Rev Colomb Cienc Pecu 2018; 31(3):213-222

Revista Colombiana de Ciencias Pecuarias

Original articles

213

In vitro rumen biohydrogenation kinetics of mixed linoleic and alfa-linolenic acids¤

Cinética de biohidrogenación ruminal in vitro de los ácidos linoleico y alfa-linolénico mezclados

Cinética de biohidrogenação ruminal in vitro dos ácidos linoleico e alfa-linolênico misturados

Julián A C Vargas1,2*, Quím, M.Sc, Dr.Sc.; Martha Olivera-Angel2, MV, Dr.Sc.Agr.; Claúdio V D M Ribeiro3, Zoot, M.Sc, PhD; Edgar E Daza C4, Quím, Dr.Sc.

1Universidad Nacional de Colombia, Departamento de Ciencias para la Producción Animal, Carrera 30 No. 45-03, Bogotá D.C, Colombia.

2Universidad de Antioquia, Carrera 75 No. 65-87, Medellín, Colombia.

3Universidade Federal da Bahia, Av. Ademar de Barros 500, Salvador, BA, Brazil.

4Grupo de Química Teórica, Departamento de Química, Universidad Nacional de Colombia, Bogotá, Colombia.

(Received: March 13, 2017; accepted: December 10, 2017)

doi: 10.17533/udea.rccp.v31n3a06

¤ Tocitethisarticle:VargasJAC,OliveraAM,RibeiroCVDM,DazaCEE.In vitro rumen biohydrogenation kinetics of mixed linoleic and alfa-linolenic acid. RevColombCiencPecu2018;31(1):213-222.

* Correspondingauthor:JuliánACVargas.UniversidadNacionaldeColombia,DepartamentodeCienciasparalaProducciónAnimal,Carrera30No.45-03,BogotáD.C.Tel.:3165000.E-mail:jcastillov@unal.edu.co

Abstract

Background:Dietary linoleic (LA) and alpha-linolenic (LN) acids are extensively isomerized andhydrogenatedbyrumenmicrobes,andthisactivitycanfurthercontributetothefattyacidprofileofruminant-derived food products. Objective: ToevaluatetheeffectsofLA:LNratioinlipidsupplementsontherumenbiohydrogenationkineticsofLAandLN,aswellasonthetrans-vaccenic acid (VA) production, using an in vitro system. Methods:Rumenfluidwascollectedfromafistulatedsteer,dilutedwithincubationbuffer,andthenincubatedwith500mgofkikuyugrass(Cenchrus clandestinus)supplementedwith16.3mgofdifferentLA:LNmixtures(100:0,75:25,50:50,25:75or0:100).Incubationswereperformedintriplicateforaperiodof0,2,4,6,8or16hours.Differencesbetweentreatmentswereevaluatedinacompletelyrandomizeddesign.Alternatively,computationalchemistrywasusedtodeterminethechangesintheGibbsfreeenergy(ΔGrxn) at 39°CfortheprincipalstepsofLAandLNruminalbiohydrogenation.Results: Partial replacement of LA by LNdecreasedtheVAconcentrationanditsaccumulationrate;italsoincreasedthestearicacidconcentrationandtheratesoftransferfromLAtoconjugatedlinoleicacid(CLA),andfromCLAtoVA.TheconversionfromCLAtoVA(ΔGrxn =-2.65kJ/mol)wasmorespontaneousthanthatfromtrans-11, cis-15octadecadienoicacid(TA)toVA(ΔGrxn =-0.29kJ/mol).Conclusion:TheLA:LNratioinlipidscanmodulateLAandLNbiohydrogenation(BH)kinetics,aswellastheVAproductionintherumen.

Keywords: Fatty acids, kinetics of biohydrogenation, lipid supplement, multi-compartmental model, thermodynamics of biohydrogenation.

