Marcadores fisiológicos y estado óxido-reductor durante las fluctuaciones térmicas en la dorada (Sparus aurata)

[spa] La acuicultura es una industria en crecimiento que proporciona cada vez más una gran cuantía de productos pesqueros para el consumo humano, siendo la dorada (Sparus aurata) una de las especies más producidas en el área del Mediterráneo. Pese a los grandes avances que se han hecho en la alimentación y el crecimiento de los cultivos, todavía existen algunas dificultades. La energía de la dieta y las fluctuaciones de temperatura afectan al estado de salud de los peces, causando grandes pérdidas en la producción de pescado durante el invierno. La presente tesis trata de analizar los efectos de la reducción del contenido lipídico de la dieta de un 18% a un 14% durante un período de fluctuación de temperatura tratando de simular el período invernal. Los animales fueron sometidos 30 días a 22°C (Pre-Frío), a continuación 50 días a 14°C (Frío) y posteriormente 35 días a 22°C (Recuperación). Para comprender mejor el período improductivo del invierno y su recuperación, evaluamos en condiciones controladas el crecimiento de los animales, parámetros de osomolaridad, la actividad del metabolismo intermediario, metabolitos plasmáticos, las defensas antioxidantes enzimáticas, glutatión, lípidos oxidados, proteínas oxidadas, expresión de proteínas de choque térmico (HSPs), la expresión de los sistemas ubiquitina-proteasoma y lisosomal, y también la respuesta hipofisaria a la fluctuación térmica y al contenido lipídico de la dieta. La reducción del contenido lipídico en la dieta a 22°C no afectó significativamente a la tasa de crecimiento estándar (SGR) ni a la tasa de conversión del alimento (FCR), tampoco a los niveles de glutatión ni a la actividad enzimática global, sin embargo, redujo la acumulación de lípidos oxidados. Esta situación sugiere la posibilidad de reducir la energía de la dieta durante los meses previos al invierno. En respuesta a una mayor cantidad de lípidos en la dieta, la expresión de los marcadores hipofisarios se reguló al alza antes de la ingesta matutina, en cada período térmico propuesto. A bajas temperaturas, los animales disminuyeron la ingesta, y el análisis de los metabolitos plasmáticos y de las actividades enzimáticas del hígado demostraron una reorganización del metabolismo hepático, principalmente en los peces con una dieta baja en lípidos. Además, el hígado mostró una falta de adaptación de la actividad enzimática con función antioxidante que repercutió en una alteración del ciclo oxido-reductor del glutatión, independientemente de la dieta. Los niveles de glutatión también disminuyeron en todas las dietas a 14ºC. Las principales vías proteolíticas también evidenciaron una menor expresión inducida por frío. La suma de estos efectos provocó la acumulación de proteínas oxidadas en el hígado de los animales alimentados con más lípidos tras 50 días exposición a 14°C. Sin embargo, la hipófisis reguló positivamente su actividad, a pesar de la depresión de los tejidos periféricos y la detención del crecimiento. La recuperación de las bajas temperaturas ocasionó una adaptación lenta de los animales y un incremento del potasio plasmático, y parece ser necesario un mayor contenido de lípidos en la dieta para restablecer una condición hepática óptima. Las actividades metabólicas y antioxidantes se restauraron gradualmente, y la expresión de las vías proteolíticas se recuperó lentamente hasta sus valores anteriores al frío, sin embargo, el ciclo oxido-reductor del glutatión no se restauró. La recuperación gradual de la temperatura y de la ingesta revirtieron la actividad hipofisaria. Por lo tanto, estos resultados sugieren que el contenido de lípidos en la dieta debería reducirse para hacer frente a las bajas temperaturas, haciendo necesaria la alimentación con dietas más energéticas y reforzadas durante el periodo de recuperación de la temperatura. Esta información puede ser útil para investigadores y productores de pescado.[eng] Aquaculture is a growing industry that is increasingly providing a sizable proportion of fishery products for human consumption. Dietary energy and temperature fluctuations affect fish health and may even trigger mortality, causing great losses in fish production during winter. The present thesis analysed the effects of reducing dietary lipid content from 18% to 14% during a temperature fluctuation period simulating winter period: 30 days at 22ºC (Pre-Cold), 50 days at 14ºC (Cold) and then 35 days at 22ºC (Recovery). To better understand this unproductive period we evaluate at indoor conditions fish growth, osomolarity parameters, intermediary metabolism activity, plasma metabolites, enzymatic antioxidant defences, glutathione, oxidised lipids, oxidised proteins, heat-shock proteins expression, ubiquitin-proteasome and lysosomal expression, and also the pituitary response to temperature and dietary lipids. Decreasing dietary lipid content at 22ᵒC did not significantly affect the standard growth rate or FCRs, glutathione levels or enzyme activities, but did reduce the amount of oxidised lipids. In response to higher dietary lipids, pituitary expression was up-regulated before the morning ingesta, at each thermal period. At low temperatures, animals reduced feed intake and the analysis of plasma metabolites and liver enzyme activities demonstrated a liver metabolism reorganisation, mainly in fish on a low-lipid diet. Liver showed a lack of adaptation of antioxidant enzyme activities, and a subsequently altered glutathione ratio, irrespective of diet. Main proteolytic pathways also evidenced a cold induced depression in its expression. Thus oxidised proteins were accumulated in D16 and D18 livers after 50 days at 14ºC. However, pituitary up-regulate its expression, despite the drastic fall of the peripheral tissues responses and the growth arrest. The recovery from low temperatures was delayed. Metabolic and antioxidant enzyme activities were gradually restored, and also expression of proteolytic pathways were late recovered to pre-cold values, but the glutathione redox cycle was not restored. Temperature and intake recovery reverted pituitary transitory up-regulated activity. Thus, these results suggest that dietary lipid contents should be reduced to cope with low temperatures and that improved diets with higher lipid content are required for recovery from low temperatures, which might be useful information for fish producers and researchers

