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- Amino acid metabolism in gilthead seabream is affected by the dietary protein to energy ratiosPublication . Teodósio, Rita; Aragão, Cláudia; Conceicao, Luis; Dias, Jorge; Engrola, SofiaThe dietary protein to energy ratio (P/E) has proven to influence protein utilization and/or growth in several fish species. This study intended to unravel the bioavailability and metabolic fate of lysine and methionine in gilthead seabream (Sparus aurata) juveniles fed plant diets with different P/E ratios. Seabream juveniles were fed two isonitrogenous diets (45% crude protein) differing in crude lipids (20 and 14%): LowP/E (P/E ratio=20.0 mg protein kJ-1) and HighP/E (P/E ratio=21.4 mg protein kJ-1). After three weeks, fish (11.6 +/- 4.3 g) were tube-fed the respective diet labelled with C-14-protein (L-amino acid mixture), C-14-lysine, or C-14-methionine. Protein, lysine, and methionine utilization were determined based on the proportion of C-14-amino acid evacuated, retained in the free or protein-bound fraction of liver and muscle, or catabolized. This study revealed that a decrease in P/E ratio resulted in lower amino acid evacuation (p < 0.05), contributing to a more efficient amino acid uptake. Results indicate that amino acids are retained as protein in the liver and not only temporarily available in the free pool. The amount of free amino acids retained in the muscle of LowP/E fed fish was significantly higher than in HighP/E fish (p < 0.05) due to a simultaneous higher retention of lysine and methionine, without affecting the overall protein retention. Methionine catabolism was significantly lower than lysine or protein independently of the P/E ratio (p < 0.05), reinforcing that this amino acid is preferentially spared for metabolic functions and not used as energy source. In contrast, increasing the dietary P/E ratio decreased lysine catabolism and increased its availability for growth. The bioavailability and metabolism of individual amino acids should be considered when optimizing P/E ratios in diets for gilthead seabream juveniles. Formulating diets with optimum P/E ratios will improve diet utilization and fish performance.
- Phaeodactylum tricornutum biomass in microdiets enhances Senegalese sole (Solea senegalensis) larval growth performance during weaningPublication . Barreto, André; Pinto, Wilson; Rodrigues, Andreia; Rocha, Rui J. M.; Unamunzaga, Carlos; Silva, Tome; Dias, Jorge; Conceicao, LuisMicroalgae are one of the most promising groups of functional ingredients for inclusion in microdiets for Senegalese sole (Solea senegalensis) larvae, supplying components with anti-oxidant effects that may be important during an accelerated stage of development where excessive reactive oxygen substance production leading to oxidative stress may occur. The diatom Phaeodactylum tricornutum complies with such potential since it is rich in fucoxanthin, a carotenoid with demonstrated antioxidant activity. This study aimed at determining the effect of dietary P. tricornutum supplementation on the growth performance, survival and levels of oxidative stress of Senegalese sole postlarvae. Four dietary treatments were tested: a commercial microdiet, used as control, and three experimental variants where P. tricornutum was included (1) whole cells at 5%-WP5, (2) broken cells at 5%-BP5 and (3) broken cells at 10%-BP10. At the end of the trial, no significant differences were found in growth performance of larvae fed diets where P. tricornutum was included at 5% (WP5 and BP5) and the commercial control. However, larvae from the BP10 treatment had a significantly higher dry weight than larvae from the remaining treatments. Moreover, the experimental diets had no significant effects on oxidative stress. Nonetheless, lipid peroxidation levels were significantly higher in smaller larvae fed diets where P. tricornutum whole cells were included, relatively to the commercial control. The results of the present study demonstrate that P. tricornutum broken cell biomass has the potential for inclusion in weaning microdiets for Senegalese sole, increasing larval growth performance at an inclusion level of 10 %.