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Resumen

Antecedentes:losácidoslinoleico(LA)yalfa-linolénico(LN)deladietasonextensivamenteisomerizadosybiohidrogenadosporlosmicroorganismosruminales,locualpuedecontribuiralperfildeácidosgrasosdelosproductos derivados de rumiantes. Objetivo: evaluarelefectodelarelaciónLA:LNensuplementoslipídicossobrelacinéticadebiohidrogenaciónruminaldelLAyLN,comotambiénsobrelaproduccióndelácidotrans-vaccénico (VA), usando un sistema in vitro. Métodos:secolectófluidoruminaldeuntorofistulado,elcualfuediluidoconbufferdeincubaciónyposteriormenteincubadocon500mgdepastokikuyo(Cenchrus clandestinus) suplementadocon16,3mgdediferentesmezclasdeLA:LN(100:0,75:25,50:50,25:75,o0:100).Lasincubacionesfuerondesarrolladasentriplicadodurante0,2,4,6,8o16horas.Diferenciasentretratamientos fueron evaluadas mediante un modelo completamente al azar. Alternativamente, se determinaron loscambiosenenergíalibredeGibbs(ΔGrxn)a39°CparalospasosprincipalesdelabiohidrogenacióndelLAyLN,usandoquímicacomputacional.Resultados: lasustituciónparcialdeLAporLNdisminuyólaconcentracióndeVAysutasadeacumulación,comotambiénincrementólaconcentracióndeácidoesteáricoylastasasdetransferenciadeLAparaácidolinoleicoconjugado(CLA)ydeCLAparaVA.Laconversiónde CLA para VA (ΔGrxn =-2,65kJ/mol)fue másespontáneaque laconversióndelácido trans-11, cis-15octadecadienóico(TA)paraVA(ΔGrxn =-0,29kJ/mol).Conclusiones:larelaciónLA:LNenlípidospuedemodularlacinéticadebiohidrogenación(BH)delLAyLNylaproduccióndeVAenelrumen.

Palabras clave: ácidos grasos, cinética de biohidrogenación, modelo multi-compartimentalizado, suplemento lipídico, termodinámica de biohidrogenación.

Resumo

Antecedêntes:oácidolinoleico(LA)ealfa-linolênico(LN)dadieta,sãoextensivamenteisomerizadosebiohidrogenadospelosmicroorganismosdorúmen,oquepodecontribuiraoperfildeácidosgraxosdosprodutos derivados de ruminantes. Objetivo: avaliaroefeitodarelaçãoLA:LNemsuplementoslipídicossobreacinéticadebiohidrogenaçãoruminaldoLAeLNcomotambémsobreaproduçãodoácidotrans-vaccênico (VA), utilizando um sistema in vitro. Métodos:coletou-sefluidoruminaldeumnovilhofistulado,oqualfoidiluídocomtampãodeincubaçãoe,emseguida,incubadocom500mgdepastokikuyu(Cenchrus clandestinus) suplementado com16,3mgdediferentesmisturasLA:LN (100:0, 75:25 , 50:50, 25:75ou0:100).Asincubaçõesforamdesenvolvidasemtriplicata,durante0,2,4,6,8ou16horas.Diferençasentretratamentos foram avaliadas utilizando-se um delineamento inteiramente casualizado. Alternativamente, foramdeterminadasasmudançasemenergia livredeGibbs (ΔGrxn) a 39 °C para as principais etapas da biohidrogenaçãodoLAeLN,utilizando-sequímicacomputacional.Resultados:asubstituiçãoparcialdeLAporLNdiminuiuaconcentraçãodeVAesuataxadeacumulação,comotambémaumentouaconcentraçãodeácidoesteáricoeastaxasdetransferênciadoLAparaoácidolinoleicoconjugado(CLA)edoCLAparaVA.AconversãodoCLAparaVA(ΔGrxn=-2,65kJ/mol)foimaisespontâneaqueaconversãodoácidotrans-11, cis-15octadecadienóico(TA)paraVA(ΔGrxn =-0,29kJ/mol).Conclusões:arelaçãoLA:LNemlipídeospodemodularacinéticadebiohidrogenação (BH)doLAeLNeaproduçãodeVAnorúmen.

Palavras-chave: ácidos graxos, cinética de biohidrogenação, modelo multi-compartimentalizado, suplemento lipídico, termodinâmica de biohidrogenação.

