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Methionine and taurine are amino acids (AA) that are usually deficient when fish meal is replaced by plant proteins. In this study, three diets were tested in juvenile meagre (initial weight: 13.4 g) for 8 weeks. The D1 diet had 0.2% methionine and 1% taurine supplementation; the D2 and D3 diets had 0.6% methionine and 1% and 2% taurine supplementation, respectively. The results showed that meagre fed the D1 diet had lower specific growth rate (2.2 to 2.5), lower feed efficiency (0.9 to 1.2) and higher food conversion rate (FCR, 1.1 to 0.8) as well as a lower activity of the alanine aminotransferase (ALAT) enzyme. Furthermore, a higher recruitment of muscle fibres (46% compared to 36%) as well as a higher fibre density was observed (1019 compared to 870 fibres mm(-2)). This study shows that meagre requires a sufficient quantity of methionine in plant-based diets to avoid a reduction in fish performance. Furthermore, taurine supplementation in the D1 diet was not able to mitigate the effects of methionine deficiency. A higher taurine supplementation did not improve meagre performance. MAR02.01.01 FEAMP-0175 info:eu-repo/semantics/publishedVersion

The sustainability of the Aquaculture industry relies on optimising diets to promote nitrogen retention and maximise fish growth. The aim of this study was to assess how different dietary formulations influence the bioavailability and metabolic fate of distinct amino acids in gilthead seabream juveniles. Amino acids (lysine, tryptophan, and methionine) were selected based on their ketogenic and/or glucogenic nature. Seabream were fed practical diets with different protein (44 and 40%) and lipid contents (21 and 18%): 44P21L, 44P18L, 40P21L, and 40P18L. After three weeks of feeding, the fish were tube-fed the correspondent diet labelled with 14C-lysine, 14C-tryptophan, or 14C-methionine. The amino acid utilisation was determined based on the evacuation, retention in gut, liver, and muscle, and the catabolism of the tracer. The metabolic fate of amino acids was mainly determined by their nature. Tryptophan was significantly more evacuated than lysine or methionine, indicating a lower availability for metabolic purposes. Methionine was more retained in muscle, indicating its higher availability. Lysine was mainly catabolised, suggesting that catabolism is preferentially ketogenic, even when this amino acid is deficient in diets. This study underpins the importance of optimising diets considering the amino acids’ bioavailability and metabolic fate to maximise protein retention in fish. info:eu-repo/semantics/publishedVersion

Commercial 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 dl-Met 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 remethylated. 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.

The 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 k J − 1 ) and HighP/E ( P / E ratio = 21.4 mg protein k J − 1 ). After three weeks, fish ( 11.6 ± 4.3 g ) were tube-fed the respective diet labelled with 14C-protein (L-amino acid mixture), 14C-lysine, or 14C-methionine. Protein, lysine, and methionine utilization were determined based on the proportion of 14C-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 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.

To support the expected increase in aquaculture production during the next years, a wider range of alternative ingredients to fishmeal is needed, towards contributing to an increase in production sustainability. This study aimed to test diets formulated with non‐conventional feed ingredients on gilthead seabream (Sparus aurata) growth performance, feed utilization, apparent digestibility of nutrients and nutrient outputs to the environment. Four isonitrogenous and isoenergetic diets were formulated: a control diet (CTRL) similar to a commercial feed and three experimental diets containing, as main protein sources, plant by‐products, glutens and concentrates (PLANT); processed animal proteins (PAP); or micro/macroalgae, insect meals and yeast (EMERG). Diets were tested in triplicate during 80 days. The EMERG treatment resulted in lower fish growth performance, higher FCR and lower nutrient and energy retentions than the other treatments. The lowest protein digestibility was found for the EMERG diet, which caused increased nitrogen losses. The PLANT and PAP treatments resulted in better fish growth performance, higher nutrient and energy retentions, and lower FCR than the CTRL treatment. The significant improvement in FCR found for fish fed PLANT and PAP diets and the high protein digestibility of these diets contribute towards minimizing the environmental impacts of seabream production. FCT: UID/Multi/04326/2019/ PTDC/MAR‐BIO/3034/2014/ DL57/2016/CP1361/CT0033. info:eu-repo/semantics/publishedVersion

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Gilthead 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. European UnionEuropean Commission [727315]; Portuguese Foundation for Science and Technology (Ministry of Science and Higher Education, Portugal) [DL 57/2016/CP1361/CT0033]

Welfare in aquaculture is a pressing topic. One of the main measures to improve the life of farmed fish is implementing environmental enrichment, which can include the addition of structural covers to rearing tanks. Here we test the effects of tank covers (fully covered, 50 % covered and uncovered) in an all-male population (N = 75 tagged individuals: 5 individuals per tank, 5 tanks per treatment) of farmed Nile tilapia using behavioural and physiological welfare indicators. Video recordings were performed over 21 days to evaluate overall swimming (swim bursts, freezing on the bottom or hovering in the water column) and aggression events (bites and chases). At the end of the experiment, blood was collected to assess circulating cortisol. Results showed a clearly lower welfare in the partially covered tanks regarding behavioural and physiological variables. The present study highlights the need to correctly study 1) the ethology of the farmed species and 2) the enrichment strategy to be implemented, before any measure is taken. While environmental enrichment is undoubtedly a much needed area of research and a promising solution to improve the welfare of farmed fish, a proper ethological assessment must be made a priori to avoid detrimental effects of poorly devised approaches. UID/Multi/04326/2020; IF/00482/2014/CP1217/CT0005; DL 57/2016/CP1361/CT0033 info:eu-repo/semantics/publishedVersion

