The use of chestnuts (Castanea sativa Mill.) as a source of resistant starch in the diet of the weaned piglet

ContentslistsavailableatSciVerseScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

The

use

of

chestnuts

(Castanea

sativa

Mill.)

as

a

source

of

resistant

starch

in

the

diet

of

the

weaned

piglet

R.S.

Ribeiro

_,

_M.

_Pinho

_,

_L.

_{Falcão-Cunha}

_,

_J.P.B.

_Freire

a_{CEER,InstitutoSuperiordeAgronomia,UniversidadeTécnicadeLisboa,TapadadaAjuda,1349-017Lisboa,Portugal}

b_{CIISA,FaculdadedeMedicinaVeterinária,UniversidadeTécnicadeLisboa,AvenidadaUniversidadeTécnica,1300-477Lisboa,Portugal}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21January2013

Receivedinrevisedform12April2013 Accepted15April2013 Available online xxx Keywords: Bacterialactivity Chestnuts Digestibility Intestinalmorphology Piglets Resistantstarch

a

b

s

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Twenty-fourpiglets,withameanweightof6.11kg,wereallocatedtothreeexperimental diets(8pigletsperdiet).Thestarchcontentoftheexperimentaldietswas39%consisting eitherof100%wheatstarch(CD),50%wheatstarchand50%chestnutstarch(CN50)or100% chestnutstarch(CN100).Thepigletsreceivedthesedietsfor35days.Totaltractapparent digestibility(TTAD)wasmeasuredwhenpigletswereaged32–39daysand46–53days. Afterwards,theanimalsweresacrificed,theirgastrointestinalcompartmentsweighed,and thecontentsoftheircaecumandcoloncollected.Dietdidnotinfluencethefeedintake orgrowthrateofthepiglets,buthadasignificanteffectontheirfeedconversionratio whichwas1.27,1.30and1.37fordietsCD,CN50andCN100,respectively(P=0.042).Piglets receivingtheCN50dietexhibitedlowerfaecalscorescomparedtoCDandCN100groups (2_{=39.31,P<0.001).Therewasatrend(P=0.067)towardsanincreaseintheTTADofdry}

matter(DM)inthetwochestnut-containingdiets,butonlywhenpigletswereyounger (32–39days).TheTTADofcrudeprotein(CP)wasdecreased(P<0.001)inCN100group rel-ativetoCDandCN50groups,independentlyofage.RelativetotheCDgroup,thecoefficient ofTTADofNDFwasincreasedby0.18unitsintheCN50groupand0.25unitsintheCN100 group,whenthepigletswereaged32–39days.Thesedifferenceswerereducedto0.09and 0.17units,respectively,whenthepigletswereaged46–53days.Thereweretendencies (P<0.10)towardsincreasesinthefullandemptyweightsofthelargeintestineinCN100 group.Moreover,diethadnosignificanteffectonvillusheightandwidth.Inthejejunum, cryptdepthwasreducedinCN100group(P=0.006),whilethevillusheight:cryptdepthratio wasincreasedwiththeCN50andCN100diets(P=0.018).Diethadnosignificanteffecton DMandpHofthecaecalorcoloniccontents.Inthecaecum,theconcentration(mmoll−1_)of

propionicacidwas5.15,9.43and14.7(P=0.042)fordietsCD,CN50andCN100,respectively. Theconcentration(mmoll−1)ofbutyricacidwas3.50,4.90and9.84(P=0.025).Conversely, diethadnosignificanteffectontheconcentrationofshortchainfattyacidsinthecolon. Inconclusion,chestnutscouldbeusedasanalternativesourceofRSinthedietofweaned piglets,althoughtheirfeedconversionratio(FCR)maybeworsened.

© 2013 Elsevier B.V. All rights reserved.

Abbreviations: ADFom,aciddetergentfibreexpressedexclusiveofresidualash;ADL,aciddetergentlignin;aNDFom,neutraldetergentfibreassayed withheatstableamylaseandexpressedexclusiveofresidualash;CD,controldiet;CN50,dietcontaining50%wheatstarchand50%chestnutstarch;CN100, dietcontaining100%chestnutstarch;CP,crudeprotein;CTTAD,coefficientoftotaltractapparentdigestibility;DM,drymatter;FCR,feedconversionratio; LR,Landrace;NDF,neutraldetergentfibre;NSP,non-starchpolysaccharides;RDS,rapidlydigestiblestarch;RS,resistantstarch;SCFAs,shortchainfatty acids;SD,standarddeviation;SDS,slowlydigestiblestarch;TDF,totaldietaryfibre;TTAD,totaltractapparentdigestibility.

∗ Correspondingauthor.Tel.:+351213653408. E-mailaddress:[email protected](J.P.B.Freire).

0377-8401/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved.

1. Introduction

Thechestnut(CastaneasativaMill.)isatraditionalnutfromEuropeanMediterraneancountries,andoneofthemost popularintheworld.InEurope,Portugalisoneofthebiggestproducers;in2010,itproducedapproximately22,000tonnes, representing3%ofworldproduction(INE,2011).

Starchaccountsfor50–80%ofthedrymattercontentofEuropeanchestnutscultivars,whileproteinrepresentsbetween 6.0and8.6%(Míguelezetal.,2004).AccordingtoPizzoferratoetal.(1999),21.5%ofrawchestnutstarchtakestheformof rapidlydigestiblestarch(RDS),20.9%isslowlydigestiblestarch(SDS),and57.6%canbetermedresistantstarch(RS).