Introduction

Fattyacids(FAs)metabolismhasamajorinfluenceon the FAs composition of ruminant-derived food products.UnsaturatedFAs, including linoleic acid(LA; c9,c12-18:2) and alpha-linolenic acid (LN;c9,c12,c15-18:3),areabundantingrassandcertainother ruminant feedstuffs (Buccioni et al., 2012). However,LAandLNarepresentatlowconcentrationsin milk and meat, because they are transformed into numerous isomers, including trans-vaccenic acid (VA;

t11-18:1)andconjugatedlinoleicacid(CLA;c9,t11-18:2) (Hur et al.,2017). Consumption of CLA has beenassociatedwithhumanhealthbenefits(Ferlay et al.,2017).

The main ruminal biohydrogenation (BH)pathwaysofLAandLNhavebeendescribedsincethe 60s (Kepler andTove, 1967) and researchershave focused on better understanding their intricate pathways (Laverroux et al., 2011; Ferlay et al., 2017).Researchershavealsostudiedwhichmicrobe

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communities (Kimet al., 2003; Maia et al., 2010) and dietary FAs (Buccioni et al., 2012; Prieto et al., 2013)playmajorrolesinincreasingVAsynthesisintherumen.ThemaininterestinVAisbecauseitisthemain endogenous precursor of CLA in milk and meat (Kayet al.,2004;Rezamand et al.,2014).

Some in vitro studies suggested that increasing levelsofLNdecreasetheBHrateofVA,resultinginenhanced levelsofVAin rumenfluid (Jouanyet al., 2007;Honkanenet al., 2012). On the other hand, Ribeiro et al. (2007)reportedthatBHrateofLAis twiceasfastasBHrateofLN,consequentlyproducingmoreVAfromLAthanfromLN.ThesecontradictoryresultsmaybeduetotheuseofLAandLNinesterifiedforms(Hoffmannet al.,2015)andthepresenceofotherFAsinfatsupplements,whichcouldinterfereLAandLNruminal BH (Pashaei et al.,2016).

The scientific literature is still inconclusiveregarding the effects of LA or LN levels onVAruminalconcentration.Therefore,theaimofthisstudywas toevaluate theeffectsofLA:LNratio in lipidsupplements on the rumen biohydrogenation kinetics ofLAandLN,aswellasonthetrans-vaccenic acid (VA) production, using an in vitro system.

Material and Methods

Ethical considerations

All procedureswere approvedby theBioethicsCommittee of Facultad de Medicina Veterinaria y de Zootecnia,UniversidadNacionaldeColombia(Act001 of 2010).

Forage collection, animal, and preparation of rumen inoculum

SamplesofKikuyugrass(Cenchrus clandestinus) with 60days of regrowthwere harvested byhandplucking(Cook,1964).Foragesamplesweredriedat60°Candgroundtopassa1-mmsieve(Romer Labs,Getzersdorf,Austria).

Onerumen-fistulatedHolsteinsteerwasusedasdonorofrumenfluid.Theanimalwaskeptinapenduring nighttime and received kikuyu grass, clean

freshwater,andmineralblockad libitum for 30 d beforetherumenfluidwascollected.Inday30(at07:00 h), 1,500mLof rumenfluidwere obtainedfromthesteerbeforefeeding.Therumenfluidwasfiltered through four layers of gauze, placed intoaflask preheated at 39°C, transferred to separatorfunnels, and gassed with CO2, recovering the intermediatephase.ThecollectedphasewasmixedwithMcDougall buffer 1:4 (McDougall, 1948),gassedwithCO2,andaddedwithurea(1g/Lbuffer),producing the rumen inoculum.

Diets, incubation procedures, and sample collections

Fiftymillilitersof rumen inoculumpre-warmedat 39 °Cwereplaced into100mL incubation tubescontaining500mggroundkikuyugrass(1mm)plus16.3mg/tube(3.3%offoragequantitytoavoidtoxiceffectsof excessive unsaturated FAs addition on cellulolytic population (Messana et al., 2013)) ofdifferentLA:LNratios(100:0,75:25,50:50,25:75or0:100).Then,thefilledtubesweregassedwithCO2,sealedwithone-holerubber stoppers (Fisherbrand,Pittsburgh,PA,USA)and incubatedinawaterbathat39°C(BlueSlandIllinois,USA) (Tilley andTerry, 1963).Extrakikuyu tubeswithout the additionofLA:LN ratiomixtureswereassignedascontroltreatment(CTRL).

