Lipid Metabolism in the rumen: new insights on lipolysis and biohydrogenation with an emphasis on the role of endogenous plant factors.

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AnimalFeedScienceandTechnology174 (2012) 1–25

ContentslistsavailableatSciVerseScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Review

Lipid

metabolism

in

the

rumen:

New

insights

on

lipolysis

and

biohydrogenation

with

an

emphasis

on

the

role

of

endogenous

plant

factors

Arianna

Buccioni

a

_{, Mauro}

_Decandia

b

_{, Sara}

_Minieri

a

_{, Giovanni}

_Molle

b

_,

_Andrea

_Cabiddu

b,∗

a_Dipartimento_di_Scienze_Zootecniche,_Università_di_Firenze,_Via_delle_Cascine,_5,₅₀₁₄₄_Firenze,_Italy b_Dipartimento_per_la_Ricerca_nelle_Produzioni_Animali,_Agris,_Località_Bonassai,₀₇₀₄₀_Olmedo_Sassari,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received10March2011

Receivedinrevisedform15February2012 Accepted15February2012 Keywords: Biohydrogenation Lipolysis PUFA Freshforage Endogenousfactor Invitrostudy

a

b

s

t

r

a

c

t

Dietcompositionisthemajorfactorinfluencingthefattyacidcompositionofmeatand milkfromruminantsbecausethefattyacids(FA)whichreachtheduodenumare,atleast inpart,ofdietaryoriginaswellastheresultofrumenmicrobialbiohydrogenation(BH)of dietarylipids.Inthisreview,effectsofsynthesisofconjugatedlinoleic(CLA)andlinolenic (CLNA)acidisomersintherumen,effectsofthelipidsinherbage,andplantendogenous factorsonsynthesisofnutraceuticalfattyacidsarediscussed.DiscoveryofbeneficialFAin ruminantproducts,suchasCLAandother␻-3FA,stimulatedmanystudiesinthelast20 years,includingthoseontherolesofminorFAintermediatesonrumenBHandmammary glandmetabolism.Muchofthisresearchwastargetedatidentifyingtheintermediates formedduringBHaswellastherumenmicrobialecologyinvolvedintheseprocesses. However,shiftingtheresearchtofeedstuffendogenousfactorswhichinfluencelipolysis (LP)andlossesofpolyunsaturatedFAintherumenmaybeofinterestinidentifying nutri-tionalstrategiestomanipulateFAprofilesinruminantproducts.ThepresenceofFAwith healthfulpropertiesinmilkormeatfromruminantscanbeenhancedbyinclusionoffresh foragesintheirdiet.Hence,thereisincreasinginterestinthecrucialroleofendogenous LP,plantsecondarymetabolites(PSM)andpolyphenoloxidase(PPO)onruminalBH.To betterunderstandthepathwaysthroughwhichPSMorPPOimpactFAmetabolism, char-acterizationoflipidsinfreshforagessuggeststheimportantroleofthedietmatrixonthe ruminalfateoflipids.AcriticaldiscussionoftheroleofoddchainbranchedFA(OBCFA) isalsoreported,includingpotentialimpactsonrumenmicrobialmetabolism.Finally,new insightsintolipidmetabolismfrominvitrotechniquesarediscussed.

Abbreviations:BH,biohydrogenation;CLA,conjugatedLA;CLNA,conjugatedLNA;DAPA,diaminopimelicacid;DHA,docosahexaenoicacid;DHS, dihy-drosphingosine;DPG,diphosphatidylglycerol;DM,drymatter;EPA,eicosapentanoicacid;ER,endoplasmicreticulum;FA,fattyacid;FAME,fattyacid methylesters;F/C,forage/concentrateratio;FFA,freeFA;FO,ﬁshoil;GA,galactolipids;GL,glycolipids;GPL,glycerophospholipids;GPSL, glycophos-phosphingolipids;GSC,glucosylceramide;LA,linoleicacid;LAB,bacteriaassociatedwithliquidphase;LA-I,linoleicisomerase;LNA,linolenicacid;LP, lipolysis;MFD,milkfatdepression;MGDG,monogalactosyldiacylglicerol;ML,membranelipids;MUFA,monounsaturatedFA;NFA,neutralFA;OA,oleic acid;OBCFA,oddbranchedchainFA;OCFA,oddchainFA;PA,palmiticacid;PB,purinebases;PBP,proteinboundphenols;PC,phosphatidylcholine;PE, phosphatidylethanolamine;PG,phosphatidylglycerol;PHS,phytosphingosine;PI,phosphatidylinositol;PL,phospholipids;PSM,plantsecondary metabo-lites;PUFA,polyunsaturatedFA;PPO,polyphenoloxidase;PS,phosphatidylserine;RA,rumenicacid;RNA,rumelenicacid;SA,stearicacid;SAB1,bacteria looselyattachedtofeedparticles;SAB2,bacteriaﬁrmlyattachedtofeedparticles;SFA,saturatedFA;SHL,sphingolipids;SL,plantsulpholipids;SPH, glyco-sphingolipids;SQDG,1,2diacylglicerol-3-(6-sulpho-␣-d-quinovopyranosyl)-snglycerol;TAG,triacylglycerides;TFA,transFA;UFA,unsaturatedFA;VA, vaccenicacid;VFA,volatilefattyacids;VNA,vaccelenicacid.

∗ Correspondingauthor.Tel.:+390792842349;fax:+39079389450. E-mailaddress:acabiddu@agrisricerca.it(A.Cabiddu).

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6. Oddandbranchedchainfattyacids:anewfrontierinthestudyofrumenmicrobialmetabolism... 16

7. Futureresearchneeds... 18

7.1. Interactionbetweenpurecultureandpuresubstrate ... 18

7.2. InteractionbetweenPPO,PSM,endogenouslipolysis,andPUFAbiohydrogenation... 19

8. Conclusions... 20

Acknowledgements... 20

References... 21

1. Introduction

Therumenenvironmentischaracterizedby1010_bacteria,₁₀7 _protozoa,₁₀6 _fungi_and_yeasts_per_ml_of_live_liquor,_a temperatureof38–39◦C,anormalrangeofpHbetween6.0and6.7,andaredoxpotentialof−150–350mV.Anydeviation fromtheseconditionsmayinﬂuencethemicrobialpopulationanditsfermentationproducts.Whilefeediswithintherumen asmallamountofdietaryfattyacids(FA)isabsorbedandcatabolisedtovolatileFA(VFA)ortoCO2.However,microorganisms areabletosynthesizeremarkableamountsofFAdenovofromprecursorcarbohydratesandtohydrogenateunsaturatedFA (UFA).ThusFAwhichreachtheduodenumareofdietaryoriginaswellastheresultofmicrobialactivity.Inrecentdecades manyauthors(e.g.,Harfoot,1978;PalmquistandJenkins,1980;Jenkins,1993)havestudiedthefateofdietarylipidsduring rumenfermentation,emphasizingthetwomajorprocessesinwhichesteriﬁedlipidsareinvolved(i.e.,lipolysis(LP)and biohydrogenation(BH)).ThismicrobialactivityleadstosynthesisoflongchainFAasthepoolforconjugatedlinoleicacid (CLA),conjugatedlinolenic(CLNA)isomersandotherBHintermediates.Whilesomeofthesearefoundinlowconcentrations intherumenliquor,theyaccumulateintissuelipidsandmilkfat(Destaillatsetal.,2005;Akraimetal.,2007).Interesting informationhasbeenobtainedfromadoptionofinvitrofermentationtechniquesintheseinvestigations.

TheaimofthisreviewistopresentanoverviewoflongchainUFAmetabolismpathwaysintherumen,factorsinfluencing synthesisofnutraceuticalFA,andresearchneedsinthefieldofruminallipidmetabolism.RuminalmetabolismofFAfrom freshforageisalsoreviewed,withanupdateonrecentresearchintoplantendogenousmechanismsinvolvedinLP,andtheir influenceonlossofPUFAintherumen.

2. Lipolysisandbiohydrogenation

LipolysisresultsinreleaseoffreeFA(FFA)fromesterstoallowBH,whichisreductionofthenumberofdoublebondson thecarbonchainoftheFA.Sincesubsequenthydrogenationcanonlyhappenifthecarboxylmoietyisfree,LPisanecessary stepinBH.ThusifonlysmallquantitiesofdietaryPUFAreachtheduodenum,thismaybeduetoamissingLP,andmay bewhatdeterminestherateofhydrogenationintherumen.Thisgroupofreactionsappearstobeaprocessutilisedby microorganismstoprotectthemselvesfromtoxiceffectsofUFA(Dehority,2003).

Afteringestion,dietaryesterifiedlipidsarehydrolysedtoFFAandglycerolaswellastosmallamountsofmono-and diglyceridesbymicrobiallipases,whichareextra-cellularenzymesassembledinsmallbeads(Jenkins,1993).Thenumberof microorganismscapableofhydrolysingestersislow,andtheiractivityishighlyspecific(Henderson,1971;Fayetal.,1990). VariousbacterialstrainsofButyrivibriofibrisolvensandAnaerovibriolipolyticaarecapableofhydrolysingtheesterbond,but B.fibrisolvenslipasehydrolysesphospholipids,A.lipolyticahydrolysesonlytri-anddi-glycerides,andtheirratesofhydrolysis differ.Lipaseactivityalsooccursinciliataeprotozoa,butnotinfungi(Dehority,2003),althoughtheircontributionisless thanthatofbacteria.

FFAmayalsoarisefromhydrolysisofplantgalactolipidsandphospholipidscatalysedbyseveralbacterialgalactosidases andphospholipases(e.g.,phospholipaseAandphospholipaseC),producedbyrumenmicrobes(Jenkins,1993).

ThehalflifeoffreeUFAisrelativelyshortduetotheirrapidhydrogenationbyrumenmicrobesintothecorresponding saturatedconﬁguration.BHhasbeenestimatedat600and900g/kgofPUFA,andonlycontributestoasmallextenttothe recy-clingofmetabolichydrogenbecauseonly1–2%ofitisusedforthispurpose(Czerkawski,1984).HarvatineandAllen(2006) completedaninvivoexperimentwithlactatingdairycowstodetermineratesofFAbiohydrogenationoffatsupplements withdifferentgradesofunsaturation,anddevelopedakineticmodelofruminalBH.Basedontheirresults,theyshowedthat passageratesofC16:0,C18:0andtotalC18-carbonFAlinearlydecreasedasUFAincreased,whilethetransC18:1fractional

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A.Buccionietal./AnimalFeedScienceandTechnology174 (2012) 1–25 3

Fig.1. Lipolysisandbiohydrogenationscheme. FromJenkins(1993),modiﬁed.

passageratewasaffectedquadraticallywithamaximumratefortheintermediatetreatment.IncreasingUFAincreasedthe extentofC18:2andC18:3biohydrogenation,anddecreasedtheextentofC18:1andtransC18:1biohydrogenation.

