The microbiology of bovine semen and the antimicrobial activity of bovine seminal plasma

242 Pages·2012·7.77 MB·English

by  Schollum, Linda May

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THEM ICROBIOLOOGFY B OVINES EMEN
AND
THEAN TIMICROBIALAC TIVITYO F BOVINES EMINALP LASMA
A thesisp resenteidn partiaflu lfilment
of ther equirementfso rt hed egreeo f
Doctoro f Philosophiyn Microbioloagty
MasseUyn iversity.
LindaM ayS chollum
December1, 977.
ii
ABSTRACT
Microbial populaitni soenmse n fr1o8m b ullsu sedf ora rtificial
3
breedingv,a riebde twee3n0 and5 600,00o rganis/mcsm, witha meano f
3
3,300/cm Wided ay,t o dayf luctuatiooncsc urreidn them icrobial
•
contenotf semena ndt herew asn o correlatiboent weetnh et otalm icrobial
countst,h ep roporiotn of theseo rganismsse nsitivteo bovinese minal
plasmai nhibitioann dt hem easureabalmeo unto f antibacteriaaclt ivitiyn
a givens emens ample. Alls emens amplesh ada ntiabcteriaalc tivity, the
amount being relaticvoenlsyt anfto ra givena nimal;m arkedd ifferences
occurrebde tweebnr eeds. Sixty-ei'gphetrcento f theb acteriias olated
froms emen( representtienngg eneraw)e rei nhibitebdy s�minapll asma.
Togethewri tho therd ata,t hiss upporttsh ev iewt hatm osts emenm icro­
organismasr et ransiencto ntaminanftrso mo utsidet her eproductitvre� ct,
derivedm ainlyf romt hep reputiacla vity.
Them ostc ommono rganismisn bovines emenw eres pecieosf Bacillus,
CorynebacterainudmM icrococcubsu,t t welveo therb acteriagle neraa ndt wo
yeasts peciewse rea lsor epresentameodn g the isolatesM.o sts emen
microorganiwsemrse s ensitivteo severaaln tibioticwsi,t hp seudomonads,
coliformasn dy eastpsr ovintgh em ostr esistant.T hec ontroolf these
organismiss importansti ncet heym ayg rowi n extendesde mena ndh aveb een
implicatiend bovineg enitadli s9rders.C hlorampheniacndo ls treptomycin
arer outineluys edi n semene xtenderasn dh aveb eenh ighlye ffectivien
controllibnagc teriaglr owth;p enicillhians b eenl esse ffective.
At leastt hreea ntimicrobiaaclt ivitiheasv eb eeno bsevredi n bovine
o
seminapll asma:( i)a n antiviraalc tivity(,i i)a heatl abile( 60C for
60 minutesa)n tibacteriaaclt ivitayn d (iii)a heats tablea ntibcaterial
activity.R esearchha sc entereodn theh eat stabflrea ctiownh ichi s
sensitivteo proteolyteincz ymebsu tn ott o periodatter eatmentT.h ish eat
stablef ractiocna nb e purifiebdy adsorptioonnt oh ighlyp olymerised
deoxyribonuclaeciicd ( DN)A. It doesn otr esemblel ysozymep,o lylysine,
salminoer spermidiinne antimicrobiaslp ectrume,l ectrophoreptriocp erties
or amino acidc ompositionD.a taf romd ialysisp,o lyacrylamgiedle
electrophoresuilst,r afiltratainodnu ltracentrifugaitnidoinc atteh att he
activem oietyo ccuris n varyinmgo leculawre ighta ggregatecsa,u sebdy
selfa ssociatiaonnd i nteractiwoint ho therm icrosolutperse senitn its
environmentA.t leastt woo f thea ggregate(sc ationaitc pH8.6)a rea nti-
bacteriably theo verlatye chniquweh ichd etectbsi ologicaaclt ivitiyn
electrophorepsreodt eins.H oweveri,t migrateass a singleb and( molecular
iii
weight2 5,000)i n sodiumd odecyslu lphat(eS D)S polyacrylamgiedle
electrophoresainsd,h asa singleN terminaalm inoa cid. Whenc ompared
witha ntibactericaolm pounddse scribebdy otherw orkers,t hea nti­
microbiaplr oteinfsr omb ovine seminapll asmaw eref oundt o havea unique
chemicaclo mposition.
