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Patterns of sperm damage in Chernobyl passerine birds
suggest a trade-off between sperm length and integrity
Ignacio G. Hermosell, Terje Laskemoen, Melissah Rowe, Anders P. Møller, Timothy A. Mousseau,
Tomás Albrecht and Jan T. Lifjeld
Biol. Lett. 2013 9, 20130530, published 2 October 2013

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Evolutionary biology

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Research
Cite this article: Hermosell IG, Laskemoen T,
Rowe M, Møller AP, Mousseau TA, Albrecht T,
Lifjeld JT. 2013 Patterns of sperm damage in
Chernobyl passerine birds suggest a trade-off
between sperm length and integrity. Biol Lett
9: 20130530.
http://dx.doi.org/10.1098/rsbl.2013.0530

Received: 8 June 2013
Accepted: 7 September 2013

Subject Areas:
evolution
Keywords:
acrosome, radiation,
sperm evolution, sperm size

Author for correspondence:
Jan T. Lifjeld
e-mail: j.t.lifjeld@nhm.uio.no

Patterns of sperm damage in Chernobyl
passerine birds suggest a trade-off
between sperm length and integrity
Ignacio G. Hermosell1, Terje Laskemoen2, Melissah Rowe2, Anders P. Møller3,
Timothy A. Mousseau4, Tomáš Albrecht5,6 and Jan T. Lifjeld2
1

Departamento de Anatomı́a, Biologı́a Celular y Zoologı́a, Universidad de Extremadura, Avenida de Elvas s/n,
06071 Badajoz, Spain
2
Natural History Museum, University of Oslo, PO Box 1172, Blindern, 0318 Oslo, Norway
3
Laboratoire d’Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362,
91405 Orsay Cedex, France
4
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
5
Academy of Sciences of the Czech Republic, Institute of Vertebrate Biology, Kvetna 8, Brno 603 65,
Czech Republic
6
Faculty of Sciences, Charles University in Prague, Vinicna 7, Praha 12844, Czech Republic
Interspecific variation in sperm size is enigmatic, but generally assumed toreflect species-specific trade-offs in selection pressures. Among passerine
birds, sperm length varies sevenfold, and sperm competition risk seems todrive the evolution of longer sperm. However, little is known about factors
favouring short sperm or constraining the evolution of longer sperm. Here,
we report a comparative analysis of sperm head abnormalities among
11 species of passerine bird in Chernobyl, presumably resulting from chronic
irradiation following the 1986 accident. Frequencies of sperm abnormalities
varied between 15.7 and 77.3% among species, more than fourfold higher
than in uncontaminated areas. Nonetheless, species ranked similarly insperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic
factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome
damage consistent with premature acrosome reaction. Sperm length, but
not sperm competition risk explained variation in sperm damage among
species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm
length and sperm integrity affecting sperm evolution in passerine birds.

1. Introduction

Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2013.0530 or
via http://rsbl.royalsocietypublishing.org.

Spermatozoa display tremendous diversity in size and shape across the animal
kingdom [1]. Fertilization mode and post-copulatory sexual selection are twomain factors assumed to shape this diversity among taxa [1– 3]. Passerine
birds have spermatozoa adapted to internal fertilization and sperm storage,
with a sevenfold variation in length, i.e. approximately 40– 290 mm [2,4,5].
Post-copulatory sexual selection seems to promote the evolution of longer
sperm [2,4,6] although the adaptive function of longer sperm in sperm competition is not well understood. Longer sperm have longer mid-pieces, whichcontain the fused mitochondrion and hence more energy resources [7]. However, energy does not seem to translate directly into higher swimming speeds[6,7], so other energy-dependent traits like longevity, could be important.
Post-copulatory sexual selection also increases total investment in sperm production [8,9], which is manifested in a disproportionate increase in sperm
number compared with the increase in sperm size across species [2]. Thissuggests that there is no clear trade-off between sperm size and number in& 2013 The Author(s) Published by the Royal Society. All rights reserved.