- Linking fearfulness and coping styles in fishPublication . Martins, Catarina I. M.; Silva, Patricia I. M.; Conceicao, Luis E. C.; Costas, Benjamin; Hoglund, Erik; Overli, Oyvind; Schrama, Johan W.Consistent individual differences in cognitive appraisal and emotional reactivity, including fearfulness, are important personality traits in humans, non-human mammals, and birds. Comparative studies on teleost fishes support the existence of coping styles and behavioral syndromes also in poikilothermic animals. The functionalist approach to emotions hold that emotions have evolved to ensure appropriate behavioral responses to dangerous or rewarding stimuli. Little information is however available on how evolutionary widespread these putative links between personality and the expression of emotional or affective states such as fear are. Here we disclose that individual variation in coping style predicts fear responses in Nile tilapia Oreochromis niloticus, using the principle of avoidance learning. Fish previously screened for coping style were given the possibility to escape a signalled aversive stimulus. Fearful individuals showed a range of typically reactive traits such as slow recovery of feed intake in a novel environment, neophobia, and high post-stress cortisol levels. Hence, emotional reactivity and appraisal would appear to be an essential component of animal personality in species distributed throughout the vertebrate subphylum.
- The supplementation of a microdiet with crystalline indispensable amino-acids affects muscle growth and the expression pattern of related genes in Senegalese sole (Solea senegalensis) larvaePublication . Canada, Paula; Engrola, S.; Mira, Sara; Teodósio, Rita; Fernandes, Jorge M. O.; Sousa, Vera; Barriga-Negra, Lúcia; Conceicao, Luis; Valente, Luisa M. P.The full expression of growth potential in fish larvae largely depends on an efficient protein utilization, which requires that all the indispensable amino acids (IAAs) are provided at an optimum ratio. The effect of supplementing a practical microdiet with encapsulated crystalline-AA to correct possible IAA deficiencies was evaluated in Senegalese sole larvae. Two isonitrogenous and isoenergetic microdiets were formulated and processed to have approximately the same ingredients and proximate composition. The control diet (CTRL) was based on protein sources commonly used in the aquafeed industry. In the supplemented diet (SUP) 8% of an encapsulated fish protein hydrolysate was replaced by crystalline-AA in order to increase the dietary IAA levels. The microdiets were delivered from mouth-opening upon a co-feeding regime until 51 days after hatching (DAH). The larvae capacity to utilize protein was evaluated using an in vivo method of controlled tube-feeding during relevant stages throughout development: pre-metamorphosis (13 DAH); metamorphosis climax (19 DAH) and metamorphosis completion (25 DAH). Somatic growth was monitored during the whole trial. A possible effect on the regulation of muscle growth was evaluated through muscle cellularity and the expression of related genes (myf5, myod2, myogenin, mrf4, myhc and mstn1) at metamorphosis climax (19 DAH) and at a juvenile stage (51 DAH). The SUP diet had a negative impact on larvae somatic growth after the metamorphosis, even though it had no effect on the development of Senegalese sole larvae capacity to retain protein. Instead, changes in somatic growth may reflect alterations on muscle growth regulation, since muscle cellularity suggested delayed muscle development in the SUP group at 51 DAH. Transcript levels of key genes regulating myogenesis changed between groups, during the metamorphosis climax and at the 51 DAH. The group fed the SUP diet had lower dnmt3b mRNA levels compared to the CTRL group. Further studies are needed to ascertain whether this would possibly lead to an overall DNA hypomethylation in skeletal muscle. (C) 2016 Elsevier B.V. All rights reserved.
- Dietary lipid level affects growth performance and nutrient utilisation of Senegalese sole (Solea senegalensis) juvenilesPublication . Borges, Pedro; Oliveira, Beatriz; Casal, Susana; Dias, Jorge; Conceicao, Luis; Valente, Luisa M. P.Over the last few years, several aspects of Senegalese sole (Solea senegalensis) culture have been developed and optimised but the dietary lipid level for optimal growth has never been determined. Hence, five isonitrogenous diets (56 % dietary protein) with increasing dietary lipid levels (4, 8, 12, 16 and 20 % DM) were fed to satiation to triplicate groups of twenty fish (mean initial weight 10 g). Fifteen tanks were randomly assigned one of the five diets. Feed was distributed using automatic feeders, and fish were fed over a 16-week period. At the end of the experiment the fish fed on diets containing the two lowest dietary lipid levels (4 and 8 %) showed a 3-fold body-weight increase with a significantly higher daily growth index than fish fed higher lipid levels (1-2 v. 0-8). Moreover, these fish displayed a significantly lower dry feed intake (12g/kg per d) and feed conversion ratio (1-0) compared with fish fed higher lipids levels (16-19 g/kg per d; feed conversion ratio 2-0). Low dietary lipid levels (< 12 %) significantly improved nutrient retention and gain and hence growth, without major effects on whole-body composition. Despite the slight alteration in n-3 PUFA muscle content in the fish fed low-fat-diets, this fish fed low dietary lipid still remains a rich n-3 PUFA product and generally maintained its nutritional value. These results evidenced a low lipid tolerance of Senegalese sole juveniles and suggest a maximal dietary inclusion level of 8 % lipids for both optimal growth and nutrient utilisation without compromising flesh quality.
