The Auk 116(1):184-193, 1999

FOAM PRODUCED BY MALE COTURNIX QUAIL: WHAT IS ITS FUNCTION?

ELIZABETH ADKINS-REGAN • Departmentof Psychologyand Section of Neurobiologyand Behavior, Cornell University, Ithaca, New York 14853, USA

ABSTRACT.--Malesof the Old Worldquail genusCoturnix are unique among birds in pos- sessinga well-developedproctodeal gland. The gland and associatedcloacal musculature are sexuallydimorphic, androgen dependent, and producea foamy substancethat is intro- ducedinto the femalealong with semenduring copulation, suggesting that the foamplays somerole in increasingmale reproductive success. I experimentally tested three hypotheses aboutthe functionof this foam in JapaneseQuail (Coturnixjaponica): (1) foam functionsin spermcompetition, (2) foamreduces the female'sreceptivity to a secondmale, and (3) foam increasesthe probabilityof fertilizationwhen a hard-shelledegg is presentin the uterus (hypothesis3 was originallyproposed by Chenget al. 1989a).Insemination shortly before ovipositionfertilized fewer eggs than inseminations earlier in theday, but only if maleshad a reducedfoam complement,supporting the third hypothesis.The othertwo hypotheses were not supported.Copulation reduced female receptivity, but this effectwas not due to the male'sfoam. Received 2 February 1998, accepted 22 June1998.

IN MOSTAVIAN SPECIES,males either have no "whipped" into foam by the action of the phallus or have a rudimentarynon-intromit- sphinctercloacae (Ikeda and Tajii 1954,Fujii tent phallus (Briskieand Montgomerie1997). and Tamura 1967, Seiwert and Adkins-Regan Thus,mating is accomplishedwithout the elab- 1998).The foam gland complexis markedly orate genitalia characteristicof many mam- sexuallydimorphic (large in malesbut rudi- mals, reptiles,and insects.Recent interest has mentaryin females),is well developedin males developedregarding the possibilitythat birds onlyduring the breeding season, and is highly have evolvedspecial anatomical and physio- androgendependent (Nagra et al. 1959,Beach logicalmechanisms to manipulatefertilization, and Inman 1965, Sachs1967, Adkins and Adler specificallysperm competition or femalesperm 1972, Siopesand Wilson 1975). manipulation(Birkhead and Moller 1992). During copulation,the male introducesnot Malesof the Old World quail genusCoturnix only semenbut also a large quantity of foam are unique among birds in possessinga well- into thefemale. The rhythmic movements of the developedproctodeal gland (McFarlandet al. sphinctercloacae that areknown to "whip up" 1968, Klemm et al. 1973, Slopes and Wilson the foam increasegreatly in frequencyas soon 1975, Bakst and Cecil 1985). In domesticated as the male detectsthe female, in advanceof ac- JapaneseQuail (Coturnixjaponica), a large,red tual mountingand cloacalcontact (Seiwert and protuberanceis presentimmediately dorsal to Adkins-Regan1998). These observations sug- the cloacalopening (Fig. 1). Thisprotuberance, gest that the foam functionsto increasethe the foam gland complex,is distinctlydifferent from the cloacalprotuberances of somepasser- male'sreproductive success by promotingfer- ines (it does not contain the coiled vas defer- tilization(Ogawa et al. 1974,Fujihara 1992). ens) and is not a sperm-storageorgan. It con- Female JapaneseQuail are unusual in that sistsof an enlargementof the dorsalportion of they lay eggsin the late afternoon(Wilson and the sphinctercloacae muscle plus the procto- Huang 1962)rather than in the morningas in deal glandproper, the unitsof whichare inter- manyother bird species(Skutch 1952). Females digitatedwith sphinctercloacae muscle fibers also are more sexuallyreceptive behaviorally (Klemm et al. 1973).The proctodealgland se- in the afternoon(Delville et al. 1986).Cheng et cretes a viscous mucoprotein that is then al. (1989a, b) proposedthat foam enabled a male to fertilize a female even when a hard- shelledegg was in the uterusduring mating, E-mail: [email protected] whichwould often be the casefor matingsoc- 184 January1999] QuailFoam and Fertilization 185

FIc. 1. Externalview of the foamgland complex (feathers removed) of a reproductivelyactive male Jap- aneseQuail. Arrow indicatesthe cloacalopening. Photograph by Marie Read. curringin the afternoonduring the female's Cauterizationis a potentially confounding peak of receptivity.In supportof this hypoth- manipulation,however, because the interdigi- esis,Cheng et al. (1989a,b) reportedthat foam tation betweenthe proctodealgland and the prolongedsperm rnotility in vitroand thatmale sphinctercloacae muscle makes it impossible JapaneseQuail whosefoam glandshad been to destroythe gland without damagingthis destroyedby cauterizationfertilized few or no muscle.Because the sphinctercloacae is essen- eggs. tial for copulation(i.e. for cloacalcontact and 186 ELIZABETHADKINS-REGAN [Auk,Vol. 116 ejaculation;King 1981,Seiwert and Adkins-Re- few seconds.If the male is lesscompetent or the fe- gan 1998),it is likely that cauterizedmales are male is unreceptive,repeated chasing, mounting, defectivein componentsof copulationcritical and attemptedcloacal contact occur, which some- for fertilizationsuccess other than foam pro- timesbut not alwaysresults in successfulejaculation duction. Also, neither in vitro observationsnor and insemination.Foam is depositedin the