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Keywords:

  • copulation duration;
  • mating behaviour;
  • multiple intromissions;
  • repeated ejaculation;
  • sexual conflict;
  • sexual selection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

The copulatory behaviour of male mammals is characterized by striking diversity in patterns of copulatory stimulation and ejaculation frequency. We conducted comparative analyses of rodents to investigate the potential influence of sperm competition in the evolution of copulatory behaviour. We found that increasing sperm competition is associated with faster rates of stimulation and earlier ejaculation among species in which males perform multiple intromissions prior to ejaculation, but with no overall change in the number of intromissions per ejaculation. Increasing sperm competition is also associated with a higher frequency of repeated ejaculations with the same female, and with shorter refractory periods between repeated copulations. Increasing sperm competition level thus appears to select for male ability to ejaculate more rapidly and more frequently with each female mated. As prolonged copulations are known to reduce female remating rates, these findings indicate that males may experience opposing selection pressures on copulatory behaviour with respect to offensive and defensive aspects of sperm competition. We conclude that sperm competition is likely to be an important selection pressure explaining diversity in mammalian copulatory behaviour.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Sperm competition occurs when the ejaculates of two or more males compete for fertilization of a given set of ova (Parker, 1970, 1998), and is a highly influential selection pressure in the evolution of male reproductive traits across diverse taxonomic groups (Birkhead & Møller, 1998). It has been hypothesized that selection pressure on males to increase fertilization success under sperm competition can influence copulatory behaviour in a variety of ways. For example, prolonged copulation in insects can function to displace the sperm of rival males, to increase the number of sperm transferred under sperm competition, or to guard females and prevent remating (Parker, 1970; Simmons, 2001). In reptiles, prolonged copulation duration can yield multiple fitness benefits for males under sperm competition, such as increased sperm transfer and plug formation (Olsson & Madsen, 1998; Olsson, 2001). It also seems likely that the very frequent copulations observed within pairs of many bird species (up to several hundred copulations per clutch), function to increase sperm transfer and so act as a paternity guard under sperm competition risk (Birkhead, 1998).

The copulatory behaviour of male mammals is characterized by striking diversity in patterns of copulatory stimulation and ejaculation frequency (Dewsbury, 1972, 1975; Dixson, 1995, 1998; Eberhard, 1996). Copulatory patterns involving prolonged stimulation of females and multiple ejaculations are especially common in rodents, primates and felids, although selection pressures promoting diversity in copulatory behaviour among these and other mammalian taxa are poorly understood (Dewsbury, 1972, 1981, 1988; Short, 1977, 1979; Eaton, 1978; Parker, 1984; Dixson, 1995, 1998; Eberhard, 1996, 1998). Prolonged copulatory stimulation performed by male mammals most frequently takes the form of multiple penile insertions, known as intromissions, during which no sperm are transferred (Beach, 1956; Dewsbury, 1972). Differences in the number and rate of intromissions delivered prior to each ejaculation accounts for much of the variation in copulatory behaviour observed across mammalian taxa (Dewsbury, 1972, 1975; Eaton, 1978; Huck & Lisk, 1986; Dixson, 1998). The copulatory behaviour of rodents has been studied extensively in this context, and the stimulation provided by multiple intromissions is typically found to be a requirement for normal sexual function. For example, copulatory stimulation can be necessary to facilitate sperm transport within the female reproductive tract, stimulate ovulation and/or maintain pregnancy (e.g. Wilson et al., 1965; Adler, 1969; Chester & Zucker, 1970; Dewsbury & Estep, 1975; Milligan, 1975; Gray et al., 1977; Davis & Connor, 1980).

