Behavioral Ecology Advance Access published July 30, 2008
Behavioral Ecology
doi:10.1093/beheco/arn068

Females affect sperm swimming performance:a field experiment with barn swallows
Hirundo rustica
A.P. Møller,a T.A. Mousseau,b and G. Rudolfsenc
Laboratoire de Parasitologie Evolutive, CNRS UMR 7103, Université Pierre et Marie Curie, Bât. A,
7ème étage, 7 quai St. Bernard, Case 237, F-75252 Paris, Cedex 05, France, bDepartment of Biological
Sciences, University of South Carolina, Columbia, SC 29208, USA, and cDepartment of Biology,
University of Tromsø, N-9037 Tromsø, NorwayaSexual conflict over fertilization may in animals with internal fertilization initiate an evolutionary arms race that causes thefemale reproductive tract to become hostile to sperm performance if females with more selective reproductive tracts have theireggs fertilized by sperm of superior quality. This hypothesis suggests that sperm should perform better when tested in a physiologically neutral cell culture medium (neutral medium) than in the same ‘‘neutral medium’’ to which fluid derived from thefemale reproductive tract has been added (female medium). We tested this prediction using in vitro tests on sperm collectedfrom barn swallows Hirundo rustica by recording sperm performance on video in neutral medium and ‘‘female medium.’’ Spermperformance differed significantly among males but also between neutral and female medium. Sperm performed less well infemale medium compared with neutral medium in terms of velocity. A principal component (PC) analysis of the 12 sperm
parameters produced 4 PCs that explained 86% of the variance. The difference in the second and the third PC between neutral
and female medium, reflecting sperm with a high degree of lateral head displacement and absence of straight and linear
movement, many static sperm, and a small fraction of sperm with medium velocity, respectively, was positively related to anindicator of female quality: tail length. The latter result may suggest that high-quality females differentially affected the relative
performance of sperm in their reproductive tract, consistent with the theory of sexual conflict. Key words: sexual conflict, sexual
selection, sperm, swimming performance. [Behav Ecol]ametes constitute the means by which sexually reproducing organisms transfer their genetic material to the next
generation, but the mere fact that sperm are much more abundant than eggs makes the question of which sperm eventually
fertilizes an egg crucial. The evolution of anisogamy sets thescene for competition among sperm for fertilization of rare
eggs (Queller 1997). This has consequences for optimal timing and allocation of sperm to females, with effects on certainty of paternity (Parker 1984). Therefore, the fitness gains
from paternal care are reduced relative to those from matesearch (Queller 1997). Particularly, attractive males will enjoy
a greater reproductive rate than the average male, making it
more beneficial for such males to invest their reproductive
effort in mating rather than parental effort (Queller 1997).
Simultaneously, females will enjoy a selective advantage fromhaving their eggs fertilized by high-quality sperm, given therelatively small number of eggs and the large number ofsperm. Many studies have suggested that females have evolved
different kinds of obstacles to fertilization to achieve fertilization by sperm of superior quality (Eberhard 1996). Examples
of such potential obstacles include female ejection of ejaculates (Baker and Bellis 1995), elongation of the female tract
increasing the distance that sperm have to travel to reach thesite of fertilization (Gomendio and Roldan 1993), the chemical environment in which sperm have to swim (e.g., Yanagimachi

G

Address correspondence to A.P. Møller. E-mail: amoller@snv.
jussieu.fr.
Received 20 November 2007; revised 15 May 2008; accepted 20
May 2008.
 The Author 2008. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org

1994), and leukocytes actively killing sperm (e.g., Johnson
1973; review in Birkhead, Møller, and Sutherland 1993). However, there are no experimental studies demonstrating thatfemale ‘‘obstacles’’ evolved to provide high-quality males witha fertilization advantage. In sperm competition games, malepartners, male nonpartners and females may have different
evolutionary interests. Male nonpartners may differ in interests from male partners because male partners prevent fertilization of the eggs of their female by extrapair males.
Extrapair males may likewise attempt to fertilize eggs rather
than allowing the male partner of a female performing fertilization. Such a conflict may be mediated by females developing immune reactions to the sperm of their partners, but not
to sperm of male nonpartners that have not yet deposited
sperm in the female reproductive tract. Females and malepartners may also differ in their evolutionary interests because
females may not necessarily attempt to have their eggs fertilized by their partner, perhaps causing females to produce
obstacles to fertilization by their partners. Finally, females
and male nonpartners may also be in conflict over fertilization
because females may avoid fertilization by unmated males ofpoor quality while such males may have no other reproductive
options than attempting to fertilize the mates of other males.
Again, this should result in females producing obstacles tofertilization by poor-quality males and such males attempting
to overcome such obstacles. The observations and arguments
listed above are evidence of sexual conflict over fertilization(Parker 1979; Rowe and Arnqvist 2005), suggesting that thefemale reproductive tract and its anatomical, physiological,
chemical, and immunological properties may have evolved asmeans of imposing stringent selection on sperm from one ormore males for eventually reaching the site of fertilization.