female's proctodeum(Cheng et al. 1989a),and spermare not cauterizationexperiments provide a direct test foundin the femaleunless foam is alsopresent (Ad- of the hypothesisthat foam increasesfertiliza- kins 1974).Thus, inseminationcan be confirmedby tion successmainly when a hard-shelledegg is examinationof the female(the foamis clearlyvisible present. in the proctodeum)or, if tactilecontact with the fe- The experimentsreported here use a lessin- male'scloaca might introduce an experimentalcon- vasivemanipulation to test the abovehypoth- found, by closeobservation of the male'sbehavior esisand alsotest two additionalhypotheses for (cloacalcontact followed by suddencessation of the the function of foam. One is that foam enables copulatoryattempt followed by featherfluffing and/ a male to reducethe abilityof a secondmale to or strutting).For data validating the use of themale's fertilize the female; that is, that it functions in behaviorto judge successfulinsemination, see Ad- kins-Regan(1995). sperm competition.Foam could function as a Dependingon the experiment,females were al- mechanical device analogousto the sperm lowedto copulatewith a male eitherin their home plugsof someother vertebrates, or it couldact cages(21 x 18 x 15 cm hardwarecloth), a smalltest- chemically,for example,by enabling a male's ing cage (38 x 24 x 18 cm), or a large testing cage spermto remainmotile longerthan a compet- (183 x 91 x 77 cm). Cage size has no effecton the ing male'ssperm. The secondhypothesis is that likelihoodthat an inseminationwill fertilize eggsor the foam reduces the female's receptivity on the number of eggs fertilized (Adkins-Regan (again decreasingthe likelihoodthat a second 1995).If the male did not begincopulating immedi- male would be able to fertilize the eggs),but ately,he was replacedwith anothermale. The male was removed after insemination,and except when througha differentmechanism, the female'sbe- measuringfemale receptivity,only resultsfrom mat- havior.Both of thesehypotheses assume that ing trials in which the male achievedinsemination femalesmight mate with multiple males. Al- were analyzed.Unless stated otherwise, mating tri- thoughlittle is knownabout the matingsystem als were conducted between 1130 and 1500 EST, a of wild JapaneseQuail, extrapair copulations time when femalesare likely to be receptive. havebeen observed in feral JapaneseQuail in a Most eggswere laid between 1600 and 2000. Eggs seminaturalenvironment (Nichols 1991), and were collecteddaily in the morning (beginningthe field observationsof the closelyrelated Com- morningafter mating)for 11 days,which is the max- mon Quail (Coturnixcoturnix) suggest that sit- imum sperm-storageinterval in this speciesand in uations in which females switch mates, or in birds in this laboratory (Sittman and Abplanalp 1965, Adkins-Regan 1995). Eggs were stored at 13øC whichmore than one male attempts to copulate for a maximumof two weeksprior to incubationat with a female,occur rather frequently (Rodri- 38øCand 60% relative humidity. Eggs were broken go-Ruedaet al. 1997). open after one week of incubationand recordedas fertile if any embryonicdevelopment had begun.It METHODS is possiblefor eggsto be fertilized,in the sensethat spermare attachedto the outer perivitellinelayer, Birds,mating trials, and fertilization measures.--The without any embryonicdevelopment being initiated quailused in thisstudy were hatched from eggspur- (Birkheadet al. 1994).Such outcomes do not, how- chasedfrom Truslow Farms, Chestertown,Mary- ever, contribute to a male's reproductive success. land, and were housedindividually after the age of Therefore,because my experimentstest hypotheses four weeks(sexual maturity beginsat ca. five to six abouthow foam influencesmale reproductivesuc- weeks).All birds were at leasteight weeksold and cess,all eggswith no embryonicdevelopment were not more than eight monthsold during the experi- categorizedas infertile, and "successfullyfertilized" mentsand werehoused on a 16:8light: dark cycle.A shouldbe takento mean fertilized suchthat embry- new set of birdswas used for eachexperiment, and onic developmenttakes place. Early embryonic maleswere sexuallyexperienced prior to use. deathsoccurred at a low rate (in eachexperiment In this species,ejaculation and insemination occur constituting6% or lessof all eggsrecorded as fertile) with the first cloacalcontact if themale is competent and wereunrelated to experimentalcondition. They and the femaleis receptive.Immediately after ejac- were recordedas fertile eggs,but resultsare not dif- ulation, the male dismounts and often fluffs his ferentif theseeggs are excludedfrom analysis.Data feathersand struts.The entire sequencelasts only a frompoor layers (i.e. females that laid fewerthan five January1999] QuailFoam and Fertilization 187 unbrokenhard-shelled eggs in the 11 daysfollowing seminationwas judgedby observationof the target the matingtrial) were excludedfrom analysis. male's behavior. Egg fertility data were analyzedby nonparametric Hypothesis2: Foam reducesfemale receptivity.