Context-dependent Effects of Amino Acid Supplementation on Nestling Growth and Immune Function

Context-dependent Effects of Amino Acid Supplementation on Nestling Growth and Immune Function | 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 Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 29 August 2025 V1 Latest version Share on Context-dependent Effects of Amino Acid Supplementation on Nestling Growth and Immune Function Authors : Ashetu Terefa 0009-0006-8564-1836 [email protected] , Arne Hegemann 0000-0002-3309-9866 , Zoltán Németh 0000-0001-6164-9952 , and Adam Lendvai Authors Info & Affiliations https://doi.org/10.22541/au.175647838.81630768/v1 275 views 183 downloads Contents Abstract Introduction Materials and methods Results Discussion Disclosures Data Accessibility Statement References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Growth and immune development—fundamental parts of any organism’s ontogeny—depend critically on nutrient availability, particularly on essential amino acids that support protein synthesis and physiological processes. However, environmental variation can reduce prey diversity and nutrient quality, producing suboptimal diets that lack essential amino acids, potentially constraining development. In this study, we experimentally supplemented great tit (Parus major) nestlings from forest and suburban environments with methionine, leucine, or tap water (control) from day 4 to day 7 post-hatching and investigated the effects on growth and immune function across each habitat. We assessed nestling’s growth using body mass, tarsus length, and wing length, and immune function by quantifying complement activity (measured as lysis) and natural antibody titers (measured as agglutination). Supplementation significantly increased body mass gain in forest nestlings, particularly in smaller individuals, suggesting enhanced protein synthesis efficiency, although its effects on tarsus and wing length were limited. Methionine significantly improved lysis activity, suggesting enhanced innate immune function, while agglutination was not notably affected. Suburban nestlings showed limited responses to supplementation, suggesting broader nutritional constraints beyond individual amino acid deficiencies. Habitat and initial body mass significantly influenced growth rate but their effects depended on the treatment. These findings highlight the complex, condition-dependent effects of amino acid supplementation on nestling development and emphasize the importance of considering habitat-specific nutritional limitations when assessing avian developmental plasticity. Context-dependent Effects of Amino Acid Supplementation on Nestling Growth and Immune Function Ashetu Debelo Terefa 1,2,3* , Arne Hegemann 4 , Zoltán Németh 1 and Ádám Z. Lendvai 1 1. Department of Evolutionary Zoology and Human Biology, Debrecen University, 4032 Egyetem tér 1, Debrecen, Hungary 2. Pál Juhász-Nagy Doctoral School of Biology and Environmental Sciences, Debrecen University, Debrecen University, 4032 Egyetem tér 1, Debrecen, Hungary 3. Department of Biology, Ambo University, 71 Ambo, Ethiopia 4. Department of Biology, Lund University, 223 62 Lund, Sweden *Corresponding author, Email: [email protected] ABSTRACT Growth and immune development—fundamental parts of any organism’s ontogeny—depend critically on nutrient availability, particularly on essential amino acids that support protein synthesis and physiological processes. However, environmental variation can reduce prey diversity and nutrient quality, producing suboptimal diets that lack essential amino acids, potentially constraining development. In this study, we experimentally supplemented great tit ( Parus major ) nestlings from forest and suburban environments with methionine, leucine, or tap water (control) from day 4 to day 7 post-hatching and investigated the effects on growth and immune function across each habitat. We assessed nestling’s growth using body mass, tarsus length, and wing length, and immune function by quantifying complement activity (measured as lysis) and natural antibody titers (measured as agglutination). Supplementation significantly increased body mass gain in forest nestlings, particularly in smaller individuals, suggesting enhanced protein synthesis efficiency, although its effects on tarsus and wing length were limited. Methionine significantly improved lysis activity, suggesting enhanced innate immune function, while agglutination was not notably affected. Suburban nestlings showed limited responses to supplementation, suggesting broader nutritional constraints beyond individual amino acid deficiencies. Habitat and initial body mass significantly influenced growth rate but their effects depended on the treatment. These findings highlight the complex, condition-dependent effects of amino acid supplementation on nestling development and emphasize the importance of