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Predietors of fertilization in the Japanese quail, Coturnix japonica
,&itn.
Behuv.,
1995, 50, 1405-1415
Predictors of fertilization in the Japanese quail, Coturnix japonica ELIZABETH
ADKINS-REGAN
CorneN (Received final
University
4 October 1994; initial acceptance 26 January acceptance 25 March 1995; MS. number: A7116)
1995;
To better understand the relationship between mating behaviour, insemination and fertilization in birds, 233 mating trials were conducted with Japanese quail to determine the percentage of eggs laid that were fertile following a single confirmed or putative insemination. Fertilization successof single inseminations was highly variable. Insemination did not guarantee fertilization of any eggs; following one-third of the trials, no fertile eggs were laid. Only 3% of inseminations fertilized as many aseight eggs,making it unlikely that a single insemination could fertilize an entire clutch in this species. Forced copulations had the same fertilization successas other copulations, and thus could be a successfulmale reproductive strategy in this species.Voiding of foamy material containing sperm by the female shortly after copulation did not reduce fertilization successin any consistently significant manner. Fertilization successwas unaffected by the presence of a hard-shelled egg in the uterus at the time of insemination. Trials in which the female ran from the male’s initial approach resulted in fewer fertile eggs following insemination; that is, this behaviour predicted low fertilization success.Fertilization successwas highly correlated and thus predictable if the same female was mated again with the same male, but was uncorrelated if she was mated with a different male or if the same male was mated with different females. These two predictors of fertilization success(female behaviour and male-female combination) are interpreted as evidence for female control of paternity via sperm selection (postcopulatory female choice). 0 1995 The Association for the Study of Animal Behaviour Abstract.
Understanding the reproductive consequences of mating behaviour is critically important for the development and testing of evolutionary hypothesesabout mating systems.In specieswith internal fertilization, the likelihood that a male mating with a fertile female will father any of her offspring depends both on whether transfer of sperm to the female’s reproductive tract occurs during the act of mating (insemination success)and on whether those sperm fertilize any eggs (fertilization success).In birds, a key taxon in contemporary mating systemsresearch, increasing evidence suggests that mating behaviour is not a reliable guide to paternity (Westneat et al. 1990; Birkhead & Moller 1992; Dunn & Lifjeld 1994). Not all matings result in successful insemination. Extrapair copulations may occur whose successmay differ from pair copulations. In both polygynous and monogamous species that engage in extrapair copulations, competition between sperm Correspondence:E. Adkins-Regan, 218 Uris Hall, Cornell University, Ithaca, NY 14853-7601,U.S.A. (email:[email protected]). 0003-3472/95/l
11405+
11 $12.00/O
from different males may determine paternity outcomes. Birds are also an important group for studying the relationship between mating and fertilization success because of the relatively monomorphic external genitalia in most species and the ovulation and oviposition cycle of females. Females of many species lay a clutch of eggs, ovipositing a single egg approximately daily. In the best known cases,ovulation of the egg to be laid the next day occurs 15-30 min after oviposition, and each egg is fertilizable for only 15-30 min after ovulation (Lake 1984; Birkhead 1988; Bakst 1993). Females store sperm for days or even weeks in sperm storage tubules (SSTs), presumably to ensure fertilization of each egg in spite of the rather short period of fertilizability (Shugart 1988; Birkhead & Msller 1992). Remarkably little is known about the fertilization successof a single natural (as opposed to artificial) insemination in birds. The experiments reported here examine fertile egg outcomes of single inseminations in Japanese quail. This speciesoffers severaladvantages for such research.
c 1995 The Association
1405
for the Study
of Animal
Behaviour
Animal
1406
Behaviour,
In common with other gallifonn birds, wild Japanese quail lay large clutches (8-12 eggs; Johnsgard 1988), and domesticated quail in the laboratory lay an egg nearly every day for an indefinite period. Ovulation occurs 15-30 min after oviposition (Doi et al. 1980). Females store sperm in SSTsand lay fertile eggs for a maximum of 10-11 days following separation from males (Sittman & Abplanalp 1965; Birkhead & Fletcher 1994). Males possess a well-developed foamproducing gland (the glandula proctodealis; Klemm et al. 1973), and foam is introduced into the female along with the semen, providing a visible marker of successfulinsemination (Adkins 1974). This foam has been hypothesized to prolong sperm motility and increase a male’s ability to fertilize eggs even when a hard-shelled egg is in the female’s uterus (Cheng et al. 1989). Forced copulations occasionally occur, and females sometimes void part of the ejaculate following unforced or forced copulation, which could influence fertilization success. The experiments were designed to answer the following questions. (1) How many eggs are fertilized by a single insemination, and how does this number compare with the clutch size of wild quail? (2) How does voiding of sperm by the female affect fertilization success?(3) How does the fertilization success of forced copulations compare with that of unforced copulations? (4) Do other male or female behaviour patterns occur during a mating encounter that predict fertilization success?(5) Does fertilization success depend on the female’s location in her oviposition and ovulation cycle? (6) Can a female be successfully fertilized by a male when she has a hard-shelled egg in the uterus? GENERAL
METHODS
Subjects
’ All subjects were hatched from eggs purchased from Truslow Farms, Chestertown, Maryland, and were reared in the laboratory. Female subjects’ ages ranged from 2 months (reproductive maturity begins at 5-6 weeks) to 13 months, with most females no older than 8 months; male subjects’ ages ranged from 2 to 8 months. Within these age ranges egg laying, male fertility and percentage of eggs fertilized are unrelated to age in this laboratory (unpublished data). Only
50, 5
females that were laying regularly were used. Birds were housed on a 16:8 h 1ight:dark cycle from hatching, with lights on at 0700 hours. On this light cycle, most females lay between 1600 and 2000 hours. All birds were housed individually in the same room. Procedures Unless stated otherwise, all birds mated between 1200 and 1800 hours, when females are most likely to be receptive (Delville et al. 1986), and mated in the female’s home cage by introduc. ing the male into her cage. In a typical mating sequence in this species, the male immediately runs towards the female, grabs the feathers at the back of her head, mounts, and performs a cloaca1 contact movement in which he leans backward with outstretched wings while mounted and briefly apposes his cloaca to the female’s. If the male is competent and the female receptive, ejaculation and insemination occur with the first cloaca1 contact, and the male immediately dismounts. After copulating, the male often fluffs his feathers, struts and growls (a distinctive vocalization). The entire sequence lasts but a few seconds. If the male is less competent or the female unreceptive, the male attempts copulation with the same speed and vigour as otherwise, but chasing of the female and repeated head-grabbing, mounting and cloaca1 contact attempts occur. Eventually the male either ceasesthe attempt or achieves cloaca1 contact with ejaculation even though the female appears to be quite unreceptive and continually tries to run away from him (here referred to as forced copulation). The female’s proctodeum (cloaca1 chamber) is the normal site of foam deposition (Cheng et al. 1989). Sperm are never found in the female following mating unless foam is also present, and vice versa (Klemm et al. 1973; Adkins 1974). If a series of cloaca1contacts occurs during the same copulation attempt, sperm and foam are not found in the female until after the last cloaca1 contact preceding the male’s dismount (Adkins 1974). Thus foam in the female’s reproductive tract, which is usually visible in the cloaca itself, indicates with a high degree of accuracy that transfer of sperm to the female (successfulinsemination) has occurred, providing a less disruptive alternative to cloaca1 lavage for confirming insemination; the male’s behaviour (sudden
Adkins- Regan: Japanese quail j>rtilization
djsmounting plus strutting, growling, or feather thdIing following a firm cloaca1 contact) provides an additional cue to insemination. Because the purpose of these experiments was to determine the fertilization consequences of a single ejaculation and insemination, I conducted mating trials as follows: (I) if the male did not attempt copulation within 1 min, he was removed and replaced; (2) all trials in which the male attempted copulation but did not appear to achieve cloaca1 contact with ejaculation were eliminated from analysis; (3) the male and female were left together only long enough for a single cloaca1 contact with ejaculation to take place, whereupon the male was removed. I noted forced copulations whenever they occurred. For all copulations, immediately after the male was removed, I examined the female by gently pulling open the cloaca1lip and checking for visible foam. If foam was visible, the trial was regarded as a putatively successfulinsemination. Post-copulatory voiding of foamy material by the female was noted, either by direct observation or by examining the drop pan under her cage. Eggs were collected daily in the morning for I I days following mating (day 1I is the last day that any fertile eggs could be expected given the maximum sperm storage interval). Eggs were stored at 13°C for a maximum of 2 weeks prior to incubation at 38°C and 60% relative humidity. I broke open eggs after I week of incubation and recorded them as fertile if any embryonic development had begun. The incidence of very early mortality was quite low, and such caseswere included with other fertile eggs in analysing the results. Most subjects participated in more than one mating trial. For females, the mimimum interval between successivetrials was 2 weeks. in a preliminary experiment, the ability of males to fertilize eggs was not related to recency of ejaculation over intervals ranging from 1 h to 2 weeks since the last ejaculation (unpublished data), consistent with what is known about rate of sperm production in this species(Clulow & Jones 1982). Therefore, males usually mated no more than once per 24 h, and never less than I h since the previous mating. Data Analysis
1 eliminated from analysis data from IO trials (lessthan 5% of the total number) where females
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laid fewer than five viable (unbroken, hardshelled) eggs. Data were summarized as the percentage of females laying at least one fertile egg (analysed with Fisher’s exact probability tests or chi-squared tests) and as the median percentage of eggs laid that were fertile (analysed with Mann Whitney U-tests). Correlations were obtained using Spearman’s rank correlation coefficient corrected for ties. All P-values are two-tailed. I treated trials using the same male or female as independent (seeexperiment I results establishing the statistical independence of such trials).