--In statistics(X 2 or Mann-Whitney U-tests)because the two experiments,similar procedures for introducing distribution of outcomesis bimodal (Adkins-Regan foam into a female as described above (foam-donor 1995); about one-third of inseminationsdo not fer- males and syringed foam) were used to determine tilize any eggs. In analyzing numbers and percent- theeffect of foamon a female'sreceptivity with a tar- agesof fertile eggs,trials usingthe samemale or fe- get male. In both experiments,21 femaleswere male were consideredstatistically independent. In paired with the sametarget male (thus,21 target prior work from our laboratory,trials were conduct- males) on four consecutivedays. The same target ed usingseveral different cohorts of quailover a pe- male was used for a given female to reduce day-to- riod of severalyears (Adkins-Regan1995), and fer- day variationin a female'sbehavior toward a target tilization outcomesusing the same male or female male.These tests took place in the largetesting cage were uncorrelated.Only if both the male and the fe- to allow the femaleto run or fly from the male. On malewere the same(which was not the casein any two of the four daysthe femalewas testedwithout experimentreported here) were the outcomescor- any prior manipulation,on oneday the femalere- related. All P-values are two-tailed. ceivedfoam (via a male donor or via syringe)prior Hypothesis1: Foamis a spermcompetition device.- to the test with the target male, and on one day the Thishypothesis was tested by introducingfoam into femalereceived a controlmanipulation prior to the a female'sproctodeum and determiningits effecton testwith the targetmale. The orderof the testswas a male'sability to fertilizeher eggs. Males in thiscase counterbalanced. will be referred to as "target" males. Two proce- In the firstexperiment, females received foam pri- dures were used to introduce foam into the females. or to testingby copulatingin theirhome cages with One was to pair the femalewith a malehoused on a foam donorswho had the normal complementof shortphotoperiod (8:16 light: darkcycle) and receiv- foam in the foam glandcomplex; the testwith the ing androgenreplacement (daily injectionwith 0.5 target male began one minute after the donor fin- mg testosteronepropionate plus 0.5 mg dihydrotes- ishedcopulating. Because behavior (receptivity) was tosteronepropionate dissolved in sesameoil). Such the dependentvariable rather than egg fertilization maleshave a developedfoam gland complex,pro- success, foam-donor males did not need to be infer- duce a normal amount of foam, and copulate,but tile and so were untreated males housed on long theyhave regressed testes and produceno sperm.I days.For the controlmanipulation, the femalesalso refer to thesemales as "foam donors."Forty females copulatedin theirhome cages and were tested with were allowedto copulatewith oneof 20 targetmales the target male one minutelater, but the home-cage and 9 foam donorsin the small testingcage so as to copulationswere with donormales whose foam had yield four kinds of matingtrials: (1) foam donorim- beenmanually expressed immediately prior to mat- mediately followed by target male, (2) target male immediatelyfollowed by foam donor,(3) target male ing. Manualexpression greatly reduces the amount only,and (4) foam donor only. Trials of type 4 were of foamthat the donor places in thefemale; if females included to confirm that the donors introduced foam copulateimmediately following manual expression of foam, no foam can then be seen in the female's into femalesbut could not fertilize any eggs. Suc- cessfulinsemination was judged by observationof proctodeum.This manipulation thus controls for ef- the male'sbehavior and, for trials of type 4, by suc- fectsof copulationon subsequent receptivity that are due to factors other than foam, such as tactile stim- cessfulfoam donationas confirmedby examination ulation from cloacal contact itself. In the second ex- of the female,sproctodeum. The secondprocedure for introducingfoam into periment,foam from two donor males was intro- femaleswas to expressfoam from oneof 20 normal ducedinto the proctodeumvia syringe,and the test fertile males,place it in a 5-ccplastic, blunt-tipped with the targetmale began one minute later. For the syringe,and injectthe foam into the female'sproc- controlmanipulation, the empty syringewas intro- todeum.Twenty-nine females and 10 maleswere al- duced.For both experiments in the testswith thetar- lowedto copulatein thefemale,s home cage to yield get male,receptivity was scored on a four-pointsys- two kinds of matingtrials: (1) experimentaltrials in tem:0 = highly unreceptive,constantly moves away whichfoam was introduced into the femalevia a sy- from male; 1 = unreceptivebut not markedlyso; 2 ringe immediatelybefore mating, and (2) controltri- = ambiguousreceptivity; and 3 = clearly receptive, als in whichthe tip of the empty syringewas intro- squatsand doesnot movewhile mounted.If the fe- ducedinto the female,sproctodeum for 2 s imme- male,sreceptivity status changed during the copu- diately before mating. Alternate femaleswere as- lationattempt, the mean of the initial andfinal scores signedto the experimentaland controltrials. Two was used. The number of times the female ran from weeks later the femaleswere allowed to copulate the male or took flight during the test was alsore- again,reversing the trial assignments.Successful in- corded.For analysis,the meanof the two target-only 188 ELIZABETHADKINS-REGAN [Auk, Vol. 116 testswas compared with eachof theother two kinds TABLE1. Testof the hypothesisthat foamis a sperm of tests.Results were analyzedby signtests. competition device in JapaneseQuail based on Hypothesis3: Foamincreases fertilization when a hard- foam-donormales and basedon syringedfoam. shelledegg is present.