Selected and shared hematological responses to apnea in elite human free divers and northern elephant seals (Mirounga angustirostris).

Despite elite human free divers achieving incredible feats in competitive free diving, there has yet to be a study that compares consummate divers, (i.e. northern elephant seals) to highly conditioned free divers (i.e., elite competitive free-diving humans). Herein, we compare these two diving models and suggest that hematological traits detected in seals reflect species-specific specializations, while hematological traits shared between the two species are fundamental mammalian characteristics. Arterial blood samples were analyzed in elite human free divers (n = 14) during a single, maximal volitional apnea and in juvenile northern elephant seals (n = 3) during rest-associated apnea. Humans and elephant seals had comparable apnea durations (∼6.5 min) and end-apneic arterial Po2 [humans: 40.4 ± 3.0 mmHg (means ± SE); seals: 27.1 ± 5.9 mmHg; P = 0.2]. Despite similar increases in arterial Pco2 (humans: 33 ± 5%; seals: 16.3 ± 5%; P = 0.2), only humans experienced reductions in pH from baseline (humans: 7.45 ± 0.01; seals: 7.39 ± 0.02) to end apnea (humans: 7.37 ± 0.01; seals: 7.38 ± 0.02; P < 0.0001). Hemoglobin P50 was greater in humans compared to elephant seals (29.9 ± 1.5 and 28.7 ± 0.6 mmHg, respectively; P = 0.046). Elephant seals overall had higher carboxyhemoglobin (COHb) levels (5.9 ± 2.6%) compared to humans (0.8 ± 1.2%; P < 0.0001); however, following apnea, COHb was reduced in seals (baseline: 6.1 ± 0.3%; end apnea: 5.6 ± 0.3%) and was slightly elevated in humans (baseline: 0.7 ± 0.1%; end apnea: 0.9 ± 0.1%; P < 0.0002, both comparisons). Our data indicate that during static apnea, seals have reduced hemoglobin P50, greater pH buffering, and increased COHb levels. The differences in hemoglobin P50 are likely due to the differences in the physiological environment between the two species during apnea, whereas enhanced pH buffering and higher COHb may represent traits selected for in elephant seals.NEW & NOTEWORTHY This study uses similar methods and protocols in elite human free divers and northern elephant seals. Using highly conditioned divers (elite free-diving humans) and highly adapted divers (northern elephant seals), we explored which hematological traits are fundamentally mammalian and which may have been selected for. We found differences in P50, which may be due to different physiological environments between species, while elevated pH buffering and carbon monoxide levels might have been selected for in seals.

Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.

Improvement in Outcomes After Cardiac Arrest and Resuscitation by Inhibition of S-Nitrosoglutathione Reductase.

BACKGROUND: The biological effects of nitric oxide are mediated via protein S-nitrosylation. Levels of S-nitrosylated protein are controlled in part by the denitrosylase, S-nitrosoglutathione reductase (GSNOR). The objective of this study was to examine whether GSNOR inhibition improves outcomes after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). METHODS: Adult wild-type C57BL/6 and GSNOR-deleted (GSNOR-/-) mice were subjected to potassium chloride-induced CA and subsequently resuscitated. Fifteen minutes after a return of spontaneous circulation, wild-type mice were randomized to receive the GSNOR inhibitor, SPL-334.1, or normal saline as placebo. Mortality, neurological outcome, GSNOR activity, and levels of S-nitrosylated proteins were evaluated. Plasma GSNOR activity was measured in plasma samples obtained from post-CA patients, preoperative cardiac surgery patients, and healthy volunteers. RESULTS: GSNOR activity was increased in plasma and multiple organs of mice, including brain in particular. Levels of protein S-nitrosylation were decreased in the brain 6 hours after CA/CPR. Administration of SPL-334.1 attenuated the increase in GSNOR activity in brain, heart, liver, spleen, and plasma, and restored S-nitrosylated protein levels in the brain. Inhibition of GSNOR attenuated ischemic brain injury and improved survival in wild-type mice after CA/CPR (81.8% in SPL-334.1 versus 36.4% in placebo; log rank P=0.031). Similarly, GSNOR deletion prevented the reduction in the number of S-nitrosylated proteins in the brain, mitigated brain injury, and improved neurological recovery and survival after CA/CPR. Both GSNOR inhibition and deletion attenuated CA/CPR-induced disruption of blood brain barrier. Post-CA patients had higher plasma GSNOR activity than did preoperative cardiac surgery patients or healthy volunteers ( P<0.0001). Plasma GSNOR activity was positively correlated with initial lactate levels in postarrest patients (Spearman correlation coefficient=0.48; P=0.045). CONCLUSIONS: CA and CPR activated GSNOR and reduced the number of S-nitrosylated proteins in the brain. Pharmacological inhibition or genetic deletion of GSNOR prevented ischemic brain injury and improved survival rates by restoring S-nitrosylated protein levels in the brain after CA/CPR in mice. Our observations suggest that GSNOR is a novel biomarker of postarrest brain injury as well as a molecular target to improve outcomes after CA.

