Following the Chernobyl nuclear power plant accident, large amounts of radionuclides were emitted and spread in the environment. Animals living in such contaminated areas are predicted to suffer fitness costs including reductions in the quality and quantity of gametes.
We studied whether aspermy and sperm quality were affected by radioactive contamination by examining ejaculates from wild caught birds breeding in areas varying in background radiation level by more than three orders of magnitude around Chernobyl, Ukraine. The frequency of males with aspermy increased logarithmically with radiation level. While 18.4% of males from contaminated areas had no sperm that was only the case for 3.0% of males from uncontaminated control areas. Furthermore, there were negative relationships between sperm quality as reflected by reduced sperm velocity and motility, respectively, and radiation.
Citation: Møller AP, Bonisoli-Alquati A, Mousseau TA, Rudolfsen G (2014) Aspermy, Sperm Quality and Radiation in Chernobyl Birds. PLoS ONE 9(6): e100296. https://doi.org/10.1371/journal.pone.0100296
Editor: Shree Ram Singh, National Cancer Institute, United States of America
Received: April 15, 2014; Accepted: May 22, 2014; Published: June 25, 2014
Copyright: © 2014 Møller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. ESM files.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
The primary function of sperm is to ensure fertility and enhance paternity, and consequently strong selection acts on both qualitative and quantitative sperm traits . Recent studies of sperm behavior have revealed a close correlation between qualitative sperm traits and fertilization success in a wide range of taxa –. Furthermore, it is now clear that the production of high-quality ejaculates may represent significant costs of reproduction for males –, and that sperm quality and production are related to several factors –, including external factors –.
Following the Chernobyl nuclear power plant accident in 1986, large quantities of radionuclides were emitted and spread in the environment. Previous studies have reported reproductive failure, such as increased infertility, early impotence, and reduced total number of sperm including reduced number of motile sperm among clean-up workers and men living in contaminated regions , , , –. Furthermore, high doses of x-rays lead to permanent sterility in humans , , while low doses do not . However, persistence of 137Cs contamination raises questions regarding long-term effects on reproduction and fertility. Possible mechanisms underlying the effect of radiation on sperm parameters are direct damage to DNA integrity  or indirectly through excess reactive oxygen species (ROS) , . Ionizing radiation can cause oxidative stress by increasing the production of ROS, which renders antioxidants incapable of defense against increasing amounts of free radicals , .
Increased rates of DNA damage and mutation rates have been reported from Chernobyl , –. Admittedly, the literature reports heterogeneous results on DNA damage and mutation rate in organisms living in areas around Chernobyl with low levels of contamination –. However, the susceptibility of sperm to DNA damage stems partly from the down-regulation of DNA repair systems during late spermatogenesis . As a consequence, ejaculated sperm may exhibit genetic damage to both nuclear and mitochondrial genomes , .
High seminal ROS levels lead to oxidative stress that in turn can reduce sperm quality and increase sperm DNA fragmentation rates –. The susceptibility of sperm cells to oxidative stress is partly due to their limited antioxidant machinery and high metabolic activity , –. In addition, oxidative stress stimulates a lipid peroxidation cascade in the plasma membrane that in particular influences sperm motility –.
Previous studies of barn swallows Hirundo rustica from a variety of locations in Ukraine have revealed a positive correlation between background radiation level and the frequency of sperm with abnormal morphology and impaired sperm motility , . There is also empirical evidence for birds breeding in heavily contaminated areas around Chernobyl having increased mutation rates, higher oxidative stress and higher incidence of morphological aberrations –, –. Hence, results from field studies on free-living birds seem to be consistent with the theoretical background on the effects of radiation on sperm production and performance.
The potential fertility of individuals can be assessed from measurements of semen quantity and quality, and such analyses provide an umbrella endpoint for a wide spectrum of processes reflecting the functional state of spermatogenesis. In addition, gametogenesis is known to be sensitive to radiation , and damage in the germ line can impair fitness. Consequently, both sperm production and sperm motility act as an interesting endpoint when examining the effect of radiation on wild populations. We recorded aspermy (absence of sperm in the seminal fluids) and sperm behavior with a computer assisted sperm analyzer (CASA) in a large sample of birds under field conditions at Chernobyl. The biological effects of radiation may vary among taxonomic groups , and that is also the case for sperm parameters , . In addition, bird species are found to vary in sperm motility , . Thus, we predicted that both presence of sperm production (lack of aspermy) and sperm behavior would be negatively associated with background radiation.
