Master thesis (30 credits) Hao Zhang & Haibo Wang. Department of Animal and Aquacultural Science Norwegian University of Life Science.

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3 Enrichment of plnt-protein sed diets for Nile tilpi (Oreochromis niloticus) with krill protein hydrolyste with high concentrtion of phospholipids rich in n-3 ftty cids Mster thesis (30 credits) Ho Zhng & Hio Wng Deprtment of Animl nd Aquculturl Science Norwegin University of Life Science Ås 2012

4 Acknowledgements We would like to show our pprecition to those who help us finish the thesis nd experiment: Firstly we would like to thnk Professors Trond Storekken nd Mgny S. Thomssen, our supervisors, from the ottom of our hert. Your ptience, kindness nd experience helped us overcome ll the prolems. Also, our thnks go to Frnk Sundy, Nin Asper nd Bjørn Reidr Hnsen. Without their outstnding guidnce nd support, we could not successfully hve crried out this experiment. We re lso grteful tht thee 20 pnelists in the tilpi tste test, for your efforts to help us. Lst ut not lest, we pprecite the support from our fmilies. You cheer us up when we re confused nd lose our confidence. You give us courge to continue our study. We cn lwys find pece nd motivtion from your words when we re upset. Thnk you nd we love you. We lso wnt to sy thnks to ech other. We re tem, without either of us, we cnnot triumph. Thnk you. UMB, Ås, Ho Zhng nd Hio Wng II

5 Astrct 28-dy experiment ws conducted to investigte the effects of dietry krill (Euphusi super) hydrolyste with high concentrtion of phospholipids rich in n-3 ftty cids (KHPL) on growth performnce, ody composition, fillet ftty cid profile nd sensory chrcteristics of Nile tilpi (Oreochromis niloticus). Three diets were produced with pre-geltinized strch nd plnt protein nd lipid sources in pst mchine. Plnt diet ws formulted with no krill product. Two diets were formulted with 5 nd 10% KHPL, respectively. Ech diet ws fed to triplicte groups of 25 fish of 43.2±0.9 g (men ± SE.) initil ody weight. The Tilpis were fed for 28 dys t 26.7±0.5 C in L cylinder tnks with re-circulted system whose wter flow rte of out 5.6 l min. The tilpis were therefter kept in clen wter to reduce muddy off-flvor, nd fter 6 dys of purging they were served to consumer tste pnel consisting of 20 untrined no-smoking judges (10 Chinese, 10 Cucsin, 5 femles nd 5 mles within ech ethnic group). The judges were served pieces of ked unslted Tilpi fillet nd were sked to judge fishy tste/smell, muddy tste/smell nd other tste/smell, nd to rnk the fish on scle from 1 to 3, where 1 represented lest ttrctive nd 3 represented the most preferred. Dietry inclusion of KHPL hd no significnt effect (P>0.05) on growth rtes, feed conversion or ody composition of the tilpi. The men finl ody weight of fish fed y ech diet ws ± ±2.67 g. The men feed conversion rte (FCR) ws 1.30±0.06 kg ingested per kg gin. The contents of whole ody lipid were from 12.6% to 13.7%, whole ody protein rnged from 14.3% to 14.8%, nd the sh levels were etween 9.9% nd 10.4%. The contents of fillet protein were from 18.9% to 19.7%, lipid levels of fillet vried from 1.6% to 2.0%. Significnt differences were found in the content ftty cid in the fillet. Incresing dietry KHPL resulted in decresing concentrtion of monoenic ftty cids, while polyenes, EPA, DHA nd totl n-3 ftty cids, s well s the of n-3/n-6 rtio incresed with incresing KHPL. The sum of n-3 ftty cids rnged from 5 to 10 mg (kg fillet). Tilpi fed the diet with most KHPL tended to e preferred y the judges, followed y the fish fed the diet with 2% krill product nd the fish fed plnt mterils only. A significnt gender difference ws found in smell (P=0.032). Femles seemed more sensitive thn mle in detecting differences in smell, while mles seemed more perceptive to tste thn femles. The Chinese judges discriminted fishy tste more efficient thn the Cucsins (P< ), while the Cucsins seemed more sensitive thn the Chinese oth differentiting muddy tste (P=0.012) nd other tste (P=0.0065). III

6 Contents 1. Introduction Mterils nd methods Fish popultion Krill hydrolyste with high concentrtion of phospholipids (KHPL) Feed formultion nd processing Feeding tril Body composition nd ftty cid nlysis: Smple preprtion Preprtion of ftty cid profile nlysis Whole ody nd fillet nutrition nlysis Sensory nlysis Sttistic nlysis Results Growth nd survivl Body composition Ftty cid profile of Tilpi fillet nd KHPL Sensory nlysis Discussion growth tril Effects of feed ingredient on growth Effect of feeding strtegy on growth Effect of KHPL on ody composition Ftty cid profile Effect of KHPL on ftty cid profile Effect of KHPL on n-3/n-6 rtio Tilpi sensory nlysis Effect of KHPL on sensory nlysis Effect of gender on sensory nlysis Effect of ethnicity on sensory nlysis Conclusion References IV

7 1. Introduction Polyunsturted ftty cid (PUFA) hs een highly pid ttention to for its use on humn crdiovsculr disese prevention nd infnt rin development (Olsen et l., 1990; Cstell et l., 1994; Steffens, 1997; Enser et l., 2000; Chou et l., 2001; Cllero et l., 2002). Numerous studies hve een done on the min resource of PUFA (Srgent et l., 1999; Steffens, 1997; Htlen et l., 2012): fish oil (Al-Souti et l., 2012; Teoh et l., 2011), nd its vegetle sustitutions (Nsopoulou nd Zetkis, 2012; Krpngiotidis et l., 2007; Bhurmiz nd Ng, 2007). Krill is shrimp-like crustcen. Antrctic Krill (Euphusi super nd E. crystllorophis) re the min krill species. Lipid of Antrctic krill is %, phospholipids minly, nd is higher thn tht of E. pcific nd Thysnoess spinifer (5%-20%, minly glycerol), nd krill in the North Pcific (Ju.S.-J. et l., 2009). Pierce et l showed the lipid content of krill (36%-48%) outnumered tht of red cr (Gecrcoide ntlis) (Pierce et l., 1969). Antrctic krill hs higher lipid content (11.3%) thn orientl river prwn (Mcrorchium nipponense), gresy-ck shrimp (Metpeneus ensis), nd Chinese white prwn (Peneus chinensis) (4.9%~6.7%) (Li et l., 2011). As newlydeveloped feed resource, krill is of gret undnce nd is regenertive resource with dditionl enefits (Colomo-Hixson et l., 2011). The yield of krill kept t 120 thousnd tons from 1992 to 2009 nd incresed to more thn 200 thousnd tons in 2010 (Nicol et l., 2012). Recently, krill hydrolyste with high content of phospholipids (KHPL) hs ecome ville, trgeting humn helth products. In 1988, T. Storekken reviewed the potentil of krill s feed for slmonid (Storekken, 1988). Krill mel ws used s nturl stxnthin pigment efore synthetic stxnthin ws introduced (Yoshitomi et l., 2006) nd oth krill hydrolyste nd mel hve een climed useful s feed ttrctnt (Kolkovski et l., 2000; Olsen et l., 2006). Allhpichy nd Shimizu hve determined tht non-muscle krill mel ws more efficient thn whole-krill mel in feeding (Allhpichy nd Shimizu, 1984). In 1

