N-acetyl cysteine as a potential treatment for persistent

N-acetylcysteineasapotentialtreatmentforpersistentbreeding-inducedendometritisinthemare

by

MichelleDianeCaissie

AThesispresentedto

TheUniversityofGuelph

Inpartialfulfillmentofrequirementsforthedegreeof

DoctorofVeterinarySciencein

PopulationMedicine

Guelph,Ontario,Canada

©MichelleDianeCaissie,August,2019

ii

ABSTRACT

N-acetylcysteineasapotentialtreatmentforpersistentbreeding-inducedendometritisinthemare

MichelleCaissieAdvisor:UniversityofGuelph,2019Dr.TraceyChenier

Persistentbreeding-inducedendometritis(PBIE)isamajorcauseofinfertilityinmares.

PBIEsusceptiblemaresdonotclearinflammationpost-breedinginatimelyfashion,ultimately

resultinginearlyembryonicdeath.N-acetylcysteine(NAC),amucolytic,hasanti-inflammatory

properties, affects inflammatory cytokines and is a mild inhibitor of nitric oxide synthase.

Increased nitric oxide (NO) levels and alterations in inflammatory cytokines have been

documented inPBIEmares.Theobjectiveofthisstudywastodetermine ifNACtreatmentof

PBIE-susceptiblemareswouldlowerNOandinflammatorycytokinelevels,therebyresultingin

the resolution of clinical signs associatedwith PBIE. The effects of NAC histologically on the

endometriumwerealsodetermined.Arandomized,blinded,cross-overdesignclinicaltrialwas

performedutilizingPBIEsusceptiblemares(n=9). Intrauterineinfusionof180mlsof3.3%NAC

wasperformed12hourspriortoinsemination.Maresweresampledforendometrialcytology

and intra-uterine fluid to determine interleukin-6 concentration (ELISA) and nitric oxide

(Colorimetric assay) levels at 12 and 60 hours post-insemination. Endometrial biopsies were

takenatthesametimepointspost-inseminationforscoringbasedonthedegreeofinflammation

Michelle Caissie

iii

presentandtodeterminegeneexpressionofinflammatorycytokinesbyqPCR.Clinicalsignsof

endometrialedemaandintra-uterinefluidvolumeswereassessedat12andthenevery24hours

postbreeding.StatisticalanalysiswasperformedusingarepeatedmeasuresANOVAandaMann

WhitneyWilcoxonTest.Pre-breedingintrauterinetreatmentwithNACdidnotimproveclinical

signs in PBIE-affectedmares, nor did it affectNO levels, IL-6 concentration or cytokine gene

expression.Differencesacrosstimepointspost-AIwerenotedforIL-6geneexpression,which

was highest at 12 hours post-AI (p=0.003). Expression of other cytokines evaluated did not

changeovertime.TreatmentwithNACdidhaveasignificanteffectonthedegreeofendometrial

inflammation noted histologically, with treated cycles displaying more diffuse and severe

neutrophil infiltrationcomparedtocontrolcycles.TheeffectsofNAContheendometriumof

PBIE-susceptiblemaresseeninthisstudyisconcerning.Furtherresearchisrequiredtoevaluate

thesafetyofNACinthetreatmentofPBIE-susceptiblemares.

Michelle Caissie

iv

ACKNOWLEDGEMENTS

Firstly, I would like to thankmy supervisor, Dr. Tracey Chenier for her guidance and

endless support throughout theDVScprogram.Researchwasanewerconcept tomewhen I

began,butDr.ChenierprovidedmewitheverythingIneededtocompletethisproject.Starting

withthebasics,progressingthroughtothemoredifficultelements,shehelpedmetogrowasa

researcher.Shewasconsistentlyavailableforfeedbackandassistance,evenlateatnightoron

weekends.Shehadauniqueabilitytocreateopenandconstructivebrainstormingsessionswhile

makingmefeelcomfortabletoaskquestions,howeversimpletheymayhavebeen.Whenthe

programbecameoverwhelming,sheknewexactlywhattosaytopushmeoutsideofmycomfort

zoneandtomovemyprogressforward.Dr.Chenierwasnotonlyagreatmentorforresearch,

shealsowasagreatmentorasaclinicianandasateacher.Duringmyfirstseasonasaresident

inTheriogenology,Dr.ChenierallowedmetogrowasaclinicianasIlearnedtomanagemyown

researchmares,whileprovidingmewith theguidanceand support I needed. Having sucha

knowledgeableandskilledcliniciantolearnfromwasaninvaluableopportunityforme,andone

thatIwillbeforevergratefulfor.Ithoroughlyenjoyedworkingwithher,bothintheclinic,and

onthisresearchproject.

Thepastthreeyearscouldalsonothavebeenpossiblewithoutmyothermentor,Dr.

CathyGartley.Dr.Gartleyhasbeenthereforme,providingvaluableinsightoneverythingfrom

tohowtodealwiththemostdifficultofcases,tofeedbackregardingmyresearchand,even

adviceonlifeoutsidetheclinic.Shehasbeenencouraging,approachable,andextremely

knowledgeable.Iwasalwaysexcitedtoseecaseswithher,notonlyforthelearning

v

opportunitiesbutalsoforthebyproductsofconversationandlaughterthatwouldemergefrom

spendingtimetogetherintheclinic.Dr.Gartleyalsotookthetimetodiscussvariousconditions

withmeoutsidetheclassroomandagreedtocoordinateaspecialtopicscoursetohelpprepare

meforboards.Icannotexpressmygratitudeforherdedicationtoteaching.

InadditiontothementorshipthatbothDr.ChenierandDr.Gartleyhaveprovidedme,I

willforevercherishtheirfriendship.

Totheothermembersofmythesiscommittee,Dr.ElizabethScholtz,Dr.JoanneHewson

andDr.RonJohnson,Ioweagreatdealofthanks.Fromtheverybeginningofthisprogram,they

wereencouraging,enthusiastic,insightfulandprovidedmewithinvaluableguidancethroughthe

completionofthisresearchproject.Iamespeciallygratefultothemforhelpingmeredesignmy

researchprojectandforalltheirhardworkduringtheeditingphaseofthisthesis.

ManythanksgotoDr.MonicaAntenosforteachingmeeverythingthereistoknowabout

pipettingandrunningELISAs.Iwouldnothavebeenabletocompletethissectionofmyresearch

projectwithout her. I thoroughly valued her teaching, knowledge, opinion, and patience. I

enjoyedmyexperienceworkingwithherandamforeverthankfulforherhelp.Aspecialthank

youtoDr.AllisonMacKayandDr.LizSt.Johnfortheirencouragementandhelpwhileworkingin

thelabaswell.

IowemygratitudetoDr.DorotheeBienzleandDr.MaryEllenClarkforperformingthe

qPCR analysis for this project. Special thanks goes to Dr. Rob Foster for his insight on the

endometrialbiopsyanalysis,forbeingonmyqualifyinganddefenseexaminationcommittee,and

forallowingmeattendhisgraduatereproductivepathologycourse.Also,thankyoutoDr.Paula

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MenziesandDr.TerriO’Sullivanforteachingmeaboutsmallruminantsandswinereproduction.

MygratitudetoWilliamSearsforrunningandbeingpatientduringthestatisticalanalysis.

IamgratefultoKarenDaPonteasafriend,ateacher,andatherapist.Shehasbeena

wealthofknowledgethroughoutmyDVSc.Karenhasbeenapillarofsupportthroughdifficult

cases,challengesoftheprogram,andinlife.Shewillalwaysholdaspecialplaceinmyheart,and

willforeverbemyfriend.ToSarahDyck,athankyouforbeingafantasticresearchassistantand

friendduringtheclinicalphaseofmyresearchproject.Iwouldhavenotbeenabletogetover

thelearningcurvewithouther,andforthatIamgrateful.ThankyoualsotothestaffattheArkell

Research Facility, most notably Danielle Watson, for their help with the mares during this

researchproject.AspecialthankstoStephanieBourgonforhelpingmewithmyELISAs,andto

PaigeVeyseyforhelpingmewithmyresearchandforbeingagoodresidentmate.

IwouldalsoliketothankDr.FirdousKhanforallhishelpatthebeginningofmyprogram,

andforprovidingmewithinvaluableinsightonmyresearchproject.Iappreciatehis,Dr.Mariana

DieldeAmorimandDr.RasaLevstein’sencouragementandguidancethroughoutmypreparation

fortheboardexam.

This project would not have been possible without the generous contributions from

EquineGuelphandtheOntarioMinistryofAgriculture,FoodandRuralAffairs(OMAFRA)andto

themIowemanythanks.

Finally,Iwouldliketothankmyfamilyfortheirendlesssupport.Therearenotenough

words to express how much they mean to me. To Blaire, my partner, who provided

encouragementwhenthingsgotdifficultandwhocelebratedsuccesseswithme.Thepastthree

yearshavenotbeeneasyforhimeither,buthislove,patienceandsupportneverwavered.Not

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onlydidhelivethroughthedaytodaywithme,healsohelpedmemasterMicrosoftWordand

Excelandhelpedmewithmywork,sowecouldspendtimetogether.Tomyparents,Daveand

Diane,theyhavebeentherefromthebeginning:fromencouragingmetofollowmydreams,to

talkingmethroughpanickingaboutanundergraduatephysics100course,totalkingmethrough

thefinalweeksofmyDVScandtellingmetonevergiveup.Iamespeciallyappreciativetomy

dad,whoremindedmetotakethingsdaybyday,andtomymomforalwayslookingoutformy

well-being,andtellingmeit’sokaytotakeabreak.Tomysiblings,DanielleandMatt,mybrother-

in-lawRick,andmynieceandnephews,Spencer,HaileyandKoen,thankyouforunderstanding

thatIcouldnotcomehometospendasmuchtimewithyouasIwanted.Tomypartner’sfamily,

theMacKenzies,Bill,Heather,Laura,Peter,Candace,EmmaandGrace,thankyouforbeinga

greatsupportsystemandforgivingmeahomeawayfromhome.Lastly,Iamthankfulformy

furryfriends,CooperandOreowhohavebeenmyconstantstudycompanions.

viii

DECLARATIONOFWORKPERFORMED

IdeclarethatIperformedalltheworkdescribedinthisthesiswiththeexceptionofthefollowing:

BacterialcultureofendometrialswabswasperformedattheAnimalHealthLaboratoryatthe

UniversityofGuelph.

Real-time quantitative PCR on endometrial tissue was performed by Dr. Bienzle’s research

laboratoryattheUniversityofGuelphintheDepartmentofPathobiology.

The preparation and staining with Hematoxylin and Eosin (H&E) of endometrial tissues for

histopathologicalevaluationwasperformedbytheAnimalHealthLaboratoryattheUniversityof

Guelph.

Histological evaluation and scoring of inflammation for these sampleswas performed byDr.

TraceyChenierintheDepartmentofPopulationMedicine,withtheinputofDr.RobFosterinthe

DepartmentofPathobiologyattheUniversityofGuelph.

ix

TABLEOFCONTENTSABSTRACT......................................................................................................................................ii

ACKNOWLEDGEMENTS.................................................................................................................iv

DECLARATIONOFWORKPERFORMED......................................................................................viii

LISTOFFIGURES...........................................................................................................................xii

LISTOFTABLES............................................................................................................................xiv

LISTOFAPPENDICES....................................................................................................................xv

LISTOFABBREVIATIONS.............................................................................................................xvii

CHAPTERONE:LITERATUREREVIEW............................................................................................1

1.1INTRODUCTIONANDSIGNIFICANCEOFPERSISTENTBREEDINGINDUCEDENDOMETRITITS(PBIE)..............................................................................................................1

1.2THEIMMUNESYSTEM&THENORMALINFLAMMATORYRESPONSE...............................2

1.3RISKFACTORSFORPBIE......................................................................................................7

1.4PATHOPHYSIOLOGYOFPBIE.............................................................................................11

1.4.1HumoralandInnateImmunity...................................................................................11

1.4.2UterineClearance.......................................................................................................15

1.4.3MucociliaryApparatusandLymphatics.....................................................................19

1.4.4CytokineProfilesofSusceptibleandResistantMares..............................................21

1.5DIAGNOSISOFPBIE...........................................................................................................23

1.6TREATMENTOFPBIE..........................................................................................................25

1.6.1EcbolicTherapy...........................................................................................................25

1.6.2UterineLavage............................................................................................................27

1.6.3Immunomodulation....................................................................................................28

1.6.4N-AcetylCysteine........................................................................................................32

1.7RESEARCHOBJECTIVES......................................................................................................35

1.8REFERENCES.......................................................................................................................36

CHAPTERTWO:THEEFFECTSOFTREATMENTWITHN-ACETYLCYSTEINEONCLINICALSIGNSINPBIESUSCEPTIBLEMARES...........................................................................................................57

2.1ABSTRACT..........................................................................................................................57

2.2INTRODUCTION..................................................................................................................58

2.3MATERIALSANDMETHODS..............................................................................................61

2.3.1SelectionofPBIESusceptibleMares..........................................................................62

x

2.3.2ClinicalTrial.................................................................................................................62

2.3.3InflammationasAssessedbyEndometrialBiopsies..................................................64

2.3.4EndometrialCytology(%Neutrophils).......................................................................65

2.3.5StatisticalAnalyses.....................................................................................................65

2.4RESULTS.............................................................................................................................66

2.4.1BiopsyInflammationScore........................................................................................66

2.4.2EstimatedIntra-UterineFluidVolumes(IUF).............................................................67

2.4.3EndometrialEdema....................................................................................................68

2.4.4EndometrialCytology(%Neutrophils).......................................................................68

2.4.5InteractionsBetweenTreatmentCyclesandHoursPost-AI(Appendix1)...............68

2.5DISCUSSION.......................................................................................................................68

2.6REFERENCES.......................................................................................................................76

CHAPTERTHREE:THEEFFECTOFTREATMENTOFPBIESUSCEPTIBLEMARESWITHN-ACETYLCYSTEINEONNITRICOXIDELEVELSANDINFLAMMATORYCYTOKINEPROFILES.....................93

3.1ABSTRACT..........................................................................................................................93

3.2INTRODUCTION..................................................................................................................94

3.3MATERIALSANDMETHODS..............................................................................................97

3.3.1MareSelection,StudyDesignandSampling.............................................................97

3.3.2Real-TimeQuantitativePCR.......................................................................................99

3.3.3Intra-UterineFluidCollection.....................................................................................99

3.3.4ELISADeterminationofInterleukin-6ConcentrationinUterineFluid....................100

3.3.5NitricOxideConcentration.......................................................................................100

3.3.6StatisticalAnalyses...................................................................................................101

3.4 RESULTS......................................................................................................................102

3.4.1EffectofIntra-UterineTreatmentof3.3%NACinPBIESusceptibleMares............102

3.4.2ChangesinEndometrialInflammationinResponsetoAIOverTime.....................102

3.4.3IL-6ConcentrationasMeasuredbyELISA................................................................103

3.4.4Nitricoxide................................................................................................................103

3.5DISCUSSION.....................................................................................................................103

3.6REFERENCES.....................................................................................................................110

CHAPTERFOUR:GENERALDISCUSSION....................................................................................122

4.1POSSIBLELINKSBETWEENMRNAEXPRESSIONANDPBIECLINICALSIGNS...................122

4.2LIMITATIONS....................................................................................................................124

xi

4.3OVERALLCONCLUSIONSANDFUTUREDIRECTIONS.......................................................127

4.4REFERENCES.....................................................................................................................132

APPENDICES...............................................................................................................................134

xii

LISTOFFIGURESCHAPTERONE

Figure1:Simplifiedflowchartdisplayingcytokineinteractions

CHAPTERTWO

Figure1:Graphicalrepresentationoftheclinicaltrialtimeline.

Figure2(a-d):PhotomicrographsofendometrialbiopsiesofPBIE-susceptiblemaresassignedan

inflammatorybiopsyscorerangingfrom1-4.

Figure3:DifferencesinmeanbiopsyinflammationscoreofPBIE-susceptiblemaresintreatedand

controlcycles.

Figure4:Photomicrographofendometrialbiopsydisplayingerosionoftheendometrialluminal

epitheliumfollowingintra-uterinetreatmentwith3.3%NAC.

Figure5:Photomicrographofendometrialbiopsydisplayingneutrophilinfiltrationofthestratum

spongiosumandglandularlumenfollowingintra-uterinetreatmentwith3.3%NAC.

Figure6:MeanbiopsyinflammationscoreofPBIE-susceptiblemaresat12and60hourspost-AI.

Figure7:MedianIUFofPBIE-susceptiblemaresintreatedandcontrolcycles.

Figure8:MedianIUFofPBIE-susceptiblemaresat12,36,60and84hourspost-AI.

Figure9:MeanendometrialedemascoreofPBIE-susceptiblemaresintreatedandcontrolcycles.

Figure10:MeanendometrialedemascoreofPBIE-susceptiblemaresat12,36,60and84hours

post-AI.

Figure11:Mean%neutrophilsofPBIE-susceptiblemaresintreatedandcontrolcycles.

Figure12:Mean%neutrophilsofPBIE-susceptiblemaresat12and60hourspost-AI.

xiii

CHAPTERTHREE

Figure1:MedianIL-6concentrationofPBIE-susceptiblemaresintreatedandcontrolcycles.

Figure2:MediannitricoxideofPBIE-susceptiblemaresintreatedandcontrolcyclespost-AI.

Figure3:MedianIL-6mRNAexpressionat12and60hourspost-AI.

Figure4:Median IL-6mRNAexpression ina subsetofPBIE-susceptiblemaresatbaseline,12

hoursand60hoursintreatedandcontrolcycles.

Figure5:MedianIL-1βmRNAexpressioninasubsetofPBIE-susceptiblemaresatbaseline,12


Figure6:MedianIL-10mRNAexpressioninasubsetofPBIE-susceptiblemaresatbaseline,12


Figure7:MedianTNF-αmRNAexpressioninasubsetofPBIE-susceptiblemaresatbaseline,12


Figure8:MedianIL-6concentrationofPBIE-susceptiblemaresat12and60hourspost-AI.

Figure9:MediannitricoxideofPBIE-susceptiblemaresat12and60hourspost-AI.

xiv

LISTOFTABLESCHAPTERONE

Table1:mRNAexpressionof select cytokines inequineendometritis literature,organizedby

study.

Table2:mRNAexpressionof select cytokines inequineendometritis literature,organizedby

cytokine.

CHAPTERTWO

Table1:InflammationbiopsyscoreassignmentforeachPBIE-susceptiblemareintreatedand

controlcyclesat12and60hourspost-AI.

CHAPTERTHREE

Table1:PairsofreferencegenesusedinclinicaltrialforeachPBIE-susceptiblemare.

Table2:Primersequencesforamplificationofreferencegenesandselectedcytokines.

Table3:MedianmRNAexpressionofIL-6,IL-10,TNF-αandIL-1βfortreatedandcontrolcycles

inPBIE-susceptiblemares.

Table4:MedianmRNAexpressionofIL-6,IL-10,TNF-αandIL-1βat12and60hourspost-AIin

PBIE-susceptiblemares.

Table5:MedianmRNAexpressionofIL-6,IL-10,TNF-αandIL-1βatbaseline,12and60hours

post-AIforasubsetofPBIE-susceptiblemares.

xv

LISTOFAPPENDICESCHAPTERTWO

Appendix1:Interactionsbetweentreatmentcyclesandhourspost-AIforoutcomesmeasured

inchapter2

Figure1:Meanbiopsyinflammationscoreintreatedandcontrolcyclesat12and60hours

postAI.

Figure2:MedianIUFintreatedandcontrolcyclesat12,36,60and84hourspostAI.

Figure3:Meanendometrialedemascoreintreatedandcontrolcyclesat12,36,60and84

hourspostAI.

Figure4:Mean%neutrophilsintreatedandcontrolcyclesat12and60hourspostAI.

CHAPTERTHREE

Appendix2:IL-6validationprocess

Table1:CalculatedvaluesofpercentrecoveryandlinearityforfinalIL-6validation.

Appendix3:Maineffectsoftreatmentandhourspost-AIoncytokinesevaluated

Figure1:MedianIL-6mRNAexpressioninPBIE-susceptiblemaresintreatedandcontrol

cycles.


cycles.

Figure3:MedianTNF-αmRNAexpressioninPBIE-susceptiblemaresintreatedandcontrol

cycles.

Figure4:MedianIL-1βmRNAexpressioninPBIE-susceptiblemaresintreatedandcontrol

cycles.

xvi


cyclesat12and60hourspost-AI.

Figure6:MedianIL-10mRNAexpressioninPBIE-susceptiblemaresat12and60hours

post-AI.

Figure7:MedianTNF-αmRNAexpressioninPBIE-susceptiblemaresat12and60hours

post-AI.

Figure8:MedianIL-1βmRNAexpressioninPBIE-susceptiblemaresat12and60hours

post-AI.

CHAPTERFOUR

Appendix2:IL-6validationprocess

Table1:CalculatedvaluesofpercentrecoveryandlinearityforfinalIL-6validation.

Appendix4:ProcessesforvalidationattemptsforIL-10,TNF-αandIL-1βELISAkits

xvii

LISTOFABBREVIATIONSAHL AnimalHealthLaboratory IgG ImmunoglobulinGANOVA AnalysisofVariance IgM ImmunoglobulinMAIC AkaikeInformationCriterion Ig(T) ImmunoglobulinTAI ArtificialInsemination iNOS InducibleNitricOxideSynthaseBSA BovineSerumAlbumin IFN-γ Interferon-γCDE ChronicDegenerative

EndometritisIL-1α Interleukin-1α

COPD ChronicObstructivePulmonaryDisease

IL-1RA Interleukin1ReceptorAgonist

CUI ChronicUterineInfection IL-1RN Interleukin1ReceptorAgonistGene

CD4+ ClusterofDifferentiation4 IL-6 Interleukin-6CD8+ ClusterofDifferentiation8 IL-8 Interleukin-8

C3 ComplementComponent3 IL-10 Interleukin-10C3b ComplementComponent3b IUF Intra-UterineFluidVolumeC5 ComplementComponent5 KCI PotassiumchlorideC5a ComplementComponent5a LRS LactatedRinger’sSolution

C5b ComplementComponent5b LPS LipopolysaccharidescDNA ComplementaryDNA LL LowerConfidenceLimitCT CycleThreshold MHC MajorHistocompatibilityComplexCOX-2 Cyclooxygenase-2 mRNA MessengerRNACRISP-3 CysteineRichSecretory

Protein3mOsm Milliosmole

DNA Deoxyribonucleicacid MS MultipleSclerosis

DUC DelayedUterineClearance MCWE MycobacterialCellWallExtractEMG Electromyography NAC N-acetylCysteineeNOS EndothelialNitricOxide

SynthaseNET NeutrophilExtracellularTrap

ELISA Enzyme-LinkedImmunosorbentAssay

nNOS NeuronalNitricOxideSynthase

EeF2 EukaryoticTranslationElongationFactor2

NO NitricOxide

FBS FetalBovineSerum NOS NitricOxideSynthase

GAPDH Glyceraldehyde3-phosphatedehydrogenase

NF-κB NuclearFactor-κB

H&E HematoxylinandEosin NOD NucleotideoligomerizationdomainhCG HumanChorionic

GonadotropinPBIE PersistentBreedingInduced

EndometritisHpf HighPowerField(400x) PBS PhosphateBufferedSaline

xviii

PRP PlateletRichPlasma TEM TransmissionElectronMicroscopyPMN Polymorphonuclear

neutrophilsTX TreatmentCycle

PGF2α ProstaglandinF2αTNF-α TumorNecrosisFactor-α

PGFM ProstaglandinF2αmetabolite SEM ScanningElectronMicroscopyRQ RelativeQuantification SE StandardErrorqPCR(qRT-PCR)

ReverseTranscriptaseQuantitativePCR

SDHA SuccinateDehydrogenaseComplexFlavoproteinSubunitA

RNA RibonucleicAcid UL UpperConfidenceLimitRPS23 RibosomalProteinS23

1

CHAPTERONE:LITERATUREREVIEW

1.1INTRODUCTIONANDSIGNIFICANCEOFPERSISTENTBREEDINGINDUCED

ENDOMETRITITS(PBIE)

In2010,horsescontributed19.6billiondollarstotheCanadianeconomy(Evans2012).At

thattime,thehorsepopulationinCanadawas963500,ofwhich41%werepartofthebreeding

sector.Twenty-twopercentofthenationalequinepopulationresidesinOntario,representing

thesecondlargesthorsepopulationwithinCanada,followingAlberta.InOntario,14%ofhorses

arewithin thebreedingsector (Evans2012).Thehealthof thebreeding industry is therefore

criticaltotheoverallsuccessofthehorseindustry.Despitethis,reproductiveefficiencyinhorses

is generally low, for a variety of reasons, including infertility caused by uterine disease.

Endometritis is the third most common medical condition encountered overall in equine

veterinary practice (Traub-Dargatzet al. 1991), and approximately 60%ofmares affectedby

endometritisarebarren(Overbecketal.2011).Causesofendometritisincludeacutebacterial

infections,chronic infections,andpersistentbreeding-induced inflammation.Estimatesof the

prevalenceofPBIEvarydependinguponthestudyandthemethodusedtodefinethecondition.

Whendefinedas“anyfluidaccumulationwithintheuterusonedayfollowingbreeding”,PBIE

was present in 13.7% of intensively managed Thoroughbred broodmares (Zent et al. 1998).

AnotherstudyidentifiedPBIEin43%ofamixedpopulationofmares(Newcombe1997).Given

thefairlylargenumberofhorsesinthebreedingsectorbothinOntarioandCanadaasawhole,

endometritis and more specifically PBIE, causes a significant economic impact to both the

CanadianandOntariobreedingindustries.

2

Maresaffectedbyendometritishaveimpairedfertility,leadingtodecreasedpregnancy

rates and the requirement for repeat breedings. Endometritis impairs fertility by two

mechanisms:prematureluteolysiscausedbyinflammatory-drivenprostaglandinrelease,andby

thedirect toxiceffectsofuterine inflammationon theembryo (Neelyetal. 1979;Troedsson

1997;Troedssonetal.2001b).Ashort-livedinflammatoryreactionisanormalresponsefollowing

breedinginallmares(Kotilainenetal.1994;Katilaetal.1995)andisnecessarysincenon-viable

spermatozoa,inflammatorydebrisandothercomponentsoftheejaculatemustbeclearedfrom

thefemale’sreproductivetractpriortoembryodescentonday5or6post-breeding(Betteridge

et al. 1982; Freeman et al. 1992). A subset of mares fail to clear the normal inflammatory

responseinatimelyfashion,leadingtopersistentinflammationandearlyembryonicdeathat

the time of embryo descent.Mares inwhich this occurs are considered susceptible to PBIE.

Understanding this post-breeding immune response and susceptibility to PBIE is key to

understandingmethodsthatmayreducetheimpactofPBIEonmarereproductiveperformance.

1.2THEIMMUNESYSTEM&THENORMALINFLAMMATORYRESPONSE

Theimmunesystemisdividedintotwobroadcategories:theinnateimmunesystemand

theadaptiveimmunesystem.Responsebytheinnateimmunesystemdoesnotrequireprevious

exposuretoanantigen;thesameresponsewilloccurregardlessoftheincitingcause.Theinnate

immune system consists of physical barriers, phagocytic cells and plasma proteins. By

comparison,theadaptiveimmunesystemhasamemorycomponent,whichprimesitsresponse

tospecificagentssuchasacertainspeciesofbacteriaorvirus,followinginitialexposure(Tizard

2013).Adaptiveimmunityisfurtherdividedintohumoralandcell-mediatedimmunity,wherethe

3

formeristargetedagainstextracellularpathogensandthelatteristargetedagainstintracellular

pathogens.Intheuterus,theinnateimmunesystemplaysthemostimportantroleinthedefense

againstequineendometritis (Snyder2017). This canbeexplainedby the fact that the innate

immuneresponseallowsforamorerapidresponsethantheadaptiveimmuneresponse.While

the adaptive immune response does play a small role in uterine defense, it requires diligent

regulationsincetheformationofantibodiestowardsantigenicmaterial,inthisinstancesemen,

wouldbedisastrousforfuturefertility(Troedsson2006;Lyle2011;Foster2017).

Researchersfirstbelievedthatendometritiswasduetobacterialcontaminationatthe

timeofbreeding(PycockandAllen1990;Asburyetal.1980;Mitchelletal.1982;Liuetal.1985;

Watsonetal.1987a;Watsonetal.1988;Williamsonetal.1987;Troedsson1991).Pycocketal.