considering habitat-specific nutritional limitations when assessing avian developmental plasticity. Keywords: methionine, leucine, chicks, great tit, urbanisation, innate immunity 26 August 2025 Ashetu Debelo Terefa Debrecen University Egyetem tér 1, Debrecen, Hungary Email: [email protected] Editor-in-Chief Ecology and Evolution Dear Editor, on behalf of my coauthors, I am pleased to submit our manuscript entitled “Context-dependent effects of amino acid supplementation on nestling growth and immune function” for consideration for publication in Ecology and Evolution . Our study investigates how amino acid supplementation (leucine, methionine, or control) influences growth and immune function in great tit ( Parus major ) nestlings in relation to habitat (forest vs. suburban) and initial nestling size. We show that supplementation affected body mass growth and complement activity (lysis scores) in a habitat- and condition-dependent manner: smaller forest nestlings exhibited compensatory growth, whereas larger forest nestlings showed relatively growth retardation. In contrast, suburban nestlings did not benefit in terms of growth, but supplementation especially methionine enhanced their immune function (lysis scores). These findings provide novel insights into how nutritional constraints and ecological context shape developmental trajectories and physiological change in wild bird populations. We believe this work will be of particular interest to readers of Ecology and Evolution , especially those working in avian ecology, physiological ecology, and evolutionary biology. We confirm that the manuscript has not been published elsewhere and is not under consideration by any other journal. All authors have read and approved the submission, and we declare no conflicts of interest. Thank you in advance for considering our work. We look forward to your response. Best regards, Ashetu Debelo Terefa Introduction In developing animals, resources must be allocated simultaneously to multiple functions—particularly growth and development of physiological systems, including vital immune defenses—and this allocation is strongly influenced by nutrient availability and quality (Zera and Harshman, 2001). In birds, rapid growth and immune system maturation are especially critical, as nestlings must reach adequate body condition and immunocompetence within a short developmental window. For instance, body size at fledgling often predicts survival, with fledglings possessing shorter wings experiencing increased mortality (Aastrup et al. 2023; Gerritsma et al. 2022; Jones et al. 2017; Jones and Ward, 2020; Martin et al. 2014; Martin, 2015; Morrison et al. 2009), and nestlings with weaker immune function may be more susceptible to infection (Merino et al. 2000). Among nutrients, amino acids play a central role in supporting these developmental processes (Alagawany et al. 2021; Herring et al. 2021; Liu et al. 2022; Ndunguru et al. 2024a, 2024b). Amino acids, as the building blocks of proteins, are essential to support tissue development, regulate enzymatic and metabolic functions, and modulate the immune defenses as well as the maintenance of physiological homeostasis (Alagawany et al. 2021). As such, insufficient amino acid levels can impair growth, health and compromise body composition, while excessive intake of certain amino acids can be toxic or lead to metabolic imbalances (Swennen et al. 2007). Amino acid requirements vary substantially depending on diet types (Langlois and McWilliams, 2022; Nicolson, 2007; Wendeln et al. 2000). Insectivorous birds heavily rely on arthropods, which are rich in protein and essential amino acids and can meet avian nutritional demands even when consumed in relatively small amounts (Finke, 2015; Ramsay and Houston, 2003). However, any reduction or change in prey availability whether due to lower biomass or decreased nutritional quality can consistently lead to slower mass gain during critical stages of nestling development, while also affecting immune function and overall physiological condition (Greenberg, 1995; Johnson et al. 2005; Lacombe et al. 1994; Mägi et al. 2009; Senécal et al. 2021). Among the nine essential amino acids, methionine and leucine are particularly important for promoting early development in nestlings. Methionine plays a key role in protein synthesis, cellular metabolism, and is a key precursor of glutathione, a major antioxidant that protects cells from oxidative stress (Grimble and Grimble, 1998; Langlois and McWilliams, 2022; Liu et al. 2022). Methionine is also involved in methylation reactions that regulate gene expression, and limited methionine intake can reduce immune responses and impair growth (Fagundes et al. 2020). In many birds, methionine is often the first limiting amino acid, and its scarcity reduces growth rates in nestlings and negatively affects reproductive traits, such as egg size and production