Improving the Aerobic Capacity in Fingerlings of European Sea Bass (Dicentrarchus labrax) through Moderate and Sustained Exercise: A Metabolic Approach.

Sustained swimming induces beneficial effects on growth and energy metabolism in some fish species. However, the absence of a standardized exercise regimen that guarantees an optimal response to physical activity is due to the anatomical, behavioral, and physiological differences among species, and the different conditions of tests applied, which are especially notable for the early stages of cultured species. The objective of this study was to assess the growth and metabolic responses of European sea bass submitted to continuous and moderate exercise exposure, selecting a practical swimming speed from swimming tests of groups of five fingerlings. The exercise-effects trial was carried out with 600 sea bass fingerlings (3-5 g body weight) distributed in two groups (control: voluntary swimming; exercised: under sustained swimming at 1.5 body lengths·s-1). After 6 weeks, growth parameters and proximal composition of both muscles were not altered by sustained swimming, but an increased synthetic capacity (increased RNA/DNA ratio) and more efficient use of proteins (decreased ΔN15) were observed in white muscle. The gene expression of mitochondrial proteins in white and red muscle was not affected by exercise, except for ucp3, which increased. The increase of UCP3 and Cox4 protein expression, as well as the higher COX/CS ratio of enzyme activity in white muscle, pointed out an enhanced oxidative capacity in this tissue during sustained swimming. In the protein expression of red muscle, only CS increased. All these metabolic adaptations to sustained exercise were also reflected in an enhanced maximum metabolic rate (MMR) with higher aerobic scope (AMS) of exercised fish in comparison to the non-trained fish, during a swimming test. These results demonstrated that moderate sustained swimming applied to sea bass fingerlings can improve the physical fitness of individuals through the enhancement of their aerobic capacities.

Cysteamine improves growth and the GH/IGF axis in gilthead sea bream (Sparus aurata): in vivo and in vitro approaches.

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed.

Muscle regeneration in gilthead sea bream: Implications of endocrine and local regulatory factors and the crosstalk with bone.

Fish muscle regeneration is still a poorly known process. In the present study, an injury was done into the left anterior epaxial skeletal muscle of seventy 15 g gilthead sea bream (Sparus aurata) juveniles to evaluate at days 0, 1, 2, 4, 8, 16 and 30 post-wound, the expression of several muscle genes. Moreover, transcripts' expression in the bone (uninjured tissue) was also analyzed. Histology of the muscle showed the presence of dead tissue the first day after injury and how the damaged fibers were removed and replaced by new muscle fibers by day 16 that kept growing up to day 30. Gene expression results showed in muscle an early upregulation of igf-2 and a downregulation of ghr-1 and igf-1. Proteolytic systems expression increased with capn2 and ctsl peaking at 1 and 2 days post-injury, respectively and mafbx at day 8. A pattern of expression that fitted well with active myogenesis progression 16 days after the injury was then observed, with the recovery of igf-1, pax7, cmet, and cav1 expression; and later on, that of cav3 as well. Furthermore, the first days post-injury, the cytokines il-6 and il-15 were also upregulated confirming the tissue inflammation, while tnfα was only upregulated at days 16 and 30 to induce satellite cells recruitment; overall suggesting a possible role for these molecules as myokines. The results of the bone transcripts showed an upregulation first, of bmp2 and ctsk at days 1 and 2, respectively; then, ogn1 and ocn peaked at day 4 in parallel to mstn2 downregulation, and runx2 and ogn2 increased after 8 days of muscle injury, suggesting a possible tissue crosstalk during the regenerative process. Overall, the present model allows studying the sequential involvement of different regulatory molecules during muscle regeneration, as well as the potential relationship between muscle and other tissues such as bone to control musculoskeletal development and growth, pointing out an interesting new line of research in this group of vertebrates.

Myomaker and Myomixer Characterization in Gilthead Sea Bream under Different Myogenesis Conditions.

Skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes' expression, including the fusogens myomaker and myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates, whereas Myomixer is a micropeptide that is moderately conserved. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules can provide a deeper understanding of myogenesis regulation in fish.

The Emerging Role of Long Non-Coding RNAs in Development and Function of Gilthead Sea Bream (Sparus aurata) Fast Skeletal Muscle.

