Age-related changes of oxidative status and immune function in a long-lived seabird

Age-related changes of oxidative status and immune function in a long-lived seabird | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Journal of Avian Biology This is a preprint and has not been peer reviewed. Data may be preliminary. 3 September 2025 V1 Latest version Share on Age-related changes of oxidative status and immune function in a long-lived seabird Authors : Beatrice Berardi 0009-0006-1988-3271 [email protected] , Giacomo Dell'Omo , Gianluca Damiani 0000-0001-6225-6309 , Gábor Czirják 0000-0001-9488-0069 , Silvia Filippi , Claudio Carere , and David Costantini Authors Info & Affiliations https://doi.org/10.22541/au.175686843.34545156/v1 Published Journal of Avian Biology Version of record Peer review timeline 369 views 179 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Experimental studies in humans and laboratory species have shown that the decline of the immune system with age (immunosenescence) and the accumulation of oxidative damage to macromolecules are two key contributors to the onset and progression of the ageing process. Although laboratory models have provided important insights, the physiological basis of ageing in natural populations remains comparatively understudied, constraining our mechanistic understanding of the ageing process. The complexity of age-related physiological changes increases further in long-lived species, which appear to possess unique adaptations reducing immunosenescence and oxidative damage. However, studies investigating the underlying physiological mechanisms in long-lived birds have yielded contrasting results. In this study, we compared four markers of oxidative status and eight immune markers between younger and older breeders of a long-lived seabird, the Scopoli’s shearwater (Calonectris diomedea), to identify potential physiological signatures of ageing. Regardless of sex, older individuals exhibited higher levels of blood antioxidant enzymes, natural antibodies, and lymphocytes compared to younger birds, while levels of DNA damage and cellular effectors of innate immunity did not differ between age classes. These findings suggest that older shearwaters may upregulate antioxidant enzyme activity, possibly to cope with increased basal production of reactive oxygen species, in line with the oxidative stress theory of ageing. Alternatively, the higher antioxidant levels of older birds might reflect selective mortality of birds with reduced protection. In contrast to the oxidative status, the observed immune patterns do not support the immunosenescence hypothesis 1. Introduction Ageing in natural animal populations has historically been studied within an evolutionary framework, focusing on the two major components of fitness: survival probability (actuarial senescence) and reproduction (reproductive senescence) (Gaillard & Lemaître 2020). By contrast, the physiological underpinnings of ageing have received far less attention, limiting our mechanistic understanding of this complex biological process. Identifying the cellular and molecular changes responsible for the physiological decline of organisms over time is central to explaining the variation in ageing patterns observed both within and between species (Selman et al. 2012, Peters et al. 2019). Among the mechanisms proposed for physiological ageing, oxidative stress (Birch-Machin & Bowman 2016) and immunosenescence (Pawelec 2018) are supposed to play important roles in determining the onset and rate of this complex biological process. Oxidative stress results from an imbalance between the production and accumulation of reactive oxygen species (ROS) and antioxidant defences that causes oxidative damage to body constituents and loss of cellular homeostasis (Luo et al. 2020). Oxidative stress is expected to increase with age due to reduced investment in somatic maintenance, protection, and repair (e.g. oxidative stress theory of ageing; Metcalfe & Alonso-Alvarez 2010). On the other hand, immunosenescence refers to the