--Fifty-eightfemales copulated % Trails with 25 malessuch that eachmale copulatedtwice with Median % on consecutivedays, once with the normal comple- No. of fertile eggs ment of foam and onceimmediately following man- Type of mating trials egg fertilea ual expressionof foam. Order of male conditions Foam-donor males (foam vs. reducedfoam) was counterbalanced.Cop- ulations occurred between 0830 and 1800, with most Foam donor + target 13 77 50 (57) occurringbefore 1400. Successfulinsemination was Target+ foam donor 11 64 20 (40) judged from the maids behavior.The experiment Targetonly 24 63 33 (50) Foam donor only 8 0 0 wasthen repeated with a new setof birds(53 females and 22 males)but with all copulationsconducted be- Syringed foam tween 1800and 1900,shortly before but prior to ex- Foam + target 24 79 29 (33) pectedoviposition. Cages were checkedfor eggpro- Syringe + target 28 89 40 (40) duction between 1800 and 2000 and then again the • Valuesin parenthesesexclude trials that yielded no fertileeggs. next morning, and those few trials without subse- quent oviposition were eliminated from analysis. Eggsfound the morningfollowing mating were not- ed but were not incubated for fertilization determi- P = 0.28).This was also the casewhen target + nation becausethese eggs are never fertilized (Ad- foam donor trials were comparedwith target kins-Regan1995). only trials (% trials with a fertile egg, X2 < In order to obtain additional information about 0.001, df = 1, P > 0.9; median % eggs fertile how quicklya male canregenerate a normalcomple- including trials yielding no fertile egg, U = ment of foam following manual expressionof the 119, P > 0.5; median % eggsfertile excluding contentsof the foam gland complex,manual expres- trials yieldingno fertile egg, U = 67, P = 0.30). sion was performedon five additional males (seven Wheneggs laid the day aftermating, which are monthsold) twice daily for six days suchthat the the firsteggs that could possibly be fertile,were first and secondexpressions (time 1 and time 2) were analyzedseparately (excluding trials with no 2.5, 5, 10, 20, or 40 min apart. The order of time in- tervals for the different males was counterbalanced fertile egg), againno significantdifferences oc- acrossdays, and time 1 was at the sametime of day curred between donor + target and target for all males. Expressedfoam was weighed to the alone trials (85 vs. 87% fertile, X2 = 0.02, df = nearestmg and the differencewas determinedbe- 1, P > 0.8) or target + donor and target alone tweenthe weightsproduced at times1 and2 on each trials (100 vs. 87% fertile, X2 = 0.88, df = 1, P > day. Data were analyzed by an analysisof variance 0.3).Using the alternativeprocedure of placing for repeated samplesfollowed by post-hoct-tests foam in femalesvia syringe (Table 1), again with a Bonferroni correction. therewas no effectof the foamon theability of target males to fertilize females,i.e. no signif- RESULTS icant differencebetween foam + target and sy- ringe + target (control)trials (% trials with a Experimentstesting whether foam is a sperm fertile egg, X2 = 0.39, df = 1, P > 0.5; median competitiondevice.--The results of the first ex- % eggsfertile including trials yielding no fer- periment,in whichfoam was introducedinto tile egg, U = 233, P = 0.057;median % eggs femalesby donormales housed on shortdays, fertile excludingtrials yielding no fertile egg, are shownin Table 1. Copulationswith foam U = 168, P = 0.1). Even if foam + target and donorsonly, as expected,fertilized no eggs. syringe+ targetoutcomes are comparedwith There were no effects of the foam donor on the Wilcoxonmatched-pairs tests, the differences egg fertilizationsuccess of targetmales. When are not significant(all Ps > 0.1). foamdonor + targettrials were compared with Experimentstesting whether foam reduces female target-onlytrials, no significantdifferences oc- receptivity.--Copulatingwith a foam versusa curred in the percentwith a fertile egg (X2 = no-foammale prior to testingwith a target 0.8, df = 1, P > 0.3) or in the median percent malehad no significanteffect on receptivityor of eggsfertile (includingtrials yielding no fer- on running and flying behavior by females tile egg, Mann-Whitney U = 114, P = 0.17;ex- (signtests; foam male vs. no male,P = 0.34;run cludingtrials yielding no fertile egg, U = 55.5, + fly following foam male vs. no male, P = January1999] QuailFoam and Fertilization 189

