The use of Prevotella bryantii 3C5 for modulation of the ruminal environment in an ovine model

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

The

use

of

Prevotella

bryantii

3C5

for

modulation

of

the

ruminal

environment

in

an

ovine

model

Martín

Fraga

_,

_Sofía

_Fernández

_,

_Karen

_Perelmuter

_,

_Nicolle

_Pomiés

_,

Cecilia

Cajarville

_,

_Pablo

_Zunino

a_{InstitutodeInvestigacionesBiológicasClementeEstable,DepartamentodeMicrobiología,Montevideo,Uruguay}

b_{InstitutoNacionaldeInvestigaciónAgropecuaria,INIA-LaEstanzuela,PlataformadeInvestigaciónenSaludAnimal,Colonia,Uruguay}

c_{UniversidaddelaRepública,FacultaddeVeterinaria,InstitutodeProducciónAnimal,DepartamentodeNutriciónAnimal,SanJosé,}

Uruguay

a

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t

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f

o

Articlehistory:

Received24April2018 Accepted17July2018

Availableonline25August2018 AssociateEditor:MilianeSouza

Keywords: Probiotics Rumen Fermentation Prevotellabryantii Ovinemodel

a

b

s

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t

IntheSouthernHemisphere,ruminantsaremostlyraisedingrazingsystemswhereanimals consumeforageandaresupplementedwithlowamountsofconcentrates.Concentrates are usually given separatelyand are rapidly ingested. Thispractice leads tochanging rumenenvironmentconditionsduringtheday,mayaltertherumenmicrobialmetabolism andcouldaffecthostperformance.ThenativeruminalPrevotellabryantiistrain3C5was administeredevery48htowethersunderexperimentalconditionssimulating Southern-Hemisphere feedingtoevaluate its potentialas arumen fermentationmodulator. The inoculumpotentialwasassessedonday17.Theammonianitrogen(NH3-N),volatilefatty

acids and ruminal pHweremonitored ona 24-hbasis 19 days afterthe beginningof theexperiment,andthemicrobialcommunitystructurewasassessedbypyrosequencing. TheadministrationofP.bryantiimodifiedthefermentationproductsanddailypHvalues comparedtothecontrol.TheNH3-Nconcentrationintherumenoftreatedanimalswas

significantlyhigherthanthatoftheuntreatedanimals.Modificationoftheruminal envi-ronmentandfermentationpathwayswasachievedwithoutalteringthegeneralstructure ofthemicrobialcommunityorthepotentialmethaneproduction.P.bryantii3C5couldbe consideredinpotentialprobioticformulationsforruminantsinsemi-intensivesystems.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Ruminantsare raised inintensive and semi-intensive pro-duction systems that include the supplementation with protein-energy concentrates. These practices can generate

∗ _{Correspondingauthorat:PlataformadeSaludAnimal,INIA-LaEstanzuela,InstitutoNacionaldeInvestigaciónAgropecuaria,Ruta50,}

km11,CP70006Colonia,Uruguay. E-mail:mfraga@inia.org.uy(M.Fraga).

a misbalance of rumen microorganisms’ metabolism, and therefore, production yields are usually affected. To over-cometheselimitations,feedadditivessuchasantibioticsor ionophores havebeen extensively used.1 _{Due to the}

emer-gence risks of resistant strains and antibiotic residues in animalproductsandbyproducts,theuseofantibioticsasfeed

https://doi.org/10.1016/j.bjm.2018.07.004

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

additiveshasbeenprogressivelyrestricted.Theuseof antibi-oticshasbeenbannedintheEuropeanUnionsinceJanuary 2006(Directive1831/2003/CEE,EuropeanCommission2003). Therefore,alternativestotheuseofantibioticshavebeen pro-posed,includingtheuseofprobiotics.2,3

