Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine

21 Pages·2016·3.2 MB·English

RESEARCHARTICLE
Intense Exercise and Aerobic Conditioning
Associated with Chromium or L-Carnitine
Supplementation Modified the Fecal
Microbiota of Fillies
MariaLuizaMendesdeAlmeida1☯,WalterHeinzFeringer,Ju´nior2,Ju´liaRibeiro
GarciaCarvalho2,IsadoraMestrinerRodrigues2,LilianRezendeJordão3,Mayara
Gonc¸alvesFonseca3¤,AdalgizaSouzaCarneirodeRezende3,AntoniodeQueirozNeto2,
J.ScottWeese4‡,Ma´rcioCarvalhodaCosta4‡,ElianaGertrudesdeMacedoLemos1☯,
a11111 GuilhermedeCamargoFerraz2☯*
1 DepartmentofTechnology,FaculdadesdeCiênciasAgra´riaseVeterina´rias,UNESPUnivEstadual
Paulista,Laborato´riodeBioqu´ımicadeMicrorganismosePlantas,Jaboticabal,SãoPaulo,Brazil,
2 DepartmentofAnimalMorphologyandPhysiology,FaculdadesdeCiênciasAgra´riaseVeterina´rias,
UNESPUnivEstadualPaulista,Laborato´riodeFarmacologiaeFisiologiadoExerc´ıcioEquino(LAFEQ),
Jaboticabal,SãoPaulo,Brazil,3 DepartmentofAnimalSciences,EscoladeVeterina´ria,Universidade
FederaldeMinasGerais,BeloHorizonte,MinasGerais,Brazil,4 DepartmentofPathobiology,Ontario
VeterinaryCollege,UniversityofGuelph,Ontario,Canada
OPENACCESS
Citation:AlmeidaMLMd,FeringerWH,Ju´nior, ☯Theseauthorscontributedequallytothiswork.
CarvalhoJRG,RodriguesIM,JordãoLR,Fonseca ¤ Currentaddress:DepartmentofAnimalMorphologyandPhysiology,FaculdadesdeCiênciasAgra´riase
MG,etal.(2016)IntenseExerciseandAerobic Veterina´rias,UNESPUnivEstadualPaulista,Laborato´riodeFarmacologiaeFisiologiadoExerc´ıcioEquino
(LAFEQ),Jaboticabal,SãoPaulo,Brazil.
ConditioningAssociatedwithChromiumorL-
‡Theseauthorsalsocontributedequallytothiswork.
CarnitineSupplementationModifiedtheFecal
*[email protected]
MicrobiotaofFillies.PLoSONE11(12):e0167108.
doi:10.1371/journal.pone.0167108
Editor:GunnarLoh,MaxRubner-Institut, Abstract
GERMANY
Received:July7,2016 Recentstudiesperformedinhumansandratshavereportedthatexercisecanalterthe
intestinalmicrobiota.Athletichorsesperformintenseexerciseregularly,butstudiescharac-
Accepted:November7,2016
terizinghorsemicrobiomeduringaerobicconditioningprogramsarestilllimited.Evidence
Published:December9,2016
hasindicatedthatthismicrobialcommunityisinvolvedinthemetabolichomeostasisofthe
Copyright:©2016Almeidaetal.Thisisanopen
host.Researchonergogenicsubstancesusingnewsequencingtechnologieshavebeen
accessarticledistributedunderthetermsofthe
limitedtotheintestinalmicrobiotaandthereisaconsiderabledemandforscientificstudies
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and thatverifytheeffectivenessofthesesupplementsinhorses.L-carnitineandchromiumare
reproductioninanymedium,providedtheoriginal potentiallyergogenicsubstancesforathletichumansandhorsessincetheyarepossibly
authorandsourcearecredited.
abletomodifythemetabolismofcarbohydratesandlipids.Thisstudyaimedtoassessthe
DataAvailabilityStatement:Allsequencingfiles impactofacuteexerciseandaerobicconditioning,associatedeitherwithL-carnitineorchro-
areavailablefromtheNCBISequenceRead
miumsupplementation,ontheintestinalmicrobiotaoffillies.Twelve“MangalargaMarcha-
Archivedatabase(accessionnumber
PRJNA308944)(https://www.ncbi.nlm.nih.gov/ dor”filliesintheincipientfitnessstageweredistributedintofourgroups:control(no
bioproject/PRJNA308944/).Allrelevantdataare exercise),exercise,L-carnitine(10g/day)andchelatedchromium(10mg/day).Inorderto
withinthepaperanditsSupportingInformation
investigatetheimpactofacuteexerciseoraerobicconditioningonfecalmicrobiotaallfillies
files.