Long non-coding RNAs (lncRNAs) are an emerging group of ncRNAs that can modulate gene expression at the transcriptional or translational levels. In the present work, previously published transcriptomic data were used to identify lncRNAs expressed in gilthead sea bream skeletal muscle, and their transcription levels were studied under different physiological conditions. Two hundred and ninety lncRNAs were identified and, based on transcriptomic differences between juveniles and adults, a total of seven lncRNAs showed potential to be important for muscle development. Our data suggest that the downregulation of most of the studied lncRNAs might be linked to increased myoblast proliferation, while their upregulation might be necessary for differentiation. However, with these data, as it is not possible to propose a formal mechanism to explain their effect, bioinformatic analysis suggests two possible mechanisms. First, the lncRNAs may act as sponges of myoblast proliferation inducers microRNAs (miRNAs) such as miR-206, miR-208, and miR-133 (binding energy MEF < -25.0 kcal). Secondly, lncRNA20194 had a strong predicted interaction towards the myod1 mRNA (ndG = -0.17) that, based on the positive correlation between the two genes, might promote its function. Our study represents the first characterization of lncRNAs in gilthead sea bream fast skeletal muscle and provides evidence regarding their involvement in muscle development.

Amino Acids and IGF1 Regulation of Fish Muscle Growth Revealed by Transcriptome and microRNAome Integrative Analyses of Pacu (Piaractus mesopotamicus) Myotubes.

Amino acids (AA) and IGF1 have been demonstrated to play essential roles in protein synthesis and fish muscle growth. The myoblast cell culture is useful for studying muscle regulation, and omics data have contributed enormously to understanding its molecular biology. However, to our knowledge, no study has performed the large-scale sequencing of fish-cultured muscle cells stimulated with pro-growth signals. In this work, we obtained the transcriptome and microRNAome of pacu (Piaractus mesopotamicus)-cultured myotubes treated with AA or IGF1. We identified 1228 and 534 genes differentially expressed by AA and IGF1. An enrichment analysis showed that AA treatment induced chromosomal changes, mitosis, and muscle differentiation, while IGF1 modulated IGF/PI3K signaling, metabolic alteration, and matrix structure. In addition, potential molecular markers were similarly modulated by both treatments. Muscle-miRNAs (miR-1, -133, -206 and -499) were up-regulated, especially in AA samples, and we identified molecular networks with omics integration. Two pairs of genes and miRNAs demonstrated a high-level relationship, and involvement in myogenesis and muscle growth: marcksb and miR-29b in AA, and mmp14b and miR-338-5p in IGF1. Our work helps to elucidate fish muscle physiology and metabolism, highlights potential molecular markers, and creates a perspective for improvements in aquaculture and in in vitro meat production.

Interaction between the Effects of Sustained Swimming Activity and Dietary Macronutrient Proportions on the Redox Status of Gilthead Sea Bream Juveniles (Sparus aurata L.).

The combination of physical exercise and a balanced diet presents substantial health benefits and could improve fish production. However, the redox balance can be affected by training regimen, dietary macronutrient ratio and their interaction. In this study, we conjointly evaluated the effects of physical activity (by voluntary swimming (VS) or sustained swimming as exercise (Ex)) and diet composition (by high-protein (HP) or high-lipid (HE) commercial diets) after 6 weeks on oxidative stress status in liver, white muscle and red muscle of gilthead sea bream juveniles. The HE diet increased the biochemical redox markers' thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products (AOPP) and reduced thiols (-SH) in the different tissues. Exercise increased AOPP and -SH levels in liver but reduced TBARS levels in white muscle. Regarding the expression of oxidative stress, chaperones and apoptosis-related genes, the VSHE group showed the highest values and the VSHP the lowest, whereas the application of sustained swimming partially equalized those differences. Diet composition modulated the enzyme activity, prioritizing the superoxide dismutase and catalase in the HE-fed groups and the glutathione-related enzymes in the HP groups. Exercise also altered enzyme activity, but in a tissue-dependent manner. Overall, the redox balance in gilthead sea bream juveniles can be affected by diet composition and sustained swimming. However, the response will partly depend on the interaction between these factors and the tissue studied. Therefore, the combination of an adequate diet and sustained exercise could be used in fish production to improve the physiological redox status.

Recombinant Bovine Growth Hormone-Induced Metabolic Remodelling Enhances Growth of Gilthead Sea-Bream (Sparus aurata): Insights from Stable Isotopes Composition and Proteomics.

