acid on calcium and phosphorus metabolism of growing pigs Effects of a reduced calcium, phosphorus and protein intake and ofbenzoic Animal Feed Science and Technology

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ContentslistsavailableatScienceDirect

Animal Feed Science and Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Effects of a reduced calcium, phosphorus and protein intake and of benzoic acid on calcium and phosphorus metabolism of growing pigs

A. Gutzwiller

^a,∗

, H.D. Hess

^a

, A. Adam

^b

, D. Guggisberg

^a

, A. Liesegang

^c

, P. Stoll

^a

aAgroscopeLiebefeld-PosieuxResearchStationALP,CH-1725Posieux,Switzerland

bEcoleNationaleSupérieureAgronomiquedeToulouse,F-31326Castanettolosan,France

cInstituteofAnimalNutrition,VetsuisseFaculty,UniversityofZurich,CH-8057Zurich,Switzerland

a rt i c l e i nf o

Articlehistory:

Received11February2010

Receivedinrevisedform23March2011 Accepted25March2011

Keywords:

Phosphorus Benzoicacid Pig Bone Metabolism

a b s t ra c t

Inordertominimiseenvironmentalpollution,manypigfeedscontainlowphosphorusand proteinconcentrationsaswellasbenzoicacid(BA),anadditivewhichreducesammonia formationintheslurry.SincebothalowPintakeandmetabolicacidosiscompromisebone mineralisation,theeffectofadietwithalowconcentrationofcalcium(Ca),phosphorus (P)andcrudeprotein(CP)andtheeffectofBAonCaandPmetabolismwereexaminedina 2×2two-factorialexperimentusingpigsfrom13to64kgbodyweight(BW).Comparedto thecontrolpigletandgrowerdiets(8.7and6.9gCa;6.9and5.3gP;172and156gCPper kg,respectively),theintakeofthelownutrientpigletandgrowerdiets(5.3gCa;4.3and 4.0gP;154and147gCPperkg,respectively,bothsupplementedwith1500U/kgmicrobial phytase)reduced(P<0.01)CaandPretentionby27%and24%,respectively,reduced(P<0.05) thegrowthrateofthepigletsby7%,anddecreased(P<0.05)thebonebreakingstrength andbonemineralcontent(P<0.01)by5%intheanimalswhichwereslaughteredat64kg BW.Benzoicacid(5and10gperkgpigletandgrowerdiet,respectively)didnotinﬂuence (P>0.05)theapparentdigestibilityofCa,increasedtheapparentdigestibilityofP(P<0.05)by 5%andincreasedtheurinaryCaandPoutput(P<0.01)by70%and83%,respectively,buthad noeffect(P>0.05)ontheproportionofingestedCaandPwhichwasretained.Furthermore, BAincreased(P<0.01)theserumactivityoftheboneformationmarkeralkalinephosphatase at25and40kgBWby17%and13%,respectivelyanddecreased(P<0.01)theconcentration oftheboneresorptionmarkerserumcrosslapsat25kgBWby12%,implyingthatBAaffected bonemetabolismat25and40kgBW.SinceBAneitheraffectedthebloodvariablesat60kg northebonebreakingstrengthandbonemineralcontent,anypossiblenegativeeffectof BAonbonemetabolismofthepigletsandoftheyounggrowinganimalsthusseemstohave disappearedduringthelastperiodofthegrowerperiod.Inconclusion,theslightmetabolic acidosiscausedbyBAhadnolastingnegativeeffectsonthebonesofthegrowingpigs.

Abbreviations:ADG,averagedailyweightgain;ALT,alanineaminotransferase;AP,alkalinephosphatase;AST,aspartateaminotransferase;BA,benzoic acid;BMC,bonemineralcontent;BW,bodyweight;CP,crudeprotein;DE,digestibleenergy;dEB,dietaryelectrolytebalance;DM,drymatter;GGT, gamma-glutamyltransferase;Mc3,Mc4,3rd,4thmetacarpalbone;N,Newton;NL,nutrientlevel;OC,osteocalcin;SCL,serumcrosslaps(epitopeofthe carboxyterminaltelopeptideoftypeIcollagen).

∗ Correspondingauthor.Tel.:+41264077223;fax:+41264077300.

E-mailaddress:andreas.gutzwiller@alp.admin.ch(A.Gutzwiller).

