Fitness correlates of blubber oxidative stress and cellular defences in grey seals (Halichoerus grypus): support for the life-history-oxidative stress theory from an animal model of simultaneous lactation and fasting.

Life-history-oxidative stress theory predicts that elevated energy costs during reproduction reduce allocation to defences and increase cellular stress, with fitness consequences, particularly when resources are limited. As capital breeders, grey seals are a natural system in which to test this theory. We investigated oxidative damage (malondialdehyde (MDA) concentration) and cellular defences (relative mRNA abundance of heat shock proteins (Hsps) and redox enzymes (REs)) in blubber of wild female grey seals during the lactation fast (n = 17) and summer foraging (n = 13). Transcript abundance of Hsc70 increased, and Nox4, a pro-oxidant enzyme, decreased throughout lactation. Foraging females had higher mRNA abundance of some Hsps and lower RE transcript abundance and MDA concentrations, suggesting they experienced lower oxidative stress than lactating mothers, which diverted resources into pup rearing at the expense of blubber tissue damage. Lactation duration and maternal mass loss rate were both positively related to pup weaning mass. Pups whose mothers had higher blubber glutathione-S-transferase (GST) expression at early lactation gained mass more slowly. Higher glutathione peroxidase (GPx) and lower catalase (CAT) were associated with longer lactation but reduced maternal transfer efficiency and lower pup weaning mass. Cellular stress, and the ability to mount effective cellular defences, could proscribe lactation strategy in grey seal mothers and thus affect pup survival probability. These data support the life-history-oxidative stress hypothesis in a capital breeding mammal and suggest lactation is a period of heightened vulnerability to environmental factors that exacerbate cellular stress. Fitness consequences of stress may thus be accentuated during periods of rapid environmental change.

Consequences of in vitro benzyl butyl phthalate exposure for blubber gene expression and insulin-induced Akt activation in juvenile grey seals.

Plastic and plasticiser pollution of marine environments is a growing concern. Although phthalates, one group of plasticisers, are rapidly metabolised by mammals, they are found ubiquitously in humans and have been linked with metabolic disorders and altered adipose function. Phthalates may also present a threat to marine mammals, which need to rapidly accumulate and mobilise their large fat depots. High molecular weight (HMW) phthalates may be most problematic because they can accumulate in adipose. We used blubber explants from juvenile grey seals to examine the effects of overnight exposure to the HMW, adipogenic phthalate, benzyl butyl phthalate (BBzP) on expression of key adipose-specific genes and on phosphorylation of Akt in response to insulin. We found substantial differences in transcript abundance of Pparγ, Insig2, Fasn, Scd, Adipoq and Lep between moult stages, when animals were also experiencing differing mass changes, and between tissue depths, which likely reflect differences in blubber function. Akt abundance was higher in inner compared to outer blubber, consistent with greater metabolic activity in adipose closer to muscle than skin, and its phosphorylation was stimulated by insulin. Transcript abundance of Pparγ and Fasn (and Adipoq in some animals) were increased by short term (30 min) insulin exposure. In addition, overnight in vitro BBzP exposure altered insulin-induced changes in Pparγ (and Adipoq in some animals) transcript abundance, in a tissue depth and moult stage-specific manner. Basal or insulin-induced Akt phosphorylation was not changed. BBzP thus acted rapidly on the transcript abundance of key adipose genes in an Akt-independent manner. Our data suggest phthalate exposure could alter seal blubber development or function, although the whole animal consequences of these changes are not yet understood. Knowledge of typical phthalate exposures and toxicokinetics would help to contextualise these findings in terms of phthalate-induced metabolic disruption risk and consequences for marine mammal health.

Partial pressure of oxygen in adipose tissue and its relationship with fatness in a natural animal model of extreme fat deposition, the grey seal.

