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- How tryptophan levels in plant-based aquafeeds affect fish physiology, metabolism and proteomePublication . Cerqueira, Marco; Schrama, Denise; Silva, Tome S.; Colen, R.; Engrola, Sofia; Conceicao, Luis E. C.; Rodrigues, Pedro; Farinha, Ana PaulaFish meal replacement by plant-protein sources is a priority in aquaculture feeds. Within this framework, dietary supplementation with essential amino acids (EAA), as tryptophan (TRP), is strategic to ensure that the individual nutritional needs are met, besides promoting enhanced immunological status. The purpose of this study was to examine the beneficial effects of TRP incorporation in plant-protein source diets on fish growth performance and nutritional status. We tested diets with 20% lower (LTRP) and 27% higher (HTRP) of the putative requirements of TRP for seabream (Sparus aurata) and assessed its impact on fish physiology and liver metabolism and proteome. After 12 weeks, growth performance, body proximate, hepatic composition and liver metabolic profiling were similar between diets. Nevertheless, liver proteome analysis indicated a higher accumulation of proteins involved in acute-phase responses, typically triggered by infection, inflammation or trauma, in fish fed with HTRP diet as compared with those fed with LTRP. The overall results obtained suggest a potential beneficial effect of TRP supplementation in terms of immune stimulation, without compromising growth or feed intake. Moreover, proteomics and metabolic profiling demonstrate to be valuable tools in this endeavour. Significance: Nutritional needs are hard to assess in aquaculture fisheries, and many times controversial depending on the methodology employed. The estimated amino acid requirements depend on both fish species and stage development, making it extremely hard to standardise. On the other hand, the substitution of fish-based to plant-based protein sources diets towards a sustainable aquaculture, may imbalance these requirements, being necessary further studies to assess the impact on fish growth and development. Finally, the incorporation of crystalized amino acids such as TRP into diets aims global better performance both at fish health/immune condition and growth development. This work focused on the potential beneficial effects of TRP supplementation into diets with a plant-based protein source, addressing the effects on the liver metabolism and proteome, and on growth performance of Gilthead seabream juveniles, a species with special relevance and economical importance in the Mediterranean region. The present study by employing proteomics together with metabolic profiling shows that TRP supplementation at the tested doses, does not compromise growth performance, and seems to stimulate the immune system. Our findings can contribute to the development of new feed formulations for Gilthead seabream species, therefore, reinforcing the resilience and competitiveness of the on-growing aquaculture industry and impact directly the sustainability of living resources with the decrease of the fisheries needs to fulfil the human search for quality proteins consume.
- Optimizing diets to decrease environmental impact of Nile tilapia (Oreochromis niloticus) productionPublication . Teodósio, Rita; Engrola, Sofia; Colen, R.; Masagounder, Karthik; Aragão, CláudiaAquaculture is one of the most thriving animal production sectors, and Nile tilapia (Oreochromis niloticus) farming represents 8% of total finfish culture. However, the industry sustainability depends on the development of cost‐effective and environmental friendly feeds. This study aimed to reduce dietary protein levels in diets for juvenile tilapia and to minimize diet environmental impact while maximizing biological efficiency. A growth trial was performed using five isoenergetic plant protein‐based diets with decreasing levels of crude protein: 360, 340, 320, 300 and 280 g/kg diet (D360, D340, D320, D300 and D280, respectively). Dietary protein utilization was assessed by metabolic trials using a radiolabelled amino acid mixture. Tilapia in all treatments showed similar growth performance and feed intake. Feed conversion ratio was significantly higher in fish fed the D280 than the D360 diet, while no differences were found for other treatments. Protein retention was significantly higher in tilapia fed the D300 than the D360 diet. Amino acid catabolism increased in fish fed the D360 diet, though without significant differences in muscle amino acid retention. This study demonstrates that dietary protein levels can be reduced to 300 g/kg diet without hindering tilapia growth and feed conversion ratio, while reducing environmental nitrogen losses.
