The effects of restricting enzyme supplementation in wheat-based diets to broilers

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ContentslistsavailableatSciVerseScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

The

effects

of

restricting

enzyme

supplementation

in

wheat-based

diets

to

broilers

A.A.

Figueiredo

a,1

_,

_B.A.

_Correia

a,1

_,

_T.

_Ribeiro

b

_,

_P.I.P.

_Ponte

b

_,

_L.

_Falcão

a

_,

_J.P.

_Freire

a

_,

_J.A.M.

Prates

b

_,

_L.M.A.

_Ferreira

b

_,

_C.M.G.A.

_Fontes

b,∗

_,

_M.M.

_Lordelo

a

a_Instituto_Superior_de_Agronomia,_Tapada_da_Ajuda,_1349-017_Lisboa,_Portugal

b_CIISA_–_Faculdade_de_Medicina_{Veterinária,}_Pólo_{Universitário}_do_Alto_da_Ajuda,_Avenida_da_Universidade_Técnica,_1300-477_Lisboa,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20October2010 Receivedinrevisedform 29December2011 Accepted3January2012 Keywords: Broiler Enzyme Wheat Xylanase

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Theperformanceofbroilersfedonxylanasesupplementeddietsforpartorallofthe pro-ductioncyclewasevaluatedintwoexperiments.Inapreliminaryexperiment,1-day-old chickswerefedawheat-baseddietsupplementedwithacommercialxylanasefortheentire durationoftheexperimentof28days,orduringthelast21,14,or7daysofthetrial.In experiment2,thegrowthperiodwasextendedto36days,andbirdswerefedxylanase sup-plementeddietsfortheentiredurationoftheexperiment,orduringthelast27,18,or9days ofthetrial.Inbothexperiments,anadditionalgroupwasfedanon-supplementedcontrol diet.Xylanase-supplementedbroilersoutperformednon-supplementedbirds.Significant differencesinbodyweightemergedat21–28daysofage,suggestingthattheresponseto xylanasesupplementationoccursprimarilyduringthelatterstagesofbroilergrowth. Broil-ersfedxylanase-supplementeddietsfortheentireexperimentpresentedsimilargrowth performance,intestinalviscosity,gastrointestinalenzymeactivityandorgansizeswhen comparedwithbirdssupplementedwiththeexogenousxylanaseforthelast14 (experi-ment1)orthelast18days(experiment2)ofthetrials.Birdsexposedtoexogenousxylanase fortheentiredurationoftheexperimentshowed,however,anon-significanttrend(P<0.1) towardsanimprovedfinalbodyweightandfeedconversionratio.Nevertheless,itappears thatanearlyexposuretotheexogenousxylanaseisofmarginalimportanceinobtaininga significantresponsetoxylanasesupplementationbybroilersfedwheat-baseddiets.

1. Introduction

Theuseofexogenousenzymesinsimple-stomachanimalnutritionhasbeenextensivelyinvestigatedduetotheirrole inattenuatingtheanti-nutritivepropertiesofsolublenon-starchpolysaccharides(NSP)presentincereals.Wheat-based dietsusuallycontainsigniﬁcantlevelsofsolublearabinoxylans,aformofNSP,whichmayimpairfeeddigestionandbird performance(Steenfeldtetal.,1998;Prestonetal.,2001).Theadditionofmicrobialxylanasestothesedietsleadstoa reductionindigestaviscosity,resultinginmoreefﬁcientnutrientdigestionandabsorption(Steenfeldtetal.,1998;Choct etal.,1999;Prestonetal.,2001).Asaconsequence,thereisanoverallimprovementinbroilerperformanceasreviewedby

Abbreviations: BW,bodyweight;FCR,feedconversionratio;GI,gastrointestinal;NSP,non-starchpolysaccharide.

∗ Correspondingauthorat:FaculdadedeMedicinaVeterinária,UniversidadeTécnicadeLisboa,AvenidadaUniversidadeTécnica,1300-477Lisboa, Portugal.Tel.:+351213652876;fax:+351213652889.

