Behavioral Ecology Advance Access originally published online on March 7, 2008
Behavioral Ecology 2008 19(4):695-702; doi:10.1093/beheco/arm158
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Polyandry, sperm competition, and reproductive success in mice
Centre for Evolutionary Biology, School of Animal Biology (M092), University of Western Australia, 35 Stirling Highway, Nedlands, Western Australia 6009, Australia
Address correspondence to R.C. Firman. E-mail: rcfirman{at}cyllene.uwa.edu.au.
Received 8 May 2007; revised 4 December 2007; accepted 5 December 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Previous studies have attempted to assess the reproductive benefits of polyandry in mammals but have failed to account for variation in mating frequency across experimental treatments. Recently, it has been shown that multiply sired litters are common in natural populations of house mice, suggesting that female mice are actively polyandrous and sperm competition occurs in this species. Here we show that female house mice mated to 3 males (polyandry) had greater postbirth pup survival than females mated 3 times to the same male (monandry). Remote behavioral observations revealed that copulation frequency did not differ across our experimental treatments. We discuss this result with reference to genetic benefit hypotheses for the evolution of polyandry. Furthermore, by genotyping parents and offspring in the polyandrous treatment, we assessed which male reproductive traits contributed to paternity. We found that the first principal component describing variation in 10 male reproductive variables explained a significant proportion of the variation in paternity when males were second to mate. This component was weighted predominantly by measures of sperm length, suggesting that males with short sperm may be more successful when engaging in a disfavored role of sperm competition.
Key words: genetic incompatibility, intrinsic sire effects, multiple mating, offspring viability, paternity, sperm size.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Female reproductive success is typically constrained either by the number of ova that can be produced or by the number of offspring that can be supported during gestation and/or following parturition. A single mating should therefore be adequate to maximize female fitness (Bateman 1948
; Trivers 1972). Nevertheless, mating with multiple males (polyandry) is a taxonomically widespread mating pattern (Smith 1984; Birkhead and Møller 1998; Simmons 2001), and evidence of multiple paternity within litters suggests polyandry is a common mating strategy of mammals (Berteaux et al. 1999; Kraaijeveld-Smit, Ward, and Temple-Smith 2002; Dean et al. 2006; Holleley et al. 2006). However, because females incur substantial costs from acquiring and copulating with multiple males (Daly 1978; Arnqvist and Rowe 2005), accounting for the evolution and maintenance of polyandry presents a complex problem in evolutionary biology.
Where females do not benefit directly from polyandry, they may solicit multiple mates to accrue indirect genetic benefits for their offspring (Yasui 1998; Jennions and Petrie 2000). It has been suggested that females may seek multiple mates to generate competition between the ejaculates of rival males (Parker 1970). If sperm competitiveness and offspring fitness were genetically correlated, enticement of sperm competition could ensure that males of high genetic quality fertilize a female's ova and enhance the viability of her offspring and/or the sperm competitiveness of her sons (Keller and Reeve 1995; Yasui 1997). Alternatively, the genetic incompatibility hypothesis argues that polyandry provides an opportunity for females to bias paternity toward the most compatible male genotype and avoid reproductive failure (Zeh JA and Zeh DW 1996, 2001; Tregenza and Wedell 2000).
There are an increasing number of studies of invertebrates that provide evidence for genetic benefits associated with polyandry. Polyandrous females have been reported to produce embryos of higher viability than monandrous females (Zeh JA and Zeh DW 1997; Tregenza and Wedell 1998; reviewed in Simmons 2005), and there is evidence to suggest that both intrinsic sire effects (García-González and Simmons 2005) and genetic incompatibilities (Tregenza and Wedell 2002; Evans and Marshall 2005) can account for variation in embryo viability. Vertebrate taxa have also been shown to accrue benefits from polyandry (Madsen et al. 1992; Fisher, Double, Blomberg, et al. 2006). However, the experimental investigations of polyandry using mammals have yielded mixed results; in some species, polyandrous females were reported to have increased reproductive success (Hoogland 1998; Keil and Sachser 1998; Fisher, Double, and Moore 2006), whereas others reported no effect (Schwagmeyer 1985; Wolff and Dunlap 2002). Unfortunately, these studies are mostly confounded by variation in the number of mating events, with polyandrous females receiving more copulations than monandrous females. This problem has been surmounted in studies of invertebrates by using an experimental protocol in which the number of copulations are experimentally controlled (Tregenza and Wedell 1998; reviewed in Simmons 2005).
