A novel Seahorse XF approach to estimate how marine and terrestrial mammals respond to hypoxia

In seals, the robust dive response (bradycardia and peripheral vasoconstriction) helps to redirect blood flow to vital organs, while terrestrial species increase local blood flow to peripheral tissues. Because kidneys undergo significant vasoconstriction when seals are submerged, we utilized kidney epithelial cells to investigate the effect of hypoxia among species with distinct diving behaviors (shallow divers: bottlenose dolphin (Tursiops truncatus), shallow/medium depth divers: California sea lion (Zalophus californianus), deep diver: Weddell seal (Leptonychotes weddellii)) and terrestrial mammals (cactus mice (Peromyscus eremicus), lowland deer mice (Peromyscus maniculatus), humans (Homo sapiens), and chipmunks (Tamias amoenus)). We leveraged the transient sealed microchamber formed by the sensor probe during Seahorse XF measurement steps to manipulate the hypoxia exposure. The normal oxygen consumption in sealed wells generates a hypoxic environment by extending the measurement time to one hour. With this novel approach, we aim to investigate whether marine and terrestrial mammals exhibit distinct bioenergetic signatures upon transient hypoxia exposure entirely within the Seahorse XF analyzer. Hypoxia exposure significantly increases basal mitochondrial respiration rates in all species examined, suggesting increased metabolic demand. Increased ATP demand possibly indicates enhanced cellular stress in response to hypoxia. Mitochondrial coupling efficiency (mitochondria’s efficiency of converting energy to ATP) consistently decreases following hypoxia in all species, suggesting increased proton leak (oxygen consumption not coupled to ATP production) and/or a reduction in maximal respiration (maximal mitochondrial capacity to transport and oxidize substrates). Proton leak consistently increased across all species after hypoxia exposure, suggesting distinct adaptive processes. Weddell seals and lowland deer mice show a significant increase in maximal respiration, implying preserved ETC efficiency. In cells from sea lions, dolphins, and cactus mice, the increase in proton leak is not followed by a significant change in maximal respiration, resulting in decreased coupling efficiencies. In human and chipmunk cells, maximal respiration is decreased, indicative of ETC impairment, and together with increased membrane proton permeability, possibly resulting in lower ATP supply for cellular functions. Notably, however, resting ATP-linked respiration (oxygen consumption coupled with ATP production), shows no significant difference between normoxia and hypoxia across species, with the exception of increases in human cells (F 1 , 10 =8.53, p=0.0149). These results indicate that human cells after hypoxia exposure have higher demands of ATP. Overall, our results show a gradient of mitochondrial resilience where Weddell seals and lowland deer mice exhibit a rapid mitochondrial recovery, sea lion, dolphin, and cactus mice show partial recovery, and humans and chipmunks appear more susceptible to hypoxia and reoxygenation stress. Funded by NSF 2020706 and Company of Biologists JEBTF25051907 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.