Behavioral Ecology

2

Numerous studies have indicated that sperm perform differently on different female backgrounds and that the female reproductive tract affects sperm performance (e.g., Eisenbach
2004; Sun et al. 2005; Satake et al. 2006; Suarez and Pacey
2006). However, there are no studies investigating whether
females may reduce the performance of sperm and whether
such female behavior is related to female fecundity(Birkhead, Møller, and Sutherland 1993; Satake et al. 2006;
Birkhead and Brillard 2007). If the environment in which
sperm move to achieve fertilization of an egg is hostile tosperm, we can predict that sperm movement will be hampered in an environment characteristic of the female tract,
whereas it will be facilitated in a physiologically neutral medium. This simple prediction has to the best of our knowledge
never previously been tested experimentally. Furthermore, we
can make the prediction that differences in sperm performance between the environment of a female reproductive
tract and a neutral environment should depend on maleand female characteristics if some females are more fecund
than others and if some males have superior abilities to fertilize eggs. Both these latter assumptions are well supported byempirical evidence (differences in fecundity: e.g., Roff 2001;
differences in fertilizing ability: Birkhead and Møller 1998).
This leads to the third prediction. Performance of spermshould be relatively worse in a female medium from the most
fecund females if such females are particularly picky about
which sperm fertilizes their eggs and therefore produce particularly strong barriers to sperm. There are no studies thathave examined these factors so far, and the mechanisms involved in these processes are still unknown (but for a recent
review of female factors affecting fertilization, see Birkhead
and Brillard 2007).
The objectives of the present study were to test 1) whether
males are consistent in sperm performance, 2) whether sperm
swim less well in female than in neutral medium, and
3) whether female medium from the highest quality females isrelatively more effective in hindering sperm performance.
Sperm performance may suffer if the female can modulate
her reproductive tract or its biochemical and immunologicalenvironment to choose sperm of specific males. Therefore,
we predict that sperm will perform less well in the most fecund
females compared with the least fecund females. To this end,
we conducted in vitro tests of sperm performance in the field,
using the barn swallow Hirundo rustica as a model organism
and using tail length of females as a proxy for quality andhence fecundity (see below).
The barn swallow is a approximately 20-g passerine, semicolonial migratory bird with a well-known life history. Spermcompetition is common as reflected by frequent extrapair
copulations (Møller 1985) and extrapair paternity reaching
up to a third in different populations (Møller and Tegelström
1997; Saino et al. 1997; Møller et al. 1998, 2003). Testes
size and hence sperm production is large for the body sizeof the species (Møller 1991; Birkhead, Briskie, and Møller
1993; Møller and Briskie 1995). Males that gain extrapair
copulations and fertilizations have long tails (a secondary
sexual character; Møller 1988, 1992; Saino et al. 1997).
Females with long tails are of superior phenotypic quality
in terms of laying date, clutch size, number of clutches,and survival (Møller 1993; Cuervo et al. 2003). Therefore,
tail length is an external indicator of phenotypic quality inboth sexes (Møller 1994). Because a third of all females
produce extrapair offspring and hence engage in extrapair
copulations, there is reason to believe that the female tract
has been subject to selection for discrimination among
sperm but also for sperm from different males to competeby overcoming any such female discrimination for successful
fertilization.

