Ruminal methanogens and bacteria populations in sheep are modified by a tropical environment

(1)

HAL Id: hal-02454663

https://hal.uca.fr/hal-02454663

Submitted on 27 May 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Ruminal methanogens and bacteria populations in sheep

are modified by a tropical environment

Moufida Rira, Diego Morgavi, Milka Popova, Carine Marie-Magdeleine,

Tatiana Silou-Etienne, Harry Archimède, Michel Doreau

To cite this version:

Moufida Rira, Diego Morgavi, Milka Popova, Carine Marie-Magdeleine, Tatiana Silou-Etienne,

et al..

Ruminal methanogens and bacteria populations in sheep are modified by a tropical

environment.

Animal Feed Science and Technology, Elsevier Masson, 2016, 220, pp.226-236.

(2)

Contents lists available atScienceDirect

Animal

Feed

Science

and

Technology

journal homepage:www.elsevier.com/locate/anifeedsci

Ruminal

methanogens

and

bacteria

populations

in

sheep

are

modiﬁed

by

a

tropical

environment

Mouﬁda

Rira

a,1

_,

_Diego

_P.

_Morgavi

a

_,

_Milka

_Popova

a

_,

_Carine

_{Marie-Magdeleine}

b

_,

Tatiana

Silou-Etienne

b

_,

_Harry

_Archimède

b

_,

_Michel

_Doreau

a,∗

a_INRA,_VetAgro_Sup,_UMR1213_Herbivores,_F-63122_{Saint-Genès-Champanelle,}_France b_INRA,_UR143_URZ,₉₇₁₇₀_Petit-Bourg,_Guadeloupe,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received11May2016

Receivedinrevisedform13July2016 Accepted9August2016

Keywords: Rumenmicrobiota Volatilefattyacids Temperatevstropicalsite Sheepbreed

Forage Methane

a

b

s

t

r

a

c

t

Microbialfermentationofcarbohydratesintherumenislargelyresponsibleforthe emis-sionofmethanebyruminants.Ruminantsfedtropicalforagesusuallyproducemoreenteric methanethanruminantsfedtemperateforages.Therelativeinfluenceofforagetype,breed andtemperatevstropicalenvironmentonrumenmicrobialpopulationsisnotknown.This experimentaimedtoseparatetheseeffects.Wedesignedtwoparallelexperimentsinsheep intwosites:temperate(France)andtropical(FrenchWestIndies),usingineachsitetwo breeds,Texel(temperateorigin),andBlackbelly(tropicalorigin)fedthesametemperate forages(C3carbonfixation,permanentgrasslandsofhighandlowquality)andtropical for-ages(C4carbonfixation,permanentgrasslandsofhighandlowquality).Wedetermined dietdigestibility,ruminalend-productsoffermentationandmicrobialgroups:total pro-tozoa,methanogensandbacteria,andselectedfibrolyticbacteria.Drymatterdigestibility coefficientwashigherintropicalsite(612vs580g/kgonaverage,P=0.004)butno differ-encewasobservedbetweenC3andC4forages.TherewasnoeffectofsiteontotalVFA concentration,buttheacetate:propionateratiowashigherforthetropicalsite(4.30vs3.93 onaverage,P=0.007).Theacetate:propionateratiowasalsoaffectedbyforagetypewith highervaluesforC3thanC4forage(4.24vs3.99onaverage,P=0.03).Concentrationoftotal rumenbacteriaandmethanogenswasdeterminedbyqPCRtargeting,respectively,therrs (16SribosomalRNAsubunit)andmcrA(methylcoenzyme-Mreductase)genes.Forboth groups,thenumberofgenecopiespergramofDMrumencontentwashigherinthe trop-icalsite(P<0.001).Forcellulolyticbacteria,highernumberofrrscopiespergramofDMof rumencontentweredetectedforFibrobactersuccinogenesinthetemperatesite(P<0.001), whereasnodifferenceswereobservedforRuminococcusflavefaciensorRuminococcusalbus numbersbetweensites,breedsandforagetype.Protozoanumbersdeterminedbycounting didnotvarybetweensites,foragesorbreeds,butasite×forageinteractionwasobserved (P=0.01):thereweremoreprotozoaandR.albusintropicalsitesfortropicalforages.Our resultssuggestthatrumenmicrobiotawasmainlyinfluencedbyenvironment (temper-atevstropical)andthatforagetype(C3vsC4)andbreedhadminoreffects.However,an interactionbetweenenvironmentandforagetypewasobservedforsomevariables.

Abbreviations:CH4,methane;VFA,volatilefattyacid;DM,drymatter;H2,hydrogen;CO2,carbondioxide;H,high;L,low;NDF,neutraldetergentﬁbre assayedwithoutaheat-stableamylaseandexpressedinclusiveofresidualash;ADF,aciddetergentﬁbreexpressedinclusiveofresidualash;OM,organic matter;NH3,ammonia;PBS,phosphatebuffersaline;DGGE,denaturinggradientgelelectrophoresis;TAE,Tris-Acetate-EDTA;MFS,methylgreenformalin saline.

∗ Correspondingauthorat:INRA,VetAgroSup,UMR1213Herbivores,F-63122Saint-Genès-ChampanelleFrance. E-mailaddresses:mouﬁdar@yahoo.fr(M.Rira),michel.doreau@clermont.inra.fr(M.Doreau).

1 _Present_address:_Ecole_Nationale_Supérieure_de_{Biotechnologie,}_Ali_Mendjli,_BP_E66,₂₅₁₀₀_Constantine,_Algeria.

(3)

M.Riraetal./AnimalFeedScienceandTechnology220(2016)226–236 227

1. Introduction

Entericmethane(CH4)fromruminantsaccountsfor39%oflivestocksectorgreenhousegasesemissions(Gerberetal.,

2013).SeveralstudieshavetesteddifferentstrategiesforCH4abatementinruminants(Hristovetal.,2013;Martinetal.,

2010).However,althoughruminantdietsarebasedonforage,thereisscantinformationontheeffectofforagetypeon

methanogenesis.Ameta-analysis(Archimèdeetal.,2011)showedthatentericCH4productionperkgofdrymatter(DM)

intakemaybehigherforruminantsfedtropicalforagesthanforthosefedtemperateforages.ThesedifferencesinCH4

productionmaybeduetotheconstitutionalvariationinthechemicalstructureoftropicalandtemperateforages,which

displayC4andC3carbonﬁxationpathways,respectively.However,differencescouldalsobeduetotheambientenvironment

ortoanimalbreed.Experimentscarriedoutwithtropicalforageshavealwaysbeenconductedinthetropicswithbreeds

originatingfromandadaptedtoatropicalenvironment;likewise,experimentswithtemperateforageshavebeenrunin

temperateareaswithbreedsoriginatingfromtheseareas.

Intherumen,CH4 production resultsfrommicrobialfermentationofcarbohydrates.Themainend productsofthis

fermentationarevolatilefattyacids(VFA),hydrogen(H2)andcarbondioxide(CO2).Alargediversepopulationof

microor-ganismsdrivesthisprocess,especiallybacteriaandprotozoa.TheH2producedismainlyusedbymethanogenicarchaeato

reduceCO2toCH4.Theactivity,diversityandconcentrationofmicrobesharbouredintherumenareinﬂuencedbydiet,

breed,andtheenvironment(Kingetal.,2011).However,toourknowledge,therelationshipslinkingthesethreefactorsand

theirrelativeimportancehavenotbeenstudied.

