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figure supplement 1. Plasma metabolites of European starlings associated with flight-training and in relation to diet quality. Change in circulating levels of (a) Beta-hydroxybutyrate, and (b) Triglycerides in plasma of European starlings over the 15-day flight-training for Trained birds (dark triangles and dashed lines) and over the same 15-day period for Sedentary birds that were not flight-trained (gray triangles, solid lines) in Experiment II. Plasma samples were collected in the early morning after an overnight without food on Day O (Pre-training), before the start of flight-training, on Day 15 immediately after the birds longest flight (Post-flight), and in the early morning after an overnight without food on Day 17 (1.5 days after recovery from the last flight). Body mass and date of measurement were included as a fixed covariate so that we report the results as least square means (LSM). Comparisons between Pre-training and Recovery indicate changes associated with the 15-day flight-training, whereas those between Recovery and Post-flight indicate changes associated with the longest flight on Day 15. Means with different letters across the three timepoints, and for the main effect of diet, are significantly different (p<0.05).
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Elite human and animal athletes must acquire the fuels necessary for extreme feats, but also contend with the oxidative damage associated with peak metabolic performance. Here, we show that a migratory bird with fuel stores composed of more omega-6 polyunsaturated fats (PUFA) expended 11% less energy during long-duration (6 hr) flights with no chan...
Citations
... Based on the observation that a peak in metabolic activity such as sustained endurance is not necessarily followed by an immediate rise in oxidative damage (e.g. [82]), we hypothesize that, in most cases, the ROS release accompanying reproduction, which also increases metabolic activity, is not of mitochondrial origin. Oxidative stress may not be an obligatory issue of high mitochondrial respiration. ...
The allocation of resources in animals is shaped by adaptive trade-offs aimed at maximizing fitness. At the heart of these trade-offs, lies metabolism and the conversion of food resources into energy, a process mostly occurring in mitochondria. Yet, the conversion of nutrients to utilizable energy molecules (adenosine triphosphate) inevitably leads to the by-production of reactive oxygen species (ROS) that may cause damage to important biomolecules such as proteins or lipids. The ‘ROS theory of ageing’ has thus proposed that the relationship between lifespan and metabolic rate may be mediated by ROS production. However, the relationship is not as straightforward as it may seem: not only are mitochondrial ROS crucial for various cellular functions, but mitochondria are also actually equipped with antioxidant systems, and many extra-mitochondrial sources also produce ROS. In this review, we discuss how viewing the mitochondrion as a regulator of cellular oxidative homeostasis, not merely a ROS producer, may provide new insights into the role of oxidative stress in the reproduction–survival trade-off. We suggest several avenues to test how mitochondrial oxidative buffering capacity might complement current bioenergetic and evolutionary studies.
... Currently available observational and experimental studies on this topic provide inconsistent conclusions regarding whether flight exercise or migration induces oxidative stress in birds. This question was primarily investigated experimentally using birds exposed to flight exercise (Costantini et al. 2008, Dick and Guglielmo 2019, McWilliams et al. 2020, and correlatively by comparing the physiological status of birds at different stages of migration (e.g. before migration or after recovery at stopover sites, e.g. ...
... If the energetic expense of flight induces the overproduction of ROS, we can hypothesize that traits associated with higher flight energy demand will be associated with elevated oxidative damage and/or will drive the evolution of more efficient antioxidant defences to prevent potential damages. Alternatively, physiological adaptations of birds to a life with high metabolic demands, including the resistance to oxidative stress, ROS production, metabolic management, the high density of mitochondria functioning as sink for ROS, or the composition of fatty acids of the energy reserve during migration may mitigate the oxidative cost of aerial locomotion across species (Hickey et al. 2012, McWilliams et al. 2020. ...
... We detected increased levels of UA in species with longer migration distances, suggesting that the role of this endogenous non-enzymatic antioxidant in oxidative defence increases with flight distance. Alternatively, because UA is a product of protein catabolism, our results could also indicate that longer migratory distances necessitate protein breakdown to fuel strenuous flights as compared with sedentary or shortdistance migratory species (McWilliams et al. 2020). MDA also showed positive evolutionary covariation with migration distance, which indicates that the evolution of increased UA concentrations cannot neutralise the increased oxidative challenge faced by long-distance migrants, ultimately leading to lipid damage. ...