Aquaculture 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. FCT: UID/Multi/04326/2019/ IF/00482/2014/CP1217/CT0005/ DL57/2016/CP1361/CT0033. info:eu-repo/semantics/publishedVersion

The regulatory role of taurocholic acid (TC) and taurolithocholic acid (TLC) in ion transport was investigated with the short-circuit current (Isc) technique in the intestine of Senegalese sole (Solea senegalensis). Under voltage clamp conditions, Isc was stable for over 3 h and averaged −15.42 ± 2.75 μA/cm2 and −65.28 + 7.69 μA/cm2 in the anterior and posterior intestine, respectively. Only bumetanide (200 μM), a loop diuretic that inhibits Na+/K+/2Cl− co-transporters, inhibited the absorptive Isc, and apical application of NPPB (200 μM), EIPA (200 μM), glibenclamide (200 μM) and DIDS (200 μM) were without effect in the absorptive current. Apical application of TC and TLC induced rapid (

Recent studies reported positive effects of taurine supplementation to plant protein-based diets on fish growth performance and feed conversion ratio but the mechanisms involved remain unclear. The objective of this work was to provide a first insight on the importance of taurine on dietary protein and lipid absorption and utilisation, in Senegalese sole (Solea senegalensis) juveniles fed plant-based diets. Seven practical diets were tested: a fishmeal-based diet (FM) and a plant protein-based formula (PP85) to which taurine was added in graded levels: 0.2% (TAU1), 0.3% (TAU2), 0.4% (TAUS), 0.7% (TAU4) and 1.5% (TAU5). Short-term metabolic trials were performed by tube-feeding the different experimental diets labelled with different tracers: C-14-taurine, C-14-triolein (as tracer of dietary triacylglycerol) and a mixture of C-14-amino acids. Feeding sole juveniles with the PP85 diet affected taurine metabolism, since a higher proportion of dietary taurine was directed to gallbladder and probably used for bile salt synthesis. Consistent with this, fish receiving the plant-based low taurine-content diets (PP85 and TAU1) displayed a poor triacyglycerol digestion capacity. A higher proportion of C-14-amino acids was retained in fish muscle when fed the TAU5 compared to the PP85 diet. These short-term trials suggested that taurine supplementation to a plant-based diet might be beneficial to sole performance by enhancing bile salt synthesis and triacylglycerol digestion as well as muscle amino acid retention. However, this dietary supplementation should be tested in long-term trials to verify the effect on fish growth and to assess the beneficial effects on protein and lipid metabolism suggested here. Statement of relevance in the general field of aquaculture (limited to 60 characters): Adding taurine to a plant-based diet is beneficial for sole (C) 2016 Elsevier B.V. All rights reserved. National Funds through Fundacao para a Ciencia e Tecnologia [PTDC/MAR/117047/2010] Fundacao para a Ciencia e Tecnologia, Portugal [SFRH/BDP/65578/2009] info:eu-repo/semantics/acceptedVersion

Broodstock rearing conditions and handling procedures should be optimized in aquaculture species in order to benefit fish welfare and guarantee optimal conditions for spawning. In teleosts, basal cortisol levels display daily rhythms, oscillating along the 24 h of the day. In this sense, handling fish at different moments of the day may lead to different stress responses. The present study aimed at investigating the optimal rearing conditions for Senegalese sole broodstock, considering ambient light intensity and handling time. The optimal light intensity (50, 100 or 200 lx) was investigated by measuring fish cortisol levels and monitoring locomotor activity rhythms under each intensity tested. Results showed a significant increase in cortisol levels of fish exposed to 200 lx, when compared to values obtained under 100 lx, accompanied by changes in locomotor activity rhythms in both tanks under study. These results suggested that 200 lx may be too high as light intensity for this species, whereas 100 lx seems to be more adequate. Also, daily rhythms of stress response were investigated in breeders from different origins (Wild and first generation, G1). Basal cortisol levels and cortisol stress response after an acute stressor (air exposure) were monitored at two distinct moments of the day (Mid-Light and Mid-Dark). Basal levels were higher during the day in the wild group, while G1 fish seemed to have lost the daily fluctuations in basal cortisol plasma levels, as well as their daily rhythms of locomotor activity. Both groups showed lower stress responses during night-time, an indication that this is an adequate period of the day to handle this species. Senegalese sole breeders born in captivity presented more pronounced stress responses when compared to wild fish, reflecting their different life history in terms of stress challenges. FP7/SME/2008/1; UID/Multi/04326/2019; DL 57/2016/CP1361/CT0007; DL 57/2016/CP1361/CT0033 info:eu-repo/semantics/publishedVersion

Nutrition shapes the individual physiological pathways prior to hatching, resulting in long-term effects on postnatal growth and physiological functions. The environment, including nutrition, determines the rate of myogenesis, the number and size of muscle fibres, the composition of sub-cellular organelles, the patterns of gene expression, influences protein turnover and the efficiency of protein deposition, among others. Moreover, protein retention efficiency and fish adaptive fitness have been said to be negatively correlated. In addition, high mortalities are normally observed in the marine larval stages in optimized farming conditions (70–80%) affecting fish production. The consequences and implications of early nutrition for aquaculture production is an important challenge for the future. Likewise, optimized feeds need to be provided in sufficient quantities and in a manner adequate to feeding behaviour, to fully express growth potential, while avoiding deterioration of water quality or disease problems caused by excessive feeding. Understanding the mechanisms that control early development and growth and their relation with nutrition are critical for the identification of time windows in development that introduce growth variation, impact growth potential, and affect viability and quality of juveniles.