Theaimofthisexperimentwastocomparerumenfermentationvariablesandmicrobialcommunitystructuresoftwo

breedsofsheep(TexelvsBlackbelly)fedC3andC4forages(permanentgrasslandsofhighandlowquality)inatemperate

andatropicalsite.Specialattentionwaspaidtomethanogensandhydrogen-producingprotozoa,whichareassociatedwith

methanogenesisincrease.Totalbacteriawerealsostudied,withafocusoncellulolyticbacteria,whichplayanimportant

roleincellwalldegradationandsocanfavourCH4production.

2. Materialsandmethods

2.1. Animals,diet,managementandexperimentaldesign

Thestudywasconductedinparallelintworesearchsiteslocatedintemperateandtropicalareas.Thetemperatesitewas

inAuvergne,France,at45.70◦Northlatitudeand3.03◦Westlongitude.Wheretheanimalswerehoused,theaveragedaily

temperaturerangedbetween11.2◦Cand15.9◦C,andtheaveragerelativehumidityrangedbetween32%and46%during

theexperiment.ThetropicalareawasintheFrenchWestIndiesat16.16◦Northlatitudeand61.30◦Westlongitude.The

averagedailytemperaturerangedbetween21.0◦Cand25.0◦Candtheaveragerelativehumidityrangedbetween83%and

88%duringtheexperiment.

Ineachsite,4Texelwethers(temperateorigin)and4Blackbellyrams(tropicalorigin)wereusedintwo4×4Latinsquare

designs.Sheepwereborninthesitewheretheexperimenttookplace.Sheepwere2yearsoldandwereﬁttedwitharumen

cannula.Theirbodyweightwas60.2±1.5kgfortheTexelsheepand51.3±4.3kgfortheBlackbellysheepinthetemperate

site;and44.7±0.7kgfortheTexelsheepand44.4±2.1kgfortheBlackbellysheepinthetropicalsite.Managementof

experimentalanimalsfollowedtheguidelinesforanimalresearchoftheFrenchMinistryofAgricultureandotherapplicable

guidelinesandregulationsforanimalexperimentationintheEuropeanUnion(EuropeanCommission,2010).

Inbothsites,sheepwerefedthesameforagefrompermanentgrasslands,onegrowninthetemperateareaandone

growninthetropicalarea.Foreachforage,thereweretwomaturitystagesthatdeterminedforagequality,high(H)andlow

(L),sothatatotalof4forageswerestudiedineachsite.Temperateforagewasasemi-mountainpermanentgrassland,ﬁrst

cycle,ﬂoweringstageharvestedinlateJune(L),andsecondcycleharvestedinlateAugust(H),bothfromthesameparcel.

ThetropicalforagewaspermanentgrasslandrichinDichanthiumspp;regrowthsof36days(H)and91days(L),harvestedin

October.Transportofforagefromonesitetotheotherwasbyship.ChemicalcompositionofforagesispresentedinTable1.

Eachexperimentalperiodlasted4weeks:2weeksforadaptationtotheforage,1weekforCH4measurements,and1

weekfordigestibilityandrumensampling.DetailedresultsondigestibilityandCH4entericproductionsarisingfromthis

studyhavebeenreportedinapreliminarycommunication(Archimèdeetal.,2013)andwillbefullypublishedinasecond

paper(Archimèdeetal.,unpublished).

Table1

Averagedrymattercontentandchemicalcompositionofexperimentalforagesa_.

TempH TempL TropH TropL

Drymatter,g/kgfreshmatterb ₈₇₅ ₈₇₉ ₈₆₆ ₈₇₈

Organicmatter,g/kgdrymatter 878 926 912 929

NDF,g/kgdrymatter 586 623 742 742

ADF,g/kgdrymatter 410 370 470 540

Crudeprotein,g/kgdrymatter 134 83 120 69

a_Temp_H₌_temperate_forage_high_quality,_Temp_L₌_temperate_forage_low_quality,_Trop_H₌_tropical_forage_high_quality,_Trop_L₌_tropical_forage_low_quality. b_Dry_matter_content_of_forages_was_on_average_1.3_percentage_unit_higher_in_temperate_site_than_in_tropical_site.

(4)

Sheepwerefedadlibitumtwicedailyat07:00and19:00andwerehousedinaclosedbarninthetemperatesiteandin asemi-openbarninthetropicalsite.Animalsweretiedinboxesequippedforseparationbetweenurineandfaecesinmale sheep.Theyhadfreeaccesstowaterandsaltblockatalltimes.

2.2. Measurementoffeedintakeanddigestibility,andrumensampling

Feedintakewasmeasuredbyweighingdailyofferedandrefusedforagesfor5consecutivedays.A200-gsampleof offeredandrefusedforageswastaken,andDMcontentwasdeterminedbyoven-dryingat60◦Cuntilconstantweight. Digestibilitycoefﬁcientwasmeasuredbytotalcollectionoffaecesfor5consecutivedays.Afterweighingandmixing,10% ofdailycollectionoffaecesweretakeneveryday,andDMcontentwasdeterminedbyoven-dryingat60◦Cuntilconstant weight.

Approximately250gofwholerumencontentswerecollectedthroughthecannulajustbeforeand3hafterthemorning feeding.Asubsampleofexactly30gwasusedformicrobialanalysis.Theremainingsubsamplewasstrainedthrougha polyestermonoﬁlamentfabric(250␮mmeshsize).ThepHwaspromptlymeasuredusingaportablepH-meter(CG840, electrodeAg/AgCl,SchottGeräte,Hofheim,Germany).

2.3. Fermentationcharacteristicsanalysis

ForVFA,0.8mLofrumenﬂuidﬁltratewasmixedwith0.5mLofasolutioncontaining4mg/mL(w/v)crotonicacid and20mg/mL(w/v)metaphosphoricacidin0.5mol/LHCl,keptat4◦Cfor2handcentrifuged(16,500×g,10min,4◦C). Thesupernatantwasstoredat−20◦_C _until_analysis._Ruminal_VFA_were_determined_in_the_rumen_liquid _phase_by_gas

chromatography(Ottenstein andBartley,1971).Thegaschromatographwasa HewlettPackard 5889equippedwitha

StabilwaxDA(30m×0.53mmi.d.)columnmaintainedat125◦C.ThecarriergaswasH2(34.6mL/min).Forruminalammonia

(NH3),1mLofliquidphasewasaddedto0.1mLofH3PO40.8Mandfrozenat−20◦Cuntilanalysis.Ruminalammoniawas

assayedbycolorimetryusingthephenol-hypochloritemethod(Weatherburn,1967).

2.4. Microbialanalysis

Thirtygramsofwholerumencontentswasdilutedwith15mLofice-coldphosphatebuffersaline(PBS)pH6.8and

homog-enizedinthree1mincycleswith1minintervalsonice,usingaPolytrongrindingmill(KinematicaGmbH,Steinhofhalde

Switzerland).Approximately0.5gwastransferredtoa2-mLEppendorftubeandstoredat−80◦_C_until_DNA_extraction.

TotalDNAwasextractedfromapproximately200mgoffrozenrumensampleusingtheQIAampDNApuriﬁcationkit

(Qiagen,Hilden,Germany)(YuandMorrison,2004).DNAconcentrationandqualitywerecheckedbytheA260andA280

absorbanceratioinaNanoQuantPlateonaspectrophotometer(Inﬁnity,Tecan,Männedorf,Switzerland).Theextracted

DNAwasdilutedto10ng/␮LandusedasatemplateforPCRtoamplifytherrsgeneoftotalbacteriaandthemcrAgene

formethanogens.PrimersusedinthisstudyarelistedinTable2(Edwardsetal.,2007;Muyzeretal.,1993).WhenPCR

productswereusedfordenaturinggradientgelelectrophoresis(DGGE),forwardprimersincludedaGCclamp(_∼40nt).All

PCRreactionswereperformedinaT100ThermalCycler(Bio-RadLaboratories,Marnes-la-Coquette,France)(Popovaetal.,

2011;Sadetetal.,2007).PCRproductswereanalyzedbyelectrophoresison20g/Lagarosegel(w/v)tochecktheirsizeand

estimatetheirconcentrationusingaLowDNAMassLadder(Invitrogen,Carlsbad,CA).