EXPERIMENT FEMALE’S
1: MA’rINGS IN THE HOME CAGE
Procedures
In experiment 1, I conducted 146 mating trials with 33 males and 54 females as described above. Subjects were sexually inexperienced at the time of their first mating trial. Results and Discussion
Following I I9 (82%) of the mating trials, foam was clearly visible in the female’s proctodeum, indicating that successful insemination had occurred. The fertilization outcomes of these I I9 inseminations are shown in Fig. la. Three features of these results are notable. First, outcomes were highly variable, ranging from no eggs fertilized to nearly all eggs fertilized. Second, the distribution of outcomes was bimodal. Following 33% of the inseminations, no eggs were fertile, but following 28% of the inseminations, more than 50% of the eggs laid were fertile. Third, even if day I eggs are ignored (see Fig. la legend), only one of these inseminations fertilized all of the eggs laid, and only three fertilized as many as eight eggs. Patterns of fertile egg laying by individual females across the I l-day post-mating period examined were highly variable as well. Fertile eggs did not necessarilyoccur on successivedays, but could be separated by days with infertile eggs. Thus successfulinsemination (transfer of sperm to the female’s reproductive tract) failed to guarantee fertilization, and fertilization success could not be predicted with any certainty. The bimodal distribution of outcomes, with one peak at 0% fertilized, suggests two underlying
Animal
1408
Behaviour,
(a)
40 35 30 25 20 15
lr
10 5 0
8 7--
nn (b)
65432l0
processes, one determining whether any eggs are fertilized, and a second determining how many eggs are fertilized. The variability in percentage of eggs fertilized was not due to experience. Once insemination was achieved (as it was for all trials shown in Fig. la), fertilization successdid not tend to differ on any measure when trials with inexperienced versus experienced birds were compared. The variability in percentage of eggs fertilized was not due to individual differences in fertility or fertilizability. The number of eggs laid by females (range S-11) was not related to the percentage fertilized. When the percentage of eggs fertilized was compared for all possible pairs of trials involving the same male but different females, the correlation between the pairs of percentages was low and insignificant (r,=O.ll, P=O.l; N=32 males, 114 trials and 83 comparisons);
n
25
50, 5
that is, the
outcome of one trial did not predict the outcome of a subsequent trial. Male 9D’s fertilization successprovides a prototypical example. He successfully inseminated eight females over 4 months. The percentages of eggs fertilized from these eight
Cc)
trials were: 0, 75, 9, 0, 56, 44, 40, 0. The same result was obtained when pairs of trials involving
the same female but different males were com-
J
pared (r,=0.17,
P=O.3;
N=23
females, 52 trials
and 29 comparisons). Figure 1. Percentage of eggs laid during the 11 days
following a single insemination that were fertilized,
(e)
0 l-9
10-19 3&39 5&59 7&79 9&99 2&29 4&49 60-69 8049 100
Percentage of eggsfertilized
showing the number of inseminations (trials) producing the different percentages. Outcomes of 100% fertilized would not be expected because the day 1 egg (collected the morning of the day after mating, but usually laid on the day of mating) cannot be fertile. (a) Results for 119 confirmed inseminations in experiment 1 (home cage mating trials). Median % fertilized was 29% (45% excluding trials yielding no fertilized eggs). (b) Trials in experiment 1 in which the female voided foam I 1 min after insemination (see Table I). (c) Results for 53 putative inseminations in experiment 2 (large testing cage mating trials). Median % fertilized was 20% (50% excluding trials yielding no fertilized eggs). (d) Trials in experiment 2 in which the female voided foam < 1 min after putative insemination (see Table I). (e) Forced copulation trials from experiments 1 (m) and 2 (0). In experiment 1, 63% of these trials resulted in at least one fertilized egg; in experiment 2, 60% of these trials resulted in at least one fertilized egg. Median % fertilized was 23% (55% excluding trials yielding no fertilized egg) for experiment 1; medians for experiment 2 were 22% and 40%, respectively.
Adkins-Regan:
Japanese quail fertilization
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Table 1. Voiding of foam by the female and fertile eggs laid
Experiment 1 Voided 2 I min* All other trials Experiment 2 Spontaneous void7 Induced voidt All other trials Experiment 3a Voided I 1 min* All other trials Experiment 3b Voided 2 1 min* All other trials
No. of trials
% Trials with a fertile egg
% Trials with > 50% fertile
Median % eggs fertile
20 99
65 68
5t 33
12 (33)§ 30 (50)
6 8 39
83 63 51
33 25 23
43 (50) 35 (50) 9 (50)
5 26
80 13
20 38
40 (45) 35 (56)
5 21
60 85
0 26
25 (36) 44 (45)
*Female voided foam 1 min or less after insemination. tFemale voided foam during the mating trial (spontaneous, < 1 min after putative insemination) or while being carried back to her home cage (induced). $x2=5.23, PcO.05 compared with all other trials in experiment 1. DMedians in parentheses exclude trials yielding no fertile eggs.
The correlation between trial outcomes (percentage of eggs fertilized) was high, positive and significant, however, when pairs of trials involving the same male and the same female were” compared (r,=0.83, WO.02; N= 10 birds of each sex, 20 trials and 10 comparisons). Thus fertilization successis predicted by the particular male-female combination but not by either the male or the female alone. Another predictor of fertilization successwas whether the female voided foam shortly after insemination (Fig. lb and Table I). If the female voided foam 1 min or less after insemination, there was a significantly lower probability that more than half the eggs would be fertilized. For three of these 20 trials, a sample of the voided foamy material was examined under a microscope; all three samples contained motile sperm. Eight (5%) of the 119 trials in Fig. la were forced copulations (also shown separately in Fig. le). The distribution of outcomes after these copulations was similar to the distribution for other trials (most of the trials in Fig. la were unforced copulations), and no measures differed significantly when forced copulation trials were compared with other trials. Females were not more likely to void foam following forced copulations than other copulations, but the small number of forced copulations made it difficult to test such a hypothesis (that females might be more
likely to void an ejaculate following forced copulation) adequately. Following 18 (12%) of the 146 total trials in experiment 1 (18 trials that are not shown in Fig. l), no foam was seen in the female, even though the male’s behaviour seemed to indicate cloaca1 contact with ejaculation. Only five (28%) of these 18 trials resulted in any fertile eggs (x2=8*72, PcO.01, compared to 67% of the 119 trials in Fig. la for which foam was seen in the female). If these 18 trials are added to the 119 trials where females had visible foam (N= 137), the overall percentage of trials yielding fertile eggs was 62%, compared to 67% for the 119 trials in Fig. la, a nonsignificant difference. Thus a few successful inseminations occur without visible foam in the female, possibly because the foam has been placed too high in the oviduct to be seen by cloaca1 examination, but the incidence of these errors (failures to correctly detect successful insemination) is low, trivially so with a large sample size. For the remaining nine of the 146 experiment 1 trials (trials not shown in Fig. l), foam was seen on the female’s exterior (usually the cloaca1 lip) instead of or in addition to its presence in the proctodeum. Only two of these trials (22%) yielded fertile eggs (x2= 5.54, P
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Animal
Behaviour,
back of the female while making cloaca1contact, resulting in incomplete or interrupted ejaculations. Two possible explanations for the appealante of foam on the female’s exterior are either that the male places foam in the female first, and then semen, or that depositing a reduced amount of foam in the proctodeum lowers fertilization success.Experimental reduction of foam quantity has no effect on fertilization success (Seiwert 1994), suggesting that the first explanation is more likely. Because trials ending in foam on the exterior of the female had lower egg fertility, such trials were not included in the analyses in the subsequent experiments.
