Influence ofoxytocininfusionduringoestrus and theearly
lutealphaseonprogesteronesecretion and the
establishment ofpregnancyin ewes
D. C. Wathes1, V. J.Ayad1,C. L. Gilbert1, S. A. McGoff1 and
C. M. Wathes2
1Department ofAnatomy,SchoolofMedicalSciences,Bristol BS8 1TD, UK and2Department of
AnimalHusbandry, University ofBristol, Langford,Bristol BS18 7DU, UK
Summary.InExperiment 1, an osmoticminipump containing oxytocinwasimplanted
s.c. in ewes for 12days beginningonDay10 of the oestrouscycle, producing \m=~\100pg
oxytocin/mlin theplasma.Twodaysafter the start ofinfusion,all ewes wereinjected
with 100\g=m\gcloprostenolandplacedwith a fertile ram. Atslaughter22days later,9
(75%)of the 12 control(saline-infused)ewes werepregnant comparedwith 1(11%)of
the 9 ewes infused withoxytocin.In the controlgroup, midcycle plasmaconcentrations
ofoxytocinweresignificantly higherinnonpregnantthan inpregnantewes.
InExperiment 2, an infertile ram was usedthroughoutto avoidany possibleeffects
ofpregnancyandoxytocininfusions weregivenat differentstagesof the oestrouscycle.
Otherwise theprotocolwas similar to that inExp.1.Oxytocininfusionduringluteo-
lysisand theearlyfollicularphasehad no effect on thesubsequent progesteronese-
cretionpattern,but infusionsbeginningthedaybeforecloprostenol-induced luteolysis
andlastingfor 7 or 12daysand infusionsbeginningon thedayof oestrus for 4daysall
delayedthesubsequentrise inplasma progesterone by\m=~\3\p=n-\4days.In theseanimals,the
cycletended to belonger.
It was concluded that anappropriate oxytocinsecretionpattern maybenecessary
for the establishment ofpregnancyin ewes and that ahigh circulating oxytocinconcen-
trationduringtheearlylutealphase delaysthedevelopmentoftheyoung corpusluteum.
Keywords: oxytocin; progesterone; sheep; pregnancy
Oxytocinis amajor productof thecorpusluteum in ruminants(Wathes& Swann, 1982),but its
roleduringthe oestrouscycleis far from certain. Whilst there isgoodevidence for an involvement in
luteolysis (seeAuletta & Flint, 1988 andWathes,1989 forreviews),meancirculating oxytocin
concentrations arehighestin theearlyto midlutealphase (Webbet ai, 1981;Schams et ai, 1982)and
receptorconcentrations in thereproductivetractpeakon thedayofoestrus(Sheldrick& Flint, 1985;
Ayad& Wathes, 1989; Ayad et ai, 1990a).Ewesactivelyimmunizedagainst oxytocinfailed to
establishpregnancywhen mated with a fertile ram(Wathesetai, 1989), suggestingthatoxytocin
mayhave an additional, uncharacterizedaction(s)in theregulationoffertility. In cows, daily
oxytocin injectionsin theearly partofthecycleinterfere with the normaldevelopmentof thecorpus
luteum (Armstrong & Hansel, 1959), although similar protocols in ewes have not had
*Present address: AFRC Institute ofAnimalPhysiology&Genetics Research,BabrahamHall, CambridgeCB24AT,
tPresent address: AFRC Institute ofEngineeringResearch, Wrest Park, Silsoe,Bedford MK454HS,UK.
majorinfluence on luteal function(Milne, 1963; Hatjiminaoglouet ai, 1979).Incontrast,oxytocin
infusionstartingin the midlutealphaseextends luteallifespan, presumably by interferingwith the
mechanismsnormally regulating luteolysis (Flint& Sheldrick, 1985).
Thepurposeof the firstexperiment reportedhere was to see whetheroxytocininfusion
influenced the establishment ofpregnancyin ewes. In the secondexperiment,weinvestigatedthe
effect of treatment withoxytocinat differentstagesin the oestrouscycleon luteal function.
Materials and Methods
Twoexperimentswereconductedduringthe1988/89breeding season, usingmature Clun Forest eweskeptatpasture,
withhay providedas asupplementaryfeed.
