Muscle Of Sea Bream Research Articles

Characterisation and expression of myogenesis regulatory factors during in vitro myoblast development and in vivo fasting in the gilthead sea bream (Sparus aurata)

The aim of this study was to characterise a primary cell culture isolated from fast skeletal muscle of the gilthead sea bream. Gene expression profiles during culture maturation were compared with those obtained from a fasting–refeeding model which is widely used to modulate myogenesis in vivo. Myogenesis is controlled by numerous extracellular signals together with intracellular transcriptional factors whose coordinated expression is critical for the appropriate development of muscle fibres. Full-length cDNAs for the transcription factors Myf5, Mrf4, Pax7 and Sox8 were cloned and sequenced for gilthead sea bream. Pax7, sox8, myod2 and myf5 levels were up-regulated during the proliferating phase of the myogenic cultures coincident with the highest expression of proliferating cell nuclear antigen (PCNA). In contrast, myogenin and mrf4 transcript abundance was highest during the differentiation phase of the culture when myotubes were present, and was correlated with increased myosin heavy chain (mhc) and desmin expression. In vivo, 30days of fasting resulted in muscle fibre atrophy, a reduction in myod2, myf5 and igf1 expression, lower number of Myod-positive cells, and decreased PCNA protein expression, whereas myogenin expression was not significantly affected. Myostatin1 (mstn1) and pax7 expression were up-regulated in fasted relative to well-fed individuals, consistent with a role for Pax7 in the reduction of myogenic cell activity with fasting. The primary cell cultures and fasting–feeding experiments described provide a foundation for the future investigations on the regulation of muscle growth in gilthead sea bream.

Microbiological evolution of gilthead sea bream (Sparus aurata) in Canary Islands during ice storage.

This study analyses the microbiological changes with traditional methods for total mesophilic aerobic, psychrotrophic, Aeromonas sp., Pseudomonas sp., Shewanella putrefaciens, Enterobacteriaceae, sulfide-reducing Clostridium and Photobacterium phosphoreum in muscle, skin and gills of whole ungutted gilthead sea bream (Sparus aurata) stored in ice during 18days. The muscle tissue showed the minor grade of contamination, followed by the skin and the gills, with statistic significance (p < 0.001). The most prominent microorganisms in the different tissues and at the end of the storage were Pseudomonas sp. (7.76, 10.11 and 10.40 log CFU/g), Aeromonas sp. (7.49, 8.24 and 9.02 log CFU/g) and S. putrefaciens (8.05, 7.49 and 8.05 log CFU/g) in sea bream harvested in the temperate water of the Canary Islands. The results obtained from this study can contribute to the improvement of microbiological knowledge of gilthead sea bream (Sparus aurata) by determining the evolution of microorganisms responsible for spoilage and their counts in different tissues such as muscle, skin, and gills during iced storage.

Effect of oregano and rosemary essential oils on lipid oxidation of stored frozen minced gilthead sea bream muscle

The study aimed to determine the effect of oregano and rosemary essential oils addition to minced gilthead sea bream muscle on lipid oxidation during frozen storage. Each essential oil was added to minced fish muscle at 500 mg/kg concentration which then was stored at −22 °C for 12 months. Oregano essential oils inhibited the formation of conjugated diene and triene hydroperoxides and thiobarbituric reactive substances when compared with untreated samples after the first month and rosemary essential oils after the first and third month of frozen storage. Oregano and rosemary essential oils could be used as ingredients to prevent oxidation in stored frozen minced gilthead sea bream muscle, when long term preservation is not needed. The rate of lipid oxidation depends on antioxidant source. The rosemary essential oil proved to be more efficient in preventing lipid oxidation of minced gilthead sea bream muscle than oregano essential oil.

Insulin-like growth factors effects on the expression of myogenic regulatory factors in gilthead sea bream muscle cells

Gilthead sea bream (Sparus aurata) is a widely cultured fish; however, muscle development regulation is poorly known. Myogenesis can be activated by the myogenic regulatory factors (MRFs: MyoD, Myf5, myogenin and MRF4) and by endocrine signals from the growth hormone (GH)/insulin-like growth factors (IGFs) axis. We cultured gilthead sea bream myocytes to better understand the role of IGFs in muscle growth and differentiation through the regulation of MRFs expression. First, we studied the expression pattern during culture development of IGFs and IGF-I splice variants. The expression of igf-II was highest at the beginning of the culture and decreased when the cells started to differentiate, similarly to that observed for total igf-I. Igf-Ib showed a paralleled expression pattern as that of total igf-I, whereas igf-Ic was more stable during culture progression. Next, we analyzed the expression of IGFs and MRFs after incubation of cells at day 4 with GH, IGF-I, IGF-II and combinations of them at 3, 6 and 18h. IGF-II increased myod2 and myf5 expression, genes involved in early muscle cell proliferation. Moreover, IGF-I caused an increase on mrf4 and myogenin expression, both involved in the later stages of development corresponding to differentiation. Regarding the regulation of IGFs expression, igf-I was stimulated by GH and IGF-II alone and combined, whereas igf-II expression was increased in response to IGF-I, suggesting a nice model of crossed regulation. Overall, the present model could be very useful to understand the different regulatory roles of these endocrine and transcription factors on fish myogenesis.