Growth hormone and insulin-like growth factors (GH/IGF axis) regulate somatic growth in mammals and fish, although their action on metabolism is not fully understood in the latter. An intraperitoneal injection of extended-release recombinant bovine growth hormone (rbGH, Posilac®) was used in gilthead sea bream fingerlings and juveniles to analyse the metabolic response of liver and red and white muscles by enzymatic, isotopic and proteomic analyses. GH-induced lipolysis and glycogenolysis were reflected in liver composition, and metabolic and redox enzymes reported higher lipid use and lower protein oxidation. In white and red muscle reserves, rBGH increased glycogen while reducing lipid. The isotopic analysis of muscles showed a decrease in the recycling of proteins and a greater recycling of lipids and glycogen in the rBGH groups, which favoured a protein sparing effect. The protein synthesis capacity (RNA/protein) of white muscle increased, while cytochrome-c-oxidase (COX) protein expression decreased in rBGH group. Proteomic analysis of white muscle revealed only downregulation of 8 proteins, related to carbohydrate metabolic processes. The global results corroborated that GH acted by saving dietary proteins for muscle growth mainly by promoting the use of lipids as energy in the muscles of the gilthead sea bream. There was a fuel switch from carbohydrates to lipids with compensatory changes in antioxidant pathways that overall resulted in enhanced somatic growth.

Diet and Exercise Modulate GH-IGFs Axis, Proteolytic Markers and Myogenic Regulatory Factors in Juveniles of Gilthead Sea Bream (Sparus aurata).

The physiological and endocrine benefits of sustained exercise in fish were largely demonstrated, and this work examines how the swimming activity can modify the effects of two diets (high-protein, HP: 54% proteins, 15% lipids; high-energy, HE: 50% proteins, 20% lipids) on different growth performance markers in gilthead sea bream juveniles. After 6 weeks of experimentation, fish under voluntary swimming and fed with HP showed significantly higher circulating growth hormone (GH) levels and plasma GH/insulin-like growth-1 (IGF-1) ratio than fish fed with HE, but under exercise, differences disappeared. The transcriptional profile of the GH-IGFs axis molecules and myogenic regulatory factors in liver and muscle was barely affected by diet and swimming conditions. Under voluntary swimming, fish fed with HE showed significantly increased mRNA levels of capn1, capn2, capn3, capns1a, n3, and ub, decreased gene and protein expression of Ctsl and Mafbx and lower muscle texture than fish fed with HP. When fish were exposed to sustained exercise, diet-induced differences in proteases' expression and muscle texture almost disappeared. Overall, these results suggest that exercise might be a useful tool to minimize nutrient imbalances and that proteolytic genes could be good markers of the culture conditions and dietary treatments in fish.

Mitochondrial Adaptation to Diet and Swimming Activity in Gilthead Seabream: Improved Nutritional Efficiency.

Sustained exercise promotes growth in different fish species, and in gilthead seabream we have demonstrated that it improves nutrient use efficiency. This study assesses for differences in growth rate, tissue composition and energy metabolism in gilthead seabream juveniles fed two diets: high-protein (HP; 54% protein, 15% lipid) or high energy (HE; 50% protein, 20% lipid), under voluntary swimming (VS) or moderate-to-low-intensity sustained swimming (SS) for 6 weeks. HE fed fish under VS conditions showed lower body weight and higher muscle lipid content than HP fed fish, but no differences between the two groups were observed under SS conditions. Irrespective of the swimming regime, the white muscle stable isotopes profile of the HE group revealed increased nitrogen and carbon turnovers. Nitrogen fractionation increased in the HP fed fish under SS, indicating enhanced dietary protein oxidation. Hepatic gene expression markers of energy metabolism and mitochondrial biogenesis showed clear differences between the two diets under VS: a significant shift in the COX/CS ratio, modifications in UCPs, and downregulation of PGC1a in the HE-fed fish. Swimming induced mitochondrial remodeling through upregulation of fusion and fission markers, and removing almost all the differences observed under VS. In the HE-fed fish, white skeletal muscle benefited from the increased energy demand, amending the oxidative uncoupling produced under the VS condition by an excess of lipids and the pro-fission state observed in mitochondria. Contrarily, red muscle revealed more tolerant to the energy content of the HE diet, even under VS conditions, with higher expression of oxidative enzymes (COX and CS) without any sign of mitochondrial stress or mitochondrial biogenesis induction. Furthermore, this tissue had enough plasticity to shift its metabolism under higher energy demand (SS), again equalizing the differences observed between diets under VS condition. Globally, the balance between dietary nutrients affects mitochondrial regulation due to their use as energy fuels, but exercise corrects imbalances allowing practical diets with lower protein and higher lipid content without detrimental effects.