EXPERIMENT 2: MATING BEHAVIOUR IN A LARGE NEUTRAL CAGE
Mating trials in experiment 1 were conducted in the female’s home cage. These are standard quail housing cages, but because they are rather small (0.01-0.02 m3) they do not permit the full range of social behaviour during a mating encounter. Such behavioural restriction may have contributed to the large number of unfertilized eggs. The brief cloaca1manipulation of the female, while less invasive than lavage, may also have influenced the results, for example, by artificially stimulating oviducal or cloaca1 movement. Some females who voided foam did so while being examined. Therefore in experiment 2, I conducted mating trials in a large neutral cage, and the criteria for insemination were behavioural observations refined in the course of experiment 1 rather than examination of the female’s proctodeum. In addition, I recorded the behaviour patterns of the male and female during the mating trial. Procedures
I conducted 53 mating trials in which observation of behaviour indicated cloaca1contact with probable ejaculation with 22 males and 3 1 females in a 2-m” hardware cloth cage. As in experiment 1, subjects were sexually inexperienced at the time of their first mating trial. For each trial, I introduced the female first, then added the male within 10 s and removed him 30 s to 1 min after ejaculation,
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Table II. Behaviour recorded in experiment 2 Male
behaviour
Latency (s) to first head grab Number of struts before ejaculation Number of struts after ejaculation Number of feather fluffs Number of growls Ejaculation, judging from male’s behaviour (yes/no) Female
hehaviour
Run from the male’sinitial approach (yes/no) Approach to within 4 cm of the male (yes/no) Peck male (yes/no) Receptivity (1 = definitely unreceptive, 2 =slightly but not fully receptive, 3=fully receptive). A fully receptive female squats and does not locomote
during attempted copulation Number of feather fluffs Voiding of foam during the trial (yes/no). If the female voided foam while being carried back to her home cage, this was noted separately
allowing a slightly longer post-ejaculatory interaction than in experiment 1 but still preventing a second ejaculation. Females were then returned to the home cage. I conducted no cloaca1 examination, but closely monitored females for voiding of foam not only during testing but also during transport back to the home cage, and also checked the testing cage itself and the floor beneath for voided foam. Beginning from the introduction of the male until his removal, I recorded behaviour patterns from both sexes with a computerized event recorder (Table II). Results and Discussion
As in experiment 1, fertilization success was highly variable; i.e. a substantial number of inseminations did not fertilize any eggs (Fig. lc). The distributions in Figs la and ic are quite similar, and the results for the two experiments did not differ on any measure. Becausethe behavioural criteria for insemination do produce some errors, a few of the trials that failed to produce fertile eggs may have been unsuccessful inseminations. The percentage of trials with no fertile eggs was in fact slightly higher for experiment 2 (43% compared with 33% in experiment l), but this difference is not significant, and the error rate for the behavioural criteria is unlikely to account for most or all of the trials without fertile eggs. Thus insemination fails to guarantee fertilization and
Adkins- Regun: Jupanese yuuil fertilixtion
Jmost never fertilizes all of the eggs laid even yhen mating occurs in a more spacious setting that allows a richer array of social behaviour. In experiment 2, unlike experiment I, voiding of foam by the female did not reduce the number of fertile eggs laid (Fig. Id and Table I). Forced copulations occurred in five (9%) of the 53 trials (Fig. le). As in experiment 1, these trials did not differ from other trials according to any outcome measure. One female behaviour did predict fertilization success.If the female ran from the male’s initial approach, copulation was less likely to result in any fertile eggs, that is, there were more trials with 0% eggs fertilized (13 of 2 1 trials, or 62X, with 0% eggs fertilized if the female ran versus 9 of 32 trials, or 28X, if the female did not run; x2=4.65, PcO.05). If trials with no fertilized eggs are excluded, then the percentage of eggs fertilized did not differ for trials with females that ran (median 43%) versus trials with females that did not run (median 50%), suggesting that it is the process underlying whether any eggs are fertilized, rather than the process determining how many are fertilized, that is predicted by this behaviour. These females were not necessarilyunreceptive once the male began head-grabbing, nor did they continue to run from the male. Neither receptivity itself nor any of the other behavioural measures was related to fertilization success.Experienced females (those with one or two previous trials) were somewhat lesslikely than inexperienced females to run from the male’s approach (running from the male occurred in 11 of 33 trials with experienced females, compared to 10 of 20 trials with inexperienced females), but this difference was not significant (x1=0.83, DO.3). nor was experience by itself related to any outcome measure, consistent with the lack of any effect of experience in experiment 1.
EXPERIMENT 3: TIME OF DAY AND OVIPOSITION STATUS In experiments 1 and 2 females mated in the afternoon when they are more receptive and have not yet oviposited. Thus it is possible that the large number of inseminations producing no fertile eggs reflects interference with fertilization successby a hard-shelled egg in the uterus. Experiment 3 examined the fertilization successof single
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inseminations as a function of time of day and of time relative to oviposition. Procedures In experiment 3a, I conducted 31 trials with insemination with 22 females and 14 males as described in General Methods, 15 between 0830 and 0930 hours (morning matings) and 16 between 1530 and 1730 hours (afternoon matings). All afternoon matings occurred prior to oviposition. In experiment 3b, I conducted 32 trials with insemination with 32 females and 25 males in the afternoon (between 1500 and 1830 hours) either before (18 trials) or after (14 trials) oviposition. A female that laid an egg mated as soon as possible after oviposition and never more than I.5 h after oviposition. Within 5 min of this mating another female that had not yet oviposited also mated, so that pre-oviposition and postoviposition matings would be matched for time of day. In both experiments, all females that had not yet laid were palpated to confirm the presenceof a hard-shelled egg in the uterus, and whenever possible, the exact time of subsequent oviposition was determined. In both experiments, insemination was confirmed by examination of the female’s proctodeum for foam. Results and Discussion In experiment 3a the median percentage 01‘ fertile eggs (including trials with no fertile eggs) tended to be somewhat lower following late afternoon inseminations compared with morning inseminations, but this difference was not signifcant (C/=75, PcO.1; Table 111). Thus, although males might be lesslikely to succeedat inseminating females in the morning (because of the females’ lower receptivity), a successfulinsemination at this time would not be less effective at fertilizing eggs. In experiment 3b, there were no differences on any measures between outcomes of preoviposition versus post-oviposition inseminations. Of the eight trials where a female oviposited less than 3 h after mating, four resulted in fertile eggs. including one mating that occurred only 15 min before oviposition. Clearly an insemination can fertilize future eggs even when a hard-shelled egg is present in the lower reproductive tract. In experiments 3a and 3b, as in experiment 2. there were no significant differences between trials
Animal
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Behaviour,
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Table III. Fertile eggs laid as a function of time of day or time relative to oviposition Mating time Experiment 3a Morning Afternoon Experiment 3b Pre-oviposition Post-oviposition
No. of trials
% Trials with a fertile egg
% Trials with > 50% fertile
Median % fertile*
15 16
87 63
47 25
50 (56) 16 (33)t
18 14
83
28
19
14
40 (45) 18 (35)
*Medians in parentheses exclude trials yielding no fertile eggs. tPcO.1.
followed by voiding of foam and other trials (Table I). As in experiment 1, the likelihood that more than 50% of the eggs would be fertile was lower if the female voided foam, but this difference was not significant even when the results of experiments 3a and 3b were combined for analysis.