Probiotics,livemicroorganismsthatconferbenefitstothe host,4_{couldbedesignedtomodulatetherumenmicrobiota,}

improving feed utilization. This goal could be achieved by enhancingfiberandstarchdigestion,promotingvolatilefatty acid(VFA)synthesisanddiminishingorbufferinglactate accu-mulationtoavoidacidificationofruminalcontents.2,5 _Some

oftheseissueshavebeenassessedinpreviousstudiesusing differentnativeandexogenousmicroorganismswithdiverse results.6–8

Prevotella isthemostrepresented bacterialgenusinthe

rumen,9_{andinparticular,Prevotellabryantiistrainshavebeen}

testedonhigh-producingdairycowstoevaluatethe poten-tialofthestrainstopreventsubacuteacidosis.10,11_Although

P.bryantiiwasnoteffectiveatpreventingruminalacidosis,the

authorsobservedthatitsadministrationcouldimprovethe ruminalenvironment.

IntheSouthernHemisphere,ruminantsaremostlyraised ingrazingsystems.Animalsusuallyconsumeforageallday longandaresupplementedwithlowamountsofconcentrates generallyprovidedseparatelyfromforage,onceortwiceaday. Thispracticeleadstodailyvariationsofrumenenvironment conditions.12,13_{Therefore,intheseconditions,theuseof}

pro-bioticscouldhelptostabilizetherumenenvironmentandfeed digestion.

Theobjectiveofthisworkwastoevaluatetheabilityofa nativeP.bryantiistraintomodulatetheruminalenvironment andfermentationandthebacterialmicrobiotastructureina modelbasedonsheepconsumingforageandsupplemented twiceadaywithahigh-starchconcentrate.

Materials

and

methods

Bacterialculturesandinoculumpreparation

P.bryantii3C5,usedintheadministrationtrial,waspreviously

isolatedfromtherumenofanonlypasture-consumingcow attheExperimentalStationatLibertad(SanJosé,Uruguay), Faculty ofVeterinary, University ofUruguay,and identified bysequencing ofthe 16SribosomalRNAgene(JQ674698.1). Thisstraingrewinaculturemediumcontaininglactateas thesole carbon sourceand exhibitedantimicrobialactivity againstEscherichiacoliandStreptococcusbovis.Bacterialcultures weregrownusingamodifiedrumen-fluid-freebrothaccording toCaldwellandBryant14_{(Table1).Forinoculumpreparation,}

Pb3C5wasinoculatedinCO2-gassedculturebottles

contain-ing50mLofbrothand incubatedat39◦Cuntiladensityof 1×108_{cells/mLwasachieved.}

Animals,feedingandpreparationofsamples

Animalswere cared forand handled accordingtothe pro-cedures approvedby the Honorary Commission ofAnimal Experimentation(CHEA)oftheUniversityofUruguayandby theNationalCommissionofAnimalExperimentation(CNEA,

Table1–Mediumcomposition.

Component %(m/v) Yeastextract 0.05 Trypticase 0.2 Hemin 0.0001 Glucose 0.05 Cellobiose 0.05 Solublestarch 0.05 Mineralsolutiona_{(mL) 3.75} VFAmixtureb_{(mL) 0.31} Na2S·9H2O 0.025 Cysteinehydrochloride 0.025 Na2CO3 0.4

a _{Mineral solution composition (g/L): NaCl, 5.4; KH}

2PO4, 2.7;

CaCl2·2H2O, 0.159; MgCl2·6H2O, 0.12; MnCl2·4H2O, 0.06;

CoCl2·6H2O,0.06;(NH4)2SO4,5.4.

b _{VFA mixture: acetic acid, 5.80mM; propionic acid, 1.60mM;}

butyricacid,0.86mM;n-valericacid,0.18mM;andisovalericacid,

0.18mM.