(b)

2

0.8

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frequency of abnormal sperm in
Chernobyl

(a)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
frequency of abnormal sperm in control areas

50

100

150
200
250
sperm length (µm)

300

Figure 1. Frequency of sperm abnormality in Chernobyl passerine species plotted against (a) the frequency of sperm abnormality in uncontaminated areas and(b) mean sperm length for the species. The line in (a) is y ¼ x.
Table 1. PGLS analyses of the proportion of abnormal sperm in relation to total sperm length and sperm competition risk (CVbm of sperm length) among passerine
species in Chernobyl, Ukraine and uncontaminated areas (Norway and Czech Republic). In all tests, the phylogenetic parameter (l ) was approximately 0 andsigniﬁcantly different from 1, suggesting there was no strong phylogenetic signal in the relationships.
area

predictor

slope

s.e.

t

p

Chernobyl (n ¼ 11 species)

total sperm length

1.02

0.24

4.32

0.002

uncontaminated (n ¼ 10 species)

sperm competition risk
total sperm length

0.08
0.16

0.09
0.29

0.89
0.55

0.40
0.60

sperm competition risk

0.04

0.07

0.63

0.54

passerine birds, owing to some significant cost or constraint
to the evolution of longer sperm.
Here, we report a comparative study of sperm abnormalities in passerine birds providing evidence for reduced
functional performance of longer sperm. Radioactive contamination following the Chernobyl accident in 1986 had aseries of negative environmental effects, including sperm
damage in mammals [10] and birds [11]. We examined thefrequency of abnormal sperm among 11 passerine species
sampled around Chernobyl in 2010 and 2011. We alsoscored sperm abnormalities in samples from 10 of these
species from uncontaminated areas elsewhere in Europe.
We tested whether the patterns of sperm damage covariedwith species-specific sperm lengths and sperm competition
risk, as sperm competition enhances both sperm quantityand quality among passerine birds [9]. Thus, we predictedthat species with a high risk of sperm competition shouldhave more robust sperm, in this case lower frequencies ofradiation-induced damage. As sperm length is positively
correlated with sperm competition risk [2,4,6], we would
also expect species with longer sperm to have less damaged
sperm. Finally, we used scanning electron microscopy(SEM) to study the nature of sperm abnormalities.

2. Material and methods
We obtained sperm samples from 102 individuals belonging to 11
species of passerine birds in Chernobyl, Ukraine, during May–
June 2010–2011, and 84 individuals from 10 of the same species
in Norway and the Czech Republic (uncontaminated areas)
during 2007–2013. A detailed sample list is given in the electronic
supplementary material, table S1. Ejaculates were obtained by cloacal massage, diluted in saline and immediately fixed in 5%
formaldehyde [6]. From each sample, an approximately 3 ml aliquot

was spread on a microscope slide and air-dried, then gently rinsed
with distilled water and air-dried again. Digital images of the spermatozoa were captured (160 magnification) using a Leica DFC420
digital camera attached to a LEICA DM6000 B microscope, and analysed with Leica APPLICATION software. We typically examined 100
spermatozoa per sample, unless fewer sperm were available, andscored them as ‘normal’ or ‘abnormal’ (i.e. different head shape or
reduced head length). All images were scored by one person(I.G.H.) to reduce observer error. Additional scoring of images
from 10 samples (one sample randomly drawn from each species
in uncontaminated areas), done blindly with respect to the first scorings, gave a high repeatability of the scored frequencies of abnormal
sperm (R ¼ 0.98, F9,10 ¼ 101.6, p , 0.001).
For SEM, we selected samples from three species with relatively high frequencies of abnormal sperm. Formalin-fixed
spermatozoa were prepared and examined as in Lifjeld et al.
[12]. Mean sperm lengths for each species were obtained fromthe avian sperm collection database at the Natural History
Museum, Oslo, only using measurements of sperm with nosign of damage from the control populations (10 species) or
Chernobyl (Luscinia luscinia). The coefficient of variation inmean sperm length (CVbm) among males from the same populations was used as an index of sperm competition risk [4].
Species-specific values for sperm length and sperm competition
risk are listed in the electronic supplementary material, table S2.
Comparative analyses of the proportion of abnormal sperm
were conducted using a generalized least-squares approach controlling for phylogenetic relatedness among species (i.e. PGLS;
electronic supplementary material, methods). For all parametrictests, proportions were arcsine square-root transformed, andsperm lengths were log10-transformed to approach normality.

