Comparative Biochemistry and Physiology, Part A 159 (2011) 105–112

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Comparative Biochemistry and Physiology, Part A
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

The effects of radiation on sperm swimming behavior depend on plasma oxidative
status in the barn swallow (Hirundo rustica)
Andrea Bonisoli-Alquati a,b,⁎, Anders Pape Møller c,d, Geir Rudolfsen e, Nicola Saino a, Manuela Caprioli a,
Shanna Ostermiller b, Timothy A. Mousseau baDipartimento di Biologia, Università degli Studi di Milano, via Celoria 26, I-20133 Milano, Italy
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
Laboratoire d'Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362, F-91405 Orsay Cedex, FrancedCenter for Advanced Study, Drammensveien 78, NO-0271 Oslo, NorwayeNorwegian Radiation Protection Authority, Dep. Environmental Radioactivity, FRAM — High North Research Centre on Climate and the Environment NO-9296 Tromsø, Norwaybca r t i c l e

i n f oArticle history:
Received 20 October 2010
Received in revised form 25 January 2011
Accepted 25 January 2011
Available online 2 February 2011
Keywords:
Antioxidant capacity
DNA damage
Oxidative stressRadioactive contamination
Reactive oxygen species
Sperm motilitya b s t r a c t
Sperm are highly susceptible to reactive oxygen species (ROS) that can damage sperm DNA and structure,
resulting in reduced fertilizing capacity. Exposure to radioactive contamination can also impair sperm
swimming behavior and fertilizing ability, both through a reduction of sperm DNA integrity and via an
increased generation of reactive oxygen species (ROS). However, the relationship between individual
oxidative status and sperm swimming behavior has never been investigated in any wild population of animals
exposed to radioactive contamination. We studied the motility of sperm collected from barn swallows,
Hirundo rustica, breeding under different levels of radioactive contamination following the Chernobyl
accident in 1986, in relation to individual oxidative status. We tested the hypothesis that the degree of
impairment of sperm swimming behavior by radioactive contamination depended on plasma antioxidant
capacity, the level of reactive oxygen metabolites (ROMs) and oxidative stress (sensu Costantini et al. 2006), a
better oxidative status being associated with higher sperm motility. Sperm behavior parameters were
subjected to principal component (PC) analysis, which extracted four PCs explaining 86% of the variance insperm motility. PC2, representing sperm with high track velocity and ample lateral head displacement, wassigniﬁcantly predicted by the interaction between radiation level and either oxidative damage or oxidative
stress. Contrary to our predictions, the highest values of PC2 were associated with relatively high radiationlevels, particularly for high levels of either ROMs or oxidative stress. In addition, there was a tendency forvalues of PC3 (representing the percent of motile sperm) and PC4 (representing slow sperm with high beatcross frequency) to depend on the interaction between radiation level and total plasma antioxidant
protection. Our results conﬁrm the importance of oxidative status in determining the genetic andphysiological outcome of exposure to radioactive contamination, complementing previous studies relating
sperm abnormality to circulating levels of speciﬁc antioxidants. Our results also complement previous
evidence that oxidative damage of sperm was negatively related to sperm motility, thus indicating a possible
trade-off in quenching pro-oxidant compounds in the plasma and the seminal ﬂuid.
© 2011 Elsevier Inc. All rights reserved.

1. Introduction
Reactive oxygen species (ROS), a class of pro-oxidants derived
from O2 that naturally occur as a result of oxidative metabolism, candamage macromolecules when left unquenched by the antioxidant
system (Halliwell and Gutteridge, 2007). Sperm are particularly
susceptible to the attack by ROS, owing to their limited antioxidant
machinery, their high metabolic activity and the high polyunsaturated
fatty acid (PUFA) content of their membranes (Sikka, 2001; Surai⁎ Corresponding author at: Department of Biological Sciences, University of South
Carolina, Columbia, SC 29208, USA.
E-mail address: alquati@mailbox.sc.edu (A. Bonisoli-Alquati).
1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2011.01.018

et al., 1998, 2001; Tremellen, 2008). Oxidative insult to sperm candamage the mitochondria, the cytoskeleton and the sperm axoneme,
resulting in a reduction of energy availability and sperm motility(MacLeod, 1943; Jones et al., 1979; Wishart, 1984; de Lamirande andGagnon, 1992; de Lamirande et al., 1997, 1998). As a result of thisdamage, less sperm reach the oocyte for fertilization (Kao et al., 2008).
In addition, ROS can also prevent fertilization through a reduction inacrosin activity (Zalata et al., 2004) and/or an impairment of spermoocyte fusion (Aitken et al., 1989; Iwasaki and Gagnon, 1992).
Conversely, levels of antioxidants have been associated with anumber of indices of sperm quality (review in Tremellen, 2008), andantioxidants supplementation is known to improve the quality ofsperm in men (reviews in Agarwal et al., 2005; Tremellen, 2008), birds