decline of major immune functions with increasing age, which can be associated, among other effects, to a low-grade chronic inflammation known as “inflamm-ageing” (Fulop et al. 2018). Adding to the complexity, age-related inflammation in turn may generate oxidative stress, further accelerating ageing in a multifaceted and interdependent process (oxidative-inflammatory theory of ageing; Romero Cabrera 2016). Studying long-lived species is particularly relevant to research on physiological ageing, as their slow senescence may reflect unique physiological adaptations (Stenvinkel & Shiels 2019). Most of our current knowledge about the factors contributing to longevity comes from studies on humans (Bauer & De la Fuente 2014), short-lived species whose lifespan has been experimentally extended (Holtze et al., 2021), and comparative studies between long- and short-lived species (Delhaye et al. 2016, Marasco et al. 2017, Jové et al. 2023). The latter indicate that, in general, long-lived species experience lower levels of oxidative stress, exhibit higher antioxidant capacity, and show fewer signs of reproductive or immune decline, as they invest more in self-maintenance compared to short-lived species (Xia & Moller 2018, Peters et al. 2019). Studies focusing on age-related variation in specific physiological markers within long-lived species yielded inconsistent results. For example, in long-lived birds, a decline in immune function has been reported for Leach’s storm-petrels ( Oceanodroma leucorhoa ) (Haussmann 2005), while innate and acquired immunity remain unchanged in common terns ( Sterna hirundo ) (Bichet et al. 2022). By contrast, European shags ( Phalacrocorax aristotelis ) exhibit an increase in oxidative damage with advancing age (Herborn et al. 2016), unlike budgerigars ( Melopsittacus undulatus ), which retain exceptional cellular resistance throughout their lifespan (Ogburn et al. 2001). The question of which set of hallmarks of ageing are truly more characteristic of long-lived species therefore remains open. Here, we compared four markers of oxidative status and eight immune markers between younger and older adults of a long-lived seabird, the Scopoli’s shearwater ( Calonectris diomedea ), to identify potential physiological signatures of ageing. We carried out the study during the breeding season because it is a particularly energy-demanding period for this species (Becciu et al. 2012), thus signatures of ageing might be more evident than during other less demanding stages of their life cycle. Specifically, we expected older individuals to exhibit higher DNA damage and haptoglobin (inflammatory marker), and lower antioxidant defences and immune markers, both innate and adaptive, compared to younger individuals, reflecting a reduction in somatic function with age. We also expected that signatures of ageing would be stronger in females due to their higher investment into reproduction compared to males (Gomes et al. 2019). 2. Materials & methods 2.1 Study birds and sampling The Scopoli’s shearwaters investigated for this study breed on Linosa Island, in the south-western Mediterranean Sea (35°52′ N, 12°52′ E). We collected blood samples in May 2024, during the egg-laying period, from breeding individuals of two age groups: younger (N=21; 5-9 years) and older (N=22; 17-36 years), as determined by their ringing history. Each group included a nearly equal number of females and males. Upon handling at the nest, we collected 400 µl of blood from the tarsal vein. We transferred the blood into heparinized Eppendorf tubes and maintained the tubes in a cool box until centrifugation. A drop of blood of each sample was smeared onto a glass microscope slide for leukocyte counts. We centrifuged the tubes (4000 RPM for 5 min) two hours after collection to separate the plasma from red blood cells and then we stored the tubes at -20°C in the field lab for two weeks and then at -80°C at the laboratory facilities until laboratory analyses. 