Paper 2 Sociosexual behaviors during the transition from non- receptivity to receptivity in rats housed in a seminatural environment

Paper 2

Sociosexual behaviors during the transition from non- receptivity to receptivity in rats housed in a seminatural

environment

Paper 2

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Behavioural Processes

j ou rn a l h o m epa ge :w w w . e l s e v i e r . c o m / l o c a t e / b e h a v p r o c

Sociosexual behaviors during the transition from non-receptivity to receptivity in rats housed in a seminatural environment

Xi Chu

, Anders Ågmo

DepartmentofPsychology,UniversityofTromsø,Norway

a r t i c l e i n f o

Articlehistory:

Received20October2014

Receivedinrevisedform4December2014 Accepted5January2015

Availableonline6January2015

Keywords:

Seminaturalenvironment Sociosexualbehavior Behavioralestrus Transition Rat

a b s t r a c t

Femalebehavioralestrusisdefinedastheperiodbetweenthefirstlordosisdisplayedduringtheestrus cycletothelordosisthatisnotfollowedbyanotherwithin60min.Inaseminaturalenvironment,an estrousfemaleconsistentlydisplayslordosisinresponsetoeverymalemountfromthestartofbehav- ioralestrusuntiltheendofit.Thismeansthatthefemalesuddenlychangesfromastateofcomplete non-receptivitytofullreceptivityandthenabruptlychangesbacktonon-receptivity.Itisunlikelythat theseabruptchangesarecausedbysuddenchangesinserumconcentrationofovariansteroids.Here,we presenttheresultsofadetailedstudyofsociosexualbehaviorsduringthetransitionfromnon-receptivity toreceptivityandviceversa.Thefrequencyofmalemountingwasclosetozerobeforeandafterestrus.It remainedataconstant,highlevelthroughoutestrus.Femaleparacopulatorybehaviorandmalepursuit ofthefemaleincreaseddrasticallyfromaverylowlevelbeforeestrustoahighlevelduringestrus.They returnedtolowlevelsimmediatelyafterestrus.Noneofthemanyotherbehaviorpatternsregistered changedduringthetransitions.Itappearsthatthesuddenincreaseinmalepursuitandfemaleparacop- ulatorybehaviorcanexplainthebeginningofbehavioralestrus,andtheirequallysuddendisappearance causesittoend.Theneurochemicalmechanismsbehindthesealmostinstantaneousbehavioralchanges areunknown.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction

In a strict sense,sexual behavior in female rats consists of assumingalordosispostureinresponsetoamale’smount.This postureischaracterizedbyaconcaveflexionoftheback,extension oftheneck,elevationofthehindquartersandrumpanddeflec- tionofthetailtooneside.Afemaledisplayingthis behaviorin response tomale mounting is frequently described as sexually receptive.Inadditiontolordosis,areceptivefemalemaydisplay someotherstereotyped motorpatterns.These include running, darting,hopping,andearwiggling(Beach,1976;Erskine,1989).

Theyareoftengroupedtogetherunderthelabelsproceptivebehav- iororsolicitation.However,sincethefunctionofthesebehaviorsis somewhatunclear,ithasbeensuggestedthatamoreappropriate labelwouldbeparacopulatorybehaviors(BlausteinandErskine, 2002;Blausteinetal.,2009).

Ovariansteroids are necessary for thedisplay of female rat sexualbehaviors.Ovariectomizedfemalesneverdisplaylordosis

∗Correspondingauthorat:DepartmentofPsychology,UniversityofTromsø,Hug- inbakken32,9037Tromsø,Norway.Tel.:+4777649213;fax:+4777645291.

E-mailaddresses:[email protected](X.Chu),[email protected](A.Ågmo).

orparacopulatorybehavior, andestrogentreatmentcanrestore thesebehaviorstothelevelseeninintactfemales(e.g.,Bolingand Blandau,1939;Meyerson,1964;Zipseetal.,2000).Progesterone, givenseveralhoursafterestradiol,enhancestheresponsetoestra- diol(e.g.,Beach,1942),andisbelievedtobeparticularlyimportant forthestimulationofparacopulatorybehaviors(Fademetal.,1979;

Fryeetal.,1998).Thefemalerat’sresponsetoestrogentreatment isdosedependent.Atlowdoses,thefemaledisplayslordosisin responsetoonlysomemounts,andshemayalsorepeatedlyreject themountingmale.Withincreasingestrogendose,theproportion ofmountsactivatinglordosisalsoincreasesandthefrequencyof rejectionsgetsreduced(e.g.,SpiteriandÅgmo,2006).Eventually thefemalewilldisplaylordosistoeverymount.Inintactfemales, theonsetofsexualreceptivityisgradual,inthesensethatonlya fractionofthemale’smountsactivateslordosis(e.g.,Madlafousek andHlinak,1978).Thisisalsothecaseattheend oftheperiod ofsexualreceptivity.Onlyforafew hoursinthemiddleofthis periodthefemalerespondswithlordosistoallmalemounts(e.g., Hardy,1972).Whenmanualstimulationisusedtoactivatelordo- sis,itappearsthatthestimulationneedstobemoreintenseatthe beginningandtheendoftheperiodofsexualreceptivitythaninthe middleofthisperiod(Blandauetal.,1941).Theobservationscon- cerningthegradualappearanceanddisappearanceofthelordosis http://dx.doi.org/10.1016/j.beproc.2015.01.001

0376-6357/©2015ElsevierB.V.Allrightsreserved.

X.Chu,A.Ågmo/BehaviouralProcesses113(2015)24–34 25

responsecoincidewithdataonchangesinserumconcentrations oftheovarianhormones,particularlyprogesterone(Söderstenand Eneroth,1981).Thereisagradualincreaseinserumconcentration ofprogesterone,whichprecedestheappearanceofsexualbehav- iorwithafewhours.Interestingly,theendofreceptivityseemsto beunrelatedtoreducedserumconcentrationofovarianhormones (SöderstenandEneroth,1982).

