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Pinnipeds spend large portions of their lives at sea, submerged, or hauled-out on land, often on remote off-shore islands. This fundamentally limits access by researchers to critical parts of pinniped life history and has spurred the development and implementation of a variety of externally attached telemetry devices (ETDs) to collect information about movement patterns, physiology and ecology of marine animals when they cannot be directly observed. ETDs are less invasive and easier to apply than implanted internal devices, making them more widely used. However, ETDs have limited retention times and their use may result in negative short- and long-term consequences including capture myopathy, impacts to energetics, behavior, and entanglement risk. We identify 15 best practice recommendations for the use of ETDs with pinnipeds that address experimental justification, animal capture, tag design, tag attachment, effects assessments, preparation, and reporting. Continued improvement of best practices is critical within the framework of the Three Rs (Reduction, Refinement, Replacement); these best practice recommendations provide current guidance to mitigate known potential negative outcomes for individuals and local populations. These recommendations were developed specifically for pinnipeds; however, they may also be applicable to studies of other marine taxa. We conclude with four desired future directions for the use of ETDs in technology development, validation studies, experimental designs and data sharing.

Wildlife researchers must balance the need to safely capture and handle their study animals to sample tissues, collect morphological measurements, and attach dataloggers while ensuring their results are not confounded by stress artifacts caused by handling. To determine the physiological effects of research activities including chemical immobilization, transport, instrumentation with biologgers, and overnight holding on a model marine mammal species, we collected hormone, blood chemistry, hematology, and heart rate data from 19 juvenile northern elephant seals (Mirounga angustirostris) throughout a translocation experiment. Across our six sampling timepoints, cortisol and aldosterone data revealed a moderate hormonal stress response to handling accompanied by minor changes in hematocrit and blood glucose, but not ketone bodies or erythrocyte sedimentation rate. We also examined heart rate as a stress indicator and found that interval heart rate, standard deviation of heart rate, and apnea-eupnea cycles were influenced by handling. However, when seals were recaptured after several days at sea, all hormonal and hematological parameters had returned to baseline levels. Furthermore, 100 % of study animals were resighted in the wild post-translocation, with some individuals observed over four years later. Together, these findings suggest that while northern elephant seals exhibit measurable physiological stress in response to handling, they recover rapidly and show no observable long-term deleterious effects, making them a robust species for ecological and physiological research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Po 2 [humans: 40.4 ± 3.0 mmHg (means ± SE); seals: 27.1 ± 5.9 mmHg; P = 0.2]. Despite similar increases in arterial Pco 2 (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 P 50 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 P 50 , greater pH buffering, and increased COHb levels. The differences in hemoglobin P 50 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 P 50 , 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.

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat ( Rousettus aegyptiacus ) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

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.

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (Copyright © 2021 American Physiological Society. All rights reserved.)