Bovines eminapll asmap roteinhsa da rapidl ethale ffecto n both
"
Gram-positiavned G ra�negativbea cteriaas wella s ureaplasmas and
mycoplasmasB.a cteriarle sistancteo bovines eminapll asmae xhibitead
degreeo f correlatiwoint hr esistantcoe antibiotiacfsf ectincge llm embrane
integrity.D etectioonf cytoplasmilce akagel,o sso f accumulatleadb elled
compoundsa ndf ailurteo accumulaat el abelleadm inoa cidb y bacteriian
contacwti ths eminapll asmas upporttsh eh ypothestihsa tm embrandea magei s
inducebdy bovines eminapll asmaa ntimicrobiparlo teins.R esistance of
Pseudomonaaesr uginotsoa bovines eminapll asmaa ppeartso be duet o the
bacteriacle llw allp reventiancgc esso f thea ctivem oietyto thec ell
membrane.
Bovines eminapll asmaa ntimicrobicaalt ionipcr oteinmsa yw ell"
contributtoe an efficienptr imarnyo nspecifdiecf encmee chanisomf the
genitatli ssues.F ewm icroorganicsomlso nisteh eu pperr eproductitvrea ct;
ascendinign fectiofnrso mt hel oweru rethraan dp reputiacla vityw,h ere
bacteriaplo pulatioanrse l arge,a reu ncommon.O nlyb acterisah ownt o be
resistnta to antimicrobial fapcrteosresn ti n bovines eminapll asmah ave
beeni mplicatiend bovinem aler eproductitvrea ct infections.D uring
coitust hea ntimicrobiaaclt ivitmya ya lsop rotectth ef emaleg enitatlr act
fromi nfection.
iv
ACKNOWLEDGEMENTS
I wouldl iket o personaltlhya nkt hef ollowing:
ProfessoDr. F.B aconan d Dr B.D.W.J arvisf,o rt heirs upervision
andg uidance.
P. Shannoann dD r K. MacMillaonf, theN ewZ ealanDda iry
Mr
BoardA rtificiBarle edinSge rvicef,o rt hep rovisioonf semena nd
seminapll asmaa,n d fotrh eire ncouragemeanntdf riendship.
Dr J. Clarkef,o rh isw orko n thea ntiviraplr opertioefs bovine
seminapll asmaa,n df orh isi nterestth roughothuet thesis. ,... .
Dr G. Midwintefro,r t hea minoa cida nalysiosf materiails olated
fromb ovine seminal plasma.
Dr C. Moore, fora ssistancwei thu ltracentrifugatteicohnn iques.
ProfessoNr. P.M arkham,O tagoU niversitfyo,r t hep rovisioonf
Ureaplassmtar ains.T heV eterinaPrayt hologDye partmenMta,s sey
Universitfyo,r L eptospiproam onaa ndL . hardjoc ulturesa,n dt he
BiochemistSreyc tiofno rt he loano f electrophoreesqiusi pment.
My parentsf,o rt heira ssistanwciet h photographfsi gaunrde sa,n d
fort heirn ever-endisnugp port.
Thanksa lsot o allm y otherc olleaguefsa,m ilayn df riendst,o o
numeroutso name,b utw hoh elpeda nde ncouragmeed throughotuhti st hesis.
v
TABLE of CONTENTS.
Page Number.
ABSTRACT ii
ACKNOWLEDGEMENTS iv
TABLE OF CONTENTS
v
LIST OF TABLES xi
LIST OF FIGURES xiv
LIST OF;, PLA,!'ES xviii
INTRODUCTION 1
CHAPTER1 : THE MICROBIOLOGYO F BOVINE SEMEN 2
1.1Mi crobialP opulationsi n Bovine Semen 2
1.2Th e Importanceo f Total MicrobialN umbersi n
Bovine Semen 5
1.3Th e Compositiono f the MicrobialF lora of Bovine
Semen 6
1.4Th e Significanceo f CertainM icroorganismsi n
Bovine Semen, thReoilre in Infertilitya nd the
9
Likelihoodo f their Transmissiont o ServicedC ows
1.5Th e Value of AntibioticA dditionst o Bovine Semen
for use in ArtificialB reeding 15
CHAPTER2 THE NATURALA NTIMICROBIAL ACTIVIOTFY B OVINE SEMEN 18
CHAPTER 3 THE AIM OF THIS INVESTIGATION 20
MATERIALA ND METHODS 21
CHAPTER4 : GENERAL EXPERIMENTALT ECHNIQUES 21
4.1Se men and SeminalP lasma Samples 21
4.2De velopmento f an Assay System to Detect and
Measuret he Antibacterial Activiitny B ovine
GenitalS ecretions 21
4.3Th e StandardM ethod for the Assay of Antibacterial
Activityi n Bovine SeminalP lasma 23
4.4Th e StandardR eferenceA ssay for Comparisono f
the Inhibitory Activity of Bovine Seminal Plasma
and Selected Antimicrobial Agents 25
/ .