The gradualincrease in thepropensity torespondwithlor- dosiseithertoamale’smountsortotheexperimenter’sfingers hasbeenestablishedinexperimentsin whichthefemaleswere subjectedtoshorttestsatregularintervalsvaryingfrom30min to6h.Whenmaleswereusedforactivatinglordosis,theywere preselectedforintensecopulatorybehavior,andamalefailingto mountwasimmediatelyreplacedwithamorevigorousone.When manualstimulationwasemployed,theexperimenterassuredthat it was appropriatelyapplied, regardlessof whether thefemale offeredresistanceornot.Thesetestingproceduresarequitedif- ferentfromwhatoccursintherat’snaturalcontextinwhichmales andfemalessharethesamehabitat,sometimeseventhesamebur- row(Calhoun,1962).Itappearsthatthemalesrarelytrytomate withnon-receptivefemales,whereasanumberofmalespursueand copulatewithanysexuallyreceptivefemale(RobitailleandBouvet, 1976).Likewise,whenfemalesarenon-receptive,theyresistthe raremountingattemptsfrommales,therebyavoidingthereceiptof thetactilestimulationneededforactivationoflordosis.Theforced sexualinteractionoccurringintheshorttestsmentionedabove seemstobeoflittleecologicalrelevance,andmightnotofferan appropriatedescriptionofthefemale’ssexualdealingswithmales duringtheestruscycle.

Weobservedgroupsofintact,cyclingfemaleshousedtogether withmalesinaseminaturalenvironmentforseveraldays.These femalesdidnotshowagradualincreaseinresponsivenesstothe males’mounts.Assoonasthefemaleenteredbehavioral estrus (definedastheperiodbetweenthefirstlordosisdisplayedandthe lordosisthatwasnotfollowedbyanotheronewithin60min)she respondedwithlordosistoeverymountuntiltheendof estrus (ChuandÅgmo,2014a).Thus,insteadofagradualincreaseinlor- dosisresponsesatthebeginningofestrusandagradualdeclineat theendofestrus,thefemalesintheseminaturalenvironmentsud- denlychangedfromcompletenon-receptivitytofullreceptivity,

andthenfromfullreceptivitytonon-receptivity.Thisobservation suggeststhatthegradualchangesinserumconcentrationofovar- ianhormonesarenotassociatedwithanygradualchangesinsexual responsivenessatthebeginningandend ofbehavioral estrusin aseminaturalenvironment.Insteaditseemsthatanon-receptive femaleisnotattractivetomales,andshedisplaysnolordosissimply becausesheisnotmountedbeforebeingfullyreceptive.Insup- portofthisnotiontherearedatasuggestingthatfemaleattractivity requiresmoreovarianhormonesthantheactivationofreceptivity (Albertetal.,1991).Consequently,assoonasafemaleisableto attractamale,sheisreadyforperforminglordosisinresponseto allhismounts.This,somehow,wouldmeanthatthedurationof behavioralestrusinaseminaturalenvironmentisdeterminedby themales’responsestothefemaleasmuchasormorethanthe female’sresponsestothemale.

Thepurposeofthepresent studywastoanalyzechangesin femaleandmalesociosexualbehaviorsaroundthetransitionfrom non-receptivitytoreceptivityandviceversa.Eventhoughsuchan analysisdoesnotdirectlyenhanceourunderstandingoftheneu- robiologicalmechanismsbehindthesechanges,itwouldmakeit possibletodeterminetherelativeimportanceofmaleandfemale behavior.

2. Method 2.1. Subjects

Male(300guponarrival)andfemale(250guponarrival)Wistar ratswereobtainedfromCharlesRiverWIGA(Sulzfeld,Germany).

Animalswere housedinsame sexpairs in Macrolon^® IVcages ina roomwithcontrolledtemperature(21±1^◦C)andhumidity (55±10%)anda 12:12hlight/darkcycle(lightson0800).Com- mercialratpelletsandtapwaterwereprovidedadlibitum.

2.2. Apparatus

The seminatural environment used in this study has been described indetail elsewhere(Chu &Ågmo, 2014a,b).Briefly,it measured2.8×2.4mandconsistedofacomplexburrowsystem and an openarea (see Fig.1).There werefour smallopenings

Fig.1. Schematicdiagram(A)andphotograph(B)oftheseminaturalenvironmentusedinthisstudy.Forfurtherdetails,seetext.

Table1

Descriptionofregisteredbehaviors(ChuandÅgmo,2014a,b).

Maleandfemalebehavior Datacollectedas Behaviordescription

Sniffing Durationandnumber Theratplacesitssnoutclosetoanybodypart,excepttheanogenitalregion,ofanotherrat whileitswhiskersmovebriskly.

Anogenitalsniffing Durationandnumber Theratsniffs,occasionallygroomsandlicks,anotherrats’anogenitalregion.

Pursuit Durationandnumber Theratrunscloselybehindanotherrat.

Nose-off Durationandnumber Facinganotherrateitherstandingon4legsorwhilerearing;itincludesboxingandteeth showing.

Flee Number Theratrunsawayfromanotherratafteranagonisticinteraction.

Malecopulatorybehavior

Mount Number Theratstandsonitshindlegsandplacesitsforepawsonanotherrat’srumpfrombehind

anddisplayspelvicthrusting.

Femalebehavior

Paracopulatorybehavior Durationandnumber Approachtoamalefollowedbyrunaway,oftenassociatedwithhops,darts,earwiggling.

Lordosis Number Femalestandsimmobilewiththebackarcheddownwardandtherumppushedupward

whilethetailisdeflectedtotheside.

Rejection Number Theratkicks,bitesorturnsaroundagainstitssuitor.

(8×8cm)betweentheburrowandtheopenarea.Alight-blocking wallofextrudedpolyethylenefoamwasusedtodividetheroom inwhichtheenvironmentwasinstalledintotwoparts,thereby providingthepossibilitytovarythelightintensityintheopenarea whilemaintainingtheburrowincompletedarkness.Videocameras werecenteredabovetheopenareaandtheburrow,respectively.

2.3. Procedure

Beforeagroupofanimalswasintroducedintotheseminatural environment,thefloorwascoveredwithaspenwoodshavings.A fewwoodsticksandplasticshelterhutswereprovidedintheopen area,andnestbuildingmaterialwasputinthenestboxes.About 3kgofpelletswereprovidedinacorneroftheopenarea,and4 waterbottleswerealsofreelyavailableinthatcorner.Thesub- jectsweremarkedintwoways:differentareasofthebackwere shavedandthetailwasmarkedwithdifferentnumbersofblack stripes, makingiteasy toidentifyeach individualsubject.Both markswereclearlyvisibleduringtheentireobservationperiod.

The12:12hlight/darkcyclewaspreservedintheopenarea.Dur- ingthedarkphase,theintensityofthelightreflectedfromthefloor coveringwasabout1lx.Itwasabout180lxduringthelightphase.

Theburrowwasmaintainedintotaldarknessfortheratsbutillumi- natedwithinfraredlightforthevideocamera.Thevideorecorders wereactivatedwhenintroducingtheanimalsat13:00onday0.

Recordingwasthencontinuousforaperiodof8days.Additional proceduraldetailscanbefoundinChuandÅgmo,2014a,b.