Influence of supplemental maslinic acid (olive-derived triterpene) on the post-mortem muscle properties and quality traits of gilthead seabream

Maslinic acid, a natural triterpene, was evaluated as a dietary supplement to modulate glycogen post-mortem mobilization in gilthead seabream muscle. For this purpose, a multidisciplinary trial was undertaken, where flesh quality criteria, as well as biochemical and histological parameters, enzymatic activities and protein expression in the muscle were assessed. Supplementing gilthead seabream diets with maslinic acid mainly resulted in hypertrophy of muscle fibres and inhibition of cathepsin B activity, with no observed differences in terms of glycogen and ATP content of the muscle, as well as glycogen phosphorylase activity. Proteomic analysis showed a low impact of maslinic acid supplementation on muscle metabolism, with most changes reflecting increased stress coping capacity and muscle hypertrophy in maslinic acid-fed fish. As a finishing strategy to improve the muscle's energetic status, the usefulness of maslinic acid seems limited. However, it shows potential for promoting muscle growth in this species.

Effect of seasonal variation in seawater dissolved mercury concentrations on mercury accumulation in the muscle of red sea bream (Pagrus major) held in Minamata Bay, Japan

Japanese stingfish (Sebastiscus marmoratus) and Bambooleaf wrasse (Pseudolabrus japonicas) are monitored annually for mercury pollution in Minamata Bay, Japan. The average total mercury concentration in the muscle of these two species in Minamata Bay was 0.36 mg kg(-1) wet weight and 0.20 kg(-1) wet weigh, respectively, between 2008 and 2010. This is higher than levels elsewhere in Japan (0.125 mg kg(-1) wet weight and 0.038 mg kg(-1) wet weight, respectively). The FDA (2001) and EPA (2004) suggested that a proportion of mercury accumulated in fish is derived from seawater. We reared young red sea bream (Pagrus major) over a 2-year period in Minamata Bay and Nagashima (control) to evaluate the uptake of mercury from seawater and dietary sources. Fish were fed a synthesized diet that did not contain mercury. There was no difference in mercury accumulation in the muscle of red sea bream between Minamata Bay and Nagashima. Thus, our results suggest that the majority of mercury accumulated in fish muscle is not from seawater.

경남 거제해역에 서식하는 감성돔(Acanthopagrus schlegeli)의 연간 RNA/DNA 및 혈액학적 특성 변화

The monthly variations of blood characteristics and RNA/DNA of black sea bream, Acanthopagrus schlegeli, habituated in Geojae costal area were analysed to determine health condition of natural stocks in terms of gonad maturation and spawning season from March 2010 to February 2011. Spawning season determinated by gonadosomatic index is from June to August. RNA/DNA ratio of black sea bream muscle was strongly correlated with spawning season. During the gonad maturation RNA/DNA ratio in dorsal muscle tissue was decreased contrast to rapid increase during spawning season. Blood composition factors increased in terms of gonad maturation are aspartate aminotransferase, cholesterol, triglyceride, total protein, glucose, globulin, alkaline phosphatase and inorganic phosphate. Other blood factors increased during spawning season are alanine aminotransferase, blood urea nitrogen, uric acid and lactate dehydrogenase.

Differences in lipid distribution and expression of peroxisome proliferator-activated receptor gamma and lipoprotein lipase genes in torafugu and red seabream

