Effects of GnRH vaccination in wild and captive African Elephant bulls (Loxodonta africana)

21 Pages·2017·26.2 MB·English

RESEARCHARTICLE
Effects of GnRH vaccination in wild and
captive African Elephant bulls (Loxodonta
africana) on reproductive organs and semen
quality
ImkeLueders1,2*,DebbieYoung3,LianaMaree4,5,GerhardvanderHorst4,5,IlseLuther4,5,
StephanBotha6,BrendanTindall7,GeoffreyFosgate8,Andre´Ganswindt2,Henk
J.Bertschinger8
a1111111111
a1111111111 1 GEOlifes-AnimalFertilityandReproductiveResearch,Frohmestr.7,Hamburg,Germany,2 Endocrine
ResearchLaboratory,DepartmentofAnatomyandPhysiology,FacultyofVeterinaryScience,Universityof
a1111111111
Pretoria,Onderstepoort,SouthAfrica,3 AfricanElephantResearchUnit,PlettenbergBay,SouthAfrica,
a1111111111
4 DepartmentofMedicalBiosciences,UniversityoftheWesternCape,Bellville,SouthAfrica,5 Department
a1111111111 ofResearchandScientificServices,NationalZoologicalGardensofSouthAfrica,Pretoria,SouthAfrica,
6 AfricanLionSafari,Cambridge,ON,Canada,7 RobbergVeterinaryClinic,56Longships,PlettenbergBay,
SouthAfrica,8 DepartmentofProductionAnimalStudies,FacultyofVeterinaryScience,Universityof
Pretoria,Onderstepoort,SouthAfrica
*[email protected]
OPENACCESS
Citation:LuedersI,YoungD,MareeL,vander
HorstG,LutherI,BothaS,etal.(2017)Effectsof Abstract
GnRHvaccinationinwildandcaptiveAfrican
Elephantbulls(Loxodontaafricana)on
reproductiveorgansandsemenquality.PLoSONE
Objectives
12(9):e0178270.https://doi.org/10.1371/journal.
pone.0178270 AlthoughtheAfricanelephant(Loxodontaafricana)isclassifiedasendangeredbytheInterna-
Editor:GovindhaswamyUmapathy,Centrefor tionalUnionforConservationofNature(IUCN),insomeisolatedhabitatsinsouthernAfrica,
CellularandMolecularBiology,INDIA contraceptionisofmajorinterestduetolocaloverpopulation.GnRHvaccinationhasbeen
Received:December18,2016 promotedasanon-invasivecontraceptivemeasureforpopulationmanagementofoverabun-
dantwildlife.Wetestedtheefficacyofthistreatmentforfertilitycontrolinelephantbulls.
Accepted:May10,2017
Published:September15,2017
Methods
Copyright:©2017Luedersetal.Thisisanopen
Intotal,17maleAfricanelephantsthatweretreatedwithaGnRHvaccinewereexaminedin
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which twogroups.Intheprospectivestudygroup1(n=11bulls,ages:8–36years),semenquality,
permitsunrestricteduse,distribution,and thetestes,seminalvesicles,ampullaeandprostate,whichwereallmeasuredbymeansof
reproductioninanymedium,providedtheoriginal
transrectalultrasound,andfaecalandrogenmetaboliteconcentrationsweremonitoredover
authorandsourcearecredited.
athree-yearperiod.Eachbullintheprospectivestudyreceived5mlofImprovac®(1000μg
DataAvailabilityStatement:Allrelevantdataare
GnRHconjugate)intramuscularlyafterthefirstexamination,followedbyaboostersix
availablefromfigshare(https://figshare.com/
weekslaterandthereafterevery5–7months.Inaretrospectivestudygroup(group2,n=6,
articles/Raw_data_ELE_GnrH_xlsx/5313388).
ages:19–33years),oneexaminationwasperformedonbullswhichhadbeentreatedwith
Funding:I.LuedersandGvDHreceivedagrant
GnRHvaccinefor5–11years.