undergoingtheconditioningprogramwereanalyzedasaseparatetreatment.Thefillies
Funding:TheauthorsarethankfultoFundac¸ãode
underwenttwoincrementalexercisetestsbeforeandaftertrainingonatreadmillfor42days
AmparoaPesquisadoEstadodeSãoPaulo
at70–80%ofthelactatethresholdintensity.Fecalsampleswereobtainedbeforeand48h
(FAPESP),processnumbers2013/14703-3and
2013/15215-2.ThanksarealsoduetotheNational afteracuteexercise(incrementalexercisetest).Bacterialpopulationswerecharacterized
PLOSONE|DOI:10.1371/journal.pone.0167108 December9,2016 1/21
IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
ScientificandTechnologicalDevelopmentCouncil bysequencingtheV4regionofthe16SrRNAgeneusingtheMiSeqIlluminaplatform,and
(CNPq)(EditalMCT/CNPq14/2012,Process 5,224,389sequenceswereobtainedfrom48samples.Theresultsshowedthat,overall,the
number482811/2012-9)BrazilianAssociationof
twomostabundantphylawereFirmicutes(50.22%)followedbyVerrucomicrobia(15.13%).
MangalargaMarchadorHorseBreeders
(Associac¸ãoBrasileiradeCriadoresdoCavalo ThetaxawiththehighestrelativeabundanceswereunclassifiedClostridiales(17.06%)and
MangalargaMarchador-ABCCMM)fortheir "5genusincertaesedis"fromthephylumVerrucomicrobia(12.98%).Therewasadecrease
operationalandfinancialsupport.
inthephylumChlamydiaeandinthegenusMycobacteriumafterthesecondincremental
CompetingInterests:Theauthorshavedeclared exercisetest.Intenseexercisechangedthecommunity’sstructureandaerobicconditioning
thatnocompetinginterestsexist.
wasassociatedwithchangesinthecompositionandstructureoftheintestinalbacterialpop-
ulationoffillies.Theintra-groupcomparisonshowedthatchromiumorL-carnitineinduced
moderatechangesinthefecalmicrobiotaoffillies,butthemicrobiotadidnotdifferfromthe
controlgroup,whichwasexercisedwithnosupplementation.FecalpHcorrelatedpositively
withSimpson’sindex,whileplasmapHcorrelatednegatively.Ourresultsshowthatexercise
andaerobicconditioningcanchangeinthemicrobiotaandprovideabasisforfurtherstudies
enrollingalargernumberofhorsesatdifferentfitnesslevelstobetterunderstandtheeffects
ofexerciseandtrainingontheintestinalmicrobiotaofhorses.
Introduction
Theintestinalmicrobiotaisacomplexpolymicrobialenvironmentinvolvingcloseinteractions
betweenthehostandmicroorganisms,particularlybacteria.Inhorses,theintestinalmicro-
biotacanbemodifiedbyvariousfactors,suchasgastrointestinaldiseases[1],changesindiet
[2],theuseofantibiotics[3,4]andgeneralanesthesia[5].Arecentstudyfoundthatchangesin
theintestinalmicrobiotamightpredictthedevelopmentofpostpartumgastrointestinaldisease
[6],indicatingthatalterationsinthemicrobiotaresultingfromvariousphysiologicalevents
mightaltertheriskofsubsequentdisease.Recentstudiesusinggeneticsequencingtechniques
reportedthatexercisemodifiedthestructureoftheintestinalmicrobiotainmice[7,8,9,10].
However,thereislittleinformationintheliteratureregardingathletehorses,atermthat
describeshorsesundergoingapracticeofintenseexerciseandconditioningprograms,and
possiblechangesintheirintestinalmicrobiota.
Manyhorsesroutinelyperformintenseexercise(exercise-inducedmetabolicacidosisby
increasingmuscleproductionofhydrogenions).Ifoneconsidersexerciseastimulusthatalters
homeostasis,asingleeffortsessioncanpromotereversiblechangesinseveralphysiological
variables,andthesealterationscanbeconfirmedbyquantifyingclinicallaboratoryvariables.
