2006年SCI不同盐度对南美白对虾生长和脂肪酸组成的影响及HUFA对其调节

Effect of hypo-and hyper-saline conditions on osmolarity and fatty acid composition of juvenile shrimp Litopenaeus vannamei(Boone,1931)fedlow-and high-HUFA dietsMiguel A Hurtado1,Ilie S Racotta1,Olivia Arjona1,Mo nica Herna ndez-Rodr|guez2,Ernesto Goytortu a1,Roberto Civera1&Elena Palacios11Centro de Investigaciones Biolo gicas del Noroeste(CIBNOR),La Paz,BCS,Me xico2Centro de Investigacio n Cient|¢ca y de Educacio n Superior de Ensenada(CICESE),Ensenada,BC,Me xicoCorrespondence:E Palacios,Centro de Investigaciones Biolo gicas del Noroeste(CIBNOR),Mar Bermejo195,Col.Playa Palo de Santa Rita, La Paz,BCS23090,Me xico.E-mail:epalacio@cibnor.mxAbstractLitopenaeus vannamei(Boone)grown in ponds are ex-posed to salinities of less than5g LÀ1during inland shrimp culture or to more than40g LÀ1from eva-poration and reduced water exchange in dry,hot climates.However,dietary requirements for shrimp grown in low or high salinities are not well de¢ned, particularly for fatty acids.Feeding shrimp post-larvae with highly unsaturated fatty acids(HUF A) enhances tolerance to acute exposure to low salinity, as a result of better nutritional status,or/and speci¢c e¡ects of HUFA on membrane function and osmo-regulation mechanisms.This study analysed the ef-fect of HUF A supplementation(3%vs.34%)on L.van-namei juveniles reared for21days at low(5g LÀ1), medium(30g LÀ1)and high salinities(50g LÀ1). Juveniles grown at5g LÀ1had lower survival com-pared with controls(30g LÀ1)or shrimp grown at 50g LÀ1,but no signi¢cant e¡ect on survival was ob-served as a result of HUFA enrichment.In contrast, growth was signi¢cantly lower for shrimp grown at 50g LÀ1,but this e¡ect was compensated by the HUFA-enriched diet.Osmotic pressure in haemo-lymph was a¡ected by salinity,but not by HUFA enrichment.Shrimp fed HUF A-enriched diets had signi¢cantly higher levels of eicosapentaenoic acid and docosahexaenoic acid in hepatopancreas and gills.These results demonstrate that growth at high salinities is enhanced with diets containing high HUFA levels,but that HUF A-enriched diets have no e¡ect on shrimp reared at low salinities.Keywords:DHA,diet,EP A,lipid,nutrition,osmo-regulation,salinityIntroductionPart of the life cycle of penaeid shrimp is spent in estuaries,where they are exposed to drastic environ-mental variations in salinities and temperature (Dall1981;Bouaricha,Thuet,Charmantier-Daures& Trilles1991).Shrimp grown in ponds are exposed to similar environmental variations from rain and evaporation or because of increasing inland shrimp culture that uses well water.Also,very high salinities can be found in some areas of aquaculture,for example,northwestern Mexico,where high summer temperature and reduced water exchange can result in salinities reaching44g LÀ1or more in ponds(Mart|nez-Cordova,Villareal,Porchas-Cornejo, Naranjo-Paramo&Aragon-Noriega1997;Ponce-Palafox,Mart|nez-Palacios&Ross1997). Presently,because of disease problems and land costs,Litopenaeus vannamei is grown successfully in low-salinity(less than5g LÀ1)inland waters,espe-cially when shrimp are adequately acclimated from the postlarval stages(Samocha,Lawrence&Pooser 1998;Laramore,Laramore&Scarpa2001;Davis, Saoud,McGraw&Rouse2002;McGraw,Davis, Teichert-Coddington&Rouse2002;Garza de Yta, Rouse&Davis2004).Successful acclimation of postlarvae can be a¡ected by age(Ogle,Beaugez&Aquaculture Research,2006,37,1316^1326doi:10.1111/j.1365-2109.2006.01568.xr2006The