Uruguay). Ten eleven-month-old Corriedale wethers (Ovis

aries; 33.8±4.3kg) witha rumen cannulawere individually

housedinmetaboliccagesattheExperimentalStationat Lib-ertad(SanJosé,Uruguay),FacultyofVeterinary,Universityof Uruguay.Animalshadaccesstowaterandwerefedadlibitum

on alfalfahay.Theywere supplementedwithcracked corn grain(1%metabolicweightbasis)intwosupplements,at10 AM and 4PM. Theaverageintake was1347gper day, and theforage/concentrateratiowas4.4/1onadrymatterbasis. Animals were randomly assigned to the Control or Pb3C5 group. Every 48h, wethers in the Pb3C5 groupreceived an intra-ruminaldoseof1×109_{Pb3C5cellsin100mLofculture}

medium,whileanimalsintheControlgroupreceived100mL ofsterilemedium.Thisexperimentalprocedurewasdesigned afterChiquetteetal.15

Wetherswereadaptedtohousing,dietandtreatmentsfor 16days.Onday17,samplesweretakenforinoculumpotential experiments.Ondays19and20,ruminalfluidsampleswere takentomeasurepH,ammonianitrogen(NH3-N)andVFAfor

a24hperiod.Additionally,onday19,ruminalfluidwastaken fromeachwethertoevaluatethemicrobialcommunity.

Inoculumpotentialassayandmethaneproduction

TheeffectofP.bryantii3C5onthefermentativepotentialof ruminal fluid was assessed using an in vitro fermentation assay basedongasproductionexperiments.16 _Experiments

wereperformedin125mLfermenterscontainingasubstrate, buffersolution,17_{reductivesolutionandfreshruminalfluid}

from each wether. The substrate (0.5g per fermenter) was a mix ofalfalfa hay (70%)and corn (30%), ground to pass a 1mm sieve, with 90% dry matter,93.4% organic matter, 34.8%neutraldetergentfiber,21.4%aciddetergentfiber,and 14.6% crudeprotein.Thissubstratewasallowedtohydrate with thebuffer solution (38mL)and the reductivesolution (2mL)for18hat4◦C(solutionsfromOeztuerketal.17_)inside

thefermenters.Then,10mLoffreshruminalfluid,obtained frombothgroupsofanimals,wasaddedtoeveryfermenter. TheheadspacewassaturatedwithCO2,andthefermenters

batches.Todeductthegasproductionoftherumenfluidof thedonors,twowereincubated withoutsubstrateaddition. Incubationwasperformedat39◦C,andtheinternalpressure wasmeasuredwithamanualmanometerD1005PS(Ashcroft®, Stratford,USA) coupledto a0.6mm needle.Measurements weretakenat2,4,8,10,12,18,24,48,72and96hafter inoc-ulation,andgaswasventedafterpressurereadings.Attimes of4,8and24h,headspacegassamplesfromthefermenters wereobtainedformeasuringmethaneconcentrationbyGCas describedbefore.18

Ruminalenvironmentmeasures

Onday19,ruminalfluidsamplesweretakenevery2h, cover-ingthefirst12hofthedayandthenevery4h,completingthe whole24hperiod.ThepHwasmeasuredimmediately.ForVFA determination,thesamplesweremixed 1:1with0.1M per-chloricacid.ForNH3-Nanalysis,rumen fluidsampleswere

preservedwithH2SO4,50%(v/v)ina100:1relation.Samples

werestoredat−20◦_{Cuntiltheywereanalyzed.}

Lacticacid andVFA (acetic,propionicand butyricacids) were quantified by HPLC separation.19 _{The chromatogram}

peaks were integrated at 210nm.20 _{The concentration of}

NH3-Nwasdetermined usingaselectiveelectrode(Thermo

ScientificOrion)accordingtomanufacturer’sinstructions.

Structureoftheruminalbacterialmicrobiota

TotalDNAwasextractedfrom10gofcontentsfromeach ani-malusingthemethodproposedbyZhouetal.21_toassessthe

ruminalbacterialcommunityofwethersbymassive sequenc-ing.ExtractedDNAwasusedtoamplifyandsequencethe16S rDNAV1-V2 regioninaGSFLXTitaniumXLR70 asinAllai etal.22_{The16SrDNAsequencesgeneratedbypyrosequencing}

weresubsequentlyanalyzedrunningtheQuantitativeInsights intoMicrobialEcology(QIIME,version1.8.0)perscripted mod-ules and workflow scripts.23 _{Thesequences were trimmed}

andthenfilteredbylength(≥150bp),quality(≥25score),and thecontentofeitheroneormoreambiguousbasesoralong homopolymer(>6).Operationaltaxonomicunits(OTU)were generatedbyaligningthereadstotheGreenGenesdatabase24

and clustered at 97% sequence identity using the PyNAST tool23_{andtheUCLUSTalgorithm,}25_{respectively.Ananalysis}

ofsimilarity wasperformedwithQIIMEusingANOSIMand ADONISanalysis.

