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- Nutrition, quality and performance in marine fish larvaePublication . Conceição, L. E. C.; Alves-Martins, D.; Engrola, S.; Gavaia, Paulo J.; Richard, Nadège; Aragão, C.O cultivo de larvas de peixes marinhos é caracterizado por altas mortalidades e por problemas de qualidade, nomeadamente malformações esqueléticas. A solução de parte destes problemas passa por uma melhor compreensão dos requisitos nutricionais. Este trabalho pretende fazer uma revisão de trabalhos recentes que demonstram como os aminoácidos, os ácidos gordos e a vitamina K, mas também o regime alimentar, podem influenciar a qualidade e a performance das larvas de peixes marinhos.
- Novel methodologies in marine fish larval nutritionPublication . Conceição, L. E. C.; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo J.; Mira, Sara; Dias, J.Major gaps in knowledge on fish larval nutritional requirements still remain. Small larval size, and difficulties in acceptance of inert microdiets, makes progress slow and cumbersome. This lack of knowledge in fish larval nutritional requirements is one of the causes of high mortalities and quality problems commonly observed in marine larviculture. In recent years, several novel methodologies have contributed to significant progress in fish larval nutrition. Others are emerging and are likely to bring further insight into larval nutritional physiology and requirements. This paper reviews a range of new tools and some examples of their present use, as well as potential future applications in the study of fish larvae nutrition. Tube-feeding and incorporation into Artemia of 14C-amino acids and lipids allowed studying Artemia intake, digestion and absorption and utilisation of these nutrients. Diet selection by fish larvae has been studied with diets containing different natural stable isotope signatures or diets where different rare metal oxides were added. Mechanistic modelling has been used as a tool to integrate existing knowledge and reveal gaps, and also to better understand results obtained in tracer studies. Population genomics may assist in assessing genotype effects on nutritional requirements, by using progeny testing in fish reared in the same tanks, and also in identifying QTLs for larval stages. Functional genomics and proteomics enable the study of gene and protein expression under various dietary conditions, and thereby identify the metabolic pathways which are affected by a given nutrient. Promising results were obtained using the metabolic programming concept in early life to facilitate utilisation of certain nutrients at later stages. All together, these methodologies have made decisive contributions, and are expected to do even more in the near future, to build a knowledge basis for development of optimised diets and feeding regimes for different species of larval fish.
- Changes in the soluble bone proteome of reared white seabream (Diplodus sargus) with skeletal deformitiesPublication . Silva, Tomé S.; Cordeiro, O.; Richard, Nadège; Conceição, L. E. C.; Rodrigues, PedroOne of the main constrains for commercial aquaculture production of white seabream (Diplodus sargus) is the high incidence of skeletal malformations in reared fish. The purpose of this study was to obtain a better understanding of the mechanisms involved in the development of these types of skeletal malformations by comparative proteomic analysis of the vertebral column of normal and deformed fish using 2DE for protein separation and MS for protein identification. We observed a 3.2 and 3.4-fold increase in the expression of two tropomyosin isoforms, one of which (tropomyosin-4) is essential for the motility and polarization cycles of osteoclasts. Furthermore, a 1.6, 1.7 and 1.8-fold increase in three parvalbumin spots was detected, suggesting a cellular response to increased intracellular Ca2+ levels. These results can be interpreted as signs of increased cellular activity in the bone of white seabream with skeletal deformities coupled to a higher degree of calcium mobilization, which elicits further studies into the use of these proteins as indicators of skeletal metabolic state.