E-mailaddress:[email protected](C.M.G.A.Fontes).

1 _Equal_{contribution.}

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Table1

Compositionandcalculatedanalysisofthewheat-baseddietusedinexperiments1and2.a

Ingredient g/kgofdiet Wheat 580.0 Soybeanmeal,470g/kgCP 165.2 Soy(full-fat) 200.0 Sodiumchloride 2.5 Calciumcarbonate 11.5 Dicalciumphosphate 17.3 dl-Methionine 1.5

Mineralandvitaminpremixb _2.0

Fillerc _20.0

Calculatednutrientcontentd

Metabolisableenergy(MJ/kg) 12.62 Crudeprotein 219.2 Lysine 11.6 Methionine 4.5 Calcium 9.2 Availablephosphorus 4.4

a_In_experiment₁_treatments_consisted_of_a_wheat-based_diet_supplemented_with_enzyme_for_the_entire₂₈_days,_the_last₂₁_days,_the_last₁₄_days,_the

last7daysornotsupplemented.Inexperiment2treatmentsconsistedofawheat-baseddietsupplementedwithenzymefortheentire36days,thelast 27days,thelast18days,thelast9daysornotsupplemented.

b _{Mineral–vitamin}_premix_provided_the_following_per_kilogram_of_diet:_biotin_0.5_mg,_calcium_pantothenate₁₀_mg,_{cholecalciferol}_0.05_mg,

cyanocobal-amin0.12mg,folicacid0.5mg,menadione2mg,nicotinicacid30mg,pyridoxine1.7mg,retinol2.7mg,thiamin1mg,␣-tocopherol20mg,riboﬂavin, 4.2mg,Co0.2mg,Cu10mg,Fe80mg,I1mg,Mn100mg,Se0.3mg,Zn80mg.

c _The_ﬁller_consisted_of_ground_wheat._When_xylanase_{supplementation}_was_provided,_the_ﬁller_was_used_to_mix_the_enzyme_into_the_diet._When_no

xylanasesupplementationwasprovided,theﬁllerwasaddedalone.

d _Calculation_of_nutrient_content_based_on_Ingredient_Analysis_Table₍_Dale_and_Batal,₂₀₀₉_).

Bedford(2000).Inaddition,supplementationwithexogenousenzymesallowstheincorporationofalternativesourcesof energythatmaybecheaperthancornincertaingeographicalareas.

Somereportsindicatethatchickensbenefitmostfromenzymeadditioninthegrowerorfinishingperiods,whereas othersstatethatthebenefitsaremoreapparentatanearlierage(Duseletal.,1998;Fontesetal.,2004;Gaoetal.,2008). Fontesetal.(2004)suggestedthattheeffectsofxylanasesupplementationaremostprominentduringthelatterstagesof growth,basedontheirstudyoftwoxylanaseswithdifferentmoleculararchitectures.Duseletal.(1998)agreedthatenzyme additionimprovesbirdperformanceprimarilyattheendofthefinishingperiod.Othersclaimthatenzymesupplementation resultsinmorepronouncedimprovementsingrowthfrom7to21daysofagethanfrom22to49daysofage(Gaoetal., 2008).Inaddition,Cowiesonetal.(2005)demonstratedthatimprovementsinperformanceoccurduringthestarterand growerperiods.Itshouldbenotedthatineachoftheabovementionedstudies,birdswerefedwithenzymesupplemented dietsthroughouttheproductioncycle.Itisthereforedifficulttodisentangletheimpactofsupplementationatdifferentages. Therearelogicalreasonswhyenzymesupplementationshouldbebeneficialtobirdsbothyoungandold.Intheyoung chick,theproductionofendogenousdigestiveenzymesisscarceandmaylimitfeeddigestion(Nitsanetal.,1991;Dunnington andSiegel,1995).Thus,theadditionofexogenousenzymescanenhancethedigestivecapacityoftheyoungbirdby com-plementingitsrepertoireofintestinalenzymes.Asthebirdages,itsdigestiveabilityandmicrobiotacapacityincrease,and themicrofloraroutebecomesmoreimportantinmediatingthebeneficialeffectsofexogenousenzymes(Bedford,2000).

