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Mutation Research 650 (2008) 210–216

Impaired swimming behaviour and morphology of spermfrom barn swallows Hirundo rustica in Chernobyl
A.P. Møller a,∗ , T.A. Mousseau b , C. Lynn b , S. Ostermiller b , G. Rudolfsen ca 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, Franceb Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USAc Department of Biology, IBG, University of Tromsø, N-9037 Tromsø, Norway

Received 30 April 2007; received in revised form 20 November 2007; accepted 8 December 2007
Available online 23 December 2007

Abstract
We investigated the motility and morphology of live sperm from barn swallows Hirundo rustica breeding in radioactively contaminated areas
around Chernobyl and control areas in Ukraine in order to test the hypothesis that swimming behaviour and morphology of sperm was impairedby radioactive contamination. We obtained sperm samples from 98% of sampled birds, thus avoiding sampling bias due to the fraction of males
not producing sperm samples. Analyses of within- and between-sample repeatability revealed significant and intermediate to large estimates for all
sperm parameters. There were significant differences between the Chernobyl area and the control area for two of 11 sperm behaviour parameters,
and significant interactions between area and year for six of these parameters. The proportion of sperm with abnormal morphology was elevatedin barn swallows from Chernobyl. A principal component (PC) analysis revealed four significant axes that explained 88% of the variance insperm behaviour parameters. One of these principal components differed between areas, and three components showed significant year by areainteractions. PC2 representing the frequency of slow sperm increased with increasing radiation in one year, but not another. PC3 representing spermwith high linearity, small amplitude of lateral head displacement and low track velocity decreased with increasing background radiation level. PC4
reflecting a large proportion of static sperm with high beat cross frequency increased with increasing background radiation level. Sperm behaviour
as reflected by principal components was predictable among years from information on level of radiation, and it was predictable among sites indifferent years. These results are consistent with the hypothesis that sperm behaviour and morphology have been affected by radiation due to theChernobyl accident.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Abnormal sperm; Age; Barn swallow; Hirundo rustica; Radiation; Sperm tracking

1. Introduction
Fertilization is a major process affecting reproductive successin sexually reproducing organisms. Hence, factors that determine the performance of sperm are supposed to be under intense
selection because of their close correlation with fertilization
success. A number of different factors have been suggested toaccount for fertilization probability including sperm size, sperm
motility and swimming speed [see, e.g., 1–6]. Given such evidence we may also expect that features of importance for sperm∗ Corresponding author. Tel.: +33 1 44 27 25 94; fax: +33 1 44 27 35 16.

E-mail address: amoller@snv.jussieu.fr (A.P. Møller).
1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2007.12.006

performance would be highly canalized, thus allowing males to
produce sperm with a high probability of success under a rangeof different environmental conditions.
Sperm morphology and performance can be affected by radiation. Studies of the barn swallow Hirundo rustica have revealeda strong positive correlation between background level of radiation in different parts of Ukraine and the frequency of spermwith abnormal morphology [7], increasing from <5% in control
regions to >40% in the most contaminated areas. In addition,that study revealed a possible mechanism underlying this effectof radiation on sperm morphology, because levels of antioxidants such as carotenoids and vitamins A and E in blood andliver showed strong, inverse correlations with the frequency ofabnormal sperm. Since antioxidants may prevent damage caused

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A.P. Møller et al. / Mutation Research 650 (2008) 210–216