Deoxynivalenol (DON), a Fusarium mycotoxin, is one of the most prevalent mycotoxins in aquafeeds. The toxicokinetics of DON are rarely studied in aquatic species. The present study used juvenile rainbow trout (Oncorhynchus mykiss) with a mean initial body weight of 7.72 ± 1.42 g in order to evaluate the pharmacokinetic behaviour and the metabolization of radiolabelled DON ([3H]-DON). In a first trial, 30 fish were tube-fed with four pellets containing a total of 125 ± 0.019 ng of [3H]-DON. At different sampling time points after feeding (1 h, 3 h, 6 h, 12 h or 24 h), the tissue distribution of the [3H]-DON was assessed by liquid scintillation counting. In a second trial, five fish were tube-fed four pellets containing a total of 663 ng of unlabelled-DON. Twenty-four hours after feeding, metabolites of DON excreted into the water were analysed by LC-MS/MS. [3H]-DON was detected in fish liver one hour after tube-feeding, indicating a rapid absorption of DON. In the first hour, [3H]-DON achieved its maximum in the gastro-intestinal tract (GIT) (20.56 ± 8.30 ng). However, 6.19 ± 0.83 ng of [3H]-DON was also detected in the water at this sampling time point. The fast excretion of [3H]-DON (above the average gastric emptying time of trout) might be related to its high-water solubility and consequent excretion with the fluid phase of the chyme. The amount of [3H]-DON in the GIT was stable during the first six hours. Such long transit time of DON through the GIT increases the potential for damage and absorption. The period between six and twelve hours seems to be the turning point in terms of DON excretion. Twelve hours after tube-feeding, the trout excreted 50.71 ± 22.17% of the tube-fed DON amount into water, while at the previous sampling time point (six hours) only 11.03 ± 6.09% were detected. These data suggest that an effective method for gastrointestinal DON detoxification in trout requires a period of action lower than six hours. In the present trial, no DON metabolites were detected in water.

Apparent digestibility coefficients (ADCs) of processed agro-food by-products were assessed in European seabass (Dicentrarchus labrax). Each experimental diet was obtained by replacing 300 g/kg of a commercial-based diet used as reference (REF) with a test ingredient: wheat germ (GERM), okara meal (OKA), poultry by-product meal (POULT), steam hydrolysed (FeHY) and enzyme-treated feather meal (FeENZ), beta-lactoglobulin (β-Lg) and peptide fractions >3,000 Da obtained from brewer's yeast (YeastP) and fish by-products (FishP). Dry matter ADC was highest in β-Lg (95%) and lowest in OKA (40%). Protein ADCs were high in β-Lg, FishP, GERM and POULT (>93%); intermediate in FeHY and FeENZ (85%–88%); and moderate in OKA and YeastP (70%–78%). The essential amino acids' ADC mean was above 91% in POULT, β-Lg, GERM and FishP, 84%–89% in FeHY and FeENZ and 73%–76% in YeastP and OKA. Energy ADC was highest in POULT and β-Lg (89%–95%) and lowest in YeastP and OKA (61%–64%). Lipid ADC was highest for POULT and GERM (100%). Phosphorus ADC was lowest in GERM (19%) and highest in β-Lg (88%). FeHY, FeENZ, POULT, GERM, FishP and β-Lg are highlighted as protein sources for European seabass.

In Zambia, every year some parts of the maize fields are abandoned post-planting. Reasons for this are not clearly known. In this paper, we examine the influence of soil and climatic factors on crop abandonment using a six-year (2007–2012) panel data by modeling the planted-to-harvested ratio (a good indicator of crop abandonment) using a fractional and linear approach. Therefore, for the first time, our study appropriately (as supported by the model specification tests that favour fractional probit over linear) models the fractional nature of crop abandonment. Regression results, which are not very different between the two specifications, indicate that, more than anything, high rainfall immediately after planting and inadequate fertilizer are the leading determinants of crop abandonment. In the agro-ecological region where dry planting takes place, low temperature during planting months negatively affects the harvested area. The results have implications on the sustainability of farming systems in the face of a changing climate.