Resistantstarchprovidesafermentablesubstrateforcaecalandcolonicmicroflora.Thus,includingRSinthedietwill influencemicrobialmetabolisminthelargeintestine;increasingthesizeofthebifidobacteriaandlactobacillipopulations (Birdetal.,2007).Recently,itwasshownthatincluding7%ofrawpotatostarch(asasourceofRS)inthedietofweanedpiglets improvedtheirfaecalscores,andmodifiedtheBacteroidetes,FirmicutesandProteobacteriapopulationsinthecolonbutnot intheileum(Bhandarietal.,2009).Theseprebioticactionsarecomparabletothoseofestablishedprebioticoligosaccharides, notablytheinsulin-typefructans(Robertfroid,2001).

Inthelargeintestineofpiglets,theconcentrationofshortchainfattyacids(SCFAs)–inparticularbutyrate–increaseswith theinclusionofRSinthediet(HedemannandBachKnudsen,2007).Moreover,dietaryRShasbeenshowntopreventpathogen infectionsanddiarrhoeainmonogastricanimals(Williamsetal.,2001),andtoimprovecolonicmucosalmorphologyand integrity(HedemannandBachKnudsen,2007;Nofraríasetal.,2007).

Inpigs,dietaryRSincreasesfaecalnitrogenexcretionanddecreasesthetotaltractapparentdigestibility(TTAD)ofcrude protein(CP)(Rideoutetal.,2008).Seemingly,theincreaseinfaecalnitrogenafterRSconsumptionisnotduetoanyeffect onthesecretionofmucinorendogenousnitrogeninthesmallintestine(Moreletal.,2005).Instead,itcanbeattributedto anincreaseinbacterialnitrogenthroughthestimulationofbacterialgrowthinthehindgut(HeijnenandBeynen,1997).

Inthepresentstudy,chestnutswereusedasanalternativesourceofRSinthedietofweanedpiglets.Controlanimals werefedastandarddietcontaining100%wheatstarch,whileexperimentalanimalsreceivedoneoftwoalternativediets. Onedietcontained50%wheatstarchand50%chestnutstarch,whiletheothercontained100%chestnutstarch.Theobjective wastodeterminetheeffectofthesedietsongrowthperformance,totaltractapparentdigestibility,smallintestinemucosal morphologyandhindgutmicrobialactivity.

2. Materialsandmethods

2.1. Animalsandhousing

Twenty-fourmalepiglets(Duroc×LR),weanedat3weeksofage,withameanweightof6.11kg(SD=0.49kg)were used.Pigletswereassignedto3groups(8pigletspergroup),accordingtoliveweight.Eachgroupreceivedoneofthree experimentaldiets.Animalswereindividuallyhousedinmetaboliccages(110cm×80cm)equippedwithstainlesssteel platesforseparatecollectionoffaecesandurine.Roomtemperaturewasinitiallysetat28◦C,andwasdecreasedby2◦C eachweekuntilitreachedafinaltemperatureof24◦C.

Afteranacclimatizationperiodofthreedays,thepigletscompleted5weeks(35days)ofexperimentalobservation. Pigletswereweighedatthebeginningofeachweek,andfaeceswerecollectedduringtwoperiodsof7days(secondand fourthexperimentalweeks)tocalculateTTAD.Theconsistencyofthefaecalmatterwasrecordeddaily,accordingtothe scaleproposedbyMarquardtetal.(1999):0=normal,1=softfaeces,2=moderatediarrhoeaand3=liquiddiarrhoea.

Attheendoftheexperiment,thepigletsweresacrificedinthemorning(08:00)byelectrocutionfollowing12hoffood deprivation.Theweightofthepancreas,theliver,andthecompartmentsofthegastrointestinaltractwerethenrecorded. Thelengthofthesmallandlargeintestinewasalsomeasured.Thecontentsofthecaecumandcolonwereimmediately collectedfortheanalysisofpH,DMcontent,microbialenzymaticactivityandlevelsofSCFAs.Tissuesamplesfromthree segmentsofthesmallintestinewerealsocollected:theduodenum(10cmfromthepylorus),jejunum(5.5mfromthe pylorus)andileum (60cmbeforetheileo-cecalvalve).Thetissue sampleswerefixedin 10%neutral buffered forma-lin,embeddedinparaffinwax,andthensectionedandmountedformicroscopicexaminationoftheintestinalvilliand crypts.

2.2. Chestnutspreparationanddietsformulation

Freshchestnutswereobtainedandmechanicallypeeledtoremovethetegumentandpericarp.Thechestnutswere thendriedat40◦Cfor48h.Thisreducedtheirmoisturecontentfromaround50%to10–12%.Thedriedchestnutswere milledthrougha2mmscreenbeforetheywereaddedtothediets.TheirchemicalcompositionisreportedinTable1.Three experimentaldietswereprepared(Table2).Thestarchcontentoftheexperimentaldietswas39%;consistingofeither100% wheatstarch(CD),50%wheatstarchand50%chestnutstarch(CN50),or100%chestnutstarch(CN100).Thedietswere formulatedaccordingtotheNRC(1998)recommendationsfor5–20kgpiglets.Theywerepelleted(3mm)andgiventothe pigletsonasinglemealperdaybasis.Animalshadaccesstowateradlibitum.

Table1

Chemicalcompositionofwheatandchestnuts(gkg−1_).

Wheat Chestnutsa Drymatter 863 881 Crudeprotein 99.3 60.1 Starch 586 625 TDFb _{123 141} aNDFomc _{121 170} ADFomd _{24.3 32.6} a_Driedat40◦_C.

b_{TDF=totaldietaryfibre,determinedusingagravimetric-enzymaticprocedureandexpressedexclusiveofproteinandash.} c_{aNDFom=neutraldetergentfibreassayedwithheatstableamylaseandexpressedexclusiveofresidualash.}

d_{ADFom=aciddetergentfibreexpressedexclusiveofresidualash.} 2.3. Chemicalanalysis

Aftercollection,faecesweredriedat70◦Cfor48h.Driedfaecesandfeedsamplesweremilledthrougha1mmscreen priortoanalysis.Forbothfaecesandfeedsamples,DMcontentwasdeterminedbyovendryingat104◦Cfor24h.Ashcontent wasdeterminedbyburningovernightat550◦C.CPwasmeasuredusingtheKjeldahlmethod(976.95;AOAC,1995).