Incubationtimeswere0,2,4,6,8,or16hours.Extratubeswithonly50mLofpre-warmedrumeninoculumwereusedasblanks.Threereplicationswereprepared per treatment. Incubationswere stoppedby adding500µLof 20g/Lmercury (II) chloride(Merck®,Kenilworth,NJ,USA)solution, and placing thetubesinanicebath.Thecontentofthetubeswasfreeze-dried (Alpha1-4 Christ® plus LO lyophilizer), ground (Romer Labs,Getzersdorf,Austria), andstoredat-60°Cuntilanalyseswereperformed.

Laboratory analysis

Foragesampleswereanalyzedforcrudeprotein(182g/kgDM)(AOAC,2006,method:984.13),ash(117g/kgDM)(AOAC,2006,method:942.05),andetherextract(30.0g/kgDM)(AOAC,2006,method:930.09).Neutraldetergentfiber(609g/kgDM)wasdetermined using alpha-amylasewithout sodiumsulfiteaddition,accordingtoVanSoestet al. (1991).

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Aciddetergentfiber(322g/kgDM)wasdeterminedusing themethoddescribed byGoering andVanSoest(1970).

The FAs in forage samples and incubatedtubeswere extracted andmethylated accordingto Garcés andMancha (1993). The FAs in theLA:LN ratio mixtures were methylated usinga 0.2 N solution of (m-trifluoromethylphenyl)trimethylammonium hydroxide (MethprepTM I)(AlltechTM,Nicholasville,KY,USA).MethylatedFAsofforageandLA:LNratiomixtures(Table1)aswellas the methyl esters of principal FAs intermediaries ofLAandLNruminalBHwerequantifiedbyGLC-FIDusingaShimadzuGC-2014gaschromatograph(ShimadzuManufacturing,Inc.,Canby,OR,USA).Thecolumnwasafusedsilicacapillary(Rt-2560,100mx0.25mmi.d.x0.2μmfilmthickness;Restec®,Inc,Belefonte,PA,USA).Heliumwasusedasthecarriergas.Detectorandinjectortemperatureswere260and270 °C, respectively, and the split ratiowas 30:1.Oventemperaturewas140°Cfor5min,increasedby4°C/minto220°C,heldfor5min,increasedby2.0°C/minto240°C,andheldfor10min.TheFAsin sampleswere identified by comparison of theirretention timeswith thoseobserved incommercialstandardsasNu-Chek®Prep(Elysian,MN,USA),andquantifiedbydirectcomparisonofthepeakareas.

Thermodynamic analyses of LA and LN ruminal BH by computational chemistry

ThermodynamicanalysesofLAandLNruminalBHwere performed using theMolecularOrbital

Table 1. Fatty acid composition of forage, and c9,c12-18:2 (LA) and c9,c12,c15-18:3 (LN) ratio mixtures.

LA:LN ratiosFatty acid composition (g/100 g FAs) Forage 100:0 75:25 50:50 25:75 0:10012:0 0.9 -1 - - - -

14:0 1.4 - - - - -

16:0 33.4 - - - - -

18:0 5.1 - - - - -

20:0 1.6 - - - - -

c9-18:1 2.5 - - - - -

c9,c12-18:2 11.6 100.0 75.7 49.3 23.9 0.30

c9,c12,c15-18:3 43.0 - 24.3 50.7 76.1 99.7

c5,c8,c11,c14-18:4 0.5 - - - - -

1Not detected.

PackageSoftware (MOPAC) (Version8.289L).Theoptimizationmethod (PM6)parameterwasused toestablishtheGibbsfreeenergyofformation(ΔGf)at39°CoftheFAsproducedduringLAandLNruminalBH.TheseΔGf valueswereusedtocalculateΔGrxn of the mainstepsofLAandLNruminalBH(Stewart,2007).TheΔGrxnforeachstepwascalculatedasfollow:

∆Grxn=∑(∆Gfproducts)-∑(∆Gfreagents) (1)

Where:

∑(∆Gfproducts)and∑(∆Gfreagents)arethesumof∆Gfof products and reagents, respectively.