The research complied with requirements for research on birds in Ukraine, and permission was given by the administration of the Chernobyl Exclusion Zone. All sampling was approved in an ethical review by the University of South Carolina Institutional Animal Care and Use Committee. All birds were handled briefly, and none died or showed signs of suffering during the short examination. All individuals flew upon release. The field studies did not involve endangered or protected species.
Study Sites and Bird Capture
We captured a total of 566 males out of a total of 1076 birds using 35 12 m mist nets placed in woodland habitat and farms in 8 different sites around Chernobyl, Ukraine during 25 May-5 June 2010 and 30 May-9 June 2011. Each site was used for capture of birds for two consecutive days covering both early morning and late evening, except for barns where a single capture session was performed for barn swallows. Sites were chosen to cover a wide range of deposition of radionuclides while still being located in the vicinity of the most contaminated areas in the exclusion zone of Chernobyl (Fig. 1). A total of 566 passerine males were examined for sperm. We measured background radiation levels at the exact capture sites of each individual by using a hand-held dosimeter (Inspector, SE International, Summertown, Tennessee, USA), which is sensitive to low levels of alpha, beta, gamma, and x-rays. Although there are significant differences in background radiation among sites (F = 1253, d.f. = 7, 279, P<0.001), there is considerable variation within each site (Table S2). Thus, we used background radiation at the exact trapping position of each individual bird as a predictor of external exposure, and radiation levels varied from 0.02 to 137.9 µSv/h. Two sites were assigned as control sites because all trapping positions had ≤0.01 µSv/h as background radiation. Previous studies have shown a strong positive relationship between internal and external dose of birds in this study area accounting for more than two thirds of the variance .
Adapted from European Union .
Morphological Characters, Sex and Age
We sexed and aged birds according to standard criteria , although the lack of reliable aging criteria prevented us from aging all species. Body mass was recorded with a Pesola spring balance to the nearest 0.1 g while keel length (as a measure of structural body size) was recorded with a digital caliper to the nearest 0.01 mm.
Subsequent to capture of males APM gently massaged the cloacal protuberance of 566 passerine males of which 490 provided an ejaculate and then released the birds following measurements and blood sampling . Sperm were collected in micro-capillaries for easy handling. Ejaculates can be collected from free-living wild birds by gentle massage resulting in contraction of the muscles surrounding the cloaca, and a small ejaculate emerging can subsequently be collected in a micro-capillary tube. There is a strict temporal coincidence of the contraction of muscles and the appearance of the ejaculate. In the absence of semen, a second and a third attempt were made with males still showing no sign of sperm being classified as cases of aspermy. This was justified because in the cases where we found no sperm in an ejaculate, there was still a clear contraction of muscles. When APM made a second or a third attempt, there was still an obvious contraction of the cloacal muscles and any substance collected from the cloaca in most cases still did not contain sperm. When such collection of ejaculates was made in uncontaminated areas near Chernobyl or in Denmark or Spain, ejaculates were almost invariably collected in the first attempt. The number of individuals with and without sperm are listed in Table S1.
When there was sperm in the sample, the ejaculate was immediately diluted in 20–100 µl, depending on the size of the ejaculate, preheated Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA). Within 1 min after ejaculation, about 4–5 µl of the diluted sperm were pipetted onto a preheated standard microscopic count slide (20 µm depth two-chamber, Leja, Nieuw-Vennep, The Netherlands) mounted on a MiniTherm slide warmer (Hamilton Thorne Inc., Beverly, MA) kept at a temperature of 39°C, which is close to the mean temperature of 41.8°C (SE = 0.06). Sperm behavior was then recorded for multiple independent frames for each slide to increase the number of sperm measured for each male using 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 10x objective. The recordings were later analyzed with computer-assisted sperm analysis (CASA, HTM-CEROS sperm tracker, CEROS v.12, Hamilton Thorne Research, Beverly, MA, USA), which allows the recording of several standard motility measurements, including (1) VSL (straight line velocity), (2) VCL (track velocity), (3) VAP (curvilinear velocity), (4) ALH (amplitude of lateral head displacement), (5) BCF (beat cross frequency), (6) LIN (linearity, ratio of VSL/VCL) and (7) STR (Straightness = ratio of VSL/VAP). These measurements were used to estimate the percentage of static sperm, and sperm with slow, medium and high velocity. In total this provides 13 descriptors of sperm behavior.
