Experimental evidence that EPA and DHA are dietary requirements in a migratory shorebird, but they do not affect muscle oxidative capacity.

Dietary n-3 long chain polyunsaturated fatty acids (LCPUFAs) are hypothesized to be natural doping agents in migratory shorebirds, enabling prolonged flight by increasing membrane fluidity and oxidative capacity of the flight muscles. Animals can obtain n-3 LCPUFAs from the diet or by conversion of dietary α-linolenic acid, 18:3 n-3. However, the capacity to meet n-3 LCPUFA requirements from 18:3 n-3 varies among species. Direct tests of muscle oxidative enhancement and fatty acid conversion capacity are lacking in marine shorebirds that evolved eating diets rich in n-3 LCPUFAs. We tested whether the presence and type of dietary fatty acids influence the fatty acid composition and flight muscle oxidative capacity in western sandpipers (Calidris mauri). Sandpipers were fed diets low in n-3 PUFAs, high in 18:3 n-3, or high in n-3 LCPUFAs. Dietary fatty acid composition was reflected in multiple tissues, and low intake of n-3 LCPUFAs decreased the abundance of these fatty acids in all tissues, even with a high intake of 18:3 n-3. This suggests that 18:3 n-3 cannot replace n-3 LCPUFAs, and dietary n-3 LCPUFAs are required for sandpipers. Flight muscle indicators of enzymatic oxidative capacity and regulators of lipid metabolism did not change. However, the n-3 LCPUFA diet was associated with increased FAT/CD36 mRNA expression, potentially benefitting fatty acid transport during flight. Our study suggests that flight muscle lipid oxidation is not strongly influenced by n-3 PUFA intake. The type of dietary n-3 PUFA strongly influences the abundance of n-3 LCPUFAs in the body and could still impact whole-animal performance.

Canada jays (Perisoreus canadensis) balance protein and energy targets simultaneously in both consumed and cached food.

Food scarce periods pose serious physiological challenges for birds, especially in energetically demanding conditions. For species in the northern hemisphere, a decrease in available resources during winter adds further physiological stress to the energetic demands of life at low temperatures. Some species cache food to provide a reliable energy and nutrient resource during scarcity. Canada Jays are a year-round food-caching resident of the North American boreal forest. Canada Jays also rear their young prior to spring green up, making food caching not only essential for adult winter survival, but also potentially important for meeting the requirements of growing offspring in late winter and early spring. We examined the diet choices of Canada Jays immediately prior to winter, and the macronutrient composition of the foods Canada Jay consumed and cached at this time. We found that Canada Jays cache the same relative amounts of macronutrients as they consume but did not vary macronutrients seasonally. The similarities in the macronutrient proportions cached and consumed suggest a consistent nutrient intake pattern, and that Canada Jays are foraging to simultaneously meet similar minimum energy and minimum protein targets for both the present and future. These simultaneous targets constrain the caching decisions of jays when presented with dietary choices.

Metabolic partitioning of sucrose and seasonal changes in fat turnover rate in ruby-throated hummingbirds (Archilochus colubris).

Hummingbirds fuel their high energy needs with the fructose and glucose in their nectar diets. These sugars are used both to fuel immediate energy needs and to build fat stores to fuel future fasting periods. Fasting hummingbirds can deplete energy stores in only hours and need to be continuously replacing these stores while feeding and foraging. Whether and how hummingbirds partition dietary fructose and glucose towards immediate oxidation versus fat storage is unknown. Using a chronic stable isotope tracer methodology, we examined whether glucose or fructose is preferentially used for de novo lipogenesis in ruby-throated hummingbirds (Archilochus colubris). Potential seasonal changes were correlated with variation in the overall daily energy expenditure. We fed ruby-throated hummingbirds sucrose-based diets enriched with 13C on either the glucose or the fructose portion of the disaccharide for 5 days. Isotopic incorporation into fat stores was measured via the breath 13C signature while fasting (oxidizing fat) during the winter and summer seasons. We found greater isotopic enrichment of fat stores when glucose was labelled compared with fructose, suggesting preference for glucose as a substrate for fatty acid synthesis. We also found a seasonal effect on fat turnover rate. Faster turnover rates occurred during the summer months, when birds maintained lower body mass, fat stores and exhibited higher daily nectar intake compared with winter. This demonstrates that fat turnover rate can substantially vary with changing energy expenditure and body composition; however, the partitioning of sucrose towards de novo fatty acid synthesis remains constant.

