Efficacy and phosphorus equivalency values of two bacterial phytases (Escherichia coli and Citrobacter braakii) allow the partial reduction of dicalcium phosphate added to the diets of broiler chickens from 1 to 21 days of age

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ContentslistsavailableatScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Efﬁcacy

and

phosphorus

equivalency

values

of

two

bacterial

phytases

(Escherichia

coli

and

Citrobacter

braakii)

allow

the

partial

reduction

of

dicalcium

phosphate

added

to

the

diets

of

broiler

chickens

from

1 to

21 days

of

age

V. Ribeiro

Jr.

a,∗

_,

_S.C.

_Salguero

a

_,

_G.

_Gomes

b

_,

_V.R.S.M.

_Barros

a

_,

_D.L.

_Silva

a

_,

S.L.T.

Barreto

a

_,

_H.S.

_Rostagno

a

_,

_M.I.

_Hannas

a

_,

_L.F.T.

_Albino

a

a_Department_of_Animal_Science,_Universidade_Federal_de_Vic¸_osa,_MG,_Brazil b_AB_Vista_Feed_Ingredients,_Marlborough,_Wilts,_UK

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received25May2016 Receivedinrevisedform 10September2016 Accepted15September2016 Keywords: Efﬁcacy Equivalency Phosphorus Broilerchickens

a

b

s

t

r

a

c

t

Theobjectiveofthisstudywastodeterminetheefﬁcacyandthephosphorus

equiva-lencyoftwobacterialphytases(Citrobacterbraakii-derivedphytaseorgenetically-modiﬁed

Escherichiacoliphytase)inthedietsofbroilerchickensfrom1to21daysofage.Atotalof

2100malebroilerchickenswererandomlydistributedin10treatmentswith10replicates

of21chickseach.Abasaldietwasformulatedcontaining1.8gperkgofnon-phytate

phos-phorusand9gperkgoftotalcalcium(T1).Fortreatments2–4,dicalciumphosphatewas

addedtogive0.9,1.8,or2.7gperkgofadditionalinorganicphosphorus.Thetreatments

from5to7received500,1000,or2000phytaseunitsofCitrobacterbraakii(FYT)perkg

whilethetreatmentsfrom8to10received250,500,or1000phytaseunitsofEscherichiacoli

(FTU)perkg.Increasinglevelsofinorganicphosphorusfromdicalciumphosphateat0.9,

1.8,and2.7gperkgimproved(P<0.05)feedintakeby52,71,and80%;weightgainby41,

75,and32%;tibiaashweightby64,156,and185%;andtibiaphosphorusby110,323,and

378%,respectively.IncreasinglevelsofCitrobacterbraakii-derivedphytaseat500,1000,

and2000FYTperkgcontributedtoasigniﬁcantimprovement(P<0.05)of52,55,and65%

forfeedintake;37,56,and65%forweightgain;52,87,and133%fortibiaashweight;and

96,173,and273%fortibiaphosphorus,respectively.IncreasinglevelsofEscherichia

coli-derivedphytaseat250to500,and1000FTUperkgincreased(P<0.05)feedintakeby50,

61,and70%;weightgainby49,60,and76%;tibiaashweightby80,103,and164%;and

tibiaphosphorusby128,198,and330%.Linearregressionequations(P<0.05)wereused

toestimatephosphorusequivalencyvaluesofthetwophytases.TheCitrobacter

braakii-andEscherichiacoli-derivedphytasescanbeusedinthedietsofbroilerchickensfrom1

to21daysofagetopartiallyreducetheadditionofdicalciumphosphateasaphosphorus

source.Thesupplementationof500,1000,and2000phytaseunitsofCitrobacterbraakiiper

kgweredeterminedtobeequivalenttotheaverageadditionof0.625,1.091,and2.024g

ofinorganicphosphorusfromdicalciumphosphateperkginbroilerdiets,respectively.

Thesupplementationof250,500,and1000phytaseunitsofEscherichiacoliperkgwere

Abbreviations:Ca,calcium;CBP,Citrobacterbraakii-derivedphytase;ECP,Escherichiacoli-derivedphytase;FI,feedintake;F:G,feed-to-gainratio;WG, weightgain;P,phosphorus;iP,inorganicphosphorus;DCP,dicalciumphosphate;nPP,non-phytatephosphorus;FYT,phytaseunitsofCitrobacterbraakii; FTU,phytaseunitsofEscherichiacoli.

∗ Correspondingauthor.

E-mailaddresses:[email protected],[email protected](V.RibeiroJr.). http://dx.doi.org/10.1016/j.anifeedsci.2016.09.008

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determinedtobeequivalenttotheaverageadditionof0.763,1.307,and2.395gofinorganic

phosphorusfromdicalciumphosphateperkginbroilerdiets,respectively.

1. Introduction

Inpoultryproduction,inorganicphosphorussourcesasdicalciumphosphate(DCP)areaddedtothefeedtomeetthe dietaryphosphorus(P)requirements,butthispracticeincreasesthecostoffeedingaswellasthePexcretion,which con-tributestoenvironmentalissuessuchaseutrophicationinaquaticecosystems(Smithetal.,1999;Waldroup,1999;Yan etal.,2003).

Therefore,thedevelopmentofexogenousphytaseisoneofthemostimportantdiscoveriesforpoultrynutritioninthe pastfewdecades,astheyallowforabetteruseofPinnon-ruminantanimalsthatconsumedietscontainingvegetalproducts (Cromwell,2009).Also,theuseofPhytaseinanimaldietsiscurrentlywidespreadandrepresentsoneofthemosteconomic waysofmeetingpartofthedietaryphosphorusrequirements(SelleandRavindran,2007).

ThereareseveraltypesofPhytasethatarecommerciallyavailable;someofthosearefungal-derivedPhytasesuchas fromAspergillusﬁcumandAspergillusniger,whicharegenerallytype3Phytase(EC3.1.3.8)astheinitialreleaseofphosphate radicalsispreferredintheC3positionofthephytate(SelleandRavindran,2007).Othercommercialphytasearederived fromthefungiPeniphoralyciiorfromthebacteriaEscherichiacoli,whichbeginthephytatedegradationatposition6(EC 3.1.3.26)ofthemyo-inositolhexaphosphate(BedfordandPartridge,2010).Moreover,phytasefromdifferentsourcesmay havedifferentcharacteristics,suchasapHfortheoptimalenzymaticactivity,resistancetodegradationintheguttract,and thermalstability(Onyangoetal.,2004).ThesecharacteristicscanaffectthePrelease,andstudiestodeterminetheefﬁcacy ofnewphytaseshouldbeongoing.