doi:10.1016/j.anifeedsci.2011.03.015

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1. Introduction

Pigslurrycontributestothepollutionoftheenvironmentwithphosphorus(P).TominimisePoutput,thePconcentration offeedsforgrowingpigsisfrequentlyreducedtothelowestlevelsnecessaryformaximalgrowthrate.SincethePrequirement formaximalbonemineralisationishigherthantherequirementformaximalgrowthrate(NRC,1998),suchfeedsmayhave anadverseeffectonbonemineralisation.FeedswithareducedPconcentrationusuallycontainalowCaconcentrationas well,sinceawideCa/PratiodecreasesPabsorption(NRC,1998).Ammoniaemissionfromanimalmanureisanadditional environmentalproblemchallengingpigproducers.Theuseoffeedswithalowcrudeprotein(CP)concentrationandadded benzoicacid(BA)reducesammoniaemission.Afterabsorption,benzoicacidistransformedinthelivertohippuricacid whichisexcretedintheurine andreducesmicrobial ureadecomposition.Benzoicand hippuricacidcontributetothe metabolicacidloadofthebody.Chronicacidosisstimulatesboneresorptionbyosteoclastsandmaythuscompromisebone mineralisation(Arnett,2003).Itisunclearwhetheraslightincreaseindietaryacidloadisofpracticalrelevanceforbone mineralisationinpigs.WhereasBuddeandCrenshaw(2003)failedtodetectanynegativeeffectofmetabolicacidosiscaused bychlorideintakeontheskeletonofpiglets,theintakeofBAsigniﬁcantlydecreasedtheboneashconcentrationingrowing pigsinabalancestudy(Saueretal.,2009),andtendedtoreduceboneashconcentrationingrowing-fatteningpigs(Bühler etal.,2010).OneobjectiveofthepresentexperimentwastodetermineifcommerciallyavailableSwissfeedsformulatedto minimiseenvironmentalpollutionaffectthebonemineralisationandthuscompromisethewelfareofgrowingpigs.Forthis purposeexperimentalpigletandgrowerdietswithCP,Pandphytaseconcentrationsasfoundinthesecommercialfeeds wereformulated,andtheeffectsofthesedietswerecomparedtotheeffectsofdietswhichwereformulatedtocontainthe nutrientconcentrationsrecommendedinSwitzerlandbyALP(2004)andwhichdidnotcontainaddedphytase.Inaddition, thehypothesiswastestedthattheincreaseddietaryacidloadprovidedbyBAaffectsCaandPmetabolism,relatedblood serumparametersandbonemineralisationofpigs.TheeffectsofdietarynutrientconcentrationandofBAonCaandP metabolism,growthandbonetraitsofpigswereexaminedinabalancestudyandafeedingtrial.Asthefocuswasseton theperiodofrapidleanbodymassaccretion,i.e.theperiodofhighCaandPrequirementsandthehighestriskofCaandP deﬁciency,theanimalswereslaughteredafterattaining60kgbodyweight(BW)inordertostudythebonetraitsattheend ofthegrowerperiod.

2. Animals,materialsandmethods

Thebalancestudyandthefeedingtrialwereapprovedbytheanimalwelfaredepartmentofthecompetentgovernment authority(approvalnumberFR77/06).

2.1. Experimentaldesignandfeedformulation

Theeffectsoftwofactors,dietarynutrientlevel(Ca,PandCP)andBAsupplementation,wereexaminedinabalance studyusing16pigsandinafeedingtrialusing64pigs.Groupsoffourlittermatesofthesamegenderwithasimilarbody weight(BW)wereblocked.Eachpigletwithinablockwasrandomlyassignedtooneofthefourdietarytreatments.

Theexperimentaldietscontainedeitherahigh(H)oralow(L)Ca,PandCPconcentration.Benzoicacidwasaddedto thedietsH+andL+,whereasthedietsH−andL−containednoBA.Thepelletedpigletandgrowerdiets(Table1)were formulatedaccordingtotherecommendationsforpigletsweighing20kgandforgrowingpigsweighing40kg(ALP,2004), exceptfortheCa,PandCPcontentofdietsL+andL−,whichcorrespondedtothelevelsfoundincommerciallyavailableSwiss pigfeedsformulatedwiththeaimtominimisePandnitrogeneffluent.TheCa/Pratioofalldietswasfixedat1.3/1.Thefeed formulationprogrammeAllix2(A-SystemsSA,Versailles,France)usedtoformulatetheexperimentaldietscontainedthe analyseddrymatter,crudeprotein,crudefat,ashandcrudefibredataoftheingredientsandfeedtabledataoftheirmineral contentandtheiraminoacidcomposition.Phytase(1500U/kgfeed;Natuphos5000G,BASF,Ludwigshafen,Germany)was addedtodietsL+andL−.Fiveand10gBA(VevoVitall,DSMNutritionalProductsLtd.,Basel,Switzerland)wereaddedperkg oftherespectivepigletandgrowerdiets(H+andL+),whichcorrespondstothedoserecommendedbythemanufacturer.

2.2. Animalsandhusbandry

LargeWhitepiglets,whichhadbeenweanedattheageoffiveweeksandhadreceivedapigletdietcontaining180g CP,11gCaand7gPperkgfeedduringthefirsttwotothreeweeksafterweaning,wereselectedfortheexperimentsatan averageBWof13kg.Theywerehousedinaclimatecontrolledbuildingandwerefedtheexperimentalpigletdietsadlibitum until25kgBW.Thereaftertheyreceivedthegrowerdietsinamountsallowingforanaveragedailygainof850gduringthe growing-finishingperiod.Drinkingwaterwasconstantlyavailable.

Thesixteencastratedmalepigletsweighing13.5±1.8kgatthestartofthebalancestudywereindividuallyhousedin penswithasurfaceof2.6m².

AtaBW of12.9±2.6kgthe36 femaleand28castratedmalepiglets usedinthefeedingtrialwereequippedwith transpondersandweretransferredtofourpens(oneforeachtreatment)with7m²ofslattedﬂoorand10m²ofconcrete ﬂoorwithstrawbedding.Eachpenwasequippedwithacomputercontrolledfeedingstation(Schauer,Prambachkirchen, Austria)whichregisteredtheamountoftheexperimentalfeedconsumedbyeachanimalandallowedforindividualfeed

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Table1

Ingredientsandcontentoftheexperimentaldiets(g/kgfeedwithadrymattercontent0.88g/g,unlessotherwiseindicated).