ForDGGEanalysis,theamountofPCRproductloadedongelswasadjustedto100ngforbothbacterialrrsandmethanogen

mcrAgenes.Gelshada6–8(w/v)polyacrylamidegradientandadenaturantgradientof30–55%forrrsand10%to30%formcrA.

Electrophoresiswasperformedin0.5×Tris-Acetate-EDTA(TAE)buffer(TAEBuffer1×,40mmol/LTrisbase,40mmol/Lglacial

aceticacid,1mmol/LEDTA)at200Vand60◦Cfor5h.Gelsweresilverstainedusingacommercialkit(Bio-RadLaboratories)

andanalyzedusingGelComparII(AppliedMaths,Kortrijk,Belgium).Thepeakareaofallbands(N)andofeachband(ni)

andthenumberofbandsSwereusedtocalculatethecommunitybiodiversityusingthreeindices:theShannonindex(H)

calculatedasH=−(ni/N).ln(ni/N),Simpson’sdominanceindex(␭)calculatedas␭=(ni/N)2,andtheevennessindex(e)

calculatedase=H/lnS(Heipetal.,1998).

Thequantitative(q)PCRfortotalbacteria(rrsgene)andmethanogens(mcrAgene)wascarriedout(Morgavietal.,2013).

TheslopeandefﬁciencyforrrsandmcrAprimerswere:_−3.45and95.6%,and_−3.43and95.1%,respectively,withR2_being

greaterthan0.99inbothcases.ThecellulolyticbacteriaFibrobactersuccinogenes,Ruminococcusﬂavefaciens,andRuminococcus

albuswerequantiﬁedwithprimerstargetingtherrsgene(DenmanandMcSweeney,2006;KoikeandKobayashi,2001).The

slopeandefﬁciencyforeachprimerpairwere,respectively,−3.19and105.8%,−3.52and92.3%,and−3.54and91.5%.For

eachrumencontentsample,resultswereexpressedasthemeanofthreereplicatesinrrsormcrAlog10copiespergramof

DMofrumencontents.

Forprotozoacounting,3mLofrumenﬂuidwasaddedto3mLofmethylgreenformalinsaline(MFS)solution(35mL/L

formaldehyde,0.14mmol/LNaCl,0.92mmol/Lmethylgreen)andstoredinthedarkatroomtemperature.Rumenﬂuid/MFS

solutionsweredilutedinanequalvolumeofPBSbeforeprotozoacountsunderamicroscope(×400)inaNeubauerchamber

(5)

M. Rira et al. / Animal Feed Science and Technology 220 (2016) 226–236 229 Table2

OligonucleotidesusedasprimersforPCR-DGGEandqPCRanalysis.

Oligonucleotides Oligonucleotidesequences Target Amplicon

length(bp)

Usein Primerreferences

534R− 5_-ATT_ACC_GCG_GCT_GCT_GG _rrs_Bacteria ₁₉₃ _PCR-DGGE _Muyzer_et_al.₍₁₉₉₃₎

341f-GC 5_-(GC)-CC_TAC_GGG_AGG_CAG_CAG

mcrAr 5‘-TTCATTGCRTAGTTWGGRTAGTT mcrAMethanogens 460–490 PCR-DGGE Lutonetal.(2002)

mcrAf-GC 5-(GC)40-GGTGGTGTMGGATTCACA

CARTAYGCWACAGC

qmcrA-R 5_{-GBARGTCGWAWCCGTAGAATCC} _mcrA_Methanogens ₁₄₀ _qPCR _Denman_et_al.₍₂₀₀₇₎

qmcrA-F 5_{-TTCGGTGGATCDCARAGRGC}

799R2 5-AACAGGATTAGATACCCTG rrsBacteria 280 qPCR Edwardsetal.(2007)

520F 5_{-AGCAGCCGCGGTAAT}

FS586-f 5_{-GTTCGGAATTACTGGGCGTAAA} _rrs_Fibrobacter_succinogenes ₁₂₁ _qPCR _Denman_and

McSweeney(2006)

FS706-r 5_{-CGCCTGCCCCTGAACTATC}

RF96-f 5-CGAACGGAGATAATTTGAGTTTACTTAGG rssRuminococcusﬂavefaciens 132 qPCR Denmanand

McSweeney(2006)

RF220-r 5-CGGTCTCTGTATGTTATGAGGTATTACC

RA1281f 5_-CCC_TAA_AAG_CAG_TCT_TAG_TTC_G _rrs_Ruminococcus_albus ₁₇₅ _qPCR _Koike_and_Kobayashi

(2001)

(6)

Table3

Drymatter(DM)intakeanddigestibility,andrumenfermentationcriteriabeforefeedinginTexel(T)andBlackbelly(B)sheepfedfourforagesintemperate andtropicalsite.a

TempH TempL TropH TropL SEM _P_valuec

T B T B T B T B Site Forage Sitex

Forage C3vsC4 DMintake,g/kgBWb Temperatesite 28.9 21.8 26.3 25.2 16.8 17.0 12.9 15.1 1.87 0.95 <0.001 0.17 <0.001 Tropicalsite 25.8 22.8 21.9 23.7 14.8 22.8 13.8 18.7 DMdigestibilitycoefﬁcient Temperatesite 0.622 0.668 0.554 0.564 0.618 0.625 0.476 0.514 0.0209 0.004 <0.001 0.02 0.27 Tropicalsite 0.637 0.652 0.542 0.576 0.666 0.662 0.604 0.555 pH Temperatesite 6.45 6.38 6.33 6.40 6.53 6.46 6.52 6.37 0.107 0.22 0.73 0.26 0.56 Tropicalsite 6.62 6.64 6.56 6.48 6.40 6.44 6.39 6.50 VFAb_,_mmol/L Temperatesite 92.7 90.5 87.5 91.0 73.1 73.8 93.0 94.4 6.72 0.79 0.02 0.30 0.05 Tropicalsite 81.3 83.1 78.6 95.5 75.3 89.9 85.9 98.9 Acetate,mmol/mol Temperatesite 711 717 751 754 722 718 711 696 12.5 0.02 <0.001 0.02 0.002 Tropicalsite 743 748 734 734 747 741 723 716 Propionate,mmol/mol Temperatesite 192 177 174 173 185 187 200 187 8.8 0.02 0.07 0.16 0.11 Tropicalsite 163 173 179 178 158 170 185 187 Butyrate,mmol/mol Temperatesite 76 80 60 60 79 83 101 97 8.0 0.13 0.002 0.08 0.01 Tropicalsite 75 69 68 69 74 75 75 79

MinorVFAb_,_mmol/mol

Temperatesite 20 25 13 11 14 12 16 20 3.10 0.65 0.30 0.01 0.06 Tropicalsite 19 10 19 19 20 14 17 18 Acetate:Propionate Temperatesite 3.72 4.06 4.35 4.37 3.94 3.86 3.43 3.73 0.260 0.007 0.02 0.10 0.03 Tropicalsite 4.58 4.42 4.24 4.18 4.78 4.40 3.96 3.85 Ammonia,mmol/L Temperatesite 4.56 8.12 6.17 8.42 3.89 4.17 4.43 6.41 1.590 0.30 0.28 0.36 0.69 Tropicalsite 6.69 5.28 8.49 6.59 7.95 5.99 8.11 5.83

a_Temp_H₌_temperate_forage,_high_quality,_Temp_L₌_temperate_forage,_low_quality,Trop_H₌_tropical_forage,_high_quality,_Trop_L₌_tropical_forage,_low quality.

b _BW:_body_weight._VFA:_volatile_fatty_acids._Minor_VFA_are_the_sum_of_isobutyrate,_valerate,_isovalerate_and_caproate. c _Breed_effect,_breed_x_site_and_breed_x_forage_interactions_were_not_signiﬁcant_for_all_measured_parameters.