2.4. Design

Fivegroupswereused.Eachgroupconsistedof4femalesand3 males.Allsubjectswereintactandsexuallynaïve.Subjectsinthe samegroupcamefromdifferentcagestoensurethattheywere unknowntoeach otherat thebeginning of observation. Inthe periodof8days,eachfemalewouldenterintoestrusatleastonce.

Thissuppositionwasbasedonthefactthatmostfemaleshavean estruscycleof4or5days(LongandEvans,1922;Nequinetal., 1979).

2.5. Behavioralobservations

From thevideo record, we registered thebehaviors defined inTable1duringfourspecificperiods.Firstwe determinedthe momentatwhichafemaledisplayedherfirstlordosis.Thiswas consideredasthebeginningofbehavioralestrus.Thenwedeter- minedthemomentatwhichalordosiswasnotfollowedbyanother onewithin60mininthatsamefemale.Thiswasconsideredthe

endofbehavioralestrus.Thetimeelapsedfromthefirsttothelast lordosiswasthedurationofbehavioralestrus.Thisdefinitionof estrusdurationhasbeenemployedinourearlier studiesin the seminaturalenvironment(ChuandÅgmo,2014a,b).Itvariedfrom 4.05to10.87h,withamean±SEMof7.41±0.49h.Foreachfemale, wedividedtheperiodofbehavioralestrusin20equalintervals, meaningthateachintervalcorrespondedto5%ofestrusduration.

Observationsofsociosexualbehaviorsduringthepreestrusinter- valstartedatatimecorrespondingto5%oftheestruslengthbefore thefirstlordosis.Observationwasendedat5%ofestruslengthfol- lowingthislordosis(firstestrousinterval).Attheendofestrus,we observedthesecondestrousinterval,correspondingtothetime from5%ofestruslengthbeforethelastlordosisuntilthatlordosis.

Thepostestrusintervallastedfromthefinallordosisuntil5%ofthe estruslengthafterit(seeFig.2forillustration).Forexample,ifa femalehadabehavioralestruslastingfor400min,5%ofthisdura- tionis20min.Thus,thepreestrusintervalstarted20minbefore thefirstlordosis,andthefirstestrusintervalwentfromthefirst lordosisuntil20minafterit.Thelastestrusintervalstarted20min beforethefinallordosis,andthepostestrusintervalended20min afterit.

Thereasonforbasingobservationlengthonunitsof5%ofestrus durationratherthanaprefixedtimewasthatweneededtoknow thebehaviorintheinitialandfinalpartsofbehavioralestrus.Since thedurationofestrusvariedconsiderablybetweenfemales,afixed timewouldhaverepresentedhighlydifferentproportionsofestrus.

Afterthefact,weknowthatthiswasnotofanyimportance,because thebehavioralchangesoccurredveryclosetotheexactmomentof transition.

2.6. Datapreparationandstatistics

Before analysis,the number of occurrences and/orthe total durationofbehaviorsduringanintervalweredividedbytheactual lengthoftheinterval.Thus,behaviorswereeitherexpressedasthe numberofoccurrencesperminuteorasduration(s)perminute ofobservation.Inadditiontocomparethefourintervalsdefined above,weneededtodetectrapidchangesinbehavioraroundthe transitionsfromnon-receptivitytoreceptivityandtheopposite.

Thereforewedividedthe8minperiodsprecedingandfollowing thefirstandlastlordosisintooneminutesegments.Carefulexam- inationofthedatasuggestedthatan8minperiodwassufficiently longfordetectingrelevantchangesinbehavior.Atthesametime, repeatedmeasuresanalysisof8oneminintervalsisperfectlyfeasi- ble.Onlybehaviorsshowingchangesbetweenestrusintervalswere subjectedtothisanalysis.

X.Chu,A.Ågmo/BehaviouralProcesses113(2015)24–34 27

Fig.2. Illustrationoftheinterval,periodandsegmentaroundthefirstandlastlordosisemployedintheanalyses.Comparisonswereconductedbetween(A)preestrus interval,thefirstestrusinterval,lastestrusinterval,andpostestrusinterval,eachofintervalswasaslongas5%ofestrusduration;(B)the8minperiodbeforeandafterthe firstandlastlordosis,respectively;(C)the60ssegmentbeforeandafterthefirstandlastlordosis,respectively.

As it turned out, most of the transition-associated changes occurredintheoneminsegmentimmediatelyprecedingorfollow- ingthefirstandlastlordosis.Therefore,wedividedtheseminutes in30ssegmentsforfurtheranalysis.The4segmentswere−60s to−30s,−29to0s(0wasthemomentoflordosis),0–29s,and 30–60s.

TheShapiro–Wilktest showedthat mostdatadeviated from anormaldistributionandtheMauchly’stestrevealedthatsome variables violated the assumption of sphericity, thereby mak- ingparametricrepeatedmeasuresANOVAinappropriate(Girden, 1992).Therefore,weemployednonparametrictestsfordetermin- ing statisticalsignificance. Dependent data wereanalyzed with Friedman’sANOVA.Afterasignificantresult,posthoctestswere correctedformultiplecomparisonsaccordingtoConover(1999).

TheMann–Whitney Utestwasusedfor analyzing independent data.Allprobabilitiesgivenintext,tablesandfiguresaretwo-tailed.

3. Results 3.1. General

Threeofthe20 femalesdidnotenter intobehavioral estrus duringthe8daysofobservation.Thesefemalesbelongedtotwodif- ferentgroups.Amongthosedisplayingestrus,7females’hadpartly overlappingperiodswith1or2otherfemales.Thedurationofthe preestrusandpostestrusintervalswas22±1minwitharangeof 12–33min.

3.2. Malebehaviortowardsestrousandnonestrousfemaleswhen bothwereavailable

Therewasalmostnomountingoffemalesneitherbeforethe beginningof estrusnoraftertheend ofestrus(seeTable2).In fact, only 1 female was mounted once by each of two males during the interval preceding estrus. Interestingly, 3 females

had theirpreestrus interval overlapping withanother female’s estrus. While the estrous females received a mean±SEM of 0.15±0.07mounts/minfromthe9malesinthe3groupswhere suchanoverlapwasfound,thepreestrousfemalesdidnotreceive any mount at all. The difference in male mounting between theestrousandnonestrousfemalesissignificant(U=0,p=0.04).