We studied the effect of changes in dietary methionine (Met) supply in broodstock and first-feeding rainbow trout fry (offspring). Three plant-based diets differing in Met level (deficient, adequate or in excess of the established requirement) were fed to the broodstock (male and female) for 6 months prior to spawning (diets BD, BA and BE, respectively). The offspring from the parental Met-groups was then challenged in turn with the different Met fry-diets (FD, FA and FE, respectively) for 3 weeks from first-feeding. At spawning, females fed diet BD had significantly higher plasma total and LDL-cholesterol and slightly lower plasma triacylglycerol. Diet BD reduced female (but not male) growth, weight of spawn and egg size, but had no effect on relative fecundity. The free amino acid profile of oocytes was modified, with levels of Met and Cys correlating positively with the Met-levels of broodstock diets. SAM and SAH levels in oocytes followed the same pattern, as opposed to SAM/SAH ratio. At the swim-up stage, no significant effect of parental diet on fry weight was noted, whereas survival was the highest in fry from BE-broodstock. The subsequent 21-day fry feeding with different Met levels highly affected the daily growth index with a significant interaction between the parental-diet and fry-diet effects. The expression of a number of genes regulating sulfur amino acid metabolism was modified either directly by the dietary Met supply in both broodstock liver and in whole fry (e.g. BHMT1, GR, GST pi, MsrA1) or indirectly by the parental Met intakes as seen in the swim-up fry (e.g. BHMT1, MTR, GST pi, MsrA1). Importantly, long-lasting parental effects linked to broodstock Met-intake were seen in the fry, 21-days after first-feeding and irrespective of the fry diet (CTH, MsrA1, MsrB2, SOD2). Similarly, parental effects were noted on the gene expression of both NPY and POMC feeding peptides in fry prior to exogenous feeding which persisted for POMC in the 21-day fry. Parental effects were also demonstrated on the key myogenic gene Myog, on fMHC and GDH in swim-up fry, which persisted for GDH in 21-day fry. In summary, our results demonstrate that dietary Met levels of rainbow trout broodstock affect various traits in the offspring, some of which persisted during the first weeks of exogenous feeding. Further studies need to evaluate the long-term persistence of the parental effects over time and to elucidate the mechanisms, whether epigenetic or not. Statement of relevance: Determining the multiple effects of dietary methionine levels on reproductive, growth performance and metabolism in offspring will help improve formulations of low fish meal feeds for rainbow trout at sensitive life cycle stages. (C) 2016 Elsevier B.V. All rights reserved. EU [288925] F.C.T., Portugal - Fundo Social Europeu (POCH) and National Funds [SFRH/BPD/108389/2015] info:eu-repo/semantics/publishedVersion

Senegalese sole farming is gaining “momentum” and the development of an optimised diet is pivotal to ensure its competitive and sustainable commercial culture. The lower fish performance often observed when replacing large amounts of dietary fishmeal by plant protein sources may result from an unbalanced supply of selected nutrients, such as taurine, which is absent in plants but abundant in fish. However, taurine is highly soluble in water and sole has a passive feeding behaviour. Therefore, the aim of this study was to test different forms of taurine inclusion in plant protein-based diets, in order to guarantee a reduced leaching of this nutrient to the environment and to ensure a reliable delivery at fish tissues. Two basal diets were formulated: a fishmeal-rich (FM) and a low fishmeal (PP85) diet, in which vegetable protein sources replaced 85% of marine-derived proteins. Based on the PP85 formulation, diets were supplemented with: crystalline l -taurine (FreeTau), encapsulated taurine microparticles (EncTau), or delayed-release taurine microcapsules (DRTau). Leaching experiments were performed for each diet and post-prandial taurine kinetics was analysed in plasma samples of Senegalese sole tube-fed with the experimental diets. Results showed that taurine losses in the FreeTau diet increased exponentially after 5 min of water immersion, reaching 100% after 15 min. Taurine encapsulation significantly reduced taurine losses after 15 min to 25% (EncTau diet) or 40% (DRTau diet). Taurine was rapidly absorbed in FM and FreeTau diets, attaining a peak in plasma at approximately 1 h after feeding. Taurine kinetics was quite different in fish fed with the encapsulated diets, as plasma levels were maintained elevated during an extended period of time. Maximal levels in diet EncTau and DRTau were attained at 1 and 6 h after feeding, respectively, demonstrating the successful production of sustained and delayed-release microcapsules. Taking into account its passive feeding behaviour, this study indicates that taurine should be previously encapsulated in diets for Senegalese sole. Depending on the objective of the study and on the feeding strategy adopted, encapsulated or delayed-release taurine microparticles should be used.

Senegalese sole was one of the earliest identified candidate species with high potential for aquaculture diversification in the south of Europe.íts culture has been possible, and commercially attempted, for several decades, but intensive production has been slow to take off. This has been explained mostly by serious disease problems, high mortality at weaning, variable growth and poor juvenile quality. However, a strong and sustained research investment that started in the eighties has led to a better understanding of the requirements and particularities of this species. More recently, better management and technical improvements have been introduced, which have led to important progress in productivity and given a new impetus to the cultivation of Senegalese sole. As a result, the last 5 years have marked a probable turning point in the culture of sole towards the development of a knowledge-driven, competitive and sustainable industry. This review will focus on the main technical improvements and advances in the state of knowledge that have been made in the last decade in areas as diverse as reproductive biology, behaviour, physiology, nutritional requirements, modulation of the immune system in response to environmental parameters and stress, and characterization and mitigation of the main disease threats.ít is now clear that Senegalese sole has important particularities that differentiate it from other current and candidate marine aquaculture species, which bring about important challenges, some still unsolved, but also notable opportunities (e.g. a nutritional physiology that is better adapted to dietary vegetable ingredients), as will be discussed here. © 2014 Wiley Publishing Asia Pty Ltd., SM holds a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO) and is supported by the European Commission Marie Curie Actions (FP7-PEOPLE-2010-RG, Project No. 274184). BC and SE are supported by Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BPD/77210/2011 and SFRH/BPD/49051/2008, respectively).