Analysisofneutraldetergentfibre(aNDFfom),aciddetergentfibre(ADFfom)andaciddetergentlignin(ADL)was per-formedsequentiallyusingthecruciblessystem,andexpressedwithoutresidualash.Forfeedsamples,athermo-stable ␣-amylasewasused(VanSoestetal.,1991).Totaldietaryfibre(TDF)inthewheatandchestnutswasdeterminedwith agravimetric-enzymaticprocedure(AOAC,2000,991.43),using␣-amylase,protease,andamyloglucosidasetreatments (MegazymeInt.IrelandLtd.,Wicklow,Ireland).Thestarchcontentofthediets,wheatandchestnutswasanalyzed enzymat-icallyaccordingtothemethoddescribedbyThivendetal.(1965).

ThepHoftheintestinalcontentswasmeasuredwithaglasselectrodepHmetre(Metrohm744m).SCFAs(aceticacid (C2),propionicacid(C3)andbutyricacid(C4))andbranchfattyacids(isobutyricacid(IC4)andisovalericacid(IC5))were quantifiedinthecaecalandcoloniccontentsbygaschromatography,followingthemethodsoutlinedbyFreireetal.(2003). Bacterialfibre(cellulolytic,xylanolyticandpectinolytic)degradationinthecaecalandcoloniccontentsweremeasuredas describedbyFalcão-e-Cunhaetal.(2004),andexpressedasthequantityofsugars(mg)liberatedafteronehourofincubation at37◦C.

Table2

Compositionoftheexperimentaldiets.

Diets CD CN50 CN100 Ingredients(gkg−1) Wheat 661 341 – Chestnutsa _{– 310 631} Soybeanmeal48 160 160 160 Driedwhey 50.0 50.0 50.0 Fishmeal 70.0 80.0 100 Soybeanoil 30.0 30.0 30.0 l-Lysine 4.0 4.0 4.0 dl-Methionine 2.0 2.0 2.0 Calciumcarbonate 7.0 7.0 7.0 Dicalciumphosphate 9.0 9.0 9.0 Sodiumchloride 2.0 2.0 2.0 MVCb _{5.0 5.0 5.0} Chemicalcomposition(gkg−1) Drymatter 909 911 919 Organicmatter 853 854 856 Ash 56.0 57.4 60.9 Crudeprotein 225 214 213 aNDFomc _{104 123 110} ADFomd _{32.2 33.0 37.5} ADLe _{7.8 10.6 9.0} Starch 390 395 394 a_Driedat40◦_C.

b_{Mineralandvitamincomplex.Compositionbykgoffeed:Vit.A:25000UI;Vit.D3:2000UI;Vit.E:20UI;Vit.C:200mg;Vit.B1:1,5mg;Vit.B2:} 5mg;Vit.B3:30mg;Vit.B5:15mg;Vit.B6:2,5mg;Vit.B9:0,5mg;Vit.B12:0,03mg;Vit.K3:1mg;Vit.H2:80mg;coline:300mg;I(potassiumiodate): 1mg;Mn(manganousoxide):50mg;Fe(ferrouscarbonate):120mg;Zn(zincoxide):140mg;Cu(cupricsulfate):160mg;Se(sodiumselenite):0.3mg; Co(cobaltcarbonate):0.5mg.

c_{aNDFom=neutraldetergentfibreassayedwithheatstableamylaseandexpressedexclusiveofresidualash.} d_{ADFom=aciddetergentfibreexpressedexclusiveofresidualash.}

Table3

Effectofdietaryproportionofchestnutsongrowthperformanceandfaecalscoresofpigletsfeddifferentamountsofchestnutsfor35dayspost-weaning.

Diet SEMA _X2A _PA CD CN50 CN100 Initialweight(kg) 6.05 6.10 6.19 0.10 – 0.840 Finalweight(kg) 17.3 18.1 16.9 0.34 – 0.425 Intake(gd−1) 509 558 520 10.0 – 0.169 Growthrate(gd−1) 402 428 384 10.7 – 0.271 FCRB _1.27a _1.30a,b _1.37b _{0.02 – 0.042} FaecalscoreC _{1.43 0.86 1.03 0.04 39.31 <0.001}

A_{X2:Pearsonchi-squaredtest.Pvaluesfortheeffectofdiet.SEM:standarderrorofthemean(n=24forgrowthperformances,n=840forfaecalscore).} a,b:inthesameline,valueswiththesameletterarenotsignificantlydifferent(P>0.05).

B _{Feedconversionratio.}

C _{0=normal,1=softfaeces,2=moderatediarrhoeaand3=liquiddiarrhoea.}

Microscopicexaminationandmeasurementofvilliheightsandwidthsandcryptdepthswereperformedin7␮mthick tissuesections,stainedwithhaematoxylin–eosin.AnOlympusBX511microscopeequippedwith4×and10×lenseswas used.ImagesweredigitallycapturedwithanOlympusDP11camera.Theheightandwidthofthevilliandthedepthof thecryptsweremeasuredusingtheOlympusDP-Softsoftware.Tenintactandcorrectlyorientedvilliandcryptsfromeach intestinalregionwereselectedforeachpiglet.