Similarly, the postestrusinterval in 4 females overlapped with other females’ estrus. The postestrous females received only 0.01±0.01mounts/minfromthe9malesdistributedin3groups whereas the female or females remaining in estrus received 0.19±0.09mounts/min.Again, thedifference in thenumber of mounts receivedwas significant(U=2, p=0.047). Thefact that themalesalmostexclusivelymountedtheestrousfemales dur- ingintervalsinwhichbothestrousandnon-estrousfemaleswere simultaneouslyavailableshowsnotonlythatthemalescoulddis- tinguishestrousfemalesfromfemalesnotinbehavioralestrusbut alsothatonlyfemalesinestrusincitedthemalestomount.

3.3. Changesinbehaviorbetweenpreestrus,estrusandpostestrus

Femalepursuitofthemaleswasanextremelyrarebehavior.

Thelongestmeanduration,0.08±0.03s/min,wasinthepreestrus intervalandtheshortest,0.003±0.003s/min,wasinthepostestrus interval. The frequency of female fleeing from the males and the duration of female anogenital sniffing of the males were also extremely low. Actually very few females displayed these behaviors atall.Similarly, thenumber and/orduration ofmale nonaffiliativebehaviors(fleeingfrom,noseoff,fighting)towards thefemaleswereverylow.Noneofthesebehaviorscould,there- fore, besubjectedto anymeaningful analysisand data arenot shown.

Thedurationandfrequencyofparacopulatorybehaviorsvar- iedbetweenintervals.Itwaslowbeforethebeginningandafter theendofbehavioralestrusandhighduringtheintervalfollowing thefirstlordosisaswellasduringtheintervalprecedingthelast.

Table2

Changesinbehaviorintransitionsbetweenpreestrus,estrusandpostestrus.

Transitionnon-receptive–receptive Transitionreceptive–non-receptive

Behaviorsinduration(s/m) Nonreceptive Receptive Receptive Nonreceptive FriedmanANOVA

Paracopulatorybehavior 2.09±0.49^*a 7.87±1.11 9.21±2.33 1.29±0.63^{*b 2}(3)=26.96,p<0.001 Malepursuitoffemales 1.88±0.47^*a 5.84±0.93 4.88±1.36 0.53±0.18^{*b 2}(3)=31,73,p<0.001

Femalenose-offmales 0.64±0.34 1.28±0.60 1.25±0.59 1.13±0.56 ²(3)=5.65,p=0.13

Femalesniffingmales 2.38±0.52 3.13±0.38 1.33±0.27 1.41±0.73 ²(3)=12.80,p=0.005

Malesniffingoffemales 3.02±0.60 4.15±0.94 3.65±1.22 2.30±0.96 ²(3)=6.39,p=0.09

Maleanogenitalsniffingoffemales 0.80±0.23 1.11±0.37 2.89±1.00 0.72±0.40^{*b 2}(3)=9.52,p=0.02 Behaviorsinfrequency(times/m)

Mountsreceived 0.01±0.01^*a 0.26±0.06 0.48±0.23 0.00±0.00^{*b 2}(3)=36.26,p<0.001

Paracopulatorybehavior 0.32±0.07^*a 0.98±0.12 0.67±0.11 0.11±0.03^{*b 2}(3)=36.18,p<0.001 Malepursuitoffemales 0.53±0.13^*a 1.06±0.17 0.81±0.23 0.12±0.03^{*b 2}(3)=31.17,p<0.001

Rejection 0.22±0.08 0.26±0.07 0.12±0.05 0.06±0.04 ²(3)=1.20,p=0.75

Femalenose-offmales 0.16±0.07 0.23±0.05 0.19±0.07 0.14±0.06 ²(3)=5.85,p=0.12

Femalesniffingmales 0.53±0.10 0.77±0.06 0.30±0.05 0.17±0.04 ²(3)=25.54,p<0.001

Malesniffingoffemales 0.72±0.12 0.93±0.14 0.49±0.10 0.27±0.08 ²(3)=21.39,p<0.001

Maleanogenitalsniffingoffemales 0.14±0.04 0.21±0.05 0.26±0.08 0.10±0.05 ²(3)=9.13,p=0.03

*aDifferentfromthereceptivestateintransitionnonreceptive–receptive.

*bDifferentfromthereceptivestateintransitionreceptive–nonreceptive(correctedformultiplecomparisonstest,ps<0.05).

Maleanogenitalsniffingofthefemaledidnotdifferbetweenthe intervalprecedingestrusandtheintervalfollowingthefirstlordo- sis,buttherewasasignificantdeclinefromtheintervalpreceding theendofestrustothepostestrusinterval.Thedurationandfre- quencyofmalepursuitofthefemalesvariedbetweenintervals.It increasedfromtheintervalprecedingestrustotheintervalfollow- ingitsbeginning,anditwasmuchreducedfromthelastintervalof estrustothepostestrusinterval.Whereastherewasnodifference betweenthefirstandlastintervalsoftheestrusperiod,itturned outthattheamountofpursuitwasloweraftertheendofestrus thanbeforeitsbeginning(Table2).

Therewasnodifferencebetweenintervalswithregardtorejec- tionofthemalesornose-off.Femalesniffingofthemalesdidnot changeduringthetransitionperiods.However,thefemalessniffed themalesmorearoundthebeginningofestrusthanaroundtheend.

Tothecontrary,malesniffingofthefemalesdidnotvarybetween intervals.

In the males, the only behaviors varying according to the females’estrousstateweremountingandpursuit.Maleanogenital sniffingappearstobeunrelatedtothebeginningofthefemales’

behavioral estrus, but declined sharplyin thepostestrus inter- val.Inthefemales,onlyparacopulatorybehaviorswereassociated

Fig.3. Paracopulatorybehaviorandmalepursuitofthefemaleduringthe8minperiodprecedingandfollowingthefirstlordosisofbehavioralestrus.A:duration;B:

frequency.Dataaremean+SEM.*differentfromtheminutebeforeorafterlordosisinmalepursuit,⁺differentfromtheminutebeforelordosisinparacopulatorybehavior, correctedformultiplecomparison,p<0.05.

X.Chu,A.Ågmo/BehaviouralProcesses113(2015)24–34 29

Fig.4. Paracopulatorybehaviorandmalepursuitofthefemaleduringthe8minperiodprecedingandfollowingthelastlordosisdisplayedduringbehavioralestrus.A:

duration;B:frequency.Dataaremean+SEM.*differentfromthelastminutebeforethelordosisinmalepursuit,⁺differentfromthelastminuteperiodinparacopulatory behavior,correctedformultiplecomparison,p<0.05.

withchanges betweenreceptivityandnon-receptivity.Onlythe behaviorsthatvariedbetweenintervalsweresubjectedtofurther analysis.