An inevitable consequence of polyandry is that sperm from rival males will compete to fertilize available ova (Parker 1970). A male's paternity will be determined both by his success in sperm competition (Parker 1970) and by any bias in sperm use that the female may impose (Eberhard 1996). Polyandry is thus predicted to favor ejaculate characteristics in males that provide them with a fertilization advantage. Comparative and correlational studies of different taxa suggest that polyandry and sperm competition have been important selective pressures on the evolution of testis size and sperm number (Harcourt et al. 1981; Moller and Briskie 1995; Stockley et al. 1997), sperm size and form (Gomendio and Roldan 1991; Gage 1994; Anderson and Dixson 2002; Byrne et al. 2003), and accessory gland size (Ramm et al. 2005).
Behavioral observations indicate that female house mice are actively polyandrous (Rolland et al. 2003) and will mate with both dominant and subordinate males (Busser et al. 1974; Oakeshott 1974). The frequency of mixed paternity in litters from natural populations of house mice has been shown to be approximately 23% (Dean et al. 2006). However, the incidence of multiple mating was estimated to be much higher (45–70%), suggesting that sperm competition is common in this species (Dean et al. 2006). Laboratory experiments can restrict natural precopulatory mate choice decisions that play a major role in house mouse reproductive ecology (Potts et al. 1991). Nevertheless, polyandry creates opportunities for postcopulatory sexual selection, and controlled conditions are essential for manipulative mating experiments designed to investigate postcopulatory mechanisms (Kraaijeveld-Smit, Ward, Temple-Smith, and Paetkau 2002). The aim of this experiment was to assess postcopulatory processes associated with polyandry in house mice. We compare the reproductive success of females mated to 3 different males (polyandry) and females mated 3 times to the same male (monandry). Moreover, we use microsatellite markers to assign paternity to females mated polyandrously and use paternity data to determine which male reproductive traits influence success in sperm competition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Outbred, wild-derived mice were received from a colony held at the Animal Resources Centre (ARC). At the ARC, the colony is maintained via a random mating design that minimizes inbreeding and maximizes genetic variation (Poiley 1960
). Animals were outbred for a single generation and the offspring used in this experiment. Mice were maintained in a controlled temperature room at 25 °C with a reversed 14:10 light:dark regime. Mice were housed in cages (12 x 12 x 30 cm) lined with chaff. Rodent feed and water were provided ad libitum. Nesting females were left undisturbed for 2 weeks following parturition (except to top up feed and water). All animals experienced the same husbandry routine throughout the duration of the experiment.
Mating design
At 8 weeks of age, 60 virgin females and males were randomly assigned to either a monandrous (same male) or a polyandrous (3 different males) mating treatment. In natural populations of house mice, a maximum of 2 sires per litter was observed (Dean et al. 2006). However, estimates of multiple paternity often underestimate the frequency of multiple mating (Dean et al. 2006). Consequently, females in the polyandrous mating treatment were mated with 3 mates. In the monandrous treatment, there were 30 pairs (1 female and 1 male), and in the polyandrous treatment, there were 10 blocks of 3 females and 3 males. In the polyandrous treatment, the same 3 males mated to 3 different females and each male had the opportunity of mating first, second, or third. Male mice can become sperm depleted when they mate with more than one female on a given day (Huber et al. 1980). Therefore, males in the polyandrous treatment were rested for at least 24 h after mating. Because females will only mate when in estrus, they had to be offered their 3 mates on the same day. As such, we could not rest males used in the monogamous treatment so that these females may have received less total ejaculate.
