n.. n.. n.. n.. n.. n.. Non-essentil Serine A * * * * B * * * * Glutmi i * * * Glutmine * * * * * * * * Proline * * * * * * * * Glyine A * * * e * B * * * e * Alnine * * * * * * * * Tyrosine Asprti i * * * * * * Hyroxyproline * Nitrogenous ompouns -Alnine * * * * * * * 1-Methyl-histiine * * * * * * * * Crnosine * * Anserine B * * * * A * * Turine * * * * * * * * * O-phosphoethnolmine Ure * * * * * L--mino * * * * N-utyri i Ammoni * * * * * * * * Ornithine * * * * * * ΣFree mino is Unlike upper se, supersripts enote signifint ifferenes etween ploiy on (t-test, P < 0.05); unlike lower se, supersripts enote signifint ifferenes etween ll experimentl groups on (SNK, P < 0.05); sterisks enote signifint ifferenes within ploiy etween n (one-wy ANOVA, P < 0.05); n.., not etetle.

13 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses ft tht iplois h higher SGRs n mm y 1 t 16 C ompre to 10 C in the present stuy, while triplois only iffere in mm y 1 etween the tempertures my point to tht the optimum temperture for growth in freshwter is ifferent etween iplois n triplois. Growth in freshwter hs often een reporte superior in triplois ompre to iplois (Fjelll & Hnsen 2010; Lelerq et l. 2011; Tylor et l. 2012; Tylor et l. 2013), n in the present stuy, triplois were signifintly hevier ompre to iplois t experimentl strt. This is likely ue to generlly lower temperture in freshwter rering tht is minly within the preferre rnge of triplois. Future stuies shoul fous on etermining the optiml temperture for growth in freshwter in triploi Atlnti slmon. In the first perio fter sewter trnsfer, growth rtes were lower in ll groups ompre to freshwter n lso lower ompre to the seon perio in se wter. Wheres groups t 10 C grew moertely, groups t 16 C only mintine (iplois) or even lost weight (triplois). Deresing onition ftors in Atlnti slmon in the weeks efore n fter smoltifition n our n re ttriutle to metoli justments (Clrke, Shelourn & Brett 1981; Sherin 1989) n inrese skeletl growth (Hor 1988). Initil sewter performne in triplois hs een ientifie to e inferior ompre to iplois (Lelerq et l. 2011; Tylor et l. 2012). Aitionlly, the poor performne fter sewter trnsfer in groups rere t 16 C ws onomitnt with high mortlity, espeilly in triplois. One reson my e insuffiient gill NKA tivity prior to sewter trnsfer n osmoregultory isturnes in osmollity n hlorie homeostsis in se wter. Therefore, ll groups t 16 C were remove fter the first sewter perio n fish kille. Similrly, Hneln et l. (2000) hve reore higher mortlity uring the first 18 ys in se wter in iploi out of seson Atlnti slmon t 18.9 C (18%) ompre to oler tempertures. In the sme stuy, slmon trnsferre t 18.9 C exhiite similr plsm hlorie onentrtions s in the present stuy n higher onentrtions to groups rere t oler tempertures. Ion perturtions my lso e ue to osmoregultory ysfuntions in other osmoregultory orgns n hnges in epithelil permeility. In ontrst to growth n smoltifition t 10 n 16 C uring the lst 6 weeks in freshwter in the present stuy, erlier stuies hve shown: (i) 1207 norml growth (SGR ~ 1.5%; Grini et l. 2011) n low mortlity (0 1.3%; Grini 2008) t oth tempertures uring the first 6 weeks fter trnsfer to se wter, when the fish were mintine t the sme tempertures fter trnsfer; (ii) no mortlity uring the first 96 h in se wter t oth tempertures, when the fish were trnsferre to 8.9 C se wter (Fjelll, Hnsen & Hung 2011); n (iii) no mortlity n norml growth (SGR %) t 16 C in the first 6 weeks in se wter, when the fish were rere t 8.9 C (Fjelll et l. 2011). Further, Frser et l. (2012) trnsferre uneryerling iploi n triploi Atlnti slmon smolts from 16 C n LD24:0 to 9 C se wter n i not experiene mortlity ue to insuffiient hypo-osmoregultory ility. In ontrst, groups t 10 C isplye typil gill NKA peks (~10 12 lmol ADP * mg protein 1 * h 1 ) efore sewter trnsfer with vlues omprle to Hneln et l. (2004), suggesting tht the fish were trnsferre within the smolt winow. No mortlities in either ploiy support this notion. During the first weeks fter sewter trnsfer, growth n e impire ue to metoli justments. The SGR in the present stuy of groups rere t 10 C ws % for the first 6 weeks in se wter, ut higher (>1% y 1 )in the weeks therefter until termintion. These oservtions re in orne with Hneln, Arnesen & Stefnsson (2003) who mesure lower growth rtes in first month fter sewter trnsfer (~0.35%); however, growth rtes more thn oule uring the seon month t se wter. As the fish in the present stuy were only smple 6 weeks fter sewter trnsfer, it is possile tht growth rtes hve reovere erlier. Tylor et l. (2012) hve foun tht uneryerling triploi Atlnti slmon n unergo smoltifition up to 4 weeks sooner thn iplois, wheres yerling smolts rehe smoltifition simultneously espite triploi fish were lrger thn iplois (Boeuf et l. 1994). In the present stuy, triploi n iploi yerling smolts t 10 C seem to hve similr smolt evelopment in ontrst to those t 16 C; espite similr gill NKA vlues n plsm ion onentrtions, triplois h signifintly higher mortlity thn iplois uring the first perio fter hnge to se wter. It hs een emonstrte tht Atlnti slmon nee pproximtely 250 egree-ys efore >90% of the smolts reh pek gill NKA enzyme tivity (Hneln et l. 2004). From the experimentl strt until sewter

