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The effect of linseed oil supplementation on performance, fatty acid composition and oxidative status of rabbits

The effect of linseed oil supplementation on performance, fatty acid composition and oxidative... doi:10.2478/v10014-011-0028-2 COBISS:1.01 Agriscategorycode:L51 TinaTrEBuSAK1,AlenkaLEvArT2,mojcavOLJC3,urskaTOmAZIn4,TatjanapIrmAn5 receivedOctober22,2011;acceptednovember15,2011. delojeprispelo22.oktobra2011,sprejeto15.novembra2011. The effect of linseed oil supplementation on performance, fatty acid composition and oxidative status of rabbits The objectiveof thepresent studywas todetermine the effectoflinseedoilsupplementationonperformance,fattyacid compositionandoxidativestatusofrabbits.TwelvemaleSIKA rabbitsweredividedintotwogroups.Thecontrolgroup(n=4) received commercial diet and the linseed group (n=8) receivedcommercialdietcontaining9%oflinseedoil,whichwas sprayed onto the pellet. rabbits were slaughtered at 115 days of age, 52 days after the start of the experiment. Live weight, weightgain,feedintakeandfeedefficiencywererecorded.The fattyacidcompositionofmuscle,adiposetissueandliverwas determined by the in situ transesterification method and gas chromatography-flame ionisation detection (GC-fId). In order to evaluate the oxidative status of rabbits, the malondialdehyde(mdA)concentrationinplasma,liverandmusclewere measured.Theresultsshowthatitispossibletoenhanceproportion of n-3 polyunsaturated fatty acids (pufA) in rabbit's muscle,adiposetissueandliverbyaddinglinseedoilinthediet withoutdetrimentaleffectonproductiveperformance.Linseed oil addition improved fatty acid composition in all tissues by increasedpufAproportionanddecreasedproportionofsaturatedandmonounsaturatedfattyacid.Linseedoilalsoreduced n-6/n-3pufAratioinalltissues.However,linseedoiladdition ledtosignificantlyhighermdAconcentrationsinplasma,liver and muscle. Since pufA are highly susceptible to oxidation, further research is needed to focus on protecting animal and theirproductsfromlipidoxidationbyaddingvariousnatural antioxidantstothediet. Key words: rabbits / animal nutrition / feed additives / linseedoil/fattyacidcomposition/oxidativestatus/performance Vpliv dodatka lanenega olja v krmo na proizvodne lastnosti, mascobnokislinsko sestavo in oksidacijki status kuncev v poskusu smo preucevali vpliv dodatka lanenega olja v krmo na proizvodne lastnosti, mascobnokislinsko sestavo in oksidacijski status kuncev. dvanajst kuncev slovenske mesnelinijeSIKAsmorazdelilivdveskupini.Kontrolnaskupina (n=4)jeuzivalastandardnokrmo,poskusnaskupina(n=8) pastandardnokrmozdodatkom9%lanenegaolja,kismoga nanesli na pelete. Kunce smo zaklali pri starosti 115 dni, 52 dnipozacetkuposkusa.vcasuposkusasmozapisovalimaso zivali,prirast,zauzivanjeinizkoristekkrme.mascobnokislinskosestavomisic,mascobnegatkivainjetersmodolocilizin situtransesterifikacijoinkapilarnoplinskokromatografijo.Za dolocitev oksidacijskega statusa kuncev smo izmerili koncentracijomalondialdehida(mdA)vplazmi,jetrihinmisici.rezultatisopokazali,dalahkozdodatkomlanenegaoljavkrmo kuncev povecamo delez n-3 veckrat nenasicenih mascobnih kislin (vnmK) v misici, mascobnem tkivu in jetrih brez negativnih ucinkov na proizvodne lastnosti. dodatek lanenega oljajeizboljsalmascobnokislinskosestavospovecanjemdeleza vnmKinzmanjsanjemdelezanasicenihinenkratnenasicenih mascobnihkislin,karjeznizalotudirazmerjen-6/n-3vnmK vvsehtkivih.dodateklanenegaoljajeznacilnopovecalkoncentracijomdAvplazmi,jetrihinmisici.KersovnmKzelo podvrzeneoksidaciji,sopotrebnenadaljnjeraziskave,vkaterih se bo potrebno osredotociti na zascito zivali in njihovih produktovpredlipidnooksidacijozdodajanjemrazlicnihnaravnihantioksidantovvkrmo. Kljucne besede:kunci/prehranazivali/krmnidodatki /lanenoolje/mascobnokislinskasestava/oksidacijskistatus/ proizvodnelastnosti univ.ofLjubljana,Biotechnicalfac.,dept.ofAnimalScience,Groblje3,SI-1230domzale,Slovenia,e-mail:tina.trebusak@bf.uni-lj.si Sameaddressas1,ph.d.,e-mail:alenka.levart@bf.uni-lj.si Sameaddressas1,e-mail:mojca.voljc@bf.uni-lj.si Sameaddressas1,e-mail:urska.tomazin@bf.uni-lj.si Sameaddressas1,Ass.prof.,ph.d.,e-mail:tatjana.pirman@bf.uni-lj.si Acta argiculturae Slovenica, 98/2, 119­125, Ljubljana 2011 INTRODUCTION Inthesedayspeoplearemoreandmoreawareofthe importance of healthy diet. polyunsaturated fatty acids (pufA),especiallyn-3pufA,areknownfortheirbeneficialeffectsonhumanhealth.Westerndietshavealow intakeofn-3pufAandarelativelyhigherconsumption ofn-6pufAandthatleadstoaveryhighn-6/n-3pufA ratio.Accordingtothiswecanspeculatethatpopulation inthedevelopedworlddonotconsumeenoughessential fatty acids with food. recent recommendations for human diets suggest increased n-3 pufA consumption anddecreasedthen-6/n-3pufAratio.deficiencyofn-3 pufAincreasestheriskofdevelopingvariousdiseases, especiallycardiovasculardiseases.Therefore,interestin obtaininganimalproductswithhighlevelsofn-3pufA has increased. The fatty acid composition of meat and otheranimalproducts(eggs,milk),intendedforhuman consumption,canbeimprovedbyspecificdietarystrategies(Leskanickandnoble,1997;raeset al.,2004;Wood et al, 2003). Because of its high -linolenic acid (C18:3 n-3)content,linseedorlinseedoilisasuitableandfrequentlyusedplantsourceofn-3pufA.-linolenicacid istheprecursorfattyacidforthesynthesisofeicosapentaenoicacid(EpA;C20:5n-3)anddocosahexaenoicacid (dHA;C22:6n-3),whichhavebeneficialcardiovascular andalsoanti-inflammatoryproperties(Connor,2000). The beneficial effects of pufA on human health are well known and have been documented by numerousstudies(reviewofWilliams,2000;reviewofriediger et al., 2009). Great numbers of experiments have been madeinordertoincreasepufAcontentinanimalproducts(Leskanickandnoble,1997;Bernardiniet al.,1999; Koubaet al.,2003;raeset al.,2004;Woodetal,2003). rabbit meat is often recommended by nutritionists becauseofitslowlipidandcholesterollevelsandhighcontentofpufAcomparedtoothermeat(dalleZotte,2004). fattyacidcompositionofrabbitmeatisbettercompared to other meat, because rabbit's diet usually contains alfalfawithhighn-3pufAcontent.Withtheadditionof linseedoilinthediettheproportionofn-3pufAinmeat increases and also affects the reduction of the n-6/n-3 pufAratio.Westerndietsaredeficientinn-3pufAand haveexcessiveamountsofn-6pufA.Thisleadstoavery highn-6/n-3pufAratiorangingfrom15/1to20/1.The optimalratioisthoughttobefrom5/1to10/1infavour of the n-6 pufA. According to Simopoulos (2002) the optimalratiobetweenn-6andn-3pufAwouldbe4/1or evenlower.dalBoscoet al.