8 2005, Ger determined the replcement of krill mel for soyen mel s protein source for juvenile Nile tilpi (Ger, 2005). The utiliztion of three different crustcen mels, from T. inermis, E. super nd Themisto liellul, hs een evluted on fillet qulity of Atlntic slmon (Slmo slr) ( Suontm et l., 2007). Beencor et l. evluted the use of three different krill y-products in feed of se rem (Sprus urt) lve (Betncor et l., 2012). The sustitution percent of krill mel (E. super mel) for fish mel should e lower thn 20% (Yoshitomi et l., 2006). Zhng lso mentioned using krill mel or wter-solule krill y-product in plnt-sed feed to increse feed intke (Zhng, 2011). Yet, the effect of KHPL on tilpi hsn t een investigted. Tilpi, group of cichlids, is cultured minly in Asi nd South Americ, mking it one of the fstest growing quculture species, nd in most countries where tilpi is produced, they re min species for consumption ( Normn-López nd Bjørndl, 2009). Most species of tilpi re omnivorous nd cn reproduce nd grow rpidly. Considerle genetic reserch nd selective reeding on tilpi hve een done (Thodesen et l., 2012; Yn et l., 2012; Bentsen et l., 2012; Pierce et l., 2012; Srker et l., 2012; Eknth et l., 1993). Consumers re willing to uy food with high PUFA for the enefits to humn helth (Foxll et l., 1998). The nutrient composition nd requirement of tilpi hve een investigted (Gonzles nd Brown, 2006; El-Syed nd Teshim, 1992; Grduño-Lugo et l., 2007). Tilpi hs potentility of eing n-3 enriched functionl food (Al-Souti et l., 2012). To dte, vriety of feed resources hve een proved tht they cn e utilized in tilpi feeding (Hussein et l., 2012; Al-Souti et l., 2012; Tonil et l., 2012; Fll et l., 2012). Normlly, whole-ody lipid content of tilpi is lower thn 8% (Clement nd Lovell, 1994). The fillet is len, nd y eing fed with commercil diet, fillet of red hyrid tilpi contined lipid even lower thn 1% (Grduño-Lugo et l., 2003). Tonil et l. (2012) tested incresing dietry mounts of flxseed oil- nd sunflower-enriched feed, nd chieved incresed fillet totl lipid from 2.4% to 10% (Tonil et l., 2012). Fitzsimmons suggested energy need of tilpi could e met y crohydrte resources in order to decrese level of dietry lipid in tilpi feed (Fitzsimmons et l., 1997). Concentrtions of n-3 PUFA in tilpi fillet will descend y using vegetle oils in feed (Ng nd Chong, 2004). 2

9 Feed will influence the ody composition of n niml (Visentiner, 2005), nd lso lter the sensory chrcteristics of niml products. Sensory nlysis is used to evlute the qulity in severl food (Grduño-Lugo et l., 2007; Plmeri et l., 2008; Dhnpl et l., 2010) nd diet hit (Munoz nd Chmers, 1993; Bryhni, 2002; Ryffel et l., 2008; Ng nd Bhurmiz, 2009) experiments. Sensory nlysis cn otin provide detiled informtion on product ttriutes (Rødotten et l., 1997). There re multiple exmples of significnt gender difference in the results of sensory nlysis. For exmple, women showed clerer preference thn men for fruits nd vegetles (Blnck et l., 2008). This ws lso found in study with senior citizens (Bker nd Wrdle, 2003). Another exmple showed tht Swiss men descried sheepy nd goty cheese more nimlitic thn women (Ryffel et l., 2008). Doty et l demonstrted tht women performed etter thn men in odor identifiction ility (Doty et l., 1985), nd mles preferred got yoghurt thn femles regrdless of flvor (Young et l., 2012). Guinrd et l. found gender difference in strong tste eer mong Americn popultion (Guinrd et l., 2000). In nother everge study, it ws proved tht men were more sensitive thn women in retro nsl correct response rte (Hollis nd Hlpern, 2012). Yet in some food flvor sensory nlysis, gender difference ws not significnt: no sexul difference on flvor of flx seed-dded gels or muffins (Alini et l., 2012; Rmchritr et l., 2005). Gender difference lso influence other nlysis sed on humn eings. In 2005, Komiym et l. found tht femles couldn t tke pin which ws stimulted y electricity s much s mles could (Komiym et l., 2005). In his ook, Schulkin pointed out tht women hve higher clcium preference thn men do (Schulkin, 2001). Femles re lso more sensitive thn mles on odor fmilirity (Severino-Perez et l., 2012). Ethnic differences my lso cuse mjor effects in the sensory nlysis. Ethnic difference my e cused y diet hit, culture/trdition or/nd genetic differences. Doty et l lso reported tht in n odor identifiction ility experiment, Koren Americn rnked the first, followed y lck nd white Americn nd ntive Jpnese (Doty et l., 1985). In children nd snck experiment, Boyer et l hs determined tht energy of Cucsin children intke 3

10 from snck is higher thn tht of Asin kids (Boyer et l., 2012). In Americns, ethnic difference ppered in dietry intke in Africn Americn nd Hispnic fmilies. Also the ethnicity plys vitl role on the choice of food: not only on food itself, lso on the idels nd identities (Devine et l., 1999). Reserches in Oslo showed tht food from the host country lcked ttrction to the femle immigrnts from non-europe countries or the immigrnts who chnged their diet fter immigrtion (Grnweidner et l., 2012; Wndel et l., 2008). Anywy, ethnic difference on food existing in the immigrnts will fde wy genertion y genertion (Jml, 1998). But in kiwifruit experiment, ethnic difference showed no significnt differences (Wismer et l., 2005). The first im of this experiment ws to find out if KHPL-enrichment of plnt-protein sed diet ffected feed intke, growth nd utiliztion of the feed in Nile tilpi. The second im ws to quntify how efficient the n-3 ftty cid rich KHPL ws in modifying the ftty cid profile of the edile prts of Nile tilpi, nd to find out how efficient fillets from tilpis fed KHPL-enriched feed could e to stisfy the requirement for n-3 ftty cids in humns. The third im ws to find out if KHPL enrichment of the feed influenced the sensory chrcteristics of tilpi, nd if differences in sensory chrcteristics cn e scried to gender or ethnicity. 2. Mterils nd methods 2.1 Fish popultion Tilpi from the 12 th genertion of selection in the GIFT (Geneticlly Improved Frmed Tilpis) progrm ( Eknth et l., 1993) ws htched nd fed to dequte size t UMB. During the 28 dys of feeding, 225 fish were kept in L PVC tnks in which the wter ws circulted nd mintined t 27. 4