- Nutritional mitigation of winter thermal stress in gilthead seabream: associated metabolic pathways and potential indicators of nutritional statePublication . Richard, Nadège; Silva, Tome S.; Wulff, Tune; Schrama, Denise; Dias, Jorge P.; Rodrigues, Pedro; Conceicao, LuisA trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4-7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state.Significance: Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with "nutritional state" could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling. (C) 2016 Elsevier B.V. All rights reserved.
- Fish larval nutrition and feed formulation: knowledge gaps and bottlenecks for advances in larval rearingPublication . Hamre, Kristin; Yufera, Manuel; Ronnestad, Ivar; Boglione, Clara; Conceicao, Luis E. C.; Izquierdo, MarisolDespite considerable progress in recent years, many questions regarding fish larval nutrition remain largely unanswered, and several research avenues remain open. A holistic understanding of the supply line of nutrients is important for developing diets for use in larval culture and for the adaptation of rearing conditions that meet the larval requirements for the optimal presentation of food organisms and/or microdiets. The aim of the present review is to revise the state of the art and to pinpoint the gaps in knowledge regarding larval nutritional requirements, the nutritional value of live feeds and challenges and opportunities in the development of formulated larval diets.
- Dietary protein: lipid ratio and lipid nature affects fatty acid absorption and metabolism in a teleost larvaPublication . Morais, S.; Koven, W.; Ronnestad, I.; Dinis, Maria Teresa; Eugénio Castanheira Conceição, LuisStudies with teleost larvae have reported poor performance associated with quantitative lipid imbalances in the diet. The present study examined the effect of dietary protein:neutral lipid ratio on fatty acid (FA) absorption efficiency and metabolism in larval Senegalese sole. In addition, the effect of lipid class (triolein (TRI) and L-3-phosphatidylcholine-1,2-di-oleoyl (PC)), carbon number and degree of saturation of the labelled NEFA, stearic acid (SA), oleic acid (OA) and DHA) was tested. FA absorption was determined by tube feeding [1-C-14]-labelled lipids and NEFA after a single meal of either non-enriched Artemia (NEA) or Artemia enriched on a soyabean oil emulsion (EA), or after feeding these diets over an extended period of time (18 d). The tested dietary protein:lipid ratios had no short-term influence but long-term feeding of a diet higher in neutral lipid (EA) increased lipid accumulation within the gut epithelium and resulted in lower FA absorption (higher label evacuation and lower retention of dietary FA), which may partially explain the trend for lower growth observed with this diet. The lipids and NEFA, showed different digestive and metabolic properties, independent of feeding regime. FA absorption increased with unsaturation, being lowest for SA, followed by OA, and highest for DHA. In addition, sole larvae had a lower capacity to digest and absorb FA esterified to TRI, compared with PC, with the order of decreasing absorption being NEFA > PC >> TRI. Moreover, larvae appeared to discriminate between the source of OA, as this FA in the free form or esterified to PC was catabolised less than TRI.