2.2 Quantification of blood-based markers of oxidative status To assess the oxidative status of birds, we relied on established methods (e.g. Costantini et al. 2013, Cheron et al. 2022, Giovani et al. 2022). Briefly, we measured in haemolysates (N=41) the activity of three antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). We also measured nuclear DNA damage in cells as a further marker of oxidative stress (N=37). We quantified the levels of (i) SOD and GPx (expressed in units/mg of proteins) using the RANSOD and RANSEL assays (Randox Labs., Crumlin, UK), respectively; (ii) CAT (expressed in units/mg of proteins) using the OxiSelect CAT Activity Assay (Euromedex, France); (iii) DNA damage (length of strand breaks in µm) using the Comet assay (single-cell gel electrophoresis). All assays were performed in duplicate, and the mean of each duplicate was included in the statistical models. 2.3 Immunological assays To analyze the immune function of birds, we relied on established methods (e.g. Matson et al. 2005, Messina et al. 2025). Briefly, we determined (i) the plasma concentration of haptoglobin (in mg/ml) (N=40) using the commercial kit ”PHASE”TM Haptoglobin Assay (Tridelta, Ireland), (ii) the plasma concentration of immunoglobulin Y (IgY, in µg/ml) (N=41) using a sensitive ELISA with commercial anti-chicken antibodies, iii) the haemagglutination (HA) titer (N=39) using the haemagglutination assay, and (iv) the number of different white blood cell types by fixing blood smears (N=34) prepared while on the field with Wright-Giemsa Stain Solution (Reinoso-Pérez et al. 2025). Smears were examined under the microscope at 100x magnification with oil immersion, and the relative number and types of leucocytes were assessed by counting 100 leucocytes. The number of white blood cells of different types was expressed per 10 4 red blood cells (RBC) (Prüter et al. 2020). All haptoglobin, ELISA, and cell count measurements were performed in duplicate, and the mean of each duplicate was included in the statistical models. 2.4 Statistical analyses We fitted Generalized Linear Models (GLM) to test the effect of age class on immune and oxidative status markers (R package lme4 , v. 1.1.36, Bates et al. 2015). In preliminary analyses, we also run models entering age as a continuous variable. These models yielded similar results to those obtained with age class entered as a discrete variable. Thus, in each model, we entered Age class (older vs. younger) and Sex as predictors, and the interaction between age and sex to examine whether the effect of age differed between females and males. We ran GLMs with Gamma family and log link function for the four oxidative status markers, haptoglobin and IgY, and negative binomial GLMs for the white blood cell counts. Full models with non-significant interactions were simplified through stepwise model reduction until the minimal adequate model (MAM) was obtained, including only significant variables. To compare changes in model deviance during the stepwise process, we applied a likelihood-ratio test using the package lmtest (v. 0.9-40, Zeileis & Hothorn 2002). 3. Results We found significant differences between younger and older individuals for SOD, GPx, CAT, haemagglutination titer and lymphocyte counts (Table 1). Specifically, compared to younger individuals, older individuals had higher levels of SOD (+ 0.56 ± 0.25 U/mg, p = 0.03) (Fig. 1a), GPx (+ 0.79 ± 0.26 U/mg, p = 0.01) (Fig. 1b), CAT (+ 0.79 ± 0.28 U/mg, p = 0.01) (Fig. 1c), haemagglutination titer (+ 0.13 ± 0.06, p = 0.04) (Fig 2a) and absolute number of lymphocytes (+0.33 ± 0.10 cells/10 4 RBC, p < 0.01) (Fig. 2d). Younger and older individuals had similar levels of all the other markers measured, for both females and males (Figs. 1 and 2). Finally, we found significant differences between females and males in CAT and SOD, with higher levels of both enzymes in males (Table 2). 