- Dietary creatine supplementation in gilthead seabream (Sparus aurata): comparative proteomics analysis on fish allergens, muscle quality, and liverPublication . Schrama, Denise; Cerqueira, Marco; Raposo de Magalhães, Cláudia; Rosa Da Costa, Ana; Wulff, Tune; Gonçalves, Amparo; Camacho, Carolina; Colen, R.; Fonseca, Flávio; Rodrigues, PedroThe quality of fish flesh depends on the skeletal muscle's energetic state and delaying energy depletion through diets supplementation could contribute to the preservation of muscle's quality traits and modulation of fish allergens. Food allergies represent a serious public health problem worldwide with fish being one of the top eight more allergenic foods. Parvalbumins, have been identified as the main fish allergen. In this study, we attempted to produce a low allergenic farmed fish with improved muscle quality in controlled artificial conditions by supplementing a commercial fish diet with different creatine percentages. The supplementation of fish diets with specific nutrients, aimed at reducing the expression of parvalbumin, can be considered of higher interest and beneficial in terms of food safety and human health. The effects of these supplemented diets on fish growth, physiological stress, fish muscle status, and parvalbumin modulation were investigated. Data from zootechnical parameters were used to evaluate fish growth, food conversion ratios and hepatosomatic index. Physiological stress responses were assessed by measuring cortisol releases and muscle quality analyzed by rigor mortis and pH. Parvalbumin, creatine, and glycogen concentrations in muscle were also determined. Comparative proteomics was used to look into changes in muscle and liver tissues at protein level. Our results suggest that the supplementation of commercial fish diets with creatine does not affect farmed fish productivity parameters, or either muscle quality. Additionally, the effect of higher concentrations of creatine supplementation revealed a minor influence in fish physiological welfare. Differences at the proteome level were detected among fish fed with different diets. Differential muscle proteins expression was identified as tropomyosins, beta enolase, and creatine kinase among others, whether in liver several proteins involved in the immune system, cellular processes, stress, and inflammation response were modulated. Regarding parvalbumin modulation, the tested creatine percentages added to the commercial diet had also no effect in the expression of this protein. The use of proteomics tools showed to be sensitive to infer about changes of the underlying molecular mechanisms regarding fish responses to external stimulus, providing a holistic and unbiased view on fish allergens and muscle quality.
- Metabolic and nutritional responses of Nile tilapia juveniles to dietary methionine sourcesPublication . Teodósio, Rita; Engrola, Sofia; Cabano, Miguel; Colen, R.; Masagounder, Karthik; Raquel Cêa de Aragão Teixeira, CláudiaCommercial diets for tilapia juveniles contain high levels of plant protein sources. Soybean meal has been utilised due to its high protein content; however, soy-based diets are limited in methionine (Met) and require its supplementation to fulfil fish requirements. DL-Methinone (DL-Met) and Ca bis-methionine hydroxyl analogue (MHA-Ca) are synthetic Met sources supplemented in aquafeeds, which may differ in biological efficiency due to structural differences. The present study evaluated the effect of both methionine sources on metabolism and growth of Nile tilapia. A growth trial was performed using three isonitrogenous and isoenergetic diets, containing plant ingredients as protein sources: DLM and MHA diets were supplemented on equimolar levels of Met, while REF diet was not supplemented. Hepatic free Met and one-carbon metabolites were determined in fish fed for 57 d. Metabolism of DL-Met and MHA was analysed by an in vivo time-course trial using 14C-labelled tracers. Only DLMet supplementation significantly increased final body weight and improved feed conversion and protein efficiency ratios compared with the REF diet. Our findings indicate that Met in DLM fed fish follows the transsulphuration pathway, while in fish fed MHA and REF diets it is reme thylated. The in vivo trial revealed that 14C-DL-Met is absorbed faster and more retained than 14C-MHA, resulting in a greater availability of free Met in the tissues when fish is fed with DLM diet. Our study indicates that dietary DL-Met supplementation improves growth performance and N retention, and that Met absorption and utilisation are influenced by the dietary source in tilapia juveniles.
- Dietary antioxidant supplementation promotes growth in senegalese sole postlarvaePublication . Xavier, Maria J; Engrola, Sofia; Conceição, Luis E C; Manchado, Manuel; Carballo, Carlos; Gonçalves, Renata; Colen, R.; Figueiredo, Vera; Valente, Luisa M PSomatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 μm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 (myod2) and the myomaker (mymk) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1. These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes.