(1990)characterized the inflammatory response inmares followingbacterial inoculationwith

Streptococcuszooepidemicus.Amassiveinfluxofneutrophilsintotheuterinelumenwithin30

minutes of inoculation occurred in all mares. Since that study, the inflammatory pathway

following bacterial inoculation has been further elucidated. An increase in arachidonic acid

metabolitesleadstoincreasedvascularpermeability,causingneutrophilinfiltration(Watsonet

al.1987b;LeBlancandCausey2009).ProstaglandinE2, leukotrieneB4aswellasother serum

proteins arepresentwithin theuterine lumenat this time andmay act as chemoattractants

(Watsonetal.1987a;Watsonetal.1987c;Watsonetal.1988;Pycocketal.1990).Antibodies,

suchasIgGorcomplement(C3)thenattachtothesurfaceofthebacteria,providingbindingsites

for neutrophil attachment, which allows opsonisation to occur. This leads to bacterial

phagocytosisbytheneutrophils.Complement (C3)and IgG levelsdecreasewithinthefirst24

hours, which is likely a result of increased consumption during the inflammatory response

4

(Troedsson1991).Thisinitialresponseisfollowedbyasecondarypeakinneutrophilnumbers

twelve hours following inoculation. This secondary peak is without a concurrent increase in

serumproteins,butinsteadisaccompaniedbyanincreaseinlocalimmunoglobulinsecretions

(IgA) (Williamson et al. 1987). Immunoglobulins identified in the uterus of mares following

bacterialinoculationconsistofIgA,IgG,IgG(T)andIgM(KenneyandKahleel1975;Asburyetal.

1980),where IgA isproducedbytheendometriumandIgGpassivelydiffuses intotheuterine

lumenfromtheperipheralcirculation(Mitchelletal.1982;Liuetal.1985).Inflammatoryexudate

is removed within 48 hours following inoculation via uterine contractions caused by

prostaglandinF2αrelease,asevidencedbylackofintra-luminalfluidvisibleonultrasound.Excess

fluidisalsoremovedvialymphaticsfollowingclosureofthecervix.

The inflammatory response to deposition of semen in the uterus is believed to be

essentiallyidenticaltothatdescribedabove,andhasbeenbettercharacterized(Kotilainenetal.

1994; Katila et al. 1995). Spermatozoa cause post-breeding inflammation via neutrophil

chemotaxisandcomplementactivation(Troedsson1995).Morespecifically,oncespermenter

theuterus,theyactivatecomplementpresentwithinuterinesecretions.Thisleadstocleavage

ofC5factorintoC5aandC5b,whichcreatesachemotacticsignaltoneutrophils.Asinbacterial

endometritis, prostaglandins, leukotriene B4 and other serum proteins may also act as

chemoattractants(Watsonetal.1987a;Watsonetal.1987c;Watsonetal.1988;Pycocketal.

1990). Neutrophils enter the uterine lumen, become activated and bind to sperm via

complementC3bfactororcomplement-independentfactors,allowingforthe initiationofthe

inflammatoryresponse.Furthermore,neutrophilextracellulartraps(NETs)andtraditionalligand

binding may enhance this neutrophil attachment, ultimately leading to phagocytosis of the

5

spermatozoa by neutrophils (Dahms and Troedsson 2002; Alghamdi and Foster 2005).

Concurrently, prostaglandin F2α, a metabolite of arachidonic acid via the cyclooxygenase

pathway, is released within the uterine lumen, initiating uterine contractions to assist with

physical evacuation of fluid and debris (Troedsson et al. 1995). Activated neutrophils are

responsibleforprostaglandinrelease,andthisispartofthenormalresponsetobreedinginan

attempt to clear the uterus of physiologic inflammation prior to embryo descent (Troedsson

1991).

Theimmediatecontractileresponsefollowingbreedingismediatedviaadrenergicnerves

andlocallyviaprostaglandinsinseminalplasma(Baeetal.2001).Concurrentoxytocinrelease

furtherheightenstheresponseofthemyometrium.Alexanderetal.(1995)demonstratedthat

oxytocinreleaseoccursfollowinginteractionwithastallion,includingstallionvocalizationand

physicalcontact,aswellasfollowingartificialinsemination.Madilletal.(2000)showedthatsuch

breedingstimulialsocausesuterinecontractionsinteasedmares.Asmeasuredbymyometrial

electrodes and a Grass polygraph, these contractions were found to be almost immediate

following the stimuli, andalmost always coincidedwithoxytocin release (Madilletal. 2000).

Oxytocin is released from the pituitary gland and binds to endometrial receptors,which are

upregulatedwhileestrogenlevelsriseandprogesteronelevelsdecrease,ondays14-17ofthe

estruscycle(StullandEvans1986;Jonesetal.1991).Therefore,theresponsetooxytocinrelease

isgreatestduringestrus.Thisisalsotruewhenexogenousoxytocinisadministeredduringestrus

anddiestrus.Myometrialcontractionsaremorefrequentbutshortlivedinestruscomparedto

diestrus,likelyduetoanincreasednumberofreceptors(Troedssonetal.1995).Oxytocinbinding

toup-regulatedreceptorsleadstoprostaglandinsynthesisandcontributestoincreaseduterine

6

contractions(Sharpetal.1997).Inaddition,prostaglandinF2αisknowntoup-regulateoxytocin

receptors, creating a positive feedback loop. Interestingly, oxytocin can lead tomyometrial

contractionsviaactivationofdirectandindirectpathways.Whenactivationtakesplaceviathe

direct pathway, myometrial contractions occur regardless of prostaglandin levels; however,

prostaglandinlevelsmustbeelevatedformyometrialcontractionstotakeplaceviatheindirect

pathway. This was demonstrated through the administration of phenylbutazone to block

prostaglandin synthesis. Normal mares treated with oxytocin and phenylbutazone did have

myometrial contractions but these were without concurrent prostaglandin F2α metabolite

(PGFM)plasmaelevations.Additionally,whenthesemaresweretreatedwithphenylbutazone

only,theyshoweddecreaseduterineclearance(Cadarioetal.1995).Therefore,phenylbutazone

blocksprostaglandin-mediateduterinecontractions,butdoesnothaveaneffectonoxytocin-

mediatedclearance.Conflictingresultswerenotedbythesameresearchersin1999,whenPGFM

levelswereonlyelevatedinmareswithintra-uterinefluidfollowingtreatmentwithoxytocin,but

notinnormalmares.Theseworkershypothesizedthatuterineinflammationprimedtheuterus

forPGF2αrelease(Cadarioetal.1999).

Regardless of themechanisms involved,myometrial contractions lead to clearance of

inflammatoryproductsfromtheuterus.Uterinecontractions,incombinationwithuterinefolds,

contributetoclearanceviathecervix.Inaddition,clearancevialymphaticsisdependentupon

myometrialcontractions(LeBlancetal.1995).Ifthepressurewithintheuterusisnotsufficient,

lymphaticdrainagecannotoccur,leadingtofluidcollectioninthelumeninsteadofitsremoval

viathelymphatics(Causey2006).Amucociliaryapparatusalsoexistswithintheuterus,where

7

mucusisproducedbyendometrialcellsandthenpropelledbycilia.This,combinedwithuterine

contractions,minimizesbacterialattachmentandmaximizesremovalofinflammatorycellsand

debris from the uterus (Causey 2007). The combination of innate immune response and

mechanicalclearanceresultintheinflammatoryresponsebeingshort-livedfollowingbreeding

innormalmares(Troedsson1999).

1.3RISKFACTORSFORPBIE

Factors associated with susceptibility to PBIE in mares include poor perineal

conformation,age,anatomiclocationoftheuterus,andfluidretentionduringdiestrus(Ricketts

andAlonso1991;Troedssonetal.1993a;LeBlancetal.1994a;LeBlancetal.1998;Brinksoetal.

2003; Woodward et al. 2012; Canisso et al. 2016). Reproductive anatomy is an important

componentofthemare’suterinedefensesystem.Thethreeanatomicalbarriersthatprotectthe

uterus fromexternal contamination are: apposition and vertical angulation of the vulvar lips

(perineal conformation), the vestibulo-vaginal sphincter and the cervix (Pascoe 2007; Pycock

2009;Bergfelt2009).Mareswithnormalperinealconformationhaveatleasttwo-thirdsofthe

vulvarlengthbelowthepelvicbrim,lessthan10-degreeanteriortilttothevulva,andvulvarlips

thatarewellapposedtopreventfecalcontaminationofthevagina(Pascoe2007;Pycock2009

McCue2008;Canissoetal.2016).Perinealconformationmaybealteredfromnormalinsome

instances.Forexample,leanathleticmareswithverylittleperinealfatmayhavearecessedanus

leadingtocranialanglingofthevulva,thereforepullingmorethanone-thirdofthevulvaabove

thepelvicbrim (Hurtgen2006;Pascoe2007). Similar changesmayoccur inoldermareswith

increasedparity as seenby lengtheningof the vulvaand increase in theangleofdeclination

8

(Pascoe1979).Thevestibulo-vaginalsphincterisformedbymucosalfoldsatthedemarcationof

thevestibulefromthevaginaattheleveloftheexternalurethralorifice(Bergfelt2009).Mares

with an incompetent vestibulo-vaginal sphincter aspirate air into the vagina (pneumovagina)

during exercise, or following urination, a term commonly referred to as “wind-sucking”.

Pneumovagina leads to irritation and inflammation of the vagina, vestibule and cervix,

predisposingthemaretosecondarybacterialinfectionandendometritis(Pascoe2007;McCue

2008;Canissoetal.2016).

Abnormalitiesaffectingthecervixincludefibrosis,tortuosityandincompetence.Cervical

malfunctioncanoccurinoldermaidenmares,mareswithrepeatedembryorecoveryattempts

or following abortion or dystocia-induced damage (Pycock 2007; McCue 2008; LeBlanc and

Causey 2009). An inability to dilate the cervix following breeding, as seenwith fibrosis post

dystociaorinoldermaidenmares,canleadtodecreaseduterineclearance(Pycock2007).Reilas

etal. (2016) showed thatocclusionof thecervixbywayofacatheter for25hours following

artificialinsemination(AI)leadstoincreasedintra-uterinefluidvolumesandincreasedneutrophil

numberinnormalmares.Inaddition,theysawaworseningoffibrosisonendometrialbiopsies

instudymareswherefluidwasretainedforeither25hoursor24hourswithsomefluidremoved

at6hours,andspeculatedthatretentionof fluid intheuterusmay leadtoalterations inthe

inflammatoryresponse.

Other anatomical abnormalities include stretching of the broad ligamentwith cranial

displacementoftheuterusoverthepelvicbrim,whichleadstodecreaseduterineclearancedue

to simple gravitational effects (LeBlanc and Causey 2009). In fact, uterine clearance of a

radiocolloidasassessedbyscintigraphydemonstratedthatmareswithamoreventraluterine

9

cervicalanglecleared<30%of theradiocolloid intwohours,comparedtonormalmaresthat

cleared >50%. Mares with delayed clearance also tended to be over the age of 12, were

pluriparousandhadahistoryofendometritis(LeBlancetal.1998).Therefore,manyoftheserisk

factorsareaffectedbothbyageofthemareandparity.

Carnevale andGinther (1992) first showed thatolderponymares indiestrushad less

uterine contractility as assessed by trans-rectal ultrasound prior to and following breeding,

compared to youngermares under the same conditions. The oldermares also had a higher

incidenceofendometritis,asassessedbyendometrialbiopsy,andincreasedintra-uterinefluid.

Woodwardetal.(2012)examinedtheassociationbetweenmareage,uterinefluidretentionand

endometrial biopsy scores. In their study, mares over 9 years of age were more likely to

accumulatefluidthanyoungermaresandevenmoresowhenmareswereolderthan17years.

AhigherpercentageofoldermaresalsohadKenneybiopsyscoresofIIborIII.Ageistherefore

likelyassociatedwithamoresevereendometrialbiopsyscore.Ageandbiopsyscoreappearto

leadtoincreasedfluidretentionandPBIEsusceptibility.However,thisstudydidnotassessthe

effectsofparityasaconfoundingvariable,asthereproductivehistoryofthemaresinthisstudy

wasunknown.SimilarresultswerenotedformareageinastudybyBarbacinietal.(2003),where

theincidenceofuterinefluidaccumulationpost-breedingwashigherinmaresaged10-16years

oldcomparedtoyoungermares.Thisstudyalsorevealedthatbarrenmaresweremorelikelyto

demonstrateuterinefluidretentioncomparedtomaidenandpost-parturientmares.Increased

levels of uterine fluid (>2cm) post-AI were associatedwith lower pregnancy rates; per cycle

pregnancyrateswere41.9%inmareswithfluidretentioncomparedto56.2%inmareswithout

fluid. Clearly, pluriparous mares tend have an increased likelihood of abnormal perineal

10

conformation,anincompetentvestibulo-vaginalsphincterandafibroticcervix,allofwhichcan

contributetosusceptibilitytoPBIE(Hurtgen2006;LeBlancandCausey2009;Christoffersenand

Troedsson2017).

The association between uterine biopsy grade and susceptibility to chronic uterine

infections(CUI)wasassessedbyTroedssonetal.(1993a).Inthisstudy,maresweredividedinto

four categories based on severity of histologic findings (1=normal to 4=severe) and were

challengedwith a bacterial inoculation of Streptococcus zooepidemicus during estrus.Mares

were considered susceptible to CUI if endometrial cytology revealed >1 neutrophil/5hpf and

endometrialculturehad>20bacterialcoloniesat96hourspostchallenge.MareswithgradeI

endometrialbiopsiesweremorelikelytoberesistanttotheinoculation,whereasmareswitha

biopsy score of grade IIIweremore likely to be susceptible to CUI, indicating an association

betweenseverityofendometrialbiopsyscoreandsusceptibilitytoCUI.However,therewasno

associationbetweenbiopsygradesIIaorIIbandsusceptibility,limitingtheapplicationofbiopsy

gradetopredictsusceptibility.InasimilarstudybyWilliamsonetal.(1989),mareswereclassified

assusceptibleorresistanttoCUIbasedonreproductivehistoryandendometrialbiopsyscores.

Followinginoculation,mareswerere-classifiedbasedontheirabilitytoclearaninoculationof

Streptococcus zooepidemicus after 10 days. Susceptibility was more highly correlated with

reproductivehistorythanendometrialbiopsy.However,astudydesignlimitationexistedinthat

mares were only classified as susceptible ten days following bacterial inoculation, allowing

considerable time for less susceptiblemares toclear the infectionprior to thesampling time

point.Inaddition,biopsyscoreswerenotdefinedusingthecurrentendometrialbiopsygrading

11

system described by Kenney and Doig (1986), limiting the capability to compare with other

studies.

1.4PATHOPHYSIOLOGYOFPBIE

1.4.1HumoralandInnateImmunity

Muchresearchhas focusedonmaresusceptibilityversus resistance toacutebacterial

endometritisandPBIE.Possibledifferencesbetweenmareswithrespecttotheirabilitytoclear

intra-uterineinfectionwerefirstdescribedbyHughesandLoy(1969)andPetersonetal.(1969),

where some mares appeared to be resistant to infections, while others did not. The initial

inflammatoryreactionappearedtobethesameamongstthesemares,butdifferedwithtimein

thatsomemares(susceptible)maintainedthe infection,andothers (resistant)cleared it.This

suggestedthatanalteredinflammatorydefensemechanismmaybeinvolved.Subsequentwork

focusedonpossibledifferencesinthehumoralimmuneresponse,specificallydifferinglevelsof

immunoglobulinsbetweensusceptibleandresistantmares.Asburyetal.(1980)foundincreased

intra-uterinelevelsofIgA,IgGandIgTinmaressusceptibletoendometritisfollowingbacterial

inoculationwithStreptococcuszooepidemicus,comparedtonormalmares.Inaddition,Mitchell

etal.(1982)notedhighertotalimmunoglobulin,IgAandIgGinsusceptiblemares,asdefinedby

endometrialbiopsies.Similarly,Williamsonetal. (1983)foundelevated levelsof IgA inmares

withactiveinfections,comparedtoresistantmares.Conversely,Troedssonetal.(1993c)found

nosignificantdifferencesbetweensusceptibleandresistantmares.ThisstudyexaminedIgA,IgG

andcomplementcleavagefactorC3expressioninmaresthatwereeitherresistantorsusceptible

toabacterialinoculationofStreptococcuszooepidemicus.Inbothgroups,levelsofIgA,IgGand

12

C3decreasedinthefirst24hoursfollowinginoculation.LevelsofIgGandC3roseinresistant

mares by 36 hours, however they remained low in susceptiblemares, suggesting continued

bacterial opsonisation. No differences were detected in IgA levels between groups. The

susceptibilityofendometritiswasthusnotattributedtodeficienciesinimmunoglobulins.

Watson and Dixon (1993) showed upregulation of MHC Class II expression in the

endometrialepitheliumofsusceptiblemares,comparedtonormalmaresinestrus.Themajority

ofcellswithMHCClassIIexpressionconsistedofTlymphocytes,butalsoincludedsomeepithelial

cells. When assessed histologically, endometrial T lymphocytes were increased in non-bred

mares susceptible to endometritis, compared to resistantmares, both in estrus and diestrus

(Fumosoetal.2007).Followingartificialinsemination,PBIE-susceptiblemareshadhigherlevels

of T lymphocytes in diestrus only, compared to resistantmares (Fumosoet al. 2007). This is

interesting to the understanding of PBIE, since no differences in T lymphocyte numberwere

observedinnormalmaresregardlessofstageofcycle(WatsonandThomson1996).Tunonetal.

(1999)characterizedthetypeofTlymphocyteexpressioninnormalmares.TheyfoundthatCD4+

Tcellswereelevatedat6hoursfollowinginsemination,butnotat48hours.Nodifferencesin

CD8+Tcellswerenotedateithertimepoint.SusceptiblemaresalsohadmoreCD4+expression

thanCD8+expressionandmoreCD8+cellscomparedtoresistantmares(WatsonandThomson

1996),likelyduetopersistentantigenicstimulationinaffectedmares.

Alterations in uterineneutrophil chemotaxis, phagocytosis, andbactericidal activity in

susceptiblemareshavebeenotherareasofresearchinunderstandingPBIE.Decreasedopsonins

fromuterinesecretionsofmaressusceptibletobacterialendometritiswasnotedbyAsburyet

al.(1982).In1985,Liuetal.demonstratedthatuterinesecretionsofmaresinfusedwithbacterial

13

inoculum,withgradeIIIuterinebiopsiesandahistoryofuterineinfections,hadlessneutrophil

elasticity and chemotaxis compared tomareswith grade I biopsies and normal reproductive

histories.Theythenshowedthatneutrophilmigrationwasdelayedfollowing inoculationwith

Streptococcuszooepidemicusinsusceptiblemarescomparedtoresistantmares.Inafollow-up

study, Lui et al. (1986) found that neutrophil levels in susceptiblemareswere elevated and

remained as such from 15 to 25 hours post inoculation compared to resistant mares.

Interestingly,neutrophilmigrationwasdecreasedinsusceptiblemaresat20hours;however,by

25 hours, migration had resumed in these mares. Watson et al. (1987c) showed further

neutrophil dysfunction, in that uterine-derived neutrophils displayed decreased phagocytic

capabilities in susceptible mares compared to resistant mares pre-inoculation. Following

induction of inflammation with 1% oyster glycogen, susceptible mare neutrophils displayed

decreasedbactericidalactivitywhenincubatedfor24hourswithagenitalstrainofStreptococcus

zooepidemicus,asassessedbypercentsurvivalofbacteria.Themechanismsbywhichneutrophil

functioncouldbeaffectedinsusceptiblemareswereinvestigatedbyTroedssonetal.(1993d),

who compared phagocytic ability of neutrophils in susceptible and resistantmares following

bacterialinoculationinestruswithStreptococcuszooepidemicus.Theauthorsconcludedthatthe

decreasedphagocyticabilityofneutrophilsseeninsusceptiblemareswascausedbydifferences

inuterinesecretionsandnotbydysfunctionalityoftheneutrophilsthemselves.Thiswasmade

evident by the demonstration of adequate phagocytosis when neutrophils from susceptible

mares were placed in an optimal environment. In addition, susceptible mares in this study

actuallyhadimprovedchemotaxiscomparedtoresistantmares.ThisisincontrasttoLiuetal.

(1985) who did not see chemotaxis improvement when uterine lavage fluid containing

14

neutrophils taken from mares with grade III biopsies were placed in the presence of

chemoattractants. Troedson (1993d) suggested that differences in components of uterine

secretionscouldresultinvariationsinopsonisationbycomplementandantibodies(Troedsson

etal.1993d).Thiswassupportedbythedecreasedbactericidalactivityofneutrophilsseenin

vitrowhenfactorsrequiredfortheclassicalandalternatecomplementpathwayswereremoved

(Watsonetal.1988).Troedsson(1999)suggestedthatstationaryageduterinefluid,asisoften

the case with PBIE-susceptible mares, can lead to inactivation of complement proteins,

decreasingneutrophilphagocytic success. Furthermore,neutrophilsmusthavea surface (the

endometrium)onwhichtoperformphagocytosis,andthissurfacemaynotbeavailablewhen

there is intra-luminal fluid present (Troedsson 1999). In summary, susceptible mares have

functional neutrophils and the same rate of phagocytosis as resistant mares; however, the

functionalandphagocyticabilitiesofthoseneutrophilsappeartobereducedwhenexposedto

the altered uterine fluid of PBIE-susceptible mares. Susceptible mares also have increased

numbers of neutrophils for prolonged periods of time following induction of inflammation,

comparedtoresistantmares(Liuetal.1986;Watsonetal.1987c;NikolakopoulosandWatson

1997;WoodwardandTroedsson2013).Therefore,theuterinefluidofsusceptiblemarescauses

alteredneutrophilfunctionality,whichcouldcontributetodifferencesinboththetimingoftheir

presencewithintheuterinelumenandtheirlevels.

Increased intra-uterine neutrophil number and altered uterine secretions can have a

directeffectonspermatozoa,furthercontributingtodecreasedfertility.Spermatozoaincubated

intheuterinesecretionsofinflameduteriinvitrodemonstratedalteredvelocityandprogressive

motility, which was attributed to the presence of neutrophils binding to the spermatozoa

15

(Alghamdietal.2001).Someoftheseeffectscouldbemediatedbythepresenceorabsenceof

seminal plasma,whose effects on fertility and the post-breeding inflammatory response are

controversial.Inlargeconcentrations,seminalplasmahasbeenshowntobedetrimentaltothe

qualityofthawedsemen(Morrelletal.2014);however,italsomayshortenthedurationofpost-

breedinginflammationwhenpresentinsmallervolumes(Troedssonetal.2000;Troedssonetal.

2001). Mares inseminated with sperm, milk extender and seminal plasma demonstrated

decreaseduterinecontractionsandincreasedintrauterineneutrophilconcentrations,compared

toAIwithspermandextenderalone.Thissuggestsbothanincreasedintensityanddurationof

inflammation post-breeding when seminal plasma is used (Portus et al. 2005). However,

Troedssonetal.(2000)showedthatseminalplasmadecreasesneutrophilphagocytosisofsperm

and Alghamdi et al. (2004) demonstrated that seminal plasma suppresses neutrophil sperm

bindinginadose-dependentmanner,invitro.Fifteen-daypregnancyrateswerealsohigherin

maresinseminatedwithspermatozoaresuspendedinseminalplasmasuspensionwithoutsemen

extender compared to spermatozoa resuspended in semen extender alone (Alghamdi et al.

2004). It has thus been suggested that seminal plasma or some of its components could be

beneficialinthemanagementofPBIEinsusceptiblemares(Troedssonetal.2000;Alghamdiet

al.2004).

1.4.2UterineClearance

Failure to clearuterine fluidpost-breedinghasbecome themost important andmost

commonlyuseddefinitionforPBIE inmares(Brinskoetal.2003). This inabilitytoclear intra-

uterinefluidischaracteristicofthecondition,andoccursduetodefectsinthenormaluterine

clearancemechanisms,whichvarybystageoftheestrouscycle.Evansetal.(1986)demonstrated

16

the influence of reproductive steroid environment on uterine clearance in anestrus maiden

maresusinguterineinfusionofStreptococcuszooepidemicus,chromium-labeledmicrospheres,

andcharcoal. Maresinthestudyweretreatedwitheitherestradiolorprogesteroneandwere

infused with Streptococcus zooepidemicus, chromium-labeled microspheres, and charcoal.

Estrogen-treatedmareshadrapiduterineclearanceofmicrospheresandbacteriaby3dayspost-

inoculation. By comparison, uterine clearance of progesterone-treatedmareswas poor and

produced persistent bacterial infections following inoculation, demonstrating the inhibitory

effectsofprogesteroneonuterineclearance.Subsequently,in1987hisgroupdemonstrateda

correlation between decreased uterine clearance and age, where youngmares cleared non-

antigenicmarkersmoreefficientlythanoldmareseveninanestrogen-dominatedenvironment

(Evansetal. 1987). Troedssonand Liu (1991)demonstrated thatmares resistant tobacterial

infections cleared a significantly greater amount of intra-uterine non-antigenic chromium-

labelled microspheres (51Cr) than susceptible mares. Mares were classified as potentially

susceptibleorresistanttouterineinfectionbasedonreproductivehistory,biopsyscoreandper

rectum examination. They were challenged with an intra-uterine infusion of Streptococcus

zooepidemicusandinoculatedwithmicrospheres.Resistantmareshadanegligiblenumberof

microspheresrecoveredbyuterinelavage24hourspostinoculationwhereasalargenumberof

microsphereswererecoveredinsusceptiblemaresat96hourspostinoculation.

Uterine contractility also appears to be different between susceptible and resistant

mares.Troedssonetal.(1993b)usedelectromyography(EMG)followingsurgicalimplantationof

myometrialelectrodestoassessmyometrialactivity.Resistantmaresshowedgreaterintensity,

durationandfrequencyofmyometrialcontractionscomparedtosusceptiblemares, following

17

intrauterine bacterial challenge. Furthermore, LeBlanc et al. (1994a) infused the uterus of

resistantandsusceptiblemareswitha radiocolloidandmonitoredclearancevia scintigraphy.

Resistantmaresclearedtheproductwithintwohoursof infusion,whereassusceptiblemares

displayeddelayedclearance.Thesamegroup (Neuwirthetal.1994) showed that susceptible

maresonlyclearedlessthan15%oftheradiocolloidwithintwohoursofinfusion,comparedto

over50%oftheproductclearedbythistimepointinresistantmares.Thesefindingscouldinpart

beexplainedbysusceptiblemarespossiblyhavinginherentlyabnormalpatternsofpropagation

of myometrial contractions, leading to retention of the fluid. Mares with delayed uterine

clearance (DUC) weremore likely to have an opposing pattern of propagation compared to

normalmares.Morespecifically,insteadoftheuterinecontractionscommencingintheuterine

hornsfollowedbytheuterinebody,contractionsinaffectedmareswereinitiatedintheuterine

bodyandthentheuterinehorns (vonReitzensteinetal.2001;LeBlancandCausey2009). In

addition, Rigby et al. in 2001 showed that susceptible mares may have decreased uterine

clearanceduetoan intrinsiccontractiledefectof themyometrium.Reproductive tractswere

removedfromyoungnulliparousmaresaswellasfromolderresistantandsusceptiblemares,

and both longitudinal and circular strips of myometrium were utilized in isometric tension

studies.StripswereexposedtoKCl,oxytocinandPGF2αandresponseswerecomparedbetween

thethreegroups.Resultsofthisin-vitrostudyshowedthatthemyometriumofsusceptiblemares

generatedlesstensionthanthatofbothyoungnulliparousandolderresistantmares;however,

theywerenotabletodeterminetheexactmechanismbywhichthisoccurred.