Although copulatory stimulation is often required by females for normal sexual function, experimental studies of both domestic and wild rodents in laboratory and semi-natural environments suggest that males do not always copulate as if aiming to stimulate females at an optimal pace to initiate pregnancy (e.g. McClintock & Adler, 1978; Gilman et al., 1979; Erskine & Baum, 1982; Fadem & Barfield, 1982; Webster et al., 1982; Huck & Lisk, 1985; Martinez & Paredes, 2001). Sex differences in preferred copulatory pace are particularly well documented for the Norway rat Rattus norvegicus. Numerous studies show that when female laboratory rats are able to control copulatory activity by moving to an area of a mating arena that is inaccessible to males, they prefer longer intervals between intromissions than are imposed under male control, and require fewer intromissions to initiate pregnancy (Edmonds et al., 1972; Gilman et al., 1979; Erskine & Baum, 1982; Erskine, 1985; Erskine et al., 1989; Martinez & Paredes, 2001).

Why should male rodents attempt to copulate at a pace that requires more than the minimum number of intromissions necessary to achieve successful pregnancy? One as yet unexplored possibility is that males may gain an advantage in sperm competition by increasing the pace of copulation to a level above that preferred by females. In mammals, there is evidence that male fertilization success increases with the number of sperm inseminated relative to competitors (e.g. Beatty, 1960; Stockley, 1997). Hence, an increased pace of copulation may increase fertilization success under sperm competition by allowing males to ejaculate more rapidly, and/or more frequently with a given female, thereby providing benefits of increased sperm transfer. Alternatively or additionally, males may prefer a faster pace of copulation because it enables them to increase intromission frequency prior to ejaculation, which in turn may allow them to ejaculate more sperm (Toner & Adler, 1986), or promote increased sperm transport within the female reproductive tract (Chester & Zucker, 1970; Toner & Adler, 1986).

The reason why male mammals ejaculate repeatedly with the same female is also uncertain, although several authors have suggested that multiple ejaculations may be adaptive under conditions of sperm competition (Lanier et al., 1979; Dewsbury & Hartung, 1980; Dewsbury, 1981; Parker, 1984; Ginsberg & Rubenstein, 1990; Dixson, 1995; Rice, 1998). Theoretical analyses indicate that spreading delivery of multiple ejaculates across a female's oestrus period could be beneficial if the timing of ovulation is unpredictable, and a large proportion of the sperm in each ejaculate die or are lost during the oestrus period (Parker, 1984). Under such conditions, repeated inseminations would usually result in a larger sperm population in female oviducts at the point of ovulation (Parker, 1984). Similarly, if seminal fluid proteins function to promote sperm competition success via short-term effects on female behaviour or physiology, then males might also benefit by spreading repeated ‘doses’ of ejaculate throughout the oestrus period (Rice, 1998). Alternatively, multiple ejaculations may function primarily to increase the total number of sperm transferred to a female rather than to spread the timing of ejaculate delivery (Lanier et al., 1979; Dewsbury & Hartung, 1980; Oglesby et al., 1981; Parker, 1984; Ginsberg & Rubenstein, 1990).

Here, we investigate relationships between sperm competition level and male copulatory behaviour in comparative analyses of rodents. Rodents are the largest mammalian order, and exhibit very wide diversity in copulatory behaviour, including variation in several key variables (number of ejaculations, intromissions, and presence or absence of locking) identified by Dewsbury (1972) as characterizing diversity in mammalian copulatory patterns. Rodents are also the only mammalian group in which details of male copulatory behaviour have been studied extensively, principally by Dewsbury and co-workers (see data sources in Supplementary Material), thus providing a reliable source of data for comparative analyses. Specific objectives of the present study are to determine whether increasing sperm competition level leads to: (i) a faster pace of copulation, as predicted if observed sex differences in preferred pace of copulation result from selection on males to increase intromission and/or ejaculation frequency in response to increasing sperm competition level, (ii) an increase in the number of intromissions per ejaculation, as predicted if multiple intromissions function to promote increased sperm transfer under sperm competition, and (iii) an increase in the number of ejaculations delivered to each female mated, as predicted if multiple ejaculations function to spread the delivery of ejaculates or increase sperm transfer under sperm competition. Results of the comparative analyses presented provide new insight into the selection pressures shaping diversity in mammalian copulatory behaviour.

