The University of MaineDigitalCommons@UMaine

Electronic Theses and Dissertations Fogler Library

Spring 5-11-2018

Improving Techniques to Study Equine CervicalMucociliary ClearanceMelissa A. HawkesUniversity of Maine, melissa.hawkes@maine.edu

Follow this and additional works at: https://digitalcommons.library.umaine.edu/etd

Part of the Animal Structures Commons, Cells Commons, Large or Food Animal and EquineMedicine Commons, Other Animal Sciences Commons, Urogenital System Commons, and theVeterinary Anatomy Commons

This Open-Access Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in ElectronicTheses and Dissertations by an authorized administrator of DigitalCommons@UMaine. For more information, please contactum.library.technical.services@maine.edu.

Recommended CitationHawkes, Melissa A., "Improving Techniques to Study Equine Cervical Mucociliary Clearance" (2018). Electronic Theses andDissertations. 2859.https://digitalcommons.library.umaine.edu/etd/2859

ii

IMPROVINGTECHNIQUESTOSTUDYEQUINE

CERVICALMUCOCILIARYCLEARANCE

By

MelissaHawkes

B.S.UniversityofMaine,2016

ATHESIS

SubmittedinPartialFulfillmentofthe

RequirementsfortheDegreeof

MasterofScience

(inAnimalSciences)

TheGraduateSchool

TheUniversityofMaine

May2018

AdvisoryCommittee:

RobertCausey,ProfessorofAnimalandVeterinaryScience,Advisor

MartinStokes,ProfessorofAnimalandVeterinaryScience

MaryTyler,ProfessorofZoology

ii

Copyright2018MelissaHawkes

IMPROVINGTEHCNIQUESTOSTUDYEQUINE

CERVICALMUCOCILIARYCLEARANCE

ByMelissaHawkes

ThesisAdvisor:Dr.RobertCausey

AnAbstractoftheThesisPresented

inPartialFulfillmentoftheRequirementsfortheDegreeofMasterofScience

(inAnimalSciences)

May2018

Bacterialuterineinfectionsinflictmajorlossesontheequinebreedingindustry.Theseinfections

usuallyarisefrombacteriaintroducedatbreeding.Micro-currentspropelledbyciliatedcellsbetween

thefoldsoftheuterusandcervixhavebeenproposedasameansbywhichcontaminantsareexpelled.

Previousdatahaveshownpossibleciliarymicro-currentspropellingcarbonparticles,occasionally

rotating,throughcervicalfolds.However,adherencetotheepitheliummayhaveinterferedwith

movementofcarboninthesestudies.Therefore,wetestedpotentiallynon-adherentsubstancesto

revealciliarymicro-currentsontheequinecervixunderhighmagnificationvideo-endoscopy.We

hypothesizedthatpolyethylenegreenmicrospheres1-5μmand70μmindiameter,wouldbesuperior

tocarboninrevealingmicro-currentsonthecervicalepitheliumandthat70μmhemisphericallycoated

bichromalmicrosphereswoulddisplayrotation.Asuspensioncontainingthesemicrospheresandcarbon

wasdepositedontothecervixof5estrousmaresandmovementofeachtypeofparticlewasrecorded

underhighvideoendoscopyforapproximately10-20minutes.Inmorethan50%ofeachvideo

recordingthefieldofviewwasobscuredbycameramovementorpoorpositioning.However,the

remainderoftherecordingcontainedshortsegmentsinwhichtheinteractionsbetweentheepithelium

andparticleswereobservable.Allobservablesegmentswerethenanalyzedforthepresenceorabsence

ofdesired(visibility,motion,androtation)andundesired(aggregation)criteriaforeachtypeofparticle.

Particleswerescoredfortheprevalenceofthesedesiredandundesiredcriteriaaccordingtothe

numberofvideosegmentsinwhichtheywereobserved.DatawereanalyzedbytheKruskal-Wallistest.

Backwardrotationofbichromalsphereswasinterpretedasevidenceofciliaryactivity.Overall,carbon

scoredequaltoorhigherthanthemicrospheres,leadingtorejectionofthehypothesis.Ofthe

microspheres,thebichromalmicrospheresscoredhighestoverall,buttendedtoaggregatemorethan

thegreenmicrospheres,aggregationbeingundesirable.Subjectiveassessmentconcludedthatcervical

movementwascloselyrelatedtorespiratorymovementsofthemare,andthattheconstantlymoving

cervicalfoldshelpedclearthedepositedparticles.Thesedatamayimprovedetectionofmucociliary

clearanceinthecervixanduterusandmayassistfuturestudiestodetectimpairedmucociliaryclearance

ininfertilemares.

iii

ACKNOWLEDGEMENTS

IwouldliketothankmyadvisorDr.RobertCauseyforallofhissupportandguidancethroughoutthis

process.WithouthimIwouldnotbewhereIaminmyknowledgeofscience.Iwouldalsoliketothank

Dr.MaryTylerforherconstantwordsofencouragement,andDr.MartinStokesforthewritingskillshe

hashelpedmewithovertheyears.

IwouldalsoliketoshowmyappreciationforAlecToothacker,AnnaRichards,andChelsieOldfieldforall

oftheirassistanceoverthepasttwoyears.Manythankstoalloftheindividualsthathavebeenpresent

andmorethanhappytohelpwithhorsehandlingthroughoutthisresearch.Iwouldalsoliketothankmy

familyandfriendsfortheirsupportandencouragement.

iv

TABLEOFCONTENTS

ACKNOWLEDGEMENTS...............................................................................................................................iii

LISTOFTABLES............................................................................................................................................vi

LISTOFFIGURES.........................................................................................................................................vii

1.INTRODUCTION.....................................................................................................................................1

1.1.TheimportanceofCiliaandMucociliaryClearance...................................................................2

1.2.MucociliaryClearanceintheRespiratoryTractofHorses.........................................................3

1.3.PreviousStudiesandPreliminaryData.......................................................................................4

1.4.MicrospheresandMicroparticlesinBiology..............................................................................8

1.4.1.Gold...............................................................................................................................9

1.4.2.Silver..............................................................................................................................9

1.4.3.PolyethyleneMicrospheres.........................................................................................10

1.4.3.1.BariumCoatedMicrospheres.........................................................................10

1.4.3.2.HemisphericallycoatedBichromalMicrospheres..........................................10

1.5Objectives..................................................................................................................................11

2.MATERIALSANDMETHODS................................................................................................................13

2.1.InVitroAssessment..................................................................................................................13

2.2.ParticleSolutionPreparation....................................................................................................14

2.3.Videoendoscope.......................................................................................................................15

2.4.MarePreparation.....................................................................................................................15

2.5.InVivoVideoAnalysis...............................................................................................................16

3.RESULTS..............................................................................................................................................17

3.1.InVitro......................................................................................................................................17

3.2.InVivo.......................................................................................................................................19

v

4.DISCUSSION........................................................................................................................................21

5.CONCLUSION......................................................................................................................................29

REFERENCES...............................................................................................................................................30

APPENDIX.INDIVIDUALINVIVOSCORETABLES........................................................................................33

BIOGRAPHYOFTHEAUTHOR.....................................................................................................................36

vi

LISTOFTABLES

Table1. Assessmentguidelinesofparticlesduringmicroscopicandendoscopicportion

oftheinvitrostudy................................................................................................14