Differing levels of nitric oxide (NO) may further contribute to decreased myometrial

contractilityand thereforedecreaseduterine clearance (Alghamdietal. 2005).Nitricoxide is

18

formedfromL-arginineviathenitricoxidesynthase(NOS)enzyme.ThreeisoformsofNOShave

been identified and includeneuronalNOS (nNOS), endothelialNOS (eNOS) and inducibleNO

(iNOS).BothiNOSandeNOShavebeenidentifiedintheuterusandtheirpatternsofexpression

vary; eNOS is constitutively produced whereas iNOS is induced by inflammatory conditions

(Moncada and Higgs 1993). It is likely that the persistence of inflammation seen in PBIE

susceptiblemares leads to increased levels ofNO (Woodwardet al. 2013b).Nitric oxide is a

smooth muscle relaxant, and high levels could account for the diminished myometrial

contractility seen inPBIE-susceptiblemares. In supportof this theory, susceptiblemareshad

higherlevelsofNOintheiruterinesecretions13hoursfollowingartificialinsemination,aswell

ashigherNOSmRNAexpressionintheirbiopsies,comparedwithresistantmares(Alghamdiet

al.2005).Differencesinnitricoxidelevelsbetweensusceptibleandresistantmareswerefurther

classifiedbyWoodwardetal. (2013b).Specifically, total intrauterineNO insusceptiblemares

increasedatsixand12hourspost-AI,whereaslevelsremainedstableinresistantmaresover24

hours. Uterine iNOSmRNA expression was not different between susceptible and resistant

maresatanytimepoint.Khanetal.(2018)performedinvitroexperimentswherebymyometrium

obtainedfromsusceptiblemareswastreatedwithanitricoxidesynthaseinhibitortodetermine

itseffectsoncontractility.Resultsshowedthatmyometrialcontractilityimprovedwhentreated

withtheinducibleNOsynthaseinhibitor1,400Wdihydrochloride.

Additional mechanisms for the delayed uterine clearance in susceptible mares may

include abnormalities in the electrical pattern for contractility, smooth muscle fatigue, and

unsuitablehormonesignaling(LeBlancandCausey2009).MareswithGradeIIIbiopsyscoreshave

upto40%lessuterineperfusioncomparedtonormalmares,duetoendometrialvesselelastosis,

19

leading to decreased amounts of hormones, neurotransmitter and inflammatory mediators

deliveredtotheuterus(Esteller-Vicoetal.2007).In1999,NikolakopoulosandWatsonshowed

that susceptible and resistant mares have similar patterns of systemic oxytocin secretion

followingAI;however,susceptiblemareshavelowerlevelsofPGFMcomparedtoresistantmares

followingbreeding.Prostaglandinmetabolite(PGFM)isusedasamarkerforlevelsofPGF2α,and

decreasedlevelsareattributedtoapossibledefectinitsproductionorreleaseattheoxytocin

receptors.Asnotedabove,whenadministeredexogenousoxytocin,susceptiblemaresproduce

minimallevelsofPGFMcomparedtoresistantmares.Itisthereforepossiblethatdifferencesin

prostaglandinplayaroleintheintrinsicabnormalitiesinuterinecontractilityandthatthesemay

becausedbydecreaseduterineperfusion.

1.4.3MucociliaryApparatusandLymphatics

Somemares susceptible toPBIEmayhaveanalteredmucociliary apparatusandpoor

lymphaticdrainage.Freemanetal. (1990)showedthatmareswithurinepooling,asourceof

inflammation,havegreaterhistologicalstainingforcarboxylatedacidmucins intheuterus,as

comparedtonormalanestrusandtransitionalmares.Furthermore,Causeyetal.(2000)showed

thatmareswithDUChaveincreasedmucusproductioncomparedtonormalmares,asassessed

by endometrial biopsy staining. Increasedmucusproduction is believed tobe in response to

uterineinflammationorirritation(Freemanetal.1990).Thismucusalsotendstobelocalizedin

fibroticnestsandinareasof increasedinflammation.This is important,asmucusflowcanbe

affectedbyincreasesinvolumeandduetoanincreaseinviscosity.Dilutionofmucuswithintra-

luminalfluidcansimilarlyimpacttheeffectivenesswithwhichmucusisabletobindandmove

particles(Causey2007).Therefore,whileexcessmucuscanbedetrimental, insufficientmucus

20

allowsadhesionofsubstratestotheendometrialepithelium,leadingtodecreasedclearance.The

issueisfurthercompoundedasincreasedfluidvolumewithintheuterusleadstoseparationof

theendometrialfolds,whichnotonlyleadstoapoorenvironmentforneutrophilphagocytosis,

butalsoaffectsmucociliaryclearance.Causey(2006,2007)speculatedthatmucociliaryflowis

bestachievedwhentheendometrialwallsapposeeachotherclosely,andthattheciliabecome

ineffectivewhentheyarefurtherapartduetofluidaccumulations.Somemaressusceptibleto

endometritisdemonstrate lossofcilia fromtheendometrialepithelium, leadingtodecreased

movement of particles and fluid (Ferreira-Dias et al. 1994; Ferreira-Dias et al. 1999). More

specifically,mareswithgradeIIuterinebiopsiesdemonstratedpatchyareasofmicrovilliloss,and

mareswithgradeIIIuterinebiopsieshadareastotallydevoidofmicrovilluscellsandciliatedcells

onscanningelectronmicroscopy(SEM)andtransmissionelectronmicroscopy(TEM)(Ferreira-

Diasetal.1994;Ferreira-Diasetal.1999).Mareswithimpairedlymphatics,apendulousuterus,

or with impaired cervical drainage show increased fluid volumes within the uterus, further

contributingtotheproblem(LeBlancetal.1995).Whiletheexactmechanismsbywhichuterine

mucociliaryclearanceaffectsPBIEhavenotbeenelucidated,itisevidentthatitlikelycontributes

significantly.

Themovementofspermatozoatotheoviductfollowingbreedingiscausednotonlyby

uterine contractions, but also by endometrial epithelial ciliary flow (Troedsson et al. 2005).

Failureofciliarymovementcouldalsoaffectspermtransport,furthercontributingtodecreased

fertility.Infact,Causey(2006)statedthatspermtransportisalteredintheoviductsofsusceptible

mares,leadingtolowernumbersofspermatozoainthecaudalisthmus,anothermechanismby

whichfertilitymaybediminishedinsusceptiblemares.

21

1.4.4CytokineProfilesofSusceptibleandResistantMares

Cytokines are cell-derived polypeptides involved in inflammatory mediation, which

recently have been found to be different between PBIE susceptible and resistant mares.

Cytokinesareproducedatsitesofinflammationbystromal,endothelialandimmunecells,and

oncepresent,managetheinflammatoryresponse(FeghaliandWright1997;Tizard2013).They

can be categorized into pro-inflammatory cytokines, which as their name implies, promote

inflammation, and anti-inflammatory cytokines, which control inflammation and prevent its

persistence.Cytokinesmediatethetypeofinflammationthattakesplace,howlongitlastsand

whatother cellsmaybecome involved,byhavingbothagonisticandantagonist functionson

differenttypesofcells(FeghaliandWright1997).Someofthemostcommonlyidentifiedpro-

inflammatorycytokinesintheuterusincludethoseoftheinterleukin-1family(IL-1αandIL-1β),

interferon-γ(IFN-γ),tumornecrosisfactor-α(TNF-α)andinterleukin8(IL-8).IL-1αandIL-1βare

involved in therecruitmentofotherpro-inflammatorycytokines,suchasTNF-α. IFN-γaids in

neutrophilmigration from thevasculature into the siteof inflammation.TNF-α is involved in

prostaglandinproduction,apoptosisandaidsneutrophils,whichhavebeenrecruitedbyIL-8,in

their ability to neutralize pathogens (Tizard 2013). Anti-inflammatory cytokines such as

interleukin-10(IL-10)andinterleukin-1RA(IL-1RA),where interleukin-1RN(IL-1RN) isthegene

whichencodes for IL-1RA,are important for theregulationof inflammation. In fact, IL-10has

beenshowntoinhibittheproductionofIL-1αandIL-1β,IL-6,IL-8andTNF-αinhumans(Tizard

2013). Finally, interleukin-6 (IL-6) can act as both a pro-inflammatory and anti-inflammatory

cytokine (Tizard 2013) and canbe present in the uterus ofmares following an inflammatory

22

stimulus(Woodwardetal.2013a).Asimplifiedschematicofinteractionsbetweencytokinescan

befoundinFigure1.

DifferinglevelsofendometrialmRNAexpressionofthesecytokinescanbenotedbetween

resistant and susceptible mares at different time points following insemination or bacterial

inoculation (summarized in Tables 1 and 2). Briefly, PBIE-susceptible mares appear to have

elevatedbaselinemRNAexpressionofpro-inflammatorycytokines(IL-1β,IL-6,IL-8andTNF-α)

anddecreasedmRNAexpressionofanti-inflammatorycytokines(IL-10) inestruscomparedto

normalmares(Fumosoetal.2003;Fumosoetal. 2007).FollowingAI,PBIEsusceptiblemares

havepersistentelevationsofpro-inflammatorycytokineexpression(IL-1β,IL-8andTNF-α)atday

7post-AI(Fumosoetal.2003;Fumosoetal.2007).IncreasedmRNAexpressionofIL-8at3hours

post-inoculationwithE.coliwasfoundinnormalmarescomparedtoPBIEmares(Christoffersen

etal.2012),andmRNAexpressionoftheanti-inflammatorycytokine,IL-10waselevatedbyday

7post-AIinsusceptiblemares(Fumosoetal.2007).Bycomparison,however,Woodwardetal.

(2013a)foundthatsusceptiblemareshadlowerlevelsofanti-inflammatoryIL-1RNandIL-10and

pro-inflammatory IL-6 at six hours post-AI, and found no differences at 24 hours post-AI

compared to resistantmares. In contrast to Fumosoet al. (2007), other studies did not find

differences in IL-8 expression between susceptible and resistant mares. Disparities amongst

studiescouldbeattributedtodifferencesinthestudydesignincludingmareinclusioncriteria,

samplesizeandtimepoints.IncreasedmRNAexpressionofpro-inflammatorycytokinesoccurs

innormalandsusceptiblemares followingAIaswellaspost infusionofextender,phosphate

bufferedsaline(PBS)andseminalplasmainnormalmares(Palmetal.2008).

23

1.5DIAGNOSISOFPBIE

AsunderstandingofPBIEpathophysiologyevolvedoverthepastfourdecadesofscientific

investigation,sohasthedefinitionanddiagnosisofthiscondition.Earlystudies(HughesandLoy

1969;Petersen1969)identifiedthatprolongedandexcessivefluidretentionoccurredfollowing

bacterial inoculation of somemares. These studies highlighted the impact of acute bacterial

endometritisonthefertilityofbroodmares.However,mareswithPBIEmayormaynothavea

bacterialcomponenttothedisease(Kotilainenetal.1994;Troedsson1995).Overtime,failure

ofuterineclearancebecame the focusof study,and the termDUCwasused todescribe the

condition(Evansetal.1986;LeBlancetal.1998;Rigbyetal.2001).Methodsto identifyDUC

mares included failure to clear radiocolloid, India ink or chromium-labelled microspheres.

However,thesemethodswereexpensiveandimpracticalforidentifyingmaresinclinicalpractice.

Brinsko(2003)recommendedthatmaresbedefinedashavingDUC/PBIEiftheyretainedgreater

than2cmofintrauterinefluidasassessedbytrans-rectalultrasoundexaminationat72hours

post-AI.Forthefirsttime,practitionershadapracticalmethodavailabletoidentifyPBIEmares

for treatment. However, this method only identified a lack of clearance and not persistent

inflammationwithintheuterus,alsotypicalofPBIE,limitingitsclinicalapplication.Troedssonet

al. (1993a)demonstrated correlationsbetweenhistological endometrial lesions andamare’s

clinical response to challenge with Streptococcus zooepidemicus, suggesting mares could be

identifiedas“potentiallysusceptible”onthebasisofanendometrialbiopsyscoreofIII.However,

correlationofsusceptibilitytoPBIEwithgradesofIIaorIIbwerepoor.Woodwardetal.(2013a,

2013b)andAlghamdietal. (2005)demonstratedasimilarapproachusing inseminationwith

killedorlivesperm,respectively.Mareswerefirstscreenedas“potentiallysusceptible”onthe

24

basisofbiopsyscore(Woodwardetal.2013a;Woodwardetal.2013b)aswellasaclinicalhistory

of low fertility rates, poor perineal conformation, and endometritis (Alghamdi et al. 2005).

Selected mares were inseminated and then monitored by trans-rectal ultrasound for fluid

retention up to 96 hours later. In addition, endometrial cytology at 96 hours was used to

demonstratepersistenceof the inflammatory response.Onlymareswithbothpersistenceof

fluidandcytologicalevidenceof inflammationat96hourspost-inseminationweredefinedas

beingsusceptibletoPBIE.Thisapproachhassincebeenusedinseveralresearchstudiestoselect

andclassifymaressusceptibletoPBIE,althoughthetimepointusedfordeterminationhasvaried

between 48, 72 and 96 hours across studies (Troedsson et al. 1991; Alghamdi et al. 2005;

Woodwardetal.2013a;Woodwardetal.2013b;Khanetal.2018).Fromaclinicalstandpoint,

thisstrictcriterionmightexcludemareswithlessseverediseasethatwouldbesubfertileand

mayprogresstomoreseverediseaseovertime.Inaddition,inseminationwithadoseofkilled

semen prior to breeding to diagnose PBIEmay define the condition clearly for experimental

studies,howeverit isnotpracticalforpractitionersinthefield. Inclusionofadditionalcriteria

such as perineal conformation, persistent endometrial edema (Pycock 2009), and breeding

history(subfertility,repeatedendometritisandhistoryoffluidaccumulationinpreviouscycles)

would likely further improve this definition and classification methodology. In other words,

mares could be classified as likely to be susceptible based on their reproductive history and

perineal conformation. Thiswould allow practitioners to plan for increasedmonitoring post-

breedingandtohaveapossibletreatmentplanprepared intheeventthatthemareshowed

signs of PBIE such as any accumulation of fluid, persistence of edema near ovulation or the

developmentofedemapost-ovulation.

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1.6TREATMENTOFPBIE

TreatmentofPBIEcanbedividedintothreebroadcategories:improvingmechanicaluterine

clearance,dilutingandremovingdebris,andmodifyingandimprovingtheimmuneresponse.

1.6.1EcbolicTherapy

Ecbolic therapies including oxytocin and prostaglandin are shown to increase uterine

clearanceby increasingmyometrial activity (Troedsson 1997; LeBlanc andCausey 2009). The

effectsofoxytocinonuterineclearancewereshownwhenbothsusceptibleandresistantmares

cleared >90% of radiocolloid following its administration (20U IV). This is in comparison to

susceptible mares only clearing negligible amounts and normal mares clearing 50-80% of

radiocolloidwhennot treatedwithoxytocin (Leblancetal.1994b).Oxytocin isawidelyused

treatmentforPBIE(LeBlancandCausey2009),withrecommendeddosagesrangingfrom10-25U

giveninthefirst48hourspost-breeding.Cadarioetal.(1999)showedthatbothsusceptibleand

resistantmareshavedose-dependentresponsestooxytocinadministration,withdosesof10U

resultinginthestrongestandmostprolongeduterinecontractionsascomparedto5Uand2.5U.

Administration of oxytocin results in uterine pressure changes within 30-65 seconds of

administration, and high amplitude uterine contractions for 30 minutes, resulting in almost

immediatevoidanceoftheuterus(Cadarioetal.1999;LeBlancetal.2009).Propulsionofuterine

fluidwasdescribedbyAllen(1991)inonemarefollowingtreatmentinestruswithoxytocin(5U

IV).Intra-uterinefluidmovedfromthecranialuterus,tothecaudaluterusandintothecranial

vagina by 1-hour post administration, as assessed by trans-rectal ultrasound. Furthermore,

administrationofoxytocin(10USC,5UIV;10UIV)intheperi-ovulatoryperioddoesnotresultin

areductionoffertility(Allen1991).However,duetoitsshortdurationofaction,oxytocinmust

26

begiveneveryfewhours.Unfortunately,somemaresdonotrespondtooxytocinadministration,

andcontinuetoaccumulateuterinefluiddespitetreatment(Hurtgen2006;LeBlancandCausey

2009).Factorssuspectedtoaffecttheresponsetooxytocintreatmentincludedecreasedornon-

functional oxytocin receptors in the endometrium, a pendulous uterus, or a closed cervix

(LeBlanc and Causey 2009).Moreover, inappropriately high dosages of oxytocin result in an

abnormalpropagationofuterinecontractions,therebyrenderingthetreatmentunsuccessful.In

fact,CampbellandEngland(2002)determinedthatdosesabove30Ucauseddecreaseduterine

contractions compared to lower doses. In addition, Rasch et al. (1996) determined that

pregnancyrateswereimprovedwhen15UofoxytocinwasadministeredIVcomparedto25UIV

seven to eight hours following AI (78% versus 52%) in mares with any intra-uterine fluid

accumulationpriortobreeding.

Oxytocin treatment causes prostaglandin release from the endometrium, potentially

prolonging itsdurationofaction.Administrationofprostaglandinssuchascloprostenolalone

alsopromotesuterineclearance,withweakerbutmoreprolongedcontractionsnotedfollowing

its administration compared to oxytocin (LeBlanc and Causey 2009). Low amplitude uterine

contractionslastuptofourorfivehours,whichnotonlyleadstophysicalclearanceofthefluid

butalsoaidswithlymphaticflow(Combsetal.1996;Troedsson1997;LeBlancandCausey2009).

Prostaglandins may also cause cervical relaxation, further aiding mechanical clearance.

ProstaglandinE2(misoprostol)appliedtothecervixhasalsobeenadministeredinthetreatment

ofPBIE,asittooleadstodilationofthecervixduetoitsdirectabilitytobreakdowncollagenand

decreasetotalcollagencontentinthecervix(Tangetal.2007;LeBlancandCausey2009).Care

mustbetakenwhenadministeringprostaglandinsforthetreatmentofPBIE.Ifgiveninthefirst

27

48hours followingovulation,mares couldhavedecreasedpregnancy rates, due to luteolytic

effects and decreased progesterone production from the early developing corpus luteum

(Troedssonetal.2001a;Nieetal.2002;Brendemuehl2002;Nieetal.2003).However,when

prostaglandinwasgivennolessthan12hourspriortoovulation,noeffectsonluteal lifespan

wereseen.Therefore,ifPBIEmaresaretobetreatedwithprostaglandins,treatmentshouldbe

limitedtowithinthistimeframe(Troedssonetal.2001a;Nieetal.2003).

1.6.2UterineLavage

The use of ecbolic drugs is often combinedwith therapeutic uterine lavage to dilute

irritants, as well as to clear inflammatory debris and excessive fluid from the uterus. Post-

breeding lavage induces uterine contractions, attracts newneutrophils and stimulates serum

opsoninstoaidintheinflammatoryresponse,duetothemildlyirritatingnatureoftheinfusion

(Brinskoetal.1990).Performinguterinelavageat>4hourspost-breedingisnotdetrimentalto

fertility(Brinskoetal.1990;Brinskoetal.1991)andhasbeenshowntoincreasepregnancyrates

inPBIEsusceptiblemares,whencombinedwithecbolictherapy4-8hourspost-breeding(Knutti

etal.2000).Furthermore,pregnancyratesdoubledinthegrouplavagedat4-8hourscompared

to those lavaged at 18-20 hours post-breeding; although this effect was not statistically

significant.

Theeffectonfertilityofpre-breedinguterinelavagewithLactatedRinger’sSolution(LRS)

immediately prior to inseminationwith fresh-chilled semenwas explored byVanderwall and

Woods(2003).Inthisstudy,sixoutoftenmareshadresidualfluidasdetectedbytrans-rectal

ultrasound immediately following the procedure and five out of six of thesemares became

pregnant. Three of the four mares that did not accumulate fluid following lavage became

28

pregnant.Noneofthepregnantmareshadresidualfluidanygreaterthan39mmindepth.This

study thereforeprovidesanotheroption for the treatmentofPBIE,especially in caseswhere

morethanonebreedingisperformedinanattempttoclearanyinflammationcausedbythefirst

breeding.

1.6.3Immunomodulation

AdditionaltreatmentsforPBIEincludeapproachestocorrectthealteredinflammatory

responseofsusceptiblemares.Theseincludeadministrationofanti-inflammatoryagentssuchas

glucocorticoids, and intrauterine infusion of agents such as Mycobacterial cell wall extract

(MCWE), platelet rich plasma (PRP) and chelating agents such as tris-EDTA (LeBlanc 2010;

WoodwardandTroedsson2015;Canissoetal.2016).

Twomainstudiesevaluatedtheefficacyofglucocorticoidtreatmentontheclinicalsigns

ofPBIE (Dell’Aqua Jretal. 2006;Buccaetal. 2008). Fivedosesof0.1mg/kgofprednisolone

acetate every 12 hours, beginning in estrus,with the last treatment given at the timeof AI,

resulted in reductionofuterine fluid volumeand improved clarity inmareswithahistoryof

uterinefluidaccumulation.However,areductionofuterineneutrophilfunctioninallmareswas

also seen (Dell’ Aqua Jr et al. 2006). Similarly, treatment with a single dose of 50mg of

dexamethasoneIVatthetimeofbreedingdecreaseduterineedema,uterinefluidvolumeand

uterinefluidturbidityinsusceptiblemares(Buccaetal.2008).Inbothstudies,pregnancyrates

wereimprovedintreatedmareshavingahistoryofintra-uterinefluidaccumulation(Dell’Aqua

Jretal.2006)orthosemareswithmorethanthreePBIEriskfactors(Buccaetal.2008),butnot

innormalmares.Interestingly,nodifferencesinneutrophilfunctionwerenotedbetweencontrol

andtreatedcycles(Buccaetal.2008).Reductionofneutrophilfunctionmayoccurbyblocking

29

thecyclo-oxygenaseand5-lipoxygenasepathwaysofinflammation(WoodwardandTroedsson

2015).Glucocorticoidsmaybebeneficial due to their effectsonNOproductionand cytokine

expression,astheyreducemRNAexpressionofIL-1,whichplaysaroleintheactivationofiNOS

(Barnes1998);however,whenmeasuringthedirecteffectsofdexamethasone inPBIEonNO

production, no effect was noted in both iNOS mRNA expression and total intrauterine NO

(Woodwardetal.2013b).Woodwardetal.(2015)showedadecreaseinIL-1βmRNAexpression

following treatment with dexamethasone in susceptible mares, but no differences in other

cytokineswerenoted.Overall,itappearsthatthepost-breedinginflammatoryresponseisless

severeandresolvesmorerapidlywhenglucocorticoidsareused.

Other treatments aimed at altering the inflammatory response include MCWE or

Propionibacteriumacnes,whichenhancestheimmuneresponsebycausingcytokinereleaseand

byactivatingimmunecells(PhillipsandFilion2001;Fumosoetal.2003;Woodwardetal.2013b;

Woodwardetal.2015).TreatmentofPBIEsusceptiblemareswithMCWEreturnedintra-uterine

pro-inflammatorycytokinelevelstothosemoretypicalofresistantmares,morespecificallywith

significantdown-regulationofIL-1β(Fumosoetal.2003;Woodwardetal.2015).Treatmentof

PBIE-susceptiblemareswithMCWE24hourspriortobreedingalsodecreasedintrauterineNO

levelscomparedtonon-treatedcontrolmares(Woodwardetal.2013b).Thiscanbeexplained

by the fact that reducing the up-regulation of pro-inflammatory cytokines leads to less

inflammationandthereforefewerinflammatoryproducts,suchasNO.

Morerecently,intrauterineinfusionofPRPhasbeeninvestigatedasapotentialtherapy

forPBIE.Plasmaisthoughttoenhancephagocytosisbyincreasingtheamountofcomplement

presentinuterinefluid(LiuandTroedsson2008).Metcalfetal.(2012)examinedtheeffectof

30

PRP treatment prior to AI on endometrialmRNA expression of cytokines in PBIE susceptible

mares.Interleukin-1β,IL-6andIL-8weredown-regulatedandiNOSexpressionreduced,inPRP-

treatedcycles,comparedtountreatedcycles. Theseresultswereattributedto therenewing

effectsofPRPbytheformationofgrowthfactors,cytokinesandchemokines.TheeffectofPRP

onpregnancyrateswasassessed inPBIE-susceptiblebarrenmares(Metcalf2014).Treatment

with PRP resulted in decreased retention of intra-luminal fluidwhen compared to untreated

cycles,whichmayhaveledinparttosignificantlyincreasedpregnancyratesduringtreatment

cycles(67%pregnancyrate inthetreatedgroupcomparedto19%inthenon-treatedgroup).

Reghinietal. (2016) examined theeffect of PRP treatment fourhourspost-AI inmareswith

chronicdegenerativeendometritis (CDE).Percentageofneutrophilsaswellasdepthof intra-

uterinefluidaccumulationswerebothreduced24hourspost-AIinCDE-affectedmarestreated

withPRPwhencomparedtocontrolcycles.However,nosignificanteffectsofPRPtreatmenton

NO levels were noted in this study. Effects of treatment with PRP prior to AI compared to

treatmentfollowingAIinPBIE-susceptiblemareswasassessedinastudybySegabinazzietal.

(2017). Both treatmentprotocols resulted in decreasedneutrophils on endometrial cytology,

higherconceptionratesandlowerCOX-2immunostainingwhencomparedtountreatedcycles.

However,noeffectofPRPtreatmentonintra-uterinefluidlevelswasobserved.Althoughthere

aresomeinconsistenciesinresultsbetweenstudies,theoveralleffectthatPRPdecreasesuterine

inflammationisclear.Morestudiesarerequiredinordertodeterminetheexactmechanismsof

actionandoptimaltreatmentprotocol.

Two proteins identified in seminal plasma also appear to have beneficial effects on

breeding-inducedinflammation:lactoferrinandCRISP-3(Dotyetal.2011;Troedssonetal.2014),

31

andresearchhasfocusedonwhethertheseproteinscouldbeofbenefitinthetreatmentofPBIE.

Morespecifically,lactoferrinhasbeenshowntoenhanceneutrophilphagocytosisofnon-viable

spermatozoa, assisting with clearance of sperm from the uterus prior to embryo descent

(Troedssonetal.2014).Incontrast,CRISP-3decreasesneutrophilbindingtolivespermatozoa,

therebyimprovingchancesoffertilization(Dotyetal.2011).Fedorkaetal.(2016)examinedthe

effects of infusionof onebillion spermatozoa alone, or in combinationwith seminal plasma,

CRISP-3orlactoferrinonmRNAexpressionofselectcytokinesinPBIEmares.Asimilarstudywas

repeated in PBIE-resistantmares (Fedorka et al. 2017). Results showed that only lactoferrin

decreasedIL-1RNmRNAexpressionwhencomparedtoothertreatmentsandnoothereffectsof

infusionwithlactoferrinorCRISP-3werenotedinPBIE-susceptiblemares(Fedorkaetal.2016).

However, infusion of PBIE-resistantmareswith seminal plasma and spermatozoa resulted in

increasedmRNAexpressionof thepro-inflammatory cytokines IL-8 and IL-1β, anddecreased

levelsofTNF-α(Fedorkaetal.2017).InfusionoflactoferrinalonesuppressedTNF-αexpression,

butnoeffectsofCRISP-3werenoted.TheseresultswereincomparisontothemRNAexpression

notedintheothertreatmentcycles.Duetothepromisingeffectsoflactoferrinoninflammatory

cytokineexpression, itsuseasapost-breedingtreatmentwasexplored(Fedorkaetal.2018).

Intra-uterine infusion of three different doses of human recombinant lactoferrin (50µg/ml,

250µg/mland500µg/mldilutedin10mlofLRS)inPBIEmares6hoursfollowingAIresultedina

decreasedneutropil:epithelial cell ratioat18hourspost treatment,butdidnotchangepost-

breeding intra-uterine fluid accumulation compared to the control cycle. Increased mRNA

expressionofIL-1RNanddecreasedmRNAexpressionofIFNγwerealsoseenintreatedcycles.

32

Giventheseresults,treatmentwithlactoferrinseemspromising,yetmoreresearchisneeded

priortoitsclinicalusage.

