Dietary fatty acids and flight-training influence the expression of the eicosanoid hormone prostacyclin in songbirds.

Diet shifts can alter tissue fatty acid composition in birds, which is subsequently related to metabolic patterns. Eicosanoids, short-lived fatty acid-derived hormones, have been proposed to mediate these relationships but neither baseline concentrations nor the responses to diet and exercise have been measured in songbirds. We quantified a stable derivative of the vasodilatory eicosanoid prostacyclin in the plasma of male European Starlings (Sturnus vulgaris, N = 25) fed semisynthetic diets with either high (PUFA) or low (MUFA) amounts of n6 fatty acid precursors to prostacyclin. Plasma samples were taken from each bird before, immediately after, and two days following a 15-day flight-training regimen that a subset of birds (N = 17) underwent. We found elevated prostacyclin levels in flight-trained birds fed the PUFA diet compared to those fed the MUFA diet and a positive relationship between prostacyclin and body condition, indexed by fat score. Prostacyclin concentrations also significantly decreased at the final time point. These results are consistent with the proposed influences of precursor availability (i.e., dietary fatty acids) and regulatory feedback associated with exercise (i.e., fuel supply and inflammation), and suggest that prostacyclin may be an important mediator of dietary influence on songbird physiology.

Concerted phenotypic flexibility of avian erythrocyte size and number in response to dietary anthocyanin supplementation.

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.

Measurements of body temperature and oxidative stress reveal differential costs associated with humoral immune function in a passerine bird.

Eco-immunology considers resistance to antigens a costly trait for an organism, but actual quantification of such costs is not straightforward. Costs of the immune response are visible in impaired coloration and reduced growth or reproductive success. Activation of the humoral immune response is a slow, complex and long-lasting process, which makes the quantification of its energetic cost a potential losing game. We implemented near-continuous measurements of body temperature in zebra finches (Taeniopygia guttata) as a proxy for the energetic cost, with a particular focus during activation of the humoral immune response until the peak of antibody release several days later. At the peak of the antibody release we additionally measured oxygen consumption (open-flow respirometry) and markers of oxidative stress (dROMs, OXY). Birds with an activated immune response maintained a higher night-time body temperature during the first 4 nights after an immune challenge in comparison to controls, implying increased night-time energy use. At peak antibody production, we did not find differences in night-time body temperature and oxygen consumption but observed differentiated results for oxygen consumption during the day. Immune-challenged females had significantly higher oxygen consumption compared with other groups. Moreover, we found that activation of the humoral immune response increases oxidative damage, a potential cost of maintaining the higher night-time body temperature that is crucial at the early stage of the immune response. The costs generated by the immune system appear to consist of two components - energetic and non-energetic - and these appear to be separated in time.

The effects of training, acute exercise and dietary fatty acid composition on muscle lipid oxidative capacity in European starlings.

Migratory birds undergo seasonal changes to muscle biochemistry. Nonetheless, it is unclear to what extent these changes are attributable to the exercise of flight itself versus endogenous changes. Using starlings (Sturnus vulgaris) flying in a wind tunnel, we tested the effects of exercise training, a single bout of flight and dietary lipid composition on pectoralis muscle oxidative enzymes and lipid transporters. Starlings were either unexercised or trained over 2 weeks to fly in a wind tunnel and sampled either immediately following a long flight at the end of this training or after 2 days recovery from this flight. Additionally, they were divided into dietary groups that differed in dietary fatty acid composition (high polyunsaturates versus high monounsaturates) and amount of dietary antioxidant. Trained starlings had elevated (19%) carnitine palmitoyl transferase and elevated (11%) hydroxyacyl-CoA dehydrogenase in pectoralis muscle compared with unexercised controls, but training alone had little effect on lipid transporters. Immediately following a long wind-tunnel flight, starling pectoralis had upregulated lipid transporter mRNA (heart-type fatty acid binding protein, H-FABP, 4.7-fold; fatty acid translocase, 1.9-fold; plasma membrane fatty acid binding protein, 1.6-fold), and upregulated H-FABP protein (68%). Dietary fatty acid composition and the amount of dietary antioxidants had no effect on muscle catabolic enzymes or lipid transporter expression. Our results demonstrate that birds undergo rapid upregulation of catabolic capacity that largely becomes available during flight itself, with minor effects due to training. These effects likely combine with endogenous seasonal changes to create the migratory phenotype observed in the wild.