Statistics

Cumulated gas volume along time was compared among treatments(controlandPb3C5).Forthispurpose,thegas pro-ducedataspecifictimewasconsideredtobedependenton theprecedingtime.Consequently,thisvariablewasanalyzed asarepeatedmeasureoverthefermentersubstrate,according tothemodel:

Yijk=+SI+Tj+(S∗T)ij+εijk

whereYijkisthevolumeofgasproduced,istheoverallmean,

SIistheeffectofPb3C5treatment(I=Control,Pb3C5)ink repli-cates(2fermentersperwether),Tjisthefixedeffectoftime

(j=2,4,6,8,10,18,26,48,72and96h),(S*T)ijistheinteraction

betweenthestrainandthetimeandεijkistheresidualerror.

AlldatasetsofpH,NH3-N andVFAinruminalcontents

weretestedbeforestatisticalanalysistoensurethatallthe assumptionsofanalysisofvariance(additivemodel, indepen-denceoferrors,datanormalityandhomoscedasticity)were met.After,theywereanalyzedusingtheMIXEDprocedureas repeated measures,usingthewetherasthesubjectforthe repeatedmeasurement,accordingtothefollowingmodel: Yijk=+Si+hj+(S∗h)ij+εik

whereYijkisthevariable,isthegeneralmean,Si thefixed

effectofthe treatment,hj isthe fixedeffectofthetimeof

measurement,(S*h)ijistheinteractionbetweentreatmentand

timeandεikistheresidualerror.

ThedatawereanalyzedusingSASsoftware(version8.2; SAS 185 Institute, Cary, NC, USA), and differences among meanswithp<0.05wereconsideredstatisticallysignificant.

Familyandgenusabundancevalueswerecomparedusing thenon-parametricKruskal–WallisANOVAtest.

Results

Inoculumactivityandpotentialmethaneproduction

Nosignificantdifferenceswereobservedbetweenthegroups

inin vitrofermentationgasproductionassociatedwiththe

addition ofruminalfluid oftreatedand untreatedanimals (ptreatment=0.0622).Themethane concentrationsin the

fer-mentersweresimilarinbothgroups(p>0.05,datanotshown).

Influenceofaddedbacteriaontheruminalparameters (VFA,pH,NH3-N)

Total VFA levels (considering acetic, propionic and butyric acids)tendedtobehigher(p=0.0622)intheruminalfluidof thetreatedanimals. Aceticandbutyric acidconcentrations weresignificantlyhigherinthePb3C5-treatedanimalsthanin thecontrolgroup(p=0.0187andp=0.0199,respectively;Fig.1). Nodifferenceswereobservedwhenpropionicacidlevelswere comparedbetweenthetwogroups(p>0.05).Lacticacidwas notdetectedinanysample.

TheruminalpHofthetreatedanimalswassignificantly lower than that of the controls during most of the day (ptreatment=0.006),andthetreatment*timeinteractionwasnot

significant(p>0.05;Fig.2).

The N-NH3 concentration in the rumen ofthe animals

treatedwithPb3C5wasalsosignificantlyhigherthanrecorded valuesintheruminalfluidofthecontrolanimals(p=0.05);the treatment*timeinteractionwasnotsignificant(p>0.05,Fig.3).