organization (Lott 1991). The sexually dimorphic foam gland, unique to the Coturnix genus, also hints at a genetically non-monogamous mating system. The order Galliformes includes several species with lek mating systems.It would be interesting to know how the fertilization success of single inseminations in these speciescompares with the results for Japanese quail in the present study. If GENERAL DISCUSSION successis similar (that is, relatively low), then it is highly unlikely that females visit the lek only once. It has been proposed that one insemination is Alternatively, greater selection for fertilizing sufficient to fertilize an entire avian clutch, even a ability of single inseminations in lekking species large clutch (Lake 1975; Birkhead 1988). The may have produced important speciesdifferences results of these experiments suggest that this is even within the order Galliformes (Lake 1975). highly unlikely in Japanese quail. The clutch size Forced inseminations had the same fertilization is 8-12, yet only eight (3%) of the 233 successful successas other inseminations even though male inseminations (confirmed or putative) in these quail lack a developed phallus. Comparable data experiments fertilized as many as eight eggs. The are not available for other species, although results also suggest caution in assuming that any forced copulations by ducks, which do have a eggs have been fertilized by a single mating, even phallus, can fertilize eggs (Burns et al. 1980). The if insemination has occurred and even in the success of forced copulation by male quail absence of competition from another male’s suggests that this behaviour could be part of a secondary or alternative reproductive strategy for sperm. The copulatory pattern of the male Japanese males of this species. Such successalso indicates quail is multiple ejaculation (Birkhead & Msller that females are not rejecting these ejaculates (see 1992; terminology of Dewsbury 1979). This below), but females would not always be expected behaviour pattern is consistent with the relatively to evolve counter-strategies to forced copulation low fertilization successof single inseminations (Westneat et al. 1990). and rhay be a male adaptation to ensure that the Whether the female had a hard-shelled egg in entire clutch does get fertilized (Hunter et al. her uterus had surprisingly little influence on the 1993). ability of an insemination to fertilize future eggs. The mating system of wild Japanesequail is not Yet evidence from other speciessuggeststhat the yet known. While some reports suggest monoga- female’s location in her oviposition and ovulation mous pairings (Nichols 1991), there are also cycle should affect fertilizability (Birkhead 1988). anecdotal reports of non-monogamous pairings In turkeys, MeIeagris gallopavo, an egg in the (Johnsgard 1988), and other speciesof quail show oviduct reduces sperm entry into the sperm considerable within-species variability in social storage tubules (SSTs; Bakst 1994). In chickens,
Adkins-Regan:
Japanese quail fertilization
Gallus gabs, artificial insemination from 1 h before to 1 h after oviposition results in a lower percentage of SSTs with spermatozoa than artificial insemination at other times (Brillard 1993), and in muscovy ducks, Cairina moschata, artificial insemination results in lower fertilization if carried out 4 h before to 1 h after oviposition than when carried out at other times (Raud & Fame 1990). In Bengalese finches, Lonchura striata, insemination time relative to ovulation influences how many sperm are stored (Birkhead 1992). Cheng et al. (1989) pointed out that female quail differ from many other birds in laying in the late afternoon rather than the early morning, and therefore are more likely to be copulating with a hard-shelled egg in the uterus. They hypothesized that one function of the male quail’s foam might be to hold sperm in the proctodeum and thus allow fertilization to occur even with a hard-shelled egg in the uterus. The present results confirm that such fertilization does occur, but further work would be necessarybefore concluding that it is the foam that permits fertilization with a hard-shelled egg in the uterus. For example, it would be interesting to know whether fertilization just before oviposition is possible in other avian species, which lpck foam. What are the likely mechanisms underlying the bimodal distribution of fertilization successoutcomes (Fig. l)? Whether any eggs are fertilized may depend on such related variables as how well the male and female cloacae are apposed, how many sperm are ejaculated, where they are placed in the female’s tract, and whether any of them reach the SSTs. How many eggs are fertilized (if any eggs are fertilized) may depend on how many sperm are stored, in which SSTs, and how and when they are releasedfrom the tubules (Birkhead & Merller 1992; Birkhead & Fletcher 1994). In both chickens and quail, stored sperm are released continuously and in a declining manner (Wishart 1987; Birkhead & Fletcher 1994) but the number of fertile eggs laid by quail is not determined solely by the rate of decline, because a fertile egg can follow one or even several infertile eggs. These intervening processesbetween ejaculation and fertilization introduce some elements of randomness into the outcome which may account for some of the characteristics of the data in Fig. 1. They also present possibilities for the female to
1413
intluence the outcome and thus control the paternity of her offspring (Birkhead & Mallet 1992; Birkhead et al. 1993; Oring et al. 1993). Such influence could arise through her behaviour (for example, how she positions her cloaca and oviduct during copulation), her oviducal physiology and anatomy (for example, regulation of sperm storage and release), and her gametic or genetic compatibility with the male (for example, selection by the ovum for individual sperm). In both passerinesand galliforms, only a very small percentage of sperm actually gets stored in the SSTs, and so statistically there is clearly very strong sperm selection in the female reproductive tract (Birkhead 1992; Briskie & Montgomerie 1992; Brillard 1993; Birkhead & Fletcher 1994). Birkhead et al. (1993) referred to the ‘hostility’ of the female tract to sperm and proposed that it is a form of adaptive sexual selection. Thus the low fertilization success of single inseminations may reflect substantial sperm selection by the females, who did not get to choose the males, rather than a neutral character of no cost to the female because of the male’s multiple ejaculation mating pattern. Two other features of the results also suggestthat some form of selection by the female could be occurring based on sperm acceptance or rejection. First, females that ran from the male’s initial approach were lesslikely to lay fertile eggs following insemination, as if running indicates behavioural rejection of the male and can be followed through with physiological rejection of his sperm. Second, fertilization success was highly correlated when females mated with the same male twice, but was uncorrelated when females mated with different males. This feature of the results could reflect anatomical, physiological, or genetic compatibility between the male and female, or male adjustment of sperm numbers according to his mate preferences, in addition to or instead of female preference/ rejection. Voiding of sperm by the female shortly after insemination is another potential mechanism for female selection (Wagner 1991; Birkhead & Msller 1992), but the present results lend only weak support to this hypothesis. In only one of the three experiments did voiding of sperm significantly reduce the number of fertile eggs laid, and some of these voidings may have been induced by examining the female’s proctodeum. Because sperm are present in the voided material, it is
Animal
1414
Behaviour.
likely that there is at least some small effect of sperm voiding, but such an effect could be difficult to detect if other mechanisms for rejection are simultaneously operating. There is good evidence for post-copulatory sperm selection or rejection by female insects (Eberhard 1985; LaMunyon & Eisner 1993), a phenomenon that has been termed ‘cryptic female choice’ (Thornhill 1983). The hypothesis that female birds engage in post-copulatory selection, and can thereby escape male detection and retaliation, deservesserious investigation in light of the present results. As Eberhard (1985) put it, ‘females, because fertilization takes place within their bodies, generally have the last say in reproduction’ (page 107).
ACKNOWLEDGMENTS
I thank Mary Ascenzi for technical assistanceand Robert Johnston for reviewing the manuscript. Supported by NSF grant BNS 8809441.
REFERENCES Adkins, E. K. 1974. Electrical recording of copulation in quail. Physiol. Behav., 13, 475477. Bakst, M. R. 1993. Oviducal sperm storage in poultry: a review. Reprod. Fert. Devel., 5, 595-599. Bakst, M. R. 1994. Fate of fluorescent stained sperm following insemination: new light on oviducal sperm transport and storage in the turkey. Biol. Reprod., 50, 987-992. Birkhead, T. R. 1988. Behavioral aspects of sperm competition in birds. In: Advances in the Study of Behavior, Vol. 18 (Ed. by J. S. Rosenblatt, C. Beer, M.-C. Busnel & P. J. B. Slater), pp. 35-72. San Diego: Academic Press. Birkhead, T. R. 1992. Sperm storage and the fertile period in the Bengalese finch. Auk, 109, 62CM25. Birkhead, T. R. & Fletcher, F. 1994. Sperm storage and the release of sperm from the sperm storage tubules in Japanese quail Coturnixjaponica Zbis, 136, 101-105. Birkhead, T. R. & Moller, A. P. 1992. Sperm Competition in Birds. London: Academic Press. Birkhead, T. R., Moller, A. P. & Sutherland, W. J. 1993. Why do females make it so difficult for males to fertilize their eggs? J. theor. BioZ., 161, 51-60. Brillard, J. P. 1993. Sperm storage and transport following natural mating and artificial insemination. Pot&. Sci., 12, 923-928.
Briskie, J. V. & Montgomerie, R. 1992. Sperm size and sperm competition in birds. Proc. R. Sot. Ser. B, 247, 89-95.
50, 5
Burns, J. T., Cheng, K. M. & McKinney, F. 1980. Forced copulation in captive mallards. I. Fertilization of eggs. Auk, 97, 875-879. Cheng, K. M., McIntyre, R. F. & Hickman, A. R. 1989. Proctodeal gland foam enhances competitive fertilization in domestic Japanese quail. Auk, 106, 28629 1. Clulow, J. & Jones, R. C. 1982. Production, transport, maturation, storage and survival of sperm in the male Japanese quail, Coturnix coturnix. J. Reprod. Fert., 64, 259-266. Delville, Y., Sulon, J. & Balthazart, J. 1986. Diurnal variations of sexual receptivity in the female Japanese quail (Coturnix coturnix japonica). Horm. Behav., 20, 13-33. Dewsbury, D. A. 1979. Description of sexual behavior in research on hormone-behavior interactions. In: Endo. crine Control of Sexual Behavior (Ed. bv C. Bever). pp. 3-32. New”York: Raven Press: . - ” Doi, O., Takai, T., Nakamura, T. & Tanabe, Y. 1980. Changes in the pituitary and plasma LH, plasma and follicular progesterone and estradiol, and plasma testosterone and estrone concentrations during the ovulatory cycle of the quail (Coturnix coturnix japonica). Gen. camp. Endocrinol., 41, 156-163. Dunn, P. 0. & Lifjeld, J. T. 1994. Can extra-pair copulations be used to predict extra-pair paternity in birds? Anim. Behav., 47, 983-985. Eberhard, W. G. 1985. Sexual Selection and Animal Genitalia. Cambridge, Massachusetts: Harvard University Press. Hunter, F. M., Petrie, M., Otronen, M., Birkhead, T. & Moller, A. P. 1993. Why do females copulate repeatedly with one male? Trends Ecol. Evol., 8, 21-26. Johnsgard, P. A. 1988. The Quails, Partridges, and Fruncolins of the World. Oxford: Oxford University Press. Klemm, R. D., Knight, C. E. & Stein, S. 1973. Gross and microscopic morphology of the glandula proctodealis (foam gland) of Coturnix c. japonica (Aves). J. Morph., 141, 171-184. Lake, P. E. 1975. Gamete production and the fertile period with particular reference to domesticated birds. Symp.
zool.
