Despite the importance of oxidative damage measures to understanding tradeoffs during migration, relatively few studies have measured the acute effects of flight on byproducts of RS production. Moreover, these studies have been spread across different study systems and measures of oxidative damage. As outlined below, the effects of acute flight on oxidative damage are more consistent in experimental studies, where flight time and speed are controlled, compared to field studies of free-living birds, and the extent of reported damage seems related to the extent of antioxidant system response. A commonly used measure of oxidative damage has been the d-ROMs test (Costantini, 2016; Skrip and McWilliams, 2016), which is most closely associated with lipid peroxidation but can reflect hydroperoxides of any origin (Davies, 2016; Ito et al., 2017). This assay is responsive to changes in oxidative status over a relatively short time scale (e.g., minutes to hours) and uses blood serum, making it both non-destructive and a more general indicator of circulating lipid peroxidation products than other more tissue-specific measures (e.g., in muscle; Abuja and Albertini, 2001; Colombini et al., 2016) and compared to other specific assays (e.g., MDA, see below).

Oxidative damage to lipids has usually been found to not change or has increased after flight across the ca. dozen studies to date, and this variation in response can be explained in part by the extent of response of the antioxidant system to the physiological challenges of the flight. In one of the first studies to investigate the acute effects of flight, pigeons (Columbia livia) flown for long durations (310 ± 31 min) had 54% higher lipid damage than in birds flown short durations (80 ± 15 min) or not flown (Costantini et al., 2008). In contrast, flight duration (range: 65–205 min) was not related to d-ROMs levels in Northern Bald Ibis on a managed migration (Geronticus eremita; Bairlein et al., 2015). Similarly, several wind-tunnel studies on starlings (S. vulgaris) that involved weeks of flight training leading to long final flights up to 6 h have not found any change in d-ROMs associated with flight, either within individuals pre- and post-flight or between individuals that were either flown or not flown (McWilliams et al., 2020; Frawley et al., 2021). In contrast, starlings involved in a similar 2-week wind-tunnel flight training decreased d-ROMs after flight compared to pre-flight indicating a potential hormetic response (DeMoranville, 2020). Zebra Finches (Taeniopygia guttata) that underwent 3 h of short-burst flight, a flight type that is more aerobically intense than sustained flight (Nudds and Bryant, 2001), showed a significantly greater increase in d-ROMs over those 3 h than unflown birds (Costantini et al., 2013). Furthermore, birds flown at the greatest speed intensity (0.9 km/h) had the highest levels of d-ROMs and birds flown at a slower speed (0.3 km/h) had intermediate levels (Costantini et al., 2013). Over a longer period of flight training, Zebra Finches flown for 2 h at 2.4 km/h per day consistently showed no acute changes in oxidative damage to lipids on a given day of flight training, and no difference in d-ROMs was detected between trained and untrained individuals over 1.5 months of daily flying (Cooper-Mullin et al., 2019). However, these same individuals exhibited a consistent increase in an antioxidant enzyme (GPx) that may have protected them against oxidative damage. Finally, correlational field studies of free-living, migrating Garden Warblers (S. borin; Skrip et al., 2015) and Nathusius’ bats (Pipistrellus nasthusii; Costantini et al., 2018) found that d-ROMs were highest in plasma samples taken most recently after migratory flight, while a study that manipulated the effort of flight by plucking wing feathers of Great Tits (Parus major; Vaugoyeau et al., 2015) found no difference in d-ROMs between handicapped and control individuals. Some of this inconsistency in results between studies seems related to the extent of flight costs and duration. Notably, the level of oxidative damage measured by the d-ROMs test in both European Starlings and Yellow-rumped Warblers (Setophaga coronata) flown for known durations in wind-tunnel experiments was highest in individuals flown for longer and that had higher energy expenditure during flight (Dick and Guglielmo, 2019; DeMoranville, 2020). Likewise, free-living Red-eyed Vireos had lower oxidative damage compared to Blackpoll Warblers caught on an island stopover site (Block Island and Rhode Island), presumably because the warblers had to fly farther to reach the stopover site (Skrip et al., 2015).