. .
vi
TABLE of CONTENTS (contd)
CHAPTER 5 TECHNIQUESU SED IN THE INVESTIGATIONO F MICROBIAL
POPULATIONSI N BOVINE SEMEN 26.
5.1Me dia used for the Determinationo f Total Microbial
Counts in Bovine Semen 26
5.2T echniquef or Total Viable Counts of Micro-
organismsi n Bovine Semen 26
5.M3a terialsa nd Methodsu sed to test the Sensitivity
of Semen Microorganismts o CommerciallyA vailable
Antibiotics 26
5.4Me thod for DifferentiatinSge men Microorganisms
Sensitivet o or Resistantt o the NaturalA nti�
microbialA ctivityi n Bovine SeminalP lasma 27
CHAPTER 6 MEDIA AND METHODSU SED IN THE IDENTIFICATIOONF
SEMEN MICROORGANISMS 28
6.1Me dia for GeneralM icrobiologicaIln vestigations 28
6.2Me dia and Methodsf or SpecificB iochemicalT ests 28
CHAPTER 7:T ECHNIQUESU SED IN THE CHEMICALA ND PHYSICAL
CHARACTERISATIONO F THE INHIBITORYF ACTORSI N BOVINE
SEMINALP LASMA 32
7.1Ma terialsu sed to Comparet he Characteristicosf
Bovine SeminalP lasma Antimicrobial Activiwtiyt h
Those of SelectedA ntimicrobial Agents 32
7.2Me thod for the Investigationo f the Effect of
ProteolyticE mzymeso n the AntibacterialA ctivity
of Bovine SeminalP lasma and SelectedA ntimicrobial
Agents 32
7.3Me thod for the Investigationo f the Effect of
PeriodateT reatmento n Bovine SeminalP lasma Anti­
BacterialA ctivitya nd the Activityo f Selected
AntimicrobialA gents 32
7.4Ul trafiltratioTne chnique 33
7.N5a tive PolyacrylamideG el Electrophoresis 33
7.6Lo cationo f AntibacterialA ctivityi n ProteinB ands
Separatedb y Native PolyacrylamideG el Electro-
phoresis 33
CHAPTER8 TECHNIQUESU SED TO DETERMINET HE SPECTRUMO F
ANTIMICROBIALA CTIVITYO F BOVINE SEMINALP LASMA 35
8.1Ma terialsu sed to compare the ActivityS pectrumo f
Bovine SeminalP lasma with those of Human Seminal
Plasma and SelectedA ntimicrobialA gents 35
8.2Te chniquesf or Determiningt he Bacterial
SensitivityS pectrumo f Bovine and Human Seminal
Plasmasa nd SelectedA ntimicrobialA gents 35
8.3Te chniquesf or Determiningt he Sensitivityo f
Mycoplasmast o Bovine and Human SeminaPll asmas
and SelectedA ntimicrobialA gents 35
/
vii
TABoLfEC ONTE(NcToSn td
8.T4e chnitqoDu eetmsei rnweh etBhoevri Sneem inal
Plmaasi sB acitoeasrttiocrB acteriocida3l6
CHAP9T ETRE CHNIQUIENST HIEUOS SLEADT OIFOA NNA NTI­
MICROFBAICATLFO RROB MO VISNMEEI NPALLA SMA 37
9.T1h Keej lda-hGlu nnDiinsggte iaonnCd r ismer
moidfiVeadSn l ykHei llPerro cefdouArrmm eo nia
&
Determi;nu asteaidso� n m esauroeft hper otein
cont(em nitc.r moN glrcapml maas) o f crude