Dependingontheeffortaswellasthedurationandintensityoftheexercisesession,these
changescanbeobservedforphysiologicalvariablesinbothhumansandhorses[11,12].For
example,thesystemic,reversible,increasedglycolyticrateandhydrolysisofATPduringthe
contractionofskeletalmusclefiberswiththeinductionofmetabolicacidosis,associatedwith
increasedplasmalactateproduction,istheclassicalresponsetotheintenseincrementalexer-
cisetest[12].Inaddition,monitoringtheactivityofserumenzymessuchascreatinekinase
(CK)andaspartateaminotransferase(AST)afterandduringacuteeffortinconditioningpro-
gramsmayrevealchangesinthepermeabilityofthesarcolemma.Theseenzymesareconsid-
eredbiomarkersofthefunctionalstateofthemusclefiber[13].
Arecentstudyexaminingtheimpactofexerciseassociatedwithvarieddietaryintakeiden-
tifiedincreasedintestinalmicrobiotadiversityandapositivecorrelationwithCKactivityin
PLOSONE|DOI:10.1371/journal.pone.0167108 December9,2016 2/21
IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
high-performancerugbyathletes[14,15].However,correspondingdataforhorsesarelacking
despitethecommonalityofintenseexerciseandgastrointestinaldiseasesinbothspecies.
Manyoralsupplementsaremarketedforhumanandequineathletesasameanstoimprove
athleticperformance,oftenwithlittleevidenceoraclearmechanism.Chromium(Chr)andL-
carnitine(LC)arepotentialergogenicsubstancesforathletesthatcanincreasetheenergy
availableforexerciseanddelaytheonsetoffatigue[16,17].Chronicoralsupplementationwith
ChrorLCduringanaerobicconditioningprogramproducedmoderatechangesintheenergy
metabolismbiomarkersoffillies[18].Whethersupplementssuchasthesealsoalterthemicro-
biotaorexertexercise-associatedmicrobiotachangesisunclear.
Thus,theobjectivesofthisstudyweretoinvestigatetheimpactofintenseexercise(incre-
mentalexercisetesting)andanaerobicconditioningprogramontheintestinalmicrobiotain
filliesandtoassesstheeffectsofsupplementationwithChrorLConexercise-andaerobic
conditioning-associatedmicrobiotaalterations.
MaterialandMethods
ThestudyfollowedtheEthicalPrinciplesinAnimalExperimentationadoptedbytheBrazilian
CollegeofAnimalExperimentationandapprovedbytheEthicsCommitteeonAnimalUse
(CEUA–FCAV/UnivEstadualPaulista)underprotocol016842/13.
Animalsandadaptationperiod
TwelveMangalargaMarchadorfillies(MM),2.5to3yearsold,withanaveragebodyweightof
330±30kgandanincipientstageoffitnessunderwentanadjustmentperiodthatlasted34
daysintheLaboratoryofPharmacologyandEquineExercisePhysiology(UnivEstadualPau-
lista/FCAV/DMFA/LAFEQ).Duringthisperiod,thefillieswereacclimatizedtotheresearch
facilityandaccustomedtothemanagement,diet,trainingfacilities,andequipment:atreadmill
(Gallopertreadmill,SahincoLTDA,Palmital,Brazil)andanautomaticwalker(Gallopertread-
mill,SahincoLTDA,Palmital,Brazil).Inthefirstfivedays,theanimalsweredewormed(Equi-
max—VirbacSau´deAnimal,Jurubatuba,SãoPaulo,Brazil)andunderwentroutine
managementproceduressuchashooftrimminganddentalassessment.Noanimalsreceived
antibioticsoranti-inflammatorydrugs,andgastrointestinaldiseasehadnotbeenreportedin
theprevious6-monthperiod.Allfillieswerehousedinpaddockswithpastures.
Diet
Dietconsistedofaconcentrateat2.5%ofbodyweightdaily,basedondrymatter[19]and
roughage(approximately4kgofTifton85hayandPanicummaximumcv.Massaipasture)
(Table1).Theconcentrateconsistedof68%corn,12%soybeanmeal,15%wheatbran,3%
Table1. Percentageofcrudeprotein(CP),neutral(NDF)andacid(ADF)detergentfiber,mineralmatter(MM),calcium(Ca),phosphorus(P),digest-
ibleenergy(DE),totaldigestiblenutrients(TDN)basedondrymatter(DM)andinMcal/kgoffeedsuppliedduringthetrial.