11 2.2 Krill hydrolyste with high concentrtion of phospholipids (KHPL) Krill hydrolyste with high concentrtion of phospholipids rich in n-3 ftty cids (OlyMeg, KHPL) used in this experiment ws produced on ord the vessel in Antrctic y Emerld fishery Co., Ålesund, Norwy. The hydrolyste ws processed immeditely fter ctch y rinsing with freshwter, enzymtic hydrolysis, shell removl y decnting, heting, repeted decnting, nd vcuum drying (Storekken, pers. comm.). The nutrient composition, s specified y the producer, is shown in Tle 1. Tle 1. Nutrient composition of the KHPL Totl dry mtter, % 92.5 Crude protein, % 44.9 Crude ft, % 48.1 Ash, % Feed formultion nd processing Three diets were produced. In the experiment, the grdients of KHPL were set s 0% (plnt diet, common group), 5% (KHPL 5% diet) nd 10% (KHPL 10% diet). Min protein sources in ll 3 diets were soyen mel, sunflower seed mel, rpeseed mel, nd pe protein nd corn gluten. Strch ws provided from whet nd lipid ws offered y KHPL nd rpeseed in different rtios. MCP, vitmin C nd severl essentil mino cids were supplemented to fulfill tilpi requirements. Y 2 O 3 ws pplied s inert indictor for digestiility ssessment, nd sodium lginte ws dded s inder. Tle 3 shows the composition of diets while Tle 4 shows nutrients composition. The dry ingredients were ground (1 mm) on lortory mill (MCP ws ground y pestle nd mortr), mixed, dded 30% hot (80 o C) wter, pssed 3 times through pst mchine, nd dried to trget dry mtter content of 90% in hot ir drying cinet set t 65 o C. 5

12 Tle 2. Formultion of diets KHPL, g kg 0 Soyen mel, g kg Sunflower seed mel, g kg Rpeseed mel, g kg 64.9 Pe protein, g kg 45.1 Corn gluten, g kg Pre-extruded whet, g kg Rpeseed oil, g kg 70 Mono clcium phosphte, g kg 20 Lysine, g kg 11.2 Threonine, g kg 7.7 Methionine, g kg 12.7 Phenyllnine, g kg 0.00 Turine, g kg 3.9 Tryptophn, g kg 1.2 Vitmin premix, g kg 10 Y 2 O 3, g kg 0.1 Vit-C 35%, g kg 0.10 Sodium lginte, g kg 20 Plnt diet KHPL 5% diet KHPL 10% diet Totl Tle 3. Nutrient composition of the diets Plnt diet KHPL 5% diet KHPL 10% diet Moisture, g kg Dry mtter, g kg Protein, g kg Lipid, g kg Strch, g kg Ash, g kg

13 2.4 Feeding tril During the 28-dy feeding tril, feed ws delivered y utomtic nd feeders. Feeders rn for 30 min, 4 times per dy, t 0900, 1300, 1700 nd 2100, respectively. Feeding rte ws set t 0.04 initilly nd ws ltered every 3 dys to keep uneten feed up to 10% ody weight. Uneten feed were recorded y collection of uneten feed s descried y Choudhury (Chowdhury, 2011). Fish ws weighed t the eginning, middle nd the end of experiment. 2.5 Body composition nd ftty cid nlysis: Smple preprtion Fifteen fish (3 5) were smpled s initil smples efore experiment nd killed y eing frozen. Fish smples (10 fish per tnks) were tken from ech tnk t the end the feeding tril nd were terminted y shrp low to the hed nd susequent freezing. Five fish per tnk were filleted for ftty cid profile. The rest of smples were ground whole used for nlysis of whole-ody composition Preprtion of ftty cid profile nlysis The ftty cid profile ws nlyzed y IHA, UMB. The smples were extrcted y chloroform-methnol method (Folch et l., 1957). Semi-frozen smples were for ftty cid profile, nd muscle ws seprted from skin. Fillets from ech tnk were squshed on the cutting ord following homogeniztion with fork. Before extrction, smples were freezedried for 6 dys. Afterwrds, smples were weighed into flsks, nd 20 ml chloroformmethnol (1:1) ws dded into ech flsk, nd the flsks were left in the fridge overnight. The next dy, the extrct ws seprted from dry mtter y running smples from funnel 7

14 with filter. A 4 ml NCl solution (0.9%) ws dded fter filtering: t first 1 ml ws dded nd mixed for 5 sec, then nother 3 ml ws dded nd mixed. The solution stood on ench overnight until the extrct ws cler. The upper lyers (qu lyer) were removed. A 5 ml liquot from the orgnic lyer ws evported in wter th (50 ) under nitrogen flux, nd weighed. Menwhile, 2 ml heptne ws dded to ech smple to dissolve the lipids. To strt methyltion, ech smple ws dded 25µl 2M sodium metoxid (NOCH 3 ). Smples were mixed 10 sec nd left for 30 sec nd mixed for nother 10 sec. Then, 0.4 ml 3 N methnolic-hcl ws dded to ech smple. Smples were put into 85 wter th for 15 min, nd the smples were shken twice during the time in the wter th. The smples were cooled under running tp wter. After 5 min with 3,000 RPM centrifugtion, 1ml smples were trnsferred into GC tue nd suject to GC nlysis Whole ody nd fillet nutrition nlysis Fish smples were hlf-thwed, cut into pieces nd weighed. Then, smples were smshed y met grinder. The ground met ws freeze-dried for 6 dys. Freeze-dried met ws homogenized in lender with dry ice. Energy (diet nd whole ody of Tilpi) ws determined in n Automtic Isoperiol Clorimeter (Prr Instrument Compny, USA). The whole ody nd fillet composition were nlyzed y the AOAC officil methods (Horwitz nd ltimer, 2005). The contents of dry mtter were mesured y drying the smples of whole ody, fillets nd diets in 105 overnight. The contents of sh were mesured y muffle furnce, t 550 overnight. Lipid ws quntified y the Accelerted solvent extrction method. Protein ws mesured y utomtic Kjeldhl-N method; nd crude protein ws clculted y multiplying totl N with Sensory nlysis The remining fish were purged to reduce off flvor components efore sensory nlysis. During the 6-dy purging period, tilpi were pooled y dietry tretment, into 3 tnks 8