(2004)demonstratedthatthe additionof8%linseedinrabbit'sdietincreasedconcentrationofn-3pufAanddecreasedn-6/n-3pufAratio inrabbitmeat. However,thehighercontentofpufAcouldleadto 120 ahighersusceptibilitytolipidoxidation.Thismightaltertissueoxidation,whichleadstotheformationoffree radicals, lipid peroxides, aldehydes (malondialdehyde (mdA)e.g.)andfurtheroxidationproductswhichhavea negativeeffectonthedieteticvalueoffatandareharmful totheorganism.SuchenrichmentofpufAmightreduce shelf-lifeofmeatproductsanditcanalsonegativelyaffectanimalsandtheirhealth.Inmeatandotheranimal products,oxidationmightreduceitsstability,nutritional andsensoryquality(Grayet al.,1996).Itisthereforevery importantthatwesupplementanimalswithantioxidants inordertopreventlipidoxidationcausedbyfeedinghigh levelsofpufA. Theaimofthisstudywastoinvestigatetheeffectof linseed oil supplementation on performance, fatty acid compositionandoxidativestatusinrabbits.Inorderto determine to what extent we can change the fatty acid compositionofmuscle,adiposetissuesandliverofrabbits, we made an experiment in which we added high proportionoflinseedoiltothecommercialrabbit'sdiet. Inordertoevaluatetheoxidativestress,theplasma,liver andmusclemdAconcentrationsweremeasured. MaTeRIals aND MeThODs All procedures were performed according to current legislation on animal experimentation in Slovenia. AnimalsusedinthisexperimentwererearedandslaughteredatthedepartmentofAnimalScience,Biotechnical faculty(Ljubljana,Slovenia). 2.1 AnImALSAnddIETS TwelvemaleSIKArabbits(63daysold,2284g)were randomlydividedintotwogroupsandassignedintotwo differentdietarytreatmentsfor52days:acontrol(n=4) andalinseeddiet(n=8).Thecontroldietwascommercialdietandthelinseeddietwascommercialdietcontaining9%oflinseedoil,whichwassprayedontopellets. Theexperimentaldietwasprepareddaily.Animalshad freeaccesstofeedandwater(nippledrinkers)andwere housedindividuallyinstandardcages.dietsampleswere takenduringtheexperimentforthepurposeofchemicalanalyses.Thefattyacidcompositionofthedietswas analysedusingagaschromatographicmethodafterthe insitutransesterificationoflipids.Thechemicalandfatty acidcompositionofthedietsarepresentedinTable1. 2.2 ExpErImEnTALprOCEdurEAndSAmpLE COLLECTIOn Eachdayanimalsreceivedweigheddailymealand the residue from the day before was weighed and discarded. Body weights were recorded each week during theexperimentalperiodandjustbeforeslaughter.After 52daysoftreatment,rabbitswereslaughteredat115days of age, by electric stunning and exsanguination. After sacrificing the animals, blood, liver, adipose tissue and legmusclesampleswerecollected.Bloodsamples,forthe purpose of measuring mdA concentration in plasma, werecollectedinto6mlevacuatedtubescontainingEdTAK2anticoagulant(367864,Bd-plymouth,uK).plasma was separated by centrifugation (1000g for 15 min at4 °C),transferredintoEppendorftubesandstoredat -70°Cuntilanalysis.Thewholeliver,partofthelegmuscleandadiposetissueweretaken,weightedandstoredat -70 °Cuntilanalyseswereperformed. 2.3 dETErmInATIOnOffATTyACIdCOmpOSITIOn Thefattyacidcompositionofmuscleandadiposetissuessampleswereanalysedusingagaschromatographic methodaftertheinsitutransesterificationoflipids.Each samplewasanalysedinduplicate.methylestersoffatty acidswerepreparedaccordingtotheprocedureofpark andGoins(1994).Abriefsummaryoftheprocedureis asfollows:frozenmuscleoradiposetissuesampleswere homogenized (Grindomix homogenizer, retsch Gmbh & Co, Haan, Germany) and approximately 0.5­0.7g (muscle) or 0.1g (adipose tissue) of the homogenized samplewasweigheddirectlyinatubewithstopperand mixedwith3ml0.5msodiumhydroxideinmethanol and 0.3 ml methylene chloride. In situ transesterification was performed by heating samples at 90ºC for 10 minintheclosedtube.Aftercooling,3mlof14%boron trifluorideinmethanolwasaddedandheatingat90ºC was continued for 10 min in the closed tube. Samples were cooled, and the fatty acid methyl esters (fAmEs) wereextractedinto1mlhexane.AnalysisoffAmEswas performedbygaschromatographyusinganAgilent6890 seriesgaschromatograph(AgilentTechnologies,Wilmington,dE,uSA)equippedwithanAgilent7683AutomaticLiquidSampler,asplitinjector,aflameionization detectorandafusedsilicacapillarycolumnOmegawax 320(Supelco,uSA).Theinjectionvolumewas1l.The chromatogramswereevaluatedbytheAgilentGCChem Stationsoftware.SeparatedfAmEswereidentifiedbyretentiontime.resultsareexpressedasapercentageofthe totalfattyacids. 2.4 mALOndIALdEHydE(mdA)dETErmInATIOn The methodology of Wong et al. (1987) modified byChirico(1994)andfukunagaet al.(1995)wasused tomeasuretheconcentrationofmdAinbloodplasma using HpLC. A brief summary of the procedure is as follows: 100l of the sample was mixed with 100 l of 0.44mphosphoricacid(H3pO4)and10lof0.2%ethanolic butylhydroxytoluene (BHT) in Eppendorf microcentrifuge tubes, left for 15 minutes, added 300l ethanolandthancentrifuged(15000g,for15minutesat 4ºC).Thesupernatant(350l)wasmixedwith1.5mlof 0.44mH3pO4,0.5mlof0.6%thiobarbituricacid(TBA) and0.9mlofmilliQdeionisedwaterinatubewithstopperandheatedat90ºCfor60minutes.Aftercooling,the sampleswerefilteredthroughmilliporefilters(poresize 0.22m)intoautosamplervials.ThemdAconcentration inliverandmusclesampleswasdeterminedfollowingthe method of vila et al. (2002) with minor modifications. Briefly,frozenliverormusclesampleswerehomogenized (Grindomix homogenizer, retsch Gmbh & Co, Haan, Germany)andapproximately0.3gofthehomogenized sample was mixed with 1.5ml of 2.5% trichloroacetic acid(TCA)inEppendorfmicrocentrifugetubes,leftfor 10minutesandthancentrifuged(15000g,for15minutes at 4ºC). 