2.5. Statisticalanalysis

Todetermineeffectsofsite,breedandforageonfeedintake,digestibility,pH,VFAconcentrationandcomposition,NH3

concentration,protozoanumbers,genecopynumbersanddiversityindices,dataunderwentanalysisofvarianceusingPROC

MIXED(SAS,2008).Themodelincludedsite(n=2),forage(n=4),breed(n=2),interactionsbetweenforageandsite,forage

andbreedandbreedandsiteasﬁxedeffectsandanimalnestedwithinbreedasrandomeffect.DifferencesbetweenC3and

C4forageswerealsoanalyzedusingorthogonalcontrasts.EffectsweredeclaredsigniﬁcantatP<0.05.Linearregressions

wereestablishedbetweenCH4emissionperkgDMandthedifferentpopulationsofmicrobes.

3. Results

3.1. Feedintake,digestibilityandruminalfermentationcharacteristics

Drymatterintakeexpresseding/kgBWdidnotvarybetweensitesandwashigherforC3foragesthanforC4forages.

Drymatterdigestibilitydifferedbetweensites,andasitexforageinteractionwasobserved,digestibilitybeinghigherin

tropicalsitethanintemperatesiteonlyforC4forages.Drymatterdigestibilitydifferedbetweenforages,butthisdifference

wasduetoforagequalitywithinC3andwithinC4forages,butdidnotvarybetweenC3andC4forages.Noeffectofbreed

wasobservedonfeedintakeanddigestibility.RuminalpHandNH3concentrationdidnotvarybetweensites,breedsor

forages,andinteractionswerenotsigniﬁcantbefore(Table3)orafterfeeding(TableS1).TotalVFAmolarconcentration

beforefeedingdidnotvarywithsite,butwashigherwithtemperateforagesthanwithtropicalforages(P<0.05).After

feeding,VFAconcentrationwashigherinthetemperatesitethaninthetropicalsite(P<0.05),andthedifferencebetween

temperateandtropicalforagesremained.Beforeandafterfeeding,theproportionofacetatewashigherfortropicalforage

inthetemperatesiteandlowerfortemperateforageinthetropicalsite,andtheproportionofpropionateandbutyrate

washigherforsheepfedtropicalforageinthetemperatesiteandlowerfortropicalforageinthetemperatesite(Table3

(7)

Table4

ProtozoalpopulationinTexel(T)andBlackbelly(B)sheepfedfourforagesintemperateandtropicalsite.a

TempH TempL TropH TropL SEM Pvaluec

Forage

C3vsC4

Total,log10cells/mL

Temperatesite 6.21 6.26 6.13 6.23 6.16 5.97 6.37 6.36 0.109 0.41 0.62 0.01 0.50

Tropicalsite 6.46 5.95 6.34 6.16 6.46 6.35 6.31 6.13

Isotricha,log10cells/mL

Temperatesite 5.16 4.69 2.68 2.74 4.35 3.46 5.27 3.92 0.970 0.77 0.16 0.41 0.11

Tropicalsite 4.11 3.03 4.25 2.48 4.70 4.00 5.23 3.16

Dasytricha,log10cells/mL

Temperatesite 5.59 5.25 5.08 3.53 5.56 5.44 5.94 5.49 0.475 0.35 0.05 0.14 0.52

Tropicalsite 4.82 4.37 5.33 4.54 5.43 5.46 4.70 5.43

LargeEntodiniomorphsb_,_log

10cells/mL

Temperatesite 5.85 6.02 6.00 6.16 5.85 5.65 5.87 6.19 0.138 0.14 0.67 0.41 0.30

Tropicalsite 6.31 5.76 6.12 6.02 6.34 6.21 6.15 5.68

SmallEntodiniomorphsb_,_log

10cells/mL

Temperatesite 5.47 5.34 5.04 5.05 5.04 4.87 5.54 5.35 0.085 0.01 <0.001 <0.001 <0.001

Tropicalsite 5.01 4.97 5.27 5.08 5.04 5.01 5.17 5.17

a_Temp_H₌_temperate_forage,_high_quality;_Temp_L₌_temperate_forage,_low_quality;_Trop_H₌_tropical_forage,_high_quality,_Trop_L₌_tropical_forage,_low quality.

b_Large_{Entodiniomorphs:}_>100_␮m,_Small_{Entodiniomorphs:}_<100_␮m.

c_Breed_effect,_breed_x_site_and_breed_x_forage_interactions_were_not_signiﬁcant_for_all_measured_parameters.

Table5

QuantiﬁcationofrumenbacteriaandmethanogensinTexel(T)andBlackbelly(B)sheepfedfourforagesintemperateandtropicalsite.a

TempH TempL TropH TropL SEM Pvalueb

Forage

C3vsC4

Totalbacteria,log10rrscopynumber/gDM

Temperatesite 11.14 11.64 11.41 11.46 11.47 11.49 11.17 11.26 0.089 <0.001 0.03 0.001 0.14 Tropicalsite 11.59 11.51 11.80 11.61 11.74 11.71 11.65 11.62

Fibrobactersuccinogenes,log10rrscopynumber/gDM

Ruminococcusalbus,log10rrscopynumber/gDM

Temperatesite 8.76 8.76 7.43 8.19 7.81 8.33 7.67 7.86 0.302 0.99 0.40 <0.001 0.92 Tropicalsite 7.62 7.57 8.48 8.07 8.47 8.45 8.17 8.00

Ruminococcusﬂavefaciens,log10rrscopynumber/gDM

Temperatesite 8.78 8.57 8.57 8.47 8.50 8.64 8.23 8.19 0.185 0.30 0.10 0.39 0.22

Tropicalsite 8.63 8.48 8.77 8.58 8.68 8.72 8.49 8.54 Totalmethanogens,log10mcrAcopynumber/gDM

a_Temp_H₌_temperate_forage,_high_quality;_Temp_L₌_temperate_forage,_low_quality;_Trop_H₌_tropical_forage,_high_quality._Trop_L₌_tropical_forage,_low quality.

b_Breed_effect,_breed_x_site_and_breed_x_forage_interactions_were_not_signiﬁcant_for_all_measured_parameters.

interactionsite×forageafterfeeding(TableS1).NoeffectofbreedwasshownforVFAconcentrationorpattern,exceptfor

totalVFAmolarconcentrationafterfeeding,whereabreed×siteinteraction(P<0.05)wasfound.

3.2. Ruminalmicrobiota

Protozoanumbersdidnotvaryamongsites,foragesorbreeds,butasite×forageinteractionwasobserved:protozoa

populationwashigherforC3forageinthetemperatesite,andforC4forageinthetropicalsite(Table4).Small

entodin-iomorphs(<100␮m)weremoreabundantinthetemperatesiteandinsheepfedC3thaninthosefedC4(P<0.05).Large

entodiniomorphs(>100␮m),DasytrichaandIsotrichawerenotdifferentbetweensites,foragesorbreeds.