15 contining tp wter which ws pre-heted to 25 o C nd erted with ir stones. The purging wter ws totlly replced every dy. A consumer tste pnel consisting of 20 non-smoking untrined judges ws estlished. Both ethnicity rtio (Chinese: Cucsin) nd gender rtio within ech ethnic group were 1:1. Judge No 10 were Chinese, while No were Cucsin. Before the tste test, ll purged fish were killed y shrp low to the hed nd led y cutting gill rches on one side of the hed. Afterwrds viscers were removed nd fish were put into 10-L oxes. A totl of 12 fish per dietry tretment were suject so sensory nlyses. Then ech fish ws weighed nd wrpped into luminum-foil. In preprtion for the sensory nlysis, the tilpis were ked in t 200 for 10 min. All smples were ked unslted. Tilpis were ked nd served to the pnelists in three susequent rounds within the sme dy. Ech pnelist received three smples of fish from ech dietry tretment (front, middle, nd ck piece of fillets from different tilpis). Chinese pnelists got left side from smples while Cucsin pnelists got right side from smples. In ech round, one pnelist ws served with the sme position from 3 different fish, one from ech dietry tretment. Immeditely fter tsting, judges nswered questionnire. The questions were s follows: 1. Do you smell or tste ny difference mong the 3 pieces? 2. If yes, cn the difference e chrcterized y differences in smell, tste or oth? 3. Is the difference due to intensity of fishy smell/tste, intensity of muddy smell/tste or intensity of other smell/tste? The judges nswered question 1 nd 2 y grding 1(yes) nd 0 (no) nd question 3 y giving grde (1/lest preferred, 2/medium or 3/most preferred) to ll smples ccording to their mouth feel. 9

16 2.7 Sttistic nlysis The dt of growth, whole ody nd fillet composition nd ftty cid profile were nlyzed y one-wy ANOVA. Duncn s new multiple test ws used to nlyze the comprisons etween tretments. The level of significnce ws set t P<0.05. One-wy ANOVA ws conducted y Microsoft Excel, while SPSS 16.0 ws used to nlyze Duncn s new multiple test. The results of sensory nlysis were checked fctoril ANOVA y SAS Results 3.1 Growth nd survivl During the 28 dys experiment, ll tilpi survived. Tle 4 shows the verge growth nd feed conversion of tilpi fed with ech diet. No sttisticlly significnt differences were seen Tle 4. Initil nd finl weight (men±se), weight gin (men±se) nd FCR (men±se) of tilpi in the 28-dys feeding tril. Plnt diet KHPL 5% diet KHPL 10% diet P-vlue Survivl rte 100% 100% 100% Initil weight, g fish 42±2.2 43±1.0 42± Finl weight, g fish 125± ± ± Weight gin, g fish 83±7.4 85±1.7 81± FCR 1.30± ± ± Feed conversion rtio, dry mtter intke (g) / Weight gin (g) 10

17 3.2 Body composition The ody compositions of tilpi efore nd fter feeding tril re shown in Tle 5. Tle 5. Initil nd finl whole ody nd fillet composition (% in wet weight) (men±s.e) Initil Finl KHPL 10% P-vlue Plnt diet KHPL 5% diet diet Whole ody, % Moisture ± ± ± Protein ± ± ± Lipid ± ± ± Ash ± ± ± Fillet, % Moisture ± ± ± Protein ± ± ± Lipid ± ± ± Aprt tendency for whole ody lipid content (p<0.10), KHPL hd no effect on the whole ody composition of tilpi (p>0.05). Fish fed with plnt diet tended to hve the highest content of lipid, 13.7±0.2%, the second ws fish with KHPL 5% diet, 13.0±0.2%; Fish fed with KHPL 10% diet contined the lowest lipid, 12.6±0.5%. The fillet compositions of tilpi fed with the different diets were t the sme levels. 11

18 3.3 Ftty cid profile of Tilpi fillet nd KHPL The ftty cid profile ws expressed s the weight percentge of ech ftty cid reltive to the totl ftty cids (%). The ftty cid profile of the KHPL is descried in Tle 6, while tht of the fish is descried in Tle 7. In tilpi fillet, oleic cid (C18:1n-9) nd plmitic cid (C16:0) were the most undnt ftty cids in ll 3 diets group. The proportions of oleic cid in groups were 22.0%-32.0% while the quntities of plmitic cid were 20.7%-26.1%. In overll view, diets impcted ftty cid profile gretly. Sturted ftty cids in fish fed the different diets were significntly different (p<0.0001), nd contents were 30.1±0.3 % (plnt diet), 33.0±0.2% (KHPL 5% diet) nd 36.2±0.2% (KHPL 10% diet). Monounsturted ftty cids lso decresed gretly (p<0.01), nd contents from plnt diet to KHPL 10% diet were 42.3±2.0%, 37.4±0.5% nd 32.0±0.5%, respectively. Content of C18:2- nd 3n-6 ftty cids lso decresed significntly (p<0.001): 11.1±0.1% (plnt diet), 10.5±0.2% (KHPL 5% diet) nd 9.4±0.0% (KHPL 10% diet). Contents of C20:1/C18:3n-3 from plnt diet to KHPL 10% diet presented declined trend: contents in plnt diet (3.0±0.1%) nd 2 (2.9±0.1%) showed no difference (p>0.05) while content in KHPL 10% diet (2.3±0.0%) showed significnt difference ginst plnt diet nd KHPL 5% diet (p<0.01). oth of EPA nd DHA contents hd clerly incresing trend: difference ws significnt (p<0.0001) in EPA, contents from plnt diet to KHPL 10% diet were 0.3±0.0 3%,0.5±0.02% nd 0.8± 0.1%, respectively. DHA contents in plnt diet nd KHPL 5% diet wsn t significntly different (p>0.05), while content in KHPL 10% diet differed drsticlly from plnt diet (p<0.001) nd KHPL 5% diet (p=0.01). DHA contents in ech diet (from plnt diet to KHPL 10% diet) were 1.9±0.3%, 3.0±0.4% nd 4.6±0.2%. The totl mount of n-3 HUFA vried gretly (p<0.001) nd contents were, in turn of plnt diet to KHPL 10% diet, 2.8± 0.4%, 4.3±0.4% nd 6.7±0.1%. However, contents of sum of n-6 HUFA were 1.5±0.2%, 1.1±0.1% nd 1.2±0.1%, showing no difference (p>0.05). Rtio of n-3 HUFA /n-6 HUFA ws significntly different (p<0.01). 12