fromtheU.S.FishandWildlifeService-African
elephantFund#AFE0705;I.Luedersreceived
fundingthroughtheGermanAcademicExchange Results
Service(DAAD)withinthePostdoctoralFellowship
Inallbullsofgroup1,testicularandaccessorysexglandsizesdecreasedsignificantlyafter
Program,Grant#D/11/45084;I.Luedersreceived
projectfundingthroughtheInternationalElephant thethirdvaccination.Insixmalesexaminedpriortovaccinationandagainaftermorethan
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 1/21
ElephantbullGnRHvaccination
FoundationandtheBirminghamZoo,USA;HJB fivevaccinations,thetestissizewasreducedby57.5%.Meantesticularheightandlength
receivedadditionalfundingthroughtheTechnology decreasedfrom13.3±2.6cmx15.2±2.8cmatthebeginningto7.6±2.1cmx10.2±1.8
InnovationAgency(Pretoria,SouthAfrica).The
cmattheendofthestudy.Postpubertalbulls(>9years,n=6)examinedpriortovaccination
fundershadnoroleinstudydesign,datacollection
andanalysis,decisiontopublish,orpreparationof producedejaculateswithviablespermatozoa(volume:8–175ml,spermconcentration:
themanuscript. 410-4000x106/ml,totalmotility:0–90%),whileafter5–8injections,only50%ofthesebulls
Competinginterests:Theauthorshavedeclared producedejaculateswithasmallnumberofimmotilespermatozoa.Theejaculatesofgroup
thatnocompetinginterestsexist. 2bulls(vaccinated>8times)weredevoidofspermatozoa.Faecalandrogenmetabolitecon-
centrationsmeasuredincaptivemalesdecreasedsignificantlyafterthefourthvaccination.
Noneofthemalesenteredmusthduringthetreatmentperiod.
Conclusions
Ourresultsshowedamarkeddecreaseinsemenquality,testicleandsecondarysexgland
sizesfollowingrepeatedGnRHvaccinations.After2–4yearsofcontinuoustreatmentevery
5–7months,theeffectsweresimilartosurgicalcastration.
Introduction
Gonadotrophinreleasinghormone(GnRH)issynthesizedinspecializedneuroendocrinecells
inthehypothalamusandtransportedtotheanteriorpituitaryviathehypothalamic-pituitary
portalsystem,whereitinducessynthesisandreleaseofthegonadotrophichormones,follicle
stimulatinghormone(FSH)andluteinizinghormone(LH)[1].Bothgonadotrophichormones
areimportantfortheregulationofmaleandfemalegonadalfunction(gametogenesisandste-
roidogenesis).Inmales,thisincludestheproductionofspermatozoaandtestosteroneinthe
testes.TheconceptofGnRHvaccinesisbasedupontheinterruptionofthehypothalamic-pitu-
itary-gonadal(HPG)axisbystimulatinganti-GnRHantibodyproduction.Theseantibodies
neutralizeendogenousGnRHand,subsequently,disruptthedownstreamreleaseofgonado-
trophichormones.BeingabletotargetGnRHinordertosuppresstesticularfunction,without
hormonalorsurgicalintervention,appearstobeanelegantmethodofcontraception.Thisis
oftenreferredtoasdown-regulationoftesticularfunctioninthepeer-reviewedliterature.Effi-
cacyis,however,dependentontheachievedhumoralimmunityandmayvarybetweenand
withinspeciesaswellasGnRHvaccineformulations[2].Forinstance,ithasbeenshownthat
olderstallionsaremoredifficulttosupressthanyoungerones[3,4].Whilemaleratsshowa
strongresponsewithalmosta100%arrestinspermatogenesis,inmaledogsadecreaseofonly
5%inspermatogenicactivitywasreported[5].
CommercialGnRHvaccineshavebeendesignedfordomesticanimalsandtestedinboth
sexesofpigs[6],cattle[7]andhorses[4,8].GnRHvaccineshavebeensuggestedasmeansof
pestorferalanimalcontrol,andhavebeentestedinseveralwildlifespecies,suchaswhite-
taileddeer(Odocoileusvirginianus,[9]),bison(Bisonbison,[10])andwildboars(Susscrofa,
[11])withmoderatetogoodsuccess.RecentstudiesindicatedthatGnRHvaccinescouldbe
usefulforthemanagementofandrogendrivenbehavioursinelephantsandthehormone
mediatedmusthconditionseeninadultAfrican(Loxodontaafricana)andAsianelephant(Ele-
phasmaximus)bulls[12–14].TheGnRHvaccineImprovac1(ZoetisAnimalHealth,South
Africa)hasbeenfrequentlyusedasanextra-labeldruginsouthernAfricatocontrolandro-
gen-relatedaggressivebehaviourandmusthinapproximately45bullsoverthepast10years
[13].Independentcasereportsonamale[15]andafemaleAsianelephant[16]suggestthat
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 2/21
ElephantbullGnRHvaccination
GnRHvaccinesarecapableofsuppressinggonadalfunctionaftermultiplemonthlyinjections.