Table2. Assessmentofparticlesduringmicroscopicandendoscopicportionsofthe

invitrostudy...........................................................................................................18

Table3. Assessmentofmicrospheresandcarbon...............................................................20

Table4. Mare1(Sara)..........................................................................................................33

Table5. Mare2(Blisstex).....................................................................................................34

Table6. Mare3(Laney)........................................................................................................34

Table7. Mare4(Susie).........................................................................................................35

Table8. Mare5(Gina)..........................................................................................................35

vii

LISTOFFIGURES

Figure1. Axoneme:microtubule-basedcytoskeletoninsidestructureofmotileciliaand

flagella.......................................................................................................................3

Figure2. Carbonparticlebeingpropelledbyciliatedepitheliuminvitro................................5

Figure3. Carbonparticlerotatingonepithelium.....................................................................7

Figure4. Translocationofcarbonparticles..............................................................................8

Figure5. Predictionoftwopossibletypesofrotationthatmaybevisible............................11

Figure6. Appearanceofseveralparticlesundervideoendoscopy.........................................19

Figure7. Particlerotationgivingrisetoflashesoflight........................................................23

Figure8. Mucusstainingeffectof1-5µmfluorescentgreenpolyethylenemicrospheres......25

Figure9. Fluidflowthroughbloodandlymphaticvessels.....................................................26

Figure10. Mucussheetsmovinginopposingdirections,inanalternatingpattern................27

Figure11. Self-clearingofthecervicalepitheliuminunder10minutes..................................28

1

CHAPTER1

INTRODUCTION

Bacterialinfectionsintheuterusofmaresareamajorcauseoflossesintheequinebreeding

industryaffectingbetween25-60%ofbarrenmares(1,4,9).Theseinfectionsusuallyarisefrom

bacterialcontaminantsintroducedatbreeding.Thefirstlineofdefenseagainsttheseinfectionshas

alwaysbeenconsideredphagocytosisbyneutrophils.Whenbacteriaarepresentintheuterusthereis

indeedaninfluxofproteinsandneutrophils(28).Thisinfluxleadstoacuteinflammationwhichusually

quicklyresolves.Mostbacteriaandinflammatorycellscanbeexpelledfromtheuterusthroughuterine

contractions.However,ifapooruterineenvironmentpersistsduetofailuretovoidfluid,neutrophils

mayshowimpairedabilitytophagocytizebacteria.Becauseneutrophilfunctionalsomaybe

fundamentallyimpairedinthemucosalimmunesystemoftheequineuterus,researchershave

thereforequestionedtheabilityofphagocytosistocleartheuterusofallbacteria(22).

Weakerthannormaluterinecontractions,auterusthathangslowinthemare’sabdomen

(oftenreferredtoasapendulousuterus),andabnormalperinealconformationareamongthereasons

foranincreasedlikelihoodofinfectionandpoorintrauterineenvironment(18).Susceptibilityto

infectionsisalsocorrelatedwithreproductiveinjury.Inpost-mortemexaminations,itwasfoundthat

mareswhohadscarring,hemorrhage,granulationandlossoffoldsinthereproductiveandurinarytract

lining,orfibroustissuearoundthecervixweremoresusceptibletoinfectionsduringtheirlives(26).

Whethertheseanatomicalabnormalitiescauseincreasedinfectionsorareaconsequenceof

upregulatedinflammatorymediatorsduetoinfectionremainsunclear.

Mucociliaryclearance(MCC)istheself-clearingofamucousmembranebythepropulsionofa

mucusblanketbyciliatedepithelialcells.Mucociliaryclearancehasbeenproposedtobeasecondary

defenseagainstbacteriaintheequinegenitaltract.Observationsofamucuslayerrestingonciliated

2

cellsinuterinebiopsiesindicatethatthekeycomponentsofamucociliaryapparatusarepresent(6).In

addition,thereisextensivelongitudinalfoldingoftheuterineandcervicalepithelia.Suchfoldsof

ciliatedepitheliumontheequinecervixmayincreasetheefficiencyofthemucociliaryapparatus,and

suggestthatmicro-currentscouldbemaximizedatthesesites(12).Currentsmayassistinremoving

bacteria,inflammatorycells,andnon-viablespermatozoa.Directvisualizationofmicrocurrentsand

mucociliaryclearanceinlivemaresisstillnecessarytounderstandhowtheuterusfightsinfection,and

howadysfunctionalmucociliaryclearancemayimpactamare’sreproductivehealth.

Inthisstudy,wesoughttooptimizeinvivovisualizationofmucocilaryclearanceinhealthy

maresinordertohelpdetermineifmucociliaryclearanceactsasadefenseagainstbacteria.

1.1. TheimportanceofCiliaandMucociliaryClearance

Cilia,thepluralofthesingularnouncilium,comesfromtheLatinwordfor"eyelashes."Ciliaare

organellesfoundasextracellularprojectionsoneukaryoticcells(13).Theyappearassmallprojections

fromalargercellbody.Therearetwotypesofciliacommonlyfoundinthebody,motileciliaandnon-

motile,orprimarycilia.Primaryciliaappeartoactsolelyasasensoryorganelle,anexamplebeingthe

dendriticknoboftheolfactoryneuronwheresmellsareconvertedintoactionpotentials.Recently,

sensoryproteinshavebeenidentifiedinmotilecilia.Theseproteinsdetectfluidflow,bittertaste,and

sexhormones(2).Motileciliaandflagellamakeupagroupoforganellesreferredtoasundulipodia(19).

Undulipodiaareaclassof9+2organelles,whichgettheirnamefromthemicrotubule-based

cytoskeletoninsidestructureofmotileciliaandflagella.Thisstructureisreferredtoastheaxoneme.In

primarycilia,theaxonemeappearsasaringofnineoutermicrotubuledoublets;inmotileciliaand

flagellatherearetwoadditioncentralmicrotubulesinglets(Figure1)(11,24).The9+2motileciliary

ultrastructureisconservedthroughouttheanimalkingdom(20).

3

RichardAllen'sImageCollection:Chapter18:Tetrahymena

Figure1.Axoneme:microtubule-basedcytoskeletoninsidestructureofmotileciliaandflagella.Motile

cilia9+2microtubule-basedcytoskeleton.Axonemearethenineoutermicrotubuledoubletsfoundinall

cilia,andthecentermicrotubulesingletsfoundonlyinmotileciliaandflagella.

1.2. MucociliaryClearanceintheRespiratoryTractofHorses

Mucociliaryclearanceisawell-establisheddefensemechanismoftherespiratorytract.A

disturbanceinthisprocessintherespiratorytractofhorses(oranymammal)canleadtomanydiseases,

suchaspneumoniaorchronicsinusitis.Whendiscussingthehealthofthelungsthereisacomplex

relationshipbetweenmucusstructureanditspropulsionbytheairwaycilia(10).Oneofthesestudiesin

horsestestedtherateatwhichmucociliaryclearancechangedwhenloweringandraisingthehead.It

wasfoundthatloweringtheheadincreasedmovementofmucus,whileraisingtheheadslowed

movementofmucusandcausedacollectionofmucusatthebottomofthelungs(23).Therehavealso

beenstudiesonthemucociliaryclearanceofsedated,diseased,andexercisedhorsestodeterminehow

4

themucociliaryclearanceratesdiffer(29).However,scientistshaveneglectedtostudyothersystems

wheremucociliaryclearancemaybepresent,suchasthereproductivesystem.