Flight training and dietary antioxidants have mixed effects on the oxidative status of multiple tissues in a female migratory songbird.

Birds, like other vertebrates, rely on a robust antioxidant system to protect themselves against oxidative imbalance caused by energy-intensive activities such as flying. Such oxidative challenges may be especially acute for females during spring migration, as they must pay the oxidative costs of flight while preparing for reproduction; however, little previous work has examined how the antioxidant system of female spring migrants responds to dietary antioxidants and the oxidative challenges of regular flying. We fed two diets to female European starlings, one supplemented with a dietary antioxidant and one without, and then flew them daily in a windtunnel for 2 weeks during the autumn and spring migration periods. We measured the activity of enzymatic antioxidants (glutathione peroxidase, superoxide dismutase and catalase), non-enzymatic antioxidant capacity (ORAC) and markers of oxidative damage (protein carbonyls and lipid hydroperoxides) in four tissues: pectoralis, leg muscle, liver and heart. Dietary antioxidants affected enzymatic antioxidant activity and lipid damage in the heart, non-enzymatic antioxidant capacity in the pectoralis, and protein damage in leg muscle. In general, birds not fed the antioxidant supplement appeared to incur increased oxidative damage while upregulating non-enzymatic and enzymatic antioxidant activity, though these effects were strongly tissue specific. We also found trends for diet×training interactions for enzymatic antioxidant activity in the heart and leg muscle. Flight training may condition the antioxidant system of females to dynamically respond to oxidative challenges, and females during spring migration may shift antioxidant allocation to reduce oxidative damage.

Dietary antioxidants attenuate the endocrine stress response during long-duration flight of a migratory bird.

Glucocorticoids (GCs) are metabolic hormones that promote catabolic processes, which release stored energy and support high metabolic demands such as during prolonged flights of migrating birds. Dietary antioxidants (e.g. anthocyanins) support metabolism by quenching excess reactive oxygen species produced during aerobic metabolism and also by activating specific metabolic pathways. For example, similar to GCs' function, anthocyanins promote the release of stored energy, although the extent of complementarity between GCs and dietary antioxidants is not well known. If anthocyanins complement GCs functions, birds consuming anthocyanin-rich food can be expected to limit the secretion of GCs when coping with a metabolically challenging activity, avoiding the exposure to potential hormonal detrimental effects. We tested this hypothesis in European starlings (Sturnus vulgaris) flying in a wind tunnel. We compared levels of corticosterone, the main avian GC, immediately after a sustained flight and at rest for birds that were fed diets with or without an anthocyanin supplement. As predicted, we found (i) higher corticosterone after flight than at rest in both diet groups and (ii) anthocyanin-supplemented birds had less elevated corticosterone after flight than unsupplemented control birds. This provides novel evidence that dietary antioxidants attenuate the activation of the HPA axis (i.e. increased secretion of corticosterone) during long-duration flight.

Does selection for behavioral and physiological performance traits alter glucocorticoid responsiveness in bank voles?

One of the key elements of an animal's Darwinian fitness is its ability to adequately respond to and cope with challenging situations. Glucocorticoid hormones, such as corticosterone, affect an organism's ability to overcome such challenges. We hypothesized that changes in the glucocorticoid response curve contribute to the evolution of increased performance during challenging conditions, and tested it on bank voles (Myodes glareolus) from a multidirectional artificial selection experiment, which involves lines selected for high aerobic exercise metabolism achieved during swimming (A - Aerobic), predatory behavior towards a cricket (P - Predatory) and ability to maintain body mass on a low-quality herbivorous diet (H - Herbivorous), as well as unselected control lines (C - Control). We elicited a glucocorticoid response either by restraining the animal or by maximum pharmacological stimulation, and measured plasma corticosterone levels at baseline, during the response and during the recovery phase. Response-level corticosterone was higher in females, and recovery from maximal level was faster than that of males. Selection did not affect baseline or stress-induced corticosterone levels, but it decreased the maximum corticosterone level in Aerobic and Predatory lines, reducing the difference between stress-induced and maximum levels. Recovery from restraint-induced corticosterone level tended to be slower in the Herbivorous than in the other lines, an effect that was stronger in females than in males. In conclusion, successful selection for increased performance in challenging conditions was not associated with changes in absolute values of the glucocorticoid response to stress, but can affect other characteristics of the glucocorticoid response curve.

