High-throughput mitochondrial aerobic metabolism measurement: assessing a plate-reader approach using avian blood cells

Mitochondria power biological processes, driving interest in measuring mitochondrial aerobic metabolism to understand individual variation in performance and fitness. High-resolution respirometry (HRR) is the gold standard for its sensitivity and versatility, but is time-consuming and low throughput, limiting large-scale studies. We assessed the validity of a high-throughput, fluorescence-based plate reader by comparing it to HRR. In a first experiment, we measured mitochondrial respiration in blood cells from two bird species (quails and chicken) across three temperatures (hypo-, normo-, and hyperthermia). The two methods were moderately correlated and revealed similar species effects; however, temperature effects differed between approaches, particularly under hyperthermic conditions, indicating that the plate-reader approach is best suited to stable, normothermic conditions. In a second experiment on sex differences in quail at normothermia, the two methods agreed quantitatively for proton leak and oxidative control efficiency and qualitatively for oxidative phosphorylation, although the plate-reader estimate was marginally significant (p=0.060 vs. p=0.046 for HRR). The plate reader's main limitation is lower sensitivity and greater measurement error, which could be offset by increasing sample sizes. Following protocol optimisation for each study system, this approach may offer a useful high-throughput complement to HRR for studies prioritising larger sample sizes.