19 Tle 6. Ftty cid profile of KHPL (men percentge in smples) used in diets fed for tilpi for 28-dy feeding tril Sturtes Monoenes 18:2-nd 3n6 20:1 Sum of n-6 HUFA Sum of n-3 HUFA EPA DHA KHPL Tle 7. Content of ftty cid profile in fillet (percentge in totl ft) of tilpi (Men ±SE) Plnt diet KHPL 5% diet KHPL 10% diet P-vlue Sturtes 30.1± ± ±0.4 < Monoenes c 42.3± ± ± C18:2-&3n-6 c 11.1± ± ± C20:5n-3 0.3± ±0.02 c 0.8±0.1 < C22:6n-3 1.9± ± ± C20:1/C18:3n-3 3.0± ± ± Sum n-3 HUFA 2. 8± ±0.7 c 6.7± Sum n-6 HUFA 1.5± ± ± Rtio n-3/n-6 PUFA 1.9± ±0.4 c 5.5± Difference level ws set t P<0.05 Men±SE with the sme superscripts in line were not significntly different t 5% significnce level c Contents of lipid nd ftty cid profile in fillet were lso clculted (Tle 8). Totl fillet lipid content did not show significnt differences: 20.3±2.1 g/kg from fish fed with plnt diet, 16.6±1.3 g/kg fish fed with KHPL 5% diet nd 15.9±0.3 g/kg fish fed with KHPL 10% diet. Ftty cid contents, however, were significntly ffected y KHPL inclusion. Differences in monounsturted, C18:2-nd 3n-6 nd totl mount of n-6 HUFA were significnt (p<0.05): contents of monounsturted ftty cid, from plnt diet to KHPL 10% diet, were 86.6±11.7mg/kg, 62.1±5.5 mg/kg nd 50.9±1.6 mg/kg, mong which fish in plnt diet differed from fish in KHPL 5% diet nd KHPL 10% diet (p<0.01) while there were no difference etween KHPL 5% diet nd KHPL 10% diet (p>0.05). Contents of C18:2-nd 3 n-6 in fish fed with plnt diet to KHPL 10% diet were 22.5±2.3 mg/kg, 17. 5±1.5 mg/kg nd 15.0±0.3 mg/kg, respectively, nd lso fish fed with KHPL 10% diet showed difference from fish in plnt diet (p<0.05) ut neither did fish fed with plnt diet nd KHPL 5% diet, 13

20 nor fish fed with KHPL 5% diet nd KHPL 10% diet showed difference (p>0.05). Contents of n-6 HUFA from fish fed with ech diet differed from ech other gretly (p<0.01): 2.9±0.3 mg/kg (plnt diet), 1.9±0.1 mg/kg (KHPL 5% diet) nd 1.9±0.1 mg/kg (KHPL 10% diet), mong which fish in plnt diet differed from fish in KHPL 5% diet nd KHPL 10% diet gretly (p<0.01) wheres fish in KHPL 5% diet nd KHPL 10% diet hd no difference (p>0.05). Contents of n-3 HUFA, DHA nd EPA hve gret significnce (p<0.01). Contents of n-3 HUFA in tilpi fed with different diets hd significntly incresing trend (p<0.0001): 5.5±0.5 mg/kg (fish in plnt diet), 7.0±0.1 mg/kg (fish in KHPL 5% diet) nd 10.6±0.1 mg/kg (fish in KHPL 10% diet). Fish fed with different diets contined DHA nd EPA showed highly significnt difference (p<0.01): contents of EPA in fish in plnt diet, KHPL 5% diet nd KHPL 10% diet were 0.7±0.1 mg/kg, 0.7±0.1 mg/kg nd 1.3±0.1 mg/kg, respectively. Fish in KHPL 10% diet showed significnt difference from fish in plnt diet nd KHPL 10% diet (p<0.01) whilst fish fed with plnt diet nd KHPL 10% diet showed no difference (p>0.05). Moreover, contents of DHA in fish fed with different diets showed clerly incresing tendency: 3.8±0.3 mg/kg (fish in plnt diet), 4.9±0.2 mg/kg (fish in KHPL 5% diet) nd 7.3±0.3 mg/kg (fish in KHPL 10% diet). However, contents of sturted ftty cid nd C18:3n-3/C20:1 hd no difference in fish fillet (p>0.05): fish from plnt diet to KHPL 10% diet contined 61.3±7.1 mg/kg, 54.7±4.1 mg/kg nd 57.6±0.7 mg/kg, respectively. Contents of C18:3n-3/C20:1 in fish fed with ech diet were 6.1±0.9 mg/kg, 4.9±0.6 mg/kg nd 3.6±0.1 mg/kg, respectively. However, content of C18:3n-3/C20:1 in fish in KHPL 10% diet ws significntly difference from tht of fish in plnt diet (p<0.05) while contents in fish plnt diet nd KHPL 5% diet s well ws fish in KHPL 5% diet nd KHPL 10% diet were not different (p>0.05). 14

21 Tle 8. Content of lipid nd ftty cid profile in fillet of tilpi (in wet weight, men ±SE) Lipid in fillet, g kg 20.3±2.1 Ftty cids, mg kg Plnt diet KHPL 5% diet KHPL 10% diet P-vlue 16.6± ± Sturted ftty cids 61.3± ± ± Monounsturted 86.6± ±5.5 C18:2-&3 n ± ±1.5 C20:5 n-3 0.7± ±0.1 C22:6 n-3 3.8± ±0.2 C20:1/C18:3 n-3 6.1± ±0.6 Sum n-3 HUFA 5.5± ±0.1 Sum n-6 HUFA 86.6± ±5.5 c 50.9± ± ± ± ±0.1 c 10.6± ±1.6 c < Difference level ws set t P<0.05 Men±SE with the sme superscripts in line were not significntly different t 5% significnce level 3.4 Sensory nlysis In the sensory nlysis, tilpi fed with KHPL 10% diets ws the most preferred y the pnelists (n=28), followed y plnt diet (n=14) nd with fish fed with KHPL 5% diet (n=12). Four questionnires (1 Chinese femle, 1 Chinese mle, 2 Cucsin femles) were not suitle for further processing, ecuse they were not le to identify differences mong the smples. Ethnicity ws lnced while gender ws unlnced. The gender difference ws found in smell (P=0.032) ut not in tste (P=0.052). The femle is more sensitive thn mle in smell ( =0.67, =0.48). Ethnic difference ws shown y Chinese eing more sensitive thn Cucsins towrds fishy tste (Chinese=2.39, Cucsin =0.49) (P< ). On the other hnd, Cucsins re sensitive thn Chinese oth in muddy tste (Chinese=1.65.Cucsin =1.19) (P=0.012) nd other tste (Chinese=0.47.Cucsin =0.097) (P=0.0065). 15

22 Tle 9. Result of tilpi sensory nlysis Diet Plnt diet KHPL 5% KHPL 10% P-vlue Prefference Ethnicity Cucsin Chinese 0.52 c Smell Tste Fishy tste/smell Muddy tste/smell Other tste/smell < Gender Femle Mle Smell Tste Fishy tste/smell Muddy tste/smell Other tste/smell Difference level ws set t P<0.05 Men±SE with the sme superscripts in line were not significntly different t 5% significnce level. 16