in adults (Keshavarz, 2003; Sekiz et al. 1975). Shortages in methionine can lead to trade-offs between growth and immunity (Brommer, 2004; Mirzaaghatabar et al. 2011; Soler et al. 2003; Zhang et al. 2024). Leucine, a branched-chain amino acid, promotes growth by stimulating muscle protein synthesis via the mTOR signaling pathway and supports translation of mRNA into proteins ensuring efficient nutrient utilization during rapid growth phases (Li et al. 2009; Nagao et al. 2015; Palii et al. 2009; Wu, 2013). Leucine also has immunomodulatory functions and may reduce protein breakdown during periods of stress or nutritional deficiency (Bonvini et al. 2018). Leucine supplementation has been shown to promote muscle growth, improve feed efficiency and modulate the immune response in domestic birds (Izumi et al. 2004; Xie et al. 2019). However, despite their known importance, the effects of methionine and leucine availability on growth and immune function in free-living bird species remain underexplored. Supplementation studies in wild birds offer a promising approach to investigate nutritional limitations and potential trade-offs between growth and immune function. Most previous studies have focused on general food supplementation, such as providing mealworms or caterpillars (e.g., Seress et al. 2020), or the effects of carotenoids or antioxidants (Biard et al. 2006; Tschirren et al. 2005), but only a few have manipulated specific amino acid intake. In this context, Tschirren et al. (2005) and Wegmann et al. (2015) found reduced or no effect of methionine supplementation on growth in great tit ( Parus major ) nestlings, but reported enhanced T-cell-mediated immune competence. Similarly, studies on other species, such as blue tits ( Cyanistes caeruleus ) and magpies ( Pica pica ), have shown that methionine supplementation can improve immunocompetence, although often at the cost of reduced growth (Brommer, 2004; Soler et al. 2003). In this study, we experimentally tested the effects of methionine and leucine supplementation on nestling growth and on components of baseline innate immune function in great tits. We hypothesized that methionine and leucine supplementation would influence nestling growth and innate immune function by altering resource allocation between competing physiological demands. Given their essential roles in protein synthesis, metabolism, and immune regulation, we predicted that these amino acids may act as limiting resources during early development. However, based on previous studies, we also expected that supplementation could mediate physiological trade-offs. In addition, we considered the possibility that responses to supplementation may be context-dependent, i.e. may be influenced by environmental factors that affect diet quality (habitat type), nestlings’ initial body mass, and nestling age. Materials and methods 2.1. Field and Experimental treatment protocol We monitored breeding of great tits in 218 nest boxes installed in three clusters in Debrecen city, Hungary, between March and June, 2022. Nest boxes were located in (1) a contiguous oak-dominated forest (n = 72), in (2) the Botanical Garden, a large, semi-managed area with high canopy cover and limited human disturbance (n = 102) or (3) open urban parks interspersed with buildings, pedestrian and vehicle traffic within the campus of University of Debrecen (n = 54). Based on vegetation structure, degree of habitat fragmentation, and human activity, we categorized the forest and Botanical Garden as ‘forest’ habitats, and the university parks as a ‘suburban’ habitat. Starting in the second week of March, nest boxes were checked twice a week for signs of nest building. To determine the precise dates of egg-laying and hatching, occupied nest boxes were visited three times a week from the date of occupancy onwards, while empty nest boxes were checked once a week until they became occupied. Once final clutch size was reached, we skipped visits for ten consecutive days to avoid disturbance and minimise risk of nest abandonment. Two days before the anticipated hatching date (i.e. 10 days after a clutch was complete), we checked the nest boxes once per 24 hours to determine the hatching date (day 0). On day 4 post-hatching, we individually marked all nestlings. While we primarily used metal rings for identification, for nestlings too small to accommodate a ring (n=17), we applied temporary colour markings (violet, red, green, and orange) on their head until they reached a size when they could be ringed with a numbered metal ring. At the day of first marking, i.e. day 4-post-hacthing, we also weighed all nestlings (n =168 in 28 broods) to the nearest 0.01g using a portable electronic balance. Based on their body mass, we ranked and grouped them into blocks of three, starting with the three heaviest nestlings. Using a randomized block design, we assigned each of the three nestlings in the first block randomly to one of the three amino acid supplementation treatments (methionine, leucine and control, see below). All broods contained at least one block., and for broods that contained more than five nestlings, we reversed the order of treatment in the subsequent block. This procedure all repeated until all nestlings were assigned to a treatment group. In total, we formed 73 blocks; 26 first, 26 second, 18 third and 3 fourth blocks. 