0.77; no-foam male vs. no male, P = 0.11; run A [] Receptivity + fly followingno-foam male vs. no male,P = :[• Run+ Fly 0.39).Nor did the female'sbehavior with target males differ as a function of whether the first 2.$- (donor) male had a normal complementof foam (foam vs. no-foam male, P = 0.18; run + 2- fly followingfoam vs. no-foammale, P = 0.75). 1.5- When thesetwo kindsof tests(foam male and no-foammale) were combinedfor analysisby 1 takingthe meanof the two tests,it wasevident thatfemales were less receptive when there had 0.5 beena prior male (seeFig. 2A). Thus,copulat- 0 ing with onemale reduced a female'sreceptiv- No male Foam male No-foam male ity to a subsequentmale, but thisreduction was not due to the first male's foam. In the second experiment(Fig. 2B), mostdifferences between test types were not significant(sign test, Ps > 0.3). Femalestended to run and fly morewhen [] Receptivity foamhad beenintroduced via syringeprior to B testing,but the differencebetween these tests :r• Run+ Fly and testswith no syringewas not significant(P I = 0.076).When syringe+ foam and syringe I I testswere combined(by taking their means) and comparedwith no syringetests, it wasev- identthat theintroduction of a syringeinto the femaleincreased her tendencyto run and fly in the presenceof the target male (Fig. 2B), but againthis effect was not dueto thepresence of foam. Experimentstesting whether foam increasesfer- tilizationin thepresence of a hard-shelledegg.- No syringe Syringe Syringe + foam Across all trials, fertilization successof trials FIG.2. (A) Femalebehavior with a targetmale af- conductedat thetwo timesof day(1800 to 1900 ter mating with a male with normal foam (foam vs. 0830 to 1800)did not differ (% of trials pro- male), a male whose foam has been manually ex- ducingany fertile eggs,47 vs. 61%, X2 = 1.72, pressed(no-foam male), or no prior male.(B) Female df = 1, P > 0.1; % eggsfertile includingtrials behavior with a target male after introduction of yielding no fertile eggs,U = 1,227,P = 0.076; foam into the female'sproctodeum via syringe(sy- % eggsfertile excludingtrials yielding no fer- ringe + foam), introductionof empty syringe (sy- ringe), or no prior manipulation(no syringe).*, P < tile eggs,U = 560, P = 0.19).Successful fertil- 0.05 when the two conditions indicated are com- ization was possibleeven shortlybefore ovi- bined and comparedwith no male tests(A: signtest, position.For example,one femaleoviposited 5 P = 0.012)or with no syringetests (B: sign test, P = min after mating,yet 50% of her subsequent 0.036). eggswere fertilized.Across all trials,fertiliza- tion success was lower with a reduced foam complement(40% of trials with reducedfoam fore oviposition.When the two matingtimes vs. 70%of trialswith normalfoam produced at were analyzed separately,the differencebe- leastone fertile egg; X 2 = 9.44,P < 0.01;% eggs tweennormal and reducedfoam trials was sig- fertile includingtrials yieldingno fertile eggs, nificant for 1800 to 1900 but not for 0800 to 1800 U = 1,170,P = 0.032;% eggsfertile excluding (Table2, Fig. 3). Thus,reduced foam affected trials yielding no fertile eggs, U = 398, P = the outcomemainly at the time whena greater 0.40).This negativeeffect of reducedfoam on number of females would have had a hard- eggfertilization was most pronounced for trials shelledegg in the uterus.This effectwas not conductedbetween 1800 and 1900,shortly be- specificto the first potentiallyfertilizable egg 190 ELIZABETHADKINS-REGAN [Auk,Vol. 116

TABLE2. Testof the hypothesisthat foamincreases 50- fertilizationwhen a hard-shelledegg is presentin the oviductof femaleJapanese Quail. 40- Median % No. of eggs 30- Type of mating Time of day trials fertilea Normal foam 1800 to 1900 25 22 (44) 20- Reducedfoam 1800 to 1900 30 0 (50)b Normal foam 0800 to 1800 29 33 (50) Reducedfoam 0800 to 1800 28 26 (50) • Valuesin parenthesesexclude trials that yieldedno fertileeggs. bMann-whitney U - 507, P = 0.032compared with normalfoam 0 matingsat 1800to 1900.See Figure 3 for the percentageof trialswith a fertile egg. -10 2.5 5 10 20 40 followingmating. When eggslaid the after- noonof the dayafter mating (i.e. the first eggs Interval (min) thatcould have been fertilized) were analyzed separatelyfor matingsconducted between 1800 FIG.4. Foamrecovery by malesfollowing manual and 1900, fertilization success tended to be expression.Foam was expressedand weighed at lower with reducedfoam (25% fertilized with times 1 and 2. The X-axis is the interval between time reducedfoam vs. 43% with normal foam), but 1 and time 2, and the Y-axis is the mean -+ SE of the in contrastto the analysisof all eggs,this dif- mass at time 1 minus the mass at time 2. Thus, a dif- ferencewas not significant(X 2 = 1.02,df = 1, P ferenceof 0 indicatesthat the male has completely recoveredhis foam complement,and a difference > 0.3). greaterthan 0 meansthat he hasnot yet recovered The resultsof the experimentdetermining his foamcomplement. The overalleffect of time in- how quickly males generatea normal foam terval was significant(F = 2.96, P = 0.047). Scoresat complementfollowing manual expressionof intervals5 min and 40 min were significantlydiffer- ent (P < 0.05), and scoresat interval 40 min were not significantlydifferent from 0 (thatis, by 40 min,time [] 1800-1900 h 1 and time 2 massesno longer differed, P < 0.05).

[] 0800-1800 h foam are shownin Figure4. Most maleshad fully regeneratedtheir foam complement by 40 min, but very few had done so before10 min (Fig.4). Thevariability in thesedata may result not only from individual differencesin the speedof foam recovery,but alsofrom whether maleshad spontaneouslyreleased foam short- ly beforefoam was expressedfor weighingat time 1, time 2, or both.Males normally deposit a blob of foam on eachpile of excreta(Ikeda andTajii 1954),and theyvoid theirexcreta fre- Foam No foam quently.