ThereisanEscherichiacoli6-phytase(phytase;EC3.1.3.26)thatisproducedbyageneticallymodifiedstrainofTrichoderma reseiiwhoseefficacyandequivalencyrelativetoPfromdicalciumphosphateshouldbeevaluatedbasedonthemethodology describedbypreliminarystudies(Adedokunetal.,2004;Jendzaetal.,2006;Adeola,2010).Additionally,thereisanew 6-phytasethatisproducedbyastrainofAspergillusoryzaeandexpresses2syntheticgenesthatbothmimicaphytasegene fromastrainofCitrobacterbraakii,andpreviousstudieswereconductedtoevaluateitsefficacy(Cowiesonetal.,2014) anditsequivalencyrelativetoPfromdicalciumphosphate(Vieiraetal.,2015).However,thosepaststudiesjustoffered experimentaldietstobirdsfrom8to21daysofage.Additionally,theyevaluatedtwoorlesslevelsofphytaseunits(500or 1000FYT/kg).Thus,studiesevaluatingtheefficacyofbothenzymeswithmoreanddifferentlevelsofphytaseunitsaswell astheirequivalencyrelativetoPfromDCPinbirdsfedtheexperimentaldietsfrom1to21daysofagearestillneeded.

TheobjectiveofthisstudywastodeterminetheefﬁcacyaswellastheequivalencyvaluesrelativetoPfromdicalcium phosphateofanewCitrobacterbraakii-derivedphytase(expressedinA.oryzae)andagenetically-modiﬁedEscherichiacoli phytase(expressedinTrichodermareseii)topartiallyreducetheadditionofdicalciumphosphateinthedietsofbroiler chickensfrom1to21daysofage.

2. Materialsandmethods

TheexperimentwasconductedattheExperimentalPoultryFarmoftheAnimalScienceDepartment,Vic¸osa,Brazil.Animal careproceduresthroughoutthestudyfollowedprotocolsapprovedbytheInstitutionalAnimalCareandUseCommittee (IACUC)guidelinesattheUFVnumber52/2013.

2.1. Birdsandexperimentaldesign

Twothousandandonehundred1-d-oldmaleCobb500broilerchickenswithaninitialweightof40Gwereusedin a21-dexperimenttoinvestigatetheeffectivenessofaCitrobacterbraakii-derivedphytase(CBP)expressedinA.oryzae, whichis availableasa commercialproductunderthenameofRonozyme® _HiPhos_(Ronozyme_HiPhos_GT,_Novozymes

A/S,Bagavaerd,Denmark),andamodiﬁedEscherichiacoli-derivedphytase(ECP)expressedinTrichodermareseii,whichis availableasacommercialproductunderthenameofQuantum®_Blue_(AB_Vista_Feed_Ingredients,_Marlborough,_Wilts,_UK).

Birdswererandomlydistributedinto10treatmentswith10pensof21chickseach.Eachpencontaining21chickswas consideredasanexperimentalunitandconsistedofoneboxwithaconcreteﬂoor,withdimensionsof1.25×1.80manda totalof2.25m2_._The_animals_were_handled_in_masonry_sheds_that_were₃_m_high_with_cement_asbestos_shingles,_low_walls_of

50cm,andahalfinchscreen(1”/2)thatwasadaptedforanimalexperimentation.Poultrylitterconsistingofnewsawdust wasutilized.Birdswereofferedfeedandwateradlibitum.

2.2. Dietsandtreatments

AP-lowcorn-andsoybean-basedbasaldiet(Treatment1)with1.8gperkgofnon-phytatephosphorus(nPP)and9.0gper kgoftotalcalcium(Ca)wasformulated,whilecontainingalloftheothernecessarynutrientstomeetthebirds’requirements accordingtoRostagnoetal.(2011)aslistedinTable1.

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Table1

Ingredientsandnutrientcompositionofthediets(g/kgdietas-fedbasis).

Ingredients Basaldiet Dicalciumphosphate-addeddiets

Groundcorn 565.6 565.6 565.6 565.6

Soybeanmeal,46% 324.1 324.1 324.1 324.1

Cornglutenmeal,60% 40.0 40.0 40.0 40.0

Soybeanoil 26.3 26.3 26.3 26.3 Commonsalt 4.9 4.9 4.9 4.9 dl-Methionine,99% 2.8 2.8 2.8 2.8 l-LysineHCl,79% 3.3 3.3 3.3 3.3 l-Threonine,98% 0.5 0.5 0.5 0.5 l-Valine,96.5% 0.3 0.3 0.3 0.3 Mineralsupplementa _1.1 _1.1 _1.1 _1.1 Vitaminsupplementb _1.1 _1.1 _1.1 _1.1 Salinomycin(12%–66ppm) 0.6 0.6 0.6 0.6 Cholinechoride60% 1.0 1.0 1.0 1.0 Antioxidant(BHT)c _0.1 _0.1 _0.1 _0.1 Dicalciumphosphate 3.9 8.9 13.7 18.5 Limestone 16.9 14.7 13.6 9.3 Sandd _7.7 _4.9 _1.2 _0.6

Calculatednutrientcomposition(g/kgdietas-fedbasis)e

Crudeprotein(g/kg) 219.5 219.5 219.5 219.5

Apparentmetabolizableenergy(AMEn,MJ/kg) 12.8 12.8 12.8 12.8

Calcium(g/kg) 9.0 9.0 9.0 9.0

Non-phytatephosphorus(g/kg) 1.8 2.7 3.6 4.5

Na(g/kg) 2.09 2.09 2.09 2.09

Digestiblelysine(g/kg) 12.19 12.19 12.19 12.19

Digestiblemethionine(g/kg) 5.78 5.78 5.78 5.78

Digestiblemethionine+cysteine(g/kg) 8.67 8.67 8.67 8.67

Digestiblethreonine(g/kg) 7.85 7.85 7.85 7.85

Digestibletryptophan(g/kg) 2.27 2.27 2.27 2.27

Calculatedtotalphosphorus(g/kg) 4.14 5.06 5.95 6.84

Analyzednutrientcomposition

Crudeprotein(g/kg) 218.8 218.4 218.1 218.6

Calcium(g/kg) 9.21 9.12 8.93 9.32

Totalphosphorus(g/kg) 4.07 5.00 5.88 6.77

a_Mineral_supplement_(supply_per_kg_diet):_broilers_in_pre-starter_phase:_manganese_(MnSO

4·H2O),88mg;iron(FeSO4·H2O),62.5mg;zinc(ZnO),

81.3mg;copper(CuSO4·5H2O),12.5mg;iodine(KI),1.25mg;selenium(Na2SeO3),0.375mg;broilersinstarterphase:manganese(MnSO4·H2O),77mg;

iron(FeSO4·H2O),55mg;zinc(ZnO),71.5mg;copper(CuSO4·5H2O),11mg;iodine(KI),1.10mg;selenium(Na2SeO3),0.33mg.

b _Vitamin_supplement_(supply_per_kg_diet):_broilers:_Pre-starter_phase:_Vitamin_supplement_(supply_per_kg_diet):_vitamin_A_{(trans—retinyl}_acetate),₉₃₇₅

IU;vitaminD3(cholecalciferol),2375IU;vitaminE(all–rac–tocoferolacetate),35IU;vitaminK(bisulfatemenadionecomplex),1.88mg;vitaminB1.