1 ml of supernatant was mixed with 1.5ml of 0.6%TBAand1mlofmilliQdeionisedwaterinatube with stopper and heated at 90ºC for 60 minutes. After thesampleswerecooled,theywerefilteredthroughmilliporefilters(poresize0.22m)intoautosamplervials. AWatersAlliance2690(Waters,milford,mA)equipped with Waters 474 scanning fluorescence detector was used to determine plasma, liver and muscle mdA. for thepurposeofseparationareversed-phaseHpLCchromatographycolumn(HyperClone5uOdS(C18)120A, 4.6×150 mm 5 micron; phenomenex Inc., uSA) and C18OdSguardcolumn(4mm×30mm;phenomenex Inc.,uSA)wereused.Themobilephaseconsistedof65% 50mmol/lKH2pO4buffer(pH6.9)and35%methanol. Themobilephaseflowratewas1.0ml/min.Theresultsof theanalysiswereevaluatedusingthemillenium32Chromatographymanagerprogram. 2.5 STATISTICALAnALySIS ThedatawereanalysedbytheGeneralLinearmodels(GLm)procedureoftheSAS/STATmodule(SAS8e, 2000;SASInc.,Cary,nC,uSA),takingintoconsiderationthedietastheonlymaineffect.differencesbetween groupsweredeterminedonthebasisofTurkey'smultiple comparisonstest.Theresultsinthetablesarepresented asleastsquaremeans(LSm)±standarddeviation.Ifnot statedotherwise,aleastsignificantdifferenceof0.05was usedtoseparatetreatmentmeans. 3.2 BOdyWEIGHTGAInAnddIETInTAKE There were no significant differences in the initial and final body weight and also in the weight gain (Table2).Somedifferencesamongtreatmentswereobserved infeedintakeandfeedefficiency.Animalsingroupthat had diet enriched with linseed oil showed significantly lowerfeedintake(forabout41g/day)andconsecutively hadsignificantlyhigherfeedefficiency.Thatmeansthat animalswithsupplementeddietconsumedsignificantly (p = 0.0006) less feed (4.44 kg) than control animals (5.82kg)for1kgweightgain.Thesedifferenceswereexpectedbecauseofthemuchhigherfat(energy)content inthelinseeddiet. Table 2: Body weight (BW), weight gain and diet intake Preglednica 2: Telesna masa zivali, prirast in zauzivanje krme Controlgroup Linseedgroup p-value InitialBW(g) finalBW(g) Weightgain(g/day) feedintake(g/day) feedefficiency(%) 2343±129 3963±322 31.8±4.0 183.4±13.0 17.3±1.7 2256±175 3903±202 32.3±3.7 142.4±9.5 22.7±2.0 0.4026 0.6979 0.8232 <0.0001 0.0010 ResUlTs aND DIsCUssION Theanimalsadaptedwelltotheexperimentalconditions.duringtheexperiment,theanimalshadnohealth orotherproblems. 3.1 CHEmICALAndfATTyACIdCOmpOSITIOnOfTHEdIETS ThechemicalandfattyacidcompositionofthedietsarepresentedinTable1.Asexpected,theadditionof linseedoilincreasedtheproportionofcrudefatfrom4.2 to12.2%.Thefattyacidcompositionofthedietsdiffered accordingtotheingredients.Thelinseeddiethadhigher proportionofn-3pufAandtotalpufA,andlowerproportionofsaturatedfattyacids(SfA)andmonounsaturatedfattyacids(mufA)comparedtothecontroldiet. The linseed diet had lower proportion of linoleic acid (C18:2 n-6) and higher proportion of -linolenic acid (C18:3 n-3). Therefore, the linseed diet had lower n6/n-3ratio(0.63vs.6.30)comparedtothecontroldiet. Table 1: Chemical and fatty acid composition of the diets Preglednica 1: Kemijska in mascobnokislinska sestava krme Controldiet Chemicalanalysis(%) Crudeprotein Crudefat Crudefibre Crudeash C16:0 C18:0 C18:1 C18:2n-6 C18:3n-3 SfA mufA pufA n-3pufA n-6pufA n-6/n-3pufA 16.4 4.2 15.0 9.4 14.31 3.25 21.13 50.26 7.93 19.64 22.05 58.32 7.99 50.33 6.30 14.9 12.2 13.6 8.5 7.85 3.56 19.37 25.85 40.74 12.60 20.81 66.59 40.74 25.85 0.63 Linseeddiet mainfattyacids(%ofthetotalfattyacids) Our results are in general agreement with other studiesshowingthatdietshighinn-3pufAdidnotsignificantly influence productive performances in rabbits (Bernardini et al., 1999; dal Bosco et al., 2004; Kouba et al., 2008; Bianchi et al., 2009). differences occurred indietintakeanddietefficiencybetweenourandtheirs results. These differences were expected because of the higherfatcontentinthedietinthelinseedgroupinour experiment, while other had isoenergetic diets. On the contrary,Bianchiet al.(2006)foundoutthat8%oflinseed supplementation determined a lower daily weight gainaswellasalowerfinalliveweightofrabbitscomparedtothegroupwithoutlinseedaddition. 3.3 fATTyACIdCOmpOSITIOnOfTISSuES AndOxIdATIvESTATuSOfrABBITS dietsupplementedwithlinseedoilledtoahigher proportionoftotalpufA,duetoareductionoftotalSfA andmufAlevelsinmuscle,adiposetissueandliver.As expected,thelinseedoiladditionsignificantlyincreased (p<0.0001)proportionofthe-linolenicacidandsome othern-3pufAinalltissues(Table3).Thelongchain n-3 pufA content was at much higher level in muscle and liver compared to adipose tissue. The main differencesbetweentissueswereobtainedinproportionofli- SfA ­ saturated fatty acids; mufA ­ monounsaturated fatty acids; pufA­polyunsaturatedfattyacids Table 3: Fatty acid composition of muscle, adipose tissue and liver (% of total fatty acids) Preglednica 3: Mascobnokislinska sestava misice, mascobnega tkiva in jeter (% od vseh mascobnih kislin) muscle Control C16:0 C16:1n-7 C18:0 C18:1 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C22:5n-3 C22:6n-3 SfA mufA pufA n-3pufA n-6pufA n-6/n-3 21.86a 3.46a 6.90a 23.48 Adiposetissue Linseed 15.04b 1.53b 6.62a 21.74 Liver Linseed 12.31b 0.93b 4.79b 23.23 Control 21.96a 2.68a 5.43a 24.98 Control 21.25a 1.40a 16.28a 17.66 Linseed 12.20b 0.22b 22.85b 11.01b 32.88b 9.53b 5.37a 0.32b 1.27b 0.49b 36.66b 11.55b 51.78b 11.92b 39.85a 3.41b 28.98a 3.30a 3.49 25.77b 20.17b 2.45 34.47a 4.72a 0.15 27.41b 28.02b 0.09 29.16a 1.55a 4.96 0.08a 0.57a 0.10 27.83 0.30b 1.37b 0.16 23.77 0.01a 0.05a 0.01 0.03b 0.11b 0.01 24.72 0.04a 0.29a 0.09 33.64a 24.86b 31.35a 28.65 19.34b 40.22a 20.03 38.53a 4.17a 34.32 51.37b 22.28b 29.06 40.00a 4.80a 35.14 55.93b 28.19b 27.70 39.75a 2.16a 37.54 8.22a 1.31b 7.31a 0.98b 17.43a