The effects of diet-shifting from invertebrates towards fruit on the condition of autumn-migrant Catharus thrushes.

Migration is an energetically challenging and risky life history stage for many animals, but could be supported by dietary choices en route, which may create opportunities to improve body and physiological condition. However, proposed benefits of diet shifts, such as between seasonally available invertebrates and fruits, have received limited investigation in free-living animals. We quantified diet composition and magnitude of autumn diet shifts over two time periods in two closely-related species of migratory songbirds on stopover in the northeastern U.S. (Swainson's thrush [Catharus ustulatus], long-distance migrant, N = 83; hermit thrush [C. guttatus], short-distance migrant, N = 79) and used piecewise structural equation models to evaluate the relationships among (1) migration timing, (2) dietary behavior, and (3) morphometric and physiological condition indices. Tissue isotope composition indicated that both species shifted towards greater fruit consumption. Larger shifts in recent weeks corresponded to higher body condition in Swainson's, but not hermit thrushes, and condition was more heavily influenced by capture date in Swainson's thrushes. Presence of "high-antioxidant" fruits in fecal samples was unrelated to condition in Swainson's thrushes and negatively related to multiple condition indices in hermit thrushes, possibly indicating the value of fruits during migration is related more to their energy and/or macronutrient content than antioxidant content. Our results suggest that increased frugivory during autumn migration can support condition, but those benefits might depend on migration strategy: a longer-distance, more capital-dependent migration strategy could require stricter regulation of body condition aided by increased fruit consumption.

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.

Flight and Dietary Antioxidants Influence Antioxidant Expression and Activity in a Migratory Bird.

Ecologically relevant factors such as exercise and diet quality can directly influence how physiological systems work including those involved in maintaining oxidative balance; however, to our knowledge, no studies to date have focused on how such factors directly affect expression of key components of the endogenous antioxidant system (i.e., transcription factors, select antioxidant genes, and corresponding antioxidant enzymes) in several metabolically active tissues of a migratory songbird. We conducted a three-factor experiment that tested the following hypotheses: (H1) Daily flying over several weeks increases the expression of transcription factors NRF2 and PPARs as well as endogenous antioxidant genes (i.e., CAT, SOD1, SOD2, GPX1, GPX4), and upregulates endogenous antioxidant enzyme activities (i.e., CAT, SOD, GPx). (H2) Songbirds fed diets composed of more 18:2n-6 PUFA are more susceptible to oxidative damage and thus upregulate their endogenous antioxidant system compared with when fed diets with less PUFA. (H3) Songbirds fed dietary anthocyanins gain additional antioxidant protection and thus upregulate their endogenous antioxidant system less compared with songbirds not fed anthocyanins. Flight training increased the expression of 3 of the 6 antioxidant genes and transcription factors measured in the liver, consistent with H1, but for only one gene (SOD2) in the pectoralis. Dietary fat quality had no effect on antioxidant pathways (H2), whereas dietary anthocyanins increased the expression of select antioxidant enzymes in the pectoralis, but not in the liver (H3). These tissue-specific differences in response to flying and dietary antioxidants are likely explained by functional differences between tissues as well as fundamental differences in their turnover rates. The consumption of dietary antioxidants along with regular flying enables birds during migration to stimulate the expression of genes involved in antioxidant protection likely through increasing the transcriptional activity of NRF2 and PPARs, and thereby demonstrates for the first time that these relevant ecological factors affect the regulation of key antioxidant pathways in wild birds. What remains to be demonstrated is how the extent of these ecological factors (i.e., intensity or duration of flight, amounts of dietary antioxidants) influences the regulation of these antioxidant pathways and thus oxidative balance.

Dietary vitamin E reaches the mitochondria in the flight muscle of zebra finches but only if they exercise.

Whether dietary antioxidants are effective for alleviating oxidative costs associated with energy-demanding life events first requires they are successfully absorbed in the digestive tract and transported to sites associated with reactive species production (e.g. the mitochondria). Flying birds are under high energy and oxidative demands, and although birds commonly ingest dietary antioxidants in the wild, the bioavailability of these consumed antioxidants is poorly understood. We show for the first time that an ingested lipophilic antioxidant, α-tocopherol, reached the mitochondria in the flight muscles of a songbird but only if they regularly exercise (60 min of perch-to-perch flights two times in a day or 8.5 km day-1). Deuterated α-tocopherol was found in the blood of exercise-trained zebra finches within 6.5 hrs and in isolated mitochondria from pectoral muscle within 22.5 hrs, but never reached the mitochondria in caged sedentary control birds. This rapid pace (within a day) and extent of metabolic routing of a dietary antioxidant to muscle mitochondria means that daily consumption of such dietary sources can help to pay the inevitable oxidative costs of flight muscle metabolism, but only when combined with regular exercise.

