Cycles
of antler and testicular
growth
in
an
aseasonal
tropical
deer
(Axis
axis)
A.
S.
I.
Loudon and
J. D.
Curlewis
M. R.C.
¡A.
F.R.C.Comparative Physiology
ResearchGroup,
Instituteof Zoology,
Zoological
Society
of
London,
Regent's
Park, London,
NW14RY, U.K.Summary.
Antler and testicularcycles
werestudied inagroupoffreeranging
axis deerstags
kept
outof doors in southernEngland.
Within theherd,
therewaslittle evidence ofaclear seasonalsynchrony
in the antlercycle.
Detailed information obtained from4stags
indicated that therewas a fixedrelationship
betweenstageof the antlercycle
and testisdiameter;
minimum testis diameter occurred1\p=n-\2
months after antlercasting
whereas maximum testis diameter occurred whenstags
werein hard antler.Changes
inbody weight,
circumference of the neck andplasma
testosteroneconcentrationslargely
paralleled
thoseof testis diameter. Motile spermatozoa were collectedat all stages of the testiscycle.
Six animals in theearly
stagesof antlergrowth
wereselected from the herd inMay
and 3 of these wereimplanted
with 1g melatonin in a Silastic rubberenvelope.
Each animal wascaptured
on3subsequent
occasionsatmonthly
intervals. Melatoninwaswithout effectontherateof increase in size of thetestis,
circumference of the neck orgrowth
andcleaning
of the antlersalthough
1 of the treated animals failedto casthis antlersattheexpected
time 8 months aftercleaning.
We conclude from thisstudy
that there is littleor noseasonalphotoperiodic
entrainment of the antler and testicularcycles
of males in thispopulation
of axisdeer.Keywords:circannualcycles;antlers;aseasonalreproduction;axis deer Introduction
In
seasonally breeding
deer from temperateregions
the seasonalcycle
ofrutting
behaviour andgrowth
andcasting
of the antlers isassociated withacycle
ofgrowth
and secretion oftestosteroneby
the testis(Lincoln,
1971;
Lincoln etal.,
1972;
Bubenik etal.,
1975; Leader-Williams, 1979;
Sempere
&Biosson,
1981).
Photoperiod
is themajor
environmentalcuewhich entrains the seasonal antlercycle (Jaczewski,
1954; Goss,
1969; Pollock,
1975)
andmorerecent work indicates that theeffects of
photoperiod
are mediatedby
thepineal gland (Plotka
etal., 1981;
Lincoln,
1985).
Further,
treatmentwith thepineal
hormone melatonincandisrupt
the normaltiming
of seasonal antlergrowth
in deer(Bubenik 1983;
Lincolnetal.,
1984).
Severalspecies
of deer live in thetropical
or
sub-tropical regions
where there arecomparatively
minor annualchanges
inphotoperiod.
For thesespecies
there is little informationonbreeding biology
and the antlercycle.
In this paper, we describe aspects of the sexual and antlercycle
of the male axisdeer(Axis
axis,
aspecies
of the Indiansubcontinent)
while maintained as afree-ranging
herd in thetemperate
zone and describe the effects ofasubcutaneous melatoninimplant
onantlergrowth
and velvetcleaning.
Materialsand Methods
Animals. Thestudywascarriedoutwith the herd of axis deerlivingoutof doorsatWhipsnadePark, Bedfordshire, 5 3 . Theprecise geographical originof the herd from the Indian subcontinent is unknown and the animals have »Present address: MRCReproductive BiologyUnit,37ChalmersStreet,EdinburghEH39EW,U.K.
been maintained in Britain forover50years.The deerwerekeptinalargegrasspaddockand offeredsupplementary
hayandconcentratesin the winter.
Throughout
theyear,the animalshadaccesstoanunheated shelter. Thecompo¬sition of the herd
changed
duringthestudybutalways comprised10-14 adultmales, 12-15 adult females and10^15
calves andsub-adults.Calving
in this herd is aseasonal withasimilar number of birthsoccurringin the winter and summermonths(Kirkwoodetai, 1987)although mortalityishighlyseasonal and calves born in the winter seldom survive(seeTable 1).Table 1. The
calving
patternof the axisdeer herdatWhipsnade,
1975-1985(data
on seasonof births from Kirkwoodetal.,
1987)
JFMAMJJASOND
No. born 14 10 11 18 6 7 10 12 12 12 13 10
No.dyingin thefirstmonth
oflife 13 10 8502003458
Antlercycleswithin the herd. To obtain further information abouttheantlercycle,the herdwascheckedonceevery
2 weeks fromJanuary1985to
September
1986 and records made of antlerstate(velvet, cleaning,hard antlerorcast)of all adultstags.