Diets Pigletdiets Growerdiets

Nutrientlevel H L H L

Ingredients

Maize 200 250 200 250

Barley 137 193 206 226

Wheat 100 50 250 150

Wheatstarch 100 125 51 85

Wheatmiddlings 4 4 4 4

Oats 64

Fat(tallowandlard) 17 13 3 7

Potatoprotein 71 98 32 96

Expelledsoybeanmeal 76 12

Expelledcanolameal 30

Wheypowder 50 50

Casein 10 10

Driedbeetpulp 50 100 50 71

Driedapplepomace 50 74 50 82

l-lysine–HCl 2.8 4.2 3.0 2.9

dl-methionine 1.3 2.1 0.8 0.9

l-threonine 0.9 1.5 0.9 0.6

l-tryptophan 0.4 0.7 0.2 0.4

Dicalciumphosphate 18.4 8.6 11.5 6.1

Calciumcarbonate 7.1 4.9 8.1 6.3

Sodiumchloride 3.0 4.0 2.7 4.8

Premix^a 4.0 4.0 4.0 4.0

Phytase,units(U)^b 1500 1500

Pelletbindingaid^c 3.0 3.0 3.0 3.0

Nutrients

Crudeprotein^d 172 154 156 147

Lysine^e 12.1 12.1 10.2 10.2

Methionine+Cysteine^e 7.5 7.5 6.5 6.5

Tryptophan^e 2.4 2.4 2.0 2.0

Crudeﬁbre^d 42 42 41 42

Crudefat^d 36 26 25 25

Ash^d 52 40 48 42

Ca^d 8.7 5.3 6.9 5.3

P^d 6.9 4.3 5.3 4.0

K^e 5.8 4.3 5.8 3.7

dEB(mEq/kg)^e,f 121 82 133 82

Phytaseactivity,U/kg^d 140 1600 220 1580

Digestibleenergy(DE)^e,MJ/kg 13.9 13.9 13.6 13.6

Vevovitall(DMS,Geleen,Netherlands)wasaddedtothebenzoicacidcontainingdiets(5g/kgpigletdietand10g/kggrowerdiet)inthefeedmillduring thefeedblendingprocess.

aProvidingthefollowingamountsoftraceelementsandvitaminsperkgofpiglet/growerdiet:40/20mgFe;0.15/0.15mgI;6/4mgCu;10/10mgMn;

75/55mgZn;0.2/0.15mgSe;8000/4000IUA;1000/400IUD3;25/65mgE;3/1mgK3;2/2mgthiamine;5/3mgriboﬂavin;0.1/0.05mgbiotin;20/15mg niacin;15/15mgpantothenicacid300/200mgcholine;4/3mgB6;0.5/0.5mgfolate;0.02/0.02mgB12.

bNatuphos5000G(BASF,Ludwigshafen,Germany).

cPellan(Mikro-Technik,Bürgstadt,Germany).

dAnalysed.

eCalculatedusingtablevaluesforeachingredient.

fdEB:dietaryelectrolytebalance,expressedinmilliequivalents(mEqK⁺+mEqNa⁺−mEqCl⁻).

rationing.Afterattaining25kgBW,thepigsusedinthefeedingtrialweretransferredtofouradjacentidenticallyequipped penswheretheywerefedthegrowerdietsuntiltheywereslaughteredat64±2.5kgBW.

2.3. Experimentalprocedures 2.3.1. Balancestudy

Attheendofeachexperimentalweekeachpigwasweighedanditsfeedintakeduringthatweekwasrecorded.When theanimalswereweighing25,40and55kg,theywereplacedintothemetabolismcrates.Fromthefirstcollectionperiod onwards,theexperimentaldietswereofferedinamountsallowingforanaveragedailygainof850gduringthegrowing- finishingperiod.Duringthecollectionperiodslastingfivedays,faeces,urineandrefusedfeedwerecollectedquantitatively onceaday.Faecesandurinesampleswerestoredat−20^◦C.Attheendofthecollectionperiods,samplesoffaeces,urine andrefusedfeedwerepooledacrossperiodforeachanimal.Faeceswerelyophilisedfor48h,andrefusedfeedandfaeces weregroundinaBrabenderlaboratorymillusinga1mmscreen.

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2.3.2. Feedingtrial

Thedailyfeedintakewasrecordedbythecomputercontrolledfeedingstations.Theanimalswereweighedeachweek, andbloodwasdrawnfromthejugularveinofeachanimalwhenithadattained25,40and60kgBW.Thebloodsamples withoutaddedanticoagulantwerecentrifugedwithin1haftercollection,andserumwasstored at−20^◦C untilit was analysed.The3rdand4thmetacarpalbonesandthetibiaofthelefthalfofeachcarcasswerecollectedwithinhalfaday afterslaughter,andtheadjacenttissuesweremanuallyremoved.Theboneswerestoredinsealedplasticbagsat−20^◦C untiltheywereanalysed.

2.4. Laboratoryprocedures

CrudeproteinwasanalysedusingtheDumasmethodonaLecoFP-2000analyser(Leco,Mönchengladbach,Germany).

Crudefibre,crudefatandashwereanalysedaccordingtotheVDLUFAmethods6.1.4,5.1.1and8.1.Phytaseactivityinthe feedswasmeasuredusingtheISO30024method.Briefly,thesampleswereincubatedwithsodiumphytate,andthereaction wasstoppedwithacidmolybdate–vanadate.Thecomplexformedbyinorganicphosphateinthepresenceofmolybdateand vanadatewasmeasuredphotometricallyat415nm.The3rdmetacarpalboneswerecrushed,defattedwithacetoneanddried at105^◦Cforthedeterminationofthefat-freedrymatter(DM).Samplesofdefattedbones,feedandfaeceswereashedina mufflefurnaceat550^◦Cuntilaconstantweightwasattained.Urineanddryashedfeed,faecesandbonesweresolubilisedin 10Mnitricacid,andtheirCaandPconcentrationswereanalysedaccordingtoEN15510:2007usinganinductivelycoupled plasmaopticalemissionspectrometer(ICP-OES,Optima2000DV,Perkin-Elmer,Schwerzenbach,Switzerland).

