A whole blood approach improves speed and accuracy when measuring mitochondrial respiration in intact avian hematocytes

Abstract

ABSTRACTUnderstanding mitochondrial biology and pathology is key to understanding the evolution of animal form and function. However, mitochondrial measurement often involves invasive, or even terminal, sampling, which can be difficult to reconcile in wild models or in longitudinal studies. Non-mammal vertebrates contain mitochondria in their red blood cells, which can be exploited for minimally invasive mitochondrial measurement. Several recent bird studies have measured mitochondrial function using isolated blood cells. Isolation adds time in the laboratory and might be associated with physiological complications. Inference may also be constrained on biological grounds by lack of tissue context. We developed and validated a protocol to measure mitochondrial respiration in bird whole blood. Endogenous respiration was comparable between isolated blood cells and whole blood. However, oxidative respiration was higher in whole blood, and whole blood mitochondria were better coupled and had higher maximum working capacity. Whole blood measurement was also more reproducible than measurement on isolated cells for all traits considered. Measurements were feasible over a 10-fold range of sample volumes, though both small and large volumes were associated with changes to respiratory traits. The protocol was compatible with long-term storage: after 24 h at 5 °C without agitation all respiration traits but maximum working capacity remained unchanged, the latter decreasing by 14%. Our study suggests that whole blood measurement provides faster, more reproducible, and more biologically (tissue context) and physiologically (mitochondrial integrity) relevant assessment of mitochondrial respiration. We recommend future studies to take a whole blood approach unless specific circumstances require the use of isolated blood cells.