MATERIALS AND METHODS
General field procedures
Wecapturedbarnswallowsin4differentfarmsaroundChernobyl,
Borispil, and Kanev in Ukraine during early June 2006. Using
mist nets placed across window and door openings, we
captured wild birds at the peak of reproduction just before
and during laying for most males. Birds were placed in cloth
bags before being measured and weighed. Subsequently,
we collected at least 2 sperm samples from all males using
a simple massage technique. Sperm were collected in microcapillaries for easy handling. We collected sperm from malebarn swallows and recorded their swimming performance ina physiologically neutral cell culture medium (neutral medium) and in medium derived from the reproductive tractof female swallows (see below for details on medium), randomly alternating the order of the 2 tests for each male,while recording sperm performance on video, and using
a computer-assisted sperm analysis (CASA) system for performance measurements. G.R. recorded sperm performance
blindly with respect to the phenotype of males and females,
and G.R. also derived statistics for sperm performance byanalysis of video recordings blindly with respect to male and female identities. It is important even to record sperm performance
blindly because otherwise bias could arise from when therecording was initiated. We have previously shown that spermmorphology and behavior is related to level of backgroundradiation in Ukraine (Møller et al. 2008). However, this effectwas not important for the interpretation of the present study
because sperm performance was compared within males.
We recorded 12 standard measurements of external morphological traits with a ruler (length of right and left wing
and left and right outermost tail feathers, length of central tailfeathers, and wingspan) or a digital caliper (length, height,
width of beak, length of tarsus, and length of keel), and wetraced the surface of the right stretched wing on a piece of paper. These measurements are accurate and repeatable, as
shown by repeat measurements of a number of individuals
in the same breeding season (Møller 1994). In the analyses
presented here, we only use tail length estimated as the meanof the right and the left outermost tail feather, body mass, andtarsus length. The justification for using these 3 variables wasthat tail length can be considered a secondary sexual character, body mass an estimate of body condition (especially whenentering tarsus length as an additional variable), and tarsus
length a measure of structural body size. We checked all individuals for broken or damaged tail feathers by determining
whether the tip was rounded, as it is the case for undamaged
feathers. Likewise, we checked the base of all tail feathers forany signs of feather sheaths that are present on feathers still
growing. A total of 2 individuals with damaged or growing
feathers were excluded from the analyses.
We did not assign individuals to nests and hence did notknow their exact breeding status, although checks of nests
revealed nest contents to mainly be eggs and small nestlings.
This time of the breeding cycle is when males copulate with
their own females but also with neighboring females (Møller
1985, 1994).
A.P.M. attempted to collect sperm from all males, and wewere successful for 190 of 193 males. When we had collected
sperm from a male, we randomly chose a female captured at
the same time and site and then collected fluid from her
reproductive tract. We used the same massage technique on
females as used for collecting sperm from males, with themuscles of the cloaca contracting repetitively before a sample
could be collected. We repeatedly attempted to collect fluid
from the female tract in the absence of muscle contractions,and we were never successful in more than 20 attempts, thus

Møller et al. • Sperm behavior and female quality

excluding the possibility that this fluid was alimentary canalmucus. This procedure produced approximately 1–2 lL oftransparent vaginal fluid from the outermost 1 mm of the cloaca; this was subsequently used for the tests. If the fluid wascontaminated with feces, which was easily detected from thewhite and/or brown color of the sample, we discarded the sample and collected a new one. We deliberately placed female
fluid in the same video system as described below to searchfor sperm and fecal matter. There were no sperm present infemale fluid in any of the 10 samples that we investigated, thus
excluding the possibility that the results were confounded bysperm from more than one male being present in the test tube.
Likewise, there was no fecal matter present in any of these 10
samples. Due to the logistic problems of conducting this kindof study under field conditions, the total number of males forwhich we were able to record videos in the 2 different media
was 20, with each male being tested with fluid from a different
female because the amount of fluid extracted was insufficientfor multiple tests on a single female. We used 166 males to testfor repeatability of sperm performance among recordings,
and we used 10 males to test for repeatability of sperm performance among ejaculates. Repeatability (R) is a standard estimate of consistency in phenotypic traits among measurementsthat allows partitioning of the phenotypic variance within
and among individuals, ranging from 0 (no consistency) to1 (all values remain constant) (Falconer and Mackay 1996).
Repeatability also has the important property that repeatability sets an upper limit to heritability (Falconer and Mackay1996). We estimated repeatability and its standard error(SE) using the equations in Becker (1984).
Sperm behavior
We quantified the behavior of sperm by adding sperm either toa neutral medium or to a mixture of fluid from the cloaca ofa female and neutral medium, followed by video recording under a microscope. Whereas a third treatment with no neutral
medium might have been desirable, this procedure was impossible because of the extremely high concentration of sperm in
the ejaculate (we estimated tens of millions of sperm in lessthan 1 lL of ejaculate), completely hindering free sperm movement and hence recording of sperm behavior. We deliberately
avoided initial dilution of all sperm samples because such a general treatment would add another layer of uncontrolled treatment effects to the entire study. The design with 2 treatmentsthat we used ensured that the treatments had the same components with the exception of fluid from the female reproductive tract. Furthermore, the amounts of ejaculate and fluid
from the female reproductive tract were so small that both
treatments were highly diluted, causing the proportion of neutral medium to exceed 80% in the 2 treatments. This makes it
unlikely that factors such as viscosity would differ between
treatments.
The sperm sample was diluted in a one-step procedure byplacing an aliquot of undiluted sperm on a prewarmed (37C) microscope slide with a 80-mm deep chamber (Hamilton
Thorne Research, Beverly, MA) and then added either neutral
medium (Dulbecco’s modified eagle medium [D-MEM]) or
female fluid and D-MEM. D-MEM contains 4500 mg/L glucose,
4 mM L-glutamine, and 110 mg/L sodium pyruvate and is
manufactured by Gibco. Temperature was maintained constant using a heating aggregate. Immediately after that, weadded 9 lL neutral medium, or 1–2 lL female fluid medium
and 9 lL neutral medium, and placed the cover glass over the
sample. We added 9 lL of neutral medium to both treatments
to ensure that the difference in performance was due to the
1–2 lL female fluid rather than any difference in neutral
medium. Sperm motility and images were recorded immedi-