Flight can be highly energy demanding, but its efficiency depends largely on flight style, wing shape and wing loading, and a range of morphological and lifestyle adaptations that can modify the cost of sustained flight. Such behavioural and morphological adaptations can also influence the physiological costs associated with migration. For instance, during intense flight and catabolism of reserves, lipid damage induced by pro‐oxidants increases, and to keep oxidative physiological homeostasis under control, the antioxidant machinery can be upregulated. Studies on the oxidative physiology of endurance flight have produced contradictory results, making generalization difficult, especially because multispecies studies are missing. Therefore, to explore the oxidative cost of flight and migration, we used samples collected during the breeding season from 113 European bird species and explored the associations of measures of antioxidant capacity (total antioxidant status, uric acid and glutathione concentration) and oxidative damage of lipids (malondialdehyde) with variables reflecting flight energetics (year‐round or specifically during migration) using a phylogenetic framework. We found that none of the traits predicting year‐round flight energy expenditure (flight style, wing morphology and flight muscle morphology) explained any measures of oxidative state. Our results suggest that birds endure their everyday flight exercise without or with low oxidative cost. However, oxidative damage to lipids and one component of the endogenous antioxidant system (uric acid), measured after the end of spring migration on breeding adult birds, increased with migration distance. Our results suggest that migration could have oxidative consequences that might be carried over to subsequent life‐history stages (breeding).
... This question was primarily investigated experimentally using birds exposed to flight exercise (e.g. Costantini et al. 2008, Dick and Guglielmo 2019, McWilliams et al. 2020, and correlatively by comparing the physiological status of birds at different stages of migration (e.g. before migration or after recovery at stopover sites, e.g. ...
... If the energetic expense of flight induces the overproduction of ROS, we can hypothesize that traits associated with higher flight energy demand will be associated with elevated oxidative damage and/or will drive the evolution of more efficient antioxidant defences to prevent potential damages. Alternatively, physiological adaptations of birds to a life with high metabolic demands, including the resistance to oxidative stress, ROS production, metabolic management, the high density of mitochondria functioning as sink for ROS, or the composition of fatty acids of the energy reserve during migration may mitigate the oxidative cost of aerial locomotion across species (Hickey et al. 2012, McWilliams et al. 2020. ...
... We detected increased levels of UA in species with longer migration distances, suggesting that the role of this endogenous non-enzymatic antioxidant in oxidative defence increases with flight distance. Alternatively, because UA is a product of protein catabolism, our results could also indicate that longer migratory distances necessitate protein breakdown to fuel strenuous flights as compared with sedentary or short-distance migratory species (McWilliams et al. 2020). MDA also showed positive evolutionary covariation with migration distance, which indicates that the evolution of increased UA concentrations cannot neutralise the increased oxidative challenge faced by long-distance migrants, ultimately leading to lipid damage. ...
Flight can be highly-energy demanding, but its efficiency depends largely on flight style, wing shape and loading, and a range of morphological and lifestyle adaptations that can modify the cost of sustained flight. Such behavioural and morphological adaptations can also influence the physiological costs associated with migration. For instance, during intense flight and catabolism of reserves, lipid damage induced by pro-oxidants increases, and to keep oxidative physiological homeostasis under control, the antioxidant machinery is upregulated. Studies on the oxidative physiology of endurance flight have produced contradictory results, making generalization difficult, especially because multispecies studies are missing. Therefore, to explore the oxidative cost of flight and migration, we explored the association between three measures of the antioxidant capacity (total antioxidant status, uric acid and glutathione concentration) and one measure of oxidative damage of lipids (malondialdehyde) with variables reflecting flight energetics (year-round or specifically during migration) across 113 European bird species using a phylogenetic framework. We found that none of the traits predicting year-round flight energy expenditure, including flight style, wing morphology and flight muscle morphology explained any measures of oxidative state measured during the energy demanding breeding period, suggesting that birds endure their everyday exercise without or low oxidative cost. However, oxidative damage to lipids and one component of the endogenous antioxidant system (uric acid), measured after the end of spring migration on breeding adult birds, increased with migration distance. Our results suggest that migration might have oxidative consequences that are carried over to subsequent life history stages (breeding).