23 4. Discussion 4.1 growth tril Effects of feed ingredient on growth Vrious studies hve een demonstrted diet formultion influences weight gin nd FCR of tilpi (Clement nd Lovell, 1994; Hussein et l., 2012; Fll et l., 2012; Al-Souti et l., 2012). In present study, dietry KHPL did not significntly ffect tilpi feed intke, growth, survivl, or feed conversion. This cn e concluded s the incresing content of n-3 PUFA nd decresing content of n-6 ftty cids in KHPL cnnot promote fster growth of tilpi, when the essentil ftty cid requirement lredy is met from plnt oild. Freshwter fish, such s tilpi, hs greter need of n-6 lipid compred to tht of n-3 lipid (NRC, 1993). The n-6 ftty cids re climed s the only ftty cids tht were needed y tilpi ccording to Knzw et l. (Knzw et l., 1980; Knzw et l., 1980). High content of n-3 lipids hs previously een demonstrted not to enhnce growth in tilpi (Tkeuchi et l., 1983), or even negtively ffects growth (Hung et l., 1998). This conclusion is lso supported y Ng et l., they reported growth of tilpi ws significntly depressed y cod liver oil diet of which the FER ws 0.82 g wet gin /g dry weight of feed while FERs of other lipid resources were from 0.97 to 1.12 wet weight gin/g dry feed weight (Ng et l., 2001). Also, Hnley confirmed 5%2% dietry lipid with up to 7.2% yellow grese dditive hd no effect on tilpi growth: neither weight gin nor FCR showed sttisticl difference (Hnley, 1991). However, the evidences of the eneficil effect of n-3 PUFA on growth re reported y severl other experiments. Chou et l. stted tht oth of n-3 nd n-6 PUFA were needed to get the mximum growth rte of hyrid tilpi (Chou nd Shiu, 1999). Lter, they reported up to 5% cod liver oil which is rich in n-3 PUFA doesn t slow down growth of hyrid tilpi (Chou et l., 2001). Sntigo nd Reyes found tht cod liver oil improved growth ut reduced rehydrolystion of roodstock of tilpi, compred to other lipid resources, e.g. corn oil nd soyen oil (Sntigo nd Reyes, 1993). 17

24 4.1.2 Effect of feeding strtegy on growth Growth rte in this experiment is higher nd FCR more efficient thn those in erlier experiments. Jckson et l. (1982) verified lower weight gin (15 g fish ) nd higher FCR on tilpi fed with fish oil-enriched feed ( Jckson et l., 1982). In nother juvenile Nile tilpi growth tril (Kumr et l., 2012), FCR ws from , while FCR oserved t this experient ws 1.3. Aprt from feed ingredients (KHPL versus fish oil/plnt oil), genetic fish mteril, nd feeding strtegy (frequent feeding, feeding to stition without stressing the fish), nd frming environment (resirted nd erted wter) my hve contriuted to the high feed intke, rpid growth, nd efficient feed conversion in our experiment. In present test, feeding rte ws djusted once every 3 dys nd rte of overfeeding ws kept t 10% totl weight. In 2012, Al-Souti et l. conducted n experiment of fish oil-enriched feed on tilpi (Al-Souti et l., 2012). In this feeding tril, grdients of fish oil were from 0%, 4%, 8%, nd 12%. The weight gin vried from 42.5 g fish to 46.1 g fish nd the FCR were from 1.56 to The fish ws rised from proximte 15.5g to out 60g. Compred with the Al-Souti et l test, the present results showed much higher weight gin nd lower FCR. Firstly, the flexiility of feeding rte in KHPL tril contriuted to these results. In the current experiment, the feeding rte ws djusted to keep uneten feed up to 10%. The flexile feeding rte ws supported y Chowdhury (2011), who conducted series of experiment to investigte the est feeding rte for tilpi. In one of those experiments, 4 declining feeding rtes (16%-, 14%-, 12%-, nd 10%- to 8%) nd 1 fixed feeding rte were set. It turned out the fixed feeding rte hd the lowest weight gin (300%) menwhile the highest gin ws otined y feeding rte 14-8% (377%), the rest were from 330.8% to 364.2%. the difference ws significnt ( Chowdhury, 2011). However, too high feeding rte my increse weight gin ut survivl rte my drop drmticlly (Sntigo et l., 1987). Secondly, the difference of weight gin nd FCR compred to other experiments ws lso from feeding frequency. Generlly, 4-time-feeding system (0900, 1300, 1700 nd 2100) ws pplied in the present study so fish hd sufficient time to sor nutrients. Growth will scend when fish is fed in higher feeding frequency ecuse of the higher feed intke (Gryton nd Bemish, 1977). In fish oil-enriched test, fish ws fed 2 times dy (0700 nd 18

25 1800) nd feed volume ws determined y fish ppetite. This cused n inefficient digestion ecuse the diet hit my ffect enzymes to digest nutrients, such s protein, crohydrte (Tung nd Shiu, 1991). Tung nd Shiu found significnt difference on weight gin when fed more frequently (6 times) thn feeding 2 times (Tung nd Shiu, 1991). Another feeding experiment on 0-yer-old hyrid sun fish (Lepomis mcrochirus) lso ccord with the current experiment: SGRs in 4-time feeding regime ws significntly higher thn tht of 1- time feeding (Wng et l., 1998). On nother hnd, Estury Grouper (Epinephelus kr) ws found tht hve higher growth nd food conversion rte t one feeding every two dys, rther thn one or two feedings per dy (Thi-Eng nd Seng-Keh, 1978). 4.2 Effect of KHPL on ody composition In present study, KHPL showed no significnt effects on ody composition, except for lipid composition. This ws in keeping with previous oservtions. But crude plm oil, linseed oil nd soyen oil, regrdless of sole or their lends, re showed no significnt difference on the ody composition of tilpi s prtil replcement of fish oil (Ng et l., 2012). In 2001, Ng et l. lso hs proved tht lipid sources mde no difference on tilpi ody composition. Fish fed with vrious oils hve nutrients t the sme level: protein t 15%, sh t 4% nd lipid t 5.5% (Ng et l., 2001). Contents of fillet protein nd lipid were similr to those in other experiments. A similr ody composition ws demonstrted y Puwstien et l in tilpi (Puwstien et l., 1999). Justi et l reported lower fillet lipid composition (round 1.08%) in tilpi when fish ws fed with flxseed feed enriched with n-3 ftty cid for dys (Justi et l., 2003) while much higher lipid content, 2.26%, ws reported y Izquierdo et l (Izquierdo et l., 2000). 19

26 4.3 Ftty cid profile Effect of KHPL on ftty cid profile Ftty cid profile represents to wht extent feed ingredient, KHPL in this cse, impcts fish lipid composition. In this tril, effect of KHPL on fillet ftty cid ws gret. The result of ftty cid profile in current test is greed with de Souz et l: the mjor lipids were oleic cid (18:1n-9), plmitic cid (16:0) nd linoleic cid (18:2n-6). In his experiment, flxseed oil ws used s tretment nd sunflower seed s common group. After 5-month feeding tril, fish ws slughtered nd the ftty cid composition ws nlyzed y FAME, the contents of oleic cid, plmitic cid nd linoleic cid were %, nd %, respectively (de Souz et l., 2007). Hung et l lso proved content of linoleic cid of tilpi fed with soyen oil-included feed ws more thn 50% (Hung et l., 1998). KHPL contined high content of n-3 PUFA. Compred to the control group, content of n- 3 PUFA incresed s content of KHPL incresed. The dietry lipid of fish ody is ffected y dietry lipid of feed (Hung et l., 1998). In the current test, KHPL hs similr effect on tilpi ftty cid composition s fish oil does. This is totlly greed with y Al-Souti: tilpi fed with 12% fish oil hs the sme FA profile s KHPL-enriched tilpi (Al-Souti et l., 2012), e.g., plmitic cid, oleic cid nd DHA re three the most undnt ftty cids in fillet, nd contents of DHA ( %) nd EPA (5.86.1%) mplified s fish oil incresed while contents of n-6 PUFAs decresed. In ddition, Krpngiotidis et l. found the sme trend: they investigted effect of fish oil, plnt oil, nd their lend. The composition of DHA nd EPA in fish oil feed ws significntly higher thn other lipid resources, (Krpngiotidis et l., 2007). Incresed n-3 PUFA nd decresed n-6 PUFA were lso found in se rem nd se ss when fed with fish oil (Mourente et l., 2005). 20