BacteriaarethoughttobemainlyresponsibleforBH,whilethecontributionofprotozoaisregardedasnegligible(Singh andHawke,1979).Thisactivityismainlyassociatedwithbacteriaattachedtofeedparticles,ratherthanwiththosein freeliquid.FreeUFAareadsorbedontofeedparticlesurfacesandhydrogenated(Harfootetal.,1975;Gersonetal.,1988). However,NamandGarnsworthy(2007)studiedtheBHrateoflinoleicacid(C18:2cis-9cis-12,LA)bymixedrumenfungi inaninvitroexperimentandnotedthatrumenfungicanbiohydrogenateLA,butthattheirBHislowerthanthatofrumen bacteria.Theendproductoffungalbiohydrogenationisvaccenicacid(C18:1trans-11,VA),asitisforrumenbacteria,and Orpinomycesisthemostactivebiohydrogenatingfungus.

ThefeedstuffswhichcompriseruminantdietsoftencontainLA,C18:3cis-9cis-12cis-15(linolenicacid,LNA)andC18:1 cis-9(Oleicacid,OA).MostbacteriainvolvedinBHarecellulolyticwiththemostimportantbeingB.fibrisolvens,identified byKepleretal.(1966)asabletoBHtheLA.Atpresentmorethan32strainsofthisbacteriumhavebeenidentifiedand characterized.Clostridiumproteoclasticum,reclassifiedasButyrivibrioproteoclasticus(Moonetal.,2008),istheonlybacteria isolatedfromtherumencapableofconvertingPUFAtoSFA.ThismicroorganismconvertsC18:1substratestoC18:0while Propionibacteriumacneshydratescis-9C18:1andtrans-11C18:1to10-OH-C18:0,whichisfurtheroxidizedto10-O-C18:0 (Kimetal.,2008;McKainetal.,2010).Fig.1showstheBHschemeofFAesters.

Whentheﬁbrecontentofthedietislowered,andhigherlevelsofconcentratesareusedinthediet,thereisareductionin thenumberofcellulolyticbacteriaintherumen(Lathametal.,1972;DoreauandFerlay,1994;Kalscheuretal.,1997;Loor etal.,2004).Thusthiskindofdietfavourslipidswhichpasstherumenwithoutbeingreduced,especiallyOAandLA(Chilliard etal.,2007).Howeverwhenthedietishighinconcentrates,otheralternativeBHpathwaysoccurwiththeappearanceof sometransfattyacids(TFA),characterizedbyatransdoublebondinthecarbonchain(GriinariandBauman,2006).

OtherdietaryfactorswhichdiminishLPandrumenBHareadvancedmaturityofforageand/orforagewhichhasbeen groundintoextremelyfineparticles(Gersonetal.,1986,1988).Inthelattercasetheadherenceofbacteriatofeedparticle surfacesispoorandthetransitratethroughtherumenisincreased,therebyreducingtimeofexposuretomicrobialactivity. TheamountandtypeoffataddedtothedietcaninfluencetheBHoflipidsintherumen.Forexample,theFAprofileof marineoilisrichinC20:5n-3(eicosapentaenoicacid,EPA)andC22:6n-3(docosahexaenoicacid,DHA)whileoilseedssuch assoybean,sunflower,canolaandlinseedmainlycontainC18PUFAandMUFA.TheseFAarehydrogenatedintherumen andinducesynthesisofBHintermediatessuchasconjugatedandtransisomers(Chilliardetal.,2007).

RumenbacterialpopulationswhichLPandBHcanbedividedintothreemaingroupsbeing:bacteriaassociatedwith theliquidphase(LAB)andbacteriaeitherloosely(SAB1)orﬁrmly(SAB2)attachedtofeedparticles(Czerkawski,1986; Legay-CarmierandBauchart,1989).Bessaetal.(2009)studiedtheFAcompositionofLAB,SAB1andSAB2withtheaimof identifyingthesebacteriafromtheirFAproﬁle,andfoundmarkeddifferencesinFAcompositionbetweenLABandSAB(i.e., detachableplusundetachablesolidassociatedbacteria)populations,consistentwithothers(Bauchartetal.,1990a;Kim etal.,2005;Vlaemincketal.,2006b).LABhadalowerFAcontentandahigherproportion(g/kgFA)ofoddbranchedchainFA (OBCFA)thandidSAB,whichhadahigherproportionofC18biohydrogenationderivedFA(C18:0;especiallytransC18:1and CLA).Theratiobetweenbranched-chainandodd-linear-chainFAwasalsohigherinLAB(2.26)thaninSAB(1.46).Bessaetal. (2009)observeddifferencesintheproportionsofFAbetweenSAB1andSAB2.ComparedtoSAB1,theSAB2compartment hadlowerproportionsofodd-chainFAandofsomebranched-chainFA(iso-C14:0andiso-C16:0andanteiso-C17:0),butthe

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Fig.2.Biohydrogenationoflinoleicacid. FromShingﬁeldetal.(2010),modiﬁed.

ratiobetweenbranched-chainandoddlinear-chainFAwasthesame.SAB2alsohadhigherproportionsoftransC18:1,CLA andparticularlyof18:2n-6and18:3n-3thanSAB1.

3. Conjugatedfattyacidisomerssynthesisintherumen

3.1. ConjugatedLinoleicAcidisomerssynthesisintherumen

MicroorganismsmayuseBHasameansofdefenceagainstUFAtoxicity,andLAisomerizationisoneofthestepswhich makesFAinoffensiveinthisregard.CLAisapoolofgeometricalandpositionalisomersofLA(cis-9,cis-12C18:2)which containaconjugatedbondsystemlocatedfrompositions2to17ofthemolecularstructure.Eachpositionalisomerhasfour geometricisomersbeing:cis,trans;trans,cis;cis,cis;trans,trans,foratotalof56possibleisomers.Thedoublebondpositions ofCLAisomersactuallyidentifiedintherumenandinmilkfatrangefrom6,8-to13,15-C18:2inmostpossiblegeometrical configurations,foratotalof32isomers(Bessaetal.,2000;Krameretal.,2004;Shingfieldetal.,2008).

Rumenicacid(RA;cis-9,trans-11C18:2isomer)issynthesizedinsmallamountsintherumenbyLAisomerization,mostly inthemammaryglandby9_desaturation_of_VA_in_lactating_ruminants₍_Griinari_and_Bauman,_1999;_Kramer_et_al.,_1999; Griinarietal.,2000;Mosleyetal.,2006;ShingﬁeldandGriinari,2007;Bernardetal.,2009).Palmquistetal.(2004)provided datatosuggestthatthisalsooccursintheadiposetissueoflambs.

ThefirststepoftheLABHpathwayconsistsofenzymaticisomerizationbycis-12,trans-11isomerase,whichturnsthe cis-12bondintoatrans-11(RA,Fig.2).Thisisomerasehasaspecificrequirementforfreecarboxylicgroups,andparticularly oneswiththeisolateddienecharacterizedbyageometry“cis-9,cis-12”.Linoleicisomerase(LA-I)doesnotneedacofactor,or anyactivatingfunctionalgroups,asitisbondedtothebacterialcellwallandhasbeenpartiallypurifiedandcharacterizedin alimitednumberofbacteriainstudiesofKeplerandTove(1967)andKepleretal.(1970)andYokoyamaandDavis(1971). Afterthebondinthetrans-11positionhasbeenformed,hydrogenationtothecis-9positioniseffectedbya microbial reductaseofB.fibrisolvens(Hughesetal.,1982).Theseinitialstepstakeplacerapidly,whiletheBHofVAtostearicacid(SA) ismuchslower,andresultsinVAaccumulationintherumen.Thuswhendifferentgroupsofmicroorganismsareinvolved, VAhydrogenationseemstobethestepwhichdeterminestherateofBH.

KempandLander(1984),classiﬁedbacteriainvolvedinBHintotwogroupsbeing:groupAbacteriawhichcanisomerize andhydrogenateLAtoVA(i.e.,B.ﬁbrisolvens),andgroupBbacteriawhichcansaturateVAtoSA(i.e.,Fusocillusspp.,C. proteoclasticum).In2003,VandeVossenbergandJoblin(2003)isolatedastrainofButyrivibriohungateifrombovinerumen whichwasabletoformSAfromLA,a rareexampleofbacteriumwhich,actingalone,candirectlyreducePUFAtothe correspondingsaturatedFA.

Inthe1980s,theliteraturesuggestedthatthecontributionofprotozoatoBHwasduetoactivityofingestedbacteria (SinghandHawke,1979).HoweverDevillardetal.(2004)observedthattheCLAandVAcontentofrumenprotozoalcells was4–5foldhigherthanthatinbacteria,whichsuggeststhatprotozoamayalsobeamajorpoolofCLAandVAintherumen. Yà ˇnez-Ruizetal.(2006,2007),usinganapproachbasedonrealtimePCRquantifiedthecontributionofprotozoatoduodenal FAflowconfirmingthatthesemicroorganismcontributealmost400g/kgofRA,300–360g/kgoftrans-10,cis-12CLAand 400g/kgoftheVAleavingtherumenbypassage.ThesimplestexplanationisthatprotozoadonotformCLAandVA,but thattheyareveryefficientinincorporatingintermediatesofbacterialBH(Devillardetal.,2006).Howeveritisdifficultto calculatetherealcontributionofprotozoatoFAsupplybecausestudiesreportedbyDehority(2003)showedthatprotozoa tendtoberetainedintherumen,asoutflowdoesnotalwaysreflectruminalconcentrations.Incontrast,Yà ˇnez-Ruizetal.

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(2006)showedthattheFAwithinprotozoacontributeasigniﬁcantproportionoftheCLAandVAreachingtheduodenum ofruminantsandthattheprotozoalFAproﬁledependsonthechemicalcompositionoftheanimals’diet.

RumenisomerizationalsoinvolvesotherUFA,suchusOA,whichhasbeenproposedasarumenprecursoroftransC18:1 isomers.SelnerandSchultz(1980)observedanincreaseofC18:1transacidsinmilkfatfromcowsfeddietsrichinOA, andMosleyetal.(2002)showedthatthiscouldbeattributedtorumenisomerizationofOA.Howeverseveralstudieshave shownthatOAistheruminalprecursorofhydroxystearicacid(10-OH-C18:0)andofketostearicacid(10-O-C18:0),whose accumulationinruminalcontentsofcattleisdirectlyrelatedtothequantityofOAintherumen(Jenkinsetal.,2006).In addition,McKainetal.(2010)demonstratedthatP.acnesDSM1897isthemainmicroorganismresponsibleforOAhydration to10-OH-C18:0,whichisfurtheroxidizedto10-O-C18:0.