... Multiple studies have found that diet composition influences the fatty acid composition of songbird tissues and particularly the composition of essential or conditionally essential polyunsaturated fatty acids (PUFAs, contrast with monounsaturated fatty acids, or MUFAs; McCue et al., 2009;McWilliams et al., 2022;Pierce and McWilliams, 2014;Twining et al., 2016). Furthermore, related work has found that the content of certain n-6 PUFA in fat stores can influence metabolism in songbirds, including effects on peak metabolic rate Pierce et al., 2005;Price and Guglielmo, 2009) and sustained energy expenditure McWilliams et al., 2020). Together, these results suggest that certain dietary PUFA act as micronutrients, but evidence for how they affect metabolism remains inconclusive. ...
... This study took place as part of a broader experiment on the exercise physiology of songbirds and additional details on methods as well as results concerning whole-animal energetics and oxidative status can be found in McWilliams et al. (2020). All animal care procedures and experimental techniques were approved by the University of Rhode Island IACUC (Protocol #AN08-02-014) and the government of Upper Bavaria, Germany (AZ 55.2-1-54-2532(AZ 55.2-1-54- -216-2014. ...
... Polyunsaturated fats include essential or conditionally essential nutrients for vertebrates (Klasing, 1998;Stevens, 2004), and studies have consistently found that diet fatty acid composition strongly influences body PUFA composition in songbirds, McCue et al., 2009;McWilliams et al., 2020;Twining et al., 2016), closely matching trends in other taxa (Cherian et al., 2009;Russo, 2009;Toft et al., 2000). These changes in body composition have been linked to corresponding changes in eicosanoid signaling for mammals (Schmitz and Ecker, 2008;Zhou and Nilsson, 2001), as well as poultry (Cherian et al., 2009;Watkins, 1991), but until now those downstream effects have not been documented in non-domesticated birds. ...
... Thus, in a largemouth bass Micropterus salmoides short-term hypoxic stress (<40 min at 3-4 mg O 2 L −1 ) led to a significant upregulation of oxygen-and redox-sensitive transcription factors (Hypoxiainducible factor 1-alpha HIF-1α, Nuclear factor erythroid 2related factor 2 Nrf2, and kelch-1ike ECH-associated protein l Keap1) and elevated expression of catalase and glutathione peroxidase, but suppressed the expression of superoxide dismutase (Xin et al., 2022). The upregulation of antioxidants is considered important for prevention of the oxidative damage to proteins, lipids, and DNA during hypoxia (Hermes-Lima et al., 2015;Xin et al., 2022) but can incur significant energy cost to the organism (Hou and Amunugama, 2015;McWilliams et al., 2020). A need for the energy investment into the cellular antioxidant protection might conflict with the necessity to conserve energy during hypoxia in fish (Buck and Hochachka, 1993;Hochachka, 1995;Hou and Amunugama, 2015). ...
Oxygen fluctuations are common in freshwater habitats and aquaculture and can impact ecologically and economically important species of fish like cyprinids. To gain insight into the physiological responses to oxygen fluctuations in two common cyprinid species, we evaluated the impact of short-term intermittent hypoxia on oxidative stress and metabolic parameters (including levels of prooxidants and oxidative lesions, antioxidants, mitochondrial enzyme activities, mitochondrial swelling, markers of apoptosis, autophagy and cytotoxicity) in silver carp Hypophthalmichthys molitrix and gibel carp Carassius gibelio. During hypoxia, gibel carp showed higher baseline levels of antioxidants and less pronounced changes in oxidative and metabolic biomarkers in the tissues than silver carp. Reoxygenation led to a strong shift in metabolic and redox-related parameters and tissue damage, indicating high cost of post-hypoxic recovery in both species. Species-specific differences were more strongly associated with oxidative stress status, whereas metabolic indices and nitrosative stress parameters were more relevant to the response to hypoxia-reoxygenation. Overall, regulation of energy metabolism appears more critical than the regulation of antioxidants in the response to oxygen deprivation in the studied species, and further research is needed to establish whether prioritizing metabolic over redox regulation during H/R stress is common in freshwater cyprinids.