Odds-ratio statistics with 95% confidence intervals were obtained from JMP  and were used to compare age and frequency of aspermy in contaminated areas versus controls. We predicted the presence or absence of sperm using logistic regression models with species, radiation level, age, body mass and keel length as predictors. When both body mass and keel length are entered into the same model, the body mass effect can be interpreted as a measure of body condition. We compared absence of sperm in different samples using contingency analyses. We used Welch’s ANOVA of unequal variances and Levene’s test  to assess differences in mean and variance in radiation between males with and without sperm.
Prior to our analysis of sperm behavior, we excluded species that were only trapped at a single site (see Table S2). Furthermore, all sperm traits were quantified as the mean value for each male across several video frames, weighted by the total number of cells for the actual frame. As different sperm traits reported from CASA are strongly correlated, we quantified sperm traits by using a principal component analysis of the 13 ejaculate parameters described above, relying on the varimax approach on the correlation matrix with Kaiser normalization . This reduced the correlated variables to three statistically independent axes that reflected different aspects of overall sperm traits (Table S3). Repeatabilities of the three principal components and the associated statistics are reported in Table S4.
Relationships between the three principal components of sperm and levels of background radiation were analyzed in mixed models with log-transformed external radiation as a fixed factor and species as a random factor. As sperm traits among different bird species are known to vary , , we assumed that their response to radiation could also vary. Consequently we followed standard guidelines  and ran both a model where each species could vary both in terms of intercept and slope, and a simpler model where species could only vary in their intercept. Model fitting and estimates were obtained with JMP  using restricted maximum likelihood estimates (REML). The significance of including the varying slope parameters was tested using log-likelihood ratio statistics, with the use of AICc (Akaike’s Information Criterion) . Finally, the model fit was verified using visual examination of normal probability plots and residual plots.
A total of 76 out of 566 male birds that were examined for sperm had no sperm, corresponding to 14.0%. In control areas 5 out of 164 (3.0%) individuals suffered from aspermy, while 74 out 402 (18.4%) individuals did so in contaminated areas. Odds ratio calculation suggests that it is 8.9 (3.0–35.7, 95% CI, P<0.001) times more likely to find aspermy in males caught in contaminated areas compared to uncontaminated control areas (Fig. 2). Indeed, both mean and variance in exposure differed between males with aspermy and sperm (Welch ANOVA for unequal variances: F = 109.64, d.f. = 1, 143.29, P<0.0001; Levene’s test: F = 104.70, d.f. = 1, 564, P<0.0001). Young birds were more common in contaminated than in control areas (61.3% of 256 individuals in contaminated areas, 49.2% of 128 individuals in control areas, odds ratio = 2.2 (1.4–3.4, 95% CI, P<0.001)). However, the main predictor of absence of sperm was background radiation level, showing a decrease in the proportion of males having sperm with increasing radiation level (Table 1; Fig. 3). The effects of species and age were not significant (χ2 = 37.05, d.f. = 31, P = 0.21, χ2 = 0.0003, d.f. = 1, P = 0.99, respectively). A full model that also included keel length and body mass to account for effects of body condition in addition to radiation showed an overall highly significant model that accounted for 20% of the variance (Table 1 model 2). The most important predictor was radiation implying that males without sperm were more common at higher levels of radiation. In addition, there was a weak effect of body mass, suggesting that males in poor condition more often had no sperm (Table 1 model 2).
Values are medians, quartiles, 5- and 95-percentiles and extreme values.
Sperm Behavior and Radiation
We obtained recordings of live sperm from 399 males belonging to 24 different species and in at least two different sites for each species, and these two sites were always an uncontaminated and a contaminated site (Table S2). Species with captures at only a single site are listed in Table S2. A principal component analysis of the CASA measurements of sperm behavior reduced the number of variables to three uncorrelated principal components accounting for 75.9% of the variance (Table S3). PC1 was highly positively correlated with all sperm velocity and motility variables. Furthermore, this principal component had strong loadings for sperm that were swimming straight and linearly (Table S2). PC2 reflected ejaculates having linear movements, and were motile, progressive and with a high fraction of rapid, medium and slow sperm thus reflecting ejaculates with motile sperm, but reduced velocity (Table S3). PC3 represented sperm with a high beat cross frequency and a high degree of straight swimming and many sperm with medium, slow and static sperm (Table S3). Thus, PC3 represented ejaculates with strongly reduced forward motion.