Theresponsetoxylanasesupplementationtendstobegreaterinolderthanyoungerbroilersduetoaninteractionbetween viscosityandbacteria(BedfordandMorgan,1996).Thismayresultfromanimprovedefﬁciencyofmicrobialfermentation inthecaecum,orthroughchangesincaecalmicrobialpopulations.Thelattereffectmightincludeadecreaseinthesizeof detrimentalmicrobialpopulationsthatcolonisetheﬁnalcompartmentsofthegastrointestinal(GI)tractduringthelatter stagesofthebird’sgrowth(ApajalahtiandBedford,1999).

Takentogetherthedatadiscussedaboveindicatethatthereareseveralunresolvedissuesconcerningthemosteffective periodfordietsupplementationwithexogenousenzymesincereal-baseddietsforbroilers.Inthepresentstudy,wehave evaluatedtheimpactofsupplementingwheat-baseddietswithexogenousxylanasesfortheentireorforonlypartofthe broilerproductioncycle,bothingrowthperformance,intestinalviscosityandorgansizes.

2. Materialsandmethods

2.1. Preliminaryexperiment(experiment1)

Apreliminarytrialwasconductedfrom0to28daysofage.Onehundredandsixtyone-day-oldRoss308malebroiler chickswereweighedandassignedto40pensof4birdseach,suchthatthebodyweight(BW)uniformityofeachpenwas similar.Chickswerewing-bandedforindividualidentiﬁcation.The40penswererandomlyassignedto5treatmentgroups consistingofabasalpelleteddiet(Table1)supplementedwithacommerciallyavailablexylanasemixture,fromTrichoderma longibrachiatumandBacillussubtilis(DaniscoAnimalNutrition,Marlborough,UK)ataninclusionrateof0.5g/kg(w/w),for

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theentire28daysoftheexperiment(treatmentA1–28),thelast21days(treatmentA7–28),thelast14days(treatment A14–28)orthelast7days(treatmentA21–28).Afifthgroupwasfedabasaldietwithoutxylanase(treatmentA0).The enzymemixturewasheatresistantandcontainedaminimumof2500U/gofendo-1,4-␤-xylanaseand800U/gofprotease, asspecifiedbythemanufacturer(oneunitofenzymeactivityisthequantityofenzymerequiredtoproduceone␮mol ofreducingsugarequivalentperminute).Thebasaldiet(Table1)containedapproximately600g/kgofwheatandwas formulatedtoensureanutrientavailabilityasdefinedbyNRC(1994).Throughouttheexperiment,chicksweregivenfree accesstowaterandfeed,whichwereprovidedwithdrinkingnipplesandhangingfeeders,respectively.Brooderswere raisedinwiredfloorpensthatwerelocatedinanenvironmentallycontrolledroomadjusteddailytotherecommended temperatures,accordingtostandardbroodingpractices.FeedconsumptionandBWwasdeterminedweeklythroughoutthe experiment,andmortalitywasrecordeddaily.Feedconversionratiowascorrectedformortality.Atday28,onebirdperpen waskilledbyanintravenousinjectionofanaqueousisotonicsolutionof125mgThiopental(Braun,Barcelona,Spain).The emptyweightofthecrop,gizzardandliver,andthelengthoftheduodenum,jejunum,ileumandcaecumweremeasured. DigestasampleswerecollectedfromthevariousGIcompartmentstodeterminexylanaseactivityandcontentviscosityas describedinSection2.3.