4. Discussion Our study simultaneously assessed age- and sex-related variation in oxidative status and immune markers in a long-lived bird species. Contrary to our expectations, older shearwaters exhibited higher levels of antioxidant enzymes and similar levels of DNA damage compared to younger shearwaters. It might be that older shearwaters upregulated levels of their antioxidant enzymes to cope with a higher basal production of ROS as expected by the oxidative stress theory of ageing. The upregulation of SOD with age is consistent with prior longitudinal and cross-sectional studies on captive zebra finches (Marasco et al. 2017), indicating that exposure to certain free radicals might increase with age as shown by research on laboratory strains (Costantini 2024). Alternatively, a selective mortality of individuals with lower antioxidant protection might occur, which would explain the higher antioxidant protection of older shearwaters. In species that undergo reproductive ageing, it has been proposed that increased antioxidant defences would represent an adaptive response to the oxidative challenges associated with ageing (Alonso-Alvarez et al. 2010). In a previous longitudinal work, we found negligible ageing of reproductive success. Thus, it might be that protective physiological mechanisms are upregulated in older individuals to shield their offspring from the harmful effects of oxidative stress (Blount et al. 2015). Regardless of age, males had higher levels of SOD and CAT than females, but similar levels of GPx and DNA damage. In oviparous vertebrates, females tend to have higher oxidative stress than males (Costantini 2018). The reasons for such sexual differences have not been clearly identified yet. One explanation for these differences might originate from sex-specific differences in sexual hormones, which have different effects on the cellular oxidative status (López-Arrabé et al. 2018). Another explanation lies in sex-specific reproductive costs. In shearwaters, females reach their lowest body mass during the egg-laying period, indicating a high energetic demand associated with producing and laying the egg, which may come at a cost to their self-maintenance systems (Lin et al. 2022). Conversely, during the egg-laying period, males are at their best body condition than in other phases of their life cycle. We do not know if sexual differences in oxidative status markers would also emerge in other less demanding phases of their life cycle. Further work is needed to address this point. Current investigations into how immune function changes with age in natural animal populations have primarily reported declines in adaptive immune traits and increases or no change in innate immunity (Bichet et al. 2022, Těšický et al. 2021). Our results on adaptive immunity diverge from this general pattern. Indeed, we found an age-related increase in the number of lymphocytes and similar levels of the immunoglobulin Y (IgY) between younger and older individuals. To our knowledge, this is the first documented case of an age-associated increase in lymphocytes in a wild bird population. Previous studies have examined lymphocytes mainly across developmental stages (e.g., chicks vs. adults; Jakubas et al. 2015). We hypothesize that an efficient antigen-specific memory could be key to the survival of these birds, enabling only highly immunocompetent individuals to reach advanced age (Lavoie 2006, Minias 2019). An alternative explanation is that differences in lymphocyte counts reflect the history of exposure to pathogens and parasites, which should increase with age (Hill et al. 2016, Bichet et al. 2022). Innate immunity in shearwaters, conversely, aligns with the general pattern described above for other bird species. Specifically, we found that haemagglutination titer - reflecting circulating levels of natural antibodies (NAbs) - increased with age, whereas the number of white blood cell types showed no age-related changes. Since NAbs can bind to and clear self-apoptotic and necrotic cells (Reyneveld et al. 2020), the observed increase in haemagglutination could potentially imply an anti-ageing or anti-inflammatory defence (Bichet et al. 2022). At the same time, the lack of age-related variations in white blood cells can be attributed to the crucial role of innate immunity