2.4. Statisticalanalysis

Dataweresubjectedtoanalysisofvariance(ANOVA),withdietasthemainfactor.Allstatisticalanalyseswereconducted withtheSAS®_{statisticalsoftwarepackage(SAS,1991).DigestibilitydatawereanalyzedwiththeMIXEDprocedureofSAS,to} considerrepeatedmeasuresintime(Littelletal.,1998).Thefaecalscoreswerecomparedwithachi-squaredtest,usingthe FREQprocedureofSAS.AllotherdatawereanalyzedwiththeGLMprocedureofSAS,accordingtoacompleteblockdesign. WhentheF-valueintheANOVAwassignificant,leastsquaresmeanswerecomparedbytheleastsignificantdifference.A differencewasconsideredsignificantiftheP-valuewaslessthan0.05.

3. Results

3.1. Growthperformanceandconsistencyoffaeces

AsshowninTable3,themeaninitialweightofthepigletswas6.11kgandthemeanfinalweightwas17.44kg.Diethad nosignificanteffectonthedailyfeedintakeandthedailygrowthrate.Themeanvalueofthesetraitswas529gand404g, respectively.

ThepigletsintheCN100groupweretheleastefficientusersoffeed;thefeedconversionratio(FCR)was0.10points higherintheCN100grouprelativetotheCDgroup(P=0.042).

Averagefaecalscorewas1.43,0.86and1.03forpigletsfromgroupsCD,CN50andCN100,respectively.Thus,therewasa trendtowardslowerfaecalscoresamongstpigletsreceivingtheCN50dietcomparedtotheCDandCN100diets,indicating abetterconsistencyoffaecesandalowerincidenceofsoftfaecesintheseanimals.

3.2. Totaltractapparentdigestibility(TTAD)

TheeffectsofdietandageonTTADaredepictedinTable4.Therewasatendency(P=0.067)towardsanincreaseinthe TTADofDMinthetwochestnut-containingdietsrelativetothecontroldiet,butonlywhenthepigletswereyounger(aged 32–39days).Consequently,therewasasignificantdiet×ageinteractioneffectontheTTADofDM(P=0.019).

Onthecontrary,theTTADofCPwasreduced(P<0.001)intheCN100grouprelativetotheCDandCN50groups, indepen-dentlyofage.Thecoefficientsoftotaltractapparentdigestibility(CTTAD)ofCPwere0.84,0.83and0.79ingroupsCD,CN50 andCN100,respectively,forpigletsaged32–39days.Thesecoefficientswere0.87,0.82and0.77forpigletsaged46–53days. TheTTADofNDFandADFwerebothincreasedwiththeinclusionofchestnutsinthediet,althoughthesedifferenceswere greateramongsttheyoungerpiglets.RelativetotheCDgroup,thecoefficientofTTADofNDFwasincreasedby0.18unitsin theCN50groupand0.25unitsintheCN100group,whenthepigletswereaged32–39days(periodA1).Thesedifferences werereducedto0.09and0.17units,respectively,whenthepigletswereaged46–53days(periodA2).Similarly,the coef-ficientofTTADofADFwasincreasedby0.24unitswiththeCN50dietandby0.40unitswiththeCN100dietduringperiod A1.DuringperiodA2,thesefigureswere0.12unitsand0.28units,respectively.Finally,theTTADofthemajorconstituents ofthedietswerenotmodifiedsignificantlybytheageofthepiglets.TheCTTADofDMwas0.90and0.89,andtheCTTADof CPwas0.79and0.77forpigletsaged32–39daysand46–53days,respectively.

Table4

Effectofdietaryproportionofchestnutsandageontotaltractapparentdigestibility.

DietA _{CD CN50 CN100 P-value}D

AgeB _{A1 A2 A1 A2 A1 A2 SEM}C _{Diet Age DxA}

CTTADE

Drymatter 0.87 0.90 0.90 0.90 0.90 0.89 0.003 0.067 0.164 0.019

Crudeprotein 0.84 0.87 0.83 0.82 0.79 0.77 0.007 <0.001 0.873 0.128

NDFF _{0.65 0.70 0.83 0.79 0.90 0.87 0.014 <0.001 0.416 0.003}

ADFG _{0.46 0.54 0.71 0.65 0.86 0.82 0.022 <0.001 0.743 0.009}

A_{CD:controldietcontaining100%wheatstarch,CN50:dietcontaining50%wheatstarchand50%chestnutstarch,CN100:dietcontaining100%chestnut} starch.

B_{A1:pigletsaged32–39days.A2:pigletsaged46–53days.} C_{SEM:Standarderrorofthemean(n=48).}

D_{Pvaluesfortheeffects:diet,ageandinteractiondiet×age.} E_{Coefficientsoftotaltractapparentdigestibility.}

F_{Neutraldetergentfibre.} G_{Aciddetergentfibre.}

3.3. Weightofthegastrointestinalcompartments

TheeffectsoftheexperimentaldietsonthedevelopmentofthegastrointestinalcompartmentsareoutlinedinTable5. Theinclusionofchestnutsinthedietshadnosignificanteffectontheweight(gkg−1liveweight)ofthefulloremptystomach orthesmallintestine.Nevertheless,therewasatendencytowardsanincreaseintheweightofthesmallintestinecontents whenpigletswerefedwithchestnuts,especiallyintheCN100group(P=0.090).

Therewerealsotendencies(P<0.10)towardsincreasesinthefullandemptyweightsofthelargeintestineintheCN100 group.Theincreaseinfullweightwasroughly19%relativetotheCDgroup,and15%relativetotheCN50group.Forthe emptyweight,thesevalueswereabout11%and21%,respectively.

Finally,theexperimentaldietshadnosignificanteffectontherelativeweightsofthepancreasorliver. 3.4. Morphologyofthesmallintestinemucosa

Theexperimentaldietshadnosignificanteffect(P>0.05)onvillusheightandwidthintheduodenum,jejunumorileum ofthepiglets(Table6).Similarlythecryptdepthandthevillusheight:cryptdepthratiointheduodenumorileumwerenot significantlyaffectedbytheexperimentaldiets.However,cryptdepthinthejejunumwasdecreasedintheCN100group (P=0.006);itwasreducedby26%relativetotheCDgroupand19%relativetotheCN50group.Therewasalsoatendency towardsdecreasedcryptdepthintheduodenumintheCN100group(P=0.059);itwasreducedby12%relativetotheother experimentaldiets.