preaptairoonfas n a ntimiccroombpiofaurlno dm
bovinsee mipnlasalm a 37
9.T2e chunseii qnI oEnx chaCnhgreo matoogfr aphy
BoviSnmeei nPallma as 38
CHAP1T0ER T ECHNIUQSUETEDOSI OSLAATNECD H ARACITSEAERN
ANTIMICFRAOCBTIIOASLRLOA TFEORDM B OVISNMEEI NAL
PLASBMYAA D SPOTRIOONNT DOE OXYRIBOANCUICDL 4E3I C
1.01D etermionfat thOiepo tni Cmoudnmi tifoonrs
Adsorpotfti hoAenn timicFraocbtoionart�D o N A 43
1.02Pr otDeeitnme irnatbiyMo ent hoofLd o wertya l
(19,5uls)etdom euarsteh per otceoinnt oefn t
materiisaaoltl efdr obmo visnmeei npallma asb y
adsorpotnitoon DNA 43
1.03Di snctoinuDoiuiaslf traotfiD oN-nAsi QlaAtnetdi ­
MicroMbatiearlit aorl e mocvireta tieo nfsr om
solution 44
1.04A ssatyo D etertmhiAenm euon otf C itrPartseee nt
inS amepsol fA nitmicroMbaeitrailia osll taefdr om
BoviSnmeei nPallma asb yA dsorpotnitDooNn A 44
1.05So iduDmo deScuyllp h(aSt)DeP S olyacryGlealm ide
Eltercorpehosis 45
RESU:LP TASR T ONE
THMEI CROBIOOFBL OOVGIYSN EEM AENNID TRSE LATIOTNOTS HHAEIN PT IBIAACLT ER
ACTIVPIRTEYS IENNT TH ISSE ECTRI.O N
CHAPTER 11 P:O PMUILCAIRTNOIB BOOINVASISL NE EM EN 47
11.1T hTeo tMailc roPboipaaultl ioonfBs o viSnmeee n 47
1.12Es mtaitiooftn h Vea rioeftM yi ocorrganisms
OccurirniE nagcS hme eSna mple 48
1.13Th Tepy eosf M icroosrmgcsao onmmniliyos ltaedo mf r
BoviSnmeee n 48
1.14T hAen tibiSoetinitsicit voyf M iocorrgsamnsi
iosladt feroBmo viSnmeee n 49
CHAP1T2E:R T HAEN TIBACATCETRIIVAOILFBT OYV ISNEEM EN 51
.., ....
1.21Th Oec curroefAn ncbteai cteArcitailvi int y
IndiviSdeumaSelanp m les 51
I .. . .
------- ------- ----------- -----
viii
TABoLfEC ONTE(NcToSn td)
12.T2h Eef feocftt h Nea tuArnatli tbearciAaclt ivity
inB oviSneem oennt hTeto laM icrobial
Pouplaotnii nS meen 52
12.T3h Eef feocfNt a tuArnatli tbearciAaclt ivoint y
thNeu embro fM icroiomsrsgS anenistitvoea nd
Resitsanttot hsiA ctiviinIt nyvd iiduSamele n
Sapmles 52
1.24T hEef feocftt h Aen titbearciAaclt ivoifBt oyv ine
SmeinPallma aso nS eveGreanle rBaa cotfe ria
iosladt feroBmo viSnmeee n 53
RESU:LP TASR TTW O
THNEA TUORFTE H AEN TIOMBIICAARCLIT VYII TNB VOIENS EMAENNBD O VISNEENM AIL
PUS�
CHAP1T3E RT HCEH EMICCHAALTR EACRISAOTFI OTANHN ET IBACTERIAL
ACTIVIINBT VOYI NSEME NIAPLL ASMA 55
13.T1h See intsiyvo iftB oviSneem iPnlamalas A nti­
BacteArcitailvt ioPt ryo teoElnyzetyi,smncad a
Copmariwsiotnth h Aec vtiitioefSs le etceAdn ti-
MicroAbgieanlt s 55
1.32T hEef feocfPt e irodaotne- tAhnet itbearcial
ActivoifBt oyv iSnmeei nPallma asa ntdh soeo f
SleeecdtA nmtiicroAbgieanlt s 55
133.T-hHee aSte intsivoiftB yo viSnmeei nPallma as
