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Trabajo presentado en el 5th International Symposium on Genomics in Aquaculture, celebrado en Albufeira (Portugal) del 21 al 23 de marzo de 2018

Trabajo presentado en el 18th International Symposium on Fish Nutrition and Feeding (ISFNF), celebrado en Las Palmas de Gran Canaria (España), del 3 al 07 de junio de 2018, The concept of maintaining animal health through the best possible nutrition is well-accepted in modern animal farming, and amino acids appear to be good candidates as functional additives to improve health. The present study aimed to explore short and long-term effects of dietary amino acids (AA) supplementation on the gilthead seabream growth performance and immune status, in the context of a challenging fishmeal-free formulation. Triplicate groups of fish (13.3±0.3g) with a fishmeal diet background were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the CTRL diet with a 2% or a 9% surplus of histidine, threonine and tryptophan (HTW2 and HTW9, respectively). After 2 and 12 weeks of feeding, fish samples of head-kidney, liver and muscle were collected for gene expression, whereas plasma was suited for humoral immune parameters. There were no differences in final body weight among treatments. However, there was a tendency for an impaired growth performance in fish fed HTW9 after 12 weeks, which may be linked to an increased FCR at early stages in this group. The expression of a panel of 29-32 genes specific for muscle, liver or head-kidney confirms an effect due to the treatments across time. A two-way ANOVA analysis revealed that 15-24 genes vary significantly depending on the tissue, and the multivariate analysis of all shows that around 83% of variation is explained by the two first principal components (PC). In this regard, PC1 (60.3%) justified the effect of individual variability within diet mostly due to HTW9 at early stages, whereas PC2 (22.8%) represents a clear time effect. Particularly in the case of HK, fish fed HTW9 displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, with only an increase in antiprotease and peroxidase activities after 12 weeks regardless of dietary treatment. Overall, dietary AA supplementation might improve the seabream immune status over a short-term period. Hence, the use of nutraceutical feeds is especially relevant before/during/after exposure to stress, taking into account that these putative advantageous effects seem to disappear after a long-term feeding period.

Trabajo presentado en el 5th International Symposium on Genomics in Aquaculture, celebrado en Albufeira (Portugal) del 21 al 23 de marzo de 2018, Prediction of growth potential and the effect of genetic, environmental and nutritional effects on growth is a central concern in aquaculture research. On the other hand, objective assessment of growth potential usually relies on mid- to long-term growth trials, which carry very high costs. In this presentation, we will discuss the estimation of long-term growth (t = 3 months) in gilthead seabream (Sparus aurata) based on gene expression measurements performed at an early stage (t = 2 weeks), comparing it with results from a similar previous trial in Atlantic salmon (Salmo salar). For this trial, gilthead seabream juveniles were reared in 21 tanks (1000 L) over the course of 3 months, being fed with a wide range of diets that differed in terms of nutritional composition, type of ingredients and performance/quality. At 2 weeks after the start of the trial, some fish were sampled to obtain liver, muscle and head kidney samples for gene expression analysis. At the end of the 3 month period, all remaining fish were bulk weighed and per-tank performance indicators estimated. Gene expression measurements were performed using reverse transcription and real-time qPCR protocols, with over 30 genes being measured per tissue. These datasets were used to calibrate linear models of growth performance measures using L1-regularized linear regression, which were then further refined by AIC-guided stepwise variable removal. Results show that this approach seems feasible in general, as previously seen with Atlantic salmon, though the prediction quality was not as high as for salmon (R2 = 0.6, rather than R2 = 0.9). The reasons identified for this difference in prediction are threefold: first, the fact that individual measurements of gene expression were performed (as opposed to pooled measurements); second, the wider range of tested dietary formulations (which increases model robustness but decreases its apparent predictive power); third, the relative diversity of ontological categories of genes, which was reduced in this trial. In conclusion, while this study confirms the feasibility of this type of approach, it underlines the challenges of obtaining a robust prediction model for fish growth potential.

Trabajo presentado en Aquaculture Europe 17, celebrado en Dubrovnik (Croacia), del 17 al 20 de octubre de 2017, Fishmeal (FM) and fish oil (FO) are the reference protein and fat sources in aquafeeds, but replacing them is industry priority due to sustainability and future availability issues. However, FM and FO are high-quality raw materials and their replacement can lead to impaired fish growth and immune status (e.g., Conceição et al. 2012). FM and FO in addition to balanced amino acid and fatty acid profiles, also bring micronutrients which are often not present in the most used alternative ingredients, e.g. from plant origin. Phaeodactilum is a microalgae rich in micronutrients and bioactive compounds such as fucoxanthin and polyunsaturated fatty acids (PUFA) particularly eicosapentaenoic acid (EPA) (Gilbert-López et al., 2014). This study intends to evaluate the effects of dietary supplementation with Phaeodactilum tricornutum incorporated in feed by two different processes, either as whole cells (integral cell wall) or broken cells (disrupted cell wall) on fish health status and performance, in a feed formulation scenario of a zero fish meal, reduced fish oil., This work was supported by EU MIRACLES project (7th Framework Program - Grant Agreement No. 613588) and by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT – Foundation for Science and Technology, under the project UID/Multi/04423/2013. LRP, SE and BC were supported by FCT (PD/BDE/114436/2016, IF/00482/2014 and IF/00197/2015, respectively).