a,b valueswithdifferentsubscriptswithineachtissuearesignificantlydifferent(p<0.05);SfA­saturatedfattyacids;mufA­monounsaturated fattyacids;pufA­polyunsaturatedfattyacids noleicacid(n-6pufA).Theproportionoflinoleicacid significantlydecreasedinmuscleandadiposetissuebut increasedinliver.Thedifferencesalsooccurredinproportionof-linolenicacid,whichincreasedinalltissues but it was at much higher level in muscle and adipose tissuecomparedtotheliver.despitethesedifferencesthe highercontentofn-3pufAinlinseeddietledtoasignificantly(p < 0.0001)lower n-6/n-3pufAratioinall tissues. Our results are in general agreement with other studies. Bernardini et al. (1999) found that addition of 160ggroundlinseed/kgdietsignificantlyincreasedthe proportionof-linolenicacidanddecreasedthen-6/n-3 pufAratiocomparedtothestandardandfishoildiet(60 gfishoil/kgdiet).Similarresultswereobtainedwhenapproximately30gofextrudedlinseed/kgdietwasadded. Thelinseeddietincreasedthelevelof-linolenicacidin muscle,perirenalfat,andinrawandcookedmeat.Linseed addition also increased the long chain n-3 pufA contentsinmeatandconsecutivelycausedasignificant decreaseinthen-6/n-3pufAratio(Koubaet al.,2008). decreased n-6/n-3 pufA ratio in rabbit's muscle, adipose tissues and liver can provide the "functional food"forhumanconsumption.Intakeofsuchfoodcontributestoabetterratioofn-6ton-3pufAinthehuman dietandthuscontributestoimprovedhumanhealth. ThemdAconcentrationsinplasma,liverandmuscleweresignificantlyinfluencedbythediet(Table4).The linseedgroup,whichwassupplementedwithlinseedoil highinpufA,showedsignificantlyhigherlevelsofmdA in plasma, liver and muscle compared to the control grouThat showed that supplementing diet with linseed oil increased the oxidation. from the nutritional point of view these results are unwanted, because of reduced shelf-lifeofmeatproductsandnegativeeffectonanimal health. Table 4: MDA concentration in plasma, liver and muscle Preglednica 4: Koncentracija MDA v plazmi, jetrih in misici Control plasmamdA(nmol/ml) LivermdA(nmol/g) musclemdA(nmol/g) 0.26 0.47 0.40 Linseed 0.36 1.09 1.24 p-value 0.0059 0.0319 0.0015 Oxidativestressisdefinedasapersistentimbalance betweenantioxidantsandpro-oxidantsinfavourofthe latter. namely, oxidation can be reduced if we provide enough antioxidants to prevent the formation of free radicals. factors affecting the extent of oxidation are: pufA content in the membranes, the amount of free radicals and the levels of antioxidants which can be of endogenousorexogenousorigin(Breneset al.,2008).We couldpreventoxidationusingdifferentdietarystrategies. vitaminEisthemostfrequentlyusedantioxidant.There are some studies dealing with feeding and nutritional valueofrabbit'smeat,butthereisalackofthemdealing withrabbitplasmaandothertissuesmoreinvolvedinthe animal'smetabolism,suchastheliver,atthesametime (Treset al.,2009). Bianchiet al.(2006)indicatedthattheadditionof 35%alfalfawith200mgvitaminE/kgfeedincreasedthe proportionof-linolenicacidwithouthavinganegative impactontheoxidativestabilityofmeat.Simultaneous additionof8%linseedhasastronginfluenceonthefatty acidcompositionofmeat,withincreasingproportionof pufA, especially -linolenic acid, which has a positive effectontheratioofn-6andn-3pufA.Similarresults were also demonstrated in a recent study about the effectsofdifferentinclusionratesofwholelinseedinthe diettothequalityofmeat,wheretheproportionofpufA also increased at the expense of SfA (Bianchi et al., 2009).Theyfoundthatinclusionof3%linseedindiets forgrowingrabbitsprovidedfavourablefattyacidcomposition and also meat quality. dal Bosco et al. (2004) indicated that the addition of 8% linseed with 200 mg ofvitaminE/kgfeedincreasedtheconcentrationofn-3 pufA and narrowed the ratio of n-6 and n-3 pufA in rabbitswithoutnegativeeffectontheoxidativestability ofmeat.Itisofnotethattheseauthorsusedmuchhigher quantities of vitamin E when linseed oil was added inthedietcomparedtothecontroldiet(200mg/kgvs. 50mg/kg).petracciet al.(2009)foundthatthedietary inclusionfrom3to6%oflinseedtogetherwith200mg ofvitaminE/kgmightbeconsideredasawaytoincrease theproportionof-linolenicacidintherabbitmeatand alsoensuresufficientoxidativestabilityoftheproduct. Theadditionof3%linseedoilinthediethadabeneficialeffectonfattyacidcompositionofrabbitmeatas it reduced the proportion of total SfA and the level of cholesterol and increased the proportion of n-3 pufA. TheadditionofvitaminEpreventedoxidationprocesses (Bielanski and Kowalska, 2008). Zsédely et al. (2008) studiedtheeffectofsunfloweroilandlinseedoilincombinationwithvariousconcentrationsofvitaminEonfattyacidcompositionandoxidativestabilityofrabbitmeat. It was expected that addition of oil would significantly increase the proportion of pufA due to the reduction of SfA and mufA and decrease the ratio between n-6 andn-3pufA.Inaddition,vitaminEimprovedoxidativestabilitycomparedtothegroupswithoutvitaminE addition. plasma and liver fatty acids composition also reflectedfeed'sfattyacidprofile(Treset al.,2009). 124 CONClUsIONs nowadays consumers are more concerned about theirhealthandthenutritionalvalueoftheirfood.Therefore, interest in obtaining animal products with better nutritionalvaluehasincreased.Ourresultsrepresentto what extent we can improve fatty acid composition of rabbitstissueswithlinseedoilsupplementation.Tosummarize,theoverallfattyacidcompositionwasinfluenced bydietarylinseedoilinclusionwithoutsignificanteffect onproductiveperformance.ThelinseedoilcausedalowercontentoftotalSfAandmufAandahighercontent ofpufA.Linseedoilalsoreducedn-6/n-3pufAratioin alltissues.However,thehighercontentofpufAintissuesledtohighersusceptibilitytolipidoxidation.Such enrichmentmightreducetheshelf-lifeofmeatproducts anditcanalsonegativelyaffectanimalsandtheirhealth. Therefore,furtherresearchisneededtoprotectanimals andtheirproductsfromlipidoxidationbyaddingvariousnaturalantioxidantstothediet. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Agriculturae Slovenica de Gruyter