Calculations and statistical analysis

RuminalBHofLAandLN for each treatment(with exception ofCTRL treatment because FAsconcentrationofBHintermediarieswereverylow)wasmodeledbyusingamulti-compartmentalmodel(Figure1).TheratesoftransferbetweenFAspoolswereestimatedusingtheSAAMIIsoftware(SAAM,1997).Trans-9octadecenoicacid(t9-18:1),trans-6octadecenoic acid (t6-18:1), cis-11 octadecenoic acid (c11-18:1), and cis-6 octadecenoic acid (c6-18:1)(i.e.,sum18:1)werenotincludedinthemodelas independent pools because their concentrations were very low.Therefore, just one poolwas usedto represent t9-18:1, t6-18:1, c11-18:1, c6-18:1,according to Ribeiro et al. (2007).Rumelenic acid (RU;c9,t11,c15-18:3) (i.e.,producedby thecis-12doublebondisomerizationofLN)wasnotdeterminedintheincubationsystems,thusitwasnotincludedin

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themodel.Therateoftransferfromstearicacid (SA; 18:0)tooleicacid(OA;c9-18:1)(dehydrogenation)wasalsoincludedinthemodelbecauseitsinclusionproduced the lowestAIC (Akaike informationcriterion) and because ruminal fungus P. communis can dehydrogenate SA to OA using a ruminal ∆9-desaturase(Kempet al.,1984;Ferlay et al.,2017). TheRosenbrock integratormethod (SAAM,1997)wasusedandoptimizedwithavariancemodelbasedonrelativedataandtheforwardderivatives.

TheVA accumulation rate (r-VA) for eachtreatmentwas calculated by fitting theGompertzmodel(Gompertz,1825)usingtheNLINprocedureofSAS,andtheirinitialparametersweredeterminedby Curve Expert Basic (Version 1.4). The fullmathematicalmodelusedwas:

V(t) = a×exp(-1×(exp(b - (c×t))) + ei (2)

Where:

V is the VA concentration at t hours.

Figure 1.Proposedmainpathwaysoflinoleic(LA;c9,c12-18:2)andalpha-linolenic(LN;c9,c12,c15-18:3)acidsbiohydrogenation.

aistheconcentrationat16hours.

b is thedifferencebetweenVAconcentrationsat0and16hours.

c is the r-VA.

ei is the residual error of the model.

ThevaluesofLA,LN,VA,andSAconcentrationswereanalyzedasacompletelyrandomizeddesignin a 6×6factorialarrangementoftreatmentandtimeusing theMIXEDprocedureofSAS.Thestatisticalmodelincludedfixedeffectsoftreatment,hourofsampling,and hour of sampling × treatment (Snedecor and Cochran,1989).Ther-VAandtheratesoftransferof the multi-compartmental modelswereanalyzedasacompletelyrandomizeddesign.Differencesbetweentreatmentsweresignificantatp<0.05.

R esults

TheLAandLNconcentrationdecreased(Figure2(a)and(b))inalltreatments,whereasVAandSAincreased

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(a) (b)

(c) (d)

Figure 2. Fattyacidsconcentrationinincubationsystemswithout(CTRL),andwiththeadditionoflinoleic(LA;c9,12-18:2),andalpha-linolenic(LN;c9,c12,c15-18:3)acidsratiomixtures(LA:LN:100:0,75:25,50:50,25:75,and0:100);(a)c9,c12-18:2;(b)c9,c12,c15-18:3;(c) trans-vaccenicacid(VA;t11-18:1);and(d)stearicacid(SA;18:0);barsrepresentthestandarderrorofthemean.Thefattyacidsconcentrationdiffersbetweentreatments(p<0.05).

Table 2. Trans-vaccenic acid accumulation rates (r-VA) when linoleic and alpha-linolenic acid ratios mixtures (LA:LN) plus kikuyu grass were incubated in rumen fl uid during 16 h.