Theserumanalyteswereassayedusingcommerciallyavailablekits:kit1489216for Ca(Roche,Basle,Switzerland;

calcium-o-cresolphthaleincomplexonformation);kit61571forP(BioMérieux,Marcyl’étoile,France;absorbanceinUVofa phosphomolybdatecomplex);kit2172933foralkalinephosphatase(AP;Roche;cleavageofp-nitrophenylphosphate);kit 2016788forgamma-glutamyltransferase(GGT;Roche;formationof5-amino-2-nitrobenzoatefroml-gamma-glutamyl-3- carboxy-4-nitroanilideandglycylglycine);kit63212foraspartateaminotransferase(AST;Biomérieux;oxaloacetateformed fromaspartatereactingwithNADH2);kit63312foralanineaminotransferase(ALT;Biomérieux;pyruvateformedfroml- alaninereactingwithNADH₂);OsteometerBiotech(Copenhagen,Denmark;ELISA)forserumcrosslaps(SCL,epitopeofthe carboxyterminaltelopeptideoftypeIcollagen);Quidel(SanDiego,CA,USA;ELISA)forosteocalcin(OC).

Thetotalbonemineralcontent(BMC)ofthetibiawasmeasuredat50%tibialength(midshaft)andat10%tibialength (distalmetaphysis)usingperipheralquantitativecomputertomography(pQCTStratecXCT960ABoneScanner;Stratec MedizinaltechnikGmbH,Pforzheim,Germany).The4thmetacarpalbonesweretransferredfromthefreezertoarefrigerator 12hbeforetheirbreakingstrengthwasdeterminedwithaZwickRoelltestingmachine(ZwickRoell,UlmGermany)using thethree-pointbendingtest.Theboneswereheldbytwosupportsspaced35mmapartandwerebrokenbyawedgelowered onthecentreoftheboneataspeedof2mm/s.Theforcewasmeasuredbyapressure-sensitivecell,andpeaksofmaximum force(fmax)wererecorded.

2.5. Statisticalanalysis

ThedatawerestatisticallyanalysedwiththeANOVAprocedureofthestatisticspackageNCSS2007(Hintze,Kaysville, Utah,USA)usingthegenerallinearmodel.Themodelincludeddietarynutrientlevel(H,L),BA(+,−)andtheirinteractionas ﬁxedfactors,andblockasrandomfactor.Theindividualpigwasconsideredtheexperimentalunit.Carcassweightservedas acovariateintheANOVAanalysingtheresultsofthebonebreakingtest.Whentheinteractionbetweenthedietarynutrient level(NL)andBAhadaPvalue<0.05,themeansofthefourtreatmentswerecomparedusingtheNewman–Keulstest.The digestibilityandretentiondataofthebalancestudyaswellastheserumCa,P,APandSCLdataofthefeedingtrial,which weredeterminedforeachanimalonthreeoccasions,werefurtheranalysedwiththerepeated-measuresANOVAusingthe mixedmodel.

Pearson’slinearcorrelationcoefﬁcientrwascalculatedtodeterminetheassociationamongtheserumconcentrationof AP(aboneformationmarker)andcrosslaps(aboneresorptionmarker).DifferencesatP<0.05wereconsideredstatistically signiﬁcant,whereasdifferenceswith0.05≤P<0.10wereconsideredtendencies.

3. Results

Theanalysednutrientcontentoftheexperimentaldiets(Table1)correspondedtotheexpectedcontent.Thedietary electrolytebalance(dEB)waslowerindietsLthanindietsH,mainlybecausedietsLcontainedlesspotassium(K)andmore chloride(Cl)thandietsH.

3.1. Balancestudy 3.1.1. Feedintake

DuetohigherfeedrefusalsofthepigletsreceivingdietsL,feedintakeduringtheﬁrstbalanceperiodat25kgBWwas13%

lower(P<0.05,Table2)withdietsLcomparedtodietsH.At40and55kgBW,dietarynutrientlevelhadnoeffect(P>0.05)

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Table2

Dailyfeedintake(kgfeedwithadrymattercontent0.88g/g)andurinarypHofpigsat25,40and55kgbodyweight(BW)feddietswithhighorlow calcium,phosphorusandproteincontentwithorwithoutbenzoicacidaddition(balancestudy,n=4)^a.

Nutrientlevel(NL) High Low S.E.M Pvalues

Benzoicacid(BA) − + − + NL BA NL×BA

Feedintake

25kgBW 1.05 1.14 0.88 1.01 0.05 0.01 0.05 0.61

40kgBW 1.47 1.51 1.24 1.47 0.08 0.14 0.14 0.28

55kgBW 1.93 1.98 1.80 1.90 0.06 0.09 0.24 0.69

pHofurine

25kgBW 5.29âb 5.46âb 5.72â 5.15^b 0.12 0.57 0.12 0.01

40kgBW 6.51 5.70 5.85 5.13 0.25 0.03 0.01 0.87

55kgBW 6.71 5.97 6.21 5.57 0.26 0.11 0.02 0.85

aWithinrow,meanvaluescarryingnocommonletterdiffer(P<0.05).

onfeedrefusals.Benzoicacidtendedtoreduce(P=0.05)feedrefusalsat25kgBW,buthadnoeffect(P>0.05)at40and55kg BW.