Antler andreproductive cycles.Observationsweremadeon4 adultstagsfromSeptember 1983toOctober 1984.
Eachanimalwascapturedat4-weeklyintervalsusingacapturerifle loaded withadartcontaining3 mgetorphine hydrochlorideina 1-5ml volume and 10mgacepromazine (Immobilon:C-Vet, BuryStEdmonds, Suffolk, U.K.).
Sedationswerereversed usinganequivalentdose ofdiprenorphine (Revivon: C-Vet). Immediatelyafter immobiliz¬
ation,bloodwascollectedbyvenepunctureandmeasurementsweremade of testis diameter andlength,neck circum¬
ference,antlerstateand body weight.Testicular volumeswerecalculatedusingtheformula,V=
l/6nW2L,
whereV=volume inml,W=meantesticular diameter andL=lengthincm.Onanumber ofoccasions,attemptswere
madeto
electroejaculate
sedatedstagswitha2-5 30cmrectalprobewithtwolateralstripelectrodes andaportablebattery-poweredstimulator
operating
at20-80cyclesand 2-12 V. Immediatelyaftercollection,spermatozoaweretreated with BWW diluent(Biggersetal., 1971)and maintainedat20-30°C for 2-4 h.Spermatozoawerescored for
motility, live/deadratio and concentrationusingahaemocytometer.
Histology ofthe testisand
epididymis.
Twostagswereculledfromthe herd in Mayand October 1986. These 2animalswerejudgedtobeat the nadir andpeak of their testiscycle(65daysaftercastingand 80daysafter velvet
cleaning
respectively).
Thetesteswerefixed in Bouin's fluid(20timesvolumeof thetissue)for 48 h and thenprocessedto
paraffin
wax.Sections of5 µ werecuton aLeitzrotarymicrotome and stainedbyHeidenhain's ironhaematoxy-lintechnique,counterstained withvanGieson's
picro-fuchsin.
Mean tubule diameterwasderived frommeasurements of tubular cross-sectional area, performed by image analysis(using
a System 3, Analytical Measuring System,SaffronWaldon, Essex,U.K.)of the circular
profile
of 20 tubules selectedatrandom from each testis. The relative stageof thespermatogenic cyclewasassessedusingcriteria establishedbyHolt(1977).Effect ofmelatoninonthe antlercycle.InMay1985,6stagswereselected from the main herd forastudyof the
effects of melatoninonantlergrowthandcleaning.All6animalswereadult and had antlers thatwereinan
early
stageofgrowthandatasimilar relativestageofdevelopmenttored deeratthis time of year(Lincoln,1971).Three animals weresedated inMay1985 and received subcutaneous Silastic rubberimplants(500-1 sheeting,0013cmthickness,
surfacearea36
cm2;
DowCorning,Midland, MI,U.S.A.)containing1 g melatonin(SigmaChemicals, Poole, Dorset,U.K.).Three control animalswerealsosedated,but didnotreceiveimplants.On 3subsequentoccasionsatintervals of 4 weeks between 08:00 and 10:00 h from JunetoAugust 1985 all 6 animalswerecaptured,bloodwascollected and
themeasurementsdescribedabovewerecarriedout.Noattemptwasmadetocollectsemen.Implantswereremoved
from the animals in October and November 1985.