Hudsonetal.(1995)isolatedtwobacterialstrainswhichconvertOAto10-OH-C18:0insheeprumenliquor.One,the 10-hydroxystearic-acid-producingbacterialgroup,consistedoftwostrainsofananaerobicgram-negativemicroorganism whosemorphologyallowedittobeidentifiedasSelenomonasruminantium.Theother,identifiedasEnterococcusfaecalis, consistedoftwostrainsofafacultativeanaerobicgrampositivechain-formingcocci.Theauthorsshowedthattherewere severaldifferencesinrepresentativecoloniesfromrumenfluidofsheepandcows.Forexample,whileinbothsheepand cowsthestrainsofStreptococcusbovis,LactobacillusandStaphylococcuswerethemostnumeroushydration-positiveisolates, sheeprumenfluidalsocontainedhydration-positivestrainsofEnterococcusandPediococcuswhichcanhydrateUFA.Overall, thesefindingssuggestthatlacticacidbacteriaarethemajorUFAhydratingbacteriaintherumen(Hudsonetal.,2000).

TheamountofVAhydrogenatedtoC18:0isaffectedbyconditionsintherumen,thetypeandtheconcentrationofdietary PUFA,andbydietarypolyphenolicsubstancessuchastanninswhichirreversiblyinhibittheprocess(Harfootetal.,1973). Vastaetal.(2009a,b)suggestedthattanninscanreduceruminalBHbecausetheyinhibitactivityofrumenmicroorganisms. Indeedtheyfoundthatthepresenceofthesepolyphenolicsubstancesinrumenliquor(above0.6mg/mlrumenfluid)results ina23%increaseofVA,a16%decreaseofSA,butnochangeintheproportionofCLA,whichcomesfromLAbymeansofan isomerizationcatalysedbyLA-I,anditsactivityisonlyreducedbythepresenceoftannins.Theauthorssuggestedthatseveral changesinBHproductionoccurbecausetanninsactselectivelyonbacterialstrains.Mooreetal.(1969)suggestedthatlarge amountsoffreeLAstopthesecondstageofBH,althoughthisdoesnotoccurwhenLAisintheesterifiedform.Chilliardetal. (2001,2003)reviewedtheeffectsofnutritionalfactorsaffectingmilkfatcompositionofruminantsandsuggested,onthe basisofliteraturedata,thatsupplementingdietswithmarineoildecreasesruminalBH.Chowetal.(2004)showedthatfish oil(FO)hasnoinfluenceonthedegreeofLPorapparentBHofLAandLNA.Thesimultaneousdecreaseintheamountof SA,andtheincreaseinVAandtrans-11,cis-15,C18:2insubstrates,whenFOisfedindicatesthatthissupplementinhibits thefinalstepofLAandLNABH.SimilarresultswerereportedbyShingfieldetal.(2003,2010)andShingfieldandGriinari (2007)invivo,whoalsoconcludedthatFOenhancestheRAcontentinmilkfatduetotheincreaseinVA.

Wasowskaetal.(2006)showedthatFOaddedtoruminantdietsisabletomodifytherateofBHbydecreasingthe initialisomerizationrateofLAandLNA,andsimultaneouslyincreasingVAaccumulation.ThesedataagreewithShingfield etal.(2003),whousedtheomasalsamplingtechniquetomeasurerumenoutflowofFFA.Theauthorsalsohighlighted theinhibitingeffectoffeedsupplementscontainingEPAorDHAongrowthofB.fibrisolvens,andonitsisomeraseactivity. SimilarresultswerenotfoundwithFO,whichdoesnotshowanyeffectsontheactivityofB.fibrisolvensinspiteofitshigh proportionofEPAandDHA.ThusFOinhibitsruminalBHthroughamechanismwhichisnotcompletelybasedoneffectson B.fibrisolvens.Huwsetal.(2010)notedthatincludingFOinthedietchangesthenumberofseveralrumenbacterialstrains, suchasA.lipolytica,F.succinogenesandR.flavefaciens.TheyalsoshowedthattheDNAconcentrationfromB.proteoclasticus strains,whicharetheonlybacterialstrainswhichhavebeenisolatedfromtherumencapableofbiohydrogenatingPUFAto C18:0,hasaweakrelationshipwithC18:0flowtotheduodenum,suggestingthatotherbacteriamayplayaroleinBHwhen FOisfedtocows.ThesedatawereconfirmedinotherstudieswhichexaminedthespecificroleofB.proteoclasticusstrains (Kimetal.,2008;Huwsetal.,2011).

MartinandJenkins(2002)demonstratedthatsomeenvironmentalfactors,suchasruminalpH,appeartohaveagreat inﬂuenceonproductionoftransC18:1andCLAisomers.TheseauthorssuggestedthatruminalpHhastobemaintained above6.0ifsynthesisofCLAistobemaximisedbecausecellulolyticbacteriaaresensitivetoacidiccondition.

Inthe1990s,GriinariandBaumanpostulatedthattherewasashiftinthenormalBHpathway(Fig.3)whenruminantswere fedhighconcentrate/lowfibrediets(Griinarietal.,1997,1998;GriinariandBauman,1999).Thistheorywascorroborated byseveralstudieswhichexaminedlowfibredietssupplementedwithplantoilsandFO(Piperovaetal.,2002;Looretal., 2004;Shingfieldetal.,2005).Thepresenceoffatinadietwithrapidlyfermentablecarbohydratesand/orahighproportion ofstarchaltersruminalconditionstoinducechangesintherumenbacterialpopulation.Asaresult,itisverylikelythat alternativepathwaystometabolizeFAareactivated.IthasbeendemonstratedthatLAmaybeisomerizedtoRAortrans-10, cis-12CLA(GriinariandBauman,2006),butthatisomerizationofLAtotrans-10,cis-12CLAconvertsthecis-9bondtoa trans-10throughactivityofacis-9,trans-10isomerase.Thisstepmayoccurviaanionicreactionassuggestedbydiscoveryof thecis-9,trans-10isomerasestructureanditsmechanismofactioninastudybyLiavonchankaetal.(2006).Inthenextstep, C18:2isomerswithtrans-10,cis-12doublebondsandtrans-10C18:1areformed.Reductionsthenoccuruntilthecarbon chainiscompletelysaturated.McKainetal.(2010)demonstratedthatB.fibrisolvensJW11metabolizetrans-10,cis-12C18:2 totrans-10C18:1,whileB.proteoclasticusP-18doesnotgrowinthepresenceoftrans-10,cis-12C18:2,butgrowsinmedium containingtrans-9,trans-11C18:2toformC18:0.TheyalsoshowedthatP.acnes,aruminalspecieswhichisomerizeslinoleic acidtotrans-10,cis-12C18:2,doesnotfurthermetabolizeCLAisomers,andthatB.fibrisolvensisabletometabolizesmall amountsoftrans-10C18:1.

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Fig.3.Shiftoflinoleicacidbiohydrogenation. FromGriinariandBauman(1999),modiﬁed.

Trans-10,cis-12CLA(Griinarietal.,1997,1998;GriinariandBauman,1999)isinvolvedinthemilkfatdepression(MFD) syndromeinruminants.AstudybyLocketal.(2007)suggestedthattheroleoftrans-10C18:1inMFDhastobereconsidered, andrecentstudiesconﬁrmedthatthisisomermaydecreasemammarylipogenesis(Shingﬁeldetal.,2009).Discrepancies inresultsamongstudiesmayberelatedtothedose-dependenteffect,asKadegowdaetal.(2008)suggestedthattrans-6, trans-7andtrans-8isomersofC18:1mightbemoreimportantthantrans-10C18:1inMFD,andshowedthattrans-10, cis-12andtrans-7,cis-9CLAweretheisomersmoststronglynegativelycorrelatedtomilkfatproportion,whichimpliesthat trans-7,cis-9CLAhasapossibleroleinMFD.

Howeverseveralstudieswithdairycowshaveidentifiedotherputativeinhibitorsofmilkfatsynthesis,suchastrans-9, cis-11andcis-10,trans-12C18:2,althoughthesynthesismechanismhasyettobeconfirmed(Sæbøetal.,2005;Perfield etal.,2007).AstronglynegativeeffectwasdetectedforC18:1trans-10withrespectCLAtrans-9,cis-11indairysheep (Cabidduetal.,2009a).Wallaceetal.(2007)incubatedruminaldigestawithLAandobtained9,11CLAisomerswithfour possiblecombinationsofstructuralgeometry(cis/trans,trans/cis,trans/transandcis/cis)and10,12CLAisomerswiththe geometrytrans/cisandcis/cis.TheyalsoincubatedpureculturesofB.fibrisolvens,ButyrivibrioproteoclasticumandP.acnes G449isolatedfromruminaldigestawithLAindeuteriumoxide-containingbufferandfoundthefirsttwobacterialstrains behavedsimilarly,producingmostlyRAwithsmallamountsofother9,11isomers,alllabelledinC-13.Notraceof10,12 isomersoccurred,exceptinculturescontainingP.acneswhichappearedtobespecializedinproducing10,12CLAisomers. TheseresultsconfirmedthehighspecificityofbacteriastrainsinsynthesisingCLAisomers,andthatdifferentmechanismsare involved.Wallaceetal.(2007)proposedthatLA-IactsbyformingaradicalintermediatewheretheHonC-11ofLAisremoved leavingaradicalwhichisthermodynamicallylessfavourablethanaconjugateddoublebondsystemwheretheradicalis locatedonC-13.Thusmovementofthedoublebondfromcarbonatoms12and13occurstomaintainthermodynamic stability.Ahydrogenatomisthenremovedfromwaterinordertocompletethereaction.Withrespecttoformationofother 9,11conjugatedisomers,whicharecertainlylessfavourableintermsofenergywithrespecttothestabilityofRAradical intermediate,theirsynthesisprobablyoccurssimultaneouslywiththatofRA,anddoesnotinvolvesubsequentcis–transor trans–cisisomerizationofthecis-9trans-11isomer.

ThekineticproﬁleofCLAproductiondiffersamongisomers,andisinﬂuencedbytheinoculatedsubstrate.Theliterature agreesinshowingthatminorisomersreachtheirmaximumconcentrationinrumenliquorlaterthanmainisomers(Buccioni etal.,2006,2008,2009;Wallaceetal.,2007).