3.1.2. pHofurine

AdditionofBAreduced(P<0.05,Table2)theurinarypHofthepiglets(at25kgBW)receivingdietLandofthegrower pigs(at40and55kgBW)receivingdietsLandH(P<0.05).TheintakeofdietsLlowered(P<0.05)urinarypHat40kgBW.

3.1.3. Mineralbalance

Overallthreecollectionperiods,thepigsreceivingdietsLingested34%lessCaand35%lessPandhadalower(P<0.01, Tables3and4)faecalCaandPoutputthanthepigsondietsH,buttheyretainedahigherproportionoftheingestedminerals (P<0.01).Despitethehigherretentionrate(gretained/gingested),thepigsondietsLretained27%lessCaand24%lessP (P<0.01)thanthepigsondietsH.Overallthreecollectionperiods,dietsLtendedtodecreaseurinaryCaexcretion(P=0.05) andat25kgBWtheydecreased(P<0.05)urinaryPexcretion.Overall,supplementationwithBAincreased(P<0.01)urinaryCa excretionbuthadnoeffect(P>0.05)ondigestibilityandretentionofCa.UrinaryPexcretionwasalsoincreased(P<0.01)byBA supplementation.IncontrasttoCa,apparentdigestibilityandretentionofPwereincreased(P<0.05)byBAsupplementation,

Table3

Intake,faecalandurinaryexcretion,retention(g/d)andapparenttotaltractdigestibilityofCaofpigsat25,40and55kgofbodyweight(balancestudy, n=4).

25kgBW

Intake 9.47 9.62 4.73 5.37 0.327 <0.01 0.26 0.46

Faecaloutput 4.61 4.84 1.21 1.87 0.216 <0.01 0.07 0.35

Urinaryoutput 0.08 0.12 0.03 0.06 0.022 0.12 0.36 0.58

Retention 4.79 4.66 3.48 3.44 0.259 <0.01 0.81 0.84

Digestibility 0.51 0.50 0.75 0.66 0.026 <0.01 0.07 0.18

Retention/intakeratio^a 0.50 0.48 0.74 0.64 0.026 <0.01 0.06 0.18

40kgBW

Intake 10.37 10.65 7.13 7.75 0.527 <0.01 0.41 0.76

Faecaloutput 5.10 4.92 2.26 2.46 0.246 <0.01 0.95 0.46

Urinaryoutput 0.09 0.17 0.05 0.12 0.024 0.10 0.01 0.75

Retention 5.19 5.56 4.82 5.17 0.428 0.40 0.42 0.98

Digestibility 0.51 0.54 0.69 0.68 0.021 <0.01 0.58 0.49

55kgBW

Intake 13.45 13.86 10.15 9.87 0.353 <0.01 0.84 0.36

Faecaloutput 6.54 6.61 3.27 3.32 0.303 <0.01 0.85 0.98

Urinaryoutput 0.10 0.13 0.08 0.24 0.052 0.39 0.08 0.21

Retention 6.81 7.12 6.80 6.31 0.270 0.16 0.75 0.17

Digestibility 0.51 0.52 0.68 0.66 0.001 <0.01 0.86 0.46

Overall

Intake 11.10 11.38 7.21 7.67 0.365 <0.01 0.32 0.81

Faecaloutput 5.35 5.40 2.21 2.52 0.194 <0.01 0.33 0.47

Urinaryoutput 0.09 0.14 0.06 0.11 0.025 0.05 <0.01 0.91

Retention 5.66 5.84 4.93 5.00 0.233 <0.01 0.59 0.82

Digestibility 0.52 0.52 0.71 0.67 0.014 <0.01 0.28 0.12

aTheretention/intakeratiowascalculatedbydividingtheamountofnutrientretainedbytheamountofnutrientingestedperday.

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Table4

Intake,faecalandurinaryexcretion,retention(g/d)andapparenttotaltractdigestibilityofPinpigsat25,40and55kgofbodyweight(balancestudy, n=4)^a.

25kgBW

Intake 7.08 7.59 3.71 4.17 0.251 <0.01 0.09 0.98

Faecaloutput 3.22 3.31 0.85 1.01 0.185 <0.01 0.49 0.81

Urinaryoutput 0.35 0.58 0.17 0.40 0.005 0.01 <0.01 0.81

Retention 3.51 3.69 2.69 2.76 0.206 <0.01 0.54 0.85

Digestibility 0.55 0.56 0.77 0.76 0.028 <0.01 0.98 0.55

Retention/intakeratio^b 0.50 0.49 0.73 0.66 0.027 <0.01 0.21 0.35

40kgBW

Intake 7.65 7.84 4.95 5.79 0.359 <0.01 0.18 0.38

Faecaloutput 3.80 3.55 1.34 1.38 0.172 <0.01 0.55 0.39

Urinaryoutput 0.18 0.37 0.20 0.41 0.068 0.66 <0.01 0.89

Retention 3.67 3.93 3.41 4.00 0.329 0.79 0.22 0.65

Digestibility 0.50 0.55 0.73 0.76 0.024 <0.01 0.16 0.80

55kgBW

Intake 10.07 10.31 7.16 7.49 0.253 <0.01 0.29 0.87

Faecaloutput 4.77 4.41 1.93 1.59 0.223 <0.01 0.15 0.98

Urinaryoutput 0.49^b 0.55^ab 0.44^b 1.04^a 0.125 0.11 0.02 0.05

Retention 4.81 5.34 4.79 4.86 0.218 0.27 0.20 0.30

Digestibility 0.53 0.57 0.74 0.79 0.023 <0.01 0.05 0.87

Overall

Intake 8.27 8.58 5.17 5.82 0.261 <0.01 0.08 0.53

Faecaloutput 3.89 3.72 1.33 1.31 0.144 <0.01 0.40 0.51

Urinaryoutput 0.34 0.50 0.26 0.57 0.054 0.88 <0.01 0.19

Retention 4.13 4.51 3.59 4.00 0.187 <0.01 0.04 0.96

Digestibility 0.53 0.57 0.75 0.77 0.017 <0.01 0.04 0.65

aDifferentlowercaseandcapitallettersassuperscriptswithinarowindicatedifferencesatP<0.05and<0.01,respectively.