Itisknownthatbacteriapresentintheuterusleadtoaninfluxofproteinsandneutrophils(27).

Asmentioned,thisinfluxmayleadtoinflammationthateventuallyimpedesthephagocyticfunctionsof

theneutrophils.Mucus,however,lackstheabilitytosupportphagocytosisduringthesetimesof

inflammation.Mucociliaryclearancethereforemayaidintheremovalofbacterialandinflammatory

cellsfromthemucosalsurface(5).Studieshavealsoshownneutrophilactivityishigherintissuesand

circulationoutsideoftheuterus,suggestingthatneutrophilactivityinsidetheuterusisnotthemainline

ofdefense,andthatbacterianeedtobeexpelledviaanotherroute(27).Therearemanyother

mechanismswhichcouldcontributetouterinedefensebutthepresenceofmucusandciliaonthe

equinegenitaltractstronglysuggestthatmucociliaryclearancemayplayarole.

Inlivemares,micro-particlesmayserveasamodelforbacteria.Micro-particleshavethe

potentialtoshowthemovementofmotilecilia,andwhetherornottheyarebeingtrappedinmucus

andexpelled.Thiscouldallowthedevelopmentoftechniquestodemonstratetheexistenceofa

functionalmucociliaryapparatusinthegenitaltractofthelivemare,andserveasabasisforfurther

research.

1.3. PreviousStudiesandPreliminaryData

Earlystudiesshowedthatwhencarbonwasinfusedintotheuterusitdidnotadheretothe

intactepitheliumduetoalayerofmucusproducedbycellsliningtheendometrium(7).Subsequent

researchexaminedsamplesofuterineepitheliumobservedunderaninvertedmicroscope,havingbeen

obtainedinasimilarmethodtoauterineembryowash.Theuterinecellscollectedshowedclear

indicationsofmotilecilia,bytherotationalmovementoffloatingraftsofepithelialcellswithbeating

cilia(Figure2).Carbonwasaddedtothesedishestosimulatebacteriaintheuterusandtobetter

5

visualizeciliarymovement.Thecarbonwaschosendueititsvisibilityandvaryingsize.Thisstudyledto

examinationofmucociliaryclearanceinthelivemare.

Figure2.:Carbonparticlebeingpropelledbyciliatedepitheliuminvitro.Stillscreenscapturedfroma

videoclipofciliateduterinecellsinapetridishobservedwithaninvertedmicroscope.Depictshow

carbonparticlesaretrappedinmucus(unstained)andmovedbythecilia.Magnificationis

approximately400x(unpublisheddatafromRobertCausey).

Subsequentstudiesconductedbymembersofourresearchteam(headedbyDr.RobertCausey)

attemptedtodeveloptechniquesforvideoendoscopicdetectionofmucociliaryclearanceinthelive

6

mare.However,animalmovementmakesdirectobservationofactivemucociliaryclearancedifficult,

sinceneitherthetissuenortheendoscopeareimmobilized.Therefore,tominimizemovement,a

techniquewasdevelopedtosecureanendoscopebyintroducingitthroughanendotrachealtube

anchoredbyaninflatablecuffattheinternalcervicalos,theepitheliumbeingexposedthrougha

windowcutinthetube(8).Inthispriorstudy(seepreliminarydata),microscopicmovementofcarbon

particlesplacedonthecervicalepitheliumwasobservable.Movementofindividualparticlesthrough

stationaryfolds,paststationarypoints,inopposingdirections,atdifferentspeeds,orwithtumbling

weredeemedindicativeofpossiblemucociliarycurrents.However,thecarbontendedtoaggregateand

adheretotheepithelium,whichmayhaveobscuredorinterferedwithmicroscopicmovement.Also,

becausedirectobservationofbeatingciliawasnotpossible,itisstillnecessarytoshowthatciliary

activityisresponsibleforthesemicro-currents.

Intheseearlyinvivotrials,carbonwasinfusedontothecervixof3maresandinteraction

betweenthecarbonandtheepitheliumwasrecordedbyhighmagnification-videoendoscopy(8).Upon

review,movementofthemare,includingbreathing,interferedwithobservation.Therefore,fixed

landmarkswererequiredtoserveasareferencepointtodemonstratetranslocationofcarbon.

However,theepithelialsurfaceoftenlackedsufficientcontrasttobeausefulmarker.Fortunately,

adheredcarbonprovidedafixedbackgroundonwhichtoobservemovementofisolatedfreecarbon

particles.Therefore,movementofsolitaryparticlespastgroupsofstationarycarbonandtranslocation

inthespacebetweentwofoldsweredeemedconsistentwithmicro-currentspossiblycausedbyciliary

motion.Inaddition,itappearedthatsomecarbonfragmentsweretumbling(Figure3)whenthey

contactedtheepithelialsurface,whichisconsistentwiththeexpectedeffectofciliaryactiononafree

particle.Themovementofcarbonparticlesrelativetoeachotherorpastanatomicallandmarkswas

thereforeanobservationthatdemonstratedmicro-currentsacrosstheepithelium(Figure4),andthe

7

tumblingofcarbonparticlesapproachingtheepithelialsurfacepotentiallyindicatesthatciliaryactionis

responsibleforthesecurrents.

Figure3.Carbonparticlerotatingonepithelium.Apparentrollingofatileshapedcarbonparticleasit

approachestheepithelium.InAandCthetwoalternatefacesoftheparticleareseenwhileinBthe

particleisseen“edgeon”.

Thesestudiesprovidedevidenceofmucociliaryclearanceinthemare,butthatcarbonmaynot

bethebestmaterialforsuchademonstration.Itwasthenproposedthatnon-adherentfluorescent

microspheresandmetallicpowders(i.e.silverandgold)mightbesuperiortocarboninassessmentof

equinecervicalmucociliaryclearanceusinghighmagnificationvideoendoscopy.Inaddition,

hemisphericallycoatedbichromalmicrospheresmightrevealmicro-currentsonthecervicalepithelium

throughtheirvisiblerotation.Forthisreason,itbecamenecessarytocomparewithcarbonthe

feasibilityofvariousothermicroparticlesandmicrospheresinrevealingmucociliarycurrents.