Metabolic reduction after long-duration flight is not related to fat-free mass loss or flight duration in a migratory passerine.

Migratory birds catabolize large quantities of protein during long flights, resulting in dramatic reductions in organ and muscle mass. One of the many hypotheses to explain this phenomenon is that decrease in lean mass is associated with reduced resting metabolism, saving energy after flight during refueling. However, the relationship between lean body mass and resting metabolic rate remains unclear. Furthermore, the coupling of lean mass with resting metabolic rate and with peak metabolic rate before and after long-duration flight have not previously been explored. We flew migratory yellow-rumped warblers (Setophaga coronata) in a wind tunnel under one of two humidity regimes to manipulate the rate of lean mass loss in flight, decoupling flight duration from total lean mass loss. Before and after long-duration flights, we measured resting and peak metabolism, and also measured fat mass and lean body mass using quantitative magnetic resonance. Flight duration ranged from 28 min to 600 min, and birds flying under dehydrating conditions lost more fat-free mass than those flying under humid conditions. After flight, there was a 14% reduction in resting metabolism but no change in peak metabolism. Interestingly, the reduction in resting metabolism was unrelated to flight duration or to change in fat-free body mass, indicating that protein metabolism in flight is unlikely to have evolved as an energy-saving measure to aid stopover refueling, but metabolic reduction itself is likely to be beneficial to migratory birds arriving in novel habitats.

Glucose transporter expression and regulation following a fast in the ruby-throated hummingbird, Archilochus colubris.

Hummingbirds, subsisting almost exclusively on nectar sugar, face extreme challenges to blood sugar regulation. The capacity for transmembrane sugar transport is mediated by the activity of facilitative glucose transporters (GLUTs) and their localisation to the plasma membrane (PM). In this study, we determined the relative protein abundance of GLUT1, GLUT2, GLUT3 and GLUT5 via immunoblot using custom-designed antibodies in whole-tissue homogenates and PM fractions of flight muscle, heart and liver of ruby-throated hummingbirds (Archilochus colubris). The GLUTs examined were detected in nearly all tissues tested. Hepatic GLUT1 was minimally present in whole-tissue homogenates and absent win PM fractions. GLUT5 was expressed in flight muscles at levels comparable to those of the liver, consistent with the hypothesised uniquely high fructose uptake and oxidation capacity of hummingbird flight muscles. To assess GLUT regulation, we fed ruby-throated hummingbirds 1 mol l-1 sucrose ad libitum for 24 h followed by either 1 h of fasting or continued feeding until sampling. We measured relative GLUT abundance and concentration of circulating sugars. Blood fructose concentration in fasted hummingbirds declined (∼5 mmol l-1 to ∼0.18 mmol l-1), while fructose-transporting GLUT2 and GLUT5 abundance did not change in PM fractions. Blood glucose concentrations remained elevated in fed and fasted hummingbirds (∼30 mmol l-1), while glucose-transporting GLUT1 and GLUT3 in flight muscle and liver PM fractions, respectively, declined in fasted birds. Our results suggest that glucose uptake capacity is dynamically reduced in response to fasting, allowing for maintenance of elevated blood glucose levels, while fructose uptake capacity remains constitutively elevated promoting depletion of blood total fructose within the first hour of a fast.

The Metabolic Flexibility of Hovering Vertebrate Nectarivores.

Foraging hummingbirds and nectar bats oxidize both glucose and fructose from nectar at exceptionally high rates. Rapid sugar flux is made possible by adaptations to digestive, cardiovascular, and metabolic physiology affecting shared and distinct pathways for the processing of each sugar. Still, how these animals partition and regulate the metabolism of each sugar and whether this occurs differently between hummingbirds and bats remain unclear.

Dietary polyunsaturated fatty acids influence flight muscle oxidative capacity but not endurance flight performance in a migratory songbird.