Aftertwotothreetreatments,adecreaseinserumtestosteroneandfaecalandrogenmetabolite
concentrationwasfoundinmaleAsian[17]andAfricanelephants[14],respectively.Thiswas
accompaniedbybehaviouralimprovementand/orattenuationorpreventionofmusth.Arise
inanti-GnRHantibodytitrescouldbeconfirmedafter3–5monthlyboostersinonefemale
[16]andafter2–4monthlyboostersinsixmaleAsianelephants[17].Onlyoneoftheabove
studiesinvestigatedtheeffectsofGnRHvaccinesonthereproductiveorgansinasingleAsian
elephant[15]andnonemonitoredtheeffectonsemenqualityineitherspecies.
AlthoughAfricanelephantpopulationsareunderpressure,insouthernAfricafertilitycon-
troliswarrantedascapacitiesinsmaller,fencedgamereservesareexceeded.Duetotheintra-
abdominallocationofthetestes,surgicalcastrationisimpracticalandexpensiveintheselarge
animals,especiallywhenconductedinthewild.Laparoscopicvasectomyhasalsobeenper-
formed,butrequiresspecializedoperatorsandexpensiveequipmentandisnotwithoutrisks
fortheanimal[18].Vasectomyleadstoirreversibleinfertilityinelephantbulls,whileandro-
gen-drivenbehavioursremainunaffected.Theaimofthisstudywastotesttheeffectsofthe
commerciallyavailableGnRHvaccineImprovac1(Zoetis,SouthAfrica)ontheHPG-axisin
elephantbulls.Thus,wehypothesiseacastration-likeeffecttooccurontheinternalreproduc-
tiveorgans,semenqualityandfaecalandrogenmetaboliteconcentrationsinadolescentand
matureAfricanelephantbulls.
Materialsandmethods
Studyanimals
Captive(n=13)andwild(n=4)Africanelephantbullswereusedforthestudyandexamined
betweenMarch2011andApril2014(Table1).Intheprospectivegroup1,consistingofnine
captiveandtwowildelephants,theeffectsoftheGnRHvaccinewerestudiedfrompriortoor
soonaftertheprimaryvaccination,andthereafterevery5to12monthsforaperiodof3years.
Seven(fivecaptive,twowild)ofthesebullswerenaïveatthetimeofthefirstexamination.The
firstfollow-upexaminationtookplaceeitherafterprimaryandfirstboosterimmunizations
(n=5)orafterthreeinjections(n=6,primary,firstboosterafter6weeksandsecondbooster
fivemonthslater,Table1).Theanimalsingroup2,consistingoffourcaptiveandtwowild
bullsthathadpreviouslybeentreatedwiththeGnRHvaccineinthesamemannerfor5–11
years(primaryimmunisation,firstboosterafter4–6weeksfollowedbyboostersevery5–7
monthsuntilthedateofexamination),wereexaminedonceonlytoassesslong-termeffectsof
thevaccineonsemenqualityandthereproductiveorgans.Age,shoulderheight,calculated
weight,treatmentstatusandnumberofvaccinationsandexaminationsforallbullsarepro-
videdinTable1.Bodyweightswerecalculatedasameanoftwopreviouslypublishedformulas
onweight/heightrelationshipofAfricanelephantbulls[19,20].Thisstudywascarriedoutin
strictaccordancewithrecommendationsintheGuidefortheCareandUseofLaboratoryAni-
malsoftheNationalInstitutesofHealth.TheresearchproposalwasapprovedbytheAnimal
UseandCareCommitteeoftheUniversityofPretoria(Certificate#V016-12).
GnRHvaccinations
Thetreatmentprotocolforbothgroupsconsistedofaprimaryimmunization,followedbya
firstboosterafter6weeks,andthereafterboostersevery5–7months.Ingroup1,animalswere
treatedovertheexaminationperiodoftwotothreeyears,ingroup2for5–11years.Thevac-
cinewasadministeredbydeepintramuscularinjectionintothemusclemassofthehindleg.In
captiveelephants,thiswasmostlydeliveredbyhandinjectionandoccasionallywithadart
gun.Wildbullswereinjectedbyhandunderanaesthesia(firstinjection)orremotelywitha
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 3/21
ElephantbullGnRHvaccination
Table1. Overviewanddetailsofallelephantstreatedandexaminedduringthecourseofthestudy.