14 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses trnsfer, there ws evition of 220 egree-ys etween groups t 10 n 16 C tht is likely to hve resulte in the ifferene in smoltifition sttus n hene the oserve inequte hypoosmoregultory ility t 16 C. Inrese freshwter temperture efore smoltifition hs shown to elerte smoltifition n susequently esmoltifition (Duston, Suners & Knox 1991). It is therefore possile tht those fish hve smolte efore the first mesurement of gill NKA tivity. Overll, the high histiine iet h positive effet on the onition ftor in oth ploiies n tempertures, suggesting enefiil effets on the metolism. The high mortlity of 49 69% uring the first perio in se wter mong the triplois t 16 C shows tht speil re shoul e tken when rering triploi smolts t elevte temperture suh s in reirultion systems. There is prominent nee for evelopment of est prtie protools for triploi smolt proution in orer to voi suh ig lossesinommerilfrming.thereisninresing proution of triploi smolts in Norwegin quulture in onjuntion with more smolt frms hnging from flow through to reirultion. Temperture In the present stuy, groups rere t 16 C evelope more prevlent n severe trts uring the lst weeks in freshwter, in ontrst to groups tht only evelope negligile trts rere t 10 C. Among oth tempertures, triplois were generlly more ffete thn iplois, ut within oth ploiies n tempertures, the iet with higher onentrtion of histiine h enefiil effet. Severe trt outreks in freshwter hve rrely een oserve in Atlnti slmon (Bjerks et l. 1996, 2001), most likely ue to low or moerte wter tempertures until trnsfer to se wter. The optimum temperture for growth in Atlnti slmon post-smolts is C for iplois of g (Hneln, Imsln & Stefnsson 2008). At higher temperture, feeing will ese s oxitive pressure inreses, ssoite with shifts in the ntioxint efene system ue to higher metoli tivity n energy emn. It my e possile tht inlusion of ietry histiine in the low histiine iet ws insuffiient for fish t 16 C ue to inrese metoli tivity. The severe inrese in lens opities in groups t 16 C uring the finl weeks in freshwter my 1208 e onnete to osmoregultory prolems, ut my lso e onnete to ifferenes in metolism t the two tempertures. Elevte tempertures up to the therml optimum result in higher growth rtes, n previous stuies hve shown positive orreltion etween fish size or growth rte n trt sore, when the severity ws mil or moerte (Wgø et l. 1996; Bjerks & Sveier 2004; Brek et l. 2005; Tylor et l. 2015). In the present stuy, the ltere growth rte etween iplois rere t 10 n 16 C my hve ontriute to the higher trt sore, wheres triploi groups i not grow s fst s iplois t 16 C, lthough evelope more trts ompre to the respetive groups t 10 C. The groups rere t 16 C performe poorly fter sewter trnsfer n were ffete y high mortlity within the first 6 weeks. Brek & Sveier (2001) trnsferre two groups of Atlnti slmon in 4-week intervl into se wter n foun the lter group not only to perform less well fter trnsfer, ut ws lso more ffete y lens opities. It is likely tht not only growth performne, ut lso the high prevlene of trts in these groups is influene y osmoregultory stress. Defetive osmoregultion uring smoltifition in se wter hs een suspete to inue trtogenesis (Bjerks et l. 2003), n this my explin the high prevlene of trts in the groups rere t 16 C in the present stuy. In prllel, istintion in the trt sore etween ploiies ws evient t eh smpling point with triplois isplying higher trt sores uner the sme tretment s iplois. Stuies ompring trts etween iploi n triploi Atlnti slmon re sre (Wll & Rihrs 1992; Lelerq et l. 2011; Tylor et l. 2013, 2015), ut show the sme tren of triplois eveloping more n greter severity of trts uner the sme onitions s iplois. Lelerq et l. (2011) n Tylor et l. (2015) trnsferre iploi n triploi Atlnti slmon silings s off-seson smolts (<50 g) to se wter in Jnury n i not oserve trts until the first summer, when the trt sore ws higher in triplois thn iplois. This supports the importne of wter temperture, espeilly uring the perios efore n fter trnsfer to se. Further, it ws foun tht histiine-enrihe iet (17.4 vs g kg 1 ) hlte trt progression in ult triploi slmon from April to Septemer (8 15 C) in the seon yer t se when pplie in Ferury of the sme yer, wheres the