- A nutritional strategy to promote gilthead seabream performance under low temperaturesPublication . Teodósio, Rita; Aragão, Cláudia; Colen, R.; Carrilho, Raquel; Dias, Jorge; Engrola, SofiaGilthead seabream (Sparus aurata) is vulnerable to low water temperature, which may occur in the Southern Europe and Mediterranean region during Winter. Fish are poikilothermic animals, therefore feed intake, digestion, metabolism and ultimately growth are affected by water temperature. This study aimed to evaluate growth performance, feed utilisation, nutrient apparent digestibility, and nitrogen losses to the environment in gilthead seabream juveniles reared under low temperature (similar to 13 degrees C). Three isolipid and isoenergetic diets were formulated: a diet similar to a commercial feed (COM) that contained 44% crude protein and 27.5% fishmeal, and two experimental diets with a lower protein content of 42% (ECO and ECOSup). In both ECO diets fishmeal inclusion was reduced (10% in ECO and 7.5% in ECOSup diet) and 15% poultry meal was included. Additionally, the ECOSup diet was supplemented with a mix of feed additives intended to promote fish growth performance and feed intake. The ECO diets presented lower production costs than the COM diet, whilst incorporating more sustainable ingredients. Gilthead seabream juveniles (+/- 154.5 g initial body weight) were randomly assigned to triplicate tanks and fed the diets for 84 days. Fish fed the ECOSup diet attained a similar final body weight than fish fed the COM diet, significantly higher than fish fed the ECO diet. ECOSup fed fish presented significantly higher hepatosomatic index than COM fed fish, most likely due to higher hepatic glycogen reserves. The viscerosomatic index of ECOSup fed fish were significantly lower compared to COM fed fish, which is a positive achievement from a consumer's point of view. ECOSup diet exhibited similar nutrient digestibility than the COM diet. Moreover, feeding fish with the ECO diets resulted in lower faecal nitrogen losses when compared to COM fed fish. The results suggest that feeding gilthead seabream with an eco-friendly diet with a mix of feed additives such as the ECOSup diet, promoted growth and minimised nitrogen losses to the environment. Nutritional strategies that ultimately promote feed intake and diet utilisation are valuable tools that may help conditioning fish to sustain growth even under low temperatures.
- Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomicsPublication . Farinha, Ana Paula; Schrama, Denise; Silva, Tome; Conceicao, Luis E. C.; Colen, Rita; Engrola, Sofia; Rodrigues, Pedro; Cerqueira, MarcoPlant protein sources play an essential role in aquaculture by reducing the use of fish meal to sustainable levels, although further supplementation is needed to fulfill fish nutritional requirements. This work addressed fish growth performance and proteome changes to dietary methionine in European seabass juveniles. A dose-dependent response to methionine (Met) was observed on fish growth consistent with proteomic analyses, suggesting Met requirement >= 0.9% (w/w). Fish fed at 0.77% (w/w) exhibited reduced growth and an enrichment in proteins involved in cellular homeostasis. Proteomics data suggest an optimal nutritional status at 1.36% Met (w/w), together with putative beneficial effects on the immune system up to 1.66% Met (w/w). The response to dietary Met involved the convergence of different metabolic and signalling pathways implicated in cell growth and immune response e.g., mTOR, Hedgehog or the T Cell receptor signalling, coupled with a fine-tuning regulation of amino acid metabolism and translation.
- Taurine supplementation to Plant-Based Diets improves lipid metabolism in Senegalese SolePublication . Raquel Cêa de Aragão Teixeira, Cláudia; Teodósio, Rita; Colen, R.; Richard, Nadège; Rønnestad, Ivar; Dias, Jorge; Conceição, Luís E. C.; Ribeiro, LauraTaurine is a sulphur-containing amino acid with important physiological roles and a key compound for the synthesis of bile salts, which are essential for the emulsion and absorption of dietary lipids. This study aimed to evaluate the effects of taurine supplementation to low-fishmeal diets on the metabolism of taurine, bile acids, and lipids of Senegalese sole. A fishmeal (FM) and a plant-protein-based (PP0) diet were formulated, and the latter was supplemented with taurine at 0.5 and 1.5% (diets PP0.5 and PP1.5). Diets were assigned to triplicate tanks containing 35 fish (initial weight ~14 g) for 6 weeks. Fish from the PP0 treatment presented lower taurine and bile-acid concentrations compared with the FM treatment, and a downregulation of cyp7a1 and abcb11 was observed. Triolein catabolism decreased in PP0-fed fish, resulting in increased hepatic fat content and plasma triglycerides, while no effects on plasma cholesterol were observed. Taurine supplementation to plant-based diets resulted in a higher taurine accumulation in fish tissues, increased bile-acid concentration, and upregulation of cyp7a1 and abcb11. Hepatic fat content and plasma triglycerides decreased with increasing dietary taurine supplementation. Taurine supplementation mitigated part of the negative effects of plant-based diets, leading to better lipid utilisation.