Matings were conducted in mouse cages (12 x 12 x 30 cm) during the dark phase under a red light. Matings were initiated at the onset of the dark phase with the introduction of a female into a male's home box. Females were checked regularly (every 2 h) to detect estrus (Champlin et al. 1973). When females were in estrus, they were inspected half-hourly for the presence of a mating plug. We used the presence of a mating plug as an indicator of a complete, successful mating event (Rugh 1968). Mating plugs can be dislodged by subsequent mates when females have mated during postpartum estrus (Dewsbury 1988); however, it is not known whether mating plugs inhibit subsequent copulations during normal estrus periods. Because we were investigating postcopulatory processes associated with polyandry (and not the efficiency of males to dislodge mating plugs), we removed plugs between mating events to ensure that subsequent copulations were not obstructed. Plugs were removed by gently pressing the female against the side of the handling bin and dislodging the plug with a blunt probe. Although plug removal may affect sperm transport within the female tract, mating plugs were removed after all mating events and in both experimental treatments. Thus, all animals were treated identically. Importantly, plug removal has no impact on litter size, indicating that conception is unaffected by this procedure (Firman RC, Simmons LW, unpublished data).
In the monandrous treatment, once a mating plug was observed, it was removed and the female was then repaired with her mate. This was repeated once a second plug was observed. Once a third plug was observed, it was removed and the female placed in a clean box with shredded newspaper for nesting. In the polyandrous treatment, when females had mated with the first male, the plug was removed and the female was introduced to her second mate. Once a second mating was achieved, the plug was removed and the female was placed with a third male for her final mating. Again, after her third mating, the plug was removed and the female placed in a clean box with shredded newspaper for nesting. In summary, all polyandrous females had 3 different mating partners, mating once with a previously unmated male, once with a male that had mated once previously, and once with a male that had mated twice previously. Monandrous females mated 3 times with a single mating partner.
In a subsidiary experiment, 9 monandrous and 9 polyandrous treatments were filmed to enable remote observations of copulatory behavior. Matings were conducted exactly as described above; however, modified wire lids were used to allow a clear view of each pair. From hour 3 of the dark phase, an overhead camera (night vision) recorded 6 mating pairs simultaneously. As in the main experiment, females were checked regularly for estrous condition. Once a mating plug was observed, it was removed and females were then paired with their same (monandrous) or next partner (polyandrous). Once a second mating plug was observed, it was removed and females were paired for their final mating. Playback of the video footage allowed us to score the number of intromissions and thrusts within each mating event and the duration of each mating event. Intromissions were scored when a male successfully mounted the female and exhibited pelvic thrusts. Ejaculations were characterized by an insertion with no thrusting (Dewsbury 1984).
In both the main experiment and the subsidiary behavioral observations, mating sessions lasted between 1.5 and 6 h, with mating usually beginning around hour 7 of the dark phase. In mice, ovulation usually occurs at approximately the midpoint of the dark phase (hour 5) (Rugh 1968). Therefore, it was assumed that all females had ovulated prior to mating.
Reproductive success
Female boxes were checked for pups beginning 18 days after mating. Litter size (number of pups) was recorded at birth. At the time of weaning (21 days after birth), or at 22 days after mating without producing a litter, females were euthanased by intraperitoneal injection of Euthal and the reproductive tract removed and preserved (10% buffered formalin saline). Ovaries were embedded in paraffin wax and serial sectioned (10 µm). The number of corpora lutea on each ovary was determined by observing every 10th section and following individual corpora lutea through the ovary. The total number of corpora lutea represented the number of ova shed during the cycle in which the female became pregnant (Stockley 2003).
Prebirth reproductive success was calculated as the proportion of ova that were fertilized and resulted in births (litter size/ova number). Postbirth reproductive success was determined as the proportion of pups that survived to weaning (number of pups weaned/litter size).
In comparing the reproductive success of monandrous with polyandrous females, we analyzed 5 reproductive variables: prebirth success, the length of gestation, litter size at birth, postbirth success, and pup weight at weaning. In considering type II errors, we follow the recommendations of Nakagawa (2004) and present effect sizes (Cohen's d) and their 95% confidence intervals (CIs) in assessing the strength of the effects we observe. Noncentral CIs were iterated using the SPSS script for the F distribution provided by Smithson (2001).
