Physiopathological responses of sole (Solea senegalensis) subjected to bacterial infection and handling stress after probiotic treatment with autochthonous bacteria.

This study aimed to evaluate the protective effects of four autochthonous bacteria isolated from juvenile sole (Solea senegalensis) intestine as dietary probiotic supplement against bacterial pathogen infection and handling/transport stressors. Growth performance and immune responses were evaluated after 85 days of feeding trial. Sole (IBW = 16.07 ±â€¯0.11 g) were fed six experimental diets, a control diet (CTRL, without the dietary probiotic supplementation), and five diets supplemented with probiotic bacteria: PB1 (Shewanella hafniensis), PB2 (Enterococcus raffinosus), PB3 (Shewanella hafniensis + Arthrobacter soli), PB4 (Pseudomonas protegens + Arthrobacter soli) and PB5 (Shewanella hafniensis + Arthrobacter soli + Enterococcus raffinosus). All bacteria were selected based on their in vitro antimicrobial activity. After the growth trial, fish were submitted to a stress factor (transport) and then each dietary group was divided in two additional groups: non-infected (placebo) and infected with Photobacterium damselae subsp. piscicida. Immune and antioxidant responses were evaluated at day 10 post-infection. In infection trial A, fish were infected on the same day of transport, whereas in trial B fish were infected after a 7-day recovery from the transport stress. At the end of the feeding trial, fish fed with PB2 and PB4 showed lower final body weight when compared with the other dietary groups. Respiratory burst activity and nitric oxide production were not affected by probiotic supplementation. Fish fed with PB5 presented lower peroxidase activity compared to CTRL. Lysozyme and alternative complement pathway activity (ACH50) showed no significant differences between treatments. The innate immune responses were significantly affected after handling stress and bacterial infection. In trial A, the ACH50 levels of infected fish were significantly lower than the placebo groups. On the other hand, in trial B fish infected with Pdp demonstrated higher ACH50 levels when compared to placebos. Peroxidase levels were strongly modulated by bacterial infection and handling stress. In trials A and B, infection had a clear downgrade effect in peroxidase levels. Lipid peroxidation, catalase, glutathione S-transferase and glutathione reductase were altered by both bacterial infection and transport. Overall, dietary probiotic supplementation did not influence growth performance of sole. The immune and oxidative defenses of sole responded differently to infection depending on the probiotic and the synergy between pathogen infection and transport.

Innate immune response, intestinal morphology and microbiota changes in Senegalese sole fed plant protein diets with probiotics or autolysed yeast.

The effects of using plant ingredients in Senegalese sole (Solea senegalensis) diet on immune competence and intestine morphology and microbial ecology are still controversial. Probiotics or immunostimulants can potentially alter the intestinal microbiota in a way that protects fish against pathogens. The current study aimed to examine the intestine histology and microbiota and humoral innate immune response in juvenile sole fed diets with low (35 %) or high (72 %) content of plant protein (PP) ingredients supplemented with a multispecies probiotic bacteria or autolysed yeast. Fish fed the probiotic diet had lower growth performance. Lysozyme and complement activities were significantly higher in fish fed PP72 diets than in their counterparts fed PP35 diets after 17 and 38 days of feeding. At 2 days of feeding, fish fed unsupplemented PP72 showed larger intestine section area and longer villus than fish fed unsupplemented PP35. At 17 days of feeding, fish fed unsupplemented PP72 showed more goblet cells than the other dietary groups, except the group fed yeast supplemented PP35 diet. High dietary PP level, acutely stimulate fish innate immune defence of the fish after 2 and 17 days of feeding. However, this effect does not occur after 73 days of feeding, suggesting a habituation to dietary treatments and/or immunosuppression, with a reduction in the number of the goblet cells. Fish fed for 38 days with diets supplemented with autolysed yeast showed longer intestinal villus. The predominant bacteria found in sole intestine were Vibrio sp. and dietary probiotic supplementation caused a reduction in Vibrio content, regardless of the PP level.

Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma.

Tenacibaculum maritimum, the aetiological agent for marine tenacibaculosis, is one of the most significant pathogens that threaten Senegalese sole, Solea senegalensis (Kaup), aquaculture. Because no immersion challenge with T. maritimum has been reported previously for this flatfish species, this study aimed to optimize bacterial yields as well as to establish a challenge model for tenacibaculosis induction. Several approaches were performed to optimize bacterial culture conditions, including treatment with non-ionic surfactants, detergents, cellulase hydrolysis and strong shaking. A prolonged bath challenge was performed for 24 h under two different temperatures, 16 and 23 °C. Moreover, mucus and plasma bactericidal activities against T. maritimum were also assessed. Culturing bacteria with strong shaking and continuous shaking provided suitable culture conditions to obtain higher bacterial yields without aggregation and fluctuation, contrary to most other treatments that showed a huge amount of bacterial aggregates. A prolonged bath method for 24 h, without skin or gill scarification, was considered suitable for disease induction with high mortality rates. Moreover, data regarding mucus and plasma bactericidal activities suggested that there is a lack of host innate immune response against T. maritimum or that this particular pathogen presents evading strategies against Senegalese sole.