Authors 1316Journal Compilation r2006Blackwell Publishing LtdLotz1992;Laramore et al.2001;McGraw et al.2002; Garza de Yta et al.2004),the ionic composition of water(Davis et al.2002;McGraw&Scarpa2003; McNevin,Boyd,Silapajarn&Silapajarn2004;Zhu, Dong,W ang&Huang2004)and genetic di¡erences (Bray,Lawrence&Leung-Trujillo1994;Samocha et al.1998;Chim,Bouveret,Lemaire&Martin2003). The e¡ect of diet on performance of juveniles at low salinity has been tested,particularly in relation to protein and carbohydrate levels(Rosas,Cuzon,Gax-iola,Le Priol,Pascual,Rossignyol,Contreras,Sanchez &V an W ormhoudt2001;W ang,Shen,Shuanglin& Mei2004)and Mg,K,phospholipids and cholesterol (Gong,Jiang,Lightner,Collins&Brock2004).Shrimp fed highly unsaturated fatty acids(HUFA)have en-hanced tolerance to acute exposures to low salinities, as reported for postlarvae(Rees,Cure,Piyatiratitivor-akul,Sorgeloos&Menasveta1994;W outers,V anhau-waert,Naessens,Pedrazzoli&Lavens1997;Palacios, Bonilla,Pe rez,Racotta&Civera2004)and juveniles (Chim,Lemaire,Delaporte,Le Moullac,Galois& Martin2001).Increased tolerance could be a result of enhanced nutritional status(Palacios,Bonilla, Luna&Racotta2004),speci¢c e¡ects of HUFA on os-moregulation mechanisms(Palacios,Bonilla,Pe rez et al.2004)or a combination of both.Several studies have analysed the e¡ect of HUFA or other lipids on juvenile growth in L.vannamei(Lim,Ako,Brown& Hahn1997;Gonza lez-Fe lix,Gatlin,Lawrence&Per-ez-V elazquez2003)and in other penaeid shrimp (Chen&Jenn1991;Deering,Fielder&Hewitt1997; Merican&Shim1997).However,no work has been carried out to test whether HUFA supplementation can enhance the performance of juveniles during grow-out at di¡erent salinities.This study analysed the e¡ect of HUF A supplementation on the growth, survival and osmotic pressure of L.vannamei juve-niles reared under hyposaline(5g LÀ1)and hyper-saline(50g LÀ1)conditions over21days.Material and methodsExperimental dietsTwo isonitrogenous and isolipidic diets,termed low or high HUF A(Table1),were formulated based onthe nutritional requirements of L.vannamei(Akiyama &Dominy1989;Camba,Pedrazzoli,Y aguachi& Akiyama1993)using MIXIT-WIN s software(Agricultur-al Software Consultants,San Diego,CA,USA).Both diets contained sardine meal and soybean paste as the main protein sources.Each diet was enriched with ICES Standard Reference Emulsions(ICES1994)with two HUF A proportions,0%and50%for low-and high-HUFA diets,respectively,resulting in2.9%and34%of total HUF A on a per cent of total fatty acids(Table2). Before preparing the diets,all ingredients were ground in a hammer mill and passed through a250-m m mesh Table1Ingredient and proximate composition of experi-mental dietsIngredients(g100gÀ1diet)Low HUFA High HUF A Wheat mealÃ40.040.0 Soybean pasteÃ30.430.4 Sardine mealÃ15.8115.81 Grenetine(binder)w 4.00 4.00 Vitamin premix zz,‰ 1.80 1.80 Sodium phosphate dibasic‰ 1.20 1.20 Mineral premix‰,z0.500.50 Choline chloride k0.200.20 Ascorbic acidÃÃ0.090.09BHT ww0.0040.004 Soybean lecithin zz 2.00 2.00 ICES emulsion‰‰(0%HUFA) 4.00–ICES emulsion‰‰(50%HUFA)– 4.00 Proximate composition(g100gÀ1dry matter,except moisture, meanÆSD)Crude protein36.6Æ0.136.2Æ0.3 Ether extract7.9Æ0.47.9Æ0.5 Ash 6.3Æ0.04 6.2Æ0.01 Crudefibre 2.8Æ0.01 2.8Æ0.01 NFE zz46.4Æ0.246.9Æ0.3 Moisture7.4Æ0.67.6Æ0.1 Gross energy(kcal gÀ1) 4.6Æ0.1 4.6Æ0.1ÃPromotora Industrial Acuasistemas(S.A.de C.V.,La Paz,BCS, Me xico).w Semillera de La Paz(La Paz,BCS,Me xico).z Vitamin premix(except where units are given,values are in mg kgÀ1diet):vitamin A(Retinol)Ã,15000IU;vitamin D3, (Cholecalciferol)Ã,7500IU;vitamin E(a-Tocopherol)Ã,400;vita-min K3(Menadione)Ã,20;thiamin(B1)3,150;ribo£avin(B2)Ã, 100;pyridoxin(B6)Ã,50;pantotheic acidÃ,100;niacinÃ,300; biotin3,1;inositol3,500;folic acidÃ,20;cyanocobalamin(B12)Ã, 0.1;celuloseÃ,1000.