bTheretention/intakeratiowascalculatedbydividingtheamountofnutrientretainedbytheamountofnutrientingested.

butthisdidnotinﬂuence(P>0.05)theproportionofingestedPwhichwasretained(retention/intakeratio).Aninteraction (P=0.05)betweennutrientlevelandBAwasobservedforurinaryPexcretionat55kgBW,indicatingthattheeffectofBAon urinaryPoutputwasmoremarkedatthelowdietarynutrientlevel.

3.2. Feedingtrial

3.2.1. Animalperformance

TheintakeofdietsLreducedthegrowthrate(P<0.05)ofthepigletsby7%,butneitheraffectedthegrowthrateduring thefatteningperiod(Table5)northedailyweightfromthebeginningoftheexperimentuntilslaughter(P>0.05,datanot shown).Neitherfeedintakenorfeedconversionwasinﬂuencedbyanytreatment.

3.2.2. Serumanalytes

TheANOVAforrepeatedmeasuresshowsthattheintakeofdietsLgloballyincreased(P<0.01)theserumCaconcentration andtheactivityoftheboneformationmarkerAPandtendedtodecrease(P=0.06)theconcentrationoftheboneresorption

Table5

Effectsofnutrientlevelandbenzoicacidintakeonanimalperformance(feedingtrial,n=16)).

Nutrientlevel(NL) H L S.E.M. Pvalues

Piglets(13–25kgBW)

InitialBW,kg 12.9 12.9 13.0 12.8 0.35 0.96 0.69 0.77

Feedintake,kg/d 0.94 0.94 0.91 0.91 0.026 0.30 0.88 0.88

ADG,g 594 591 539 562 17 0.02 0.57 0.45

Feedconversion,kg/kg 1.68 1.66 1.67 1.73 0.044 0.43 0.55 0.32

Growingpigs(25–60kgBW)

Feedintake,kg/d 1.66 1.79 1.68 1.71 0.049 0.54 0.15 0.32

ADG,g 784 818 814 806 13 0.51 0.32 0.12

Feedconversion,kg/kg 2.13 2.18 2.05 2.13 0.046 0.21 0.21 0.84

BW:bodyweight;ADG:averagedailyweightgain.

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Table6

Effectsofnutrientlevelandbenzoicacidintakeonserumanalytesandbonetraits(feedingtrial,n=16)^a.

Nutrientlevel(NL) H L S.E.M. Pvalues

Serumanalytes Overall

Ca,mmol/l 2.75 2.78 2.85 2.82 0.022 <0.01 0.79 0.07

P,mmol/l 3.15^B 3.29^A 3.18^B 3.20^B 0.036 0.26 <0.01 0.01

AP,U/l 170 179 177 207 7 0.01 <0.01 0.12

SCL,ng/l 591 569 559 547 19 0.06 0.24 0.71

25kgBW

Ca,mmol/l 2.79 2.82 2.91 2.88 0.027 <0.01 0.94 0.26

P,mmol/l 3.24 3.35 3.22 3.26 0.044 0.21 0.09 0.46

AP,U/l 198^B 211^B 196^B 249^A 10 0.08 <0.01 0.05

SCL,ng/l 545 456 511 476 20 0.76 <0.01 0.23

40kgBW

Ca,mmol/l 2.74 2.75 2.83 2.78 0.023 0.01 0.38 0.21

P,mmol/l 3.19^b 3.33^a 3.21^b 3.19^b 0.035 0.09 0.11 0.02

AP,U/l 167 182 171 201 6 0.07 <0.01 0.23

SCL,ng/l 556 611 544 564 20 0.21 0.11 0.44

60kgBW

Ca,mmol/l 2.71 2.77 2.79 2.80 0.02 0.01 0.11 0.17

P,mmol/l 3.02^b 3.20â 3.11âb 3.13âb 0.03 0.91 <0.01 0.02

AP,U/l 149 150 157 167 7 0.06 0.43 0.48

SCL,␮g/l 673 630 620 614 30 0.18 0.33 0.47

Bonetraits,tibia

BMC,midshaft,mg/cm 232 226 212 221 5 0.02 0.79 0.14

Breakingstrength,N 770 734 704 717 17 0.04 0.56 0.21

AP:alkalinephosphatase;SCL:serumcrosslaps.

aDifferentlowercaseandcapitallettersassuperscriptswithinarowindicatedifferencesatP<0.05and<0.01,respectively.

markerserumcrosslaps(SCL)intheserum(Table6).DietsL+lowered(P<0.01)theserumPconcentrationcomparedtodiets H+.BenzoicacidincreasedserumAPactivity(P<0.01)andincreasedtheserumPconcentrationofthepigletsreceivingdiets H(P<0.01).TheeffectofBAontheboneturnovermarkersdecreasedwithadvancingbodyweight:Benzoicacidloweredthe SCLconcentrationat25kgBW(P<0.01)onlyandincreasedtheAPactivityat25andat40kgBW(P<0.01),butnotat60kg BW.TheAPactivityandtheSCLconcentrationofthe64pigletswerenegativelycorrelatedat25kgBW(r=−0.36;P<0.01), butnotat40kgBW(r=0.16;P=0.22)andat60kgBW(r=−0.04;P=0.77).Therewasnotreatmenteffectontheconcentration oftheboneformationmarkerOCandontheactivitiesoftheenzymesAST,ALTandGGT(datanotshown).