FIG. 3. Testof the hypothesisthat foam increases DISCUSSION fertilizationwhen a hard-shelledegg is present.Fe- males copulatedwith males possessinga normal Theseresults provide further evidencethat foamcomplement (foam) or a reducedfoam comple- ment (no foam)shortly prior to oviposition(1800 to male JapaneseQuail can successfullyfertilize 1900)or earlierin the day (0830to 1800).See Table 2 future eggseven when the femalehas a hard- for other measures(*, X2 = 6.45, df = 1, P < 0.02, shelledegg in the uterusduring mating, and comparedwith normalfoam copulationsat 1800to theyconfirm Cheng et al.'s(1989a, b) hypoth- 1900). esisthat the male'sproctodeal gland foam in- January1999] QuailFoam and Fertilization 191 creases the likelihood of successful fertilization sperm-storagetubules (SSTs) at the uterovagi- at this time. The experimentalmanipulation nal junctionand alsoat theinfundibulum in the usedhere, manual expression of foamfrom the upper oviduct, the site of ovum fertilization male prior to mating,does not interferewith (Lake 1975,Friess et al. 1978,Hatch 1983,Shu- copulationor with ejaculationof sperm.It does gart 1988,Bakst 1993b, Birkhead and Fletcher not completelyeliminate all foam, but the re- 1994).If ovipositionoccurs before the sperm suits of the experimentto determine recovery have reachedthe SSTs,foam might help the of a normal foam complementindicate that spermremain motile until the egghas passed foam was substantiallyreduced at the time of and they can resumetheir journeyto the tu- mating, immediatelyafter manual expression. bules,or it might help preventthe spermfrom Chenget al. (1989a,b) implied that a foam stickingto the eggshell or in someother way gland evolvedin male Coturnixbecause of the being displacedand preventedfrom entering specialproblem created by the temporalcoin- the SSTs(Cheng et al. 1989b;Bakst 1993a,b). cidence between imminent oviposition and Foammanipulation affected the percentageof maximumsexual receptivity in the female.Al- trialsyielding fertile eggs but not the percent- thoughit is not possiblebased on currentin- ageof eggsfertilized if any werefertilized (Ta- formation to know which suite of characteris- ble 2, Fig. 3). Furthermore,the first potentially tics came first, the male's or the female's, one fertilizable eggs were not disproportionately implicationof this scenariois that in species affectedby the foam manipulation.Taken to- that do not produce foam, successfulfertiliza- gether,these findings suggest that foaminflu- tionat thistime would be unlikelyeven if males enceswhether sperm reach the SSTsin suffi- could achieve insemination. Fertilization suc- cient numbers to be used at all, rather than how cess of artificial insemination of domesticated quicklythey reachthe SSTs. fowl (Gallusgallus), turkeys (Meleagrisgallopa- The other two hypothesesof foam function vo), and Muscovy Ducks (Cairina moschata) were not supported.Foam from one male did shortlybefore oviposition is indeed low (Birk- not reduce the fertilization success of a second head et al. 1996). It is not clear, however, male. Copulatingwith one male reducedsub- whetherthis is the samephenomenon as seen sequent receptivity toward a second male in JapaneseQuail mating with a reducedfoam somewhat, but this effect was not due to the complement,for two reasons.First, in the stud- male'sfoam. Exactly how longa female'srecep- ies discussedby Birkheadet al. (1996), fertil- tivity is reducedfollowing copulation has not ization success was low when insemination oc- been determined,although it is clear that re- curred shortly after and before oviposition. ceptivity is fully recoveredby the following Second, in those studies the reduction in fertil- day. Reductionor terminationof femalerecep- izationsuccess applied mainly to the next po- tivity following copulation is a widespread tentiallyfertilizable egg, not to all eggs,in con- phenomenon(Greenberg and Noble 1944,Ad- trastto thepresent results in whichthe next po- ler and Allen 1983, Whittier and Tokarz 1992, tentially fertilizable eggs were not dispropor- Erskine 1995).Receptivity in domesticatedtur- tionately affected. keyshas been shown to be causedby eversion By what mechanismdoes foam aid fertiliza- of the oviduct in responseto tactile stimuli tion in male JapaneseQuail? The foam has in- (Scheinand Hale 1965).Its functionhas usually terestingphysical and chemicalproperties, ei- beenassumed to be the sameas mate guarding ther of which could be involvedin buffering by males,but this doesnot addressthe ques- sperm during oviposition.The foam is a vis- tion of why femalesshould reduce their recep- cousfluid consistingof mucopolysaccharidestivity followingcopulation, especially females and/or mucoproteins(Fujii and Tamura1967, that mate multiple times. An alternativeexpla- Klemmet al. 1973)into whichbubbles of air are nationis that reducedreceptivity is a mecha- whippedby the actionof the sphinctercloacae nism wherebyfemales control sperm usage by, muscle (Seiwert and Adkins-Regan1998). Its for example,letting the first male's sperm reach foamy consistencyis quite stable,at least out- the SSTs before interference from a second side of the animal (pets.obs.). Foam prolongs male canoccur, or by preventingone male from spermmotility in vitro(Cheng et al. 1989b).Jap- copulatingtwice in closesuccession and thus anese Quail like many other birds, have filling all of the SSTs.Observations of wild 192 ELIZABETHADKINS-REGAN [Auk,Vol. 116