Evaluating mucus exudation dynamics through isotopic enrichment and turnover of skin mucus fractions in a marine fish model.

Fish skin mucus is composed of insoluble components, which form the physical barrier, and soluble components, which are key for interrelationship functions. Mucus is continuously secreted, but rates of production and exudation are still unknown, as are the underlying mechanisms. Using stable isotope analysis, here, we evaluate skin mucus turnover and renewal in gilthead sea bream, separating raw mucus and its soluble and insoluble fractions. Isotopic abundance analysis reveals no differences between mucus and white muscle, thus confirming mucus samples as reliable non-invasive biomarkers. Mucus production was evaluated using a single labelled meal packaged in a gelatine capsule, with both 13C and 15N, via a time-course trial. 13C was gradually allocated to skin mucus fractions over the first 12 h and was significantly (4-fold) higher in the soluble fraction, indicating a higher turnover of soluble mucus components that are continuously produced and supplied. 15N was also gradually allocated to mucus, indicating incorporation of new proteins containing the labelled dietary amino acids, but with no differences between fractions. When existent mucus was removed, dietary stable isotopes revealed stimulated mucus neoformation dependent on the components. All this is novel knowledge concerning skin mucus dynamics and turnover in fish and could offer interesting non-invasive approaches to the use of skin mucus production in ecological or applied biological studies such as climate change effects, human impact, alterations in trophic networks or habitat degradation, especially of wild-captured species or protected species.

Proteomic characterization of primary cultured myocytes in a fish model at different myogenesis stages.

Myogenesis is a complex two-phase process of proliferation and differentiation, which seems to be greatly conserved in vertebrates. For the first time in fish, we identify the changes that occur in the proteome during this process in a gilthead sea bream (Sparus aurata) myocyte primary cell culture (on days 4, 8 and 12), using 2-D gel electrophoresis and LC-MS/MS. A significant increase of myogenin expression at day 8 marked the transition from proliferation to differentiation. Of the 898 spots in the proteome analysis, the 25 protein spots overexpressed on day 4 and the 15 protein spots overexpressed on day 8 indicate the end of proliferation and the beginning of differentiation, respectively. Proliferation was characterized by enrichment of proteins involved in actin cytoskeleton remodelling and in cellular metabolic processes (transcription, ubiquitination, response to stress and glucose metabolism). During differentiation, 41 proteins were overexpressed and 51 underexpressed; many of them related to biosynthetic processes (RNA and protein synthesis and folding, and pentose pathways), terminal myotube formation and muscle contraction. The main cellular processes of both phases of muscle development in fish are similar with those observed in mammals but extended in time, allowing sequential studies of myogenesis.

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.

A long-term growth hormone treatment stimulates growth and lipolysis in gilthead sea bream juveniles.

The enhancement of the endocrine growth hormone (GH)/insulin-like growth factor I (IGF-I) system by the treatment with a sustained release formulation of a recombinant bovine GH (rBGH), is a good strategy to investigate growth optimization in aquaculture fish species. To further deepen into the knowledge of rBGH effects in fish and to estimate the growth potential of juveniles of gilthead sea bream, the present work evaluated rBGH injection on growth, GH/IGF-I axis and lipid metabolism modulation, and explored the conservation of GH effects provoked by the in vivo treatment using in vitro models of different tissues. The rBGH treatment increased body weight and specific growth rate (SGR) in juveniles and potentiated hyperplastic muscle growth while reducing circulating triglyceride levels. Moreover, the results demonstrated that the in vivo treatment enhanced also lipolysis in both isolated hepatocytes and adipocytes, as well as in day 4 cultured myocytes. Furthermore, these cultured myocytes extracted from rBGH-injected fish presented higher gene expression of GH/IGF-I axis-related molecules and myogenic regulatory factors, as well as stimulated myogenesis (i.e. increased protein expression of a proliferation and a differentiation marker) compared to Control fish-derived cells. These data, suggested that cells in vitro can retain some of the pathways activated by in vivo treatments in fish, what can be considered an interesting line of applied research. Overall, the results showed that rBGH stimulates somatic growth, including specifically muscle hyperplasia, as well as lipolytic activity in gilthead sea bream juveniles.