- Impact of dietary protein hydrolysates on skeleton quality and proteome in Diplodus sargus larvaePublication . Vareilles, Mahaut; Richard, Nadège; Gavaia, Paulo J.; Silva, Tomé S.; Cordeiro, O.; Guerreiro, Inês; Yúfera, M.; Batista, I.; Pires, C.; Pousão-Ferreira, P.; Rodrigues, Pedro; Rønnestad, I.; Fladmark, K. E.; Conceição, L. E. C.In order to investigate the effects of dietary protein hydrolysates (PH) on larval growth performance, skeleton quality and proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding with live feed and three microencapsulated diets differing in the molecular weight of their PH fraction (Control – inclusion of CPSP-90; H – inclusion of a high amount in 0.5–30 kDa hydrolysates; L – inclusion of a high amount in <0.5 kDa hydrolysates). At 15 days after hatching (DAH), proteome expression changes were assessed in entire larvae by twodimensional gel electrophoresis and the quality of larval skeleton was analysed at 28 DAH through double staining of cartilage and bone. Dietary PH fractions tested affected growth, the larvae fed diet L being significantly larger than those fed diet H, but it did not affect the incidence of deformed larvae, nor the number of deformities per fish. Two-dimensional analysis of larvae proteome allowed the detection and the comparative quantification of a total of 709 protein spots having a pI between 4 and 7, around half of which had an expression significantly affected by dietary treatment, the main difference being between proteome of Control larvae with those of both groups L and H. From these spots, 52 proteins involved in diverse processes such as cytoskeletal dynamics, energetic, lipoprotein, amino acid (AA), and nucleotide metabolisms, protein chaperoning and degradation, and signal transduction, were identified. This study revealed that the molecular weight of the dietary protein hydrolysate fraction had a minor impact on skeletal deformities in white seabream larvae, but affected growth performance and had a strong impact on larvae whole body proteome.
- Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variationsPublication . Silva, Tomé S.; Costa, Ana M. Rosa da; Conceição, L. E. C.; Dias, Jorge P.; Rodrigues, Pedro; Richard, NadègeFarmed gilthead seabream is sometimes affected by a metabolic syndrome, known as the "winter disease", which has a significant economic impact in the Mediterranean region. It is caused, among other factors, by the thermal variations that occur during colder months and there are signs that an improved nutritional status can mitigate the effects of this thermal stress. For this reason, a trial was undertaken where we assessed the effect of two different diets on gilthead seabream physiology and nutritional state, through metabolic fingerprinting of hepatic tissue. For this trial, four groups of 25 adult gilthead seabream were reared for 8 months, being fed either with a control diet (CTRL, low-cost commercial formulation) or with a diet called "Winter Feed" (WF, high-cost improved formulation). Fish were sampled at two time-points (at the end of winter and at the end of spring), with liver tissue being taken for FT-IR spectroscopy. Results have shown that seasonal temperature variations constitute a metabolic challenge for gilthead seabream, with hepatic carbohydrate stores being consumed over the course of the inter-sampling period. Regarding the WF diet, results point towards a positive effect in terms of performance and improved nutritional status. This diet seems to have a mitigating effect on the deleterious impact of thermal shifts, confirming the hypothesis that nutritional factors can affect the capacity of gilthead seabream to cope with seasonal thermal variations and possibly contribute to prevent the onset of "winter disease".
- Recent advances in nutrition of fish larval | Avanços recentes em nutrição de larvas de peixesPublication . Conceição, L. E. C.; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo J.; Mira, Sara; Dias, J.Os requisitos nutricionais de larvas de peixes são ainda mal compreendidos, o que leva a altas mortalidades e problemas de qualidade no seu cultivo. Este trabalho pretende fazer uma revisão de novas metodologias de investigação, tais como estudos com marcadores, genómica populacional, programação nutricional, génomica e proteómica funcionais, e fornecer ainda alguns exemplos das utilizações presentes e perspectivas futuras em estudos de nutrição de larvas de peixes.