as the first line of defence against pathogens (De Coster et al. 2010), coupled with its relatively low energetic maintenance cost (Nebel et al. 2013). In conclusion, our study provides evidence in a free-living longevous bird species that, irrespective of their sex, older individuals have higher levels of blood antioxidant enzymes, natural antibodies and lymphocytes than younger individuals while levels of DNA damage and the other leucocytes were similar between age classes. Furthermore, our results highlight the importance of simultaneously considering multiple physiological markers to detect and reveal the complexity of biological processes occurring throughout ageing. We propose that maintaining high levels of antioxidants, natural antibodies and lymphocytes into old age might be key to achieve a longer life and, possibly, a higher reproductive success due to the shielding of chicks against intergenerational transfer of oxidative damage or pathogens. Since this study is cross-sectional, longitudinal data are needed to disentangle the relative contributions of within- and between-individual changes to the underlying mechanisms of ageing in long-lived species. Conflict of interest The authors declare no conflict of interest. Ethic statement Fieldwork was carried out under the authorizations issued by the Regione Siciliana, Assessorato Agricoltura e Foreste with letter Prot. 17233 dated December 1, 2010, and subsequent communications Prot. 2452 dated February 01, 2018, and Prot. 48759 dated May 22, 2023. Funding statement This study was funded by a doctoral fellowship from the University of Tuscia to B.B and from the association Ornis italica. Field work was financially supported by Ornis italica and Berta maris . Acknowledgments We thank Eleonora Dell’Omo for her support in the field, and Katja Pohle for technical support during laboratory analyses. References Alonso-Alvarez, C., Pérez-Rodríguez, L., García, J. T., Vinuela, J. & Mateo, R. 2010. Age and breeding effort as sources of individual variability in oxidative stress markers in a bird species. Physiological and Biochemical Zoology , 83 : 110-118 . Bates D., Mächler M., Bolker B. & Walker S. 2015. Fitting Linear Mixed-Effects Models using lme4. Journal of Statistical Software , 67 : 1–48. https://doi.org/10.18637/jss.v067.i01. Becciu P, Massa B & Dell’Omo G. 2012. Body mass variation in Scopolis shearwater Calonectris diomedea breeding at Linosa Island. Pages 16-18. In: Ecology and Conservation of Mediterranean Seabirds and other bird species under the Barcelona Convention, Proceedings of the 13th Medmaravis PanMediterranean Symposium. Alghero (Italy) 14-17 Oct. 2011. Eds: Ysou P, Baccetti N & Sultana J. Bauer, M. E. & De la Fuente, M. 2014. Chapter 4 - Oxidative Stress, Inflammaging, and Immunosenescence, Editor(s) : Irfan Rahman, Debasis Bagchi. Inflammation, Advancing Age and Nutrition , Academic Press, Pages 39-47. https://doi.org/10.1016/B978-0-12-397803-5.00004-6 Bichet, C., Moiron, M., Matson, K. D., Vedder, O. & Bouwhuis, S. 2022. Immunosenescence in the wild? A longitudinal study in a long‐lived seabird. Journal of Animal Ecology , 91 : 458-469. https://doi.org/10.1111/1365-2656.13642 Birch‐Machin M.A. & Bowman A. 2016. Oxidative stress and ageing. British Journal of Dermatology , 175: 26–29. https://doi.org/10.1111/bjd.14906 Blount, J. D., Vitikainen, E. I. K., Stott, I. & Cant, M. A. 2015. Oxidative shielding and the cost of reproduction. Biological Reviews of the Cambridge Philosophical Society, 91: 483–497. https://doi.org/10.1111/brv.12179 Cheron, M., Costantini, D., Angelier, F., Ribout, C. & Brischoux, F. 2022. Aminomethylphosphonic acid (AMPA) alters oxidative status during embryonic development in an amphibian species. Chemosphere , 287 , 131882. https://doi.org/10.1016/j.chemosphere.2021.131882 Costantini D., Monaghan P. & Metcalfe N. 2013. Loss of integration is associated with reduced resistance to oxidative stress. The Journal of Experimental Biology , 216: 2213-2220. https://doi.org/10.1242/jeb.083154 Costantini, D. 2018. Meta-analysis