by free radicals in DNA and other molecules [8,9], this raises
the possibility that sperm abnormality is caused by mutations
arising from the action of free radicals. A few studies in humans
have shown changes in ultra-structure of the sperm head when
exposed to high levels of radiation [10], and the frequency oftranslocations in mouse spermatozoa increased with radiationdose rate [11–13]. In addition, the total number of sperm andthe number of motile sperm has been shown to be lower in
Chernobyl victims than in controls [14].
The objective of the present study was to test the extent
to which sperm swimming behaviour and morphology wereaffected by background radiation around Chernobyl, using thebarn swallow as a model system. Because sperm morphology
may change in response to increased radiation levels [7,10], we
also tested if sperm swimming behaviour changed in responseto the level of radiation after controlling for differences insperm morphology. Furthermore, we tested in two different
ways whether sperm behaviour could be predicted from priorknowledge of the relationship between sperm performance andradiation. This was done by determining whether the relationshipbetween sperm behaviour and radiation in one year could be used
to predict sperm behaviour of different birds in another year. Furthermore, we tested explicitly whether mean sperm performancewas consistent among years for particular sites with specificlevels of radiation. In order to do so we captured adult malebarn swallows around Chernobyl and in control areas, sampledejaculates from these males and assessed sperm for swimming
behaviour and morphological abnormalities. We adopted a computer assisted sperm analysis (CASA) approach because studiesof sperm quality in humans and animals have recently shown thatsperm swimming speed and motility are prime determinants offertilization success [1–5]. Likewise, CASA studies of chicken
sperm performance have indicated that sperm velocity is a major
determinant of fertility [3].
Barn swallows are small, insectivorous, migratory passerines that breed commonly in farms and other human habitations[15]. Males and females are relatively similar in appearance,with the exception of the outermost tail feathers that on average are 20% longer in males than in females in our Ukrainianstudy populations [16]. Males with long tails enjoy several mating advantages including earlier reproduction and higher mating
success [15]. Previous studies of barn swallows in Ukraine haveshown that males in contaminated areas have paler color thanmales in control areas [17]. Partial albinism in the plumage iselevated in the Chernobyl region [18], and such albinism is
associated with reduced survival prospects [19]. Furthermore,
the phenotypic characters of males that are most suppressedin Chernobyl compared with control areas are those that are
most important for mating success [16]. Male barn swallows
in Chernobyl have elevated frequencies of abnormal spermand greatly suppressed levels of antioxidants [7]. Finally, thefrequency of non-reproducing adults is elevated, reproductive
success is suppressed, and adult survival rate is reduced in
Chernobyl compared with control areas [20]. Hatching failure due to infertile eggs increased from 1.4% in control areas
to 5.9% in contaminated areas, a more than four-fold increase
[20].

211

Fig. 1. Geographical location of the Ukrainian study sites in relation to background radiation level. Red-colored regions indicate radionuclide levels greaterthan 5 Ci/km2 [27]. Green asterisks indicate contaminated sampling areas, while
black asterisks indicate control sampling areas. The radioactivity symbol indicates the location of the Chernobyl reactor. For interpretation of the references
to color in this figure legend, the reader is referred to the web version of thearticle.

2. Material and methods
2.1. General ﬁeld procedures
We captured barn swallows in a number of different farms around Chernobyl,
Borispil and Kanev in Ukraine during late May and early June 2005 to 2006
(Fig. 1). Using mist nets placed across windows and doors 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, weighed and assessed
for ectoparasites. Then blood was sampled and feather samples were collected.
Subsequently, we collected one or two sperm samples from all males, using asimple massage technique. Sperm were collected in micro-capillaries for easy
handling.
Our field measurements of background radiation at the ground level usinga hand-held Inspector dosimeter (Model: Inspector, SE International, Inc.,
Summertown, TN, USA) revealed levels of mainly ␥ radiation of on average
0.390 mR/h (S.E. = 0.317) at 14 breeding sites in the Chernobyl region. As control areas we chose Kanev, about 120 km southwest of Kiev, and Ghovtnere,
about 35 km southeast of Kiev. Both had relatively low levels of radiation: meanlevels were 0.025 mR/h (S.E. = 0.002) at five breeding sites near Kanev, and
0.006 mR/h (S.E. = 0.003) at Ghovtnere near Borispil. We cross-validated ourradiation data with officially published measurements [21]. The latter were estimated as the mid-point of the ranges from this published source. This analysis
revealed a very strong positive relationship (linear regression on log–log transformed data: F = 159.46, d.f. = 1,18, r2 = 0.89, P < 0.0001, slope (S.E.) = 1.28
(0.10), suggesting that our field estimates of radiation were comparable withother estimates.
We have ringed barn swallows in Ukraine since 2000, and we assigned aminimum age to birds. When annual ringing effort is high, as it has been in our
Ukrainian study sites [20], we can assume that unringed birds are yearlings. The
reasons are that (1) adults hardly ever move from one site to another once they
have chosen a breeding site [15], and (2) among 415 local recruits ringed as
nestlings in Spain, Italy and Denmark 413 were first captured as breeders when
1 year old, with the two remaining birds first being captured breeding when 2
years old [15,22, A.P. Møller unpublished data]. Because annual survival rate ofadult barn swallows is in the range 0.3–0.5 [20], very few reach an age of morethan a couple of years.
We did not assign individual birds to nests and hence did not know their exact
breeding status, although checks of nests revealed the contents to mainly be withfeather lining but no eggs, or with eggs or small nestlings. An indirect measureof stage of breeding can be obtained from the presence or absence of mud on thebeak. Male (and female) barn swallows build nests out of mud, with building inAuthor's personal copy