Sot.
Land.,
35,225-244.
Lake, P. E. 1984. The male in reproduction. ology
and Biochemistry
of the Domestic
In: Physi-
Fowl,
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(Ed. by B. M. Freeman), pp. 381405. London: Academic Press. LaMunyon, C. W. & Eisner, T. 1993. Postcopulatory sexual selection in an arctiid moth (Utetheisa ornatrix).
Proc.
natn.
Acad.
4692. Lott, D. F. 1991. Intraspecific Systems
of Wild
Vertebrates.
Sci.
U.S.A.,
Variation
90, 4689in the Social
Cambridge: Cambridge
University Press. Nichols, C. R. 1991. A comparison of the reproductive and behavioural differences in feral and domestic Japanese quail. Master’s thesis, University of British Columbia. Oring, L. W., Reed, J. M., Alberico, J. A. R. & Fleischer, R. C. 1993. Female control of paternity: more than meets the eye. Trends Ecol. Evol., 8, 259.
Adkins-Regan:
Japanese
Raud, H. & Fame, .I. M. 1990. Rhythmic occurrence of sexual behaviour and egg laying activity of muscovy ducks. Br. Poult. Sci., 31, 23-32. geiwert, C. M. 1994. The neuromuscular system controlling foam production in Japanese quail: an investigation of structure and function. Ph.D. thesis, Cornell University. Shugart, G. W. 1988. Uterovaginal sperm-storage glands in sixteen species with comments on morphological differences. Auk, 105, 379-383. Sittman, K. & Abplanalp, H. 1965. Duration and recovery of fertility in Japanese quail Coturnix cofurnix japonica.
Br. Poult.
Sci., 6, 245-250.
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Thornhill, R. 1983. Cryptic female choice and its implications in the scorpionfly Harpobittacus nigriceps. Am. Nat.,
122,765-788.
Wagner, R. H. 1991. Evidence that female razorbills control extra-pair copulations. Behaviour, 118, 157-169. Westneat, D. F., Sherman, P. W. & Morton, M. L. 1990. The ecology and evolution of extra-pair copulations in birds. In: Current Ornithology, Vol. 7 (Ed. by D. M. Power), pp. 331-369. New York: Plenum Press. Wishart, G. J. 1987. Regulation of the length of the fertile period in the domestic fowl by numbers of oviductal spermatozoa as reflected by those trapped in laid eggs. J. Reprod. Fert., 80, 493498.
Behuv.,
1995, 50, 1405-1415
Predictors of fertilization in the Japanese quail, Coturnix japonica ELIZABETH
ADKINS-REGAN
CorneN (Received final
University
4 October 1994; initial acceptance 26 January acceptance 25 March 1995; MS. number: A7116)
1995;
To better understand the relationship between mating behaviour, insemination and fertilization in birds, 233 mating trials were conducted with Japanese quail to determine the percentage of eggs laid that were fertile following a single confirmed or putative insemination. Fertilization successof single inseminations was highly variable. Insemination did not guarantee fertilization of any eggs; following one-third of the trials, no fertile eggs were laid. Only 3% of inseminations fertilized as many aseight eggs,making it unlikely that a single insemination could fertilize an entire clutch in this species. Forced copulations had the same fertilization successas other copulations, and thus could be a successfulmale reproductive strategy in this species.Voiding of foamy material containing sperm by the female shortly after copulation did not reduce fertilization successin any consistently significant manner. Fertilization successwas unaffected by the presence of a hard-shelled egg in the uterus at the time of insemination. Trials in which the female ran from the male’s initial approach resulted in fewer fertile eggs following insemination; that is, this behaviour predicted low fertilization success.Fertilization successwas highly correlated and thus predictable if the same female was mated again with the same male, but was uncorrelated if she was mated with a different male or if the same male was mated with different females. These two predictors of fertilization success(female behaviour and male-female combination) are interpreted as evidence for female control of paternity via sperm selection (postcopulatory female choice). 0 1995 The Association for the Study of Animal Behaviour Abstract.
Understanding the reproductive consequences of mating behaviour is critically important for the development and testing of evolutionary hypothesesabout mating systems.In specieswith internal fertilization, the likelihood that a male mating with a fertile female will father any of her offspring depends both on whether transfer of sperm to the female’s reproductive tract occurs during the act of mating (insemination success)and on whether those sperm fertilize any eggs (fertilization success).In birds, a key taxon in contemporary mating systemsresearch, increasing evidence suggests that mating behaviour is not a reliable guide to paternity (Westneat et al. 1990; Birkhead & Moller 1992; Dunn & Lifjeld 1994). Not all matings result in successful insemination. Extrapair copulations may occur whose successmay differ from pair copulations. In both polygynous and monogamous species that engage in extrapair copulations, competition between sperm Correspondence:E. Adkins-Regan, 218 Uris Hall, Cornell University, Ithaca, NY 14853-7601,U.S.A. (email:[email protected]). 0003-3472/95/l
11405+
11 $12.00/O
from different males may determine paternity outcomes. Birds are also an important group for studying the relationship between mating and fertilization success because of the relatively monomorphic external genitalia in most species and the ovulation and oviposition cycle of females. Females of many species lay a clutch of eggs, ovipositing a single egg approximately daily. In the best known cases,ovulation of the egg to be laid the next day occurs 15-30 min after oviposition, and each egg is fertilizable for only 15-30 min after ovulation (Lake 1984; Birkhead 1988; Bakst 1993). Females store sperm for days or even weeks in sperm storage tubules (SSTs), presumably to ensure fertilization of each egg in spite of the rather short period of fertilizability (Shugart 1988; Birkhead & Msller 1992). Remarkably little is known about the fertilization successof a single natural (as opposed to artificial) insemination in birds. The experiments reported here examine fertile egg outcomes of single inseminations in Japanese quail. This speciesoffers severaladvantages for such research.