The present study aimed to assess the effects of increased availability of dietary amino acids (AA) on brain monoamine neurotransmitters and the metabolic processes resulting from stressful situations in fish. Senegalese sole ( Solea senegalensis ) juveniles (24.2 ± 0.4 g wet mass) were weekly subjected to an acute handling stressor (HDLG) or remained undisturbed (CTL). Additionally, both treatments were fed a control or a high protein (HP) diet (CTL, CTL HP, HDLG and HDLG HP). The HP diet slightly increased the levels of digestible indispensable AA, together with tyrosine and cysteine. Repeated handling induced a stress response after 14 and 28 days in fish held at both HDLG and HDLG HP treatments. While dietary treatment and handling stress activated the serotonergic system at 14 days, these effects were not observed after 28 days. In addition, the HP diet minimized the decrease in plasma indispensable AA due to repeated handling stress after 28 days. It was concluded that HP diet decreased post-stress plasma glucose and lactate levels in HDLG HP specimens only at 14 days of treatment. Moreover, dietary treatment was also effective in stimulating DA synthesis and release, thus dietary phenylalanine supplementation can increase DA biosynthesis in fish.

Item does not contain fulltext Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26 degrees C. This study assessed the effects of temperature on sole metabolic status, in particular in what concerns plasma free amino acid changes during thermal acclimation. Senegalese sole maintained at 18 degrees C were acclimated to either cold (12 degrees C) or warm (26 degrees C) environmental temperatures for 21 days. Fish maintained at 18 degrees C served as control. Plasma concentrations of cortisol, glucose, lactate, triglycerides, proteins, and free amino acids were assessed. Cold acclimation influenced interrenal responses of sole by increasing cortisol release. Moreover, plasma glucose and lactate concentrations increased linearly with temperature, presumably reflecting a higher metabolic activity of sole acclimated to 26 degrees C. Acclimation temperature affected more drastically plasma concentrations of dispensable than that of indispensable amino acids, and different acclimation temperatures induced different responses. Asparagine, glutamine and ornithine seem to be of particular importance for ammonia detoxification mechanisms, synthesis of triglycerides that may be used during homeoviscous adaptation and, to a lesser extent, as energetic substrates in specimens acclimated to 12 degrees C. When sole is acclimated to 26 degrees C taurine, glutamate, GABA and glycine increased, which may suggest important roles as antioxidant defences, in osmoregulatory processes and/or for energetic purposes at this thermal regimen. In conclusion, acclimation to different environmental temperatures induces several metabolic changes in Senegalese sole, suggesting that amino acids may be important for thermal acclimation. 01 juli 2012

Physiological responses after an acute handling stress and their subsequent effects on innate immune parameters, plasma free amino acids (AA) and liver energy substrates were assessed in Senegalese sole (Solea senegalensis). Eight groups of six specimens (136.1 ± 58.4 g wet weight) were maintained undisturbed, while other eight groups of six specimens were used for acute stress challenge (air exposed during 3 min). A group of six specimens was sampled for blood and head-kidney collection immediately after air exposure (time 0), while the remaining groups were sampled at 5 and 30 min, 1, 2, 4, 6 and 24 h. Undisturbed fish were sampled at the same times and used as control. Fish were fasted for 24 h prior to air exposure and sampling. Plasma cortisol, glucose, lactate and osmolality levels increased immediately after stress peaking at 1 h in air exposed fish. Changes in plasma free AA were also observed at 1 and 24 h after stress. In liver, glycogen levels significantly decreased at 30 min and 1 h, while triglycerides values significantly increased at 1, 2 and 4 h in air exposed fish. In addition, total AA levels in liver augmented significantly at 2 h holding high until 24 h in air exposed specimens. The respiratory burst of head-kidney leucocytes from air exposed fish was significantly higher than that from control groups at 2 and 6 h after air exposure. On the other hand, plasma lysozyme activity significantly decreased at 4 h after acute stress in air exposed fish, while plasma alternative complement pathway followed an inverse linear relationship with respect to cortisol showing the lowest value at 1 h after air exposure. The present study suggests that Senegalese sole presents a stress response comparable to that observed in other teleosts. While some indispensable AA may be used for the synthesis of compounds related to the stress response or fatty acid transport, dispensable AA were probably mainly employed either as energy sources or in gluconeogenesis. Moreover, results from non-specific immune parameters assessed suggest that cortisol may act as regulator of the innate immune system.

The effects of prolonged feed deprivation were assessed on blood plasma metabolites and free amino acid levels in Solea senegalensis. Juvenile specimens were maintained at two experimental conditions (24 h fasted and 21 days feed-deprived). In feed-deprived fish, relative growth rate and hepatosomatic index as well as plasma triglycerides and proteins levels were significantly lower. However, plasma cortisol levels were higher in feed-deprived fish, while plasma glucose and lactate values were not significantly different between treatments. Furthermore, feed-deprived fish showed higher levels of total plasma free amino acids than 24 h fasted fish. In 21 days feed-deprived sole, an increase in plasma cortisol levels may suggest a functional role in mobilizing energy due to the catabolic action of this hormone in teleosts. Higher levels of glutamine, arginine and ornithine in 21 days feed-deprived fish may be indicative of a dual role for these amino acids: ammonia detoxification and carbon source for gluconeogenesis. The increased plasma glucogenic and branched-chain amino acid levels, together with the maintenance of plasma glucose in 21 days feed-deprived sole, suggest active liver gluconeogenic processes supported by tissue proteolysis.