3. ResultsFrequencies of abnormal sperm varied from 15.7 to 73.3%
among Chernobyl species (N ¼ 11), with an overall mean

Biol Lett 9: 20130530

0

3

(a)

acrosome

nucleus

5 µm(b)acrosome

nucleus(c)nucleusmidpiece

flagellum

acrosome
5 µm(d)nucleusmidpiece flagellum

5 µm

acrosome
midpiece flagellum

4. Discussion
nucleus
5 µm(f)nucleusflagellum
midpiece
5 µm(g)nucleusflagellum

acrosome

midpiece

5 µm

Figure 2. Scanning electron micrographs of normal and abnormal spermatozoaof three passerine species: (a,b) barn swallow Hirundo rustica, (c,d) common
blackbird Turdus merula and (e–g) common chaffinch Fringilla coelebs.

Biol Lett 9: 20130530

flagellum
5 µm

(e)

Our study documented three novel patterns of sperm
damage. First, all studied passerine birds in the Chernobyl
area have elevated frequencies of sperm abnormality, even
25 years after the radioactive fallout. Background radiationin this environment has had significant negative impact on
many birds and other taxa [14,15]. Studies of local barn swallow Hirundo rustica populations have revealed a series ofphysiological and morphological defects [16,17], including
increased sperm abnormalities and reduced sperm swimming
speed [11,18]. Our results indicate that most passerine birds
in the area are significantly affected by radiation-induced
sperm damage.
Second, our SEM analyses identified premature acrosome
reaction as the likely mechanism of the observed sperm
damage. Correct timing of the acrosome reaction is crucial
for fertilizing success of an ejaculate, and spermatozoa without
an intact acrosome are unable to penetrate the inner perivitelline layer of the ovum [13]. Our sperm samples were fresh
ejaculates, so the conditions causing the defect must be related
to the physiology of the male. Several factors are known to
induce premature acrosome reaction in birds and mammals,
including mutations [19], extracellular calcium and lead
[20,21], and oxidative stress [22]. Interestingly, a link between
oxidative stress and poor sperm performance has alreadybeen indicated in barn swallows from Chernobyl [11,23], so

midpiece

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frequency of 39.6%. Among uncontaminated populations of
the same species (N ¼ 10), frequencies varied from 0.1 to
21.1%, with an overall mean frequency of 8.6%. The frequency
of abnormal sperm was always higher in Chernobyl than thatin uncontaminated populations (paired t9 ¼ 7.68, p , 0.001;
figure 1a), although for one outlier species, Coccothraustes coccothraustes, frequencies were quite similar. Sperm abnormalities
tended to be positively correlated between contaminated anduncontaminated areas (Pearson r ¼ 0.629, p ¼ 0.051, n ¼ 10;
figure 1a, with the outlier species excluded: r ¼ 0.928, p ,0.001, n ¼ 9), which implies an intrinsic, species-specific factor
underlying the consistent variation in sperm abnormalities
between environments.
Variation in sperm abnormalities among Chernobyl
populations was significantly associated with sperm length,
i.e. species with longer sperm had more abnormal sperm(table 1 and figure 1b). By contrast, there was no significant
association with sperm competition risk (table 1). Among
uncontaminated populations, neither sperm length nor
sperm competition risk explained significant variation in
sperm abnormalities (table 1), but sperm length had a significant effect when the outlier was excluded (PGLS, t ¼ 2.77,
n ¼ 9, p ¼ 0.028).
SEM images of spermatozoa (figure 2) revealed that
abnormalities were restricted to the acrosome, i.e. the anterior
part of the head. The types of damage varied between a bent
acrosomal tip (figure 2b), a reduced acrosome and acrosomal
microtubule helix (figure 2g), an ‘emptied’ acrosome withremnants of the plasma membrane (figure 2f ) and an entirely
missing acrosome (figure 2d). These changes reflect the various stages of the acrosome reaction, which normally takes
place when the sperm penetrates the inner perivitelline
layer of the ovum [13]. Hence, we interpret the sperm headabnormalities as evidence of premature acrosome reaction.