c 1995 The Association
1405
for the Study
of Animal
Behaviour
Animal
1406
Behaviour,
In common with other gallifonn birds, wild Japanese quail lay large clutches (8-12 eggs; Johnsgard 1988), and domesticated quail in the laboratory lay an egg nearly every day for an indefinite period. Ovulation occurs 15-30 min after oviposition (Doi et al. 1980). Females store sperm in SSTsand lay fertile eggs for a maximum of 10-11 days following separation from males (Sittman & Abplanalp 1965; Birkhead & Fletcher 1994). Males possess a well-developed foamproducing gland (the glandula proctodealis; Klemm et al. 1973), and foam is introduced into the female along with the semen, providing a visible marker of successfulinsemination (Adkins 1974). This foam has been hypothesized to prolong sperm motility and increase a male’s ability to fertilize eggs even when a hard-shelled egg is in the female’s uterus (Cheng et al. 1989). Forced copulations occasionally occur, and females sometimes void part of the ejaculate following unforced or forced copulation, which could influence fertilization success. The experiments were designed to answer the following questions. (1) How many eggs are fertilized by a single insemination, and how does this number compare with the clutch size of wild quail? (2) How does voiding of sperm by the female affect fertilization success?(3) How does the fertilization success of forced copulations compare with that of unforced copulations? (4) Do other male or female behaviour patterns occur during a mating encounter that predict fertilization success?(5) Does fertilization success depend on the female’s location in her oviposition and ovulation cycle? (6) Can a female be successfully fertilized by a male when she has a hard-shelled egg in the uterus? GENERAL
METHODS
Subjects
’ All subjects were hatched from eggs purchased from Truslow Farms, Chestertown, Maryland, and were reared in the laboratory. Female subjects’ ages ranged from 2 months (reproductive maturity begins at 5-6 weeks) to 13 months, with most females no older than 8 months; male subjects’ ages ranged from 2 to 8 months. Within these age ranges egg laying, male fertility and percentage of eggs fertilized are unrelated to age in this laboratory (unpublished data). Only
50, 5
females that were laying regularly were used. Birds were housed on a 16:8 h 1ight:dark cycle from hatching, with lights on at 0700 hours. On this light cycle, most females lay between 1600 and 2000 hours. All birds were housed individually in the same room. Procedures Unless stated otherwise, all birds mated between 1200 and 1800 hours, when females are most likely to be receptive (Delville et al. 1986), and mated in the female’s home cage by introduc. ing the male into her cage. In a typical mating sequence in this species, the male immediately runs towards the female, grabs the feathers at the back of her head, mounts, and performs a cloaca1 contact movement in which he leans backward with outstretched wings while mounted and briefly apposes his cloaca to the female’s. If the male is competent and the female receptive, ejaculation and insemination occur with the first cloaca1 contact, and the male immediately dismounts. After copulating, the male often fluffs his feathers, struts and growls (a distinctive vocalization). The entire sequence lasts but a few seconds. If the male is less competent or the female unreceptive, the male attempts copulation with the same speed and vigour as otherwise, but chasing of the female and repeated head-grabbing, mounting and cloaca1 contact attempts occur. Eventually the male either ceasesthe attempt or achieves cloaca1 contact with ejaculation even though the female appears to be quite unreceptive and continually tries to run away from him (here referred to as forced copulation). The female’s proctodeum (cloaca1 chamber) is the normal site of foam deposition (Cheng et al. 1989). Sperm are never found in the female following mating unless foam is also present, and vice versa (Klemm et al. 1973; Adkins 1974). If a series of cloaca1contacts occurs during the same copulation attempt, sperm and foam are not found in the female until after the last cloaca1 contact preceding the male’s dismount (Adkins 1974). Thus foam in the female’s reproductive tract, which is usually visible in the cloaca itself, indicates with a high degree of accuracy that transfer of sperm to the female (successfulinsemination) has occurred, providing a less disruptive alternative to cloaca1 lavage for confirming insemination; the male’s behaviour (sudden
Adkins- Regan: Japanese quail j>rtilization
djsmounting plus strutting, growling, or feather thdIing following a firm cloaca1 contact) provides an additional cue to insemination. Because the purpose of these experiments was to determine the fertilization consequences of a single ejaculation and insemination, I conducted mating trials as follows: (I) if the male did not attempt copulation within 1 min, he was removed and replaced; (2) all trials in which the male attempted copulation but did not appear to achieve cloaca1 contact with ejaculation were eliminated from analysis; (3) the male and female were left together only long enough for a single cloaca1 contact with ejaculation to take place, whereupon the male was removed. I noted forced copulations whenever they occurred. For all copulations, immediately after the male was removed, I examined the female by gently pulling open the cloaca1lip and checking for visible foam. If foam was visible, the trial was regarded as a putatively successfulinsemination. Post-copulatory voiding of foamy material by the female was noted, either by direct observation or by examining the drop pan under her cage. Eggs were collected daily in the morning for I I days following mating (day 1I is the last day that any fertile eggs could be expected given the maximum sperm storage interval). Eggs were stored at 13°C for a maximum of 2 weeks prior to incubation at 38°C and 60% relative humidity. I broke open eggs after I week of incubation and recorded them as fertile if any embryonic development had begun. The incidence of very early mortality was quite low, and such caseswere included with other fertile eggs in analysing the results. Most subjects participated in more than one mating trial. For females, the mimimum interval between successivetrials was 2 weeks. in a preliminary experiment, the ability of males to fertilize eggs was not related to recency of ejaculation over intervals ranging from 1 h to 2 weeks since the last ejaculation (unpublished data), consistent with what is known about rate of sperm production in this species(Clulow & Jones 1982). Therefore, males usually mated no more than once per 24 h, and never less than I h since the previous mating. Data Analysis
1 eliminated from analysis data from IO trials (lessthan 5% of the total number) where females
1407
laid fewer than five viable (unbroken, hardshelled) eggs. Data were summarized as the percentage of females laying at least one fertile egg (analysed with Fisher’s exact probability tests or chi-squared tests) and as the median percentage of eggs laid that were fertile (analysed with Mann Whitney U-tests). Correlations were obtained using Spearman’s rank correlation coefficient corrected for ties. All P-values are two-tailed. I treated trials using the same male or female as independent (seeexperiment I results establishing the statistical independence of such trials).
EXPERIMENT FEMALE’S
1: MA’rINGS IN THE HOME CAGE
Procedures
In experiment 1, I conducted 146 mating trials with 33 males and 54 females as described above. Subjects were sexually inexperienced at the time of their first mating trial. Results and Discussion
Following I I9 (82%) of the mating trials, foam was clearly visible in the female’s proctodeum, indicating that successful insemination had occurred. The fertilization outcomes of these I I9 inseminations are shown in Fig. la. Three features of these results are notable. First, outcomes were highly variable, ranging from no eggs fertilized to nearly all eggs fertilized. Second, the distribution of outcomes was bimodal. Following 33% of the inseminations, no eggs were fertile, but following 28% of the inseminations, more than 50% of the eggs laid were fertile. Third, even if day I eggs are ignored (see Fig. la legend), only one of these inseminations fertilized all of the eggs laid, and only three fertilized as many as eight eggs. Patterns of fertile egg laying by individual females across the I l-day post-mating period examined were highly variable as well. Fertile eggs did not necessarilyoccur on successivedays, but could be separated by days with infertile eggs. Thus successfulinsemination (transfer of sperm to the female’s reproductive tract) failed to guarantee fertilization, and fertilization success could not be predicted with any certainty. The bimodal distribution of outcomes, with one peak at 0% fertilized, suggests two underlying
Animal
1408
Behaviour,
(a)
40 35 30 25 20 15
lr
10 5 0
8 7--
nn (b)
65432l0
processes, one determining whether any eggs are fertilized, and a second determining how many eggs are fertilized. The variability in percentage of eggs fertilized was not due to experience. Once insemination was achieved (as it was for all trials shown in Fig. la), fertilization successdid not tend to differ on any measure when trials with inexperienced versus experienced birds were compared. The variability in percentage of eggs fertilized was not due to individual differences in fertility or fertilizability. The number of eggs laid by females (range S-11) was not related to the percentage fertilized. When the percentage of eggs fertilized was compared for all possible pairs of trials involving the same male but different females, the correlation between the pairs of percentages was low and insignificant (r,=O.ll, P=O.l; N=32 males, 114 trials and 83 comparisons);
n
25
50, 5
that is, the
outcome of one trial did not predict the outcome of a subsequent trial. Male 9D’s fertilization successprovides a prototypical example. He successfully inseminated eight females over 4 months. The percentages of eggs fertilized from these eight
Cc)
trials were: 0, 75, 9, 0, 56, 44, 40, 0. The same result was obtained when pairs of trials involving
the same female but different males were com-
J
pared (r,=0.17,
P=O.3;
N=23
females, 52 trials
and 29 comparisons). Figure 1. Percentage of eggs laid during the 11 days
following a single insemination that were fertilized,
(e)
0 l-9
10-19 3&39 5&59 7&79 9&99 2&29 4&49 60-69 8049 100
Percentage of eggsfertilized
showing the number of inseminations (trials) producing the different percentages. Outcomes of 100% fertilized would not be expected because the day 1 egg (collected the morning of the day after mating, but usually laid on the day of mating) cannot be fertile. (a) Results for 119 confirmed inseminations in experiment 1 (home cage mating trials). Median % fertilized was 29% (45% excluding trials yielding no fertilized eggs). (b) Trials in experiment 1 in which the female voided foam I 1 min after insemination (see Table I). (c) Results for 53 putative inseminations in experiment 2 (large testing cage mating trials). Median % fertilized was 20% (50% excluding trials yielding no fertilized eggs). (d) Trials in experiment 2 in which the female voided foam < 1 min after putative insemination (see Table I). (e) Forced copulation trials from experiments 1 (m) and 2 (0). In experiment 1, 63% of these trials resulted in at least one fertilized egg; in experiment 2, 60% of these trials resulted in at least one fertilized egg. Median % fertilized was 23% (55% excluding trials yielding no fertilized egg) for experiment 1; medians for experiment 2 were 22% and 40%, respectively.
Adkins-Regan:
Japanese quail fertilization
1409
Table 1. Voiding of foam by the female and fertile eggs laid
Experiment 1 Voided 2 I min* All other trials Experiment 2 Spontaneous void7 Induced voidt All other trials Experiment 3a Voided I 1 min* All other trials Experiment 3b Voided 2 1 min* All other trials
No. of trials
% Trials with a fertile egg
% Trials with > 50% fertile
Median % eggs fertile
20 99
65 68
5t 33
12 (33)§ 30 (50)
6 8 39
83 63 51
33 25 23
43 (50) 35 (50) 9 (50)
5 26
80 13
20 38
40 (45) 35 (56)
5 21
60 85
0 26
25 (36) 44 (45)
*Female voided foam 1 min or less after insemination. tFemale voided foam during the mating trial (spontaneous, < 1 min after putative insemination) or while being carried back to her home cage (induced). $x2=5.23, PcO.05 compared with all other trials in experiment 1. DMedians in parentheses exclude trials yielding no fertile eggs.