Given the susceptibility of PUFA to oxidative damage (Cooper-Mullin and McWilliams, 2016; Skrip and McWilliams, 2016), some studies use the concentration of malondialdehyde (MDA) to assess oxidative damage because it is a relatively stable peroxidation byproduct of long-chain PUFAs that are common in cell membranes (Halliwell and Gutteridge, 2007; Ayala et al., 2014). MDA levels were elevated in blood samples taken from Budgerigars (Melopsittacus undulatus) 24 h after completing 5 days of mild escape-flight training, although this effect was largely dependent on whether or not individuals had been supplemented with antioxidants (Larcombe et al., 2008). Conversely, natural variation in aerobic activity (including flight) of Budgerigars was unrelated to MDA concentrations (Larcombe et al., 2015). Likewise, MDA concentration did not differ between Common Blackbirds presumed to be year-round residents versus presumed to have recently completed a migratory flight (Eikenaar et al., 2017). Therefore, studies that examine oxidative damage to lipids (i.e., d-ROMs and MDA) show varied responses to flight, likely due to variation in flight intensity and the extent to which a bird’s antioxidant system responds in preparation for and during flight.

Oxidative damage to protein, measured as the concentration of protein carbonyls (Halliwell and Gutteridge, 2007; Davies, 2016), has also been assessed in response to flight given the potential for muscle damage (Guglielmo et al., 2001; Dick and Guglielmo, 2019). Plasma protein carbonyls were significantly higher in Zebra Finches flown for 3 h at 0.9 km/hr compared to individuals flown at 0.3 km/hr or unflown individuals (Costantini et al., 2013). Similarly, concentration of protein carbonyls in the flight muscle of Yellow-rumped Warblers was positively related to the duration of flight in a wind tunnel and was significantly higher in individuals flown for 6 h than for unflown individuals (Dick and Guglielmo, 2019). Meanwhile, in a field study on European Robins (Erithacus rubecula) during migration, plasma concentrations of protein carbonyls were highest among birds caught at night and therefore presumably directly out of endurance flight (Jenni-Eiermann et al., 2014).

In sum, studies on the acute effects of flight on oxidative damage provide evidence for an increase in damage after flight (as assessed using d-ROMs, MDA, and protein carbonyls) but certainly not in all cases. Only protein carbonyls have consistently increased in response to flight, although there are few studies to date that have used this measure. Clearly, levels of oxidative damage are dynamic and seem responsive to a host of ecologically relevant factors, including duration of flight and diet, which complicates comparisons between studies especially when different assays are used.

Birds can protect themselves from exercise-induced increases in RS production using a robust endogenous antioxidant system (Figure 1). Whether a bird depletes its antioxidant capacity, or increases it in response to flight, may depend on ecologically relevant factors such as individual condition (e.g., fat stores and oxidative damage levels), access to dietary antioxidants, and the amount of exercise. Enzymatic and non-enzymatic antioxidants may also respond differently to acute oxidative challenges, indicating that these components may serve different or complimentary roles in the antioxidant system.

Enzymatic antioxidants, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), are the most commonly assayed enzymes in avian studies and have been shown in most cases to rapidly increase in activity in response to flight. For example, European Robins caught during nocturnal migratory flight had higher GPx activity compared to birds on stopover (Jenni-Eiermann et al., 2014), and Yellow-rumped Warblers flown in a wind tunnel had higher SOD after flight than individuals at rest (Dick and Guglielmo, 2019). The response of enzymatic antioxidants in flight-trained European Starlings to an approximately 3-h flight in a wind tunnel depended on availability of dietary antioxidants (Frawley et al., 2021). Specifically, starlings fed a diet not supplemented with anthocyanin, a dietary antioxidant, had decreased GPx activity immediately after flight, whereas GPx activity did not change after flight for starlings fed a diet supplemented with anthocyanins (Frawley et al., 2021). Furthermore, Zebra Finches flown for short bursts had similar serum GPx, CAT, and SOD activity after flight compared to unflown control birds (Costantini et al., 2013). Zebra Finches flown for 2 h had elevated GPx activity immediately after flight, and this acute increase in GPx activity was consistently observed even after ca. 2 and 6 weeks of daily flight, indicating that birds were able to rapidly downregulate GPx activity when the oxidative challenge had passed (Cooper-Mullin et al., 2019). Interestingly, daily flight by Zebra Finches over 1.5 months increased coordination between the enzymatic (GPx) and non-enzymatic components of the antioxidant system (Cooper-Mullin et al., 2019). Furthermore, the inclusion of dietary precursors for these enzymatic antioxidants (e.g., selenium for GPx) can increase the activity of the enzyme during flight (Zigo et al., 2017). The common theme that emerges from these studies is that the activity of enzymatic antioxidants rapidly changes during bird flight, but the extent of these changes is related to the availability of dietary antioxidants and the response of other components of the antioxidant system.