Elephant Wildor Numberof Study Age Shoulder Calculated Numberof Totalnumber
number captive examinations type rangea height1 bodyweight1 GnRHvaccinationsatfirst ofGnRHvaccinationsat
(years) (cm) (kg) examination lastexamination
1 captive 4 group1 24–27 293–296 4172–4336 2 7
2 captive 4 group1 24–27 276–287 3595–4025 2 7
3 captive 4 group1 17–20 240–265 2401–3197 2 7
4 captive 4 group1 12–15 230–256 2124–3232 2 7
5 captive 3 group1 8–12 210–212 1566–1657 0 4
6 captive 4 group1 10–13 225–237 1994–2316 0 8
7 captive 4 group1 23–26 290–308 4147–4935 0 8
8 captive 4 group1 23–26 292–310 4230–5029 0 8
9 captive 4 group1 23–26 297–306 4443–4843 0 8
10 wild 4 group1 30–33 302 4396 0 5
11 wild 4 group1 15–18 247 2483 0 5
12 captive 1 group2 26 293 4034 >5 >8
13 captive 1 group2 19 248 2662 >5 >8
14 wild 1 group2 28 317 5046 >8b >8b
15 wild 1 group2 33 336 5815 >8b >8b
16 captive 1 group2 20 263 3033 >8b >8b
17 captive 1 group2 30 336 5815 >8b >8b
Group1:multipleexaminationsovertime;Group2:singleexaminationoflong-termtreatedelephants
aelephantswereexaminedoveraperiodoftwotothreeyears,thereforeage,shoulderheightandbodyweightaregivenasrangesastheyincreasedover
thecourseofthestudy
bexactnumberofvaccinationsfortheseanimalsisnotknown,buteachbullhadmorethan8injectionsatthetimeofexamination
https://doi.org/10.1371/journal.pone.0178270.t001
dartgunfromahelicopter,inwhichcasetheglutealmusclemasswastargeted.Eachimmuni-
zationconsistedof5mlImprovac1(ZoetisAnimalHealth,SouthAfrica)containing1000μg
GnRH-proteinconjugate.
Anaesthesia
Inallbutonecaptivebullprocedureswerecarriedoutunderstandingsedationwithmedeto-
midineandbutorphanol,aspreviouslydescribed[21].Onecaptiveandallfourwildbullswere
fullyimmobilizedusingacombinationof12–15mgetorphine(M99,NovartisAnimalHealth,
Isando,SouthAfrica)and10–20mgdetomidine(Domosedan,ZoetisAnimalHealth,South
Africa),SouthAfrica)accordingtoestimatedweightanddeliveredremotelyfromadartgun
firedfromahelicopter(wildbulls)ortheground(captivebull).Reversalswereachievedwith
100–150mgdiprenorphine(M5050,NovartisAnimalHealth,Isando,SouthAfrica)andatipa-
mezoleHCL(Antisedan,ZoetisAnimalHealth,SouthAfrica)administeredintravenously.
Ultrasoundexamination
Priortoultrasoundexaminationandsemencollectionofstandingorrecumbentbulls,faeceswere
removedmanuallyafterwhichtherectumwasflushedwithwaterusingahosepipe.Trans-rectal
ultrasoundexaminationswereperformedaspreviouslydescribed[22,23].Aportable,battery-
drivenultrasoundmachine(Logice,GeneralElectric(GE)HealthcareGmbH,42655Solingen,
Germany)equippedwitha2–7MHzconvexprobewasused.Inelephantsupto11yearsofage,
thetestes,whicharelocatedcaudaltothekidneys,couldbevisualizedusingahand-heldultra-
soundprobe.Inolderbulls,anextensionhandlewasnecessarytoreachthetestes.Theextension
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 4/21
ElephantbullGnRHvaccination
Fig1.Convexultrasoundprobeandsteelextensionusedfortransrectalexaminationofintra-
abdominallysituatedtesticlesinelephantbulls.
https://doi.org/10.1371/journal.pone.0178270.g001
handleconsistedofa42cmlonghollowsteelpipewithaT-shapedhandleandagrooveatthe
otherendtoholdtheultrasoundprobe,whichwasheldinplacewithducttape.Theprobeendof
theextensionwasangledat45˚tofacilitatethelocationofeachtestis(Fig1).