Analysis of the first principal component explaining 39.6% of the variance and that strongly correlated with sperm velocity and progressive sperm showing that there were significant species-specific effects (Table 2). There was no significant main effect of radiation (Table 2), while there was a significant interaction between species and radiation level and a significant year effect (Table 2). This interaction effect implies that sperm behavior was not consistently negatively correlated with radiation across all species; some species showed no significant radiation effect while others showed strong radiation effects.
Analysis of the second principal component that accounted for 24.2% of the variance and that correlated with sperm having medium velocity, showed a significant species-specific slope in relation to radiation level (Table 2). We found a significant effect for PC2 in relation to radiation (Table 2). In addition, there was a significant interaction between radiation and species (Table 2) and an effect of year (Table 2). This interaction effect implies that sperm behavior was not overall negatively correlated with radiation in all, but only in some species.
The third principal component, explaining 12.0% of the variance in sperm traits, representing ejaculates with strongly reduced forward motion, showed a species-specific response to radiation (Table 2). However, there was no significant main effect of radiation (Table 2), nor a significant year effect (Table 2). The interaction between species and background radiation was statistically significant (Table 2). This interaction effect implies that sperm behavior was not overall negatively correlated with radiation in all but only in some species.
The main findings of our study were, firstly, that aspermy is more common in birds living in contaminated areas near Chernobyl than in uncontaminated control areas. Second, among males that produce sperm there was a general decrease in sperm velocity and motility in relation to radioactive contamination around Chernobyl.
This study is by definition correlational. A similar experimental study under field conditions would not be possible for ethical and logistic reasons. However, this is by far the largest study of sperm in birds under field or laboratory conditions, and we are unaware of even a single other study investigating the effects of ionizing radiation on sperm in birds. Our study was conducted on free-living birds that live under more challenging natural conditions such as common food and nutrient restriction and the adverse effects of predation, parasitism and competition, as compared to the often benign and sheltered conditions experienced by animals in the lab.
There were 79 males that did not have any sperm either due to sperm depletion or aspermy. Levels of background radiation predicted aspermy for birds caught in the surroundings of Chernobyl, with more males without sperm being found at high levels of background radiation. We found no significant heterogeneity among species, suggesting that these severe effects of radiation on aspermy had a common ecological background. This is surprising given that the sex ratio is more male biased at higher levels of radiation . Hence, males in contaminated areas should on average compete more strongly for mates, but have fewer mating opportunities and thus less frequently have been depleted of sperm. This is opposite to the observed effect.
The high level of aspermy of up to 40% at the highest radiation levels is by any measure very large when compared to natural levels. There are only few comparable data for other organisms including humans. Lifjeld et al.  showed that 4% of willow warblers Phylloscopus trochilus and 2% of bluethroats Luscinia svecica had no sperm in the seminal glomera during the breeding season, which is similar to what we found in the uncontaminated control areas. In addition, the total number of sperm and the number of motile sperm has been found to be lower in Chernobyl victims than in controls .
Sperm behavior in birds living in the vicinity of Chernobyl appears to be affected by radiation, and our results are consistent with previous studies of sperm traits in birds at Chernobyl . In addition, survival rate of adult birds is reduced in Chernobyl compared to nearby control areas suggesting that only males of high quality survive . Thus, the estimated effect of radiation on sperm quality and quantity should be considered as conservative. Despite this likelihood, the sperm velocity was reduced among 24 species that were sampled in at least two different locations. This result is unlikely to be due to differences in reproductive timing as there is no indication of differences in laying date between Chernobyl and Ukrainian control areas .
We found large year effects for PC1 and PC2, but not for PC3. These year effects may arise for a number of reasons including differences in population density, food abundance or climate among years. We have no possibility for discriminating among these explanations since none of these factors appeared to vary consistently among years.
The species-specific effect of radiation on all three components of sperm behavior seems to follow a pattern that bird species having relatively high frequency of poorly performing sperm in areas with low background radiation are less sensitive to radiation than species that have low frequency of such sperm in areas with low background radiation. We are the first to report such an effect on sperm behavior among individuals from wild populations, although the result is in line with other studies showing that there may be species-specific differences in biological effects of radiation . However, the standard errors indicate large intraspecific variation (Table 2) and thus care should be taken when interpreting the slope. However, the present result is consistent with previous findings for large samples of barn swallows from Chernobyl .