Non-enzymatic endogenous antioxidants include sacrificial molecules such as glutathione (GSH) and uric acid, which arise from a diverse set of metabolic pathways and cycles and have varying metabolic uses (Tsahar et al., 2006; Carro et al., 2012; Cooper-Mullin and McWilliams, 2016). Uric acid, a byproduct of protein catabolism and a powerful antioxidant, has been found to consistently increase after flight in several field and lab studies. European Starlings flown in wind tunnels had elevated serum uric acid immediately after up to 6 h of flight (Carter et al., 2020; McWilliams et al., 2020; Frawley et al., 2021). White-crowned Sparrows (Zonotrichia leucophrys) that were flown at high speeds in a hop/hover wheel (Tsahar et al., 2006) and pigeons flown for 4 h (Gannes et al., 2001) also had increased uric acid concentrations after flight. Serum uric acid was also higher in Garden Warblers, Pied Flycatchers (Ficedula hypoleuca), and European Robins on active migration compared to birds at rest or refueling on stopover (Jenni-Eiermann and Jenni, 1991). Given that it is a byproduct of protein catabolism during exercise, uric acid may serve as a particularly important antioxidant for flying birds.

The complexity of interpreting uric acid measurements as a marker of non-enzymatic antioxidant capacity can be avoided by using the OXY-adsorbent test, a common serum-based assay that provides a general snapshot of a bird’s ability to quench RS. Generally, OXY has been shown to decrease after flight, indicating that birds deplete antioxidant capacity in response to an acute challenge. Pigeons showed a 19% drop in serum antioxidant capacity after being flown for 200 km (ca. 310 ± 31 min, Costantini et al., 2008), and Zebra Finches depleted antioxidant capacity immediately after 2 h of flight and then increased OXY after getting a reprieve from regular daily flying (Cooper-Mullin et al., 2019). European Starlings also reduced OXY (and oxidative damage, noted above) immediately after flights in a wind tunnel, and the extent of this change was more apparent in birds that expended more energy during their longest flights (DeMoranville, 2020). OXY also changed in European Starlings immediately after flights, although the direction and extent of the change depended on season (fall vs. spring migration) and whether diets were supplemented with antioxidants (Frawley et al., 2021).

In summary, there is some evidence that enzymatic antioxidants, particularly GPx, may serve as a first line of defense for birds faced with an oxidative challenge, although research is scarce on the acute responses of other major antioxidant enzymes, particularly CAT and SOD. Likewise, non-enzymatic endogenous antioxidants such as uric acid consistently increase after flight, suggesting they may be functionally important. In contrast, other components of non-enzymatic antioxidant capacity as measured with the OXY test usually decrease after flight, and the extent of this acute change in OXY after flight depends on the acute response of enzymatic antioxidants such as GPx when regular daily flying is prolonged (Cooper-Mullin et al., 2019). Consumption of dietary antioxidants by European Starlings affects the response of enzymatic antioxidants as well as non-enzymatic antioxidants (DeMoranville et al., 2021; Frawley et al., 2021) and has a multitude of related effects: antioxidant consumption reduces corticosterone production in flight-trained birds, and thus potentially protects against the costs of high glucocorticoids (Casagrande et al., 2020), and enhances the breeding condition of males (Carbeck et al., 2018). The role of corticosterone during preparation for migration, in departure during migration (e.g., from stopover sites), and in support of sustained migratory flights has been recently reviewed by Bauer and Watts (2021). Importantly, Cooper-Mullin et al. (2021) used stable isotope-labeled vitamin E to reveal that consumed dietary antioxidants are absorbed and reach muscle mitochondria of Zebra Finches but only when they are regularly exercised (2 h of regular daily flying). Further studies are needed that simultaneously measure multiple components of the antioxidant system and the contribution of dietary antioxidants to better understand how birds respond to the oxidative demands of flight and migration.

