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Protein is the most expensive part of fish diets and supplies amino acids (AA) for energy, growth, protein synthesis and as substrates for key metabolic pathways. Functional AA is a term used to describe AA that are involved in cellular processes apart from protein synthesis. A deficiency, or imbalance, in functional AA may impair body metabolism and homeostasis. Recent years have seen an increased interest in AA to increase disease resistance, immune response, reproduction, behavior and more. This has led to a boost of commercially available functional fish feeds that aim to optimize fish performance and quality of the product. This review aim to collect recent findings of functional AA and of how they may improve fish health and welfare. It will focus on functional properties of some of the most studied AA, namely arginine, glutamine, glutamate, tryptophan, sulfur amino acids (methionine, cysteine and taurine), histidine and branched chain amino acids. Where information is not available in fish, we will point towards functions known in animals and humans, with possible translational functions to fish.

One of the many functions of taurine is to protect cells against oxidation, by protecting mitochondrial integrity and respiration. Taurine metabolism has attracted much attention in fish nutrition due to the fact that as plant ingredients replace fishmeal, dietary taurine has declined. As the endogenous synthesis of taurine might be too low to protect cells against oxidative stress and apoptosis, the present study aimed to test whether taurine may protect liver cells from apoptosis. Liver cells isolated from Atlantic salmon (Salmo salar) were grown in media supplemented with a physiological concentration of taurine (25 (se 0·5) mm) or without any taurine supplementation (14 (se 3) μm) for 3 d. To increase oxidation in the mitochondria and maximise any cellular response of taurine supplementation, 100 μm-CdCl2 was added or not added to the cells at day 3. At day 4, cells were harvested and assessed for viability. As expected, the addition of CdCl2 decreased cell viability without showing any interaction with taurine supplementation. Cells grown in the taurine-supplemented media had lower protein abundance of active caspase-3. In addition, the protein abundance of phosphorylated mitogen-activating phosphokinase (P-p63, P-p42/44 and P-p38) as well as cytochrome P450 were reduced when taurine was added to the media. Cells grown without taurine supplementation had a more condensed chromatin and more smeared DNA, also pointing to a higher apoptosis in these cells. In conclusion, taurine attenuated apoptosis in primary liver cells isolated from Atlantic salmon, and as such, taurine may be conditionally indispensable in Atlantic salmon. [ABSTRACT FROM PUBLISHER]

To be able to study nutrient requirement and utilization in any species, a diet supporting normal feed intake and growth equally well as a traditional fish meal-based diet is needed. Additionally the formulation of the diet should allow low levels of the nutrient under study. When studying the amino acid metabolism and requirements, one cannot rely on the fish meal-based diets as fish meal are nicely balanced according to requirements. Therefore the current study aimed to develop a plant protein-based diet (with low fish meal inclusion) to be used in the nutritional studies of Senegalese sole juveniles supporting feed intake and growth close to that obtained in a fish meal-based control feed. Two experiments were conducted to evaluate whether Senegalese sole juveniles would accept and utilize diets containing high plant protein inclusion. For testing the acceptance of high plant protein inclusion, two diets were formulated: a reference diet that contained fish meal as the main protein source (450 g kg−1 dry matter) whereas in the test diet, fish meal was substituted by a mixture of plant ingredients (soybean meal, corn and wheat gluten) withl-lysine supplementation. In order to improve the palatability, 50 g kg−1 squid meal was added to both diets. The indispensable amino acids (IAA) profile of the test diet was made similar to the control diet by adding crystalline amino acids. Further, automatic feeders were used to improve the feed intake. Fish (24 g initial body weight) were fed the diets for a period of 4 weeks. As fish accepted both diets equally well, a second study was undertaken to test the growth performance. Fish (6 g initial BW) were fed the diets for a period of 12 weeks. The use of automatic feeders to deliver the feed and the addition of both squid and balancing the indispensable amino acids resulted in growth performance and accretion not differing from the fish meal fed control. It can be concluded that juvenile Senegalese sole are able to grow and utilize high plant-protein diets when both diet composition and feeding regime are adequate for this species. [ABSTRACT FROM AUTHOR]