Feed DM(%) CP(%) NDF(%) ADF(%) MM(%) Ca(%) P(%) DEMcal/Kg TDN(%)
PasturePanicummaximumcv.Massai 31.4 11.8 80.7 37.5 7.45 0.42 0.37 - 59.5
HayTifton85(Cynodonspp.) 94.9 9.4 80.7 39.9 10.02 0.48 0.27 2.50 56.0
Concentrate 90.2 14.2 13.9 5.4 8.00 1.32 0.55 3.15 -
Mineralsalt* 100.0 - - - 100.00 17.5 6.0 - -
*Compositionperkgofmineralsalt:Calcium175g;Phosphorus60g;Magnesium13.6g;Sulfur12g;Sodium120g;Zinc2.200mg;Copper1.200mg;
Manganese970mg;Iron2.000mg;Cobalt21mg;Iodine125mg;Selenium10mg;Fluor(max.)600mg.
doi:10.1371/journal.pone.0167108.t001
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IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
calciticlime,1%dicalciumphosphateand1%mineralsalt(CoequiPlus1),aswellas40mLof
soybeanoilperday.Inaddition,mineralsalt(CoequiPlus–Tortuga,SãoPaulo,Brazil)and
waterwereprovidedfreely.
Threefilliesweresupplementedwith10gperdayofL-carnitinepowder(SweetMix1,Soro-
caba,SãoPaulo,Brazil),andthreeanimalsweresupplementedwith10mgperdayofchelated
chromium(Tortuga,SãoPaulo,Brazil),bothofwhichwereofferedwiththeconcentrate.Body
weightwasdeterminedusingadigitalscalebeforethefirstincrementalexercisetesting(IET)
andondays14,32andimmediatelybeforethesecondIET.
Incrementalexercisetesting
Theincrementalexercisetestingconsistedofa10-minutewarm-up(5minat1.7ms-1and5
minat3.5ms-1withoutinclination)[18].Afterthewarm-up,thetreadmillwasraisedtoa3%
slopeataspeedof4ms-1,andtheprogressivespeedphasebegan,duringwhichspeedwas
increasedby1ms-1everythreeminutes.Exercisecontinueduntiltheanimalsreachedfatigue,
whichwasidentifiedbythefillies’inabilitytofollowthebeltspeedevenundervoicecommand
(incentive)tocontinue(fatigue).Thefatiguewasdefinedasthemomentinwhichahorse
couldnolongerkeepupwiththetreadmilldespitehumaneencouragement.Thecooldown
lasted10minutes(5minat3.5ms-1and5minat1.7ms-1).Bloodsampleswerecollecteddur-
ingthelast30secondsofeachstagethroughacatheter(BD™Insyte14GA,Cha´caraSanto
Antonio,Brazil)andwandsysteminsertedintotheexternaljugularvein.Plasmalactatecon-
centrationvs.velocitystagedatawereusedtodeterminethelactatethreshold(LT).
Conditioningprotocol
AnoverviewofthestudydesignisshowninFig1.Afirstincrementalexercisetesting(IET-1)
onthetreadmillwasconductedafteranadaptationperiodtoestablishtheLTfromtheplasma
lactateconcentrationforeachfilly.TheLTisdeterminedatthepointwherethelinearincrease
oftheplasmalactate-velocitycurve(Fig2)showsanabruptandexponentialincreaseofplasma
lactateconcentration(deflectionpoint).Thedeflectionpointrepresentsthestartofanimbal-
ancebetweenlactateproductionandremoval/metabolismandwasusedasaguidetodetermine
therelatedvelocity(VLT)onthetreadmillduringtheconditioningperiod.Thus,theinitial
runningintensity(VLT)wassetcorrespondingto70%VLT.Theaerobicconditioningperiod
lasted42days.Afterthisperiod,thefilliesunderwentanotherincrementalexercisetest(IET-2).
Theconditioningprotocoldesignconsistedofusingthetreadmillandautomaticwalker
alternatelysixdaysperweekandrestonMondays.Thetreadmillexerciseconsistedofa
5-minutewarm-upwalk(1.7ms-1)withoutaslope,followedby30minutesofexercisewitha
3%slopeandspeedat70%VLTduringthefirsttwoweeksand75%VLTduringthethirdand
fourthweeks.Therecoveryperiodconsistedofa5-minwalk(1.7ms-1)withoutaslope.Inthe
fifthandsixthweeks,theprotocolconsistedofa5-minutewarm-upwalk(1.7ms-1)withno
inclination,followedbyanintervalworkoutwitha3%slopefor20minutes,whichwasbroken
downinto4minutesat70%VLT,4minutesat75%VLT,4minutesat80%VLT,4minutesat
75%VLT,and4minutesat70%VLT.Therecoveryconsistedofa5-minwalk(1.7ms-1)with-
outinclination.Thehorsestrainedontheautomaticwalkerandtreadmillonalternatedays;
exerciseonthewalkerconsistedofa60-minwalkat2ms-1whilerotationwasreversedevery
10minutes[18].