When a given flight is associated with an acute increase in oxidative damage and an acute decrease in antioxidant capacity, then the interpretation is relatively straightforward: higher metabolic rate during exercise increases the escape of radicals from the electron transport chain, RS diffuse from the mitochondria to the cytosol and intercellular spaces, and then they are quenched by certain aspects of the antioxidant system. Here we discuss possible mechanisms that can explain other results (e.g., no oxidative damage or change in certain components of the antioxidant system).

When no acute changes in oxidative damage or antioxidant capacity are observed following flight, one possible interpretation is that flight did not increase the amount of radicals escaping from the electron transport chain. There are several, not mutually exclusive, ways that this could be achieved: production of electrons (in the form of reduced nicotinamide adenine dinucleotide, NADH) by the citric acid cycle could be limited by reduced mass-specific activity of the cycle (Tretter and Adam-Vizi, 2005). Alternately, electrons are less likely to escape and form radicals if they are rapidly combined with protons and oxygen to form water. In addition, uncoupling proteins may prevent the formation of large gradients that might produce a scarcity of protons to combine with electrons (Cooper-Mullin and McWilliams, 2016). Electron escape and RS production might also be regulated by the concentration and activity of cytochrome oxidase, which catalyzes the combination of electrons, protons and oxygen (Bode et al., 2013). Although these factors have not been studied extensively in birds or in relation to aerobic activity, variation in RS production across species and tissues could help explain the range of acute responses to flight in birds documented across studies to date.

Inconsistent responses of oxidative damage and antioxidant capacity to flight may also be the result of complex interactions between RS production and the antioxidant system of birds. As a general model, we might assume that RS are continuously produced in animal tissues at a certain rate and that this rate increases during flight (Halliwell and Gutteridge, 2007). Simultaneously, RS and byproducts of RS damage may be neutralized at a certain rate through the actions of antioxidant enzymes, sacrificial molecules, and dietary compounds (Figure 1; Cooper-Mullin and McWilliams, 2016). Combined, this means that RS, and therefore measured concentrations of damaged compounds, will accumulate and increase when the rate of RS production is greater than the rate of neutralization and decrease when the rate of neutralization is greater. If the rate of RS neutralization is static, then damage will accumulate throughout the period of flight and then decrease during the following recovery period. However, it is well established that the antioxidant system can flexibly respond and can increase capacity by upregulating the expression of antioxidant enzymes, upregulating the production of sacrificial molecules, and increasing the transport of stored antioxidants into target tissues (Cooper-Mullin and McWilliams, 2016; Costantini, 2019). Moreover, it is reasonable to expect the rate of RS neutralization to be upregulated proportionally with the accumulation of RS and RS byproducts (Dröge, 2002).

In Figure 4, we present a conceptual model that demonstrates how the extent of measurable oxidative damage associated with a given flight may depend on the pace and extent of antioxidant upregulation, and the intensity of the flight. In particular, a simple exponential response of neutralization to RS concentration can limit the accumulation of RS during flight and lead to more rapid reductions following flight. Meanwhile, the addition of a lag in response, due to the time required for signaling and the synthesis of antioxidants (Ji et al., 2006; Sthijns et al., 2016), is enough to create a simple hormetic response where accumulated damage decreases in the middle of the flight [Figure 4, High RS – AOX (lag)]. Hormesis can also depend on the intensity of an oxidative challenge (Nikolaidis et al., 2012; Costantini, 2014), meaning that milder activity can, at times, produce a muted antioxidant response and therefore higher levels of accumulated RS byproducts [Figure 4, Low RS – AOX (lag)]. All of these relatively simple factors can interact to produce considerable variation in measurement of damage, which will depend on the timing of sample collection, the tissue collected, the intensity of activity, and the regulatory responsiveness of the study species. Furthermore, there is evidence that the antioxidant response may differ between seasons and tissues, and that access to dietary antioxidants may boost or attenuate certain antioxidant responses (Frawley et al., 2021) and this can depend on regular exercise (Cooper-Mullin et al., 2019, 2021). In sum, the antioxidant response is not static, and acquiring a better understanding of the pace and extent of antioxidant up- and down-regulation under ecologically relevant situations is needed.