Selection should act on traits important for fitness, and consequently males should allocate resources and trigger mechanisms underlying sperm function aimed at avoiding oxidative stress and preventing oxidative damage to sperm structures. Barn swallows in Chernobyl do not seem to protect their sperm from oxidative stress and they have elevated levels of ROS . The observed effect of radiation on sperm reported in this study is likely attributable to production errors during spermatogenesis, may result from increased perioxidation of the sperm membrane after formation , or may be due to acrosome damage . The most likely mechanism would be increased exposure to radiation due to the Chernobyl accident.
The negative effect of high doses of radiation on intra- and intercellular ROS levels is well known , while the low dose effect of radiation at the individual level is less understood . The observed negative effect of radiation on reproductive traits in birds at the individual levels may help explain the observed decline in wild bird populations in the most contaminated areas in Chernobyl . In addition, absence of sperm from males inhabiting the most radioactively contaminated areas will prevent the spread of mutations from such areas. Mutation rates around Chernobyl are elevated by up to more than a factor twenty . The underlying mechanisms responsible for the inter-specific response of sperm to radiation may help to understand mechanisms that influence sperm production errors and maintain sperm integrity under extreme environmental perturbations. Finally, knowledge about the species-specific response to radiation may be important for management following nuclear accidents. In summary, the effect of radioactive contamination on sperm shows how external environmental perturbations could impair traits that are crucial for reproductive success and hence fitness.
Bird species and number of individuals with aspermy and with sperm around Chernobyl.
Bird species and number of individuals sampled for sperm around Chernobyl.
Principal component analysis of 13 sperm parameters recorded with CASA. Rotation method: Varimax with Kaiser Normalization. Loadings above 0.4 are shown in bold.
Repeatability (R) of the three principal components of sperm behavior.
We are grateful for logistic help during our visit to Ukraine from G. Milinevsky, L. Milinevski, I. Chizchevsky, A. Peklo, A. Rozhok, I. Kozeretska, S. Taykova, W. Mardal and E. Flensted-Jensen. We received funding from the CNRS (France), University of South Carolina College of Arts & Sciences, NATO, the Fulbright Program, the Samuel Freeman Charitable Trust, the National Geographic Society and Norwegian Radiation Protection Authority in support of this research. Support and sperm tracking equipment was kindly provided by Ivar Folstad, University of Tromsø.
Conceived and designed the experiments: APM GR. Performed the experiments: APM ABA TAM GR. Analyzed the data: APM GR. Contributed reagents/materials/analysis tools: GR. Contributed to the writing of the manuscript: APM GR.
Birkhead TR, Møller AP (1998) Sperm Competition and Sexual Selection. London: Academic Press.
- 2. Birkhead TR, Martinez JG, Burke T, Froman DP (1999) Sperm mobility determines the outcome of sperm competition in the domestic fowl. Proc R Soc Lond Ser B - Biol Sci 266: 1759–1764.
- 3. Gage MJG, Macfarlane CP, Yeates S, Ward RG, Searle JB, et al. (2004) Spermatozoal traits and sperm competition in Atlantic salmon: Relative sperm velocity is the primary determinant of fertilization success. Curr Biol 14: 44–47.
- 4. Hunter FM, Birkhead TR (2002) Sperm viability and sperm competition in insects. Curr Biol 12: 121–123.
- 5. Laskemoen T, Fossøy F, Rudolfsen G, Lifjeld JT (2008) Age-related variation in primary sexual characters in a passerine with male age-related fertilization success, the bluethroat Luscinia svecica. J Avian Biol 39: 322–328.
- 6. Laskemoen T, Kleven O, Fossøy F, Robertson RJ, Rudolfsen G, et al. (2010) Sperm quantity and quality effects on fertilization success in a highly promiscuous passerine, the tree swallow Tachycineta bicolor. Behav Ecol Sociobiol 64: 1473–1483.
- 7. Levitan DR (2000) Sperm velocity and longevity trade off each other and influence fertilization in the sea urchin Lytechinus variegatus. Proc R Soc Lond Ser B - Biol Sci 267: 531–534.
- 8. Malo AF, Garde JJ, Soler AJ, Garcia AJ, Gomendio M, et al. (2005) Male fertility in natural populations of red deer is determined by sperm velocity and the proportion of normal spermatozoa. Biol Reprod 72: 822–829.
