Dietary fat quality did not affect oxidative status (H2)
Our study does not provide evidence to support H2 that migratory songbirds fed diets composed of more 18:2n-6 are more susceptible to oxidative damage and thus must increase antioxidant capacity compared to when fed diets with less 18:2n-6. This hypothesis was informed by the biochemistry and oxidative susceptibility of PUFA (Halliwell and Gutteridge 2007, Skrip and McWilliams 2016) and the demonstrated responsiveness of the antioxidant system in migratory birds (Jenni-Eiermann et al. 2014, Skrip et al. 2015, Dick and Guglielmo 2019). Additionally, 18:2n-6 has been shown to stimulate antioxidant enzymes in fish (Li et al. 2013, Zengi̇n and Yilmaz 2016) but not in rats (Tou et al. 2011). Dietary long-chain n-3 or n-6 PUFA did not affect oxidative damage or enzymatic antioxidants in the pectoralis of Yellow-rumped warblers (Dick and Guglielmo 2019), and a companion study (citation redacted for initial review) demonstrated that antioxidant gene expression and enzyme activities (GPx, SOD, CAT) did not increase in these same starlings consuming more 18:2n-6. The lack of change in oxidative parameters among diets suggest that dietary 18:2n-6 composition did not oxidatively challenge birds even after flight training. It is possible that a bird’s antioxidant system is equipped to combat reactive species associated with consuming any amount of PUFA which would explain the lack of differences among diet groups. For example, White-throated sparrows (Zonotrichia albicollis ) fed diets with more 18:2n-3 and 18:2n-6 PUFA had higher levels of d-ROMs, but similar ratios of oxidative damage: antioxidant capacity (Alan and McWilliams 2013), and Common blackbirds (Turdus merula ) caught during migration had higher circulating total n-3 and n-6 PUFAs and had higher non-enzymatic antioxidant capacity and similar damage levels compared to resident birds (Eikenaar et al. 2017, Jensen et al. 2020). Alternatively, Common blackbirds may instead mediate total unsaturation levels in their diet and hence fat stores to minimize the oxidative challenge of consuming more PUFA (Jensen et al. 2020) rather than relying on their antioxidant system for protection. A companion study revealed that increased 18:2n-6 elevated the eicosanoid hormone prostacyclin which mediates potential downstream effects on energetics and aerobic performance, and this could be another alternative explanation for the lack of an effect of dietary PUFA on oxidative parameters (citation redacted for initial review). In sum, lipid peroxidation likely remains a relevant challenge for migratory songbirds that rely on 18:2n-6 and other fats to fuel migratory flights (Pierce et al. 2004, Pierce and McWilliams 2005, Price et al. 2008, Smith and McWilliams 2010), and birds seem able to modulate their antioxidant system in response to the oxidative challenges fats pose and thereby successfully protect against damage. Whether or not these adjustments to the antioxidant system have metabolic tradeoffs or tradeoffs with immunity remains unknown (Costantini 2019). Thus, future studies that compare the effect of different amounts of dietary 18:2n-6 on a migratory bird’s physiological status, (e.g. oxidative, metabolic, and immune statuses) will better elucidate the direct effects of dietary 18:2n-6 on the potential tradeoffs among physiological systems.