2.2. Experiment2

Theprotocolforthisexperimentwassimilartoexperiment1,exceptthat120birdswereassignedto40pensof3birds eachandthedurationofthetrialwas36days.The40penswererandomlyassignedto5treatmentgroupsconsistingofa basalpelleteddiet(Table1)supplementedwiththesamexylanasemixtureata0.5g/kg(w/w)inclusionratefortheentire 36days(treatmentB1–36),thelast27days(treatmentB9–36),thelast18days(treatmentB18–36)orthelast9daysof theexperiment(treatmentB27–36).Onegroupwasfedanon-supplementedbasaldiet(treatmentB0).Performancewas measuredasdescribedpreviously.Attheendoftheexperimentalperiod,onebirdperpenwaskilledtomeasurethesizeof thevariousGIcompartmentsandtocollectdigestasamplesfortheanalysisofxylanaseactivityandviscosity.

2.3. Analyticalprocedures

Priortothedetectionofxylanaseactivity,digestasampleswerecentrifugedat13,000×gfor10minat4◦C andthe supernatantwascollectedforanalysis.QualitativeanalysisofxylanaseactivityinGIcontentswasperformedinagarplates usingpuriﬁedwheatarabinoxylan(Megazyme,Wicklow,Ireland)ata0.1%(w/v)ﬁnalconcentration,in10mMTris–HClpH 7.0.Catalyticactivitywasdetectedaftera16hincubationperiodat37◦CthroughtheCongoRedassayplate,asdescribed byPonteetal.(2004)andMourãoetal.(2006).

Priortomeasuringtheviscosityofwheat,15gofthecerealwasgroundedthrougha1mmsieve,towhich15mLofa 50mMPhosphatebuffersolution,pH7.0,wasaddedandcentrifugedfor10minat10,867×g.Thesupernatantwasthen removedtoaneppendorftube,placedinawaterbathfor30minat40◦Candcentrifugedfor5minat3354×g.Viscositywas thenmeasuredat6rpmusingaviscometer(ModelLVDVCP-II,BrookﬁeldEngineeringLaboratories,Middleboro,MA)with acupmaintainedat24◦C,asdescribedbyRibeiroetal.(2011).

Tomeasuretheviscosityofsmallintestinecontents,sampleswerecollectedfromtheduodenumplusjejunumandileum. Sampleswerecentrifugedfor10minat10,867×gandtheviscosityofthesamplesupernatantwasmeasuredat6rpmusing aviscometerwithacupmaintainedat24◦C.

2.4. Statisticalanalysis

DatarelatedtobirdperformancefromeachexperimentweresubjectedtoANOVAaccordingtothegenerallinearmodels procedureofSAS(SASInstitute,2001).TheLeastSquaredMeansprocedurewasusedtodetectsigniﬁcantdifferencesbetween treatmentgroupsinasetofcontrasts.DifferenceswereconsideredsigniﬁcantwhenP<0.05.

3. Results

3.1. Preliminaryexperiment(experiment1)

Birdsfedthedietsupplementedwithxylanasesthroughouttheexperimentalperiod(A1–28)wereheavier(P<0.05) thanbirdswithoutsupplementationorbirdssupplementedduring21–28days(Table2).Thisdifferencewasobserved bothatdays21and28(P<0.05).Incontrast,birdsfedthexylanase-supplementeddietforthelast14(A14–28)or21days (A7–28)oftheexperimentreachedaﬁnalBWthatwasnotdifferentfromthefully(A1–28)supplementedbirds(P>0.05). Feedintakewasunaffectedbytreatment(Table2).However,theglobalandlastweek’sfeedconversionrate(FCR)ofbirds supplementedinthelastone,two,threeorfourweeksoftheexperimentwasreduced(P<0.05)whencomparedwithbirds fedanon-supplementeddiet(Table2).Themortalityratewas4.38%andnotinﬂuenced(P<0.05)bydietarytreatment.