23 páginas, 3 figuras, 4 tablas., The effect of dietary taurine supplementation on growth performance, metamorphosis success and amino acid metabolism of Senegalese sole (Solea senegalensis) larvae was investigated. These parameters were assessed in larvae fed control and taurine supplemented microcapsules during the pelagic phase. Subsequently, a similar evaluation was carried out in newly settled larvae fed upon Artemia, in order to verify the effect of earlier dietary taurine supplementation in larvae reared under improved feeding conditions. Results showed that dietary taurine supplementation did not affect larval growth performance and metamorphosis during the pelagic phase. However, by the end of the trial, Senegalese sole previously fed taurine supplemented microcapsules had a significantly higher growth performance and metamorphosis completion success than larvae fed control microcapsules. These differences were likely related to the improvement of feeding conditions upon settlement, which probably helped revealing the positive effects of earlier dietary taurine supplementation on Senegalese sole performance. Additionally, Senegalese sole may have benefited from taurine antioxidant properties during metamorphosis, since larval antioxidant defences may saturate at this stage. Furthermore, results from metabolic trials have shown that dietary taurine supplementation significantly increased amino acid retention in Senegalese sole larvae when a concomitant increase of taurine body levels was found. Therefore, an increase in larval growth potential and metamorphosis success was observed under dietary taurine supplementation and these results may help understanding why dietary taurine supplementation has been reported to simultaneously increase taurine body levels and growth performance in other fish species, leading to a better comprehension on the role of taurine during fish development., This work was supported by project POCI/CVT/60176/2004—SULFAAR, financed by POCI 2010 (FCT, Portugal), which is co-financed by FEDER. Wilson Pinto and Cláudia Aragão benefited from grants SFRH/BD/27889/2006 and SFRH/BPD/37197/2007 (FCT, Portugal), respectively. Manuel Yúfera contribution was funded by Consejería Innovación, Ciencia y Empresa—Junta de Andalucía (Spain) which is co-financed by FEDER (project P06-AGR-01697). This publication benefits from participation in LARVANET COST action FA0801.

Fish held at high stocking densities are generally exposed to chronic stress situations that impose severe energy demands and may predispose the fish to infection. Senegalese sole Solea senegalensis (Kaup) juveniles (78.8±18.9 g body weight) were maintained at low (LSD; 4 kg m−2 at the end of the experiment), medium (MSD; 9 kg m−2) and high (HSD; 14 kg m−2) stocking densities during a period of 63 days. Although disease outbreaks were observed in fish reared at HSD, growth and food consumption did not vary among different treatments. Results from plasma cortisol and free amino acids (FAAs) showed significant differences among different rearing densities pointing to HSD as stressful rearing condition. However, higher plasma glucose and osmolality levels indicated that fish held at MSD may also be under stress. The higher usage of FAAs from the HSD group may be due to the higher demand for energy production in order to cope with stressful rearing conditions, higher rate of protein synthesis or due to synthesis of other important metabolites related to stress response. Therefore, results from the present study point out to HSD as a stressful rearing condition and suggest that crowding stress may affect amino acid requirements.

Um modelo mecanístico que simule o crescimento durante a fase larvar de peixes deverá contribuir para um melhor conhecimento do processo de crescimento e respectivo metabolismo, com a intenção de elucidar as suas necessidades alimentares e desenvolver estratégias de alimentação adequadas. O modelo desenvolvido simula o crescimento e a evolução da composição bioquímica das larvas ao longo do tempo. O modelo depende essencialmente do consumo de alimento, sendo os nutrientes absorvidos usados para a produção de energia ou em processos de biossíntese e é baseado na estequiometria do metabolismo intermediário. Simulações utilizando este modelo sugerem que se deve prestar mais atenção a possíveis desequilíbrios no perfil de aminoácidos (AA) e a eventuais excessos de lípidos no alimento usado em cultivo larvares, de forma a optimizar o crescimento. Estudos usando marcadores isotópicos têm sido utilizados para estudar o metabolismo de AA e de lípidos em larvas de peixes e técnicas de modelação foram aplicadas de forma a permitir uma melhor compreensão dos resultados obtidos nestes estudos. A capacidade digestiva, bem como a utilização energética de diferentes AA, ácidos gordos e classes de lípidos, tem sido estudada utilizando a administração de nutrientes marcados (normalmente com 14C) directamente no tubo digestivo, através de um microcapilar. Este método permite a quantificação do nutriente marcado presente nas fezes, retido nos tecidos da larva e catabolizado. Um outro método, combinando a utilização de alimento vivo cujas proteínas estão marcadas com um isótopo estável com uma técnica espectroscópica/espectrométrica, permite a determinação do enriquecimento isotópico de AA individuais e pode ser usado para estimar o perfil ideal de AA indispensáveis na dieta. Em conjunto, estes dois tipos de técnicas de análise de fluxos de nutrientes têm permitido avanços importantes na compreensão da fisiologia da nutrição e crescimento de larvas de peixes. A mechanistic simulation model of fish larval growth should contribute to a better understanding of growth and metabolism of fish larvae. It should also assist in studying the nutritional requirements and developing feeding strategies. A model was developed that simulates growth and biochemical composition in time. The model is driven by feed intake, with absorbed nutrients being used for energy production and biosynthesis, based on the stoicheometry of intermediary metabolism. Model simulations suggest that imbalanced dietary amino acid (AA) profiles and dietary lipid excess may lead to suboptimal growth in fish larvae culture. Tracer studies have therefore been used to study AA and lipid metabolism in fish larvae. In addition, modeling techniques have been also used in these studies, to better interpret results obtained. Digestive capacity, as well as utilization of different AA, fatty acids and lipid classes, has been studied using radiolabeled nutrients (normally with 14C) using a tube-feeding technique. This method allows quantification of the tracer that is present in faeces, retained in tissues and catabolised. Another method, combining the use of live food in which proteins are labeled with a stable isotope and a spectroscopic/spectrometric technique that allows determination of the isotopic enrichment in individual AA, can be used to estimate ideal dietary indispensable AA profiles. Together these two types of techniques that allow the study of nutrient fluxes have lead to important advances in the understanding of fish larvae nutritional physiology and growth.