Stress coping and evolution of aerobic exercise performance: corticosterone levels in voles from a selection experiment.

The locomotor performance achieved in a challenging situation depends not only on physiological limitations, such as the aerobic exercise capacity, but also on behavioral characteristics, such as adequate coping with stress. The stress response is mediated largely by the hypothalamic-pituitary-adrenal (HPA) axis, through modulated release of glucocorticoids. We used a unique experimental evolution model system to test the hypothesis that the evolution of an increased aerobic exercise performance can be facilitated by modification of the glucocorticoid-related stress-coping mechanisms. Bank voles (Myodes glareolus) from 'aerobic' (A) lines, selected for 22 generations for high maximum swim-induced rate of oxygen consumption (V̇O2,swim), achieved a 64% higher V̇O2,swim than those from unselected, control lines. The temporal pattern of exercise during the swimming trial also evolved, and the A-line voles achieved V̇O2,swim later in the course of the trial, which indicates a modification of the stress response characteristics. Both V̇O2,swim and the average metabolic rate measured during the trial tended to increase with baseline corticosterone level, and decreased with the post-exercise corticosterone level. Thus, increased baseline corticosterone level promotes high metabolic performance, but a high corticosterone response to swimming acts as an inhibitor rather than stimulator of intense activity. However, neither of the corticosterone traits differed between the A-selected and control lines. Thus, the experiment did not provide evidence that evolution of increased aerobic performance is facilitated by the modification of glucocorticoid levels. The results, however, do not exclude the possibility that other aspects of the HPA axis function evolved in response to the selection.

Contrasting response of haematological variables between long-term training and short exercise bouts in zebra finches (Taeniopygia guttata).

Physical aerobic activity is oxygen demanding, but - particularly for birds - there is still little understanding of how blood contributes to oxygen supply under various activity levels. In a two-factorial experimental design, we investigated the long-term effect of daily flight training and the immediate effect of a short exercise bout on a set of haematological variables: haemoglobin (Hb) content, haematocrit (Hct), and red blood cell number (RBCcount) and size (RBCarea) in zebra finches (Taeniopygia guttata). For a period of 6 weeks, birds were either trained daily for 3 h in a flight arena or remained untrained. Subsequently, half of each group was blood sampled either in the resting condition or after a 5 min exercise bout in a flight-hover wheel. We found significantly lower Hb content, Hct and RBCcount compared with that in untrained controls in response to training, while RBCarea did not differ between treatments. Response to an exercise bout revealed the opposite pattern, with significantly higher Hb content and Hct compared with that in non-exercised birds. Additionally, RBCarea was significantly smaller immediately after exercise compared with that in non-exercised birds, and such short-term flexibility represents a novel finding for birds. This contrasting response in erythrocyte characteristics with respect to long-term training and short exercise bouts appears as a clear pattern, presumably underlain by changes in water balance. We infer alterations of blood flow to be involved in adequate oxygen supply. During an exercise bout, RBCarea flexibility may not only enhance oxygen delivery through improved erythrocyte surface area to volume ratio but also improve blood flow through a compensatory effect on blood viscosity.

More than just the numbers-contrasting response of snake erythrocytes to thermal acclimation.

Acclimation to lower temperatures decreases energy expenditure in ectotherms but increases oxygen consumption in most endotherms, when dropped below thermoneutrality. Such differences should be met by adjustments in oxygen transport through blood. Changes in hematological variables in correspondence to that in metabolic rates are, however, not fully understood, particularly in non-avian reptiles. We investigated the effect of thermal acclimation on a snake model, the grass snakes (Natrix natrix). After 6 months of acclimation to either 18 °C or 32 °C hematocrit, hemoglobin concentration, erythrocyte number, and size were assessed. All variables revealed significantly lower values under warm compared to cold ambient temperature. Our data suggest that non-avian reptiles, similarly as birds, reduce erythrocyte fraction under energy-demanding temperatures. Due to low deformability of nucleated erythrocytes in sauropsids, such reduced fraction may be important in decreasing blood viscosity to optimize blood flow. Novel findings on flexible erythrocyte size provide an important contribution to this optimization process.