Inaddition,thevillusheight:cryptdepthratiointhejejunumofpigletsintheCDgroupwas22%and73%lowerthanin theCN50andCN100groups,respectively(P=0.018).WiththeCN100diet,theincreaseinthevillusheight:cryptdepthratio intheduodenumwasclosetoreachstatisticalsignificance(P=0.055).

Table5

Effectofdietaryproportionofchestnutsontherelativeweight(gkg−1_{ofliveweight)ofthegastrointestinaltract,pancreasandliverofpiglets.}

DietA _SEMB _PB CD CN50 CN100 Stomach Full 12.5 13.0 14.3 0.51 0.358 Empty 8.55 9.12 8.77 0.17 0.394 Content 3.95 3.89 5.51 0.46 0.280 Smallintestine Full 54.9 54.8 58.5 1.17 0.346 Empty 45.3 43.7 44.7 0.64 0.626 Content 9.57 11.1 13.9 0.82 0.090 Length(m) 15.2 15.5 15.4 0.21 0.842 Largeintestine Full 39.9 41.3 47.6 1.45 0.067 Empty 22.0 20.2 24.5 0.76 0.059 Content 17.9 21.1 23.2 1.21 0.221 Length(m) 2.83 2.49 2.54 0.07 0.125 Pancreas 2.37 2.29 2.21 0.07 0.634 Liver 27.3 26.7 29.8 0.86 0.314

A_{CD:controldietcontaining100%wheatstarch,CN50:dietcontaining50%wheatstarchand50%chestnutstarch,CN100:dietcontaining100%chestnut} starch.

Table6

Effectofdietaryproportionofchestnutsonsmallintestinalmucosamorphology.

DietA _SEMB _PB CD CN50 CN100 Villusheight(␮m) Duodenum 414 407 421 9.25 0.875 Jejunum 374 433 385 13.3 0.109 Ileum 324 325 319 11.2 0.985 Villuswidth(␮m) Duodenum 199 229 202 7.10 0.125 Jejunum 166 179 172 3.83 0.327 Ileum 168 189 182 5.45 0.209 Cryptdepth(␮m) Duodenum 516 520 453 10.2 0.059 Jejunum 411a ₃₇₆a ₃₀₆b _{12.3 0.006} Ileum 386 343 325 11.6 0.106

Villusheight:cryptdepthratio

Duodenum 0.80 0.79 0.93 0.02 0.055

Jejunum 0.92a _1.17b _1.26b _{0.05 0.018}

Ileum 0.85 0.95 0.99 0.04 0.297

A_{CD:controldietcontaining100%wheatstarch,CN50:dietcontaining50%wheatstarchand50%chestnutstarch,CN100:dietcontaining100%chestnut} starch.

B _{Pvaluesfordieteffect.SEM:standarderrorofthemean(n=24).}

a,b:inthesameline,valueswiththesameletterarenotsignificantlydifferent(P>0.05). Table7

Effectofdietaryproportionofchestnutsondrymatter,pH,bacterialenzymaticactivityandshortchainfattyacidsinthecaecalcontentsofthepiglets.

DietA _SEMB _PB

CD CN50 CN100

Drymatter(gkg−1_{) 122 121 86.3 8.51 0.159}

pH 6.69 6.47 6.33 0.07 0.144

Bacterialenzymaticactivity(mgsugarh−1g−1DM)

Xylanolytic 258 206 140 21.7 0.083 Pectinolytic 369 312 437 25.1 0.114 Cellulolytic 96.1 115 129 11.7 0.547 SCFA(mmoll−1₎C C2 11.9 17.7 25.9 2.47 0.063 C3 5.15a _9.43ab _14.7b _{1.58 0.042} C4 3.50a _4.90a _9.84b _{1.05 0.025} IC4 1.13 1.89 3.65 0.47 0.075 IC5 1.33 1.54 3.10 0.34 0.062 Total 23.0a _35.5ab _57.2b _{5.85 0.048} Molarproportion C2:C3 2.40a _1.93b _1.82b _{0.09 0.008} C2:C4 4.04a _3.75a _2.73b _{0.22 0.031} C3:C4 1.75 1.98 1.53 0.12 0.276 C2:total 0.53a _0.50a _0.46b _{0.01 0.001} C3:total 0.23 0.26 0.26 0.01 0.069 C4:total 0.14 0.14 0.17 0.01 0.051

A_{CD:controldietcontaining100%wheatstarch,CN50:dietcontaining50%wheatstarchand50%chestnutstarch,CN100:dietcontaining100%chestnut} starch.

B _{Pvaluesfordieteffect.SEM:standarderrorofthemean(n=24).}

a,b:inthesameline,valueswiththesameletterarenotsignificantlydifferent(P>0.05). C _{Shortchainfattyacids:C2:acetic,C3:propionic,C4:butyric,IC4:isobutyric,IC5:isovaleric.}

3.5. Chemistryandbiochemistryofcaecalcontents

TheeffectsoftheexperimentaldietsonthechemistryandbiochemistryofthecaecalcontentsarereportedinTable7.Dry matter,pHandbacterialfibredegradation(cellulolytic,xylanolyticandpectinolytic)inthecaecumwerenotsignificantly affectedbythecompositionoftheexperimentaldiets.Averagedacrossthethreediets,meanDMcontentwas109.8gkg−1, andmeanpHwas6.50.TherewasatendencytowardslowerxylanolyticactivityinthepigletsfedtheCN100diet(P=0.083). Thetotalreplacementofwheatwithchestnuts(CN100group)produceda2.5-foldincreaseintheconcentrationofSCFAs inthecaecumofthepiglets,ascomparedtotheCDgroup.Thisincreasecanbeattributedtohigherlevelsofacetic(C2), propionic(C3)andbutyric(C4)acids;theconcentrationofC2wasdoubled(P=0.063)andtheconcentrationsofC3andC4 werealmosttrebled(P=0.042andP=0.025,respectively)relativetotheCDgroup.Moreover,theratiosofC2:C3acids,C2:C4 acidsandC2:totalacidswereallreducedintheCN100grouprelativetotheCDgroup;by25%,32%and13%,respectively.