3.2. Conjugatedlinolenicacidisomerssynthesisintherumen

BHintermediatesofLNAwereﬁrstobservedbyReiser(1951),whofoundthatafterlinseedoilwasincubatedwithrumen sheepliquor,theLNAconcentrationdecreasedwhiletheproportionofC18:2andC18:1increased.Shorlandetal.(1955) subsequentlyobservedthatthedepotfatofgrazingruminantshadaFAproﬁlelowinLNA,whichisthemainFAingrass. BoththeseauthorshypothesizedthattherumenBHactivitycouldplayafundamentalroleinreducingLNAreduction.

Inthe1960s,studiesofWardetal.(1964),WildeandDawson(1966)andKepleretal.(1966)wereimportantmilestones inourknowledgeofrumenBHpathways.In1967,KeplerandTovepurifiedcis-12,trans-11isomerasefromB.fibrisolvens andconcludedthatthisenzymemightbeactiveonLAandLNAisomers.ThefirststepinbiohydrogenationofLNAmight beisomerizationoftheC12bond,withthepossibleyieldofintermediatescis-9,trans-11,cis-15C18:3andcis-9,trans-13, cis-15C18:3.

ThishypothesiswassupportedbyidentificationinmilkfatoftwonovelC18:2isomers(i.e.,trans-11,cis-15C18:2and cis-9,trans-13C18:2)byUlberthandHenninger(1994).Destaillatsetal.(2005)characterizedcis-9,trans-11,cis-15C18:3 andcis-9,trans-13,cis-15C18:3andconcludedthatthepresenceoftheseFAinrumenfluid,whichdefinitelyoriginatein smallquantitiesfromrumenmetabolism,confirmthepathwayreportedinFig.4.Thesameauthorsproposed“rumelenic acid”(RNA),“isorumelenicacid”and“vaccelenicacid”(VNA)astrivialnamesrespectivelyforcis-9,trans-11,cis-15C18:3 andcis-9,trans-13,cis-15C18:3andtrans-11,cis-15,C18:2byreferencetoRA,LNAandVA.Looretal.(2004,2005b)founda positivecorrelationbetweenvaccelenicacidand␣-LNAlevelsinmilkfatofdairycowsfeddietssupplementedwithlinseed

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Fig.4.Ruminalbiohydrogenationoflinoleicand˛-linolenicacids. FromDestaillatsetal.(2005),modiﬁed.

orsunfloweroil,whichwasconfirmedbyWasowskaetal.(2006)whoshowedthattheconcomitantpresenceofLNAand FOinruminalfluidinducesaccumulationofRNAandVNA.

GriinariandBauman(1999)proposedanalternativepathwayofLABHinvolvingacis-9,trans-10isomerasewhichwas analogouswiththefateofLAwhenrumenfermentationinvolvesruminantlowﬁbrediets(Fig.5).Itwaspostulatedthat thetrans-10intermediatesmayalsoplayaroleinMFD,andoriginatefromtheBHofLNAinawaysimilartoC18:2isomers withatrans-10doublebond.

Looretal.(2005a,b),completedaninvivoexperimentonmilkcompositionofcowsfedahighconcentratediet (con-centrate/forageratio650:350ondrymatter(DMbasis))supplementedwithlinseedoilat30g/kgDMandfoundadrastic MFDassociatedwithincreasedyieldoftrans-11,cis-15C18:2,andtransisomersofC18:3,withareductioninyieldofC18:0 pluscis-9C18:1.Theynoted,inparticular,thathighlevelsofdietaryconcentratereducedmilkfatyieldscomparedtodiets withalowconcentratecontent(i.e.,−90g/dofmilkfat)andthisdecreasewasworsened−400g/dofmilkfatwhenalow concentratedietwassupplementedwithoil.ThesechangeswereaccompaniedbychangesinthemilkFAproﬁle.

Thetrans-10C18:1 contentofmilkfatasa consequenceoffeedinghighlevelsofconcentrateplus unsaturatedoil accountedfor360g/kgoftotaltrans-C18:1inthestudyofGriinarietal.(1998),for590ginPiperovaetal.(2000),for 670ginOfferetal.(2001),for430ginPetersonetal.(2003),andfor240ginLooretal.(2005a).Thedifferentproportions oftrans-10C18:1inmilkfatinthesestudiesmaybeduetotherelativelylowlevelsofdietarystarch,230g/kgDM,inLoor etal.(2005a)andbecausethedietsweresupplementedwithpolyunsaturatedoils.

Offeretal.(1999),Donovanetal.(2000),Whitlocketal.(2002)andShingﬁeldetal.(2003)provideotherevidencefor alinkbetweencis-11-C18:1andFO-inducedMFD.DietaryFOresultedina7-foldincreaseintheproportionoftrans-9, cis-11-C18:2intherumen,whichsuggeststhatthisFAmaybearuminalprecursorofcis-11-C18:1.

4. Lipidfractionsinherbage,theirmanipulationandeffectsonPUFAruminalbiohydrogenation

4.1. Glycolipids

Thelipidcompositionofherbage(Figs.6and7)isbasedontwoclassesofpolarlipidswhichareusuallyassociatedwith cellularmembranes,andthusarealsocalledmembranelipids(ML):glycolipids(GL)andphospholipids(PL).Incontrast, theseedlipidproﬁlemainlyconsistsofnon-polarlipids,triacylglycerides(TAG;WeeninkquotedbyGarton,1960;Hawke, 1973).InGLlipidstheheadofthepolargroupisacarbohydrate,inPLitisbasedonphosphate,andTAGhavenopolar group.Herbagelipidaccountsfor33,135and832g/kgoftotalFAinTAG,FFAandMLrespectively(Garton,1960).Since TAGisanessentialandcrucialenergysourceduringearlystagesofseedlinggrowth,itisassumedthatFAmobilization duringseedgerminationislinkedtoplanttissuedevelopment.HoweverleaflipidFAaremainlybasedonGL,withalow energycontentanda25%higherPUFAcontentthanTAG,whichisconnectedtoGL’smetabolicandstructuralrolesin photosynthesis(DörmanandBenning,2002).ThehigherlevelsofPUFAinplantleavesaremainlyduetoLNA,whichis positivelycorrelatedwithtotallipidcontent(Crombie,1958)andrateofphotosynthesisAllakhverdievetal.(2001),whose

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Fig.5.Alternativepathwayoflinolenicacid. FromGriinariandBauman(1999),modiﬁed.

resultsconﬁrmthoseofpreviousstudiesshowingthatwatersolublecarbohydratestendtoincreaseduringdaylight(Orr etal.,1997).Photosynthesistakesplaceinthethylakoidmembranewhichisaﬂuidmosaiccontainingproteins,lipidsand pigments(HeinzandSiefermann-Harms,1981).Thethylakoidmembranecontainsabout400goflipids/kgDMandmainly consistsofGLandPL,whilelipidslocalizedinleafchloroplastsaccountforabout200–250g/kgDM(RoughanandBatt,1969;

Fig.6. Commontypesofstoragesandmembranelipids.Allthelipidtypesshownhaveeitherglycerolorsphingosineasthebackbone,towhichareattached oneormorelongchainalkylgroupsandapolarheadgroup.Intriacylglycerols,glycerophospholipids,galactolipids,andsulpholipids,thealkylgroupsare fattyacidsinesterlinkage.

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Fig.7.Structureofmembraneglycerolipids.Inhigherplantsthenon-phosphorousglycolipidsMGDG,DGDGandSQDGarethepredominantlipidclasses inchloroplast.Phospholipids(i.e.,PC,PE,andPG)areabundantinextraplastidicmembranes.Additionalphospholipids(i.e.,PA,PS,NAPEDPG,andPI)are minorcomponentsofplantmembranesandarerestrictedtospeciﬁcorganellesoraccumulateunderonlysomeenvironmentalconditions.

FromDörman(2005),modiﬁed. Table1

Lipidcompositionofryegrassleaves(g/kgoflipids).

Glycolipids (308g/kgoflipids) Phospholipids (368g/kgoflipids) Simplelipids (234g/kgoflipids) Pigment(92g/kgof lipids) DGDG 105 PE 44 Diglycerides 88 Chlorophyls 141

MGDG 105 PC 35 Fattyacids 84 Carotenes 34

SQDG 44 PI 13 Sterols 32 Tocopherols 3

Esteriﬁedsterylglycosides 38 Sterylesters,waxes 30

Componentfattyacids(415g/kgoflipids)

C16:0 C18:0 C18:1 C18:2 C18:3 Other

159 10 23 120 620 68

AdaptedandcalculatedfromHudsonandWarwick(1977).

PC=phosphatidylcholine; PE=phosphatidylethanolamine; PG=phosphatidylglycerol; PI=phosphatidylinositol; PS=phosphatidylserine; DPG=diphosphatidylglycerolMGDG=monogalactosyldiacylglycerol;DGDG=digalactosyldiacylglycerol;SQDG=sulphoquinovosyldiacylglycerol.

Hawke,1973;HeinzandSiefermann-Harms,1981;Jarrigeetal.,1995).Dependingontheirfunction,TAGsynthesistakes placeontheendoplasmicreticulum(ER)whilethatofpolarlipidsoccursinthethylakoid(Gounarisetal.,1986;Fig.8). MostGLinfreshforage(Table1)aremonogalactosyldiacylglicerol(MGDG)anddigalactosyldiacylglycerol(DGDG),also calledgalactolipids(GA)andwereﬁrstdiscoveredbyCarteretal.(1956)andsubsequentlyconﬁrmedbyWeenink(1961).

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Fig.8.Synthesisdenovoofpalmitate,stearateandoleatetakesplaceintheplastidstroma.Therelativecontributionofplastidorextra-plastidgenerated diacylglycerolvariesamongplantspecies.Theelongationreactionsforformationofverylongchainfattyacidsareshownherewithacyl-CoAsubstrates,but othersubstratesmaybeusedindifferenttissues.Forclarity,routesoffattyacidandacyllipidsynthesisareshownseparately,buttheyareinterdependent tosomeextent.

FromGounarisetal.(1986),modiﬁed.

Shorland(1961)andWeenink(1962)laterfoundthattheFAprofileofGLwerealmostentirelycomposedofLNA(Table2; O’BrienandBenson,1964;RoughanandBatt,1969)andthattheneutralfattyacid(NFA)andFFAfractionshaddifferentFA profiles(Table3).Plantsulpholipids(SL)arespecificallyassociatedwiththephotosyntheticmembranesofhigherplants, wheretheirstructureisbasedon1,2diacylglicerol-3-(6-sulpho-␣-d-quinovopyranosyl)-snglycerol(SQDG;Figs.6and7). PlantSLarethethirdcomponentofGLfreshherbagelipidsafterGAandsphingolipidswithlessLAaswellasmorepalmitic (PA)andSAthanMGDGandDGDG(O’BrienandBenson(1964),inagreementwithRoughanandBoardman(1972)who postulatedthehigherratioofSFAinSL.TheSQDGlevelisabout40–50g/kgoftotallipidinplantleaves(Roughanand Boardman,1972;HudsonandWarwick,1977;Table4).