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.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4 -/- mouse model of Leigh syndrome, continuously breathing 11% O 2 (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% O 2 . Compared to WT control mice, Ndufs4 -/- mice breathing air have reduced brain O 2 consumption as evidenced by an elevated partial pressure of O 2 in IJV blood (P ijv O 2 ) despite a normal PO 2 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 P ijv O 2 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% O 2 ) 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., Competing Interests: Declaration of Competing Interest V.K.M. and W.M.Z. are co-inventors on a patent application submitted by Massachusetts General Hospital on the use of hypoxia as a therapy. V.K.M. owns equity stake in Raze Therapeutics and is a paid advisor for Janssen Pharmaceuticals and 5 AM Ventures. R.S. holds equity in BlueBird Bio., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diving physiology has received considerable scientific attention as it is a central element of the extreme phenotype of marine mammals. Many scientific discoveries have illuminated physiological mechanisms supporting diving, such as massive, internally bound oxygen stores and dramatic cardiovascular regulation. However, the cellular and molecular mechanisms that support the diving phenotype remain mostly unexplored as logistic and legal restrictions limit the extent of scientific manipulation possible. With next-generation sequencing (NGS) tools becoming more widespread and cost-effective, there are new opportunities to explore the diving phenotype. Genomic investigations come with their own challenges, particularly those including cross-species comparisons. Studying the regulatory pathways that underlie diving mammal ontogeny could provide a window into the comparative physiology of hypoxia tolerance. Specifically, in pinnipeds, which shift from terrestrial pups to elite diving adults, there is potential to characterize the transcriptional, epigenetic, and posttranslational differences between contrasting phenotypes while leveraging a common genome. Here we review the current literature detailing the maturation of the diving phenotype in pinnipeds, which has primarily been explored via biomarkers of metabolic capability including antioxidants, muscle fiber typing, and key aerobic and anaerobic metabolic enzymes. We also discuss how NGS tools have been leveraged to study phenotypic shifts within species through ontogeny, and how this approach may be applied to investigate the biochemical and physiological mechanisms that develop as pups become elite diving adults. We conclude with a specific example of the Antarctic Weddell seal by overlapping protein biomarkers with gene regulatory microRNA datasets., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Immune responses to nitrogen gas bubbles, particularly activation of inflammation via the complement cascade, have been linked to the development of symptoms and damage associated with decompression sickness (DCS) in humans. Marine mammals were long thought not to be susceptible to such dive-related injury, yet evidence of DCS-like injury and new models of tissue nitrogen super-saturation suggest that bubbles may routinely form. As such, it is possible that marine mammals have protective adaptations that allow them to deal with a certain level of bubble formation during normal dives, without acute adverse effects. This work evaluated the complement response, indicative of inflammation, to in vitro nitrogen bubble exposures in several marine mammal species to assess whether a less-responsive immune system serves a protective role against DCS-like injury in these animals. Serum samples from beluga (Delphinapterus leucas), and harbor seals (Phoca vitulina) (relatively shallow divers) and deep diving narwhal (Monodon monoceros), and Weddell seals (Leptonychotes weddellii) were exposed to nitrogen bubbles in vitro. Complement activity was evaluated by measuring changes in the terminal protein C5a in serum, and results suggest marine mammal complement is less sensitive to gas bubbles than human complement, but the response varies between species. Species-specific differences may be related to dive ability, and suggest moderate or shallow divers may be more susceptible to DCS-like injury. This information is an important consideration in assessing the impact of changing dive behaviors in response to anthropogenic stressors, startle responses, or changing environmental conditions that affect prey depth distributions.

Marine mammals have highly specialized physiology, exhibited in many species by extreme breath-holding capabilities that allow deep dives and extended submergence. Cardiovascular control and cell-level hypoxia tolerance are key features of this phenotype. Identifying genomic signatures tied to physiology will be valuable in understanding these natural model species, which may generate translational opportunities to human diseases arising from hypoxic stress or tissue injury. Genomic analyses have now been conducted in dolphins, river dolphins, minke whales, bowhead whales, and polar bears, with multispecies studies exploring evolutionary signals across marine mammal lineages, encompassing extinct and extant divers. Single-species genome studies for sirenians do not yet exist. Extant marine mammals arose in three lineages from separate aquatic recolonizations. Their physiological specializations, along with these independent origins create an interesting case to examine convergent evolution. Although molecular mechanisms of hypoxia tolerance are not universally apparent across marine mammal genomic studies, altered evolutionary rates have been identified for genes linked to oxygen binding and transport (e.g., MB , HBA , and HBB ), blood pressure control (e.g., endothelin pathway genes), and cell protection in multiple species. Despite convergent phenotypes across clades, instances of identical molecular convergence have been uncommon. Given the inherent logistical and regulatory difficulties associated with functional genetic experiments in marine mammals, several avenues of further investigation are suggested to enable validation of candidate genes for hypoxia tolerance: leveraging phylogeny to better understand convergent phenotypes; ontogenic studies to identify regulation of key genes underlying the elite, adult, hypoxia-tolerant physiology; and cell culture manipulations to understand gene function.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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.

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.