Lipid content is one of the major determinants of the meat quality in fish. However, the mechanisms underlying the species-specific distribution of lipid are still poorly understood. The present study was undertaken to investigate the mechanisms associated with lipid accumulation in two species of fish: torafugu (a puffer fish) and red seabream. The lipid content of liver and carcass were 67.0% and 0.8% for torafugu, respectively, and 8.8% and 7.3% for red seabream, respectively. Visceral adipose tissue was only apparent in the red seabream and accounted for 73.3% of its total lipid content. Oil red O staining confirmed this species-specific lipid distribution, and further demonstrated that the lipid in the skeletal muscle of the red seabream was mainly localized in the myosepta. We subsequently cloned cDNAs from torafugu encoding lipoprotein lipase 1 (LPL1) and LPL2, important enzymes for the uptake of lipids from blood circulation system into various tissues. The relative mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) and the LPLs of torafugu were determined by quantitative real-time PCR together with their counterparts in red seabream previously reported. The relative mRNA levels of PPARγ and LPL1 correlated closely to the lipid distribution of both fish, being significantly higher in liver than skeletal muscle in torafugu, whereas the highest in the adipose tissue, followed by liver and skeletal muscle in red seabream. However, the relative mRNA levels of LPL2 were tenfold lower than LPL1 in both species and only correlated to lipid distribution in torafugu, suggesting that LPL2 has only a minor role in lipid accumulation. In situ hybridization revealed that the transcripts of LPL1 co-localized with lipids in the adipocytes located along the myosepta of the skeletal muscle of red seabream. These results suggest that the transcriptional regulation of PPARγ and LPL1 is responsible for the species-specific lipid distribution of torafugu and red seabream.

短波長域用および長波長域用の携帯型近赤外分光計によるマダイの背肉およびウマヅラハギの肝臓の脂質測定

短波長域用および長波長域用の携帯型近赤外分光計によるマダイの背肉およびウマヅラハギの肝臓の脂質測定

Development Temperature Has Persistent Effects on Muscle Growth Responses in Gilthead Sea Bream

Initially we characterised growth responses to altered nutritional input at the transcriptional and tissue levels in the fast skeletal muscle of juvenile gilthead sea bream. Fish reared at 21–22°C (range) were fed a commercial diet at 3% body mass d−1 (non-satiation feeding, NSF) for 4 weeks, fasted for 4d (F) and then fed to satiation (SF) for 21d. 13 out of 34 genes investigated showed consistent patterns of regulation between nutritional states. Fasting was associated with a 20-fold increase in MAFbx, and a 5-fold increase in Six1 and WASp expression, which returned to NSF levels within 16h of SF. Refeeding to satiation was associated with a rapid (<24 h) 12 to 17-fold increase in UNC45, Hsp70 and Hsp90α transcripts coding for molecular chaperones associated with unfolded protein response pathways. The growth factors FGF6 and IGF1 increased 6.0 and 4.5-fold within 16 h and 24 h of refeeding respectively. The average growth in diameter of fast muscle fibres was checked with fasting and significant fibre hypertrophy was only observed after 13d and 21d SF. To investigate developmental plasticity in growth responses we used the same experimental protocol with fish reared at either 17.5–18.5°C (range) (LT) or 21–22°C (range) (HT) to metamorphosis and then transferred to 21–22°C. There were persistent effects of development temperature on muscle growth patterns with 20% more fibres of lower average diameter in LT than HT group of similar body size. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups, whereas there were no differences in the expression of MAFbx and Six1. It was concluded that myogenesis and gene expression patterns during growth are not fixed, but can be modified by temperature during the early stages of the life cycle.

Multidisciplinary analytical investigation of phospholipids and triglycerides in offshore farmed gilthead sea bream (Sparus aurata) fed commercial diets

In this work, a quantitative characterisation of lipid (both triglycerides and phospholipids) rearrangements in the muscle of offshore-raised gilthead sea bream was carried out as a function of fish growth between April and September. Relative percentages of lipid classes and fatty acids/acyls composition of the commercial feeds and fish dorsal muscles were assessed by means of an interdisciplinary analytical approach. A combination of preparative chemistry and experimental results from NMR spectroscopy, GC, 3D-TLC as well as proximate analysis permitted the observed growth parameters in key metabolic events to be linked with fish fattening and lipid turnover. While defined effects of feed composition on fatty acid profiles of fillets were ascertained, the relative increase of fatty acyls in triglycerides and phospholipids were also estimated enabling detailed evaluation of TAG:PL ratio in adult offshore-farmed gilthead sea bream. NMR was also used to quantify PUFA regiospecific distribution in TAG and PL.

Dietary Tools To Modulate Glycogen Storage in Gilthead Seabream Muscle: Glycerol Supplementation

The quality and shelf life of fish meat products depend on the skeletal muscle's energetic state at slaughter, as meat decomposition processes can be exacerbated by energy depletion. In this study, we tested dietary glycerol as a way of replenishing muscle glycogen reserves of farmed gilthead seabream. Two diets were tested in duplicate (n = 42/tank). Results show 5% inclusion of crude glycerol in gilthead seabream diets induces increased muscle glycogen, ATP levels and firmness, with no deleterious effects in terms of growth, proximate composition, fatty acid profile, oxidative state, and organoleptic properties (aroma and color). Proteomic analysis showed a low impact of glycerol-supplementation on muscle metabolism, with most changes probably reflecting increased stress coping capacity in glycerol-fed fish. This suggests inclusion of crude glycerol in gilthead seabream diets (particularly in the finishing phase) seems like a viable strategy to increase glycogen deposition in muscle without negatively impacting fish welfare and quality.