Hormone determinations. Plasmatestosteronewasextracted with 10 volumes ofdiethylether and testosterone
concentrationwasmeasuredby radioimmunoassay usingantiserum raised insheep againsta
testosterone-3-carboxy-methyloxime-bovineserumalbuminconjugate. The antiserum(No. 505)was
supplied
bythe MRCReproductive
BiologyUnit,
Edinburgh,
andadetaileddescriptionof itspreparation
anduseappearselsewhere(Webbetai, 1985).Tritiated testosterone
([1,2,6,7-3H];
sp. act. 82Ci/mmol) was supplied by Amersham International, Amersham,Berks.,U.K. Testosterone standard (SigmaChemicalCo.,StLouis, MO, U.S.A.)was addedatconcentrations of
6-25-400pg/tube.Free and boundtestosteronewere
separated
usingcharcoal-dextran. The antiserum bound 36% ofthe labelledtestosterone atafinal dilution of 1:127 000 and 50% of the bound labelwasdisplaced by46pgtestoster¬
onestandard.Sensitivity,definedasthe first
point
onthe standardcurvesignificantly
different fromzerobinding,was6-25pg/tube
(0-3ng/ml
plasma). Cross-reactivity at 50% displacementwas as follows: 5a-dihydroxytestosterone,42%; androstenedione, 5-6%; progesterone, 0-4%; oestradiol-17ß,0-4%; androstanediol, 0-3%, andandrosterone, <01%.
Recoveryof
[3H]testosterone
addedtoplasmawasdeterminedforeach assay and allsampleswerecorrected for these losses.Mean recoveryover4 assayswas81-5%(+1%,s.e.m.).Serial dilution ofplasmafrom4animals showedparallelismwithtestosteronestandards. Toassessthe accuracy oftheassay,testosterone(0-5-4ng/ml plasma)was
addedtoplasmawithalowtestosteroneconcentrationasmeasuredby
radioimmunoassay.
Recoveryof addedtestos¬teronewas76-7%(+5%).Theintra-assaycoefficient of variationwasestimatedbymeasurement(n=8)oftestoster¬
oneconcentration in
plasma
samples containing7-1 and 1·
1ng/mloftestosteroneandwas3-6 and 4-9%respectively.
Theinterassaycoefficient of variation for thesamelevels oftestosteronein5 assayswas6-1 and 9-2%respectively.
Plasma melatonin concentrations weredetermined according to the methodoriginallydescribed by Fraseretal.
(1983)andsubsequentlymodifiedby Webleyetal.(1985).Thesensitivityof theassaywas10pg/mland theintra-assay
coefficient of variationwas41%.
Results Antler
cycles
within the herdThe antler
cycles
of all adult males in the herdareshowninFig.
1. Of the 3 animals forwhich2 consecutivecasting
dateswereobtained,
themeaninterval fromcasting
tocasting
basedon2-week observationswas53 weeks(range
45-60weeks);
in 2animalsforwhichconsecutivecleaning
dates wereavailable,
the mean interval was 49-5 weeks. In 7animals,
the duration of the hard antlerphase
was30-2 weeks(range
23-37weeks),
while in 7animals,
the interval from thecasting
of the old antlertothecleaning
of the velvet from thenewantlerwas25-5 weeks(range
17-30weeks).
Of the 4stags(Nos 8-11)
studied in detail over1983-84,
cleaning
of the velvet occurred inJanuary,
March,
June andJuly.
Theremaining
stags
inthe herdcleanedtheir antlers in the winteror summermonths with most animals
cleaning
betweenMay
andSeptember.
Several of thestags
whichcleaned velvet in the winter months wereobservedtocarry theremnantsof dead and
dying
velvet from their antlersforconsiderableperiods
and in these animalsprecise
estimates ofcleaning
date aretherefore difficulttoassess.Noneofthe animals in the herdwasobservedtoshowanyseasonalchange
in the colourorform ofcoat. Antler andreproductive cycle
Dataon
changes
intestosteroneconcentration,
testiculardiameter,
neckgirth
andbody weight
in relation tothe antlercycles
of 4stags
areshown inFig.
2. Since each antlercycle
was out ofphase,
withcasting
inSeptember,
October,
December andJanuary,
thedata have been normalized around the timeofantlercasting.
Anexample
ofanindividualantlercycle
fromonestag
is shown inFig.
3. In allanimals,
therewere marked fluctuations in testis size with mean testis diameterranging
from a minimum of 231-6mm(±10,
s.e.m.)
to a maximum of 42-6mm(+1-2, s.e.m.).
Estimated mean maximum and mean minimum testis volumes were 87-2 ml
(±6-6)
and 17-8 ml(±1-8) respectively.