Nine experimental feeds were prepared, having two different digestible energy levels (DE, 19.5 and 21.5 MJ kg−1) and three different amino acid levels (90%, 100% and 110%), and formulated with two alternative strategies (protein and fat vary freely or only protein varies freely). The design was not balanced and thus the 110% amino acid profile was only studied at 19.5 MJ kg−1 DE. Atlantic salmon ( Salmo salar) with a mean body weight of 449±19 g were distributed in 24 tanks and were fed each of the experimental diets for a period of 90 days. The fish were fed three times daily and uneaten feed was collected. Increasing the dietary DE energy from 19.5 to 21.5 MJ kg−1 and the amino acid level from 90% to 100% of the requirement increased the feed intake and growth performance in Atlantic salmon. Increasing the amino acid level from 90% to 100% had a positive effect on the feed intake, growth and protein accretion. Also, the mRNA levels of insulin-like growth factor I in muscle tissues pointed to this positive effect when the amino acid level increased. High probabilities were associated with a negative effect on the feed intake of reducing the amino acid level regardless of the DE level. It is recommended that the balanced amino acid level should not be adjusted by the same percentage rate as dietary energy, and possibly should not be reduced at all in order to maintain optimum performance and profitability. [ABSTRACT FROM AUTHOR]

Abstract: Fish fillet is a source of dietary protein with a well-balanced amino acid composition that also contains high levels of B-vitamins and high levels of the trace elements selenium and iodide. In the present study, we elucidated whether there was a partitioning of the micro-and macronutrients between the soluble fractions (fish protein hydrolysate, FPH) and the insoluble fractions obtained after the enzymatic hydrolysis of fish-raw materials. The FPHs from cod, salmon and saithe contained high levels of taurine, potassium and B-vitamins. Salmon FPH was particularly rich in niacin and pantothenic acid. All FPHs were low in the amino acid tryptophan. The cod and saithe insoluble peptide fractions contained high levels of the indispensable amino acids including tryptophan and of the trace elements selenium, iron and zinc. Iodide levels were high in both the soluble and insoluble fractions. Potential explanations for the segregation of macro- and micronutrients between the FPHs and their corresponding insoluble peptide fractions are discussed. These differences may have nutritional impact when testing different FPHs or insoluble peptide fractions in biological systems. [Copyright &y& Elsevier]

Abstract: Two trials (I and II) were conducted to finally determine the Lys requirement in the Atlantic salmon during the fast growing period in seawater. First three test diets with well balanced AA''s as well as a IAA:DAA ratio close to 1, but differing in amount and source of an attractant, were developed allowing for both a reduction of dietary Lys and a reasonable performance when supplementing Lys. The diets were based on our previous study in which all fish meal was replaced by plant proteins, of which resulted in both inferior body weight gain and lipid deposition ([Espe, M., Lemme, A., Petri, A., El-Mowafi, A. 2006. Can Atlantic salmon grow on diets devoid of fish meal? Aquaculture 255, 255–262]). Basically, the amino acid profiles of all test diets did not differ (p >0.05) from the control fish meal based diet although 90% of the fish meal was replaced by plant proteins. The diets were fed to Atlantic salmon of BW 327 g for a period of 85 days. The test diet supporting both growth and deposition of protein and lipid not differing (p >0.05) from the fish meal control finally consisted of plant proteins supplemented with 5% fish meal, 5% fish soluble and 3% squid hydrolysate. This diet then was used in a dose response experiment aiming to determine Lys requirement in the fast growing salmon. Atlantic salmon with BW of 642 g were fed graded amounts of Lys (2.85 to 9.19 g Lys/16 g N) for a period of 85 days. Dietary Lys did not affect growth (p >0.05), but the protein accretion suggested an optimum dietary Lys supply of 5.04 g /16 g N (corresponding to 0.12 g Lys/fish/day). This response was accompanied with reduced fat accretion. To obtain weight gain to the level present in the fish meal control diet 3.17 g Lys/16 g N was sufficient. Thus lean growth rather than body weight gain should be the response parameter to determine Lys requirement in salmon. [Copyright &y& Elsevier]