Redox Balance Affects Fish Welfare

Aquaculture is a growing industry that is increasingly providing a sizable proportion of fishery products for human consumption. Thus, in the last years, several efforts are made in improving fish welfare. As well as in the rest of vertebrates, fish welfare is sensible to a balanced redox status. Numerous inputs like diet and environmental factors could alter this balance. In this sense, the last feeding strategies are focused on developing a more sustainable aquaculture, trying to maintain a redox balance. On the other hand, under culture conditions, animals cannot migrate to more favourable conditions, and environmental stress is one of the most relevant inputs that could compromise redox balance. This chapter is focused on the review of last works in redox balance analysis in Mediterranean aquaculture species and is organized as follows: (1) redox reactions on poikilotherms versus homeotherms; (2) effect of feeding strategies and environmental stress in fish redox balance; and (3) wide vision in fish redox balance

Modulation of pituitary response by dietary lipids and throughout a temperature fluctuation challenge in Gilthead Sea Bream

Low temperatures provoke drastic reductions in gilthead sea bream (Sparus aurata) activity and nourishment, leading to growth arrest and a halt in production. However, scarce data exist concerning the implications of central core control during the cold season. The aim of this work was to study the effects of low temperature and recovery from such exposure on the pituitary activity of sea bream juveniles fed 18% or 14% dietary lipid. A controlled indoor trial was performed to simulate natural temperature fluctuation (22 ◦C to 14 ◦C to 22 ◦C). Meanwhile, we determined the regulatory role of the pituitary by analyzing the gene expression of some pituitary hormones and hormone receptors via qPCR, as well as plasma levels of thyroidal hormones. In response to higher dietary lipids, hormone pituitary expressions were up-regulated. Induced low temperatures and lower ingesta modulated pituitary function up-regulating GH and TSH and thyroid and glucocorticoid receptors. All these findings demonstrate the capacity of the pituitary to recognize both external conditions and to modulate its response accordingly. However, growth, peripheral tissues and metabolism were not linked or connected to pituitary function at low temperatures, which opens an interesting field of study to interpret the hypothalamus–pituitary–target axis during temperature fluctuations in fish.info:eu-repo/semantics/publishedVersio

Skin mucus metabolites in response to physiological challenges: A valuable non-invasive method to study teleost marine species