- 9. Rudolfsen G, Figenschou L, Folstad I, Kleven O (2008) Sperm velocity influence paternity in the Atlantic cod (Gadus morhua L.). Aquacult Res 39: 212–216.
- 10. Dewsbury DA (1982) Ejaculate cost and male choice. Am Nat 119: 601–610.
- 11. Folstad I, Skarstein F (1997) Is male germ line control creating avenues for female choice? Behav Ecol 8: 109–112.
- 12. Parker GA, Pizzari T (2010) Sperm competition and ejaculate economics. Biol Rev 85: 897–934.
- 13. Wedell N, Gage MJG, Parker GA (2002) Sperm competition, male prudence and sperm-limited females. Trends Ecol Evol 17: 313–320.
- 14. Cornwallis CK, Birkhead TR (2006) Social status and availability of females determine patterns of sperm allocation in the fowl. Evolution 60: 1486–1493.
- 15. Cornwallis CK, Birkhead TR (2007) Changes in sperm quality and numbers in response to experimental manipulation of male social status and female attractiveness. Am Nat 170: 758–770.
- 16. Møller AP, Mousseau TA, Rudolfsen G, Balbontin J, Marzal A, et al. (2009) Senescent sperm performance in old male birds. J Evol Biol 22: 334–344.
- 17. Pizzari T, Cornwallis CK, Froman DP (2007) Social competitiveness associated with rapid fluctuations in sperm quality in male fowl. Proc R Soc B - Biol Sci 274: 853–860.
- 18. Fischbein A, Zabludovsky N, Eltes F, Grischenko V, Bartoov B (1997) Ultramorphological sperm characteristics in the risk assessment of health effects after radiation exposure among salvage workers in Chernobyl. Environ Health Perspect 105: 1445–1449.
- 19. Goncharov NP, Katsiya GV, Kolesnikova GS, Dobracheva GAD, Todua TN, et al. (1998) Endocrine and reproductive health status of men who had experienced short-term radiation exposure at Chernobyl. Int J Androl 21: 271–276.
- 20. Helfenstein F, Losdat S, Møller AP, Blount JD, Richner H (2010) Sperm of colourful males are better protected against oxidative stress. Ecol Lett 13: 213–222.
- 21. Meistrich ML (1982) Quantitative correlation between testicular stem-cell survival, sperm production, and fertility in the mouse after treatment with different cyto-toxic agents. J Androl 3: 58–68.
- 22. Rudolfsen G, Figenschou L, Folstad I, Tveiten H, Figenschou M (2006) Rapid adjustments of sperm characteristics in relation to social status. Proc R Soc B - Biol Sci 273: 325–332.
- 23. Sakharov IY, Evdokimov VV, Atochina EA (1996) Sperm angiotensin-converting enzyme activity in Chernobyl victims and patients with chronic prostatitis. Androl 28: 259–260.
- 24. Sawyer DE, Roman SD, Aitken RJ (2001) Relative susceptibilities of mitochondrial and nuclear DNA to damage induced by hydrogen peroxide in two mouse germ cell lines. Redox Report 6: 182–184.
- 25. Sawyer DE, Mercer BG, Wiklendt AM, Aitken RJ (2003) Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutat Res -Fundamental Mol Mech Mutagen 529: 21–34.
- 26. Birioukov A, Meurer M, Peter RU, Braunfalco O, Plewig G (1993) Male reproductive-system in patients exposed to ionizing irradiation in the Chernobyl accident. Arch Androl 30: 99–104.
- 27. Chekhovich AV, Pomerantzeva MD, Ramaiya LK, Shevchenko VA (1993) Genetic damages in laboratory mice exposed in the Chernobyl nuclear power station zone 4 years after injury. Genetika 29: 312–322.
- 28. Goncharov NP, Katsiya GV, Kolesnikova GS, Dobracheva GAD, Todua TN, et al. (1998) Endocrine and reproductive health status of men who had experienced short-term radiation exposure at Chernobyl. Int J Androl 21: 271–276.
- 29. Pomerantseva MD, Ramaiya LK, Chekhovich AV (1997) Genetic disorders in house mouse germ cells after the Chernobyl catastrophe. Mutat Res -Fundamental Mol Mech Mutagen 381: 97–103.
- 30. Ash P (1980) The influence of radiation on fertility in man. Br J Radiol 53: 271–278.