3

ately after activation using a Sony CCD black and white video
camera (XC-ST50CE PAL, Sony, Tokyo, Japan) at 50 Hz vertical frequency, mounted on an external negative phasecontrast microscope (Olympus CH30, Olympus, Tokyo,
Japan) with a 310 objective. Video recordings that allowedmagnification were stored on mini-DV tapes. The recordings
were later analyzed using CASA (HTM-CEROS sperm tracker,
CEROS version 12, Hamilton Thorne Research). The image
analyzer was set at frame rate 50 Hz, number of frames 25,
minimum contrast of 30, and minimum cell size 20 pixels.
Cells having a smoothed path velocity (VAP) ,10 lm/s and a
VSL ,5 lm/s were considered to be static. Each motility measurement lasted 0.5 s. Between 7 and 20 replicate measurements for each of 12 different sperm performance, variables
were made from each ejaculate (depending on the number ofmales ‘‘queuing’’ for being tested) (see below for details of thedifferent variables). Variation in number of replicate measurements for each male was due to variation in amount of sperm.
For each male, we quantified sperm motility within 2 min after
extraction of the sperm, although most were recorded within
1 min, and any effect of storage was tested explicitly usingorder of treatment as a factor (see below). We kept both males
and females until the very moment when female medium had
to be used, then extracted the sperm and the fluid, and added
this immediately to the test chamber.
We recorded 12 different measures of sperm performancewith CASA. Because there is only very limited information
about the sperm performance factors determining fertilizationin birds, even in chickens, with sperm motility and swimming
speed being the only exceptions (Birkhead et al. 1999; Froman
et al. 1999), and none of these having been based on extensive video recordings, we decided to be prudent and obtaindata on each of 12 different variables. We recorded meanvalues of VAP (smoothed path velocity [microns/second]),
VSL (straight-line velocity [microns/second]), VCL (track velocity [microns/second]), ALH (amplitude of lateral headdisplacement [microns]), BCF (beat cross frequency [Hertz]),
LIN (linearity [VSL/VCL]), and STR (straightness [VSL/
VAP]) for each sample. The actual numbers of cells arecounted in all field of analysis. As the velocity measurements
are calculated for each cell, we were able to estimate percentage of static sperm cells (fraction of all cells that are notmoving at all), sperm with slow velocity (fraction of all cells
moving with VAP ,10 lm/s or VSL ,5 lm/s), with medium
velocity (fraction of all cells moving with VAP between 10 and
50 lm/s), sperm with rapid velocity (fraction of all cells moving with VAP .50 lm/s), and the percentage of progressive
sperm (fraction of all sperm that moved with STR .80 and
VAP .50 lm/s) (see Technical Guide, Hamilton Thorne Biosciences, Beverly, MA, for more information on variables andtheir definitions).
Statistical analyses
The proportion of progressive, static, rapid, medium, and slow
sperm was square root arcsine transformed before analyses. We
analyzed sperm performance in neutral medium from different frames of video recordings using analyses of variance(ANOVAs), with each frame containing different sperm. Male
identity was entered as a fixed factor in this analysis. For the 20
males analyzed in 2 kinds of medium, we tested for order oftreatment effects (neutral medium first, followed by neutral
medium or the reverse) on sperm performance in 1-way
ANOVAs, with order as a factor, and none of these tests for order effects reached statistical significance. Hence, this factor
was subsequently eliminated. Finally, in analyses of sperm behavior in neutral medium, we included date to control for differences among days and time of day recorded to the nearest

Behavioral Ecology

4

Table 1
PC analysis of sperm traits, with the 5 first components accounting
for 85.8% of the variance
Variable

PC1

PC2

PC3

PC4

Eigenvalue
Percentage of variance
VAP
VSL
VCL
ALH
BCF
STR
LIN
Progressive
Rapid
MediumSlow
Static

5.51
45.95
0.39
0.41
0.22
0.13
20.08
0.29
0.32
0.38
0.37
20.32
20.18
20.08

2.27
18.89
0.17
0.03
0.50
0.50
0.02
20.43
20.41
0.07
0.22
0.04
20.08
20.21

1.50
12.53
0.14
0.10
0.24
0.02
0.12
20.10
20.07
20.26
20.25
20.41
20.20
0.73

1.01
8.46
20.04
20.04
0.02
20.19
0.95
20.02
20.07
0.11
0.09
20.12
0.11
20.12

Loadings exceeding 0.30 were considered as being important andthey are shown in bold.