2.2. Amino acid solutions and oral administration of the treatments We prepared a leucine solution (19 mg/ml) by mixing L-leucine powder (CAS No 61-90-5 (Prod No W329703), purity: ≥ 99%, Sigma Aldrich), and a methionine solution, (30 mg/ml), by mixing L-methionine powder (CAS No. 63-68-3 (prod, No. 64319 ), BioUltra, > 99.5%, Sigma Aldrich) with distilled water. The solution’s concentration was close to each amino acid’s respective maximum solubility in water. The methionine and leucine treatment groups received 100 µl of each solution, respectively, and the same volume of tap water was substituted for the control group. An automated pipette was used to administer the solutions (methionine, leucine, distilled water) orally to all nestlings within the respective treatment groups. Nestlings received the amino acid solutions or the water once daily between 6:00 – 17:00 from day 4 to day 7 of age. 2.3. Measurements of growth and blood sampling After each supplementation (see above), we recorded the body mass of each nestling to the nearest 0.01g using an electronic balance. On day four (i.e. first day of the supplementation) and on day 8 (i.e. one day after the last supplementation), we also measured tarsus and wing length using callipers (±0.1 mm) and rulers (±1 mm), respectively. Blood samples were collected on day 8 from the brachial wing vein of nestlings using capillary tubes, temporarily stored on ice in the field. Returning to the lab the same day, blood samples were centrifuged at 1431 RCF for 5 minutes. Plasma and red blood cells were separated and stored at−20 °C until subsequent laboratory analysis. 2.4. Haemagglutination and haemolysis assay To assess natural antibody and complement activity in nestling plasma, we used a hemagglutination and hemolysis assay as per Matson et al. (2005). We choose this assay as natural antibody titers and complement activity have been shown to develop during the nestling phase (e.g. Aastrup and Hegemann, 2021; Killpack et al. 2013; Mauck et al. 2005), are sensitive to environmental and individual conditions of nestlings (Aastrup et al. 2023; Nwaogu et al. 2023; Wemer et al. 2021) and have predictive capacity of survival (Hegemann et al. 2015; Roast et al. 2020). We centrifuged fresh rabbit red blood cells (RBCs) stored with EDTA were centrifuged at 1431 RCF for 5 minutes to separate cellular components from the supernatant. We then, measured the hematocrit value of the RBCs and used it to prepare a 1% RBC working solution in a phosphate buffered saline (PBS) for the assay. The assay was conducted on 96-well, round-bottom microplates. Control wells were established by adding 12.5 µL of PBS as negative controls and 12.5 µL of pooled Japanese quail ( Coturnix japonica ) plasma as positive controls. Plasma samples (12.5 µL each) of great tit nestlings were loaded into a plate, and a serial dilution was performed by sequentially aspirating, mixing, and transferring 12.5 µL across columns using a multichannel pipette. Finally, 12.5 µL of 1% rabbit RBC suspension was added to each well. Plates were sealed, incubated at 37°C for 90 minutes, dried at a 45° angle for 20 minutes, and photographed for agglutination scoring. After another 70 minutes of horizontal drying, plates were photographed for lysis scoring. Scoring was done following Matson et al. (2005) and blindly with respect to treatment category. 2.5. Data analysis We analyzed the effects of amino acid supplementation on nestling growth and immune function in linear mixed-effects models using R (version 2023-10-31). We analyzed both the repeated raw measurements (tarsus, wing length and body mass) and the growth rates (delta values). This allowed us to test the existence of any pre-treatment (day 4) differences in the measured traits, while simultaneously analyze treatment effects and if its effect varied by age, environment or developmental conditions. or to estimate the direction of change during the experiment. To calculate growth rates (delta values), we subtracted the post-treatment values (day 8) from the pre-treatment values (day 4) for tarsus and wing length. For body mass, we used slope estimates from best linear unbiased predictions (BLUPs) as growth rate (delta values). To analyze the effect of amino acid supplementation on structural development (body mass), we used linear mixed models using the lmer function from the lmerTest package (Kuznetsova et al. 2017) using raw repeated data as response