2.50mg;vitaminB2,6.25mg;vitaminB6,3.5mg;pantothenicacid(D–calciumpanthenate),12.5mg;biotin,0.088mg;nicotinicacid,37.5g;folicacid,

0.875mg;vitaminB12(cyanocobalamin),0.015mg;broilersinstarterphase:Vitaminsupplement(supplyperkgdiet):vitaminA(trans—retinylacetate),

8250IU;vitaminD3(cholecalciferol),2090IU;vitaminE(all–rac–tocoferolacetate),31IU;vitaminK(bisulfatemenadionecomplex),1.65mg;vitamin

B1.2.20mg;vitaminB2,5.5mg;vitaminB6,3.08mg;pantothenicacid(D–calcuimpanthenate),11mg;biotin,0.077mg;nicotinicacid,33mg;folicacid,

0.77mg;vitaminB12(cyanocobalamin),0.013mg. c _Butylated_{hydroxytoluene.}

d _Phytases_replaced_sand_in_the_diets.

e_Based_on_ingredients_composition_of_the_Brazilian_Tables_for_Poultry_and_Swine_(2011).

Fortreatments2–4,thebasaldietwassupplementedwith0.9,1.8,or2.7gperkgofinorganicphosphorus(iP)fromDCP tocreatedietswith2.7,3.6or4.5gperkgofP,respectively.Fortreatments5–7,thebasaldietwassupplementedwith500, 1000,or2000phytaseunitsofCBPperkg.Thebasaldietwassupplementedwith250,500,or1000phytaseunitsofECPper kgfortreatments8–10.ForCBP,Vieiraetal.(2015)deﬁnedaunitofphytase(FYT)asthequantityofenzymethatliberates 1␮molofiPmin−1from5.0mMsodiumphytateatpH5.5and37◦C.ForECP,Adedokunetal.(2004)reportedthattheunit ofphytase(FTU)isdeﬁnedasthequantityofenzymethatliberates1␮molofiPmin−1from5.1mMsodiumphytateatpH 5.5and37◦C.

2.3. Measurementsandslaughter

Theevaluatedperformanceparameterswerefeedintake(FI,g/bird),weightgain(WG,g/bird),andfeed-to-gainratio (F:G,g/g).At21daysofage,4birdsperexperimentalunit,whichbodyweightwasclosetotheaverageweightofeachpen, wereselectedtobeslaughtered.Eachbirdwaseuthanizedandtherighttibiawasremovedtoevaluateash(g/kg)andP (g/kg)contents.

2.4. Chemicalanalysis

TibiasampleswerecollectedtodetermineashandPcontent.FeedsampleswerecollectedtoperformanalysesofNitrogen (N),Ca,P,andtheenzymeactivityofphytases.Tibiaandfeedsamplesweredriedat55◦Cinaforced-draftovenfor3days. Tibiaandfeedsamplesweregroundthrougha1-mmscreen.SampleswerethenusedtodetermineDMcontentbyoven dryingat105◦Cfor24h.Ashcontentofthesampleswasdeterminedaccordingtothemethod942.05(AOAC,2000).Nitrogen

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contentofthedietswasdeterminedbythecombustionmethod990.03(AOAC,2000;modelFP2000,LecoCorp.,St.Joseph, MI).CalciumandPweredeterminedafterwet-ashdigestionwithnitricandperchloricacidsaccordingtothemethod935.13 (AOAC,2000).Calciuminwet-ashedsampleswasdeterminedbytheatomicabsorptionspectrophotometricmethod968.08 (AOAC,2000)usinganatomicabsorptionspectrometer(AAnalyst300,PerkinElmer,Norwalk,CT).Phosphorusconcentration wasdeterminedusingacolorimetricassay(FiskeandSubbarow,1925).AcidmolybdateandFiske’sSubbaRowreducer solutionwereaddedtowet-ashsamplestoperformaphosphor-molybdenumcomplex.Colorintensitywasproportional toPconcentrationandwasdeterminedwithaspectrophotometerusingabsorbanceat620nm(SpectraCount,Model# AS1000,Packard,Meridian,CT).TheanalyseswereperformedattheAnimalNutritionLaboratoryoftheDepartmentof AnimalScience,FederalUniversityofVic¸osa.

ThephytaseactivitiesweredeterminedinaprivateLabusingthemethodISO30024:2009(Gizzietal.,2008)andthe methoddescribedbyBasuetal.(2007).

ThemethodISO30024:2009hasbeenpublished(Gizzietal.,2008)andrecognizedasaharmonizedphytasemethod byInternationalStandardsOrganization(BedfordandPartridge,2010).Thismethod(ISO30024:2009)isconsideredtobe aderivedversionoftheonedescribedbyEngelenetal.(2001),astheassaydeterminesphytaseactivityunderverysimilar invitroconditions:itisatthesamepHandtemperature,usesessentiallythesamereagents,buffers,substratepreparation, thesamedetectionmechanism,etc.Althoughthereareotherproceduraldifferences(thetimeoftheassayisreducedfrom 60to30minandthenewassayisstandardizedwithaphosphatestandardcurveinsteadofanenzymestandardcurve,for example),oneofthemaindifferencescomparedwiththeEngelenmethodisintheextractionprocedure(Sheehan,2010).

TheE.coliphytaseproduct,QuantumTM_Phytase,_has_shown_problems_with_detection_by_these_methods_(Sheehan,_2010).

Generally,phytaseiseasilyrecoveredfrommashdietsbytheEngelenorISOmethodsbutafterpelleting,problemshave arisen.AspecializedextractiontechniqueatpH10.0isthereforerequiredinordertofullysolubilizetheenzymeintothe extractionbuffer(Basuetal.,2007).Asnewphytasescometomarket,furtherextractionandassaymodifycationsmaybe necessary,sothatatrulyuniversalmethodmaynotbepossible(Sheehan,2010).