Sixtotensecondlongultrasoundvideorecordings,eachoftheampullae,prostate,seminal
vesiclesandtestes,weremadeforretrospectiveanalysis.Frozenimageswereusedtomeasure
thelengthandheightofeachorganusingthelargestdimensionsineachcase.Thediameter
wascalculatedasameanofbothvaluesforstatisticalanalyses.Sinceaccessorysexglandsare
mostlyasymmetricaloramorphous-shapedorgans,thecircumferencewasalsomeasuredby
circumscribingtheorgan.Theoutlined2-Dimageswerethenusedtocalculatetherespective
areasforstatisticalanalyses.Sincetestesandaccessorysexglandsarepairedorgans,themeans
ofthecombinedmeasurementswerecalculated.
Semencollection
Twosemencollectionmethodswereinitiallyusedandthenconsistentlyappliedinsubsequent
examinationsofindividualbulls.Semencollectionofbullsunderstandingsedationwasper-
formedbytrans-rectal,manualmassageoftheprostateandampullaeofthevasdeferentiaas
previouslydescribedfortrained[24]orsedatedAsianelephants[25].Thepositionofthe
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 5/21
ElephantbullGnRHvaccination
ampullaeandprostatewaslocatedbymeansofultrasound.Oncesemenstarteddrippingfrom
thepenis,itwascollectedin50mlpolypropylenetubes(Greinercentrifugetubes,GreinerBio-
OneInternationalGmbH,4550Kremsmu¨nster,Austria)attachedtofunnelsmadefromplastic
rectalsleeves.Thetubeswerechangedregularlytocollectejaculatoryfractionsandtoavoid
possibleurinecontaminationofsemensamples.
Themethodusedforelectro-ejaculationofonecaptiveandfourwildimmobilisedbullswas
similartotheonedescribedbyHowardetal.[26].Aportable,batterydrivencattleelectro-
ejaculator(ElToro3,ElectronicResearchGroup,Midrand,Johannesburg,SouthAfrica)
equippedwithahand-heldprobe,specificallydesignedforelephants,wasused.Theprobe
whichwas15cmindiameterwasfittedwiththreeventralelectrodes,each20cmby3cmand
spaced3cmapart.Theprobewasplacedintotherectumdorsaltotheurethraatthedepthof
theampullaeandprostate.Lowvoltagestimuliwereappliedandgraduallyincreasedas
describedpreviously[27].Inourcase,however,muchlowervoltages,notexceeding16V,
resultedinejaculation.Thenumberanddurationofstimuliwasadaptedaccordingtothe
responseofeachmale(urethralcontractions,erection,mushroomingoftheglanspenis)and
usuallyconsistedof102–4sstimulipercyclewith30sintervalsin-betweencycles.Thestimuli
ofthefirstcyclewerejustsufficienttocausemildcontractionoftheanalsphincter.Thereafter,
witheachsubsequentcycle,thevoltagewasincreasedstepwiseuntilejaculationcommenced.
Atthisstage,thevoltagewaskeptmoreorlessconstantbutstimulusdurationwasincreasedto
approximately10s.Ejaculatesorejaculatoryfluidswereusuallyobtainedwithin20minofthe
firstcycleandwerecollectedin50mlGreinertubesasdescribedabove.
Semenanalysis
Totalvolumewasrecordedafteraddingallfractionstogether.Ejaculatoryfractionsofeach
bullwereassessedindividuallyforthepresenceofspermatozoa.Analiquotofeachfraction
wasimmediatelyassessedfortotalandprogressivemotilityandsemensmearsweremade.
Spermconcentrationandpercentagemotilespermweredeterminedusingautomatedimage
analysistechniques.ThisanalysisinvolvedaNikonEclipse50imicroscope(IMP,Johannes-
burg,SouthAfrica)withatemperaturecontrolledstageat37˚C(HS-50,IMP,Johannesburg,
SouthAfrica)andfittedwithaBasler312fcdigitalcamera(MicropticSL,Barcelona,Spain).
Spermconcentrationandmotilitywereassessedusinga10xnegativephaseobjectiveandana-
lysedwiththeSpermClassAnalyser(SCA1)(MicropticSL,Barcelona,Spain)version4.1.0.0
or5.4.0.0at50framespersecond.ChamberedLeja1slides(Leja1slide,Leja1ProductsB.