- Can Senegalese sole post-larvae effectively grow on low dietary DHA and lipid levels during weaning?Publication . Pinto, Wilson; Engrola, S.; Santos, André; Bandarra, Narcisa M.; Dias, Jorge; Conceicao, LuisSenegalese sole (Solea senegalensis) is a highly-valuable flatfish species with interest for diversification of aquaculture in Mediterranean countries. Unlike other fish species known to have a high dietary DHA requirement during the larval stage, several studies have suggested dietary DHA could play a minor role for Senegalese sole during the early developmental stages. This suggestion was recently supported by studies that indicated an ability of Senegalese sole to biosynthesise DHA from its precursors during the larval period. However, it is still uncertain if Senegalese sole post-larvae can effectively grow on low dietary DHA levels during weaning. Moreover, recent studies focusing on the juvenile phase of Senegalese sole have suggested a possible impairment of this species to efficiently digest or absorb high dietary lipid levels. Yet, if a reduction of dietary lipid levels would affect a normal development of Senegalese sole during the early stages remains to be established. Therefore, this study aimed at determining if a reduction of dietary DHA and lipid levels would affect Senegalese sole performance and lipidme-tabolism duringweaning and post-larval period. In this study, Senegalese sole post-larvae were reared according to four dietary treatments: low lipid levels (treatments LFAT and LFAT_DHA) and moderate lipid levels (MOD-FAT and MODFAT_DHA). DHA levels in LFAT_DHA and MODFAT_DHA diets were higher than in LFAT and MOD-FAT diets, respectively. Subsequently, effects of experimental diets on Senegalese sole post-larval growth, survival, fatty acid composition and histopathological condition of intestinal and liver tissues were evaluated. Results showed that feeding on diets with low DHA and lipid levels significantly reduced the growth performance of Senegalese sole post-larvae. However, at the end of the experimental period, no significant differences were observed for post-larval survival in response to dietary treatments. In overall, this study disadvises that weaning diets for Senegalese sole post-larvae should contain low DHA and lipid levels, as a reduction of these levels may compromise post-larval growth performance.Statement of relevance: Determining essential fatty acid requirements in marine fish larvae is of utmost importance for the aquaculture industry, contributing to fulfil the growth potential of farmed species. In the case of Senegalese sole, a highly valued species farmed in Mediterranean countries, it may have the capacity to biosynthesise DHA from its precursors during the larval stage. However, the inclusion of DHA-rich oils in weaning diets is costly, considerable savings could be achieved by replacing DHA-rich oils by common fish oils in weaning diets for this species. Furthermore, Senegalese sole has also been shown a poor ability to deal with high dietary lipid levels during the juvenile stage. However, no studies have addressed this capacity in Senegalese sole larvae. This study comprises an evaluation on the effects of reducing dietary DHA and lipid levels during Senegalese sole weaning by integrating an assessment of these effects on post-larval growth, survival, fatty acid composition and histopathological condition. (C) 2016 Elsevier B.V. All rights reserved.
- A new method for the study of essential fatty acid requirements in fish larvaePublication . Morais, Sofia; Conceicao, LuisThis study describes a methodology with potential application in the estimation of essential fatty acid (EFA) requirements of fish larvae. Senegalese sole (Solea senegalensis) larvae were fed, from 16 days after hatching (DAH), on Artemia enriched with different oils, inducing graded dietary concentrations of DHA: (1) soyabean oil, containing no measurable amounts of DHA (NDHA); (2) fish oil, inducing a medium DHA level (MDHA, 3 g DHA/100 g fatty acids); and (3) a mixture of Easy DHA Selco and Microfeed, resulting in high DHA content (HDHA, 8 g/100 g). At 28 DAH a metabolic trial was conducted where larvae were tube fed [1-(14) C]DHA, in order to determine its absorption, retention in the gut and body tissues, as well as its oxidation. At 23 DAH the HDHA treatment induced a significantly higher larval growth, while at 32 DAH significant differences were only found between the NDHA and HDHA treatments. The absorption of tube-fed [1-(14) C]DHA was extremely high (94-95%) and independent of feeding regime. However, in larvae fed NDHA Artemia, a significantly higher amount of label was retained in the gut compartment and a concurrently lower retention was measured in the body. A significantly higher proportion of the absorbed DHA label was oxidized in larvae fed HDHA, compared to NDHA. Based on these results, we suggest that increasing dietary supply of DHA above the larval requirement level results in its increased oxidation for energy purposes and we propose potential applications of the tube feeding methodology using radiolabelled EFA in conjunction with dose-response studies.