2.5. Statisticalanalysis

Alldatawereanalyzedwithone-wayANOVAusingtheGLMprocedureofSASstatisticalpackage(SASInstitute,Inc., 2010).

Thestatisticalmodelisdescribedasfollows: yij = +i+εij;i =1,...,a;j =1,...,n;

where:

y_ij=observationjingrouportreatmenti. =theoverallmean.

i=theﬁxedeffectofgrouportreatmenti(denotesanunknownparameter).

εij=randomerrorwithmean0andvariance2.

BasedonthemethodologydescribedbyAdedokunetal.(2004),orthogonalpolynomialcontrastswereusedtoevaluate theperformanceandtibiacharacteristicswhensupplementallevelsofiPfromDCP(0.9,1.8,or2.7gperkg),phytaseunits ofCBP(500,1000,or2000FYTperkg),orphytaseunitsofECP(250,500,or1000FTUperkg)wereaddedtothebasaldiet, whichwasusedaslevel0.0forrunningorthogonalpolynomialcontrastsforDCPorCBPorECP.

ThelinearresponsefunctionsthatbestﬁtthedatawerederivedfromtheDCP,CBP,orECPlevelsandarelistedasfollows: Forlinearregressionequations:

Ys = as+bsxs(Linearregressionequationforgperkgof iPfromDCP)(1);

Yc = ac +bcxc(LinearregressionequationforFYTofCBPperkg)(2);

Ye = ae +bexe(LinearregressionequationforFTUof ECPperkg)(3);

Eq.(1) = Eq.(2):as+bsxs=ac +bcxc;

Or

Eq.(1) = Eq.(3):as+bsxs=ae +bexe.

WhereYistheresponsecriterion(performanceorbonetraits);xsisthesupplementediPfromDCP(gkg−1 diet),xc is thesupplementedFYTperkgfromCBP,andx_eisthesupplementedFTUperkgfromECP;a_sistheinterceptofthelinear regressionforsupplementediPfromDCP(gkg−1diet),a_cistheinterceptofthelinearregressionforsupplementedFYTper kgfromCBP,anda_eistheinterceptofthelinearregressionforsupplementedFTUperkgfromECP;b_sistheslopeofthe responsecriteriontothesupplementediPfromDCP,bcistheslopeoftheresponsecriteriontothesupplementedFYTper kgfromCBP,andb_eistheslopeoftheresponsecriteriontothesupplementedFTUperkgfromECP.Thelinearresponse

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Table2

Performanceandtibiacharacteristicsofbroilersfeddietswithinorganicphosphoruslevels(g/kg)fromdicalciumphosphateorgradedlevelsofFYT/kgof CitrobacterbraakiiorgradedlevelsofFTU/kgofEscherichiacolifrom1to21daysofagea_.

Item Basaldiet SupplementaliPfromDCP,g/kg Supplementalphytaseunits fromCBP,FYT/kg

Supplementalphytaseunits fromECP,FTU/kg

SEMb

0.9 1.8 2.7 500 1000 2000 250 500 1000

FeedIntake,g/birdc, e, g, h, i, k, l, m, n ₆₅₉ ₉₉₉ ₁₁₂₉ ₁₁₈₄ ₁₀₀₃ ₁₀₁₉ ₁₀₉₀ ₉₈₉ ₁₀₆₀ ₁₁₂₀ _15.13

Weightgain,g/birdc,e,g,h,i,k,l,m,n ₄₃₁ ₆₁₀ ₇₅₆ ₇₈₆ ₅₉₃ ₆₇₂ ₇₁₄ ₆₄₂ ₆₈₉ ₇₅₈ _13.62

F:G,g/gd,f,j _1.537 _1.647 _1.498 _1.510 _1.691 _1.522 _1.530 _1.546 _1.545 _1.480 _0.028

Tibiaash,g/kgc,g,h,k,l ₃₀₅ ₅₀₀ ₇₈₀ ₈₆₉ ₄₆₄ ₅₇₀ ₇₁₀ ₅₄₈ ₆₁₈ ₈₀₆ _26.33

TibiaP,g/kgc, g,h,k,l _18.0 _37.8 _75.9 _85.8 _35.2 _49.1 _66.9 _40.9 _53.6 _77.3 _3.35

Mortality(%) 7.6 3.3 0.5 0.5 3.8 0.0 0.0 2.4 0.0 0.0 1.17

a_P₌_phosphorus;_iP₌_Inorganic_P;_ECP₌_Escherichia_coli-derived_phytase;_FTU₌_phytase_units_from_ECP;_CBP₌_Citrobacter_{braakii-derived}_phytase;

FYT=phytaseunitsfromCBP;basaldiethadnoiPorsupplementalphytase;thebasaldietwasusedaslevel0.0torunorthogonalpolynomialcontrastsfor DCPorCBPorECP;F:G=feed-to-gainratio.

b _SEM₌_Standard_pooled_error_of_the_means;_(sample_size₌_100). c _Linear_Effect_of_DCP_(P_<_0.01). d _Linear_Effect_of_DCP_(P_<_0.05). e_Quadratic_Effect_of_DCP_(P_<_0.01). f _Quadratic_Effect_of_DCP_(P_<_0.05). g_Linear_Effect_of_CBP_(P_<_0.01). h _Linear_Effect_of_CBP_(P_<_0.05). i _Quadratic_Effect_of_CBP_(P_<_0.01). j _Quadratic_Effect_of_CBP_(P_<_0.05). k _Linear_Effect_of_ECP_(P_<_0.01).

l _Linear_Effect_of_ECP_(P_<_0.05). m _Quadratic_Effect_of_ECP_(P_<_0.01).

n _Quadratic_Effect_of_ECP_(P_<_0.05).

equationsforsupplementediPfromDCPandthoseforsupplementedFYTperkgfromCBPorFTUperkgfromECPwereset tobeequal,andweresolvedforPequivalencyvaluesfortheirrespectivevariable.

3. Results

Inthecurrentstudy,thephytase-supplementeddietswereformulatedtocontain500,1000,or2000FYT/kgofCBPor250, 500,or1000FTUperkgofECP.Theanalysestestsweredeterminedtobe545,1380,and4210FYT/kginCBP-supplemented dietsand377,559,and1360FTU/kginECP-supplementeddiets.Theseresultswerehigherthanexpected,particularlyfor CBP-supplementeddiets,buttheydidnotcompromisetheresultssincetheincreaseinphytaseunitslevelswerekept.