- Dietary indispensable amino acids profile affects protein utilization and growth of Senegalese sole larvaePublication . Canada, Paula; Engrola, S.; Richard, Nadège; Lopes, Ana Filipa; Pinto, Wilson; Valente, Luísa M. P.; Da Conceição, Gisele CristinaIn diet formulation for fish, it is critical to assure that all the indispensable amino acids (IAA) are available in the right quantities and ratios. This will allow minimizing dietary AA imbalances that will result in unavoidable AA losses for energy dissipation rather than for protein synthesis and growth. The supplementation with crystalline amino acids (CAA) is a possible solution to correct the dietary amino acid (AA) profile that has shown positive results for larvae of some fish species. This study tested the effect of supplementing a practical microdiet with encapsulated CAA as to balance the dietary IAA profile and to improve the capacity of Senegalese sole larvae to utilize AA and maximize growth potential. Larvae were reared at 19 A degrees C under a co-feeding regime from mouth opening. Two microdiets were formulated and processed as to have as much as possible the same ingredients and proximate composition. The control diet (CTRL) formulation was based on commonly used protein sources. A balanced diet (BAL) was formulated as to meet the ideal IAA profile defined for Senegalese sole: the dietary AA profile was corrected by replacing 4 % of encapsulated protein hydrolysate by CAA. The in vivo method of controlled tube-feeding was used to assess the effect on the larvae capacity to utilize protein, during key developmental stages. Growth was monitored until 51 DAH. The supplementation of microdiets with CAA in order to balance the dietary AA had a positive short-term effect on the Senegalese sole larvae capacity to retain protein. However, that did not translate into increased growth. On the contrary, larvae fed a more imbalanced (CTRL group) diet attained a better performance. Further studies are needed to ascertain whether this was due to an effect on the voluntary feed intake as a compensatory response to the dietary IAA imbalance in the CTRL diet or due to the higher content of tryptophan in the BAL diet.
- Inclusion of protein hydrolysates in the diet of white seabream (Diplodus sargus) larvae: impacts on skeleton quality and larvae proteome expressionPublication . Richard, Nadège; De Vareilles, M.; Gavaia, Paulo J.; Silva, Tomé S.; Cordeiro, O.; Yúfera, M.; Rodrigues, Pedro; Conceição, L. E. C.In order to investigate the effects of dietary protein hydrolysate on larval skeleton quality and larvae proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding onwards with live feed and three microencapsulated diets ir differing only on the molecular weight of their protein hydrolysate fraction.
- Two-dimensional proteomics as a tool to evaluate nutritional effects in farmed fishPublication . Richard, Nadège; Gavaia, Paulo J.; De Vareilles, M.; Silva, Tomé S.; Cordeiro, O.; Rodrigues, Pedro; Conceição, L. E. C.The use of proteomics in fish research is at a relatively early stage compared to terrestrial vertebrates. It has already been applied in the field of fish nutrition, enabling to point out metabolic changes occurring in response to dietary manipulations such as a variation in energy content, dietary nitrogen compostion, lipid composition or the incorporation of plant protein sources in aquafeeds.
- Metabolic molecular indicators of chronic stress in gilthead seabream (Sparus aurata) using comparative proteomicsPublication . Alves, Ricardo N.; Cordeiro, O.; Silva, Tomé S.; Richard, Nadège; De Vareilles, M.; Marino, G.; Di Marco, Patrizia; Rodrigues, Pedro; Conceição, L. E. C.The aim of this study was to identify possible metabolic molecular indicators of chronic stress in gilthead sea bream Sparus aurata. Two potential stressful conditions were tested: repeated handling and crowding at high stocking density. Gilthead seabream kept under optimized rearing conditions were used as control fish. Cortisol was measured as primary stress indicator and the liver proteome of stressed fish was compared to that of control fish using comparative proteomics. Plasma cortisol levels in sea bream repeatedly handled and crowded at high stocking density were significantly higher than in undisturbed control fish. A total of 560 spots were detected and the statistical analysis revealed a differential expression in about 50% of all detected proteins. Spots with greater than 2-fold or lower than 0.5-fold changes were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Proteins like fatty acid binding protein (lipid transport and antioxidant role), heat shock cognate protein (chaperoning), calmodulin (Ca2+ signaling), mitochondrial porine — voltage-dependent anion channel (lipid oxidation), glutamine synthetase (ammonia metabolism), cofilin and beta-tubulin (cytoskeleton), hemoglobin and several other proteins involved in carbohydrate metabolism (triose-phosphate isomerase, pyruvate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alfa-enolase) were differentially expressed in fish under chronic stress. Some of these proteins may be used in the future as chronic stress and/or part of a panel of welfare biomarkers, after validation studies using RT-PCR and ELISA assays.
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