Therelativeweightandlengthofthebird’sGIcompartmentswereunaffectedbyxylanasesupplementation(Table3). Digestaviscositiesinthehindgutandforegutwerealsonotdifferentbetweenbirdsfedthedifferentdiets(Table3).Finally, xylanaseactivitywasdetectedalongtheentireGItractofmostbirdsfedxylanase-supplementeddiets.Thiswasthecase

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Table2

Bodyweight,feedintakeandfeedconversionrateofbroilersfedawheat-baseddietsupplementedwithenzymeinspeciﬁcperiods(experiment1).

Treatmenta _Contrasts

A0 A21–28 A14–28 A7–28 A1–28 SEM A0vsA1–28 A21–28vsA1–28 A14–28vsA1–28 A7–28vsA1–28

Bodyweight(g) 7d 158 148 161 158 167 1.57 0.0780 <0.0001 0.2371 0.1142 14d 426 413 430 424 439 4.23 0.3720 0.0476 0.5428 0.3574 21d 759 758 788 781 828 7.62 0.0041 0.0029 0.0882 0.0618 28d 1206 1225 1282 1259 1330 12.15 0.0009 0.0046 0.1847 0.0727 Feedintake(g) 0–7d 165 143 160 160 170 3.71 0.6089 0.0221 0.3568 0.3403 7–14d 394 367 376 382 381 5.17 0.4344 0.4172 0.7480 0.9463 14–21d 606 581 608 609 616 7.18 0.6800 0.1422 0.7276 0.7559 21–28d 904 880 897 869 882 15.18 0.6179 0.9476 0.7407 0.7601 0–28d 2070 1972 2041 2020 2050 22.10 0.7856 0.2834 0.8966 0.6778

Feedconversionrate(g/g)

0–7d 1.46 1.37 1.37 1.44 1.39 0.0153 0.1425 0.7602 0.7067 0.2897

7–14d 1.49 1.41 1.42 1.47 1.42 0.0235 0.8651 0.1949 0.2944 0.5974

14–21d 1.88x _1.76xy _1.72yz _1.71yz _1.61z _0.0227 _0.0001 _0.0273 _0.1213 _0.1682

21–28d 2.12x _1.90y _1.85y _1.84y _1.77y _0.0299 _<0.0001 _0.0774 _0.2044 _0.2747

0–28d 1.73x _1.61y _1.59y _1.62y _1.55y _0.0159 _0.0002 _0.1576 _0.3104 _0.1117

Meansinthesamelinewithdifferentlettersuperscripts(x,y,z)aresigniﬁcantlydifferent(P<0.05).

a_Treatments_consisted_of_a_wheat-based_diet_not_supplemented_(A0)_or_supplemented_with_enzyme_for_the_last₇_days_(A21–28),_the_last₁₄_days

(A14–28),thelast21days(A7–28)ortheentire28days(A1–28).

evenforbirdsfromtreatmentsreceivingtheenzymeatthelaterexperimentalstages(datanotshown).Viscosityofwheat usedinbothexperimentswas4.05cpo.

3.2. Experiment2

Toinvestigatetheeffectsofrestrictingenzymesupplementationinanextendedexperimentalperiod,birdsinexperiment 2werekilledatday36.FinalBW wasnotsigniﬁcantlydifferentbetweenbirdsfedenzymesupplementeddietsduring theentireduration oftheexperiment(B1–36), thelast 27days(B9–36), orthelast18 days(B18–36)(Table4).Birds supplementedthroughouttheexperimentwereheavier(P<0.05)thannon-supplemented(B0)birdsorthosesupplemented forthelast9daysoftheexperiment(B27–36)(Table3).Asobservedinexperiment1,feedintakewasunaffectedbytreatment (Table4).TotalFCRwasnotdifferent(P>0.05)betweenbroilersfedenzymesupplementeddietsduringthetotalduration oftheexperiment(B1–36)andthosereceivingenzymeinthelast27days(B9–36)orthelast18days(B18–36)ofthetrial

Table3

Relativeweightandlengthofgastrointestinaltractcompartmentsandviscosityofdigestasamplesofbroilersfedawheat-baseddietsupplementedwith enzymesinspeciﬁcperiods(experiments1and2).a