sperm also have longer acrosomes. Regardless of the actual
mechanism underlying the inferred premature acrosome reaction of longer sperm, the pattern reflects a possible trade-off
between sperm length and sperm integrity, and a possible
explanation for why there is no run-away selection for longer
sperm in passerine birds as compared with other taxa [2].
Acknowledgement. We thank various field assistants for help with sperm
sampling and Antje Hoenen for help with SEM analyses.

Funding statement. The work was supported by grants from theReferences
1.

2.

3.

4.

5.

6.

7.

8.

9.

Pitnick S, Hosken DJ, Birkhead TR. 2009 Spermmorphological diversity. In Sperm biology: anevolutionary perspective (eds TR Birkhead,
DJ Hosken, S Pitnick), pp. 69 –149. Oxford,
UK: Elsevier.
Immler S, Pitnick S, Parker GA, Durrant KL, Lüpold
S, Calhim S, Birkhead TR. 2011 Resolving variationin the reproductive tradeoff between sperm size
and number. Proc. Natl Acad. Sci. USA 108,5325–5330. (doi:10.1073/pnas.1009059108)
Snook RR. 2005 Sperm in competition: not playing
by the numbers. Trends Ecol. Evol. 20, 46 –53.
(doi:10.1016/j.tree.2004.10.011)
Lifjeld JT, Laskemoen T, Kleven O, Albrecht T, Robertson
RJ. 2010 Sperm length variation as a predictor of
extrapair paternity in passerine birds. PLoS ONE 5,
e13456. (doi:10.1371/journal.pone.0013456)
Jamieson BGM. 2006 Avian spermatozoa: structure
and phylogeny. In Reproductive biology andphylogeny of birds. Part A. (ed. BGM Jamieson),pp. 249 –511. Enfield, NH: Science Publishers Inc.
Kleven O, Fossøy F, Laskemoen T, Robertson RJ,
Rudolfsen G, Lifjeld JT. 2009 Comparative evidence
for the evolution of sperm swimming speed bysperm competition and female sperm storage
duration in passerine birds. Evolution 63, 2466 –
2473. (doi:10.1111/j.1558-5646.2009.00725.x)
Rowe M, Laskemoen T, Johnsen A, Lifjeld JT. 2013
Evolution of sperm structure and energetics inpasserine birds. Proc. R. Soc. B 280, 20122616.
(doi:10.1098/rspb.2012.2616)
Pitcher TE, Dunn PO, Whittingham LA. 2005 Spermcompetition and the evolution of testes size inbirds. J. Evol. Biol. 18, 557–567. (doi:10.1111/j.
1420-9101.2004.00874.x)
Rowe M, Pruett-Jones S. 2011 Sperm competition
selects for sperm quantity and quality in the

10.

11.

12.

13.

14.

15.

16.

17.