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A. Bonisoli-Alquati et al. / Comparative Biochemistry and Physiology, Part A 159 (2011) 105–112(Biswas et al., 2007) and ﬁshes (Pike et al., 2010). An imbalance
between the production of ROS and their neutralization by the
antioxidant system, resulting in oxidative stress (sensu Costantini,2008), is a leading cause of loss of fertility in humans (Aitken 1999;
Tremellen, 2008) and domestic animals (Wishart, 1984; Surai et al.,1998, 2001).
In addition, oxidative stress can damage nuclear and mitochondrial
DNA of sperm (Moody and Hassan, 1982; Imlay and Linn, 1988; Saleh
et al., 2003; Li et al., 2006), increasing the frequency of aberrant sperm
and adverse pregnancy outcomes (Shen and Ong, 2000; Aitken andKrausz, 2001; Morris et al., 2002). Sperm are likely to carry DNA damage
owing to the down-regulation of their DNA repair systems and to theloss of the ability to undergo apoptosis during spermatogenesis (Sawyer
et al., 2001, 2003; Lewis and Aitken 2005; Marchetti and Wyrobek,2008). Consequently, ejaculated sperm may exhibit unusually highlevels of damage to the nuclear and the mitochondrial genomes (Sawyer
et al., 2001, 2003; Morris et al., 2002), with important transgenerational consequences (Aitken et al., 2009). Consistent with this
scenario, antioxidant supplementation was also shown to improvesperm DNA integrity (Greco et al., 2005; Menezo et al., 2007), as well as
the chances of fertilization (Suleiman et al., 1996; Tremellen et al.,2007).
Exposure to radiation can impair sperm swimming behavior,through a damaging effect on DNA integrity. Radiation has been shown
to reduce the total number of sperm and the number of motile spermin humans (Cheburkov and Cheburkova, 1993; Sakharov et al., 1996;
Fischbein et al., 1997). Likewise, studies on rodents have demonstrated
that high doses of radiation can severely damage spermatogonial stem
cells (Meistrich, 1982) and increase the frequency of translocations inspermatocytes (Pomerantseva et al., 1997).
Radiation may also have an indirect, negative effect on sperm DNA
via the production of an excess of ROS. Studies of humans and wild
populations of animals in the Chernobyl region, where an accident at
the local nuclear power plant in 1986 gave rise to the largest anthropogenic release of radionuclides in history, have shown a decreased
abundance of antioxidant resources (Neyfakh et al., 1998; Ivaniota
et al., 1998; Kumerova et al., 2000; Møller et al., 2005a, 2008a; Møller
and Mousseau, 2007) and an increase in oxidative stress (BonisoliAlquati et al., 2010a), DNA damage (Frenzilli et al., 1998, 2001;
Bonisoli-Alquati et al., 2010b) and mutation rate (Ellegren et al., 1997;
Møller et al., 2005a; reviews in Møller and Mousseau, 2006; Yablokov
et al., 2009; but see Furitsu et al., 2005).
Previous studies have shown that the frequency of sperm
abnormalities in barn swallows (Hirundo rustica) from the Chernobyl
region is negatively correlated with circulating and stored levels of
speciﬁc antioxidants (i.e. vitamins C and E, carotenoids; Møller et al.,2005a), thus pointing to a role of antioxidants in protecting the spermfrom the damaging effects of radiation. Importantly, we have shown
also that radioactive contamination following the Chernobyl accident
predicted features of sperm swimming behavior and that sperm
motility was predictable in subsequent years based on radiation levels(Møller et al., 2008b).
However, the relationship of sperm swimming behavior with a more
integrated index of individual oxidative status, rather than with theconcentration of individual antioxidants, has never been addressed inany species exposed to different levels of radioactive contamination.
This is despite the demonstration that oxidative stress is increased inbarn swallows inhabiting areas where radioactive contamination isrelatively high (Bonisoli-Alquati et al., 2010a). In this study we tested
whether individual plasma oxidative status predicts the effects ofradiation on sperm swimming behavior. To this aim, we measured
overall plasma antioxidant capacity and levels of ROS metabolites, andused the ratio between these two measures to assess individual
oxidative stress (Iorio, 2004; Costantini, 2008; see also Bonisoli-Alquati
et al., 2010a). We correlated these data to previously collected
information on sperm swimming behavior and radiation level, expect-