Effect of Storage Conditions on Total Volatile Base Nitrogen Determinations in Fish Muscle Extracts

Total volatile base nitrogen (TVBN) is a widely used fish spoilage indicator. Because TVBN is typically measured from muscle extracts, analytical laboratories would benefit from understanding how different conditions for storing extracts affect TVBN results. To examine this question, extracts from the muscle of gilthead sea bream (Sparus aurata) were prepared and stored. Different storage temperatures (4, −20, and −80°C) and storage periods (7 days to 2 months) were tested. To compare how well the different storage conditions worked for different TVBN levels, the study took advantage of the fact that TVBN values change depending on how long the gilthead sea bream is stored before extracts are prepared. Extracts were prepared from fish maintained in ice for 0, 4, and 10 days postmortem. Comparing the storage conditions tested, all offered statistically identical results independent of the storage period and temperature. Nevertheless, only the extract maintained at 4°C for 7 days achieved statistically equal values to not stored extracts.

Effects of Preslaughter Stress Levels on the Post-mortem Sarcoplasmic Proteomic Profile of Gilthead Seabream Muscle

Fish welfare is an important concern in aquaculture, not only due to the ethical implications but also for productivity and quality-related reasons. The purpose of this study was to track soluble proteome expression in post-mortem gilthead seabream muscle and to observe how preslaughter stress affects these post-mortem processes. For the experiment, two groups of gilthead seabream (n = 5) were subjected to distinct levels of preslaughter stress, with three muscle samples being taken from each fish. Proteins were extracted from the muscle samples, fractionated, and separated by 2DE. Protein identification was performed by MALDI-TOF-TOF MS. Analysis of the results indicates changes on several cellular pathways, with some of these changes being attributable to oxidative and proteolytic activity on sarcoplasmic proteins, together with leaking of myofibrillar proteins. These processes appear to have been hastened by preslaughter stress, confirming that it induces clear post-mortem changes in the muscle proteome of gilthead seabream.

Confirmatory development and validation of HPLC‐DADmethod for the determination of tetracyclines in gilthead seabream (Sparus aurata) muscle tissue

A confirmatory high-pressure liquid chromatographic method for the determination of nine tetracyclines in Sparus aurata (gilthead seabream) muscle tissue is developed and presented herein. Tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, chlortetracycline, 4-epi-chlortetracycline, doxycycline, methacycline and demeclocycline were separated on a Kromasil, C18 (250 mm × 4 mm, 5 μm) analytical column by gradient elution with a mobile phase consisting of 0.001 M ethylenediaminetetraacetic acid/sodium salt and acetonitrile at 25°C. Diode array detection with monitoring at 280 nm (for the determination of chlortetracycline, 4-epi-chlortetracycline, methacycline and demeclocycline) and 355 nm (for tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline and demeclocycline) was applied for peak identification and quantification of analytes. Examined antibiotics were isolated from gilthead seabream tissue by leaching using a citrate buffer (pH 4.0) and purified by solid phase extraction using Oasis HLB(200 mg/6 mL) cartridges. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity according to the European Union Decision 2002/657/EC.

Maslinic acid, a natural triterpene, and ration size increased growth and protein turnover of white muscle in gilthead sea bream (Sparus aurata)

Maslinic acid (MA) is a pentacyclic triterpene used, at experimental level, as an additive to stimulate growth, protein-turnover rates and hyperplasia in fish. In this experiment we have studied the effects of feeding with MA at daily fixed ration of 10 g kg−1 of body mass, on growth, protein-turnover rates and nucleic acid concentration in white muscle of gilthead sea bream grown in a local fish farm. The experimental groups contained 0 (control), 50 (MA50) and 100 (MA100) mg MA per kg of diet. Two groups were provided feed ad libitum (control AL and MA100AL), while three were given a fixed ration (control R, MA50R, and MA100R). We have determined the following: growth rate; nucleic acid and protein levels; relative and absolute protein-accumulation (KG, AG), synthesis (KS, AS) and degradation (KD, AD) rate, protein-synthesis capacity (CS), protein-synthesis efficiency (KRNA), protein-synthesis rate per DNA (KDNA) and protein-retention efficiency. At the end of the experiment, higher body weights and muscle growth rates were found in both groups of fish fed with 100 mg MA per kg of diet. Feed-efficiency rate, protein-efficiency ratio and protein productive value, were higher in the MA100AL group than in control AL. Fractional and absolute protein-synthesis and degradation rates in white muscle of MA100AL and MA100R fish were higher than in the control, resulting in a higher protein-accumulation rate and tissue growth. Total DNA content, indicated cell hyperplasia, was higher in MA100AL and MA100R than its respective controls. Studies of optical and electronic microscopy corroborated these results. These findings indicate that MA added to the diet can stimulate the growth, white-muscle protein-turnover rates and tissue hyperplasia in gilthead sea bream.