Table8

Effectofdietaryproportionofchestnutsondrymatter,pH,bacterialenzymaticactivityandshortchainfattyacidsinthecoloniccontentsofthepiglets.

DietA _SEMB _PB

CD CN50 CN100

Drymatter(gkg−1_{) 213 189 185 7.36 0.248}

pH 6.95 6.65 6.62 0.20 0.106

Bacterialenzymaticactivity(mgsugarh−1g−1DM)

Xylanolytic 439a ₃₉₉a ₂₂₄b _{35.4 0.023} Pectinolytic 325a ₄₃₉ab ₅₇₇b _{41.2 0.035} Cellulolytic 220 264 362 29.7 0.132 SCFA(mmoll−1₎C C2 21.3 25.6 24.8 1.59 0.525 C3 9.17 10.4 11.5 0.62 0.327 C4 11.4 12.4 12.0 0.72 0.865 IC4 3.01 3.55 3.65 0.19 0.359 IC5 5.17 4.91 4.55 0.33 0.768 Total 50.0 56.8 56.5 3.09 0.621 Molarproportion C2:C3 2.37 2.41 2.19 0.07 0.365 C2:C4 1.93 2.06 2.08 0.07 0.714 C3:C4 0.83 0.86 0.97 0.04 0.388 C2:total 0.43 0.44 0.44 0.01 0.724 C3:total 0.18 0.19 0.20 0.01 0.189 C4:total 0.23 0.22 0.21 0.01 0.463

A_{CD:controldietcontaining100%wheatstarch,CN50:dietcontaining50%wheatstarchand50%chestnutstarch,CN100:dietcontaining100%chestnut} starch.

B_{Pvaluesfordieteffect.SEM:standarderrorofthemean(n=24).}

a,b:inthesameline,valueswiththesameletterarenotsignificantlydifferent(P>0.05). C_{Shortchainfattyacids:C2:acetic,C3:propionic,C4:butyric,IC4:isobutyric,IC5:isovaleric.}

Finally,therewasatendencytowardsanincreaseintheC3:totalandC4:totalratios(P=0.067andP=0.051,respectively)in theCN50andCN100groupscomparedtotheCDgroup.

3.6. Chemistryandbiochemistryofcoloniccontents

Inthecoloniccontents,meanDMcontentwas196gkg−1,andmeanpHwas6.74(Table8).Dietarycompositionhadno effectonthesetraits.

Thetotalreplacementofwheatwithchestnuts(CN100group)produceda50%reductioninbacterialxylanolyticactivity inthecoloniccontents,butthepartialreplacementofwheat(CN50group)hadnosignificanteffectonxylanolyticactivity. Theinclusionofchestnutsinthedietincreasedbacterialpectinolyticactivityinthecolon,butagainthiseffectwasonly significantintheCN100group.Onthecontrary,diethadnosignificanteffectonbacterialcellulolyticactivityinthecolonic contents.

Finally,dietarycompositionhadnoeffect(P>0.05)onthelevelsormolarproportionsofSCFAsinthecolon.Themean levelsofC2,C3andC4acidsinthecolonwere23.9;10.4and11.9mmol1−1,respectively.Themeanmolarproportionsof C2:total,C3:totalandC4:totalwere0.44,0.19and0.22,respectively.

4. Discussion

ThefreshchestnutsusedinthisstudyhadaninitialdryDMcontentof50%,sotheyweredriedat40◦C(toreachaDM contentexceeding88%)beforetheyweremilledandmixedintotheexperimentaldiets.Starchgelatinizationtakesplaceina rangeoftemperaturesthatdependsonthestarchsource,moisturecontent(Chiotellietal.,2002)andamylose:amylopectin ratio(CorreiaandBeirão-da-Costa,2012).ArecentstudyofEuropeanchestnutcultivars(C.sativaMill.)concludedthat,at amoisturecontentof50%,thegelatinizationtemperatureofchestnutstarchrangesfrom68to81◦C(Moreiraetal.,2012). Hence,itissafetoassumethatthedryingprocedureadoptedinthepresentstudydidnotmodifythecrystallinestructure ofthestarchorlevelofRSinthechestnuts.

4.1. Growthperformanceandfrequencyofdiarrhoea

Thepartialortotalreplacementofwheatwithchestnutsdidnotmodifyfeedintake,suggestingthatthechestnutswere wellacceptedbythepigletsandtherewerenoproblemsconcerningpalatability.Nevertheless,pigletsfedthecontroldiet showedabetterFCRthanpigletsfedtheCN100diet.Thisresultindicatesthatthechestnutswereusedlessefficientlyby thepigletscomparedtothewheat.

It isknown that theconcentrationof SCFAs inthe largeintestine of pigletsincreases withtheinclusion of RS in thediet (HedemannandBach Knudsen,2007).Consistentwiththis,higher levelsof SCFAswerefoundinthecaecum ofpigletsfedwithchestnuts (Table7).Relativetoglucose,SCFAshavea lowenergyvalueforswine(Jorgensenetal., 1997;BachKnudsen,2011).Hence,resistantstarchesarelowenergyingredientsforpigs(Cervantes-Pahmetal.,2009). Morerecently,Gerritsetal.(2012)reportedthatretrogradedcornstarch,usedasaresistantstarch,hasonly53%ofthe digestibleenergyvalue,73%ofthemetabolizableenergyvalue,and83%ofthenetenergyvalueofenzymaticallydegradable starch.

