Haemolysis overestimates plasma oxidative stress biomarkers in free-ranging roe deer

Quantifying oxidative stress has garnered extensive interest in evolutionary ecology and physiology since proposed as a mediator of life histories. However, while the theoretical framework of oxidative stress ecology is well-supported by laboratory-based studies, results obtained in wild populations on oxidative damage and antioxidant biomarkers have shown inconsistent trends. We propose that red blood cell lysis could be a source of bias affecting measurements of oxidative stress biomarkers, distorting the conclusions drawn from them. Using an experimental approach consisting of enriching plasma from roe deer with lysed red blood cells, we show that the values of commonly used oxidative stress biomarkers linearly increase with the degree of haemolysis - assayed by haemoglobin concentration. This result concerns oxidized proteins (carbonyls) and lipids (TBARS), as well as enzymatic (superoxide dismutase) and non-enzymatic (trolox assay, OXY assay) antioxidant markers. Based on 707 roe deer blood samples collected in the field, we next show that the occurrence of haemolysis in plasma samples is negatively related to age. Finally, we illustrate that considering the variance explained by age-related haemolysis improves explanatory models for inter-individual variability in plasma oxidative stress biomarkers, without substantially altering the estimates of the parameters studied here. Our results raise the question of the veracity of the conclusions if the degree of haemolysis in plasma is not considered in animal models such as roe deer, for which the occurrence and severity of haemolysis vary according to individual characteristics. We recommend measuring and controlling for the degree of haemolysis be considered in future studies that investigate the causes and consequences of oxidative stress in ecophysiological studies.