Lipid content and fatty acid profile of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles as affected by feed containing different amounts of plant protein sources.

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5-10.8% of fat) followed by fins (3.4-6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4-4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n-6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n-6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish - the muscle.

Dietary lipid levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (Solea senegalensis Kaup).

In Senegalese sole (Solea senegalensis Kaup), growth is negatively correlated to dietary lipid levels. To understand the molecular basis of this effect a molecular toolbox of 12 genes, including fgf6, fst, mstn1, myf5, mrf4, myod1, myod2, myog, myHC, mylc2, igf1r and insr, was developed. The expression profiles of these genes were investigated in white muscle and liver of fish fed with three dietary lipid levels (4%, 12% and 20%). The expression of igf-I and igf-II was also examined. MRFs and myosins were only expressed in the muscle and, except for myf5, the general trend was a decrease in expression with an increase in dietary lipids. Fgf6 was identified for the first time in liver and its expression augmented in hepatic tissues with increasing dietary lipid levels. A similar tendency was observed for mstn1 and igf-I. The opposite was observed for igf1r expression in muscle and liver. Myog, mrf4, mylc2 and igf1r were highly correlated with growth and nutrient utilisation indices. In addition to its practical implications, this work provides a valuable contribution towards our understanding of the genetic networks controlling growth in teleosts.

Expression of the myosin light chains 1, 2 and 3 in the muscle of blackspot seabream (Pagellus bogaraveo, Brunnich), during development.

Previous studies on the histochemistry and immunoreactivity of fibres in lateral muscle of blackspot seabream indicated that there is a developmental transition in the composition of myofibrillar proteins, which presumably reflects changes in contractile function as the fish grows. We hypothesize that the phenomenon underscores age and spatial differences in the expression of myosin light chains (MLC), not studied yet in this species. In this study, we examined selected stages in the post-hatching development of the muscle of blackspot seabream: hatching (0 days), mouth opening (5 days), weaning (40 days) and juveniles (70 days). The spatial expression of embryonic MLC 1 (MLC1), 2 (MLC2) and 3 (MLC3) was studied by in situ hybridization. Overall, MLC expression patterns were overlapping and restricted to the fast muscle. At hatching and mouth opening, all MLC types were highly expressed throughout the musculature in fast muscle. The expression levels in fast muscle remained high until weaning when germinal zones appeared on the dorsal and ventral areas. The germinal zones were characterized by small-diameter fast fibres with high levels of MLC expression. This pattern persisted up to day 70, when the germinal zones disappeared and expression of MLCs was observed only in the smaller cells of the fast muscle mosaic. These results support our hypothesis and, together with previous imuno- and histochemistry results, allow a better understanding of the mechanism of muscle differentiation and growth in fish beyond larval stages, and form- the basis for further comparative and experimental studies with this economically relevant species.

Dietary protein content influences both growth and size distribution of anterior and posterior muscle fibres in juveniles of Pagellus bogaraveo (Brunnich).

Muscle cellularity was studied in Pagellus bogaraveo juveniles fed on diets with different protein contents. Measured in transversal body sections, at both post-opercular and post-anal locations, the morphometric variables estimated were: total muscle area (A), total number of fibres (N), number of fibres per unit area of muscle (N (A)) and cross-sectional fibre area (a) of the two main muscle fibre types. At the end of the experiment fish fed on diets having more than 40% of protein displayed significantly higher body weight. Fish fed on protein-rich diets exhibited greater a and N. For fish fed on 30 and 50% protein diets the morphometric parameters measured grew linearly with the fish weight. High-protein diets favoured muscle hyperplasia. When comparing rostral and caudal locations, a greater N and a smaller a of posterior red fibres were the consistent differences found-a fact, to our knowledge, so far unreported for fish.

Hyperplastic and hypertrophic growth of lateral muscle in blackspot seabream Pagellus bogaraveo from hatching to juvenile.