minute to account for diurnal variation in copulation activity
and hence age of sperm. The latter is known to peak in the earlymorning with a much smaller peak in the evening (Møller
1985). None of these variables entered as significant predictors.
Because the number of sperm performance parameters waslarge, we reduced these to a small number of independent measures in a principal component (PC) analysis. This analysis was
based on the transformed original variables (as describedabove), using the correlation matrix and orthogonal rotation(Table 1). Four PCs with eigenvalues exceeding 1.00 were subsequently identified and used for further analyses. We included
all 4 PCs because they accounted for considerable amounts ofvariation (PC4 accounted for more than 8% of the variance andcould thus potentially be important). These PCs could readily
be interpreted based on the factor loadings of the originalvariables (Table 1), assuming that loadings exceeding 0.30
are significant (Tabachnick and Fidell 1996). More specifically,
PC1 had positive loadings exceeding 0.30 for VAP, VSL, LIN,
and the proportion of progressive and rapid sperm and a negative loading on sperm with medium velocity. This PC is thus an
indicator of high straight-line velocity with progressive movement. PC2 had strong positive loadings for VCL and ALH andstrong negative loadings for STR and LIN. Therefore, this PC
reflected sperm with a high degree of lateral head displacement and absence of straight and linear movement. PC3 hada strong positive loading for the proportion of static sperm anda negative loading for the proportion of sperm with medium
velocity. Thus, this PC reflected sperm with slow or static movement. Finally, PC4 had a strong positive loading on BCF, implying sperm with high beat cross frequency.
We rescaled estimates of the PCs relative to medium by rotating the coordinate system 45 to the right (see Figure 1), so
the Y axis coincided with the line where performance in the 2
media was similar (i.e., Y ¼ X). We subsequently calculated
orthogonal deviations of the 4 PCs from this new Y axis as
estimates of relative performance in the 2 media. The nullassumption is that mean deviation should be zero for all 4
PCs and that deviations for the 4 PCs should constitute a homogeneous sample. Thus, we tested if means differed fromzero in 1-sample t-tests, and we tested whether deviations differed among the 4 PCs in a 1-way ANOVA with post hoc comparisons of means.
We used the difference in sperm parameters between theneutral medium and the female medium in tests designed

Figure 1
Mean performance measured as the first PC (PC1) for sperm of
20 male barn swallows in neutral and female fluid medium. The line
Y ¼ X indicating that the sperm perform equally well in the 2 media
is shown.

to investigate the relationships between difference in sperm
performance and male and female external morphology. In
these analyses, we included male and female tail length, bodymass, and tarsus length (a measure of body size) as predictor
variables. We used the Akaike information criterion (AIC) andchange in AIC to identify the best-fit models (for procedures,
see Burnham and Anderson 2002), assuming that a difference
between models of AIC .2.00 is biologically significant.
All values reported are means (SE), and we used a significance level of 5%.

RESULTS
Sources of variance of sperm performance
We were able to obtain sperm from 190 of 193 male barn swallows, or 98.4%. Repeatability of sperm parameters within andamong samples was moderate with only one value not being
statistically significant (Table 2). Whereas all within-sample
repeatabilities ranged from 0.07 to 0.48, they were muchsmaller at 0.01 to 0.33 for repeatability among different samples taken from the same male (paired t-test, t ¼ 6.25, degrees
of freedom [df] ¼ 15, P , 0.0001). These results imply thatsperm parameters were repeatable based on multiple estimates of the same sample but also that individual males wererepeatable in their sperm behavior among ejaculates.
Sperm performance in different media
Sperm performed less well in female than in neutral medium
with respect to velocity. Rescaled PC1 differed significantly
from rescaled PC2, PC3, and PC4 (Tukey post hoc tests, P ,0.0001), whereas all other differences were not significant(Tukey post hoc tests, P . 0.10). The rescaled estimate of
PC1 relative to medium was 21.36 (SE ¼ 0.33), differingsignificantly from zero (1-sample t-test: t ¼ 24.16, df ¼ 19,
P ¼ 0.0005), whereas the means for PC2, PC3, and PC4 did
not differ significantly from zero. Sperm from 16 of 20 males
performed better in neutral medium (Figure 1). For comparison, similar data for PC2 demonstrate no clear difference insperm performance between neutral and female medium forthis component of sperm behavior (Figure 2). According toMøller et al. • Sperm behavior and female quality

5

Table 2
Mean (mean of means estimated for each sequence of video analyzed and SE) and within- and among-sample repeatability ofsperm parameters from barn swallows from Ukraine
Variable