Comparative data set

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Data on copulatory behaviour were collated for 60 rodent species (see Supplementary Material for data and sources). Most of the information included in the data set comes from studies of copulatory behaviour conducted under laboratory conditions. Such studies have two main advantages in the context of the present study. First, they permit detailed recording of male copulatory behaviour that would not be possible under natural conditions, and available evidence indicates that basic copulatory patterns of male rodents do not vary qualitatively in captivity (Dewsbury, 1975). In addition, investigations of variation in copulatory pace indicate that behaviour recorded under laboratory conditions is more likely to reflect male interests (McClintock & Adler, 1978; Erskine, 1989). This is advantageous in the present context, as we are interested in how sperm competition level may affect optimal copulatory behaviour of males, which is likely to be more apparent when female influence on copulatory pace is reduced.

As copulatory patterns often change across a series of multiple ejaculations, we have focused on data for the first ejaculatory series, to permit comparisons across species with single and multiple ejaculations. Data for wild species were used in preference to those collected for domesticated or laboratory strains where available (e.g. data for wild house mice Mus musculus and Norway rats R. norvegicus were used in preference to those for laboratory strains). A potential disadvantage of using data from laboratory studies is that variation in measures of male copulatory behaviour may arise because of differences in female oestrus condition (Dewsbury, 1990). That is, measures of copulatory behaviour can be quantitatively different when females are in natural oestrus (cycling or post-partum) compared with when oestrus is artificially induced with hormone injections, although such patterns often vary between different species (Dewsbury, 1990). In the present study, we have maximized the number of species in the comparative data set by including data for females in both natural and artificially induced oestrus. Where sources report data for the same species using females with different types of oestrus (cycling, artificial, post-partum), we have used an average of all reported values. Females in artificial oestrus are commonly used in studies of copulatory behaviour, and to exclude them from the data set would result in a prohibitively small sample size. However, as far as we have been able to determine, there is no systematic bias between oestrus type and any of the variables under investigation. Hence, variation in modes of oestrus induction are unlikely to explain relationships between sperm competition level and patterns of copulatory behaviour.

Definitions of copulatory behaviour

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Data for several standard measures of copulatory behaviour were used: (i) ejaculation number – the mean number of ejaculations achieved with a single female before attainment of some satiety criterion – usually defined as a period of 30 min with no copulations; (ii) ejaculation latency – the mean latency from the first intromission to the end of the first (or only) ejaculation; (iii) post-ejaculatory interval – the interval between the end of the first ejaculation and the beginning of the next intromission, for species with multiple ejaculations; (iv) intromission number – the number of intromissions preceding the first ejaculation; and (v) inter-intromission interval – the mean interval separating the intromissions of the first copulatory series, for species with multiple intromissions. Note that the mean inter-intromission interval is typically calculated by dividing ejaculation latency by intromission number, and is thus a measure of the rate of intromissions (Beach & Jordan, 1956). For all species in the data set, the presence or absence of a copulatory lock was noted. Species with locks were included in the main data set as having a single intromission per ejaculation. However, as this copulatory pattern may be functionally distinct from those of other species in which males typically ejaculate on a single intromission, we also repeated certain analyses with locking species excluded. Where data for copulatory behaviour variables were reported from more than one source, average values were used. In a few cases (2% of total data points), measures of copulatory behaviour were reported as ‘less than’ or ‘greater than’. For example, average ejaculation latency for Cavia porcellus is reported as less than 60 s. Rather than exclude these species from the analyses, we have included the data with an arbitrary but consistent alteration of 10% in the appropriate direction (i.e. 0.54 s ejaculation latency for C. porcellus).