Abstract: The present experiment tested whether Atlantic salmon accept, grow and utilise diets devoid of fish meal. Four diets were prepared, one reference diet in which 49% of the diet was fish meal and three diets not containing any fish meal. The amino acid composition in the test diets not containing fish meal was made similar to the reference diet by balancing with crystalline amino acids. All plant protein diets also contained 5% fish protein hydrolysate and, additionally, two diets were added with either stick water or squid hydrolysate in order to improve the palatability and thus feed acceptance. The experimental diets were fed to Atlantic salmon of 300 g body weight for a period of 3 months during which period no fish died. Fish fed the plant protein diets showed reduced feed intake as compared to fish fed the reference fish meal diet resulting in lower growth in the former. Feed conversion did not differ between treatments. Although the fish fed the fish meal diet showed higher growth than those fed the alternative three diets, protein accretion was not significantly different. In contrast, fat retention was significantly higher in the fishmeal treatment suggesting both a higher retention of dietary fat which was higher in the fish meal diet and a better utilisation of the dietary energy by salmon fed the vegetable diets due to a highly balanced dietary amino acid profile. The use of up to 10% crystalline amino acids in the diet increased the amino acid digestibility in the plant protein diets but utilisation of the dietary protein was not affected. The latter indicate that crystalline amino acids are utilised as well as protein bound amino acids at the level of 10% addition. Taking all together, particularly the diet containing squid hydrolysate might be an alternative to the fish meal diet in order to produce salmon. [Copyright &y& Elsevier]

It has previously been demonstrated that soya protein, which contains isoflavones and low methionine???glycine and lysine???arginine ratios, has a hypocholesterolaemic effect. In the present study, the hypocholesterolaemic effects of an isoflavone-enriched casein diet (HDI) and a single-cell protein-based diet (SCP) devoid of isoflavones but with low methionine???glycine and lysine???arginine ratios were investigated in obese Zucker rats after 6 weeks of feeding. The control diet contained casein, which has high ratios of methionine???glycine and lysine???arginine. HDI and SCP feeding reduced the concentrations of total cholesterol and cholesteryl esters in plasma and liver, and changed the fatty acid composition of the hepatic cholesteryl esters. Faecal cholesterol and bile acid levels were markedly higher in SCP-fed rats than in controls, whereas HDI feeding had only minor effects. However, both HDI and SCP feeding increased the hepatic gene expression of cholesterol 7?? hydroxylase. In contrast, the hepatic acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase activities and the gene expression of the LDL receptor were increased by HDI, but not by SCP feeding. The present results suggested that the cholesterol-lowering effect of SCP was related to the enterohepatic circulation, whereas HDI seemed to lower the plasma cholesterol via the circulation. Plasma homocysteine level was reduced in rats fed HDI and SCP compared to rats fed casein. In summary, diets enriched in isoflavones or containing proteins with low methionine???glycine and lysine???arginine ratios lowered the plasma cholesterol and homocysteine levels, changing the plasma profile from atherogenic to cardioprotective. [ABSTRACT FROM PUBLISHER]