27 Tilpi need moderte mount of LNA (18:2n-6) nd lesser mount of LA (18:3n-3) s they re precursors for ll PUFAs in freshwter fish. In this experiment, content of C18:3n- 6 nd C18:2n-6 ws significntly higher in the control group which ws mde of plnt resources, while it decresed in KHPL 5% diet nd KHPL 10% diet s content of KHPL incresed. The reltively high content of C18:3n-6 nd C18:2 n-6 ws resulted in ssimiltion nd esterifiction of dietry lipid (Henderson, 1996), especilly plnt lipid. This ws indicted tht ody lipid composition is ffected y dietry lipid (Ferreir et l., 2011). In the resulting ftty cid profile of the fish, the C18:3n-3/C20:1 is of miguity. This cn e explined s C18:3n-3 nd C20:1(C20:1 (n-7) + (n-9)) couldn t e differentited y GC mchines due to uncertin resons. Generlly, C18:3n-3 constntly exists in plnt resource while C20:1 is undnt in mrine resource. In current experiment, plnt diet hd no mrine resource so the content of C18:3n-3/C20:1 should e mostly contriuted to C18:3n-3, which ws 3.0%. In KHPL 5% diet, rtio of rpeseed oil nd KHPL ws 1:1 so content of C18:3n- 3 outnumered C 20:1. In KHPL 10% diet, KHPL content incresed significntly, so C 20:1 took mjor proportion of C18:3n-3/C20:1. This indicted lck of C18:3n-3 in KHPL. The fct tht content of C18:3n-3/C20:1 decresed gretly from 2.9% to 2.3% ws oserved in KHPL 5% diet nd KHPL 10% diet. Menwhile, contents of EPA nd DHA incresed significntly ( % nd %, respectively). The reson should e the incresing contents of KHPL in diets led to decresing content of C18:3n-3, menwhile C18:3n-3 is the precursor to EPA nd DHA. As the ccumultion of EPA nd DHA, content of C18:3n- 3 kept dropping. This ws greed with y Clelnd et l.: they found inverse reltionship etween linolenic cid nd EPA/DHA, mening higher linolenic cid content results in lower EPA nd DHA contents (Clelnd et l., 1992). The sme trend ws reported y severl other reserch groups (Grg et l., 1990; McMurchie et l., 1990; Jmes et l., l991; Krpngiotidis et l., 2007; Krpngiotidis et l., 2007). In ddition, vrition of contents of sturted ftty cid nd monounsturted ftty cid ws different from the results of other experiments. Content of sturted ftty cid incresed while monounsturted ftty cid decresed due to different content of these two groups in KHPL. In Ng et l test, tilpi fed with plm oil showed stle content of monounsturted 21

28 ftty cid s plm oil increse (Ng et l., 2006). The incresed sturted ftty cid with incresed KHPL ws greed with tilpi fed with fish oil (Al-Souti et l., 2012). However, KHPL-enriched tilpi cn contriute smll mount to humn dietry intke of n-3 nd n-6 ftty cids. Simopoulos (2000) reported recommendtion on dily intke of DHA nd EPA where 650 mg/d ws the minimum for n dult while either of DHA nd DPA should e more thn 220 mg/d (Simopoulos, 2000). However, the mximum of DHA+EPA in KHPL-enriched tilpi is less thn 9 mg/kg (fish fed with KHPL 10% diet). This indictes consumers must consume other foods to fulfill dily need. In nother reserch on reltion of n-3 ftty cid intke nd occurrence of Alzheimer disese (AD), higher dily intke of n-3 ftty cid decrese incidence of AD to 20-30% (Morris et l., 2003). Compred to PUFA intke in Alzheimer disese reserch, tilpi in current experiment cn hrdly offer sufficient PUFA to the ged Effect of KHPL on n-3/n-6 rtio The use of mrine resource modified PUFA of tilpi fillet: n-3 incresed while n-6 decresed. N-3 /n-6 rtio is criticl index of PUFA composition for humn helth (Mourente et l., 2005; Nsopoulou nd Zetkis, 2012; Tonil et l., 2012; Mourente nd Bell, 2006). In the present study, N-6/n-3 rtio depresses when KHPL increses. In humn dietry history, plnt is new choice on menu since yers go. Before vegetrin diet, humn eings te met nd wild fruit which figured our dietry hit geneticlly (Simopoulos, 2002). Now, 17% plnt offers 90% food supply (Krger, 1995). But cerel grins re rich in n-6 ftty cid nd low in n-3 ftty cid. In Cucsin diet, rtio of n-6/n-3 is 10-20:1 while the recommended rtio is 1-2:1 (Simopoulos, 1999). A investigtion reported high rtio of n-6 nd n-3 leds to high frequency of thromus nd theroms (Simopoulos, 2002). In feeding trils, plnt resources give fish more n-6 ftty cid while mrine resources give more n-3 PUFA. Krpngiotidis et l reported fish oilfed tilpi ws 2.0, rnking the first on n-3/n-6 rtio, followed y linseed oil-fed Tilpi (1.5), while mixture of plm oil nd fish oil/linseed oil (1.2 nd 0.9, repectively) nd coconut oil hs significntly dverse effect on n-3/n-6 rtio (0.3) (Krpngiotidis et l., 22

29 2007). Compred with the result ove, 10% KHPL hs similr effect on n-3/n-6 rtio s linseed oil nd weker thn fish oil. In nother humn-relted experiment, linseed oil decresed n-6/n-3 rtion in plsm of crnivorous consumers down to reltive higher vlue, 3.8 (Weill et l., 2002). 4.4 Tilpi sensory nlysis The effect of KHPL on sensory nlysis of tilpi is focused on in this experiment. The sensory pnel could identify difference in smell nd tste Effect of KHPL on sensory nlysis KHPL used in this experiment ws rich in phospholipids. KHPL gve tilpi specil olfctory response compred to feed with plnt resource. Phospholipids contin much more unsturted ftty cids thn triglyceride. In cooking process, high level of unsturted ftty cids presents desirle smell y eing oxidized nd increses totl mount of voltile compounds (Slter et l., 1988). The phospholipids in tilpi is elieved put forth quenching effect on the sum of heterocyclic Millrd compounds during food prepring (Whitfield et l., 1988). Olfctory difference is the result of diverse lipid composition nd polr extent, lso lipid interction in the Millrd rection plys vitl role in flvor formultion (Whitfield et l., 1988). Deficiency of phospholipids ffected rom of rost eef gretly (Mottrm nd Edwrds, 1983; Mottrm, 1998). Also, hypothesis tht lipid optimized level of sulfur compounds which gve food sulfurous odors ffirmed the role of phospholipids in cooked met (Mottrm, 1998). In respect of tste modifiction, up to 1.5 mg/g long-chin polyunsturted ftty cid worsened the slmon flvor with metl tste nd itterness ccording to Refsgrd et l. (Refsgrd et l., 2000). Also, totl phospholipids s well s ftty cid content mong phospholipids were highly linked to flvor uniqueness of eef (Lrick nd Turner, 1990). Igene nd Person demonstrted tht phospholipids donted much more thn triglyceride did 23