Experiment1b _Experiment₂b

A0 A21–28 A14–28 A7–28 A1–28 SEM P B0 B27–36 B18–36 B9–36 B1–36 SEM P

Weight(g/kgofBW) Crop 4.2 3.3 3.4 3.1 3.0 0.148 NS 3.8 2.9 2.7 3.0 3.3 0.144 NS Gizzard 10.7 9.3 9.9 9.4 9.2 0.231 NS 8.5 8.8 10.0 9.1 7.8 0.280 NS Liver 28.1 30.3 30.9 28.2 28.8 0.661 NS 31.2 28.6 27.4 28.5 27.0 0.760 NS Length(cm/kgofBW) Duodenum 18.0 18.7 18.5 18.3 18.4 0.336 NS 27.4 27.5 26.3 27.8 28.4 0.564 NS Jejunum 46.3 46.9 51.8 49.1 46.3 1.171 NS 67.6 71.0 68.2 68.8 73.9 1.094 NS Ileum 49.6 48.8 49.3 48.7 46.8 1.137 NS 70.8 69.4 69.8 65.1 73.6 1.116 NS Caecum 13.0 11.9 12.4 12.5 12.1 0.264 NS 20.2 17.3 17.9 17.6 17.5 0.578 NS

Contentviscosity(cpo)c

Duodenum+Jejunum 4.07 3.55 3.54 3.55 3.53 0.122 NS 3.05 3.11 3.05 3.04 2.82 0.072 NS

Ileum 6.23 6.18 5.43 5.09 5.44 0.284 NS 5.33x _4.53xy _4.36xy _4.84xy _3.90y _0.167 _0.044

Meansinthesamelinewithdifferentlettersuperscripts(x,y,z)aresigniﬁcantlydifferent(P<0.05).

a_Measurements_were_taken_at₂₈_days_of_age_in_experiment₁_and₃₆_days_in_experiment_2._N₌_40.

b _In_experiment₁_treatments_consisted_of_a_wheat-based_diet_not_supplemented_(A0)_or_supplemented_with_enzyme_for_the_last₇_days_(A21–28),_the_last

14days(A14–28),thelast21days(A7–28)ortheentire28days(A1–28).Inexperiment2treatmentsconsistedofawheat-baseddietnotsupplemented (B0)orsupplementedwithenzymeforthelast9days(B27–36)thelast18days(B18–36),thelast27days(B9–36),ortheentire36days(B1–36).

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Table4

Bodyweight,feedintakeandfeedconversionrateofbroilersfedwheat-baseddietsupplementedwithenzymeinspeciﬁcperiods(experiment2).

Treatmenta _Contrasts B0 B27–36 B18–36 B9–36 B1–36 SEM B0vsB1–36 B27–36vsB1–36 B18–36vsB1–36 B9–36vsB1–36 Bodyweight(g) 9d 216 211 216 222 215 1.63 0.7145 0.6010 0.7630 0.1283 18d 638 639 660 648 661 5.59 0.1974 0.1749 0.9672 0.4704 27d 1207 1173 1248 1247 1263 10.92 0.1020 0.0092 0.6676 0.6509 36d 1731 1709 1855 1862 1878 20.44 0.0243 0.0065 0.7117 0.8065 Feedintake(g) 0–9d 218 223 216 218 218 2.64 0.9660 0.9773 0.8310 0.5232 9–18d 623 620 635 620 629 6.13 0.7499 0.6459 0.7732 0.6415 18–27d 1050 995 1066 1066 1076 13.20 0.5287 0.0558 0.7983 0.7959 27–36d 1149 1111 1181 1164 1153 14.94 0.9206 0.3933 0.5734 0.8276 0–36d 3041 2963 3091 3073 3077 27.93 0.6756 0.2178 0.8828 0.9629

Feedconversionrate(g/g)