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Denmark taking place between 24 and 2 days before start of egg laying (A. P.
Møller unpublished information on more than 200 pairs observed for 1 h daily
during their entire breeding cycle in 1984 to 1990). This period coincides withthe period when almost all copulations take place [23]. Barn swallows building
nests have fresh or dried mud on their beaks, while birds that are not building do
not. Therefore, any male with mud on its beak will be copulating intensely withits mate, while that is not the case for males without mud. Males that are moreadvanced in their breeding cycle do still copulate with their mates, but copulate
more frequently with other females [23]. We recorded the presence of mud on
the beak (22 of 101 males) and used this dichotomous character in the analyses
as a proxy for stage in the breeding cycle.

2.2. Sperm behaviour and morphology
The first sperm sample was transferred to a microscope chamber, without
the experimenter having any knowledge of the individual male. Hence, all measurements obtained from the video equipment were obtained blind with respect
to identity of the individual male. For each male we quantified sperm motility
within 2 min following extraction of the sperm.
The sperm sample was diluted in a one-step procedure, by placing an aliquotof undiluted sperm on a pre-warmed (37 ◦ C) microscope slide with an 80-mm
deep chamber (Hamilton Thorne Research, Beverly, MA, USA). Immediately
afterwards, 9 ml Dulbecco’s Modified Eagle Medium (D-MEM; Invitrogen,
Carlsbad, California) was added and a cover glass was placed over the sample.
D-MEM (Gibco) contains 4500 mg/l glucose, 4 mM l-glutamine and 110 mg/l
sodium pyruvate. Sperm motility and images were recorded with a Sony CCD
black-and-white video camera (XC-ST50CE PAL, Sony, Tokyo, Japan) at 50 Hz
vertical frequency, mounted on an external negative phase-contrast microscope(Olympus CH30, Olympus, Tokyo, Japan) with a 10× objective. Video recordings were stored on a Sony TRV900 mini-DV camcorder. Several different video
frames of sperm motility from each sperm drop were recorded, which allowed
us to capture a larger number of sperm cells from each ejaculate. The recordings were later analyzed using computer-assisted sperm analysis (HTM-CEROS
sperm tracker, CEROS version 12, Hamilton Thorne Research, Beverly, MA,
USA). The image analyzer was set at frame rate of 50 Hz, number of frames
25, minimum contrast 30, and minimum cell size 20 pixels. Each motility
measurement lasted 0.5 s. Several motility measurements were made for each
ejaculate.
We recorded mean values of (1) VAP (smoothed path velocity; ␮m/s), VSL(straight line velocity; ␮m/s), (2) VCL (track velocity; ␮m/s), (3) ALH (amplitude of lateral head displacement; ␮m), (4) BCF (beat cross frequency; Hz),
(5) LIN (linearity, VSL/VCL) and (6) STR (straightness, VSL/VAP) for eachsample. Cells having a VAP < 10 ␮m/s and a VSL < 5 ␮m/s were excluded fromthe motility analysis, and counted as static. The measurements were used to estimate (7) the percentage of static sperm (the percentage of sperm that remained
static), (8) the percentage of sperm with slow velocity (i.e. <10 ␮m/s), (9) thepercentage of sperm with medium velocity (10–50 ␮m/s), (10) the percentage of
sperm with high velocity (>50 ␮m/s) and (11) the percentage progressive sperm(the percentage of all sperm that moved with STR > 80 and VAP > 50 ␮m/s).
Images were downloaded from the video recordings for analysis to a PC
via a firewire connection and stored in DV-AVI before conversion to MPEG-2
format for storage on DVD-ROM. Still images were grabbed from video files
using Nero ShowTime software on a PC.
Sperm morphology was recorded using image analysis of still images. We
measured head length and total length to the nearest 0.8 ␮m. These two measurements were used to estimate tail length as total sperm length minus sperm headlength. Measurements were made from all males, with repeat measurements forten males that were sampled twice. These measurements were conducted blindwith respect to origin of the samples. The number of sperm measured per maleranged from 5 to 106, depending on availability of high-quality still images. The
repeatability of measurements of the length of 126 individual sperm was 0.82.