1.6.4N-AcetylCysteine

N-acetyl cysteine (NAC) is a mucolytic agent used in the treatment of bacterial

endometritiswhenthepresenceofabiofilmissuspected,orincaseswhereexcessuterinemucus

is present inhorses (Ferris et al. 2016).N-acetyl cysteinedisruptsdisulphidebondsbetween

mucinpolymers, reducingmucusviscosity (LeBlanc2010). Inadditionto itsmucolyticeffects,

NAC has demonstrated antioxidant and anti-inflammatory properties in multiple models of

diseaseinbothhumanandveterinarymedicine.Thismaybeinpartduetoitsmetabolism,asit

isde-acetylatedtocysteine.Cysteineisaprecursorofglutathione,whichdecreasesfreeradical

formation (Bisselinget al. 2004). Increased levels of glutathionewere detected in circulating

neutrophilsinahumanclinicaltrialevaluatingNACforthetreatmentofcysticfibrosis.Airway

neutrophilcountssignificantlydecreasedfollowinghigh-doseNACtreatmentinthesepatients

(Tirouvanziametal.2006). Inahumanchronicobstructivepulmonarydisease(COPD) invitro

model,NACwasevaluatedforbothitsmucolyticpropertiesandroleasananti-oxidant(Cazzola

etal.2017).Thestudyfoundthathigherconcentrationsofthedrugreducedpro-oxidativefactors

andenhancedanti-oxidativefactors,inadditiontoinhibitingpro-inflammatorycytokines(IL-1β,

Il-8,TNF-α),suggestingadose-dependenteffectofNAC.

N-acetyl cysteine affects cytokine and LPS-inducedproductionofNO in rat peritoneal

macrophages, C6 glial cells and primary astrocytes (Pahan et al. 1997). This in vitro study

demonstratedthatwhenthesecellswereincubatedwithNACtwohourspriortotheadditionof

anLPSstimulus,NOproductionwas inhibitedat the levelof iNOS.Thiswasattributedtothe

33

inhibitionofnuclearfactorkappa-light-chainenhancerofactivatedBcells(NF-κB).Similareffects

were noted in a murine LPS-induced preterm labor model, where multiple aspects of the

inflammatory response were reduced by NAC pre-treatment (Paintlia et al. 2008). The anti-

inflammatoryeffectsofNACwerefurtherinvestigatedinaratmultiplesclerosismodel.Here,

treatmentwithNACblockedtheinductionofmonocyte/macrophageproducedTNF-α,IFN-γ,IL-

1βandiNOSaswellassplenocyteproductionofIFN-γ.Inaddition,splenocyteproductionofthe

anti-inflammatorycytokineIL-10wasincreasedintheNAC-treatedgroup,demonstratingaclear

impactofNAConcytokineproduction(Stanislausetal.2005).Finally,studieshaveexaminedthe

effectsofNAConNOproductionspecifically(Bergaminietal.2001;Majanoetal.2004).Inboth

studies, inflammation was induced by LPS in rat models. Treatment with NAC resulted in

inhibitionofiNOS,andMajanoetal.(2004)wereabletodemonstratethatthisinhibitionmay

haveoccurredpartiallyatthelevelofNF-κB.

N-acetyl cysteine has been effective for treatment of inflammation in horses in

experimentalstudies,specificallyincasesofmucosalinjuryintherightdorsalcolon,andcorneal

ulceration(Haffneretal.2003;Rottingetal.2003).ThesestudiesshowthatNAChasmorethan

justmucolyticeffects.Itsabilitytoaltercytokineexpression,iNOSinductionandneutrophillevels

providesevidencethatitmaybeusefulincasesofuterineinflammation,suchasPBIE.

MultiplerecentstudieshaveexaminedtheutilityofNACfortreatmentofendometritis

(Witteetal.2012;Gores-Lindholmetal.2013;Melkusetal.2013).Potentialadverseeffectsof

NAC on the uterus have been assessed (Witte et al. 2012; Melkus et al. 2013). First, oral

administration of NAC was evaluated in normal mares during estrus. The effects on mucus

viscosityweredeterminedone-andfive-daysfollowingtreatment,inadditiontoevaluationof

34

endometrial epithelial integrity, neutrophil infiltration and COX-2 staining on endometrial

biopsiestakenondayfive.OralNACtreatmenthadnoeffectonendometrialepithelialintegrity

ormucus viscosity, but did result in decreased infiltration of neutrophils and COX-2 staining

comparedtountreatedmares (Witteetal.2012).Furtherstudiesconsistedofhealthymares

treatedwith intra-uterine infusionof5%NACduringestrus (Melkusetal.2013).Endometrial

biopsiesweretakenat24-and72-hours followingtreatment inorder todetermineepithelial

integrity, neutrophil infiltration and COX-2 staining. Results showed that no damage to the

epitheliumwassustained,andthatat72hourspost-treatment,fewerneutrophilsandlessCOX-

2stainingwaspresentintreatedmarescomparedtocontrols.ThisdemonstratedthatNACwas

safe,andthatitmayalsohaveananti-inflammatoryeffectintheuterus(Melkusetal.2013).In

amoreclinicalstudy,Gores-Lindholmetal. (2013)assessedendometrialstructure,neutrophil

activityandreproductiveperformanceinbothfertileandbarrenmarestreatedwithintra-uterine

NAC.Theyshowedthatperipheralneutrophilreleaseofoxygenradicalswasreducedby3.0%

NACsolution,therebypossiblydiminishingexcessiveintra-uterineneutrophil-induceddamage,

andthatendometrialepithelialarchitecturewasmaintained.Decreasedmucuswasnotedatthe

histological level in fertile mares but not in barren mares treated with NAC. These results

conflicted with those by Witte et al. (2012) and could be explained by differing routes of

administrationofNAC.Thesestudiessuggestthatintrauterineinfusionororaltreatmentwith

NACisnotdetrimentalontheendometrium,mayormaynotaffectmucusproductioninfertile,

healthy mares, and likely reduces inflammation in the uterus, as evidenced by decreased

neutrophilinfiltrationandCOX-2staining.

35

Basedonitsanti-inflammatory,anti-oxidantandNOmodulatingeffects,theutilityofNAC

as anadjunct for the treatmentofPBIE in susceptiblemares isworthyof further study. This

researchwasthereforeundertakenwiththegoaloffurtheringourunderstandingoftheeffects

ofusingNACinthemanagementPBIEinmares.

1.7RESEARCHOBJECTIVES

Thespecificobjectivesofthisresearchwere:

1. TodetermineifmaressusceptibletoPBIEtreatedwithN-acetylcysteinewillresolvetheir

clinicalsignsmorerapidlyintreatedcyclescomparedtocontrolcycles,and

2. TodetermineifmaressusceptibletoPBIEwillhavelowerconcentrationsofnitricoxideand

a modified inflammatory cytokine profile following treatment with N-acetyl cysteine

comparedtocontrolcycles.

36

1.8REFERENCES

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122. Troedsson,M.H.T.(1999).Uterineclearanceandresistancetopersistentendometritisin

themare.Theriogenology.52,461–471.

123. Troedsson,M.H.T. (2006).Breeding-inducedendometritis inmares.Vet.Clin.Equine22,705-712.

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(2001a).EffectofprostaglandinF2αonpregnancyratesandlutealfunctioninthemare.


125. Troedsson, M.H.T., de Morales, M.J. and Liu, I.K.M. (1993a). Correlations between

histologicendometriallesionsinmaresandclinicalresponsetointrauterineexposurewith

Streptococcuszooepidemicus.Am.J.Vet.Res.54,570-572.

126. Troedsson,M.H.T.,Desvousges,A.,Alghamdi,A.S.,Dahms,B.,Dow,C.A.,Hayna,J.,Valesco,

R., Collahan, P.T.,Macpherson,M.L., Pozor,M. and Buhi,W.C. (2005). Components in

seminalplasmaregulatingspermtransportandelimination.Anim.Reprod.Sci.89,171–186.

127. Troedsson,M.H.T.,Esteller-Vico,A.,Scoggin,K.E.,Woodward,E.M.,Squires,E.L.,Ball,B.A.

andMaxwell,H. (2014). Equine seminal plasmaderived lactoferrin regulatesbindingof

polymorphonuclearneutrophils(PMNs)tospermatozoa.J.EquineVet.Sci.34,49.

128. Troedsson,M.H.T.,Lee,C.-S.,Franklin,R.andCrabo,B.G.(2000).Theroleofseminalplasma

inpost-breedinguterineinflammation.J.Reprod.Fertil.Suppl56,341-349.

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betweenequinesemenandtheendometrium:theinflammatoryresponsetosemen.Anim.Reprod.Sci.68,273-278.

49

130. Troedsson,M.H.T.andLiu,I.K.M.(1991).Uterineclearanceofnon-antigenicmarkers(51Cr)

inresponsetoabacterialchallengeinmarespotentiallysusceptibleandresistanttochronic

uterineinfections.J.Reprod.Fertil.Suppl44,283–8.

131. Troedsson,M.H.T., Liu, I.K.M., Ing,M. and Pascoe, J. (1995). Smoothmuscle electrical

activityintheoviduct,andtheeffectofoxytocin,prostaglandinF2α,andprostaglandinE2

onthemyometriumandtheoviductofthecyclingmare.Biol.Reprod.Mono.1,475-488.

132. Troedsson,M.H.T.,Liu,I.K.M.,Ing,M.,Pascoe,J.andThurmond,M.(1993b).Multiplesite

electromyography recordings of uterine activity following an intrauterine bacterial

challengeinmaressusceptibleandresistanttochronicuterineinfection.J.Reprod.Fertil.99,307–313.

133. Troedsson,M.H.T.,Liu, I.K.M.andThurmond,M.(1993c). Immunoglobulin(IgGandIgA)

and complement (C3) concentrations in uterine secretion following an intrauterine

challenge of Streptococcus zooepidemicus in mares susceptible to versus resistant to

chronicuterineinfection.Biol.Reprod.49,502-506.

134. Troedsson,M.H.T.,Liu,I.K.M.andThurmond,M.(1993d).Functionofuterineandblood-

derived polymorphonuclear neutrophils in mares susceptible and resistant to chronic

uterineinfection:phagocytosisandchemotaxis.Biol.Reprod.49,507-514.

135. Tunon,A.-M.,Nummijarvi,A.,Katila,T.,Magnusson,U.andRodriguez-Martinez,H.(1999).

Distribution of T cells in the endometrium of themare 6 and 48 h after insemination.

Reprod.Dom.Anim.34,443-444.

136. Vanderwall,D.K.andWoods,G.L. (2003).Effecton fertilityofuterine lavageperformed

immediatelypriortoinseminationinmares.J.Am.Vet.Med.Assoc.222,1108-1110.

137. vonReitzenstein,M.,Callahan,M.A.,Hansen,P.J.andLeBlanc,M.M.(2002).Abberations

inuterinecontractilepatternsinmareswithdelayeduterineclearanceafteradministration

ofdetomidineandoxytocin.Theriogenology.58,887-898.

138. Watson, E.D. and Dixon, C.E. (1993). An immunohistological study of MHC class II

expressionandTlymphocytesintheendometriumofthemare.EquineVet.J.25,120-124.

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139. Watson, E.D. and Thomson, S.R.M. (1996). Lymphocyte subsets in the endometriumof

genitallynormalmaresandmaressusceptibletoendometritis.EquineVet.J.28,106-110.

140. Watson,E.D.,Stokes,C.R.andBourne,F.J.(1987a).Cellularandhumoralmechanismsin

maressusceptibleandresistanttopersistentendometritis.Vet.Immunol.Immunopathol.16,107–21.

141. Watson, E.D., Stokes, C.R. and Bourne, F.J. (1987b). Influence of arachidonic acid

metabolites in vitro and in uterinewashings onmigration of equine neutrophils under

agarose.Res.Vet.Sci.43,203-207

142. Watson E.D., Stokes, C.R. and Bourne, F.J. (1988). Concentration of immunoreactive

leukotrieneB4inuterinelavagefluidfrommareswithexperimentallyinducedandnaturally

occurringendometritis.J.Vet.Pharmacol.Therap.11,130-134.

143. Watson,E.D.,Stokes,C.R.,David,J.S.E.,andBourne,F.J.(1987c).Concentrationsofuterine

luminalprostaglandinsinmareswithacuteandpersistentendometritis.EquineVet.J.19,31-37.

144. Williamson,P.,Dunning,A.,O’Connor,J.andPenhale,W.J.(1983).Immunoglobulinlevels,

proteinconcentrationsandalkalinephosphataseactivityinuterineflushingsfrommares

withendometritis.Theriogenology.19,441-448.

145. Williamson, P.,Munyua, S.Martin, R. and Penhale,W.J. (1987). Dynamics of the acute

uterine response to infection, endotoxin infusion and physical manipulation of the

reproductivetractinthemare.J.Reprod.Fert.Suppl35,317-325.

146. Williamson, P., Munyua, S.J.M. and Penhale, J. (1989). Endometritis in the mare: A

comparisonbetweenreproductivehistoryanduterinebiopsyasatechniqueforpredicting

susceptibilityofmarestouterineinfection.Theriogenology.32,351-357.

147. Witte, T.S.,Melkus, E.,Walter, I., Senge, B., Schwab, S., Aurich, C. and Heuwieser,W.

(2012). Effects of oral treatment with N-acetylcysteine on the viscosity of intrauterine

mucusandendometrialfunctioninestrousmares.Theriogenology.78,1199-1208.

51

148. Woodward,E.M.,Christoffersen,M.,Campos,J.,Betancourt,A.,Horohov,D.,Scoggin,K.E.,

Squires,E.L.andTroedsson,M.H.T.(2013a).Endometrialinflammatorymarkersoftheearly

response inmares susceptibleor resistant topersistentbreeding-inducedendometritis.

Reproduction.145,289-296.

149. Woodward,E.M.,Christoffersen,M.,Campos,J.,Horohov,D.W.,Scoggin,K.E.,Squires,E.

andTroedsson,M.H.T.(2013b).Aninvestigationofuterinenitricoxideproductioninmares

susceptibleandresistanttopersistentbreeding-inducedendometritisandtheeffectsof

immunomodulation.Reprod.Dom.Anim.48,554–561.

150. Woodward, E.M., Christoffersen, M., Campos, J., Squires, E.L. and Troedsson, M.H.T.

(2012). Susceptibility to persistent breeding-induced endometritis in the mare:

Relationship to endometrial biopsy score and age, and variations between seasons.


151. Woodward, E.M., Christoffersen,M., Horohow, D., Squires, E.L. and Troedsson,M.H.T.

(2015). The effect of treatment with immune modulators on endometrial cytokine

expressioninmaressusceptibletopersistentbreeding-inducedendometritis.EquineVet.J.47,235-239.

152. Woodward,E.M.andTroedsson,M.H.T.(2013)Equinebreeding-inducedendometritis.J.EquineVet.Sci.33,673–82.

153. WoodwardE.M.andTroedsson,M.H.T.(2015).Inflammatorymechanismsofendometritis.


154. Zent, W.W., Troedsson, M.H.T. and Xue, J.-L. (1998). Postbreeding uterine fluid

accumulationinanormalpopulationofThoroughbredmares:Afieldstudy.Proc.AnnualConventionAAEP.44,64-65.

52

Figure1:Simplifiedflowchartdisplayingcytokineinteractions(Tizard2013)

53

Study TimePoints Mares Cytokines Results

Fumosoetal.2003 Baseline(estrus&diestrus),24hourspostAI&7dayspostAI

PBIEsusceptible&resistantmares

IL-1β,IL-6&TNFα

Baseline:IL-1β,IL-6,TNFαelevatedinsusceptiblemaresinestrus;IL-1β&TNFαelevatedinsusceptiblemaresindiestrus24hourspostAI:nodifferencesbetweensusceptible&resistant7dayspostAI:IL-1β&TNFαelevatedinsusceptiblemares

Fumosoetal.2007 Baseline(estrus&diestrus),24hourspostAI&7dayspostAI


IL-8&IL-10 Baseline:IL-8elevated&IL-10reducedinsusceptiblemaresinestrus;IL-8&IL-10elevatedinsusceptiblemaresindiestrus24hourspostAI:IL-8elevated&IL-10reducedinsusceptiblemares7dayspostAI:IL-8&IL-10elevatedinsusceptiblemares

Palmetal.2008 Pre-treatment(baseline)&12hoursfollowingtreatment(PBS,seminalplasma,skim-milkbasedsemenextender)

Normalmares IL-1β,IL-6&TNFα

IL-1β,IL-6&TNFαallincreasedpostAIforalltreatmentscomparedtopre-treatmentlevels

Nashetal.2010 12hourspre-treatment&24hours(frozen/thawedsemen)

Normalmares IL-8 IL-8levelsremainedthesamepre&posttreatment

Christoffersenetal.2010

0,3,12,24,48&72hoursfollowingintra-uterineinoculationwithE.coli

Normalmares IL-1β,IL-8,IL-10&TNFα

IL-1β,IL-8,IL-10&TNFαincreasedat3hourspostinoculation

Woodwardetal.2013

Baseline,2,6,12&24hourspostAI


IL-1β,IL-6,IL-8,IL-10,IFN-γ,IL-1RN&TNFα

IL-6,IL-1RN&IL-10decreasedat6hourspostAIinsusceptiblemares

Table1:mRNAexpressionofselectcytokinesinequineendometritisliterature,organizedbystudy.TableadaptedfromWoodwardandTroedsson(2015).

54

Cytokine Result TimePoint StudyType Authors

IL-1β ↑insusceptible Baselineestrus PBIEsusceptible&resistantmares

Fumosoetal.2003↑insusceptible BaselinediestrusNodifferences 24hourspostAI↑insusceptible 7dayspostAI↑foralltreatmenttypes 12hourspostAI

comparedtopre-treatment

Pre-treatment(baseline)&12hoursfollowingtreatment(PBS,seminalplasma,skim-milkbasedsemenextender)innormalmares

Palmetal.2008

↑ 3hourspostinoculation 0,3,12,24,48&72hoursfollowingintra-uterineinoculationwithE.coliinnormalmares


Nodifferences Baseline,2,6,12&24hourspostAI


Woodwardetal.2013

IL-6 ↑insusceptiblemares Baselineestrus PBIEsusceptible&resistantmares

Fumosoetal.2003Nodifferences BaselinediestrusNodifferences 24hourspostAINodifferences 7dayspostAI↑foralltreatmenttypes 12hourspostAI



Palmetal.2008

55

↓insusceptiblemares 6hrspostAIinsusceptiblemares


Woodwardetal.2013

Nodifference Baseline,2,12&24hourspostAI

TNFα ↑insusceptiblemares Baselineestrus PBIEsusceptible&resistantmares

Fumosoetal.2003↑insusceptible BaselinediestrusNodifferences 24hourspostAI↑insusceptiblemares 7dayspostAI↑foralltreatmenttypes 12hourspostAI



Palmetal.2008

↑ 3hourspostinoculation 0,3,12,24,48&72hoursfollowingintra-uterineinoculationwithE.coliinnormalmares




Woodwardetal.2013

IL-8 ↑insusceptiblemares Baselineestrus PBIEsusceptible&resistantmares

Fumosoetal.2003↑insusceptiblemares Baselinediestrus↑insusceptiblemares 24hourspostAI↑insusceptiblemares 7dayspostAINodifferences 12hourspre&24hours

postAI12hourspre-treatment&24hours(frozen/thawedsemen)innormalmares

Nashetal.2010

↑ 3hourspostinoculation 0,3,12,24,48&72hoursfollowingintra-


56

uterineinoculationwithE.coliinnormalmares



Woodwardetal.2013

IL-10 ↓insusceptiblemares Baselineinestrus PBIEsusceptible&resistantmares

Fumosoetal.2003↑insusceptiblemares Baselineindiestrus↓insusceptiblemares 24hourspostAI↑insusceptiblemares 7dayspostAI↑ 3hourspostinoculation 0,3,12,24,48&72

hoursfollowingintra-uterineinoculationwithE.coliinnormalmares


↓insusceptiblemares 6hourspostAI PBIEsusceptible&resistantmares

Woodwardetal.2013Nodifferences Baseline,2,12&24

hourspostAIIFN-γ Nodifferences Baseline,2,6,12&24

hourspostAIPBIEsusceptible&resistantmares

Woodwardetal.2013

IL-1RN ↓insusceptiblemares 6hourspostAI PBIEsusceptible&resistantmares

Woodwardetal.2013

Nodifferences Baseline,2,12&24hourspostAI


Woodwardetal.2013

Table2:mRNAexpressionofselectcytokinesinequineendometritisliterature,organizedbycytokine

57

CHAPTERTWO:THEEFFECTSOFTREATMENTWITHN-ACETYLCYSTEINE

ONCLINICALSIGNSINPBIESUSCEPTIBLEMARES

2.1ABSTRACT

Persistentbreedinginducedendometritis(PBIE)isamajorcauseofinfertilityinmares.

Uterine inflammationat the timeof embryonicdescentultimately results inearlyembryonic

death.Asevereendometrialbiopsygrade(IIborIII)hasbeenidentifiedasariskfactorforPBIE.

Intra-uterinefluidaccumulation(>2cmindepth),pathologicendometrialedemaandelevated

intra-uterine neutrophil levels are all clinical features of PBIE. N-acetyl cysteine (NAC), a

mucolyticusedtotreatbacterialendometritisinmares,hasanti-inflammatoryproperties,and

was investigated to determine if intra-uterine infusion before breeding would result in the

improvement of clinical signs associated with PBIE. In addition, the effects of NAC at the

histological level on the endometrium were determined. A randomized, blinded, cross-over

designclinicaltrialwasperformedutilizingPBIEsusceptiblemares(n=9).Intrauterineinfusionof

3.3% NAC was performed 12 hours prior to insemination, and endometrial cytology and

endometrialbiopsysampleswereobtainedat12and60hourspost-insemination.Endometrial

biopsieswerescoredbasedonthedegreeofinflammation(neutrophils)present.Clinicalsignsof

endometrialedemaandintra-uterinefluidvolumeswereassessedbytrans-rectalultrasoundat

12andthenevery24hourspostbreeding.StatisticalanalysisutilizedrepeatedmeasuresANOVA

andaMannWhitneyWilcoxonTest.TreatmentwithNACdidnotimproveclinicalsignsinPBIE-

affected mares. However, treatment with NAC had a significant effect on the degree of

endometrialinflammationnotedhistologically,withtreatedcyclesdisplayingmorediffuseand

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severeneutrophilinfiltrationcomparedtocontrolcycles.TheeffectsofNAContheendometrium

of PBIE-susceptible mares seen in this study is concerning. Further research is required to

evaluatethesafetyofNACinthetreatmentofPBIE-susceptiblemares.

2.2INTRODUCTION

Uterineinflammationisanormalresponsetobreedingandisbelievedtocleartheuterus

ofdebris,spermatozoaandinflammatoryproductspriortoembryodescent.Insomemares,the

inflammatoryresponseisprolonged,leadingtoearlyembryonicdeathandinfertility.Affected

maresareconsideredsusceptibletopersistentbreeding-inducedendometritis(PBIE)(Troedsson

2006;WoodwardandTroedsson2015;ChristoffersenandTroedsson2017).

ConsiderableresearchhasfocusedonthereasonssomemaresaresusceptibletoPBIE,

withthemajorityofstudiesfocusingonthe immunesystemandmechanicalclearanceofthe

uterus(NikolakopoulosandWatson,1999;Watson2000;LeBlancandCausey2009;Troedsson

2006;WoodwardandTroedsson2015;ChristoffersenandTroedsson2017).Multipleaspectsof

immune dysfunction have been investigated as potential contributors to PBIE. An altered

humoralimmuneresponse,specificallydifferinglevelsofimmunoglobulinsbetweensusceptible

andresistantmares,hasbeenidentified(Asburyetal.1980;Mitchelletal.1982;Williamsonet

al.1983;Troedsson1991).Incontrast,Troedssonetal(1993b)foundthatPBIE-susceptiblemares

didnothaveimmunoglobulindeficiencyat24hoursfollowingAI,disprovingthepreviousbelief

that immunoglobulin alterationswere the cause of PBIE. Troedsson et al. (1993c) and other

researchersdemonstratedthatPBIE-susceptiblemareshadelevatednumbersof intra-uterine

neutrophilswithreducedfunctionalityandphagocyticproperties(Luietal.1986;Watsonetal.

59

1987;Watsonetal.1988;NikolakopoulosandWatson1997;Troedssonetal.1993c;Woodward

andTroedsson2013).Additionalstudieshaverecentlyfurtheredourunderstandingoftherole

of inflammation inPBIE. Differences inmRNAexpressionofbothpro-andanti-inflammatory

cytokineshavebeenidentifiedeitheratbaselinelevels,oratmultipletimepointsfollowingAI,

betweennormalandaffectedmares.Persistentexpressionofpro-inflammatorycytokines for

prolonged periods of time, as well as reduced expression of anti-inflammatory cytokines

followingbreeding,maycontributetothemanifestationofPBIE(Fumosoetal.2003;Fumosoet

al. 2007; Christoffersenet al. 2012;Woodwardet al. 2013a; Christoffersen and Troedsson,

2017).PBIE-susceptiblemaresarealsoknowntohaveanunderlyingmyometrialdefect,causing

decreaseduterine contractility (Troedssonetal. 1993a; LeBlancetal. 1994a;Neuwirthetal.

1994;Rigbyetal.2001;vonReitzenseinetal.2001;LeBlancandCausey2009).Inaddition,intra-

uterinenitricoxidelevelsareelevatedinPBIE-susceptiblemares,furthercontributingtoreduced

uterine contractions (Alghamdi et al. 2005; Woodward et al. 2013b; Khan et al. 2018). A

reductioninuterinecontractilityleadstodecreaseduterineclearance,afeatureofPBIE.From

these studies, it is clear that themechanisms involved in uterine health following breeding,

includingtheimmuneresponse,persistentinflammationanduterineclearance,arecomplexand

interdependent.DysregulationofanyofthesemechanismshavethepotentialtoleadtoPBIEin

mares.

ClinicalsignsofPBIEincludeabnormalendometrialedema(Samper2007),persistenceof

a>2cmdepthofintra-uterinefluidbeyond48hourspost-AIdetectedontrans-rectalultrasound

(Brinsko 2003; Pycock 2007), presence of inflammatory cells on endometrial cytology and

60

endometrial histopathology consistent with uterine inflammation (LeBlanc 2010; Troedsson

2011;Canissoetal.2016).

Treatmentmodalities for PBIE do exist, yet these are notwithout limitations. Ecbolic

therapies, including oxytocin and prostaglandin, increase uterine clearance by increasing

myometrialactivity,andcanbehighlyeffectiveat improvinguterineclearance(LeBlancetal.

1994b, Combs et al. 1996; Troedsson 1997; Cadario et al. 1999; LeBlanc and Causey 2009).

However, some mares are refractory to oxytocin and continue to accumulate fluid despite

therapy(Hurtgen2006;LeBlancandCausey2009).Additionally,prostaglandinsadministeredas

ecbolic therapy post-ovulationmay interfere with progesterone production from the corpus

luteum, potentially resulting in early embryonic death. Prostaglandins are therefore only

recommendedinthepre-ovulatoryperiod,limitingtheiruseintreatmentofPBIE(Troedssonet

al. 2001; Nie et al. 2002; Brendemuehl 2002; Nie et al. 2003). Immunomodulators, such as

glucocorticoids,areuseful inmareswithsterile inflammationbutshouldbeavoided inmares

with concurrent bacterial involvement or othermedical conditions such as laminitis (LeBlanc

2010).Therefore,alternativetreatmentmodalitiesforPBIEareneeded.

N-acetylcysteine(NAC)isamucolyticagentthatdisruptsdisulphidebondslinkingmucin

polymers(LeBlanc2010)andhasanti-inflammatoryandanti-oxidantproperties(Bergaminietal.

2001;Majanoetal.2004;Cazzolaetal.2017).N-acetylcysteineinhibitedtheenzymeresponsible

forNOsynthesisandblockedpro-inflammatorycytokineproductioninamultiplesclerosis(MS)

model using rats, and enhanced anti-oxidative factors in in vitro human chronic obstructive

pulmonarydisease(COPD)research(Stanislausetal.2005).N-acetylcysteineiscurrentlyused

as an intra-uterine infusion inmareswith excessive uterinemucus production or in cases of

61

bacterial endometritis complicated by a biofilm (LeBlanc and Causey, 2009; LeBlanc 2010;

Troedsson2012).InfusionofNACimprovedfertilityratesofbarrenmareswhenusedduringthe

priorcycleor48hourspre-breeding(Gores-Lindholmetal.2009).Oral(Witteetal.2012)and

intra-uterine(Melkusetal.2013)NACadministrationdecreasedneutrophilnumberandCOX-2

staininginuterinehistologicalsamplesinnormalmares.N-acetylcysteineappearstobesafefor

use in horses: it was reported to cause no adverse effects on normal mare endometrium

following intra-uterine infusion of either a 3.3% or 5% solution (Gores-Lindholmet al. 2013;

Melkusetal.2013)orfollowingoraladministration(10mg/kg)(Witteetal.2012).Nostudiesto

datehaveexaminedtheeffectsofNAContheresolutionofclinicalsignsofPBIEinsusceptible

mares. Based on its anti-inflammatory and anti-oxidant effects, we investigated NAC as a

potentialtreatmentforPBIEinsusceptiblemares.

