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d-Old Age

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d-Old Age

    d-Old Age

    AgriculturalSciencesinChina

    2011,1o(2):304311

    Availableonlineatwwwsciencedirect.com

    ScienceDirectFebruary2011

    NonphytatePhosphorusRequirementandEfficacyofaGenetically

    EngineeredYeastPhytaseforYellowBroilersat22--to42--d.-OldAge

    JIANGZong-yong,ZHOUGuilian,LINYingcai,JIANGShouqun,LIDe

    zhong,XIPeng-binandZHANG

    Han-xing

    KeyLaboratoryofAnimalNutritionandFeedScience(SouthChina).MinistryAgricult"reInstn"teofAnimalScienceGuangdong

    AcademyofAgriculturalSciencesGuangzhou510640,P.R.China

    Anexperimentwasconductedtoinvestigatetherequirementofnonphytatephosphorus(nPP)andefficacyofagenetically

    engineeredyeastphytase(PHYA)forLingnanyellowbroilersfrom22-to42-d

    oldage.Atotalof13201-d-oldmalechicks

    wererandomlydividedinto11dietarytreatmentgroups,whichconsistedof4replicatefloorpenswith30birdsperpen.

    Thecontrolgroup(treatment1)wasfedwithbasaldietofnPP0.08%withoutdicalciumphosphateorphytase

    supplementation.DietarylevelsofnPPwere0.16,0.24,0.32,0.40,0.48,and0.56%,respectively,fortreatments2to7,

    throughadditionofdicalciumphosphate(chemistrygrade)tothebasaldiet.Dietsoftreatments8to11weresupplemented

    withPHYAat200,400and600Ukg

    ,acommercialphytaseproduct(PHYB)at400Ukg'1level,respectively.Thebirds

inO.32

    0.56%nPPgroupsgainedmorethanthoseoftheothergroupsfP<0.05).ThenPPsupplementationsignificantly

    improvedfeedintakeP<0.05).Thefeedgainratiowassignificantlydecreasedby0.40%nPPdietcomparedtothecontrol

    birdsrJP<O.05).Thelevelof0.48%nPPwasrequiredforoptimumtibiadevelopment.TheadditionsofPHYAat400and

    600Ukg.ilevelandPHYBallsignificantlyimprovedADG(

    <0.05),ADFI(P<0.o5),anddrydefattedtibiaweight(<0.05).

    Similarly,thepercentageoftibiaashwasincreasedby600Ukg.PHYAsupplementationrP&lt;0.05).TherequirementofnPP

    formaximalADGandhighestpercentagetibiaashbothwasO.40%.ThephosphorusequivalencyvalueofPHYAwas

    estimatedas685Ukg.1formaleyellowbroilersof22?to42-d?oldage. Keywords:nonphytatephosphorus,growth,tibiaash,geneticallyengineeredyeastphytase,phosphorusequivalency

    value,yellowbroilers

    lNTRODU(TION

    Nutrientrequirementsareimportanttechnologicalindi

    cesinmodempoultryindustryandmajorbasisforfeed

    formulation.Phosphorusisnotonlyacomponentof

    bonebutalsoisanessentialmineralelementthatplays

    avitalroleinvariousphysiologicalprocesses(Soares

    1995).Inrecentyears.therehavebeenseveralpub-

    lishedresearchreportsaboutmetabolizableenergy,crude

    proteinandaminoacidsrequirementsofyellowbroil

    ers(Cheneta1.1998;Jiangeta1.2003;Xieta1.2008;

    Zhengeta1.2008;Zhoueta1.2004).However.there

    arefewreportsaboutphosphorusrequirementofyel

lowbroilersandtheonlyresultiSaboutavailablephos

    phorusrequirementofShiqiyellowbroilersreported byChengeta1.(2008).Manyresearchesindicated thatthebestwayisaddingexogenousphytasetophos

    ThispaperistranslatedfromitsChineseversioninScientiaAgriculturaSinica-

    CorrespondenceJIANGZong-yong,Professor,Tel:+86

    20-87596262,Fax:+86?20?87503358,E-mail:jiangz38@hotmailcorn

    @2011.CAASAllfightsreselvadPublishedbyE帕?