V.,TheNetherlands)withadefinedvolume(3μl)anddepth(20μm)wereloadedbymicropi-
petteforautomatedanalysis.Alldisposablessuchasslidesandpipettetipsusedweremain-
tainedat37˚C.Wherespermconcentrationwastoohighforautomatedanalysis,thesemen
wasdilutedinHam’sF10culturemedium(Sigma-Aldrich,Johannesburg,SouthAfrica).At
least500spermatozoawereanalyzedformotilitywithSCAforeachelephant.Thebestquality
semenfractionwasusedtoreporttotalmotility,progressivemotility,spermconcentration
andspermmorphologyforeachbull.SemensmearswereprocessedandstainedwithSperm-
Blue1(MicropticSL,Barcelona,Spain)accordingtovanderHorst&Maree[28]andeosin-
nigrosin(SectionReproduction,VeterinaryFaculty,UniversityofPretoria,Pretoria,S.A.)
within30minofcollectiontoassessspermmorphology.Theeosin-nigrosinsmearwasmade
bymixingpre-heated(37˚C)stainandsemenonapre-heatedmicroscopeslideata2:1(stain:
semen)ratio.Thestainedspermatozoawereevaluatedformorphologyusingbrightfield
microscopyat1000xmagnification.Evaluationandclassificationofspermmorphologyof100
spermatozoaperelephantbullwascarriedoutaccordingtothecriteriaoriginallydescribedfor
thedomesticcattle[29,30].
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 6/21
ElephantbullGnRHvaccination
Faecalsamplingandandrogenmetaboliteassay
Faecalsampleswerecollectedwithin6hoursofdefecationfromthe11captivebullstwice
monthlyandoneweekpriorto,duringandoneweekaftereachexamination.Faecalmatter
wasthoroughlymixedduringcollectionandstoredat-20˚Cuntilfurtherprocessing.Frozen
faecalsampleswerelyophilized,pulverized,andputthroughanylonsievetoseparatecourse
material[31].Aweighedamount(approximately0.05g)ofdriedfaecalpowderwasthen
extractedwith80%ethanolinwater(3ml)byvortex-mixingfor10–15minandsubsequent
centrifugationfor5minutes.Thefaecalextractswereassayedforimmunoreactiveandrogen
metabolitesusinganenzyme-immunoassayfor5α-Androstan-3β-ol-17-on(Epiandrosterone)
whichhasbeenvalidatedinseveralmammalianspecies[32,33]includingAfricanandAsian
elephantbulls[34,35]formeasuringfaecalandrogenmetabolite(fAM)concentrations.Serial
dilutionsoffaecalextractsgavedisplacementcurvesthatwereparalleltotherespectivestan-
dardcurve.Sensitivityoftheassayat90%bindingwas3pg/wellandtheintra-andinter-assay
coefficientsofvariationrangedbetween2.4and11.9%.Theassaywasperformedonmicrotiter
platesasdescribedbyGanswindtetal.(34)andconductedattheEndocrineResearchLabora-
toryattheFacultyofVeterinaryScience,UniversityofPretoria.
Statisticalanalyses
Fortheultrasoundrelateddata,descriptivestatisticswerecalculated,histogramswereplotted,
andtheAnderson-Darlingtestfornormalitywasperformed(MINITABStatisticalSoftware,
Release13.32,MinitabInc,StateCollege,PA,USA)toassessthenormalityassumption.Data
weretransformedusingthenaturallogarithmtoimprovethedistributionalformpriortosta-
tisticalanalysis.ThenumberofGnRHvaccinationswastransformedintoanordinalvariable
forstatisticalmodellingbasedonthefollowingcriteria:noGnRHvaccinations,2vaccinations,
3vaccinations,4vaccinations,and5ormorevaccinations.Correlationsbetweenquantitative
datawereassessedbycalculatingSpearman’srho.TheeffectofmultipleGnRHvaccinations
wasestimatedwithinagenerallinearmodellingapproachtoadjustfortherepeatedsampling
ofindividualelephants.Elephantagewasdichotomizedbasedonlessthan20yearsofageand
heightwasdichotomizedbasedonthemedianofthedata(287cm).Arandomeffecttermwas
includedforelephantandafirstorderautoregressivecorrelationstructurewasusedtoadjust
forrepeatedmeasurements.ModelsincludedafixedeffectsfornumberofpreviousGnRH
vaccinations,age,heightattheshoulder(usedtoapproximatebodyweightbyformulas
describedpreviously),andtimesincefirstvaccination.Post-hocpairwisecomparisonswere
adjustedusingtheBonferronimethod.Statisticalanalyseswereperformedincommercially
availablesoftware(IBMSPSSStatistics,Version23,InternationalBusinessMachinesCorp.,
Armonk,NY,USA)andresultsinterpretedatthe5%levelofsignificance.Valuesarepresented
asmean±standarddeviation(SD),unlessstatedotherwise.