Excessive adiposity is associated with altered oxygen tension and comorbidities in humans. In contrast, marine mammals have high adiposity with no apparent detrimental effects. However, partial pressure of oxygen (Po2 ) in their subcutaneous adipose tissue (blubber) and its relationship with fatness have not been reported. We measured Po2 and temperature at different blubber depths in 12 healthy juvenile grey seals. Fatness was estimated from blubber thickness and morphometric parameters. Simultaneously, we monitored breathing pattern; heart rate and arterial blood saturation with a pulse oximeter; and relative changes in total hemoglobin, deoxyhemoglobin, and oxyhemoglobin in blubber capillaries using near-infrared spectroscopy (NIRS) as proxies for local oxygenation changes. Blubber Po2 ranged from 14.5 to 71.4 mmHg (39.2 ± 14.1 mmHg), which is similar to values reported in other species. Blubber Po2 was strongly and negatively associated with fatness (LME: p < 0.0001, R2marginal = 0.53, R2conditional = 0.64, n = 10), but not with blubber depth. No other parameters explained variability in Po2 , suggesting arterial blood and local oxygen delivery did not vary within and between measurements. The fall in blubber Po2 with increased fatness in seals is consistent with other animal models of rapid fat deposition. However, the Po2  levels at which blubber becomes hypoxic and consequences of low blubber Po2 for its health and function, particularly in very fat individuals, remain unknown. How seals avoid detrimental effects of low oxygen tension in adipose tissue, despite their high and fluctuating adiposity, is a fruitful avenue to explore.

Shining new light on sensory brain activation and physiological measurement in seals using wearable optical technology.

Sensory ecology and physiology of free-ranging animals is challenging to study but underpins our understanding of decision-making in the wild. Existing non-invasive human biomedical technology offers tools that could be harnessed to address these challenges. Functional near-infrared spectroscopy (fNIRS), a wearable, non-invasive biomedical imaging technique measures oxy- and deoxyhaemoglobin concentration changes that can be used to detect localized neural activation in the brain. We tested the efficacy of fNIRS to detect cortical activation in grey seals (Halichoerus grypus) and identify regions of the cortex associated with different senses (vision, hearing and touch). The activation of specific cerebral areas in seals was detected by fNIRS in responses to light (vision), sound (hearing) and whisker stimulation (touch). Physiological parameters, including heart and breathing rate, were also extracted from the fNIRS signal, which allowed neural and physiological responses to be monitored simultaneously. This is, to our knowledge, the first time fNIRS has been used to detect cortical activation in a non-domesticated or laboratory animal. Because fNIRS is non-invasive and wearable, this study demonstrates its potential as a tool to quantitatively investigate sensory perception and brain function while simultaneously recording heart rate, tissue and arterial oxygen saturation of haemoglobin, perfusion changes and breathing rate in free-ranging animals. This article is part of the theme issue 'Measuring physiology in free-living animals (Part I)'.

Predicting consequences of POP-induced disruption of blubber glucose uptake, mass gain rate and thyroid hormone levels for weaning mass in grey seal pups.

Persistent organic pollutants (POPs) are endocrine disruptors that alter adipose tissue development, regulation and function. Top marine predators are particularly vulnerable because they possess large fat stores that accumulate POPs. However, links between endocrine or adipose tissue function disruption and whole animal energetics have rarely been investigated. We predicted the impact of alterations to blubber metabolic characteristics and circulating thyroid hormone (TH) levels associated with polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and organochlorine pesticides (OCPs) on suckling mass gain and weaning mass in wild grey seal pups. Glucose uptake by inner blubber was a strong predictor of whole animal mass gain rate, which in turn, resulted in heavier weaning mass. Weaning mass was predicted to increase by 3.7 ± 1.59 (sem) %, through increased mass gain rate, in the absence of the previously reported suppressive effect of dioxin-like PCB (DL-PCBs) on blubber glucose uptake. PBDEs were, conversely, associated with faster mass gain. Alleviation of this effect was predicted to reduce weaning mass by 6.02 ± 1.86% (sem). To better predict POPs effects on energy balance, it is crucial to determine if and how PBDEs promote mass gain in grey seal pups. Weaning mass was negatively related to total T3 (TT3) levels. A 20% (range = 9.3-31.7%) reduction in TT3 by DL-PCBs partially overcame the effect of DL-PCB -mediated reduction in blubber glucose uptake. Overall, DL-PCBs were thus predicted to reduce weaning mass by 1.86 ± 1.60%. Organohalogen impacts on whole-animal energy balance in grey seal pups appear to partially offset each other through opposing effects on different mechanisms. POP effects were generally minor, but the largest POP-induced reductions in weaning mass were predicted to occur in pups that were already small. Since weaning mass is positively related to first-year survival, POPs may disproportionately affect smaller individuals, and could continue to have population-level impacts even when levels are relatively low compared to historical values. Our findings show how in vitro experiments combined with measurements in vivo can help elucidate mechanisms that underpin energy balance regulation and help to quantify the magnitude of disruptive effects by contaminants and other stressors in wildlife.