Dietary linoleic acid, antioxidants, and flight training influence the activity of oxidative enzymes in European Starlings (Sturnus vulgaris).

Multiple studies have demonstrated that diet (e.g., fatty acid composition, antioxidants) and exercise training affect the metabolic performance of songbirds during aerobic activity, although the physiological mechanisms that cause such an effect remain unclear. We tested the hypothesis that elevated proportions of dietary linoleic acid (18:2n6) and amounts of dietary anthocyanins (a hydrophilic antioxidant class) influence the activity and protein expression of oxidative enzymes in flight and leg muscle of European Starlings (Sturnus vulgaris N = 96), a subset of which were flown over 15 days in a wind tunnel. Carnitine palmitoyl transferase (CPT) and citrate synthase (CS) activity displayed 18:2n6-dependent relationships with soluble protein concentration. Lactate dehydrogenase (LDH) was similarly related to protein concentration although also dependent on both dietary anthocyanins and flight training. 3-Hydroxyacyl CoA Dehydrogenase (HOAD) activity increased throughout the experiment in flight muscle, whereas this relationship was dependent on dietary anthocyanins in the leg muscle. Soluble protein concentration also increased throughout the experiment in the flight muscle, but was unrelated to date in the leg muscle, instead being influenced by both dietary anthocyanins and flight training. Training also produced additive increases in CPT and leg muscle HOAD activity. FAT/CD36 expression was related to both dietary 18:2n6 and training and changed over the course of the experiment. These results demonstrate a notable influence of our diet manipulations and flight training on the activity of these key oxidative enzymes, and particularly CPT and CS. Such influence suggests a plausible mechanism linking diet quality and metabolic performance in songbirds.

Flight training in a migratory bird drives metabolic gene expression in the flight muscle but not liver, and dietary fat quality influences select genes.

Training and diet are hypothesized to directly stimulate key molecular pathways that mediate animal performance, and flight training, dietary fats, and dietary antioxidants are likely important in modulating molecular metabolism in migratory birds. This study experimentally investigated how long-distance flight training, as well as diet composition, affected the expression of key metabolic genes in the pectoralis muscle and the liver of European starlings (Sturnus vulgaris, n = 95). Starlings were fed diets composed of either a high or low polyunsaturated fatty acid (PUFA; 18:2n-6) and supplemented with or without a water-soluble antioxidant, and one-half of these birds were flight trained in a wind-tunnel while the rest were untrained. We measured the expression of 7 (liver) or 10 (pectoralis) key metabolic genes in flight-trained and untrained birds. Fifty percent of genes involved in mitochondrial metabolism and fat utilization were upregulated by flight training in the pectoralis (P < 0.05), whereas flight training increased the expression of only one gene responsible for fatty acid hydrolysis [lipoprotein lipase (LPL)] in the liver (P = 0.04). Dietary PUFA influenced the gene expression of LPL and fat transporter fatty acid translocase (CD36) in the pectoralis and one metabolic transcription factor [peroxisome proliferator-activated receptor (PPAR)-α (PPARα)] in the liver, whereas dietary antioxidants had no effect on the metabolic genes measured in this study. Flight training initiated a simpler causal network between PPARγ coactivators, PPARs, and metabolic genes involved in mitochondrial metabolism and fat storage in the pectoralis. Molecular metabolism is modulated by flight training and dietary fat quality in a migratory songbird, indicating that these environmental factors will affect the migratory performance of birds in the wild.

The effects of dietary linoleic acid and hydrophilic antioxidants on basal, peak, and sustained metabolism in flight-trained European starlings.

Dietary micronutrients have the ability to strongly influence animal physiology and ecology. For songbirds, dietary polyunsaturated fatty acids (PUFAs) and antioxidants are hypothesized to be particularly important micronutrients because of their influence on an individual's capacity for aerobic metabolism and recovery from extended bouts of exercise. However, the influence of specific fatty acids and hydrophilic antioxidants on whole-animal performance remains largely untested. We used diet manipulations to directly test the effects of dietary PUFA, specifically linoleic acid (18:2n6), and anthocyanins, a hydrophilic antioxidant, on basal metabolic rate (BMR), peak metabolic rate (PMR), and rates of fat catabolism, lean catabolism, and energy expenditure during sustained flight in a wind tunnel in European starlings (Sturnus vulgaris). BMR, PMR, energy expenditure, and fat metabolism decreased and lean catabolism increased over the course of the experiment in birds fed a high (32%) 18:2n6 diet, while birds fed a low (13%) 18:2n6 diet exhibited the reverse pattern. Additionally, energy expenditure, fat catabolism, and flight duration were all subject to diet-specific effects of whole-body fat content. Dietary antioxidants and diet-related differences in tissue fatty acid composition were not directly related to any measure of whole-animal performance. Together, these results suggest that the effect of dietary 18:2n6 on performance was most likely the result of the signaling properties of 18:2n6. This implies that dietary PUFA influence the energetic capabilities of songbirds and could strongly influence songbird ecology, given their availability in terrestrial systems.