Paternity analysis
Animals in the polyandrous treatment were genotyped to assign paternity (number of litters = 23, number of pups = 115). DNA was extracted from approximately 1 mm3 of ear tissue using the Easy DNA high-speed extraction kit (Fisher Biotec, Subiaco, Australia). We genotyped animals at 4, 6, 8, or 10 microsatellite loci (D1Mit17, D2Mit1, D4Mit1, D6Mit138, D10Mit14, D11Mit4, D13Mit1, D14Mit132, D16Mit1, and D18Mit17) (Dietrich et al. 1992; Blouin et al. 1996; Dean et al. 2006). Labeled forward primers were obtained from Geneworks (Adelaide, Australia) (FAM) and Applied Biosystems (Melbourne, Australia) (NED, PET, and VIC) and unlabeled primers from Geneworks. Single 10-µl reactions were run in a PTC-0200 DNA Engine (Geneworks) and contained 1x polymerase chain reaction (PCR) buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl) (Invitrogen, Melbourne, Australia), 1.5 mM MgCl2 (Invitrogen), 200 µM deoxynucleotide triphosphates (Invitrogen), 0.25 µM of forward labeled primer, 0.25 µM of reverse primer, 0.5 units platinum Taq polymerase (Invitrogen), and 
200 ng of template DNA. The thermocycling profile for all 10 loci was as follows: 5 min denature at 95 °C, 50 cycles of 90 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, followed by 72 °C for 3 min. PCR products (1.5 µl) were combined and run on a ABI 3730 sequencer, sized using Genescan-500 LIZ size standard, and genotyped using Genemapper software (v3.0).
Paternity was unambiguously assigned for 97 pups by manual exclusion. For 18 pups, paternity could not be unambiguously assigned after screening 10 loci. Therefore, paternity was assigned via a maximum likelihood approach using Cervus (v2.0) (Marshall et al. 1998). These families were analyzed with a known parent (mother) and 3 candidate parents (sires). Cervus assigned paternity with >95% confidence for 13 pups and >80% confidence for 5 pups.
Male reproductive traits
Following their final mating, males were euthanased and frozen. Males were later thawed and the reproductive organs and accessory glands removed for measurement. Measurements included weight of the testes, seminal vesicles, coagulating gland, and prostate gland; total epididymal sperm number; sperm head length, sperm midpiece length, sperm tail length; and total sperm length.
Sperm was retrieved from the epididymis by repeatedly cutting the cauda epididymis with fine scissors and placed in an Eppendorf tube with 1 ml of dH2O. The tube was vortexed for 2 min, and an aliquot of the sperm suspension was preserved in a 4% formaldehyde solution. Sperm counts were obtained by counting sperm cells in an improved Neubauer hemocytometer. Total epididymal sperm number was determined by taking the mean of 2 counts and incorporating the dilution factor. Aliquots of sperm solution (20 µl) were smeared on slides, air-dried, and stained with Coomassie brilliant blue (0.22% Coomassie blue, 50% methanol, 10% acetic acid, and 40% dH2O) (Larson and Miller 1999). Images of stained sperm were taken at x400 and x1000 magnification using Image Pro Plus (v4.5). Initially, 5 sperm per male were measured using the image analysis application ImageJ (v1.32). All sperm measurements were highly repeatable (P values < 0.001, repeatability estimates > 0.891). Nevertheless, we doubled the sample size of sperm measured (10 sperm per male) because this yielded greater repeatability (P values < 0.001, repeatability estimates > 0.940). Freezing and/or vortexing sperm in dH2O could potentially generate hypo-osmotic shock and impact our measures of absolute sperm morphology. To examine the reliability of our procedures, we compared the sperm measures we obtained using the above method, with measurements made on sperm from fresh semen samples. Fresh semen samples were obtained for 28 nonexperimental males immediately after they were euthanased. Sperm were obtained from the epididymis as above, vortexed in an isotonic sperm medium (modified Tyrode's solution, Murase and Roldan 1996), and then treated exactly as per sperm from our experimental males. There was no difference in sperm head length (mean: fresh = 8.19 µm, frozen = 8.26 µm; F1,54 = 1.017, P = 0.318), sperm midpiece length (mean: fresh = 22.07 µm, frozen = 22.24 µm; F1,54 = 2.069, P = 0.156), or sperm tail length (mean: fresh = 114.73 µm, frozen = 115.58 µm; F1,54 = 2.899, P = 0.094) for samples prepared using frozen material and those prepared using fresh material.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Reproductive success
Analysis of video footage from the subsidiary experiment revealed that there was no difference in the number of intromissions received by females in the 2 mating treatments. Data were analyzed with a single-factor (mating treatment) repeated measures (the 3 sessions on which each female was exposed to a male) analysis of variance (ANOVA) (mating treatment: F1,16 = 0.010, P = 0.920; mating session: F2,32 = 0.329, P = 0.722). The same was true of the number of thrusts (mating treatment: F1,16 = 0.080, P = 0.781; mating session: F2,32 = 0.851, P = 0.437) received by females in the 2 mating treatments and the duration of mating events (mating treatment: F1,16 = 0.063, P = 0.805; mating session: F2,32 = 0.080, P = 0.923). Males ejaculated only once in each mating event. Therefore, mating frequencies were consistent across the polyandrous and monandrous treatments.