ThesupplementalPlevelsfromDCPresultedinlinearandquadraticeffectsonbroilerWG,FI,andF:G.Tibiaashandtibia PwereonlylinearlyaffectedbyDCPlevels(P<0.05,Table2),buttibiaashshowedatendencyforquadraticeffect.Increasing PlevelsfromDCPof0.9,1.8,and2.7g/kgimproved(P<0.05)FIby52,71,and80%;WGby41,75,and32%;tibiaashweightby 64,156,and185%;andtibiaPby110,323,and378%,respectively.TheincreasingFYTlevelsofCBPperkgresultedinlinear andquadraticeffectsonWGandFI,butonlyaquadraticeffectonF:GandalineareffectontibiaashandtibiaP(P<0.05). TheCBPsupplementationof500,1000,and2000FYT/kgcontributedtoanincrease(P<0.05)of52,55,and65%forFI;37, 56,and65%forWG;52,87,and133%fortibiaash;and96,173,and273%fortibiaP,respectivelyinrelationtothebasal diet.TheincreasingFTUlevelsofECPperkgresultedinlinearandquadraticeffectsonWGandFI,butonlyalineareffecton tibiaashandtibiaP(P<0.05).TheECPadditionof250,500,and1000FTU/kgincreased(P<0.05)FIby50,61,and70%;WG by49,60,and76%;tibiaashweightby80,103,and164%;andtibiaPby128,198,and330%.

Inthisstudy,linearregressionequations(Table3)wereusedtodeterminePequivalencyvaluesofCBPandECP,when DCPisusedasaninorganicPsourceinbroilerdietsbasedonthemethodologydescribedbypaststudies(Adedokunetal., 2004;Adeola,2010).Also,equivalencyPequationswereusedtodeterminePequivalencyvaluesofCBPandECP,whenDCP isusedasaninorganicPsourceinbroilerdiets.

Phosphorus equivalency values for phytase units of Citrobacter braakii per kg (Table 4)and for phytase units of EscherichiacoliperkgwerecalculatedbyusingequivalencyequationsforWG,FI,tibiaash,andtibiaPresults,exceptfor F:G,whichshowedalowr2_and_regression_slopes_that_were_not_signiﬁcant_(P₌_0.081,_for_regression_slope_of_DCP;_P₌_0.236,

forregressionslopeofCBP;andP=0.086,forregressionslopeofECP).Thelevelsof500,1000,and2000FYT/kgofCBP weredeterminedtobeequivalenttotheaverageadditionof0.625,1.091,and2.024gofiPfromDCPperkginbroilerdiets, respectively.Meanwhile,250,500,and1000FTU/kgofECPweredeterminedtobeequivalenttotheaverageadditionof 0.763,1.307,and2.395gofiPfromDCPperkginbroilerdiets,respectively.

4. Discussion

Inthisstudy,DCPwaschosenasaniPsource.PaststudieshaveusedmonosodiumphosphateasaPsource(Adedokun etal.,2004;Jendzaetal.,2006;Adeola,2010)orDCP(Vieiraetal.,2015).Cowiesonetal.(2014)reportednodifference inbroilerperformancewhenevaluatingthreesourcesofP(phosphatesofmonocalcium,dicalcium,andtricalciumwith

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Table3

Linearregressionequationsestimatedtoeachvariableonresponseofinorganicphosphoruslevels(g/kg)fromdicalciumphosphateorgradedlevelsofFYT perkgfromCitrobacterbraakii-derivedphytaseorgradedlevelsofFTUperkgfromEscherichiacoli-derivedphytasea_.

Item Psource Regressionequationb _Standard_error_of_the_intercept _Intercept_Probability _Standard_error_of_the_Slope _Slope_Probability _r2_of_equationc

Feed DCP Yˆ=737.50+189.31X 21.788 <0.01 12.940 <0.01 0.849 intake, CBP Yˆ=782.48+0.183X 27.808 <0.01 0.024 <0.01 0.599 g/bird ECP Yˆ=779.53+0.41X 26.124 <0.01 0.045 <0.01 0.676 Weight DCP Yˆ=464.20+134.36X 15.201 <0.01 9.028 <0.01 0.854 gain, CBP Yˆ=487.61+0.131X 15.041 <0.01 0.013 <0.01 0.725 g/bird ECP Yˆ=501.66+0.29X 17.323 <0.01 0.030 <0.01 0.712 Tibia DCP Yˆ=317.64+219.16X 24.156 <0.01 14.347 <0.01 0.859 ash, CBP Yˆ=341.0+0.196X 18.544 <0.01 0.016 <0.01 0.794 g/kg ECP Yˆ=364.80+0.47X 22.389 <0.01 0.039 <0.01 0.790 Tibia DCP Yˆ=18.13+26.86X 3.233 <0.01 1.920 <0.01 0.837 P, CBP Yˆ=21.29+0.024X 2.290 <0.01 0.002 <0.01 0.791 g/kg ECP Yˆ=22.42+0.06X 2.360 <0.01 0.004 <0.01 0.836

a_P₌_phosphorus;_DCP₌_dicalcium_phosphate;_iP₌_inorganic_P;_CBP₌_Citrobacter_{braakii-derived}_phytase;_FYT₌_phytase_units_from_CBP;_ECP₌_Escherichia

coli-derivedphytase;FTU=phytaseunitsfromECP.

b_Linear_regression_equations_were_estimated_to_each_variable_using_individual_observations_instead_the_means. c_Coefﬁcient_of_{determination.}

Table4

Equivalencyequationsandphosphorus(P)equivalencyvalues(g/kg)ofCitrobacterbraakii-derivedphytaseandEscherichiacoli-derivedphytasea_. Phytase Itemc _Equivalency_equationsb

EquivalencyPvaluesd_,_g/kg

500 1000 2000

CBP Feedintake,g/bird 737.50+189.31(iP,g/kg)=782.48+0.183(FYT/kg) 0.720 1.204 2.171

Weightgain,g/bird 464.20+134.36(iP,g/kg)=487.61+0.131(FYT/kg) 0.662 1.149 2.124

Tibiaash,g/kg 317.64+219.16(iP,g/kg)=341.0+0.196(FYT/kg) 0.554 1.001 1.895

TibiaP,g/kg 18.13+26.86(iP,g/kg)=21.29+0.024(FYT/kg) 0.564 1.011 1.905

Mean 0.625 1.091 2.024

250 500 1000

ECP Feedintake,g/bird 737.50+189.31(iP,g/kg)=779.53+0.41(FTU/kg) 0.763 1.305 2.388