Common Quail and feral captive Japanese ACKNOWLEDGMENTS Quail suggestthat mate switchingand extra- I thankCynthia Seiwert for valuablediscussion of pair copulationsdo occur (Nichols1991, Rod- quail foamand for assistancewith oneof the exper- rigo-Ruedaet al. 1997).Although the foam it- imentsreported here. The research was supported by self doesnot seemto function as a spermcom- NSF grant BNS 88-09441. petition device,other opportunitiesfor sperm competitionare likely to occurin this species. LITERATURE CITED Such opportunitiesconceivably could occur evenduring the immediatepostcopulatory pe- ADKINS,E. K. 1974.Electrical recording of copulation riod, becausemale JapaneseQuail engage in in quail. Physiologyand Behavior13:475-477. forced copulationswith unreceptivefemales. ADK1NS,E. K., AND N. t. ADLER. 1972. Hormonal con- These forced copulations,although less suc- trol of behaviorin the JapaneseQuail. Journalof Comparativeand PhysiologicalPsychology 81: cessfulat achievinginseminations than other 27-36. copulations,have the samefertilization success ADKINS-REGAN, E. 1995. Predictors of fertilization in asother copulations if inseminationis achieved the JapaneseQuail (Coturnixjaponica). Animal (Adkins-Regan1995). In other words, the fe- Behaviour 50:1405-1415. male'sefforts to controlsperm usage might not ADLER,N. T., ANDt. O. ALLEN.1983. The origin of always be successful.The testis size of male sexualbehavior: A functionalanalysis. Pages JapaneseQuail alsosuggests that considerable 475-510 in Handbook of behavioral neurobiol- spermcompetition occurs in this species. ogy,vol. 6 (E. Satinoffand P. Teitelbaum,Eds.). The hypothesestested here do not exhaust Plenum Press, New York. the possiblefunctions of foamin maleJapanese BAKST,M. R. 1993a.The anatomyof reproductionin birds,with emphasison poultry.Pages 15-28 in Quail. Eachpile of excretaof a breedingmale Manipulationof the avian genome(R. J. Etches has a blob of white foam on top; thesepiles are and A.M. V. Gibbins,Eds.). CRC Press,Boca Ra- deposited throughout the day as the male ton, Florida. moves about. A domesticated male housed in BAKST,M. R. 1993b.Oviducal sperm storage in poul- the laboratorydeposits about 1.5 g of foamper try: A review. Reproduction,Fertility and De- day on its excreta (Adkins-Regan unpubl. velopment 5:595-599. data), which could entail somephysiological BAKST,M. g., AND H. C. CECIL.1985. A microscopic cost.Schleidt and Shalter(1972) suggested that evaluationof the male turkeyproctodeal gland. foam functions in territory maintenanceby vi- Journalof Morphology186:361-368. BEACH, F. g., AND N. G. INMAN. 1965. Effects of cas- suallymarking the male'sarea. Although more tration and androgenreplacement on matingin recent observations cast doubt on the idea that male quail. Proceedingsof the National Acade- Coturnixare territorial (Nichols1991, Rodrigo- my of SciencesUSA 54:1426-1431. Rueda et al. 1997), a signalingfunction of the BIRKHEAD,T. R., E. J. A. CUNNINGHAM, AND K. M. male's foam cannot be ruled out at this time. CHENG.1996. The inseminationwindow pro- Althoughthe foam gland in JapaneseQuail vides a distortedview of spermcompetition in is highly sexuallydimorphic, androgen depen- birds. Proceedingsof the RoyalSociety of Lon- dent, and increases fertilization success when don Series B 263:1187-1192. oviposition is imminent, little evidence exists BIRKHEAD,t. g., ANDF. FLETCHER.1994. Sperm stor- age and the releaseof sperm from the sperm to suggestthat sexual selectionhas played a storagetubules in JapaneseQuail Coturnixjapon- role in its evolution.The existingobservations ica. Ibis 136:101-105. of mating behaviorsuggest the potential for BIRKHEAD,t. R., AND A. P. MOLLER.1992. Sperm sexualselection in this species,however, which competition in birds: Evolutionary causesand may have played a role in the evolutionof two consequences.Academic Press, London. other striking male characters:crowing and B1RKHEAD,t. g., B. C. SHELDON, AND E FLETCHER. testissize. Females are attractedto playbacksof 1994. A comparativestudy of sperm-egginter- the male'scrow, which is a loud sexuallydi- actions in birds. Journal of Reproductionand Fertility 101:353-361. morphic signal given frequently during the BRISKIE,J. V., AND g. MONTGOMERIE.1997. Sexual se- breeding season(Goodson and Adkins-Regan lectionand the intromittentorgan of birds.Jour- 1997), and male Coturnixhave relativelylarge nal of Avian Biology28:73-86. testesfor their body size comparedwith other CHENG, K. M., g. g. HICKMAN, AND C. g. NICHOLS. birds (Moller 1991). 1989a.Role of theproctodeal gland foam of male January1999] QuailFoam and Fertilization 193