reveals that reproductive strategies are associated with sexual differences in oxidative balance across vertebrates. Current Zoology , 64 : 1-11. https://doi.org/10.1093/cz/zox002 Costantini D. 2024. The role of organismal oxidative stress in the ecology and life-history evolution of animals. Fascinating Life Sciences, Springer, 2024. De Coster, G., De Neve, L., Martín-Gálvez, D., Therry, L. & Lens, L. 2010. Variation in innate immunity in relation to ectoparasite load, age and season: a field experiment in great tits ( Parus major ). Journal of Experimental Biology , 213 : 3012-3018. https://doi.org/10.1242/jeb.042721 Delhaye J, Salamin N, Roulin A, Criscuolo F, Bize P & Christe P. 2016. Interspecific correlation between red blood cell mitochondrial ROS production, cardiolipin content and longevity in birds. Age, 38:433–43. https://doi.org/10.1007/s11357-016-9940-z Fulop, T., Larbi, A., Dupuis, G., Le Page, A., Frost, E. H., Cohen, A. A., … & Franceschi, C. 2018. Immunosenescence and inflamm-aging as two sides of the same coin: friends or foes? Frontiers in Immunology , 8 : 1960. https://doi.org/10.3389/fimmu.2017.01960 Gaillard, J. M. & Lemaître, J. F. 2020. An integrative view of senescence in nature. Functional Ecology , 34 : 4-16. https://doi.org/10.1111/1365-2435.13506 Giovani, G., Filippi, S., Molino, C., Peruffo, A., Centelleghe, C., Meschini, R., & Angeletti, D. 2022. Plastic additive di (2-ethylhexyl) phthalate (DEHP) causes cell death and micronucleus induction on a bottlenose dolphin’s (Tursiops truncatus) in vitro-exposed skin cell line. Frontiers in Marine Science , 9 : 1-12. https://doi.org/10.3389/fmars.2022.958197 Gomes, A. D. S., Vieira, J. L. F. & da Silva, J. M. C. 2019. Sexual differences in oxidative stress in two species of Neotropical manakins ( Pipridae ). Journal of Ornithology, 160: 1151–1157. https://doi.org/10.1007/s10336-019-01673-8 Jakubas, D., Wojczulanis-Jakubas, K. & Kośmicka, A. 2015. Factors affecting leucocyte profiles in the little auk, a small Arctic seabird. Journal of Ornithology, 156: 101–111. https://doi.org/10.1007/s10336-014-1101-5 Jové, M., Mota-Martorell, N., Fernandez-Bernal, A., Portero-Otin, M., Barja, G. & Pamplona, R. 2023. Phenotypic molecular features of long-lived animal species. Free Radical Biology and Medicine , 208 : 728-747. https://doi.org/10.1016/j.freeradbiomed.2023.09.023 Haussmann, M. F., Winkler, D. W., Huntington, C. E., Vleck, D., Sanneman, C. E., Hanley, D. & Vleck, C. M. 2005. Cell-mediated immunosenescence in birds. Oecologia , 145 : 269-274. https://doi.org/10.1007/s00442-005-0123-3 Herborn, K. A., Daunt, F., Heidinger, B. J., Granroth‐Wilding, H. M., Burthe, S. J., Newell, M. A. & Monaghan, P. 2016. Age, oxidative stress exposure and fitness in a long‐lived seabird. Functional Ecology , 30 : 913-921. https://doi.org/10.1111/1365-2435.12578 Hill, S. C., Manvell, R. J., Schulenburg, B., Shell, W., Wikramaratna, P. S., Perrins, C., … & Pybus, O. G. 2016. Antibody responses to avian influenza viruses in wild birds broaden with age. Proceedings of the Royal Society B: Biological Sciences , 283 : 20162159. https://doi.org/10.1098/rspb.2016.2159 Holtze, S., Gorshkova, E., Braude, S., Cellerino, A., Dammann, P., Hildebrandt, T. B., … & Sahm, A. 2021. Alternative animal models of aging research. Frontiers in Molecular Bioscience 8: 1-26. https://doi.org/10.3389/fmolb.2021.660959 Lavoie, E. T. 2006. Avian immunosenescence. Age , 27 : 281-285. doi 10.1007/s11357-005-4561-y Lin, Y., Patterson, A., Jimenez, A. G. & Elliott, K. 2022. Altered oxidative status as a cost of reproduction in a seabird with high reproductive costs. Physiological and Biochemical Zoology , 95 : 35-53. https://doi.org/10.1086/717916 López-Arrabé, J., Monaghan, P., Cantarero, A., Boner, W., Pérez-Rodríguez, L. & Moreno, J. 2018. Sex-specific associations between telomere dynamics and oxidative status in adult and nestling pied flycatchers. Physiological and Biochemical Zoology , 91 : 868-877. https://doi.org/10.1086/697294 Luo, J., Mills, K., le Cessie, S., Noordam, R. & van