Experimentalgroupsandfecalcollection
Attheendoftheadaptationperiod,threefillieswererandomlydistributedintoacontrol
groupthatreceivedthesamefeedingregimenbutdidnotexercise.
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IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
Fig1.Workflowoftheexperimentalprotocol.C(controlgroup,didnotexercise);E=onlyconditioning;Car=conditioningplusL-carnitine
supplementation;Chr=conditioningpluschromiumsupplementation;ET(exercisetotal:allfilliesundergoingtheconditioningwereanalyzedasa
separatetreatment).IET,incrementalexercisetest(IET-1andIET-2,beforeandafteraerobicconditioning,respectively);VLT(velocityrelatedtolactate
threshold)wasusedasaguidetodeterminethetrainingintensity,70–80%VLT;fs,fecalsample;mb,musclebiomarkers(plasmalactateandpH,CKand
AST).FatigueintheIETswasidentifiedbythefillies’inabilitytofollowtreadmillspeed.
doi:10.1371/journal.pone.0167108.g001
TheremainingninefilliesweredistributedaccordingtotheLTvelocitiesthatwere
obtainedindividuallyinthefirstincrementalexercisetest(IET-1)tobalancethegroupsin
termsoftheirconditioningdegree.Todistributetheexperimentalgroups(exercise,L-carni-
tine,andchromium),apreliminarydrawbasedonindividualVLTwasemployed,withfillies
classifiedintolow(4filliesbetween4.23and4.83ms-1),moderate(2filliesbetween5.00and
5.83ms-1),andhigh(6filliesbetween6.67and7.00ms-1)speed.Subsequently,thefillieswere
randomlydistributedintheexperimentalgroups.
Intotal,thepresentstudyhadfourexperimentalgroups:control(C,withoutexerciseor
supplementation,n=3),exercise(E,exercisewithoutsupplementation,n=3),L-carnitine
(Car,exerciseandsupplementationwithL-carnitine,n=3)andchromium(Chr,exerciseand
supplementationwithchromium,n=3).
FecalsampleswerecollectedimmediatelybeforeIET-1(C1-0,E1-0,Car1-0andChr1-0)
and48hafterIET-1(C1-48,E1-48,Car1-48,andChr1-48)andimmediatelybeforeIET-2
(C2-0,E2-0,Car2-0andChr2-0)and48hafterIET-2(C2-48,E2-48,Car2-48,andChr2-48).
Inordertoinvestigatetheimpactofacuteexerciseandaerobicconditioningonfecalmicro-
biota,allfilliesthatunderwentattheexercisewereanalyzedasaseparatetreatment(totalexer-
cisegroup).Thus,ETgroupswereformed,comprisingallfilliesthatunderwentacuteexercise
andaerobicconditioning(n=9):ET1-0,ET1-48,ET2-0,andET2-48.
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IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
Fig2.Typicalrelationshipbetweenplasmalactateconcentrationandvelocity(lactate-velocitycurve)in
incrementalexercisetest(IET)involvingaseriesofstages.Noteanabruptandexponentialincreaseofplasma
lactateconcentration(deflectionpoint-lactatethreshold(LT)).Thevelocityatlactatethreshold(VLT)hasbeenused
toelaboratethephysicalconditioningprotocoloffillies.
doi:10.1371/journal.pone.0167108.g002
Finally,toevaluatetheeffectsofsupplementationandaerobicconditioning,thesamples
takenatthebeginningoftheconditioningperiod(C0,E0,Car0andChr0)werecompared
withsamplestakenaftertheconditioningperiod(C42,E42,Car42,andChr42).
FecalsamplingandpHmeasurement
Fecalsampleswerecollectedfromalltwelvefilliesatthesametimeofdaydirectlyfromthe
rectum,viarectalpalpation,bythesameresearcher(J.R.G.Carvalho),immediatelybeforeand
48haftertheIETs.FecalpHwasdeterminedimmediatelyusingacalibratedindustrialpH
meter(Digimed—DM22,SãoPaulo,Brazil)in30goffecesdilutedin30mLofdeionized
water.Aliquotsofapproximately10gwerestoredinafreezerat-80˚CuntilDNAextraction
wasperformed.