LA:LN ratiosCTRL 100:0 75:25 50:50 25:75 0:100 SEM p-value

r-VA (%/h) 0.353a 0.335a 0.232b,c 0.245b 0.184c 0.185c 0.014 <0.001

Values with diff erent superscript letters (a, b, c) within a row diff er statistically (p<0.05).SEM: standard error of the mean.p-value: diff erences in parameters between treatments.

(Figure 2(c) and (d)) as incubation time increased (p<0.01). In addition,VAconcentration decreased(Figure2 (c)),whereasSAconcentration increased(Figure2(d))from100:0to0:100LA:LNratios,at16-hourincubation(p<0.01).ItwasnoteworthythatLAandLNconcentrationsunexpectedlyincreasedfrom2to4h,whereasSAconcentrationunexpectedlydecreasedinthesameperiodfortreatmentsinwhichLA:LNratiosmixtureswereadded.

Ther-VAdecreasedastheLNproportionincreasedin the LA:LN ratiomixtures (Table 2; p<0.01).However,r-VAdidnotdifferbetweenthefollowingtreatments:CTRLandLA:LN100:0,LA:LN75:25andLA:LN50:50, andLA:LN25:75 andLA:LN0:100(Table2;p>0.05).

PartialsubstitutionofLAbyLNincreasedtherateof transfer from LA to CLA, from CLA to VA, and

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fromCLAtosum18:1(Figure1;Table3;p<0.05).Also,therateoftransferfromLNtotrans-11, cis-15octadecadienoicacid(TA;t11,c15-18:2)differedbetween 100:0 and 25:75 LA:LN ratiomixture,aswell as between 75:25 and 25:75LA:LN ratiomixtures, and concomitantly, the rates of transfer fromSAtoOAjust increasedfrom75:25to25:75LA:LNratiosmixtures(Figure1;Table3;p<0.01).Fortheotherratesoftransfer,comparisonbetweentreatmentswerenotsignificantlydifferent(Figure1;Table3;p>0.10).

TheΔGrxn at 39 °C for the conversion from CLA (c9,t11-18:2) toVA (t11-18:1) (-2.65 kJ/mol)waslowerthanthatfromTA(t11,c15-18:2)toVA(-0.29kJ/mol)indicatingthatconversionfromCLAtoVAwasmorespontaneousthanthatfromTAtoVA.Inaddition,ΔGrxn at 39 °C for the conversion from VA toSA(+15.9kj/mol)presentedlowspontaneityunderruminal conditions (Figure 3).

Table 3. Rates of transfer (%/h) between intermediates of linoleic (LA; c9,c12-18:2) and alpha-linolenic (LN; c9,c12,c15-18:3) acids biohydrogenation when LA:LN ratios mixtures and kikuyu grass, were incubated in rumen fl uid during 16 h.

LA:LN ratios mixtures

Reaction 100:0 75:25 50:50 25:75 0:100 SE p-value

BH of Linoleic acid

c9,c12-18:2c9,t11-18:2 0.059a 0.057a 0.054a 0.068a,b 0.075b 0.005 0.044

c9,t11-18:2t11-18:1 0.76a 0.58a 0.59a 1.81b 4.17c 0.261 0.001

c9,t11-18:2sum 18:11 0.041a 0.044a 0.038a 0.21b 1.90a,b 0.013 0.037

BH of Linolenic acid

c9,c12,c15-18:3t11,c15-18:2 0.076a 0.076a 0.076a,b 0.10b 0.096a,b 0.018 <0.001

t11,c15-18:2t11-18:1 0.24 0.31 0.24 0.13 0.17 0.065 0.114

t11,c15-18:2sum 18:1 0.10 0.020 0.24 0.039 0.040 0.054 0.466

Other BH pathways

t11-18:118:0 0.078 0.072 0.053 0.057 0.075 0.026 0.894

18:0c9-18:1 0.0020a,b 0.000039a 0.0027b 0.32c 0.080a,b,c 0.001 0.003

c9-18:118:0 0.043 0.032 0.047 1.90 2.04 0.339 0.095

sum 18:1118:0 0.054 0.027 0.045 0.21 0.16 0.020 0.597

Values with diff erent superscript letters (a, b, c) within a row diff er statistically (p<0.05).p-value refers to diff erences in parameters between treatments.1Sum 18:1= 18:1-t9 + 18:1-t6 + 18:1-c6 + 18:1-c11.