- Modulation of dietary protein to lipid ratios for gilthead seabream on-growing during summer temperature conditionsPublication . Aragão, Cláudia; Cabano, Miguel; Colen, R.; Teodósio, Rita; Gisbert, Enric; Dias, Jorge; Engrola, SofiaGilthead seabream (Sparus aurata) tend to increase fat deposition during summer farming conditions in the Mediterranean, which may negatively affect productive performance and consumers' quality perception of the final product. Therefore, this study evaluated the impacts of protein to lipid ratios in low fishmeal/fish oil diets on growth performance, body composition, feed conversion and nutrient utilization of seabream on-grown during summer temperature conditions. The experimental diets contained low levels of fishmeal, fish oil, and crude protein (39%), differing in crude lipid content: 16% (MF diet) or 12% (LF diet). A growth trial was per-formed with seabream (initial weight: 100 & PLUSMN; 7 g) from August to October (water temperature: 23.1 & PLUSMN; 2.2 & DEG;C). A digestibility trial was also performed (at 23 & DEG;C). Key performance indicators, whole-body composition and ac-tivities of digestive enzymes were evaluated at the end of the experiment (64 days). Low dietary lipid levels negatively affected lipid, energy, and amino acid digestibility, and as a result, fish fed the LF diet presented higher nitrogen faecal losses. Still, the decrease in nutrient digestibility was not related to dietary effects on the digestive enzyme activities. The experimental diets did not compromise the activity of pancreatic, gastric, and intestinal digestive enzymes nor feed utilization, but a slight growth impairment was observed in fish fed the LF diet, probably due to the lower amino acid and lipid digestibility. However, a potential benefit of this dietary treatment towards reducing fat accumulation in seabream during summer was observed. Nevertheless, the environmental impact of the nitrogen losses during seabream on-growing should be considered when estimating the sustainability of the production. This study demonstrated that the optimisation of diet formulations should account for the environmental conditions, especially in Mediterranean aquaculture, so the economic and envi-ronmental impacts may be correctly evaluated towards a more sustainable fish production.
- Dietary methionine supplementation improves the European seabass (Dicentrarchus labrax) immune status following long-term feeding on fishmeal-free dietsPublication . Machado, Marina; Engrola, Sofia; Colen, R.; Conceicao, Luis E. C.; Dias, Jorge; Costas, BenjaminMethionine is a limiting amino acid (AA) in fish diets, particularly in those containing high levels of plant protein (PP), and is key in the immune system. Accordingly, outcome on the fish immune mechanisms of methionine-deficient and methionine-supplemented diets within the context of 0 % fishmeal formulation, after a short and prolonged feeding period, was studied in European seabass (Dicentrarchus labrax). For this, seabass juveniles were fed a (i) fishmeal-free diet, meeting AA requirements, but deficient in methionine (MET0·65); (ii) as control, the MET0·65 supplemented with L-methionine at 0·22 % of feed weight (CTRL); (iii) two diets, identical to MET0·65 but supplemented at 0·63 and 0·88 % of feed weight of L-methionine (MET1·25 and MET1·5, respectively); and (iv) a fishmeal-based diet (FM), as positive control. After 2 and 12 weeks of feeding, blood and plasma were sampled for leucocyte counting and humoral parameter assays and head-kidney collected for gene expression. After 2 weeks of feeding, a fishmeal-free diet supplemented with methionine led to changes in the expression of methionine- and leucocyte-related genes. A methionine immune-enhancer role was more evident after 12 weeks with an increased neutrophil percentage and a decreased expression of apoptotic genes, possibly indicating an enhancement of fish immunity by methionine dietary supplementation. Furthermore, even though CTRL and FM present similar methionine content, CTRL presented a reduced expression of several immune-related genes indicating that in a practical PP-based diet scenario, the requirement level of methionine for an optimal immune status could be higher.