Bloodsamplinganddeterminationofplasmaandserumvariablesof
muscleactivityandbodyweight
BloodsampleswereobtainedviacatheterbeforetheIET,andafterreachingfatigue,andtrans-
ferredtocollectiontubescontainingsodiumfluoridetodeterminelactateconcentrationsby
electro-enzymaticallymethod(YSI2300analyzer,YSIInc.,YellowSprings,Ohio,USA).
VenouspHwasdeterminedusingaportablechemistryanalyzer(i-STATcartridgesEC8+;
HeskaCorporation,FortCollins,Colorado).Bloodaliquotswerecollectedimmediatelybefore
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IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
and6h,12h,and24haftertheIETsin10-mLtubesbothwithandwithoutanticoagulantand
wereusedtoevaluateCKandASTenzymeactivitiesbyspectrophotometry(QuickLabChem-
istryAnalyzer,Hameln,Germany).
DNAextractionandsequencing
DNAwasextractedfromfecalsamplesusingtheFastDNASPINkitforsoil(Mpbio1).DNA
wasquantifiedandqualifiedbyspectrophotometry(NanoDrop1,ThermoFisherScientific).
TheV4regionofthe16SrRNAgenewasamplifiedinareactioncontainingthefollowing:
25μLofKapa2GFastHotStartReadyMix2X(KapaBiosystems,USA),1.3μLofMgCl
2
(50mM)(Invitrogen,USA),1.0μLofBSA(2mg/mL)(Bio-Rad),16.7μLofnucleasefree
water,2μLofDNAand2μLofforward(S-D-Bact-00564-a-S-1550-AYTGGGYDTAAAGNG-
30)andreverse(S-D-Bact-0785-b-A-1850-TACNVGGGTATCTAATCC-30)primers(10
pMol/μL)asdescribedbyKlindworthetal.(2013)[20].TheoligonucleotidesusedinthePCR
weredesignedsuchthattheycontainedIlluminaadaptersandindexes,accordingtothemanu-
facturer’srecommendations.PCRwascarriedunderthefollowingconditions:3minat94˚C
forinitialdenaturation,followedby25cyclesof45sat94˚Cfordenaturation,1minat50˚C
forannealingand90sat72˚Cforextension,andfinally10minat72˚Cforafinalribbonexten-
sionbeforeholdingat4˚C.Ampliconlibrarieswerepurifiedwithmagneticbeads(Agencourt
AMPureXP,BeckmanCoulterInc.,Mississauga,ON)bymixing72μLofmagneticbeadswith
20μLoftheamplifiedlibraries,and,afterincubationatroomtemperaturefor5min,samples
werewashedwith80%ethanoltwiceandelutedin40μLofnuclease-freewater.Thepurified
sampleswerequantifiedinaspectrophotometer(NanoDrop,ThermoFisherScientific)and
qualifiedona1%agarosegel.Afterthis,thesamplesweredilutedto5ng/μL.Sequencingwas
performedattheUniversityofGuelph’sAdvancedAnalysisCentre,usinganIlluminaMiSeq
platform(IlluminaRTAv1.17.28;MCSv2.2)withthe2x250V2reagents.Thesedataareavail-
ableintheNCBISequenceReadArchiveunderaccessionnumberPRJNA308944.
Bioinformaticandstatisticalanalyses
BioinformaticanalysiswasperformedusingMothursoftware(version1.34.4)[21].Good-
qualitysequenceswereclassifiedas"operationaltaxonomicunits"(OTUs),groupedat97%
similarityandclassifiedbasedontheRibosomalDatabaseClassifier(RDP,March2012)[22].
ChimeradetectionwasperformedusingUchime[23].
Sub-samplingwasperformedbasedforthesamplewiththeminimumnumberofreadsto
reducetheerrorsinnon-uniformsequences.Toensuresub-samplingrepresentation,Good’s
coverageandrarefactioncurvesthatrepresentedthenumberofOTUsbythenumberofreads
foreachsamplewereevaluated.Thediversityofeachsamplewasestimatedbycalculatingthe
inverseofSimpson’sindex,andrichnesswascalculatedusingCatchallsoftware[24].Bar
graphsrepresentingtherelativeabundanceofthemainphylaandgenerapresentineach
groupatthedifferenttimepointsweregenerated,andtheSteel-Dwasstestwasusedtocom-
parerelativeabundancesbetweengroups,controllingformultiplecomparisonserror.The
ANOVAtestwasusedtocomparetheresultsfromCatchallandtheinverseofSimpson’s
indexcalculatedforeachgroup(p<0.05significancelevel).