In the main experiment, of the 30 mated females in each treatment, 23 monandrous females and 24 polyandrous females produced litters. Length of gestation (F1,45 = 0.102, P = 0.750, d [95% CI] = 0.016 [0.000, 0.634]), litter size at birth (F1,58= 0.262, P = 0.610, d [95% CI] = 0.035 [0.000, 0.772]), and pup weight at weaning (F1,41 = 2.587, P = 0.115, d [95% CI] = 0.414 [0.000, 1.965]) did not differ between treatments. A preliminary repeated measures ANOVA confirmed that there was no significant block effect in the polyandrous treatment on prebirth (litter size/ova produced) or postbirth (pups weaned/litter size) reproductive success (P values > 0.990, degrees of freedom = 28,23), and so data for each female were treated independently in our comparison with the monandrous treatment. To analyze variation in litter size at birth and thus prebirth reproductive success, we used a generalized linear model (GLM) with a logit link function and binomial error distribution with number of ova released as the binomial denominator. The data were overdispersed, so we estimated the dispersion parameter from the scaled deviance and present F-tests rather than 
2 (Crawley 1993). There was no effect of monandrous and polyandrous treatments on the numbers of ova produced that resulted in live births (F1,58 = 0.483, P = 0.490, d [95% CI] = 0.065 [0.000, 0.906]) (Figure 1). To analyze litter size at weaning and thus postbirth pup survival success, we again used a GLM to analyze the number of pups weaned with litter size at birth as the binomial denominator. There was a significant effect of treatment on postbirth pup survival (F1,45 = 5.916, P = 0.019, d [95% CI] = 0.901 [0.015, 2.879]). Pups born to females in the polyandrous treatment had a significantly greater probability of survival to weaning than pups born to females in the monandrous treatment (Figure 2).
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Contrasting the 95% CIs for the effect sizes of the 5 variables examined suggests that effects range from close to zero to moderate in the case of length of gestation and litter size at birth and very large in the case of pre- and postbirth survival and pup weight at weaning (Cohen 1988
). The range of effect sizes suggests that with very large samples, additional effects may have proved statistically significant, in particular pup weight at weaning.
Paternity and male traits
Due to complete reproductive failures (i.e., only 24 polyandrous females produced litters), we did not have paternity data to contrast all 30 males mating in all 3 sequence positions (first, second, and third to mate). Preliminary ANOVAs showed no significant effect of block (we had 10 blocks of 3 males competing for fertilizations with 30 females) on P1 (F9,13 = 1.07, P = 0.443), P2 (F9,13 = 1.39, P = 0.284), or P3 (F9,13 = 0.37, P = 0.93). The raw data suggest a mating-order effect on paternity (Figure 3), and to maximize sample size and thus statistical power, we conducted 2 separate repeated measures analyses, one contrasting P1 with P2 (18 males) and one contrasting P2 with P3 (18 males). Males for which we had both P1 and P2 data did not differ significantly from one another in their paternity (between subject F17,18 = 0.779, P = 0.695), which was greater when they mated first (within subject F18,17 = 5.053, P = 0.038). Males for which we had both P2 and P3 data tended to have greater repeatability of paternity in these disfavored roles (between subject F17,18 = 2.098, P = 0.064), and paternity did not depend on whether they mated second or third (within subject F18,17 = 0.249, P = 0.624).