Weightgain,g/bird 464.20+134.36(iP,g/kg)=501.66+0.29(FTU/kg) 0.818 1.358 2.437

Tibiaash,g/kg 317.64+219.16(iP,g/kg)=364.80+0.47(FTU/kg) 0.751 1.287 2.359

TibiaP,g/kg 18.13+26.86(iP,g/kg)=22.42+0.06(FTU/kg) 0.718 1.277 2.394

Mean 0.763 1.307 2.395

a_P₌_phosphorus;_iP₌_Inorganic_P;_DCP₌_dicalcium_phosphate;_CBP₌_Citrobacter_{braakii-derived}_phytase;_FYT₌_phytase_units_from_CBP;_ECP₌_Escherichia

coli-derivedphytase;FTU=phytaseunitsfromECP.

b_Equivalency_equations_were_calculated_for_each_variable_when_linear_regression_of_DCP_and_linear_regression_of_CBP_are_equal_or_linear_regression_of

DCPandlinearregressionofECPareequal.

c_All_variables_were_used_to_estimate_P_equivalency,_excepting_to_feed-to-gain_ratio_that_showed_low_r2_._There_was_not_{signiﬁcance}_for_regression_slope

ofDCP(P=0.081),regressionslopeforECP(P=0.086),andregressionslopeofCBP(P=0.236).

d_Equivalency_P_values_(g_per_kg)_relative_to_iP_from_DCP.

potassiumphosphateofinorganicphosphorus)inthediets,buttibiaash(%)washigherinbirdsthatwerefedMCPthan thosethatwerefedeitherDCPorTCP.However,DCPwasusedbecauseitiscurrentlyaddedtobroilerdietsinBrazil.

ThesupplementaliPlevelsfromDCPresultedinlinearandquadraticeffectsonperformanceandboneparametersof broilerchickens.Theseresultsareinaccordancewiththoseobservedinseveralstudies(Adedokunetal.,2004;Jendza etal.,2006;Adeola,2010;Vieiraetal.,2015),whichreportedlinearorquadraticinﬂuencesonperformanceandbone mineralizationofbroilerchickensthatwerefeddietswithsupplementaliPfromdifferentinorganicsources.

Inthecurrentstudy,theresultsshowedthatCBPandECPincreasetheavailabilityofphosphorusinP-lowcorn-and soybean-basedbasaldiets.Similarresultswerereportedbypaststudies(Adeola,2010;Shawetal.,2011;Cowiesonetal., 2014;OlukosiandFru-Nji,2014).Phytasecanpartlyreplacedicalciumphosphateasaphosphorussource,accordingtothe FTUorFYTlevelsinthediet(Nelsonetal.,1968).Simonsetal.(1990)reportedthatmorethan65%ofthePwasreleased bytheadditionofphytaseinbroilerdiets.Denbowetal.(1995)showedthatPreleasedbyPhytaserangedfrom31to58% for250to1000Uphytaseperkgoffeed.Yietal.(1996)calculatedthatupto37%ofthephytatePinsoybeanmealwas releasedbytheadditionof1000Uofphytase/kgofdiet.Waldroupetal.(2000)indicatedthatapproximately50%ofthe phytate-boundPinacorn–SBMdietwasreleasedbyphytase.

Thesupplementationof500,1000,and2000FYT/kgofCBPshowedaverageimprovementsof60.5,92.75,and134% comparedtothebasaldiet.Meanwhile,thesupplementationof250,500,and1000FTU/kgofECPshowedaverage improve-mentsof76.75,105.5,and160%comparedtothebasaldiet.Severalstudieshavereportedimprovementsonperformance associatedwiththeuseofPhytaseinbroilerdiets.Jendzaetal.(2006)reportedan87%increaseintheWGofbirdsat42days oldwhentheyweresupplementedwith500FTU/kgphytasefromE.coli.Onyangoetal.(2005a)observedanincreaseof17 and20%intheWGofbroilerchickensat8and22daysold,respectively,whentheywerefedaP-lowdietsupplemented with500and1000FTU/kgphytaseofasecondgenerationE.coliphytase.Dilgeretal.(2004)reportedanincreaseof6and

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19%,respectively,inWGofbroilerchickensfrom8to22daysoldwhentheyweresupplementedwith500and1000FTU/kg phytasederivedfromE.coli.

ThelinearregressionequationswereusedtodeterminePequivalencyvaluesofCBPandECP,whenDCPisusedasan inorganicPsourceinbroilerdietsaccordingtothemethodologydescribedbypaststudies(Adedokunetal.,2004;Adeola, 2010).ThoseauthorsveriﬁedbothlinearandquadraticresponsestogradedlevelsofiPandunitsofphytase,buttheyused linearequationsratherthanquadraticequationstodeterminePequivalencyvalues.Additionally,thelinearregression equa-tionscorrectlydeterminePequivalencyvaluesandrepresentasimplemethodcomparedtoquadraticregressionequations orotherpolynomialdegrees.

Paststudieshaveconsideredhigherr2_values_as_a_reference_for_choosing_performance_or_bone_{mineralization}_criterion_to

estimatePequivalencyvalues(Adedokunetal.,2004;Adeola,2010;Vieiraetal.,2015).KornegayandQian(1996)relayed thattheresponsecriterionthatarechosenindataanalysescaninﬂuencetheresultsthatareobtainedinthetrial.However, severalauthorshavedevelopedregressionsbyusingtreatmentmeansratherthanindividualobservations,whichmayhave resultedinelevatedr2_values_(Jendza_et_al.,_2006)._Therefore,_in_this_study,_the_regression_equations_were_developed_by_using

individualobservationsasrecommendedbyJendzaetal.(2006).AimingtoestimatethePequivalencyvalues,wechosethe responsecriterionthatprovidedbestr2_values_considering_regression_equations_developed_by_using_individual_observations

insteadtheaveragevalues.Thus,ther2 _values_should_be_reported_as_use_of_averages_or_individual_replicates_on_ongoing

studies.