Shining new light on mammalian diving physiology using wearable near-infrared spectroscopy.

Investigation of marine mammal dive-by-dive blood distribution and oxygenation has been limited by a lack of noninvasive technology for use in freely diving animals. Here, we developed a noninvasive near-infrared spectroscopy (NIRS) device to measure relative changes in blood volume and haemoglobin oxygenation continuously in the blubber and brain of voluntarily diving harbour seals. Our results show that seals routinely exhibit preparatory peripheral vasoconstriction accompanied by increased cerebral blood volume approximately 15 s before submersion. These anticipatory adjustments confirm that blood redistribution in seals is under some degree of cognitive control that precedes the mammalian dive response. Seals also routinely increase cerebral oxygenation at a consistent time during each dive, despite a lack of access to ambient air. We suggest that this frequent and reproducible reoxygenation pattern, without access to ambient air, is underpinned by previously unrecognised changes in cerebral drainage. The ability to track blood volume and oxygenation in different tissues using NIRS will facilitate a more accurate understanding of physiological plasticity in diving animals in an increasingly disturbed and exploited environment.

Discontinuing Treatment Against Medical Advice: The Role of Perceived Autonomy Support From Providers in Relapsing-Remitting Multiple Sclerosis.

BACKGROUND: Long-term medication adherence is problematic among patients with chronic medical conditions. To our knowledge, this was the first study to examine factors associated with nonadherence among patients with relapsing-remitting multiple sclerosis who discontinue disease-modifying treatments against medical advice. PURPOSE: To examine differences in perceived provider autonomy support between disease-modifying treatment-adherent relapsing-remitting multiple sclerosis patients and relapsing-remitting multiple sclerosis patients who discontinued disease-modifying treatments against medical advice. METHODS: Self-report questionnaires and a neurologic exam were administered to demographically matched adherent (n = 50) and nonadherent (n = 79) relapsing- remitting multiple sclerosis patients from the Midwest and Northeast USA. RESULTS: Adherent patients reported greater perceived autonomy support from their treatment providers, F(1, 124) = 28.170, p < .001, partial η2 = .185. This difference persisted after controlling for current multiple sclerosis healthcare provider, education, disease duration, Expanded Disability Status Scale, perceived barriers to adherence, and prevalence of side effects, F(1, 121) = 9.61, p = .002, partial η2 = .074. Neither depressive symptoms, F(1, 124) = 1.001, p > .05, partial η2 = .009, nor the occurrence of a major depressive episode, χ2(1, N = 129) = .288, p > .05, differed between adherent and nonadherent patients. CONCLUSIONS: Greater perceived autonomy support from treatment providers may increase adherence to disease-modifying treatments among patients who discontinue treatment against medical advice. Results may inform interventions for patients who discontinue treatment against medical advice.

Persistent Organic Pollutant Burden, Experimental POP Exposure, and Tissue Properties Affect Metabolic Profiles of Blubber from Gray Seal Pups.