Vanishing benefits - The loss of actinobacterial symbionts at elevated temperatures.

Only a few insect species are known to engage in symbiotic associations with antibiotic-producing Actinobacteria and profit from this kind of protection against pathogens. However, it still remains elusive how widespread the symbiotic interactions with Actinobacteria in other organisms are and how these partnerships benefit the hosts in terms of the growth and survival. We characterized a drastic temperature-induced change in the occurrence of Actinobacteria in the gut of the terrestrial isopod Porcellio scaber reared under two different temperature (15 °C and 22 °C) and oxygen conditions (10% and 22% O2) using 16S rRNA gene sequencing. We show that the relative abundance of actinobacterial gut symbionts correlates with increased host growth at lower temperature. Actinobacterial symbionts were almost completely absent at 22 °C under both high and low oxygen conditions. In addition, we identified members of nearly half of the known actinobacterial families in the isopod microbiome, and most of these include members that are known to produce antibiotics. Our study suggests that hosting diverse actinobacterial symbionts may provide conditions favorable for host growth. These findings show how a temperature-driven decline in microbiome diversity may cause a loss of beneficial functions with negative effects on ectotherms.

Environmental cues and dietary antioxidants affect breeding behavior and testosterone of male European starlings (Sturnus vulgaris).

Environmental cues, such as photoperiod, regulate the timing of major life-history events like breeding through direct neuroendocrine control. Less known is how supplementary environmental cues (e.g., nest sites, food availability) interact to influence key hormones and behaviors involved in reproduction, specifically in migratory species with gonadal recrudescence largely occurring at breeding sites. We investigated the behavioral and physiological responses of male European starlings to the sequential addition of nest boxes and nesting material, green herbs, and female conspecifics and how these responses depend on the availability of certain antioxidants (anthocyanins) in the diet. As expected, cloacal protuberance volume and plasma testosterone of males generally increased with photoperiod. More notably, testosterone levels peaked in males fed the high antioxidant diet when both nest box and herbal cues were present, while males fed the low antioxidant diet showed no or only a muted testosterone response to the sequential addition of these environmental cues; thus our results are in agreement with the oxidation handicap hypothesis. Males fed the high antioxidant diet maintained a constant frequency of breeding behaviors over time, whereas those fed the low antioxidant diet decreased breeding behaviors as environmental cues were sequentially added. Overall, sequential addition of the environmental cues modulated physiological and behavioral measures of reproductive condition, and dietary antioxidants were shown to be a key factor in affecting the degree of response to each of these cues. Our results highlight the importance of supplementary environmental cues and key resources such as dietary antioxidants in enhancing breeding condition of males, which conceivably aid in attraction of high quality females and reproductive success.

High oxidative stress despite low energy metabolism and vice versa: Insights through temperature acclimation in an ectotherm.

Reactive oxygen species (ROS) are inescapable byproducts of energy metabolism and may cause costly damage to biomolecules. Organisms have evolved different means to counter oxidative stress, such as modulation of ROS production, neutralization of produced ROS through free radical scavenging and the repair or removal of the damaged structures. A positive relationship between metabolic rate and ROS production is commonly expected, but the oxidative burden of aerobic metabolism remains poorly understood. We investigated whether acclimation to ambient temperatures imposing variation in standard metabolic rate (SMR) is mirrored in the oxidative status of an ectotherm. Grass snakes (Natrix natrix) acclimated for six months to warm (32 °C) conditions revealed seven times higher SMR compared to cold-acclimated (18 °C) individuals. In contrast to SMR, the amount of damage measured as reactive oxygen metabolites test (dROMs) and abundance of micronucleated erythrocytes was significantly lower in warm-acclimated individuals, while non-enzymatic antioxidant capacity of plasma was unaltered by thermal acclimation. Our results support that high SMR may allow snakes to better cope with oxidative stress, possibly through tissue repair or removal of damaged tissues that also requires energy costs. The reversed association between self-maintenance metabolism and oxidative damage to biomolecules provides novel rational for temperature dependent life histories of ectotherms. How oxidative stress may contribute to the known reduced rates of ectotherm growth or reproduction under cold temperatures or if oxidative stress may even drive such life history trait declines are now important challenges to be addressed.