Low guanylyl cyclase activity in Weddell seals: implications for peripheral vasoconstriction and perfusion of the brain during diving.

Nitric oxide (NO) is a potent vasodilator, which improves perfusion and oxygen delivery during tissue hypoxia in terrestrial animals. The vertebrate dive response involves vasoconstriction in select tissues, which persists despite profound hypoxia. Using tissues collected from Weddell seals at necropsy, we investigated whether vasoconstriction is aided by downregulation of local hypoxia signaling mechanisms. We focused on NO-soluble guanylyl cyclase (GC)-cGMP signaling, a well-known vasodilatory transduction pathway. Seals have a lower GC protein abundance, activity, and capacity to respond to NO stimulation than do terrestrial mammals. In seal lung homogenates, GC produced less cGMP (20.1 ± 3.7 pmol·mg protein-1·min-1) than the lungs of dogs (-80 ± 144 pmol·mg protein-1·min-1 less than seals), sheep (-472 ± 96), rats (-664 ± 104) or mice (-1,160 ± 104, P < 0.0001). Amino acid sequences of the GC enzyme α-subunits differed between seals and terrestrial mammals, potentially affecting their structure and function. Vasoconstriction in diving Weddell seals is not consistent across tissues; perfusion is maintained in the brain and heart but decreased in other organs such as the kidney. A NO donor increased median GC activity 49.5-fold in the seal brain but only 27.4-fold in the kidney, consistent with the priority of cerebral perfusion during diving. Nos3 expression was high in the seal brain, which could improve NO production and vasodilatory potential. Conversely, Pde5a expression was high in the seal renal artery, which may increase cGMP breakdown and vasoconstriction in the kidney. Taken together, the results of this study suggest that alterations in the NO-cGMP pathway facilitate the diving response.

Muscular apoptosis but not oxidative stress increases with old age in a long-lived diver, the Weddell seal.

Seals experience repeated bouts of ischemia-reperfusion while diving, potentially exposing their tissues to increased oxidant generation and thus oxidative damage and accelerated aging. We contrasted markers of oxidative damage with antioxidant profiles across age and sex for propulsive (longissismus dorsi) and maneuvering (pectoralis) muscles of Weddell seals to determine whether previously observed morphological senescence is associated with oxidative stress. In longissismus dorsi, old (age 17-26 years) seals exhibited a nearly 2-fold increase in apoptosis over young (age 9-16 years) seals. There was no evidence of age-associated changes in lipid peroxidation or enzymatic antioxidant profiles. In pectoralis, 4-hydroxynonenal-Lys (4-HNE-Lys) levels increased 1.5-fold in old versus young seals, but lipid hydroperoxide levels and apoptotic index did not vary with age. Glutathione peroxidase activity was 1.5-fold higher in pectoralis of old versus young animals, but no other antioxidants changed with age in this muscle. With respect to sex, no differences in lipid hydroperoxides or apoptosis were observed in either muscle. Males had higher HSP70 expression (1.4-fold) and glutathione peroxidase activity (1.3-fold) than females in longissismus dorsi, although glutathione reductase activity was 1.4-fold higher in females. No antioxidants varied with sex in pectoralis. These results show that apoptosis is not associated with oxidative stress in aged Weddell seal muscles. Additionally, the data suggest that adult seals utilize sex-specific antioxidant strategies in longissismus dorsi but not pectoralis to protect skeletal muscles from oxidative damage.