8

Figure4.Translocationofcarbonparticles.LeftPanel-A,B,C:Translocationofcarbonparticle(red

circle)paststationarycarbonadheredtoepithelium.RightPanel-D,E,F:Differentratesofcarbon

translocationofafastparticle(redcircle)overtakingaslowparticle(bluecircle)bothmovingrelativeto

stationarycarbon.Arrowindicatesdirectionoftranslocationineachpanel

1.4. MicrospheresandMicroparticlesinBiology

Inconsideringwhichparticlestoevaluatefordetectingmucociliaryclearanceintheequine

uterus,particlesneedtomeetthefollowingcriteria.First,theyneedtobenon-toxicinthemare:the

mareuteruscandevelopadramaticinflammatoryresponsetocertainirritants,anditwasnecessaryto

pickparticleswhichwerelikelytobenon-irritating.Second,theyneededtobevisibleagainstthepink

9

mucosalsurface.Third,theyshouldbesmallenoughthatmucociliaryclearancewouldlikelybe

effective;therefore,asizeofapproximately50μmorlessseemedappropriate.Fourth,theyshouldnot

aggregateintolargerclumps;iftheydothentheresultingmaterialmaybetoolargefortransportby

mucociliaryclearance.Fifth,theyshouldbeabletoshowrotation,whichcouldbeusedasevidencefor

propulsionofonesideoftheparticlebyactivelybeatingcilia.Thefollowingmicroparticleswerefeltto

bepossiblecandidatesforuseinvideoendoscopystudiesofmucociliaryclearanceinthemare.

1.4.1. Gold

Onestudyexaminedtheuptakeandpossibletoxicityofgoldparticlesinhumanleukemiacells.

Thegoldparticleswerenotcytotoxicandreducedthelevelofdestructiveoxidativespeciesinthecell.

However,whengoldsaltscoatedinacationicmaterialwereused,theycausedtoxiceffects,butonlyat

anatomicconcentrationof10nMorhigher(21).Goldcoatedpolystyrenesphereshavebeenusedin

respiratoryexperimentsinvolvingmucociliaryclearance.Thesphereswereusedtotrackthespeedof

mucociliaryclearanceintracheaeexcisedfromchickenembryos(16).Toourknowledgegoldhasnot

beenusedtorevealmucociliaryclearanceinthereproductivetract.

1.4.2. Silver

Alesscostlyoptiontogold,butwithmanyofthesameadvantages,mightbesilver.Aconcern

associatedwiththeuseofsilverpowderisitsaffinitytoclumpinanaqueousenvironment.Inorderto

decreasethiseffect,applicationsofadispersionagentareverycommon.Oneofthesemethodsisto

reactsilvernitrateoverheatwithascorbicacidandgumarabic,followedbyheatingwithammonia.

Whenthereactioniscomplete,thesilverpowderisfilteredoutandusedforvariousstudies(25).For

example,thispowderwasusedtotrackimaginginanelectronicchip,andthoughitdidreduce

clumping,itraisesconcernsaboutirritationtoanepithelialsurface.Thepossibilityofaddinganon-

10

irritatingdetergenttobreakthesurfacetensionoftheseparticlescouldleadtoaviableoptionofinvivo

studiesofmucociliaryclearance.

1.4.3. PolyethyleneMicrospheres

Fluorescentpolyethylenemicrospheresmaybeanalternativetocarboninstudiesof

mucociliaryclearance.Polyethylenemicrosphereshavebeenusedforthedemonstrationofmucociliary

activityintherespiratoryepitheliumofotherspecies(14,15,17).Advantagesoffluorescent

microspheresarethattheyarenon-toxic,inert,andhighlyvisible.Itwasoneofthegoalsofthepresent

study,therefore,totestsuchmicrospheresinmucociliaryclearanceexperimentsinthemareinvivo.In

particularwewishedtodetermineiftherewereanoptimalmicrospherediameterforthistypeofstudy,

andifitwerepossibletoobservemicrospheresinvivousingthelowerpowermagnificationof

commonlyavailablevideo-endoscopes.Availablesizesincluded1-5μm,and70μm.

Inaddition,thefollowingspecial-applicationmicrosphereshaveotherqualitiesthatmight

conferspecificadvantages:

1.4.3.1 BariumCoatedmicrospheres

Thebariumservestwopurposes—oneistoincreasetheweightoftheparticlesoitisheavier

thanthefluidinwhichitissuspended.Theparticlesaremorelikelytofalltothesurfaceandbe

propelledalongthetissueofthemare’suterus.Thesecondpurposeforusingbariumcoated

microsphereshasbeentoincreasevisibilitywithintheuterusofthemare.Thesespheresarecommonly

usedtodetectanddiagnoseproblemsinthedigestivetractbecauseoftheirhighvisibilitywithinabody

cavitywhenusingradiographictechniques(3).

1.4.3.2 HemisphericallyCoatedBichromalMicrospheres

Hemisphericallycoatedbichromalmicrospheresmaybeanoveltechniquetorevealciliary

activitybyshowingrotationofspheres.Thebottomofthespheremovesinthedirectionofthecurrent

11

ifpropelledfrombelowbycilia,whereasthebottomofasphererollingoverastationarysurfacemight

appeartobestationary,ormoveintheoppositedirection.Itcanthereforebehypothesizedthat

directionofrotationofthespheremaybeusedtopredictthepresenceorabsenceofciliarypropulsion

(figure5).Ifresultswarrant,microspherescouldthenbeusedtostudytheeffectofdiseaseortherapy

onmucociliaryclearancerates,andmayleadtoamoreeconomicalmethodtoevaluatemucociliary

activityinaclinicalsetting.

Figure5:Predictionoftwopossibletypesofrotationthatmaybevisible.Predictedrotationof

hemisphericallycoatedmicrospheresduetoA)CiliarycurrentorB)passiveflow.

1.5 Objectives

Theoverallobjectiveofthisstudy,therefore,wastodeterminethebestparticlefor

videoendoscopytodetectevidenceofmucociliaryclearancemovingforeignmaterialoverthemare’s

cervix.Itwashypothesizedthatpolyethylenegreenmicrospheres1-5μmand70μmindiameter,

12

wouldbesuperiortocarboninrevealingmicro-currentsonthecervicalepitheliumandthat50μm

hemisphericallycoatedbichromalmicrosphereswoulddisplayrotation.

13

CHAPTER2

MATERIALSANDMETHODS

2.1.InVitroAssessment

Thefollowingparticleswerestudiedbyinvitroanalysis:silverandgoldpowder,1-5µmgreen

fluorescentspheres,70µmgreenfluorescentmicrosphere,and50µmwhitefluorescentbariumcoated

microspheres(Cospheric®,SantaBarbara,CA).

Onemgofeachparticlewasplacedintoseparatewellsofa96-wellplate.Eachwellthen

received250µLofdistilledwaterandwasviewedunderaninvertedmicroscope(Amscope®Irvine,CA)

andvideorecorded.Currentwascreatedtosimulatemotionofmucosalfluidintheuteruscreatedby

thecontractionoftheuterus.Thiswassimulatedbyrepeatedlyaspiratingandexpellingtheliquidwitha

10µLpipettetogeneratemicrocurrents.Detergent(1µL)wasaddedtodisruptthesurfacetensionand

betterdistributetheparticles.Recordingsofeachsessionwerelaterobservedandassessedforvisibility,

degreeofclumping,andmobilityofeachkindofsphere.

Thenextportionoftheinvitrostudywastoobservetheparticlesinasix-wellplatewithan

endoscope,thelargerwellsbeingrequiredtoaccommodatetheendoscope.Intoeachwell,2mgof

eachparticlewassubmergedin5mLofdistilledwaterinseparatewells.The1-5µmgreen

microspheres,70µmgreenmicrospheres,50µmbariummicrospheresalsoreceived10µLofTween20

detergenttoaiddispersion.Currentwascreatedineachwellbyusinga1mLpipette.Eachwellwas

videorecordedusinghigh-magnificationvideoendoscopyfor5minutes.Recordingswerereviewedand

particlesassessedforvisibility,theirdegreeofclumping,andtheirmobility.Thewellcontaininga

mixtureofallparticleswasalsoobservedforhowtheparticlesinteractedwithoneanother(Table1).