Trabajo presentado en Aquaculture Europe 17, celebrado en Dubrovnik (Croacia), del 17 al 20 de octubre de 2017, In the last decade, many studies have focused on improving aquafeeds with the purpose of reducing the inclusion level of fishmeal (FM), replacing it with vegetable proteins. A major concern is that this can lead to feeds with imbalanced amino acid (AA) profiles, impairing feed conversion rates and immune response of farmed fish (Conceição et al. 2012). These deficiencies, often marginal, may be restored by supplementing feeds with crystalline AA to achieve a desirable metabolic efficiency, growth performance, and/or health status (Li et al., 2009). Histidine and its derivatives act as antioxidants or can mitigate the impact of oxidative stress (Bellia et al., 2008). Threonine is involved in many physiological and biochemical processes, including immune functions. Tryptophan is a versatile AA being a precursor to produce bioactive molecules and neurotransmitters such as serotonin, kynurenine and melatonin (Li et al., 2009), which affect the immune system. The present studied aimed to explore the effects of the inclusion of a combination of these three essential amino acids on gilthead seabream growth and immune status, in the context of a challenging fishmeal-free basal formulation., This work was supported by Project ALISSA (reference ALG-01-0247-FEDER-3520), financed by Portugal and the European Union through FEDER, COMPETE 2020 and CRESC Algarve 2020, in the framework of Portugal 2020, and through the COMPETE and Operational Human Potential Programmes and national funds through FCT (Portugal). L. Ramos-Pinto, M. Machado, R. Azeredo, B. Reis and B. Costas benefited were supported by FCT, Portugal (PD/BDE/114436/2016, SFRH/BD/108243/2015, SFRH/BD/89457/2012, PD/BDE/129262/2017 and IF/00197/2015, respectively)

Trabajo presentado en Aquaculture Europe 17, celebrado en Dubrovnik (Croacia), del 17 al 20 de octubre de 2017, Measurement of gene expression in the tissues of fish is increasingly a common practice in aquaculture and fish nutrition research trials, due to the underlying assumption that important information on the physiological, nutritional and immunological state of fish can be recovered from such measurements. In this study, we seek to determine whether it is possible to go beyond a qualitative assessment of fish nutritional state, and obtain a quantitative measure of fish growth potential (i.e. prediction of fish growth) from gene expression measurements (in liver, muscle, head kidney and red blood cell samples) obtained at the end of a short feeding trial with distinct diets, using Atlantic salmon as animal model.

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

Gilthead seabream is the main aquaculture species produced in Portugal, as well as in the Mediterranean. However, the winter disease syndrome, a series of metabolic and immune system disorders caused by low temperatures, affects seabream culture, leading to important industrial losses, either as mortalities or growth decreases during spring. These effects can be minimized through dietary optimisation, either improving diets for the end of summer season, or feeding seabream with a fortified diet during autumn/winter. However, the development of a fortified diet for autumn/winter is hindered by the lack of tools to evaluate fish metabolic and physiological condition. Nevertheless, the recent development of new generation molecular tools provides good perspectives to supply molecular indicators of condition that would support this task.

Post-mortem flesh deterioration is dependent on the energy reserves present at the time of death. Early depletion of muscle glycogen leads to the buildup of lactate and to the early onset of rigor mortis, resulting in the activation of endogenous proteases and the degradation of myofibrillar proteins, and consequent muscle softening. The purpose of this study was to modulate the energy status of the muscle at the time of death through the use of dietary muscle buffering compounds, namely glycerol and maslinic acid. Four fish groups of gilthead seabream (in duplicate) were fed for three months with four different diets. The four diets were obtained by starting from a commercial diet formulation and applying a 2×2 factorial design, with two levels of glycerol supplementation (0 and 5%) and two levels of maslinic acid supplementation (0 and 0,025%). The diets were formulated to be isonitrogenous and isolipidic. Fish were slaughtered by immersion in ice-salt water slurry and muscle samples were immediately obtained from three fish of each tank, for a total of six muscle samples per treatment. Sarcoplasmic proteins were extracted from each muscle sample, separated/quantified by 2D-DIGE and identified by peptide fragment fingerprinting using MALDI-TOF MS. Preliminary analysis of the results shows an effect of the diets on muscle parameters such as measured pH and onset of rigor mortis. At the proteome level, the addition of glycerol and maslinic acid to the diets seemed to have affected the abundance of proteins related to oxidative stress (HSC70, HSC71, peroxiredoxin, transferrin), aldehyde toxification (ALDH1A2, ALDH6A1, ALDH7A1), energy homeostasis (adenylate kinase, nucleoside-diphosphate kinase), cytoskeleton (cofilin-2) and signalling (phosphohistidine phosphatase).

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.

In aquaculture, skeleton deformities are a serious problem as they affect growth, morphology, survival of fish and also increase productions costs and decrease market value of the final products. Since vitamin K is known for playing and important role in bone metabolism, the aim of this study was to investigate the effects of dietary vitamin K1 supplementation on skeleton deformities and on the expression of proteins involved involved in skeleton development of Senegalese sole.