Hepatic Mitochondrial Isolation Protocol in the Seahorse Hippocampus erectus Using Differential Centrifugation v1

Mitochondrial isolation is a critical step in respirometry studies that aim to elucidate cellular bioenergetics and mitochondrial function. In aquatic organisms such as fish, measurements of mitochondrial respiration offer valuable insights into how physiological performance is shaped by environmental factors such as temperature changes and oxygen availability. The liver, with its high metabolic activity and mitochondrial density, is an optimal tissue for these analyses. Here, we describe a protocol for isolating hepatic mitochondria from Hippocampus erectus, a tropical seahorse species of ecological interest. Approximately 0.2 g of liver tissue was collected from individuals weighing 12.26 ± 0.25 g, resulting in mitochondrial suspensions with protein concentrations of 6 - 10 mg/mL. At these concentrations, oxygen flux can be reliably measured using high-resolution respirometry (HRR) with the OxygraphTM O2k (Oroboros Instruments, Innsbruck, Austria), which has a minimum detectable flux of approximately 5 pmol O₂·s⁻¹·mL⁻¹. The protocol covers tissue dissection, HRR system calibration, differential centrifugation for mitochondrial isolation, and subsequent measurement of mitochondrial respiration. This work provides essential methodological and physiological insights that advance the growing field of bioenergetic research in non-model marine species.