... Although starvation poses a significant risk to all animals (McCue 2010), fasting is a widespread phenomenon among various taxa. For example, hibernating mammals (such as polar bears, Robbins et al. 2012;ground squirrels, Krilowicz 1985;bats, Boyles et al. 2006), migratory birds (McWilliams et al. 2020), and estivating amphibians (Moreira et al. 2020) undergo long periods of fasting in the wild. Many teleost fishes also engage in fasting; for instance, cichlids fast during reproduction because of their mouth breeding behaviour (Faber-Hammond et al. 2019), Mekong giant catfish undergo periodical fasting during the wet season (Medo et al. 2020), and catadromous anguillid eels and anadromous salmonids fast during spawning migration from their feeding habitats (Rutter 1903;van Ginneken et al. 2005). ...
Although starvation poses a serious risk of death, it is a common phenomenon among anadromous salmonids that fast after returning to the river following oceanic feeding migration. To address the effects of geographic environmental factors on their feeding, we examined the river feeding patterns and condition of sea-run migrants of white-spotted char (Salvelinus leucomaenis) in rivers with latitudinal variation from 38°N to 46°N along the Sea of Japan. The river feeding patterns showed a significant latitudinal trend: char were observed to feed at lower latitudes and fast at higher latitudes. In contrast, the condition factor did not exhibit any latitudinal trends. These findings suggest that the environmental conditions encountered by individuals prior to and after river entry may influence their river feeding. Active feeding by the southern sea-run migrant char may be an adaptive strategy to maintain their body condition in response to the local conditions. This study highlights the importance of rivers not only as spawning and growth habitats for juveniles, but also as feeding habitats for certain anadromous salmonids.
... Findings from subsequent laboratory studies in which birds were fed specific diets provide more mixed findings about the relative roles of n-3 versus n-6 fatty acids as well as the exact mechanisms through which such fats might enhance flight performance (e.g. Dick & Guglielmo, 2019a, 2019bMcWilliams et al., 2020;Nagahuedi et al., 2009;Price, 2010;Price & Guglielmo, 2009). For instance, a recent study by McWilliams et al. (2020) found that during endurance flight, European starlings Sturnus vulgaris expend 11% less energy when fed diets containing more of the n-6 and n-3 PUFA LIN and ALA compared with diets containing more of the MUFA oleic acid (18:1n-9), but birds consuming more n-6 PUFA also experienced greater oxidative damage. ...
... Dick & Guglielmo, 2019a, 2019bMcWilliams et al., 2020;Nagahuedi et al., 2009;Price, 2010;Price & Guglielmo, 2009). For instance, a recent study by McWilliams et al. (2020) found that during endurance flight, European starlings Sturnus vulgaris expend 11% less energy when fed diets containing more of the n-6 and n-3 PUFA LIN and ALA compared with diets containing more of the MUFA oleic acid (18:1n-9), but birds consuming more n-6 PUFA also experienced greater oxidative damage. In contrast, Dick and Guglielmo recently found that dietary n-3 altered muscle phospholipid fatty acid composition and decreased muscle oxidative capacity in yellow-rumped Warblers Setophaga coronata but that dietary PUFA content and composition (i.e. ...
... PUFAs are thought to be particularly efficient fuels for migratory flight in songbirds (e.g. McWilliams et al., 2020) and because migratory blackbirds have been shown to have higher levels of PUFA compared with residents (Jensen et al., 2020). Therefore, we expected that birds from migratory populations would vary more in PUFA (i.e. ...
Seasonal migration is a physiologically demanding endeavour that animals prepare for by finding and storing energy. Species and populations vary in their tendencies to migrate and the energetic demands of migration likely determine the degree and type of preparation that different animals undergo.