Preparation ofosmoticminipumps.Bothexperimentsinvolved infusion ofoxytocinvia Alzetminipumps (Alza
Corporation,PaloAlto, CA, USA)filled with either vehicle(saline,0-9%w/vNaClcontaining001% aceticacid)or
oxytocin (Hoechst, Frankfurt. WestGermany)dissolvedin the same vehicle. Solutions were filtered (0-22µ filters,
Millipore Corp.,Bedford, MA, USA)before use and loaded into thepumpsunder sterileconditions. InExp.
were stored desiccatedovernightat 4"C before insertion. Thefollowing morning theywereincubated for 4 h at 37°C
in 15 ml sterile saline and thenimplanteds.c. over thé chest wall behind the left elbow under localanaesthesia. InExp.2
pumpswereincubatedovernightin sterile saline at 4UC and then transferred to fresh saline for incubation for 1 h at
37°C thefollowing morningbeforeimplantations.c. on the dorsal surface of theneck.
Experiment1.Twenty-oneewes were divided at random into 3 treatmentgroups: (1) no infusion (n
vehicle infusion(«=5)and(3) oxytocininfusion(n=9, 1-25mg oxytocin/mlin Alzetminipumps 2ML2). The start
of the treatment wasphasedover 7daysto avoid all animalscominginto oestrus at the same time. All ewes were run
initiallywith araddled,vasectomized ram and raddle marks were noteddaily.Tendaysafteroestrus,minipumpswere
implantedand 2dayslater(Day12 ofthecycle)all ewes weregivenasinglei.m.injectionof 100µg cloprostenol
(prostaglandinF-2aanalogue(PG);CoopersAnimal Health Ltd.Crewe, Cheshire, UK)andtransferred to apaddock
containinga raddled, fertile ram. Thepumpswere left inplacefor 12daysand then removed underlocal anaesthesia.
After a further 12days (22 daysafter the PGinjection),ewes wereslaughteredand thereproductivetracts were
removed. Structures visible on the ovaries were noted and the tracts wereopenedto examine the number, size and
apparent viabilityofany embryos.
Bloodsamples (10ml) were collectedby jugular venepuncture3 times a week from the first oestrus until PG
treatment and thereafterdaily. Following centrifugationat 4°C and 2000# separate aliquantsofplasmawere stored
Experiment2. Theprotocolwas similar to that ofExp. 1, but aninfertile ram was usedthroughoutto avoidany
pregnancy treatment interactions. Ewes(n
came into oestrusnaturally,suchthat the start of treatment wasphasedover 15days.All animals receivedaninjection
of PG onDay12 ofthe oestrouscycle.The treatmentgroupsare illustrated inFig. 1. Group4ewes received Alzet
Minipumps2ML2containingI -23mg oxytocin/ml; groups 2, 3, 5 and 6 received AlzetMinipumps2001containing
6-15mg oxytocin/ml.Thesmaller pumpshad a slower flow, so these concentrationsproducedthe same net release of
oxytocin.All ofthe animals inGroups1-5 wereslaughtered22daysafter the PGinjectionforexamination oftheir
reproductivetracts and ovaries. A further 3 ewes(Group6) received anoxytocininfusion fromDay11 ofthecycle,
but wereslaughtered2daysafter PG for measurement ofoxytocin receptorconcentrations. The oviducts, endo-
metrium andmyometriumwere dissected out andprocessedasdescribedby Ayad& Wathes(1989)andAyadet al.
Bloodsampleswerecollected 3 times a weekby jugular venepuncturefrom the first oestrus untilpumpinsertion
forprogesteroneRIA.Thereafter,bloodsampleswere collecteddailyformeasurement ofprogesteroneandoxytocin.
petroleum-etherextraction as describedbyWathes el al. (1986).The detection limit was16pg/tube,the extraction
efficiencywas 74± 1-3% and the intra-andinterassaycoefficients of variation were 111 and 141%, respectively.