AntimicArcotbiivailt y 56
CHAP1T4E RT HEEF FEOCFST O DMI CUTIRAOTNET HAEN TIBACTERIAL
ACTIYV IOBTFO VISNMEENI APLL ASMA 58
14.T1h Eef feocfVt a ryAimonugn otfsC itr0antt eh e
InhibiZtnoieposrn do uecdb yB oviSnmeei nPallma as
AnbtaicteArcitailv ity 58
14.M2do icftaiioonft hAeg aWre lDlfi fiuosAns staoy
IncorpCotirraattee 58
CHAP1T5E:R I NVTEISGANTO IFTO HPEH IYACSLS ATTOE F TAHNIET-
MIOCBRIFAALC TIONRBS O VISNEEMA ILPN USMA 60
15.T1h Aeb sorbSapnecucemto rfB oviSnmeei nPallsa ma 60
15.D2il aysoifsB oviSnmeei nPallma as 60
1.53U ltfriarlattioofBn o viSneem iPnlamalas 60
1.54N atiPvoel yamcirdyGele Eall tercorpehosoifs
BoviSnmeei nPallma as 61
RESU:LP TASR TTH REE
THSEP EUCMTO RFA CTIVAINTMDYO DOEFA CTIOOFTN H AEN TCIRMOIBAICATLI YV IT
OFB OIVNSEE MAILPN USMA
.
/ .
.
ix
TABoLfEC ONTE(NcToSn td)
CHAP1T6E RT HSEP EUCMTO RFA NTIOMBIICAARCLT IVIINBT OYV INE
SMEINPALL ASMA 62.
1.61T hBea cteRresipasolent oB ovianneHd u man
SmeinPallma asa ntdo S eclteeAdn timiiaclr ob
Agents 62
1.62Th Eef efcotf B oviSneem iPnlasalm oan
Myocpalmsasa nVdi urses 64
CHAPT1E7R THMEO DOEFA CTIOOFTN H AEN TIMICARCOTBIIVAILT Y
OFB OVISNEEM IPNLAALS MA 65
1.71Th Mei crosEcxoapmiicn oaftB iaocnt etrrieaa ted
witBho viSnmeei nPallma as 65
1.72Ba cterivosetrasBtuaiscc t eriEoffceicdotafsl
BoviSneem iPnlamalas 66
1.73A Cpoamsroionf t hAen tcitbearAicatli voift y
BoviSnmeei nPallma asa nodf t hAec tivoifat y
Seclteioofn CeorimcamlAlvya ilAanbitlbei otic6s7
174.F urtEhveird efnocre CtehlMele mbrbaenien g
thSeti eo fA ctiofno trh Aen timiiacAlrc otbivity
inB oviSnmeei nPallma as 68
RESU:LP TASR FTO UR
THIES AOTLIAONNCD H ARACTEROIFAS NAA TNITOINM ICFRAOCBTIFOAORRLMB OVINE
SEMIPNLAALS MA
·CHAP18T EPR:R ELIMAITNTAERMYTP OIT OSSL AAT E FACRTEOSRPI OBNLSE
FOTRH AEN TIMICARCOTBIIVAOILFBT OYV ISNHEEI NAL
PLSAMA 70
1.81A ciDdi alyasnAidcs e tPorneec ipittoga itvieo n
ac ruPdree aptairoofna nA ntimicCroombpioauln d
presiennB to viSneem iPnlamalas 70
18.I2o Enx chaCnhgreo mraatpoohgfyB oviSneem inal
Plsama 71
CHAP1T9E RD EOXYRIBOANCUI(CDNDL AEI)I SCAO TLIOOFNA COMPOUND
RESPIOBNLFSEO SRO MOEFT HAEN TIOHBIICAARCLT IVITY
OFB OVISNEEM IPNLAALS MA 74
1.91C ointdiofnostr h Aed sorpotfti hoAenn btaicterial
ActivointtDyoN A 74
1.92C odntioinfso trh dei osicsaitoonf t hDeN pAr-otein
Copmlex 76
1.93Re covoeftr hyAe c tiFvrea ctfirootnmh Cei trate/
Ethasnoollu tion 77
1.94S ummoafrt yh Eex tracPtriosocsnfe otrh Ies otliaon
ofa nA ntibacterifarloBm o FvaiScnmeteio nra l
Plmaasb y AdsorpDtNiAo n onto 77
/ . .