Totestforthesuitabilityofpremixingandstoringaliquotsofparticlesuspensions,particle

mixtureswereleftintheirwellsandfrozenovernight.Theywerethenthawedafter24hoursand

14

viewedwithhigh-magnificationvideoendoscopyandassessedforanypossibledegradation,suchas

cracksorfracturing.

Table1:Assessmentguidelinesofparticlesduringmicroscopicandendoscopicportionsoftheinvitro

study.Particlesreceivedscoresbasedontheirvisibilityandmobility(1ifyes,0ifno),andaggregation

(zerorepresentsnoaggregation,while4,3,2,1represent100%,75%,50%,25%,ofthevisiblemicro-

particlesclumpingtogether,respectively.)

Criterion Results

Visibility 1or0

Aggregation 0-4

Mobility 1or0

2.2.ParticleSolutionPreparation

Differentsuspensionsofparticlesweremadetooptimizetheamountofeachparticle.Mareone

receivedasingledosemixtureof5mgofthe1-5µmgreenmicrospheres,1mgofcarbon,and2mLof

distilledwater.Maretworeceivedasingledoseof1mgofthe50µmbichromalmicrospheres,1mgof

the70µmgreenmicrosphere,1mLofdistilledwater,and10µlofTween20detergent.Maresthree,

four,andfivereceivedvaryingdoses(dependingontheamountofedema,andpossibleirritation)with4

pre-preparedsuspensionsasfollows:twosyringeswithsuspensionone:10mgofthe1-5µmgreen

microspheres,10mgofcarbon,and2mLofdistilledwater;andtwosyringeswithsuspensiontwo:

10mgofthe70µmgreenmicrospheres,10mgofthebichromalmicrospheres,2mLofdistilledwater,

and10µLofTween20detergent.

15

2.3.Videoendoscope

Avideoendoscope(Fujifilm,Wayne,NJ;EG-250WR5)wasattachedtoahigh-definition

endoscopydigitalvideoprocessorwithFICE(flexiblespectralimagingcolorenhancement)andalight

source(Fujifilm,Wayne,NJ;EPX-4440HDwithFICE).Thevideoprocessorwasthanconnectedtoa

televisionmonitor,aswellasalaptopcomputer(AppleMacbook,Suzhou,China.)Theanalogoutputof

thevideoprocessorwasconnectedtoalaptopusinganElgatovideocapture(Elgato,Munich,Germany;

10020840)inordertoconvertthefiletoacompatibledigitalformat.

2.4.MarePreparation:

ThistrialwasconductedduringJulyandAugustof2017.Becausethecervixiseasiertoexamine

whilemaresareinestrus,cervicalmucociliaryclearancewasassessedbyhigh-magnificationvideo

endoscopyin5estrousStandardbredmareswithdominantfolliclesofdiameter30mmorgreater.

Folliclesizewasdeterminedbytransrectalultrasonographyofestrousmares.Allmareswererestrained

inexaminationstocksfortransrectalultrasonographyandendoscopy.

Mares'tailswerewrappedwithasoftbandage.Sterilepreparationoftheperinealregionusing

genericbetadinescrubwasfollowedbyawaterrinsetopreventthebetadinefromenteringthevaginal

canal.Sedation(5mgofdetomidineIV)wasgivenpriortothestartofexperimentalexaminationin

ordertoreducemovement.Usingasterilegloveandsterilelubricant(FirstPriorityInc,Elgin,IL;NDC

#58829-200-05)a14mmx52cmendotrachealtube(Jorgensen,Loveland,CO;#J835Z)wasinsertedand

anchoredtotheinternalcervicalos(internalopeningofthecervix)byaninflatablecuff.A1cmx3cm

window(withedgessmoothed)waspreviouslycutintheendotrachealtube,withthelongaxisoriented

longitudinally,toallowthefoldsofthecervixtoflowintothetube.Thiscutwasmadewithascalpeland

theedgessmoothedwithabutaneflame.

16

Asuspensionofthemicrospheresandcarbonwasthendepositedonthecervicalepithelium

usingacatheterpassedthroughtheendoscope.High-magnificationvideoendoscopysessionswere

thenrecordedfor10-20minutes.

2.5.InVivoVideoAnalysis:

Followingexaminations,recordingswerereviewed.Portionsofthevideoswithaclearviewof

theepitheliumandparticles,atamagnificationof200Xorgreater,andlastinganobservableamountof

time(determinedtobetwosecondsorgreater)wereloggedforeachvideo.Withineachvideosegment,

eachtypeofparticlereceivedasinglescore(1or0)forthepresenceorabsenceinthevideosegmentof

thefollowingobservationsforeachparticle:particlevisibility,clumping,rotation,ormovement(i.e.

movementpastafixedpointofimmobilizedcarbon,twoparticlestranslocatedatdifferentspeedsorin

differentdirectionsorpastafixedanatomicallandmark,suchascapillarybranchpoint).Oncenumerical

scoreswereadded,theywerethendividedbythetotalnumberofvideosegmentsintherecordingto

determinethepercentofsegmentswhichcontainedtheobservationinquestionforeachtypeof

microparticle(e.g.percentageofsegmentscontainingcarbonmovement).Thepercentageswere

assignedascoreasfollows:0%=0;1%to25%=1;26%to50%=2;51%to75%=3;and76%to100%=

4.Scoreswereorderedsuchthatthehigherthenumberthemoredesirabletheparticle.Mediantotal

scoreswerethenanalyzedbytheKurskal-Wallistesttofindastatisticaldifferencebetweenusing

differentparticles.

17

CHAPTER3

RESULTS

3.1.InVitro

Thevariousmicroparticleswereanalyzedbyvideomicroscopyandvideoendoscopyinvitroto

determinetheirsuitabilityforinvivouseinthehorse.Thegoldpowderhadadistinctivecolorand

reflectivepropertywithlargegranuleswhichwerehighlyvisibleinfluid.However,thegoldparticles

exhibitedclumpingandaggregatedonthebottomofthewellswhenviewedbothundertheinverted

microscopeandwiththeendoscope.Whenawatercurrentwasapplied,thegoldremainedimmobile.

Silverpowderexhibitedahighdegreeofclumping.Incomparisontothegold,theparticlesofsilver

weremuchsmallerandasaresultmoreparticleswerestimulatedbythecurrentappliedtothedish.

Duetothelargedegreeofclumpingofgoldandsilverparticlestheywereconsideredunsuitableand

excludedfromsubsequentexperiments.

The1-5µmgreenfluorescentmicrospheresweredifficulttoseeduetotheirsize.Therewasno

bindingtoothermicrospheres,andtheylargelyappearedtobedispersedfreelyinthesolution.(Table

2).Whenawatercurrentwasappliedthesespheresmovedfreelythroughoutthesolution.Theywere

chosenforthenextphaseoftheexperimentduetotheircloseproximityinsizetobacteria.Ifciliaare

presentthentheviewermightbeabletoseeeachindividualsphereinteractingwiththeciliaasitmoves

throughtheuterus.