2.3. Statistical analyses
Information on sperm behaviour and morphology was quantified as meanvalue per male based on repeat estimates. We tested for consistency in estimates from the same ejaculate, from different ejaculates for the same males

collected during the same capture, and from different ejaculates for the same
males collected in different years (within-sample repeatability, among-sample
repeatability within years and among-sample repeatability among years, respectively). Repeatability estimates (standard errors) were obtained using variancecomponents from one-way ANOVAs [24], with a repeatability of zero implying no consistency in estimates of sperm parameters among measurements or
samples from the same individual, while a repeatability of one implies complete identity of estimates among measurements or samples from the same
individual.
We tested for differences in sperm parameters between areas using one-way
ANOVA. Relationships between sperm parameters and levels of backgroundradiation were analyzed by linear regression with log-transformed radiation
as the predictor variable. Because sperm behaviour may be affected by spermmorphology independent of levels of background radiation, we used multipleregression models with log-transformed radiation level, mean sperm head lengthand mean sperm tail and mid-piece (hereafter sperm tail for simplicity) length aspredictor variables. In these analyses we included mean and standard deviations
for all sperm sampled.
We quantified sperm behaviour by using a principal component analysis of
the 11-ejaculate behaviour parameters described above, by use of a varimax
approach to the correlation matrix. This reduced the correlated variables to four
statistically independent axes that reflected different aspects of overall sperm
behaviour.
We randomly excluded one sample for the males that were sampled in both
years to have each individual represented by a single observation in order toavoid pseudo-replication.
We tested for effects of age by adding age as an additional variable to these
multiple-regression models.
We identified best-fit multiple-regression models, using the software JMP[25]. The best-fit model was determined with Akaike’s information criterion as
an estimate of the improvement in fit for addition of variables [26]. We startedout by using the best-fit models and then deleting factors according to their delta
AIC values, using the criterion that a change in AIC of more than 2.00 would
be considered biologically meaningful [26].
Sample sizes differed among analyses depending on availability of data. All
values reported are means (S.E.).

3. Results
3.1. Determinants of sperm swimming behaviour
We were able to obtain sperm from 190 of 193 male barnswallows (98.4%). Repeatability of sperm parameters within
and among samples is reported in Table 1. Most variables show
moderate repeatabilities and only one value was not statistically significant. Repeatability of sperm head length, spermtail length and percentage abnormal sperm was highly significant. All among-sample repeatabilities were smaller than thewithin-sample repeatabilities (paired t-test, t = 4.69, d.f. = 10,
P = 0.0008). Among-year repeatability of sperm swimming
parameters based on a small sample of 18 males that survived
from 1 year to the next and were sampled in both years, did notreach statistical significance (F < 1.66, d.f. = 17,18, P > 0.15).
In contrast, the percentage abnormal sperm was highly repeatable among years (percentage abnormal sperm: F = 901.52,
d.f. = 17,18, P < 0.0001, R (S.E.) = 0.99 (0.002)).
A principal component analysis of the 11 sperm-behaviour
variables produced a model with four principal components that
accounted for 88% of the variance (Table 2). PC1 tended to
be positively correlated with all variables, with the strongest
correlation with the proportion of rapid sperm (Table 2). PC2
reflected the proportion of slow and medium speed sperm, thus
reflecting ejaculates with slow sperm (Table 2). PC3 represented

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213

Table 1
Mean values (mean of means and S.E.) and within- and among-sample repeatability of sperm parameters from barn swallows from Ukraine
Variable
Pct. static
Pct. slow
Pct. medium
Pct. rapid
Pct. progressive
LIN
STR
BCF (Hz)ALH (␮m)
VCL (␮m/s)
VAP (␮m/s)
Head length (␮m)
Tail length (␮m)
Pct. abnormal sperm

Mean (S.E.)