Theinclusionofchestnutsinthepiglets’dietimprovedtheconsistencyoffaecesintheCN50groupascomparedtothe CDandCN100groups.Wherewheatwastotallyreplacedbychestnuts,faecalscoreswerenotsignificantlydifferentfrom thoseofthecontrolgroup.Bhandarietal.(2009)haverecordedsimilarresults.Rawpotatostarch,aRSingredient,reduced postweaningscouringinpigletsat7%,butat14%,thereversewastrue.Theauthorsattributedthiseffecttothelargeamount ofundigestedstarchintheileum;toohighaconcentrationofdietarystarchintheileumimpairsdigestionandincreasesthe riskofscouring.Hence,theeffectofRSonfaecalconsistencyandpostweaningscouringcanbeeitherpositiveornegative, dependingonitslevelofinclusioninthepiglets’diet.

4.2. Totaltractapparentdigestibility(TTAD)

Theadditionofchestnutstothepiglets’diethadnonegativeeffectontheTTADofDM,evenwhentheyweretheonly sourceofstarchinthediet.Infact,theTTADofNDFwasactuallyhigherintheCN100group,compensatingforthelower TTADofCP.

ThechestnutsusedinthisexperimentcontainedlowerlevelsofCPthantheequivalentweightofwheat(Table1).Hence, whentheexperimentaldietswereformulated,extrafishmealwasaddedtothechestnut-containingdiets,inordertobalance thelevelsofCPacrossthethreediets.Fishmealisahighlydigestibleproteinsourceforpiglets(KimandEaster,2001),and itisimplausiblethatitcouldbelessdigestiblethantheproteinfoundinwheat.Thus,thelowerTTADofCPintheCN100 groupmustbeexplainedbylowerdigestibilityofthechestnutproteinfractionand/orbyanincreaseinendogenousnitrogen excretioninthefaecesofthepiglets,causedbytheRSfromthechestnuts.Indeed,ithasalreadybeendemonstratedthatRS supplementationincreasesfaecalnitrogenexcretioninpiglets(HeijnenandBeynen,1997).Thiseffectcanbeattributedto adecreaseinilealnitrogendigestibility(ScrijverDeetal.,1999)andanincreaseinbacterialnitrogenproduction,stemming fromthestimulationofbacterialgrowthinthegutcausedbythefermentationofundigestedRS(Birdetal.,2007andRegmi etal.,2011).ThehigherconcentrationofSCFAsinthecaecumofpigletsfedchestnutsinthisstudy(Table7)isconsistent withthishypothesis,asSCFAsarealsoproducedbyRSfermentation.

Whenchestnutswereincludedinthediet,theTTADofbothNDFandADFwereincreased,withthegreatestenhancement intheCN100group.Thisfindingcannotbeattributedtodifferingdietarylevelsofthesenon-starchpolysaccharides(NSPs), asthethreeexperimentaldietscontainedsimilaramountsofNDFandADF(Table2).Inpigs,thelargeintestineisthemain siteofNSPfermentation.␤-glucans,solublearabinoxylans,andpectinsarealldegradedinthecaecumandproximalcolon, whereasthemoreinsolubleNSPs(e.g.,celluloseandinsolublearabinoxylans)aredegradedmoreslowlyinmoredistal regionsofthecolon(BachKnudsen,2011).

Resistantstarchmaymodifythemicrobialcompositionofthelargeintestine.Forexample,itcanstimulatethegrowthof theintestinallactobacilli(Birdetal.,2007)andbifidobacteria(Regmietal.,2011)populationsinpigs.Resistantstarchmay alsoalterthedegradationofNSPsinthelargeintestine;shiftingtheirutilizationtomoredistalpartsofthecolon(Jonathan etal.,2013).Thus,thealteredTTADofNDFandADFinthepigletsfedthechestnut-baseddietsprobablyreflectstheeffect ofresistantstarchonthedegradationofspecificNSPsbythelargeintestinalmicrobiota.

4.3. BacterialenzymesandSCFAinthecaecalandcoloniccontents

Theadditionofchestnutstothepiglets’dietreducedxylanolyticbacterialactivityandincreasedpectinolyticactivity inthecolon,withtheseeffectsbeinggreaterintheCN100groupthantheCN50group.Thiswouldseemtosuggestthat theRSfromchestnuts,arrivinginthelargeintestine,isabletomodifybacterialactivityinthecolon;althoughitis impos-sibletoconcludewhichmicrobialpopulationswerestimulatedordepressed.Thesefindingsareconsistentwithprevious studieswhich haveshownthat RSfromvarioussourcescanpromotethegrowthof thebifidobacteriaand lactobacilli populationsinthecolonofthepig,withapotentialprebioticeffect(Birdetal.,2007;Bhandarietal.,2009;Regmietal., 2011).

Ontheotherhand,itisknownthatdietarynon-starchpolysaccharidescaninfluencebacterialenzymaticactivityinthe hindgutofthepig(Vareletal.,1987).Therefore,thehigherxylanolyticactivityintheCDgroupisconsistentwiththepresence ofarabinoxylansfromwheat,sincethesepolysaccharidesarethemajorhemicellulosesinthisgrain(Englyst,1989).Inthe caseoftheCN50andCN100groups,thelowerxylanolyticactivityandthehigherpectinolyticactivitycanbeexplainedbya decreaseinarabinoxylansandanincreaseinpectinswhenwheatwaspartiallyortotallyreplacedbychestnutsinthediets. Nevertheless,ourdatacannotconfirmthishypothesis,sincewehavenoinformationaboutthecompositionofnon-starch polysaccharidespresentinthechestnuts.