- 31. Clifton DK, Bremner WJ (1983) The effect of testicular x-irradiation on spermatogenesis in man - a comparison with the mouse. J Androl 4: 387–392.
- 32. Rowley MJ, Leach DR, Warner GA, Heller CG (1974) Effect of graded doses of ionizing radiation on the human testis. Radiat Res 59: 665–678.
- 33. Lanzafame FM, La Vignera S, Vicari E, Calogero AE (2009) Oxidative stress and medical antioxidant treatment in male infertility. Reprod Biomed Online 19: 638–659.
- 34. Zalata A, Hafez T, Comhaire F (1995) Evaluation of the role of reactive oxygen species in male infertility. Human Reprod 10: 1444–1451.
- 35. Mikkelsen RB, Wardman P (2003) Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms. Oncogene 22: 5734–5754.
- 36. Ward JF (1994) DNA-damage as the cause of ionizing radiation-induced gene activation. Radiat Res 138: S85–S88.
- 37. Frenzilli G, Bosco E, Antonelli A, Panasiuk G, Barale R (2001) DNA damage evaluated by alkaline single cell gel electrophoresis (SCGE) in children of Chernobyl, 10 years after the disaster. Mutat Res - Genetic Toxicol Environ Mutagen 491: 139–149.
- 38. Gemignani F, Ballardin M, Maggiani F, Rossi AM, Antonelli A, et al. (1999) Chromosome aberrations in lymphocytes and clastogenic factors in plasma detected in Belarus children 10 years after Chernobyl accident. Mutat Res - Genet Toxicol Environ Mutagen 446: 245–253.
- 39. Frenzilli G, Lori A, Panasiuk G, Ferdeghini M, Barale R (1998) Comet assay on children’s leukocytes 8 years after the Chernobyl disaster. Mutat Res - Genet Toxicol Environ Mutagen 415: 151–158.
- 40. Bonisoli-Alquati A, Voris A, Mousseau TA, Møller AP, Saino N, et al. (2010) DNA damage in barn swallows (Hirundo rustica) from the Chernobyl region detected by use of the comet assay. Comp Biochem Physiol C - Toxicol Pharmacol 151: 271–277.
- 41. Ryabokon NI, Goncharova RI (2006) Transgenerational accumulation of radiation damage in small mammals chronically exposed to Chernobyl fallout. Radiat Environ Biophys 45: 167–177.
- 42. Ellegren H (2000) Microsatellite mutations in the germline: implications for evolutionary inference. Trends Genet 16: 551–558.
- 43. Møller AP, Mousseau TA (2003) Mutation and sexual selection: A test using barn swallows from Chernobyl. Evolution 57: 2139–2146.
- 44. 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 - Biol Sci 272: 247–252.
- 45. Møller AP, Mousseau TA, de Lope F, Saino N (2007) Elevated frequency of abnormalities in barn swallows from Chernobyl. Biol Lett 3: 414–417.
- 46. Meeks HN, Wickliffe JK, Hoofer SR, Chesser RK, Rodgers BE, et al. (2007) Mitochondrial control region variation in bank voles (Clethrionomys glareolus) is not related to Chernobyl radiation exposure. Environ Toxicol Chem 26: 361–369.
- 47. Wickliffe JK, Bickham AM, Rodgers BE, Chesser RK, Phillips CJ, et al. (2003) Exposure to chronic, low-dose rate gamma-radiation at Chornobyl does not induce point mutations in Big Blue (R) mice. Environ Mol Mutagen 42: 11–18.
- 48. Garcia O, Mandina T (2005) DNA damage evaluated by the comet assay in lymphocytes of children with Cs-137 internal contamination caused by the Chernobyl accident. Mutat Res - Genet Toxicol Environ Mutagen 565: 191–197.
- 49. Furitsu K, Ryo H, Yeliseeva KG, Thuy LTT, Kawabata H, et al. (2005) Microsatellite mutations show no increases in the children of the Chernobyl liquidators. Mutat Res - Gen Toxicol Environ Mutagen 581: 69–82.
- 50. Mahfouz R, Sharma R, Thiyagarajan A, Kale V, Gupta S, et al. (2010) Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril 94: 2141–2146.
- 51. Agarwal A, Sharma RK, Nallella KP, Thomas AJ, Alvarez JG, et al. (2006) Reactive oxygen species as an independent marker of male factor infertility. Fertil Steril 86: 878–885.