Sperm competition level

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

We have used relative testes size as an index of sperm competition level, as it is well established that increasing sperm competition level selects for larger testes relative to body size and higher sperm production rates in mammals (Short, 1977, 1979; Harcourt et al., 1981; Kenagy & Trombulak, 1986; Møller, 1988, 1989; Ginsberg & Rubenstein, 1990; Preston et al., 2003). relative testes size values used were taken from Kenagy & Trombulak (1986), based on the regression equation for the relationship between log body mass and log testes mass for rodents, or calculated from the same regression equation using body and testes mass data from later published sources (Pierce et al., 1990). Using this approach, species with larger than average testes for a rodent of similar body size have a relative testes size value greater than 1, and species with smaller than average testes as those with a relative testes size value less than 1. Rattus rattus (the black rat), reported by Kenagy & Trombulak (1986) as having a relative testes size of 5.12, was an extreme outlier (approximately 3 standard deviations) in some relationships between relative testes size and copulatory behaviour. We examined scatter plots of all relationships, and excluded Rattus from analyses where it exerted disproportionate influence as an extreme outlier and/or caused violation of assumptions of the regression analyses performed using the Comparative Analysis by Independent Contrasts (CAIC) program (see below). Those cases where its exclusion affects the significance of the analyses presented are noted in the results section (Table 1).

Table 1.  Relationships between male copulatory behaviour and sperm competition level (measured as relative testes size) across rodents.
Response variableSpecies level analysesIndependent contrasts analyses
nFr2βPnFr2βP
  1. Results are for simple linear regression analyses with and without control for phylogeny (independent contrasts analyses).

  2. n, number of species or independent contrasts; r2, unadjusted r2; F, F ratio; β, slope of the regression line (forced through the origin for independent contrasts analyses); P, probability (n.s. >0.05); all species, analysis for all species in the data set; nonlocking species, analysis excludes species with copulatory locks; multiple intromission species, analysis for species with multiple intromissions only.

  3. *Not significant (P > 0.05) without exclusion of Rattus rattus as an outlier.

All species
 Ejaculation latency171.170.07−0.27n.s.140.470.03−7.18n.s.
 Post-ejaculatory interval167.290.34−0.590.017138.180.41−38.520.014
 Intromission number180.000.000.01n.s.151.640.100.67n.s.
 Ejaculation number2119.170.500.710.0001636.640.711.540.000
Multiple intromission species
 Inter-intromission interval125.610.36−0.600.039*1210.440.44−3.230.01
 Ejaculation latency145.670.32−0.570.035*118.100.45−38.500.017*
 Intromission number160.170.010.11n.s.130.150.010.17n.s.
Non-locking species
 Intromission number160.150.010.10n.s.131.530.110.66n.s.

Comparative analyses

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

The CAIC (version 2.6.9) software package (Purvis & Rambaut, 1995) was used to explore evolutionary associations between key variables in the data set when controlling for potential phylogenetic effects (Harvey & Pagel, 1991). The phylogenetic relationships used to identify independent contrasts were inferred from a variety of sources (Avise et al., 1979; Anderson, 1985; Sarich, 1985; Rogers & Engstrom, 1992; Catzeflis et al., 1993; Robinson et al., 1997; Adkins et al., 2001; Liu et al., 2001; Murphy et al., 2001), including the mammal taxonomy of Corbet & Hill (1991). Interfamilial relationships were mainly inferred from the mammalian supertree of Liu et al. (2001), based on molecular and morphological data, with information for hysricognaths from the molecular phylogeny of Adkins et al. (2001). Estimated intrafamilial phylogenetic relationships were inserted at appropriate branches. Relationships within the Cricetidae are largely based on the albumin immunological phylogeny reported in Sarich (1985), with relationships within the genus Peromyscus from taxonomic information and the phylogenies of Avise et al. (1979) and Rogers & Engstrom (1992), based on allozymic variation. Relationships within the family Arvicolidae are based on the morphological phylogeny of Anderson (1985), and those within the Muridae on the DNA–DNA hybridization phylogeny of Catzeflis et al. (1993). Estimated relationships between higher level taxa based on supertrees (Liu et al., 2001) are thus likely to be more reliable than those used for lower level relationships within families and genera. As phylogenetic relationships among rodents are uncertain and subject to persistent controversy, particularly with respect to relationships among the hystricomorphs (e.g. Cao et al., 1997; Adkins et al., 2001), branch lengths were set as equal for CAIC analyses, and we repeated the comparative analyses using three versions of the phylogeny based on available published information. The phylogeny used for the results presented is shown in the Supplementary Material provided. The alternative versions tested were more conservative; both were unresolved for the hystricomorph rodents, and one also included a less resolved phylogeny for the Cricetidae. The main results of the comparative analyses were robust across both of the alternative phylogenies used. Hence, although the accuracy of the phylogenies used is a limiting constraint in the present analyses, the results presented are relatively robust to variation in the phylogenetic information used. Comparative analyses were also performed using mean species values as independent data points, for comparison with results of the independent contrasts method (Price, 1997).