Knowledge concerning the health and welfare of fish is important to conserve species diversity. Fish mucosal surfaces, and particularly the skin, are of utmost importance to protect the integrity and homeostasis of the body and to prevent skin infections by pathogens. We performed three trials simulating different environmental and anthropogenic challenges: fish capture (air exposure), bacterial infection and fasting, with the aim of evaluating epidermal mucus as a non-invasive target of studies in fish. In this initial approach, we selected three well-known marine species: meagre (Argyrosomus regius), European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) for our study. Mucus viscosity was measured in order to determine its rheological properties, and mucus metabolite (glucose, lactate, protein and cortisol) levels were analysed to establish their suitability as potential biomarkers. Skin mucus appeared as a viscous fluid exhibiting clearly non-Newtonian behaviour, with its viscosity being dependent on shear rate. The highest viscosity (p < 0.05) was observed in sea bream. Mucus metabolites composition responded to the different challenges. In particular, glucose increased significantly due to the air exposure challenge in meagre; and it decreased during food deprivation in sea bream by a half (p < 0.05). In contrast, mucus protein only decreased significantly after pathogenic bacterial infection in sea bass. In addition, mucus lactate immediately reflected changes closely related to an anaerobic condition; whereas cortisol was only modified by air exposure, doubling its mucus concentration (p < 0.05). The data provided herein demonstrate that mucus metabolites can be considered as good non-invasive biomarkers for evaluating fish physiological responses; with the glucose/protein ratio being the most valuable and reliable parameter. Determining these skin mucus metabolites and ratios will be very useful when studying the condition of critically threatened species whose conservation status prohibits the killing of specimens.info:eu-repo/semantics/acceptedVersio

Oxidative attack during temperature fluctuation challenge compromises liver protein homeostasis of a temperate fish model

Seasonal variations in water temperature are a natural stressor of temperate fish that affect growth performance and metabolism globally. Gilthead sea bream is one of the most economically interesting species in the Mediterranean; but its liver metabolism is affected by the cold season. However, the effects of cold on protein turnover mechanisms have hardly been studied. Here, we study the relationship between liver oxidative status and protein homeostasis pathways during a 50-day low temperature period at 14 °C, and subsequent recovery at two times: 7 days (early recovery) and 30 days (late recovery). Liver redox status was determined by measuring oxidised lipids and proteins, the glutathione redox cycle and major antioxidant enzymes activities. Protein turnover was analysed via liver protein expression of HSP70 and HSP90; proteasome 26S subunits and polyubiquitination, as markers of the ubiquitin-proteasome system (UPS); and cathepsin D, as a lysosomal protease. Low temperature exposure depressed antioxidant enzyme activities, affecting the glutathione redox cycle and reducing total glutathione levels. Both the UPS and lysosomal pathways were also depressed and consequently, oxidised protein accumulated in liver. Interestingly, both protein oxidation and polyubiquitination tagging depended on protein molecular weight. Despite all these alterations, temperature recovery reverted most consequences of the cold at different rates: with delayed recovery of total glutathione levels and oxidised protein degradation with respect to enzyme activities recovery. All these findings demonstrate that protein liver homeostasis is compromised after chronic cold exposure and could be the cause of liver affectations reported in aquaculture of temperate fish

Using stable isotope analysis to study skin mucus exudation and renewal in fish

Fish skin mucus is proposed as a novel target for the study of physiological condition and to conduct minimally invasive monitoring of fish. Whereas mucus composition has been a major interest of recent studies, no practical techniques have been proposed to gain understanding of the capacity and rhythm of production and exudation. Here, we used stable isotope analysis (SIA) with a labelled meal, packaged in gelatine capsules, to evaluate mucus production and renewal in a fish model, the gilthead sea bream (Sparus aurata). Mucus 13C- and 15N-enrichment reached higher levels at 12 h post-ingestion without significant differences at 24 h. When the formation of new mucus was induced, 13C-enrichment in the new mucus doubled whereas 15N-enrichment only increased by 10%. These results indicate the feasibility of adopting SIA in mucus studies and allow us to propose this methodology as a means to improve knowledge of mucus turnover in fish and other animals

Chronic cold stress alters the skin mucus interactome in a temperate fish model

Temperate fish are particularly sensitive to low temperatures, especially in the northern Mediterranean area, where the cold season decreases fish-farm production and affects fish health. Recent studies have suggested that the skin mucus participates in overall fish defense and welfare, and therefore propose it as a target for non-invasive studies of fish status. Here, we determine the mucus interactome of differentially expressed proteins in a temperate fish model, gilthead sea bream (Sparus aurata), after chronic exposure to low temperatures (7 weeks at 14°C). The differentially expressed proteins were obtained by 2D-PAGE of mucus soluble proteins and further assessed by STRING analyses of the functional interactome based on protein-protein interactions. Complementarily, we determined mucus metabolites, glucose, and protein, as well as enzymes involved in innate defense mechanisms, such as total protease and esterase. The cold mucus interactome revealed the presence of several subsets of proteins corresponding to Gene Ontology groups. "Response to stress" formed the central core of the cold interactome, with up-regulation of proteins, such as heat shock proteins (HSPs) and transferrin; and down-regulation of proteins with metabolic activity. In accordance with the low temperatures, all proteins clustered in the "Single-organism metabolic process" group were down-regulated in response to cold, evidencing depressed skin metabolism. An interactome subset of "Interspecies interaction between species" grouped together several up-regulated mucus proteins that participate in bacterial adhesion, colonization, and entry, such as HSP70, lectin-2, ribosomal proteins, and cytokeratin-8, septin, and plakins. Furthermore, cold mucus showed lower levels of soluble glucose and no adaptation response in total protease or esterase activity. Using zymography, we detected the up-regulation of metalloprotease-like activity, together with a number of fragments or cleaved keratin forms which may present antimicrobial activity. All these results evidence a partial loss of mucus functionality under chronic exposure to low temperatures which would affect fish welfare during the natural cold season under farm conditions