ing the deleterious effects of radiation on sperm motility to be bufferedin individuals with relatively high plasma levels of antioxidants (or,conversely, to be exacerbated by relatively high levels of oxidative
stress). We analyzed sperm viability and motility based on computerassisted sperm analysis (CASA), as several studies in different animal
taxa have shown that sperm motility and swimming behavior are major
predictors of fertilization success (Froman et al., 1999; Garcia Gonzales
and Simmons, 2005; Rudolfsen et al., 2008; Gasparini et al., 2010).
The barn swallow is a small, insectivorous passerine bird that
commonly breeds in farms across Europe after migrating from thewinter quarters in sub-Saharan Africa (Møller, 1994). In our study
populations in Ukraine, males inhabiting radioactively contaminatedareas have paler plumage coloration than males from control areas(Camplani et al., 1999). The frequency of albinistic feathers was also
increased in individuals from contaminated areas (Møller andMousseau, 2003), and the albinistic phenotype was found to be atleast partly of germline origin and associated with reduced survival
prospects (Ellegren et al., 1997). Overall, mutation rate (Ellegren et al.,1997), DNA damage (Bonisoli-Alquati et al., 2010b) and oxidative stress(Bonisoli-Alquati et al., 2010a) are all increased in barn swallows fromthe heavily contaminated sites around Chernobyl.
2. Material and methods
2.1. Study species and ﬁeld procedures
We captured barn swallows (n= 88) in seven different farms in the
Chernobyl region, Ukraine, during late May and early June 2005–2006(Fig. 1), using mist nets placed across windows and doors. Birds wereplaced in cloth bags before being measured and weighed. We thencollected a blood sample in heparinized capillary tubes. Subsequently,
we collected one or two sperm samples from all males, using a simple
massage technique. Sperm were collected in micro-capillaries andimmediately transferred to the microscope chamber (see below). We
collected our ﬁeld measurements of background radiation at the groundlevel using a hand-held Inspector dosimeter (Model: Inspector,
SE International, Inc., Summertown, TN, USA). Radiation levels
were mainly due to gamma radiation and were on average 0.0379
mR/h (standard error (SE) = 0.0069; range 0.02–0.065 mR/h) at seven
breeding sites (Fig. 1). Previous analyses have shown that our ﬁeld
measurements have strong positive relationships with ofﬁciallypublished data (i.e. Shestopalov, 1996; see Møller et al., 2008b). Thus,
our estimates of radioactive contamination at each breeding colony
were comparable with other estimates. Admittedly, we do not haveinformation on internal dose of the birds involved in this study.
However, background radiation level accurately predicts averageinternal dose of individual birds from Chernobyl (Gaschak et al., 2008).
2.2. Sperm behavior analyses
The recording and analysis of sperm behavior have been describedin details elsewhere (Møller et al., 2008b; see also Supplementary
Material and Methods). Brieﬂy, the sperm sample was transferred to a
microscope chamber, without the experimenter having knowledge ofthe individual male. Here the sperm was diluted in a one-step procedure by adding 9 μL of Dulbecco's Modiﬁed Eagle Medium (D-MEM;
Invitrogen, Carlsbad, CA, USA) to the sample. We then conducted
video recording by means of a Sony CCD black-and-white video
camera (XC-ST50CE PAL, Sony, Tokyo, Japan), mounted on an external
negative phase-contrast microscope (Olympus CH30, Olympus,
Tokyo, Japan) with a 10× objective. The video recordings were thendownloaded for analysis to a PC. For each ejaculate, we recorded themean values of (1) VAP (smoothed path velocity; μm/s), (2) VSL (straightline velocity; μm/s), (3) VCL (curvilinear velocity; μm/s), (4) ALH(amplitude of lateral head displacement; μm), (5) BCF (beat crossfrequency; Hz), (6) LIN (linearity, VSL/VCL) and (7) STR (straightness,

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107

Fig. 1. Location of the study sites (1. Kanev, 2. Konongia, 3. Zalyska, 4. Ghovtnere, 5. Pisky, 6. Dytiatku, 7. Martinovichy). Adapted from De Cort et al. (1998).