Thesimilaritybetweenthebacterialpopulationsineachsamplewascomparedusingadis-
tancematrixinPHYLIPformattocalculatetheYue&Claytoncoefficient,whichtakesinto
accountthebacterialrichnessandevenness(communitystructure),aswellastheClassicJac-
cardcoefficient,whichtakesintoaccountonlybacterialrichness(membership).Thecalcula-
tionswerevisuallyrepresentedbydendrogramsthatwerebuiltusingFigtreesoftware(version
1.4.0,http://tree.bio.ed.ac.uk/).Principalcoordinateanalysis(PCoA)wasalsoperformedto
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IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
comparesamplesimilaritiesin3dimensionsusingtheJMPsoftware(SASInstituteInc.).The
similaritybetweencommunitystructureandmembershipfoundinsamplesfromeachgroup
wascomparedusingtheParsimonytestandbycalculatingtheanalysisofmolecularvariance
(AMOVA).Theparsimonymethodisagenerictestthatdescribeswhethertwoormorecom-
munitieshavethesamestructure.
Significantdifferencesovertime(pre-vs.post-conditioning)oftheenergyintake,concen-
trate,bodyweight,andplasmaandserumbiomarkers(lactate,pH,AST,andCK)were
assessedusingANOVAfollowedbytheHolm-Sidaktest.Student’st-testforpairedsamples
wasusedtodeterminetheimpactofacuteexercise(baselineIETsandfatigue).Statistical
power-analysistestwasperformed.ThePearsoncorrelationwasusedtoestimatetherelation-
shipbetweenCatchallresults,theinverseofSimpson’sindex,CKandASTenzymaticactivi-
ties,lactateconcentration,andplasmaandfecalpH.Allanalyseswereperformedatthe5%
significancelevel.
Results
Dietandbodyweight
Overthetrialperiod,thecontrolgroupconsumedanaverageof0.87±0.26%ofconcentrate
relativetobodyweight;inthefirst14days,theaverageconsumptionwas1.10±0.01%and
decreasedto0.63±0.02%.Thefilliesundergoingtheexerciseprogramconsumed
1.23±0.06%bodyweightofconcentrate.Thisdifferencewasstatisticallysignificant
(P=0.020).Fig3andthedatainS1Tableshowtheamountsofconcentrateanddigestible
energysuppliedandthebodyweights.Theconcentrateintake(kg)anddigestibleenergy
(Mcal)suppliedtofilliesinthecontrolgroupstartedtodecrease(P<0.001)intheconcentrate
(kg)anddigestibleenergy(Mcal)suppliedforfilliesinthecontrolgroupstartingfromthe14th
dayafterthebeginningofthefitnessprogram.Averagebodyweightdidnotvarysignificantly
(P=0.306)inanyoftheexperimentalgroupsovertime.
Compositionanddifferencesinmicrobialcommunities
Metrics. Afterthequalitycontrolstepsandcleaningofsequences,atotalof5,224,389
sequencesfrom48sampleswereretained(median:98,661;SD:38,979).Asub-sampleof
16,656sequencespersamplewasusedtonormalizethenumberofsequencesinallsamplesfor
subsequentcomparisons.TheaverageofGood’scoverageachievedaftersub-samplingwas
95±0.9%,indicatingadequatecoverage.TherarefactioncurvesareshowninFig4.The
mediannumberofOTUsfromthesubsampledpopulationwas1851(min1290tomax2296;
average1852±256).
RelativeAbundances:effectsofintenseexerciseandconditioning. Fig5Aand5Bshow
thephylaandgenerawithmedianrelativeabundancesgreaterthan1%.Thesequences
obtainedwereclassifiedintoatotalof25phyla,withFirmicutes(50.22%)andVerrucomicro-
bia(15.13%)beingthemostcommon.Atotalof698generawereassignedto25phyla,but
only14hadmedianrelativeabundancesequaltoorgreaterthan1%.Themostabundant
genus-levelidentificationswereaClostridialesOTUthatwasnotclassifiedatthegenuslevel
(17.06%)followedby“5genusincertaesedis”(12.98%)fromtheVerrucomicrobiaphylum.