Data were normalized with log (relative testes size, inter-intromission interval, ejaculation number, intromission number) or square root (post ejaculation interval, ejaculation latency) transformations prior to all analyses, with the exception that predictor variables were not transformed for species level multiple regression analysis. The comparative data were also tested for each of the following evolutionary and statistical assumptions of the models used by CAIC: (i) there is no relationship between the absolute values of the contrasts and the estimated nodal values for each trait, (ii) the absolute magnitude of the contrasts are independent of the standard deviation in the contrasts, and (iii) predicted values of the dependent variable are uncorrelated with the absolute values of the standardized residuals from the regression equation. In several analyses in which relative testes size was tested as a predictor variable, exclusion of R. rattus (post ejaculatory interval, ejaculation number, ejaculation latency) or R. norvegicus (inter-intromission interval) was necessary to meet these assumptions. With the exception of the relationship between relative testes size and ejaculation latency (see Table 1 and Discussion), exclusion of Rattus did not alter the overall statistical significance of the analyses.

Both species level and contrast data were analysed using simple linear regression analysis in SPSS version 10. Regressions based on contrast data were forced through the origin (Purvis & Rambaut, 1995). Because several of the predictor variables used in the multiple regression analyses were correlated (e.g. measures of copulatory pace), we also examined variance inflation factors as a measure of collinearity. Variance inflation factors were less than 2, indicating that our multiple regression models are unlikely to be affected by collinearity.

Pace of copulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Results of the comparative analyses show that increasing sperm competition level is associated with a faster pace of copulation among rodents (Table 1). For species with multiple intromissions, increases in relative testes size are significantly associated with decreasing inter-intromission intervals (Table 1), and hence an increased rate of intromissions. Ejaculation latency also decreases with relative testes size for species with multiple intromissions (Table 1), indicating that males reach the point of ejaculation more quickly in species that experience higher sperm competition level. For species with multiple ejaculations, increases in relative testes size are also associated with significantly shorter post ejaculatory intervals (Table 1; Fig. 1), indicating that species with relatively high sperm competition level resume copulating after a shorter refractory period. Increasing sperm competition level is thus associated with increased intromission rates and reduced ejaculation latencies in species with multiple intromissions, and reduced post ejaculatory intervals in species with multiple ejaculations.

image

Figure 1. Linear regression through the origin of contrasts in (a) post-ejaculatory interval and (b) ejaculation number per female, against relative testes size.

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Intromission number

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

We found no evidence that the number of intromissions per ejaculation increase with relative testes size (Table 1), suggesting that multiple intromissions do not function to promote increased sperm transfer in response to sperm competition. The same conclusion is reached when locking species are excluded from the analysis (Table 1). Whilst the faster rate of intromissions observed under higher sperm competition level might have been expected to lead to more intromissions per ejaculation, ejaculation latency is reduced with increasing sperm competition level (Table 1). Thus, males in species that experience higher sperm competition level appear to provide a similar number of intromissions, but within a shorter period.