Persistent organic pollutants (POPs) are toxic, ubiquitous, resist breakdown, bioaccumulate in living tissue, and biomagnify in food webs. POPs can also alter energy balance in humans and wildlife. Marine mammals experience high POP concentrations, but consequences for their tissue metabolic characteristics are unknown. We used blubber explants from wild, gray seal ( Halichoerus grypus) pups to examine impacts of intrinsic tissue POP burden and acute experimental POP exposure on adipose metabolic characteristics. Glucose use, lactate production, and lipolytic rate differed between matched inner and outer blubber explants from the same individuals and between feeding and natural fasting. Glucose use decreased with blubber dioxin-like PCBs (DL-PCB) and increased with acute experimental POP exposure. Lactate production increased with DL-PCBs during feeding, but decreased with DL-PCBs during fasting. Lipolytic rate increased with blubber dichlorodiphenyltrichloroethane and its metabolites (DDX) in fasting animals, but declined with DDX when animals were feeding. Our data show that POP burdens are high enough in seal pups to alter adipose function early in life, when fat deposition and mobilization are vital. Such POP-induced alterations to adipose metabolic properties may significantly alter energy balance regulation in marine top predators, with the potential for long-term impacts on fitness and survival.

Intrinsic and extrinsic factors drive ontogeny of early-life at-sea behaviour in a marine top predator.

Young animals must learn to forage effectively to survive the transition from parental provisioning to independent feeding. Rapid development of successful foraging strategies is particularly important for capital breeders that do not receive parental guidance after weaning. The intrinsic and extrinsic drivers of variation in ontogeny of foraging are poorly understood for many species. Grey seals (Halichoerus grypus) are typical capital breeders; pups are abandoned on the natal site after a brief suckling phase, and must develop foraging skills without external input. We collected location and dive data from recently-weaned grey seal pups from two regions of the United Kingdom (the North Sea and the Celtic and Irish Seas) using animal-borne telemetry devices during their first months of independence at sea. Dive duration, depth, bottom time, and benthic diving increased over the first 40 days. The shape and magnitude of changes differed between regions. Females consistently had longer bottom times, and in the Celtic and Irish Seas they used shallower water than males. Regional sex differences suggest that extrinsic factors, such as water depth, contribute to behavioural sexual segregation. We recommend that conservation strategies consider movements of young naïve animals in addition to those of adults to account for developmental behavioural changes.

Using blubber explants to investigate adipose function in grey seals: glycolytic, lipolytic and gene expression responses to glucose and hydrocortisone.

Adipose tissue is fundamental to energy balance, which underpins fitness and survival. Knowledge of adipose regulation in animals that undergo rapid fat deposition and mobilisation aids understanding of their energetic responses to rapid environmental change. Tissue explants can be used to investigate adipose regulation in wildlife species with large fat reserves, when opportunities for organismal experimental work are limited. We investigated glucose removal, lactate, glycerol and NEFA accumulation in media, and metabolic gene expression in blubber explants from wild grey seals. Glycolysis was higher in explants incubated in 25 mM glucose (HG) for 24 h compared to controls (C: 5.5 mM glucose). Adipose-derived lactate likely contributes to high endogenous glucose production in seals. Lipolysis was not stimulated by HG or high hydrocortisone (HC: 500 nM hydrocortisone) and was lower in heavier animals. HC caused NEFA accumulation in media to decrease by ~30% relative to C in females, indicative of increased lipogenesis. Lipolysis was higher in males than females in C and HG conditions. Lower relative abundance of 11-ß-hydroxysteroid dehydrogenase 1 mRNA in HG explants suggests glucose involvement in blubber cortisol sensitivity. Our findings can help predict energy balance responses to stress and nutritional state in seals, and highlight the use of explants to study fat tissue function in wildlife.

Obtaining accurate glucose measurements from wild animals under field conditions: comparing a hand held glucometer with a standard laboratory technique in grey seals.