Glyco-sphingolipids(SPH)haveasphingosinechainastheirbackbone(Figs.6,9,10),towhichareattachedaFAandone ormorecarbohydratesasthepolarheadgroup(Lehninger,1979).Inplantcells,SPHaremainlyglucosylceramide(GSC; cerebrosides)andglycosylatedinositolphosphoryl-ceramides(Fig.11),withhigherSFAcontentandlowerlevelsofcis-9

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Fig.9.Glycosylceramidesfromplants,fungiandmammals.Theceramidebackbonesofglucosylceramidesfromplants(atthetop)showgreatvariety.Their sphingoidbasescarrythreepossiblemodiﬁcationsbeing:hydroxylationortransdesaturationatC-4,andcisortransdesaturationatC-8,thecombination ofwhichresultsinsevenfrequentplantsphingoidbases.Thesesphingoidbasesarelinkedtomorethantena-hydroxyfattyacidsvaryinginchainlength andn-9desaturation.Incontrasttoplants,glucosyl-andgalactosylceramidesfromfungi(middle)haveacharacteristicfungalconsensusstructurewith onlyafewstructuralvariations.

FromWarneckeandHeinz(2003),modiﬁed.

Fig.10.Structuresofceramideglycosidesfromplants.Theceramidebackbonesofplantsphingolipidshavelargevariability.Theirlong-chainbasescarry fourpossiblemodiﬁcations(ongreybackground),whicharehydroxylationor(E)-desaturationatC-4,and(E)-or(Z)-desaturationatC-8resultinginseven frequentlongchainbases.

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C18:3 952.8 818.7 AdaptedfromO’BrienandBenson(1964).

MGDG=monogalactosyldiacylglycerol;DGDG=digalactosyldiacylglycerol;tr=trace. –=notreportedintheoriginalpublication.

Table3

Fattyacidcompositionofredcloverlipidfractions(g/kgoffattyacids).

Galactolipids Hexanesolublelipids

C8:0 – 2.2 C10:0 – tr C12:0 – 5.4 C14:0 – 19.2 C16:0 21.2 142.3 C18:2 19.2 60.1 C18:3 959.6 770.7

AdaptedandcalculatedfromWeenink(1961). –=notreportedintheoriginalpublication. Table4

Lipidcompositionofplantcellmembranes(moles/kgtotallipids).

PC PE PG PI PS DPG MGDG DGDG SQDG Sterols Glyco-sphingolipids Endomembranes Reticulum+Golgi 434.8 232.6 60 60 30 – – – – 41.5 – Tonoplast 152.8 152.8 20 59 20 – – – – 144.3 121.7 Plasmamembrane 83.6 93.2 15 16 11 – – – – 56 63 Chloroplast Outermembrane 320 100 50 – – 170 300 60 – – Innermembrane 90 10 – – 550 300 50 – – Thylakoids 70 10 – – 580 270 70 – – Mitochondria Outermembrane 520 220 30 100 – – – – – 130 – Innermembrane 370 330 20 40 – 110 – – – 130 –

AdaptedandcalculatedfromJouhetetal.(2007). –=notreportedintheoriginalpublication.

PC=phosphatidylcholine; PE=phosphatidylethanolamine; PG=phosphatidylglycerol; PI=phosphatidylinositol; PS=phosphatidylserine; DPG=diphosphatidylglycerolMGDG=monogalactosyldiacylglycerol;DGDG=digalactosyldiacylglycerol;SQDG=sulphoquinovosyldiacylglycerol.

Fig.11. Aglycolipidsbasalstructure(glycosylinositolphosphoceramide)onethesimplestlipidsinhigherplants,themostabundantsphingolipidinthe membranesofleavesoftomatoandsoybean.

FromMarkhametal.(2006),modiﬁed.

22–26-carbonmonounsaturatedFA.Thuswhileinositol-containingSPHarefoundonlyinplantsandfungi,GSCistheonly SPHwhichplants,fungiandanimalshaveincommon(WarneckeandHeinz,2003).

4.2. Phospholipids

AnalyticalresearchbyCarteretal.(1969),laterextendedbyHsiehetal.(1978),revealedthatglycophosphosphingolipids (GPSL)containN-acetyl-glucosamineandaninositolbondedviaaphosphodiesterlinktoaceramideandviaaglycosidic linktoachainofcarbohydratesofvariablecomposition,sugarssuchasmannose,arabinoseand/orfructose(Bromleyetal.,

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Fig.12.Thecommonglycerophospholipidsarediacylglycerolslinkedtohead-groupalcoholsthroughaphosphodiesterbond.Headgroupsubstituents couldbeethanolamine,choline,serine,glycerol,myo-inositol4,5biphosphateorphosphatidyl-glycerol.

FromNelsonandCox(2004),modiﬁed.

2003).Themainsphingoidbasesinplantsaredihydrosphingosine(DHS)andphytosphingosine(PHS)ofwhichithasbeen estimatedthatthereare300–400molecularspecies(Hannunetal.,2001).Thesimplestformsofawiderangeofstructures areinyeastsandfungi.Morecomplexformsareinprotozoaandinplants,(Fig.9).GPSLmakesup1g/kgofDMinleaves,while LAandLNAacidsare92and720g/kgoftotalFA,respectively.AswithGL,wherethebackboneconsistsofglycerolipids,these lipidsarecalledglycerophospholipids(GPL:Fig.12)while,ifthebackboneisasphingosinelipid,theyarecalledsphingolipids (SHL).

GPLarederivedfromphosphatidicacidand,inallthesecompounds,theheadgroupisjoinedtoglycerolthrougha phos-phodiesterbond.Theusuallevelofphospholipidsinforageisabout260g/kgofetherextract,correspondingto22g/kgof DM(Weenink,1962)withphospatidylcholinbeingthemaincomponent(450g/kgoftotalPL),followedby phospatidylglyc-erol(300g/kg),phospatidylethanolamine(180g/kg)andphosphatidylinositol(100g/kg),asreportedbyRoughanandBatt (1969).Howevertherearedifferencesintheliterature(HudsonandWarwick,1977),probablybecausethePLcomposition wasobtainedfromdifferentforagesspeciesandanalysedusingdifferentmethods.TheFAproﬁleofGPLhashigherlevels ofPA,andlowerlevelsofLAandLNA(560g/kg,210g/kg,90g/kgofFA,respectively;Weenink(1962),withforagespecies probablyhavinganeffect(Table5;RoughanandBoardman,1972).

Overall,inherbagesamplesthelipidfractionsassociatedwithdifferentFAprofilesmayinfluenceruminalLPandBH. IndeedGLdisappearedveryquicklyfromtherumenofsheepfedgrassherbageand,after8hofruminalincubation,MGDG hydrolysiswastwicethatofDGDG(Dawsonetal.,1974).ItisalsowellknownthatLNAinplantsismorecommoninthepolar fractionthaninFFAorTAG(826and470g/kgofFA,respectively)whileLAismorecommoninTAGthanFFAorthepolar fraction(230,174,50g/kgofFA,respectively).Thisdistributionofplantlipidshelpsexplainthehighertransferefficiencyof PUFAtomeatandmilkofruminantsfedfreshforageratherthanoilsandoilseeds(DoreauandFerlay,1994;Dhimanetal., 1999;Bernardetal.,2009;Leeetal.,2009a).IndeedDawsonetal.(1974)hypothesizedthattheBHrateoflinolenicacid islowerwhenitcomesfromMGDGandDGDGratherthanFFA.RecentresultsfromCabidduetal.(2010)pointedoutfor thefirsttimeinthebibliographythattheBHrateofLNAwasnegativelycorrelatedwithMLfractionlevel(Fig.13)intwo foragespeciesattwodifferentphenologicalstages,whereasViciasativahadhighervaluesofML,LNA(Fig.14)thanTrifolium incarnatum.

5. Plantendogenousfactorswhichinﬂuencefattyacidproﬁle,PUFAruminallipolysisandbiohydrogenation

Variationsinthelipidcompositionoffreshforagesareduetomanyfactors,includingplantspecies(Crombie,1958; Shorland,1961;RoughanandBatt,1969;Meloetal.,1995;Addisetal.,2005;Cabidduetal.,2005)cultivarswithinspecies

Table5

Fattyacidcomposition(g/kgtotalfattyacids)onlipidfractionsfromPeaandBeanleaves.

Pea Bean C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 MGDG 26.7 0 tr tr 90.8 881.5 26.6 0 tr tr 48.5 924.9 DGDG 167.8 0 82.7 tr 81.6 668.0 168.0 0 41.4 tr 20.4 770.2 PC 219.1 0 74.0 31.5 448.0 227.5 215.1 0 41.5 10.3 296.6 436.6 PE 205.2 0 62.1 tr 510.0 222.8 253.3 0 31.2 tr 348.8 366.7 PG 538.1 0 42.6 21.2 210.2 187.8 753.7 0 76.0 tr 32.1 138.2 PI 418.3 0 73.8 tr 291.1 216.8 548.3 0 74.7 tr 126.3 250.7 SL 340.1 0 104.8 tr 291.1 216.8 428.5 0 105.6 tr 72.9 392.9

AdaptedandcalculatedfromRoughanandBoardman(1972). tr=trace.

MGDG=monogalactosyldiacylglycerol; DGDG=digalactosyldiacylglycerol; PC=phosphatidylcholine; PE=phosphatidylethanolamine; PG=phosphatidylglycerol;PI=phosphatidylinositol;SL=sulpholipid(sulphoquinovosyldiglyceride).

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Fig.13.Fattyaciddistribution(valuesinbracketsareexpressedasg/kgoffattyacidmethylesters)ofplantswhere(a)and(b)areFAproﬁlesofmembrane lipidsofViciasativaatvegetativeandreproductivestages,respectively,and(c)and(d)areFAproﬁlesofmembranelipidsofTrifoliumincarnatumat vegetativeandreproductivestages.

FromCabidduetal.(2010),adapted.