Little is known about the amino acid (AA) requirements of larval fish and their possible ontogenetic changes. This paper aims to contribute to the study of these requirements in two common cultured species in Southern Europe: gilthead seabream (Sparus aurata) and Senegalese sole (Solea senegalensis). Moreover, it is intended to identify possible dietary AA imbalances occurring in normal hatchery conditions for both species. Fish larvae were reared following standard procedures and the normal feeding schemes used in hatcheries, which includes the use of live prey for several weeks. The experiments were finished once the fish were adapted to a dry feed. Samples were collected at different points for dry weight, total protein, and AA analysis. The A/E ratios [each indispensable AA content×(total indispensable AA content including cysteine and tyrosine)−1×1000] of fish and food were compared to identify possible dietary AA imbalances. The AA profile of both fish species changed during ontogeny, but was more stable during seabream than Senegalese sole development. This is probably linked to the marked metamorphosis observed in sole. These changes in fish larval AA profile suggest that the AA requirements change during seabream and Senegalese sole ontogenesis. Several dietary AA deficiencies were found during the ontogenetic development of both species. These deficiencies result from ontogenetic changes in the fish AA profile, but also from differences in the AA composition of the different food items. These results suggest that both the live food (rotifers and Artemia at different developmental stages) and the dry feed used in the rearing of gilthead seabream and Senegalese sole does not have an AA profile that entirely meets the requirements for the early stages of these species.

The rearing of most marine fish species still relies on live food. Amino acids (AA) are the building blocks for protein synthesis, are important energy substrates, and are involved in specific physiological functions. Thus, the AA pools (free and protein-bound) of the rotifer Brachionus rotundiformis and of Artemia parthenogenetica metanauplii were analysed, after enrichment with different diets or at different salinities. The changes in these pools were related to their impact when used as feed for the developing fish larvae. Rotifers and Artemia were enriched for 24 h in microalgae, in commercial diets, or starved for the same time period. The enrichment at different salinities was also tested using Artemia . The free AA (FAA) quantity and quality was strongly affected by the enrichment used, in both rotifers and Artemia metanauplii. The relative FAA content was significantly higher in rotifers and Artemia enriched with microalgae than in the ones enriched with commercial products, and between the different microalgae tested, the enrichment in T. chui resulted in the highest FAA content for both preys. Differences regarding the FAA quantity and quality were also found between rotifers and Artemia . With the exception of the starved preys, a higher relative FAA content was found in rotifers than in Artemia metanauplii and while the FAA pool of the rotifers was dominated by alanine, taurine was the dominant AA in the FAA pool of Artemia . Salinity had only a small effect on the FAA pool of Artemia . Concerning the protein fraction, starvation increased the relative protein content in the rotifers. Among the metanauplii, the relative protein content was affected neither by the enrichment nor by salinity, except for a significantly lower content found when the microalgae Nannochloropsis gaditana was used. The quality of the protein pool is affected by the different enrichments, but not by salinity. However, the changes in the protein-bound AA pool were less pronounced than for the FAA pool and reflect differences in the protein content and/or quality of the diet. This study showed that the AA composition of cultured zooplankton is affected by feeding regime and by salinity. The choice of the best zooplankton enrichment and/or mixture of enrichments for the different fish larval stages, should take into account the AA composition, in order to fulfil the energetic and nutritional requirements of the larvae.

The objective of this work was to evaluate the effect of a dietary amino acid imbalance, originating from the use of a soy protein concentrate (SPC) as the major protein source, on the growth performance and amino acid metabolism of Senegalese sole (Solea senegalensis) postlarvae. Senegalese sole (85.6±24.6 mg wet weight) were fed one of two experimental diets: one based on fish meal (FM) and another based on SPC. Diets were isonitrogenous (around 56% crude protein) and isoenergetic. Diet acceptability was very good and the growth rate was 6.9% day−1 for sole eating the FM diet and 6.0% day−1 for sole eating the SPC diet. Mass-specific ammonia excretion and the activities of selected amino acid metabolic enzymes (ALAT, ASAT and GDH) did not present significant differences between treatments, although this may have been due to the high variability found for these parameters in the SPC treatment. This variability may suggest different capacities of individual fish to adapt to the possible methionine dietary deficiency. The utilization of amino acids as a substrate for lipogenesis does not seem to be affected by the dietary protein source, since NAPDH-generating enzymes (G6PD and ME) had similar activities in both treatments. Amino acid metabolism in Senegalese sole postlarvae seems to be slightly affected by the dietary protein source. Nevertheless, the changes induced by the SPC diet do not seem to impair growth, at least at the high dietary protein level used in this experiment.