Figure 3. Thermodynamic description of themain stepsof linoleic (LA; c9,c12-18:2) and alpha-linolenic (LN;c9,c12,c15-18:3) acids biohydrogenation.Thevalues next tothearrowscorrespondtochangesofGibbsfreeenergyofreaction(ΔGrxn,kJ/mol)at39ºC.

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Discussion

ThegoalofthecurrentstudywastoinvestigatetheeffectsofLA:LNratioinlipidsupplementsontheBHkineticsofLAandLN,aswellasontheVAproductionin an in vitro ruminal system. Our results indicated that partialsubstitutionofLAbyLNinlipidsupplementsincreased isomerization and biohydrogenation rates of themain stepsofLAandLN ruminalBH, anddecreased VA production.

Ourdatashowedthat,regardlessofLA:LNratio,LNdisappears faster thanLA.This suggests that,regardlessofLA:LNproportioninlipidssupplements,LN tends to be biohydrogenated faster thanLA.Additionally,wefoundthatLAtendstoaccumulatein ruminal fluidwhen its concentration in lipidsupplementsisgreaterthanLNconcentration.ThisagreeswithJouanyet al.(2007),whodemonstratedthat disappearance rates of LA ranged from 78.7to85.9%,whereas thatofLNrangedfrom80.0 to91.2%,alsoshowingthatLAruminalBHproducesamajordiversityofFAsisomersthanLAruminalBH.Thus,regardlessoflipidsupplementcomposition,LNtends toescapemoreeasily thanLNfromruminalBH. In addition, if lipid supplements are rich inLA, the proportion of LA that escapes from ruminal BH increases.This informationmaybe useful fordesigning lipid supplements directed to increase LA andLNconcentrationinruminantmilkandfat.

FactorsaffectingruminalVAconcentrationhavebeen extensively studied (Jenkins et al., 2008; Hur et al.,2017),consideringthatthemajorityofCLAinruminant milk and meat derives from desaturation of VA by stearoyl-CoA desaturase in mammary gland andtissues(Kayet al.,2004;Rezamandet al.,2014).Our data revealed that partial substitution of LA by LNdecreased theVAconcentration, and increasedSA production. Additionally, partial substitution of LAbyLNdecreased the r-VA. It suggests that anincrease in LA concentration in lipid supplements could decrease the conversion from VA to SA (a commonstepofLAandLNruminalBH),increasingVAruminalaccumulationwiththerespectivedecreasein SA ruminal production. These results are in

agreementwithRibeiroet al.(2007),whofoundthattheBHrateofLAistwiceasfastastheBHrateofLN,consequentlyproducingmoreVAfromLAthanfromLN.Thus,supplementsrichinLAmaybemoredesirablethansupplementsrichinLNforenhancingCLA concentration in ruminant milk and meat.

We observed that LA and LN concentrationsdecreasedfrom2to4h,and,unexpectedly,decreasedfrom 4 to 6 h. Concomitantly, SA concentrationdecreasedfrom2to4h,and,unexpectedlyincreasedfrom4 to6h.Similar changes inLA,LN,andSAconcentrations at the same hours have been reported in other in vitro studies (Jouany et al.,2007;Sterket al., 2010), but no explanation has been proposed. Since thebestmulti-compartmentalmodel(i.e.,modelwiththe lowestAIC;datanot shown)wasobtainedwhenconversionfromSAtoOAwasincluded,andtheLA:LNratioinmixturesaffectedtherateoftransferfromSAtoOA,wesuggestthatdesaturationfromSAtoOAmayexplaintheincreaseinLAandLN,andthedecreasein SA concentration between the aforementionedincubation times. Rumen conditions does not favor FAs desaturation (Van Soest et al.,1994).However,ithasbeen observed that fungi (i.e., Piromyces communis) areabletodesaturateSAtoOA,andformCLA(Kempet al., 1984;Ferlayet al.,2017).Therefore,consideringthat FAs concentration patterns and mathematical models have biological coherence, these results may constitute evidence that ruminal microorganisms are capable of desaturate FAs, suggesting that additional studies are neededtoidentifyspecificmicrobialspeciesinvolvedin FAs ruminal desaturation.