The first evidence of potential acute recovery on stopover came from actively migrating European Robins (Erithacus rubelica) caught flying at night through a Swiss mountain pass in autumn (Jenni-Eiermann et al., 2014). Birds captured at night out of flight had higher oxidative damage to proteins (circulating protein carbonyls) than birds captured while resting during the day (Jenni-Eiermann et al., 2014). However, the timeframe over which recovery occurred was unknown, because damage was not tracked over time within individuals. The first study to track oxidative damage in relation to time after a long-distance flight found that circulating oxidative damage to lipids (d-ROMs) decreased among recaptured Garden Warblers the longer they were at a Mediterranean stopover site in spring [between 1 and 192 h (8 days)]; opportunistic intra-individual sampling (n = 2) confirmed that damage decreased within birds over a matter of days (Skrip et al., 2015). Taking ‘the next logical step’ after Skrip et al.’s (2015) cross-sectional finding, Eikenaar et al. (2020b) longitudinally tracked changes in damage within individuals and confirmed that migrating songbirds are capable of recovering from oxidative damage on stopover. Northern Wheatears caught out of spring migration in Germany and held in captivity for 3 days decreased their oxidative damage to polyunsaturated fats (MDA) and appeared to stabilize that damage at a consistent level, suggesting that there may be a threshold of low damage or ‘oxidative set-point’ (Eikenaar et al., 2020b) that birds seek to reach before taking their next long-distance flight.

Taken together, these three studies provide the first evidence that songbirds are capable of recovering from oxidative damage after flight. How they do so, however, remains an open question. Do birds repair damaged molecules, replace them, or a combination of both? Do resources available at a given stopover site or the physiological condition of an individual bird influence how birds address oxidative damage (Figure 1)? The minimum time within which birds begin to recover after a flight is also uncertain. Migrating songbirds held in handling bags for short periods before blood sampling (30 min, Costantini et al., 2007; 20–192 min, Skrip et al., 2015) exhibited no change in oxidative measures. However, Gray Catbirds (Dumetella carolinensis) caught during autumn migration and kept overnight had increased uric acid and decreased levels of residual total antioxidant capacity within at least a 12-h period (Cohen et al., 2008a).

Evidence is also mounting that birds are capable of recovering their antioxidant capacity while on stopover. Although time on stopover and circulating non-enzymatic antioxidant capacity (OXY) were not correlated in the cross-sectional analysis of Garden Warblers described above, antioxidant capacity did increase in the two Garden Warblers opportunistically resampled over the course of several days, hinting that such recovery is possible (Skrip et al., 2015). An experimental study with flight-trained European Starlings (DeMoranville, 2020) was the first to demonstrate that songbirds can recover antioxidant capacity after long-distance flight. Starlings depleted their circulating non-enzymatic antioxidant capacity (OXY) during 15 days of nearly daily flight in a wind tunnel (totaling >640 km), but within 2 days of rest (i.e., no flight), birds recovered to their baseline (pre-flight-training) levels independent of whether they were supplemented with water-soluble antioxidants (i.e., anthocyanin) (DeMoranville, 2020). There is evidence that restoration of antioxidant capacity in metabolically active tissues is mediated by the transcription of antioxidant enzyme proteins. For example, during recovery, these same flight-trained starlings had greater mRNA levels of antioxidant enzymes in the liver (CAT, SOD2, and GPX1) and pectoralis (SOD2) compared to unflown starlings, while lipid damage levels were similar (DeMoranville et al., 2021). Like recovery from damage, recovery of antioxidant capacity, however, remains to be mechanistically explained.