Heemst, D. 2020. Ageing, age-related diseases and oxidative stress: what to do next? Ageing research reviews , 57 : 1568-1637. https://doi.org/10.1016/j.arr.2019.100982 Marasco, V., Stier, A., Boner, W., Griffiths, K., Heidinger, B. & Monaghan, P. 2017. Environmental conditions can modulate the links among oxidative stress, age, and longevity. Mechanisms of Ageing and Development , 164 : 100-107. https://doi.org/10.1016/j.mad.2017.04.012 Matson, K. D., Ricklefs, R. E. & Klasing, K. C. 2005. A hemolysis-hemagglutination assay for characterizing constitutive innate humoral immunity in wild and domestic birds. Developmental & Comparative Immunology , 29: 275–286. https://doi.org/10.1016/j.dci.2004.07.006 Metcalfe, N. B. & Alonso‐Alvarez, C. 2010. Oxidative stress as a life‐history constraint: the role of reactive oxygen species in shaping phenotypes from conception to death. Functional Ecology , 24 : 984-996. https://doi.org/10.1111/j.1365-2435.2010.01750.x Messina S., Prüter H., Czirjak G.A. & Costantini D., 2025. Lower adaptive immunity in invasive Egyptian geese compared to sympatric native waterfowls. Comparative Biochemistry and Physiology Part A, 299 : 111752. https://doi.org/10.1016/j.cbpa.2024.111752 Minias, P. 2019. Evolution of heterophil/lymphocyte ratios in response to ecological and life‐history traits: a comparative analysis across the avian tree of life. Journal of Animal Ecology , 88 : 554-565. https://doi.org/10.1111/1365-2656.12941 Nebel, S., Buehler, D. M., Kubli, S., Lank, D. B. & Guglielmo, C. G. 2013. Does innate immune function decline with age in captive ruffs Philomachus pugnax ? Animal Biology, 63: 233-240. https://doi.org/10.1163/15707563-00002409 Ogburn, C. E., Carlberg, K., Ottinger, M. A., Holmes, D. J., Martin, G. M. & Austad, S. N. 2001. Exceptional cellular resistance to oxidative damage in long-lived birds requires active gene expression. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences , 56 : 468-474. https://doi.org/10.1093/gerona/56.11.B468 Pawelec, G. 2018. Age and immunity: what is “immunosenescence”? Experimental Gerontology , 105 : 4-9. https://doi.org/10.1016/j.exger.2017.10.024 Peters, A., Delhey, K., Nakagawa, S., Aulsebrook, A., & Verhulst, S. 2019. Immunosenescence in wild animals: meta‐analysis and outlook. Ecology Letters , 22:1709-1722. https://doi.org/10.1111/ele.13343 Prüter, H., Franz, M., Twietmeyer, S., Böhm, N., Middendorff, G., Portas, R., … & Czirják, G. Á. 2020. Increased immune marker variance in a population of invasive birds. Scientific Reports , 10 :1-13. Reinoso-Pérez, M. T., Dhondt, K. V., Yánez Abad, A. A., Rodríguez-García, V. M. & Dhondt, A. A. 2025. Pathogen-induced stress in wild house finches ( Haemorhous mexicanus ): leukocyte dynamics as health indicators. The Journal of Wildlife Diseases , 61 : 334-347. https://doi.org/10.7589/JWD-D-24-00164 Reyneveld G.I., Savelkoul H.F.J., Parmentier H.K. 2020. Current understanding of natural antibodies and exploring the possibilities of modulation using veterinary models. A Review. Frontiers in Immunology, 11: 1-19. doi: 10.3389/fimmu.2020.02139 Romero Cabrera, A. J. 2016. Inflammatory oxidative aging: a new theory of aging. Medcrave online Journal of Immunology , 3 : 00103. https://doi.org/10.15406/moji.2016.03.00103 Selman, C., Blount, J. D., Nussey, D. H. & Speakman, J. R. 2012. Oxidative damage, ageing, and life-history evolution: where now? Trends in Ecology & Evolution , 27 : 570-577. http://dx.doi.org/10.1016/j.tree.2012.06.006 Stenvinkel, P. & Shiels, P. G. 2019. Long-lived animals with negligible senescence: clues for ageing research. Biochemical Society Transactions, 47 : 1157-1164. http://dx.doi.org/10.1042/BST20190105 Těšický, M., Krajzingrova, T., Świderská, Z., Syslova, K., Bilkova, B., Eliáš, J., … & Vinkler, M. 2021. Longitudinal evidence for immunosenescence and inflammaging in free-living great tits. Experimental Gerontology , 154 : 1-10. https://doi.org/10.1016/j.exger.2021.111527 Xia, C. & Møller, A. P. 2018. Long-lived birds suffer less from oxidative stress. Avian Research , 9 : 1-7. https://doi.org/10.1186/s40657-018-0133-6 Zeileis A. & Hothorn T. 2002. Diagnostic checking in regression relationships. R News , 2 : 7–10. https://CRAN.R-project.org/doc/Rnews/. Table 1. Outcomes of full and reduced models fitted for four oxidative markers and eight immune markers. Models for which a significant effect of Age and/or Sex emerged are shown in bold. Reduced models were obtained through backward elimination of non-significant terms, starting with the interaction. Statistical significance is indicated as follows: 0 ‘***’, 0.001’**’, 0.05 ‘*’, ‘.’ 