Mean (SE)Within-sample F

Within-sample R (SE)Among-sample F

Among-sample R (SE)Pct. static
Pct. slow
Pct. medium
Pct. rapid
Pct. progressive
LIN
STR
BCF (Hz)ALH (lm)
VSL (lm/s)
VCL (lm/s)
VAP (lm/s)
PC1
PC2
PC3
PC4

36.25 (1.67)
5.10 (0.29)
24.29 (0.81)
34.36 (1.36)
29.84 (1.23)
64.75 (1.16)
83.33 (1.02)
21.48 (0.35)
3.85 (0.11)
53.81 (0.88)
78.85 (1.51)
57.15 (1.17)
20.00 (0.07)
0.00 (0.05)
20.00 (0.04)
0.00 (0.03)

10.21***
1.73***
4.74***
9.89***
8.98***
7.82***
5.36***
3.50***
5.30***
6.08***
7.92***
8.25***
13.09***
5.36***
9.06***
2.63***

0.48 (0.04)
0.07 (0.02)
0.27 (0.04)
0.47 (0.04)
0.44 (0.04)
0.41 (0.04)
0.30 (0.04)
0.20 (0.03)
0.30 (0.04)
0.25 (0.03)
0.41 (0.04)
0.42 (0.04)
0.52 (0.04)
0.28 (0.04)
0.42 (0.04)
0.13 (0.03)

9.81***
1.25
5.95***
8.34***
7.38***
2.01*
3.69**
3.26**
9.03***
3.66**
5.61***
5.50***
8.23***
3.65**
8.23***
2.02*

0.33 (0.02)
0.01 (0.01)
0.22 (0.02)
0.29 (0.02)
0.27 (0.02)
0.05 (0.01)
0.15 (0.02)
0.14 (0.02)
0.31 (0.02)
0.15 (0.02)
0.21 (0.02)
0.20 (0.02)
0.28 (0.02)
0.15 (0.02)
0.28 (0.02)
0.07 (0.01)

Sample size is 166 males with repeat recordings for a single ejaculate, whereas 10 males were analyzed for 2 different ejaculates. Pct. ¼ %.
*P , 0.05, **P , 0.01, ***P , 0.0001.

the loading of sperm parameters on PC1 (Table 1), theseresults for PC1 imply that smoothed path velocity, straight-line
velocity with rapid and progressive movement, and linearity of
movement were greater in neutral than in female medium.

We tested whether male and female phenotypic characters
could predict relative sperm performance in female medium
compared with neutral medium. Relying on 4 PCs, there wasno significant model retained for difference in PC1 between
neutral and female medium. Difference in PC2 was significantly positively related to female tail length (Figure 3), implying that sperm had higher amplitude of lateral headdisplacement, higher track velocity, and nonstraight and nonlinear movement in female medium compared with neutral
medium when the female had a long tail (Table 3). In otherwords, sperm moved in straighter lines in neutral medium,

especially when females had long tails. This result also suggests that the lack of difference in PC2 (and PC3) between the
2 media was because of interactions with male and female
phenotypes obscuring any main effects. For the differencein PC3 between neutral and female medium, female taillength (Figure 4), female tarsus length, and male tarsus
length entered as predictors in a model that accounted for
56% of the variance (Table 3). The difference in PC3 increased with female tail length and tarsus length and decreased with male tarsus length (Table 3). This implies thatin female medium, fewer sperm had medium velocity andmore were static (not moving) than in neutral medium
when the female medium is derived from a long-tailed female,even after controlling for female and male tarsus length(which indicates body size) (Table 3). Finally, the differencein PC4 increased with male body mass (Table 3), implyingthat beat cross frequency was greater in female than inneutral medium when the male that produced the sperm
was heavy.

Figure 2
Mean performance measured as the second PC (PC2) for sperm of
20 male barn swallows in neutral and female fluid medium. The line
Y ¼ X indicating that the sperm perform equally well in the 2 media
is shown.

Figure 3
Difference in performance measured as the second PC (PC2) for
sperm of 20 male barn swallows in neutral and female fluid medium
in relation to female tail length.

Sperm performance and adult phenotype

Behavioral Ecology

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Table 3Models of the relationship between difference in PC scores for sperm performance between neutral and female fluid medium andfemale and male external morphology
Variabledf

Sum of squares

PC2
Female tail length
Error
PC3
Male tarsus length
Female tail length
Female tarsus length
Error
PC4
Male body mass
Female tarsus length
Error

1,18
1
18
3,16
1
1
1
16
2,17
1
1
17

15.48
15.48
36.73
24.44
7.50
15.03
6.27
19.57
3.09
2.89
1.27
6.25

F

P

Slope (SE)

Effect size

Delta AIC

7.59
7.59

0.013
0.013

0.133 (0.048)

0.54

6.48

6.66
6.13
12.29
5.12

0.004
0.025
0.00290.038

20.013 (0.005)
0.133 (0.038)
0.011 (0.005)

0.53
0.66
0.49

4.48
9.39
3.55

4.20
7.85
3.47

0.033
0.012
0.080

0.014 (0.005)
0.006 (0.003)

0.56
0.41

5.59
1.71

AICs for the full models were 17.59, 7.57, and 217.25. Effect size was Pearson’s product–moment correlation coefficient. Delta AIC was thedifference between the full model and the model excluding a given variable from the model.

