Effects of storage conditions on oxidative stress biomarkers: methodological implications for ecological and evolutionary studies

Understanding oxidative stress in ecological and evolutionary contexts requires reliable biomarker quantification across taxa, tissues and experimental setups. However, storage conditions such as temperature and duration may bias these measurements. Here, we evaluated the stability of oxidative stress biomarkers, including three antioxidant enzymes (superoxide dismutase, glutathione reductase, glutathione peroxidase) and a lipid peroxidation marker (malondialdehyde) in amphibian, mammal, bird and insect samples stored under various temperature conditions (-80, -20, 4°C) from a few hours to 8 months. Storage significantly affected biomarker values depending on the marker, tissue and taxon. Notably, even long-term storage at -80°C altered some markers. In insect samples, lipid peroxidation was also influenced by triglyceride levels, indicating a potential confounding factor. Our results highlight the need to consider storage effects in oxidative stress studies. We also provide practical recommendations, aiming to improve data reliability across field and laboratory eco-evolutionary studies, as well as biomedical contexts.