Ghrelin and Its Receptors in Gilthead Sea Bream: Nutritional Regulation.

Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim of this work was to identify the sequences of preproghrelin and Ghrelin receptors (ghsrs), and to study their responses to different nutritional conditions in gilthead sea bream (Sparus aurata) juveniles. The structure and phylogeny of S. aurata preproghrelin was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of preproghrelin, ghsrs and members of the Gh/Igf-1 system were determined in key tissues. preproghrelin and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary ghsrs expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary gh expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver igf-1 expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis.

Redox Challenge in a Cultured Temperate Marine Species During Low Temperature and Temperature Recovery.

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. To better understand this unproductive winter period in aquaculture, the redox status in a cultured marine species, the gilthead sea bream, was analyzed for the first time by inducing controlled temperature fluctuations and reducing dietary lipid content. Two groups of fish (by triplicate), differing in their dietary lipid content (18% vs. 14%), were subjected to 30 days at 22°C (Pre-Cold), 50 days at 14°C (Cold) and then 35 days at 22°C (Recovery). Plasma and liver redox metabolites (oxidized lipid, oxidized protein and thiol groups), liver glutathione forms (total, oxidized and reduced) and liver antioxidant enzyme activities were measured. Reducing dietary lipid content did not affect gilthead sea bream growth, glutathione levels or enzyme activities, but did reduce the amount of oxidized lipids. A sustained low temperature of 14°C showed a lack of adaptation of antioxidant enzyme activities, mainly catalase and glutathione reductase, which subsequently affected the glutathione redox cycle and caused an acute reduction in total hepatic glutathione levels, irrespective of diet. Antioxidant enzyme activities were gradually restored to their pre-cold levels, but the glutathione redox cycle was not restored to its pre-cold values during the recovery period used. Moreover, the lower lipid diet was associated with transiently increased liver oxidized protein levels. Thus, we propose that fish should be fed a low lipid diet during pre-cold and cold periods, which would reduce oxidized lipid levels without affecting fish growth, and a higher energy diet during the recovery period. Moreover, diets supplemented with antioxidants should be considered, especially during temperature recovery.

Recombinant bovine growth hormone (rBGH) enhances somatic growth by regulating the GH-IGF axis in fingerlings of gilthead sea bream (Sparus aurata).

The growth hormone (GH)/insulin-like growth factors (IGFs) endocrine axis is the main growth-regulator system in vertebrates. Some authors have demonstrated the positive effects on growth of a sustained-release formulation of a recombinant bovine GH (rBGH) in different fish species. The aim of this work was to characterize the effects of a single injection of rBGH in fingerlings of gilthead sea bream on growth, GH-IGF axis, and both myogenic and osteogenic processes. Thus, body weight and specific growth rate were significantly increased in rBGH-treated fish respect to control fish at 6weeks post-injection, whereas the hepatosomatic index was decreased and the condition factor and mesenteric fat index were unchanged, altogether indicating enhanced somatic growth. Moreover, rBGH injection increased the plasma IGF-I levels in parallel with a rise of hepatic mRNA from total IGF-I, IGF-Ic and IGF-II, the binding proteins IGFBP-1a and IGFBP-2b, and also the receptors IGF-IRb, GHR-I and GHR-II. In skeletal muscle, the expression of IGF-Ib and GHR-I was significantly increased but that of IGF-IRb was reduced; the mRNA levels of myogenic regulatory factors, proliferation and differentiation markers (PCNA and MHC, respectively), or that of different molecules of the signaling pathway (TOR/AKT) were unaltered. Besides, the growth inhibitor myostatin (MSTN1 and MSTN2) and the hypertrophic marker (MLC2B) expression resulted significantly enhanced, suggesting altogether that the muscle is in a non-proliferative stage of development. Contrarily in bone, although the expression of most molecules of the GH/IGF axis was decreased, the mRNA levels of several osteogenic genes were increased. The histology analysis showed a GH induced lipolytic effect with a clear decrease in the subcutaneous fat layer. Overall, these results reveal that a better growth potential can be achieved on this species and supports the possibility to improve growth and quality through the optimization of its culture conditions.

Moderate and sustained exercise modulates muscle proteolytic and myogenic markers in gilthead sea bream (Sparus aurata).