Ejaculation number

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

We found that increasing sperm competition level is associated with higher ejaculation number in rodents (Table 1; Fig. 1), as predicted if multiple ejaculations function to spread the delivery of ejaculates or increase the total sperm transfer. We conducted multiple regression analysis to examine whether a higher ejaculation number might result from the increased pace of copulation observed under increasing sperm competition level (Table 2a). Results of the analysis reveal that the length of the post ejaculatory interval explains a significant proportion of the variation in ejaculation number, whereas changes in inter-intromission interval and ejaculation latency have no significant influence on ejaculation number independent of this effect (Table 2a). Hence, whilst these measures of copulatory pace are all correlated with sperm competition level, and also have the potential to regulate ejaculation number, it is the post ejaculatory interval that appears to have the strongest influence on ejaculation number. One interpretation of this analysis is that reductions in the post ejaculatory interval are the principal mechanism for achieving higher ejaculation frequencies under increasing sperm competition level. To examine this possibility, we tested relative testes size and the post ejaculatory interval as explanatory terms in a model to explain ejaculation number. We predicted that the association between relative testes size and ejaculation number (Table 1; Fig. 1) would disappear when controlling for the post ejaculatory interval, if reductions in the post ejaculatory interval were the proximate mechanism to increase ejaculation number. The results of this analysis suggest that relative testes size has an additional influence on ejaculation number beyond any effect it may have on the post ejaculatory interval, as it remains a significant predictor of ejaculation number when controlling for the length of the post ejaculatory interval (Table 2b).

Table 2.  Multiple regression analysis of male rodent ejaculation number per female, performed with and without control for phylogeny (independent contrasts analyses), to test: (a) The importance of different components of copulatory behaviour to ejaculation number; (b) whether reductions in post-ejaculatory interval are the sole mechanism by which large testes influence ejaculation number.
VariableSpecies level analysesIndependent contrasts analyses
nFr2βtPnFr2βtP
  1. n, number of species or independent contrasts; r2, unadjusted r2; F, F ratio; β, slope of the regression line; t, t statistic; P, probability (n.s. >0.05).

a) Ejaculation number255.810.44  0.004237.270.52  0.002
 Constant   4.130.000   
 Post-ejaculatory interval   −0.84−3.800.001   −0.57−2.960.008
 Inter-intromission interval   0.030.15n.s.   −0.12−0.60n.s.
 Ejaculation latency   0.331.53n.s.   −0.13−0.67n.s.
b) Ejaculation number1416.410.73  0.0001325.810.82  0.000
 Constant   3.820.002   
 Post-ejaculatory interval   −0.43−2.630.022   −0.51−3.150.009
 Relative testes size   0.573.460.005   0.503.110.010

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Results of our comparative analyses show that increasing sperm competition is associated with higher intromission rates and reduced ejaculation latencies in rodent species with multiple intromissions, but no overall increase (or decrease) in intromission number per ejaculation. Increasing sperm competition is also strongly associated with more frequent ejaculations, and shorter post-ejaculatory refractory intervals in species with multiple ejaculations. As discussed below, these findings are likely to have important implications for our understanding of diversity in mammal copulatory behaviour, including the evolution of prolonged copulatory sequences involving multiple intromissions and ejaculations.

Sperm competition and pace of copulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Females of rodent species with multiple intromissions often prefer a slower pace of copulation than is imposed under male control (e.g. McClintock & Adler, 1978; Gilman et al., 1979; Erskine & Baum, 1982; Fadem & Barfield, 1982; Webster et al., 1982; Huck & Lisk, 1985; Martinez & Paredes, 2001). Results of our analyses provide the first comparative evidence that males are selected to increase the pace of copulation in response to increasing sperm competition level. That is, among species with multiple intromissions, we found that males with relatively large testes for their body size tend to perform intromissions at a faster rate and ejaculate more rapidly.

There are several possible explanations for the positive association between intromission rate and sperm competition level reported here. It is possible, for example, that changes in intromission rate may result from selection on males to ejaculate more rapidly under sperm competition, and thereby reduce the risk of interruption or take-over by rival males (e.g. see Dewsbury, 1984). Under this scenario, an increased intromission rate could directly promote rapid ejaculation, or may instead be necessary to maintain a minimum level of stimulation required for normal sexual function when ejaculation latency is reduced via some other mechanism. Our findings are also consistent with results of previous experimental and comparative studies demonstrating a significant positive correlation between inter-intromission interval and post ejaculatory intervals, both within and across a range of rodent species (Sachs, 1978; Sachs & Dewsbury, 1978). The consistency of this relationship has led some authors to suggest that the inter-intromission interval and post ejaculatory intervals may share a common control mechanism (Beach, 1956; Sachs & Dewsbury, 1978; see also below). Hence, in the context of the present study, it is possible that selection under sperm competition is acting directly on males’ ability to resume copulating quickly, with reductions in the inter-intromission interval following as an indirect consequence.