Oxytocinwas measured after extraction of4-mlplasma sampleson C18cartridges(JonesChromatography Ltd,
Hengoed, Mid-Glamorgan, UK)with minormodifications to a method describedbyWathes et al. (1986). Columns
wereprewettedwith 3 ml80% acetonitrile (ACN)in 01% trifluoroacetic acid(TFA) followedby5 mlH20.Plasma
sampleswereprepared bydilution in anequalvolume ofphosphatebuffer(1-4 g Na2HP04.2H20,0-26g KH2P04,
8-65g NaCl/1)andcentrifugationfor 10 min at 4°C and1500#. Theywere thenapplied slowly to the columns and
washedthroughwith 20 ml 10% ACN in 01% TFA.Oxytocinwas eluted with 3 ml 80% ACN in01% TFA. The
ACN was removedby centrifugationin arotaryfilmevaporation centrifugeset at 35°C(Univap Uniscience,
Cambridge, UK)and theremainingextract was freeze dried. The extract wasredissolved in 0-5 mlphosphatebuffer
solution were measured in an RIA foroxytocin usingantiserum85/2 (Gilbert et ai, 1991). Separationwasachieved
—80°C foroxytocinand at
—20"C for progesteroneforsubsequentestimationby radioimmunoassay
=28) wereassignedat random to different treatment groupsasthey
on 0-25-ml aliquants of plasma using
1mg/mlbovine serum albumin(SigmaChemicalCo.. Poole, Dorset,RIAgrade). Aliquantsof 50µ ofthis
Fig. 1.Diagramto showprotocolfor ewes in Exp. 2: time of(D) saline infusion, (^HI of
oxytocin infusion, PG shows theinjectionof 100µg cloprostenoli.m.givento all ewes on Day
12 of thecycle.The treatmentgroupswere:(1) controls, (2) oxytocininfusion fromDay11 of
thecycleuntil 40 h after PG treatment, (3) oxytocin infusion fromDay 11 for 7days, (4)
oxytocininfusion fromDay11 for 12days, (5)oxytocininfusion onDays2-6 after PG and(6)
oxytocininfusion fromDay 11.Group6 ewes wereslaughtered2daysafterPG, all the others
22daysafter PG as shown.
using0-15 mlanti-rabbit-precipitating antibody (UCB BioProducts, Belgium).Thesensitivityof theassaywas 0-5pg
pertube and the intra- andinterassaycoefficients of variation were 8-5 and2-8%, respectively.
Receptormeasurements. Concentrations ofoxytocin receptorsinmembrane fractions were estimatedbyincubat¬
ingasingle, saturatingconcentration of[3H]oxytocin (NEN, Stevenage, Herts, UK)with asingleconcentration of
membraneproteinas describedby Ayad& Wathes(1989)andAyadet ai(1990a). Specific bindingwas measured on 2
separateoccasions for eachsampleand the mean value was obtained. Theinterassaycoefficient of variation was
Statisticalanalysis.Thedesignof bothexperimentswas ofrepeatedmeasures overtime,withoxytocininfusion as
theexperimentalfactor. All data wereanalysed byanova and means werecompared usingthe leastsignificant
difference for the time oxytocinor time pregnancyinteractions. Twoanalyseswere undertaken forExp.
effect ofpregnancyonoxytocin profilesinthe controlanimals.Groups1 and2;and(ii)the effect ofoxytocininfusion
onprogesterone profiles,with the control animals subdivided intopregnantandnonpregnant groups.Asingle analy¬
sis wasperformedinExp.2, the effect ofoxytocininfusion onprogesterone profilesinGroups1-5.
There were noapparentdifferences betweenGroup
infusion),so these animals were considered as asinglecontrolgroup.All animals(controland
oxytocin-infused)had received raddle marksbyeither the second or thirdmorningafter PG
injection.Atslaughter (22 daysafterPG),9 of the 12 control ewes werepregnant (75%).Their
reproductive organseach contained 1-3corporalutea(CL) (21+0-20)and 1-3 viableembryos
1 (no infusion)andGroup2(vehicle
Time from chloprostenol Injection (days)
Fig.2. Concentrations of(a) oxytocinand (b) progesteronein control ewes which conceived
(·, =9)or did not conceive( , =3)and inoxytocin-infusedewes which did not conceive
( , =8), Exp. 1; ( IH ) infusionperiod.Data arealignedaround the PGinjection (Day 0,
arrow) givenonDay12 of the oestrouscycle.All ewes showed oestrus 2-3daysafterinjection.
Points marked with an asterisk weresignificantlydifferent (anova)at < 005 or < 001
k(a) incomparisonbetweenpregnantandnonpregnantcontrol ewes and (b) incomparison
with thepregnantcontrol ewes. Data are means, with standard errors omitted forclarity.