    doi:101016/S1671-2927(11)6OO08-4

    NonphytatePh0sphorusRequirementan

    

    dEfficacyofaGeneticallyEngin

    

    eeredYeastPhytaseforYellowBroilers305

    phorusavailabilityinplantfeed.Thegeneencoding phytasewithhighspecificactivitywasusedtoimprove thefermentationpotencyofphytaseinrecombinanthost anddecreasetheproductioncost(Yaoela1.1998; Wangeta1.2004).Aphytasegenesequenceandits useinichiapastoriyeastbygeneengineeringtech.

    nologyhasbeenpatented(Cheneta1.2000).The growthperformance,feedefficacy,nutrient bloavallabtlmes,andbonetraitsareofwidevariation betweenyellowbroilerstrains.Therefore,thenPPre- quirementshoulddiflferfromeachother.Becausethe requirementsofphosphorusinpoultryarerelatively uncertain,thedifferenceofphosphorusconcentration andbioavailabilityoffeedingredientsisveryimportant (vanderKliseta1.1996;Fangeta1.2007),anover- doseofphosphorusoftenwasgenerallysuppliedin

    broilerdiets.Thedietaryadditionoffeedphosphates notonlyincreasesthefeedandproductioncost,but mayalsoleadtoanincreaseofsolublePinthelitter resultinginthepotentialforwatercontaminationfrom excessPinthesoil.Themaximumutilizationoffeed phosphorusbypoultrywiththeadditionofexogenous dietaryphytaseshouldhelpinreducingtheamountof dietaryNPPsupplementation.Ithasbeenwelldocu- mentedthatmicrobialphytaseiseffectiveinreleasinga significantportionofthephytatePpresentinmaizeand soyabeanmealandmakingitavailabletobroilers,but theamountofNPPthatcanbesparedwithphytase dependsonthesourcesandcharacteristicsofphytase (Wysseta1.1999).Theobjectiveofourresearchwas toevaluatetherequirementofnPPandefficacyofa geneticallyengineeredyeastphytaseinperformanceand tibiacharacteristicsinmaleLingnanyellowbroilers duringtheperiodof22to42doldage,withtheaimof

    providingatheoreticalbasisforthefeedformulation andphytaseutilizationofChineseyellowbroilers. MATERIALSANDMETHODS

    Phytase

    ThephytaseAproductusedinthisstu'lywasderived fromthechimeraofAspergillusnigerphytaseandAs

    pergillusfumigatusphytase,wasproducedin GuangdongPublicLaboratory"ofAnimalBreedingand Nutrition.China.Thechimerawerecreatedbystep wisesubstitutionsofDNAfragmentsencodingregmns 47,178237and338381fromAspergillusnigerNRRL

    3135phytasegenefortheircounter'.partsinAspergil_?

    lusfumigatusphytasegene,expressedinPichiatpastoris, andthenpurified.Theenzymeactivityofthephytase (powder)producedfromthisexperimentwas5346Ug' afterspraydrying.ThephytaseB(BASF,Mt.Olive, NJ)isarecombinantenzymefromAspergillusniger (theactivity5000U).

    Chicksandhousing

    Atotalof132022doldmalechicks(Lingnanyellow

    broiler,aChinesequalitymeattypechicken,obtained

    fromInstituteofAnimalScience,GuangdongAcad- emyofAgriculturalSciences,China)wererandomly allottedtooneOfl1dietarytreatments,whichcon

    sistedof6replicatefloorpenswith3'0birdsperpen. Eachpenwas1.3mx3.5mandthefloorwascovered

    withwoodshaving.Allthebirdswerefedthesame conventionaldietcontaining0.45%nPPand1.0%cal

    ciumfrom1to21dofage.Birdswereallowedto consumewaterandmasheddietsadlibtum.Animal carewasfollowedasperstandardpracticesofLingnan yellowbroilersrecommendedbytheInstituteOfAni

    malScience,GuangdongAcademyofAgricultural Sciences,China.

    Experimentaldiets

    Thetrialwasdesignedwithasinglefactorialarran? gement.Al1treatmentshadthesameconcentrationsof allnutrientsexceptforphosphorusorphytase.Thecon

    trolgroup(treatmentl1wasfedwiththebasaldietwith

    outdicalciumphosphateorphytasesupplementation. DietaryconcentrationsofnPPwere0.16,0.24,O_32,O.4O, O.48,and0.56%,respectively,fortreatments2,3,4,5,

    6,and7,throughadditionofdicaleiumphosphate (chemistrygrade)tothebasaldietcontaining0.08%nPP. PhytaseAat200,400and600Ukg.levelweresupplied intobasaldietrespectivelyfortreatments8,9,and10. PhytaseBof600Ukg'wasaddedintobasaldietfor treatmentl1.Nutrientlevelsofthebasaldiet(Table1, werebasedonNRCrI994)recommendations.