VSL/VAP). Cells having a VAP b 10 μm/s and a VSL b 5 μm/s were
excluded from the motility analysis, and counted as static. We used
these measurements to estimate (8) the percentage of static sperm(the percentage of sperm that remained static), (9) the percentage of
sperm with slow velocity (i.e. b10 μm/s), (10) the percentage of spermwith medium velocity (10–50 μm/s), (11) the percentage of spermwith high velocity (N50 μm/s) and (12) the percentage of progressive
sperm (the percentage of all sperm that moved with STRN 80 and
VAPN 50 μm/s).

analytical procedures (Iorio, 2004). The plasma concentration ofROMs was measured by the d-ROMs test (Diacron), according to apreviously described protocol (Bonisoli-Alquati et al., 2010a; see
Supplementary Material and Methods). The results of d-ROMs test are
expressed as mM H2O2.
The ratio of ROMs/TAC × 1000 was used as an index of overall
oxidative status (or ‘oxidative stress’; sensu Costantini et al., 2006).
Higher values indicate a higher unbalance between pro-oxidants andantioxidant resources. Oxidative stress values were log-transformed
before analyses to reach normality.

2.3. Plasma antioxidant capacity (TAC), reactive oxygen metabolites(ROMs) and oxidative stress2.4. Statistical analyses
The plasma antioxidant barrier includes both exogenous (e.g.
ascorbate, tocopherols, carotenoids) and endogenous (e.g. uric acid,enzymes) compounds. The total antioxidant capacity (TAC) of plasma
was measured using the OXY-Adsorbent test (Diacron, Grosseto, Italy)
according to a previously described protocol (Bonisoli-Alquati et al.,2010a). Brieﬂy, this test uses a colorimetric determination to quantify
the ability of the plasma antioxidant barrier to cope with the oxidant
action of hypochlorous acid (HClO), an endogenously produced prooxidant. The test reﬂects levels of circulating antioxidants including
vitamin C and E, carotenoids and uric acid, but does not account forlevels of enzymatic antioxidants (see Costantini, in press).
Reactive oxygen metabolites (ROMs) are peroxidation products(primarily hydroperoxides, ROOH) of reactive oxygen species (ROS).
They can be considered markers of early oxidative damage, as theircleavage by metals leads to the generation of two highly reactive prooxidants, the alkoxyl (R–O•) and alkylperoxyl (R–OO•) radicals,
which in turn can promote an oxidative cascade (Costantini, 2008).
ROMs are relatively more stable than ROS (that can last as short as afew seconds) and therefore they can be detected and quantiﬁed by

We conducted principal component analysis (PCA) on the 12
sperm parameters by applying the varimax rotation. We then used thescores for the principal components (PCs) as response variables inlinear mixed models. For each measure of individual oxidative status
and each principal component, we used Akaike Information Criterion(AIC) scores to compare and rank the four different models including(i) only radiation level (as a covariate); (ii) only the measure ofoxidative status (TAC, ROMs or oxidative stress; also as a covariate);(iii) both radiation level and the measure of oxidative status; or(iv) both variables together with their interaction term. Two models
are assumed to be equally informative when the difference in their
AIC scores was lower than 2.00 (Burnham and Anderson, 2002). Basedon the hypothesis that background radiation affects sperm swimming
behavior depending on individual oxidative status, we expect best-ﬁt
models to include both radiation levels and the measure of oxidative
status. Statistical analyses were performed using SPSS 13.0 (SPSS Inc.,
Chicago, IL, USA) and SAS 9.1 (SAS Institute Inc., Cary, NC, USA).
We included site and year as random factors in all analyses.

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Table 1
Principal components of the 12 sperm variables. The eigenvalues associated with eachprincipal component, together with the amount of variance explained are also
indicated. Only the principal components with an eigenvalue N1.00 were extracted.

Eigenvalue
Percent of variance
Cumulative percent of variancePct. static sperm
Pct. slow sperm
Pct. medium sperm
Pct. rapid sperm
Pct. progressive sperm
VAP
VSL
VCL
ALH
BCF
STR
LIN

PC1

PC2

PC3

PC4

5.45
45.38
45.38
− 0.54
0.14
−0.14
0.74
0.84
0.86
0.95
0.21
− 0.05− 0.25
0.85
0.92

2.05
17.07
62.45
− 0.28− 0.26
0.01
0.41
0.21
0.42
0.20
0.92
0.79
0.14
− 0.15− 0.34

1.75
14.55
77.00
− 0.78
0.35
0.92
0.40
0.35
−0.08
−0.12
−0.10
0.25
− 0.22
0.03
0.02

1.13
9.43
86.43
0.02
0.69
0.04
− 0.21−0.14− 0.14− 0.12− 0.02− 0.020.83
0.15− 0.01

