Acute- and long-term flight stimulated antioxidant protection (H1)
We found evidence that repeated bouts of flight initiated a hormetic response that activated the antioxidant system to protect against the accumulation of oxidative damage, consistent with H1. This concept of hormesis, the mild exposure to reactive species and subsequent activation of protective and repair mechanisms, has been demonstrated mainly in the skeletal muscle of humans (McArdle et al. 2002, Rattan 2008). In the present study, the immediate, acute effects of a flight included a reduction in both non-enzymatic antioxidant capacity as well as oxidative damage (PF-AF; Fig. 2A, B), consistent with the acute-effects of flight hypothesis (H1a). The coincident reduction in both antioxidant capacity and oxidative damage suggests that birds effectively avoided oxidative damage caused by short-term exercise at least in part by using the non-enzymatic component of their antioxidant system. Similarly, birds exposed to 15 days of flight-training and two days of recovery were able to decrease circulating damage to lipids (RC-BG; Fig. 2A) while untrained birds were not (long-term flight effect, H1b). The apparent over-compensation of the antioxidant system to enable a decrease in oxidative damage during a given flight (H1a) seems novel to this study, as some flight-training studies report no change in markers of circulating damage after an acute flight (Skrip et al. 2016, Cooper-Mullin et al. 2019). Most studies report increases in markers of circulating oxidative damage shortly after a migratory flight in free-living birds (Jenni-Eiermann et al. 2014, Costantini et al. 2018), after a 200 km experimental flight in homing pigeons (Costantini et al. 2008), or in the pectoralis after an experimental flight in a wind tunnel (Dick and Guglielmo 2019). We found that the reduction in both non-enzymatic antioxidant capacity as well as oxidative damage was more apparent in birds that expended more energy per unit time during their longest flight while flying at a prescribed pace (Fig. 3). Perhaps flying birds that expend less energy per unit time are less metabolically challenged, and thus do not need to deplete OXY as much to prevent oxidative damage. This conclusion is similar in concept to (Dick and Guglielmo 2019) who found less pectoralis damage in Yellow-rumped warblers with lower flight energy expenditures.
Consistent with the flight effects proposed in H1b, regular daily flying depleted non-enzymatic antioxidant capacity with an associated decrease in oxidative damage immediately after a flight; although both non-enzymatic antioxidant capacity and oxidative damage returned to baseline levels after 48 hrs of rest (without wind tunnel flying). These results are in accordance with flight-trained zebra finches that also decreased OXY during 2-hrs of acute flight and then increased OXY after birds had a reprieve from regular flying (Cooper-Mullin et al. 2019). This consistent and relatively rapid (no more than a few days) depletion and recovery of non-enzymatic antioxidant capacity suggests that stopovers during migration may be important in allowing birds to maintain their oxidative status over the course of the entire migration (McWilliams et al. 2021). In support of this, plasma d-ROMs decreased with increasing stopover duration (0-8 nights) in Garden warblersSylvia borin , and plasma non-enzymatic antioxidant capacity increased with fat stores accumulated at a stopover site in Blackpoll Warblers Setophaga striata and Red-eyed Vireos Vireo olivaceus (Skrip et al. 2015). Importantly, after 15 days of exercise training, European starlings exposed to an acute flight further depleted their non-enzymatic antioxidant capacity without an associated increase in oxidative damage. This provides additional evidence that birds during migration seem able to rapidly adjust their antioxidant system to maintain overall low levels of circulating damage.
We also found evidence in the liver and pectoralis that flight training activated the antioxidant system so that after several weeks of daily flying the non-enzymatic antioxidant capacity was maintained at levels similar to those of untrained birds, and oxidative damage was prevented in all tissues (H1c). Repeated bouts of flight apparently protected birds against lipid damage in the liver and the pectoralis (Frawley et al. 2021a), as lipid hydroperoxide concentration was lower or similar in the liver and pectoralis, respectively, of flight-trained birds compared to untrained birds. In our companion study (citation redacted for initial review), the gene expression of CAT , SOD2 , andGPX1 were upregulated in the liver of flight-trained birds, while only SOD2 was upregulated in the pectoralis, which likely explains how lower liver damage levels in flight-trained birds was achieved. This enzymatic upregulation combined with lower damage in the liver suggests that the liver is preferentially protected compared to the pectoralis, perhaps to preserve its crucial role in processing fatty acids during flight or to protect those fatty acids from degradation. There were also no differences detected in plasma OXY or liver or pectoralis hydroxyl and peroxyl scavenging capacities between flight-trained and untrained birds after 48 hrs of rest; thus birds were able to maintain constant long-term levels of antioxidant capacity when energetically challenged and avoid increases in oxidative damage. Although long-term antioxidant capacity in the tissues was unchanged, it is possible that flight-trained birds were able to prevent the accumulation of oxidative damage by utilizing non-enzymatic antioxidant capacity in tissues during flight, as also shown for OXY in the plasma, and by increasing the gene expression of enzymatic antioxidants (citation redacted for initial review).