The70µmgreenfluorescentmicrospheresscoredhighonvisibility.Therewasnosignificant

clumpingamongthespheres.Theydidhoweverappeartositonthesurfaceofthedistilledwater,for

whichreason1µLofTween20wasaddedtobreakthesurfacetension.Thisallowedforthefree-

flowingmotionofthesubmergedspheres.

18

Thebariumsphereswerenoteasilyvisibleinsolutionunlessablackbackgroundwasplaced

underthe96-welland6-wellplates.ThebariumspheresdidnotclumpinsolutionbutTween20needed

tobeaddedtoallowfordispersal.Thebariumspheresdidnotsinkinthewaterasexpected,their

densitybeingrelativelyclosetowater.Thebariumspheresappearedmobileandtraveledinwaves

followingthecurrent.Duetotheirlowscoringvisibility,thesesphereswerenotusedfortheinvivopart

oftheexperiment.

Table2:Assessmentofparticlesduringmicroscopicandendoscopicportionsoftheinvitrostudy.

Particlesreceivedscoresbasedontheirvisibilityandmobility(1ifyes,0ifno),andaggregation(zero

representsnoaggregationwhile4,3,2,1represent100%,75%,50%,25%,ofthevisiblemicro-particles

clumpingtogether,respectively.)

Criterion Gold Silver 50µmBarium

microspheres

1-5µmGreen

microspheres

70µmGreen

microspheres

Carbon

Visibility 1 1 0 1 1 1

Aggregation 4 3 0 0 0 2

Mobility 0 0 0 1 1 1

Themixtureofparticles(Figure6)wasperformedtoseeanyparticleinteractions.Particleswere

againscoredfortheirvisibility,aggregationwithotherparticles,andmobility.However,forthisportion

oftheexperiment,awatchwaskeptonpossibleadversereactionsthatcouldinjurethemare,however,

nonewereseen.

19

Figure6:Appearanceofseveralparticlesundervideoendoscopy.Appearanceundervideoendoscopyof

A:70μmgreenmicrospheres,B:Silver,gold,bariumimpregnated(50μm)andgreenmicrospheres(1-5

μm,and70μm),C:1-5μmmicrosphereswithclumping.D:Bichromalmicrospheres

3.2.InVivo

Thesuspensionsselectedweretwomixtures.Thefirstwascarbonand1-5µmfluorescent

greenpolyethylenemicrospheres,and5mlofdistilledwater;thesecondwas70µmfluorescentgreen

polyethylenemicrospheres,50µmbichromalpolyethylenemicrospheres,5mlofwaterand10µlof

Tween20detergent.Thefirstmarereceivedonedoseof1mgofthe1-5µmmicrospheresand1mgof

carbon.Thesecondmarereceivedasingledoseof5mgofeachparticleusedinthisstudy.Thethirdand

fourthmaresreceivedtwodosesof10mgofeachparticleusedinthisstudy.Thefifthmarereceived

onedoseof10mgofeachparticleusedinthisstudy.VideoanalysisresultsareshowninTable3.

Overall,carbonwassuperiortoallmicrospheres.Ofthemicrospheres,the1-5μmappearedtobethe

mostsuitableforin-vivoanalysis.

20

Table3:Assessmentofmicrospheresandcarbon.Valuesintablerepresentmedianscoresfromalltrials

onascaleof0-4,fromleasttomostdesirable.Withinrows,valueswithoutasuperscriptincommon

arestatisticallydifferent(P<0.05).aisstatisticallydifferentfrombandc;bisstatisticallydifferentfrom

canda;cisstatisticallydifferentfromaandb.

Criterion 1-5μmGreenMicrospheres

(n=5)

70μmGreenMicrospheres

(n=4)

50μmBichromalMicrospheres

(n=4)

Carbon

(n=5)

Visibility 3a 3a 2a 4b

Aggregation 3b,c 0.5a 1a,b 4c

Motion 2a 2a 2a 4b

Rotation 0a 0a 0a 2b

CombinedMedian 2a 1a 1.5a 4b

21

CHAPTER4

DISCUSSION

Itwashypothesizedthatfluorescentmicrospheres1-5μmand70μmindiameterwouldbe

superiortometallicpowders(i.e.silverandgold)andcarboninassessmentofequinecervical

mucociliaryclearanceusinghighmagnificationvideoendoscopy.Thishypothesiswasnotsupporteddue

todataobtainedduringtheinvitroportionofthisstudy.Itwasalsohypothesizedthatpolyethylene

microspherestherangeof1-70μm,(includinghemisphericallycoatedbichromalmicrospheresof50

μm)wouldallbesuperiortocarboninrevealingmicro-currentsonthecervicalepitheliumsurfacewhen

observedbyhighmagnificationvideo-endoscopyinthelivemare.Thedatafromthisportiondidnot

supportthehypothesis,either.Wefoundthatcarbonmaycurrentlybethebestoptionforobserving

mucociliarymicro-currentsonthecervicalepitheliumofthemare.

Theinvitroexperimentsshowedwhichparticleswereabletoeasilyflowthroughthewater,as

wellashowtheywouldreactwitheachother.Metallicparticlesappearedtobetooheavyanddidnot

flowinthesolution.Thesealsoaggregatedandwerethoughttobetoolargeforciliatomove.Originally

therewereissueswithproperdispersionthroughoutthesolution,suchthataddingTween20was

testedtodisruptthesurfacetensionofthewaterandallowforbetterdispersal,whichitdid.Particles

werethenabletomovemorefreelythroughthesolution.Therewassomeaggregationofthe1-5µm

fluorescentgreenmicrospheresbutnotenoughtoexcludethemfromtheinvivoexperiments.

Bariummicrospheresdidnotperformasexpected.Theyweresuppliedinaclearvialthat

showedthemaswhite,butwhenviewedinasolutiontheywerenearlytransparent,makingthem

difficulttofindandobserve,whichledtothedecisiontoexcludethemfromtheinvivoportionofthis

study.Bichromalmicrosphereswerenotviewedinvitrobecausetheywerenotavailableatthetimeand

wereveryexpensive.Theywerethesamematerialasthe70µmfluorescentgreenmicrosphere,with

22

theonlydifferencebeingthecolor.Itwasdecidedthattheseparticlescouldbeusedintheinvitro

studybasedonthedataalreadycollected.

Particlesintheinvitroportionsofthisstudywereallscoredaccordingtothesamecriteria,

howeveritwasobservedthatwhenaggregationoccurredithadlittleimpact,eitherpositivelyor

negatively,duetoanabundanceofsmallparticlesremainingvisibletoshowmicrocurrents.

Consequently,anyparticlesscoringa0invisibilityora0inmobilitywouldnotbesuitable,but

aggregationwasnotbyitselfsufficienttojudgeaparticleunsuitable.

Twosuspensionswereusedinvivo.Thefirstwas1-5µmfluorescentgreenpolyethylene

microspheres,carbon,and5mlofdistilledwater,thesecondbeingcarbonwith70µmfluorescent

greenpolyethylenemicrospheres,50µmbichromalpolyethylenemicrospheres,5mlofwaterand10µl

ofTween20detergent.Varyingamountsofparticleswereusedinthefirsttwoexaminationsofthe

studytodeterminetheoptimalamountneededforobservation.While1mgofeachparticlewas

sufficientforobservation,thelikelihoodoffindingparticleswasgreatlyimprovedbyusingtwodosesof

10mg(atotalof20mgofeachparticlepermare).