- 52. Yumura Y, Iwasaki A, Saito K, Ogawa T, Hirokawa M (2009) Effect of reactive oxygen species in semen on the pregnancy of infertile couples. Int J Urol 16: 202–207.
- 53. Tremellen K (2008) Oxidative stress and male infertility: A clinical perspective. Human Reprod Update 14: 243–258.
- 54. Tunc O, Thompson J, Tremellen K (2009) Improvement in sperm DNA quality using an oral antioxidant therapy. Reprod Biomed Online 18: 761–768.
- 55. Jones R, Mann T (1973) l-Lipid peroxidation in spermatozoa. Proc R Soc Lond Ser B - Biol Sci 184: 103–107.
- 56. Aitken RJ, Clarkson IS (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 81: 459–469.
- 57. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, et al. (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 59: 1037–1046.
- 58. Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS (1998) A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. J Cell Sci 111: 645–656.
- 59. Møller AP, Mousseau TA, Lynn C, Ostermiller S, Rudolfsen G (2008) Impaired swimming behaviour and morphology of sperm from barn swallows Hirundo rustica in Chernobyl. Mutat Res - Gen Toxicol Environ Mutagen 650: 210–216.
- 60. Bonisoli-Alquati A, Mousseau TA, Møller AP, Caprioli M, Saino N (2010) Increased oxidative stress in barn swallows from the Chernobyl region. Comp Biochem Physiol A-Mol Integr Physiol 155: 205–210.
- 61. Møller AP, Mousseau TA (2001) Albinism and phenotype of barn swallows (Hirundo rustica) from Chernobyl. Evolution 55: 2097–2104.
International Atomic Energy Agency (1992) Effects of ionizing radiation on plants and animals at levels implied by current radiation protection standards. Vienna: IAEA.
- 63. Hinton TG, Alexakhin R, Balonov M, Gentner N, Hendry J, et al. (2007) Radiation-induced effects on plants and animals: Findings of the United Nations Chernobyl Forum. Health Phys 93: 427–440.
- 64. Meistrich ML, Samuels RC (1985) Reduction in sperm levels after testicular irradiation of the mouse - a comparison with man. Radiat Res 102: 138–147.
- 65. Kleven O, Fossøy F, Laskemoen T, Robertson RJ, Rudolfsen G, et al. (2009) Comparative evidence for the evolution of sperm swimming speed by sperm competition and female sperm storage duration in passerine birds. Evolution 63: 2466–2473.
- 66. Rowe M, Pruett-Jones S (2011) Sperm competition selects for sperm quantity and quality in the Australian Maluridae. PLoS ONE 6: e15720.
- 67. Gashchak SP, Maklyuk YuA, Maksymenko AN, Maksymenko VM, Martynenko VI, et al. (2008) Radioactive contamination and abnormalities in small birds in the Chernobyl zone from 2003–2005. Radiat Biol Radioecol 28: 28–47.
Svensson L (2006) Identification guide to European passerines. Thetford, UK: British Trust for Ornithology.
Gelman A, Hill J (2007) Data Analysis using Regression and Multilevel/Hierarchical Models. New York: Cambridge University Press.
SAS (2012) JMP version 10.0. Cary, NC: SAS Inc.
- 71. Møller AP, Bonisoli-Alquati A, Rudolfsen G, Mousseau TA (2012) Elevated mortality among birds in Chernobyl as judged from skewed age and sex ratios. PloS One 7(4): e35223.
- 72. Lifjeld T, Laskemoen T, Fossøy F, Johnsen A, Kleven O (2007) Functional infertility among territorial males in two passerine species, the willow warbler Phylloscopus trochilus and the bluethroat Luscinia svecica. J Avian Biol 38: 267–272.
- 73. Hermosell IG, Lasakemoen T, Rowe M, Møller AP, Mousseau TA, et al. (2013) Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity. Biol Lett 9: 20130530.
UNSCEAR (2008) Effects of ionizing radiation. Vol. I: 1–390. New York: United Nations.
- 75. Prise KM (2006) New advances in radiation biology. Occupat Med-Oxford 56: 156–161.
- 76. Møller AP, Mousseau TA (2007) Species richness and abundance of forest birds in relation to radiation at Chernobyl. Biol Lett 3: 483–486.
European Union (1998) Atlas of Caesium deposition on Europe after the Chernobyl accident. Bruxelles: EU Publication EUR 16733.