ÃICN Biomedicals(Aurora,OH,USA).‰Sigma(St.Louis,MO,USA).z Mineral premix(g kgÀ1diet):KCl,0.5;MgSO4Á7H2O,0.5; ZnSO4Á7H2O,0.09;MnCl2Á4H2O,0.0234;CuCl2Á2H2O,0.005; KI,0.05;CoCl2Á2H2O,0.0025;Na2HPO4,2.37.k Choline chloride(65%active agent).ÃÃStay-C(L-ascorbil-2polyphosphate,35%active agent),Roche-Vitamins(Parsippany,NJ,USA).ww Butylated Hydroxytoluene.(ICN Biomedicals)zz Soybean lecithin ODONOJAI s,Distribuidora de Alimentos Naturales y Nutricionales S.A.de C.V.,Me xico,DF,Me xico.‰‰ICES Standard Reference Emulsions(ICES1994).zz NFE was calculated as:100À(%crude protein1%ether extract1%ash1%crude¢bre).HUFA,highly unsaturated fatty acids;NFE,nitrogen-free extract.Aquaculture Research,2006,37,1316^1326Salinity e¡ect on juvenile shrimp fed HUFA diets M A Hurtado et al.r2006The AuthorsJournal Compilation r2006Blackwell Publishing Ltd,Aquaculture Research,37,1316^13261317sieve(CSC Scienti¢c,Fairfax,V A,USA).The dry ingre-dients of each diet were mixed thoroughly in a food mixer before the soybean lecithin was added as de-scribed in Civera and Guillaume(1989).After the oil was dispersed,water was added(approximately40% of the total‘as is’ingredient weight)and mixed.The re-sulting mixture was pelleted using a meat grinder and a2-mm die.The pellets were dried in a forced-air oven at451C for12h.The experimental diets were analysed for dry mat-ter,crude protein(NÂ6.25),ether extract,crude ¢bre,ash and nitrogen-free extract(NFE)contents according to standard methods(AOAC1995;Table1). Gross energy values of the diets were determined with an adiabatic calorimeter(Parr Instrument, Moline,IL,USA).Lipids from pellets were extracted with chloro-form:methanol(2:1)according to Bligh and Dyer (1959),trans-esteri¢ed with boron tri£uoride^metha-nol(BF3in14%methanol;Supelco,Bellefonte, P A,USA)and the fatty acid methyl esters(F AME) were analysed in a Hewlett^Packard G1800B gas chromatograph equipped with an Omega W ax(30m lengthÂ0.25mm inner diameter,0.25-m m-thick ¢lm)fused silica capillary column(Supelco),mass se-lective detector(MSD),helium as a carrier gas and a temperature gradient of165^2201C at31C minÀ1. The identi¢cation of FAME was further veri¢ed by comparison of retention times with those of known standards(Supelco).The fatty acid composition of the two diets is shown in Table2.Feeding trial and experimental designA feeding trial was conducted in the Laboratorio de Mejoramiento de Semillas at CIBNOR(La Paz,BCS, Me xico).Juveniles of L.vannamei cultured in earthen ponds were acclimated for1week in60-L tanks (0.28mÀ2,0.7mÂ0.4m)at a density of16organ-isms per tank,25.4Æ0.61C, 5.9Æ0.1mg LÀ1 dissolved oxygen,30g LÀ1,a12:12h light:dark photoperiod and80%daily water exchange.Shrimp were fed daily5%total biomass of a commercial shrimp diet(35%protein,PIASA,La Paz,BCS,Mexi-co)before the feeding trial.The trial was performed in18tanks containing 60L of¢ltered(5m m)and ultraviolet(UV)-sterilized water at a temperature of25.4Æ0.61C.Approxi-mately80%of the water was exchanged daily.A5-HP blower(Sweetwater TM,Apopka,FL,USA),and air stones maintained dissolved