Influenceofaddedbacteriaonthestructureofruminal bacterialmicrobiota

The microbial community structure, assessed eight hours after the first feed offer on day 19, was similar in both groupsofanimals(pADONIS=0.837;pANOSIM=0.712),andwhen

comparingthestructureatdifferentphylogenetic levels,no differenceswerefound(p>0.05).Anaverage2588.6(±645.9)

0 0 0 10 20 30 40 50 60 20 15 10 Time (h) Time (h) 5 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Ptreatment = 0.062 Ptreatment = 0.833 Ptreatment = 0.019 Ptreatment = 0.020 20 40 60 80 100 120 140 160 180 200 30 0 2 4 6 8 10 12 14 16 40 50 60 70 80 90 100 110 120 [total VF A] (mM) [Propionic acid] (mM) [Acetic acid] (mM) [Butyr ic acid] (mM)

Fig.1–TotalVFA,acetic,propionicandbutyricacidconcentration.Meansfrom5animalsinControl(dashline)andPb3C5 (fullline)treatmentsforeachtimearepresented.Time0correspondstothefirstfeedintake(8AM).pfortreatmentis insertedineachgraphic.

5.5 5.7 5.9 6.1 6.3 6.5 6.7 6.9 7.1 7.3 7.5 0 5 10 15 20 pH Time (h) Ptreatment =0.006

Fig.2–DailyruminalpH.Themeansoffiveanimalsat everytimepointispresentedforthecontrolgroup(dashed line)andthePb3C5-treatedgroup(solidline).Time0 correspondstothefirstfeedintake,8AM;pfortreatmentis insertedinthegraphic.

sequencesperanimalwereanalyzedandweredesignatedto 1987OTUs.Themicrobiotaofeveryanimalwasdominated

byBacteroidetes,representingmorethan65%ofallsequences.

Firmicuteswasthesecondmostrepresentedphyluminboth

groupsofanimals,while15.8%and18.8%ofsequencesinthe controland treatedgroups, respectively, couldnot be clas-sified.Prevotellaceawas themost representedfamily inthe rumenofanimalsinbothtreatmentandcontrolgroups,with 56%and50%ofallthesequences,respectively.

0 10 20 30 40 50 0 6 12 18 24 [NH 3] m g /d L Time (h) P_treatment=0.05

Fig.3–Evolutionofruminalammoniaconcentration duringtheday.Themeansoffiveanimalsarepresentedfor eachgroup.Time0correspondstothefirstfeedintake,8 AM;pfortreatmentisinsertedinthegraphic.

Discussion

Therumenbearsacomplexecosystemand,themodulation ofruminalfermentationandthemicrobiotabyprobiotic bac-teriaisaconsiderablechallenge.However,inthiswork,the ruminalenvironmentwasmodulatedbyaddingrepeatedlow

dosesofthenativeP.bryantii3C5strain(Pb3C5). Administra-tionofthisstraineffectivelymodifiedtheVFAprofileinthe treatedanimalscomparedtothe non-treatedcontrols.The modulationoftotalVFAintheruminal contentsoftreated animalscouldreflectafermentativeshiftassociatedwiththe influenceofthe administeredbacteria.Thiseffectwas evi-dencedbyasignificantlyhigherconcentrationofaceticand butyricacidsinthePb3C5-treatedanimals.Itisimportantto note thatbutyric acid,which significantlyincreasedinthe treatedanimals,playsanimportantroleintherumen,being themostenergetic VFAalong withpropionicacid.5 _Butyric

acidisalsometabolizedintherumenmucosaeand intesti-nalepithelium7,26,27 asanenergysourceandexertsmitotic andtrophiceffectsonthehostgutepithelia.28,29_Theseeffects

mayenhancedigestionandabsorptionefficiency,whichmay contributetoabetternutrientabsorption.30

AnimalsthatbelongedtothePb3C5-treatedgroupshowed lowerruminalpHvaluesthanthoseinthecontrolgroup.This observationcouldbeassociatedwiththeincreaseinVFA con-centration.AlthoughruminalpHvalueswerelowerthaninthe controlgroup,thesevalueswereneverdangerousorharmful totheanimals.Ruminantscanshowsubclinicalacidosissigns whenpHvaluesarebetween5.2and5.6foralongtime,31_and

theyshowacuteacidosissignswhentheruminalpHreaches valueslowerthan5.2,1_{butinourconditions,thelowestmean}

ruminalpHobservedwas5.9.