Acute effects of intense exercise on the antioxidant system in birds: does exercise training help?

The acute effects of an energy-intensive activity such as exercise may alter an animal's redox homeostasis, although these short-term effects may be ameliorated by chronic exposure to that activity, or training, over time. Although well documented in mammals, how energy-intensive training affects the antioxidant system and damage by reactive species has not been investigated fully in flight-trained birds. We examined changes to redox homeostasis in zebra finches exposed to energy-intensive activity (60 min of perch-to-perch flights twice a day), and how exercise training over many weeks affected this response. We measured multiple components of the antioxidant system: an enzymatic antioxidant (glutathione peroxidase, GPx) and non-enzymatic antioxidants (measured by the OXY-adsorbent test) as well as a measure of oxidative damage (d-ROMs). At no point during the experiment did oxidative damage change. We discovered that exposure to energy-intensive exercise training did not alter baseline levels of GPx, but induced exercise-trained birds to maintain a higher non-enzymatic antioxidant status as compared with untrained birds. GPx activity was elevated above baseline in trained birds immediately after completion of the second 1 h flight on each of the three sampling days, and non-enzymatic antioxidants were acutely depleted during flight after 13 and 44 days of training. The primary effect of exercise training on the acute response of the antioxidant system to 2 h flights was increased coordination between the enzymatic (GPx) and non-enzymatic components of the antioxidant system of birds that reduced oxidative damage associated with exercise.

Dynamics of Individual Fatty Acids in Muscle Fat Stores and Membranes of a Songbird and Its Functional and Ecological Importance.

Although tissue fatty acid (FA) composition has been linked to whole-animal performance (e.g., aerobic endurance, metabolic rate, postexercise recovery) in a wide range of animal taxa, we do not adequately understand the pace of changes in FA composition and its implications for the ecology of animals. Therefore, we used a C4 to C3 diet shift experiment and compound-specific δ13C analysis to estimate the turnover rates of FAs in the polar and neutral fractions of flight muscle lipids (corresponding to membranes and lipid droplets) of exercised and sedentary zebra finches (Taeniopygia guttata). Turnover was fastest for linoleic acid (LA; 18:2n6) and palmitic acid (PA; 16:0), with 95% replacement times of 10.8-17.7 d in the polar fraction and 17.2-32.8 d in the neutral fraction, but was unexpectedly slow for the long-chain polyunsaturated FAs (LC-PUFAs) arachidonic acid (20:4n6) and docosahexaenoic acid (22:6n3) in the polar fraction, with 95% replacement in 64.9-136.5 d. Polar fraction LA and PA turnover was significantly faster in exercised birds (95% replacement in 8.5-13.3 d). Our results suggest that FA turnover in intramuscular lipid droplets is related to FA tissue concentrations and that turnover does not change in response to exercise. In contrast, we found that muscle membrane FA turnover is likely driven by a combination of selective LC-PUFA retention and consumption of shorter-chain FAs in energy metabolism. The unexpectedly fast turnover of membrane-associated FAs in muscle suggests that songbirds during migration could substantially remodel their membranes within a single migration stopover, and this may have substantial implications for how the FA composition of diet affects energy metabolism of birds during migration.

Turnover of muscle lipids and response to exercise differ between neutral and polar fractions in a model songbird, the zebra finch.

The turnover rates of tissues and their constituent molecules give us insights into animals' physiological demands and their functional flexibility over time. Thus far, most studies of this kind have focused on protein turnover, and few have considered lipid turnover despite an increasing appreciation of the functional diversity of this class of molecules. We measured the turnover rates of neutral and polar lipids from the pectoralis muscles of a model songbird, the zebra finch (Taeniopygia guttata, N=65), in a 256 day C3/C4 diet shift experiment, with tissue samples taken at 10 time points. We also manipulated the physiological state of a subset of these birds with a 10 week flight training regimen to test the effect of exercise on lipid turnover. We measured lipid δ13C values via isotope ratio mass spectrometry (IRMS) and estimated turnover in different fractions and treatment groups with non-linear mixed-effect regression. We found a significant difference between the mean retention times (τ) of neutral and polar lipids (t119=-2.22, P=0.028), with polar lipids (τ=11.80±1.28 days) having shorter retention times than neutral lipids (τ=19.47±3.22 days). When all birds were considered, we also found a significant decrease in the mean retention time of polar lipids in exercised birds relative to control birds (difference=-2.2±1.83 days, t56=-2.37, P=0.021), but not neutral lipids (difference=4.2± 7.41 days, t56=0.57, P=0.57). A larger, more variable neutral lipid pool and the exposure of polar lipids in mitochondrial membranes to oxidative damage and increased turnover provide mechanisms consistent with our results.