DISCUSSION
An important finding of this study was that sperm behavior differed between neutral and female medium but also that sperm
performance differed significantly among males. Second, notall sperm performance variables showed effects of medium, implying that this effect was not ubiquitous among sperm performance traits. Finally, the difference in sperm performance
between neutral and female medium was significantly related
to tail length of females, an indicator of female quality andother female traits, but less so to the size of male traits.
Analyses of performance of sperm collected from wild barnswallows showed significant male and treatment effects.
Repeatabilities were generally intermediate to small, withamong-ejaculate repeatabilities being significantly smaller
than within-ejaculate repeatabilities. The higher withinejaculate repeatabilities imply that we are able to estimate
sperm behavior relatively accurately, whereas the small
among-ejaculate estimates of repeatability imply that there isconsiderable variation among ejaculates, although there is also
evidence of significant consistency among males. The significant male effects imply that males are consistent in most measures of performance of their sperm that we recorded bothwhen multiple estimates were obtained for the same ejaculate
but also for different ejaculates and that males differed significantly in terms of performance of their sperm. Such consistentdifferences are necessary assumptions for evolution of ejaculate features, but they are also requirements for predictionsof fertilization ability. Recent studies suggest that size, motility,
and swimming speed of sperm may predict fertilization probability to some extent in both external and internal fertilizers(e.g., Lahnsteiner et al. 1998; Birkhead et al. 1999; Froman
et al. 1999; Levitan 2000; Kupriyanova and Havenhand 2002;
Gage et al. 2004; Garcia Gonzales and Simmons 2005;
Rudolfsen et al. 2008).
The main objective of this study was to test for differences insperm performance in neutral medium and in medium withfluid derived from the female reproductive tract. One of the
4 components of sperm performance behaved differently
among treatments. This shows that effects are not ubiquitous
for all performance traits because only one of 4 PCs showeda significant treatment effect. Closer inspection of Figure 1
revealed that the position of the line Y ¼ X deviated froma random distribution by 1.4 standard deviations (1 standard
deviation ¼ 1.47 and displacement of the line Y ¼ X to Y ¼ 22 1
X results in half of the 20 observations being below and half
above the latter line). This must by all means be considereda large effect (sensu Cohen 1988). The surprising feature ofthis result is that sperm performed ‘‘less well’’ in their ‘‘naturalenvironment’’ (fluid from the female reproductive tract) thanin artificial neutral medium, begging the question why thatshould be the case. The actual components of the female fluidthat affected sperm performance remain to be determined.
A simple explanation is that fluid taken directly from females
is more viscous than the cell culture medium. We consider
this possibility to be unlikely given the high degree of dilutionthat we used in the experiments (see Materials and Methods).
However, as the female reproductive tract in humans is able tomount effective immune responses against infectious agents
and, occasionally, sperm cells (Naz and Menge 1994), we speculate that immunoglobulins can bind to sperm in birds andhence directly influence swimming behavior of sperm (fora review of the effect of antisperm antibodies on mammalianfertility, see Chiu and Chamley 2004). A previous study of barnswallows has shown an elevated level of circulating immunoglobulins around the timing of egg laying in adult female
barn swallows, but not in males (Saino et al. 2001). The magnitude of this peak in immunoglobulins is positively related tothe tail length of the male partner, but not the tail length of

Figure 4
Difference in performance measured as the third PC (PC3) for
sperm of 20 male barn swallows in neutral and female fluid medium
in relation to residual female tail length (after controlling statisticallyfor male and female tarsus length). The line is the linear regressionline.