3.4. Changesinbehaviorduringthe8minperiodsbeforeand afterlordosis

Thenumberoflordosesdisplayedduringthefirst8minperiod ofestruswasnotdifferentfromthenumberdisplayedduringthe last8minperiod(4.29±0.70and5.71±1.83,respectively,z=0.26, p=0.795). Theanalysis ofthe displayof paracopulatory behav- iorsduringthe8minprecedingthefirstdisplayoflordosis(and almost always the first male mount) shows that these behav- iors remainedat a low level until thelast minute, when there wasasharpincreaseinbothdurationandfrequency.Friedman’s ANOVArevealedasignificantdifferencebetweenminutes(dura- tion,²(7)=40.00,p<0.001;frequency,²(7)=50.96,p<0.001).

Posthoccomparisonsshowedthattherewasnosystematicchange between–8minand–1min(0beingthemomentofthefirstlor- dosis),whereasboththedurationandfrequencyofparacopulatory behaviorswerelonger/higherduringtheminuteprecedinglordosis thanduringallotherminutes(p<0.05whencorrectedformultiple comparisons).Whenanalyzingthe8minperiodfollowingthefirst lordosisitwasfoundthatthedurationofparacopulatorybehavior declined(²(7)=15.37,p=0.030).However,posthoccomparisons betweenthe8periodsdidnotyieldanysignificantdifference.Like- wise,thefrequencyofthesebehaviorsremainedstableduringthe 8minfollowingthefirstlordosis(²(7)=11.88,p=0.110).Dataare illustratedinFig.3AandB.Theanalysisofparacopulatorybehavior precedingthelastlordosisofbehavioralestrusrevealedadifference betweenperiods(duration,²(7)=28.51,p<0.001;frequency,² (7)=32.32,p<0.001). Particularly, there wasan increase in the

frequencyofthisbehaviorduringtheminute precedingthelor- dosis,accordingtoposthoctests.The8minperiodfollowingthe lastlordosisshowedacompletelydifferentpattern.Therewasno differencebetweentheoneminperiodsaccordingtoFriedman’s ANOVA(duration,(²(7)=8.41,p=0.30;frequency,²(7)=8.18, p=0.32)(Fig.4).

Maleanogenitalsniffingofthefemalewasnotanalyzedaround thefirstlordosissincetherewasnochangeinthisbehavior.Sim- ilarly, therewasnochangeduringthe8minprecedingthelast lordosis(ps>0.90).Tothecontrary,therewasadeclineafterthelor- dosis(duration,²(7)=24.63,p=0.001;frequency,²(7)=22.92, p=0.002).However,multiplecomparisonsofthe8oneminperiods didnotrevealanysignificanteffectwhentheappropriatecorrec- tionwasused.DataareshowninFig.4.Theabsenceofanyincrease precedingthelordosissuggeststhatanogenitalsniffingisnotasso- ciatedwiththat behaviorin thewaypursuit orparacopulatory behaviorare.Thedeclinetoalmostzeroshortlyafterthelordo- sismightsuggestthatthefemalehadcompletelylosthercapacity toattractthemales.

Whenpursuitwasanalyzedminutebyminuteduringthe8min precedingthefirstlordosisitbecameapparentthattheduration aswellasthefrequencyofthisbehaviordramaticallyincreased duringthelastminute(Fig.3).Therewasasignificantdifference betweenminutes (duration,² (7)=35.34,p<0.001;frequency, ²(7)=37.41,p<0.001).Theamountofpursuitbetween–1min and thefirst lordosiswaslargerthan that inmost otherinter- vals(p<0.05,correctedformultiplecomparisons).Analysisofthe entire8minperiodfollowingthefirstlordosisshowedaneffectof time(duration,²(7)=19.52,p=0.007;frequency,²(7)=17.68, p=0.010;Fig.3).Therewasamoderatereductioninpursuit.The lastlordosis,likethefirst,wasprecededbyanincreaseinpursuit.

Therewasadifferencebetweenthe8oneminperiods(duration,²

Fig.5. Durationandfrequencyofparacopulatorybehaviorandmalepursuitofthefemaleduringtheminuteprecedingandfollowingthefirst(A)andthelast(B)lordosis ofbehavioralestrus,respectively.Theseminutesweredividedin30ssegments.Forthelastlordosis,maleanogenitalsniffingisalsoshown.Dataaremean+SEM.

(7)=21.14,p=0.004;frequency,²(7)=27.59,p<0.001).Afterthe lastlordosistherewasanimmediatedecline,andpursuitremained atastable,lowlevelthroughoutthe8min.Therewasnodifference betweentheoneminperiods(duration, ² (7)=8.05,p=0.330;

frequency,²(7)=9.16,p=0.240;Fig.4).

3.5. Changesinbehaviorduringthe60ssegmentsprecedingand followingthefirstandlastlordosisofbehavioralestrus

The four 30s segments around the first lordosis differed withregardtoparacopulatorybehavior(duration,²(3)=29.22, p<0.001;frequency,² (3)=27.59,p<0.001).Therewasasharp andsignificantriseinparacopulatorybehaviorinthe30ssegment immediatelyprecedinglordosis(Fig.5).Interestingly,thehighlevel ofparacopulatorybehaviorpersistedduringthe30ssegmentfol- lowingthefirstlordosis.Thereasonisthat13ofthoselordoses (76%) werea response toa male mount. Since malesnormally mountinbouts,mostmalesperformedadditionalmounts,andthe femalesdisplayedadditionallordoses,withinsecondsofthefirst.

Becauseofthesesupplementarymounts,paracopulatorybehavior remainedatahighlevel.However,therewasadecreasefromthe 0–29speriodtothe30–60speriodaccordingtoposthoccompar- isons(Fig.5).

The analysis of the 30s segments around the last lordosis revealedsignificantdifferences(duration,²(3)=37.18,p<0.001;

frequency,²(3)=32.19,p<0.001).Posthoccomparisonsestab- lishedthatanincreaseinparacopulatorybehavioroccurredduring

thelast30sbeforeit (Fig.5).Theeventsfollowingthelastlor- dosisturnedouttobedistinctfromthoseoccurringafterthefirst.

Therewassharpdeclineinparacopulatorybehavioralreadyduring the30ssegmentfollowingthelordosisandtherewasnodiffer- encebetween0–29sand30–60s(ps>0.27;Fig.5).Sinceonly4 (23%)ofthelastlordoseswereassociatedwithmaleejaculation andonly2(12%)withmalemissionthisimmediatedeclinecannot beattributedtomaleinactivityfollowingthesebehaviors.Infact, whenwecomparedthe11femaleswhoselastlordosiswaspro- vokedbymalemounting(9females)orsniffing(2females)withthe 6femaleshavingreceivedanejaculation(4females)oranintromis- sion(2females)withtheMann–Whitneytest,wefailedtofindany significantdifferenceatanytimesegment(ps>0.05).Thesedata areillustratedinFig.6.