Purpose: Glaucoma, a leading cause of blindness worldwide, often remains undetected until irreversible vision loss has occurred. Treatments focus on lowering intraocular pressure (IOP), the only modifiable and readily measurable risk factor. However, IOP can vary and does not always predict disease progression. MicroRNAs (miRNAs) are promising biomarkers. They are abundant and stable in biological fluids, including plasma and aqueous humor (AqH). We aimed to identify differentially expressed miRNAs in AqH and plasma from glaucoma, exfoliation syndrome (XFS), and control subjects., Methods: Plasma and AqH from two ethnic cohorts were harvested from glaucoma or XFS (often associated with glaucoma, n = 33) and control (n = 31) patients undergoing elective surgery. A custom miRNA array measured 372 miRNAs. Molecular target prediction and pathway analysis were performed with Ingenuity Pathway Analysis (IPA) and DIANA bioinformatical tools., Results: Levels of miRNAs in plasma, a readily accessible biomarker source, correlated with miRNA levels in AqH. Twenty circulating miRNAs were at least 1.5-fold higher in glaucoma or XFS patients than in controls across two ethnic cohorts: miR-4667-5p (P = 4.1 × 10-5), miR-99b-3p (P = 4.8 × 10-5), miR-637 (P = 5.1 × 10-5), miR-4490 (P = 5.7 × 10-5), miR-1253 (P = 6.0 × 10-5), miR-3190-3p (P = 3.1 × 10-4), miR-3173-3p (P = 0.001), miR-608 (P = 0.001), miR-4725-3p (P = 0.002), miR-4448 (P = 0.002), and miR-323b-5p (P = 0.002), miR-4538 (P = 0.003), miR-3913-3p (P = 0.003), miR-3159 (P = 0.003), miR-4663 (P = 0.003), miR-4767 (P = 0.003), miR-4724-5p (P = 0.003), miR-1306-5p (P = 0.003), miR-181b-3p (P = 0.004), and miR-433-3p (P = 0.004). miR-637, miR-1306-5p, and miR-3159, in combination, allowed discrimination between glaucoma patients and control subjects (AUC = 0.91 ± 0.008, sensitivity 85.0%, specificity 87.5%)., Conclusions: These results identify specific miRNAs as potential biomarkers and provide insight into the molecular processes underlying glaucoma.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Marine mammals are characterized as having physiological specializations that maximize the use of oxygen stores to prolong time spent under water. However, it has been difficult to undertake the requisite controlled studies to determine the physiological limitations and trade-offs that marine mammals face while diving in the wild under varying environmental and nutritional conditions. For the past decade, Steller sea lions (Eumetopias jubatus) trained to swim and dive in the open ocean away from the physical confines of pools participated in studies that investigated the interactions between diving behaviour, energetic costs, physiological constraints, and prey availability. Many of these studies measured the cost of diving to understand how it varies with behaviour and environmental and physiological conditions. Collectively, these studies show that the type of diving (dive bouts or single dives), the level of underwater activity, the depth and duration of dives, and the nutritional status and physical condition of the animal affect the cost of diving and foraging. They show that dive depth, dive and surface duration, and the type of dive result in physiological adjustments (heart rate, gas exchange) that may be independent of energy expenditure. They also demonstrate that changes in prey abundance and nutritional status cause sea lions to alter the balance between time spent at the surface acquiring oxygen (and offloading CO 2 and other metabolic by-products) and time spent at depth acquiring prey. These new insights into the physiological basis of diving behaviour further our understanding of the potential scope for behavioural responses of marine mammals to environmental changes, the energetic significance of these adjustments, and the consequences of approaching physiological limits.

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., (Copyright © 2016 the American Physiological Society.)

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., (Copyright © 2016 the American Physiological Society.)