variable, and treatment, age, initial body mass, and habitat type as fixed effects. We also included all possible interactions. Nest box ID and individual ID (nested within nest box ID) were included as random effects. Hatching date and clutch size were tested but excluded from the final models due to their lack of significance. Also, to analyze the effect of amino acid supplementation on growth rate (delta values), we used linear mixed models included treatment, initial body mass, and habitat type as fixed effects and all possible interactions. Nest box ID was included as random effect. Hemagglutination was evaluated using linear mixed effects model while hemolysis result was analyzed using generalized linear mixed models with negative binomial distribution (Brooks et al. 2017). In both cases, we used the same model structure as for the delta values, i.e. treatment, initial body mass and habitat type were included as a fixed effect with all their interactions, while nest ID was specified as random effect in both models. While initial body mass was included as continuous variable in the analyses (see above) to assess its potential as predictors of growth rate (Ronget et al. 2018), we categorized nestlings into three phenotypic classes light, intermediate, and heavy based on their initial body mass measured on day 4 for better visualization of how initial body mass influenced growth responses, and this is shown in the figures. This classification enabled an easier visualization of condition-dependent treatment effects. Results Amino acid supplementation significantly explained variation in body mass between day 4 and day 8 but this effect was dependent on nestling age, initial (day 4) body mass, and habitat (four-way-interaction, p = 0.002, Table 1). This result indicates that the effects of treatment on body mass gain varied depending on both the environmental context (habitat) and individual condition. In particular, treatment effects were more pronounced in lighter nestlings, and differed between forest and suburban habitats (Table 1 & Appendix 1). Analysis of the random effects revealed significant differences both among broods (\(\sigma^{2}=0.11)\) and among nestlings within a brood (\(\sigma^{2}=0.028)\). The treatment did not affect tarsus and wing length, either alone or in interaction with another variable (Appendix 2, A & B). Tarsus and wing length were only explained by initial (day 4) body mass and the interaction between habitat and age (Appendix 2, A & B), indicating that nestlings grew their tarsus and wing at a higher rate in forest than in suburban habitat. In contrast to body mass, individual identity did not explain significant variation in either tarsus or wing length (Appendix 2, A & B). These results were corroborated by growth rate (which was calculated from BLUP slope estimates) analyses, revealing that the effects of amino acid supplementation were strongly context-dependent, emerging through a significant three-way interaction between treatment, initial body mass, and habitat (Table 2). While there was no overall effect of treatment on growth rates, both methionine and leucine supplementation significantly increased growth rates in smaller nestlings (1.45 ± 0.05 g/day, mean ± SE), but this benefit was restricted to the forest habitat (Figure 1). In contrast, larger forest nestlings showed reduced growth when supplemented (0.09 ± 0.03 g/day, mean ± SE), suggesting a possible cost of excessive amino acid intake in well-developed individuals. These treatment effects were absent in the suburban habitat, where growth patterns differed: small suburban nestlings tended to grow faster than larger ones, but did not benefit from supplementation (Figure 1, Appendix 3). Agglutination was not affected by amino acid supplementation (F 2,144.3 = 0.827, p = 0.439), habitat (F 1,112.1 = 0.23, p =0.629), initial body mass (F 2,124 = 0.975, p = 0.325) or any of the interactions among these variables. In contrast, the effect of supplementation on lysis titer was dependent on habitat type and initial body mass. Lysis scores were lower in forest nestlings compared to suburban ones (z = −2.37, p = 0.017), albeit this difference got smaller in initially larger nestlings (z = 2.00, p = 0.044). Moreover, while none of the amino acid treatments affected lysis scores in the forest, nestlings in the suburbs treated with amino acids had better lysis scores, although this only reached statistical significance in the case of methionine (methionine: z = 2.22, p = 0.026, leucine: z = 1.56, p = 0.118, Figure 2). Discussion Our study demonstrates that supplementation with the essential amino acids methionine and leucine affects nestling growth and immune function in ways that depend on both habitat and individual condition. In forest habitat, both amino acids enhanced growth in initially smaller nestlings, but reduced it in initially larger ones, suggesting