30 on wrmed-over flvor (Igene nd Person, 1979). In current reserch, content of long-chin polyunsturted ftty cid in phospholipids is up to 0.01mg/g fillet, so the dverse effect could e neglected Effect of gender on sensory nlysis In spect of smell, femle hs gift of detecting odor, compred to mle. This my e ttriuted to hormones, memory to smells nd other cuses. The gond hormones re one of the resons tht led to difference etween odors detective ility of genders. They works directly on relevnt orgns or ffect stimultions of centrl nerve system (Gndelmn, 1983). The rehydrolysteive hormones impct electronic connection etween rin nd olfctory cues (Simerly, 1990). For exmple, the excretion of estrogen incresed performnce of odor identifiction during ovultion nd pregnncy (Good et l., 1976; Cruso et l., 2004). Dte ck to 1899, the perception of femle to cmphor solution ws 0.001, higher thn tht of mle to the sme solution, (Toulouse nd Vschide, 1899). In lter test, womn proved more sensitive thn mn on rtificil chemicl (Le. Mgen, 1952). The perceptivity of women s olfctory is 8 times ccurte thn tht of men s (Doty nd Cmeron, 2009).. Femles lso hve dvntge of nming nd rememering odors. According to Lehrner, women performed outweighed men on smell identifiction test which hd up-to-21-dy retention intervl (Lehrner, 1993). Women hs etter performnce on smell verl processing, i.e. memory for nming memorle odor nd clssifiction. However, mn nd womn perform eqully on primrily sensory cuity (Öerg et l., 2002). But in some studies, sexul vrition ws found in childhood when gond hormones were not secreted in high level (Koeleg nd Köster, 1974). In ddition, Lrsson et l. climed tht sed on result of n experiment in Sweden, gender hs nothing to del with odor detection nor identifiction (Lrsson et l., 2000). In current sensory nlysis, the gender rtio ws lnced. In femle group, no one ws pregnnt, so odor identifiction ility ws not ffected y estrogen. 24

31 4.4.3 Effect of ethnicity on sensory nlysis More thn n identifiction of one s irth, the word ethnicity presents group of people hving the sme ncestry. Aprt from nthropology, ethnicity includes the culture they were rised up nd lifestyle they hd, to e specific, food they got used to et. In the current sensory nlysis, ethnicity plys role of gret vitlity in smell/tste distinguish. The sensory pnelists were divided into 2 groups: Cucsin nd Chinese. Chinese hve 3000-yer history of freshwter quculture since Yin dynsty. Freshwter fish, such s common crp, silver crp nd so on, hs ecome n unchngele prt in Chinese diet, so Chinese cn er high level of the smell/tste of mud nd other smell/tste of which most were generted from on living environment of freshwter fish or the diet they hve. In Chinese sensory pnelists, 6 of them were from south costl re of Chin, y whom the men level of fishy tste/smell tolernce ws incresed. The reson ws tht mrine fish tke higher portion on diet of Chinese living in the costl re, compred to inlnd Chinese. A prove is the usge of mrine lge: due to the vilility nd price, costl dwellers consume higher portion of lge s vegetle or its sustitution thn inlnd dwellers (Bngmei nd Aott, 1987). However, fishy tste/smell endurnce of Chinese ws still lower thn tht of Cucsin. In contrst to Chinese, Cucsins re fmilir with oil-rich mrine fish, so they re not sensitive to fishy smell/tste. But due to unpopulrity of freshwter fish in Europe, Cucsins hve strong response to freshwter fish, minly on muddy smell/tste. It ws the first time tht 10 Cucsin judges et Tilpi, so they would experience nd clssify odors from smples into olfctory memory tht they hve hd. In generl, judgments of consumers to food nd the originlity of consumers re connected (Guerrero et l., 2009). It ws elieved tht odor judgment ssocited with mother s diet strted cn egin t even in pregnncy period (Hudson nd Distel, 1999). Like lnguge lerning, odor/tste dption depends on environments nd ethnicity strongly: people will ccept food with odors/tstes they re fmilir nd ttempt to conclude ll odors into their sensory memory. In Itly, children hs developed hit of drinking hot coffee which hs ecome prt of their fmily nd socil life (Rozin nd Cines, 1982). Children intke of 25

32 vegetle nd fruit ws lso in the sme hit formtion: mount of vegetle nd fruit prents te, trditionl fmily meltime nd intke-strt ge mtters in positive correltion (Cooke et l., 2003). A consumer-cceptnce reserch showed tht ethnicity hd gret influence on snck choice of Americns nd Asins: significnt difference on fish snck choice while no difference on penut snck. Asin consumers hd higher evlutions on fish snck thn Americns did, due to the fish-eting hit of Asin (Suknrk et l., 1998). Fmilirity is one of key roles in tste cceptnce nd cn explin the ltertions in crossculturl food evlution (Chung et l., 2012). 5. Conclusions The series of experiments proved the pplicility of KHPL in tilpi nd enhnced effect of KHPL on fillet sensory chrcteristics. In feeding tril, KHPL ws utilized s min lipid resource in tilpi feed t the first time nd it proved hs no effect on growth. The effect of KHPL on ody composition ws detected on whole ody lipid content only, nd hd no effect on other nutrients. However, the modifiction of ftty cid profile, especilly n-3 nd n-6 ftty cids, ws significnt. Compred to plnt diets, diets pplying KHPL lters contents of DHA, EPA nd linoleic cid. Moreover, with help of KHPL, n-3/n-6 rtio of tilpi ecomes higher thn tht of fish fed with whole-plnt feed, which ws much more eneficil to humn eing s helth. But eventully, tilpi is len fish (fillet lipid less thn 2%), so it cn hrdly stisfy dietry requirement of n-3 PUFA in humn. In sensory nlysis, higher content of KHPL proved fvorite of ssumed consumers, oth Cucsin nd Chinese. Through this test, we lso find the difference cn e ttriuted to gender nd ethnicity: due to nurture nd lerning, Chinese hs higher endure of erthy nd other smell/tste, while cnnot tolerte fishy smell/tste; u contrire, Cucsin hve higher fishy tolernce ut ws wek t ering muddy smell/tste. 26