There was no significant relationship between sperm competition level and ejaculation latency across all species in the data set. Moreover, among species with multiple intromissions, at least one, R. rattus, did not conform to the general inverse relationship between ejaculation latency and sperm competition intensity. Relatively little is known about factors which could influence ejaculation latency under natural conditions, although factors such as risk of interruption or take-over by rival males, and/or predation risk would be worthy of further investigation.

Evolution of multiple intromissions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Although previous evidence indicates that varying intromission numbers within species can influence male ejaculatory performance (Toner & Adler, 1986), we found no evidence that the number of intromissions per ejaculation increases with relative testes size across rodents, or that an increased pace of copulation under sperm competition functions to increase intromission number per ejaculation. Interspecific variation in intromission number may thus be more closely linked to female stimulation thresholds than to variation in sperm competition level per se (see also Dewsbury, 1978). Maintaining intromission number at a minimum level to achieve successful pregnancy is obviously important for males, but the amount of stimulation provided can also have important consequences for male success in sperm competition by reducing the probability that females will remate, as vagino-cervical stimulation typically inhibits subsequent female sexual activity (e.g. Goldfoot & Goy, 1970; Carter & Schein, 1971; Hardy & DeBold, 1972; Oglesby et al., 1981; Romano & Benech, 1996; review in Huck & Lisk, 1986). Importantly, male rodents may thus experience opposing selection pressures with respect to offensive and defensive aspects of sperm competition – that is, the need to increase sperm transfer with rapid and/or frequent ejaculations, as demonstrated here, while also providing sufficient copulatory stimulation to reduce the probability that their ejaculates will be diluted in subsequent copulations. Such opposing selection pressures could explain why males of many rodents such as Mesocricetus and Peromyscus species switch from a copulatory pattern involving rapid multiple ejaculations to one of slower multiple intromissions with prolonged inter-intromission intervals once the ejaculatory series are complete (e.g. Dewsbury, 1974; Huck et al., 1988).

Function of multiple ejaculations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Results of our comparative analyses indicate that ejaculation number per female is strongly positively correlated with relative testes size across rodents. This relationship is consistent with the early observations of Short (1979) in relation to overall copulation frequency and relative testes size in the great apes, and subsequent analyses of copulation frequency in primates (Dixson, 1995, 1998) and other mammals (Møller & Birkhead, 1989). Parker (1984) first raised the question of why males should partition sperm into multiple ejaculations with the same female under conditions of sperm competition, and suggested that it may be advantageous by either (i) spreading the timing of ejaculates under certain conditions involving high rates of sperm death, or (ii) increasing the number of sperm transferred. A function of multiple ejaculations in increasing the number of sperm transferred seems most consistent with results of our comparative analyses, as we found that increasing sperm competition level is associated with shorter post ejaculatory intervals, whereas the opposite result might be predicted under selection pressure to spread the timing of ejaculates throughout the oestrus period.

If multiple ejaculations function to increase sperm numbers within the female reproductive tract under conditions of sperm competition (Lanier et al., 1979; Dewsbury & Hartung, 1980; Dewsbury, 1981; Oglesby et al., 1981; Ginsberg & Rubenstein, 1990), it is likely that some constraint must exist to prevent sperm transfer in a single large ejaculate (Parker, 1984). It is possible, for example, that producing a single large ejaculate may require a relatively long ejaculation latency, such that a strategy of partitioning sperm into several smaller ejaculates could be favoured to maximize sperm transfer under conditions where there is a risk of interruption before completing the whole copulatory series. Interruption by rival males could also be important after ejaculation, as there is a critical period during which sperm transport can be disrupted by subsequent copulations (Adler & Zoloth, 1970). A risk of disruption to sperm transport by rival males may thus impose conditions analogous to the high rates of sperm death modelled by Parker (1984), which favour the partition of sperm into multiple ejaculates.