(Quartaccietal.,1995;Elgersmaetal.,2003;Cabidduetal.,2009c),plantageandphenologicalstage(Cabidduetal.,2009c), positionoftheleavesontheplant(Krebskyetal.,1996)levelofsensitivitytodrought(Quartaccietal.,1995),andgrazing orcuttingmanagement(Dewhurstetal.,2001).Nichols(1963)reportedthatlipidclassesarefoundindifferentproportions indifferentplantorgans(i.e.,leavesversusstems),withleaveshavingahigherGLcontentthanstems.Stemsareverylowin TGA,andSLcomparedtoleaveswherethepolarfractionsarethemainlipidcomponent.

Severalauthors(e.g.,Molleetal.,2004)havereportedthatdietsofgrazingruminantsareusuallyricherinleavesthan stemsleadingtotheassumptionthatleavesarethemaindietarysourceoflipidforBH,andthisissupportedbythestrong relationshipbetweentheleaﬁnessofgrazedforagesandthevaccenicacidcontentinmilkfromgrazingsheep(Cabidduetal.,

Fig.14.Relationshipbetweenmembranelipidfractionandlinolenicacid(LNA)ruminalbiohydrogenationinherbagesamplesofViciasativa(rumble symbol)andTrifoliumincarnatum(trianglesymbol)atvegetative(opensymbols)andreproductive(solidsymbols)stages.

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A.Buccionietal./AnimalFeedScienceandTechnology174 (2012) 1–25 15 Table6

Fattyacidcomposition(g/kgfattyacids)onlipidfractionsofredcloverleaves.

C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18br C18:0 C18:1 C18:2 C18:3

Triglycerides 7.1 109.5 – 2.9 174.6 6.0 tr tr 28.4 37.4 407.2 339.3

Phospholipids – 3.6 – tr 566.2 28.2 tr tr 33.8 40.2 229.7 100.3

Galactosylglycerides – 0.8 – – 16.6 tr – – 3.1 6.1 25.2 948.2

AdaptedandcalculatedfromWeenink(1962). tr=trace.

–=notreportedintheoriginalpublication. br=branched.

2004).Thelipidfractioninleavesisnotrandomlydistributed,with400g/kgofleaflipidsinchloroplasts,withGLrestricted toplastidmembranesandPL,themaincomponentsofextraplastidicmembranes(Table4).GLandPLconstitute600g/kgof leaflipids,althoughthisallocationchangeswhenchloroplasts(745versus255g/kgoflipidforGLandPL,respectively)or cytoplasms(840versus150g/kgoflipidsforPLandSL,respectively)areconsidered.

AsreportedinTable6,LAandLNAlevelsdifferamonglipidclasses(SchwertnerandBiale,1973),withahigherlevelof LAinTGAandinPL(373and250g/kgoftotalFA,respectively),whereasLNAismorecommoninGL(950g/kgoftotalFA; Weenink,1961).ItisofinterestthatthedifferentdistributionofLAandLNAinplantorgans,anddifferentFAprofileinthese lipidclasses,mayinfluencethefateofFAtoLPandoxidationloss.Almostfourdecadeshavepassedsincethefirstresults fromFaruqueetal.(1974)suggestedthathydrolysisofTGAandGAfromgrasswasdueprimarilytoplantenzymeactivity, suchasgalactolipaseswhichcatalyseMGDGandDGDG,phospholipaseswhichcatalyseGPLandsulpholipaseswhichcatalyse SL.Inrecentyearsseveralstudieshavebeencompletedtoclarifythepathwaysofendogenouslipolyticsystemsinplants (Leeetal.,2009b;VanRanstetal.,2011).Sincefreshforagecanbearichsourceof␻-3FAandCLAprecursors,considerable efforthasbeenmadetodevelopefficientenrichmentstrategiesfortheseFAinanimalproductsusingfeedingstrategies.In thelastdecadethelargeamountofresearchonplantendogenousfactorsinvolvedinruminallipidmetabolismassumed thatfreshforagecanplayanimportantroleinmakingruminantproductslessexpansive,healthierandtheirproduction moreenvironmentallysustainable.ApreliminarystudybyLeeetal.(2003)showedthatendogenouslipolyticactivityhad adetrimental(−25%)effectonMLcontent,andwasassociatedwitha180%increaseinFFA,whichwasdemonstratedby incubatingleafbladesofLoliumperenneandTrifoliumpratensewithbufferwithoutrumenliquor.Endogenouslipolysis ingrassforagespeciesreachedaplateauafter2hofinvitroincubationwhilethisplateauwasreachedveryslowlyin legumeswitharesultingdetrimentaleffectontheLAandLNABHratioofthelegumes(DewhurstandLee,2004).Leeetal. (2003)hypothesizedthatthisactionmightbeduetopolyphenoloxidase(PPO),whichhadbeenpreviouslyshowntohave aninhibitingeffectonendogenousplantproteolysis(Jonesetal.,1995).Asreportedbyseveralauthors,PPOisacopper metalloproteinwhichfunctionsinthepresenceofoxygen,andisabletoreduceLPinredcloverthroughdeactivationof lipolyticenzymesand/orthroughformationofprotein–phenol–lipidcomplexes.WhenthePPOenzymefromredcloverwas incubatedwithawiderangeofortho-diphenolsinthepresenceofoxygen,therewasrapidconversionfromthelatentto theactiveformofPPO(Leeetal.,2008a),aconversioninhibitedbyaddingascorbicacid,suggestingthatthismechanism requiresformationofortho-quinones.OtherfactorswhichinfluencetheefficacyofPPO,toproducephenol-boundprotein and/orPPOactivationincludesoxygenconcentration,thephenologicalstageoftheplantandtheplant’sNDFcontent(Lee etal.,2009b;Cabidduetal.,2011).

PolyphenoloxidaseincreaseslipidprotectionfromendogenouslipolyticactivityandrumenBHby11–22%(Leeetal., 2007b,2008a),toamaximumof33%(Leeetal.,2006,2007b).Howeveritcannotbedeﬁnitelyconcludedfromtheseresults whetherprotectionisduetolipaseinhibition,oriflipidprotectionisduetoprotein–phenoland/orlipid–phenolcomplexes (Leeetal.,2010).

SeveralstudiesbyLeeetal.(2003,2004,2006,2007b,2008a,2009b,2010)showedthatLPismediatedbyplant endoge-nouslipasesupto50%.Duringgrazing,thefeedbolusisexposedtooxygenbyruminationandthePPOsystemisactivated (Leeetal.,2009b).Inasimulatedrumenenvironment,theextentofendogenousLPafter24hofincubationrangesfrom 650g/kgoflipidsforcocksfootto820g/kgforredclover(Leeetal.,2006,2008a,2011).Bauchartetal.(1990b)foundthat lipidhydrolysiswasmuchhigherindietsenrichedwithlipidsthanindietswithoutlipidsupplements.Severalauthors alsonotedthatLPintherumenneedsapHof6–7(TammingaandDoreau,1991),asdoestherumenmicrobialpopulation andtheirlipolyticactivity(Songetal.,2008).Wheninvivoresultsfromruminantsfedredcloversilage,withhigherPPO activity,werecomparedwiththosefedgrasssilagewithlowerPPOactivity,itwasfoundthatredclovertransfersC18:2 andC18:3fromfeedtomilkmoreefficientlythangrasses(Dewhurstetal.,2003;Leeetal.,2009a;Moorbyetal.,2009). Similarresultshavebeenreportedwhenredcloverwascomparedwithwhiteclover(Steinshamn,2010),thereby support-ingthehypothesisthatPPOoffersprotectionfromplantendogenouslipolysisandfromruminalBH.Asreportedbyseveral authors,manyfactorsareinvolvedinthisprocessbecause,insuchcases,redclovermayindirectlyinfluenceruminallipolysis andbiohydrogenationthroughashiftintheruminalmicrobialpopulation(Huwsetal.,2010),orbylipidencapsulationin protein–phenolmatrices(Leeetal.,2010).Becauseruminallipolysisandbiohydrogenationarealsoindirectlyinfluenced bytherumenpassagerate,theremaybeanassociationbetweenPPOlipolysisprotection,andtheincreaseinpassagerate, asreportedbyVanhataloetal.(2007)inwhiteclover.TheremaybealackofconsistencybetweenthePPO,passagerate andlipidencapsulationtheories.ThuswesuggestthatlipolysisandBHaredirectlyinfluencedbytheassociationofPPO,

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insilagefermentationandthusreducedMLexposuretolipase(VanRanstetal.,2009).Bycontrast,thiseffectdidnotoccur forclover,probablyduetothenegativeeffectofproteinboundphenols(PBP)onmicrobiallipase(VanRanstetal.,2009). Leeetal.(2010)showedthat,inspiteoftheincreaseinPBPafterwilting,endogenouslipolysisisnotaffectedbut,after invitroincubation,LPwaslowerinwiltedcocksfootthantallfescue,duetotheinhibitingeffectofPBPonruminalmicrobial activity(Leeetal.,2011).TheDMcontent(i.e.,wiltingeffect)mayhaveadetrimentaladditiveeffectonlipolysistoamore damagingdegree(VanRanstetal.,2010).

BiohydrogenationisheavilyinfluencedbyPSM,whichincludesPPO,fattyacidoxidation(FAOP)andtannins(Leeetal., 2007a;Cabidduetal.,2009b,2010).Severalreportsontherelationshipbetweentanninandrumenmetabolismmention thenegativeeffects ofmodulationofproteinandfibredegradationondevelopmentoftherumenmicroflora.However littleinformationisavailableoneffectsofpolyphenoliccompounds,suchasLA-I,onrumenenzymeactivity(Cabidduetal., 2009b,2010).HoweverVastaetal.(2009a,b)reportedthattanninsdonotinterferewithLA-I,butdointerferewithmicrobial proliferation.Thustanninsdonotinhibittheactivityofmicrobialenzymes,butshiftthecompositionoftherumenmicrobial population.