Inthecurrentstudy,meanPequivalencyvaluesofCBPwerelowerthanthoseobservedbyVieiraetal.(2015),who reportedthat500and1000phytaseunitsprovidedaverageestimationsof1.00and1.66nPPperkg,respectively.The authorsusedabasaldietcontaining1.4gperkgofnon-phytatephosphorus(nPP)and8.0gperkgoftotalcalcium(Ca), whileweusedabasaldietwith1.8gperkgofnon-phytatephosphorus(nPP)and9.0gperkgoftotalcalcium(Ca).Probably, thehighCalevelusedinthecurrentstudycontributedtothedivergentestimates.Oneofthepossibleexplanationsfor thedifferentresponsesinstudiesevaluatingphytaseefficacyisthehighcalciumlevel(Adedokunetal.,2004),whichcan decreasetheefficacyofPhytaseinbirdsduetophytateprecipitationandtheformationofthephytate-Cacomplexinthe smallintestine(Selleetal.,2009).Applegateetal.(2003)verifiedthataCalevelupto9.0g/kgcanreducetheintestinal activityofaphytaseby9%andphytatehydrolysisby12%.Also,studiesevaluatingphytaseefficacycurrentlyvarywith regardstothePlevelsinthebroilerdietswhilekeepingCalevelsfixed,whichinfluencestheCa:tPratiointheexperimental diets.LeiandStahl(2000)reportedthatahighCa:tPratiocanreducephytatesolubility,therebyincreasingitsresistanceto hydrolysis.

ForsupplementalphytaseunitsofECPperkg,thisstudyestimatedPequivalencyvalueshigherthanthosereportedby paststudies.Adedokunetal.(2004)reportedthat500and1000FTU/kgprovideaverageestimationsof0.486and1.031gof iPperkg,respectively.Jendzaetal.(2006)observedthat500FTU/kgofECPwasdeterminedtobeequivalenttotheaddition of0.49or1.00gofiPperkgwhenusingWGandboneash,respectively.Thepaststudiesusedtoprovideexperimentaldiets atday8(Dilgeretal.,2004;Adedokunetal.,2004;Onyangoetal.,2005a;Jendzaetal.,2006).Meanwhile,weprovided experimentaldietsatday1.ThissuggeststhatbirdsfedaP-lowdietfrom1-d-olddemonstratehighereffectivenessandP equivalencyvaluesofphytaseunitsofECPperkg.BirdsfedadietwithadequatePlevelsbeforereceivingexperimentaldiets canreducetheirresponsetophytasesupplementation(Onyangoetal.,2005a,b)duetothePstoresthatareaccumulated duringtheﬁrst7d,whichmaydecreasevariabilitybymodifyingtheresponsetolowerlevelsofPinthediet(Jendzaetal., 2006).

Overall,theresultsofthisstudydemonstratedthatitispossibletoreducetheamountofDCPaddedtothefeedwhen Escherichiacoli-andCitrobacterbraakii-derivedPhytasearesupplementedtothebroilerdiets.ThemeanPequivalencyvalues ofCBPrangedbetween0.625and2.024g/kgofiPfromDCPaccordingtotheFYTlevelthatissupplemented.Thissuggests thepossibilityofreducingtheaddedDCPbetween3.378and10.940kg/tonincorn-soybeanmealbaseddietsforbroiler chickensfrom1to21daysofage.Also,themeanPequivalencyvaluesofECPrangedbetween0.763and2.395g/kgofiP fromDCPaccordingtotheFTUlevelthatissupplemented.ItalsosuggeststhepossibilityofreducingtheaddedDCPbetween 4.124and12.946kg/tonincorn-soybeanmealbaseddietsforbroilerchickensfrom1to21daysofage.However,thisstudy usedahighCaconcentrationinthebasaldiet(9.0g/kg),whichcouldaffectthephytaseactivityandunderestimatingthe results.Maybe,thepossibilityofreducingtheadditionofDCPcouldbehigherusingdietswithlowerCaconcentration.

Thus,thedietarysupplementationofthesephytasesmayreducetheuseofinorganicphosphorussourcesinbroilerdiets.

5. Conclusions

TheCitrobacterbraakii-andEscherichiacoli-derivedphytasescanbeusedinthedietsofbroilerchickensfrom1to21days ofagetopartiallyreducetheadditionofdicalciumphosphateasaphosphorussource.

Thesupplementationof500,1000,and2000phytaseunitsofCitrobacterbraakiiperkgweredeterminedtobeequivalent totheaverageadditionof0.625,1.091,and2.024gofinorganicphosphorusfromdicalciumphosphateperkginbroiler diets,respectively.

Thesupplementationof250,500,and1000phytaseunitsofEscherichiacoliperkgweredeterminedtobeequivalentto theaverageadditionof0.763,1.307,and2.395gofinorganicphosphorusfromdicalciumphosphateperkginbroilerdiets, respectively.

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Conﬂictofintereststatement

Theauthorsdeclarethattherearenoconﬂicofinterest. Acknowledgements

TheauthorsexpresstheirgratitudetotheDepartmentofAnimalScienceoftheFederalUniversityofVic¸osa,thecompany ABvista,andtheCAPES.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.anifeedsci.2016.09.008.

References

AOAC,2000.OfﬁcialMethodsofAnalysis,17thed.Assoc.Off.Anal.Chem.,Allington,VA.

Adedokun,S.A.,Sands,J.S.,Adeola,O.,2004.DeterminingtheequivalentphosphorusreleasedbyanEscherichiacoli-derivedphytaseinbroilerchicks.Can. J.Anim.Sci.84,437–444.

Adeola,O.,2010.PhosphorusequivalencyvalueofanEscherichiacoliphytaseinthedietsofWhitePekinducks.Poult.Sci.89,1199–1206. Applegate,T.J.,Angel,R.,Classen,H.L.,2003.Effectofdietarycalcium,25hydroxycholecalciferol,orbirdstrainonsmallintestinalphytaseactivityin

broilerchickens.Poult.Sci.82,1140–1148.

Basu,S.S.,Winslow,S.,Nelson,A.,Ono,M.,Betts,S.,2007.ExtractionMethodsandAssaysforFeedEnzymes.WorldIntellectualPropertyOrganization (WIPO),Geneva,Switzerland,WO/2007/002192.

Bedford,M.R.,Partridge,G.G.,2010.EnzymesinFarmAnimalNutrition,2nded.CABIPublishing,Wallingford,UK.

Cowieson,A.J.,Fru-Nji,F.,Adeola,O.,2014.DietaryphosphateequivalenceoffourformsofPicontrastedwithanovelmicrobialphytasefromCitrobacter braakiiinbroilerchickens.Anim.Prod.Sci.55,1145–1151.

Cromwell,G.L.,2009.ASAScentennialpaper:landmarkdiscoveriesinswinenutritioninthepastcentury.J.Anim.Sci.87,778–792.

Denbow,D.M.,Ravindran,V.,Kornegay,E.T.,Yi,Z.,Hulet,R.M.,1995.Improvingphosphorusavailabilityinsoybeanmealforbroilersbysupplemental phytase.Poult.Sci.74,1831–1842.

