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- Transition from endogenous to exogenous feeding in hatchery-cultured European eel larvaePublication . Benini, Elisa; Engrola, Sofia; Politis, Sebastian Nikitas; Sørensen, Sune Riis; Nielsen, Anders; Conceição, Luis E. C.; Santos, André; Tomkiewicz, JonnaThe transition from endogenous to exogenous feeding is critical during fish early life, where appropriate feed availability and timing of initiation of feeding influence survival. For European eel (Anguilla anguilla), establishing first feeding culture is at a pioneering state, where successful production of larvae has recently enabled feeding experiments. In the present study, three diets and potential benefits of early feeding during the transition from yolk-sac stage to feeding larvae were explored, including molecular analyses of genes involved in digestive functions and growth. Three consecutive trials were performed using hatchery produced eel offspring. In Feeding regimes 1 and 3, expression of npy and cck (appetite regulation) was higher, while expression of pomca (food intake) was lower in non-prefed larvae, indicating increased fasting and higher starvation risk. In contrast, Feeding regime 2 led to the highest survival ever registered for European eel larvae i.e. 20% at 20 dph, in spite that prefeeding resulted in reduced survival rate during the endogenous feeding stage. This was associated with initial hsp90 (stress/repair) upregulation in larvae receiving prefeeding, however, with subsequent downregulation during exogenous feeding. Notably, the growth related gh expression was higher in prefed larvae, indicating growth benefits of prefeeding. Likewise, prefeeding resulted in pomca as well as try, tgl, and amyl2a (digestion) upregulation, providing evidence of beneficial maturation of gut functionalities. Essentially, Feeding regime 2 demonstrated a continuous upregulation of growth, appetite and digestion related genes, which in combination with the highest survival suggest that dietary requirements were partially met. Moreover, in Feeding regime 2, gh and tgl were expressed at a higher level in prefeeding larvae than in the control, indicating that prefeeding might be advantageous in spite observed mortality, but further research is needed, including timing of feed application.
- Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free dietsPublication . Ramos-Pinto, Lourenco; Martos-Sitcha, Juan A.; Reis, Bruno; Azeredo, Rita; Fernandez-Boo, Sergio; Perez-Sanchez, Jaume; Alvar Calduch-Giner, Josep; Engrola, Sofia; Conceicao, Luis E. C.; Dias, Jorge; Silva, Tome S.; Costas, BenjaminEuropean aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 +/- 0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R-2) and predicted (Q(2)) with four components up to 93% and 78% of total variance, respectively. Component 1 (R-2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.
- Sustainable feed formulation to community‐based aquaculture: Oreochromis niloticus fingerlings performance and antioxidant statusPublication . Pires, Sílvia F. S.; Vital, Inês; Pereira, Vitória; Bustani, Henriques; Aragão, Cláudia; Engrola, Sofia; Cabano, Miguel; Dias, Jorge; Soares, Amadeu M. V. M.; Rodrigues, Andreia C. M.; Rocha, Rui J. M.Community-based aquaculture can reduce dependence on natural resources, promote biodiversity conservation, and improve local economies and food security. However, this activity is highly dependent on local conditions regarding natural resources, such as the availability of produced organisms, adequate feeds, and environmental factors. As ectothermic organisms, fish are more susceptible to temperature fluctuations in culture conditions. A set of raw ingredients (e.g., cassava and local beans) produced or available in villages from Cabo Delgado (Mozambique) with nutritional potential for fish feed were selected to produce an experimental diet. The following objectives were defined: (1) evaluate growth performance of tilapia fingerlings fed a diet produced with local ingredients, compared with a commercial-like diet; and (2) evaluate the response to thermal stress (18, 26, and 32degree celsius) by tilapia fingerlings fed with tested diets. Tilapia fed with an experimental diet presented lower growth rates, lower DNA damage, higher neurophysiological, and antioxidant activity, leading to increased oxidative stress. Regarding energy budget, tilapia fed with the experimental diet presented higher protein content at 26degree celsius and lipids at 18degree celsius, leading to greater energy available at these temperatures. Overall, local ingredients can be successfully used as an additional feed source for tilapia production in community-based aquaculture in earthen ponds.
- Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?Publication . Navarro-Guillén, Carmen; Dias, Jorge; Rocha, Filipa; Castanheira, M. F.; Martins, Catarina I. M.; Laizé, Vincent; Gavaia, Paulo; Engrola, SofiaMetabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism.
- Thermal plasticity of the miRNA transcriptome during Senegalese sole developmentPublication . Campos, C.; Sundaram, Arvind Y. M.; Valente, L. M. P.; Conceição, L. E. C.; Engrola, S.; Fernandes, Jorge M. O.Several miRNAs are known to control myogenesis in vertebrates. Some of them are specifically expressed in muscle while others have a broader tissue expression but are still involved in establishing the muscle phenotype. In teleosts, water temperature markedly affects embryonic development and larval growth. It has been previously shown that higher embryonic temperatures promoted faster development and increased size of Senegalese sole (Solea senegalensis) larvae relatively to a lower temperature. The role of miRNAs in thermal-plasticity of growth is hitherto unknown. Hence, we have used high-throughput SOLiD sequencing to determine potential changes in the miRNA transcriptome in Senegalese sole embryos that were incubated at 15°C or 21°C until hatching and then reared at a common temperature of 21°C. Results: We have identified 320 conserved miRNAs in Senegalese sole, of which 48 had not been previously described in teleosts. mir-17a-5p, mir-26a, mir-130c, mir-206-3p, mir-181a-5p, mir-181a-3p and mir-199a-5p expression levels were further validated by RT- qPCR. The majority of miRNAs were dynamically expressed during early development, with peaks of expression at pre-metamorphosis or metamorphosis. Also, a higher incubation temperature (21°C) was associated with expression of some miRNAs positively related with growth (e.g., miR-17a, miR-181-5p and miR-206) during segmentation and at hatching. Target prediction revealed that these miRNAs may regulate myogenesis through MAPK and mTOR pathways. Expression of miRNAs involved in lipid metabolism and energy production (e.g., miR-122) also differed between temperatures. A miRNA that can potentially target calpain (miR-181-3p), and therefore negatively regulate myogenesis, was preferentially expressed during segmentation at 15°C compared to 21°C. Conclusions: Temperature has a strong influence on expression of miRNAs during embryonic and larval development in fish. Higher expression levels of miR-17a, miR-181-5p and miR-206-3p and down-regulation of miR-181a-3p at 21°C may promote myogenesis and are in agreement with previous studies in Senegalese sole, which reported enhanced growth at higher embryonic temperatures compared to 15°C. Moreover, miRNAs involved in lipid metabolism and energy production may also contribute to increased larval growth at 21°C compared to 15°C. Taken together, our data indicate that miRNAs may play a role in temperature-induced phenotypic plasticity of growth in teleosts.