The correlation between trial outcomes (percentage of eggs fertilized) was high, positive and significant, however, when pairs of trials involving the same male and the same female were” compared (r,=0.83, WO.02; N= 10 birds of each sex, 20 trials and 10 comparisons). Thus fertilization successis predicted by the particular male-female combination but not by either the male or the female alone. Another predictor of fertilization successwas whether the female voided foam shortly after insemination (Fig. lb and Table I). If the female voided foam 1 min or less after insemination, there was a significantly lower probability that more than half the eggs would be fertilized. For three of these 20 trials, a sample of the voided foamy material was examined under a microscope; all three samples contained motile sperm. Eight (5%) of the 119 trials in Fig. la were forced copulations (also shown separately in Fig. le). The distribution of outcomes after these copulations was similar to the distribution for other trials (most of the trials in Fig. la were unforced copulations), and no measures differed significantly when forced copulation trials were compared with other trials. Females were not more likely to void foam following forced copulations than other copulations, but the small number of forced copulations made it difficult to test such a hypothesis (that females might be more
likely to void an ejaculate following forced copulation) adequately. Following 18 (12%) of the 146 total trials in experiment 1 (18 trials that are not shown in Fig. l), no foam was seen in the female, even though the male’s behaviour seemed to indicate cloaca1 contact with ejaculation. Only five (28%) of these 18 trials resulted in any fertile eggs (x2=8*72, PcO.01, compared to 67% of the 119 trials in Fig. la for which foam was seen in the female). If these 18 trials are added to the 119 trials where females had visible foam (N= 137), the overall percentage of trials yielding fertile eggs was 62%, compared to 67% for the 119 trials in Fig. la, a nonsignificant difference. Thus a few successful inseminations occur without visible foam in the female, possibly because the foam has been placed too high in the oviduct to be seen by cloaca1 examination, but the incidence of these errors (failures to correctly detect successful insemination) is low, trivially so with a large sample size. For the remaining nine of the 146 experiment 1 trials (trials not shown in Fig. l), foam was seen on the female’s exterior (usually the cloaca1 lip) instead of or in addition to its presence in the proctodeum. Only two of these trials (22%) yielded fertile eggs (x2= 5.54, P
1410
Animal
Behaviour,
back of the female while making cloaca1contact, resulting in incomplete or interrupted ejaculations. Two possible explanations for the appealante of foam on the female’s exterior are either that the male places foam in the female first, and then semen, or that depositing a reduced amount of foam in the proctodeum lowers fertilization success.Experimental reduction of foam quantity has no effect on fertilization success (Seiwert 1994), suggesting that the first explanation is more likely. Because trials ending in foam on the exterior of the female had lower egg fertility, such trials were not included in the analyses in the subsequent experiments.
EXPERIMENT 2: MATING BEHAVIOUR IN A LARGE NEUTRAL CAGE
Mating trials in experiment 1 were conducted in the female’s home cage. These are standard quail housing cages, but because they are rather small (0.01-0.02 m3) they do not permit the full range of social behaviour during a mating encounter. Such behavioural restriction may have contributed to the large number of unfertilized eggs. The brief cloaca1manipulation of the female, while less invasive than lavage, may also have influenced the results, for example, by artificially stimulating oviducal or cloaca1 movement. Some females who voided foam did so while being examined. Therefore in experiment 2, I conducted mating trials in a large neutral cage, and the criteria for insemination were behavioural observations refined in the course of experiment 1 rather than examination of the female’s proctodeum. In addition, I recorded the behaviour patterns of the male and female during the mating trial. Procedures
I conducted 53 mating trials in which observation of behaviour indicated cloaca1contact with probable ejaculation with 22 males and 3 1 females in a 2-m” hardware cloth cage. As in experiment 1, subjects were sexually inexperienced at the time of their first mating trial. For each trial, I introduced the female first, then added the male within 10 s and removed him 30 s to 1 min after ejaculation,
50, 5
Table II. Behaviour recorded in experiment 2 Male
behaviour
Latency (s) to first head grab Number of struts before ejaculation Number of struts after ejaculation Number of feather fluffs Number of growls Ejaculation, judging from male’s behaviour (yes/no) Female
hehaviour
Run from the male’sinitial approach (yes/no) Approach to within 4 cm of the male (yes/no) Peck male (yes/no) Receptivity (1 = definitely unreceptive, 2 =slightly but not fully receptive, 3=fully receptive). A fully receptive female squats and does not locomote
during attempted copulation Number of feather fluffs Voiding of foam during the trial (yes/no). If the female voided foam while being carried back to her home cage, this was noted separately
allowing a slightly longer post-ejaculatory interaction than in experiment 1 but still preventing a second ejaculation. Females were then returned to the home cage. I conducted no cloaca1 examination, but closely monitored females for voiding of foam not only during testing but also during transport back to the home cage, and also checked the testing cage itself and the floor beneath for voided foam. Beginning from the introduction of the male until his removal, I recorded behaviour patterns from both sexes with a computerized event recorder (Table II). Results and Discussion
As in experiment 1, fertilization success was highly variable; i.e. a substantial number of inseminations did not fertilize any eggs (Fig. lc). The distributions in Figs la and ic are quite similar, and the results for the two experiments did not differ on any measure. Becausethe behavioural criteria for insemination do produce some errors, a few of the trials that failed to produce fertile eggs may have been unsuccessful inseminations. The percentage of trials with no fertile eggs was in fact slightly higher for experiment 2 (43% compared with 33% in experiment l), but this difference is not significant, and the error rate for the behavioural criteria is unlikely to account for most or all of the trials without fertile eggs. Thus insemination fails to guarantee fertilization and
Adkins- Regun: Jupanese yuuil fertilixtion
Jmost never fertilizes all of the eggs laid even yhen mating occurs in a more spacious setting that allows a richer array of social behaviour. In experiment 2, unlike experiment I, voiding of foam by the female did not reduce the number of fertile eggs laid (Fig. Id and Table I). Forced copulations occurred in five (9%) of the 53 trials (Fig. le). As in experiment 1, these trials did not differ from other trials according to any outcome measure. One female behaviour did predict fertilization success.If the female ran from the male’s initial approach, copulation was less likely to result in any fertile eggs, that is, there were more trials with 0% eggs fertilized (13 of 2 1 trials, or 62X, with 0% eggs fertilized if the female ran versus 9 of 32 trials, or 28X, if the female did not run; x2=4.65, PcO.05). If trials with no fertilized eggs are excluded, then the percentage of eggs fertilized did not differ for trials with females that ran (median 43%) versus trials with females that did not run (median 50%), suggesting that it is the process underlying whether any eggs are fertilized, rather than the process determining how many are fertilized, that is predicted by this behaviour. These females were not necessarilyunreceptive once the male began head-grabbing, nor did they continue to run from the male. Neither receptivity itself nor any of the other behavioural measures was related to fertilization success.Experienced females (those with one or two previous trials) were somewhat lesslikely than inexperienced females to run from the male’s approach (running from the male occurred in 11 of 33 trials with experienced females, compared to 10 of 20 trials with inexperienced females), but this difference was not significant (x1=0.83, DO.3). nor was experience by itself related to any outcome measure, consistent with the lack of any effect of experience in experiment 1.