oxygen levels above 5mg LÀ1.A photoperiod of a12:12h light:dark cycle was used throughout the experiment.Shrimp were individually weighed to the nearest 0.1g using a digital balance(Mettler Toledo,Greifen-see,Switzerland).Specimens with an average initial weight of(3.5Æ0.5g)(meanÆSD)were stocked at a density of16per tank(stocking density related to bottom surface area:57shrimp mÀ2).After1week of acclimation at30g LÀ1,the commercial feed was replaced with the two experimental diets described above(low and high HUFA).Shrimp were gradually exposed to5,30(control)or50g LÀ1salinity at a rate of1.6g LÀ1hÀ1(approximately15h).These experi-mental salinities were established in a previous essay (unpublished data)as having minimal juvenile shrimp mortality in an acute1-h exposure test.To change salinities,a gravity drop system was used(Ra-m|rez,Avila&Ibarra1999).The water was prepared in advance with tap water for5g LÀ1,and dissolved unprocessed marine salt(Salinera Punta Arenas, BCS,Me xico)in seawater for50g LÀ1,heated to the Table2Fatty acids(%of total fatty acids)of low and high HUFA dietsLow-HUFA High-HUFA 14:0 5.5Æ0.4 1.0Æ0.01 16:021Æ0.0513Æ0.03 18:0 4.5Æ0.01 3.6Æ0.02 16:1n–7 1.9Æ0.01 1.5Æ0.01 18:1n–916Æ0.49.8Æ0.1 18:1nn-7 1.9Æ0.01 1.8Æ0.01 20:1n–90.4Æ0.01 1.2Æ0.01 20:1n–110.2Æ0.01 1.3Æ0.01 18:2n–639Æ0.925Æ0.4 18:3n–6 1.7Æ0.08 1.3Æ0.05 18:3n–3 2.8Æ0.04 2.0Æ0.05 20:4n–60.4Æ0.010.8Æ0.01 20:5n-3 1.0Æ0.0113Æ0.03 22:6n–3 1.5Æ0.0115Æ0.06 S SATÃ33Æ0.319Æ0.01 S MUFA w21Æ0.417Æ0.2 S PUFA z46Æ1.364Æ0.2 S HUFA‰ 2.9Æ0.0234Æ0.14 n–3/n-60.1Æ0.01 1.2Æ0.01 20:5n-3/22:6n-3 1.6Æ0.01 1.1Æ0.01 PUI z128Æ4263Æ2V alues are expressed as meanÆSE(n56).ÃSum of saturated fatty acids.w Sum of monounsaturated fatty acids.z Sum of polyunsaturated fatty acids with two or more double bonds.‰Sum of highly polyunsaturated fatty acids with four or more double bonds.z Poly-unsaturation index.Salinity e¡ect on juvenile shrimp fed HUFA diets M A Hurtado et al.Aquaculture Research,2006,37,1316^1326r2006The Authors 1318Journal Compilation r2006Blackwell Publishing Ltd,Aquaculture Research,37,1316^1326same temperature as in the experimental tanks and aerated for24h before changing salinity.Three repli-cates were used for each dietary-salinity treatment (2Â3Â3518replicates)with15shrimp per repli-cate.Shrimp were fed twice daily.Total daily feed was initially set at5%of the biomass in each tank,distrib-uted manually in two ratios(50%at09:00hours and 50%at17:00hours).Rations were adjusted daily, based on apparent consumption,where the added feed closely matched the maximum food ingested, with little or no feed remaining.Faeces,exuviae and dead shrimp were removed daily.During the trial, shrimp density was adjusted for mortality with tagged organisms,but these were not used at the end of the experiment for analyses.At the end of the trial,growth rate(relative weight gain),survival,feed allowance and feed conversion ratio were determined for each replicate of each treatment as follows:growth rate(GR%)5[(¢nal mean weight^initial mean weight)/initial mean weight]Â100;survival(S%)5(¢nal number of shrimp/initial number of shrimp)Â100;feed con-version ratio(FCR)5feed intake(g)/total weight gain,(g).Total weight gain was corrected with the fol-lowing formula:corrected total weight gain5¢nal total weight1[1/2(average initial weight1average ¢nal weight)Ânumber of dead shrimp]^initial total weight(Kitabayashi,Kurata,Shudo,Nakamura& Ishikawa1971).Apparent feed