Consumption of dietary antioxidants on stopover may contribute to recovery of antioxidant capacity, and migratory birds seem able to select diets that provide fats and antioxidants that can assist or accelerate this recovery (Sapir et al., 2004; Cohen et al., 2009). Migratory songbirds at an offshore New England stopover site during autumn preferred fruits from species with higher fat content and selected fruits rich in dietary antioxidants (Alan et al., 2013; Bolser et al., 2013). Birds can select diets using solely antioxidant content, as shown by a series of food-choice experiments involving Blackcaps (Sylvia atricapilla) (Catoni et al., 2008a, b; Schaefer et al., 2014). Given a choice of food supplemented with anthocyanins or not, Blackcaps preferred a supplemented diet (Catoni et al., 2008a; Schaefer et al., 2008). Sources of dietary antioxidants during spring migration are understudied but may include seeds (Cohen et al., 2008b; Beaulieu and Schaefer, 2013), insects (Catoni et al., 2008a; Eeva et al., 2010), leaves (Catoni et al., 2008a), and fruits (Cohen et al., 2009; Alan and McWilliams, 2013; Bolser et al., 2013). It is highly likely that birds take advantage of fruits in tropical areas prior to or during spring migration, as suggested by a geolocator study that found migrating Red-Eyed Vireos make an unexpectedly long stopover in Colombia during spring (Callo et al., 2013).

Recent experiments with actively migrating birds in autumn in New England have revealed the benefits of consuming dietary antioxidants and how antioxidant capacity and fat stores interactively influence the time passerines spend on stopover (Cooper-Mullin and McWilliams, 2021). For example, gavage-feeding dietary anthocyanins to Hermit Thrushes (Catharus guttatus) on an ad libitum diet boosted their ability to build non-enzymatic antioxidant capacity (OXY; Cooper-Mullin and McWilliams, 2021). Furthermore, birds on an ad libitum diet increased non-enzymatic antioxidant capacity (OXY) compared to birds on a diet designed to maintain their weight at capture (Cooper-Mullin and McWilliams, 2021). Studies in mammals have identified anthocyanins as enhancers of the NRF2 antioxidant pathway that activates the expression of antioxidant genes (Shih et al., 2007; Aboonabi and Singh, 2015; Tian et al., 2019; Aboonabi et al., 2020). Dietary antioxidants may also help songbirds increase enzymatic antioxidant capacity by stimulating the same pathway. For example, European Starlings supplemented with anthocyanins had higher mRNA levels of antioxidant enzymes associated with combating reactive species production in the mitochondria (SOD2) and lipid peroxidation in the peroxisome (CAT) in flight muscle compared to unsupplemented birds (DeMoranville et al., 2021). How exogenous and endogenous antioxidants interact in birds on stopover while recovering or refueling still needs more investigation.

The studies described above demonstrate that birds (evidence mostly in songbirds) can decrease oxidative damage and build antioxidant capacity rapidly, in a matter of days after a migratory flight. These studies also demonstrate that dietary anthocyanins (Catoni et al., 2008a; Schaefer et al., 2008), and flavonoids (Catoni et al., 2008b), as well as dietary PUFA (Alan and McWilliams, 2013; DeMoranville et al., 2020; McWilliams et al., 2020), influence a songbird’s oxidative status and stopover length (Cooper-Mullin and McWilliams, 2021). However, how the quantity or quality of a bird’s fat stores, the energetic demands of previous flights, and the available resources at a stopover aid or hinder this recovery is unclear. The challenge of refueling on stopover is twofold for birds: they have to accumulate fatty acids that make them more efficient fliers (Price, 2010; Ferretti et al., 2019; McWilliams et al., 2020), but they also need to protect those fats as well as recover from the previous flight (Metzger and Bairlein, 2011; Skrip et al., 2015). Future research should focus on the oxidative challenges that recovery and refueling simultaneously pose to migrating birds.

The ability to use endogenous and exogenous antioxidants varies among individuals depending on body condition, recent oxidative challenges, and ecological factors (Dick and Guglielmo, 2019; Loughland and Seebacher, 2020; Cooper-Mullin et al., 2021). This individual variation extends to the specific antioxidant strategies available to birds during oxidative challenges, as individuals may also rely on alternate antioxidants to protect against damage (DeMoranville, 2020). Future longitudinal studies that examine intraindividual antioxidant responses to oxidative challenges (e.g., flight, high PUFA diets, lack of dietary antioxidants, breeding, and temperature acclimatization) will help us understand the protective strategies available to birds during specific life history stages. Especially informative would be longitudinal studies that compare tissues that vary in metabolic rate, mitochondrial density, and potentially in their capacity to maintain oxidative balance (e.g., DeMoranville et al., 2020, 2021; Frawley et al., 2021).