The effect of linseed oil supplementation on performance, fatty acid composition and oxidative status of rabbits

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Abstract

doi:10.2478/v10014-011-0028-2 COBISS:1.01 Agriscategorycode:L51 TinaTrEBuSAK1,AlenkaLEvArT2,mojcavOLJC3,urskaTOmAZIn4,TatjanapIrmAn5 receivedOctober22,2011;acceptednovember15,2011. delojeprispelo22.oktobra2011,sprejeto15.novembra2011. The effect of linseed oil supplementation on performance, fatty acid composition and oxidative status of rabbits The objectiveof thepresent studywas todetermine the effectoflinseedoilsupplementationonperformance,fattyacid compositionandoxidativestatusofrabbits.TwelvemaleSIKA rabbitsweredividedintotwogroups.Thecontrolgroup(n=4) received commercial diet and the linseed group (n=8) receivedcommercialdietcontaining9%oflinseedoil,whichwas sprayed onto the pellet. rabbits were slaughtered at 115 days of age, 52 days after the start of the experiment. Live weight, weightgain,feedintakeandfeedefficiencywererecorded.The fattyacidcompositionofmuscle,adiposetissueandliverwas determined by the in situ transesterification method and gas chromatography-flame ionisation detection (GC-fId). In order to evaluate the oxidative status of rabbits, the malondialdehyde(mdA)concentrationinplasma,liverandmusclewere measured.Theresultsshowthatitispossibletoenhanceproportion of n-3 polyunsaturated fatty acids (pufA) in rabbit's muscle,adiposetissueandliverbyaddinglinseedoilinthediet withoutdetrimentaleffectonproductiveperformance.Linseed oil addition improved fatty acid composition in all tissues by increasedpufAproportionanddecreasedproportionofsaturatedandmonounsaturatedfattyacid.Linseedoilalsoreduced n-6/n-3pufAratioinalltissues.However,linseedoiladdition ledtosignificantlyhighermdAconcentrationsinplasma,liver and muscle. Since pufA are highly susceptible to oxidation, further research is needed to focus on protecting animal and theirproductsfromlipidoxidationbyaddingvariousnatural antioxidantstothediet. Key words: rabbits / animal nutrition / feed additives / linseedoil/fattyacidcomposition/oxidativestatus/performance Vpliv dodatka lanenega olja v krmo na proizvodne lastnosti, mascobnokislinsko sestavo in oksidacijki status kuncev v poskusu smo preucevali vpliv dodatka lanenega olja v krmo na proizvodne lastnosti, mascobnokislinsko sestavo in oksidacijski status kuncev. dvanajst kuncev slovenske mesnelinijeSIKAsmorazdelilivdveskupini.Kontrolnaskupina (n=4)jeuzivalastandardnokrmo,poskusnaskupina(n=8) pastandardnokrmozdodatkom9%lanenegaolja,kismoga nanesli na pelete. Kunce smo zaklali pri starosti 115 dni, 52 dnipozacetkuposkusa.vcasuposkusasmozapisovalimaso zivali,prirast,zauzivanjeinizkoristekkrme.mascobnokislinskosestavomisic,mascobnegatkivainjetersmodolocilizin situtransesterifikacijoinkapilarnoplinskokromatografijo.Za dolocitev oksidacijskega statusa kuncev smo izmerili koncentracijomalondialdehida(mdA)vplazmi,jetrihinmisici.rezultatisopokazali,dalahkozdodatkomlanenegaoljavkrmo kuncev povecamo delez n-3 veckrat nenasicenih mascobnih kislin (vnmK) v misici, mascobnem tkivu in jetrih brez negativnih ucinkov na proizvodne lastnosti. dodatek lanenega oljajeizboljsalmascobnokislinskosestavospovecanjemdeleza vnmKinzmanjsanjemdelezanasicenihinenkratnenasicenih mascobnihkislin,karjeznizalotudirazmerjen-6/n-3vnmK vvsehtkivih.dodateklanenegaoljajeznacilnopovecalkoncentracijomdAvplazmi,jetrihinmisici.KersovnmKzelo podvrzeneoksidaciji,sopotrebnenadaljnjeraziskave,vkaterih se bo potrebno osredotociti na zascito zivali in njihovih produktovpredlipidnooksidacijozdodajanjemrazlicnihnaravnihantioksidantovvkrmo. Kljucne besede:kunci/prehranazivali/krmnidodatki /lanenoolje/mascobnokislinskasestava/oksidacijskistatus/ proizvodnelastnosti univ.ofLjubljana,Biotechnicalfac.,dept.ofAnimalScience,Groblje3,SI-1230domzale,Slovenia,e-mail:tina.trebusak@bf.uni-lj.si Sameaddressas1,ph.d.,e-mail:alenka.levart@bf.uni-lj.si Sameaddressas1,e-mail:mojca.voljc@bf.uni-lj.si Sameaddressas1,e-mail:urska.tomazin@bf.uni-lj.si Sameaddressas1,Ass.prof.,ph.d.,e-mail:tatjana.pirman@bf.uni-lj.si Acta argiculturae Slovenica, 98/2, 119­125, Ljubljana 2011 INTRODUCTION Inthesedayspeoplearemoreandmoreawareofthe importance of healthy diet. polyunsaturated fatty acids (pufA),especiallyn-3pufA,areknownfortheirbeneficialeffectsonhumanhealth.Westerndietshavealow intakeofn-3pufAandarelativelyhigherconsumption ofn-6pufAandthatleadstoaveryhighn-6/n-3pufA ratio.Accordingtothiswecanspeculatethatpopulation inthedevelopedworlddonotconsumeenoughessential fatty acids with food. recent recommendations for human diets suggest increased n-3 pufA consumption anddecreasedthen-6/n-3pufAratio.deficiencyofn-3 pufAincreasestheriskofdevelopingvariousdiseases, especiallycardiovasculardiseases.Therefore,interestin obtaininganimalproductswithhighlevelsofn-3pufA has increased. The fatty acid composition of meat and otheranimalproducts(eggs,milk),intendedforhuman consumption,canbeimprovedbyspecificdietarystrategies(Leskanickandnoble,1997;raeset al.,2004;Wood et al, 2003). Because of its high -linolenic acid (C18:3 n-3)content,linseedorlinseedoilisasuitableandfrequentlyusedplantsourceofn-3pufA.-linolenicacid istheprecursorfattyacidforthesynthesisofeicosapentaenoicacid(EpA;C20:5n-3)anddocosahexaenoicacid (dHA;C22:6n-3),whichhavebeneficialcardiovascular andalsoanti-inflammatoryproperties(Connor,2000). The beneficial effects of pufA on human health are well known and have been documented by numerousstudies(reviewofWilliams,2000;reviewofriediger et al., 2009). Great numbers of experiments have been madeinordertoincreasepufAcontentinanimalproducts(Leskanickandnoble,1997;Bernardiniet al.,1999; Koubaet al.,2003;raeset al.,2004;Woodetal,2003). rabbit meat is often recommended by nutritionists becauseofitslowlipidandcholesterollevelsandhighcontentofpufAcomparedtoothermeat(dalleZotte,2004). fattyacidcompositionofrabbitmeatisbettercompared to other meat, because rabbit's diet usually contains alfalfawithhighn-3pufAcontent.Withtheadditionof linseedoilinthediettheproportionofn-3pufAinmeat increases and also affects the reduction of the n-6/n-3 pufAratio.Westerndietsaredeficientinn-3pufAand haveexcessiveamountsofn-6pufA.Thisleadstoavery highn-6/n-3pufAratiorangingfrom15/1to20/1.The optimalratioisthoughttobefrom5/1to10/1infavour of the n-6 pufA. According to Simopoulos (2002) the optimalratiobetweenn-6andn-3pufAwouldbe4/1or evenlower.dalBoscoet al.