intake was estimated per day per tank,but it is reported as the mean of each treatment during the length of the experiment.Sample collectionAt the end of the experimental period,shrimp were starved for24h and then sampled and weighed(ap-proximately15organism per treatment).Haemo-lymph samples were withdrawn using a10m L pipette,from the pericardial cavity,and osmolarity was assessed using a vapour pressure osmometerTable3Proportion of principal fatty acids(meansÆSE)in neutral and polar fractions of the hepatopancreas from Litopenaeus vannamei juveniles fed two diets and exposed to di¡erent salinities(5,30and50g LÀ1)Low-HUF A diet(g LÀ1)High-HUFA diet(g LÀ1)S D SÂD 5305053050Neutral fraction16:019Æ0.5b21Æ0.4a21Æ0.2a15Æ0.3c14Æ0.1c15Æ0.1c0.050.010.01 18:0 5.2Æ0.2a 4.9Æ0.1ab 5.3Æ0.3a 4.1Æ0.4b 4.6Æ0.4ab 4.5Æ0.1ab NS0.01NS 16:1n-7 3.4Æ0.2b 4.1Æ0.3ab 4.4Æ0.2a 2.5Æ0.2c 2.1Æ0.1c 2.5Æ0.1c0.050.010.05 18:1n-922Æ0.7a23Æ0.4a22Æ0.3a13Æ0.2b13Æ0.3b13Æ0.1b NS0.01NS 18:1n-7 4.9Æ0.3ab 4.7Æ0.3ab 5.4Æ0.2a 3.7Æ0.1b 3.4Æ0.1b 4.2Æ0.2ab0.010.01NS 20:1n-9 1.1Æ0.1b 1.2Æ0.1b 1.2Æ0.1b 1.9Æ0.1a 2.1Æ0.1a 2.0Æ0.1a0.050.01NS 18:2n-620Æ0.6b22Æ0.9a22Æ0.5a21Æ0.3ab20Æ0.6b21Æ0.3ab NS NS0.05 18:3n-3 1.8Æ0.1b 2.1Æ0.1ab 2.2Æ0.1a 2.1Æ0.1ab 1.9Æ0.1ab 2.0Æ0.1ab NS NS0.05 20:4n-6 1.6Æ0.3a0.9Æ0.1b 1.0Æ0.1b 1.1Æ0.1ab 1.3Æ0.2ab 1.1Æ0.1ab NS NS0.05 20:5n-3 4.3Æ0.8b 2.2Æ0.3c 2.2Æ0.3c11Æ0.4a11Æ0.6a9.9Æ0.1a0.010.010.05 22:6n-3 4.2Æ0.6c 3.0Æ0.4cd 2.3Æ0.2d12Æ0.4b14Æ0.3a13Æ0.3ab0.050.010.01 Polar fraction16:012Æ0.4a12Æ0.4a11Æ0.2ab11Æ0.4ab10Æ0.4b10Æ0.2b NS0.01NS 18:07.7Æ0.37.3Æ0.47.7Æ0.17.7Æ0.37.2Æ0.37.5Æ0.2NS NS NS 16:1n-7 1.6Æ0.1b 2.0Æ0.1a 2.1Æ0.1a 1.0Æ0.1c 1.0Æ0.1c 1.0Æ0.1c0.010.010.01 18:1n-913Æ0.3b15Æ0.2a14Æ0.3ab7.4Æ0.1c7.0Æ0.2c7.0Æ0.1c0.050.010.01 18:1n-7 3.2Æ0.1ab 3.2Æ0.2ab 3.7Æ0.1a 3.0Æ0.2bc 2.6Æ0.1c 3.1Æ0.1bc0.010.01NS 20:1n-90.9Æ0.1b 1.0Æ0.1b 1.0Æ0.1b 1.2Æ0.1a 1.4Æ0.1a 1.4Æ0.1a0.010.01NS 18:2n-623Æ1.0b28Æ1.1a28Æ0.7a11Æ0.2c12Æ0.5c11Æ0.2c0.010.010.01 18:3n-3 2.2Æ0.1b 2.9Æ0.1a 2.9Æ0.1a0.9Æ0.1c0.9Æ0.1c0.8Æ0.1c0.010.010.01 20:4n-6 4.1Æ0.2 3.5Æ0.1 4.0Æ0.2 3.1Æ0.3 3.1Æ0.1 3.3Æ0.1NS0.01NS 20:5n-312Æ0.98.4Æ0.39.2Æ0.525Æ1.023Æ0.724Æ0.30.010.01NS 22:6n-311Æ0.7ab9.1Æ0.4b7.4Æ0.5b18.6Æ0.3a22Æ0.6a21Æ0.4a0.050.010.01 The results of the bifactorial ANOV A are shown in the last three columns(n510individuals per treatment).See Fig.1for statistical analyses.HUF A,highly unsaturated fatty acids.Aquaculture Research,2006,37,1316^1326Salinity e¡ect on juvenile shrimp fed HUFA diets M A Hurtado et al.r2006The AuthorsJournal Compilation r2006Blackwell Publishing Ltd,Aquaculture Research,37,1316^13261319(Model OM230B W escor,UT,USA).After haemo-lymph sampling,the uropods were sampled to assess moulting stage(Robertson,Bray,Leung-Trujillo& Lawrence1987)and only shrimp on intermoult and early premoult stages were considered.The rest of each organism was wrapped in an aluminium foil, labelled,frozen in liquid nitrogen and stored at^701C for later fatty acid analysis of gills and hepatopancreas.Fatty acid analyses of tissuesGills and hepatopancreas were dissected on a cold plate and weighed.Lipids were extracted using chloroform:methanol2:1,butylated hydroxytolune (BHT)as an antioxidant and23:0as an internal stan-dard.Neutral and polar lipid fractions of gills were se-parated,fatty acids were trans-esteri¢ed and FAME were analysed using GC-MS as described in Palacios, Bonilla,Luna et al.