Circulating nitrate-nitrite reduces oxygen uptake for improving resistance exercise performance after rest time in well-trained CrossFit athletes

This study aimed to determine the effects of circulating nitrate plus nitrite (NOx) concentrations on resistance exercise performance, VO2 and biomarkers of muscle damage. Eleven well-trained male CrossFit athletes (29.2 ± 3.7 years, 78.9 ± 5.4 kg, 175.1 ± 6.3 cm) carried out a resistance exercise test after drinking 140 mL of beetroot juice (BJ) or placebo. The test consisted of repeating the same resistance exercise routine twice: wall ball shots plus full back squat with 3-min rest (1st routine) or without rest (2nd routine) between the two exercises. Higher NOx plasma levels were verified after BJ than placebo in the pretest and post-test (p < 0.001). A higher number of repetitions was observed after BJ intake compared to placebo in the full back squat exercise during the first routine (p = 0.004). A significantly reduced VO2 was detected after BJ intake compared to placebo during rest and full back squat execution in the first routine (p < 0.05). Plasma myoglobin concentrations were significantly increased with BJ compared to placebo (p = 0.036). These results showed that plasma NOx levels reduced VO2 after BJ intake during rest time. These reduced VO2 was a key factor for improving full back squat performance during the first routine

Subnormothermic perfusion in the isolated rat liver preserves the antioxidant glutathione and enhances the function of the ubiquitin proteasome system

The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated ratliversafter15minperfusionattemperaturesrangingfromnormothermia(37 ∘ C) to subnormothermia (26 ∘ Cand22 ∘ C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22 ∘ C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22 ∘ C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like ( 훽 5) proteasome activity was significantly increased in the 26 ∘ C (46%) and 22 ∘ C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus

Skin multi-omics-based interactome analysis: integrating the tissue and mucus exuded layer for a comprehensive understanding of the teleost mucosa functionality as model of study

From a general structural perspective, a mucosal tissue is constituted by two main matrices: the tissue and the secreted mucus. Jointly, they fulfill a wide range of functions including the protection of the epithelial layer. In this study, we simultaneously analyzed the epithelial tissue and the secreted mucus response using a holistic interactome-based multi-omics approach. The effect of the gilthead sea bream (Sparus aurata) skin mucosa to a dietary inclusion of spray-dried porcine plasma (SDPP) was evaluated. The epithelial skin microarrays-based transcriptome data showed 194 differentially expressed genes, meanwhile the exuded mucus proteome analysis 35 differentially synthesized proteins. Separately, the skin transcripteractome revealed an expression profile that favored biological mechanisms associated to gene expression, biogenesis, vesicle function, protein transport and localization to the membrane. Mucus proteome showed an enhanced protective role with putatively higher antioxidant and antimicrobial properties. The integrated skin mucosa multi-interactome analysis evidenced the interrelationship and synergy between the metabolism and the exuded mucus functions improving specifically the tissue development, innate defenses, and environment recognition. Histologically, the skin increased in thickness and in number of mucous cells. A positive impact on animal performance, growth and feed efficiency was also registered. Collectively, the results suggest an intimate crosstalk between skin tissue and its exuded mucus in response to the nutritional stimulus (SDPP supplementation) that favors the stimulation of cell protein turnover and the activation of the exudation machinery in the skin mucosa. Thus, the multi-omics-based interactome analysis provides a comprehensive understanding of the biological context of response that takes place in a mucosal tissue. In perspective, this strategy is applicable for evaluating the effect of any experimental variable on any mucosal tissue functionality, including the benefits this assessment may provide on the study of the mammalian mucosa