Glucose is an important metabolic fuel and circulating levels are tightly regulated in most mammals, but can drop when body fuel reserves become critically low. Glucose is mobilized rapidly from liver and muscle during stress in response to increased circulating cortisol. Blood glucose levels can thus be of value in conservation as an indicator of nutritional status and may be a useful, rapid assessment marker for acute or chronic stress. However, seals show unusual glucose regulation: circulating levels are high and insulin sensitivity is limited. Accurate blood glucose measurement is therefore vital to enable meaningful health and physiological assessments in captive, wild or rehabilitated seals and to explore its utility as a marker of conservation relevance in these animals. Point-of-care devices are simple, portable, relatively cheap and use less blood compared with traditional sampling approaches, making them useful in conservation-related monitoring. We investigated the accuracy of a hand-held glucometer for 'instant' field measurement of blood glucose, compared with blood drawing followed by laboratory testing, in wild grey seals (Halichoerus grypus), a species used as an indicator for Good Environmental Status in European waters. The glucometer showed high precision, but low accuracy, relative to laboratory measurements, and was least accurate at extreme values. It did not provide a reliable alternative to plasma analysis. Poor correlation between methods may be due to suboptimal field conditions, greater and more variable haematocrit, faster erythrocyte settling rate and/or lipaemia in seals. Glucometers must therefore be rigorously tested before use in new species and demographic groups. Sampling, processing and glucose determination methods have major implications for conclusions regarding glucose regulation, and health assessment in seals generally, which is important in species of conservation concern and in development of circulating glucose as a marker of stress or nutritional state for use in management and monitoring.

Navigating uncertain waters: a critical review of inferring foraging behaviour from location and dive data in pinnipeds.

In the last thirty years, the emergence and progression of biologging technology has led to great advances in marine predator ecology. Large databases of location and dive observations from biologging devices have been compiled for an increasing number of diving predator species (such as pinnipeds, sea turtles, seabirds and cetaceans), enabling complex questions about animal activity budgets and habitat use to be addressed. Central to answering these questions is our ability to correctly identify and quantify the frequency of essential behaviours, such as foraging. Despite technological advances that have increased the quality and resolution of location and dive data, accurately interpreting behaviour from such data remains a challenge, and analytical methods are only beginning to unlock the full potential of existing datasets. This review evaluates both traditional and emerging methods and presents a starting platform of options for future studies of marine predator foraging ecology, particularly from location and two-dimensional (time-depth) dive data. We outline the different devices and data types available, discuss the limitations and advantages of commonly-used analytical techniques, and highlight key areas for future research. We focus our review on pinnipeds - one of the most studied taxa of marine predators - but offer insights that will be applicable to other air-breathing marine predator tracking studies. We highlight that traditionally-used methods for inferring foraging from location and dive data, such as first-passage time and dive shape analysis, have important caveats and limitations depending on the nature of the data and the research question. We suggest that more holistic statistical techniques, such as state-space models, which can synthesise multiple track, dive and environmental metrics whilst simultaneously accounting for measurement error, offer more robust alternatives. Finally, we identify a need for more research to elucidate the role of physical oceanography, device effects, study animal selection, and developmental stages in predator behaviour and data interpretation.

HSP70 abundance and antioxidant capacity in feeding and fasting gray seal pups: suckling is associated with higher levels of key cellular defenses.

Heat shock proteins (HSPs) and antioxidants are key cellular defenses against stress. Seals routinely undergo protracted fasting, which is normally associated with physiological stress in other animals. We tested the hypotheses that (1) relative HSP70 protein abundance is higher in liver and blubber of fasting relative to suckling wild gray seal pups; (2) differences in HSP70 are mirrored in tissue superoxide dismutase (SOD) and catalase activity, as well as glutathione levels; (3) extracellular HSP70 correlates with hepatic and blubber HSP70 abundance; and (4) protein carbonylation, an index of oxidative damage, is lower in tissues with higher levels of these cellular stress markers. In contrast to our expectation, suckling pups had higher relative HSP70 abundance and glutathione levels in liver and blubber and higher hepatic catalase activity. Plasma HSP70 did not correlate with liver or blubber abundance of the protein. Suckling pups did not experience greater protein carbonylation, suggesting that cellular protective mechanisms prevent protein damage despite an apparent increase in cellular stress. SOD activity was not affected by nutritional state, but in blubber tissue, it was positively correlated with blubber thickness. Greater requirements for antioxidants and HSPs in suckling pups or in animals with thicker blubber could arise from rapid protein synthesis, high metabolic fuel availability, and/or exposure to lipophilic toxins. Developmental and nutritional changes in cellular defenses have important implications for gray seals' susceptibility to additional stress exposure.