In all animals therewas afixedrelationship
betweenstage
of the antlercycle
and testis
size;
minimum testis diameter occurred 1-2 months afterantlercasting
whereas maxi¬mum testis diameter occurred when
stags
were in hard antler. Maximum antlergrowth
rate(0-75
cm/day)
occurred 1 month after theshedding
ofthe old antler. Plasmatestosteroneconcen¬ trations werelargely
non-detectableduring
theperiod
ofantlergrowth
but,
coincicent with theperiod
of hard antler andlarge
testissize,
testosteroneconcentrations above non-detectable levels werefrequently
observed. None ofthestags
developed
a mane of hair at anystage
of the testiscycle.
The
body weights
and neck circumference of these animalschanged
in association with the testiscycle
rather than the time ofyear. Theperiod
of decline in the volume of the testis was associated with asteep
andsignificant
decline in thebody weight
of thestag
(maximum
mean vsminimummean
body weight;
t=6-5,
<001).
Themaximummeanbody weight
forthe 4stags
was 110-5
(+ 1-9) kg
and in eachcase was achieved 3-4months before antlercasting.
Minimum meanbody weight
occurredduring
the middle of theantlergrowth
phase
andwas88-7(±1-3)
kg,
some21-7kg
lower than maximumweight.
Partialcorrelation ofbody weight,
testis diameter and\Y/////////Ah-V777Ä
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10 11 - — — — — — —·— — — — — — — — — — — — — — -1-1-1 SNJMMJ SNJMMJ SNFig.
1.Cycles
ofantlergrowth
for 11adult stagsover a2-year
period. Stags
8-11 aredescribed inmoredetail inFigs
2and 3.Stags
5-7 received melatoninimplants
inMay
1985.Implants
wereremoved 5-6 months later.Stags
1-7werestudied fromJanuary
1985 toOctober1986;
Stags
8-11 were studied fromJuly
1983 to October 1985. Horizontal bars indicate rangesof transitiontovelvet(open areas)
orhard horn(cross-hatched areas).
Crossindicatesthe death ofananimal.neck circumference showed that neck circumferencewas
significantly
associated withchanges
in testis diameter(/
=2-28,
<005)
andnotwithbody weight
(t
=218,
jP>0-05).
Motile
spermatozoa
wereejaculated
at allstages
of the testiscycle
andsperm countsranged
from 2
105
to 5109
ml.Although
severalattempts
atelectroejaculation
wereunsuccessful,
therewas noassociation of
stage
oftestiscycle
with failuretocollectsemen nor wasthereasignifi¬
cant correlation between sperm
density
intheejaculate
and testis volume(r2
=0-288).
Data onspermatozoacollected
by
electroejaculation
aresummarized in Table 2.Histology of
thetestisandepididymis
The2animals culledatthe nadirand
peak
of the testiscycle
in 1986 showedsubstantial differ¬ ences inthehistological
appearanceofthe testis andepididymis (Figs
4 &5).
Meantubule cross-sectional surface area was 161(
+0-9s.e.m.)
and 25-8(±108)
3
µ 2
and estimated tubule diameterwas 163-2(±
5-3s.e.m.)
and212(±
3-7)
10'
µ 2
for theregressed
anddeveloped
testisrespectively.
Intheregressed
state(Fig.
4a),
manyofthe tubuleswereinanearly phase
ofdevelop¬
ment with a
high proportion
showing
a reducedlumen,
someprimary
spermatogonia
but fewelongating spermatids.
However,
in 10% of thetubules,
there wasevidence of activespermato-genesis
as indicatedby
the presence of youngunelongated spermatids
and anenlarged
lumen. Thesespermatids
were all round inshape
andwereabundant in certain tubules but therewere noignificant
numbers ofelongating spermatids
inanyof the tubules examined.Inthedeveloped
testis"ig. 4b),
therewasevidence ofactivespermatogenesis
inalarge
number of the tubulesexamined,
ith abundant
elongating spermatids
and anenlarged
lumen to the tubule.Large
numbers oflermatozoawere
present
in theepididymides
of bothstags
(Fig. 5).
Fig.
2.Changes
intesticular diameter(a),
testosteroneconcentration(b),
neckcircumference(c)
andbody weight (d)
for4 adult axis deerstags. Data havebeen normalized for eachanimal around the timeofantlercasting.
Thetiming
of hardhorn(hatched bar)
and antlerre-growth
(open bar)
is shown below.The
effect of
melatoninonthe antlercycle
The melatonin
implants produced high
concentrations of melatonin in theperipheral plasma.