Theobjectiveofthisstudywastodeterminetheefficacyofintra-uterineinfusionofNAC

asatreatmentforPBIEinsusceptiblemares.Wehypothesizedthatintra-uterinetreatmentof

PBIE-susceptiblemareswithNACwouldresultinmorerapidresolutionofexcessiveendometrial

edema, percent neutrophils on endometrial cytology, intra-uterine fluid volume and

inflammationasassessedbyendometrialbiopsy,comparedtountreatedcycles.

2.3MATERIALSANDMETHODS

AlltheproceduresperformedonanimalsinthisstudywereapprovedbytheUniversity

ofGuelphAnimalCareCommittee(AnimalUtilizationProtocolnumber3213),inaccordance

withtheCanadianCouncilonAnimalCareguidelines.

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2.3.1SelectionofPBIESusceptibleMares

MarespreviouslyidentifiedassusceptibletoPBIE(Khanetal.2018)wereutilizedinthis

study.MareswereselectedinamannersimilartothatofAlghamdietal.(2005),andWoodward

etal.(2013a,2013b;2015).PotentiallyPBIE-susceptiblemareswereidentifiedonthebasisofa

KenneybiopsyscoreofIIborIII,negativebacterialcultureand<2neutrophilsperhighpower

field(hpf)onendometrialcytology.StudymareswerethenconfirmedtobePBIE-susceptibleby

failuretoclearintra-uterinefluidfollowingintra-uterinechallengewithkilledspermatozoa(i.e.

persistence of greater than 2cm of intrauterine fluid 96 hours following insemination).

Immediatelypriortothecommencementoftheclinicaltrial,studymareswereagainscreened

toensuretheywerenegativeforbacterialendometritis,andpre-trialendometrialbiopsieswere

obtained.BacterialculturesweresubmittedtotheAnimalHealthLaboratory(AHL,Guelph,ON,

Canada).Onlymareswithoutbacterialendometritiswereenrolledinthetrial.

2.3.2ClinicalTrial

Ablinded,randomized,crossoverdesignedclinicaltrialwasperformedusingtenPBIE-

susceptiblemares(Figure1).MareswerehousedattheArkellEquineResearchStationatthe

UniversityofGuelphandwereofvariousbreeds(Standardbredandmixedbreed)andages(7-19

years). The order of Treatment (N-acetyl cysteine intra-uterine infusion) and Control (saline)

cycles for each mare were assigned via random number allocation, with a washout period

between experimental cycles that consisted of at least one estrous cycle. The primary

investigatorconductingclinicalexaminationsandallpersonnelinvolvedinthedailycareofthe

mareswereblindedtotreatmentandcontrolassignmentofthemaresduringdatacollection.

OnlycycleswhichfitthedefinitionofPBIE,(>2cmofintra-uterinefluidforatleast48hourspost

63

AI)wereincludedintheanalysis. Forthisreason,onemarewasremovedfromthestatistical

analysisforbothtreatmentandcontrolcycles.

Maresweremonitoredbytrans-rectalpalpationandultrasoundandestruswasinduced

with an intra-muscular dose (125mcg) of cloprostenol (Estrumate®, Intervet Canada Corp.,

KirklandQC,CAN)asneeded.Onceinestruswitha35mmorlargerfollicleidentified,2,500IUof

humanchorionicgonadotropin(hCG,Chorulon®,IntervetCanadaCorp.,Kirkland,QC,Canada)

wasadministeredintravenouslytoinduceovulation.Duringtreatmentcycles(TX),180mlofa

3.3%NACsolutionconsistingof30mlofN-acetylcysteine(AcetylcysteineInjection20%solution,

AlvedaPharmaceuticals Inc., Toronto,ON,Canada)diluted in150mlof0.9% sodiumchloride

(0.9%SodiumChlorideInjectionUSP,BaxterCorporation,Mississauga,ON,Canada)wasinfused

into the uterus using a 21-inch insemination pipette (Partnar Animal Health, Ilderton, ON,

Canada). During control cycles (CONTROL), 180ml of sterile saline (0.9% Sodium Chloride

InjectionUSP)was infused into theuterus. Uterine infusions forboth treatmentandcontrol

cycleswereperformedatthetimeasthehCGinjection.Twelvehoursfollowinginfusion,mares

wereartificiallyinseminatedwith1x109killedspermatozoain30mlofsemenextenderprepared

aspreviouslydescribed(Woodwardetal.2013a;Woodwardetal.2013b;Woodwardetal.2015;

Khanetal.2018).Maresweremonitoredviatrans-rectalultrasoundbeginning12hourspost-AI,

andthiswasrepeatedat24hourintervalsthroughtoandincluding84hourspost-AI(12,36,60

and84hourspost-AI),todetermineendometrialedemascore(0=none;1=minimal;2=moderate;

3=normalmaximaland4=excessiveorpathological;DieldeAmorimetal.2016)andintra-uterine

fluid volume (IUF). The IUF volume was calculated using the formula IUF

(ml)=length*width*height,eachmeasured incentimeters,wherethe IUFwasthemeanvalue

64

derivedfrom2-5calculationsoffluidasassessedintheuterinebodyaswellasfromwithinthe

leftandrightuterinehorns.

Endometrialcytology,endometrialfluid(thissamplewasusedforadifferentexperiment)

and endometrial biopsy samples were taken, in that order, at 12 and 60 hours post-AI.

Endometrial cytology samples were aseptically obtained using a guarded swab (Kalayjian

Industries Inc., SignalHill, CA,USA) (Khanetal. 2018).Briefly, theguarded swabwaspassed

throughthecervix,theswabwasadvancedbeyondthesheathandthenrotatedseveraltimes

beforebeingleftinplaceforaminimumof30seconds,andthentheswabwasretractedintothe

sheath.Theoutersheathwasthenrotatedtocollectendometrialcellsinthecap,andtheentire

unitwas then removed. Endometrial biopsieswere taken aseptically at thebaseof the right

uterinehorn,bytrans-rectalguidanceofaPilling-Weckbiopsypunch(JorgensenLaboratories,

Loveland, CO, USA), through the cervix, as previously described (Kenney 1978). Endometrial

biopsy samples were placed in 10% formalin solution (Surgipath® 10% Neutral Buffered

Formalin, Leica Biosystems Richmond, Inc., Richmond, IL, USA) and submitted to the Animal

HeathLaboratory(UniversityofGuelph)forembeddinginparaffinwax,sectioningandroutine

stainingwithHematoxylinandEosin(H&E).

2.3.3InflammationasAssessedbyEndometrialBiopsies

Endometrial biopsies were examined for the degree and distribution of neutrophils,

lymphocytesandplasmacells(‘inflammatorycells’)byaBoardCertifiedTheriogenologist(TC),

blindedtomaretreatmentassignments.Asemi-quantitative inflammationscorebetween1-4

(1=mild; 2=moderate; 3=marked and superficial; 4= severe and diffuse) was assigned to

endometrialbiopsies,andwasbasedonthecompilationofthreeseparateobservationsmade

65

acrossaminimumof10highpowerfields(Figure2;Table1).Thefirstobservationconsistedof

thenumberofneutrophilsperhighpowerfieldasadaptedfromAlvarengaetal.(2016)(1=0-3

neutrophils/hpf);2=(4-10neutrophils/hpf);3=10-30neutrophils/hpf);4=>30neutrophils/hpf).

Thesecondobservationconsistedofthedepthofinflammatorycellinfiltration,withscoresof1-

3 having inflammatory cells present in themost superficial areas only (stratum compactum)

whereasthosewithdiffuseinfiltrationofinflammatorycellsdeepintotheendometrium(stratum

spongiosum) were given a score of 4. The third observation consisted of the presence of

endometrial luminalepitheliumerosion,asdefinedbylossofepitheliumwithevidenceofcell

proliferationonboththeedgesandbelowtheareaoferosion(cellsbecomingthinandstretched

ontheedges;presenceofhyperchromaticcellsbelow).Thepresenceoferosioncontributedto

amoreseverescore(3or4).

2.3.4EndometrialCytology(%Neutrophils)

Endometrial cytology slideswere prepared and stainedwithmodifiedWright Giemsa

stain(PROTOCOLTM,Hema3TMStatPack,FisherBrand®,Pittsburg,PA),aspreviouslydescribed

(Khanetal.2018).Thepercentageofneutrophilstototalcellswasdetermined(endometrialcells

and neutrophils combined) across multiple fields of view, at 400x. This accounted for any

variability of cell distribution. The mean percentage of neutrophils was calculated after a

minimumof500totalcellswerecounted.

2.3.5StatisticalAnalyses

StatisticalanalyseswereperformedusingtheSASsoftwareprogram(SASInstitute,Inc.,

Version9.3,Cary,NC,USA).Differences in IUF,%neutrophilsanduterineedemascoreswere

comparedbetweenNAC-treated(TX)andsaline(CONTROL)cycles,aswellasacrosstimepoints

66

using repeatedmeasures ANOVA.Mare identification (ID) was included as a variable in the

statistical model, but was removed, as no significance was detected. Differences in biopsy

inflammationscorebetweenNAC-treated(TX)andsaline(CONTROL)aswellasacrosstimepoints

wereanalyzedusingtheMannWhitneyWilcoxontest.TheAkaikeinformationcriterion(AIC)was

utilizedtodeterminethebestfitmodel.NormalityofthedatawastestedusingtheShapiro-Wilk

test. Data for estimated intrauterine fluid volume (IUF) required logarithmic transformation,

however all other outcome parameters were normally distributed. Results are presented as

medians+/-lowerlimit(LL)andupperlimit(UL)whenlogtransformationwasrequiredandas

means+/-standarderror(SE)whennodatatransformationswereperformed.Wheretherewas

noeffectoftreatment,dataforNAC-treated(TX)andsaline(CONTROL)cycleswerecombinedto

thenexaminetheeffectofnumberofhourspost-AI.Similarly,wheretherewasnoeffectofhours

post AI, data for 12 and 60 hours post-AI were combined to then examine the effects of

treatment.Correlationbetween%neutrophilsandbiopsyinflammationscorewasanalyzedusing

the Spearman correlation coefficient, to account for the significance in the model and

distributionofthedata.CorrelationbetweenIUFanduterineedemascorewasassessedusing

the Pearson correlation coefficient, since the model had no effects and data was normally

distributed.Thepvaluewassetat<0.05forstatisticalsignificanceforallstatisticalanalyses.

2.4RESULTS

2.4.1BiopsyInflammationScore

Pre-trialbiopsieswereavailablefor7ofthe9PBIE-susceptiblemaresusedinthisstudy.

Themeanpre-trial(baseline)biopsyinflammationscorewas1.14+/-0.65.Biopsyinflammation

67

scorewassignificantlygreaterintheNACtreatedcycle(TXmean=3.0+/-0.5)comparedtothe

salinetreatedcycle(CONTROLmean=2.2+/-0.5),whenbiopsyinflammationscoresat12and

60hourspost-AIwerecombinedforeachtreatmentcycle(p=0.01)(Figure3).Theendometrial

effectsofNACincludederosionsoftheluminalepitheliumandmarkedinfiltrationofneutrophils

inthestratumcompactum(Figures2d,4and5).Oneofthedifferences incriteriabetweena

biopsyinflammationscoreof1-3comparedtoabiopsyinflammationscoreof4wasthepresence

ofdeep-seatedneutrophil infiltration.The incidenceofan inflammationscoreof4wasmuch

greaterinNACtreatedcycleswithtimepointscombined(8/18)incomparisontosalinetreated

cycles(1/18)withtimepointscombined(Table1).Therewasnosignificanttimeeffectonbiopsy

inflammationscorefrom12to60hourspost-AIwhenbothtreatmentcycleswerecombined;

mean(12hourspostAI)was2.8+/-0.5whereasmean(60hourspost-AI)was2.3+/-0.5(p=0.12)

(Figure6).

2.4.2EstimatedIntra-UterineFluidVolumes(IUF)

TreatmentwithNACdidnotaffectcalculatedIUFwhentimepointswerecombinedwithin

eachtreatmentcycle(p=0.78;TXmedian=3.9cm3;range=1.1-14.7cm3;CONTROLmedian=4.5

cm3;range=1.2-16.8cm3)(Figure7).Incontrast,theeffectoftimewassignificant(p=0.005),

withmoreintra-uterinefluidpresentat36hourspost-AI(median=9.8cm3;range=2.6-36.2cm3)

comparedto12hourspost-AI(median=3.4cm3;range=1-11.6cm3),60hourspost-AI(median

= 3.9cm3; range = 0.9-17.5cm3) and 84 hours post-AI (median = 2.5cm3; range = 0.6-9.5cm3)

(Figure8).

68

2.4.3EndometrialEdema

TreatmentwithNACdidnotaffectendometrialedemascores(p=0.90;TXmean=1.7+/-

0.6;CONTROLmean=1.7+/-0.6)(Figure9).Endometrialedemascorewassignificantlyhigher

(p=0.04)at12hours(mean=2.1+/-0.6)and36hours(mean=2.0+/-0.6)comparedto60hours

post-AI(mean=1.3+/-0.6)and84hours(mean=1.3+/-0.6)post-AI(Figure10).Theestimated

IUFanduterineedemascoreswerenotsignificantlycorrelated(r=-0.14;p=0.45).

2.4.4EndometrialCytology(%Neutrophils)

TreatmentwithNACdidnotaffect%neutrophilsonendometrialcytology (p=0.39;TX

mean = 48.4% +/- 12.5%; CONTROL mean = 40.9% +/- 12.5%) (Figure 11). Evidence of

inflammationdecreasedovertimepost-AI(p=0.005).The%neutrophilswassignificantlyhigher

at12hourspost-AI(mean=57.7%+/-12.5%)comparedto60hourspost-AI(mean=31.6%+/-

12.5%) (Figure12).Additionally,%neutrophilsandbiopsy inflammationscorewerepositively

correlated(r=0.43;p=0.006).

2.4.5InteractionsBetweenTreatmentCyclesandHoursPost-AI(Appendix1)

There was no significant interaction between treatment cycle and hours-post-AI for

biopsy inflammation score (p=0.12;Appendix1: figure1), IUF (p=0.84;Appendix1: figure2),

endometrialedema(p=0.65;Appendix1:figure3),nor%neutrophils(p=0.98;Appendix1:figure

4).

2.5DISCUSSION

Intra-uterineinfusionofPBIE-susceptiblemareswitha3.3%NACsolution12hourspre-

AIresultedinmoreinflammationonendometrialbiopsycomparedtotreatmentwithsaline12

69

hourspre-AI.ThisisincontrasttopreviousstudieswheretreatmentwithNACdidnotappearto

bedetrimental to theendometrium,andtheendometrial luminalepitheliumremained intact

(Witteetal.2012;Gores-Lindholmetal.2013;Melkusetal.2013).Melkusetal.(2013)reported

thatintra-uterineinfusionof5%NACinnormalmaresresultedinlessendometrialinflammation

onbiopsyat72hourspost-treatmentcomparedtocontrolmarestreatedwithsaline.However,

NACtreatedandcontrolnormalmaresbothhadhighernumbersofneutrophils/hpf24hours

followingtreatment(10.9neutrophils/hpfinNACgroup;8.3neutrophils/hpfinsalinegroup).This

resolved by 72 hours, with treatedmares having significantly less neutrophils than controls.

Normal mares treated orally with NAC for four days had fewer neutrophils present in the

endometriumonedayfollowingtreatment,comparedtountreatedcontrols(Witteetal.2012).

ItisinterestingthatintheWitteetal.(2012)andMelkusetal.(2013)studies,normalmaresin

estrus hadmore neutrophilswithout provocation of inflammation, such asAI. In the studies

mentionedabove,maresweresimplytreatedinestrusandbiopsieswereobtained.Untreated

maresorthosetreatedwithsalinehadgreaterthan2neutrophils/hpf,whichisconsideredtobe

indicativeofendometritis(Woodwardetal.2013a)(4.8neutrophils/hpfwithnotreatment;3.9

neutrophils/hpfwithsaline infused intra-uterineat72hoursposttreatment).Thereasonsfor

thisincreaseinsupposedlynormalmareswithoutaninflammatorychallengearenotknown,but

could include differences in the original classification as normalmares, posing as a possible

limitationtotheirstudy,ortotheirritatingnatureoftheinfusionofNACitself.Gores-Lindholm

etal. (2013)reportedthatthedegreeofacute inflammation inendometrialbiopsieswasnot

differentthreedaysfollowingintra-uterineinfusionofa3.3%NACsolutionascomparedtopre-

treatment within a mixed group of 20 mares (10 barren treated; 6 fertile treated; 6 fertile

70

control).However,maresinthatstudyweretreatedwithuterinelavagewithlactatedRinger’s

solution(LRS)dailyfortwodaysbetweentheinitialinfusionandtheprocurementofthebiopsy,

possiblyaidingresolutionofanyinflammationresultingfromtheNACinfusionbythetimethe

biopsy was taken. In contrast to the above studies, which utilized either normal or mixed

populationsofmares,ourtrialinvolvedonlyPBIE-affectedmares,selectedpartiallyonthebasis

ofpoorendometrialbiopsyscores(GradeIIbandIII).Normalmaresarelikelymorecapableof

clearing any inflammatory reaction caused by infusion of NAC or other irritating substances.

However, this inflammatory reactionmaynotbeclearedas rapidly inmares thatarealready

susceptibletouterineinflammation,suchasthoseusedinourstudy.

Inflammatoryscoresof4werealmostexclusivelyassignedtomaresintheNACtreated

cycles (Table 1). The inflammation noted in these biopsies consisted almost entirely of

neutrophils and was diffusely deep-seated, extending into the stratum spongiosum and

occasionallyintotheglandularlumen(Figures2dand5).Additionally,severelyaffectedmares,

withascoreof3or4,oftendisplayedsignsofareactive luminalepitheliumindicativeofcell

injury. The biopsies in our study also had some areas of erosion or complete loss of the

endometrialluminalepithelium(Figure4)andthesechangesweretakenintoconsiderationfor

thebiopsyinflammationscore(scoreof3or4).Thesechanges,aswellasthosementionedabove,

areallsuggestiveofamarkedendometrialresponsetotheirritatingnatureofNAC.Witteetal.

(2012) reported that treatment with NAC caused increased epithelial cell proliferation and

suggestedittobeevidenceofendometrialregeneration.Gores-Lindholmetal.(2013)mentioned

anincreaseinepithelialcellheightinsalinetreatedfertilemarescomparedtoNACtreatedfertile

mares. The changes in these studies could suggest endometrial reactivity; however, the

71

epitheliumremainedintact.Thebiopsieswithareasoferosionwereverysimilarinappearance

tothoseseeninmarestreatedwithintra-uterinekerosene,whichwaspreviouslyreportedto

causesevereinflammation(R.Foster,Personalcommunication;Bracheretal.1991).Mareswith

GradeIIIbiopsyscoresthatreceivedanintra-uterineinfusionof50mlofcommerciallyavailable

kerosene demonstrated a more prolonged and severe inflammatory response histologically,

compared to similarly treated mares with Grade I or II biopsy scores (Bracher et al. 1991).

However,inthatstudy,mononuclearcellswerethepredominantinflammatorycelltypepresent,

comparedtothepredominanceofneutrophilsseeninourstudyfollowingtheNACinfusion.One

previousstudyusingintra-uterineNAC(Gores-Lindholmetal.2013)demonstratedanincrease

in lymphocytesandplasmacellsonlyonendometrialbiopsy followingNAC infusion inbarren

mares,buttheauthorsnotedthatthesechangesweretoochronicinnaturetobeduetoNAC

infusion.

Theosmolalityof3.3%NACsolutionusedinthepresentstudywasconsiderablyhigher

thanthe0.9%salineusedinthecontrolcycles(saline=280mOsm;NAC=668mOsm).Whileno

studieshaveexaminedtheeffectsofhyperosmoticsolutionsontheendometriumspecifically,

multiple studies have shown that hyperosmotic stressors are damaging to cells and are pro-

inflammatoryinnature(Nemethetal.2002;Luoetal.2005;Schwartzetal.2008;Brockeretal.

2012). Exposure of cells to hyperosmotic solutions leads to an efflux of fluid out of the cell,

shrinkageofthecell,alterationinproteinsynthesis,anddamagetoboththecytoskeletonand

nucleus. This can lead to cellular apoptosis (Brocker et al. 2012). It is possible that the

inflammatory reaction seen in our study was further exacerbated by insemination 12 hours

followingNACinfusion,limitingtheabilityofthealreadycompromisedendometriumtoclearthe

72

inflammation. In previous studies that administered intra-uterine NAC, treatment was often

given 24-36 hours prior to breeding (LeBlanc and Causey 2009; Gores-Lindholm et al. 2009;

LeBlanc2010;Lyle2012;Troedsson2012;Gores-Lindholmetal.2013;Buczkowskaetal.2015),

perhapsallowingforsomerecoverypriortoanotherinsult.Giventhebiopsyfindingsinourstudy,

onecouldspeculatethatintra-uterineinfusionofNACmaynotbeasinertaspreviouslybelieved.

TreatmentwithNACdidnot improveclinical signsofPBIE, including IUF. Intrauterine

fluid retention occurs in PBIE mares for several reasons, including lack of cervical dilation,

pendulousuterusandpooruterinecontractility.Cervicalpatencyisknowntoimproveuterine

drainage (Pycock 2007, LeBlanc andCausey2009, Reliaset al. 2016). Some studymareshad

fibrotic and tortuous cervices that resulted fromuseas repeatedembryodonors inprevious

research studies. Therefore, somemares retained considerable fluid, leading to a significant

amountofvariabilityinIUFlevelsbetweenmares.Thismayhavelimitedtheapparentefficacyof

intra-uterineNACtreatmentinourstudy.

The PBIE-susceptible mares in our study demonstrated the expected inflammatory

responseovertime,includingchangesinIUF,uterineedemascoresand%neutrophils.IUFwas

mostelevatedat36hoursanddiminishedthereafter.Myometrialcontractionsareoneofthe

mainmechanismsofuterineclearance(Troedsson2006;LeBlancandCausey2009;Troedsson

andWoodward2016;ChristoffersenandTroedsson2017).Normalmaresclearintra-uterinefluid

within24hoursfollowingAI(Katila1995);itisconsideredabnormaltofindanyintra-uterinefluid

greater than 24 hours post-ovulation and into diestrus (Newcombe 1998). However, PBIE-

susceptiblemaresdonotdemonstratenormaluterineclearance.LeBlancetal.(1994a;1995)

found that susceptible mares cleared negligible amounts of radiocolloid 4 hours following

73

infusion;furthermore,Indiainkwasmaintainedforupto72hourspost-infusion.Furthermore,

Troedsson et al. (1993a) showed that susceptible mares inoculated with Streptococcus

zooepidemicus had a marked decrease in myometrial activity between 7-17 hours post-

inoculationcomparedtoresistantmares.Inourstudy,IUFwasgreatestat36hoursbutnotat12

hourspost-AI.Troedssonetal.(1993a)demonstratedthatthepatternofuterinecontractilityof

PBIE-susceptible and normal mares was similar initially following a challenge, however the

durationand intensityofmyometrial contractions rapidlydeclined inPBIE-susceptiblemares,

whereasinresistantmaresitdidnot.ThismayexplaintheincreaseinIUFseenfrom12to36

hoursinourstudymares.

EndometrialedemaandestimatedIUFwerenotsignificantlycorrelatedinthisstudy.This

iscontradictorytoKotilainenetal.(1994)wherepre-treatment(AI,extender,seminalplasma,

PBS)uterinefluidlevelwaspositivelycorrelatedwithuterineedema.Inaddition,Samper(2007),

foundthatpresenceoffluidatthetimeofovulationwasmorecommonlynotedinmareswith

gradethree,fourandfiveedemascores.Thesedifferencescouldbeaccountedforbythefact

thatpre-treatmentfluidlevelscouldvaryfrompost-treatmentfluidlevelsandthatthegroupof

maresinthosestudieswerenormalandnotscreenedasPBIE-susceptible.

Percent neutrophils on cytologywere highest at 12 hours post-AI in our studywhen

treatmentcycleswerecombined.Aninfluxofneutrophilsintotheendometriumoccursfollowing

infusionwithspermatozoainnormalmares,andwasfirstdocumentedbyKotilainenetal.(1994).

Innormalmares,neutrophilsappearat4hours,peakat8hoursandareabsentby48hourspost-

breeding(Katila1995).Thisresponseisverysimilartothatnotedinnormalmaressubjectedto

intra-uterineinfusionofStreptococcuszooepidemicusfourhoursfollowingbreeding(Troedsson

74

1995).AsimilarpatternofneutrophilinfiltrationoccursinPBIE-susceptiblemares,butismuch

moresevere.Neutrophilsweresignificantlyelevatedinsusceptiblemaresasearlyas2hourspost

AI, compared to 6 hours post AI in resistant mares (Woodward et al. 2013a). Endometrial

neutrophil infiltration peaked in both groups at 6 hours post AI, and decreased thereafter,

howeverneutrophilswereconsistentlyelevatedinsusceptiblemaresthroughout(Woodwardet

al.2013a).Overall,persistentinflammationwasafeatureofourPBIE-susceptiblestudymares,

consistentwithpreviousfindings.

DoseandtimingofadministrationofNACmayhaveaffectedefficacyinthepresentstudy.

A3.3%solutionwasselectedbasedonpreviousstudiesusingintra-uterineNACinmaresasa

mucolytic(LeBlancandCausey2009;LeBlanc2010;Lyle2012;Troedsson2012;Gores-Lindholm

etal.2013;Buczkowskaetal.2015;Scoggin2016).Sub-fertilemareswithahistoryofmucus

hyper-secretion that received an intrauterine infusion of 3.3% NAC pre-breeding, attained a

pregnancyrateof77%(Gores-Lindholmetal.2013).However,researchinotherfields,suchas

incysticfibrosis(humans)androdentpulmonarystudies,suggeststhatthedoseofNACneeds

tobesubstantialenoughtocreateananti-inflammatoryeffect(Pahanetal.1997;Tirouvanziam

etal.2006).ItisthereforepossiblethattheselecteddoseofNACinourstudywasadequatefor

itspreviouslyknownmucolyticeffects,butnothighenoughtocausedirectanti-inflammatory

effects.NACwasadministeredpre-breedinginourclinicaltrial,inthesamefashionasprevious

studies(Gores-Lindholmetal.2013).Ratintra-peritoneal,cellcultureandtissuemodelstudies

havedemonstratedtheanti-inflammatoryeffectsofNACwhenadministeredasapre-treatment

priortoinductionofinflammation(Pahanetal.1997;Majanoetal.2004;Cazzolaetal.2017).

Together,thisevidenceprovidedthebasisforthetimingofNACinfusioninourstudy.However,

75

since no other studies have examined NAC treatment specifically in PBIE mares, treatment

followingtheinflammatoryresponseinducedbybreedingmightprovemorebeneficial.Infact,

oralNACtreatmentofpatientsclinicallyillwithcysticfibrosisledtomodulationofthepulmonary

inflammationseenwiththiscondition(Tirouvanziametal.2006).

Inconclusion,intra-uterinetreatmentwith3.3%NACdidnotimproveclinicalsignsinPBIE

mares.NACinfusionwasassociatedwithasignificantandprolongedacuteinflammatoryreaction

oftheendometrium,whichwassuspectedtobeduetothehyperosmoticnatureofNAC.Further

studiesareneededtoexploretheeffectsofhypertonicsolutionsontheendometriuminboth

normal and PBIE-susceptible mares. Additional studies evaluating alternative non-irritating

immunomodulatory treatments such as platelet-rich plasma, which might normalize the

inflammatoryresponseofPBIEmares,wouldbeofvalue.

76

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34. Nie,G.J.,Johnson,K.E.,Wenzel,J.G.W.andBraden,T.D.(2003).Lutealfunctioninmaresfollowingadministrationofoxytocin,cloprostenolorsalineonday0,1or2post-ovulation.Theriogenology.60,1119-1125.