0–9d 1.27 1.30 1.25 1.26 1.29 0.0124 0.5816 0.7894 0.3099 0.3725

9–18d 1.53 1.47 1.48 1.48 1.46 0.0192 0.2580 0.8363 0.8363 0.7739

18–27d 1.90 1.84 1.79 1.73 1.76 0.0222 0.0502 0.2416 0.5395 0.7539

27–36d 2.13 2.11 2.02 1.92 1.80 0.0356 0.0045 0.0063 0.0525 0.2800

0–36d 1.69 1.67 1.63 1.59 1.57 0.0146 0.0067 0.0183 0.1316 0.4772

a_Treatments_consisted_of_a_wheat-based_diet_not_supplemented_(B0)_or_supplemented_with_enzyme_for_the_last₉_days_(B27–36)_the_last₁₈_days_(B18–36),

thelast27days(B9–36),ortheentire36days(B1–36).

(Table4).Fullysupplementedbirds(B1–36)showedanimproved(P<0.05)overallFCRthannon-supplemented(B0)and birdssupplementedduringthelast9days(B27–36).Mortalityratewas3.33%andnotinﬂuenced(P<0.05)bytreatment.

TheweightandlengthofthevariousGIcompartmentsrelativetoBWanddigestaviscositiesintheduodenumand jejunumwereunaffected(P<0.05)bytreatment(Table3).Viscosityintheileum,however,waslower(P<0.05)inbroilers supplementedthroughouttheexperimentthaninnon-supplementedbirds(Table3).Nodifferencesweredetectedinileum contentviscositybetweenbroilersfedenzyme-supplementeddietsduringthelast9,18,27,and36days.Asobservedin experiment1,xylanaseactivitywasdetectedalongtheentireGItractofmostanimalsfedaxylanase-supplementeddiet (datanotshown).

4. Discussion

Inthepresentstudy,weinvestigatedtheimpactofaddingexogenousxylanasesatdifferentagesontheperformance of broilers fed a wheat-based diet. Birds fed a xylanase-supplemented diet throughout theirlife outperformed non-supplementedbirds,intermsofbothBWandFCR.Thesebroilerperformancedataareconsistentwithpreviousﬁndings thatmicrobialxylanasescanimprovethenutritivevalueofwheat-baseddietsforbroilers(Choctetal.,1999;Prestonetal., 2001;Marronetal.,2001;Steenfeldtetal.,1998;Engbergetal.,2004;Wuetal.,2004).

Theprimarygoalofthisstudywastodeterminethestageoftheproductioncycleatwhichtheprimaryresponseto xylanase-supplementationoccurs.SigniﬁcantdifferencesinBWbetweenthesupplementedandnon-supplementedgroups begantoemergeasbirdsgotolder,atdays21and28inthepreliminaryexperiment,anddays27and36inexperiment2. Thesedataareconsistentwithpreviousreportsthattheresponsetoxylanasesupplementationmayoccurprimarilyduring thelatterstagesofbroilergrowth(BedfordandMorgan,1996;Duseletal.,1998).

Thepopulationof poultry’sGImicrofloraismore prolificasthe birdages and thusin olderbirds,it is likelythat feedenzymesinfluencebroilerperformancethroughaninteractionwithmicrobialpopulations.Theactionofexogenous xylanasesinthelatterstagesofgrowthmaybefacilitatedbychangesinthemicrobialpopulationscolonisingthebird’sGI tract(Bedford,2000).Asyet,theexactmechanismbehindthisinteractionremainstobeestablished.Onepossibilityisthat, byactivelycontributingtothede-polymerisationofsolublearabinoxylansintheupperregionsoftheGItract,xylanaseslead toasignificantchangeinthenutrientsthatareavailableformicrobialfermentation.Thechangeinnutrientprofileresults fromadecreaseinthepercentageofstructuralpolysaccharidesandincreaseinthelevelsofavailablexylo-oligosaccharides, whicharenotattackedinthesmallintestine(ApajalahtiandBedford,1999).Futurestudiesusingxylo-oligosaccharide supplementationinsteadofxylanasemayenableelucidatingthispossibility.