Values measured 1 month afterimplant
insertion were330,
191 and172pg/ml
for the 3 treated animals and<8pg/ml
for the control animals. BetweenMay
andAugust
thereweresignificant
increases in testisdiameter,
circumference of the neck andlength
of the antlers in bothgroups(Fig.
6;
analysis
ofvariance;
<001)
but melatonin treatment was without effect(P
>005).
Afterthe removal of the
implants
between October and December1985,
Stag
6(Fig. 1),
which hadreceivedan
implant,
remained in hard antler for 14 months fromJuly
1985through
tocasting
on3September
1986.Discussion
The results
presented
here indicate that in the axis deer stag the antlercycle
is associated withchanges
inactivity
of the testis. In all 4 animals studied indetail,
testis size reachedanadirshortly
after thecasting
of the old antler and increasedgradually
during
theperiod
of antlergrowth.
30 50 2> 20' 2 10-~ì— — — S — --110 100 - CÜ
Fig.
3. Seasonalchanges
in testis diameter(O), plasma
testosteroneconcentration( ),
neck circumference( )
andbody weight (·)
forStag
9. Thetimesofelectroejaculation
areindi¬ cated withaverticalarrow,closedarrowsindicating
successandopenarrowfailuretoproduce
a semen
sample.
The antlercycle
isindicatedas agrowth phase (velvet; open)
andhard horn(cross-hatched).
Table 2. Data on success of collection
by electroejaculation
and spermatozoadensity
andmotility
at3 differentstagesof the antlercycle
No. ofattempts No. of times Mean( +s.e.m.) Meanmotility tocollectsemen semencollected cone,(no./ml) %(range)
Antler invelvet Antler in hardhorn
(1stfourmonths)
Antlerinhard horn
(2nd
fourmonths) 16 9 15 138-2(±2-9)xl07
55 (0-80) 8 9-6(+6-6)xl07
63(20-85) 12 ll-3(±3-4)x107 68(20-80)Maximaltestissizeoccurred 2-3 monthsafterthe
shedding
ofthevelvet.Thus,
inthisspecies,
asinseasonally breeding species
of deer that live in thetemperate
zone, there is a close correlation betweenchanges
intesticularsize,
testosteronesecretion andthegrowth
anddevelopment
ofthe antler(red
deer:Lincoln, 1971;
Lincoln &Kay,
1979;
white-tailed deer: Mirachi etal.,
1975;
roe deer:Bramley,
1970;
reindeer:Leader-Williams, 1979).
Inaddition,
both the circumference of the neck andbody weight
showedpronounced
changes
inassociationwith thechange
intestis size and testosteronesecretionwith thepeak
in neck circumference andbody weight occurring
atthesame timeasmaximaltestissize. In thisrespect,
the axisstag
issimilartotheseasonally breeding
red deerstag
(Lincoln
etal., 1970; Lincoln,
1971;
Mitchelletal.,
1976).
Successful
electroejaculation
ofmotile spermatozoa was achieved bothduring
antlergrowth
and hardantler,
despite
anestimated5-foldchange
intestisvolume.Histological
evidence from the testis removedduring early
antlergrowth
indicated thatlimitedspermatogenesis
mayoccuratthe nadir of thetestiscycle
although
collection of motilespermatozoa
by electroejaculation
atallstages
ofthe antler
cycle
may reflectstorageofspermatozoa
in theepididymis
throughout
theperiod
ofFig.
4.Seminiferoustubulesfromthe testis of(a)
anadult axisstag(Stag
12,
notshowninFig.
1)
culledatabout the nadir of thecycle
in testissize,
indicating
areduced lumen volume andthe presence ofprimary spermatogonia,
and(b).
Stag
3(adult,
seeFig.
1),
culledatabout thepeak
of thecycle
intestissize,
indicating
theenlarged
lumenand abundantelongating spermatids.
Scalebar= 100µ .low
spermatogenic activity
rather thanspermatogenic
activity
of the testis. Inacomparison
of thedegree
of seasonal testicularregression
in severalspecies
of seasonaldeer,
Lincoln(1985) suggested
that a reduction in testis
weight
to below 25% of the seasonal maximum was associated withcomplete infertility
asjudged by
anarrestinspermatogenesis.