The broad phylogenetic distribution and rapid phenotypic transitions of mammalian hibernators imply that hibernation is accomplished by differential expression of common genes. Traditional candidate gene approaches have thus far explained little of the molecular mechanisms underlying hibernation, likely due to (1) incomplete and imprecise sampling of a complex phenotype, and (2) the forming of hypotheses about which genes might be important based on studies of model organisms incapable of such dynamic physiology. Unbiased screening approaches, such as proteomics, offer an alternative means to discover the cellular underpinnings that permit successful hibernation and may reveal previously overlooked, important pathways. Here, we review the findings that have emerged from proteomics studies of hibernation. One striking feature is the stability of the proteome, especially across the extreme physiological shifts of torpor-arousal cycles during hibernation. This has led to subsequent investigations of the role of post-translational protein modifications in altering protein activity without energetically wasteful removal and rebuilding of protein pools. Another unexpected finding is the paucity of universal proteomic adjustments across organ systems in response to the extreme metabolic fluctuations despite the universality of their physiological challenges; rather each organ appears to respond in a unique, tissue-specific manner. Additional research is needed to extend and synthesize these results before it will be possible to address the whole body physiology of 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., (© 2015. Published by The Company of Biologists Ltd.)

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., (Copyright © 2014 the American Physiological Society.)

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, β-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.

13-lined ground squirrels, Ictidomys tridecemlineatus, are obligate hibernators that transition annually between summer homeothermy and winter heterothermy - wherein they exploit episodic torpor bouts. Despite cerebral ischemia during torpor and rapid reperfusion during arousal, hibernator brains resist damage and the animals emerge neurologically intact each spring. We hypothesized that protein changes in the brain underlie winter neuroprotection. To identify candidate proteins, we applied a sensitive 2D gel electrophoresis method to quantify protein differences among forebrain extracts prepared from ground squirrels in two summer, four winter and fall transition states. Proteins that differed among groups were identified using LC-MS/MS. Only 84 protein spots varied significantly among the defined states of hibernation. Protein changes in the forebrain proteome fell largely into two reciprocal patterns with a strong body temperature dependence. The importance of body temperature was tested in animals from the fall; these fall animals use torpor sporadically with body temperatures mirroring ambient temperatures between 4 and 21°C as they navigate the transition between summer homeothermy and winter heterothermy. Unlike cold-torpid fall ground squirrels, warm-torpid individuals strongly resembled the homeotherms, indicating that the changes observed in torpid hibernators are defined by body temperature, not torpor per se. Metabolic enzymes were largely unchanged despite varied metabolic activity across annual and torpor-arousal cycles. Instead, the majority of the observed changes were cytoskeletal proteins and their regulators. While cytoskeletal structural proteins tended to differ seasonally, i.e., between summer homeothermy and winter heterothermy, their regulatory proteins were more strongly affected by body temperature. Changes in the abundance of various isoforms of the microtubule assembly and disassembly regulatory proteins dihydropyrimidinase-related protein and stathmin suggested mechanisms for rapid cytoskeletal reorganization on return to euthermy during torpor-arousal cycles.

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 β-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.

Apneustic hunters such as diving mammals exploit body oxygen stores while submerged; therefore, any decline in oxygen handling at advanced life stages could critically impair foraging ability. We calculated the aerobic dive limit (cADL = 17.9 ± 4.4  min SD) from blood and muscle oxygen stores and published metabolic rates of Weddell seals within (9-16 years, n = 24) and beyond peak-reproductive age (17-27 years, n = 26), to investigate (1) senescent constraints in apneustic hunting, and (2) whether mass or age primarily determines oxygen stores and ADL in older seals. We compared cADL with behavioral ADL from 5,275 free-ranging dives (bADL = 24.0 ± 5.3 min, n = 18 females). We observed no changes in Weddell seal oxygen stores, its determinants, or in ADLs late in life. Oxygen stores were better predicted by mass than age, consistent with published findings for young adults. Hematological panels (n = 6) were consistent across mass and age, though hematocrit (females > males, 6% elevation) and mean corpuscular hemoglobin content (females < males, 8% reduction) varied by sex. Whole blood viscosity was decreased with increasing mass in females and was higher than in males overall (+18%). This was largely due to elevated hematocrit in females, although plasma viscosity also varied under some conditions. Females had higher blood volume and elevated blood oxygen stores (vol% body mass), which did not translate into significantly higher cADL (18.1 vs. 17.1 min for males). Neither cADL nor bADL were mass- or age-dependent., (© 2011 Wiley Periodicals, Inc.)