Intrinsic anti-inflammatory properties in the serum of two species of deep-diving seal.

Weddell and elephant seals are deep-diving mammals, which rely on lung collapse to limit nitrogen absorption and prevent decompression injury. Repeated collapse and re-expansion exposes the lungs to multiple stressors, including ischemia-reperfusion, alveolar shear stress and inflammation. There is no evidence, however, that diving damages pulmonary function in these species. To investigate potential protective strategies in deep-diving seals, we examined the inflammatory response of seal whole blood exposed to lipopolysaccharide (LPS), a potent endotoxin. Interleukin-6 (IL6) cytokine production elicited by LPS exposure was 50 to 500 times lower in blood of healthy northern elephant seals and Weddell seals compared with that of healthy human blood. In contrast to the ∼6× increased production of IL6 protein from LPS-exposed Weddell seal whole blood, isolated Weddell seal peripheral blood mononuclear cells, under standard cell culture conditions using medium supplemented with fetal bovine serum (FBS), produced a robust LPS response (∼300×). Induction of Il6 mRNA expression as well as production of IL6, IL8, IL10, KC-like and TNFα were reduced by substituting FBS with an equivalent amount of autologous seal serum. Weddell seal serum also attenuated the inflammatory response of RAW 267.4 mouse macrophage cells exposed to LPS. Cortisol level and the addition of serum lipids did not impact the cytokine response in cultured cells. These data suggest that seal serum possesses anti-inflammatory properties, which may protect deep divers from naturally occurring inflammatory challenges such as dive-induced hypoxia-reoxygenation and lung collapse.

Brown adipose tissue: The heat is on the heart.

The study of brown adipose tissue (BAT) has gained significant scientific interest since the discovery of functional BAT in adult humans. The thermogenic properties of BAT are well recognized; however, data generated in the last decade in both rodents and humans reveal therapeutic potential for BAT against metabolic disorders and obesity. Here we review the current literature in light of a potential role for BAT in beneficially mediating cardiovascular health. We focus mainly on BAT's actions in obesity, vascular tone, and glucose and lipid metabolism. Furthermore, we discuss the recently discovered endocrine factors that have a potential beneficial role in cardiovascular health. These BAT-secreted factors may have a favorable effect against cardiovascular risk either through their metabolic role or by directly affecting the heart.

Androgen-sensitive hypertension associated with soluble guanylate cyclase-α1 deficiency is mediated by 20-HETE.

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.

Prioritization of skeletal muscle growth for emergence from hibernation.

Mammalian hibernators provide an extreme example of naturally occurring challenges to muscle homeostasis. The annual hibernation cycle is characterized by shifts between summer euthermy with tissue anabolism and accumulation of body fat reserves, and winter heterothermy with fasting and tissue catabolism. The circannual patterns of skeletal muscle remodelling must accommodate extended inactivity during winter torpor, the motor requirements of transient winter active periods, and sustained activity following spring emergence. Muscle volume in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) calculated from MRI upper hindlimb images (n=6 squirrels, n=10 serial scans) declined from hibernation onset, reaching a nadir in early February. Paradoxically, mean muscle volume rose sharply after February despite ongoing hibernation, and continued total body mass decline until April. Correspondingly, the ratio of muscle volume to body mass was steady during winter atrophy (October-February) but increased (+70%) from February to May, which significantly outpaced changes in liver or kidney examined by the same method. Generally stable myocyte cross-sectional area and density indicated that muscle remodelling is well regulated in this hibernator, despite vastly altered seasonal fuel and activity levels. Body composition analysis by echo MRI showed lean tissue preservation throughout hibernation amid declining fat mass by the end of winter. Muscle protein synthesis was 66% depressed in early but not late winter compared with a summer fasted baseline, while no significant changes were observed in the heart, liver or intestine, providing evidence that could support a transition in skeletal muscle regulation between early and late winter, prior to spring emergence and re-feeding.

Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels.

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3.

Intrinsic circannual regulation of brown adipose tissue form and function in tune with hibernation.

Winter hibernators repeatedly cycle between cold torpor and rewarming supported by nonshivering thermogenesis in brown adipose tissue (BAT). In contrast, summer animals are homeotherms, undergoing reproduction, growth, and fattening. This life history confers variability to BAT recruitment and activity. To address the components underlying prewinter enhancement and winter activation, we interrogated the BAT proteome in 13-lined ground squirrels among three summer and five winter states. We also examined mixed physiology in fall and spring individuals to test for ambient temperature and seasonal effects, as well as the timing of seasonal transitions. BAT form and function differ circannually in these animals, as evidenced by morphology and proteome dynamics. This intrinsic pattern distinguished homeothermic groups and early vs. late winter hibernators. Homeothermic variation derived from postemergence delay in growth and substrate biosynthesis. The heterothermic proteome varied less despite extreme winter physiological shifts and was optimized to exploit lipids by enhanced fatty acid binding, ß-oxidation, and mitochondrial protein translocation. Surprisingly, ambient temperature did not affect the BAT proteome during transition seasons; rather, the pronounced summer-winter shift preceded environmental changes and phenotypic progression. During fall transition, differential regulation of two fatty acid binding proteins provides further evidence of recruitment and separates proteomic preparation from successful hibernation. Abundance of FABP4 correlates with torpor bout length throughout the year, clarifying its potential function in hibernation. Metabolically active BAT is a target for treating human obesity and metabolic disorders. Understanding the hibernator's extreme and seasonally distinct recruitment and activation control strategies offers untapped potential to identify novel, therapeutically relevant regulatory pathways.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Fibroblasts as an experimental model system for the study of comparative physiology.

Mechanistic evaluations of processes that underlie organism-level physiology often require reductionist approaches. Dermal fibroblasts offer one such approach. These cells are easily obtained from minimally invasive skin biopsy, making them appropriate for the study of protected and/or logistically challenging species. Cell culture approaches permit extensive and fine-scale sampling regimes as well as gene manipulation techniques that are not feasible in vivo. Fibroblast isolation and culture protocols are outlined here for primary cells, and the benefits and drawbacks of immortalization are discussed. We show examples of physiological metrics that can be used to characterize primary cells (oxygen consumption, translation, proliferation) and readouts that can be informative in understanding cell-level responses to environmental stress (lactate production, heat shock protein induction). Importantly, fibroblasts may display fidelity to whole animal physiological phenotypes, facilitating their study. Fibroblasts from Antarctic Weddell seals show greater resilience to low temperatures and hypoxia exposure than fibroblasts from humans or rats. Fibroblast oxygen consumption rates are not affected by temperature stress in the heat-tolerant camel, whereas similar temperature exposures depress mitochondrial metabolism in fibroblasts from rhinoceros. Finally, dermal fibroblasts from a hibernator, the meadow jumping mouse, better resist experimental cooling than a fibroblast line from the laboratory mouse, with the hibernator demonstrating a greater maintenance of homeostatic processes such as protein translation. These results exemplify the parallels that can be drawn between fibroblast physiology and expectations in vivo, and provide evidence for the power of fibroblasts as a model system to understand comparative physiology and biomedicine.

The Antarctic Weddell seal genome reveals evidence of selection on cardiovascular phenotype and lipid handling.

The Weddell seal (Leptonychotes weddellii) thrives in its extreme Antarctic environment. We generated the Weddell seal genome assembly and a high-quality annotation to investigate genome-wide evolutionary pressures that underlie its phenotype and to study genes implicated in hypoxia tolerance and a lipid-based metabolism. Genome-wide analyses included gene family expansion/contraction, positive selection, and diverged sequence (acceleration) compared to other placental mammals, identifying selection in coding and non-coding sequence in five pathways that may shape cardiovascular phenotype. Lipid metabolism as well as hypoxia genes contained more accelerated regions in the Weddell seal compared to genomic background. Top-significant genes were SUMO2 and EP300; both regulate hypoxia inducible factor signaling. Liver expression of four genes with the strongest acceleration signals differ between Weddell seals and a terrestrial mammal, sheep. We also report a high-density lipoprotein-like particle in Weddell seal serum not present in other mammals, including the shallow-diving harbor seal.