(1-9 +0-20)and one ewe had one additionaldegenerating embryo.The 3nonpregnantcontrol
ewes had all reovulated. In contrast, only
slaughter (11%),with 2CL and
recently reovulated. The ovaries of the others contained either mature(n
infused ewes, eventhoughnosignsofdegenerating conceptuseswere found in theirreproductive
1 of the 9oxytocin-infusedewes waspregnant at
1 embryo.Of theremaining8nonpregnant ewes, only2 had
= 2ewes)CL. These data indicate that thelifespanof the CL was maintained in theoxytocin-
Time fromcloprostenol injection (days)
Fig.3.Progesteroneconcentrations in ewes inExp.2 treated withoxytocinat different times as
shownby ( ). (a)and(b)Group1, control(·,
=5), (b)Group4 ( , =4), Group5( , =5). Data arealignedaround the PGinjection
(Day 0, arrow) givenonDay12 of the oestrouscycle.Points marked with an asterisk were
significantlydifferent(anova)incomparisonwith the control ewes at < 005 ir or < 001
*. Data are means,with standard errors omitted forclarity.
=7), (a) Group2( , =5), Group3( ,
Because of the likelihood thatpregnancywould have influenced the endocrineprofiles,it was
considered appropriateto subdivide the control andoxytocin-infused groupsintopregnantand
nonpregnantanimalsforanalysisof the hormone data. All results arepresentedin relation to the
dayof the PGinjection (Day 0),which was either 2 or 3daysbefore oestrus. Theoxytocininfusions
produced continuouslyraisedoxytocinconcentrationsin all ewes(Fig. 2a).There was considerable
dailyvariation in the exact concentration, but all values were within therange40-252pg/ml.
Values from the oneoxytocin-infusedewe which conceived have been omitted fromFig.2 for
clarity,but were within the samerange (65-144 pg/ml).Acomparison between thepregnantand
nonpregnantcontrol ewes showed that the luteal-phase oxytocinconcentrations tended to be
higherin the control animals which did notconceive,withsignificantdifferences onDays9 and 10
(time pregnancy interaction, <005), whereas luteal-phase progesteroneconcentrations
tended to be lower(Fig. 2b),withsignificantdifferences onDays 8, 9 and 11 (time oxytocin
interaction, < 0001, Fig. 2).In thenonpregnant oxytocin-infused ewes,theprogesteronecon¬
centrations rose moreslowlyand reached lowerplateauxthan in thepregnantcontrols,differences
being significantfromDay8 onwards(Fig. 2b).Theprolongationof the lutealphasein these
animals was reflected in the maintenance ofprogesteronesecretionbeyond Day17. In the one
pregnant, oxytocin-infused animal, progesteroneconcentrations also roseslowly,buttheywere
similar to those inpregnantcontrol ewesby Day11 after PG(datanotshown).
Theoxytocininfusions were timed toprovide high oxytocinconcentrationsduring luteolysis
and theearlyfollicularphase only (Group 2); luteolysisandthroughouttheperiovulatory period
(Group 3); luteolysis,theperiovulatory periodandearlylutealphase (Group 4);and fromshortly
before ovulation for 4days (Group 5).Theplasma oxytocinconcentrationsproduced bythepumps
duringthese times were within the samerangeas forExp.1.
Of the 25 ewes,20 had received raddle marksbythe second or thirdmorningafter PGinjection.
Of theremaining5animals,2 were noted on the fourthdayand 3 were not raddled at all, but these
animals were distributed in each ofGroups 1, 3, 4 and 5. anova analysisrevealedsignificant
differences between theprogesterone profilesof the 5groups (P < 0 01) (Fig. 3). Progesterone
concentrations increased moreslowlyin each ofGroups 3, 4 and 5, with adelayof ~3^4days
comparedwith the control ewes.Although progesteroneconcentrations inGroup2 tended to be
slightlylower than in controls, the difference wassignificant onlyonDay14. However, peak pro¬
gesteroneconcentrations were not different betweengroups (3-3 ± 0-20, 2-9 + 0-53, 2-5± 0-22,
2-9±0-47 and 2-7 ±0-46ng/mlforGroups 1-5, respectively).InGroups4 and 5, lutealregression
was alsoclearly delayed (Fig. 3b).Whereas all(7/7)ofthe control ewes had reovulated atslaughter,
this was not the case foranyof theoxytocin-treated groups; 3/5 (60%), 2/5 (40%), 1/4 (25%)and
2/5 (40%)had reovulated inGroups 2-5, respectively.Eleven of theoxytocin-treatedanimals still
had mature orregressingCL in their ovaries, but inonlyone animal inGroup2 did the CL still
present appearto becystic.