15 Journl of Fish Diseses 2017, 40, F Smrus et l. Triploiy n trt Journl of Fish Diseses men trt sore in the triploi group fe the respetive other iet inrese nerly twofol uring the sme time (Tylor et l. 2015). This inites tht enrihe iets shoul preferly e pplie throughout the entire proution perio, ut t lest with suffiient time prior to risk perios in orer to rete uffer funtion. Hene, it my e possile tht the severe trt outrek in freshwter in groups t 16 C might hve een preventle, if, in prtiulr, the high histiine iet ws pplie some weeks efore the temperture ws inrese. Histiine n NAH Supplementtion of ietry histiine signifintly reue the severity of trts in oth ploiies rere t 10 n 16 C in freshwter, n in se wter for 13 n 6 weeks in groups t 10 n 16 C, respetively. These finings re in line with previous stuies showing tht histiine supplementtion reues the outrek n evelopment of trts in the slmon lens in se wter in oth iplois (Brek et l. 2005; Wgø et l. 2010; Remø et l. 2014) n triplois (Tylor et l. 2015). The ietry histiine onentrtions use in the present stuy were oth ove the requirement for growth for Atlnti slmon (8 g his kg 1 ; Sott 1998) n the high histiine iet ws similr to the requirement to minimize trt evelopment for Atlnti slmon smolt fter se trnsfer. Brek et l. (2005) fe iets with two ifferent levels of histiine (11.7 n 18.0 g kg 1 ) for the lst 6 weeks in freshwter (men temperture 12.7 C) to Atlnti slmon n foun only negligile trts n low lens NAH levels efore sewter trnsfer, irrespetive of ietry histiine level. In the present experiment, oth iploi n triploi smolt h high lens onentrtions of NAH t the strt of the experiment, showing tht the synthesis of NAH n e initite in freshwter. This my e funtion of fish size or smoltifition sttus n thus iffers from previous stuies where lens NAH onentrtion hs een investigte in freshwter (Brek et l. 2005,; Remø et l. 2014). Despite similr lens histiine onentrtions etween ploiies t the experimentl strt, triplois h signifintly lower lens NAH. During the freshwter perio, lens NAH onentrtions eline in ll groups. The sme pttern ws foun y Tylor et l. (2015) in ult iploi n triploi Atlnti slmon in se wter 1209 n my inite lower ility for etylting NAH in triplois ompre to iplois n hene mke them more prone to trt evelopment. Inee, triploi groups evelope more trts ompre to their iploi onspeifis until sewter trnsfer n lens NAH onentrtions were, with one exeption (2N16LH), lower. The present results suggest tht oth iploi n triploi slmon require high ietry histiine onentrtion to sustin high lens NAH levels n reue the risk of trt evelopment lso in freshwter when rere t high temperture. The perio fter sewter trnsfer is onsiere risk perio for trt evelopment (Brek et l. 2005); however, oth the prevlene n severity of trts n e minimize y inresing the ietry histiine onentrtion (Remø et l. 2014). After sewter trnsfer, NAH plys n importnt role s n osmolyte in the slmon lens (Brek et l. 2005). In the present stuy, there ws tren of reue lens NAH shortly fter sewter trnsfer. Rhoes et l. (2010) suggeste tht NAH is the mjor osmolyte in the slmon lens ue to positive orreltions (in vitro, in vivo) of NAH n the osmollity of the externl meium. Tr oße et l. (2009) foun tht the efflux of NAH from Atlnti slmon lenses ex vivo uner hypo-osmoti onitions is relte to the ietry histiine ontent n the protein soure. Further, NAH is the preferre soure of utiliztion ue to its superior effiieny in lens osmoregultion ompre to efflux of other mino is (Tr oe et l. 2010). Therefore, the reue onentrtions fter 2 ys in se wter my e n inition of NAH use s n osmolyte when trnsferre to se wter. Six weeks fter sewter trnsfer, lens NAH onentrtions were higher in oth ploiies fe the high ompre to low histiine iet, n higher in iplois fe the high histiine iet ompre to triplois. The triploi groups h higher trt sore ompre to the respetive iploi groups given the sme iets. These results le to the ssumption tht triplois hve higher ietry histiine requirement thn iplois ue to higher men trt sore uner the sme onitions, ssuming tht the requirements for other trt mitigting nutrients re met. Musle imizoles In Atlnti slmon, nserine is quntittively the most unnt white musle imizole. Anserine