Skeletal muscle proteomics: carbohydrate metabolism oscillates with seasonal and torpor-arousal physiology of hibernation.

The physiology of small mammalian hibernators shifts profoundly over a year, from summer homeothermy to winter heterothermy. Torpor-arousal cycles define high-amplitude tissue activity fluctuations in winter, particularly for skeletal muscle, which contributes to the energetically demanding rewarming process via shivering. To better understand the biochemistry underlying summer-winter and torpor-arousal transitions, we applied two-dimensional gel electrophoresis coupled with liquid chromatography/mass spectrometry/mas spectrometry to the soluble proteins from hindlimb muscle of 13-lined ground squirrels (Ictidomys tridecemlineatus) in two summer and six winter states. Two hundred sixteen protein spots differed by sampled state. Significantly, intrawinter protein adjustment was a minor component of the dataset despite large discrepancies in muscle activity level among winter states; rather, the bulk of differences (127/138 unequivocally identified proteins spots) occurred between summer and winter. We did not detect any proteomic signatures of skeletal muscle atrophy in this hibernator nor any differential seasonal regulation of protein metabolism. Instead, adjustments to metabolic substrate preferences dominated the detected proteomic differences. Pathways of carbohydrate metabolism (glycolysis and gluconeogenesis) were summer enriched, whereas the winter proteome was enriched for fatty acid ß-oxidation. Nevertheless, our data suggest that some reliance on carbohydrate reserves is maintained during winter. Phosphoglucomutase (PGM1), which reversibly prepares glucose subunits for either glycolysis or glycogenesis, showed apparent winter state-specific phosphorylation. PGM1 was phosphorylated during rewarming and dephosphorylated by interbout arousal, implying that glucose supplements lipid fuels during rewarming. This, along with winter elevation of TCA cycle enzymes, suggests that hindlimb muscles are primed for rapid energy production and that carbohydrates are an important fuel for shivering thermogenesis.

Dive behaviour impacts the ability of heart rate to predict oxygen consumption in Steller sea lions (Eumetopias jubatus) foraging at depth.

The predictive relationship between heart rate (f(H)) and oxygen consumption (VO2) has been derived for several species of marine mammals swimming horizontally or diving in tanks to shallow depths. However, it is unclear how dive activity affects the f(H):VO2 relationship and whether the existing equations apply to animals diving to deeper depths. We investigated these questions by simultaneously measuring the f(H) and VO2 of Steller sea lions (Eumetopias jubatus) under different activity states (surface resting or diving), types of dives (single dives or dive bouts), and depths (10 or 40 m). We examined the relationship over dives only and also over dive cycles (dive + surface interval). We found that f(H) could only predict VO2 over a complete single dive cycle or dive bout cycle (i.e. surface intervals had to be included). The predictive equation derived for sea lions resting on the surface did not differ from that for single dive cycles. However, the equation derived over dive bout cycles (multiple dives + surface intervals) differed from those for single dive cycles or surface resting, with similar f(H) for multiple dive bout equations yielding higher predicted VO2 than that for single dive bout cycles (or resting). The f(H):VO2 relationships were not significantly affected by dive duration, dive depth, water temperature or cumulative food consumed under the conditions tested. Ultimately, our results demonstrate that f(H) can be used to predict activity-specific metabolic rates of diving Steller sea lions, but only over complete dive cycles that include a post-dive surface recovery period.

Field Physiology: Studying Organismal Function in the Natural Environment.