ResultsofqPCRquantiﬁcationofthebacterialrrsandmethanogenmcrAgenecopiesaresummarizedinTable5.The

concentrationofbacterialrrscopieswashigherinthetropicalsite(P<0.05)andforHforages. TheconcentrationofF.

succinogenesrrsgenecopieswashigherinthetemperatesite(P<0.05).TheconcentrationofR.albuswasinﬂuencedby

neithersitenorforage,butaforage×siteinteractionwasobserved:higherrrscopiesweredetectedinthetemperatesite

forC3forages,whereasinthetropicalsitehigherrrscopieswereobservedforC4forages.TherewasnodifferenceinR.

(8)

Fig.1.Relationshipbetweenrumenmicrobesandmethaneentericemissionaccordingtosite(temperate(France,F)vstropical(FrenchWestIndies,FWI)) andoriginofforages(TemperateC3(Temp)vsTropicalC4(Trop)):highqualityandlowqualityforagefedtoTexelandBlackbelly.Eachpointisthemean of4individualvalues.

MethanogenconcentrationexpressedasmcrAgenecopynumberinrumencontentswashigherinthetropicalsite.Effects

offorageandinteractionbetweensiteandforagewereobserved:methanogenconcentrationswerehigherinsheepfedC4

foragesinthetropicalsiteandinsheepfedC3foragesinthetemperatesite(Table5).

DGGEanalysisshowednodifferencewithinbacterialcommunitystructureandmethanogeniccommunitystructure,and

clusteringpatternsdidnotdiffersubstantiallyamongindividualsheep(datanotshown).Diversityindiceswerecalculated

fromtherrsDNAPCR-DGGEproﬁles.Shannon,dominanceandevennessindicesdidnotvarybetweensitesorbreeds

(TableS2).However,atendentialsite×breedinteractionwasobservedfortheShannonindexinrrsDNAPCR-DGGEproﬁles

(P=0.055).Therewasnodifferencebetweenforagesineitherthenumberofbandsorthevaluesofdiversityindicesforthe

rrsDNAPCR-DGGEproﬁles(datanotshown).

3.3. Relationshipsbetweenmicrobialpopulationsandmethaneproduction

Fig.1presentsrelationshipsbetweenmicrobialabundancesandCH4productionaccordingtosite,breedandforages

(meanof4animalsforeachcombination).Signiﬁcantandpositiverelationshipswerefoundbetweenprotozoapopulation

andCH4production(r=0.53,P<0.05),andbetweenR.albusandCH4production(r=0.52,P<0.05)(Fig.1).Norelationship

wasfoundbetweentotalbacteria,R.ﬂavefaciens,F.succinogenesandCH4production(r=0.44,0.27and0.05,respectively,

P>0.05).HigherCH4productionwasassociatedwithhighermethanogensnumbersbuttherelationshipwasnon-signiﬁcant

(9)

4. Discussion

Factorsotherthananimalandfeedcharacteristicsareknowntoaffectrumendigestionincluding,amongotherfactors,

climaticconditions(Decruyenaereetal.,2009).DigestionandCH4productionbyruminantsdifferbetweentemperateand

tropicalconditions.However,astrialsareusuallycarriedoutusingfeedstuffsharvestedonsiteandasingle,locallyadapted

breed(Archimèdeetal.,2011)ithasnotbeenpossibletoassesstherelativeimportanceofthesefactors.Toourknowledge,

ourstudyistheﬁrsttosimultaneouslycompareinatemperateandinatropicalenvironment,atemperateandatropical

sheepbreedfedthesamediet,allowingaﬁnerinterpretationofallegeddifferencesbetweentemperateandtropicalsites

inCH4 production,rumenfermentation,andmicrobialecosystem.Resultsonintakeanddigestibilitywillbethoroughly

discussedinasecondpaper(Archimèdeetal.,unpublished)andarebrieﬂydiscussedinthismanuscript.

4.1. Effectofenvironmentondigestiveprocesses

IntakeparkgBWwasnotaffectedbysite.Thetemperatureinthetropicalsitewascertainlynothighenoughtoaffect

intakeassuggestedbythereviewofMorand-FehrandDoreau(2001).Thehigherdigestibilityobservedinthetropicalsite

isinaccordancewiththereviewofMorand-FehrandDoreau(2001)forawiderangeoftemperatures,forasameintake.

Independentlyofdietandanimaltype,thereislimitedexperimentalevidencetoexplaindifferencesinrumenfermentation

andmethanogenesisbetweentemperateandtropicalsites.Suchdifferencescanresultfromdifferencesintemperatureor

hygrometry.Inthisstudy,nodifferenceinVFAconcentrationbetweensiteswasobservedbeforefeeding,butafterfeeding,

theconcentrationwaslowerforthetropicalsitethanforthetemperatesite.Thismaybeexplainedbythedifferencein

temperature,asitwasreportedbyKelleyetal.(1967)incowsfedaconstantdietatdifferentcontrolledtemperatures,and

byMartzetal.(1990)incowsfedforagedietswithmoderatedifferencesinintake.Anotherexplanationcouldbethedilution

ofVFAintherumenduetohigherwaterconsumption(Silanikove,1992).However,inthisexperiment,rumenwatercontent

was87.7%onaverageandwassimilarbetweensites,whichdoesnotsupportthishypothesis.

Incontrasttoourresults,increaseintotalrumenVFAcontentandadecreaseinpHwerereported(Kadzereetal.,2002)

whenambienttemperatureincreasedbecauseVFAwereabsorbedlessefﬁciently.Inthepresentstudy,thetropicalsite

hadhigheracetateproportionthanthetemperatesitebeforeandafterfeeding;thesametendencywasobservedforthe

acetate:propionateratio.Thisresultisinlinewithapreviousstudyshowingthathighambienttemperaturewasassociated

withhigherproportionofacetate(Kelleyetal.,1967).However,thistrendisnotgeneral,asanothertrialshowedthatan

increaseintemperaturedidnotchangeVFApatternincattle,andincreasedpropionateinsheep(Lippke,1975).

TheobserveddifferencesinVFApatterncanbeduetodifferencesinmicrobialcommunitiesbetweentemperateand

tropicalsites,especiallyastherewasnoeffectofsiteondailyfeedintake,onaverage20.5gDM/kgbodyweightforboth

sites(Archimèdeetal.,2013).TheqPCRanalysisrevealedthattotalbacteriaandmethanogennumberswerehigherforthe

tropicalsite.Changeinbacterialnumbersmaybepartiallyexplainedbythelowerconcentrationofsmallentodiniomorphs

inthetropicalsite,andhenceadecreasedpredatoryactivityonbacteria.Incontrasttoourresults,asigniﬁcantchangein

thecompositionbutnotintheconcentrationofmicrobialpopulationsatelevatedambienttemperatureandhumiditywas

reported(Tajimaetal.,2007),thisshiftbeingaccompaniedbyadecreaseinconcentrationofVFAintherumen.Thetargeted

cellulolyticbacteriawerenotaffectedbysite,exceptforF.succinogenes,whichwasmoreabundantinthetemperatesite

(Table5).Thisbacteriumisamajorﬁbre-degradingspeciesintherumenthatdoesnotproduceH2andisnotassociated

withhigherCH4production(Chaucheyras-Durandetal.,2010).AhigheracetateproductionisgenerallylinkedtohigherCH4

production,buttherewasnositeeffectonCH4production(Archimèdeetal.,2013)forthisexperiment(18.7and19.4g/kg

DMintakeonaveragefortemperateandtropicalsites,respectively).Methanewasgenerallyproducedinhigherquantities

whenH2-producingcellulolyticspeciessuchasRuminococciweredominant,becauseoftheirassociationwithmethanogens,

whichconsumeH2andproduceCH4(Chaucheyras-Durandetal.,2010;Morgavietal.,2010)resultingsimultaneouslyin

higherproductionofacetate(Morgavietal.,2010;Pavlostathisetal.,1990).Thisisinlinewithourresultswherethenumber

ofRuminococciwasalsonotaffectedbyanyofthefactorstestedandnotcorrelatedwithCH4production(Fig.1).