6. Oddandbranchedchainfattyacids:anewfrontierinthestudyofrumenmicrobialmetabolism

Ruminalbacteriacontain50–90g/kg(DM)lipids,withPUFAandoddandbranchedchainfattyacids(OBCFA)representing 40g/kgand50g/kgoftotalFA,respectively(Kaneda,1991;MansbridgeandBlake,1997).OBCFAfromrumenmicroorganisms mainlyconsistsofisoC13:0,anteisoC13:0,isoC14:0,isoC15:0,anteisoC15:0,isoC16:0,anteisoC16:0,isoC17:0,anteiso C17:0(Vlaemincketal.,2004a,2006a).ThereisincreasinginterestinmilkOBCFAaspotentialdiagnostictoolsofrumen functionbecausedeterminingmilkOBCFAisnon-invasive,andthusmoreacceptablethancurrentinvivotechniqueswhich usuallyinvolveuseofruminallyfistulatedanimals(Vlaemincketal.,2006a;Bessaetal.,2009).RumenbacteriaOBCFAare mainlyC15:0isomers,with590–670g/kgoftotalOBCFA(Vlaemincketal.,2004b).Amongdietarycomponents,starchand fibreproportionsplayacriticalroleinrumenandmilkOBCFAcontent(Basetal.,2003;Vlaemincketal.,2004b;Cabrita etal.,2009)throughtheirinfluenceonthecompositionanddevelopmentofthemicrobialpopulationand,inparticular, onnumbersofbacteriaofcellulolyticbacterialstrains(Vlaemincketal.,2004a).Oddchainfattyacids(OCFA)areformed throughelongationofpropionateorvaleratewhereasprecursorsofbranchedchainfattyacids(BCFA)arethebranchedchain aminoacidsvaline,leucineandisoleucine,orthebranchedshortchaincarboxylicacids(BSCFA)isobutyricandisovalericacid (Kaneda,1991).AmultiplelinearregressionbasedonOBCFAhighlightsthepositiveinfluenceofruminalisoC15:0,anteiso C15:0andanteisoC17:0onthemolarproportionofacetate,whileC15:0wasmostimportantfortheruminalproportionof propionate(Vlaemincketal.,2004b).Sincetheruminalmicrobialpopulationisinfluencedbyawiderangeofvariablessuch asdietchemicalcomposition,ruminalpH,dietaryF/Cratio,ruminalNH3concentration,theserelationshipshaveonlybeen partiallyconfirmedbyothersstudies.Rigoutetal.(2003)andCabritaetal.(2007)foundsmallerresponsesintheC17:0 contentofmilkafterruminalinfusionofpropionicacidthaninC15:0content,whileDewhurstetal.(2007)foundlower levelsofC15:0andC17:0inmilkfromcowsfedwithhighF/CratiosthanfromcowsfeddietswithlowF/Cratios.OBCFA levelsarenegativelyinfluencedbylipidsupplementsaddedtothediet(Bauchartetal.,1990a)andalsobyPSM(Cabiddu etal.,2009b,2010),asaresultoftheirinhibitingeffectontheruminalmicrobialpopulation,directlybytoxinsorindirectly byreductionofnutrientsubstrateavailabilityforthemicrobes.Overall,OBCFAlevelsinmilkappearstobeapotentially usefultooltoevaluaterumenfermentationparameters,withoutignoringeffectsoffactorssuchasfeedparticlesizeandfeed management,whichcaninfluencetherumenVFAprofile(DehorityandOrpin,1997;Looretal.,2004).Indeed,exceptfor C17:1whichisendogenouslysynthesizedbytheactionof9_desaturase₍_Fievez_et_al.,₂₀₀₃_),_C15:0_and_C17:0_are_partially denovosynthesized(Vlaemincketal.,2006a;Cabritaetal.,2007).OBCFAhavebeenconfirmedtobeextremelyrelevant asmarkersofruminalmicrobialactivity,especiallywheneffectsofincubationtimeofsubstrateorsamplepreparation methodsofmicrobialcolonization/contaminationinvivoandinvitroneedtobeevaluated(Kimetal.,2005).Attemptsto validaterelationshipsbetweenrumenOBCFAoutputandmilkOBCFAaremixedcomparedwithmicrobialmarkerssuchas diaminopimelicacid(DAPA)andpurinebases(PB;Fievezetal.,2003).Vlaemincketal.(2005)foundthatdailymilksecretion ofOBCFAwascloselyrelatedtoduodenalflowofDAPAandPB,withverylowcoefficientsofvariation(10.1&and10.0%for DAPAandPB,respectively).Dewhurstetal.(2007)alsoseemtoindicatethatmilkOBCFAarenotalwaysrelatedtoduodenal flowoftheseFA,suggestingthatcorrelatingmilkOBCFAlevelwithrumenVFAmayidentifyaninterestingrelationship betweenisoC14:0andisoC15:0.Theseresultsalsosupporttheargumentfortherebeingarelationshipbetweendietary NDFlevelsandisoC14:0andisoC15:0levelsinruminalmicroorganisms(Vlaemincketal.,2006c).Arelationshipalso

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Fig.15.RelationshipbetweenruminalammoniacontentandsomeOBCFAintherumen. FromLooretal.(2002),Moorbyetal.(2006),Leeetal.(2008b),andDewhurstetal.(2007),adapted.

occurredbetweenlinearoddchainfattyacids(OCFA)andtheproportionofpropionateintherumenbecauseamylolytic bacteriacontainshighlevelsofOCFA(Vlaemincketal.,2004b,2006c),suggestingthatitmaybepossibletopredictrumen fermentationpatternsfrommilkOBCFA,ratherthanfromthechemicalcompositionofthediet.Inparticular,thelower valuesoftherootmeansquarepredictionerrorbetweenmilkOBCFAandruminalVFA(3.0%,9.0%and8.9%foracetate, propionateandbutyrate,respectively),duringthevalidationofthepredictionequation(Vlaemincketal.,2006c),suggest thatthisnewapproachiscorrect.

Newchallenges inestablishingthepreciseroleofOBCFA includeimprovingourunderstandingoftherelationships betweenseveralcellulosolyticbacteriastrainswhichcontainhighlevelsofodd-chainiso-fattyacidscomparedtoamylolytic bacteriawhichareparticularlyrichinOCFA(Vlaemincketal.,2006a).Vlaemincketal.(2006a)foundadecreaseinisoC14:0 andisoC15:0andisoC15:0,andanincreaseintheisoC17:0andanteisoC17:0,proportioninmilkfromcowsfedgrassversus maizesilage.Howevertheresultsdifferedamongcows,sheepandgoats,perhapsbecausemethylmalonyl-CoAsynthesis usestheFAdifferentlyamongspecies(Vlaemincketal.,2006a).Understandingthemechanismofactionoftheruminal microbialpopulationremainsaworkinprogress,althoughitisknownthatlipidsupplementsinhibitrumenbacteriain differentways,withGram+beingmoresensitivethanGram−,andthattheseeffectschangewiththeunsaturationlevels andcisversustransconfigurationoftheFA.Thereareseveralopinionsontheeffectsofsupplementingruminantdietwith lipids.Forexample,adietenrichedinLAandLNAresultedinareductionofOBCFAinmilk,whereasaFOormarinealgae supplementincreasedthetotalOBCFAcontentofmilk(Vlaemincketal.,2006a).Shingfieldetal.(2008)showedthatmilk fromcowsfeddietssupplementedwithsunfloweroils,withahighlevelofLA,linearlyincreasedOBCFAlevels(isoC15:0, C15:0andanteisoC17:0)comparedwithmilkfromtheunsupplementedgroup.Thesameresultsoccurredinvitro,where bacterialsynthesisofOBCFAincreasedwithDHAsupplementfeedinglevel(Vlaemincketal.,2008).

SomemilkOBCFAlevelsarenegativelycorrelatedwithdietarystarchlevels(isoC14:0,isoC15:0andisoC16:0)while others(isoC15:0andisoC14:0)arepositivelycorrelated.Inasimilarway,anegativerelationship(Fig.15)occurredbetween anteisoC17:0andruminalNH3asapotentialmarkerofrumenproteindegradabilityaccordingtoVlaemincketal.(2006a), whofoundalinearrelationshipbetweenduodenalmicrobialproteinflowandsecretionofOBCFAinmilk.HoweverDoreau etal.(2007)didnotconfirmtheirfindings,probablyduetothedifficultyofcorrelatingOBCFAwithduodenalmicrobial proteinflowinindividualcows,asrecentlyreportedbyGadeyneetal.(2011).Finally,isoC17:0isstronglycorrelatedwith BHintermediatessuchustransmoneneFA.OBCFAdetectioninmilkcouldbeapotentialwaytodiscriminatebetween grazingruminantswhosedietshavedifferentbotanicalcomposition,asreportedbyLourençoetal.(2007)whofound40% higherlevelsofisoC17:0intherumenoflambsgrazingnaturalpastureswithhighlevelsofplantdiversitythaninlambs grazingpasturerichinlegumesorryegrass.UnfortunatelytheseresultswerenotconfirmedbyCoppaetal.(2011)who foundnodifferencesintheisoC17:0levelinmilkwhentheycomparedcowsgrazingpastureswithdifferentbotanical compositions.ThedifficultyindescribingtheruminalbacterialpopulationsbasedontheirOBCFAprofilewasconfirmedby thehigherlevelsofanteisoC15:0insomestrainsoftheB.fibrisolvens,thelowcontentofthisfattyacidinS.ruminantium andhighcontentofisoC17:0insomestrainsofPrevotellaruminicola(Vlaemincketal.,2006b).Finally,asweshallseelater, futureevaluationsoftheprotozoa/bacterialcommunityratiomaybeausefulwayofimprovingtheaccuracyofpredictions, and/orevaluationofrumenfunction,becausethelevelofOBCFAinprotozoaisconsistent(110versus160g/kgoftotalFAin bacteriaandprotozoa,respectively),asreportedbyOr-Rashidetal.(2007).

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Fig.16.Comparisonoftwoapproachestoclassifyruminalbacterialonthebasisofbiohydrogenationpower(a)ortaxonomy(b). (a)isfromHarfootandHazelwood(1988);(b)isfromLourenc¸oetal.(2010).