To understand better the growth mechanisms in the economically important fish Pagellus bogaraveo, in terms of muscle fibre hyperplasia v. hypertrophy, the lateral muscle of this fish was studied morphometrically from hatching to juvenile comparing rostral and caudal locations. Fish were sampled at 0, 5, 23, 40, 70, 100, 140 and 180 days. Fibre types were first identified by succinate dehydrogenase (SDH) and immunostaining with a polyclonal antibody against fish slow myosin (4-96). Morphometric variables were then measured in transverse body sections, at both post-opercular and post-anal locations, to estimate the following variables: total muscle area [A (muscle)], total fibre number [N (fibres)], fibre number per unit area of muscle [N(A)(fibres, muscle)] and cross-sectional fibre area [a (fibres)] of the two main muscle fibre types (white and red). Overall, growth throughout the various stages resulted from increases both in the number and in the size of muscle fibres, paralleled by an expansion of the [A (muscle)]. Nonetheless, that increase was not significant between 0-5 days on one hand and 100-140 days, on the other hand. On the contrary, the [N(A)(fibres, muscle)] declined as the body length increased. Analysis of the muscle growth kinetics suggested that, within the important time frame studied, hyperplasia gave the main relative contribution to the increase of white muscle [A (white muscle)], whereas red muscle [A (red muscle)] mainly grew by hypertrophy, with both phenomena occurring at a faster pace posteriorly in the body. Finally, when comparing rostral and caudal locations, a greater [N (fibres)] and [A (muscle)] of the posterior white and red fibres were the consistent features. It was also observed that the proportion of the cross-sectional area of the myotomal muscle comprised of white muscle was greater in the anterior part of the fish.

Muscle differentiation in blackspot seabream (Pagellus bogaraveo, Brunnich): histochemical and immunohistochemical study of the fibre types.

The aim of this work was to gain insights into the mechanism of muscle differentiation and growth in Pagellus bogaraveo, by studying muscle fibre phenotypes identified by immunohistochemistry. At hatching, several layers of deep fast-white fibres were covered by a superficial fibre monolayer. At 5 days, slow-red fibres appeared near the lateral line nerve. At 40 days, the intermediate-pink muscle became visible, and in the slow-red and fast-white muscle layers transitions from larval myosin isoforms to the isoforms typical of adult muscle occurred. Between 70 and 100 days, small fibres with a distinct ATPase profile appeared throughout the fast-white muscle, marking the onset of "mosaic" hyperplasia. The myosin of the original superficial monolayer fibres underwent two myosin transformations, before being slowly replaced by an adult slow-red isoform. In juveniles and adults, the slow-red muscle layer could be resolved into two distinct types. The analysis of fibre phenotypes indicated that post-larval muscle growth occurred by two distinct stages of hyperplasia. This study offers a basis for further comparative and experimental studies with this economically relevant species, namely for identifying factors influencing its muscle growth dynamics and disclosing underlying mechanisms.

Annual assessment of the sea urchin (Paracentrotus lividus) humoral innate immune status: Tales from the north Portuguese coast.

Innate immune status of the sea urchin Paracentrotus lividus population from two different rocky shore beaches in the northern Portuguese coast was evaluated for a period of one year. Although some ecological studies regarding the effect of toxics on the immune parameters of the sea urchin were made in Portuguese waters, there is a current lack of knowledge concerning their immune status all over the year. In perspective of a changing ecosystem in these waters due to global warming and colonization of new species, it is important to assess the status of the major species living in the area. In this way, immune parameters such as total protein content, nitric oxide concentration, haemolytic activity, protease activity, lysozyme concentration and bactericidal activity were evaluated in the perivisceral coelomic fluid, and were correlated with the gonadal index of the population and water parameters. Also, the spawning period can upset some immune status parameters, and others such as haemolytic activity and bactericidal activity against Vibrio anguillarum, showed a clear correlation with the gonad maturation status. The knowledge of the basal immune status of the species could serve as ecological indicator of some stress agent or contaminant into the field; also, coelomic fluid is suggested as good quality marker to assess the immune status of sea urchins.

Effect of two experimental diets (protein and lipid vegetable oil blends) on the volatile profile of Senegalese sole (Solea senegalensis Kaup, 1858) muscle.

The aim of this study was to determine differences among volatile compounds composition of Senegalese sole muscle fed with extruded diets containing different plant protein (PP) and vegetable oil (VO) sources. Two set of experiments were performed on growing sole. One growth trial used a control diet containing fish meal (FM) as the main protein source and different PP-based diets. Another growth trial compared a control diet containing fish oil (FO) as the main lipid source and different VO-based diets; after a period, all sole were fed with the FO diet. Results showed that the incorporation of PP sources up to 75% allowed the production of a similar content of major volatile compounds to the control diet. In VO-based diets, some significant differences were found in the levels of some volatile compounds in sole muscle; however, no significant differences were obtained through sensory evaluation.

Effect of storage time and heat processing on the volatile profile of Senegalese sole (Solea senegalensis Kaup, 1858) muscle.