35. Neuwirth,L.,LeBlanc,M.M.,Mauragis,D.,Klapstein,E.andTran,T.(1994).Scintigraphic

measurementofuterineclearanceinmares.Vet.Radiol.Ultrasound.36,64-68.36. NewcombeJ.R.(1997).Theeffectoftheincidenceanddepthofintra-uterinefluidinearly

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extended semen reduce the antigenic challenge to the mare’s uterus compared withnaturalservice?Theriogenology.47,583-590.


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induction of NO production by endotoxin or cytokine stimulated rat peritonealmacrophages,C6glialcellsandastrocytes.FreeRadic.Biol.Med.24,39-48.

40. Paintlia, M.K., Paintlia, A.S., Singh, A.K. and Singh, I. (2008). Attenuation of

lipopolysaccharideinducedinflammatoryresponseandphospholipidsmetabolismatthefeto-maternalinterfacebyN-acetylcysteine.Pediatr.Res.64,334-339.

41. Pycock,J.F.(2007).Therapyformareswithuterinefluid,1stedn,Ed:CurrentTherapyin

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42. Rigby, S.L., Barhoumi, R., Burghardt, R.C., Colleran, P., Thompson, J.A., Varner, D.D.,

Blanchard,T.L.,Brinsko,S.P.,Taylor,T.,Wilkerson,M.K.andDelp,M.D.(2001).Mareswithdelayeduterine clearancehavean intrinsicdefect inmyometrial function.Biol.Reprod.65,740–747.

43. Samper,J.C.(1997).Ultrasonographicappearanceandpatternofuterineedematotimeof

ovulationinmares.Proc.Annul.ConventionAAEP.43,189-191.44. Samper, J.C. (2007).How to interpret endometrial edema in broodmares.Proc.Annul.

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45. Schwartz,L.,Abolhassani,M.,Pooya,M.,Steyaert,J.-M.,Wertz,X.,Isreal,M.,Guais,A.andChaumet-Riffaud, P. (2008). Hyperosmotic stress contributes to mouse colonicinflammation through the methylation of protein phosphatase A2. Am. J. Physiol.Gastrointest.LiverPhysiol.295,G934-G941.

46. Stanislaus,R.,Gilg,A.G.,Singh,A.K.,Singh, I. (2005).N-acetyl-L-cysteineamelioratesthe

inflammatory disease process in experimental autoimmune encephalomyelitis in Lewisrats.J.Autoimmun.2.


L.A. (2006). High dose oral N-acetylcysteine, a glutathione prodrug, modulatesinflammationincysticfibrosis.Proc.Natl.Acad.Sci.103,4628-4633.

48. Troedsson,M.H.T.(1991).Uterinedefensemechanismsinthemare.(thesis).49. Troedsson,M.H.T.(1995).Uterineresponsetosemendepositioninthemare.Proc.Soc.

Theriogenol.130-135.50. Troedsson,M.H.T. (1997). Therapeutic considerations formating-induced endometritis.

Pferdeheilkunde.13,516-520.51. Troedsson,M.H.T. (2006).Breeding inducedendometritis inmares.Vet.Clin.Equine22,

705-712.52. Troedsson,M.H.T.(2011).Endometritis.In:EquineReproduction.2ndedn,Ed:McKinnon,

A.O.,Squires,E.L.,Vaala,W.E.,Warner,D.D.,Wiley-Blackwell,NewJersey.pp.2608–2619.53. Troedsson, M.H.T and Woodward, E.M. (2016). Our current understanding of the

pathophysiology of equine endometritis with an emphasis on breeding-inducedendometritis.Biol.Reprod.16,8-12.

54. Troedsson,M.H.T.,Ababneh,M.M.,Ohlgreen,A.F.,Madill,S.,Vetscher,N.andGregas,M.

(2001). EffectofprostaglandinF2α onpregnancy ratesand luteal function in themare.Theriogenology.55,1891-1899.

55. Troedsson,M.H.T.,Liu,I.K.M.,Ing,M.,Pascoe,J.andThurmond,M.(1993a).Multiplesiteelectromyography recordings of uterine activity following an intrauterine bacterialchallengeinmaressusceptibleandresistanttochronicuterineinfection.J.Reprod.Fertil.99,307–313.

56. Troedsson,M.H.T.,Liu,I.K.M.andThurmond,M.(1993b).Immunoglobulin(IgGandIgA)and complement (C3) concentrations in uterine secretion following an intrauterinechallenge of Streptococcus zooepidemicus in mares susceptible to versus resistant tochronicuterineinfection.Biol.Reprod.49,502-506.

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57. Troedsson,M.H.T.,Liu,I.K.M.andThurmond,M.(1993c).Functionofuterineandblood-

derived polymorphonuclear neutrophils in mares susceptible and resistant to chronicuterineinfection:phagocytosisandchemotaxis.Biol.Reprod.49,507-514.

58. vonReitzenstein,M.,Callahan,M.A.,Hansen,P.J.andLeBlanc,M.M.(2002).Abberations

inuterinecontractilepatternsinmareswithdelayeduterineclearanceafteradministrationofdetomidineandoxytocin.Theriogenology.58,887-898.

59. Watson,E.D.(2000).Post-breedingendometritisinthemare.Anim.Reprod.Sci.60-61,221-

232.60. Watson,E.D., Stokes,C.R. andBourne, F.J. (1987).Cellular andhumoralmechanisms in

maressusceptibleandresistanttopersistentendometritis.Vet.Immunol.Immunopathol.16,107–21.

61. Watson E.D., Stokes, C.R. and Bourne, F.J. (1988). Concentration of immunoreactive

leukotrieneB4inuterinelavagefluidfrommareswithexperimentallyinducedandnaturallyoccurringendometritis.J.Vet.Pharmacol.Therap.11,130-134.

62. Williamson,P.,Dunning,A.,O’Connor,J.andPenhale,W.J.(1983).Immunoglobulinlevels,

proteinconcentrationsandalkalinephosphataseactivityinuterineflushingsfrommareswithendometritis.Theriogenology.19,441-448.

63. Witte, T.S.,Melkus, E.,Walter, I., Senge, B., Schwab, S., Aurich, C. and Heuwieser,W.

(2012). Effects of oral treatment with N-acetylcysteine on the viscosity of intrauterinemucusandendometrialfunctioninestrousmares.Theriogenology.78,1199-1208.

64. Woodward,E.M.andTroedsson,M.H.T.(2013)Equinebreeding-inducedendometritis.J.

EquineVet.Sci.33,673–82.65. WoodwardE.M.andTroedsson,M.H.T.(2015).Inflammatorymechanismsofendometritis.

EquineVet.J.47,384-389.66. Woodward,E.M.,Christoffersen,M.,Campos,J.,Betancourt,A.,Horohov,D.,Scoggin,K.E.,

Squires,E.L.andTroedsson,M.H.T.(2013a).Endometrialinflammatorymarkersoftheearlyresponse inmares susceptibleor resistant topersistentbreeding-inducedendometritis.Reproduction.145,289-296.


andTroedsson,M.H.T.(2013b).Aninvestigationofuterinenitricoxideproductioninmaressusceptibleandresistanttopersistentbreeding-inducedendometritisandtheeffectsofimmunomodulation.Reprod.Dom.Anim.48,554–561.

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68. Woodward, E.M., Christoffersen, M., Horohow, D., Squires, E.L. and Troedsson, M.H.T.

(2015). The effect of treatment with immune modulators on endometrial cytokineexpressioninmaressusceptibletopersistentbreeding-inducedendometritis.EquineVet.J.47,235-239.

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Figure1.Graphicalrepresentationoftheclinicaltrialtimeline.IntrauterineinfusionwithN-acetylcysteine(TX)orsaline(CONTROL)wasperformedattimeofhCGinjection,withAIof1x109killedspermatozoaperformed12hours later.Maresweremonitoredbyultrasound12hourspost-AIandthenevery24hours.Samplingtimepointsindicatewhenendometrialcytology,fluidsamplesandbiopsiesweretaken(Abbreviations:hCG,humanchorionicgonadotropin;NAC,N-acetylcysteine).

ß24hrsà

hCG

TXorCONTROL

ß12hrsà

AI

ß12hrsà

96hrspostTXorCONTROL

Monitorviaultrasoundevery24hrs

12hrspost-AI 36hrspost-AI 60hrspost-AI 84hourspost-AI

ß24hrsà

EndometrialcytologyIUFcollectionEndometrialbiopsy

EndometrialcytologyIUFcollectionEndometrialbiopsy

MonitorforOvulation

0hrs

ß24hrsà

84

a b

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Figure2.Photomicrographsofendometrialbiopsies fromPBIE-susceptiblemares. Inflammationbiopsyscoreswereassignedbyablinded,boardcertifiedTheriogenologist.Scoringwasbasedondegreeanddistributionofinflammatorycellsandpresenceorabsenceofendometrialepithelialerosion(a:inflammationbiopsyscoreof1;b:inflammationbiopsyscoreof2;c:inflammationbiopsyscoreof3;d:inflammationbiopsyscoreof4).HematoxylinandEosin(H&E),x200.

c

d

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Figure3.Differencesinmeanbiopsyinflammationscore(+/-SE)onhistopathologyofuterinebiopsiesfrom9PBIE-susceptiblemarestreatedbyintra-uterineinfusionwithN-acetylcysteine(TX)andsaline(CONTROL)cycles.Maximumbiopsyscorepossiblewas4.Asignificanttreatmenteffectwaspresentwhenbothbiopsy timepoints (12and60hourspost-AI)within treatmentcycleswerecombined(p=0.01).

0

0.5

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syInfla

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Figure4.Photomicrographofendometrialbiopsy (score=4)displayingerosionofendometrialluminalepitheliumfollowingintra-uterine3.3%NACadministered12hourspriortoAIinsomePBIE-susceptiblemares.HematoxylinandEosin(H&E),x200.

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Figure5.Photomicrographofendometrialbiopsy(score=4)displayingneutrophilinfiltrationofthestratumspongiosumandglandularlumenfollowingintra-uterine3.3%NACadministered12hourspriortoAIinsomePBIE-susceptiblemares.HematoxylinandEosin(H&E),x400.

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Figure6.Meanbiopsyinflammationscore(+/-SE)onhistopathologyofuterinebiopsiesfrom9PBIE-susceptiblemaresat12and60hourspostAIwhenbothintra-uterineinfusiontreatmentcycles(TX=N-acetylcysteine,CONTROL=saline)werecombinedwithineachtimepoint.Maximumbiopsyscorepossiblewas4.Nosignificanttimeeffectwaspresent(p=0.12).

Figure7.Medianintra-uterinefluidvolume(cm3)(+/-LLandUL)of9PBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhenthetimepoints12,36,60and84hourspostAIwerecombinedwithineachtreatmentcycle.Nosignificanteffectoftreatmentwaspresent(p=0.78).

0

0.5

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12HOURS 60HOURS

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syInfla

mmationScore

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TX CONTROL

IUF(cm3)

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Figure8.Medianintra-uterinefluidvolume(cm3)(+/-LLandUL)of9PBIE-susceptiblemaresat12,36,60and84hourspostAIwhentreatmentcycles(TX=N-acetylcysteine,CONTROL=saline)werecombinedwithineachtimepoint.Differingsuperscriptsbetweentimepointsweresignificantlydifferentatp<0.05.

Figure9.Meanendometrialedemascore(+/-SE)of9PBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhenthetimepoints12,36,60and84hourspostAIwere combined within each treatment cycle. Maximum edema score possible was 4. Nosignificanteffectoftreatmentwasobserved(p=0.90).

0

5

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15

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35

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12HOURS 36HOURS 60HOURS 84HOURS

IUF(cm

3 )p=0.005

A A

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TX CONTROL

Endo

metria

lede

mascore

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Figure10.Meanendometrialedemascore(+/-SE)of9PBIE-susceptiblemaresat12,36,60and84hourspostAIwhentreatmentcycles(TX=N-acetylcysteine,CONTROL=saline)werecombinedwithineachtimepoint.Maximumedemascorepossiblewas4.Betweentimepoints,differingsuperscriptsweresignificantlydifferentatp<0.05.

Figure 11.Mean% neutrophils (+/- SE) on cytology of uterine swabs obtained from 9 PBIE-susceptible mares treated by intra-uterine infusion with N-acetyl cysteine (TX) or saline(CONTROL).Nosignificanttreatmenteffectwaspresentwhenbothcytologytimepoints(12and60hourspost-AI)withintreatmentcycleswerecombined(p=0.39).

0

0.5

1

1.5

2

2.5

3


Endo

metria

lede

mascore

AA

B B

p=0.04

0

10

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40

50

60

70

TX CONTROL

%neu

trop

hils

92

Figure 12.Mean% neutrophils (+/- SE) on cytology of uterine swabs obtained from 9 PBIE-susceptiblemares at 12 and 60 hours post AI when treatment cycles (N-acetyl cysteine=TX,saline=CONTROL) were combined within each time point. Between time points, differingsuperscriptsweresignificantlydifferentatp<0.05.Mare Treatment

Cycle12hourspost-AI

60hourspost-AI

ControlCycle

12hourspost-AI

60hourspost-AI

Daisy 3 2 4 3Daphnee 4 1 2 2Gertrude 3 4 3 1BettyBoop 3 2 3 2Janie 4 4 3 1Aster 4 4 3 1Fern 2 2 2 2Dragon 2 4 1 1Leah 2 4 3 2Table1. InflammationbiopsyscoreassignmentforeachPBIE-susceptiblemare intreatedandcontrolcyclesat12and60hourspost-AI.Scoreassignmentwasperformedbyablinded,boardcertifiedTheriogenologistandwasbasedondegreeanddistributionofinflammatorycellsaswellaspresenceorabsenceofendometrialepithelialerosion.

0

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%neu

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CHAPTERTHREE:THEEFFECTOFTREATMENTOFPBIESUSCEPTIBLEMARESWITHN-ACETYLCYSTEINEONNITRICOXIDELEVELSAND

INFLAMMATORYCYTOKINEPROFILES3.1ABSTRACT

Persistentbreeding-inducedendometritis(PBIE)isamajorcauseofinfertilityinmares.

Transient uterine inflammation is a normal response to breeding, however, PBIE susceptible

maresdonotclearthis inflammation inatimelyfashion.Uterine inflammationatthetimeof

embryonicdescent fromtheoviductsultimately results inearlyembryonicdeathand is seen

clinicallyasinfertility.N-acetylcysteine(NAC),amucolyticusedtotreatendometritisinmares,

hasanti-inflammatoryproperties,affectsinflammatorycytokinespost-treatmentandisamild

inhibitor of nitric oxide synthase. Increased nitric oxide (NO) levels and alterations in

inflammatorycytokineshavepreviouslybeendocumentedinPBIEmares.Theobjectiveofthis

studywastodetermineifNACtreatmentofPBIE-susceptiblemareswouldlowernitricoxideand

inflammatory cytokine levels. A randomized, blinded, cross-over design clinical trial was

performedutilizingPBIEsusceptiblemares (n=9).Mareswerescreenednegative forbacterial

endometritispriortoenrolment inthestudy. Intrauterineinfusionof180mlof3.3%NACwas

performed12hourspriortoinsemination,whenafollicle>35mmwaspresent.Interleukin-6

concentration (ELISA) andnitric oxide level (Colorimetric assay) in intra-uterine fluid samples

were determined at 12 and 60 hours post-insemination. Gene expression of inflammatory

cytokines (Interleukin-6, Interleukin-10, Interleukin-1β and Tumor Necrosis Factor-α) were

determined by qPCR from endometrial biopsy samples taken at 12 and 60 hours post-

insemination. Statistical analysis was performed using a repeated measures ANOVA. In this

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clinical trial, pre-breeding intrauterine treatment with NAC did not affect NO levels, IL-6

concentrationorcytokinegeneexpression.Differencesacrosstimepointspost-AIwerenoted

forIL-6geneexpression,whichwashighestat12hourspost-AI(p=0.003).Expressionofother

cytokinesevaluateddidnotchangeovertime.WhiletreatmentwithNACpre-breedingdidnot

haveaneffectonthecytokinesevaluated,theassessmentofinflammatorycytokinesbothatthe

gene and protein level at different time points post artificial insemination will add to the

understandingofthealterationsininflammatorycytokinelevelsinmaressusceptibletoPBIE.

3.2INTRODUCTION

Persistentbreedinginducedendometritis(PBIE)inmaresischaracterizedbyaprolonged

inflammatory response following breeding, ultimately leading to early embryonic loss and

infertility.Anormalandtransient inflammatoryresponseoccurspost-breeding;however, ina

subset of susceptible mares, this inflammation persists (Troedsson, 2006; Woodward and

Troedsson2015;ChristoffersenandTroedsson,2017).SusceptibilitytoPBIEisassociatedwithan

altered inflammatory response as well as poor mechanical clearance of the uterus

(NikolakopoulosandWatson1999;Watson2000;Troedsson2006; LeBlancandCausey2009;

WoodwardandTroedsson2015;ChristoffersenandTroedsson,2017;Khanetal.2018).

Theinflammatoryresponseoftheendometriumcanbecharacterizedbyalterations in

cytokines.Baselinelevelsofpro-inflammatorycytokinesaredifferentbetweenPBIE-susceptible

versus resistant mares. Specifically, during estrus, PBIE-susceptible mares have elevated

endometrial mRNA expression of IL-1β, TNF-α, IL-6 and IL-8 compared to resistant mares

(Fumosoetal.2003;Fumosoetal.2007).Additionally,endometrialmRNAexpressionofthe

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anti-inflammatorycytokineIL-10islowerduringestrusinPBIE-susceptiblemares,andhigherin

diestrus, compared to normal mares (Fumoso et al. 2007). Differences in the endometrial

inflammatoryresponsebetweennormalandPBIE-susceptiblemaresarealsoevidentfollowing

artificialinsemination(AI).ExpressionofIL-1β,TNF-α,IL-8andIL-10mRNAiselevatedsevendays

post-AIinPBIE-susceptiblemarescomparedtoresistantmares(Fumosoetal.2003;Fumosoet

al. 2007). Other differences include reduced IL-6, IL-1RN and IL-10 in susceptible mares

comparedtoresistantmares6hourspostAI(Woodwardetal.2013a).

Treatment approaches used in the management of mares with PBIE include ecbolic

therapy such as oxytocin, to enhance uterine clearance, and immunomodulators such as

dexamethasoneandmycobacterialcellwallextract(MCWE)tosuppressinflammation(LeBlanc

2010;WoodwardandTroedsson2013).TheeffectofimmunomodulationonmRNAexpression

ofinflammatorycytokineshasbeenexploredinbothsusceptibleandresistantmares(Fumosoet

al.2003;Fumosoetal.2007;Woodwardetal.2015).Itappearsthatcorrectionofthealtered

inflammatoryresponseassociatedwithsusceptibilitytoPBIEmaybeaneffectiveapproachto

treatment.

In addition to an altered inflammatory response post-breeding, researchers have

demonstratedthatelevatedlevelsofnitricoxide(NO),asmoothmusclerelaxant,maycontribute

to thedecreasedmechanical clearanceof theuterus reported inPBIEmares (Alghamdietal.

2005;Woodwardetal.2013b;Khanetal.2017).Induciblenitricoxidesynthase(iNOS)isthe

enzyme responsible for theproductionof inducedNO in theequineuterus. PBIE susceptible

mareshadelevatedNOlevelsintheiruterinesecretionsandincreasediNOSmRNAexpressionat

13hoursfollowingAI(Alghamdietal.2005).Adose-dependentinhibitionofNOonmyometrial

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contractilitywasdemonstratedinvitro(Khanetal.2017),suggestingthatalteredNOlevelsin

PBIE-susceptiblemaresplayaroleinreducedmyometrialcontractilityanduterineclearance.

Given what is currently understood about the pathophysiology of PBIE, use of a

compoundexhibitingbothanti-inflammatoryandNO-suppressiveeffectsshouldbeaneffective

approachtotreatment.N-acetylcysteine(NAC)isamucolyticagentusedintreatmentofequine

bacterialendometritiswhereabiofilmissuspected,orincaseswhereexcessuterinemucusis

present (LeBlanc andCausey 2009;Gores-Lindholmet al. 2009; LeBlanc 2010). Intra-uterine

treatmentwitha3.3%NACsolutioninmareswithahistoryofendometritis,eitherduringthe

previouscycleor48hourspriortobreeding,leadstohigherthananticipatedfertilityrates(85%)

(Gores-Lindholmetal.2009;LeBlanc2010).N-acetylcysteine(NAC)reducesmucusviscosityby

disrupting disulphide bonds (LeBlanc 2010), and hasmild anti-oxidant and anti-inflammatory

effectsinmultiplemodelsofdisease(Stanislausetal.2005;Cazzolaetal.2017).Inhorses,NAC

inhibitedserum-derivedneutrophiloxidativeburstwheninfusedasa3%solutionanddidnot

haveanadverseeffectontheendometriumofeitherfertileorbarrenmares(Gores-Lindholmet

al.2013).Intra-uterineandoraladministrationofNACinmaresledtodecreasedneutrophiland

COX-2 staining on histologic endometrial samples from normal mares, confirming an anti-

inflammatoryeffect(Witteetal.2012;Melkusetal.2013).However,nostudiestodatehave

examinedeffectsofintrauterineinfusionofNAConNOlevels,cytokinemRNAexpression,oron

cytokineconcentrationinuterinefluid.

Theobjectiveofthisstudywastodetermineifintra-uterineinfusionofPBIE-susceptible

mareswitha3.3%NACsolutioninsalinewoulddecreaseNOlevelsandaltertheendometrial

cytokineprofilecomparedtocontrolcycles.

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3.3MATERIALSANDMETHODS

AlltheproceduresperformedonanimalsinthisstudywereapprovedbytheUniversity

ofGuelphAnimalCareCommittee(AnimalUtilizationProtocolnumber3213),inaccordancewith

theCanadianCouncilonAnimalCareguidelines.

3.3.1MareSelection,StudyDesignandSampling

Arandomized,blinded,crossoverdesignedclinicaltrialwasperformedwithawashout

period(minimumoneestrouscycle)betweenexperimentalcycles.Theprimaryinvestigator,who

performedallclinicalevaluations,wasblindedtomareassignment(treatedversuscontrolcycles)

duringdatacollection.Inordertorandomizetheorderoftreatmentandcontrolcycles,mares

wereassignedanumber,withthosenumberssubsequentlyrandomlysortedintotreatedand

controlcycles.

MaressusceptibletoPBIE(n=10)wereselectedforinclusionintheclinicaltrialbasedon

an endometrial biopsy score of IIb or III, normal cytology and negative bacterial culture on

endometrial swab, and persistent accumulation of intra-uterine fluid 96 hours following

challengeby inseminationwithkilledsemen(Alghamdietal. 2005;Woodwardetal. 2013a;

Woodward et al. 2013b; Woodward et al. 2015). Mares were housed at the Arkell Equine

ResearchStationattheUniversityofGuelphforthedurationofthestudyandwereofvarious

breeds(Standardbredandmixedbreed)andages(7-19years).

Mares weremonitored for estrus by trans-rectal palpation and ultrasound.Mares in

diestrus,determinedbypresenceofacorpusluteum(CL),weregivenanintra-muscularinjection

ofcloprostenol(125mcg)(Estrumate®,IntervetCanadaCorp.,Kirkland,QC,Canada)toinduce

estrus.Onceafollicle>35mmwasidentifiedduringestrus,maresweregiven2,500IUofhuman

98

chorionic gonadotropin (hCG, Chorulon®, Intervet Canada Corp., Kirkland, QC, Canada)

intravenously,toinduceovulation.Usinga21-inchinseminationpipette(PartnarAnimalHealth,

Ilderton,ON,Canada),maresweretrans-cervically infusedwitheithera3.3%solutionofNAC

consisting of 30ml of N-acetyl cysteine (Acetylcysteine Injection 200mg/ml, Alveda

Pharmaceuticals Inc., Toronto, ON, Canada) added to 150ml of 0.9% sodium chloride (0.9%

SodiumChlorideInjectionUSP,BaxterCorporation,Mississauga,ON,Canada)(treatedcycle)or

180ml of 0.9% sodium chloride (0.9% Sodium Chloride Injection USP, Baxter Corporation,

Mississauga, ON, Canada) (control cycle). Twelve hours following infusion, the mares were

inseminatedwith1x109killedspermatozoain30mlofsemenextender,aspreviouslydescribed

(Woodwardetal.2013a;Woodwardetal.2013b;Woodwardetal.2015;Khanetal.2018).

Trans-rectalultrasonographywasperformedbeginning twelvehoursafterAI,andwas

repeatedevery24hoursthereafteruntil84hourspost-AI(12,36,60and84hours),toevaluate

endometrial edema grade (0-4; 0 = no edema; 4 = pathologic edema) and endometrial fluid

volume.FluidvolumewascalculatedusingtheformulaV=length*width*depth,eachmeasured

incm.Multiplelocationsweremeasuredintheuterinebodyandhorns,andanaverageof2-5

locationswereused in the calculation. Additional samples takenat12and60hourspost-AI

consistedofanendometrial swab forcytology,collectionofuterine fluidandanendometrial

biopsy. Cytology samples were aseptically obtained using a guarded uterine swab (Kalayjian

IndustriesInc.,SignalHill,CA,USA)aspreviouslydescribedbyKhanetal.(2018).Endometrial

biopsy specimens were procured in an aseptic manner using a Pilling-Weck biopsy punch

(JorgensenLaboratories,Loveland,CO,USA),asdescribedbyKenney(1978).Theendometrial

biopsy sample was immediately placed in 655µL of RNAlater (Sigma-Aldrich®, Oakville, ON,

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Canada)andstoredovernightat4°C.Thefollowingmorning,itwasremovedfromtheRNAlater

andstoredat-80°Cuntilanalysis,asperthemanufacturer’sinstructions.

3.3.2Real-TimeQuantitativePCR

A pre-trial endometrial biopsy samplewas obtained from a subset ofmares (n=4), in

additiontoendometrialbiopsiesobtainedat12and60hourspost-AIforallmares.TotalRNA

fromendometrialtissuewasisolatedwiththeQiagenRNeasy™kit(©QIAGEN,Toronto,CAN),

accordingtothemanufacturer'sprotocol.FirststrandcDNAwassynthesizedfrom200ngofRNA

with50μmoligoprimerandreversetranscriptaseenzyme.ThereferencegenesSDHA,β-actin,

EeF2,RPS23andGAPDHwereamplifiedfromdifferentconcentrationsoftemplatecDNA.Most

reference genes were found to be inconsistent among mares, therefore, a specific set of

referencegeneswasoptimizedforeachmareandusedtodeterminetherelativeexpressionof

thegeneof interest foreachmare (Table1).Reverse transcriptionofmRNAwas initiated to

inactivate the reverse transcriptase and simultaneously activate the thermostable DNA

polymerase.cDNAwasamplifiedintriplicatebyqRT-PCRinaLightCycler480instrument(Roche

LifeScience,Laval,PQ)usingSYBRGreen(Roche)andprimerstoequineIL-1β, IL-6,IL-10,and

TNF-α (Table 2). Cycle threshold (CT) valueswere calculated and normalized to eachmare’s

specificreferencegenepair(calibratornormalizedrelativequantification).

3.3.3Intra-UterineFluidCollection

Intra-uterinefluidwascollectedinoneoftwoways.Inmareswithlow-to-moderatefluid

volume,aBivonacatheter(PartnarAnimalHealth)waspassedasepticallythroughthecervixand

into themare’suterus. After inflating the cuff, ahandheld suctionpump (NalgeneTM hand-

operatedvacuumpump,Sigma-Aldrich®Oakville,ON,Canada)wasattachedtothecatheterand

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anassistantappliedexternalsuctionuntilfluidwasobtainedinthetube.Inmareswithalarge

volumeof intra-uterine fluid, a samplewas readilyobtainedby free-flowduringendometrial

swabsampling,throughtheouterplasticguard.Thefluidsamplewasimmediatelyplacedina

polypropylene centrifuge tube (Corning® Incorporated, Corning, NY, USA) and held at room

temperatureuntilprocessing.Oncereturnedtothelaboratory,thefluidwascentrifugedat1500

x g for 12minutes to remove cellular debris. The supernatant was aspirated, placed in 1ml

aliquots (Fisherbrand®, Cryogenic Vial 1.2mL, Fisher Scientific, Markham, ON, Canada) and

storedat-20°Cforfurtheranalyses.