(2004)demonstratedthatthe additionof8%linseedinrabbit'sdietincreasedconcentrationofn-3pufAanddecreasedn-6/n-3pufAratio inrabbitmeat. However,thehighercontentofpufAcouldleadto 120 ahighersusceptibilitytolipidoxidation.Thismightaltertissueoxidation,whichleadstotheformationoffree radicals, lipid peroxides, aldehydes (malondialdehyde (mdA)e.g.)andfurtheroxidationproductswhichhavea negativeeffectonthedieteticvalueoffatandareharmful totheorganism.SuchenrichmentofpufAmightreduce shelf-lifeofmeatproductsanditcanalsonegativelyaffectanimalsandtheirhealth.Inmeatandotheranimal products,oxidationmightreduceitsstability,nutritional andsensoryquality(Grayet al.,1996).Itisthereforevery importantthatwesupplementanimalswithantioxidants inordertopreventlipidoxidationcausedbyfeedinghigh levelsofpufA. Theaimofthisstudywastoinvestigatetheeffectof linseed oil supplementation on performance, fatty acid compositionandoxidativestatusinrabbits.Inorderto determine to what extent we can change the fatty acid compositionofmuscle,adiposetissuesandliverofrabbits, we made an experiment in which we added high proportionoflinseedoiltothecommercialrabbit'sdiet. Inordertoevaluatetheoxidativestress,theplasma,liver andmusclemdAconcentrationsweremeasured. MaTeRIals aND MeThODs All procedures were performed according to current legislation on animal experimentation in Slovenia. AnimalsusedinthisexperimentwererearedandslaughteredatthedepartmentofAnimalScience,Biotechnical faculty(Ljubljana,Slovenia). 2.1 AnImALSAnddIETS TwelvemaleSIKArabbits(63daysold,2284g)were randomlydividedintotwogroupsandassignedintotwo differentdietarytreatmentsfor52days:acontrol(n=4) andalinseeddiet(n=8).Thecontroldietwascommercialdietandthelinseeddietwascommercialdietcontaining9%oflinseedoil,whichwassprayedontopellets. Theexperimentaldietwasprepareddaily.Animalshad freeaccesstofeedandwater(nippledrinkers)andwere housedindividuallyinstandardcages.dietsampleswere takenduringtheexperimentforthepurposeofchemicalanalyses.Thefattyacidcompositionofthedietswas analysedusingagaschromatographicmethodafterthe insitutransesterificationoflipids.Thechemicalandfatty acidcompositionofthedietsarepresentedinTable1. 2.2 ExpErImEnTALprOCEdurEAndSAmpLE COLLECTIOn Eachdayanimalsreceivedweigheddailymealand the residue from the day before was weighed and discarded. Body weights were recorded each week during theexperimentalperiodandjustbeforeslaughter.After 52daysoftreatment,rabbitswereslaughteredat115days of age, by electric stunning and exsanguination. After sacrificing the animals, blood, liver, adipose tissue and legmusclesampleswerecollected.Bloodsamples,forthe purpose of measuring mdA concentration in plasma, werecollectedinto6mlevacuatedtubescontainingEdTAK2anticoagulant(367864,Bd-plymouth,uK).plasma was separated by centrifugation (1000g for 15 min at4 °C),transferredintoEppendorftubesandstoredat -70°Cuntilanalysis.Thewholeliver,partofthelegmuscleandadiposetissueweretaken,weightedandstoredat -70 °Cuntilanalyseswereperformed. 2.3 dETErmInATIOnOffATTyACIdCOmpOSITIOn Thefattyacidcompositionofmuscleandadiposetissuessampleswereanalysedusingagaschromatographic methodaftertheinsitutransesterificationoflipids.Each samplewasanalysedinduplicate.methylestersoffatty acidswerepreparedaccordingtotheprocedureofpark andGoins(1994).Abriefsummaryoftheprocedureis asfollows:frozenmuscleoradiposetissuesampleswere homogenized (Grindomix homogenizer, retsch Gmbh & Co, Haan, Germany) and approximately 0.5­0.7g (muscle) or 0.1g (adipose tissue) of the homogenized samplewasweigheddirectlyinatubewithstopperand mixedwith3ml0.5msodiumhydroxideinmethanol and 0.3 ml methylene chloride. In situ transesterification was performed by heating samples at 90ºC for 10 minintheclosedtube.Aftercooling,3mlof14%boron trifluorideinmethanolwasaddedandheatingat90ºC was continued for 10 min in the closed tube. Samples were cooled, and the fatty acid methyl esters (fAmEs) wereextractedinto1mlhexane.AnalysisoffAmEswas performedbygaschromatographyusinganAgilent6890 seriesgaschromatograph(AgilentTechnologies,Wilmington,dE,uSA)equippedwithanAgilent7683AutomaticLiquidSampler,asplitinjector,aflameionization detectorandafusedsilicacapillarycolumnOmegawax 320(Supelco,uSA).Theinjectionvolumewas1l.The chromatogramswereevaluatedbytheAgilentGCChem Stationsoftware.SeparatedfAmEswereidentifiedbyretentiontime.resultsareexpressedasapercentageofthe totalfattyacids. 2.4 mALOndIALdEHydE(mdA)dETErmInATIOn The methodology of Wong et al. (1987) modified byChirico(1994)andfukunagaet al.(1995)wasused tomeasuretheconcentrationofmdAinbloodplasma using HpLC. A brief summary of the procedure is as follows: 100l of the sample was mixed with 100 l of 0.44mphosphoricacid(H3pO4)and10lof0.2%ethanolic butylhydroxytoluene (BHT) in Eppendorf microcentrifuge tubes, left for 15 minutes, added 300l ethanolandthancentrifuged(15000g,for15minutesat 4ºC).Thesupernatant(350l)wasmixedwith1.5mlof 0.44mH3pO4,0.5mlof0.6%thiobarbituricacid(TBA) and0.9mlofmilliQdeionisedwaterinatubewithstopperandheatedat90ºCfor60minutes.Aftercooling,the sampleswerefilteredthroughmilliporefilters(poresize 0.22m)intoautosamplervials.ThemdAconcentration inliverandmusclesampleswasdeterminedfollowingthe method of vila et al. (2002) with minor modifications. Briefly,frozenliverormusclesampleswerehomogenized (Grindomix homogenizer, retsch Gmbh & Co, Haan, Germany)andapproximately0.3gofthehomogenized sample was mixed with 1.5ml of 2.5% trichloroacetic acid(TCA)inEppendorfmicrocentrifugetubes,leftfor 10minutesandthancentrifuged(15000g,for15minutes at 4ºC). 