The ovaries of the 3 ewes inGroup6slaughtered2daysaftercloprostenol injectionall con¬
tainedregressing corporalutea. The concentrations ofoxytocin receptorin the oviduct, endome-
trium and myometriumwere 91 ± 12, 518±104 and 419±73fmol/mg protein, respectively
(x + sem).These are within therange reportedfor oestrous ewesby Ayad& Wathes(1989).
The treatmentregimensdescribedprovideda consistent model which could be used to examine the
effect ofoxytocininfusion around the time of ovulation and theearlylutealphasein unrestrained
ewes. The Alzetminipumps producedahigh plasma oxytocinconcentrationthroughoutthe time
theywere inplace,92% ofplasma samples beingwithin 40-180pg/ml.Whilst this ishigherthan the
mean values foundduringthe lutealphasein control ewes, such concentrations arefrequently
reachedduringthepeaksofplasma oxytocinrecorded atluteolysis (Flint& Sheldrick, 1983;
Hooperetai, 1986; Wathes et ai, 1986). However, theconsistently high oxytocinconcentrations
produced bythepumps clearlydiffered from thephysiological patternof releasethroughoutmuch
of thecycle: long pulsesof 1-3 hnormallyoccurduringlutealregression (Flint& Sheldrick, 1983;
Hooperetai, 1986; Wathes et ai, 1986)whereaslow-amplitude,shortpulsesof 1-4 min occur at
oestrus(Gilbertet ai, 1991). Despitethehigh oxytocin concentrations,there was no evidence for
downregulationof theoxytocin receptor duringthe oestrousperiod,as the 3 infused ewes killed at
oestrus 2daysafter PGinjectionall had uterinereceptormeasurements at thehighend of the
normalrangefor oestrous ewes. Similar results have beenreported byFlint & Sheldrick(1985).
Theprostaglandintreatment wasnecessaryto induce lutealregression,asoxytocininfusion
alonestartingin the midlutealphaseinhibitsluteolysisin bothsheep (Flint& Sheldrick, 1985)and
cows(Gilbertet ai, 1989).As shownbyFlint & Sheldrick(1985), exogenous cloprostenoloverrides
thisinhibition;all ewes so treatedexperiencedimmediate lutealregressionandnearlyall showed
oestrus within 2-3daysof treatment. As raddle marks wereonlynoted on adailybasis, it was not
possibletogivean accurate measure of the time from treatment to the onset of oestrus in the
variousgroups,but there was nomajorshift in thetimingof the oestrus in association with the
Themajorconclusion fromExp.1 was thatoxytocininfusion could block the establishment of
pregnancy.The tracts were not examined until 20daysafteroestrus, so we do not know at what
stagethepregnanciesfailed.Injectionsofoxytocinaround the time of oestrus increased electro-
myographic activity throughoutthereproductivetract(oviduct,uterus andcervix)for ~5-20 min
(Gilbert & Wathes, 1989; E. L. Matthews, C. Walker & V. J.Ayad, unpublished observations).One
possibilityis thatoxytocininfusion acts on thereproductivetract musculature to interfere with
sperm transportandpreventfertilization. Interaction withoxytocin receptorsin the oviduct(Ayad
et ai, 1990a, b)could also influence the rate ofeggorembryo transport.In theoxytocin-infused
animals, theplasma progesteroneconcentrations rose moreslowlyafter oestrus than in the controls
and other workers have shown that lowprogesteroneconcentrations in theearlylutealphaseare
detrimental toembryosurvival, probablyvia an effect on the uterine environment(Wilmutet ai,
1985; Ashworth et ai, 1989).In aprevious experiment (Watheset ai, 1989),we found that active
immunizationagainst oxytocinalsopreventedewes fromestablishing pregnancyand these animals
were shown to have raisedgonadotrophinconcentrations. It is clear thatoxytocinhas thepotential
to influencefertilityin a number ofwaysand further work isnecessaryto establish which are most
Additional evidence for involvement ofoxytocininfertilitywasprovided by Exp. 1, which
showed thatcirculating oxytocinconcentrations werehigherinmidcyclein the control ewes which
failed to conceive. As theoxytocin profile appearedto differ also in theprevious cyclebefore
mating,this could not be attributable to an effect ofpregnancy.This result must beregardedas
preliminary,asonly3 animals were involved, but theslightlyraisedoxytocinconcentrations occur¬
ring naturally mayhave had an adverse effect onfertility. Alternatively, theycould have been the
symptomof some otherproblemsassociated with luteal malfunction. We have shownpreviously
that gonadotrophin-releasinghormone(GnRH)-stimulatedanoestrous ewes have significantly
higher plasma oxytocinconcentrations in theearlylutealphaseif animals do not receive a7-day
progesterone priming (Hunteretai, 1989).