Phocid seal leptin: tertiary structure and hydrophobic receptor binding site preservation during distinct leptin gene evolution.

The cytokine hormone leptin is a key signalling molecule in many pathways that control physiological functions. Although leptin demonstrates structural conservation in mammals, there is evidence of positive selection in primates, lagomorphs and chiropterans. We previously reported that the leptin genes of the grey and harbour seals (phocids) have significantly diverged from other mammals. Therefore we further investigated the diversification of leptin in phocids, other marine mammals and terrestrial taxa by sequencing the leptin genes of representative species. Phylogenetic reconstruction revealed that leptin diversification was pronounced within the phocid seals with a high dN/dS ratio of 2.8, indicating positive selection. We found significant evidence of positive selection along the branch leading to the phocids, within the phocid clade, but not over the dataset as a whole. Structural predictions indicate that the individual residues under selection are away from the leptin receptor (LEPR) binding site. Predictions of the surface electrostatic potential indicate that phocid seal leptin is notably different to other mammalian leptins, including the otariids. Cloning the grey seal leptin binding domain of LEPR confirmed that this was structurally conserved. These data, viewed in toto, support a hypothesis that phocid leptin divergence is unlikely to have arisen by random mutation. Based upon these phylogenetic and structural assessments, and considering the comparative physiology and varying life histories among species, we postulate that the unique phocid diving behaviour has produced this selection pressure. The Phocidae includes some of the deepest diving species, yet have the least modified lung structure to cope with pressure and volume changes experienced at depth. Therefore, greater surfactant production is required to facilitate rapid lung re-inflation upon surfacing, while maintaining patent airways. We suggest that this additional surfactant requirement is met by the leptin pulmonary surfactant production pathway which normally appears only to function in the mammalian foetus.

Effects of handling regime and sex on changes in cortisol, thyroid hormones and body mass in fasting grey seal pups.

Survival of seal pups may be affected by their ability to respond appropriately to stress. Chronic stress can adversely affect secretion of cortisol and thyroid hormones, which contribute to the control of fuel utilisation. Repeated handling could disrupt the endocrine response to stress and/or negatively impact upon mass changes during fasting. Here we investigated the effects of handling regime on cortisol and thyroid hormone levels, and body mass changes, in fasting male and female grey seal pups (Halichoerus grypus). Females had higher thyroid hormone levels than males throughout fasting and showed a reduction in cortisol midway through the fast that was not seen in males. This may reflect sex-specific fuel allocation or development. Neither handling frequency nor cumulative contact time affected plasma cortisol or thyroid hormone levels, the rate of increase in cortisol over the first five minutes of physical contact or the pattern of mass loss during fasting in either sex. The endocrine response to stress and the control of energy balance in grey seal pups appear to be robust to repeated, short periods of handling. Our results suggest that routine handling should have no additional impact on these animals than general disturbance caused by researchers moving around the colony.

Functional analysis of the 5' flanking region of the human G6PC3 gene: regulation of promoter activity by glucose, pyruvate, AMP kinase and the pentose phosphate pathway.