Carbonexhibitedthehighesttotalscore,andthedeterminingfactorappearedtobetheability

ofcarbontorevealrotationofparticlesassmallasapproximately5µm,probablythroughtheirirregular

shapedistortingthemeniscusleadingtorepeatingflashesoflight(Figure7).Rotationcouldnotbeseen

inanyofthepolyethylenemicrospheres,exceptforthebichromalmicrospheres.However,thelatter

rarelydisplayedrotation,possiblyduetotheirlargesizeandpossiblybeingimmobilizedbymucus.

23

Figure7:Particlerotationgivingrisetoflashesoflight.Apparentrotationcapturedinthreeimagesover

approximatelyonesecond[A,B,C],probablyduetotheirregularshapeofcarbondistortingthe

meniscusleadingtorepeatingflashesoflight[B].

B

A

C

24

Withoutaggregation,the1-5µmmicrosphereswouldnothavebeenvisible,individualparticles

beingtoosmalltobeseenandthe1-5µmpolyethylenegreenmicrospheresweredifficulttoseeon

theirownduetotheirsmallsize.Whenclumpedtogetheritbecameclearthattheymaynotbeableto

adequatelyshowmucociliarymicro-currents,buttheywereobservedtobeextremelyusefulathelping

visualizethemucuspresentatlowermagnification(Figure8),almostasiftheywereactingasastain.

Thiswasoftenhelpfulinseeinggrossmovementofthemucusandgavesuperiorcontrastforviewing

otherparticles.

25

Figure8:Mucusstainingeffectof1-5µmfluorescentgreenpolyethylenemicrospheres.A)Microscopic

viewof1-5µmpolyethylenegreenmicrospheresrevealingamucusclusterandthefoldsinthecervix.

Mucuscanbeseenasagreenishfluidinwhichotherparticlesaretrapped.B)Macroscopicimageof1-

5µmpolyethylenemicrospheresrevealingmucusclustersandfoldstheytravelthroughonthecervix.

Thebichromalspheresweremucheasiertoviewontheepithelialsurface.Unfortunately,the

bichromalspheresfailedtoshowtherotationassociatedwithciliarymovement.Thisobservationmay

26

beduetotheparticlesbeingtoolarge.Movementandrotationweretypicallyseenwithsmaller

particlesofroughly10µmorless.

Uponexaminationofvideos,interestingobservationsweremade,suchasparticlesmoving

slowlyinamucusblanket,consistentwithdescriptionsofmucociliaryclearanceintherespiratorytract.

Inaddition,bloodcellsflowingthroughcapillariescouldbediscerned,alongwiththeapparentflowin

surfacelymphaticvessels(Figure9).Bloodvesselsandlymphatics(vesselsappearingtoosmallfor

passageofaredbloodcell)wereoftenusedasfixedlandmarkstovisualizetranslocationofparticles.In

someclips,itwasobservedthatdifferentregionsofthemucuslayerweremovingindependently,

possiblyduetodifferentsectionsofciliapropellingseparatemucus“sheets”(Figure10).Gross

movementofparticleswasnotexaminedclosely,butrevealedinterestingfeatureswhenstainedwith

the1-5µmpolyethylenemicrospheres.Grossmovementwasoriginallyexpectedtobeonalargescale,

duetouterinecontractionsandtheabdominalpress.

Figure9:Fluidflowthroughbloodandlymphaticvessels.Fromtheleftpaneltotherightpanel[T=0s,1s,

2s],thereisavisiblechangeinthevolumeoffluidinsidethevessels.

27

Figure10:Mucussheetsmovinginopposingdirections,inanalternatingpattern.Alternatingsheetsof

mucusmovingindifferentdirections.RedBox:Sheetofmucusmovingtotheleft.BlueBox:Sheetof

mucusmovingtotheright.

Anadditionalinterestingfindingthatwasobservedgrosslyover10-30minuteswasthatthe

epithelialsurfaceofthecervixwasabletofullycleartheparticles.Futurestudiesshouldincludeamore

detailedexaminationofthemacroscopicpathstakenbytheseparticlestwasalsoobservedthat,over

anentiresectionoftheepithelium,movementwasataconstantrateintheoppositedirectionto

gravity,afindingwhichcouldbeexplainedbyciliarymotion(Figure11).

28

Figure11:Self-clearingofthecervicalepitheliuminunder10minutes.Macroscopicviewofthecervical

epitheliumatapplicationofparticles(toppanel)andsevenminutespost-application(bottompanel)

showingself-clearing.

29

CHAPTER5

CONCLUSION

Thisstudyhasprovidedadditionalevidenceforthepresenceofmucociliaryclearanceinthe

mare'scervix.Althoughthepurposeofthisstudywastofindaparticlesuperiortocarbonforvisualizing

mucociliaryclearance,carbonseemsthebestparticletorevealmicroscopicmovement,and1-5µm

polyethylenemicrospheresforgrossmovement.Carboncanapparentlyshowrotationbydistortionof

themeniscusleadingtoflashesofreflectedlight.Futurestudiesthatwillbeconductedinclude

chromeoendoscopyandtheinclusionofvitalstainstorevealadditionalfeatures.

Basedontheseresults,amixtureof10mgcarbonand10mgof1-5µmpolyethylene

microspheresin1mLofwatermaybethebestavailableparticlemixtureforthistypeofstudy,andmay

behelpfulasadiagnostictestinidentifyingfocalareasofthecervixandendometriumwithpathologic

foci.Thegoalofallequinereproductivemucociliaryresearchistoimproveuponcurrentunderstanding

ofequineuterinedefensesinordertoimprovetreatmentanddiagnosisofequineinfertility.