Inthepresentworkwefoundlittleeffectofenvironmentalconditionsonrumenfermentationandmicrobialpopulations.

Thereareveryfewdataintheliterature:achangeinrumendiversityandstructureofmicrobiotawasobserved(

Romero-Pérezetal.,2011),suggestingthatshiftsinmicrobialpopulationsmayhavebeenduetodifferencesintemperaturesensitivity

amongmicrobialspecies,buttherangeoftemperaturetestedbytheseauthorswasbetween−5◦_C_and₋₃₀◦_C._The_rumen

anditscontentsarethermallystablebutamodestchangeanddisturbancewithintherumenmayoccurwithchangesin

ambienttemperature.Adifferenceindrinkingwatertemperaturecaninduceadifferenceinthetemporarydecreasein

rumentemperatureafterdrinking(Bewleyetal.,2008).Anotherhypothesisisthatadifferenceinambienttemperature(9

vs.26◦C)resultsinadifferenceinmeanretentiontimethroughhormonalchanges,whichmayinﬂuencerumenfermentation

(Barnettetal.,2015)andthusperhapsmicrobiota.

4.2. Effectofforagetypeondigestiveprocesses

Todate,studiesontropicalforageshavelargelyfocusedoncomparisonsofvariousplantspeciesorphysiologicalstages

(Assoumaya,2007).Thehigherintakeoftemperateforagescomparedtotropicaloneswasindependentofsiteconﬁrming

(10)

withdataontropicalforagescarriedoutintropicalareas.Inthepresentexperiment,itwasobservedalogicalreduction

indigestibilitywithpoorqualityforagesbutalsoanoticeableimprovementindigestibilityoflowerqualityC4foragesin

thetropicalsite.InC4forages,digestibilitybetweenhighandlowqualityhayisseverelyreducedcomparedtoC3and

animalsonthetropicalsiteseembetteradaptedtoextractnutrientsfromthispoorqualityfeedresource.Thecurrentstudy

showsthatgrassphysiologicaltype(temperateC3vstropicalC4)hadmoreinﬂuenceonrumenfermentationthansite.

DifferencesbetweenforagesinVFAconcentrationandpatterncouldbeexplainedbytheirquality:natureandcontentof

ﬁbreandamountoflignin.Inthepresentstudy,poorqualityforages(HvsL)resultinalowerVFAconcentrationandina

higheracetate:propionateratio,independentlyofthesite.Thisconﬁrmsliteraturedataontropicalforages:adecreasein

propionaterelativetototalVFA,whileacetateincreasedwithadvancingmaturityoftheC4grassPanicummaximumwas

observed(Assoumaya,2007;Rellingetal.,2001).ThereisnodirectcomparisonbetweenC3andC4grassesintheliterature,so

thatitisdifﬁculttoknowwhetherdifferencesbetweenC3andC4foragesobservedinthisstudyderivedfromthechemical

composition,suchasthehighercontentofsecondarymetabolitesofDichanthiumannulatum(Awadetal.,2015)present

inthemixtureofDichanthiuminthisstudy.Inameta-analysis(Assoumayaetal.,2007),ruminalOMdigestion,whichis

relatedtoVFAproduction,wassimilarbetweentropicalandtemperateforagesforthesameﬁbrecontent,suggestingthat

chemicalcompositionplaysamajorroleinthedifferencesfoundbetweentropicalandtemperateforages.Inourexperiment,

butyrateproportionwashigherforLthanforHforages,andhigherafterfeedingfortemperatethanfortropicalforages.

Thissuggeststhatbutyrateproductionisnotdirectlyrelatedtochemicalcomposition.Butyrateisgenerallyassociatedwith

protozoa(Eugèneetal.,2004),butinourstudytherewasnoeffectofforagetypeonnumbersofprotozoa,bacteriaor

methanogens.However,asigniﬁcantsite×forageinteractionbetweentemperateandtropicalwasfoundfortotalbacteria,

R.albus,methanogensandprotozoa.Thisshowsthatthesepopulationswerehighestwithtropicalforagesinthetropicalsite,

suggestingthatdegradationofpoorqualityC4foragescharacteristicofthetropicsrequiresadensermicrobialcommunity.

Foragetype(C3vsC4)hadnoeffectonthepopulationnumbersoftotalorcellulolyticbacteriaandmethanogens,whereas

aglobaleffectofforageisshownforbothtotalbacteriaandmethanogens.Thissuggestsaneffectofquality,i.e.nutritive

valueofforages,onthesepopulations.Bycontrast,astrongvariationinruminalbacteriaofyoungsteersfedeitheraC3or

aC4grasswasreported(Pittaetal.,2010).

NodifferenceinmethanogenmcrAcopy numberwasdetectedamongforages, althoughweexpectedtoﬁndmore

methanogenswithtropicalforages,becauseinthistrialtheygeneratedmoreCH4thantemperateforages:17.5and20.7g/kg

DMintakeonaverageforC3temperateforagesandC4tropicalforages,respectively(Archimèdeetal.,2013).Theseresults

areinlinewithpreviousresults(Danielssonetal.,2012;Zhouetal.,2011)whoreportedthatCH4productionwasnotrelated

tototalmethanogenpopulation.Methaneproductioncouldberelatedtomethanogenactivityratherthantheirabundance

(Popovaetal.,2011).

4.3. Effectofbreedondigestiveprocesses

Thereislittleinformationavailableontheextenttowhichrumenfermentationandmicrobesvarybetweensheepbreeds.

Inourexperimenttwocontrastingsheepbreedswerechosen:oneoftemperateoriginandtheotheroftropicalorigin.Overall,

rumenfermentationandmicrobeswerenotaffectedbybreedforanyofthevariablesmeasured.Theabsenceofchangein

ruminalVFAconcentrationbetweengenotypesagreeswithapreviouscomparisonoftwosheepbreeds:alocaloneand

animprovedone(Ile-de-FranceandChurra-da-Terra-Quente)fedthesamediet(Lourenc¸oetal.,2013).Veryfewstudies

havecomparedmorewidely-contrastingbreeds.AcomparisonbetweenlocalBostaurusbreedandaBosindicusbreedin

atropicalenvironmentshowednodifferencesindigestiveprocesses(GrimaudandDoreau,2003).Differencesindigestion

betweenbreedsmaydiffermoreforpoorqualityfeeds,forwhichlocalbreedsshouldbebetteradaptedthanimproved

breeds(Lopezetal.,2001).However,breed×forageinteractionwasnotsigniﬁcantinourstudy,andtheabsenceofbreed

effectmightbeduetotheadaptationofruminalmicrobiotaforbothTexelandBlackbellytotheenvironmentwherethe

experimenttookplace,sincetheywerebornandraisedclosetoeachexperimentalsite.Theestablishmentoftherumen

microbiotaisinﬂuencedbynutritionaland/orenvironmentalexposureinyounganimalsafterbirth.Consequently,induced

variationsinmicrobialpopulationscolonizingtherumencanleadtomodiﬁcationsinfermentationandCH4 production

(Abeciaetal.,2013).TheadaptationofTexelandBlackbellymicrobiotatothetemperatevstropicalenvironmentmayhave

reduceddifferencesbetweenbreeds.Inaddition,climateconditionsduringthistrialwerenotextremeenoughtofavor

distinctadaptiveandbehavioralresponsesbetweenbreeds.Unfortunately,theoriginandhistoryofexperimentalanimals

isnotspeciﬁedinmoststudies.Atthemicrobiallevel,qPCR analysisdidnotrevealanydifferencebetweenTexeland

Blackbelly.Theseresultscorroboratepreviousstudiesshowingthatmethanogens,bacteriaandprotozoapopulationwere

notsigniﬁcantlydifferentbetweentwocattlebreeds(Rookeetal.,2014).Incontrasttothisstudy,someauthorshaveshown

theimpactofbreedonpotentialCH4productionandrumenmicrobiotaandsuggestthatthehostanimalexertsacontrolling

effectonitsowngutmicrobiota(Danielssonetal.,2012;Kingetal.,2011).

