Dietary antioxidants increased plasma lipid damage after flight (H3)
We also did not find support for H3 that migratory songbirds fed dietary anthocyanins have increased non-enzymatic antioxidant capacity, and lower levels of lipid damage in all three tissues compared to songbirds not fed anthocyanins. Anthocyanin supplementation did not affect non-enzymatic antioxidant capacity in the plasma or the two metabolic tissues. The same dietary anthocyanin concentration positively affected immune function in European blackcaps (Catoni et al. 2008, Schaefer et al. 2008), attenuated the cortisone response to flight in European starlings (Casagrande et al. 2020), increased testosterone levels and breeding behaviors in male European starlings (Carbeck et al. 2018), and affected enzymatic antioxidant activity and lipid damage in the heart, non-enzymatic antioxidant capacity in the pectoralis, and protein damage in leg muscle (Frawley et al. 2021a). Thus, we were surprised to find that the same concentration of anthocyanins did not directly affect antioxidant capacity as measured in this study. Perhaps when dietary anthocyanins are available they are used in a tissue-specific manner for these other functions (immunity, metabolic protection) rather than directly for reactive species mitigation which then may allow any energy-cost savings to be invested in other aspects of the antioxidant system including, for example, enzymatic antioxidants or glutathione (the precursor to GPx) that are not detected in the OXY measurement. There is considerable cross-talk among inflammatory, immune, metabolic, and antioxidant pathways so that simultaneously measuring these multiple pathways seems necessary to understand potential trade-offs in response to dietary antioxidants (Costantini 2019).
Contrary to hypothesis 3, we found that d-ROM levels immediately after acute flight were lower in birds consuming diets without anthocyanins compared to birds supplemented with antioxidants. There are two potential explanations for this result. First, dietary anthocyanins prevented the accumulation of reactive species used to stimulate the endogenous antioxidant system, resulting in more oxidative damage. This preventative effect of consuming daily antioxidants (vitamin E and C in this case) decreased the expression of antioxidant enzymes and transcription factors in the skeletal muscle of humans (Ristow et al. 2009, Merry and Ristow 2016). Second, birds not consuming anthocyanins were more oxidatively challenged during flight which required them to upregulate endogenous antioxidants in red blood cells to reduce circulating damage. The circulating enzymatic antioxidant system can respond rapidly; for example, GPx activity increases in European robins during nocturnal migration and in zebra finches during experimentally-imposed 2-hr daily flights (Jenni-Eiermann et al. 2014, Cooper-Mullin et al. 2019), respectively. Given that water-soluble antioxidants such as anthocyanins are likely utilized as they are metabolized rather than stored for later use, as are lipid-soluble dietary antioxidants (Halliwell and Gutteridge 2007), perhaps this short half-life may be why we detected an antioxidant effect in the plasma in direct response to an acute oxidative challenge rather than in plasma or tissues after recovery from flight. We provide evidence that dietary anthocyanins influenced oxidative damage after an energetic challenge, yet to untangle the mechanistic underpinnings of this relationship, future studies that measure multiple antioxidant classes and evaluate the crosstalk among various physiological pathways are required.