The results ofExp.1suggestedthatoxytocininfusions could inhibit lutealdevelopment.How¬
ever, interpretationof these results was confoundedbythe difference inpregnancyrate between
control and treated ewes.Therefore, Exp.2 wasperformedto examine the effect ofoxytocinin¬
fusion at differentstagesof the oestrouscycleonprogesteroneconcentrations in unmated ewes.
This showed thatoxytocininfusiondelayedthe rise inprogesterone followingoestrusby~3-4
days.Oneexplanationcould be thatoxytocinhaddelayedovulation, butpreliminaryresults from a
subsequent experimentin which ewes wereslaughteredonDay2 after oestrus do notsupportthis
hypothesis (D.C. Wathes, V. J.Ayad& E. L. Matthews, unpublished observations).The data from
the ewes inGroup2 showed thatoxytocininfusionduringthe follicularphasealone had little effect
on thesubsequent progesterone profile.Itappears,therefore, that the influence was on theyoung
corpusluteum. It is well established thatdaily oxytocin injectionsbetweenDays3 and 6 of thecycle
have an adverse effect oncorpusluteumdevelopmentincows (Armstrong& Hansel, 1959;Hansel
&Wagner, 1960; Stapleset ai, 1961;Tsanget ai, 1990).Such treatment resulted in reducedplasma
progesteroneconcentrations from aboutDay6 onwards and inmanycases also caused the forma¬
tion oflarge,fluid-filled, cystic corporalutea. In theseexperiments,themajorityof animals treated
in thiswayreturned to oestrusprematurelyandoxytocinwasprobably causinglutealregression by
stimulatingendometrial secretion of PGF-2a(Oyedipeet ai, 1984). However, previouswork in
ewes has been lessconvincing.Neither Milne(1963)nor Schreiber & Milvae(1988)were able to
influence thelengthof the oestrouscyclein ewesby oxytocin injectionsat variousstagesof the
cycle, although Hatjiminaoglouet ai(1979)did causeprematurelutealregressionin 2 of 8 ewes
given daily oxytocin injectionsbetweenDays0 and 7. This treatment also resulted in a lower luteal
Usingtheprotocoldescribed in thepresent study, onlyone ewe returned to oestrusprematurely
Day9 of thecycle (some daysbefore theanticipated onset ofluteolysis)tended to increasecycle
length.Apossible explanationfor thedelayed regressioncould be the need to have an increased
progesteroneconcentration for a minimum of 7-10daysbefore the endometrium cansynthesize
bothoxytocin receptorsand PGF-2a, assuggested byMcCracken et ai(1984). However, our
previousstudies into the mechanism ofluteolysisin GnRH-treated anoestrous ewes(Hunteret ai,
1989) indicated that an apparently normal type ofoxytocin-PGF-2ct interaction can cause
In conclusion, oxytocininfusionduringovulation and theearlylutealphase preventsewes from
becoming pregnantanddelaysthe rise inprogesteronesecretionbythecorpusluteum. These re¬
sultssuggestthat anappropriate oxytocinsecretionpattern maybenecessaryin the establishment
We thank Mr G. Davies for care of the animals; Hoechst(Frankfurt,W.Germany)for dona¬
tion of theoxytocin;and Mrs J. Skinner and Mrs J. Hood for secretarial assistance. The work was
supported by grantsfrom the AFRC and Wellcome Trust.
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