Differencesinmicrobialpopulationswerepredominantlyattributabletodiet,withthehostbeinglessinﬂuential.In

additiontofeedcompositioneffects,theenvironmentappearstoinﬂuencetheestablishmentand developmentofthe

(11)

5. Conclusion

Thisstudyshowsthatthedifferencesobservedintherumenmicrobiotaofsheepbetweentropicalandtemperatesites

wereintrinsictothelocationandcouldnotbeattributedtolocalforages(temperateC3vstropicalC4)orbreeds.Total

bacteriaandmethanogensweremoreabundantinthetropicalsite.Thisstudyalsoshowstheadaptationcapacityofeach

breedtoitsenvironment,suggestedbythesite×breedinteraction.Futureworkshouldcharacterizethemicrobiotausing

high-throughputsequencingforphylogeneticandmetagenomicanalysistogainabetterunderstandingoftheinﬂuenceof

theenvironmentinshapingrumenpopulations.

Conﬂictofinterest

Theauthorsdeclarethattherearenotconﬂictsofinterest.

Acknowledgments

ThisworkhasbeengrantedbytheFrenchNationalAgencyforResearch(ANR)(EPADprojectANR-09-STRA-01),byRegion

GuadeloupeandotherEuropeanfunding:FEOGA,FEDER,FSE.WethankthestaffofUE1414Herbipôle,INRA(France),

especiallySébastienAlcouffe,AndréGuittardandDenisRoux,thestaffofUE1284PTEA,INRA(Guadeloupe)especiallyFred

Periacarpinforanimalsampling,andDominiqueGraviouforhelpinmicrobialanalyses.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version, at

http://dx.doi.org/10.1016/j.anifeedsci.2016.08.010.

References

Abecia,L.,Martín-García,A.I.,Martínez,G.,Newbold,C.J.,Yá ˜nez-Ruiz,D.R.,2013.Nutritionalinterventioninearlylifetomanipulaterumenmicrobial colonizationandmethaneoutputbykidgoatspostweaning.J.Anim.Sci.91,4832–4840.

Archimède,H.,Eugène,M.,Marie-Magdeleine,C.,Boval,M.,Martin,C.,Morgavi,D.P.,Lecomte,P.,Doreau,M.,2011.Comparisonofmethaneproduction betweenC3andC4grassesandlegumes.Anim.FeedSci.Technol.166,59–64.

Archimède,H.,Rira,M.,Eugène,M.,Morgavi,D.P.,Anaïs,C.,Periacarpin,F.,Calif,B.,Martin,C.,Marie-Magdeleine,C.,Doreau,M.,2013.Intake,total-tract digestibilityandmethaneemissionofTexelandBlackbellysheepfedC4andC3grassestestedsimultaneouslyinatemperateandatropicalarea.Adv. Anim.Biosci.4,285.

Assoumaya,C.,Sauvant,D.,Archimède,H.,2007.Etudecomparativedel’ingestionetdeladigestiondesfourragestropicauxettempérés.INRAProd. Anim.20,383–392.

Assoumaya,C.,2007.EtudeDesFacteursLimitantl’ingestionChezLesPetitsRuminantsValorisantDesFourragesTropicaux.PhDThesis,Agro.ParisTech., Paris,France,298pp.

Awad,M.M.,Ragab,E.A.,Atef,A.,2015.PhytochemicalinvestigationandbiologicalevaluationofDichanthiumannulatum(Forrsk).J.Sci.Innov.Res.4, 131–137.

Barnett,M.C.,McFarlane,J.R.,Hegarty,R.S.,2015.Lowambienttemperatureelevatesplasmatriiodothyronineconcentrationswhilereducingdigesta meanretentiontimeandmethaneyieldinsheep.J.Anim.Physiol.Anim.Nutr.99,483–491.

Bewley,J.M.,Grott,M.W.,Einstein,M.E.,Schutz,M.M.,2008.Impactofintakewatertemperaturesonreticulartemperaturesoflactatingdairycows.J. DairySci.91,3880–38837.

Chaucheyras-Durand,F.,Masséglia,S.,Fonty,G.,Forano,E.,2010.Inﬂuenceofthecompositionofthecellulolyticﬂoraonthedevelopmentof hydrogenotrophicmicroorganisms,hydrogenutilization,andmethaneproductionintherumensofgnotobioticallyrearedlambs.Appl.Environ. Microbiol.76,7931–7937.

Danielsson,R.,Schnürer,R.,Arthurson,V.,Bertilsson,J.,2012.MethanogenicpopulationandCH4productioninswedishdairycowsfeddifferentlevelsof forage.Appl.Environ.Microbiol.78,6172–6179.

Decruyenaere,V.,Buldgen,A.,Stilmant,D.,2009.Factorsaffectingintakebygrazingruminantsandrelatedquantiﬁcationmethods:areview.Biotechnol. Agron.Soc.Environ.13,559–573.

Denman,S.E.,McSweeney,C.S.,2006.Developmentofareal-timePCRassayformonitoringanaerobicfungalandcellulolyticbacterialpopulationswithin therumen.FEMSMicrobiol.Ecol.58,572–582.

Denman,S.E.,Tomkins,N.W.,McSweeney,C.S.,2007.Quantitationanddiversityanalysisofruminalmethanogenicpopulationsinresponsetothe antimethanogeniccompoundbromochloromethane.FEMSMicrobiol.Ecol.62,313–322.

Edwards,J.E.,Huws,S.A.,Kim,E.J.,Kingston-Smith,A.H.,2007.Characterizationofthedynamicsofinitialbacterialcolonizationofnonconservedforagein thebovinerumen.FEMSMicrobiol.Ecol.62,323–335.

Eugène,M.,Archimède,H.,Michalet-Doreau,B.,Fonty,G.,2004.Effectsofdefaunationonmicrobialactivitiesintherumenoframsconsumingamixed diet(freshDigitariadecumbensgrassandconcentrate).Anim.Res.53,187–200.

EuropeanCommission,2010.Directive2010/63/EUoftheEuropeanParliamentandoftheCouncilof22September2010ontheprotectionofanimals usedforscientiﬁcpurposes.Off.J.Europ.Union,L276/33-L276-79.Availableat:

http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32010L0063&from=EN(accessed12.06.16).

Gerber,P.J.,Steinfeld,H.,Henderson,B.,Mottet,A.,Opio,C.,Dijkman,J.,Falcucci,A.,Tempio,G.,2013.TacklingClimateChangeThroughLivestock−A GlobalAssessmentofEmissionsandMitigationOpportunities.FoodandAgricultureOrganizationoftheUnitedNations(FAO),Rome,Italy. Grimaud,P.,Doreau,M.,2003.Effectsoflevelofintakeandnitrogensupplementationondigestionbycowsinatropicalenvironment.Anim.Res.52,

103–118.