Forassessmentofthefaecalandrogenmetaboliteconcentrationingroup1,one-way
repeatedmeasureANOVAwasapplied.
Results
Inthisstudy,itwaspossibletomonitor11bullstreatedwithGnRH(Improvac1)overa
three-yearperiod.Anothersixelephants(2wild,4captive)thathadpreviouslybeentreated
withImprovac1morethaneighttimeswereexaminedonceonly(Table1).
Allbullswerehealthyandingoodbodycondition.Applicationofthevaccinebydartgun
orhand-injectionprovedequallyeffective.Onlyfourofthe84(3.4%)Improvac1injections
resultedinside-effects.Theseconsistedoflocalswelling(Fig2)and/ordiscomfortatinjection
site(n=2),stiffnessinhindlegs(n=1)andmildlamenesswhichlastedoneday(n=1).
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 7/21
ElephantbullGnRHvaccination
Fig2.Photographofanelephanthindlegshowingswelling(circleline)onedayafterGnRHvaccine
booster.
https://doi.org/10.1371/journal.pone.0178270.g002
Ultrasonographicchangesofreproductiveorgans
Sizeoforgans. Dimensionsoftestes,seminalvesicles,ampullaeandprostateatthebegin-
ning(0–2vaccinations)andendofthestudy((cid:21)5vaccinations)areshowninTable2.Inthe
sevenelephantsexaminedpriortoGnRHvaccination,testissizewassignificantlycorrelatedto
ageatthebeginningofthestudy(r=0.897,p=0.0062).Themeantesticularlengthandheight
of10bullsdecreasedsignificantlyfrom13.3±2.6x15.2±2.8cmatthebeginning(0–2injec-
tions)to7.6±2.1x10.2±1.8cmattheend(after>5injections)ofthestudy(p=0.0009,
t=6.121,pairedt-test,n=10,Table2).Similarly,thedimensionsoftheampullae,prostate
andseminalvesiclesdecreasedsignificantly(Table2).
Negativecorrelationsbetweenageandsize-adjustedorganareaatthetimeofexamination
andnumberofvaccinationswerefoundforthetestes(Spearman’srho=-0.728,p<0.001,
Fig3A),ampullae(Spearman’srho=-0.825,p<0.001,Fig3B),seminalvesicles(Spearman’s
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 8/21
ElephantbullGnRHvaccination
Table2. Comparativeultrasoundmeasurementsforreproductiveorganarea,dimesions(lengthxheight)andcircumferenceatstartversusend
ofthestudyofgroupIbulls.
StudyStart StudyEnd StudyStart StudyEnd StudyStart StudyEnd
Organ: Area(cm2) Area(cm2) Lengthxheighta(cm) Lengthxheighta(cm) Circumference(cm) Circumference(cm)
Testicle 165.6±54.6 56.5±25.5 13.3±2.6x15.2±2.8 7.6±2.1x10.2±1.8 46.6±9.3 29.3±5.5
Ampulla 9.1±3.0 2.2±0.5 2.3±0.7x5.1±1.1 1.0±0.3x3.2±0.5 nm nm
Seminal 28.2±13.7 14.2±14.5 4.1±1.6x9.0±1.3 2.2±1.4x7.1±1.7 nm nm
Vesicle
Prostate 13.2±6.1 6.8±3.3 3.6±1.1x6.2±1.2 2.1±0.8x5.5x1.8 nm nm
Resultsforn=10elephantsatstudystart(<2vaccinations)versusstudyend((cid:21)5vaccinations).
amaximumlengthxheightdimensionoforgan;nm=notmeasured
https://doi.org/10.1371/journal.pone.0178270.t002
rho=-0.448,p=0.001,Fig3C)andprostateglands(Spearman’srho=-0.436,p=0.002,Fig
3D)(n=17,Table3).