Møller et al. • Sperm behavior and female quality

the female, and female barn swallows deposit immunoglobulins into their eggs (Saino et al. 2001).
Sperm from a minority of males performed better in female
medium than in the neutral control fluid, whereas sperm frommost males performed worse in female medium (Figure 1). In
contrast, there was no such consistent difference for PC2–
PC4, suggesting that an experimental artifact did not cause
the results for PC1. Given that sperm performance was consistent among males for multiple ejaculates, this suggests thatsperm from different males varied in performance between
the 2 kinds of medium. Interpretations of this result include
coincidental significant relationships, interaction effects between males and females, or superior performance of certain
males in multiple media. We can exclude the first explanation
because we only performed a very restricted number of statistical tests to avoid problems of significance arising by chance.
Obviously, it would have been more appropriate to assess
sperm performance in medium derived from several females
by using an experimental design that crossed all males with all
females. We were unable to perform such an experiment for
logistical reasons because each male and each female at mostproduced a couple of microliters of liquid. As an ethical note,
we would also like to emphasize that all birds were left unharmed, allowing them to be released alive. Thereby, we will
be able to continue our studies on surviving individuals in theyears to come.
We predicted that sexual conflict over fertilization should
affect relative performance of sperm in neutral and female medium and that indicators of male and female phenotypic quality might predict part of this variation (see Table 2 for evidenceof differences in sperm performance among males). If theexternal phenotype of males reflected differences in performance of their sperm and differences in performance among
media, we predicted that males with large secondary sexual
characters with a condition-dependent expression shouldhave sperm with superior performance in female medium
compared with neutral medium. Such a difference in performance could be due to sperm produced by males with large
secondary sexual characters having superior overall performance. Alternatively, sperm of such males may be better able
to cope with the biochemically hostile cloacal environment offemales because past selection has produced adaptations insperm to cope with the environment of the reproductive tractof females (for a similar argument, see Birkhead, Møller, andSutherland 1993). This might especially apply to sperm frommales that are likely to be found in sperm competition situations more frequently than expected by chance, as is the casein the barn swallow (Saino et al. 1997). We found no evidence
consistent with such effects of male phenotype because maletail length did not enter as a significant predictor in any of the
4 tests. However, there was a weak positive relationship between male tarsus length and PC3 of relative sperm performance and a weak positive relationship between male bodymass and PC4 (Table 3).
If fecund females were particularly choosy in terms of fertilization of their eggs, we should expect relative sperm performance in female medium to decrease with indicators offemale quality. Tail length of female barn swallows is sucha quality indicator, reliably reflecting earliness of breeding,clutch size, annual fecundity, and viability (Møller 1993;
Cuervo et al. 2003). Indeed, the difference in PC2 and PC3
between neutral and female medium was partly explained byvariation in female phenotype. Female tail length accounted
for variation in PC2, with sperm having higher amplitude oflateral head displacement, higher track velocity, and nonstraight and nonlinear movement in female medium compared with neutral medium when the female had a long tail(Figure 3). Furthermore, female tail length accounted for7

variation in PC3, with fewer sperm with medium velocity
and more static sperm in female medium compared with neutral medium when the female medium derived from a longtailed female, suggesting that sperm performed less well in
medium derived from long-tailed females (Figure 4). This reduction in relative performance of sperm in female medium
when the female is of superior quality is as expected if fecund
females produce greater obstacles to fertilization of their eggs.
However, the mechanisms underlying such a potentiallyreduced sperm performance remain unknown (Eady 1994;
Bernasconi et al. 2002). An explanation for the results in that
long-tailed females may have produced larger volumes of
fluid, making a larger difference between neutral and female
fluid (the larger volume might actually be the way that highquality females are more effective in hindering the movementof sperm). There were no obvious consistent differencesamong females in terms of volume of fluid, although quantification of the volume of fluid must await future studies. Here
we have shown experimental evidence for individual variationin the extent to which sperm performance is reduced when
sperm swim in female medium rather than neutral medium.
These findings are consistent with published information on
extrapair paternity in barn swallows. Long-tailed males lose little paternity in their own nests, but gain paternity in neighboring nests, as shown by tail manipulation experiments (Sainoet al. 1997). In fact, long-tailed high-quality females are generally mated to long-tailed males (Møller 1993), and such females
produce no or few extrapair offspring (Møller and Tegelström
1997; Saino et al. 1997). Therefore, the findings reported here
may provide a new hypothesis for how intraspecific differencesin extrapair paternity may come about: high-quality females
mated to the most attractive males are the least likely to produce extrapair offspring, even if engaging in extrapair copulations, because such females have the strongest defenses against
fertilization of their eggs by males of poor quality.
In conclusion, we have shown that fluid from the female reproductive tract affects sperm performance under standardized in vitro conditions, with variation among sperm fromdifferent males. The reduced sperm performance due to female fluid was significantly related to an indicator of female
quality, suggesting that the most fecund females may produce
the largest obstacles to have their eggs fertilized. This provides
evidence consistent with the theory of sexual conflict.

FUNDING
University of South Carolina School of the Environment;
Samuel Freeman Charitable Trust; National Science Foundation; National Geographic Society to A.P.M. and T.A.M.
We are grateful to G. Milinevski, A. Peklo, and N. Saino for logistical
help in Ukraine. Anne Houde provided most helpful suggestions, as
did several referees.

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