Loadings with absolute value N0.50 are highlighted in bold. PC1 tended to be positively
correlated with all variables indicating higher sperm velocity and was negatively
correlated with the percent of static sperm; PC2 represented sperm with high track
velocity (VCL), but also with ample lateral displacement of the head (ALH); PC3
reﬂected highly non-static sperm, moving with medium velocity; PC4 represents slow
sperm with a high beat cross frequency (BCF). VAP (smoothed path velocity; μm/s), VSL(straight line velocity; μm/s), VCL (curvilinear velocity; μm/s), ALH (amplitude of lateralhead displacement; μm), BCF (beat cross frequency; Hz), LIN (linearity, VSL/VCL) andSTR (straightness, VSL/VAP) (see also Material and Methods).

3. Results
The PCA on the 12 sperm parameters extracted four PCs that
accounted for N86% of the variance in our data (Table 1). PC1 waspositively correlated with all variables indicating higher sperm velocity
and negatively with the percent of static sperm; PC2 represented spermwith high track velocity (VCL), but also with ample lateral headdisplacement (ALH); PC3 reﬂected highly non-static sperm, moving
with medium velocity, and PC4 represented slow sperm with high beatcross frequency (BCF) (Table 1).
For all PCs, the best-ﬁt models always included both radiation level
and the measure of oxidative status (Table 2).

Table 2
Akaike Information Criterion (AIC) values for the models of the four principal
components of sperm swimming behavior. For each of the variables of oxidative status(total antioxidant capacity or TAC; reactive oxygen metabolites level or ROMs, and theirratio or Oxidative Stress), we compared the models including (i) only radiation (as acovariate), (ii) only the oxidative status variable (as a covariate), (iii) both variables or(iv) both variables and their interaction. The AIC values of the best-ﬁt models arehighlighted in bold. Two models are assumed to be equally informative when the
difference in their AIC scores was lower than 2.00 (Burnham and Anderson, 2002; see
Statistical Analyses for more details).
PC1

PC2

PC3

PC4

Total antioxidant capacity (TAC) (n = 75)
Rad
242.0
TAC
217.9
TAC + Rad
210.0
TAC + Rad + TAC × Rad
212.1

233.6
210.1
200.1
201.5

210.8
199.6
187.2
187.0

238.2
220.6
213.7
212.6

Reactive Oxygen Metabolites (ROMs) (n = 77)
Rad
242.0
ROMs
213.0
ROMs + Rad
205.6
ROMs + Rad + ROMs × Rad
196.8

233.6
201.9
193.3
176.3

210.8
195.5
181.8
176.2

238.2
196.5
189.2
182.7

Oxidative stress (Stress) (n = 65)
Rad
StressStress + Rad
Stress + Rad + Stress × Rad

233.6
155.6
147.2
132.0

210.8
163.5
152.2
143.1

238.2
161.1
143.1
143.6

242.0
175.5
166.2
156.9

Table 3Best-ﬁt models for the principal components in relation to background radiation level(Rad), total antioxidant capacity (TAC) and their interaction (see also Table 2).
d.f.

F

P

PC1
Rad
TAC

Estimate

Coefﬁcient (SE)

1,7.3
1,70.4

1.34
1.10

0.283
0.299

− 12.23 (10.55)− 2.1 × 10− 3 (2.0 × 10− 3)

PC2
Rad
TAC

1,4.2
1,70.8

7.01
0.33

0.055
0.568

22.37 (8.45)− 1.1 × 10− 3 (1.1 × 10− 3)

PC3
Rad
TAC
TAC × Rad

1,63.2
1,68.4
1,68.7

0.31
3.52
2.80

0.582
0.065
0.099

13.10 (23.68)
8.6 × 10− 3 (4.6 × 10− 3)− 0.20 (0.11)

PC4
Rad
TAC
TAC × Rad

1,62.01,68.6
1,68.8

2.96
1.79
3.21

0.090
0.185
0.077

51.74 (30.06)
7.6 × 10− 3 (5.7 × 10− 3)− 0.25 (0.14)