The postprandial metabolism of dietary free amino acids (AA) was studied in post-larval Senegal sole, Solea senegalensis, aged 32 days after hatching (DAH). The diet was administered as a single pulse (36 nl, 43.1 mmol/l) using a micro tube-feeding technique and a dissolved mixture of crystalline AA. In four separate treatments the diet contained L [U-(14)C] tracer for two indispensable AA (IAA), lysine and arginine or two dispensable amino acids (DAA), glutamate and alanine. The post-larva absorbed all tested AA from the diet with similar efficiency (97.5%). A small fraction of the IAA was catabolised (11.5+/-1.1 and 15.1+/-3.3%, for lysine and arginine, respectively) and a high proportion was retained in the body (86.7+/-1.3 and 81.6+/-4.1%). For the DAA more were catabolised (64.9+/-5.3% and 41.4+/-7.2% for glutamate and alanine, respectively) and less were retained (32.9+/-5.1% and 56.3+/-7.2%). On this basis, it appears that post-larval Senegal sole use DAA in preference to IAA as energy substrates while the retention (assimilation efficiency) is better for the IAA. These results support other recent studies that early stages of fish have a better capacity to regulate AA catabolism than previously believed and that indispensable AA are saved for body growth.

Expansion of the aquaculture industry is limited by incomplete knowledge on fish larval nutritional requirements. Nevertheless, it is believed that dietary taurine deficiencies may be particularly critical for fish larvae. The reasons include the high taurine levels found during egg and yolk-sac stages of fish, suggesting that taurine may be of pivotal importance for larval development. Moreover, unlike aquaculture feeds, natural preys of fish larvae contain high taurine levels, and dietary taurine supplementation has been shown to increase larval growth in several fish species. This study aimed to further explore the physiological role of taurine during fish development. Firstly, the effect of dietary taurine supplementation was assessed on growth of gilthead sea bream (Sparus aurata) larvae and growth, metamorphosis success and amino acid metabolism of Senegalese sole (Solea senegalensis) larvae. Secondly, the expression of taurine transporter (TauT) was characterised by qPCR in sole larvae and juveniles. Results showed that dietary taurine supplementation did not increase sea bream growth. However, dietary taurine supplementation significantly increased sole larval growth, metamorphosis success and amino acid retention. Metamorphosis was also shown to be an important developmental trigger to promote taurine transport in sole tissues, while evidence for an enterohepatic recycling pathway for taurine was found in sole at least from juvenile stage. Taken together, our studies showed that the dependence of dietary taurine supplementation differs among fish species and that taurine has a vital role during the ontogenetic development of flatfish, an extremely valuable group targeted for aquaculture production.

Expansion of the aquaculture industry is limited by incomplete knowledge on fish larval nutritional requirements. Nevertheless, it is believed that dietary taurine deficiencies may be particularly critical for fish larvae. The reasons include the high taurine levels found during egg and yolk-sac stages of fish, suggesting that taurine may be of pivotal importance for larval development. Moreover, unlike aquaculture feeds, natural preys of fish larvae contain high taurine levels, and dietary taurine supplementation has been shown to increase larval growth in several fish species. This study aimed to further explore the physiological role of taurine during fish development. Firstly, the effect of dietary taurine supplementation was assessed on growth of gilthead sea bream (Sparus aurata) larvae and growth, metamorphosis success and amino acid metabolism of Senegalese sole (Solea senegalensis) larvae. Secondly, the expression of taurine transporter (TauT) was characterised by qPCR in sole larvae and juveniles. Results showed that dietary taurine supplementation did not increase sea bream growth. However, dietary taurine supplementation significantly increased sole larval growth, metamorphosis success and amino acid retention. Metamorphosis was also shown to be an important developmental trigger to promote taurine transport in sole tissues, while evidence for an enterohepatic recycling pathway for taurine was found in sole at least from juvenile stage. Taken together, our studies showed that the dependence of dietary taurine supplementation differs among fish species and that taurine has a vital role during the ontogenetic development of flatfish, an extremely valuable group targeted for aquaculture production.

This study evaluated the effect of dietary taurine supplementation on the growth performance and methionine metabolism of gilthead seabream larvae. For this purpose, a growth experiment was carried out in which seabream larvae were fed upon rotifers supplemented with “blank” or taurine-enriched liposomes. A complementary trial was also done in which seabream larvae fed with live prey were subsequently tube-fed a solution containing a L-[U-14C] methionine with or without a taurine supplement. Results from the growth experiment showed that rotifers were successfully enriched with taurine, but no effects were observed on larval growth performance, survival or amino acid composition. Furthermore, dietary taurine supplementation did not result in an increase of larval taurine levels, a factor that may have been determinant for the absence of effects observed on growth performance. In the tube-feeding trial, results showed that dietary taurine supplementation led to an increase of methionine retention in larvae. These findings suggest the existence of an active taurine biosynthesis pathway for gilthead seabream during the larval stage. Hence, gilthead seabream may not be dependent on dietary taurine to maintain the taurine body pool, since it may convert taurine from methionine if required. Taken together, the results from this study indicate that dietary taurine supplementation does not seem to enhance the larval growth performance in fish species able to biosynthesise taurine during this stage, which seems to be the case of gilthead seabream. However, this study also showed that in these species, dietary taurine supplementation may ultimately affect larval metabolism by increasing methionine availability for several important physiological purposes, contributing to a better understanding on the role of taurine during the early life stages of fish development.