Dilger,R.N.,Onyango,E.M.,Sands,J.S.,Adeola,O.,2004.Evaluationofmicrobialphytaseinbroilerdiets.Poult.Sci.83,962–970.

Engelen,A.J.,vanderHeeft,F.C.,Randsdorp,P.H.G.,Somers,W.A.C.,Schaefer,J.,vanderVat,B.J.C.,2001.Determinationofphytaseactivityinfeedbya colorimetricenzymaticmethod:collaborativeinterlaboratorystudy.J.AOACInt.84,629–633.

Fiske,C.H.,Subbarow,Y.,1925.Thecolorimetricdeterminationofphosphorus.J.Biol.Chem.66,375–400.

Gizzi,G.,Thyregod,P.,vonHolst,C.,Bertin,G.,Vogel,K.,Faurschou-Isaksen,M.,Betz,R.,Murphy,R.,Andersen,B.B.,2008.Determinationofphytase activityinfeed:inter-laboratorystudy.J.AOACInt.91,259–267.

Jendza,J.A.,Dilger,R.N.,Sands,J.S.,Adeola,O.,2006.EfﬁcacyandequivalencyofanEscherichiacoli-derivedphytaseforreplacinginorganicphosphorusin thedietsofbroilerchickensandyoungpigs.J.Anim.Sci.84,3364–3374.

Kornegay,E.T.,Qian,H.,1996.Replacementofinorganicphosphorusbymicrobialphytaseforyoungpigsinacorn-soybean-mealdiet.Br.J.Nutr.76, 563–578.

Lei,X.G.,Stahl,C.H.,2000.Nutritionalbeneﬁtsofphytaseanddietarydeterminantsofitsefﬁcacy.J.Appl.Anim.Res.17,97–112.

Nelson,T.S.,Shieh,T.R.,Wodzinki,R.J.,Ware,J.H.,1968.Theavailabilityofphytatephosphorusinsoybeanmealbeforeandaftertreatmentwithamold phytase.Poult.Sci.47,1842–1848.

Olukosi,O.A.,Fru-Nji,F.,2014.Theinterplayofdietarynutrientspeciﬁcationandvaryingcalciumtototalphosphorusratioonefﬁcacyofabacterial phytase:growthperformanceandtibiamineralization.Poult.Sci.93,3037–3043.

Onyango,E.M.,Bedford,M.R.,Adeola,O.,2004.TheyeastproductionsysteminwhichEscherichiacoliphytaseisexpressedmayaffectgrowth performance,boneash,andnutrientuseinbroilerchicks.Poult.Sci.83,421–427.

Onyango,E.M.,Bedford,M.R.,Adeola,O.,2005a.EfﬁcacyofanevolvedEscherichiacoliphytaseindietsofbroilerchicks.Poult.Sci.84,248–255. Onyango,E.M.,Bedford,M.R.,Adeola,O.,2005b.Phytaseactivityalongthedigestivetractofthebroilerckick:acomparativestudyofan

Escherichiacoli-derivedandPeniophoralyciiphytase.Can.J.Anim.Sci.85,61–68.

Rostagno,H.S.,Albino,L.F.T.,Donzele,J.L.,Gomes,P.C.,Oliveira,R.F.,Lopes,D.C.,Ferreira,A.S.,Barreto,S.L.T.,Euclides,R.F.,2011.BrazilianTablesfor PoultryandSwine:CompositionofFeedstuffsandNutritionalRequirements,3rded.ImprensaUniversitária,Vic¸osa

http://www.lisina.com.br/arquivos/Geral%20English.pdf.

SASInstitute,Inc,2010.SASOnlineDoc®Version9.1.3.SASInstitute,Inc.,Cary,NC,USA.

Selle,P.H.,Ravindran,V.,2007.Microbialphytaseinpoultrynutrition.Anim.FeedSci.Technol.135,1–41.

Selle,P.H.,Cowieson,A.J.,Ravindran,V.,2009.Consequencesofcalciuminteractionswithphytateandphytaseforpoultryandpigs.Livest.Sci.124, 126–141.

Shaw,A.L.,Hess,J.B.,Blake,J.P.,Ward,N.E.,2011.Assessmentofanexperimentalphytaseenzymeproductonliveperformance,bonemineralization,and phosphorusexcretioninbroilerchickens.J.Appl.Poult.Res.20,561–566.

Sheehan,N.,2010.Analysysofenzymes,principalsandproblems:developmentinenzymeanalysis.In:Bedford,M.R.,Partridge,G.G.(Eds.),Enzymesin FarmAnimalNutrition.,2nded.CABIPublishing,Wallingford,UK,pp.260–272.

Simons,P.C.M.,Versteegh,H.A.J.,Jongbloed,A.W.,Kemme,P.A.,Slump,P.,Bos,K.D.,1990.Improvementofphosphorusavailabilitybymicrobialphytasein broilersandpigs.Br.Poult.Sci.64,525–540.

Smith,V.H.,Tilman,G.D.,Nekola,J.C.,1999.Eutrophication:impactsofexcessnutrientinputsonfreshwater,marine,andterrestrialecosystems.Environ. Pollut.100,17–196.

Vieira,S.L.,Anschau,D.L.,StefanelloSeraﬁni,N.C.,Kindlein,L.,Cowieson,A.J.,Sorbara,J.O.B.,2015.PhosphorusequivalencyofaCitrobracterbraakii phytaseinbroilers.J.Appl.Poult.Res.00,1–8.

Waldroup,P.W.,Kersey,J.H.,Saleh,E.A.,Fritts,C.A.,Yan,F.,Stilborn,H.L.,2000.Nonphytatephosphorusrequirementandphosphorusexcretionofbroilers feddietscomposedofnormalorhighavailablephosphatecornwithandwithoutmicrobialphytase.Poult.Sci.79,1451–1459.

Waldroup,P.W.,1999.Nutritionalapproachestoreducingphosphorusexcretionbypoultry.Poult.Sci.78,683–691.

Yan,F.,Kersey,J.H.,Fritts,C.A.,Waldroup,P.W.,2003.Phosphorusrequirementsofbroilerchickssixtonineweeksofageasinﬂuencedbyphytase supplementation.Poult.Sci.82,294–300.

Yi,Z.,Kornegay,E.T.,Ravindran,V.,Denbow,D.M.,1996.Improvingphytatephosphorusavailabilityincornandsoybeanmealforbroilersusingmicrobial phytaseandcalculationofphosphorusequivalencyvaluesforphytase.Poult.Sci.75,240–249.