a response that depends on the growth trajectory. Conversely, in the suburban habitat, supplementation did not significantly affect growth regardless of nestling size. When examining two parameters of innate baseline immune function, methionine-supplemented suburban nestlings exhibited improved complement activity (measured as lysis), whereas no such effect was observed in forest habitat nestlings. As a result, the effects of amino acid supplementation on these traits appeared to be condition- and context-specific. Similar to our findings in suburban nestlings, previous studies have shown that methionine enhances immune responses in great tit nestlings (Brommer, 2004; Tschirren et al. 2005). Also, Soler et al (2003) observed the same pattern in methionine-supplemented magpie nestlings. However, Wegmann et al. (2015) found no effect of methionine on growth rates in great tit nestlings, which partially contradicts our findings. In blue tits, methionine improved immune responses but only in parasitized nests, where nestlings with experimentally enhanced immunocompetence grew faster (Pitala et al. 2010). Additionally, methionine combined with choline within a food significantly enhanced antibody titers in broilers (Swain and Johri, 2010). However, short-term immune benefits may not always translate into long-term advantages, as nestlings often compensate for initial growth reductions and achieve comparable body sizes before fledging. Collectively, these findings demonstrate the context-dependent and tightly regulated allocation of resources between growth and immune defense, shaped by environmental pressures and survival costs (Brommer, 2004; Pitala et al. 2010). Insights from domesticated species further illustrate the dose- and context-dependent effects of amino acid supplementation agreeing with our findings. In broilers, leucine supplementation enhanced breast meat yield and mitigated body weight loss up to an optimal dietary level, while excessive leucine impaired growth (Erwan et al. 2009). Similarly, moderate leucine supplementation in rock pigeons ( Columba livia ), improved growth, enhanced milk protein synthesis, and increased offspring survival, whereas higher leucine levels produced negative effects due to imbalances in branched-chain amino acid metabolism (Xie et al. 2019). In Japanese quail, embryonic supplementation of methionine and leucine had programmatic effects on post-natal growth, whereas nutritional treatment with the same amino acids after hatching had no detectable effect on growth (Ndunguru et al. 2024a, 2024b, Reda et al. 2025 in prep). Wild insectivore birds, like great tits, may naturally consume high amino acid levels when feeding on arthropods, which dominate their diet during peak growth (Ramsay and Houston, 2003). Thus, the relatively high supplementation levels used in our study may have exceeded optimal thresholds for promoting growth and immunity in some nestlings. Suburban nestlings showed higher complement activity following the supplementation compared to their forest counterparts. This difference suggests that suburban environments may impose greater nutritional limitations, making nestlings more responsive to dietary improvements. These findings align with previous studies showing that urbanization can create developmental constraints through poor nutritional conditions such as limited access to high-quality prey (e.g., caterpillars) and exposure to pollutants like arsenic from anthropogenic waste (Bailly et al. 2016; Biard et al. 2017; Sánchez-Virosta et al. 2018; Senar et al. 2021). Additionally, environmental and anthropogenic stressors and human disturbance may further compromise nestling development in urbanized habitats (Demeyrier et al. 2017; Kasprzykowski et al. 2014), contributing to reduced growth (Maness and Anderson, 2013) and potentially shortened lifespan (Salmón et al. 2016). In general, amino acid supplementation can enhance growth in underdeveloped individuals under natural conditions, but may carry risks in others depending on habitat context and body condition (Figure 1). Nestlings raised in higher-quality habitats tend to grow faster and exhibit better individual fitness (McKinnon et al. 2012; Stantial et al. 2021), whereas poor habitat quality has been associated with reduced reproductive success (Saulnier et al. 2022; Seress et al. 2020; Wawrzyniak et al. 2020). Structurally complex forest habitats are known to support better nutritional status in great tits (Seress and Liker, 2015), as reflected in wider feather growth bars (Catfolis et al. 2023). Also, urbanization has been linked to significant genetic differentiation between urban and forest populations of great and blue tits, potentially altering their development and reproductive outcomes (Bisikirskienė et al. 2024). A recent review also emphasizes how urban-driven shifts in vegetation, resource availability, and abiotic