3.3.4ELISADeterminationofInterleukin-6ConcentrationinUterineFluid

Intrauterine IL-6 concentration was determined using the Equine IL-6 DuoSet®ELISA

(R&DSystems™,Minneapolis,MN,USA)accordingtomanufacturerinstructions.Sampleswere

diluted1:20inreagentdiluent(1%BSAinPBS)andruninduplicate.Theintra-assaycoefficient

ofvariationwas7.4%andtheinter-assaycoefficientofvariationwas10.9%.Thekitwasvalidated

forequine intrauterine fluidbydeterminationofpercent recoveryand linearity,according to

R&DSystems™Spike,Recovery&LinearityProtocol.Thedetailedvalidationprocesscanbefound

inAppendix2.

3.3.5NitricOxideConcentration

Nitricoxideisunstableandhasashorthalf-lifeinvivo,necessitatingthemeasurementof

its metabolites, nitrate and nitrite, as alternatives. The concentration of nitrate and nitrite

presentinintrauterinefluidwasmeasuredtoprovideanestimateofNOconcentration,usingthe

Nitrate/NitriteColorimetricAssayKit(CaymanChemicalCo.,AnnHarbor,MI,USA),accordingto

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the manufacturer’s instructions. Samples were diluted 1:5 with buffer solution, and run in

duplicate.

3.3.6StatisticalAnalyses

Statistical analyseswereperformedusing the SAS softwareprogram (version9.3, SAS

Institute,Inc.,Cary,NC,USA).OnlyestrouscyclesfittingthedefinitionofPBIEwereincludedin

theanalysis(n=9salinecontrol;n=9NACtreatmentcycles).Differences inendometrialmRNA

expressionofIL-6,IL-10,TNF-αandIL-1β,aswellasIL-6andNOconcentrationsinuterinefluid,

werecomparedbetweencontrolandtreatedcyclesovertime(12hand60hpost-AI)byrepeated

measuresANOVA.TheAkaikeInformationCriterion(AIC)wasutilizedtodeterminethebest-fit

model.TheShapiro-Wilktestdeterminedthatthedatawasnotnormallydistributedandrequired

logarithmictransformationpriortoanalysis,thereforeresultsarepresentedasmedians(back

transformed)+/-upperandlowerconfidencelimits.

Interactioneffectsbetweentreatmentandtimewereexaminedinthemodel,andthep

valuewas set at <0.05 for statistical significance.When therewas no statistically significant

treatment-by-time interaction seen, thedata fromboth treatmentgroupswere combined to

examinetheeffectoftimepost-AI,anddatafrombothtimepointswerecombinedwithineach

treatmentgrouptoevaluateforanymaineffectsoftreatment.

Forthesubsetofmareswithbaselinebiopsiesavailable(T=0;n=4),differencesinIL-6,IL-

10,TNF-αandIL-1βmRNAexpressionwerealsocomparedbetweenbaseline,12hoursand60

hourspost-AIusingrepeatedmeasuresANOVA.

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3.4 RESULTS

3.4.1EffectofIntra-UterineTreatmentof3.3%NACinPBIESusceptibleMares

When both time points were combined within each treatment group, intrauterine

infusionwith3.3%NAC12hourspriortoAIdidnotaffectendometrialmRNAexpressionofIL-6

(p=0.86)(Appendix3:Figure1),IL-10(p=0.58)(Appendix3:Figure2),TNF-α(p=0.92)(Appendix

3:Figure3),orIL-1β(p=0.65)(Appendix3:Figure4)comparedtountreatedcycles(Table3).NAC

infusion had no effect on the concentration of IL-6 in uterine fluid as determined by ELISA

(p=0.083)(Figure1),nordiditaffecttheamountofNOinuterinefluid(p=0.46)(Figure2).

3.4.2ChangesinEndometrialInflammationinResponsetoAIOverTime

a) mRNAexpression–12versus60hours(n=9mares)

EndometrialIL-6mRNAexpressionwassignificantlygreaterat12hourspostAIcompared

to60hourspostAI,whenbothtreatmentgroupswerecombinedwithineachtimepoint(p=0.02)

(Figure3).Inthecontrolcycle,IL-6mRNAexpressionat12hourspostAIwassignificantlygreater

thanat60hourspostAI(p=0.02)(Appendix3:Figure5).EndometrialmRNAexpressiondidnot

changefrom12to60hoursforIL-10(p=0.73)(Appendix3;Figure6),TNF-α(p=0.86)(Appendix

3:Figure7)andIL-1β(p=0.6)(Appendix3:Figure8),whenbothtreatmentgroupswerecombined

withinineachtimepoint(Table4).

b) mRNAexpression–baselineversus12and60hourspost-AI(n=4)(Table5)

Forthesubsetoffourmareswithbaselinebiopsies,therewasanoverallsignificantmain

effectoftimeonIL-6mRNAexpression(p=0.0004)(Figure4)aswellasanoverallsignificanteffect

oftimeonIL-1βmRNAexpression(p=0.0030)(Figure5).Incontrast,mRNAexpressionofIL-10

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(p=0.1779)(Figure6)andTNF-α(p=0.0744)(Figure7)didnotchangeovertime.Nosignificant

treatmentmaineffectwasseenforthesubsetofmareswithbaselinebiopsies.

3.4.3IL-6ConcentrationasMeasuredbyELISA

IL-6concentration in intrauterinefluid (ELISA)didnotchangeovertimepostAI,when

bothtreatmentgroupswerecombinedwithinineachtimepoint(p=0.42)(Figure8).Therewas

notimebytreatmentinteraction.

3.4.4Nitricoxide

AlthoughNOincreasedfrom12to60hourspost-AI,thechangeinNOconcentrationover

timedidnotreachsignificance,whenbothtreatmentgroupswerecombinedwithinineachtime

point(p=0.056)(Figure9).Therewasnotimebytreatmentinteraction.

3.5DISCUSSION

Theanti-inflammatoryeffectsofNAC,includingcytokinemodulation,havebeenshown

inmultiplemodels of animal andhumandisease. In a human chronic obstructivepulmonary

disease (COPD) in vitro study, NAC inhibited pro-inflammatory cytokines (IL-1β, IL-8, TNF-α)

(Cazzolaetal. 2017).Similarly, inamultiplesclerosis (MS)study, treatmentwithNAC led to

inhibitionofmonocyte/macrophageinducedTNF-α,IFN-γ,IL-1βproductionandstimulatedIL-10

production(Stanislausetal.2005).However,theresultsofthecurrentstudysuggestthatthe

anti-inflammatoryeffectsofintra-uterineinfusionofa3.3%NACsolutionweretoomildtoresult

inalterationsofcytokineexpression.Bycomparison,otherimmunomodulatorytherapieshave

demonstratedthepotentialtoaltertheinflammatorycytokineprofileofPBIE-susceptiblemares.

Some treatment modalities (lactoferrin, Mycobacterium Cell Wall Extract (MCWE) and

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dexamethasone)lowerpro-inflammatorycytokinemRNAexpression,suchasTNF-α(Fedorkaet

al.2016)andIL-1β(Fumosoetal.2003;Woodwardetal.2015)andincreasemRNAexpression

of IL-10,ananti-inflammatorycytokine(Fumosoetal. 2007)to levelsmoretypicalofnormal

mares.However,manyoftheothercytokinesevaluatedinthesestudies(IL-6,IL-1RN,IL-8)were

unaffectedby these treatmentmethods. It is possible that a treatment effect followingNAC

infusionwasnotapparentduetothechoiceofcytokinesevaluatedinthecurrentstudy;further

studiesexaminingtheeffectsonotherendometrialcytokinesmayelicitadditionalinformation.

OtherpossiblereasonsforthelackoftreatmenteffectofNACincludetheselectionoftimepoints

forsampling,differencesinthedefinitionofPBIEmaresbetweenstudiesanddifferencesinqPCR

methodology,includingtheselectionofreferencegenes.

Methodologies used in qPCR differ across studies and may account for variability in

endometrialcytokinemRNAexpression.Specifically,thechoiceofreferencegene(s)appearsto

vary.Inmoststudies,onlyonereferencegeneisselectedtodeterminerelativegeneexpression,

andlimitedreportingofthevalidationprocessisgiven(Fumosoetal.2003;Fumosoetal.2007;

Christoffersenetal.2012a;Christoffersenetal.2012b;Woodwardetal.2013a;Woodwardet

al.2013b;Woodwardetal.2015;Fedorkaetal.2016;Fedorkaetal.2017;Fedorkaetal.2018).

Recently,ithasbeensuggestedthatmorethanonereferencegeneshouldbeselected,inorder

toincreasethevalidityoftheanalysis(Bustinetal.2009).Inaddition,referencegenespreviously

believedtobestablehavesincebeenshowntohavealargedegreeofvariationacrossstudies

(Kozeraet al. 2013). Therefore, validation of chosen reference genes specific to the species,

experimentaldesign,andtissueofinterestwouldimproverepeatabilityacrossstudies(Jacobet

al.2013;Kozeraetal.2013;Bustinetal.2017).Significantvariabilityisknowntooccurinequine

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geneexpressionacrossdifferenttissues(Tessieretal.2017).Inthepresentstudy,twostable

referencegeneswereselectedandvalidatedforeachmare,andsevenoftheninemaresshared

atleastonegenewithinthereferencegenepairs(EeF2)(Table1).GiventhatcytokinemRNA

expressionismeasuredrelativetothatofselectedreferencegenes,itisthereforepossiblethat

someofthedifferencesnotedacrossstudieswithrespecttorelativecytokinegeneexpressionin

equineendometritis,areduetothedifferencesinresearchmethodologiesandtheselectionof

referencegenesbetweenstudies.

Intra-uterineinfusionwithNACdidnotalterNOlevelspost-AIinPBIE-susceptiblemares.

Elevated intra-uterine NO levels in PBIE susceptible mares are suspected to play a role in

decreaseduterinecontractilityanduterineclearance(Alghamdietal. 2005;Woodwardetal.

2013b;Khanetal.2017;Khanetal.2018).Alghamdietal.(2005)showedthatPBIEsusceptible

mareshadhigherNOintheiruterinesecretionsandhigheriNOSmRNAexpression13hourspost-

AIcomparedtoresistantmares.Inaddition,Woodwardetal.(2013b)showedthatsusceptible

mareshadelevatedmRNAexpressionofiNOSat2and6hourspost-AI.Nitricoxideproduction

andiNOSsynthesiswerereducedbytreatmentwithNACinratin-vitrostudies,murinepre-term

laborstudies,andmultiplesclerosisstudies(Pahanetal.1997;Bergaminietal.2001;Majano

etal.2004;Stanislausetal.2005;Paintliaetal.2008).Mycobacteriumcellwallextract(MCWE),

animmunomodulator,givenIV24hourspriortoAIdecreaseduterineNOinsusceptiblemares.

Inthesamestudy,administrationof50mgofdexamethasoneIV24hourspriortoAI,didnot

decreaseuterineNOconcentrations inPBIE-susceptiblemares (Woodwardetal. 2013b). In-

vitro treatment of endometrial tissue sampleswith 1400W, a highly specific iNOS inhibitor,

resultedindecreasedendometrialNOSactivityinbothsusceptibleandresistantmares(Khanet

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al. 2018). ItappearsthatNACisaverymild iNOSinhibitor,andthedoseandtimingofNAC

administrationinthisstudymayexplainthelackofeffectsseen.

The preparation and dose of 3.3% NAC intra-uterine infusion was chosen for several

reasons.ThisdosewasfirstdescribedbyLeBlancandCausey(2009)asbeingmucolyticandable

todislodgeinspissateduterinesecretionsinmareswithahistoryofinfertility.Theeffectsofa

3.3%NACintra-uterineinfusionwerefurthercharacterizedbyGores-Lindolhmetal.(2009)and

LeBlanc (2010), who showed improved pregnancy rates (85%) in mares with a history of

endometritis.Further,Gores-Lindholmetal.(2013)demonstratedthattreatmentwithNACat

this dose led to decreased uterine mucus thickness, and had no adverse effects on the

endometrium in barren and healthymareswith Kenney biopsy scores ranging from I to IIb.

Pregnancy rates of 77% were attained when using this dose combined with uterine lavage,

oxytocintreatmentandas-neededintra-uterineantibiotics.Sincethattime,3.3%NAChasbeen

recommendedforcasesofmucushypersecretionorinmareswithsuspectedbiofilm(Lyle2012;

Troedsson2012;Buczkowskaetal.2015;Scoggin2016).Recently,aninvitrostudydemonstrated

that3.3%NACdisruptedE.colibiofilmandreadilykilledE.coliwithinthebiofilm(Ferrisetal.

2016).However,itispossiblethatthedosechosenwastoolowtoaffectcytokineexpressionand

NOconcentrations. Tirouvanziametal. (2006) showed thatonlyhighdosesofNACaffected

cytokine concentration in a human cystic fibrosis study. In addition, NO release from rat

peritoneal macrophages was significantly reduced when higher doses of NAC were utilized

(Pahanetal. 1997). Inmostofthesestudies,NACwasadministeredpre-breedingorpriorto

inductionof inflammation, justifying the timingofadministration inour study.However, it is

possible that treatment during an inflammatory responsemay bemore beneficial than pre-

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treatment.Infact,Tirouvanziametal.(2006)showedclinicalimprovementinalreadyillpatients

treatedwithoralNAC.

Interleukin-6wastheonlycytokineshowingsignificantdifferencesinrelativeexpression

across timepoints. Increasedexpressionof IL-6at12hourscomparedto60hourspost-AI, is

similartothefindingsofWoodwardetal.(2013a),whoshowedelevatedIL-6mRNAexpression

between2and12hourspost–AI,andFumosoetal.(2003)whoshowedhigherexpressionofIL-

6mRNAat24hourscomparedto7dayspost-AI.Whilethesetimelinesarenotidenticaltothose

choseninourstudy,thetrendsnotedbetweenstudiesaresimilarinthatIL-6mRNAexpression

iselevatedattimepointsnearestAIanddecreasesovertime.

ThelackofdifferencesobservedforendometrialIL-10,TNF-αandIL-1βmRNAexpression

between12and60hourspost-AIisincontrasttootherstudies,wheresomeofthesecytokines

changedintheirexpressionovertime.Interleukin-10waselevatedinsusceptiblemaresat2and

24hours,andIL-1βmRNAexpressionwasincreasedat2and6hourscomparedtobaselinelevels,

followingAI(Woodwardetal.2013a).Interleukin-10mRNAexpressioninsusceptiblemareswas

lowerat24hourscomparedto7dayspost-AI(Fumosoetal.2007).Interestingly,thesestudies

alsoshowednochangesforothercytokines.NochangesinTNF-αmRNAexpressionwerenoted

inWoodwardetal.(2013a),nordidIL-1βmRNAexpressionchangebetween24hoursand7days

post-AI(Fumosoetal.2003).

Baseline cytokine mRNA expression is reportedly different in PBIE susceptible mares

comparedtonormalmares(Fumosoetal.2003;Fumosoetal.2007).Comparisonstocytokine

mRNAexpressionatbaselinehavealsobeenmadeinPBIEsusceptiblemaresfollowingAI,with

IL-1β, IL-6, IL-8, IL-10and IFN-γbeingelevated frombaselineatvarious timepoints following

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insemination (Woodward et al. 2013a). In our study, although only a subset of mares had

baselinebiopsiestakenforqPCRanalysis,baselinemRNAexpressionwaslowerforallcytokines

tested,andincreasedat12hoursfollowingAI.Thiselevationincytokineexpressiondecreased

by60hourspost-AI,butdidnotreturntobaselinelevels.ThechangesobservedinIL-6andIL-1β

mRNAexpressionovertimearereflectiveoftheinflammatoryresponsetoAI.However,thelack

of significant changes across time for IL-10 and TNF-α mRNA expression from baseline was

unexpected.TumorNecrosisFactor-αispro-inflammatoryandwouldbeexpectedtorisenear

thetimeofAItoevokeendometrialinflammation.Interleukin-10isanti-inflammatoryandwould

beexpectedtorisesometimeafterAI,tomediatetheinflammatoryresponse.Ourresultsare

similartoWoodwardetal.(2013a)whofoundnodifferencesinTNF-αfollowingAIinsusceptible

mares and amuch lower expressionof IL-10 in susceptiblemares in comparison to resistant

mares.IL-10andTNF-αmayplaylessofaroleinPBIEthanpreviouslysuspected.

Despite an increase in IL-6 mRNA expression 12 hours post-AI, intra-uterine IL-6

concentrationasmeasuredbyELISA,wasnotsignificantlydifferentacrosstimepointspost-AIin

this study. However, IL-6 concentration was greatest at 12 hours post-AI, even though not

statisticallysignificant,demonstratingthatIL-6concentrationfollowsthesamepatternasmRNA

expression.Inthepresentstudy,considerablevariabilityinIL-6concentrationacrossmaresmade

detectionofdifferencesdifficult,and the lackof significancecouldbeattributed to the large

variabilityinourdata.

Inconclusion,treatmentwith3.3%NACasanintrauterineinfusion12hourspriortoAI

didnotaffectmRNAexpressionofIL-6,IL-10,TNF-αandIL-1β,nordiditaffectintra-uterineIL-6

concentration and NO levels, in PBIE-susceptible mares. In general, when comparing mRNA

109

expressiontobaselinevaluesforthesubsetofmareswithpre-trialbiopsies,itappearsthatAIin

itselfcausesanincreaseinmRNAexpressionacrosstimepointspost-AI.Subsequentdifferences

forallmaresbetween12and60hourspost-AIarelessevident,notablyforIL-10,TNF-αandIL-

1β,butareclearforIL-6. IL-6mRNAexpressionisgreatestat12hourscomparedto60hours

post-AI,implicatingitaslikelyplayingaroleinthemanifestationofPBIEinsusceptiblemares.

FuturestudiesshouldbeaimedatcharacterizingtheinflammatorycytokinemRNAexpression

patternpost-AIandhowitdiffersfromnormalinPBIEsusceptiblemares.Fromthere,whatis

knownoftheeffectofdifferenttreatmentmodalitiesonthesecytokinesatspecifictimepoints

couldbeutilizedaccordinglyintreatingPBIEsusceptiblemares.

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40. Woodward,E.M.andTroedsson,M.H.T. (2013)Equinebreeding-inducedendometritis.J.EquineVet.Sci.33,673–82.

41. Woodward,E.M.andTroedsson,M.H.T.(2015).Inflammatorymechanismsofendometritis.

EquineVet.J.47,384-389.42. Woodward,E.M.,Christoffersen,M.,Campos,J.,Betancourt,A.,Horohov,D.,Scoggin,K.E.,

Squires,E.L.andTroedsson,M.H.T.(2013a).Endometrialinflammatorymarkersoftheearlyresponse inmares susceptible or resistant to persistent breeding-induced endometritis.Reproduction.145,289-296.


andTroedsson,M.H.T.(2013b).Aninvestigationofuterinenitricoxideproductioninmaressusceptibleand resistant topersistentbreeding-inducedendometritis and theeffectsofimmunomodulation.Reprod.Dom.Anim.48,554–561.

44. Woodward, E.M., Christoffersen, M., Horohow, D., Squires, E.L. and Troedsson, M.H.T.

(2015). The effect of treatment with immune modulators on endometrial cytokineexpressioninmaressusceptibletopersistentbreeding-inducedendometritis.EquineVet.J.47,235-239.

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Figure1.MedianIL-6concentrationinintra-uterinefluid(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemares(n=9) inN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhentimepoints(12h,60hpost-AI)werecombinedforeachcycle.Differingsuperscriptsaresignificantatp<0.05.

Figure2.Mediannitricoxideinintra-uterinefluid(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemares (n=9) inN-acetylcysteinetreated(TX)andsaline (CONTROL)cycleswhentimepoints (12h,60hpost-AI)werecombinedforeachcycle.Differingsuperscriptsaresignificantatp<0.05.

0

5

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IL-6Con

centratio

n(ng/ml)

AA

0

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30

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50

60

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NO(µM)

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Figure3.MedianIL-6mRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemares(n=9)at12and60postAIwhentreatmentcycleswerecombinedwithineachtimepoint.Asignificanttimeeffectwasobserved(p=0.02).Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQ,relativequantification).

Figure4.MedianIL-6mRNAexpression(includinglowerandupperconfidencelimits)inasubsetofPBIE-susceptiblemareswithbaselinebiopsies(n=4).Differencesbetweenbaseline,N-acetylcysteinetreated(TX)at12hoursand(TX)at60hourspost-AIaredepictedbydifferinguppercaseletters.Differencesbetweenbaseline,salinetreated(CONTROL)at12hoursand(CONTROL)at60 hours are depicted by differing lower case letters. Significance was defined as p<0.05(Abbreviations:RQ,relativequantification).

0

0.2

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0.6

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12HOURS 60HOURS

Relativ

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RNAexpressio

nofIL-6

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B

p=0.02

00.20.40.60.81

1.21.41.61.82

2.2

Baseline 12hours 60hours

Treated

Control

aA

B

A a

b

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Figure5.MedianIL-1βmRNAexpression(includinglowerandupperconfidencelimits)inasubsetofPBIE-susceptiblemareswithbaselinebiopsies(n=4).Differencesbetweenbaseline,N-acetylcysteinetreated(TX)at12hoursand(TX)at60hourspost-AIaredepictedbydifferinguppercaseletters.Differencesbetweenbaseline,salinetreated(CONTROL)at12hoursand(CONTROL)at60 hours are depicted by differing lower case letters. Significance was defined as p<0.05(Abbreviations:RQ,relativequantification).

Figure6.MedianIL-10mRNAexpression(includinglowerandupperconfidencelimits)inasubsetofPBIE-susceptiblemareswithbaselinebiopsies(n=4).Differencesbetweenbaseline,N-acetylcysteinetreated(TX)at12hoursand(TX)at60hourspost-AIaredepictedbydifferinguppercaseletters.Differencesbetweenbaseline,salinetreated(CONTROL)at12hoursand(CONTROL)at60 hours are depicted by differing lower case letters. Significance was defined as p<0.05(Abbrevations:RQ,relativequantification).

0

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Figure7.Median TNF-αmRNAexpression (including lower andupper confidence limits) in asubsetofPBIE-susceptiblemareswithbaselinebiopsies(n=4).Differencesbetweenbaseline,N-acetylcysteinetreated(TX)at12hoursand(TX)at60hourspost-AIaredepictedbydifferingupper case letters. Differences between baseline, saline treated (CONTROL) at 12 hours and(CONTROL)at60hoursaredepictedbydifferinglowercaseletters.Significancewasdefinedasp<0.05(Abbreviations:RQ,relativequantification).

Figure8.DifferencesinmedianIL-6concentrationinintra-uterinefluid(includingLLandUL)ofPBIE-susceptiblemaresat12and60hourspostAI.Differingsuperscriptsaresignificantatp<0.05.

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Figure9.DifferencesinmedianNOinintra-uterinefluid(includingLLandUL)ofPBIE-susceptiblemaresat12and60hourspostAI.Differingsuperscriptsaresignificantatp<0.05.

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DAISY GERTRUDE DRAGON ASTER LEAH DAPHNEE JANIE BettyBoop

FERN

Referencegenes

EeF2/B-actin √

RPS23/SDHA √ √EeF2/GAPDH √

EeF2/RPS23 √ √ √

EeF2/SDHA √ √

Table1.PairsofreferencegenesusedinclinicaltrialforeachPBIE-susceptiblemare. ForwardPrimer ReversePrimerEeF2 CCAACGGCAAGTTCAGCAAG GCTTCTCGATCAGCTTTGCCGAPDH GTTTGTGATGGGCGTGAACC TTGGCAGCACCAGTAGAAGCSDHA GCAGAAGAAGCCATTTGAGG CCTGTCGATTACGGGTCTGTRPS23 ACGAGATCAGAAGTGGCACG ACCTTAAAGCGGACTCCAGGβ-actin GACCCAGATCATGTTTGAGACCT TGATGGAGTTGAAGGTAGTTTCG TNF-α AGCCCATGTTGTAGCAAACC CACTTTGGGGTGATCGGCIL-6 CCCGTGACCCAACTGCAA TGTTGTGTTCTTCAGCCACTCAIL-10 CCAGTCTGAGAACAGCTGCA GCTCCACTGCCTTGCTCTTAIL-1β GGGTTTGAACATGCTGCAGG CTCATCAGAAGCTGGGTGCATable2.Primersequencesforamplificationofreferencegenesandselectedcytokines.

Table3.TableofmedianmRNAexpressionwithlower(LL)andupper(UL)limitsofIL-6,IL-10,TNF-α and IL-1β in PBIE-susceptiblemares, following intra-uterine treatmentwith 3.3%NACtreated(TX)andsaline(CONTROL)cycles(n=9)whenbothtimepoints(12h,60hpost-AI)werecombinedwithineachtreatmentcycle.

Cytokine MedianmRNAExpression:NACTreatmentCycle

LL

UL MedianmRNAExpression:SalineControlCycle

LL

UL P-value

IL-6 0.28 0.11 0.7 0.26 0.1 0.64 0.86IL-10 0.018 0.008 0.04 0.024 0.01 0.052 0.58TNF-α 0.09 0.05 0.15 0.09 0.05 0.16 0.92IL-1β 0.011 0.004 0.026 0.013 0.005 0.032 0.65

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Table4.TableofmedianmRNAexpressionwithlower(LL)andupper(UL)limitsofIL-6,IL-10,TNF-α and IL-1β at12and60hourspost-AI inPBIE-susceptiblemares (n=9)when treatmentcycleswerecombinedateachtimepoint.

CytokineBaseline

mRNAExpressionMean(LL,UL)

Cycle12hpost-AI

mRNAExpressionMean(LL,UL)

60hpost-AImRNAExpressionMean(LL,UL)

IL-6 0.013(0.003,0.052) CONTROL 2.11

(0.53,8.28)0.068

(0.017,0.27)

TX 0.79(0.2,3.11)

0.07(0.02,0.30)

IL-10 0.006(0.001,0.026) CONTROL 0.03

(0.008,0.15)0.012

(0.003,0.05)

TX 0.03(0.006,0.13)

0.006(0.001,0.03)

TNF-α 0.05(0.03,0.09) CONTROL 0.08

(0.05,0.15)0.08

(0.04,0.14)

TX 0.17(0.09,0.30)

0.06(0.03,0.11)

IL-1β 0.003(0.001,0.008) CONTROL 0.04

(0.02,0.09)0.005

(0.002,0.01)

TX 0.02(0.008,0.04)

0.008(0.003,0.02)

Table5.TableofmedianmRNAexpressionwithlower(LL)andupper(UL)confidencelimitsofIL-6,IL-10,TNF-αandIL-1βatbaseline,12and60hourspost-AIfortheN-acetylcysteinetreated(TX)andsaline(CONTROL)cyclesforasubsetofPBIE-susceptiblemares(n=4).

Cytokine MedianmRNAexpression12hourspost-AI

LL

UL MedianmRNAexpression60hourspost-AI

LL

UL Pvalue

IL-6 0.5 0.2 1.25 0.14 0.06 0.36 0.02IL-10 0.023 0.01 0.05 0.019 0.009 0.04 0.73TNF-α 0.091 0.05 0.16 0.086 0.05 0.15 0.86IL-1β 0.013 0.005 0.03 0.010 0.004 0.026 0.60

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CHAPTERFOUR:GENERALDISCUSSION

4.1POSSIBLELINKSBETWEENMRNAEXPRESSIONANDPBIECLINICALSIGNS

Persistentbreeding inducedendometritis continues tobea sourceof infertility in the

equinebreedingindustry(Newcombe1997).PBIEiscausedbymechanicalfactors,suchaspoor

myometrialcontractility,andbydifferencesintheuterineimmuneresponsefollowingbreeding

(ChristoffersenandTroedsson2017).Altered inflammatorycytokineexpression isbelieved to

playaroleinPBIE,withsusceptiblemareshavingincreasedandpersistentmRNAexpressionof

pro-inflammatory cytokines and decreased mRNA expression of anti-inflammatory cytokines

followingAI(WoodwardandTroedsson2015;ChristoffersenandTroedsson2017).Manystudies

haveexaminedcytokinemRNAexpressioninbothPBIEresistantandsusceptiblemares,butnone

haverelatedmRNAexpressiontoclinicalsignsassociatedwithPBIE.N-acetylcysteine,usedasan

intra-uterinemucolyticinmares,hasalsobeenshowntohaveanti-inflammatoryproperties,such

asaffectingcytokinemRNAexpressioninothermodelsofdisease(Stanislausetal.2005;LeBlanc

2010;Cazzolaetal.2017).