Maleanogenitalsniffingofthefemalevariedaroundthelastlor- dosiswithregardtoduration(²(3)=9.16,p=0.027)butnotwith regardtofrequency(²(3)=7.79,p=0.051).Whencorrectedfor multiplecomparisons,therewasnosignificantdifferencebetween thefour30ssegments.

Aswasthecaseforparacopulatorybehaviorsinthefemales,the amountofmalepursuitchangedaroundthefirstlordosis(dura- tion,²(3)=25.63,p<0.001;frequency,²(3)=29.52,p<0.001).

Therewasanincreaseinthe30ssegmentpriortothatlordosis.

Pursuitremainedatahighlevelduringthe30ssegmentfollow- ingthefirstlordosis.Aswasthecaseforparacopulatorybehavior, thiscaneasilybeexplainedbythefactthatmanyofthemalesper- formedadditionalmountsduringthissegment.However,pursuit

X.Chu,A.Ågmo/BehaviouralProcesses113(2015)24–34 31

Fig.6. Paracopulatorybehaviorandpursuitinfemaleseitherdisplayingtheirlastlordosisofbehavioralestrusinresponsetoamountortonon-sexualstimulation(N=11) orfemalesdisplayinglordosisinresponsetoamaleintromissionorejaculation(N=6).Therewasnogroupdifferenceneitherduringthe60ssegmentfollowinglordosisnor duringthe8minperiodfollowingit.Dataaremean+SEM.

declinedsignificantlyinthesegmentbetween30and60safterthe firstlordosis.Withregardtochangesaroundthelastlordosis,they werealsosignificant(duration,²(3)=27.44,p<0.001;frequency, ²(3)=24.46,p<0.001,Fig.5B).Therewasalargeincreaseduring the30ssegmentprecedingthelastlordosis.Afterittherewasan immediatedeclineinpursuit,andnodifferencewasfoundbetween 0–29sand30–60s.

3.5.1. Relationshipsbetweenparacopulatorybehaviorandmale pursuit

In ordertoget a clearerpicture ofthe transitionfromnon- receptivity to receptivity we determined the male and female behaviorimmediatelyprecedingthefirstlordosis.Inallfemalesthe lastactperformedbeforethatlordosiswasparacopulatorybehav- ior.Inallmales,thecorrespondingactwaspursuitofthefemale.In 11ofthefemales,thedisplayofparacopulatorybehaviorpreceded malepursuit.Inthe6remainingfemales,malepursuitpreceded paracopulatorybehavior.Achisquaretestshowsthattherewas nodifferencebetweenfemaleparacopulatorybehaviorleadingto malepursuitandmalepursuitleadingtofemaleparacopulatory behavior(²(1)=1.47,p=0.225).Thelastlordosiswasalsoalways precededbyfemaleparacopulatorybehaviorandbymalepursuit ofthefemale.

4. Discussion

4.1. Changesinbehaviorassociatedwiththebeginningof behavioralestrus

Thefemalebehaviorpatterndefiningthebeginningofestrusis thedisplayoflordosis.Themainstimulusleadingtothepresenta- tionofthisbehavioristactilestimulationprovidedbythemounting male(KowandPfaff,1976).Datareportedhereshowthatthemales onlyexceptionallymountedthefemalesbeforethemountwasable toactivatealordosisresponse.Thissuggeststhatthefemalesonly hadthecapacitytostimulatethemalestomountwhentheywould respondwithlordosis.This,inturn,mustmeanthatthefemales emittedsomestimulusorstimulispecificforthestateofsexual receptivity.It iswellknownthatsexuallyreceptive femalerats produceolfactorystimuli attractivetomales.Theproductionof these stimuli ishormone-dependent, since odors fromovariec- tomized females are farless attractive than odors from intact, estrousfemalesorfromovariectomizedfemalestreatedwithestra- diol(e.g.,Carretal.,1965;Lucasetal.,1982).Whetherthereisa suddenchangeinfemaleodorassociatedwiththeinitiationofmale mountingornotispresentlyunknown.However,itappearsaquite unlikelyevent.Althoughthemechanismsunderlyingthehormone- inducedchangesinodorremainobscure,therearemanyreasonsto

believethatthey,likemostothersteroideffects,aregradualrather thansudden.Furthermore,maleratshaveneverbeenobservedto displaymountinginresponsetofemaleodors.Theydo respond witherection(Sachs,1997),releaseofgonadotropins(Grahamand Desjardins,1980Kameletal.,1977)aswellasofsometransmitters (e.g.,(Masetal.,1995),andapproachtotheodorsource(Ågmo, 2003).Nonetheless,thetactilestimulationoftheperinealregion requiredforactivatingamount(ContrerasandÅgmo,1993)cannot beprovidedbyanyodor.

In addition to odor, sexually receptive females emit high frequencyvocalizations.Thehormone-dependenceofthesevocal- izationshasbeenfirmlyestablished(Floody,1981Matochiketal., 1992),butavocalizingfemaleisnomoreattractivetoamalethan asilentfemale(SnoerenandÅgmo,2013).Moreover,whenmales havesimultaneousaccesstoseveralfemalestheirchoiceofcop- ulationpartneris independentoffemalevocalizations(Snoeren etal.,2014).Consequently, thereisnot muchreasontobelieve thatfemalevocalizationsprovokedthemalestoinitiatemount- ing.Insteadofsearchingforadditionalspecificstimuliemittedby thesexuallyreceptivefemalewetriedtofindthesuddenpresence ofabehaviorpatternthatmightincitethemaletomountand/or thesuddenabsenceofbehaviorsthatmightimpedethemaleto mount.

Thedurationor frequencyoftheprosocialbehavior ofsniff- ingandtheantisocialbehaviorsofrejectionandnose-offdidnot changebetweenintervals,i.e.,theywereatthesamelevelbefore thebeginningofestrus,duringthefirstandlastintervalsofthe estrusperiod,andaftertheendofestrus.Thus,thesebehaviorsdo notseemtobeassociatedwiththeappearanceanddisappearance ofsexualreceptivity.Incontrast,thedurationofmaleanogenital sniffingofthefemalegotmuchreducedfromthelastintervalof estrustothepostestrusinterval.Thisreductionmightberelatedto thedisappearanceofpursuitofthefemalesandmounting.Against suchaproposalitcouldbearguedthattherewasnoincreasein anogenitalsniffingassociatedwiththebeginningofestrus.More- over,wehaveearlierreportedthatthereisnorelationshipbetween theintensityoffemaleormalesexualbehaviorandsocialoranti- socialbehaviors(ChuandÅgmo,2014a).Itappearsdoubtfulthat thereductioninanogenitalsniffingisofanymajorimportancefor thecessationofsexualactivity.