Birds fuel their migratory flight using stored fat. Previous work has suggested that in addition to being energetically dense, certain lipids like polyunsaturated fatty acids (PUFAs) may be particularly efficient fuels that are preferentially mobilized for flight or serve may additional functions, such as modulating membrane fluidity or stimulating gene expression as ligands, during long‐distance migratory flight.
Using a series of behavioural and metabolic experiments in a partial transplant aviary setting of Common Blackbirds (Turdus merula) populations that range from being fully migratory to partially migratory to sedentary, we asked how migratory restlessness (Zugunruhe), weight gain and oxidation of stearic acid (18:0), oleic acid (18:1n‐9), linoleic acid (18:2n‐6) and alpha linolenic acid (18:3n‐3; ALA) varied with migratory mode and season.
We found no population‐level differences in the timing of Zugunruhe. However, before migration, a greater proportion of individuals from fully migratory populations exhibited weight gain and migratory restlessness, as well as increased oxidation of PUFAs. After Zugunruhe, only individuals from migratory populations showed reduced PUFA oxidation. All populations showed increased oxidation of saturated fat after Zugunruhe.
Our results demonstrate that certain migratory traits, like the timing of Zugunruhe are expressed similarly across populations in response to local environmental conditions, while other traits, like lipid oxidation, can vary among populations.
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... Furthermore, if this were simply a way to save weight in flight, it is unlikely that we would see a similar pattern in the resting (and fasting) birds unless it is a predictive adjustment when birds are in migratory condition as seen in ref. 53, which could be tested by measuring rates of protein catabolism in the same species in nonmigratory condition. This would be particularly interesting if rapid protein breakdown at the start of flight or fasting provides an influx of uric acid as an antioxidant in the migratory condition (55,56). By showing a reduction in the protein contribution to flight fuel over time, we also show that the protein loss, at least initially in flight, is unlikely to be solely an iterative adjustment to fuel loads throughout flight (16,26). ...
During migration, long-distance migratory songbirds may fly nonstop for days, whereas shorter-distance migrants complete flights of 6 to 10 h. Fat is the primary fuel source, but protein is also assumed to provide a low, consistent amount of energy for flight. However, little is known about how the use of these fuel sources differs among bird species and in response to flight duration. Current models predict that birds can fly until fat stores are exhausted, with little consideration of protein's limits on flight range or duration. We captured two related migratory species-ultra long-distance blackpoll warblers (Setophaga striata) and short-distance yellow-rumped warblers (Setophaga coronata)-during fall migration and flew them in a wind tunnel to examine differences in energy expenditure, overall fuel use, and fuel mixture. We measured fat and fat-free body mass before and after flight using quantitative magnetic resonance and calculated energy expenditure from body composition changes and doubly labeled water. Three blackpolls flew voluntarily for up to 28 h-the longest wind tunnel flight to date-and ended flights with substantial fat reserves but concave flight muscle, indicating that protein loss, rather than fat, may actually limit flight duration. Interestingly, while blackpolls had significantly lower mass-specific metabolic power in flight than that of yellow-rumped warblers and fuel use was remarkably similar in both species, with consistent fat use but exceptionally high rates of protein loss at the start of flight that declined exponentially over time. This suggests that protein may be a critical, dynamic, and often overlooked fuel for long-distance migratory birds.
... Flights were Dzialo et al. Frontiers in Zoology (2023) 20:9 performed during natural spring migration season of starlings living in Germany, in an advanced research wind tunnel that enables precise control of steady flight conditions [32,72] and has been successfully used in numerous flight-related studies on birds [20,32,73]. We predicted that starlings flown each day for several weeks would have reduced red blood cell number, lower haematocrit and haemoglobin content, and smaller cell size compared to more sedentary starlings. ...
... We acknowledge that the unclear flightinduced effects may be due to the potentially mild nature of the exercise, which in turn may not be sufficient to induce oxidative stress and haematological changes in starlings. However, using similar flight training regimens, previous studies have successfully simulated the exercise nature of migratory flights and its effects on bird physiology [20,32,76] including the effects on oxidative status [71,73]. Worth noting, the flying group in our study was exposed to an additional 600-700 km of continuous flapping flight compared to non-flying birds, which likely increases the risk of oxidative damage compared to sedentary birds, although it may not have been sufficient to induce acute effects on haematological variables, such as typically associated with strenuous activities. ...