7. Futureresearchneeds

7.1. Interactionbetweenpurecultureandpuresubstrate

Futureresearchaimedatbetterunderstandingruminallipidmetabolismshouldfocusontheoccurrenceof biohydro-genatedintermediatesbystudyinglipidmetabolismbypurebacterialstrainsgrowingonuniqueUFAsubstrates,quantifying impactsofprotozoalisomerizationmechanismsandtheirprotectiveeffectsonFABH,andevaluatingeffectsofplant endoge-nousfactorsonLPandBH.Inrecentyearsthefocusofresearchintolipidmetabolismshaschanged,withmoreattention beingpaidtoidentifyingthemetabolicpathwaysofsubstratesbymeansofculturingindividualmicrobialstrains.Inthis area,resultsfromdifferentgroupsproposedifferentclassificationcriteriaofbacteriainvolvedinthevariousPUFA biohy-drogenationsteps.InthecaseofButyrivibriospp.,thesecriteriaincludetheclassicalbiohydrogenationsteps(Fig.16a),and thetaxonomy(Fig.16b)alsobasedontoxiceffectoflinoleicacid.(HarfootandHazelwood,1988;Lourençoetal.,2010). AsreportedinSection3ofthispaper,rumenbacteriainvolvedinBHwereclassifiedeitherasgroupA(Hazlewoodetal., 1979asquotedbyKempandLander(1984)orgroupB(KempandLander,1984).Studyinglipidmetabolismwithspecific strains,associatedwithspecificsubstrates,couldbehelpfulbecause,asreportedbyMcKainetal.(2010),whenone com-paresvariousruminalbacteriawiththeirrelatedbiohydrogenatedintermediates,thenthereductionofCLAisomers(9,11 geometricisomers)occursviadifferentmechanismscomparedwithmetabolismofotherunsaturatedFA(Fig.17).These resultsexplainwhyinterpretingresultsofruminalmicrobialecologystudiesthroughBHproductsaremorecomplexthan originallythought.Forexample,B.proteoclasticumdidnotgrowinthepresenceofCLAcis-9,trans-11orCLAtrans-10,cis-12, andtheirC18:0productionwasalsoinfluencedbyitsgrowthphase(Wallaceetal.,2006),suggestingthatthefateofFA,both precursorsandintermediates,isstronglyinfluencedbythebacterialstrainsandtheirphysiologicalstage(i.e.,exponential versusstationary)ofthemicroorganisms.Theinteractionbetweentheprotozoalandbacterialcommunitiesassociatedwith specificsubstratesalsoappearsinterestinginrelationshiptotheirimpactonBHintermediates.Theruminalmicrobial pop-ulation(expressedasviablecells)consistsofbacteria(1010_/ml_of_ruminal_liquid),_protozoa₍₁₀4_–106_/ml_of_ruminal_liquid) andfungi(103_/ml_of_ruminal_liquid)._Bacteria_usually_has_the_highest_genetic_diversity₍_Nam_and_Garnsworthy,_2007;_Jenkins etal.,2008),anditisrecognizedthatruminalbacteriaaremainlyresponsibleforLP(Dawsonetal.,1977;Jenkinsetal.,2008; Lourençoetal.,2010)andBH(NamandGarnsworthy,2007).However,contrarytoexpectations,protozoaalsoinfluencethe contentofBHintermediatebyisomerase(Devillardetal.,2006;Or-Rashidetal.,2011;Shenetal.,2011)asdofungi(Nam andGarnsworthy,2007).ResultsofOr-Rashidetal.(2011)appearinterestinginthiscontextastheircomparisonoflinoleic acidmetabolisminbacteria,protozoaandtheirmixtureshowedthatallmetabolizelinoleicacidtodifferentCLAisomers andprotozoawereincapableofbiohydrogenatingLAand,whentestedinthebacterial/protozoalmixture,modulatedBH ofLA(Varadyovaetal.,2008).ThevaryingcapacitiesofthemicrobialgroupstometabolizeLAreportedinFigs.17and18 showthatbacterial/protozoalmixturesmayhavegreatercapabilitytobiohydrogenateCLAcis-9,trans-11toVAthan bac-teriaalone,withalowerrateofruminalbiohydrogenation.Thisprovidesnewinsightsintolipidmetabolismthroughthe newlyidentifiedintermediatestrans-11,trans-13CLAandcis-11,trans-13CLA(Or-Rashidetal.,2011;Fig.18).However thelackofSAproductionfromC18:1cis-12andfromC18:1trans-12remainstobeexamined.Theruminalbiohydrogenate intermediateprofilecanbechangedbymodulatingthehydrogenation/isomerizationrate,asobservedbyLaverrouxetal. (2011),whofoundthatisomerizationofVAappearstobeveryfastinthefirst5hofincubationwitha22.5%increaseofthe isomerizationratiowhentherumenfluidcamefromcowsfedahighF/Cdiet.Atpresent,itisnotclearwhythisincreased isomerizationoccursattheexpenseofhydrogenation,althoughitisprobablyduetochangesintheruminalbacterialand

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Fig.17.RoleofButyrivibriospp.,PropionibacteriumacnesandButyrivibrioproteoclasticumonmetabolismoftheunsaturatedfattyacidslinoleicandoleic acids.

FromMcKainetal.(2010)andWallaceetal.(2006),modiﬁed.

Fig.18.MetabolicpathwaysforbiosynthesisofCLAandrelatedcompoundsfromlinoleicacidbyrumenbacteria(B)andprotozoa(P).Solidarrowsare bacterialandprotozoalactivitiesleadingtoformationofisomersofCLAandC18:1,includingC18:0,theminordottedarrowsarethebacterialactivity leadingtoformationoftheprospectivecompounds,andthemajordottedarrowsrepresentthebacterialactivities.

FromOr-Rashidetal.(2011),modiﬁed.

protozoalcommunitiesstrains.AllthesechangesarealsofoundontheBHintermediates,andhaveimpactsofdifferent magnitudesontheruminalbacteriaandprotozoaOBCFA.

7.2. InteractionbetweenPPO,PSM,endogenouslipolysis,andPUFAbiohydrogenation

Lipidsinforagearelessdegradedthanoils,asreportedbyPerrieretal.(1992).TheseresultswereconﬁrmedbyWachira etal.(2000)whofounda15%lowerlinolenicBHratioinsheepfedonlygreenforagethaninsheepalsofedlinseed-based supplements.Thismaybeduetoplantssecondarymetabolites,suchasPPOandtannins(Vastaetal.,2009b;VanRanst etal.,2011;Cabidduetal.,2009b,2010),butmayalsobeduetoprotectionoflipidsfromLPandBHbycellularstructures, asreportedbyDoreauandFerlay(1994).TheliteraturesuggeststhatBHlevelsare820–850g/kgofLAand920–970g/kg ofLNA(Jalcetal.,2009a,b)incornandgrasssilages.AninterestingstudycompletedbyLeeetal.(2010)highlightedthe inﬂuenceofPPOonruminalBHprocessesthroughitscontrolofLP.Resultsfromseveralstudiesshowedthatendogenous

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Fig.19. RelationshipsbetweentanninscontentsandactivePPOinTrifoliumspp.(X)Plantagospp.()Medicagospp.().PPOactivitywasdeterminedas opticaldensity/gfreshweight.

FromCabidduetal.(2011),adapted.

LP,after24hofincubationinasimulatedrumenenvironment,rangedbetween650g/kgoflipidsforcocksfootand820g/kg forredclover(Leeetal.,2006,2008a,2010).TheseresultsdonotagreewithBauchartetal.(1990b),whofoundthatlipolysis washigherincowsfedlipid-supplementeddietsthaninthosefednonsupplementeddiets.Astronginteractionbetween ruminalpHandbothmicrobialandendogenouslipaseshasbeendiscovered:indeedtheruminalmicrobialpopulationhas anoptimalpHof6.0–6.7(TammingaandDoreau,1991),whileplantendogenouslypoliticsystemsneedanoptimumpH of7–8,(Songetal.,2008).SincePUFAinforageareintheformofglycolipids,morestudiesontheendogenouslipolytic systemappearrequired,whichshouldfocusonhowgalactolipaseactivityisinfluencedbyPSMlevel,ratherthanPUFA,on microbialactivity.AsreportedbyKaniuga(2008),studiesongalactolipaseactivityarescantandrelatedtoeffectsoflight andtemperature,whichrevealthatitispossibletomodulatethisactivitybygeneticallymodifyingplants.Onlyrecentlydo someauthorsmentionthatgalactolipasearecapableofhydrolysinggalactolipidsandphospholipids,butnottriglycerides (MatosandPham-Thi,2009),andthatitsactivityisstronglyinfluencedbyforagespecies(GemelandKaniuga,1987).Another endogenousfactorwhichoccursinplants,suchasChrysanthemumcoronarium,iscoronaricacid(C18:H32O3,anepoxyfatty acid)whichcanhaveinhibitoryeffectsonrumenmicroorganism(−30%ofruminalBHrespecttocontrol)asreportedby Woodetal.(2010).InadditionCabidduetal.(2006)foundthatincludingC.coronarium,adaisyplantwithahighlevelofLA, inthedietofgrazingsheepincreasedmilkCLAcis-9,trans-11by60%andC18:1trans-11by60%throughitsinhibitingeffect onruminalmicrobialactivitymediatedbyLA,andbyincreasingmammarygland9_desaturase_activity._It_is_recognized thatC.coronariumalsohasPPOactivity,withaprobablesynergisticorantagonisticeffectonLPandBH.Forthisreason,itis necessarytounderstandtheinteractionsofUFA,PPO,coronaricacidandothersPSMandclarifytheroleofeachcomponent ontheruminalbiohydrogenationpathways.PreliminaryresultsfromCabidduetal.(2011)showedthatplantPPOactivity couldbeaffectedbyspecifictanninswithaninteractionofforagespeciesandplantphenologicalstage(Fig.19).

8. Conclusions

ThestudyofrumenFAmetabolismisimportanttotheunderstandingoffactorswhichinﬂuencethetypesofFAinhuman foodproductsderivedfromruminants.FoodproductsfromruminantsarenaturallyrichinCLA,VAandCLNA,andthe variationintheproportionsoftheseFAinhumanfoodsiscloselylinkedtotheanimals’diet.

Theplantlipidfractionsassociatedwiththeseendogenousfactors(i.e.,PSM,PPO)affectsrumenLPandBH.Althoughthe criticalroleofbacteriainBHiswellknown,protozoahavealsobeenshowntointeractwithFAmetabolismtoreduceBH andincreasetheextentofisomerization.ThisimpliesthatthereareunknownintermediateFApathways.

AchallengeinthefutureistocompletestudiesonplantlipidfractionsinconjunctionwithPSMandPPOinorderto discriminatebetweeneffectsofplantlipidsonFAbiohydrogenateintermediates.Thismaybecomethebasisforachieving moresustainable,lessexpansiveandhealthierruminantderivedhumanfood.

Acknowledgements

ThisresearchwascompletedwiththesupportoftheItalianAnimalScienceandProductionAssociationcommission “Evaluationofinvitrotechniquesforthecharacterizationoffeedsandforthestudyofdigestiveprocessesofdomestic animals”.Specialthanksmustbegiventothecoordinator,PaoloBani,withoutwhosehelpitwouldnothavebeenpossible. Theresearchalsoreceivedﬁnancial supportfromPRINProject2007–prot. 200778K3KJ–“Effectsoftanninsofsome ruminantfeedsontherumendegradabilityofproteinandﬁbrefractions,onthebiohydrogenationoflipidsandonthe

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qualityofmilkandmeat”,fromtheMinistryofUniversityandResearch(MiUR).WewouldalsoliketothanktheRegione AutonomadellaSardegna,whichﬁnanceddevelopmentofSections4–7throughtheproject“studyofdietarystrategiesto improvethequalityofdairysheepproducts”.Finallytheauthorsaresincerelyindebtedtotheanonymousrefereeswhose detailedadviceplayedafundamentalroleinimprovingthemanuscript.

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