RelativeAbundances:effectsofsupplementation. Therewerenosignificantchangesin
theabundanceofthemainphylaandgenerainresponsetoconditioningandchromiumorL-
carnitinesupplementation,eitherimmediatelybeforeor48hafterIETs(allP>0.05).The
onlysignificantchangesobservedafteracuteexerciseconsistedofadecreaseinthephylum
Chlamydiae(P=0.031)andinthegenusMycobacterium(P=0.038)whencomparingE2-0
andE2-48.
PLOSONE|DOI:10.1371/journal.pone.0167108 December9,2016 8/21
IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
Fig3.(A)Concentratedigestibleenergy;(B)theamountofconcentrate;(C)meanbodyweightsof
filliessubjectedtoincrementalexercisetests(IET-1and-2)andanaerobicfitnessprogramfor42
days.*Indicatesareductionindigestibleenergyandtheamountofconcentratesuppliedtothecontrolgroup
comparedtotheotherexperimentalgroups.#indicatesareductionstartingfromthe32nddayofconditioning
withrespecttothecontrolgroup.Theaveragebodyweightofallexperimentalgroupsremainedunchanged
duringthetrial.
doi:10.1371/journal.pone.0167108.g003
PLOSONE|DOI:10.1371/journal.pone.0167108 December9,2016 9/21
IntenseExerciseandAerobicConditioningModifiedtheFecalMicrobiotaofFillies
Fig4.Meanvaluesandstandarddeviationsofrarefactioncurvescomparingthenumberof
sequencesandOTUsfoundintheDNAoffillies’stool.Groups:IET-1-0:immediatelybeforethefirst
incrementaltest(blue);IET-1-48:48hafterthefirstincrementaltest(red);IET-2-0:immediatelybeforethe
secondincrementaltest(green);IET-2-48:48hafterthesecondincrementaltest(purple).
doi:10.1371/journal.pone.0167108.g004
Populationanalysis–PhylotypeApproach:effectsofconditioningandsupplementa-
tion. Table2showstheresultsfromthecomparisonoftheexperimentalgroupsthatper-
formedphysicalactivitybeforeandaftertheconditioningperiodandergogenic
supplementation.Membershipofthecontrolgroupvariedsignificantlyovertimeaccordingto
theresultsofboththeparsimonyandAMOVA(C0vs.C42)tests.Theexercise-only(E0vs.
E42)andexercisepluschromiumsupplementation(Chr0vs.Chr42)groupsunderwentsignif-
icantchangesintheirmembershipregardlessofthestatisticaltestapplied.TheAMOVA
resultsrevealedachangeinthemembershipofthecarnitinegroup(Car0vs.Car42).There
wasnodifferencebetweenchromiumandcarnitinesupplementationcomparedtotheexercise
controlgroup(E42vs.Car42andE42vs.Chr42).
Populationanalysis–PhylotypeApproach:effectsofintenseexerciseandcondition-
ing. Table3showstheeffectsofIETandaerobicconditioningonthefecalmicrobiotacom-
munities.TheresultsoftheAMOVAtestshowedthatacuteexercise(ET1-0vs.ET1-48and
ET2-0vs.ET2-48)changedboth,communitiesstructureandmembership.Theparsimonytest
resultsalsorevealedsignificantchangesinthemembershipcomparingthebeginningwiththe
endofthetrial(ET1-0vs.ET2-48).
Dendrogramsandprincipalcoordinateanalysis(PCoA). Figs6and7showdendro-
gramsandPCoAplots,respectively,representthesimilarityofmicrobialcommunities
observedineachsample,demonstratingthatsamplescollectedbeforeandaftertheincremen-
taltestsformedwell-definedclustersintermsofmembershipandstructure.Interestingly,in
bothPCoAplots,itcanbeobservedthatthesamplescollectedatthebeginningoftheexperi-
mentweremoresimilartosamplescollectedattheendoftheconditioningperiodandafter
thelastincrementaltest,suggestingrecovery(adaptation)oftheintestinalmicrobiotaafter
conditioning.
Nosignificantdifferenceswereobservedintermsofthediversity(inverseSimpson’s)or
composition(Catchall)whencomparinganyoftheexperimentalgroups.Therawdatavalues
areshowninS2Table.
PLOSONE|DOI:10.1371/journal.pone.0167108 December9,2016 10/21