The effect of heat treatment and the presence or absence of fish skin on the volatile composition of Senegalese sole muscle was studied. The volatile profile of Senegalese sole at different storage periods was also evaluated. All samples were analysed by HS-SPME-GC-IT/MS and subjected to sensory evaluation. As expected, cooking enhanced the production/liberation of volatile compounds. Fish with the skin present, after cooking, had higher levels of sulphur compounds, 2-nonanone, ethyl octanoate and lower contents of hexanol and heptanol than skinned fish; moreover, the samples with the skin had a better overall sensory acceptability. During storage, changes on the volatile composition of Senegalese sole samples were found. The major differences were obtained after 2 weeks of storage. Compounds such as hexanal, heptanal, octanal, decanal, (E)-2-hexenal, (E)-2-decen-1-al, (E,Z)-2,6-nonadienal, benzaldehyde, 4-ethyl-benzaldehyde, 1-penten-3-ol, heptanol and (E)-2-octen-1-ol decreased after 2 weeks of storage, and other compounds, such as 3-methyl-1-butanal, 2-methyl-1-butanal, 2-heptanone, dimethyl trisulphide, dimethyl tetrasulphide and 2-methyltetrahydrothiophen-3-one increased. These differences were confirmed by sensory evaluation. Principal component analysis was applied to the chemical data.

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact.

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry.

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. II--Genetic factors related to animal performance and advances in methodology.

Selective breeding is an effective tool to improve livestock. Several selection experiments have been conducted to study direct selection responses as well as correlated responses in traits of skeletal muscle growth and function. Moreover, comparisons of domestic with wild-type species and of extreme breeds provide information on the genetic background of the skeletal muscle phenotype. Structural muscular components that differed with increasing distance in lean growth or meat quality in mammals were found to be myofibre number, myofibre size, proportions of fibre types as well as the numbers and proportions of secondary and primary fibres. Furthermore, markers of satellite cell proliferation, metabolic enzyme activities, glycogen and fat contents, the expression of myosin heavy chain isoforms, of activated AMPKα and other proteins in skeletal muscle tissue and circulating IGF1 and IGF-binding proteins have been identified to be involved in selection responses observed in pigs, cattle and/or chicken. The use of molecular methods for selective breeding of fish has only recently been adopted in aquaculture and studies of the genetic basis of growth and flesh quality traits are scarce. Some of the molecular markers of muscle structure/metabolism in livestock have also been identified in fish, but so far no studies have linked them with selection response. Genome scans have been applied to identify genomic regions exhibiting quantitative trait loci that control traits of interest, for example, muscle structure and meat quality in pigs and growth rate in chicken. As another approach, polymorphisms in candidate genes reveal the relationship between genetic variation and target traits. Thus, in large-scale studies with pigs' associations of polymorphisms in the HMGA2, CA3, EPOR, NME1 and TTN genes with traits of carcass and meat quality were detected. Other studies revealed the significance of mutations in the IGF2 and RYR1 genes for carcass lean and muscle fibre traits in pigs. Mutations in the myostatin (MSTN) gene in fish were also examined. Advances in research of the genetic and environmental control of traits related to meat quality and growth have been made by the application of holistic 'omics' techniques that studied the whole muscle-specific genome, transcriptome and proteome in relation to muscle and meat traits, the development of new methods for muscle fibre typing and the adaptation of biophysical measures to develop parameters of muscle fibre traits as well as the application of in vitro studies. Finally, future research priorities in the field are defined.

Conjugated linoleic acid in diets for large-size rainbow trout (Oncorhynchus mykiss): effects on growth, chemical composition and sensory attributes.

The effects of graded levels (0 %, 0.5 %, 0.75 and 1 %) of dietary conjugated linoleic acid (CLA) were assessed on 97 g rainbow trout. Fish were fed to satiation twice a day for 12 weeks. At the end of the experiment, all groups of fish weighed more than 250 g and no significant differences were detected in growth performance, feed conversion, nutrient or energy utilisation or body composition between treatments. A decrease in liver lipid content resulted from including CLA and was accompanied by a reduction in malic enzyme activity. The muscle saturated acid and PUFA content did not vary between dietary treatments, despite the increasing concentration of stearic acid and CLA. In the liver, however, both fractions increased significantly with dietary CLA. Moreover, the MUFA decreased significantly in both muscle and liver. CLA was incorporated into tissue lipids, with levels in flesh (2.1-4.2 %) being 2-fold higher than in liver (0.8-1.9 %). In muscle, the percentage of cis-9, trans-11 isomer ranged from 39.5 % to 41.8 % and that of trans-10, cis-12 isomer from 31.4 % to 33.4 % of total CLA. The incorporation of CLA isomers in the liver varied with dietary treatment, and the cis-9, trans-11 isomer seemed to be more efficiently incorporated than trans-10, cis-12. Sensory data indicated slight-to-moderate differences between the trout fed with and without CLA. The present results suggest that 250 g rainbow trout can incorporate CLA in both muscle and liver, contributing to the production of a functional food.