The migratory flights of birds are primarily fueled by fat; however, certain fatty acids may also enhance flight performance and the capacity to oxidize fat. The natural doping hypothesis posits that n-3 long-chain polyunsaturated fatty acids (PUFA) increase membrane fluidity and aerobic and fatty acid oxidative enzymes in the flight muscles, which enables prolonged endurance flight. Support for this hypothesis is mixed, and there is no empirical evidence for increased flight performance. We fed yellow-rumped warblers ( Setophaga coronata coronata) diets enriched in either n-3 or n-6 long-chain PUFA or low in long-chain PUFA and evaluated flight muscle metabolism and endurance performance in a wind tunnel flights lasting up to 6 h. Fatty acid profiles of muscle phospholipids confirmed enrichment of the targeted dietary fatty acids, whereas less substantial differences were observed in adipose triacylglycerol. Contrary to the predictions, feeding n-3 PUFA decreased peroxisome proliferator-activated receptors-ß mRNA abundance and muscle oxidative enzyme activities. However, changes in muscle metabolism were not reflected in whole animal performance. No differences were observed in flight performance among diet treatments in terms of endurance capacity, energy costs, or fuel composition. These measures of flight performance were more strongly influenced by body mass and flight duration. Overall, we found no support for the natural doping hypothesis in a songbird. Furthermore, we caution against extending changes in flight muscle metabolic enzymes or fatty acid composition to changes to migratory performance without empirical evidence.

Flight muscle protein damage during endurance flight is related to energy expenditure but not dietary polyunsaturated fatty acids in a migratory bird.

Migration poses many physiological challenges for birds, including sustaining high intensity aerobic exercise for hours or days. A consequence of endurance flight is the production of reactive oxygen species (ROS). ROS production may be influenced by dietary polyunsaturated fatty acids (PUFA), which, although prone to oxidative damage, may limit mitochondrial ROS production and increase antioxidant capacity. We examined how flight muscles manage oxidative stress during flight, and whether dietary long-chain PUFA influence ROS management or damage. Yellow-rumped warblers were fed diets low in PUFA, or high in long-chain n-3 or n-6 PUFA. Flight muscle was sampled from birds in each diet treatment at rest or immediately after flying for up to a maximum of 360 min in a wind tunnel. Flight increased flight muscle superoxide dismutase activity but had no effect on catalase activity. The ratio of glutathione to glutathione disulphide decreased during flight. Oxidative protein damage, indicated by protein carbonyls, increased with flight duration (Pearson r=0.4). Further examination of just individuals that flew for 360 min (N=15) indicates that oxidative damage was related more to total energy expenditure (Pearson r=0.86) than to flight duration itself. This suggests that high quality individuals with higher flight efficiency have not only lower energy costs but also potentially less oxidative damage to repair after arrival at the destination. No significant effects of dietary long-chain PUFA were observed on antioxidants or damage. Overall, flight results in oxidative stress and the degree of damage is likely driven more by energy costs than fatty acid nutrition.

Effects of experimental manipulation of hematocrit on avian flight performance in high- and low-altitude conditions.

Despite widely held assumptions that hematocrit (Hct) is a key determinant of aerobic capacity and exercise performance, this relationship has not often been tested rigorously in birds and results to date are mixed. Migration in birds involves high-intensity exercise for long durations at various altitudes. Therefore, it provides a good model system to examine the effect of Hct on flight performance and physiological responses of exercise at high altitude. We treated yellow-rumped warblers (Setophaga coronata) with avian erythropoietin (EPO) and anti-EPO to experimentally manipulate Hct and assessed flight performance at low and high altitudes using a hypobaric wind tunnel. We showed that anti-EPO-treated birds had lower Hct than vehicle- and EPO--treated birds post-treatment. Anti-EPO-treated birds also had marginally lower exercise performance at low altitude, committing a higher number of strikes (mistakes) in the first 30 min of flight. However, anti-EPO-treated birds performed significantly better at high altitude, attaining a higher altitude in a ramped altitude challenge to 3000 m equivalent altitude, and with a longer duration of flight at high altitude. Birds exercising at high altitude showed decreased Hct, increased glucose mobilization and decreased antioxidant capacity, regardless of treatment. In summary, we provide experimental evidence that the relationship between Hct and exercise performance is dependent on altitude. Future studies should investigate whether free-living birds adaptively modulate their Hct, potentially through a combination of erythropoiesis and plasma volume regulation (i.e. hemodilution), based on the altitude they fly at during migratory flight.