Within-sample F

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)
78.85 (1.51)
57.15 (1.17)
14.04 (0.11)
71.98 (0.40)
8.96 (1.17)

10.21***
1.73***
4.74***
9.89***
8.98***
7.82***
5.36***
3.50***
5.30***
7.92***
8.25***–––

Within-sample R (S.E.)

Among-sample F

Among-sample R (S.E.)

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)
0.41 (0.04)
0.42 (0.04)–––

9.81***

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)
0.21 (0.02)
0.20 (0.02)
0.77 (0.05)
0.83 (0.04)
0.76 (0.05)

1.25
5.95***
8.34***
7.38***
2.01*
3.69**
3.26**
9.03***
5.61***
5.50***
7.72***
10.82***
7.24***

Sample size is 166 males with repeat recordings for a single ejaculate, while 10 males were analyzed for two different ejaculates.
* P < 0.05.
** P < 0.01.
*** P < 0.0001.

sperm with a high degree of linearity, small amplitude of lateralhead displacement and low track velocity (Table 2). Finally,
PC4 represented static sperm with high beat cross frequency(Table 2).
Two of the 11 sperm variables differed significantly between
samples from Chernobyl and control areas, after controlling foryear effects (Table 3). Mean values for these significant differences were larger in Chernobyl than in the control areas, with the
exception of sperm tail length, which was shorter in Chernobyl
than in the control areas. Seven variables differed significantly
between years (Table 3). In addition, six of 11 variables showedsignificant area-by-year interactions (Table 3).

Analysis of the principal components revealed a significant
difference between areas for PC3, significant year effects for
all four principal components, and a significant year-by-areainteraction for PC1–3 (Table 3).
There was no significant difference in any sperm parameter
between males with and without mud on their beaks (results notshown). Hence, there was no evidence that sperm parameters
differed among males captured at different stages of the breeding
cycle.
Age did not predict sperm morphology or swimming
behaviour, with none of the 14 tests statistically significant(F < 0.67, d.f. = 1,137, P > 0.41).

Table 2
Principal component analysis of 11 sperm parameters of barn swallows

3.2. Sperm behaviour and radiation

Eigenvalue
Percent
Cumulative percentage
Pct. static Sperm
Pct. slow Sperm
Pct. medium Sperm
Pct. rapid Sperm
Pct. progressive Sperm
LIN
STR
BCF
ALH
VCL
VAP

PC1

PC2

PC3

PC4

5.26
47.77
47.77
−0.37
0.05
0.16
0.40
0.39
0.32
0.35
0.15
0.16
0.31
0.38

1.79
16.29
64.07
−0.18
0.40
0.59
−0.15
−0.21
−0.22
0.04
0.38
0.34
0.03
−0.28

1.56
14.15
78.22
−0.02
0.37
0.11
−0.08
0.07
0.42
0.26
0.12
−0.58
−0.48
−0.11

1.12
10.18
88.40
0.40
−0.33
−0.15
−0.25
−0.21
0.18
0.37
0.62
0.03
0.17
0.08

Loadings exceeding 0.40 are highlighted in bold. Interpretation of PCs: PC1:
loading on percentage of rapid sperm. This implies that PC1 represents an axis
of increasing frequency of rapid sperm. PC2: loadings on percentage of slow andmedium sperm. This axis represents the frequency of slow sperm. PC3: loadings
on sperm with high LIN (linearity) and low ALH (amplitude of lateral headdisplacement) and VCL (track velocity). This implies that PC3 represents spermwith high linearity, small amplitude of lateral head displacement and low track
velocity. PC4: loadings on static sperm with high BCF (beat cross frequency).
Therefore, PC4 represents static sperm with high beat cross frequency.