The best-ﬁt models for PC1 scores did not include any signiﬁcanteffect of radiation level, TAC, ROMs or oxidative stress, neither alone
nor in interaction with each other (Tables 3–5). In the best-ﬁt models
for PC2 scores, the scores were signiﬁcantly predicted by theinteraction between radiation level and ROMs (Table 4; Fig. 2). PC2
values were highest for highest levels of radiation and highest levels
of ROMs. Thus, sperm had highest track velocity and highest lateralhead displacement for increasing levels of radiation, in particularwhen plasma levels of ROMs were also high (Fig. 2). A qualitatively
similar effect was disclosed by the analysis of PC2 scores as a functionof radiation level and oxidative stress (Table 5), with highest PC2scores for high levels of oxidative stress and relatively high radiationlevels (Fig. 3). Thus, sperm had highest track velocity and amplest
lateral head displacement for relatively high levels of radiation andhigh levels of plasma ROMs, even after accounting for TAC of plasma(Fig. 3).
PC3 scores were only marginally signiﬁcantly predicted by theinteraction between TAC levels and background radiation level(Table 3; Fig. 4). This effect implied that the percent of motile
sperm was highest for the lowest level of background radiation andfor highest plasma levels of antioxidants (Fig. 4). PC4 scores were also
marginally non-signiﬁcantly predicted by the interaction between
background radiation levels and TAC (Table 3; Fig. 5). The coefﬁcients

Table 4Best-ﬁt models for the four principal components in relation to background radiationlevel (Rad), reactive oxygen metabolites (ROMs) levels and their interaction (seeTable 2).
d.f.

F

P

Slope (SE)

PC1
Rad
ROMs
ROMs × Rad

1,37.9
1,64.01,69.1

0.78
2.57
2.18

0.383
0.114
0.144

19.17 (21.71)
0.73 (0.45)− 16.88 (11.43)

PC2
Rad
ROMs
ROMs × Rad

1,32.3
1,69.8
1,71.3

3.96
7.90
11.29

0.055
0.006
0.001

− 41.03 (20.61)− 1.13 (0.42)
33.80 (10.06)

PC3
Rad
ROMs
ROMs × Rad

1,41.5
1,64.01,67.5

0.02
1.01
1.21

0.899
0.31880.275− 2.17 (16.93)
0.39 (0.38)− 10.37 (9.43)

PC4
Rad
ROMs
ROMs × Rad

1,31.4
1,71.81,72.0

0.16
0.06
0.00

0.693
0.813
0.984

5.47 (22.90)
0.17 (0.42)
0.21 (10.54)

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109

Table 5Best-ﬁt models for the four principal components in relation to background radiationlevel (Rad), oxidative stress levels (or Stress, as indexed by the ratio of total antioxidant
capacity to the reactive oxygen metabolites levels; see Material and Methods) and theirinteraction (see Table 2).
d.f.

F

P

Slope (SE)

PC1
Rad
StressStress × Rad

1,6.88
1,59.4
1,59.7

3.45
0.00
0.05

0.106
0.977
0.829

− 16.95 (9.13)− 0.05 (1.71)
8.83 (40.83)

PC2
Rad
StressStress × Rad

1,6.67
1,58.4
1,58.8

2.89
5.18
6.62

0.135
0.027
0.013

19.34 (11.37)− 3.11 (1.36)
84.05 (32.67)

PC3Rad
StressStress × Rad

1,9.44
1,58.9
1,58.9

6.58
0.23
0.18

0.029
0.633
0.670

−21.87 (8.53)− 0.71 (1.48)
14.99 (35.03)

PC4Rad
Stress1,7.96
1,57.9

0.18
6.58

0.670
0.029

6.99 (13.57)
0.81 (0.49)

Fig. 3. Relationship between the PC2 scores, background radiation level and oxidative
stress in the plasma. In panel (a), individual values of those individuals for which we
could determine both TAC and ROMs levels are shown (n = 65). In panel (b), a surface
was interpolated based on the coefﬁcients for the effects of background radiation level,oxidative stress levels and the interaction between the two in the best-ﬁt model. The
coefﬁcients of the model are reported in Table 5. The loadings of PC2 scores on theoriginal parameters of sperm motility and swimming behavior are reported in Table 1.

for this effect implied that slow sperm with high beat cross frequency
were most frequent for relatively high levels of radioactive contamination, particularly when plasma TAC was low (Fig. 5).
The effect of year was not signiﬁcant in any analysis (P always
N0.05). Similarly, there was no signiﬁcant effect of site in any analysis(P always N0.05). The inclusion of body mass or body condition (asindexed by the residuals of the regression of body mass on tarsus
length) did not qualitatively change the results of any of the best-ﬁt
models (details not shown).
4. Discussion

Fig. 2. Relationship between the PC2 scores, background radiation level and plasma
levels of reactive oxygen metabolites (ROMs). In panel (a), individual values are shown(n = 77). In panel (b), a surface was interpolated based on the coefﬁcients for the effectsof background radiation level, ROMs levels and the interaction between the two in thebest-ﬁt model. The coefﬁcients of the model are reported in Table 4. The loadings of PC2
scores on the original parameters of sperm motility and swimming behavior arereported in Table 1.