stressors including light and noise pollution can influence breeding and development in insectivorous birds such as great tits (Chen et al. 2023). Our findings suggest that early postnatal nutrition can modulate growth trajectories in a context- and condition-dependent manner. Nestlings with lower initial body mass (i.e. lighter nestlings) showed blunted growth rates compared to their heavier counterparts, especially in the forest habitat, but this disadvantage was partially mitigated by amino acid supplementation. In particular, both methionine and leucine enhanced the growth of small nestlings in the forest, indicating a compensatory effect under protein-limited conditions. Interestingly, these same treatments had no effect in suburban nestlings and even appeared to reduce growth in heavier forest nestlings, suggesting that amino acid availability interacts with individual condition and ecological context. This supports the idea that nutrient supplementation can alleviate developmental constraints in poorly conditioned individuals, but excess amino acids may become unnecessary (or even costly) when nutritional needs are already met. Initial body mass emerged as a strong predictor of growth trajectories in our study and this is in line with previous study that identified it as a key determinant of development and survival outcomes in birds (O’Connor, 1975; Ronget et al. 2018). Although we did not track post-treatment survival, which remains beyond the scope of this study, future work could explore how supplementation affects longer-term fitness outcomes. Variation in initial body mass likely reflects differences in pre-hatching maternal investment, particularly egg size (Krist, 2011; Schifferli, 1973). Nestling growth rate is a key determinant of post-fledging survival as the biggest and heaviest individuals at fledgling show higher survival. Traits, such as body mass index, wing length and hatching date have been shown to be reliable predictors of survival in altricial birds (Aastrup et al. 2023; Maness and Anderson, 2013). Immune traits can also provide predictive power: in laying hens, higher titers of natural antibodies are indicative of increased survival (Sun et al. 2011). In our study, body mass and lysis activity were strongly influenced by habitat type and nestlings initial (day 4) body mass, while tarsus and wing length were explained by age and initial body mass alone. In conclusion, amino acid supplementation can enhance nestlings’ development, but its effectiveness based on the individual initial body condition and ecological context. Methionine supplementation improved immune function (complement activity) in suburban nestlings, whereas no such effect was observed in forest environments. Furthermore, smaller nestlings in forest exhibited a compensatory growth when provided with methionine or leucine supplementation. Future research should explore the effect of varying concentrations of leucine, methionine, and other amino acids (such as taurine) (Arnold et al. 2007; Ramsay and Houston, 2003) and investigate long-term consequences of amino acid supplementation during early age on future survival and reproduction under different ecological conditions. Author Contributions Ashetu Debelo: research planning, conducted field and laboratory work, performed data analysis, and contributed to drafting, reviewing, and editing the manuscript. Arne Hegemann : laboratory work and data analysis, and contributed to manuscript review and editing. Zoltán Németh: planning, supervision, review and editing. Ádám Z. Lendvai: planning, supervision, laboratory work and data analysis, and contributed to manuscript review and editing. Acknowledgements We thank the management of the Botanical Garden for granting access to the study site on their property. Special thanks are due to Péter Hegedűs, Noémi Bújnoczki, Ivan Gonzalez, and Wondimu Ersino for their assistance during the fieldwork. Our thanks also go to Brigitta Csernus for assisting in sourcing rabbit RBCs and to Szemán-Nagy Gábor György and Gyula Pinczés for granting access to the hematocrit centrifuge. Funding Informations The study was funded by the National Development, Research and Innovation Fund (OTKA K139021 to ÁZL). AD received a Stipendium Hungaricum Scholarship from Tempus Public Foundation for Ph.D. studies. We acknowledge support from the University of Debrecen Program for Scientific Publication. AH was funded by a grant from the Swedish Research Council (2024-05731). Disclosures There is no conflict of interest Data Accessibility Statement All data used during the analysis in this manuscript are attached as a supportive material . Tables Table 1. ANOVA results for the effects of treatment, habitat type, initial body mass, and nestling age on great tit nestling raw body mass measurement taken between day 4 to 8 post hatching. Nestlings received 100 µl of either methionine solution, leucine