Analysis of the four principal components in relation to radiation level, year and morphology of barn swallows revealed
three significant models that included background radiation levels (Table 4). PC2 scores increased significantly with radiationlevel, with additional effects of year, and a marginally significant interaction between radiation level and year (Table 4).
PC3 scores decreased significantly with level of radiation(Fig. 2A), and this effect differed between years (Table 4). PC4scores increased significantly with level of radiation (Fig. 2B),
and this effect differed between years (Table 4). In addition, there was a marginal effect of tail length that differed
between years, with long-tailed males having low PC4 scores(Table 4).
Analysis of the four principal components in relation to radiation level, year and sperm morphology revealed two significant
models that included sperm morphology (Table 5). PC3 scores
decreased significantly with tail length of sperm, accounting for
5% of the variance (Table 5). PC4 scores decreased significantlywith the percentage of deformed sperm, and this effect accounted
for 11% of the variance (Table 5). The models for PC1 and PC2
did not reach statistical significance.

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Table 3
Tests for differences in sperm parameters between areas
Variable

F (area)

F (year)

F (area × year)

Mean (S.E.)
Chernobyl 2005

Mean (S.E.)
Chernobyl 2006

Mean (S.E.)
controls 2005

Mean (S.E.)
controls 2006

Pct. static
Pct. slow
Pct. medium
Pct. rapid
Pct. progressive
LIN
STR
BCF (Hz)ALH (␮m)
VCL (␮m/s)
VAP (␮m/s)
PC1
PC2
PC3
PC4
Head length (␮m)
Tail length (␮m)
Pct. abnormal sperm

0.33
0.23
1.20
0.00
0.26
0.23
0.09
0.37
7.49**
10.42**
2.45
0.59
0.01
9.00**
2.54
3.18
4.84*
7.94**

38.93***
2.42
13.91***
22.77***
15.19***
0.49
2.82
0.62
19.47***
14.93***
6.08*
18.51***
5.21*
4.80*
17.31***
0.63
0.24
2.78

1.81
0.33
0.80
3.84*
7.95**
18.68***
9.03**
1.04
5.06*
0.06
7.66**
5.43*
7.32**
9.70**
0.14
0.43
0.17
2.34

44.98 (2.81)
4.68 (0.53)
19.76 (1.45)
30.47 (2.38)
27.89 (2.17)
68.48 (2.07)
84.89 (1.86)
21.57 (0.66)
3.36 (0.19)
76.28 (2.63)
58.83 (2.11)-0.27 (0.31)-0.52 (0.19)
0.29 (0.17)
0.42 (0.15)
18.44 (0.19)
93.70 (0.62)
27.19 (3.00)

29.81 (2.68)
5.25 (0.51)
27.14 (1.38)
37.85 (2.26)
30.47 (2.07)
60.22 (1.97)
82.16 (1.77)
21.74 (0.62)
4.73 (0.18)
88.21 (2.50)
58.13 (2.01)
0.41 (0.30)
0.51 (0.18)-0.69 (0.16)-0.19 (0.14)
18.81 (0.26)
93.89 (0.84)
19.06 (3.75)

47.36 (3.50)
4.62 (0.66)
22.93 (1.80)
25.15 (2.96)
22.35 (2.70)
59.71 (2.57)
78.10 (2.32)
21.87 (0.82)
3.26 (0.23)
67.64 (3.27)
60.93 (2.51)-1.34 (0.39)
0.02 (0.23)
0.27 (0.21)
0.22 (0.18)
18.66 (0.24)
96.79 (0.77)
28.26 (1.28)

23.85 (3.34)
5.88 (0.63)
27.46 (1.72)
42.80 (2.82)
38.43 (2.58)
71.15 (2.46)
87.73 (2.22)
20.56 (0.78)
3.70 (0.22)
78.13 (3.12)
48.75 (2.63)
0.96 (0.37)-0.07 (0.22)
0.44 (0.20)-0.50 (0.17)
18.76 (0.25)
96.88 (0.81)
15.44 (1.69)

Sample sizes are 101 for Chernobyl and 65 for control areas, with d.f. = 1,162.
* P < 0.05.
** P < 0.01.
*** P < 0.0001.