TheintakeofchestnutstarchsignificantlyincreasedtheconcentrationofSCFAsinthecaecumbutnotinthecolon. Martinetal.(2000)foundthattheincreaseinSCFAconcentrationintheportalvenousbloodofpigsoccurred4hafter

a meal containing raw potato starch or highamylose maize starch, confirming that the SCFAs produced by RS fer-mentation are rapidlyabsorbed in the colon of these animals. In thepresent study,the contents of the colon were collected 12h afterfeeding, so it is possible that the SCFAs were largely absorbed when thecolonic contents were collectedfor analysis.Thiswould explain theapparentlackof effect oftheexperimentaldiets onSCFAlevelsin the colon.

ThetypeofSCFAformedinthehindgutdependsonthecompositionofthecarbohydratesavailableforfermentation.For example,highNSPintakeprovokesaconsiderableincreaseintheconcentrationofacetate,togetherwithsmallerincreases inlevelsofpropionateandbutyrate(Wangetal.,2004).Conversely,thefermentationofRSyieldshigherproportionsof butyricacid(Martinetal.,2000;Birdetal.,2007).Inthepresentstudy,theconcentrationofbutyratewasalmosttrebled inthecaecumofpigsreceivingtheCN100diet,andtheratioofacetatetobutyratewassignificantlyreduced.Thiswould appeartoconfirmthatRSfromthechestnutswasthemajorcarbohydrateavailableforfermentationinthelargeintestine ofthepigletsfedtheCN100diet.

DifferentSCFAsinthecolonareknowntoplaydifferentphysiologicalroles(Mosenthin,1998),withbutyratebeingthe mostimportant.Butyrateispreferentiallymetabolizedbycolonocytestosupplyenergy,andplaysaselectiveantimicrobial rolethroughitsenhancementofthebifidibacteriaandlactobacillipopulations(Birdetal.,2007;Bhandarietal.,2009;Regmi etal.,2011).SCFAsalsoactasanti-diarrhoealagents–butyratebeingthemosteffective–byenhancingsodiumandwater absorption.Thus,feedingpigletschestnutsmayimprovethephysiologicalconditionsintheirhindgut,buttheeffectwill dependonthelevelofchestnutsintheirdiet.

4.4. Morphologyofthesmallintestinalmucosaandweightofthegutcompartments

Theinclusionofchestnutsinthepiglets’dietmodifiedthemorphologyofthesmallintestinemucosa,causingadecrease incryptdepthinthejejunum.Theseresultsarenotunexpected;theinclusionof80gkg−1ofrawpotatostarchinthe dietoftheweanedpiglethasbeenshowntoincreasevillusheight,while160gkg−1wasshowntodecreasecryptdepth (HedemannandBachKnudsen,2007).Withgrowing-finishingpigs,theadditionof250or350gkg−1ofrawpotatostarch causedadecreaseinvillusheightintheileum,buthadnoeffectoncryptdepth(Nofraríasetal.,2007).Thus,itwouldseem thatRScanmodifythemorphologyoftheintestinalmucosaindifferentways,dependingonitslevelofinclusioninthe diet.Inthepresentstudy,thevillusheight:cryptdepthratiowasheightenedinthepigletsfedchestnuts,indicativeofan improvementinthedigestivecapacityofthesmallintestine(Montagneetal.,2003).

Thetotalreplacementofwheatstarchbychestnutstarchinduceda trendtowardsanincreaseinboth thefulland emptyweightofthelargeintestine.SimilarresultswerereportedbyMartinez-Puigetal.(2003)andBirdetal.(2007), usingpotatoorhigh-amylosemaize starchesassourcesofRS.BothauthorsnotedthatthetrophiceffectofRSonthe largeintestine waspartlycausedbytheelongationofthecolon,althoughinthepresentstudy,diethadnosignificant effectonthelengthofthelargeintestine.Hence,theincreasedweightmayhavereflectedanincreaseinthethicknessof theintestinalwall.SomearguethatSCFAs–themajorproductsofRSfermentationinthelargeintestine–representthe keystimuliforintestinalgrowth(ToppingandClifton,2001;Martinez-Puigetal.,2003).Othersbelievethatthe mechan-icalactionof RSitselfhasagreaterinfluenceonlargeintestine sizethanitsfermentationproductsBirdet al.(2007). Usingrawpotatostarchbaseddiets,Nofraríasetal.(2007)foundthattheincreasedweightofthehindgutwasdueto hypertrophyofthetunicamuscularis,mediatednotonlybytheweightofthehindgutcontents butalsobyitshigher butyrateconcentration.Inthepresentstudy,pigletsfedthechestnut-containingdietsshowedelevatedlevelsofSCFAs (espe-ciallybutyrate)inthecaecum.Ourresultsarethereforeconsistentwiththeproposedtrophiceffectofthesefermentation products.

5. Conclusion

Inthepresentstudy,chestnutswerewellacceptedbytheexperimentalanimalsandcouldthereforebeusedasan alternativesourceofRSinthedietofweanedpiglets.Nevertheless,whenwheatstarchwastotallyreplacedbychestnut starch,theFCRandtheTTADofCPwereimpaired.Interestingly,thearrivalofchestnut-derivedRSinthehindgutinduced atrendtowardsanincreaseinboththefullandemptyweightofthelargeintestine,andincreasedlevelsofSCFAsinthe caecum.Finally,bacterialactivityinthecolonwasmodulatedbytheinclusionofchestnutsinthediet;xylanolyticactivity wasreduced,whilepectinolyticactivitywasincreased.

Acknowledgements

WewouldliketothankCac¸adorPecuáriaSAforsupplyingthepigletsusedintheseexperiments,Reagroforproviding thefeedingredients,andDavidPritchettforhiscontributionineditingthismanuscript.

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