JapaneseQuail in natural copulations.Auk 106: plex of the JapaneseQuail. Experientia24:941- 279-285. 943. CHENG, K. M., R. E MCINTYRE, AND A. R. HICKMAN. MOLLER,A. P. 1991. Sperm competition,sperm de- 1989b. Proctodealgland foam enhancescom- pletion,paternal, and relativetestis size in birds. petitivefertilization in domesticJapanese Quail. American Naturalist 137:882-906. Auk 106:286-291. NAGRA, C. L., R. K. MEYER, AND N. BILSTAD. 1959. DELVILLE,Y., J. $ULON,AND J. BALTHAZART.1986. Di- Cloacal glands in JapaneseQuail (Coturnixco- urnal variationsof sexualreceptivity in the fe- turnix japonica):Histogenesis and responseto male JapaneseQuail ( Coturnixcoturnix japonica). sex steroids. Anatomical Record 133:415. Hormones and Behavior 20:13-33. NICHOLS,C. R. 1991.A comparisonof the reproduc- tive and behavioural differences in feral and do- MRSKINE,M. $. 1995. Prolactin releaseafter mating and genitosensorystimulation in females. En- mesticJapanese Quail. M.S. thesis,University of docrine Reviews 16:508-528. British Columbia, Vancouver. OGAWA, K., Y. NAKANISHI, H. toJo, AND g. IMANI- FRIESS,A.E., E SINOWATZ, E, K.-H. WROBEL,AND R. SCKLEK-WINNISCH.1978. The uterovaginal SHI. 1974. Effect of frothy fluid from cloacal glandon fertility in theJapanese Quail (Coturnix spermhost glands of the quail(Coturnix coturnix coturnixjaponica). Bulletin of the Facultyof Ag- japonica).Cell TissueResearch 191:101-114. ricultureof KagoshimaUniversity 24:35-40. FUJIHARA,N. 1992. Accessoryreproductive fluids RODRIGO-RUEDA,F. J., j. D. RODR•GUEZ-TEIJEIRO,M. and organs in male domestic birds. World's PUIGCERVER, AND $. GALLEGO. 1997. Mate PoultryScience Journal 48:39-56. switchingin a non-monogamousspecies? The FuJII, $., AND t. TAMURA.1967. Studies on the cloacal caseof the Common Quail (Coturnixcoturnix). gland of the quail: II. Histochemicalobserva- Ethology103:355-364. tionson secretionsin the gland.Japanese Poul- SACHS,B. D. 1967.Photoperiodic control of the clo- try Science4:194-200. acal gland of the JapaneseQuail. Science157: GOODSON,J. L., AND E. ADKINS-REGAN.1997. Play- 201-203. back of crowsof male JapaneseQuail elicitsfe- SCHEIN, M. W., AND E. B. HALE. 1965. Stimuli elicit- malephonotaxis. Condor 99:990-993. ing sexualbehavior Pages440-482 in Sex and GREENBERG,B., AND G. K. NOBLE. 1944. Social be- behavior(E A. Beach,Ed.). John Wiley and Sons, havior of the American chameleon(Anolis caro- New York. linensisVoigt). PhysiologicalZoology 17:392- SCHLEIDT,W. M., AND M.D. SHALTER.1972. Cloacal 439. foam gland in the quail Coturnixcoturnix. Ibis HATCH,$. A. 1983. Mechanismand ecologicalsig- 114:558. nificanceof spermstorage in the NorthernFul- SEIWERT,C. M., AND E. ADKINS-REGAN.1998. The mar with reference to its occurrence in other foam productionsystem of the male Japanese birds. Auk 100:593-600. Quail: Characterization of structure and func- IKEDA,K., ANDK. TAJII.1954. On thefoamy ejaculate tion. Brain, Behavior and Evolution: 52:61-80. of JapaneseQuail (Coturnixcoturnix japonica, T. SHUGART,G. W. 1988. Uterovaginal sperm-storage et S.). ScientificReports of the MatsuyamaAg- glands in sixteenspecies with commentson morphologicaldifferences. Auk 105:379-383. ricultural College3:1-4. SIOPES,t. D., AND W. O. WILSON. 1975. The cloacal KING, A. S. 1981. Cloaca.Pages 63-132 in Form and gland--An external indicator of testicularde- function in birds, vol. 2 (A. S. King and J. Mc- velopmentin Coturnix.Poultry Science54:1225- Lelland, Eds.). Academic Press,New York. 1229. KLEMM, R. D., C. E. KNIGHT, AND $. STEIN. 1973. SITTMAN, K., AND H. ABPLANALP.1965. Duration and Grossand microscopicmorphology of the glan- recoveryof fertility in JapaneseQuail Coturnix dula proctodealis(foam gland) of Coturnixc. ja- coturnixjaponica. British Poultry Science 6:245- ponica(Aves). Journal of Morphology141:171- 250. 184. SKUTCH,A. E 1952.On the hourof layingand hatch- LAKE,P.E. 1975.Gamete production and the fertile ing of birds' eggs.Ibis 94:49-61. periodwith particularreference to domesticated WHITTIER,J. g., ANDR. R. TOKARZ.1992. Physiolog- birds. Symposiaof the ZoologicalSociety of ical regulationof sexualbehavior in femalerep- London 35:225-244. tiles.Pages 24-69 in Biologyof the Reptilia,vol. LIFJELD,J. t., P.O. DUNN, D. E WESTNEAT.1994. Sex- 18E (C. Gansand D. Crews,Eds.). University of ual selectionby sperm competitionin birds: ChicagoPress, Chicago. Male-malecompetition or femalechoice? Jour- WILSON,W. O., AND R. H. HUANG.1962. A compar- nal of Avian Biology25:244-250. isonof the timeof ovipositingfor Coturnixand MCFARLAND, L. Z., R. L. WARNER, W. O. WILSON, chicken.Poultry Science 41:1843-1845. ANDF. B. MATHER.1968. The cloacalgland com- AssociateEditor: J. Ekman