3.2.3. Bonetraits

TheintakeofdietsLreducedthebreakingstrength(P<0.05)oftheMc4andtheBMCofthemidshaftofthetibiaby5%

(P<0.05;Table6),whereasBAintakehadnoeffectonanybonetrait.TherewasnotreatmenteffectontheBMCofthedistal tibialepiphysisandontheconcentrationofash,CaandPinthefatfreedrymatteroftheMc3(P>0.05,datanotshown).

4. Discussion

ThepigsreceivingthedietsLdigestedCaandPmoreefficientlythanthepigsondietsH,whichcanbeattributedmainly tothe1500UphytaseaddedperkgdietL,anamountknowntocleaveapproximately1gofPfromphytate(Düngelhoef andRodehutscord,1995;Kornegay,2001).ThePdigestibilitycoefficientof0.76indietsLdeterminedinthebalancestudy isslightlyhigherthanthedigestibilitycoefficientof0.70calculatedonthebasisoftabularvaluesfortheingredientsand phytatePhydrolysedbyphytase,butisintheorderofmagnitudeobservedbyLeietal.(1993a,b),Youngetal.(1993), AlmeindaandStein(2010)andLétourneau-Montminyetal.(2010)inpigsfedphytasesupplementeddiets.Similarly,P digestibilityofdietsHdeterminedinthebalancestudywashigherthanthecalculatedvalue(0.55vs.0.50onaverage).In viewofthehighvariabilityofPdigestibilitydataofdietscontainingphytase(JohansenandPoulsen,2003)andthefactthat Pdigestibilitydeterminedbytotalcollectionoffaeces–themethodusedinourexperiment–oftenyieldshighervalues thanmethodsusinganindigestiblemarker(Agudeloetal.,2010;Blaabjergetal.,2010),theexperimentallydeterminedP digestibilitydatacorrespondquitewelltothecalculateddata.Basedonthedigestibilityvaluesobtainedinthebalancestudy, thepigletandthegrowerdietsLcontainedapproximately0.23gand0.22gdigestiblePperMJDE,respectively.Theavailable Prequirementformaximumgrowthofpigletsweighing10–20kgandofgrowingpigsweighing20–50kgis3.2gand2.3g perkgdietcontaining14.2MJ/kgDE(NRC,1998),whichcorrespondstoapproximately0.20gand0.15gdigestiblePperMJ DE,usingthefactor0.9toconvertavailablePtodigestibleP(Jongbloedetal.,1991).ThePrequirementformaximisingbone strengthandboneashcontentishigherthantherequirementforgrowth(NRC,1998).ThedigestiblePconcentrationofthe experimentalpigletdietsLthuswasnotsufficienttomaximisebonemineralisation,whereasthegrowerdietsLcontained sufficientamountsofdigestibleP.

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ThisevaluationbasedontheNRC(1998)dataisconﬁrmedbytheresultsofthebalancestudy.ThepigletsfeddietsL retained27%lessCaand24%lessPthanthepigletsondietsH,whichimpliesthatat25kgBWCaandPrequirementsfor maximummineralaccretionwerenotmetbydietsL,whereasretentionofCaandPdidnotdifferbetweenpigsondietsH andpigsondietsLat40and55kgBW.

Benzoicacidaddedtothegrowerdietsat10g/kgsigniﬁcantlyreducedthepHofurine,indicatingthattheadditive markedlyincreasedthedietaryacidload.AdropinurinarypHwasalsoobservedwhen5g/kgBAwereaddedtothepiglet dietL,whereasthebufferingcapacityofthepigletdietHcontaininghigherconcentrationsofCa,PandCPpresumably preventedthedropinurinepH.

BenzoicacidincreasedtheapparentdigestibilityofPbuthadnoeffectontheproportionofingestedCaandPwhichwas retained(retention/intakeratio).Bycontrast,Mrozetal.(2000),whostudiedtheeffectsofformic,fumaricandbutyricacid andofCa-benzoateaddedtofeedswithadEBof280meq/kgongrowingpigs,observedthat20g/kgbenzoateincreased thedigestibilityandretention/intakeratioofCaandhadnoeffectonPdigestibility,butdecreasedtheretentionofP.Sauer etal.(2009)reportedthattheadditionof10and20g/kgofbenzoicacidtothedietofyounggrowingpigsreceivingadiets supplementedwithsodiumbicarbonate(dEB200mEq/kg)linearlyincreasedthedigestibilityandretention/intakeratioof bothCaandP.Thereasonsforthesecontradictoryobservationsareunknownbutcouldberelatedtodifferencesbetween theexperimentaldietsintheirmineralconcentrationanddEB,thepresenceoforganicacidsotherthanbenzoicacidaswell astheabsenceofmicrobialphytaseinthedietsusedbyMrozetal.(2000)andSaueretal.(2009).