Australian Maluridae. PLoS ONE 6, e15720. (doi:10.
1371/journal.pone.0015720)
Pomerantseva MD, Ramaiya LK, Chekhovich AV.
1997 Genetic disorders in house mouse germ cells
after the Chernobyl catastrophe. Mutation Res. 381,
97 –103. (doi:10.1016/S0027-5107(97)00155-3)
Møller AP, Surai P, Mousseau TA. 2005 Antioxidants,
radiation and mutation as revealed by sperm
abnormality in barn swallows from Chernobyl.
Proc. R. Soc. B 272, 247–252. (doi:10.1098/rspb.
2004.2914)
Lifjeld JT, Hoenen A, Johannessen LE, Laskemoen T,
Lopes RJ, Rodrigues P, Rowe M. 2013 The Azores
bullfinch (Pyrrhula murina) has the same
unusual and size-variable sperm morphology as theEurasian bullfinch (Pyrrhula pyrrhula). Biol. J. Linn.
Soc. 108, 677– 687. (doi:10.1111/j.1095-8312.2012.
02040.x)
Stepinska U, Bakst MR. 2006 Fertilization. In
Reproductive biology and phylogeny of birds. Part A.
(ed. BGM Jamieson), pp. 553 –587. Enfield, NH:
Science Publishers Inc.
Møller AP, Mousseau TA. 2007 Species richness andabundance of forest birds in relation to radiation at
Chernobyl. Biol. Lett. 3, 483–486. (doi:10.1098/
rsbl.2007.0226)
Møller AP, Mousseau TA. 2009 Reduced abundanceof insects and spiders linked to radiation at
Chernobyl 20 years after the accident. Biol. Lett. 5,
356 –359. (doi:10.1098/rsbl.2008.0778)
Møller AP, Mousseau TA, de Lope F, Saino N. 2007
Elevated frequency of abnormalities in barnswallows from Chernobyl. Biol. Lett. 3, 414 –417.
(doi:10.1098/rsbl.2007.0136)
Bonisoli-Alquati A, Mousseau TA, Møller AP, Caprioli
M, Saino N. 2010 Increased oxidative stress in barnswallows from the Chernobyl region. Comp.

18.

19.

20.

21.

22.

23.

24.

Biochem. Physiol. A 155, 205–210. (doi:10.1016/j.
cbpa.2009.10.041)
Møller AP, Mousseau TA, Lynnn C, Ostermiller S,
Rudolfsen G. 2008 Impaired swimming
behaviour and morphology of sperm from barnswallows Hirundo rustica in Chernobyl. Mutation
Res. 650, 210 –216. (doi:10.1016/j.mrgentox.2007.
12.006)
Brown J, Cebra-Thomas JA, Bleil JD, Wassarman PM,
Silver LM. 1989 A premature acrosome reaction isprogrammed by mouse t haplotypes during spermdifferentiation and could play a role in transmission
ratio distortion. Development 106, 769– 773.
Lemoine M, Grasseau I, Brillard JP, Blesbois E. 2008
A reappraisal of the factors involved in in vitro
initiation of the acrosome reaction in chicken
spermatozoa. Reproduction 136, 391 –399. (doi:10.
1530/REP-08-0094)
Oliveira H, Spanò M, Santos C, Pereira M. 2009 Leadchloride affects sperm motility and acrosome
reaction in mice. Cell Biol. Toxicol. 25, 341–353.
(doi:10.1007/s10565-008-9088-4)
Bansal AK, Bilaspuri GS. 2011 Impacts ofoxidative stress and antioxidants on semenfunctions. Vet. Med. Int. 2011, 686137. (doi:10.
4061/2011/686137)
Bonisoli-Alquati A, Møller AP, Rudolfsen G, Saino N,
Caprioli M, Ostermiller S, Mousseau TA. 2011 The
effects of radiation on sperm swimming
behavior depend on plasma oxidative status in thebarn swallow (Hirundo rustica). Comp. Biochem.
Physiol. A 159, 105– 112. (doi:10.1016/j.cbpa.2011.
01.018)
Pizzari T, Dean R, Pacey A, Moore H, Bonsall MB.
2008 The evolutionary ecology of pre- and postmeiotic sperm senescence. Trends Ecol. Evol. 23,
131–140. (doi:10.1016/j.tree.2007.12.003)

Biol Lett 9: 20130530

Research Council of Norway, the Czech Science Foundation (P506/
12/2472), the Samuel Freeman Charitable Trust, the University ofSouth Carolina, the Fulbright Program and NATO.

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we speculate that premature acrosome reaction is caused byoxidative stress and accelerated sperm senescence [24]. There
was no seasonal trend in the proportion of abnormal sperm(see electronic supplementary material, methods).
Finally, species with longer sperm had more damaged
sperm. This implies that longer sperm, under otherwise similar
extracellular conditions, has a lower structural integrity of theacrosome, and may be more susceptible to lipid peroxidationof the plasma membranes [22]. Oscine passerine birds arecharacterized by the acrosome being longer than the nucleus[5], and longer acrosomes could be structurally less stable.
However, it is not yet known whether species with longer