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Pairwise correlations revealed strong covariation between male reproductive traits (Table 1). Therefore, a principal component analysis (PCA) was applied to minimize the data set and summarize the variation in the 10 traits. We extracted 2 components that collectively accounted for 52.5% of the variation in male reproductive traits (Table 2). Variables that were 0.7 times as large as the largest eigenvector were considered to have contributed significantly to that principal component (PC) (Mardia et al. 1979
). The PC1 accounted for approximately 30% of the variation and was weighted predominantly by measures of sperm size (sperm head, midpiece, tail, and total length). The PC2 accounted for 23% of the variation and was weighted predominantly by the weight of the glands (seminal vesicles, coagulating, and prostrate glands) and total epididymal sperm number.
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We looked for an effect of the 2 PCs on a male's sperm competition ability when engaging in a favored (P1) or disfavored (P2) role. We used P2 rather than P3 because these measures did not differ, and we had a larger sample of males with P2 values. A GLM of the number of pups sired with litter size as the binomial denominator and a logit link function revealed no significant effect of PC1, PC2, or block on P1 (whole model: F9,11 = 1.449, P = 0.294; again F-tests were adopted due to overdispersion of the paternity data). Similarly, PC2 (F1,10 = 2.458, P = 0.148) and block (F9,10 = 2.820, P = 0.061) did not influence P2; however, PC1 (F1,10 = 20.70, P = 0.001) did have a significant effect on P2 (whole model: F11,10 = 6.321, P = 0.003) (Figure 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Studies of house mice from seminatural and natural populations have revealed that females mate polyandrously and produced litters of mixed paternity (Potts et al. 1991
; Dean et al. 2006). Our data show that polyandrously mated females have greater postbirth pup survival than monandrously mated females. Unlike previous studies of mammals (Schwagmeyer 1985; Hoogland 1998; Keil and Sachser 1998; Wolff and Dunlap 2002), we can eliminate any influence of copulation frequency on female reproductive success. Due to the constraint of our experimental design that males in the monandrous treatment could not be rested between their successive matings, polyandrously mated females may have received more total ejaculate over their 3 mating events compared with females in the monandrous treatment. There is no evidence to suggest that female mammals receive nutritional benefits from ejaculates. Indeed, female mammals secrete specific antibodies against seminal proteins suggesting that they may be costly for females (Arnqvist and Rowe 2005). Therefore, it seems unlikely that a paternal effect driven by male sperm depletion could explain our result of decreased pup survival in litters produced by females mated monandrously. Furthermore, we can eliminate the possibility that our result was generated by husbandry practices because all females were treated identically. However, it is possible that increased pup survival in polyandrous litters could be due to differential maternal investment. It could be that females mated polyandrously deemed their offspring to be of better quality than females mated to a single male and invested more in maternal care. Unfortunately, we could not determine whether increased pup mortality in the monandrous treatment was due to lack of maternal care/infanticide or as a consequence of nonviable offspring. But is there any evidence to suggest that our finding can be explained by the acquisition of indirect genetic benefits?
Polyandry has been proposed to create an opportunity for the avoidance of incompatibilities between parental genotypes by facilitating postcopulatory mechanisms that reduce reproductive failure (Zeh JA and Zeh DW 1996). Support for this hypothesis comes from empirical (Tregenza and Wedell 2002; Evans and Marshall 2005) and comparative investigations (Stockley 2003). Interestingly, we found no effect of polyandry on prebirth reproductive success; the number of ova that were released and resulted in live pups at birth did not differ between polyandrously and monandrously mated females. Instead, we found an effect of polyandry on postbirth pup survival. It is possible that unfavorable combinations of parental genes resulted in pup death after parturition. In this case, the genetic incompatibility hypothesis could explain our result of increased pup mortality within monandrous litters. Inbreeding is often viewed as a form of genetic incompatibility and is known to reduce offspring viability (Tregenza and Wedell 2000). However, studies of mice show that inbreeding does not influence the number of young reared to weaning (Margulis 1998; Firman and Simmons forthcoming). Therefore, reduced pup survival in monandrous litters seems unlikely to be explained by effects of inbreeding. Nevertheless, we cannot exclude other forms of genetic incompatibilities, such as the disruption of coadapted gene complexes and selfish genetic elements, accounting for the result we have observed.