More controlled captive-bird experiments as well as field studies are needed that test the proposed hypothesis that as birds build fat stores in preparation for migration they also build certain components of their antioxidant capacity. More specifically, does the extent of the building of antioxidant capacity during preparation for migration determine the extent of observed oxidative damage. For example, to date there has been no longitudinal study of wild songbirds to parallel the multi-season assessment of Gutiérrez et al. (2019) who sampled Hudsonian Godwits at three time periods from January to March (wintering, fuelling, and pre-departure), the latter as they prepared for their >10,000-km northbound migratory flight. Marasco et al.’s (2021) study of captive-reared migratory Common Quails reminds us that controlled experiments using photoperiod manipulations to induce migration state in birds can provide important complementary (and at times contradictory) information about how the antioxidant system changes during preparation for migration. Tracking the oxidative and metabolic status of songbirds in the months and weeks leading up to departure would be particularly valuable to understanding in situ preparation, especially in the context of available dietary fat and antioxidant resources.

It remains unknown what amount of damage is considered too much damage. Our understanding of the varying response of songbirds to oxidative stress in migration and other contexts would be greatly supported by a more robust understanding of the phenomenon of oxidative damage. In particular, the time course over which the processes of RS production, antioxidant upregulation/mobilization, and RS neutralization occur during flight remains unknown. One way to evaluate this conundrum is to conduct studies that link oxidative status (antioxidant capacity and oxidative damage) to measures of short- and long-term performance (e.g., flight efficiency, speed and timing of migration, foraging, refueling, and fecundity). Doing so within the context of important life history stages such as migration and breeding would reveal potential carryover effects (McWilliams et al., 2021). Only one study to date (Skrip et al., 2016) has examined how antioxidant status during repeated flights change antioxidant allocation during subsequent breeding. At least in that model species (Zebra finch), use of dietary antioxidants to maintain oxidative balance during repeated twice-daily flights over months affected female allocation of dietary antioxidants to eggs. No studies to date of a migratory bird have determined the consequences of such carryover effects (with dietary antioxidants serving as the currency) from one stage of the annual cycle to the next on reproductive success.

There is a considerable amount of crosstalk among major physiological pathways including those involved in metabolism (PPARs, glucocorticoid production), immunity, inflammation (NF-κB), and antioxidants (NRF2 and PPARs) due to shared/multifunctional transcription factors that respond to similar factors like exercise, RS production, and dietary antioxidants (Finck and Kelly, 2006; Syeda et al., 2019). One such recent study examined these shared transcription factors and their downstream genes in a migratory bird and found that flying over the course of 2 weeks and consuming antioxidants influenced both metabolic and antioxidant mRNA expression in a tissue specific manner (DeMoranville et al., 2020, 2021). Studies that integrate across multiple systems including the metabolic, antioxidant, immune, inflammatory, endocrine, will help assess the overall state of the birds and determine if modulating the antioxidant system, for example, leads to tradeoffs with other physiological states. One such recent study of this type suggests a possible trade-off between the immune and antioxidant systems in a migratory bird (Eikenaar et al., 2018).

No study to date has examined the extent to which dietary antioxidants are directly used to protect accumulating fat stores and/or contribute to recovery from oxidative damage incurred during flight. The recent study by Cooper-Mullin et al. (2021) used stable-isotope labeled Vitamin E to demonstrate for the first time that consumed dietary antioxidants are absorbed and transported to the mitochondria of flight muscle, but only if birds are exercising. What remains to be demonstrated is how these and other types of dietary antioxidants are used during metabolism, and the extent to which they reduce the need to upregulate more costly components of the antioxidant system such as enzymes. Metabolism of all nutrients starts with processing in and absorption through the gut, and this is influenced by the gut microbiome (Clark and Mach, 2017). The extent to which variation in the gut microbiome of birds while in migration affects the availability and utilization of ingested antioxidants is not known, although such information has important implications for both our understanding of how birds work (i.e., their physiology) and also how to ensure that conservation and management efforts provide what they require during migration (Bahrndorff et al., 2016; Trevelline et al., 2019).