Metabolic Effects of Insulin and IGFs on Gilthead Sea Bream (Sparus aurata) Muscle Cells

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and l-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, l-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K–Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and l-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K–Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Purification, immunological properties and molecular cloning of two allergenic parvalbumins from the crimson sea bream, Evynnis japonica

Parvalbumin, a calcium-binding sarcoplasmic protein of 12kDa, represents a major allergen of fish. Previous immunoblotting experiments suggested the presence of a 14kDa allergen, together with parvalbumin, in sea breams. In this study, the 14kDa allergen (PA I) and parvalbumin (PA II) were purified from the white muscle of crimson sea bream, Evynnis japonica, by gel filtration and reverse-phase HPLC. Amino acid sequencing of lysylendopeptidase peptide fragments demonstrated that both PA I and PA II were isoforms of parvalbumin. As analysed by ELISA, PA I showed weaker reactivities with IgG (monoclonal or polyclonal antibody) and serum IgE in fish-allergic patients than did other fish parvalbumins, including PA II. The amino acid sequences of PA I and PA II were elucidated by cDNA cloning. PA I was found to have more specific amino acid residues at several positions compared to the other fish parvalbumins.

Comparison of Metals in Tissues of Fish From Paradeniz Lagoon in the Coastal Area of Northern East Mediterranean

Present study was performed to examined the metal concentrations in muscle, liver, gonad and gill of gilthead seabream, European seabass, Leaping mullet and Flathead grey mullet from Paradeniz Lagoon, Mediterranean coastal area. The metal concentrations found in muscle tissue varied for Fe: 2.3-51.4, Zn: 6.63-14.8, Cu: 0.38-1.10, Mn: 0.38-1.06, Cr: 0.34-1.13, Ni: 0.58-.074, Pb: 0.32-1.02, Cd: 0.20-0.67 and Co: 0.30-0.61 mg/kg wet weight. Iron showed the highest levels in all tissues, and generally followed by zinc. On the other hand, cadmium, cobalt and lead showed lower levels than other metals. Statistically significant differences were observed in the mean metal values obtained from investigated fish species and their tissues. The levels of metals in the examined fish were well below the regulatory values of various govermental agencies.

CHANGES IN MUSCLE PROPERTIES DURING POSTMORTEM STORAGE OF FARMED SEA BREAM (SPARUS AURATA)

ABSTRACT This study assesses the effects of storage temperature on postmortem changes in textural parameters (firmness, water holding capacity), and their relationship with biochemical and electrophoretic determinations (muscle collagen content and solubility, amount and integrity of sarcoplasmic and myofibrillar proteins) in sea bream muscle. Storage at 1C resulted in a prolongation of the firmness compared with 4C, with no noticeable changes in water holding capacity. This delay in muscle softening at 1C could be partially explained by a limited proteolysis of some myofibrillar proteins, although inconsistent tendency was observed for other fractions. In contrast, the rate of muscle collagen degradation was higher at 4C compared with 1C, this becoming the main contributing factor to firmness losses. The great variety of protein fractions separated electrophoretically, and the relatively minor and contradictory changes observed suggest the unfeasibility of electrophoresis as a routine procedure for studying freshness in stored sea bream. PRACTICAL APPLICATIONSAccording to the results obtained, it is strongly recommended that the storage of sea bream (Sparus aurata) throughout the commercial chain is carried out at 1C, instead of the usual temperature of 4C. This modification clearly delays the process of muscle softening, thus improving objective quality in this species.The electrophoretic separation of muscle proteins has been previously proposed as a suitable procedure for assessing the freshness of fish during cold storage. However, the results obtained here indicate that the attempts aimed to the development of an electrophoretic procedure for detecting modifications in muscle proteins that could be correlated with flesh textural properties were unsuccessful. This is especially true if short‐term deterioration is assessed.