Enhanced reproductive success of polyandrously mated females could also be explained by an intrinsic male effect, whereby females promote sperm competition to acquire genes for their offspring that promote viability (Yasui 1997). The intrinsic male quality hypothesis assumes that genetically superior males are successful in sperm competition and sire offspring that have enhanced viability (Yasui 1997; Hosken et al. 2003; García-González and Simmons 2005). We found that the first male allowed to copulate had the advantage in gaining fertilizations. It is typical in mammals that the male mating closest to the timing of ovulation has the advantage in gaining paternity (Gomendio et al. 1998); however, the first male advantage we observed may have been emphasized by our experimental protocol. Nevertheless, we found that, collectively, the reproductive traits we measured were significant predictors of a male's ability to gain paternity when in the disfavored role of sperm competition; in particular, the PC score describing male reproductive variables was weighted most strongly by measures of sperm morphology, suggesting that disfavored males with shorter sperm were more successful in fathering offspring and thus producing mixed paternity litters. Therefore, our results could be consistent with the intrinsic male quality hypothesis if these male reproductive traits were associated with genetic quality. We are unable to distinguish between the intrinsic male quality and genetic incompatibility hypotheses with our data. Moreover, studies of female mate choice in house mice suggest that male compatibility and intrinsic genetic quality are both important components in female mating preferences, and their relative influence on mating decisions depends on the degree of variability in each trait among available males (Roberts and Gosling 2003).
Studies have shown that sperm morphology may be an important determinant of paternity (Gomendio and Roldan 1991; Stockley et al. 1997; LaMunyon and Ward 1999). Sperm competition experiments using invertebrates have shown that males with shorter sperm experience higher competitive fertilization success (Gage and Morrow 2003; García-González and Simmons 2007). Although the mechanisms by which small sperm gain a competitive advantage are unclear, it has been suggested that the evolution of sperm morphology may in part be explained by the morphology of female sperm storage organs (Briskie et al. 1997; García-González and Simmons 2007; Simmons and Kotiaho 2007). In taxa lacking sperm storage, comparative studies suggest that increased sperm length results in greater energy reserves and faster swimming speeds and thereby provides a competitive advantage (Gomendio and Roldan 1991; Balshine et al. 2001; Anderson and Dixson 2002; Byrne et al. 2003). Specifically, the size of the sperm midpiece (which contains the mitochondrial sheath and provides energy for motility) may be important in determining the outcome of sperm competition. However, we found that short sperm, including midpiece length, were associated with high P2 values. In the red deer, it has been shown that sperm with short midpieces have greater in vitro swimming velocities (Malo et al. 2006), and ejaculates with faster sperm achieve greater noncompetitive fertilization success (Malo et al. 2005). Considering the patterns of paternity observed in our experiment, it could be that mouse ejaculates containing short sperm exhibit better motility and provide males with a competitive advantage when in the disfavored role of sperm competition. Further research on house mice is necessary to investigate the relationship between sperm size and sperm swimming performance.
In conclusion, we have shown that female mice benefit from polyandry and have greater postbirth pup survival than females mated monandrously. It is now important to establish which mechanisms, maternal effects, genetic incompatibility, and/or intrinsic male quality, contribute to offspring fitness. Also, our data showed that shorter sperm confer higher competitive fertilization success, indicating that sperm morphology is important in mammalian sperm competition.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Australian Research Council (35403000); Australian Postgraduate Award (R.C.F.).
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ACKNOWLEDGEMENTS |
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This research was approved by the University of Western Australia Animal Ethics Committee (300/100/299). We thank M. Beveridge for molecular training, T. Stewart for histological sectioning, B. Roberts for performing DNA extractions, K. Taralla for mouse husbandry, and F. García-González and M. Dean for comments on the manuscript.
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