1 ml of supernatant was mixed with 1.5ml of 0.6%TBAand1mlofmilliQdeionisedwaterinatube with stopper and heated at 90ºC for 60 minutes. After thesampleswerecooled,theywerefilteredthroughmilliporefilters(poresize0.22m)intoautosamplervials. AWatersAlliance2690(Waters,milford,mA)equipped with Waters 474 scanning fluorescence detector was used to determine plasma, liver and muscle mdA. for thepurposeofseparationareversed-phaseHpLCchromatographycolumn(HyperClone5uOdS(C18)120A, 4.6×150 mm 5 micron; phenomenex Inc., uSA) and C18OdSguardcolumn(4mm×30mm;phenomenex Inc.,uSA)wereused.Themobilephaseconsistedof65% 50mmol/lKH2pO4buffer(pH6.9)and35%methanol. Themobilephaseflowratewas1.0ml/min.Theresultsof theanalysiswereevaluatedusingthemillenium32Chromatographymanagerprogram. 2.5 STATISTICALAnALySIS ThedatawereanalysedbytheGeneralLinearmodels(GLm)procedureoftheSAS/STATmodule(SAS8e, 2000;SASInc.,Cary,nC,uSA),takingintoconsiderationthedietastheonlymaineffect.differencesbetween groupsweredeterminedonthebasisofTurkey'smultiple comparisonstest.Theresultsinthetablesarepresented asleastsquaremeans(LSm)±standarddeviation.Ifnot statedotherwise,aleastsignificantdifferenceof0.05was usedtoseparatetreatmentmeans. 3.2 BOdyWEIGHTGAInAnddIETInTAKE There were no significant differences in the initial and final body weight and also in the weight gain (Table2).Somedifferencesamongtreatmentswereobserved infeedintakeandfeedefficiency.Animalsingroupthat had diet enriched with linseed oil showed significantly lowerfeedintake(forabout41g/day)andconsecutively hadsignificantlyhigherfeedefficiency.Thatmeansthat animalswithsupplementeddietconsumedsignificantly (p = 0.0006) less feed (4.44 kg) than control animals (5.82kg)for1kgweightgain.Thesedifferenceswereexpectedbecauseofthemuchhigherfat(energy)content inthelinseeddiet. Table 2: Body weight (BW), weight gain and diet intake Preglednica 2: Telesna masa zivali, prirast in zauzivanje krme Controlgroup Linseedgroup p-value InitialBW(g) finalBW(g) Weightgain(g/day) feedintake(g/day) feedefficiency(%) 2343±129 3963±322 31.8±4.0 183.4±13.0 17.3±1.7 2256±175 3903±202 32.3±3.7 142.4±9.5 22.7±2.0 0.4026 0.6979 0.8232 <0.0001 0.0010 ResUlTs aND DIsCUssION Theanimalsadaptedwelltotheexperimentalconditions.duringtheexperiment,theanimalshadnohealth orotherproblems. 3.1 CHEmICALAndfATTyACIdCOmpOSITIOnOfTHEdIETS ThechemicalandfattyacidcompositionofthedietsarepresentedinTable1.Asexpected,theadditionof linseedoilincreasedtheproportionofcrudefatfrom4.2 to12.2%.Thefattyacidcompositionofthedietsdiffered accordingtotheingredients.Thelinseeddiethadhigher proportionofn-3pufAandtotalpufA,andlowerproportionofsaturatedfattyacids(SfA)andmonounsaturatedfattyacids(mufA)comparedtothecontroldiet. The linseed diet had lower proportion of linoleic acid (C18:2 n-6) and higher proportion of -linolenic acid (C18:3 n-3). Therefore, the linseed diet had lower n6/n-3ratio(0.63vs.6.30)comparedtothecontroldiet. Table 1: Chemical and fatty acid composition of the diets Preglednica 1: Kemijska in mascobnokislinska sestava krme Controldiet Chemicalanalysis(%) Crudeprotein Crudefat Crudefibre Crudeash C16:0 C18:0 C18:1 C18:2n-6 C18:3n-3 SfA mufA pufA n-3pufA n-6pufA n-6/n-3pufA 16.4 4.2 15.0 9.4 14.31 3.25 21.13 50.26 7.93 19.64 22.05 58.32 7.99 50.33 6.30 14.9 12.2 13.6 8.5 7.85 3.56 19.37 25.85 40.74 12.60 20.81 66.59 40.74 25.85 0.63 Linseeddiet mainfattyacids(%ofthetotalfattyacids) Our results are in general agreement with other studiesshowingthatdietshighinn-3pufAdidnotsignificantly influence productive performances in rabbits (Bernardini et al., 1999; dal Bosco et al., 2004; Kouba et al., 2008; Bianchi et al., 2009). differences occurred indietintakeanddietefficiencybetweenourandtheirs results. These differences were expected because of the higherfatcontentinthedietinthelinseedgroupinour experiment, while other had isoenergetic diets. On the contrary,Bianchiet al.(2006)foundoutthat8%oflinseed supplementation determined a lower daily weight gainaswellasalowerfinalliveweightofrabbitscomparedtothegroupwithoutlinseedaddition. 3.3 fATTyACIdCOmpOSITIOnOfTISSuES AndOxIdATIvESTATuSOfrABBITS dietsupplementedwithlinseedoilledtoahigher proportionoftotalpufA,duetoareductionoftotalSfA andmufAlevelsinmuscle,adiposetissueandliver.As expected,thelinseedoiladditionsignificantlyincreased (p<0.0001)proportionofthe-linolenicacidandsome othern-3pufAinalltissues(Table3).Thelongchain n-3 pufA content was at much higher level in muscle and liver compared to adipose tissue. The main differencesbetweentissueswereobtainedinproportionofli- SfA ­ saturated fatty acids; mufA ­ monounsaturated fatty acids; pufA­polyunsaturatedfattyacids Table 3: Fatty acid composition of muscle, adipose tissue and liver (% of total fatty acids) Preglednica 3: Mascobnokislinska sestava misice, mascobnega tkiva in jeter (% od vseh mascobnih kislin) muscle Control C16:0 C16:1n-7 C18:0 C18:1 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C22:5n-3 C22:6n-3 SfA mufA pufA n-3pufA n-6pufA n-6/n-3 21.86a 3.46a 6.90a 23.48 Adiposetissue Linseed 15.04b 1.53b 6.62a 21.74 Liver Linseed 12.31b 0.93b 4.79b 23.23 Control 21.96a 2.68a 5.43a 24.98 Control 21.25a 1.40a 16.28a 17.66 Linseed 12.20b 0.22b 22.85b 11.01b 32.88b 9.53b 5.37a 0.32b 1.27b 0.49b 36.66b 11.55b 51.78b 11.92b 39.85a 3.41b 28.98a 3.30a 3.49 25.77b 20.17b 2.45 34.47a 4.72a 0.15 27.41b 28.02b 0.09 29.16a 1.55a 4.96 0.08a 0.57a 0.10 27.83 0.30b 1.37b 0.16 23.77 0.01a 0.05a 0.01 0.03b 0.11b 0.01 24.72 0.04a 0.29a 0.09 33.64a 24.86b 31.35a 28.65 19.34b 40.22a 20.03 38.53a 4.17a 34.32 51.37b 22.28b 29.06 40.00a 4.80a 35.14 55.93b 28.19b 27.70 39.75a 2.16a 37.54 8.22a 1.31b 7.31a 0.98b 17.43a a,b