An evolutionary solution of terrestrial isopods to cope with low atmospheric oxygen levels.

The evolution of current terrestrial life was founded by major waves of land invasion coinciding with high atmospheric oxygen content. These waves were followed by periods with substantially reduced oxygen concentration and accompanied by the evolution of novel traits. Reproduction and development are limiting factors for evolutionary water-land transitions, and brood care has probably facilitated land invasion. Peracarid crustaceans provide parental care for their offspring by brooding the early stages within the motherly brood pouch, the marsupium. Terrestrial isopod progeny begin ontogenetic development within the marsupium in water, but conclude development within the marsupium in air. Our results for progeny growth until hatching from the marsupium provide evidence for the limiting effects of oxygen concentration and for a potentially adaptive solution. Inclusion of air within the marsupium compensates for initially constrained growth in water through catch-up growth, and it may explain how terrestrial isopods adapted to short- and long-term changes in oxygen concentration.

Mass scaling of metabolic rates in carabid beetles (Carabidae) - the importance of phylogeny, regression models and gas exchange patterns.

The origin of the allometric relationship between standard metabolic rate (MR) and body mass (M), often described as MR=aMb , remains puzzling, and interpretation of the mass-scaling exponent, b may depend on the methodological approach, shapes of residuals, coefficient of determination (r2) and sample size. We investigated the mass scaling of MRs within and between species of Carabidae beetles. We used ordinary least squares (OLS) regression, phylogenetically generalized least squares (PGLS) regression and standardized major axis (SMA) regression to explore the effects of different model-fitting methods and data clustering caused by phylogenetic clades (grade shift) and gas exchange patterns (discontinuous, cyclic and continuous). At the interspecific level, the relationship between MR and M was either negatively allometric (b<1) or isometric (b=1), depending on the fitting method. At the intraspecific level, the relationship either did not exist or was isometric or positively allometric (b>1), and the fit was significantly improved after the analyzed dataset was split according to gas exchange patterns. The studied species originated from two distinct phylogenetic clades that had different intercepts but a common scaling exponent (OLS, 0.61) that was much shallower than the scaling exponent for the combined dataset for all species (OLS, 0.71). The best scaling exponent estimates were obtained by applying OLS while accounting for grade shifts or by applying PGLS. Overall, we show that allometry of MR in insects can depend heavily on the model fitting method, the structure of phylogenetic non-independence and ecological factors that elicit different modes of gas exchange.

Physical mechanism or evolutionary trade-off? Factors dictating the relationship between metabolic rate and ambient temperature in carabid beetles.

The tight association between ambient temperature (T) and metabolic rate (MR) is a common occurrence in ectotherms, but the determinants of this association are not fully understood. This study examined whether the relationship between MR and T is the same among individuals, as predicted by the Universal Temperature Dependence hypothesis, or whether this relationship differs between them. We used flow-through respirometry to measure standard MR and to determine gas exchange patterns for 111 individuals of three Carabidae species which differ in size (Abax ovalis, Carabus linnei and C. coriaceus), exposed to four different temperatures (ten individuals of each species measured at 6, 11, 16 and 21°C). We found a significant interaction between ln body mass and the inverse of temperature, indicating that in a given species, the effect of temperature on MR was weaker in larger individuals than in smaller individuals. Overall, this finding shows that the thermal dependence of MR is not body mass invariant. We observed three types of gas exchange patterns among beetles: discontinuous, cyclic and continuous. Additionally, the appearance of these patterns was associated with MR and T. Evolution in diverse terrestrial environments could affect diverse ventilation patterns, which accommodate changes in metabolism in response to temperature variation. In conclusion, explaining the variance in metabolism only through fundamental physical laws of thermodynamics, as predicted by the Universal Temperature Dependence hypothesis, appears to oversimplify the complexity of nature, ignoring evolutionary trade-offs that should be taken into account in the temperature - metabolism relationship.

Increased fat catabolism sustains water balance during fasting in zebra finches.