    @2011,CAASAlldghtsre3ervedPublishedbyElsevierLtd 306J1ANGZongyongetal

    Table1Ingredientandcompositionofthebasaldietforthegrowerbroilers(fedbasis)

    IngredientRatiof%1Nutrient1eve1Content

    Corn6333Metabolizableenergy(MJk)12.5O

    Soybeanmeal30.30Crudeprotein(%)19.O8

    Soybeanoil2.5OCalciumr%)270.90

    Limestone2.3OTotalphosphorusf%0.38

    Salt025Nonphytatephosphorusr%10.08

    DLmethionine012Lysine(%)1.O0

    Vitaminmineralpremix.)1.20Methioninef%1O.40 Iota1l0000Meth;onine+cvsteinef%1070

    Threonine(%)0.77

    Tryptophan(%)0.25

    "Suppliedperkilogramofdiet:vitaminA,12500IUkg'.;vitaminD3,2750IUkg'';vitaminE,0

    .019gkg'.;vitaminK3,O.005gkg..;thiamine,0.0025gkg';riboflavin

    O0075gkg;pyridoxine,0005gkg';cobalamin,O025mkg.';niacin,0.05gkg..;calciumpantot

    henate,O015gkg';folicacid,O.00125gkg;biotin,O.05mgkg';Cu, 8mgkg';I,035mgkg.';Fe,80mgkg';Mn,60mgkg';Zn,40mgkg;Se,015mgkg

    .)Valuesaremeansofanalysesinduplicate

    Measurements

    A11birdswereweighedandfeedconsumptionwas measuredperpenond22oftheexperimentandat42

    d,whentheexperimentwasended.Mortalitywas checkeddailyanddeadbirdswereweighedtoadjust feedconversionratio.At42dofage.rateoflegabnor- malitywascheckedperpen.TWobirdsperpen.rep

    resentativeofthemeanbodyweight(BW),werese. lectedandslaughteredbyseveringthecarotidartery andiugularveinafter16.hfeedwithdrawa1.Theright tibiawasremoved.weighedandstoredinanairtight plasticbagat20.Cforboneashdetermination.The righttibiasamplesofthebirdswithinapenwerepooled afterremovingthesofttissueandthenfatofthesamples wasextractedbySoxhletpetroleum.etherfor4hand driedtoaconstantweightatl00.C.Pooledtibia samplesweredriedtoashinafumaceat550.Cfor4 h.Theashfromtibiasampleswassolubilizedwith nitricandperchloricacidandthencalciumandphos

    phoruscontentwereanalysedbyspectrophotometer andatomicabsorptionspectrophotometer,respectively. Proximateanalysesoffeedingredientswereperformed byfollowingtheproceduresofA0ACr1990). Statisticalanalysis

    Penservedastheexperimentalunitforgrowth performance.whereasindividualchickwasusedas experimentalunitforbonecomposition.Statistica1 analysiswasperformedusingthegenerallinearmodels procedureofSAS(SASInstitute1996).Probability values<0.05weretakentoindicatestatistical significance.Alld~/tawereexpressedasmeans~SE. Whensignificantdifferencesamongorbetweentreat

    mentmeanswerefound,meanswereseparatedusing

    repeatedt-testsusingprobabilitiesgeneratedbytheLS meansoption.Followingtheseanalyses,nonlinearre

    gressionanalysiswasconductedusingthePROCNLIN procedureoffSASInstitute1996)toestimatethe breakppintsofselectedvariables,incorporatingtheSAS macroofRobbinseta1.r1979).Variablesthatwerefit bytheregressionanalysisincludedBWgainandbone ashcontent.Nonlinearregressionanalysiswascon

    ductedusingthePROCNLINprocedureofSAS(SAS Institute19961toestimatethephosphorusequivalency valueofrecombinantyeastphytase.Theregression mode1wasfollowedbythepredictionequationY=Bn (1.B,xe),whereYisaveragedailygain(ADG)of

    chicksandXistheconcentrationofnPPorphytasein unitsperkilogramofdiet,B0,B1,andB2wereobtained bymethoddescribedbyMarquardtr1963).