3.3. Predictability of sperm performance
We investigated whether sperm behaviour could be predicted
in two different ways. First, we tested whether we could predict sperm behaviour in 2006 based on levels of radiation andtail length, using the regression models based on principal components and levels of radiation and tail length in 2005. Onlymodels for PC2, PC3 and PC4 reached the level of significance,
Table 4Best-fit models of principal components 2, 3 and 4 (derived from a principal component analysis of 11 ejaculate parameters) in relation to background radiationlevel, year, tail length and their interactions
VariableFig. 2. The relationship between (A) PC3 score of ejaculates and backgroundradiation level (mR/h), and (B) PC4 score of ejaculates and background radiationlevel (mR/h). The relationships are based on residuals after adjusting for thevariables listed in Table 4.

Sum ofsquaresF

P

Slope (S.E.)

Delta
AIC

PC2
Radiation
Year
Radiation × year

4.81
18.17
6.38

2.83
10.71
3.76

0.09
0.00130.05

0.65 (0.39)−0.35 (0.11)−0.75 (0.39)

0.07
8.10
1.81

PC3
Radiation
Year
Radiation × year

32.26
24.53
44.89

26.21
19.93
36.47

<0.0001<0.0001<0.0001−1.69 (0.33)
0.40 (0.09)
1.99 (0.33)

30.34
38.04
31.71

PC4
Radiation
Year
Tail length
Radiation × year
Tail × year

6.57
9.41
3.25
3.39
4.88

6.77
9.70
3.35
3.50
5.03

0.010
0.002
0.07
0.06
0.026

0.85 (0.33)
0.26 (0.08)−0.015 (0.008)−0.61 (0.33)−0.018 (0.008)

2.98
17.70
3.00
1.59
3.15

The three models had the statistics F = 4.11, d.f. = 3,162, r2 = 0.07, P = 0.0077,
AIC = 91.69; F = 15.57, d.f. = 4,158, r2 = 0.22, P < 0.0001, AIC = 38.42 andF = 6.05, d.f. = 5,153, r2 = 0.17, P < 0.0001, AIC = 1.07. Delta AIC was calculated as the difference in AIC between the best-fit model and the model excluding
a specific variable.

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Table 5Best-fit models of principal components 3 and 4 (derived from a principal component analysis of 11 ejaculate parameters) in relation to background radiation level,year, sperm morphology and their interactions
Variable

Sum of squares

F

P

Slope (S.E.)

Delta AIC

PC3
Tail length

9.53

6.46

0.012

−0.05 (0.02)

4.40

PC4
Pct. deformed sperm

8.51

9.14

0.0034−1.53 (0.51)

6.85

The two models had the statistics F = 9.53, d.f. = 1,161, r2 = 0.05, P = 0.012, AIC = 58.58 and F = 9.14, d.f. = 1,157, r2 = 0.11, P = 0.0034, AIC = 3.26. Delta AIC was
calculated as the difference in AIC between the best-fit model and the model excluding a specific variable.