EXPERIMENT 3: TIME OF DAY AND OVIPOSITION STATUS In experiments 1 and 2 females mated in the afternoon when they are more receptive and have not yet oviposited. Thus it is possible that the large number of inseminations producing no fertile eggs reflects interference with fertilization successby a hard-shelled egg in the uterus. Experiment 3 examined the fertilization successof single
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inseminations as a function of time of day and of time relative to oviposition. Procedures In experiment 3a, I conducted 31 trials with insemination with 22 females and 14 males as described in General Methods, 15 between 0830 and 0930 hours (morning matings) and 16 between 1530 and 1730 hours (afternoon matings). All afternoon matings occurred prior to oviposition. In experiment 3b, I conducted 32 trials with insemination with 32 females and 25 males in the afternoon (between 1500 and 1830 hours) either before (18 trials) or after (14 trials) oviposition. A female that laid an egg mated as soon as possible after oviposition and never more than I.5 h after oviposition. Within 5 min of this mating another female that had not yet oviposited also mated, so that pre-oviposition and postoviposition matings would be matched for time of day. In both experiments, all females that had not yet laid were palpated to confirm the presenceof a hard-shelled egg in the uterus, and whenever possible, the exact time of subsequent oviposition was determined. In both experiments, insemination was confirmed by examination of the female’s proctodeum for foam. Results and Discussion In experiment 3a the median percentage 01‘ fertile eggs (including trials with no fertile eggs) tended to be somewhat lower following late afternoon inseminations compared with morning inseminations, but this difference was not signifcant (C/=75, PcO.1; Table 111). Thus, although males might be lesslikely to succeedat inseminating females in the morning (because of the females’ lower receptivity), a successfulinsemination at this time would not be less effective at fertilizing eggs. In experiment 3b, there were no differences on any measures between outcomes of preoviposition versus post-oviposition inseminations. Of the eight trials where a female oviposited less than 3 h after mating, four resulted in fertile eggs. including one mating that occurred only 15 min before oviposition. Clearly an insemination can fertilize future eggs even when a hard-shelled egg is present in the lower reproductive tract. In experiments 3a and 3b, as in experiment 2. there were no significant differences between trials
Animal
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Behaviour,
50, 5
Table III. Fertile eggs laid as a function of time of day or time relative to oviposition Mating time Experiment 3a Morning Afternoon Experiment 3b Pre-oviposition Post-oviposition
No. of trials
% Trials with a fertile egg
% Trials with > 50% fertile
Median % fertile*
15 16
87 63
47 25
50 (56) 16 (33)t
18 14
83
28
19
14
40 (45) 18 (35)
*Medians in parentheses exclude trials yielding no fertile eggs. tPcO.1.
followed by voiding of foam and other trials (Table I). As in experiment 1, the likelihood that more than 50% of the eggs would be fertile was lower if the female voided foam, but this difference was not significant even when the results of experiments 3a and 3b were combined for analysis.
organization (Lott 1991). The sexually dimorphic foam gland, unique to the Coturnix genus, also hints at a genetically non-monogamous mating system. The order Galliformes includes several species with lek mating systems.It would be interesting to know how the fertilization success of single inseminations in these speciescompares with the results for Japanese quail in the present study. If GENERAL DISCUSSION successis similar (that is, relatively low), then it is highly unlikely that females visit the lek only once. It has been proposed that one insemination is Alternatively, greater selection for fertilizing sufficient to fertilize an entire avian clutch, even a ability of single inseminations in lekking species large clutch (Lake 1975; Birkhead 1988). The may have produced important speciesdifferences results of these experiments suggest that this is even within the order Galliformes (Lake 1975). highly unlikely in Japanese quail. The clutch size Forced inseminations had the same fertilization is 8-12, yet only eight (3%) of the 233 successful successas other inseminations even though male inseminations (confirmed or putative) in these quail lack a developed phallus. Comparable data experiments fertilized as many as eight eggs. The are not available for other species, although results also suggest caution in assuming that any forced copulations by ducks, which do have a eggs have been fertilized by a single mating, even phallus, can fertilize eggs (Burns et al. 1980). The if insemination has occurred and even in the success of forced copulation by male quail absence of competition from another male’s suggests that this behaviour could be part of a secondary or alternative reproductive strategy for sperm. The copulatory pattern of the male Japanese males of this species. Such successalso indicates quail is multiple ejaculation (Birkhead & Msller that females are not rejecting these ejaculates (see 1992; terminology of Dewsbury 1979). This below), but females would not always be expected behaviour pattern is consistent with the relatively to evolve counter-strategies to forced copulation low fertilization successof single inseminations (Westneat et al. 1990). and rhay be a male adaptation to ensure that the Whether the female had a hard-shelled egg in entire clutch does get fertilized (Hunter et al. her uterus had surprisingly little influence on the 1993). ability of an insemination to fertilize future eggs. The mating system of wild Japanesequail is not Yet evidence from other speciessuggeststhat the yet known. While some reports suggest monoga- female’s location in her oviposition and ovulation mous pairings (Nichols 1991), there are also cycle should affect fertilizability (Birkhead 1988). anecdotal reports of non-monogamous pairings In turkeys, MeIeagris gallopavo, an egg in the (Johnsgard 1988), and other speciesof quail show oviduct reduces sperm entry into the sperm considerable within-species variability in social storage tubules (SSTs; Bakst 1994). In chickens,
Adkins-Regan:
Japanese quail fertilization
Gallus gabs, artificial insemination from 1 h before to 1 h after oviposition results in a lower percentage of SSTs with spermatozoa than artificial insemination at other times (Brillard 1993), and in muscovy ducks, Cairina moschata, artificial insemination results in lower fertilization if carried out 4 h before to 1 h after oviposition than when carried out at other times (Raud & Fame 1990). In Bengalese finches, Lonchura striata, insemination time relative to ovulation influences how many sperm are stored (Birkhead 1992). Cheng et al. (1989) pointed out that female quail differ from many other birds in laying in the late afternoon rather than the early morning, and therefore are more likely to be copulating with a hard-shelled egg in the uterus. They hypothesized that one function of the male quail’s foam might be to hold sperm in the proctodeum and thus allow fertilization to occur even with a hard-shelled egg in the uterus. The present results confirm that such fertilization does occur, but further work would be necessarybefore concluding that it is the foam that permits fertilization with a hard-shelled egg in the uterus. For example, it would be interesting to know whether fertilization just before oviposition is possible in other avian species, which lpck foam. What are the likely mechanisms underlying the bimodal distribution of fertilization successoutcomes (Fig. l)? Whether any eggs are fertilized may depend on such related variables as how well the male and female cloacae are apposed, how many sperm are ejaculated, where they are placed in the female’s tract, and whether any of them reach the SSTs. How many eggs are fertilized (if any eggs are fertilized) may depend on how many sperm are stored, in which SSTs, and how and when they are releasedfrom the tubules (Birkhead & Merller 1992; Birkhead & Fletcher 1994). In both chickens and quail, stored sperm are released continuously and in a declining manner (Wishart 1987; Birkhead & Fletcher 1994) but the number of fertile eggs laid by quail is not determined solely by the rate of decline, because a fertile egg can follow one or even several infertile eggs. These intervening processesbetween ejaculation and fertilization introduce some elements of randomness into the outcome which may account for some of the characteristics of the data in Fig. 1. They also present possibilities for the female to
1413
intluence the outcome and thus control the paternity of her offspring (Birkhead & Mallet 1992; Birkhead et al. 1993; Oring et al. 1993). Such influence could arise through her behaviour (for example, how she positions her cloaca and oviduct during copulation), her oviducal physiology and anatomy (for example, regulation of sperm storage and release), and her gametic or genetic compatibility with the male (for example, selection by the ovum for individual sperm). In both passerinesand galliforms, only a very small percentage of sperm actually gets stored in the SSTs, and so statistically there is clearly very strong sperm selection in the female reproductive tract (Birkhead 1992; Briskie & Montgomerie 1992; Brillard 1993; Birkhead & Fletcher 1994). Birkhead et al. (1993) referred to the ‘hostility’ of the female tract to sperm and proposed that it is a form of adaptive sexual selection. Thus the low fertilization success of single inseminations may reflect substantial sperm selection by the females, who did not get to choose the males, rather than a neutral character of no cost to the female because of the male’s multiple ejaculation mating pattern. Two other features of the results also suggestthat some form of selection by the female could be occurring based on sperm acceptance or rejection. First, females that ran from the male’s initial approach were lesslikely to lay fertile eggs following insemination, as if running indicates behavioural rejection of the male and can be followed through with physiological rejection of his sperm. Second, fertilization success was highly correlated when females mated with the same male twice, but was uncorrelated when females mated with different males. This feature of the results could reflect anatomical, physiological, or genetic compatibility between the male and female, or male adjustment of sperm numbers according to his mate preferences, in addition to or instead of female preference/ rejection. Voiding of sperm by the female shortly after insemination is another potential mechanism for female selection (Wagner 1991; Birkhead & Msller 1992), but the present results lend only weak support to this hypothesis. In only one of the three experiments did voiding of sperm significantly reduce the number of fertile eggs laid, and some of these voidings may have been induced by examining the female’s proctodeum. Because sperm are present in the voided material, it is
Animal
1414
Behaviour.
likely that there is at least some small effect of sperm voiding, but such an effect could be difficult to detect if other mechanisms for rejection are simultaneously operating. There is good evidence for post-copulatory sperm selection or rejection by female insects (Eberhard 1985; LaMunyon & Eisner 1993), a phenomenon that has been termed ‘cryptic female choice’ (Thornhill 1983). The hypothesis that female birds engage in post-copulatory selection, and can thereby escape male detection and retaliation, deservesserious investigation in light of the present results. As Eberhard (1985) put it, ‘females, because fertilization takes place within their bodies, generally have the last say in reproduction’ (page 107).
ACKNOWLEDGMENTS
I thank Mary Ascenzi for technical assistanceand Robert Johnston for reviewing the manuscript. Supported by NSF grant BNS 8809441.
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