A further possibility is that copulation rate is regulated according to the availability of mature sperm (Short, 1979; Birkhead et al., 1993; Preston et al., 2003), and that sperm production or storage is constrained (see also Preston et al., 2001). Under this hypothesis, sperm competition remains the selective force driving the evolution of rapid multiple ejaculations, but the associations between relative testes size and copulatory activity may be mechanistic in nature. High androgen production associated with relatively large testes could provide the impetus for increased sexual activity (Signoret & Balthazart, 1993; Gábor et al., 1994), thereby acting synergistically with sperm availability. Testosterone has been shown to exert a strong influence on key variables of male copulatory behaviour in a range of laboratory rodents (Malmnäs, 1977; Wood et al., 1996; James & Nyby, 2002). In rats, for example, the administration of testosterone leads to reductions in ejaculation latencies, inter-intromission intervals and post ejaculatory intervals, but has no apparent influence on intromission number (Malmnäs, 1977). Results of these studies thus indicate that potential mechanistic explanations for the relationships between relative testes size and measures of copulatory behaviour presented here are worthy of more detailed experimental investigation.

In conclusion, results of the comparative results presented for rodents indicate that sperm competition selects for male mammals to ejaculate more frequently and, in some cases, more rapidly. Our results also suggest that males may experience potentially conflicting selection pressures with respect to offensive and defensive behavioural adaptations to sperm competition. These findings thus provide support for previous suggestions that sperm competition is likely to be an important selection pressure explaining diversity in mammal copulatory behaviour (e.g. Parker, 1984; Ginsberg & Rubenstein, 1990). Further studies are needed to explore the influence of sperm competition on copulatory behaviour in natural populations, and to test the generality of the patterns reported here across broader taxonomic groups as more data become available.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

We gratefully acknowledge comments on the manuscript and/or helpful discussion from G. A. Parker, S. Ramm, F. Kraaijeveld-Smit, J. R. Clarke and A. H. Harcourt. Comments from two referees were also very helpful. This research was funded by the UK Natural Environment Research Council grant number NER/B/S/2000/00797. PS is also grateful for generous support provided by a Visiting Scholarship to St John's College, University of Oxford.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Comparative data set
  6. Definitions of copulatory behaviour
  7. Sperm competition level
  8. Comparative analyses
  9. Results
  10. Pace of copulation
  11. Intromission number
  12. Ejaculation number
  13. Discussion
  14. Sperm competition and pace of copulation
  15. Evolution of multiple intromissions
  16. Function of multiple ejaculations
  17. Acknowledgments
  18. Supplementary Material
  19. References
  20. Supporting Information

Table S1. List of species included in the analyses with information on copulatory behaviour and relative testes size. Lock: presence or absence of copulatory lock; IN: intromission number - the number of intromissions preceding the first ejaculation; EN: ejaculation number - the mean number of ejaculations achieved with a single female before attainment of some satiety criterion; III: inter-intromission interval - the mean interval separating the intromissions of the first copulatory series; PEI: post-ejaculatory interval - the interval between the end of the first ejaculation and the beginning of the next intromission; EL: ejaculation latency - the mean latency from the first intromission to the end of the first (or only) ejaculation; RTS: relative testes size - based on the regression equation for the relationship between log body mass and log testes mass for rodents in Kenagy and Trombulak (1986); (s): seconds; species data labelled (1) or (2) are for different populations.

Fig. S1. Estimated phylogenetic relationships among species included in the analyses (see Methods for sources).

FilenameFormatSizeDescription
JEB_742_sm_tableS1.doc117KSupporting info item
JEB_742_sm_figS1.doc37KSupporting info item

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