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.

Weddell seals (Leptonychotes weddellii) are large-bodied, long-lived mammals that live only in the circumpolar Antarctic. As such, they represent an ideal sentinel species for the impacts of environmental change on polar species. However, longitudinal physiological studies of marine mammals are notoriously difficult due to their largely aquatic lifestyle, such that many baseline indicators of health and condition are not well defined. Fifty known-age, adult seals (9-27 years, 24 males, 26 females) in McMurdo Sound were sampled for mass, total body fat, blubber depth and a suite of blood parameters (21 variables) to assess hydration state, nutritional plane, reproductive hormones (females only), organ function and immune status. Total body mass increased with age, whereas variation in blubber depth was best described by mass and/or sex, with an overall greater blubber thickness in female seals (p<0.001). Ten blood parameters showed a significant effect of one or more model variables, most prominently between the sexes. Serum chemistry suggested that females were better nourished and displayed lower indicators of acute stress compared to males. We noted limited age-related declines that might indicate impaired organ function. Overall, seals demonstrated clinically normal values for large mammals, even at the upper end of the age continuum. Future physiological studies in this species are strongly encouraged to incorporate potential sex effects in experimental design and analysis., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The ability to use heart rate (fh) to predict oxygen consumption rates ([Formula: see text]) in Steller sea lions and other pinnipeds has been investigated in fasting animals. However, it is unknown whether established fh:[Formula: see text] relationships hold under more complex physiological situations, such as when animals are feeding or digesting. We assessed whether fh could accurately predict [Formula: see text] in trained Steller sea lions while fasting and after being fed. Using linear mixed-effects models, we derived unique equations to describe the fh:[Formula: see text] relationship for fasted sea lions resting on land and in water. Feeding did not significantly change the fh:[Formula: see text] relationship on land. However, Steller sea lions in water displayed a different fh:[Formula: see text] relationship after consuming a 4-kg meal compared with the fasting condition. Incorporating comparable published fh:[Formula: see text] data from Steller sea lions showed a distinct effect of feeding after a 6-kg meal. Ultimately, our study illustrated that both feeding and physical environment are statistically relevant when deriving [Formula: see text] from telemetered fh, but that only environment affects the practical ability to predict metabolism from fh. Updating current bioenergetic models with data gathered using these predictive fh:[Formula: see text] equations will yield more accurate estimates of metabolic rates of free-ranging Steller sea lions under a variety of physiological, behavioral, and environmental states.

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., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Red-toothed shrews (Soricidae, subfamily Soricinae) are an intriguing model system to examine the free-radical theory of aging in wild mammals, given their short (<18months) lifespan and high mass-specific metabolic rates. As muscle performance underlies both foraging ability and predator avoidance, any age-related decline should be detrimental to fitness and survival. Muscle samples of water shrews (Sorex palustris) and sympatrically distributed short-tailed shrews (Blarina brevicauda) were therefore assessed for oxidative stress markers, protective antioxidant enzymes and apoptosis. Activity levels of catalase and glutathione peroxidase increased with age in both species. Similarly, Cu,Zn-superoxide dismutase isoform content was elevated significantly in older animals of both species (increases of 60% in the water shrew, 25% in the short-tailed shrew). Only one oxidative stress marker (lipid peroxidation) was age-elevated; the others were stable or declined (4-hydroxynonenal adducts and dihydroethidium oxidation). Glutathione peroxidase activity was significantly higher in the short-tailed shrew, while catalase activity was 2x higher in water shrews. Oxidative stress indicators were on average higher in short-tailed shrews. Apoptosis occurred in <1% of myocytes examined, and did not increase with age. Within the constraints of the sample size we found evidence of protection against elevated oxidative stress in wild-caught shrews., (2010 Elsevier Inc. All rights reserved.)

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.