(2004).Statistical analysisData are presented as meanÆstandard error.Per-centages were arcsine transformed for statistical analysis,but untransformed means are presented in tables and¢gures.A two-way factor ANOV A was ap-plied to analyse the di¡erences in growth,survival, FCR,food intake,osmolarity,moulting frequency and fatty acid composition of the gills and hepato-pancreas,using low-and high-HUF A diets as the¢rst independent variable and the three salinity treat-ments(5,30and50g LÀ1)as the second independent variable.Means were compared by a post hoc Tukey’s multiple-range test(Zar1999).Signi¢cant levels were considered at P o0.05.ResultsThe proximate composition of diets was similar (Table1),and the main di¡erence between diets was the fatty acid composition(Table2).Diets enrichedTable4Proportion of principal fatty acid(meansÆSE)in neutral and polar fraction of gills from Litopenaeus vannamei juveniles fed two diets and exposed to di¡erent salinities(5,30and50g LÀ1)Low-HUFA diet(g LÀ1)High-HUFA diet(g LÀ1)S D SÂD 5305053050Neutral fraction16:011Æ1.1a11Æ0.9a11Æ0.5a8.6Æ0.7b9.4Æ0.6b9.1Æ0.2b NS0.01NS 18:08.3Æ0.38.7Æ0.28.7Æ0.38.6Æ0.28.3Æ0.28.7Æ0.3NS NS NS 16:1n-7 1.1Æ0.2ab 1.2Æ0.3a 1.1Æ0.2ab0.7Æ0.1b0.9Æ0.1ab0.9Æ0.1ab NS0.05NS 18:1n-910Æ1.0a9.9Æ1.1a8.9Æ0.6ab 5.7Æ0.2b 6.7Æ0.4b 6.4Æ0.6b NS0.01NS 18:1n-7 2.7Æ0.2 2.8Æ0.3 2.8Æ0.2 2.5Æ0.2 2.9Æ0.2 3.1Æ0.2NS NS NS 20:1n-9 1.3Æ0.1 1.4Æ0.1 1.4Æ0.1 1.4Æ0.1 1.3Æ0.1 1.3Æ0.1NS NS NS 18:2n-614Æ0.8b16Æ0.4a15Æ0.4ab9.7Æ0.3c11Æ0.3bc11Æ0.4bc0.010.01NS 18:3n-3 1.1Æ0.1a 1.2Æ0.1a 1.1Æ0.1a0.7Æ0.1b0.7Æ0.1b0.7Æ0.1b NS0.01NS 20:4n-6 6.1Æ0.5a 5.5Æ0.5a 5.9Æ0.2a 5.0Æ0.5b 5.0Æ0.2b 5.4Æ0.2ab NS0.01NS 20:5n-319Æ1.3b18Æ1.4b18Æ0.9b27Æ1.0a25Æ0.5a25Æ0.4a NS0.01NS 22:6n-311Æ0.8b12Æ0.8b11Æ0.4b16Æ0.3a16Æ0.6a16Æ0.5a NS0.01NS Polar fraction16:015Æ0.3ab16Æ0.5a15Æ0.2ab14Æ0.3b15Æ0.5ab15Æ0.1ab NS0.01NS 18:011Æ0.2ab11Æ0.2a11Æ0.2a11Æ0.2ab11Æ0.2ab10Æ0.2b NS0.01NS 16:1n-7 1.3Æ0.1ab 1.5Æ0.1ab 1.6Æ0.1a0.9Æ0.1b 1.1Æ0.1ab 1.1Æ0.1ab0.010.01NS 18:1n-913Æ0.3a13Æ0.3a13Æ0.4a7.9Æ0.3b8.3Æ0.1b8.1Æ0.1b NS0.01NS 18:1n-7 3.6Æ0.2ab 3.4Æ0.1b 3.6Æ0.2ab 3.4Æ0.1b 3.4Æ0.1b 3.9Æ0.2a0.05NS NS 20:1n-9 1.4Æ0.1 1.5Æ0.1 1.6Æ0.1 1.5Æ0.1 1.5Æ0.1 1.5Æ0.1NS NS NS 18:2n-619Æ0.6b22Æ0.5a21Æ0.4ab11Æ0.3c12Æ0.4c11Æ0.4c0.010.01NS 18:3n-3 1.0Æ0.2bc 1.6Æ0.1a 1.3Æ0.1ab0.6Æ0.1c0.7Æ0.1c0.6Æ0.1c0.050.010.05 20:4n-6 4.2Æ0.2a 3.2Æ0.1b 3.9Æ0.2ab 3.7Æ0.2ab 3.9Æ0.2ab 4.0Æ0.1a0.05NS0.01 20:5n-314Æ0.5c11Æ0.4c13Æ0.5c23Æ0.5a21Æ0.3ab21Æ0.2ab0.010.01NS 22:6n-39.4Æ0.5b8.3Æ0.6b8.3Æ0.3b15Æ0.5a16Æ0.5a16Æ0.3a NS0.01NS The results of the bifactorial ANOV A are shown in the last three columns(n510individuals per treatment).See Fig.1for statistical analyses.HUFA,highly unsaturated fatty acidsSalinity e¡ect on juvenile shrimp fed HUFA diets M A Hurtado et al.Aquaculture Research,2006,37,1316^1326r2006The Authors 1320Journal Compilation r2006Blackwell Publishing Ltd,Aquaculture Research,37,1316^1326with 0%HUFA emulsion (low-HUFA diet)contained 3%HUFA composed of1%20:5n-3[eicosapentaenoic acid (EP A)]and 1.5%22:6n-3[docosahexaenoic acid (DHA)]and a 1.6DHA/EP A ratio.Pellets enriched with 50%HUFA (high-HUFA diet)contained 34%HUFA composed of 13%EP A and 15%DHA and a 1.1DHA/EP A ratio (Table 2).The HUFA levels in each diet a¡ected the composi-tion of fatty acids in the hepatopancreas (Table 3)and gills (Table 4).In the hepatopancreas,EP A and DHA were three-to fourfold higher in the neutral fraction and twofold or more higher in the polar fraction in shrimp fed the high-HUFA diet compared with those fed the low-HUFA diet.However,the proportions of other