solution or tap water (control group) through oral supplementation. Columns display degrees of freedom (numerator and denominator), F-values, and associated p -values. Statistically significant effects ( p - value < 0.05) are indicated in bold . Treatment 2 756.04 2.03 0.132 Habitat 1 777.53 3.07 0.080 Initial body mass 1 806.93 210.49 < 0.001 Nestlings age 1 692.63 491.29 <0.001 Habitat: initial body mass 1 806.93 2.87 0.091 Habitat: nestlings age 1 692.63 7.02 0.008 Initial body mass: nestlings age 1 687.92 3.78 0.052 Habitat: treatment 2 756.04 3.23 0.040 Initial body mass: treatment 2 756.65 2.15 0.117 Age: treatment 2 702.47 2.79 0.062 Habitat: initial body mass: nestlings age 1 687.92 5.25 0.022 Habitat: initial body mass: treatment 2 756.65 3.53 0.030 Habitat: nestlings age: treatment 2 702.47 5.86 0.003 Initial body mass: nestlings age: treatment 2 697.65 3.26 0.039 Habitat: initial body mass: nestlings age: treatment 2 697.65 6.30 0.002 Table 2. ANOVA results for fixed effects on great tit nestling body mass growth rate (BLUP slope estimates). Nestlings were randomly grouped into three treatment groups and received either methionine, leucine or tap water (control group) orally from age of day 4 to 7. Columns show degrees of freedom (numerator and denominator), F-values, and associated p -values. Significant effects ( p < 0.05) are shown in bold . Treatment 2 138.97 1.42 0.244 Habitat 1 141.48 4.28 0.040 Initial body mass 1 156.62 16.02 < 0.001 Habitat: Initial body mass 1 156.62 3.66 0.057 Habitat: treatment 2 138.97 3.06 0.050 Initial body mass: treatment 2 139.03 1.90 0.154 Habitat: initial body mass: treatment 2 139.03 3.27 0.041 Figure 1. Best linear unbiased prediction (BLUP) slopes of nestling body mass growth (delta values) across habitats (forest vs suburban) as a function of initial body mass. The BLUP slopes were derived from a linear mixed-effects model including all fixed effects and their interactions listed in Table 2. Great tit nestlings received either methionine, leucine or tap water (control group) through oral supplementation and categorized into three groups: light , intermediate , and heavy, (for illustration purposes) based on their initial (day 4) body mass measurement. Note that in the statistical model, initial body mass was treated as a continuous covariate and the categorization into three classes is done for visualization only (see statistical methods). Figure 2. Lysis (titer) of forest and suburban great tit nestlings supplemented methionine, leucine or tap water from age of day 4 to 7 post-hatching. Appendices Appendix 1. Figure showing initial day body mass and subsequent mass measurements of great tit nestlings from forest and suburban habitats. Nestlings received oral supplementation of either methionine, leucine or tap water (control groups) from day 4 to 7 of age. Appendix 2 . ANOVA results of great tit nestlings wing and tarsus length analyses. Nestlings received methionine, leucine solution or tap water (control group) from day 4 to 7 of ages. A) Wing length Treatment 2 263.89 0.002 0.998 Initial body mass 1 183.66 97.58 < 0.001 Habitat 1 169.34 4.333 0.038 Nestlings age 1 274.153 3886.15 < 0.001 Habitat: age 1 274.06 4.86 0.02 B) Tarsus length Treatment 2 144.17 0.27 0.77 Habitat 1 74.03 12.38 < 0.001 Initial body mass 1 242.37 115.76 < 0.001 Age 1 184.27 404.77 <0.001 Habitat: age 1 156.94 15.28 <0.001 Initial body mass: age 1 183.69 7.85 0.01 Appendix 3 . Great tit nestlings body mass, tarsus length and wing length growth rate within forest and suburban habitats Forest Light 1.45 ± 0.05 2.00 ± 0.03 3.81 ± 0.08 Forest Intermediate 1.45 ± 0.04 1.95 ± 0.03 3.86 ± 0.13 Forest Heavy 1.54 ± 0.03 1.91 ± 0.05 4.16 ± 0.11 Suburban Light 1.52 ± 0.05 1.89 ± 0.05 3.34 ± 0.17 Suburban Intermediate 1.60 ± 0.04 1.85 ± 0.05 3.62 ± 0.25 Suburban Heavy 1.54 ± 0.03 1.75 ± 0.04 4.57 ± 0.09 References 1. Aastrup, C., Hegemann, A., 2021. 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Collection Ecology and Evolution Keywords ecological experiment evolutionary ecology experimental evolution terrestrial vertebrate Authors Affiliations Ashetu Terefa 0009-0006-8564-1836 [email protected] University of Debrecen View all articles by this author Arne Hegemann 0000-0002-3309-9866 Lund University View all articles by this author Zoltán Németh 0000-0001-6164-9952 University of Debrecen View all articles by this author Adam Lendvai University of Debrecen View all articles by this author Metrics & Citations Metrics Article Usage 275 views 183 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Ashetu Terefa, Arne Hegemann, Zoltán Németh, et al. Context-dependent Effects of Amino Acid Supplementation on Nestling Growth and Immune Function. Authorea . 29 August 2025. DOI: https://doi.org/10.22541/au.175647838.81630768/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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