and hence we restricted the analyses to these three components.
Regression of observed PC2, PC3 and PC4 against predicted values derived from the regression models from 2005 and radiationand tail length recorded in 2006, showed significant positive
relationships (PC2: F = 3.66, d.f. = 1,85, r2 = 0.04, P = 0.046;
PC3: F = 38.71, d.f. = 1,85, r2 = 0.31, P < 0.0001; PC4: F = 8.40,
d.f. = 1,85, r2 = 0.10, P = 0.0048). Thus, we were able to validate our regression models from 2005 by using data from 2006,
showing some predictive power in the statistical models.
Second, we tested whether mean principal components wereconsistent for the nine different sites, using one-way analysis of variance. Indeed, there was a significant repeatability ofsperm performance for the different sites among years for PC2,PC3 and PC4 (PC2: F = 9.31, d.f. = 8,9, r2 = 0.89, P = 0.0015;
PC3: F = 5.63, d.f. = 8,9, r2 = 0.83, P = 0.0091; PC4: F = 8.01,
d.f. = 8,9, r2 = 0.88, P = 0.0026), while the analysis for PC1 did
not reach statistical significance (F = 3.04, d.f. = 8,9, r2 = 0.73,
P = 0.059). Thus, mean swimming performance of sperm wasconsistent among years for specific sites with specific levels ofradiation.
4. Discussion
The main findings of our study of sperm behaviour and morphology of barn swallows from Ukraine were that (i) there
were highly significant differences in sperm behaviour andmorphology among males; (ii) several sperm parameters differed between the Chernobyl region and uncontaminated control
areas; (iii) principal component scores of sperm-behaviour variables were significantly related to level of background radiation,
independent of morphology of males and male age; (iv) principal
component scores of sperm-behaviour variables were related tosperm morphology and sperm abnormality; and (v) sperm performance in a given year could be predicted from information on
sperm behaviour in a previous year and from levels of radiation.
We will briefly discuss these results.
We found clear evidence of differences in swimming
behaviour and morphology of sperm between male barn swallows from the Chernobyl region and control areas. Two out
of 11 variables differed significantly between areas, which are
more than the expected 0.7 variables under the null hypothesis of no difference (at 5% significance). The tests suggested
that swimming behaviour of sperm was inferior for male barnswallows from Chernobyl compared with males from control
areas. These differences could not be accounted for by differ-ences in timing of reproduction because the mean laying date issimilar in Chernobyl and in the control areas [20]. Likewise,
there was no evidence of age effects on the conclusions. A
larger fraction of sperm from birds at Chernobyl had abnormal morphology compared with sperm from birds in the control
areas, as we have shown before [7]. In addition, tail lengthof sperm was significantly reduced among male birds from
Chernobyl, the mean difference amounting to 3%, consistent
with the hypothesis that radiation caused an increase in mutation rate. Ellegren et al. [19] showed for micro-satellites in thebarn swallow that the frequency of germline mutations in Chernobyl was elevated by a factor two to ten compared with control
areas.
If sperm have evolved a morphology that maximizes fertilization ability, a difference in mean tail length of 3% is significant.
Consistent with this interpretation we found that sperm swimming behaviour as reflected by the third principal component
decreased with increasing tail length of sperm. Therefore, malebarn swallows with long-tailed sperm had reduced linearity,
increased lateral head movement and high track velocity. In
addition, the fourth principal component decreased as the percentage of deformed sperm increased. This implies that malebarn swallows had more static sperm with high beat frequency
when they had a large percentage of deformed sperm. These findings are consistent with the hypothesis that sperm swimming
behaviour of barn swallows is reduced in the most contaminated areas because they have short-tailed sperm and a large
fraction of sperm with abnormal morphology. Alternatively,
selective mortality could differentially affect the compositionof the population of males in the two regions. Because adultmortality rate is elevated in Chernobyl compared with the control area [20], birds from contaminated areas may be of higher
phenotypic quality with less variance in sperm morphology
than males from the control areas. If selection was important, old birds should have superior sperm morphology andbehaviour compared with young birds, but that was clearly not
the case.
Sperm behaviour and morphology showed evidence of statistical consistency among estimates obtained from the same
sample and among estimates obtained from different samples
from the same male barn swallow. The level of consistency
was low to intermediate, raising the question whether sperm
parameters are predictable. We explicitly tested whether thiswas the case by use of two different approaches. First, wedeveloped regression models based on data on sperm behaviour

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and radiation from 2005 to predict the same variables in 2006.
Although the degree of consistency between observed and predicted estimates in 2006 was not high, it was still statisticallysignificant, implying that sperm behaviour was to some extent
predictable from knowledge of background radiation levels.
Second, we tested to which extent mean sperm behaviour forparticular sites with specific levels of radiation was consistentamong years. Again, we found a high and significant degree ofconsistency among years, suggesting that sperm behaviour could
be predicted. In conclusion, we have shown that sperm behaviour
is predictable based on information on levels of background radiation, despite the fact that individual males vary considerably insperm behaviour among samples.
The implications of this study are that low-level radioactive contamination does affect ejaculate features and spermmorphology, with dose-dependent relationships. This may haveconsequences for fertilization success as suggested by reduced
hatching of eggs of barn swallows from the Chernobyl zone [20].
Acknowledgements
We are grateful to G. Milinevski, A. Peklo and E. Pysanetsfor logistical help in Ukraine. APM and TAM received funding from the CNRS (France), the University of South Carolina
School of the Environment, the Samuel Freeman Charitable
Trust, the National Science Foundation and the National Geographic Society to conduct this research. GR received funding
from the Norwegian Research Council (177744/V40).
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