PreviousstudieshaveidentifiedtheFAsintermediatesandproductsofLAandLNruminalBH,modellingtheirBH kinetics (Jenkins et al., 2008; Hur et al.,2017).However,nopreviousstudieshaveexploredtheeffectsofdifferentLA:LNratiosinlipidsupplementsontheLAandLN ruminalBHkineticsutilizing amulti-compartmentalmodel.Ourdata showed thatpartialsubstitutionofLAbyLNincreasedtheratesoftransferfrom LA to CLA, from CLA to VA, and from CLA to sum18:1.Onthecontrary,therateoftransferfromLNtoTAslightly increased,and fromTAtoVAdidnotincreaseasLNincreasedinthemixtures.Inthissense,the increaseofLN enhancedBHofLA; however,increase of LA did not enhanceBH of LN.ThissuggestthatLAandLNmayinteractduringtheirBH,

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whichisinaccordancewithTroegeler-Meynadieret al. (2003), and Jouany et al.(2007),whosuggestedthatLAandLNshare linoleate isomeraseenzyme (EC5.2.1.5)inthefirststepofLAandLNruminalBH.Thus,notonlyLAandLNconcentrationbutalsotheirproportioninlipidsupplementcouldaffecttheratesoftransferofprincipalstepsofLAandLNruminalBH.ConsideringthattransferefficiencyofFAsfromrumen to milk and ruminant meat is limited by ruminal BH (Schmidely et al.,2017),thekineticparametersderived from this study could be incorporated into mathematicalmodels to explore theeffectsofFAscomposition in lipid supplements on milk and meat FAsconcentrations.Thisinformationcouldbeusefulfor designing nutritional strategies to enhance the qualityofruminant-derivedproducts.

Inthisstudy,weexploredforthefirsttime,theuseof computational chemistry to evaluate the spontaneity of the reactions duringLA andLN ruminalBH.Ourdata showed that conversion fromCLA toVAismore spontaneous than that fromTA toVA.Thethermodynamic responsesof these reactionswere inaccordancewith their respectivekinetics responses,because the ratesof transfer fromCLA toVAweregreater than the rates of transfer fromTA toVA,irrespectiveof theLA:LNratioused.Also,ourdatashowedthatconversionfromVAtoSAmaypresentlowspontaneityunderruminalconditions.ThisexplainswhyVA tends to accumulate in the rumen, as revealed by the presentstudy,aswellasinotherstudiesexploringtheeffectsof lipidsupplementationonVAconcentration(Sterk et al., 2010; Prieto et al.,2013).Thus,kineticandthermodynamicapproacheswerebiologicallycoherent,suggesting that thermodynamics could be useful for interpretingthekineticsofLAandLNruminalBH.

Insummary,thisstudyindicatesthattheLA:LNratiocanmodulatethekineticsofLAandLNruminalBHaffectingVAproduction.Wedemonstratedthatthermodynamics contributes to the understanding of severalkineticsresponsesduringLAandLNruminalBH.Thus, an integrated overviewof kinetics andthermodynamicsofLAandLN ruminalBHcouldhelp to design future strategies for improving FAs composition of ruminant-derived food products.

Acknowledgements

TheauthorsthanktheBogotaResearchDivision(DIB)oftheNationalUniversityofColombia,andColciencias for funding this study,which derivedfrom Project “Efecto de la suplementación de vacas lactantes con fuentes ricas en ácidos grasos insaturados sobre el perfil de la leche, transcripción de enzimas lipogénicas, actividad SRBP1 y su relación con diferentes componentes del perfil de ácidos grasos en leche”(code:1115-452-21319).ThanksalsototheUniversityofAntioquia.

Conflicts of interest

The authors declare they have no conflicts ofinterestwith regard to the resultspresented in thisreport.

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