Red-toothed (soricine) shrews are consummate predators exhibiting the highest energy turnovers and shortest life spans (ca. 18 months) of any mammal, yet virtually nothing is known regarding their physiological aging. We assessed the emerging pattern of skeletal muscle senescence (contractile/connective tissue components) in sympatric species, the semi-aquatic water shrew (WS), Sorex palustris, and the terrestrial short-tailed shrew (STS), Blarina brevicauda, to determine if muscle aging occurs in wild, short-lived mammals (H(0): shrews do not survive to an age where senescence occurs), and if so, whether these alterations are species-specific. Gracilis muscles were collected from first-year (n=17) and second-year (n=17) field-caught shrews. Consistent with typical mammalian aging, collagen content (% area) increased with age in both species (S. palustris: approximately 50%; B. brevicauda: approximately 60%). Muscle was dominated by stiffer Type I collagen, and the ratio of collagen Type I:Type III more than doubled with age. The area ratio of muscle:collagen decreased with age in both species, but was considerably lower in adult STS, suggesting species-specificity of senescence. Extracellular space was age-elevated in B. brevicauda, but was preserved in S. palustris ( approximately 50 vs. 10% elevation). Though juvenile interspecific comparisons revealed no significance, adult WS myocytes had 68% larger cross-sectional area and occurred at 28% lower fibers/area than those of adult STS. We demonstrate that age-related muscle senescence does occur in wild-caught, short-lived mammals, and we therefore reject this classic aging theory tenet. Our findings moreover illustrate that differential age adjustments in contractile/connective tissue components of muscle occur in the two species of wild-caught shrews., ((c) 2009 Wiley-Liss, Inc.)

Classic aging theory postulates the absence of pronounced organismal senescence in wild animals since mortality probably occurs first. Large-bodied, long-lived mammals are a recognized exception to this tenet, yet organismal senescence has not been investigated to date in such mammals in the wild. Furthermore, oxidative stress theory of aging supports the suggestion that exercise hypoxia, as regularly incurred during apneustic foraging in diving mammals might lead to cellular dysfunction and accelerated aging. To determine if an aspect of organismal senescence occurs in wild marine mammals, we examined the pattern of skeletal muscle aging (contractile and connective tissue components of longissimus dorsi and pectoralis muscles) in free-ranging adult Weddell seals (9-26 years). The average myocyte cross-sectional area was 22% greater with age in the longissiums dorsi, but no significant increase occurred in the pectoralis. Cross-sectional area was not related to body mass. Changes in myocyte number per area were consistent with the 35-40% age-increase in extracellular space in both muscle groups. Also consistent with extracellular space remodeling, total and relative collagen contents were significantly elevated in older seals (115% in longissimus dorsi; 65% in pectoralis). The ratio of muscle myocyte to collagen declined with age (50-63%) at both sites. Additionally, a shift towards a higher ratio of type I to type III collagen occurred with advancing age in both muscle groups (79% increase in pectoralis; 49% in longissimus dorsi). We reject the classic tenet and null-hypothesis that Weddell seals do not survive to an age where muscular senescence becomes detectable.

We tested the hypothesis that immersion hypothermia enhances the diving capabilities of adult and juvenile muskrats by reducing rates of oxygen consumption (V O2). Declines in abdominal body temperature (T(b)) comparable to those observed in nature (0.5-3.5 degrees C) were induced by pre-chilling animals in 6 degrees C water. Pre-chilling did not reduce diving V O2 of any animal tested in 10 degrees C or 30 degrees C water, irrespective of the nature of the dive. Most behavioural indices of dive performance, including average and cumulative dive times, were unaffected by T(b) reduction in adults, but depressed in hypothermic juveniles (200-400 g). Hypothermia reduced diving heart rate only on short (<25s) dives (16% reduction, P=0.01), but did not affect the temporal onset of diving bradycardia. Post-immersion V O2 was higher for pre-chilled than for normothermic muskrats, but the difference became insignificant on longer (>90 s) dives. Our findings suggest that the mild hypothermia experienced by muskrats in nature has minimal effect on diving and post-immersion metabolic costs, and thus has little impact on the dive performance of this northern semi-aquatic mammal.