Seasonal and flight-related variation of galectin expression in heart, liver and flight muscles of yellow-rumped warblers (Setophaga coronata).

Galectins, a family of multifunctional glycan-binding proteins, are proposed as biomarkers of cellular stress responses. Avian migration is an energetically challenging physical stress, which represents a physiological model of muscular endurance exercises. This study assesses change in galectin gene expression profiles associated with seasonal variation in migratory state and endurance flight in yellow-rumped warblers (Setophaga coronata). Bioinformatics analysis and real-time qPCR were used to analyse the expression of galectins in flight muscle, heart and liver tissues of 15 warblers separated into three groups of winter unflown, and fall migratory flown/unflown birds. Five transcripts similar to chicken and human galectins -1, -2, -3, -4, and -8 were identified in warbler tissues. The expression of these galectins showed no seasonal changes between two experimental groups of birds maintained under unflown winter and fall conditions indicating a minor role of galectins in preparation for migration. However, endurance flight led to a significant elevation of galectin-1 and galectin-3 mRNAs in flight muscles and galectin-3 mRNA in heart tissue while no changes were observed in liver. Different changes were observed for the level of O-GlcNAcylated proteins, which were elevated in flight muscles under winter conditions. These results suggest that secreted galectin-1 and galectin-3 may be active in repair of bird muscles during and following migratory flight and serve as molecular biomarkers of recent arrival from migratory flights in field studies.

Seasonal dietary shifting in yellow-rumped warblers is unrelated to macronutrient targets.

Dietary shifting, for example from insects to fruits, is a common mechanism used in migratory songbirds to accumulate fat to fuel migratory flights. We examined a potential underlying cause of dietary shifting in yellow-rumped warblers (Setophaga coronata) by comparing energy and protein intake goals of birds during fall migration and winter. We offered captive warblers pairs of three diets differing in macronutrient composition in both the fall and winter. Using the principles of the geometric framework of nutrition we evaluated protein and energy intake to determine if consumption of the diet pairs was adjusted to meet an energy or protein intake target, and if the target differed seasonally. Regardless of season, the warblers preferred the diet with the lowest protein content and highest carbohydrate content. Total energy intake was maintained relatively constant during migration, at around 60 kJ/day, regardless of diet combination, and at about 50 kJ/day during winter. This suggests that warblers consume macronutrients available to them without protein limitations to reach their total energy intake target. When the diet combination offered allows, the warblers mixed their diet intake to consume roughly 0.5 g/day of protein, regardless of season, which suggested a constant protein target. Our findings suggest that songbirds prefer to alter non-protein energy intake proportionally to meet changing energy demand, rather than an overall increase in macronutrient intake. Additionally, they have the ability to shift their diet based on availability, resulting in high flexibility in their macronutrient intakes to maintain energy intake.

High feeding intensity increases the severity of fatty liver in the American mink (Neovison vison) with potential ameliorating role for long-chain n-3 polyunsaturated fatty acids.

BACKGROUND: Rapid body fat mobilization, obesity, and an inadequate supply of n-3 polyunsaturated fatty acids (PUFA) have been suggested to play roles in the etiology of fatty liver in the American mink (Neovison vison). This study examined the effects of feeding intensity and dietary fat source on fatty liver induced by fasting. In a multi-factorial design, 3 different fat sources (herring oil, rich in n-3 PUFA, soya oil, rich in n-6 PUFA, and canola oil, rich in n-9 monounsaturated fatty acids) were fed to mink at a low and high feeding intensity for 10 weeks, followed by an overnight or a 5-day fasting treatment to induce fatty liver. RESULTS: Fasting led to the development of fatty liver with increased severity in the mink fed at the high feeding intensity. The herring oil diet, high in long-chain n-3 PUFA, was found to decrease the severity of fatty liver in the mink at the high feeding intensity. CONCLUSION: Preventing excessive weight gain and increasing dietary intake of n-3 long-chain PUFA may help prevent excessive lipid accumulation during prolonged periods of fasting or inappetence by promoting hepatic fatty acid oxidation.