... Once hatchlings were able to feed independently, we additionally offered them live mealworms, fresh fruits and vegetables. The diet has been successfully used to hand-raise starlings at the MPIO in previous experiments [32,73,130]. From about the age of 35-100 days, the starlings were moved to outdoor aviaries, kept under a natural light cycle, and maintained on an MPIO diet that consisted of insect powder, lettuce, fresh apples and oranges, dried fruit pellets, and mealworms. ...
Background
Endurance flight impose substantial oxidative costs on the avian oxygen delivery system. In particular, the accumulation of irreversible damage in red blood cells can reduce the capacity of blood to transport oxygen and limit aerobic performance. Many songbirds consume large amounts of anthocyanin-rich fruit, which is hypothesized to reduce oxidative costs, enhance post-flight regeneration, and enable greater aerobic capacity. While their antioxidant benefits appear most straightforward, the effects of anthocyanins on blood composition remain so far unknown. We fed thirty hand-raised European starlings (Sturnus vulgaris) two semisynthetic diets (with or without anthocyanin supplement) and manipulated the extent of flight activity in a wind tunnel (daily flying or non-flying for over two weeks) to test for their interactive effects on functionally important haematological variables.
Results
Supplemented birds had on average 15% more and 4% smaller red blood cells compared to non-supplemented individuals and these diet effects were independent of flight manipulation. Haemoglobin content was 7% higher in non-supplemented flying birds compared to non-flying birds, while similar haemoglobin content was observed among supplemented birds that were flown or not. Neither diet nor flight activity influenced haematocrit.
Conclusion
The concerted adjustments suggest that supplementation generally improved antioxidant protection in blood, which could prevent the excess removal of cells from the bloodstream and may have several implications on the oxygen delivery system, including improved gas exchange and blood flow. The flexible haematological response to dietary anthocyanins may also suggest that free-ranging species preferentially consume anthocyanin-rich fruits for their natural blood doping, oxygen delivery-enhancement effects.
... The wildlife profession has a long history of raising wildlife in captivity for various research and educational purposes including rehabilitation (Escobedo-Bonilla et al. 2022), supplementing wild populations (Biggins et al. 1998), disease research (Rhyan et al. 2020), nutrition and energetic studies (McWilliams et al. 2020), and outreach (Learmonth et al. 2021). Knowledge of nutritional requirements and physiological metrics necessary to assess animal welfare are foundational to raising captive wildlife species successfully. ...
Moose (Alces alces) have been raised in captivity for research and educational purposes for decades. Past research has focused mostly on milk replacer diets to produce healthy calves, with limited research of vital signs associated with routine health checks of young animals. We hand-raised 20 calves in 4 cohorts (2009, 2012, 2019, 2021) using commercially available milk replacers mixed with water only, and measured vital signs of 11 calves in 2019 and 2021. Growth rate from birth through weaning was 0.98 ± 0.02 kg • d-1 , with maximum growth rate of ~1.3 kg • d-1 sustained for 6 weeks after weaning was initiated. Heart rate declined with age from 103.5 ± 2.6 beats • min-1 at 5 days old to 81.6 beats at 80 days old, whereas respiration rate increased from 16.3 ± 2.5 to 36.7 ± 4.4 breaths • min-1. Respiration rate increased with ambient air temperature from 11.1 ± 2.9 breaths • min-1 at 9 °C to 45.2 ± 3.2 at 26 °C. Respiration rate was highly variable after 3-week old calves began daily walks in a larger enclosure and ambient air temperature increased towards the summer maxima (July). Mean rectal temperature was 38.5 ± 0.03°C, and declined marginally with increasing vapor pressure and wind speed. Our hand-raising protocol and milk replacer diets produced calf growth rates higher than those reported previously, and similar to dam-raised calves consuming pelleted ration and available grass forage.