Swimming activity primarily accelerates growth in fish by increasing protein synthesis and energy efficiency. The role of muscle in this process is remarkable and especially important in teleosts, where muscle represents a high percentage of body weight and because many fish species present continuous growth. The aim of this work was to characterize the effects of 5 wk of moderate and sustained swimming in gene and protein expression of myogenic regulatory factors, proliferation markers, and proteolytic molecules in two muscle regions (anterior and caudal) of gilthead sea bream fingerlings. Western blot results showed an increase in the proliferation marker proliferating cell nuclear antigen (PCNA), proteolytic system members calpain 1 and cathepsin D, as well as vascular endothelial growth factor protein expression. Moreover, quantitative real-time PCR data showed that exercise increased the gene expression of proteases (calpains, cathepsins, and members of the ubiquitin-proteasome system in the anterior muscle region) and the gene expression of the proliferation marker PCNA and the myogenic factor MyoD in the caudal area compared with control fish. Overall, these data suggest a differential response of the two muscle regions during swimming adaptation, with tissue remodeling and new vessel formation occurring in the anterior muscle and enhanced cell proliferation and differentiation occurring in the caudal area. In summary, the present study contributes to improving the knowledge of the role of proteolytic molecules and other myogenic factors in the adaptation of muscle to moderate sustained swimming in gilthead sea bream.

Effects of sustained exercise on GH-IGFs axis in gilthead sea bream (Sparus aurata).

The endocrine system regulates growth mainly through the growth hormone (GH)/insulin-like growth factors (IGFs) axis and, although exercise promotes growth, little is known about its modulation of these factors. The aim of this work was to characterize the effects of 5 wk of moderate sustained swimming on the GH-IGFs axis in gilthead sea bream fingerlings. Plasma IGF-I/GH ratio and tissue gene expression of total IGF-I and three splice variants, IGF-II, three IGF binding proteins, two GH receptors, two IGF-I receptors, and the downstream molecules were analyzed. Fish under exercise (EX) grew more than control fish (CT), had a higher plasma IGF-I/GH ratio, and showed increased hepatic IGF-I expression (mainly IGF-Ia). Total IGF-I expression levels were similar in the anterior and caudal muscles; however, IGF-Ic expression increased with exercise, suggesting that this splice variant may be the most sensitive to mechanical action. Moreover, IGFBP-5b and IGF-II increased in the anterior and caudal muscles, respectively, supporting enhanced muscle growth. Furthermore, in EX fish, hepatic IGF-IRb was reduced together with both GHRs; GHR-II was also reduced in anterior muscle, while GHR-I showed higher expression in the two muscle regions, indicating tissue-dependent differences and responses to exercise. Exercise also increased gene and protein expression of target of rapamycin (TOR), suggesting enhanced muscle protein synthesis. Altogether, these data demonstrate that moderate sustained activity may be used to increase the plasma IGF-I/GH ratio and to potentiate growth in farmed gilthead sea bream, modulating the gene expression of different members of the GH-IGFs axis (i.e., IGF-Ic, IGF-II, IGFBP-5b, GHR-I, and TOR).

Growth-promoting effects of sustained swimming in fingerlings of gilthead sea bream (Sparus aurata L.).

Fish growth is strongly influenced by environmental and nutritional factors and changing culture conditions can help optimize it. The importance of early-life experience on the muscle phenotype later in life is well known. Here, we study the effects of 5 weeks of moderate and sustained swimming activity (5 BL s(-1)) in gilthead sea bream during early development. We analysed growth and body indexes, plasma IGF-I and GH levels, feed conversion, composition [proximate and isotopic ((15)N/(13)C)] and metabolic key enzymes (COX, CS, LDH, HOAD, HK, ALAT, ASAT) of white muscle. Moderate and continuous exercise in fingerlings of gilthead sea bream increased plasma IGF-I, whereas it reduced plasma GH. Under these conditions, growth rate improved without any modification to feed intake through an increase in muscle mass and a reduction in mesenteric fat deposits. There were no changes in the content and turnover of muscle proteins and lipid reserves. Glycogen stores were maintained, but glycogen turnover was higher in white muscle of exercised fish. A lower LDH/CS ratio demonstrated an improvement in the aerobic capacity of white muscle, while a reduction in the COX/CS ratio possibly indicated a functional adaptation of mitochondria to adjust to the tissue-specific energy demand and metabolic fuel availability in exercised fish. We discuss the synergistic effects of dietary nutrients and sustained exercise on the different mitochondrial responses.