TheclinicaltrialreportedinthisworkwasbasedonourhypothesisthattreatmentofPBIE

susceptiblemareswithNACwouldalterinflammatorycytokinemRNAexpression,leadingtothe

resolutionofPBIEclinicalsigns.Theresultsofourstudydidnotrevealanybeneficialeffectof

NACtreatmentoneitherPBIEclinicalsignsorcytokinemRNAexpression,makingitdifficultto

assess any improvement of clinical signs by means of altered mRNA expression, following

treatment.However,somespeculationscanstillbemaderegardingthisconceptbyexamining

patternsofcytokinemRNAexpressionandcomparingthemtotheclinicalsignsobservedatthe

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different time points post-AI. IL-6 mRNA expression was significantly greater at 12 hours

compared to 60 hours post AI. This is similar to%neutrophil levels and endometrial edema

scores,bothofwhichweresignificantlyelevatedattimepointsnearestAI(12hourspostAIfor

% neutrophils; 12 and 36 hours post AI for uterine edema scores). Similar, though slightly

different trendswerenoted for intra-uterine fluid (IUF) volume,with significantly larger fluid

volumesat36hourscomparedto12,60and84hourspostAI.Thispatternofinflammationwas

expectedasneutrophilsare recruited followingbreeding (Kotilainenetal.1994) startingat4

hoursandpeakingat8hoursafterAI (Katila1995). InPBIEsusceptiblemares, thispattern is

similarbutisevenmorepronounced(Woodwardetal.2013).Fromthis,onecouldspeculatethat

IL-6playsaroleinpromotinginflammationearlyonintheuterineresponse,andthatthisinturn

results in the clinical signs observed in PBIE mares. Interleukin-6 has both pro- and anti-

inflammatoryproperties. It is involved inthe initiationof the inflammatoryresponsebutalso

controlsinflammationbymeansofdecreasingneutrophilrecruitment(Tizard2013).Theeffects

ofIL-6onneutrophilswereapparentinthisstudy,where%neutrophilsdecreasedsignificantly

by60hourspostAI,possiblyasaresultofearlierIL-6mRNAup-regulation.

ThepersistenceofclinicalsignsinourPBIEstudymares,albeitreducedat60hoursfrom

12hours,couldbetheresultofalackofIL-10mRNAup-regulation,asrelativeexpressionofIL-

10didnotchangebetween12and60hourspost-AI.Interleukin-6promotesexpressionofIL-10,

whichisananti-inflammatorycytokine(Tizard2013).ItispossiblethatalteredIL-6expression

also ledtoaberrations in IL-10expression.Wehypothesizethat the failureof increased IL-10

expressionneededtomodulatetheinflammatoryresponsetobreeding,couldaccountforsome

of the persistence of clinical signs seen with PBIE. One would expect that under normal

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circumstances,levelsofIL-10mRNAexpressionwouldbehighestfollowinginflammation.Yet,if

thisincreaseinIL-10doesnotoccur,asissuspectedinPBIEmares,persistentinflammation,and

thereby persistence of clinical signs, results. This supports the notion that anti-inflammatory

cytokineexpressionisalteredinPBIEsusceptiblemares,andthattheremayinfactbelackofan

anti-inflammatoryresponse,notjustanexaggeratedpro-inflammatoryresponse,playingarole

inthiscondition.

Conclusionsontheeffectsofthepro-inflammatorycytokinesTNF-αandIL-1β,onclinical

signscannotbemadeduetoalackofchangenotedacrosstimepointsfollowingAI.Itispossible

thatIL-1βandTNF-αareinfactaffectedinPBIEsusceptiblemares,butthatthetimepointof

significant increase was not examined in this study. Further investigations are required to

determinehowthesecytokinesmayimpactclinicalsignsassociatedwithPBIE.Moreover,inan

attempttobetterlinkclinicaloutcomeswithcytokinemRNAexpression,futurestudiesshould

examinehow treatmentmethods known toalter cytokinemRNAexpression, suchasMCWE,

dexamethasone,plateletrichplasma(PRP)andlactoferrin,affectPBIEclinicalsigns.

4.2LIMITATIONS OneofthelargestlimitationsofourstudywastheinabilitytomeasureIL-10,TNF-α,and

IL-1β concentration in uterine fluid. While equine ELISA kits exist for these cytokines, no

validations have been published specifically for equine uterine fluid. As such, validationwas

attemptedforthesekitsbutwasunsuccessful.Themostcommonreasonforfailureofvalidation

wassuspectedtobematrixinterferenceduetoanunidentifiedcompoundorsubstancewithin

uterine fluid that caused either false increases or decreases of measured cytokine

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concentrations.Many of the sampleswere viscous in nature, possibly contributing tomatrix

interferenceoftheassay.Theviscosityofthesamplewassuspectedtobeduetothehighprotein

contentinfluidobtainedfromaninflameduterus.SomeELISAkitscanbevalidateddespitethis

by simplydiluting the sample appropriatelywith the reagentdiluentprovided in the kit (MC

personalcommunication,R&Dsystemstechnicalsupport).ThiswasthecaseforIL-6,theonly

successfully validated cytokine, as the sample was diluted 1:20; however, the appropriate

dilutionfactorvariesforeachindividualcytokine.Thedilutionfactorshouldbegreatenoughto

overcomematrixinterference,butnotsomuchsothatthecytokineofinterestcannolongerbe

detected.VariousdilutionswereattemptedforIL-10,TNF-α,andIL-1β,asreportedinAppendix

4.

Although IL-6was successfully validated, the process itself had limitations, notably in

termsofthenegativecontrol.Forvalidation,apositiveandnegativecontrolwereutilizedand

consistedofPBIE-susceptiblemaresenrolledinthestudy(positivecontrols)andnormalmares

(negative controls) (Appendix 2). Undiluted uterine fluidwas readily available for PBIE-study

mares,however,normalmaresdidnothaveanyuterinefluid.Theprocurementofendometrial

fluidfromthesemareswasthroughalow-volumelavage,ultimatelydilutingthesample.While

theexactvolumeofinfusedfluidwasknownforthisprocedure,theamountrecoveredwasnot

100%, leading toanunknowndilution factor. Thismadecomparisonsbetweennegativeand

positivecontrolmaresdifficult. Sincefluidcouldnotbecollectedinanyotherwayinnormal

mares,thisrepresentedanotherlimitationforthevalidationprocess.

Othervalidationmethodsthatcouldhavebeenattemptedincludepurificationorwashing

ofthesamplewithspincolumnsandaddingfetalbovineserum(FBS)tothereagentdiluent.The

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quantityofFBStobeaddedtothereagentdiluentvariesgreatlybasedonthetypeofsample,

andcanrangefrom10-50%(MCpersonalcommunication,R&Dsystemstechnicalsupport).FBS

andspincolumnscouldhaveeitherboundtheviscouscomponentofthesampleorfiltereditout.

Neitherofthesemethodswereperformedduetothelimitedvolumeofsamplefluidsavailable

fromthestudymares,sinceeachcytokinewouldrequireitsownvalidationattemptswitheither

FBS,spincolumnsorboth.Furtherstudiesshouldbeperformedtodetermineiftheadditionof

FBStoreagentdiluentcontributestothevalidationprocessandifso,whattheappropriateFBS

concentrationiswhenrunningthesekitsonequineuterinefluid.Inthesameregard,perfecting

themethodologyforendometrialfluidtobefilteredthroughspincolumnscouldalsobeatopic

ofresearch.

Theequine specieshasagreatdealof variability in its geneexpression (Tessieretal.

2017),makingtheselectionofreferencegenesachallenge.Consequently,eachstudymarehad

her own set of reference genes. Thismethod is valid as the reference genes selected were

expressed ina stablemanner foreach individualmare,allowing forcomparisons tobemade

acrossthem.Thismethodissuperiortochoosingtworeferencegenesforallmares,thatarenot

expressed in a stable manner. The latter could be accomplished by employing software

methodologysuchasGeNormorNormFinder,whicharedesignedtochoosethetwobestoverall

referencegenesforallsubjectsinastudy.Thiswasattemptedinourclinicaltrial,however,large

variabilityinthedatameantthatnotwobestreferencegenescouldbeidentified,leadingtothe

selectionofthebesttwoforeach individualmare.Toconfirmthatreferencegeneswerenot

affectingresultoutcomes,thevariable“referencegene”wasenteredintotherepeatedmeasures

ANOVAmodeltoconfirmthattheywerenon-significant.

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Anadditionallimitationofthisstudywastherepeatedbiopsiesandprocurementoffluid

atmultipletimepointsduringeachestrouscycle.Theeffectofprevioustimepointsamplingon

subsequent samples is unknown.WatsonandSertich (1992) andChristoffersenetal. (2012),

demonstratedthatrepeatedbiopsieswerenotdetrimentaltouterinehealthorpregnancyrates;

however,nostudieshavespecificallyexaminedtheeffectsofprocurementoffluid,endometrial

cytologyandbiopsycombinedonthelevelofinflammation48hourslater.Ideally,onesampling

time pointwould have taken place per cycle,withmultiple cycles of treatment and control,

althoughthevariabilityacrosscyclesbecomesgreaterwiththisapproach.

4.3OVERALLCONCLUSIONSANDFUTUREDIRECTIONS

Despitesignificantamountsofresearch,themechanismsunderlyingthepathophysiology

ofPBIE inmaresremainpoorlyunderstood. Thisstudyprovidedmore informationonmRNA

expressionofIL-6,IL-10,TNF-αandIL-1βinPBIE-susceptiblemaresatboth12and60hourspost-

AI.BaselinemRNAexpressionofthesecytokineswasalsoobtainedforasubsetofmares.While

statistically significant differences were not often noted between these time points, the

informationandtrendsfoundinthestudycontributedtodefiningthegeneralcytokineprofile

and alterations in the immune response of PBIE-susceptible mares. Knowing more about

differentcytokineexpressionatvarioustimepointspost-AIandhowthesedifferfromnormal

maresmayhelptargetthedevelopmentofnewtreatmentmodalities.Furthermore,integrating

theimmuneresponsewiththeclinicalpictureofPBIE,byassessingbothcytokineexpressionand

clinicalsignsatvarioustimepoints,couldhelpcompleteourunderstandingofPBIE.Forexample,

PRPshowspromiseinitseffectsonIL-6,IL-8andIL-1βandiNOSmRNAexpression24hourspost-

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AIinPBIE-susceptiblemares(Metcalfetal.2012).ElevatedNOisbelievedtoplayaroleindelayed

uterineclearance.OthertimepointscouldbeexaminedfollowingtreatmentwithPRPandclinical

impactscouldbeassessed,suchasfluidaccumulation,asithasbeenshownthatiNOSisaffected.

Furtherresearchisneededtodeterminehowothercytokineswouldbeaffectedatdifferenttime

pointsbyvarioustreatmentsandhowclinicalsignswouldtherebybeimpacted.

Woodwardetal.(2013)demonstratedthatmanycytokineschangesignificantlyintheir

mRNAexpressionat6hourspostAI.Thisisnottosayhowever,thatcytokinemRNAexpression

alsodoesnotchangeatothertimepointspostAI,orthatothercytokinesarealteredatdifferent

time-points. Up-regulation of mRNA expression of inflammatory cytokines can be extremely

short-livedandcouldeasilybemissediftheappropriatetimepointsarenotselectedforeach

cytokine of interest. Increased expression may also be extremely mild in nature, making

differencesdifficulttodetect,eveninextremeinflammatorymodels,suchassepticshock(MC

personalcommunication,Dr.Wood).Infuturestudies,additionaltimepointsbothimmediately

afterAIandlaterintheimmuneresponse,shouldbeexaminedinordertocapturechangesto

cytokine mRNA expression across a broader timeline of the immune response. Additional

cytokinesofinterestincludeIL-8,IL-1RAandIFN-γ.IL-8isinvolvedinneutrophilchemotaxisand

itiswell-knownthatneutrophilsareelevatedinPBIE-susceptiblemares.IL-1RA,encodedbyIL-

1RNgene,isananti-inflammatorycytokineinvolvedintheregulationofIL-1βwhereasIFN-γis

pro-inflammatory. IL-1RN and IFN-γ changed across certain time points in the study by

Woodward et al. (2013) and recently, PBIE-susceptible mares treated with CRISP-3 and

lactoferrin had altered endometrial IL-1RN and IFN-γ expression (Fedorka et al. 2018). The

129

additional examination of these cytokines in future studies would certainly add to our

understandingofthepathophysiologyofPBIE.

TreatmentwithNAChadnoeffectontheresolutionofclinicalsignsoronendometrial

cytokinemRNAexpression inPBIEsusceptiblemares. However,asignificantandunexpected

findingwastheseveredetrimentaleffectsoftreatmentwithNAContheendometriuminPBIE-

susceptible mares. Loss of endometrial luminal epithelium (erosion), diffuse, deep-seated

neutrophil infiltration and presence of neutrophils in the glandular lumenwere some of the

changesnotedinNACtreatedcycles,andaresimilartochangesnotedfollowingintra-uterine

irritantssuchaskerosene.TheseresultswereparticularlysurprisinggiventhatNACiswidelyused

inmares that could be susceptible to PBIE, such as thosewith bacterial endometritiswith a

biofilm or in mares with abnormal mucus accumulation. The results from this study should

prompt re-evaluation of the practice of NAC intrauterine infusion in mares with a history

suggestiveofPBIE.

ThisisthefirststudydemonstratingtheadverseeffectsofNAContheendometriumof

PBIE mares. Previous studies suggested NAC did not appear to have an effect on the

endometrium, however those studies included normal mares or mares with histories of

barrenness, but not necessarily PBIE. As was found for kerosene (Bracher et al. 1991), it is

possiblethatthedetrimentaleffectsofNACareexacerbatedinPBIEmaresorthosewithpoor

biopsygrades.Furthermore,itispossiblethatNACinitselfisnotdetrimentaltotheendometrium

histologically,butwhenitiscombinedwithAIinPBIE-susceptiblemares,theadverseeffectsare

severe,andthatsimilarchangescouldbeseenwithotherirritantscombinedwithNAC.Further

studiesareneededtoconfirmtheadverseeffectsofNAConmareswithdifferentendometrial

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biopsygradesand/orwhengivenincombinationwithAIorothertreatments.Thiswouldallow

for clinicians to better target their patients when considering NAC infusion and allow for

appropriatemeasurestobetaken,suchasperforminguterinelavagebetweenNACinfusionand

breeding when NAC treatment is necessary. It is evident that more research needs to be

undertakenbeforetreatinganyPBIE-susceptiblemarewithNAC.

Finally,muchislefttolearnregardingtheoverallpathophysiologyofPBIE.Riskfactors

for PBIE-susceptiblemares, such as endometrial biopsy grade or extensive fibrosis, could be

evaluatedfortheireffectsontheexpressionof inflammatorygenes.Studieshaveshownthat

mareswith severeendometrial fibrosisassociatedwithendometrosishavealtereduterocalin

anduteroferrinexpressionpatternsaswellasalteredhormonereceptors,suggestingthatnormal

uterinefunctionalityisalteredincasesofendometrialpathology(Lehmannetal.2011;Rebordao

etal.2014).ItispossiblethatsomeofthesechangesalsoplayaroleinthedevelopmentofPBIE.

Specific treatments could then be developed for more targeted immunologic abnormalities.

Inflammationisatightlyregulatedprocessandbeginswithrecognitionofforeignmaterial,such

asspermatozoa.Thisisfollowedbythereleaseofinflammatorymediatorsforcellularsignaling

torecruitinflammatorycells.ItiswellknownthatsomeoftheseprocessesarealteredinPBIE-

susceptiblemares, howevermuch remainsunknown. Studiesmoving forward could focuson

differences in inflammatory gene sequencing in response to AI in PBIE-susceptible mares

compared to resistant mares. Marth et al. (2015, 2016) demonstrated the importance of

pathogenrecognitionandNOD-likereceptorsaswellasinductionofchemokinesinnormalmares

followingbacterialinoculationwithE.coli(3,12,24,48and72hours).Theinflammatoryresponse

wasveryrapid,occurringwithin3hourspost-inoculation(Marthetal.2015).Similarstudiesof

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PBIEresistantandsusceptiblemaresatmultipleandearly(1-3hours)timepointsfollowingAI

wouldprovideuswithnewinsightintohowandwhyPBIEdevelops.Itispossiblethatsusceptible

maresalsohavealteredrecognitionofpathogens,inthiscasespermatozoa,leadingtotheup-

regulationofchemokines,cytokinesandothermediatorsofinflammation,suchasNO.Additional

informationonthealteredimmuneresponseofPBIEmares,includingtheinitialrecognitionof

pathogensandcellsignaling,willclarifythepathophysiologyofthisimportantcauseofinfertility.

Inconclusion,PBIEremainspoorlyunderstoodinmaresdespiteextensivepastresearch,

likelyduetothemultifacetedpathophysiologyofthisdiseaseprocessthatincludesriskfactors,

altered mechanical clearance mechanisms, potentially abnormal pathogen recognition,

persistent inflammationpost-breeding, and altered response to traditional uterine therapies.

Futureresearchtobetterunderstandtheearlypost-breedingresponseinparticularmayhelpto

identifykeytimepointsandmechanismsfortherapythatcanimpacttheimmuneresponseof

PBIE-susceptiblemares.

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4.4REFERENCES1. Bracher,V.,Neuschaefer,A.andAllen,R.W.(1991).Theeffectofintra-uterineinfusionof

keroseneontheendometriumofmares.J.Reprod.Fert.Suppl44,706-707.2. Cazzola,M.,Calzetta,L.,Facciolo,F.,Rogliani,P.andMatera,M.G.(2017).Pharmacological

investigationontheanti-oxidantandanti-inflammatoryactivityofN-acetylcysteineinanexvivomodelofCOPDexacerbation.Resp.Res.18,1-10.

3. Christoffersen,M.andTroedsson,M.H.T.(2017). Inflammationandfertility inthemare.Reprod.Dom.Anim.Suppl52,14-20.

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APPENDICESAppendix1.Interactionsbetweentreatmentcyclesandhourspost-AIforoutcomesmeasured

0

0.5

1

1.5

2

2.5

3

3.5

4

12HOURS 60HOURS

Biop

syInfla

mmationScore

TX

CONTROL

Figure1.Meanbiopsyinflammationscore(+/-SE)onhistopathologyofuterinebiopsiesfrom9PBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cyclesat12and60hourspostAI.Maximumbiopsyscorepossiblewas4.Therewasnosignificantinteractionbetweentreatmentandhours-post-AI(p=0.12).

135

0

10

20

30

40

50


IUF(cm

3 )

TX

CONTROL

Figure2.Medianintra-uterinefluidvolume(cm3)(+/-LLandUL)of9PBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cyclesat12,36,60and84hourspostAI.Therewasnotreatmentbyhours-post-AIinteraction(p=0.84).

0

0.5

1

1.5

2

2.5

3

3.5


Endo

metria

lede

mascore

TX

CONTROL

Figure3.Meanendometrialedemascore(+/-SE)of9PBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(Control)cyclesat12,36,60and84hourspostAI.Maximumedemascorepossiblewas4.Therewasnotreatmentbyhours-post-AIinteraction(p=0.65).

136

0

10

20

30

40

50

60

70

80

90

12HOURS 60HOURS

%neu

trop

hils

TX

CONTROL

Figure4.Mean%neutrophils(+/-SE)oncytologyofuterineswabsobtainedfrom9PBIE-susceptiblemaresat12and60hourspostAIinN-acetylcysteinetreated(TX)orsaline(CONTROL)treatedcycles.Nosignificanttreatmentbytimeinteractionwaspresent(p=0.98).

137

Appendix2.ValidationprocessforIL-6ELISAkit

ThevalidationprocessfortheEquineIL-6DuoSet®ELISA(R&DSystems™,Minneapolis,

MN, USA) was based on manufacturer instructions (Spike, Recovery, and Linearity & ELISA

DevelopmentGuidebyR&Dsystems).Aspikedneatcytokinesolution(A)waspreparedusingthe

cytokinestandardtoaconcentrationof45ng/ml (approximately10timestherecommended

highstandardofthestandardcurve).

Threebasesamplesof1000µLwereprepared:unspiked,spikedandcontrol.Theunspiked

sampleconsistedonlyof1000µLsamplematerial.Thespikedsampleconsistedof20µLofspiked

neatcytokinesolution(A)and980µLofsamplesolution.Thecontrolsamplehad20µLofspiked

neatcytokinesolution(A)and980µLofreagentdiluent(1%BSAinPBS),whichwasprovidedby

themanufacturer.

Serialdilutionsweremadefromtheneatsample:1:2,1:4and1:8withreagentdiluent.

Positiveandnegativecontrolsampleswereincludedinthevalidationprocess.Positivecontrols

consistedofstudymares(PBIEsusceptible)withexcessivefluid,allowingformultiplevalidation

attempts.Negativecontrolsconsistedofendometrialfluidobtainedbylow-volumelavagefrom

normalmares.Whileournegativecontrolsarenotidealduetodilutionofendometrialsamples

inorder tocollect fluid, therewasnoothermethodtocollectendometrial fluid fromnormal

maresthathavenointra-uterinefluid.

Percentrecoveryandlinearityofsamplesduringvalidationrunswerecalculatedasper

theSpike,RecoveryandLinearity&ELISADevelopmentGuideprovidedby themanufacturer

(R&D Systems Inc.) Methodology was validated when percent recovery and linearity of all

samplesfellwithin80-120%.

138

Severalvalidationrunswererequired. Thepositivecontrolmaresamples (spikedand

unspiked)wererunatdifferentdilutionfactors(1:20,1:30and1:50).The1:20dilutionfactor

allowed for percent recovery and linearity values to fall within 80-120%, validating this kit.

Samplesforthestudywerethereforeconductedata1:20dilutionwithreagentdiluent.

139

IL-6Validation Calculated%Negativecontrolrecovery 137.83%Positivecontrol(1:20)recovery 96.43%Positivecontrol(1:30)recovery 56.32%Positivecontrol(1:50)recovery 55.77%Negativecontrolspiked1:2linearity 83.16%Negativecontrolspiked1:4linearity 88.93%Negativecontrolspiked1:8linearity 93.55%Negativecontrolunspiked1:2linearity 27.05%Negativecontrolunspiked1:4linearity 45.34%Negativecontrolunspiked1:8linearity 32.66%Positivecontrol(1:20)spiked1:2linearity 100.40%Positivecontrol(1:20)spiked1:4linearity 112.78%Positivecontrol(1:20)spiked1:8linearity 111.57%Positivecontrol(1:20)unspiked1:2linearity 90.17%Positivecontrol(1:20)unspiked1:4linearity 99.37%Positivecontrol(1:20)unspiked1:8linearity 97.65%Positivecontrol(1:30)spiked1:2linearity 110.18%Positivecontrol(1:30)spiked1:4linearity 125.41%Positivecontrol(1:30)spiked1:8linearity 132.74%Positivecontrol(1:30)unspiked1:2linearity 93.24%Positivecontrol(1:30)unspiked1:4linearity 99.41%Positivecontrol(1:30)unspiked1:8linearity 77.52%Positivecontrol(1:50)spiked1:2linearity 106.78%Positivecontrol(1:50)spiked1:4linearity 117.25%Positivecontrol(1:50)spiked1:8linearity 116.92%Positivecontrol(1:50)unspiked1:2linearity 86.34%Positivecontrol(1:50)unspiked1:4linearity 81.29%Positivecontrol(1:50)unspiked1:8linearity 70.19%Spikedcontrol1:2linearity 88.95%Spikedcontrol1:4linearity 73.54%Spikedcontrol1:8linearity 67.15%Table1.CalculatedvaluesofpercentrecoveryandlinearityforfinalIL-6validation.Valuesofpercentrecoveryandlinearityforthepositivecontrol(1:20)spikedandunspikedareboldedandfallbetween80-120%.

140

Appendix3.Maineffectsoftreatmentandhourspost-AIoncytokinesevaluated

Figure1.MedianIL-6mRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhentimepoints(12h,60hpost-AI)werecombinedforeachcycle.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQ,relativequantification).

Figure2.MedianIL-10mRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhentimepoints(12h,60hpost-AI)werecombinedforeachcycle.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQ,relativequantification).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

TX CONTROL

Relativ

e(RQ)m

RNAexpressio

nofIL-6

A

A

0

0.01

0.02

0.03

0.04

0.05

0.06

TX CONTROL

Relativ

e(RQ)m

RNAexpressio

nofIL-10

A

A

141

Figure3.MedianTNF-αmRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhentimepoints(12h,60hpost-AI)werecombinedforeachcycle.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQ,relativequantification).

Figure4.MedianIL-1βmRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresinN-acetylcysteinetreated(TX)andsaline(CONTROL)cycleswhentimepoints(12h,60hpost-AI)werecombinedforeachcycle.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:relativequantification).

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

TX CONTROL

Relativ

e(RQ)mRN

Aexpressio

nofTNF-a A A

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

TX CONTROL

Relativ

e(RQ)mRN

Aexpressio

nofIL-1b

A

A

142

Figure5.MedianIL-6mRNAexpression(includinglowerandupperconfidencelimits) inPBIE-susceptiblemares inN-acetylcysteinetreated (TX)andsaline (CONTROL)cyclesat12and60hourspostAI.Nosignificanttreatmentbytimeinteractionwasseen(p=0.41).Asignificantmaineffect of timewas present (p=0.02), whereas no significantmain treatment effectwas seen(p=0.86).Differentuppercaselettersshowsignificantdifferencesbetween12and60hourspostAI.Significancewasdefinedasp<0.05(Abbreviations:RQ,relativequantification).

Figure6.MedianIL-10mRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresat12and60hourspostAI,whenTXandCONTROLcycleswerecombined.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQ,relativequantification).

0

0.01

0.02

0.03

0.04

0.05

0.06

12HOURS 60HOURS

Relativ

e(RQ)m

RNAexpressio

nofIL-10

A

A

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

12HOURS 60HOURS

Relativ

e(RQ)mRN

Aexpressio

nofIL-6

Treated

ControlB

A

143

Figure7.MedianTNF-αmRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresat12and60hourspostAI,whenTXandCONTROLcycleswerecombined.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQrelativequantification).

Figure8.MedianIL-1βmRNAexpression(includinglowerandupperconfidencelimits)ofPBIE-susceptiblemaresat12and60hourspostAI,whenTXandCONTROLcycleswerecombined.Differingsuperscriptsaresignificantatp<0.05(Abbreviations:RQ,relativequantification).

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

12HOURS 60HOURS

Relativ

e(RQ)m

RNAexpressio

nofTNF-a

AA

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

12HOURS 60HOURS

Relativ

e(RQ)mRN

Aexpressio

nofIL-1b

A

A

144

Appendix4.ProcessesforvalidationattemptsforIL-10,TNF-αandIL-1βELISAkits

ThevalidationattemptsforIL-10,TNF-αandIL-1βELISAkits(R&DSystems)followedthe

sameprinciplesasthatdescribedinAppendix1forIL-6.

Briefly, four validation attemptswere conducted for IL-10. The spiked solution had a

concentrationof92.5ng/mlofIL-10.Forthefirstvalidation,positivecontrolswerediluted1:20,

1:30andneat,basedonsuccessfulresultsfromtheIL-61:20dilutionvalidation.Resultsfellbelow

the standardcurve, therefore in the secondattempt samples froma suspected trulypositive

controlmare(normalmarepost-breeding)wereutilized,yetresultscontinuedtofallbelowthe

standardcurve.Thestandardcurvewasthusshiftedtorangefrom5ng/mlto0.078ng/mlandall

neat positive control samples were utilized. However, the IL-10 kit was never successfully

validated.

For TNF-α, four validation attempts were conducted. The spiked solution had a

concentration of 37.5ng/ml of TNF-α.The first validation consisted of neat, 1:20 and 1:30

dilutionsofpositivecontrols.Resultsweremostpromisingwithdilutionsnear1:4,resultingin

three other validation attempts of 1:4 dilutions with different positive controls being used.

Attemptswereunsuccessful.

For IL-1β, one single validation attempt was conducted. The spiked solution had a

concentrationof45ng/mlofIL-1β.Thepositivecontrolmarewasdiluted1:10,1:20and1:30for

validation.Thiswasunsuccessful.Nootherattemptscouldbepursuedassamplesweredepleted.

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