Abstract: A protein preparation labelled by incorporation of [U]14C-AA was hydrolysed to various degrees and administered to a teleost fish larva (Atlantic halibut, Hippoglossus hippoglossus L.) by tube-feeding, and its post-administration utilisation was studied. Three treatments were prepared: IntP—intact protein, PHP—pepsin-hydrolysed protein, and HHP—highly hydrolysed protein (using pepsin, trypsin, endoproteinase Glu-C, Asp-N, and Pro-C). At small doses (11.4±1.5 μg larvae−1), the intact protein (IntP) was digested and absorbed to 36±5.5%. However, the relative absorption efficiency of the intact protein was reduced as the dose increased. Absorption efficiency was higher when the protein was hydrolysed prior to feeding the larvae and was constant at 63% (R 2=98) independent of degree of proteolysis and dose (ranging from 3.5 to 35 μg larvae−1). The initial absorption rate increased with the degree of hydrolysis. Calculations based on data collected during the first 30–120 min show that the absorption of PHP and HHP into extra-intestinal body tissues was 2.2 and 3 times as fast, respectively, as that of intact protein. However, the rates of absorption did not influence the distribution of absorbed AA into either catabolism or anabolism, as all larvae, independent of which protein solution they were given, catabolised 42±7% of the absorbed AA, and accumulated 49±6% into the body tissue, during the 20 h post-feeding incubation period. Larval age and size did not influence the absorption and utilisation of the hydrolysed protein preparations. This was different from the intact protein, as significantly higher fractions of the intact protein were absorbed by the larvae at 31 days past first feeding (dpff) than by larvae at 25 dpff. Analysis of the faecal evacuation suggested that the poor protein utilisation in the younger larvae was due to enhanced faecal evacuation, which in some larvae was more than 50% only 4 h post diet administration, at a time when the process of digestion and absorption was far from complete. This indicated that faecal evacuation is a critical factor in the utilisation of slowly digested and absorbed feed components, such as intact proteins, by fish larvae. [Copyright &y& Elsevier]

A radioactive soluble model protein for studies of protein digestion, absorption and amino acid (AA) metabolism in larval fish was successfully produced in Atlantic salmon by oral administration of uniformly [U]-14C-labelled amino acids followed by blood sample withdrawal (48 h post-administration) and purification. The salmon serum protein (14C-SSP) was characterised in terms of the protein composition and specific activity of its amino acids. Most radioactivity was found in the three most abundant serum proteins, which had apparent molecular weights of 65, 75 and 120 kDa, respectively, of which labelling was found in all the amino acid residues of the SSP that were analysed. The digestibility of the 14C-SSP was tested by in vivo tube feeding using early stages of Atlantic halibut and was found to be more efficiently digested and utilised than the 14C-methylated bovine serum albumin (14C-BSA) that has been used in previous studies. This supports the notion that proteins labelled by 14C-methylation are not suitable as model proteins in metabolic studies due to modification of their 14C-methylated lysine residues. Further studies on the 14C-SSP demonstrated a digestibility of 59±13% in juvenile halibut, while at the pre-metamorphic stage, it was only 25±13%. This supports the hypothesis that there is a significant improvement in the ability to digest and utilise dietary proteins as the digestive system becomes fully developed, including a functional (acid-producing) stomach. [Copyright &y& Elsevier]

The kinetics and interaction of the in vitro uptake of l-methionine were studied in the mid-intestine in Atlantic salmon by the everted sleeve method. The uptake kinetics of l-methionine alone was measured, and also how l-methionine interacted with other dietary amino acids when present in physiological concentration within the incubation medium. Knowledge of how the uptake of l-methionine is affected by other amino acids at physiological concentrations in the gut is important as methionine is considered to be the first limiting amino acid in many proteins used in fish feeds. The carrier-mediated uptake of l-methionine showed a maximum rate of transport (Vmax) of 0.12 nmol mg−1 min−1 and the apparent affinity value (Kt) was estimated to be 3.0 mM l-methionine. The permeability coefficient (P) for the nonsaturable uptake was estimated to be 0.017 nmol mg−1 min−1 mM−1. These in vitro experiments might indicate that an efficient uptake of l-methionine in a physiological mixture of all amino acids depends on a certain concentration of l-methionine in the incubation solution (2.5 mM), as methionine uptake was inhibited at lower concentrations. At higher l-methionine concentrations (10–25 mM), the uptake of l-methionine when present alone was not different from the uptake of l-methionine measured in a mixture of all amino acids. However, when the other neutral amino acids were omitted from the incubation medium, a reduced uptake of 10 and 25 mM l-methionine was found. [Copyright &y& Elsevier]