In the reddeer,
seasonalinfertility
is associated withasignificant
reduction in Sertoli celldensity,
stemspermatogonia
number and the number ofelongating spermatids (Hochereau-de
Reviers &Lincoln,
1978).
However,
despite
a 5-fold decline in testisvolume,
testicularregression
in the axis deer isnot associated withcomplete
azoospermia.
In the herd studiedhere,
females conceivethroughout
the year; it is not known whether fertilematings
cantakeplace
with males in velvetorwhetheronly
males in hard antlerare able tocompete
foraccess to females and rutsuccessfully.
In thenon-herding
Reeves'muntjac
(Muntjiacus reevesi),
births can occurin any month ofthe year(Zuckerman,
1953;
Chapman
&Fig.
5.Proximalepididymis
of(a)
Stag
12and(b) Stag
3indicating
thepresence ofabundantspermatozoa.Bar = 100 µ .
Dansie,
1970),
but the male exhibitsaseasonalantlercycle
and iscapable
offertilemating
while the antlersareatanystageofdevelopment (Chapman
&Chapman,
1982).
In the
wild,
there is aregional
variation in thetiming
of the antlercycle
andseason of births withaxisstagsinthe north of their rangeexhibiting
aseasonal antlercycle
(Schaller,
1967; Mishra,
1982),
while in the south of the range there is littlesynchrony
in the antlercycle
or birth season(Phillips,
1928; Morris,
1935).
Within theaxis herdatWhipsnade
births occurredthroughout
the yearand the antlercycles
of malesshowedpoorsynchrony
withoneanotheralthough
themajority
ofanimalscleaned their velvet in thesummermonths. Onepossible explanation
of suchsynchrony
is that seasonalmortality
(see
Table1)
maysynchronize
antlercycles
since males within the herd would tend to achievepuberty
anddevelop
thefirst antler at a similar time ofyear. The poorsynchrony
of antlergrowth
betweenmalessuggests
thatphotoperiod
isnotinvolvedincontrolling
the antlercycle
inthisspecies.
Thiscontrastswithseasonally
breeding
deer in whichexperimental
manipulations
ofphotoperiod
can drive the antlercycle (Jaczewski,
1954; Goss,
1969).
Sincephotoperiod
manipulation
of axisstage
wasimpractical,
animalsweretreated with melatoninas an40 35 30 80' 70 60 50' 801 70 60' 50'
Á
-my
Fig.
6.Changes
in testisdiameter, neck circumferenceand antlerlength
for control(·)
andmelatonin-implanted
(O)
stags fromMay
toAugust
1985. The times ofshedding
of velvet are shown as vertical closed and open arrows for the control and melatonin-treated stagsrespectively.
alternative. The lack ofaneffect of melatoninon the
growth
of thetestis and antlercontrastswith work on white-tailed deer(Bubenik,
1983)
and red deer(Lincoln
etal.,
1984).
Treatment with melatonin eitherby implant
or oral administration 2h before dusksignificantly
advances antlercleaning,
testicularredevelopment
and testosterone secretionby
the testis in thesespecies.
Although
melatonin failed to hasten thedevelopment
of the testis and the hard antler in the axisstag, 1 of the 3 treated animals failedtocast atthe
expected
time and remained in hard antler for 14months after melatonin treatment.
Thus,
thepossibility
oflong
term effects of such melatonintreatments cannotbe
ignored.
We conclude
that,
in thesub-tropical
axisdeer,
thephotoperiodic-pineal
link to the repro¬ ductive axis may be absent oronly
able to entrain thereproductive
system
weakly. Long-term
cyclic changes
in testicularactivity
may occurin the absence ofphotoperiodic
entrainment. The role ofphotoperiod
inother seasonal deerspecies
maytherefore be toentrain rather than drivedirectly
seasonalrhythms
ofreproduction
and antlergrowth.
WethankAlison
Beasey,
R.Kock andR.Cinderey
for theirhelp throughout
thecourseof this workatWhipsnade,
Dr H.Moore,
DrW.Holt andMrD.Spratt
for assistance with collection and assessmentofsemenand thepreparation
of stained tissuesections;
and Dr A.McNeilly
for thegift
of antiserum.References
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Received 26 October 1987