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.