Althoughthevisiblebenefitsofxylanasesupplementationbegintoemergeat21–28daysofage,thisdoesnotexclude thepossibilitythatanearlyexposuretomicrobialxylanasesisbeneficial.Thedurationofthepreliminaryexperiment(28 days)waspossiblytooshorttoderiveappropriateconclusionsinthisrespect.Therefore,experiment2lastedfor36days andthedatarevealedthatbirdsreceivingexogenousenzymesthroughoutoftheexperimentreachedasimilarfinalBWto thosethatreceivedenzymesexclusivelyinthelast18days.Thus,theadditionalearlyexposuretomicrobialxylanases(until day18inexperiment2)hadonlyamarginaleffectonthefinalBWofbirds.Inaddition,FCRwassignificantlyalteredin

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birdsreceivingtheexogenousenzymethroughoutexperiment2,indays9–36anddays18–36,whencomparedwithbirds withoutenzyme.Thus,ineconomicterms,itmaybebeneﬁcialtorestrictxylanasesupplementationtohalfoftheproduction cycleinbroilerswithaproductioncycleof36days.

Dietaryfibrecaninfluencethedevelopmentandsizeofbroilers’digestiveorgans.DietswithhighlevelsofsolubleNSP induceaconsiderableenlargementofsomeportionsoftheGItract(Brenesetal.,1993),stimulatinganincreaseinprotein turnoverrates,whichismainlyinfluencedbytheincreaseindigestaviscosity(Dänickeetal.,2000).Theadditionofexogenous cellulasesandhemicellulasesdecreasesdigestaviscosityandthereforeimprovesfeedpassagerateandnutrientabsorption, whichmayleadtoadecreaseintherelativeweightofthedigestivetractandapossibleimprovementincarcassyield.Inthe presentstudyhowever,therelativeweightsofcrop,gizzardandliver,andrelativelengthsoftheduodenum,jejunum,ileum andcaecumdidnotvarysignificantlybetweentreatmentgroups.Inaddition,intestinalviscositieswererelativelymodest andwerenotinfluencedbytheadditionofexogenousenzymesineitherexperiment.Itwouldthereforeappearthatlow viscositywheat-baseddietshaveamarginalinfluenceonGItractsizeandintestinalviscosity,whicharenotsignificantly reducedbytheadditionofexogenousenzymes(Low,1989).Thus,datademonstratedrangesofdigestaviscosityinthe currentwheat-baseddietsbetween2.8and6.2cpo,whichmaybetoosmalltoinfluencethemorphologyoftheGItract. 5. Conclusion

Thecurrentstudyindicatesthatxylanasesupplementationisparticularlyeffectiveduringthelatterstagesofbroiler growth.Broilerfeedsupplementationduringtheentireproductioncyclemaynotberequiredandmaybereplacedbya restrictedsupplementationinthelasthalfoftheproductionperiodforbroilerswithaproductioncycleof36days.These observationsmaybeofsigniﬁcantimportancetocommercialfeedmanufacturersandpoultryproducers.However,response toenzymerestrictionwhenotherexogenousenzymesandcereal-baseddietsareusedremainstobeinvestigated. Acknowledgements

WethankSociedadeAgrícoladaQuintadaFreiriaSAforsupplyingthe1-day-oldchicksandReagroforprovidingthe feedingredientsusedintheseexperimentsandDavePritchettandM.NeilRedfordforthecontributioneditingthisarticle. ThisworkwassupportedbyFundac¸ãoparaaCiênciaeaTecnologia,Lisbon,Portugal(grantPTDC/CVT/103942/2008).T. RibeiroandP.I.P.PonteweresupportedbyFundac¸ãoparaaCiênciaeTecnologiathroughgrantsSFRH/BD/32321/2006and SFRH/BD/17969/2004,respectively.

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