    RESULTS

    Growthperformance

    AsshowninTable2.ADGofchicksincreasedwith increasingdietarynPPlevelfrom0.08to0.32%.Chicks fedwilh0-32.0.40,0.48,and0.56%nPPdietgainedmore thanthoseofthecontrolsandbirdsfedwith0.16%nPP dietrP<0.05).Chicksfedwith0.16%nPPdietalso hadsignificantlyhighADGthanthecontrolsr<0.05).

    ADFIOfalltreatmentswassignificantlyhigherthan thatofthecontrolsf<0.05).Feedtogainratioofthe controlbirdswashigherthanthatofchicksfedwith0.40% nPPdietfP<0.05).Nolegabnormalitywasobserved @2011CAASAlldghtsreservedPublishedbyElsevierLtd NonphytatePhosphorusRequirementandEfficacyofaGeneticallyEng

    ineeredYeastPhytas

    

    eforYellowBroilers3O7

    Meanswithinarowwithoutcommonsubscriptlettersaresignificantlydifferent(P<0.05)

    Valueswiththesamesuperscriptlettersarenotsignificantlydifferent(P>O?05)

    'Caluesaremeans~SEThesameasbelow

    inanytreatments.

    Asshownin11able3.weightgainandfeedintakeof birdssignificantlyimprovedbysupplementationof phytaseAorBrP<0.05).ADGofchicksfedwith400 and600UkgphytaseAdietswerehigherthanthatof thecontrolsandchickswith200UkgphytaseA

    fP<0.05).Chicksfedwith400UkgphytaseBalso gainedmorethanthecontrols(P<O.05).Chicksfed with400and600UkgphytaseAand400Ukg'phytase

    BdietshadgreaterADFIthanthecontrolbirdsfP<0.05). Thefeedefncienciesofchickswerenotinflencedby thephytasesourcesandlevels.

    Table3Effectofphytaseindietsongrowthperformanceofmalebroilersfrom22--to42?-d-ol

    dage

    Bonemeasurements

    AsshowninTable4,tibiaashcontentsofchickswere significantlyaffectedbydietarynPPcOncentratiOns. Thedefattedtibiaweight,percentagephosphorusof drydefattedtibiaandphosphorusoftibiaashincreased withdietarynPPc0ncentrationfP<O.05).Themaxi

    mumdefattedtibiaweight,percentagephosphorusof tibiaashwereobservedinchicksfedwith0.48%nPP diet.ChicksfedwithnPPaddeddietshadsignificantly

    higherpercentageofcalciumofdrydefattedtibiaand tibiaashcontentsthanthoseofthecontrolsfP<0.05). Nodifie:renceofcalciumcontentsoftibiaashwasob. servedamonganygroupofchicks.Themeanratioof calciumtophosphorusofdrydefattedtibiawas 2.025:1forallbirds.

    Table4Effectofnonphytatephosphoruslevelsontibiaash(drydefatted)contentofmalebroil

    ersfrom22to42doldage

    TheeffectsofphytaseontibiacharacteristicsOf chicksarepresentedinTable5.Thepercentagecalcium ofdrydefattedtibia,phosphorusofdrydefattedtibia andphosphorusoftibiaashwereelevatedbysupple. mentalphytaseAorphytaseBr<0.05).Thedefatted

    tibiaweightwasincreasedby400or600Ukgphytase

    Aor400Ukg.'phytaseBsupplementationfP<0.05). Percentagetibiaashcontentsincreasedbysupplemental 600UkgphytaseAfP<0.05).Themeanratioofcal

    ciumtophosphorusofdrydefattedtibiawas2.02:lfor allbirds.Chickswith600UkgphytaseAexhibitedsimi.

    1artibiacharacteristicswiththosefedwithphytaseB. @2011,CAASAllhtsreservedPublishedbyElsevierLtd Regressionanalysis

    Resultsofthenonlinearregressionanalysistoestimate thenPPrequirementforoptimumADGandtibiaashare showninle6.Inthepresenceofdua1.slopeinflection regression.theRvalueswere0.6870and0.5080forADG andtibiaash,respectively,morethanthoseoftheother tworegressionequations.So,therequirementofnPPfor maximalADGandthehighestpercentagetibiaashwas

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