Oneoftheremarkableresultsfoundintheseminaturalenviron- mentwasthatthedurationofparacopulatorybehaviorssuddenly increasedfromclosetozero shortlybeforethefirst lordosisto aratherhighlevelimmediatelybeforeit.Thereafter,thefemales maintainedahighlevelofparacopulatorybehaviorthroughoutthe entirebehavioral estrus(Chu and Ågmo,2014a).Ever sincethe publicationofBeach’s(1976)seminalpaperishasbeengenerally acceptedthatthesebehaviorsarepowerfulstimuliforactivating malemounting(Erskine,1989).Indeed,inmaleswithoutsexual experience,in castrated males maintainedonlow testosterone replacement,and in maleswithotherwise impairedcopulatory behavior,femaleparacopulatorybehaviorsareessentialfortheini- tiationofmounting(HlinakandMadlafousek,1977;Hlinaketal., 1987;MadlafousekandHlinak,1983;Madlafouseketal.,1976).

With regard to the male, it is known that pursuit of the female almost always precedes mounting (Ågmo, 1999). In the present study, male pursuit of the female showed a pat- tern most similar to that of paracopulatory behaviors, that is a sharp increase immediately before the first lordosis and a sharp and lasting decline after the last. The temporal coinci- dence between the increase in paracopulatory behaviors and pursuit and the beginning of behavioral estrus together with the coincidence in decline at the end of estrus may suggest a causal relationship. The exact nature of that relationship is, however, not completely clear. Considering that the males could already be engaged in copulatory activities with other

females, it is likely that the female entering estrus simply communicatedthatshealsowasreadytocopulate.Obviously,this wouldalsoholdforsituationsinwhichnootherfemalewasavail- able.Thewaythefemaleapparentlycommunicatedherreadiness waseitherbydisplayingparacopulatorybehavior,therebyinciting themaletopursueandeventuallymounther,orbyrespondingto themale’spursuitwithparacopulatorybehavior,whichwasended bythemale when heperformeda mount. Itmight beremem- beredthatthefirstlordosiswasalwaysprecededbyparacopulatory behavioronthepartofthefemaleandbypursuitonthepartof themale.

Anintriguingquestionhereiswhytheoccasionaldisplaysof paracopulatorybehaviorandpursuitduringseveralminutespre- cedingthefirstlordosisdidnotleadtomalemountingandfemale lordosis.Thedurationofeach episodeofparacopulatorybehav- iorsandpursuitdidnotincreaseshortlybeforethefirstlordosis, whereasthefrequencyoftheseepisodesdid.Inviewofthis,wepro- posethatisolateddisplaysofpursuitorofparacopulatorybehavior donotleadtosexualinteraction.Severalofthesedisplaysinrapid successionareneededforstimulatingthesubjectstoengagein copulatorybehavior. It is mostlikely that each displayof pur- suit orof paracopulatory behaviorcontributes toenhance both generalarousalandsexualmotivationinwaysoutlinedinsome detail elsewhere(Ågmo, 1999; Ågmo,2011).Thisenhancement declinesspontaneouslywithtime.Consequently,inorderforthese behaviorstoincreasearousal/motivationtothepointrequiredfor activatingcopulatorybehaviortheymustoccurwitharelatively highfrequency.Thisisexactlywhat happensinthe30speriod precedingthefirstlordosis.

Thecentral nervouseventsleading tothedramatic increase in paracopulatorybehavior and male pursuit in a time spanof 30sareentirelyunknown.Since anextensivediscussionof the manypossibleexplanationsisfarbeyondthescopeofthepresent paperwewillonlymentionsomehypotheticalmechanisms.The moststraightforwardwouldprobablybeenhancedreleaseofsome transmitter.Itisknownthatfemaleratsreleasedopamineinthe nucleusaccumbensand striatumwhenexposedtosexuallyrel- evantstimuli(JenkinsandBecker,2003;Pfausetal.,1995),and althoughtheprecopulatoryreleaseissmallerthanthatproduced bycopulationitmightcontributetotheinitiationofbehavior.How- ever,inthepreopticareadopamineisnotreleasedinresponseto aninaccessiblemale,althoughdopamineconcentrationsaremuch increasedduringcopulation(Matuszewichetal.,2000).Likewise, oxytocinisreleasedintheparaventricularnucleusduringcopula- tion(Nyuykietal.,2011),butwhetherthispeptidecouldparticipate intheinitiationofcopulationisentirelyunknown.Noradrenaline mayalsobeinvolved,becauseestrogensand progesteronehave beenshowntomodifynoradrenergicfunctionintheventrome- dial nucleus and preopticarea(Etgen et al.,2001), and release ofthistransmitterincreasesduringcopulation(VathyandEtgen, 1989).However,vaginocervicalstimulationisnecessaryforrelease (EtgenandMorales,2002),makingitunlikelythatnoradrenaline participatesintheinitiationoffemalecopulatorybehavior.There isalsoa numberofpeptidetransmittersinadditiontooxytocin thatareinvolvedinthecontrolofcopulatorybehaviorinfemale rats(reviewedinArgiolasandMelis,2013),buttherearenodata as to whetherany of them might participate in thetransition fromnon-receptivitytoreceptivity.Adifferentpossibilityisthat atransitoryincreaseinestrogenavailabilitystimulatesfast-acting membranereceptors.Theactivationofthesereceptorsmayhave behavioraleffectswithinseconds(reviewedinManietal.,2012).

Sinceestrogenreleasefromtheovariesshowconsiderablefluctua- tions(Nequinetal.,1979;SöderstenandEneroth,1981),asudden increase inextracellular estrogenconcentrationis feasible.It is alsopossiblethatestrogensand/orprogesteroneareproducedand releasedlocallyfromneurons(Corniletal.,2006).Furtherstudies

X.Chu,A.Ågmo/BehaviouralProcesses113(2015)24–34 33

areneededbeforeanyofthesespeculationscanbediscardedor confirmed.

4.2. Changesinbehaviorassociatedwiththeendofbehavioral estrus

Manyhypotheses concerningthecausefortheendof estrus havebeenlaunched.Amongthoseisthesuggestionthatintromis- sivestimulationcontributestotheendofsexualreceptivity(e.g., Erskine,1985).Sincethedurationofestrusisreducedbyexpos- ingalsoovariectomizedratstoacopulatingmale(e.g.,Lodderand Zeilmaker,1976)ortoexperimenter-providedvaginocervicalstim- ulation(e.g.,Bennettetal.,2002)itwasconcludedthatdiminished availabilityofovarianhormonescannotberelatedtothetermina- tionofbehavioralestrus.Thereislittlereasontobelievethatgenital stimulationwouldacceleratethecatabolismofovarianhormones.