In this study we assessed the covariation between sperm motility
and plasma total antioxidant capacity (TAC), oxidative damage (asindexed by ROMs level) and oxidative stress (as indexed by the ratioof the two measures; Costantini et al., 2006) in barn swallows under
different levels of radioactive contamination. Consistently with our
prediction that the deleterious effects of radiation on sperm motility
would depend on individual oxidative status, the effect of chronic
exposure to low-level radiation on different features of sperm
swimming behavior depended on the indices of antioxidant status.
In particular, sperm swimming with highest track velocity andamplest lateral head displacement (i.e. highest values on the PC2)
were associated with highest ROMs and relatively high background110

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Fig. 4. Relationship between the PC3 scores, background radiation level and plasma
total antioxidant capacity (TAC). In panel (a), individual values are shown (n = 75). In
panel (b), a surface was interpolated based on the coefﬁcients for the effects ofbackground radiation level, TAC and the interaction between the two in the best-ﬁt
model. The coefﬁcients of the model are reported in Table 3. The loadings of PC3 scoreson the original parameters of sperm motility and swimming behavior are reported inTable 1.

radiation levels. Interestingly, this result held true even whenanalyzing oxidative stress (i.e. accounting for variation in antioxidant
capacity of plasma; Fig. 3). Both track velocity and lateral headdisplacement are known to correlate with sperm penetration capacity of the cervical barrier (Aitken, 2006). In turn, the capacity ofpenetrating the cervical mucus has been linked to the probability of invitro fertilization in humans and cows (Sharara et al., 1995), as well as
to the probability of pregnancy in vivo, particularly when controlling
for the quality of the cervical mucus (Eggert-Kruse et al., 1989). Thus,
contrary to our expectations, the best performance of sperm in termsof track velocity and lateral head displacement was associated withrelatively high levels of radioactive contamination and highest
oxidative damage and stress. A trade-off in allocation of antioxidants
between blood plasma and seminal ﬂuid could account for this effect.
A number of antioxidants are known to be present in the seminal ﬂuid
of birds, including vitamins E and C, glutathione, ubiquinols, as well as
antioxidant enzymes like superoxide dismutase (SOD), glutathione
peroxidase and catalase (Sikka, 2001; Surai et al., 2001; Rowe andMcGraw, 2008). If a trade-off in scavenging ROS from different tissues
is operating, relatively high levels of ROMs and a correspondingly highoxidative stress in the plasma might then be associated with lowlevels of ROMs and stress in the seminal ﬂuid (and with correspondingly viable and motile sperm). This possibility, however, remains to
be tested. Interestingly, a recent study of great tits (Parus major) has
shown a negative relationship between sperm concentration ofFig. 5. Relationship between the PC4 scores, background radiation level and plasma
total antioxidant capacity (TAC). In panel (a), individual values are shown (n = 75). In
panel (b), a surface was interpolated based on the coefﬁcients for the effects ofbackground radiation level, TAC and the interaction between the two in the best-ﬁt
model. The coefﬁcients of the model are reported in Table 3. The loadings of PC4 scoreson the original parameters of sperm motility and swimming behavior are reported inTable 1.

malondialdehyde (MDA), a marker of lipid peroxidation (Monaghan
et al., 2009), and sperm swimming ability (Helfenstein et al., 2010).
Helfenstein et al. (2010) found that an experimental increase ofparental workload induced both an increase in MDA concentration
and a decrease in sperm motility and swimming ability. It could beargued, however, that Helfenstein et al. (2010) induced acute,transient oxidative stress, while the individuals in our study were
exposed to a mild, chronic stress. We suggest that future studies
simultaneously assess oxidative status of both plasma and seminal
ﬂuid, in order to better describe the integrated response of organisms
to radioactive contamination. A validation of our results through thecapture and analysis of birds over a wider range of radioactive
contamination levels will also be important. In fact, the range ofradioactive contamination levels reported here is narrower than therange across which we have previously sampled birds (e.g. BonisoliAlquati et al., 2010b). However, it should be noted that all the best-ﬁt
models included both radiation and the measure of oxidative status(and most of the best-ﬁt models also included the interaction
between the two covariates; see Table 2), thus indicating thatradiation level is important in explaining variation in sperm motility
across individuals in our sample.
We found that the effects of radiation on sperm motility andbehavior marginally depended on plasma antioxidant capacity. The
percent of motile sperm was highest for the lowest level of backgroundradiation and for highest plasma levels of antioxidants, thus suggestingthat antioxidants might have a role in protecting the sperm from prooxidants arising as a function of radiation exposure. Conversely, slow
sperm with high beat cross frequency were most frequent for relatively