30

REFERENCES

[1]BainAM.TheroleofinfectionininfertilityintheThorough-bredmare.TheVeterinaryRecord1996;78:168–175.[2]BrodySL.SensoryFunctionsofMotileCiliaandImplicationforBronchiectasis.FrontiersinBioscience2012;S4:3:1088–1098.[3]BurbidgeHM,RobertsonID.Prosandconsofbarium-impregnatedpolyethylenespheresingastrointestinaldisease.VeterinaryClinicsofNorthAmericaSmallAnimalPractice2000;2:449–465.[4]CanissoIF,StewartJ,CoutinhodaSilvaMA.Endometritis:ManagingPersistentPost-BreedingEndometritis.VeterinaryClinicsofNorthAmericaEquinePractice2016;32:465–480.[5]CauseyR.PathogenesisofequineendometritisduetoStreptococcuszooepidemicus.Ph.D.Thesis,LouisianaStateUniversity;1995.[6]CauseyR,GinnP,KatzB,HallB,AndersonK,LeBlancM.Mucusproductionbyendometriumofreproductivelyhealthymaresandmareswithdelayeduterineclearance.JournalofReproductionandFertilitySupplement2000;56:333–339.[7]CauseyR.MucusandtheMare:Howlittleweknow.JournalofTheriogenology2007;68:3:386–394.[8]CauseyR,ChristianE,HawkesM,DiverA,StokesM.Examinationoftheequinecervixbyhighmagnificationvideo-endoscopy.JournalofClinicalTheriogenologySupplement2016;8:345.[9]DimockWW,EdwardsPR.Pathologyandbacteriologyofthereproductiveorgansofmaresinrelationtosterility.KentuckyAgriculturalExperimentStationBulletin(ResearchBulletin)1928;286:157–237.[10]DixonPM.Respiratorymucociliaryclearanceinthehorseinhealthanddisease,anditspharmaceuticalmodification.TheVeterinaryRecord1992;131:229–235.[11]GardinerM.TheImportanceofCilia.HowardHughesMedicalInsititueBulletin2015;18:33–36.[12]GintherO.ReproductiveBiologyoftheMare.Equiservices,CrossPlainsWI,USA,1992.[13]GrayJ.CiliaryMovement.,CambridgeUniversityPress,London,1928.[14]InoueD,FurubayashiT,OgawaraK,KimuraT,HigakiK,KatsumiH,SakaneT,YamamotoA,HigashiY.Invitroevaluationofnasalmucociliaryclearanceusingexcisedratnasalseptum.BiologicalandPharmaceuticalBulletin2012;35:889–894.

31

[15]JacobS,ZhuY,KraftR,CottoC,CarmicalJR,WoodTG,EnkhbaatarP,HerndonDN,HawkinsHK,CoxRA.Physiologicandmolecularchangesinthetrachealepitheliumofratsfollowingburninjury.InternationalJournalofBurnsandTrauma2015;5:36–45.[16]KirchJ,GuentherM,DoshiN,SchaefarU.F.,SchneiderM,MitragoriS,LehrM.Mucociliaryclearanceofmicro-andnanoparticlesisindependentofsize,shapeandcharge-anexvivoandinsilicoapproach.JournalofControlledRelease2012;159:128-134.[17]LaiY,DilidaerD,ChenB,XuG,ShiJ,LeeRJ,CohenNA.Invitrostudiesofadistillateofrectifiedessentialoilsonsinonasalcomponentsofmucociliaryclearance.AmericanJournalofRhinologyandAllergy2014;28:244–248.[18]LeBlancMM,NeuwirthL,JonesL,CageC,MauragisD.Dfferencesinuterinepositionofreproductivelynormalmaresandthosewithdelayeduterineclearancedetectedbyscintigraphy.Theriogenology1998;50:49–54.[19]LouviA,GroveEA.CiliaintheCNS:thequietorganelleclaimscenterstage.Neuron2011;69:1046–1060.[20]Martin,E,Hine,R.ADictionaryofBiology.OxfordUniversityPress,Cary,NC,USA,2008.[21]MurphyCJ,GoleAM,AlkilanyAM,GoldsmithEC,BaxterSC.Goldnanoparticlesinbiology:beyondtoxicitytocellularimaging.AccountsofChemicalResearch2008;41:1721-1730.[22]PetersonFB,McFeelyRA,DavidJSE.Studiesonthepathogenesisofendometritisinthemare.ProceedingsoftheAmericanAssociationEquinePractitioners1969;15:279–287.[23]RaidalSL,LoveDN,BaileyGD.Effectsofpostureandaccumulatedairwaysecretionsontrachealmucociliarytransportinthehorse.AustralianVeterinaryJournal1996;73:45–49.[24]ScholeyJM.Intraflagellartransportmotorsincilia:movingalongthecell’santenna.JournalofCellBiology2008;180:23–29.[25]SongpingW,ShuyuanM.PreparationofultrafinesilverpowderusingascorbicacidasreducingagentanditsapplicationinMLCI.MaterialsChemistryandPhysics2005;89:423–427.[26]TroedssonMH,WoodwardEM.Ourcurrentunderstandingofthepathophysiologyofequineendometritiswithanemphasisonbreeding-inducedendometritis.ReproductiveBiology2016;16:8–12.[27]WatsonED.Post-breedingendometritisinthemare.AnimalReproductionScience2000;60:221–232.[28]WilliamsonP,MunyuaS,MartinR,PenhaleW.J.Dynamicsoftheacuteuterineresponsetoinfection,endotoxininfusionandphysicalmanipulationofthereproductivetractinthemare.JournalofReproductionandFertility1987;35:317–325.

32

[29]WilloughbyRA,EckerGL,McKeeL,RiddollsLJ.Useofscintigraphyforthedeterminationofmucociliaryclearanceratesinnormal,sedated,diseasedandexercisedhorses.CanadianJournalofVeterinaryResearch1991;55:315–320.

33

APPENDIX:Individualinvivoscoretables

Appendix:Individualassessmentofmicrospheresandcarbonaccordingto4criteria.Valuesintable

representscoresonascaleof0-4,fromleasttomostdesirable.

0=Presentin0%ofvideosegments

1=Presentin1-25%ofvideosegments

2=Presentin26-50%ofvideosegments

3=Presentin51-75%ofvideosegments

4=Presentin76-100%ofvideosegment

Table4:Mare1(Sara)

Criterion 1-5μmGreen

Microspheres

70μmGreen

Microspheres

50μmBichromal

Microspheres

Carbon

Visibility 2 - - 4

Aggregation 2 - - 4

Motion 2 - - 3

Rotation 0 - - 1

34

Table5:Mare2(Blisstex)

Criterion 1-5μmGreen

Microspheres

70μmGreen

Microspheres

50μmBichromal

Microspheres

Carbon

Visibility 3 2 2 4

Aggregation 0 1 1 2

Motion 2 2 2 4

Rotation 1 0 0 2

Table6:Mare3(Laney)

Criterion 1-5μmGreen

Microspheres

70μmGreen

Microspheres

50μmBichromal

Microspheres

Carbon

Visibility 3 3 3 4

Aggregation 3 1 1 4

Motion 2 1 1 4

Rotation 0 0 0 2

35

Table7:Mare4(Susie)

Criterion 1-5μmGreen

Microspheres

70μmGreen

Microspheres

50μmBichromal

Microspheres

Carbon

Visibility 4 3 2 4

Aggregation 4 0 2 4

Motion 4 2 2 3

Rotation 0 0 1 2

Table8:Mare5(Gina)

Criterion 1-5μmGreen

Microspheres

70μmGreen

Microspheres

50μmBichromal

Microspheres

Carbon

Visibility 3 3 2 4

Aggregation 3 0 1 3

Motion 2 2 3 4

Rotation 0 0 0 2

36

BIOGRAPHYOFTHEAUTHOR

MelissaHawkeswasborninYork,MaineonMay27,1994.ShewasraisedinKittery,Maineand

graduatedfromR.W.TraipAcademyin2012.SheattendedtheUniversityofMaineandgraduatedin

2016withaBachelor’sdegreeinAnimalScience.SheenteredtheAnimalSciencegraduateprogramat

TheUniversityofMaineinthefallof2016.Afterreceivingherdegree,Melissawillbeapplyingto

veterinaryschoolsinordertobeginhercareerinveterinarymedicine.Melissaisacandidateforthe

MasterofSciencesdegreeinAnimalSciencefromtheUniversityofMaineinMay2018.