Insteadithasbeensuggestedthatsexualstimulationleadstomod- ifiedreleaseoftransmitters,forexamplevasopressin(Södersten etal.,1983)orglutamate(Georgescuetal.,2012),andthismodi- fiedreleasewouldbringbehavioralestrustoanend.Itmayalsobe notedthatintensegenitalstimulationsometimesfailtoreducethe durationofestrus,andifitdoes,thereductionmaybeexplainedas apurelyperipheraleffect(vanderSchootetal.,1993).Themecha- nismsbehindtheendofbehavioralestrusinfemalesnotreceiving anygenitalstimulationhavereceivedlittleattention,butsinceall femalesinthepresentstudydidreceivesuchstimulationitisofno concernhere.

Bothintensestimulationfromamaleandartificialvaginocervi- calstimulationnotonlycausesareducedestrusdurationbutalso aconsiderableenhancementoffemalerejectionsofthecopulat- ingmale(Blausteinetal.,2009;Coopersmithetal.,1996;Pfaus etal.,2000).Intheseminaturalenvironmenttherewasnoincrease inrejectionsasestrusterminationapproached.Indeed,rejections remainedatalowlevelthroughouttheperiodofestrus(Chuand Ågmo,2014a).Thisfactisprobablya consequenceof thelower rateofsexualinteractionsintheseminaturalenvironment,even in situations where onlyone receptive femalewasavailable to thethree males.It is alsoimportant toobservethat there was noreductioninparacopulatorybehaviorsintheperiodpreceding estrustermination.Indeed,ineveryfemalethelastlordosiswas precededbyparacopulatorybehavior.Furthermore,themalescon- tinuedtopursuethefemaleswithundiminishedintensity,showing thattheyremainedattractive,andmalepursuitalwaysanteceded thelastlordosis.Immediatelyafterthelastlordosisthere wasa drasticreductionofparacopulatorybehaviorsandofmalepursuit.

Asimilar,short-livedreductionwasseenaftereverylordosis.After thelast,however,thisreductionbecamepermanent.Wesuggest thatthepostestruslevelofparacopulatorybehaviorsandofmale pursuit wastoolowtostimulate theanimalstoengagein fur- thercopulatoryacts.Thus,behavioralestruscametoanendwhen thefemale suddenly ceased todisplayparacopulatory behavior and/orthemaleceasedtopursueher.Malefatiguecanbeexcluded, becausewheneverotherfemalesremainedinestrus,themalescon- tinuedtocopulatewiththem.Likewise,changesinthenumber ofrejections canbeofnoimportancesincetherewerenosuch changes.

Theneurochemicalchanges associatedtheend ofbehavioral estrus are obscure. Even though increased release of gluta- matemightexplainreducedestrusdurationasobservedinpair tests, it cannot account for the end of estrus in the seminat- ural environment. A prominent effect of glutamatergic agents is to stimulate rejections and reduce paracopulatory behav- iors(GeorgescuandPfaus,2006a;GeorgescuandPfaus,2006b), somethingnotobservedinthepresentstudy.Likewise,another transmitterproposedtobeinvolvedinestrustermination,vaso- pressin, also reduces paracopulatory behavior and stimulates

rejection(PedersenandBoccia,2006).Whetheranyoftheother transmittersthatoccasionallyhavebeenreportedtoreducefemale ratsexualbehaviorisinvolvedinestrusterminationornotiscom- pletelyunknown.

5. Conclusion

Thetransitionfromastateofnon-receptivitytoreceptivityas wellastheinversehasbeenthoughttobegradual.Thisnotion wasbasedondatafromdiscretepair-tests(seeIntroduction).In thepresent experiment,inwhich groupsof ratswereobserved continuouslyduringtheestruscycle,anentirelydifferentpicture emerged. Thefemalesdidnot showagradual changeinsexual behavior,but suddenlyenteredand lefta stateofreceptivityat thebeginningandendofbehavioralestrus,respectively.Wehave earlierpointedout thatprolongedobservationofgroupsofrats inanenvironmentsharingsomecharacteristicswiththenatural environmentisessentialfortheexternalvalidityofanydescrip- tionofbehavior(ChuandÅgmo, 2014a).Therefore,wepropose thatfemaleratsnormallyshowabruptchangesinsexualbehavior duringtheestruscycle,andthatthegradualchangedescribedear- lierisanartifactoftheobservationprocedure.Wealsosuggestthat theincreaseinfemalerejectionsandreductionsinparacopulatory behaviorpriortotheendofestrusinpair-testsisanotherartifact, causedbyafrequencyofsexualinteractionsfarabovethatwehave observedintheseminaturalenvironment.Nevertheless,itispos- siblethatadifferentgroupcompositioncouldleadtoresultsmore inlinewithdatafrompairtests.

The initiation and termination of copulatory behavior were strictlyassociatedwithrapidmodificationsoftheamountoffemale paracopulatory behaviorand male pursuitof thefemale.When thesemodificationsoccuratthebeginningofbehavioral estrus, thefemaleisalreadyfullyreceptive,meaningthatshedisplayslor- dosisinresponsetoeverymount.Sinceshedidsountilthevery lastlordosis,weproposethatshewasfullyreceptivealsowhen behavioral estrusended.Thisassertionisstronglysupportedby thefactthattheamountofestrogensneededforactivatinglordo- sisislowerthanthatneededfortheparacopulatorybehaviors.The endofbehavioralestrusis,therefore,notrelatedtoreducedrecep- tivitybuttoreducedparacopulatorybehaviorand/orreducedmale pursuit.Sincethatbehaviormayeitherincitethemaletopursue andmountthefemale,orbearesponsetomalepursuitalsoleading toamount,itsabsencewilleffectivelyterminatemounting,and,in consequence,lordosis.

Wehavehereofferedabehavioralexplanationforthebeginning andendofbehavioralestrus.Theneurochemicaleventssubjacent tothesebehavioralchangesareunknown.Futureresearchneeds toemployproceduresforstudyingtransmitterreleasewithatime resolutionatthesubminutelevel.

Acknowledgements

Financial support was received from the Faculty of Health Sciences, University ofTromsø. KnutOlsen and TrulsTraasdahl assembledtheseminaturalenvironment.CarinaSørensen,Katrine HarjoandNinaLøvhaugprovidedexcellentcareoftherats.

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