0.1, ‘ ’ 1. df χ² p d.f χ² p Oxidative stress SOD ageclass 35 20.52 * 35 20.52 * sex 34 16.27 ** 34 16.27 ** ageclass*sex 33 15.13 GPx ageclass 35 18.62 ** 35 18.62 ** sex 34 18.33 ageclass*sex 33 17.24 CAT ageclass 35 26.09 ** 35 26.09 ** sex 34 22.09 * 34 22.09 * ageclass*sex 33 21.94 DNA damage ageclass 35 4.31 35 4.31 sex 34 4.25 ageclass*sex 33 4.24 Immunity Haemagglutination ageclass 37 1.43 * 37 1.43 * sex 36 1.43 ageclass*sex 35 1.41 Lymphocytes ageclass 32 118.71 *** 32 118.71 *** sex 31 118.48 ageclass*sex 30 111.84 IgY ageclass 37 0.16 37 0.16 sex 36 0.15 ageclass*sex 35 0.15 Haptoglobin ageclass 37 7.66 37 7.66 sex 36 7.66 ageclass*sex 35 7.65 Eosinophils ageclass 32 40.32 32 39.36 sex 31 39.55 ageclass*sex 30 39.36 Basophils ageclass 32 28.90 32 25.22 sex 31 28.82 ageclass*sex 30 25.74 . Heterophils ageclass 32 37.87 32 35.73 sex 31 37.79 . ageclass*sex 30 35.73 Monocytes ageclass 32 42.10 32 40.84 sex 31 42.06 ageclass*sex 30 40.97 Table 2. Estimated marginal means ± standard errors from the models for CAT and SOD. Both antioxidant enzymes showed significant differences between males and females. Values are presented on the log-transformed scale. CAT 4.65 ± 0.21 3.99 ± 0.19 0.03 SOD 1.51 ± 0.19 0.82 ± 0.17 0.01 Figure 1 Figure 2 Figure 1. Activity levels of superoxide dismutase (SOD) (a), glutathione peroxidase (GPx) (b), and catalase (CAT) (c), and quantification of DNA damage (d) in relation to the age class of shearwaters. Values (given on the log scale) are presented as estimated marginal means ± standard errors. Significant differences between younger and older individuals are indicated (* p < 0.05, ** p < 0.01). Figure 2. Haemagglutination (Ha) titer (a), levels of haptoglobin (b), and IgY (c), and number of lymphocytes (d), monocytes (e), eosinophils (f), heterophils (g) and basophils (h), in relation to the age class of shearwaters. Values are presented as estimated marginal means ± standard errors (all on the log-transformed scale except for IgY). Significant differences between young and old individuals are indicated (* p < 0.05, ** p < 0.01). Information & Authors Information Version history V1 Version 1 03 September 2025 Peer review timeline Published Journal of Avian Biology Version of Record 13 Mar 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Journal of Avian Biology Keywords ageing antioxidants immunity immunosenescence oxidative stress seabirds Authors Affiliations Beatrice Berardi 0009-0006-1988-3271 [email protected] Università degli Studi della Tuscia Dipartimento di Scienze Ecologiche e Biologiche View all articles by this author Giacomo Dell'Omo Ornis Italica View all articles by this author Gianluca Damiani 0000-0001-6225-6309 Università degli Studi della Tuscia Dipartimento di Scienze Ecologiche e Biologiche View all articles by this author Gábor Czirják 0000-0001-9488-0069 Leibniz Institute for Zoo and Wildlife Research View all articles by this author Silvia Filippi Università degli Studi della Tuscia Dipartimento di Scienze Ecologiche e Biologiche View all articles by this author Claudio Carere Università degli Studi della Tuscia View all articles by this author David Costantini Università degli Studi della Tuscia Dipartimento di Scienze Ecologiche e Biologiche View all articles by this author Metrics & Citations Metrics Article Usage 369 views 179 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Beatrice Berardi, Giacomo Dell'Omo, Gianluca Damiani, et al. 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