TheincreasedurinaryCaandPexcretionofourpigsreceivingdietssupplementedwithBAisinaccordancewiththe increasedurinaryPoutputinpigsingestingBAreportedbySaueretal.(2009)andtheincreasedurinaryoutputofeither Caalone(PatienceandChaplin,1997)orofbothCaandP(BuddeandCrenshaw,2003)inpigsfeddietsthatwereacidiﬁed withchloride.PigsthusreactsimilarlyasrodentsandhumanswhorespondtoametabolicacidosisbyincreasingurinaryCa andPexcretion(Osther,2006;Novicetal.,2008).SinceurinaryPoutputwaslowcomparedtoPintakeandfaecalPoutput, theincreasedurinaryPexcretioncausedbyBAwastoolowtoaffectPretentioninourpigs.Thereduceddailyweightgain ofthepigletsoftreatmentsLwasprobablycausedbythelowPintake,sinceadietaryPconcentrationslightlybelowNRC requirementhasbeenshowntoreducethegrowthrateofpigletswithoutaffectingtheirfeedintake(ReinhartandMahan, 1986).TheelevatedserumCaconcentrationobservedinpigsondietsLcomparedtothepigsondietsHreﬂectthemarginal Pintake,alowPstatusbeingassociatedwithanincreasedserumCaandadecreasedserumPconcentrationinpigs(Reinhart andMahan,1986;Scottetal.,1994;Liesegangetal.,2002).ItisunclearwhyinthepresenttrialtheserumPconcentration decreasedinresponsetoalowPintakeonlyinthepigsreceivingBAandwhyBAincreasedtheserumPconcentrationin pigletsreceivingdietsHexclusively.

TheenzymeAPoccursathighconcentrationsinosteoblastsandlivercells,andanelevatedserumactivityisasignof alteredosteoblastorliverfunction.TreatmenteffectsonserumAPinthepresentexperimentcanbeattributedtoaltered osteoblastfunction,sincetheactivitiesoftheenzymesALT,ASTandGGTintheserum,whichareusedasbiomarkersforliver dysfunction,wereunaffectedbythedietarytreatments.TheactivityofAPisincreasedintheserumofPdeficientgrowing pigsandisnegativelycorrelatedwithbonestrength(Boydetal.,1983;KochandMahan,1985;Epkeetal.,2002;Liesegang etal.,2002),whereastheconcentrationoftheboneresorptionmarkerSCLisincreasedinpigsfedalowPdiet(Bühleretal., 2010).TheincreasedAPactivityandthedecreasedSCLconcentrationintheserumofthepigsreceivingdietsLthusindicates thatboneformationandremodellingwereaffectedbythelowPintake.TheincreaseinserumAPactivityat25and40kgBW andthedecreasedSCLconcentrationat25kgBWcausedbyBAsuggeststhatBAalsoaffectedbonemetabolismoftheyoung animals,especiallyofthosereceivingdietsL.NotreatmenteffectonserumOCconcentrationwasobservedinourstudy.OC doesnotseemtobeasensitivebiomarkerforalteredbonemetabolisminPdeficientpigs,asinfourofsixexperimentsits serumconcentrationdidnotchangeinresponsetoPdeficiency(Nicodemoetal.,1998;Liesegangetal.,2002;Bühleretal., 2010;Létourneau-Montminyetal.,2010;vs.Carteretal.,1996;Shawetal.,2006).

TheelevatedserumAPactivityat60kgBWaswellasthereducedbonebreakingstrengthandBMCobservedinthe pigsondietsLimplythattheseanimalswereunabletofullycompensatethelowPintakeduringthepigletrearingperiod, althoughthecalculateddigestiblePcontentofthegrowerdietsLwasabovetherequirementassessedbyNRC(1998).

ThisresultsupportsthefindingofVarleyetal.(2010a,b)thatthebonemineralcontentofslaughterpigsfedinsufficient amountsofPduringtheweanerperiodisreducedevenifthefeedofferedlateroncontainssufficientamountsofP.Maximum incorporationofCaandPintothebonesduringtheweanerperiodthusseemstobeprerequisiteforsubsequentmaximum bonemineralisation.AlthoughdietsLreducedbonebreakingstrengthandBMD,theydidnotaffecttheconcentrationof ash,CaandPinthebonedrymatterofthepigs.Thesamephenomenon–asignificantincreaseinbonebreakingstrength, butnotinbonePconcentrationinpigletswhosedietwassupplementedwithphytase–wasobservedbyColumbusetal.

(2010).ThefactthatPdeficiencycausesosteopenia,thatisareductioninboththeashandtheorganicmatterofthebone (KochandMahan,1985;Hagemoseretal.,2000)decreasesthesensitivityofthebonemineral/DMratioasanindicatorofP deficiency.Boneash/volumeorboneP/volumeratiohavethereforebeenrecommendedtocharacterisebonemineralisation inPdeficientpigs(Hagemoseretal.,2000).BenzoicacidneitheraffectedAPactivityat60kgBWnorthebonetraits.Any possiblenegativeeffectofBAonbonemetabolismofthepigletsandoftheyounggrowinganimalsthusdisappearedduring thelastperiodofthegrowerperiod.ThereportedeffectsofBAonbonetraitsingrowingpigsareinconclusive.Increasing amountsofBAaddedtodietscontainingtherecommendedamountsofCPandPhadnoeffectontotalboneash,butdecreased theashconcentrationinthefemoralDMofpigsweighing40kg(Saueretal.,2009),suggestingapossiblenegativeeffectof BAonbonemineralisation.Similarly,Bühleretal.(2010)reportedthatBAtendedtoreduceashconcentrationintheDMof