Continuous physiological measurements collected in field settings are essential to understand baseline, free-ranging physiology, physiological range and variability, and the physiological responses of organisms to disturbances. This article presents a current summary of the available technologies to continuously measure the direct physiological parameters in the field at high-resolution/instantaneous timescales from freely behaving animals. There is a particular focus on advantages versus disadvantages of available methods as well as emerging technologies "on the horizon" that may have been validated in captive or laboratory-based scenarios but have yet to be applied in the wild. Systems to record physiological variables from free-ranging animals are reviewed, including radio (VHF/UFH) telemetry, acoustic telemetry, and dataloggers. Physiological parameters that have been continuously measured in the field are addressed in seven sections including heart rate and electrocardiography (ECG); electromyography (EMG); electroencephalography (EEG); body temperature; respiratory, blood, and muscle oxygen; gastric pH and motility; and blood pressure and flow. The primary focal sections are heart rate and temperature as these can be, and have been, extensively studied in free-ranging organisms. Predicted aspects of future innovation in physiological monitoring are also discussed. The article concludes with an overview of best practices and points to consider regarding experimental designs, cautions, and effects on animals. © 2021 American Physiological Society. Compr Physiol 11:1979-2015, 2021.

Sulfide catabolism ameliorates hypoxic brain injury.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Energetics of breath-hold hunting: modeling the effects of aging on foraging success in the Weddell seal.

A simulation model was developed to predict age-related changes in foraging energetics of individual Weddell seals, Leptonychotes weddellii. Aging in diving animals is interesting because their characteristic cyclic sequence of apneustic hunting and eupnea should elevate oxidative stress, possibly accelerating aging. Such a hypothesis can be evaluated by modeling energetics of constrained, time-partitioned activities with well-defined costs. Three possible consequences of physiological aging in divers were specified and appraised. The model examined the potential impacts of age-related decline in muscle contractile ability, increased buoyancy, and reduced aerobic dive limit, alone and in combination, on a daily energy budget. A uniform age effect evident in model outputs is reduced foraging efficiency. The components of this net effect are exacerbated for sub-optimal behavioral-response settings or environmental conditions. The model predicts that with advancing age, efficiency declines increase for aging scenarios in the following order: simulated 'young' adults; 'old' seals with increased buoyancy; 'old' seals with reduced aerobic dive limit; 'old' seals having reduced muscle contractile efficiency; and, 'old' seals with all three conditions. The model indicates narrowed behavioral options to maintain positive energy balance in older animals, suggesting that behavioral plasticity may not allow older animals to compensate for age-related performance constraints.

Chemical immobilization of Weddell seals (Leptonychotes weddellii) by ketamine/midazolam combination.

OBJECTIVE: To provide reliable, effective immobilization for Weddell seals under extreme field conditions using an injectable ketamine/midazolam combination. STUDY DESIGN: Observational study. ANIMALS: Thirty adult Weddell seals (12 male, 18 female) in Erebus Bay, Antarctica, body mass (mean +/- SD) 412 +/- 47 kg, aged 9-27 years. METHODS: Seals were immobilized with a target dose of 2 mg kg(-1) ketamine hydrochloride and 0.1 mg kg(-1) midazolam hydrochloride (IM), based on visually estimated body mass. When required, maintenance doses were administered at a target of 0.5 mg kg(-1) ketamine hydrochloride and 0.025 mg kg(-1) midazolam hydrochloride (IV). RESULTS: Complete immobilization was achieved in 33 of 40 injections (14 of which were repeat events on the same individual). Time to immobilization averaged 12 +/- 4 minutes, with a duration of initial immobility of 38 +/- 19 minutes. Total immobilization time varied by handling protocol, including condition assessment and muscle biopsy (Protocol 1, 60 +/- 13 minutes), condition assessment and instrument attachment (Protocol 2, 154 +/- 13 minutes), and condition assessment, muscle biopsy and instrument retrieval (Protocol 3, 48 +/- 8 minutes). Overall, a total immobilization time of 114 +/- 60 minutes was accomplished with 4 +/- 4 maintenance doses, and an average recovery time of 36 +/- 17 minutes. Most effects of the anesthetic combination were unrelated to mass, age, sex or total body fat. However, leaner seals had longer duration of initial immobility (% and kg total body fat) and recovery times (kg fat). Apnea events were uncommon and treated effectively with doxapram. No animals died. CONCLUSIONS AND CLINICAL RELEVANCE: Reliable and effective field immobilization of Weddell seals was accomplished with a low dose of ketamine hydrochloride and midazolam hydrochloride, utilizing IM injection initially and IV maintenance methods.