Heip,C.H.R.,Herman,P.M.J.,Soetaert,K.,1998.Indicesofdiversityandevenness.Océanis24,61–87.

Hristov,A.N.,Oh,J.,Firkins,J.L.,Dijkstra,J.,Kebreab,E.,Waghorn,G.,Makkar,H.P.S.,Adesogan,A.T.,Yang,W.,Lee,C.,2013.Mitigationofmethaneand nitrousoxideemissionsfromanimaloperations:I.Areviewofentericmethanemitigationoptions.J.Anim.Sci.91,5045–5069.

(12)

Kelley,R.O.,Martz,F.A.,Johnson,H.D.,1967.Effectofenvironmentaltemperatureonruminalvolatilefattyacidlevelswithcontrolledfeedintake.J.Dairy Sci.50,531–533.

King,E.E.,Smith,R.P.,St-Pierre,B.,Wright,A.D.G.,2011.DifferencesintherumenmethanogenpopulationsoflactatingJerseyandHolsteindairycows underthesamedietregimen.Appl.Environ.Microbiol.77,5682–5687.

Koike,S.,Kobayashi,Y.,2001.DevelopmentanduseofcompetitivePCRassaysfortherumencellulolyticbacteria:Fibrobactersuccinogenes,Ruminococcus albusandRuminococcusﬂavefaciens.FEMSMicrobiol.Lett.204,361–366.

Lippke,H.,1975.Digestibilityandvolatilefattyacidsinsteersandwethersat21and32◦_C_ambient_temperature._J._Dairy_Sci._58,_1860–1864.

Lopez,S.,Frutos,P.,Mantecon,A.R.,Giraldez,F.J.,2001.Comparativedigestionofherbagebytwobreedsofsheep:effectsofgrassmaturitystageandlevel ofintake.Anim.Sci.73,513–522.

Lourenc¸o,A.L.,Cone,J.W.,Fontes,P.,Dias-da-Silva,A.A.,2013.Effectsofsheepbreedandsoybeanmealsupplementationonrumenenvironmentand degradationkinetics.NJASWagen.J.LifeSci.64–65,77–85.

Luton,P.E.,Wayne,J.M.,Sharp,R.J.,Riley,P.W.,2002.ThemcrAgeneasanalternativeto16SrRNAinthephylogeneticanalysisofmethanogen populationsinlandﬁll.Microbiology148,3521–3530.

Martin,C.,Morgavi,D.P.,Doreau,M.,2010.Methanemitigationinruminants.Frommicrobestothefarmscale.Animal4,351–365.

Martz,F.A.,Payne,C.P.,Matches,A.G.,Belyea,R.L.,Warren,W.P.,1990.Forageintake,ruminaldrymatterdisappearance,andruminalbloodvolatilefatty acidsforsteersin18and32◦_C_{temperatures.}_J._Dairy_Sci._73,_1280–1287.

Morand-Fehr,P.,Doreau,M.,2001.Alimentationdesruminantssoumisàunstressdechaleur.INRAProd.Anim.14,15–27. Morgavi,D.,Forano,E.,Martin,C.,Newbold,C.J.,2010.Microbialecosystemandmethanogenesisinruminants.Animal4,1024–1036.

Morgavi,D.P.,Martin,C.,Boudra,H.,2013.FungalsecondarymetabolitesfromMonascusspp.reducerumenmethaneproductioninvitroandinvivo.J. Anim.Sci.91,848–860.

Muyzer,G.,DeWaal,E.C.,Uitterlinden,A.G.,1993.Proﬁlingofcomplexmicrobialpopulationsbydenaturinggradientgelelectrophoresisanalysisof polymerasechainreaction-ampliﬁedgenescodingfor16SrRNA.Appl.Environ.Microbiol.59,695–700.

Ottenstein,D.M.,Bartley,D.A.,1971.ImprovedgaschromatographyseparationoffreeacidsC2-C5indilutesolution.Anal.Chem.43,952–955.

Pavlostathis,S.G.,Miller,T.L.,Wolin,M.J.,1990.CellulosefermentationbycontinuousculturesofRuminococcusalbusandMethanobrevibactersmithii.Appl. Microbiol.Biotechnol.33,109–116.

Pitta,D.W.,Pinchak,W.E.,Dowd,S.E.,Osterstock,J.,Gontcharova,V.,Youn,E.,Dorton,K.,Yoon,I.,Min,B.R.,Fulford,J.D.,Wickersham,T.A.,2010.Rumen bacterialdiversitydynamicsassociatedwithchangingfrombermudagrasshaytograzedwinterwheatdiets.Microb.Ecol.59,511–522.

Popova,M.,Martin,C.,Eugène,M.,Mialon,M.M.,Doreau,M.,Morgavi,D.P.,2011.Effectofﬁbre-andstarch-richﬁnishingdietsonmethanogenicArchaea diversityandactivityintherumenoffeedlotbulls.Anim.FeedSci.Technol.166-167,113–121.

Ranilla,M.J.,Jouany,J.P.,Morgavi,D.P.,2007.Methaneproductionandsubstratedegradationbyrumenmicrobialcommunitiescontainingsingle protozoalspeciesinvitro.Lett.Appl.Microbiol.45,675–680.

Relling,E.A.,VanNiekerk,W.A.,Coertze,R.J.,Rethman,N.F.G.,2001.AnevaluationofPanicummaximumcv.Gatton.Theinﬂuenceofstageofmaturityon dietselection,intakeandrumenfermentationinsheep.S.Afr.J.Anim.Sci.31,85–92.

Romero-Pérez,G.A.,Ominski,K.H.,McAllister,T.A.,Krause,D.O.,2011.Effectofenvironmentalfactorsandinﬂuenceofrumenandhindgutbiogeography onbacterialcommunitiesinsteers.Appl.Environ.Microbiol.77,258–268.

Rooke,J.A.,Wallace,R.J.,Duthie,C.A.,McKain,N.,deSouza,S.M.,Hyslop,J.J.,Ross,D.W.,Waterhouse,T.,Roehe,R.,2014.Hydrogenandmethaneemissions frombeefcattleandtheirrumenmicrobialcommunityvarywithdiet,timeafterfeedingandgenotype.Br.J.Nutr.112,398–407.

StatisticalAnalysisSystem,2008.SAS/STAT9.2User’sGuide.SASInstitute,Cary,NC.

Sadet,S.,Martin,C.,Meunier,B.,Morgavi,D.P.,2007.PCR-DGGEanalysisrevealsadistinctdiversityinthebacterialpopulationattachedtotherumen epithelium.Animal1,939–944.

Silanikove,N.,1992.Effectsofwaterscarcityandhotenvironmentonappetiteanddigestioninruminants:areview.Livest.Prod.Sci.30,175–1794. Tajima,K.,Nonaka,I.,Higuchi,K.,Takusari,N.,Kurihara,M.,Takenaka,A.,Mitsumori,M.,Kajikawa,H.,Aminov,R.I.,2007.Inﬂuenceofhightemperature

andhumidityonrumenbacterialdiversityinHolsteinheifers.Anaerobe13,57–64.

Weatherburn,M.W.,1967.Phenol-hypochloritereactionfordeterminationofammonia.Anal.Chem.39,971–974.

Yu,Z.,Morrison,M.,2004.ImprovedextractionofPCRqualitycommunityDNAfromdigestaandfecalsamples.Biotechniques36,808–813. Zhou,M.,Chung,Y.H.,Beauchemin,K.A.,Holtshausen,L.,Oba,M.,McAllister,T.A.,Guan,L.L.,2011.Relationshipbetweenrumenmethanogensand