A. Bonisoli-Alquati et al. / Comparative Biochemistry and Physiology, Part A 159 (2011) 105–112

low levels of plasma antioxidant capacity and relatively high background radiation, thus indicating an impairment of sperm motility (anda likely decrease in fertilizing capacity; Surai et al., 1998, 2001;
Tremellen, 2008), whenever antioxidant resources are insufﬁcient toscavenge the excess of ROS generated by ionizing radiation. Thus, arelatively worse oxidative status in terms of circulating ROMs levels was
positively related to sperm velocity, particularly for higher radiationlevels, while better plasmatic antioxidant defenses were associated withhigher sperm motility, in particular when radiation was higher. Thissuggests that the antioxidant defense of some sperm traits might betraded against other sperm functions. In this case, particularly while
exposed to radiation, males might have had to produce only a few, faster
sperm, or more, motile but slow sperm. Similar to the hypothesis thatplasma oxidative status is traded against oxidative status in the seminal
ﬂuid, this explanation is speculative and awaits further tests. Admittedly, however, differences in sperm number could have contributed tothe observed pattern given that spermatogenesis entails intense
metabolic cost (Parapanov et al., 2008), and could thus increase ROS
production in the ejaculate. Consistent with this hypothesis, ROS havebeen proposed as a proximate explanation for the documented negative
association between sperm traits (e.g. Levitan, 2000; Moore et al., 2004;
Schulte-Hostedde and Millar, 2004), particularly between sperm
numbers and indices of sperm quality (Moore et al., 2004; SchulteHostedde and Millar, 2004; see Dowling and Simmons, 2009 for a
discussion).
Overall, these results complement previous evidence showing an
increase in aberrant sperm and an impaired sperm swimming
behavior with increasing background radiation levels (Møller et al.,2005a, 2008b). They also extend the ﬁnding of a negative relationshipbetween somatic concentration of speciﬁc antioxidants and sperm
aberrations under radioactive contamination to a more integrated
measure of overall oxidative status (sensu Monaghan et al., 2009).
Ultimately, rather than the level of any single antioxidant, it is theimbalance between ROS generation and available antioxidant
resources that will determine the ﬁtness outcome of oxidative insultsarising from radioactive contamination (Costantini, 2008; Monaghan
et al., 2009).
The ﬁnding of an impairment of sperm motility (and therefore
fertilizing capacity) that simultaneously depends on individual
oxidative status and background radiation level might reconcile
several features of the reproductive behavior and population
dynamics of barn swallows inhabiting the radioactively contaminatedarea around Chernobyl. Indeed, present results are consistent with apreviously documented reduction in hatching success of barn swallow
eggs in radioactively contaminated sites (Møller et al., 2005b), andmight contribute to explaining the population decline of barnswallows breeding in the region (Ellegren et al., 1997; Møller et al.,2006).
Heritable gene mutations and chromosomal translocations (Searle
et al., 1974; Russell et al., 1998), genomic instability (Dubrova et al.,1993) and cancer (Nomura et al., 2004) have all been reported astrans-generational consequences of parental exposure to ionizing
radiation (Marchetti and Wyrobek, 2005). Interestingly, an increased
mutation rates has also been described in the offspring of individuals
heavily exposed to the Chernobyl radioactive fallout (Dubrova et al.,
1996; Weinberg et al., 2001; but see Furitsu et al., 2005). Our results
suggest that trans-generational effects of exposure to ionizing
radiation due to sperm aberrations may vary in frequency dependingon the oxidative status of the male and possibly depending on thepattern of antioxidant allocation to different body functions andtissues.
Acknowledgements
The authors gratefully acknowledge the help of G. Milinevski, I.
Chizhevsky, O. Bondarenko, and the Chernobyl EcoCenter. The authors

111

acknowledge ﬁnancial support from the CNRS (France), The National
Geographic Society, the Samuel Freeman Charitable Trust, the NATO
CLG program, the Fulbright Program, and the School of theEnvironment and the College of Arts and Sciences of the Universityof South Carolina. D. Costantini provided precious suggestions for
conducting the lab tests. Two anonymous reviewers provided
valuable comments on an earlier draft of the manuscript.

Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.cbpa.2011.01.018.

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