Modificationsoftherumenenvironmentwerealsoseenin thedailyruminalNH3-Nconcentration,withahigherdaily

concentrationinthePb3C5-treatedanimalsthaninthe con-trolgroup.Theseresultsaresimilartothoseobtainedwhen theprobioticpotentialofBacillus subtilisnattowasassessed usinginvitroandinvivoapproaches.6,32_NH

3-Nisamicrobial

proteinprecursorintherumen;itisnecessaryforgood micro-bialgrowthandmicrobialproteinsynthesisandisconsidered themostimportantsourceofnitrogenforproteinsynthesis, especiallyforfibrolyticbacteria.33_{Generally,theNH}

3-Nlevel

ishighwhenproteinfeedstuffsorgoodqualityyoungforage aregiven.34_{ThemodulationinducedbyPb3C5administration}

couldbeassociatedwithabetterdigestibilityofthefeedand aninfluenceonhostnitrogenandproteinmetabolism.

Theseruminalenvironmentmodificationswereobtained withoutaltering the potentialmethane productionnor the potentialbasedoninvitrogasproductionexperiments.

Thestructure ofthe ruminal microbial communities in bothgroupsofanimalswassimilaraftertheadministration ofPb3C5.

The microbiota of all animals was dominated by Bac-teroidetes and Firmicutes phyla, and Prevotella was the most abundant genus, which is in concordance with sev-eralanalysesoftheruminalmicrobiotaperformedbyother authors.9,35,36

Thisresultcouldbeobservedasapositivetraitsincethe diversityofthe rumen microbiota wasnot affected bythe treatment.Consideringtheseresults,itisnotpossibleto asso-ciatethepredominanceortheabsenceofcertaingroupsof microorganismswiththechangesobservedintherumen envi-ronmentinducedbyPb3C5administration.Insimilarstudies performedwithhumanbeings,researchersobserved signifi-cantchangesatthecommunitytranscriptomelevel,butthey didnotfindanydifferenceinthecommunitystructure.37

Conclusion

Inthiswork,themodulationofruminalfermentationcouldbe achievedbytheadditionofrepeateddosesofanativebacterial strain.Thesedosesweresmallwhencomparedwiththewhole ruminal microbial community, but a differential fermenta-tionpatterncouldbeobserved.Pb3C5administrationmodified fermentationproducts,aceticandbutyricacids,whichcould explainthedifferencesofthedailypHvaluesinthetreated animals. Additionally, a differential pattern of NH3-N

con-centrationintherumenwasobserved.Themodificationof ruminalfermentationandenvironmentwasachievedby pro-vidingrepeatedlowdosesofPb3C5.Althoughinthisapproach, thecausesoftheruminalfermentationmodulationcouldnot beassociatedwithchangesinthemicrobiotastructure,the effectmaybeassociatedwithchangesinruminalmicrobial metabolismAdditionalstudiesshouldbeperformedtoshed lightonthespecificactivityofPb3C5anditspossibleusein probioticformulationsforruminantsinsemi-intensive sys-tems.

Funding

This study was supported byAgencia Nacional de Investi-gación(ANII,Uruguay)[grantsnumbers:PRFMV200912799; MOVCA2013110852;ANIIPOS20113374].

Authors’

contribution

MF,CCandPZinitiatedanddesignedthestudy.MF,SF,NP per-formedtheexperiments.KPcontributedwithstrains.MF,SF, CC,analyzedthedata.MF,CC,PZwrotethepaper.Allauthors read,contributedandapprovedthefinalmanuscript.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsthankLuisVázquez,MaximilianoPastorini,Paola Delgado,PaolaSanPedro,DiegoFernández,CristinaManzzi, BrunoCremella,VirginiaRamón,ErnestoRosasandElenade TorresfortheirsupportwiththeanimalexperimentsandLuis GustavoCorbelliniforhelpinginstatisticalanalysis

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