polyunsaturated fatty acids such as 18:2n-6,18:3n-3and 20:4n-6(ARA)in the neutral fraction were similar in shrimp fed both diets while in the po-lar fraction,these fatty acids were present in higher proportions in shrimp fed the low-HUF A diet.In the neutral fraction,ARA,EP A and DHA decreased with increasing salinity ,but only in shrimp fed the low-HUF A diet.A similar e¡ect was observed in the polar fraction,but only for DHA.In gills,with the excep-tion of 18:0,18:1n-7and 20:1n-9in the neutral frac-tion,and 18:1n-7,20:1n-9and ARA in the polar fraction of gills,all other fatty acids in gills of shrimp were a¡ected by diet,with a higher proportion of EP A and DHA in shrimp fed the high-HUFA diet.However,in contrast with the hepatopancreas,di¡erences be-tween diets were not so pronounced,although more evident di¡erences were observed in the polar com-pared with the neutral fraction.In relation to salinity ,a lower proportion of 18:2n-6in the neutral fraction was observed in shrimp exposed to 5g L À1compared with 30and 50g L À1.In the polar fraction,several fatty acids were a¡ected by salinity ,particularly for lower levels of ARA and higher levels of 18:3n-3at 30g L À1but only in the low-HUFA diet.10080(a)(b)6040G r o w t h (%)S u r v i v a l (%)200120S N.S.N.S.bbabaaaaaaP<0.01N.S.P<0.01P<0.05D S ×D S D S ×D 9672482405 g L −130 g L −150 g L −15 g L−130 g L −150 g L−1Figure 1Survival (a)and growth (b)of Litopenaeus van-namei juveniles grown at 5,30and 50g L À1during 21days and fed low-(white bars)and high-(hatched bars)highly unsaturated fatty acids (HUFA)diets.The results are expressed as means ÆSE of three replicates and were analysed by bifactorial ANOV A,with the signi¢cances indi-cated in ¢gure:S,salinity (5,30and 50g L À1);D,diet (low and high HUF A)and interaction (S ÂD)as main factors (P o 0.05).When the interaction was signi¢cant,means were compared with a Tukey’s post hoc test and means not sharing the same superscripts are signi¢cantly di¡er-ent.NS,not signi¢cantly di¡erent.N.S.N.S.P<0.05SDS ×D N.S.N.S.P<0.01S900O s m o t i c p r e s s u r e (m O s m /k g )F i n a l b i o m a s s (g )102030405060708090100babbca800700600100DS ×D 5 g L −130 g L −150 g L −15 g L −130 g L −150 g L −1(a)(b)Figure 2Final biomass (a)and osmolarity in haemo-lymph (b)in Litopenaeus vannamei juveniles grown at 5g L À1(206mOsm kg À1),30gwL À1(832mOsm kg À1)and 50g L À1(1547mOsm kg À1)and fed low-(white bars)and high-(hatched bars)highly unsaturated fatty acids (HUF A)diets.The results are expressed as means ÆSE.See Fig.1for statistical analyses.Aquaculture Research,2006,37,1316^1326Salinity e¡ect on juvenile shrimp fed HUFA diets M A Hurtado et al .r 2006The AuthorsJournal Compilation r 2006Blackwell Publishing Ltd,Aquaculture Research,37,1316^13261321Regardless of diet,juveniles kept for 21days at 5g L À1had lower survival compared with controls (30g L À1)and shrimp grown at 50g L À1but no sig-ni¢cant di¡erences between both diets were ob-served at any salinity (Fig.1a).In contrast,growth was signi¢cantly lower for shrimp grown at 50g L À1and fed the low-HUF A diet compared with shrimp grown at 5and 30g L À1,with intermediate values for shrimp at 50g L À1and fed the high-HUF A diet (Fig.1b).The ¢nal biomass was higher for shrimp reared at 30g L À1,with no signi¢cant di¡erences in response to diet (Fig.2a).Feed intake was inversely related to salinity but no di¡erences were observed in relation to HUFA content in the diet (Fig.3a).Feed conversion ratio was signi¢cantly lower at 30g L À1(global mean 1.3)than at 5(global mean 1.7)and 50g L À1(global mean 1.7).Although no signi¢cant e¡ect of HUFAwas observed,a signi¢cant interaction indicated that the in£uence of HUF A depends on sali-nity ,with the highest FCR in shrimp fed the low-HUFA diet and grown at 50g L À1and the lowest for shrimp grown at 30g L À1and also fed the low-HUFA diet (Fig.3b).Osmotic pressure in haemolymph ranged between574and 980mOsm kg À1.Osmotic pressure was a function of medium salinity ,with signi¢cant di¡er-ences between the three salinities,but without any in£uence of HUFA content in the diet (Fig.2b).Con-sidering water osmolarity (approximately 206mM for water with 5g L À1,832mM for 30g L À1and 1547mM for 50g L À1),the di¡erence between haemo-lymph and water osmolarity (also termed osmoregula-tory capacity )was 439Æ10mM at 5g L À1and 733Æ10mM at 50g L À1.DiscussionHighly unsaturated fatty acids requirements of L.vannamei are approximately 6%of total fatty acids(Gonza lez-Felix et al .2003)and as high as 28%for good performance (Lim et al .1997).W e found that le-vels as low as 3%(or as high as 34%)of HUFA did not a¡ect the survival and growth of shrimp grown at 30g L À1salinity over a 21-day trial.A longer trial might have had a more pronounced e¡ect,but even with levels of 0.4%ARA,1.0%EP A and 1.5%DHA in the diet,shrimps had levels as high as 4%,14%and 11%of ARA,EP A and DHA,respectively ,in the polar lipids of the hepatopancreas or gills.These results in-dicate a high capacity of retention of these essential fatty acids in juvenile L.vannamei ,in spite of theirlow levels in the diet (Gonza lez-Felix et al .2003).Synthesis from 18carbons could also contribute to high levels of essential fatty acids in tissues,although shrimp have very low synthesis rates from18-carbon precursors (Teshima,Kanazawa &Koshio 1992;Deering et al .1997).For salinity treatments,better performance or bio-mass was expected and obtained at 30g L À1,as this value is close to the isosmotic point of this species:25^26g L À1for juveniles (Castille &Lawrence 1981;Rodriguez 1981;Gong et al .2004),although the iso-smotic point does not necessarily correspond to the optimal performance (Bray et al .1994;Brito,Chimal &Rosas 2000).Nevertheless,it is assumed that shrimp near the isosmotic point will spend less energy on respiration and excretion (Rosas et al .2001),therefore exhibiting optimal growth rates and the most e⁄cient energy conversion.Additionally ,they have higher food consumption (Rosas et al .2001;W ang et al .2004).As salinity deviates from the isosmotic point,the osmotic balance in shrimp is modi¢ed,so it must expend more energy for osmo-regulation and less on growth.Accordingly ,respira-N.S.N.S.N.S.P<0.01P<0.01P<0.05S DS ×D S abcaab abab abbD S ×D 5 g L −130 g L −150 g L −15 g L−130 g L−150 g L−160(a)(b)50403020100321C o n v e r s i o n R a t i oF e e d i n t a k e (g )Figure 3Feed intake during the whole experiment (a)and feed conversion ratio (b)of Litopenaeus vannamei juve-niles grown at 5,30and 50g L À1and fed low-(white bars)and high-(hatched bars)highly unsaturated fatty acids (HUFA)diets.The results are expressed as means ÆSE.See Fig.1for statistical analyses.Salinity e¡ect on juvenile shrimp fed HUFA diets M A Hurtado et al .Aquaculture Research,2006,37,1316^1326r 2006The Authors1322Journal Compilation r 2006Blackwell Publishing Ltd,Aquaculture Research,37,1316^1326。