valueswithdifferentsubscriptswithineachtissuearesignificantlydifferent(p<0.05);SfA­saturatedfattyacids;mufA­monounsaturated fattyacids;pufA­polyunsaturatedfattyacids noleicacid(n-6pufA).Theproportionoflinoleicacid significantlydecreasedinmuscleandadiposetissuebut increasedinliver.Thedifferencesalsooccurredinproportionof-linolenicacid,whichincreasedinalltissues but it was at much higher level in muscle and adipose tissuecomparedtotheliver.despitethesedifferencesthe highercontentofn-3pufAinlinseeddietledtoasignificantly(p < 0.0001)lower n-6/n-3pufAratioinall tissues. Our results are in general agreement with other studies. Bernardini et al. (1999) found that addition of 160ggroundlinseed/kgdietsignificantlyincreasedthe proportionof-linolenicacidanddecreasedthen-6/n-3 pufAratiocomparedtothestandardandfishoildiet(60 gfishoil/kgdiet).Similarresultswereobtainedwhenapproximately30gofextrudedlinseed/kgdietwasadded. Thelinseeddietincreasedthelevelof-linolenicacidin muscle,perirenalfat,andinrawandcookedmeat.Linseed addition also increased the long chain n-3 pufA contentsinmeatandconsecutivelycausedasignificant decreaseinthen-6/n-3pufAratio(Koubaet al.,2008). decreased n-6/n-3 pufA ratio in rabbit's muscle, adipose tissues and liver can provide the "functional food"forhumanconsumption.Intakeofsuchfoodcontributestoabetterratioofn-6ton-3pufAinthehuman dietandthuscontributestoimprovedhumanhealth. ThemdAconcentrationsinplasma,liverandmuscleweresignificantlyinfluencedbythediet(Table4).The linseedgroup,whichwassupplementedwithlinseedoil highinpufA,showedsignificantlyhigherlevelsofmdA in plasma, liver and muscle compared to the control grouThat showed that supplementing diet with linseed oil increased the oxidation. from the nutritional point of view these results are unwanted, because of reduced shelf-lifeofmeatproductsandnegativeeffectonanimal health. Table 4: MDA concentration in plasma, liver and muscle Preglednica 4: Koncentracija MDA v plazmi, jetrih in misici Control plasmamdA(nmol/ml) LivermdA(nmol/g) musclemdA(nmol/g) 0.26 0.47 0.40 Linseed 0.36 1.09 1.24 p-value 0.0059 0.0319 0.0015 Oxidativestressisdefinedasapersistentimbalance betweenantioxidantsandpro-oxidantsinfavourofthe latter. namely, oxidation can be reduced if we provide enough antioxidants to prevent the formation of free radicals. factors affecting the extent of oxidation are: pufA content in the membranes, the amount of free radicals and the levels of antioxidants which can be of endogenousorexogenousorigin(Breneset al.,2008).We couldpreventoxidationusingdifferentdietarystrategies. vitaminEisthemostfrequentlyusedantioxidant.There are some studies dealing with feeding and nutritional valueofrabbit'smeat,butthereisalackofthemdealing withrabbitplasmaandothertissuesmoreinvolvedinthe animal'smetabolism,suchastheliver,atthesametime (Treset al.,2009). Bianchiet al.(2006)indicatedthattheadditionof 35%alfalfawith200mgvitaminE/kgfeedincreasedthe proportionof-linolenicacidwithouthavinganegative impactontheoxidativestabilityofmeat.Simultaneous additionof8%linseedhasastronginfluenceonthefatty acidcompositionofmeat,withincreasingproportionof pufA, especially -linolenic acid, which has a positive effectontheratioofn-6andn-3pufA.Similarresults were also demonstrated in a recent study about the effectsofdifferentinclusionratesofwholelinseedinthe diettothequalityofmeat,wheretheproportionofpufA also increased at the expense of SfA (Bianchi et al., 2009).Theyfoundthatinclusionof3%linseedindiets forgrowingrabbitsprovidedfavourablefattyacidcomposition and also meat quality. dal Bosco et al. (2004) indicated that the addition of 8% linseed with 200 mg ofvitaminE/kgfeedincreasedtheconcentrationofn-3 pufA and narrowed the ratio of n-6 and n-3 pufA in rabbitswithoutnegativeeffectontheoxidativestability ofmeat.Itisofnotethattheseauthorsusedmuchhigher quantities of vitamin E when linseed oil was added inthedietcomparedtothecontroldiet(200mg/kgvs. 50mg/kg).petracciet al.(2009)foundthatthedietary inclusionfrom3to6%oflinseedtogetherwith200mg ofvitaminE/kgmightbeconsideredasawaytoincrease theproportionof-linolenicacidintherabbitmeatand alsoensuresufficientoxidativestabilityoftheproduct. Theadditionof3%linseedoilinthediethadabeneficialeffectonfattyacidcompositionofrabbitmeatas it reduced the proportion of total SfA and the level of cholesterol and increased the proportion of n-3 pufA. TheadditionofvitaminEpreventedoxidationprocesses (Bielanski and Kowalska, 2008). Zsédely et al. (2008) studiedtheeffectofsunfloweroilandlinseedoilincombinationwithvariousconcentrationsofvitaminEonfattyacidcompositionandoxidativestabilityofrabbitmeat. It was expected that addition of oil would significantly increase the proportion of pufA due to the reduction of SfA and mufA and decrease the ratio between n-6 andn-3pufA.Inaddition,vitaminEimprovedoxidativestabilitycomparedtothegroupswithoutvitaminE addition. plasma and liver fatty acids composition also reflectedfeed'sfattyacidprofile(Treset al.,2009). 124 CONClUsIONs nowadays consumers are more concerned about theirhealthandthenutritionalvalueoftheirfood.Therefore, interest in obtaining animal products with better nutritionalvaluehasincreased.Ourresultsrepresentto what extent we can improve fatty acid composition of rabbitstissueswithlinseedoilsupplementation.Tosummarize,theoverallfattyacidcompositionwasinfluenced bydietarylinseedoilinclusionwithoutsignificanteffect onproductiveperformance.ThelinseedoilcausedalowercontentoftotalSfAandmufAandahighercontent ofpufA.Linseedoilalsoreducedn-6/n-3pufAratioin alltissues.However,thehighercontentofpufAintissuesledtohighersusceptibilitytolipidoxidation.Such enrichmentmightreducetheshelf-lifeofmeatproducts anditcanalsonegativelyaffectanimalsandtheirhealth. Therefore,furtherresearchisneededtoprotectanimals andtheirproductsfromlipidoxidationbyaddingvariousnaturalantioxidantstothediet.

Journal

Acta Agriculturae Slovenicade Gruyter

Published: Dec 1, 2011

References