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-β-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

Consequences of <i>in vitro</i> 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

Fitness correlates of blubber oxidative stress and cellular defences in grey seals (<i>Halichoerus grypus</i>):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

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 non-invasive technology for use in freely diving animals. Here, we developed a non-invasive 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

Signs of life:oxygen sensors confirm viability, measure oxygen consumption and provide rapid, effective contamination monitoring for field-based tissue culture

Understanding the ecology and evolution of wildlife and domesticated species requires knowledge of their physiological responses to environmental change and the constraints under which they operate. However, whole animal experiments are often limited in sample size and can be logistically and ethically challenging. Culture techniques represent a powerful approach, but are used infrequently in field research due to practical constraints. We used minimal tissue culture equipment in a remote field site for in vitro explant experiments using blubber from wild grey seals Halichoerus grypus. Assessing explant viability and detecting microbial contamination in remote field sites, where facilities are often small, unspecialised and more vulnerable to bacterial or fungal infection, present major challenges. We investigated whether oxygen‐sensitive planar optodes (OSPO) in closed system respirometry could be used to assess oxygen consumption by blubber explants from suckling and fasting wild seal pups as a proxy for viability. We also explored whether OSPOs could provide rapid information on whole animal relevant physiological metrics by determining whether explant oxygen consumption correlated with the nutritional state of the animal, blubber depth and other tissue metabolic properties, including glucose uptake, lactate production and lipolysis. Vials containing blubber explants consumed significant amounts of oxygen compared to controls, showing tissues were metabolically active. Oxygen consumption differed between nutritional states and blubber tissue depth. These differences were reflected in other tissue metabolic properties. Dissolved oxygen levels remained consistent over 24 hr in 94% of control vials containing only culture media. In 6% of control vials extremely rapid oxygen consumption preceded, by 2–3 days, colour changes in the phenol‐red containing media that indicate lactic acidosis from microbial metabolic activity. Oxygen use in control vials was, therefore, an effective monitoring system that provided vital early warning of media contamination, allowing stocks to be discarded, which prevented erroneous results and avoided waste of valuable field time and irreplaceable samples. OSPO are thus a useful tool for simultaneously assessing tissue oxygen consumption, investigating functional physiological differences and monitoring microbiological contamination in culture experiments, particularly in field laboratories studying live tissues from wildlife

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 &lt; 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

Degradation of mangrove tissues by arboreal termites (\u3cem\u3eNasutitermes acajutlae\u3c/em\u3e) and their role in the mangrove C cycle (Puerto Rico): Chemical characterization and organic matter provenance using bulk δ\u3csup\u3e13\u3c/sup\u3eC, C/N, alkaline CuO oxidation‐GC/MS, and solid‐state \u3csup\u3e13\u3c/sup\u3eC NMR

Arboreal termites are wood decaying organisms that play an important role in the first stages of C cycling in mangrove systems. The chemical composition of Rhizophora mangle, Avicennia germinans, and Laguncularia racemosa leaf, stem, and pneumatophore tissues as well as associated sediments was compared to that of nests of the termite Nasutitermes acajutlae. Nests gave δ13C values of −26.1 to −27.2‰ (±0.1) and C/N of 43.3 (±2.0) to 98.6 (±16.2) which were similar to all stem and pneumatophores but distinct from mangrove leaves or sediments. Organic matter processed by termites yielded lignin phenol concentrations (Λ, lambda) that were 2–4 times higher than stem or pneumatophores and 10–20 times higher than that of leaves or sediments, suggesting that the nests were more resistant to biodegradation than the mangrove vegetation source. 13C NMR revealed that polysaccharide content of mangrove tissues (50–69% C) was higher than that of the nests (46–51% C). Conversely, lignin accounted for 16.2–19.6% C of nest material, a threefold increase relative to living mangrove tissues; a similar increase in aromatic methoxyl content was also observed in the nests. Lipids (aliphatic and paraffinic moieties) were also important but rather variable chemical components of all three mangrove species, representing between 13.5 and 28.3% of the C content. Termite nests contained 3.14 Mg C ha−1 which represents approximately 2% of above ground C storage in mangroves, a value that is likely to increase upon burial due to their refractory chemical composition

Fostering Prevention of Cervical Cancer by a Correct Diagnosis of Precursors: A Structured Case-Based Colposcopy Course in Finland, Norway and UK

Simple Summary Cervical cancer prevention is presently undergoing a thorough reformation due to introduction of human papillomavirus (HPV)-testing and vaccines in primary prevention. The screening program, however, is more than a single test or preventive intervention-the possible lesion has to be found, located and treated. Colposcopy plays a major role in this management. Literature dealing with training and learning, especially with colposcopy, is extremely scarce. The aim of the European Federation of Colposcopy, EFC, is to improve the education and training in colposcopy, e.g., by organizing colposcopy courses. The aim of our prospective interventional study was to pilot this intensive participant activating EFC Basic Colposcopy Course on the short- and long-term learning of colposcopy-related knowledge, image recognition and the diagnostic confidence. High-quality colposcopy is essential in cervical cancer prevention. We performed a multicentre prospective interventional pilot-study, evaluating the effect of a six-hour case-based colposcopy course on short- and long-term learning of colposcopy-related knowledge, diagnostic accuracy levels and confidence. We recruited 213 colposcopists participating in three European Federation of Colposcopy (EFC) basic colposcopy courses (Finland, Norway, UK). The study consisted of three tests with identical content performed before, after and 2 months after the course, including ten colposcopic images, ten patient cases and scales for marking confidence in the answers. Outcome measures where mean scores in correct case-management, diagnosis (including high-grade lesion recognition), transformation-zone recognition and confidence in answers. Results were compared between the three tests and stratified according to experience. Mean test scores improved after the course for all participants. The increase was highest for beginners. Confidence in answers improved and the number of colposcopists showing high confidence with low scores decreased. A structured case-based course improves skills and confidence especially for inexperienced colposcopists; however, trainers should be aware of the risk of overconfidence. To complement theoretical training, further hands-on training including high-quality feedback is recommended. Conclusions drawn from long-term learning are limited due to the low participation in the follow-up test.Peer reviewe

Stochastic Resonance in a Dipole

We show that the dipole, a system usually proposed to model relaxation phenomena, exhibits a maximum in the signal-to-noise ratio at a non-zero noise level, thus indicating the appearance of stochastic resonance. The phenomenon occurs in two different situations, i.e. when the minimum of the potential of the dipole remains fixed in time and when it switches periodically between two equilibrium points. We have also found that the signal-to-noise ratio has a maximum for a certain value of the amplitude of the oscillating field.Comment: 4 pages, RevTex, 6 PostScript figures available upon request; to appear in Phys. Rev.