G6PC3 is a widely expressed isoform of glucose-6-phosphatase, found in many foetal and adult tissues. Mutations in this gene cause developmental abnormalities and severe neutropenia due to abolition of glucose recycling between the cytoplasm and endoplasmic reticulum. Low G6PC3 expression as a result of promoter polymorphisms or dysregulation could produce similar outcomes. Here we investigated the regulation of human G6PC3 promoter activity. HeLa and H4IIE cells were transiently transfected with G6PC3 promoter coupled to the firefly luciferase gene, and promoter activity was measured by dual luciferase assay. Activity was highest in a 453 bp segment of the G6PC3 promoter, from -455 to -3 relative to the transcriptional start site. This promoter was unresponsive to glucostatic hormones. Its activity increased significantly between 1 and 5.5 mM glucose, and was not elevated further by glucose concentrations up to 25 mM. Pyruvate increased its activity, but ß-hydroxybutyrate and sodium acetate did not. Promoter activity was reduced by inhibitors of hexokinase, glyceraldehyde phosphate dehydrogenase and the oxidative branch of the pentose phosphate pathway, but not by a transketolase inhibitor. Deletion of two adjacent Enhancer-boxes (-274 to -279 and -299 to -304) reduced promoter activity and abolished the glucose effect, suggesting they could function as a glucose response element. Deletion of an additional downstream 140 bp (-140 to -306) restored activity, but not the glucose response, suggesting the presence of repressor elements in this region. 5-Aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) reduced promoter activity, showing dependence on AMP-kinase. Regulation of the G6PC3 promoter is thus radically different to that of the hepatic isoform, G6PC. It is sensitive to carbohydrate, but not to fatty acid metabolites, and at much lower physiological concentrations. Based on these findings, we speculate that reduced G6PC3 expression could occur during hypoglycemic episodes in vivo, which are common in utero and in the postnatal period. If such episodes lower G6PC3 expression they could place the foetus or infant at risk of impaired immune function and development, and this possibility requires further examination both in vitro and in vivo.

Effects of mass and body composition on fasting fuel utilisation in grey seal pups (Halichoerus grypus Fabricius): an experimental study using supplementary feeding.

This study used supplementary feeding to test the hypothesis that fuel partitioning during the postweaning fast in grey seal pups is affected by size and composition of energy reserves at weaning, and by extra provisioning. Mass and body composition changes were measured during suckling and fasting to investigate the effect of natural differences in energy reserves at weaning on subsequent allocation of fat and protein to energy use. We fed seven pups for 5 days after weaning, to investigate the effect of increased fuel availability, and particularly protein, on fuel utilisation. After correcting for protein used during the moult, the proportional contribution of fat was 86-99% of total energy use. Pups with greater energy reserves, i.e. those that were heavier and fatter at weaning, had higher rates of fat and energy use. There was no significant relationship between adiposity at weaning and proportional contribution of fat to energy use, perhaps due to a limited sample size or range of body masses and adiposity. Supplemented individuals used energy, specifically fat, much faster and utilised proportionally less of their endogenous protein by departure than non-supplemented individuals. Fat metabolism contributed a similar percentage to daily energy use in both groups. These findings show that pups spare protein, even when energy use is dramatically increased. Pups that receive greater maternal provisioning and lay down more protein may have increased survival chances at sea. This study highlights the importance of protein reserves in first year survival of grey seal pups.

Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology.

Leptin is a multifunctional hormone, produced predominantly in adipocytes. It regulates energy balance through its impact on appetite and fat metabolism, and its concentration indicates the size of body fat reserves. Leptin also plays a vital role in stretch-induced surfactant production during alveolar development in the fetus. The structure, expression pattern, and role of leptin have not previously been explored in marine mammals. Phocid seals undergo cyclical changes in body composition as a result of prolonged fasting and intensive foraging bouts and experience rapid, dramatic, and repeated changes in lung volume during diving. Here, we report the tissue-specific expression pattern of leptin in these animals. This is the first demonstration of leptin expression in the lung tissue of a mature mammal, in addition to its expression in the blubber and bone marrow, in common with other animals. We propose a role for leptin in seal pulmonary surfactant production, in addition to its likely role in long-term energy balance. We identify substitutions in the phocine leptin sequence in regions normally highly conserved between widely distinct vertebrate groups, and, using a purified seal leptin antiserum, we confirm the presence of the leptin protein in gray seal lung and serum fractions. Finally, we report the substantial inadequacies of using heterologous antibodies to measure leptin in unextracted gray seal serum.