ThedecreaseintesticularandampullarsizewasalreadysignificantafterthesecondImpro-
vac1injection.Fortheseminalvesicleandprostateareas,however,thedifferencesonly
becamesignificantafterthethirdandfifthinjections,respectively(Table4).Onecaptivebull
inparticular(bull#9),respondedslowercomparedtotheothermalesinthestudy.Whiletes-
tissizealsodecreased,thesizeofhisseminalvesiclesandampullaeremainedrelatively
unchangedthroughoutthestudy.
Ultrasonographicappearance. Thetestesofallbullschangedfromfullandround(Fig
4A–4C)atthebeginningtoovalandamorphousattheendofthestudy(Fig4D–4H).Thetes-
ticulartissueappearedtobesofterandcompressedbytheintestines(Fig4D–4G).Theecho-
texturewasechogenicatthebeginning(Fig4A–4C)butprogressivelylostechogenicityduring
thecourseofthestudy(Fig4D–4G).Aconsistentfindinginelephantstreatedmorethanfive
timeswasadilatationofthetesticularvein(Fig4Eand4F).Thecolourdopplerflowpattern
priortotreatmentwasdiffuse(Fig4C)whereasafterrepeatedtreatmentsonecentralveinwith
lessperipheralflowbecameevident(Fig4F).Elephantsthatweretreatedmorethanfivetimes
alsohadsmallanddeformedtesticles(Fig4D–4H).Theappearanceofthetesticulartissuein
sixelephantsthathadbeentreatedmorethanseventimeswasinhomogenouswithhyperechoic
areas(Fig4Gand4H),possiblysignsofparenchymalatrophy(Fig4G)anddegeneration,
resemblingfibrosis(Fig4H).
Priortotreatment,fluid(hypoechoic)wasvisibleintheampullaeofallmatureanimals(Fig
5A).Thefluidcontentdecreasedovertimewhich,afterthreevaccinations,significantly
reducedthesizeoftheampullae.Afterfivevaccinations,fluidwasnolongervisibleinthe
ampullae(Fig5B).Thefluidwithintheseminalvesicleswasclearlyvisibleinmostbullsatthe
beginningofthestudy(Fig5C)andonlyappearedtobeaffectedaftermorethanfourvaccina-
tions.Bytheendofthestudy,seminalvesiclefluidhaddisappearedcompletelyinsevenbulls
ofgroup1.Intheremaininganimals,smallanechoicfluidfillingwasvisible(Fig5Dand5F).
Thesizeoftheprostatealsodecreased(Fig5E,pre-treatment);theeffectbecomingclearlyvisi-
bleaftermorethanfivevaccinations(Fig5F;Table4).
Semencollectionandevaluation
Penisextrusionofelephantsinstandingsedationwassometimesspontaneousandprobablyas
aresultofthesedativedrugs.Ifnot,extrusionoccurredduringthecleaningoftherectumand
trans-rectalmassaging.Inmostcases,continuoustrans-rectalmassagingoftheunderlying
organsresultedinthedevelopmentofafullyextendedpenis.Intwocases,urinedribblingwas
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 9/21
ElephantbullGnRHvaccination
Fig3.GraphsshowingthenegativecorrelationbetweennumberofGnRHvaccinationand
reproductiveorgansizeinelephantsbullsofgroup1.
https://doi.org/10.1371/journal.pone.0178270.g003
observedpriortomassaging.Whentheurethrawasstimulatedinthecorrectarea,however,
urinedribblingceasedandasemensamplewasobtained.Urinecontaminationofsomefrac-
tionswasrecordedinfourof31manualcollections.One(bull#5)ofthesevenmalesassessed
priortotreatmentwas8yearsoldand,accordingtotheappearanceofreproductiveorgans,
lowtestosteronelevelsandabsenceofspermatozoaintheejaculate,wasassessedasprepuber-
tal.Thismalewasthereforenotincorporatedinthesemenevaluationovertime.
Inuntreatedmales,transrectalmassagepriortothefirstvaccinationproducedejaculate
volumesof8–50ml.Electro-ejaculationoftheimmobilisednaïvewildbullswashighlysuc-
cessfulandproducedejaculatevolumesof43ml(bull#11)and175ml(bull#10)priortovac-
cination.Electro-ejaculationofwildbullsonlongstandingtreatment(group2;bulls#14and
#15)producedonlydropsofsperm-freeseminalplasma.
PLOSONE|https://doi.org/10.1371/journal.pone.0178270 September15,2017 10/21