Patterns of physiological flexibility in response to fasting are well established, but much less is known about the contribution of water deprivation to the observed effects. We investigated body composition and energy and water budget in three groups of zebra finches: birds with access to food and water, food-deprived birds having access to drinking water and food-and-water-deprived birds. Animals were not stimulated by elevated energy expenditure and they were in thermoneutral conditions; thus, based on previous studies, water balance of fasting birds was expected to be maintained by increased catabolism of proteins. In contrast to this expectation, we found that access to water did not prevent reduction of proteinaceous tissue, but it saved fat reserves of the fasting birds. Thus, water balance of birds fasting without access to water seemed to be maintained by elevated fat catabolism, which generated 6 times more metabolic water compared with that in birds that had access to water. Therefore, we revise currently established views and propose fat to serve as the primary source for metabolic water production. Previously assumed increased protein breakdown for maintenance of water budget would occur if fat stores were depleted or if fat catabolism reached its upper limits due to high energy demands.

Site-specific regulation of adult neurogenesis by dietary fatty acid content, vitamin E and flight exercise in European starlings.

Exercise is known to have a strong effect on neuroproliferation in mammals ranging from rodents to humans. Recent studies have also shown that fatty acids and other dietary supplements can cause an upregulation of neurogenesis. It is not known, however, how exercise and diet interact in their effects on adult neurogenesis. We examined neuronal recruitment in multiple telencephalic sites in adult male European starlings (Sturnus vulgaris) exposed to a factorial combination of flight exercise, dietary fatty acids and antioxidants. Experimental birds were flown in a wind tunnel following a training regime that mimicked the bird's natural flight behaviour. In addition to flight exercise, we manipulated the composition of dietary fatty acids and the level of enrichment with vitamin E, an antioxidant reported to enhance neuronal recruitment. We found that all three factors - flight exercise, fatty acid composition and vitamin E enrichment - regulate neuronal recruitment in a site-specific manner. We also found a robust interaction between flight training and vitamin E enrichment at multiple sites of neuronal recruitment. Specifically, flight training was found to enhance neuronal recruitment across the telencephalon, but only in birds fed a diet with a low level of vitamin E. Conversely, dietary enrichment with vitamin E upregulated neuronal recruitment, but only in birds not flown in the wind tunnel. These findings indicate conserved modulation of adult neurogenesis by exercise and diet across vertebrate taxa and indicate possible therapeutic interventions in disorders characterized by reduced adult neurogenesis.

Repeat Sampling of Female Passerines During Reproduction Reveals Surprising Higher Plasma Oxidative Damage During Resting Compared to Active State.

Traditional models of oxidative stress predict accumulation of damage caused by reactive oxygen species (ROS) production as highly correlated with aerobic metabolism, a prediction under increasing scrutiny. Here, we repeat sampled female great tits (Parus major) at two opposite levels of energy use during the period of maximum food provisioning to nestlings, once at rest and once during activity. Our results were in contrast to the above prediction, namely significantly higher levels of oxidative damage during rest opposed to active phase. This discrepancy could not be explained neither using levels of "first line" antioxidant enzymes activity measured from erythrocytes, nor from total nonenzymatic antioxidant capacity measured from plasma, as no differences were found between states. Significantly higher levels of uric acid, a potent antioxidant, were seen in the plasma during the active phase than in rest phase, which may explain the lower levels of oxidative damage despite high levels of physical activity. Our results challenge the hypothesis that oxidative stress is elevated during times with high energy use and call for more profound understanding of potential drivers of the modulation of oxidative stress such as metabolic state of the animal, and thus also the time of sampling in general.

Mitochondrial metabolism in blood more reliably predicts whole-animal energy needs compared to other tissues.

Understanding energy metabolism in free-ranging animals is crucial for ecological studies. In birds, red blood cells (RBCs) offer a minimally invasive method to estimate metabolic rate (MR). In this study with European starlings Sturnus vulgaris, we examined how RBC oxygen consumption relates to oxygen use in key tissues (brain, liver, heart, and pectoral muscle) and versus the whole organism measured at basal levels. The pectoral muscle accounted for 34%-42% of organismal MR, while the heart and liver, despite their high mass-specific metabolic rate, each contributed 2.5%-3.0% to organismal MR. Despite its low contribution to organismal MR (0.03%-0.04%), RBC MR best predicted organismal MR (r = 0.70). Oxygen consumption of the brain and pectoralis was also associated with whole-organism MR, unlike that of heart and liver. Overall, our findings demonstrate that the metabolism of a systemic tissue like blood is a superior proxy for organismal energy metabolism than that of other tissues.