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Methionine-choline-deficient ( MCD) mammals are known to accumulate liver TAG probably due to phosphatidylcholine ( PC) deficiency and thus assembly of VLDL and transport of lipids from liver to peripheral organs. To assess whether supplementation of choline could spare methionine and secure a healthy liver metabolism, by reducing the endogenous PC synthesis without interfering with lipid transport and distribution, Atlantic salmon with initial BW of 700 g were fed adequate (1.9 g Met/16 gN) or surplus methionine (2.5 g Met/16 gN) diets of which were supplemented with choline or not for a period of 19 weeks. Fish fed the low-methionine diets had reduced growth ( p = .013) due to reduced protein gain ( p = .007), while lipid gain slightly improved in fish fed the choline-supplemented diets ( p = .047). Also, feed conversion improved when fed surplus methionine ( p < .001), while choline supplementation had no impact on feed conversion. No interaction between choline and methionine on growth performance or retention existed. Phospholipid status in liver and muscle was not affected by treatments, and no liver TAG accumulation occurred at the methionine levels used. Gene expression of ApoB100 necessary for assembling VLDL or pemt necessary for endogenous PC synthesis was unaffected by treatments. Capacity of methylation ( MAT, BHMT) within the liver was not affected by treatment nor was the gene expression of enzymes in liver transsulfuration ( CBS or CDO). Methionine status within liver was unaffected by treatments, while free methionine reduced in those fish fed the low-methionine diets in muscle and plasma. Cystathionine and taurine were elevated when fed surplus methionine. Choline supplementation had no impact on sulphur amino acid metabolites in either tissue. Neither did choline supplementation improve TAG mobilization from liver to muscle. To conclude, choline does not improve endogenous phospholipid synthesis or transport of TAG from liver to muscle depot when added to diets containing 1.9 g Met/16 gN, while surplus methionine improved growth and protein retention, indicating that 1.9 g Met/16 gN is enough to support a healthy liver metabolism, but too low to support muscle protein deposition in adult salmon fed high plant protein diets for longer periods of time. [ABSTRACT FROM AUTHOR]

We previously reported that juvenile Atlantic salmon with mean initial BW 11.5 g offed a methionine deficient diet had lower weight gain due to a reduced protein accretion, while lipid gain was unaffected. Muscle of the fish fed the methionine deficient diet was depleted for sulphur amino acids, while in liver, the concentration of these metabolites was maintained within narrow limits. We speculated whether this could be due to an increased muscle proteolysis to support a prioritized liver metabolism in fish fed the low methionine diets. In this study, we assessed whether genes associated with muscle proteolysis increased under methionine deficiency. The composition of the diets was similar to those used previously containing 1.6 or 2.1 g Met/16 g N. We confirmed that the fish fed the low methionine diet gained less protein compared to fish fed the DLmethionine enriched diet (P = 0.014), but growth did not reduce significantly. Also the deficient fish maintained the concentrations of liver sulphur amino acids and reduced muscle free methionine. Several of the other free amino acids within muscle increased. Further, methylation capacity was maintained in liver but reduced in the muscle (P = 0.78 and 0.04, respectively). Gene expression of muscle IGF-1 was lower (P = 0.008) and myosin light chain 2 tended (MLC2, P = 0.06) to be reduced in fish fed low methionine diet, concurrently the activity of cathepsins B+L increased (P = 0.047) in muscle of fish fed the low methionine diet. Gene expression of the muscle-specific E3 ubiquitine ligases (Murf and MaFbx) was not affected by treatment. Thus, the lower protein gain observed in fish fed the low methionine diet may be caused by reduced protein synthesis in line with the reduced IGF-1 gene expression in the white trunk muscle. Thus, to support metabolism, the dietary protein needs to be balanced in amino acids to support metabolism in all compartments of the body and secure maximal protein gain. [ABSTRACT FROM AUTHOR]

Phosphatidylcholine is synthesized endogenously through the enzyme phosphatidylethanolamine-methyl transferase. As endogenous choline synthesis requires methyl groups from S-adenosylmethionine (SAM), the endogenous synthesis of choline may depend on SAM availability. SAM availability depends on methionine and ATP. SAM is also a precursor for cysteine and may affect taurine and glutathione concentrations. To investigate whether choline synthesis or transsulphuration is prioritized and the interactions between taurine and choline supplementation when methionine availability is low, juvenile Atlantic salmon were fed low methionine diets with and without taurine and choline supplementation. There were no differences in growth or protein accretion following treatments. Fish fed the low methionine diets did not develop a fatty liver, but choline supplementation increased the concentration of total phospholipids in liver and muscle. Taurine supplementation increased taurine concentrations in liver and muscle, but no interactions with choline were present. Liver SAM was unaffected by treatments. Two of three tanks fed the low methionine diet without choline and taurine supplementation had a higher gene expression of p38MAPK (mitogen activated phosphokinase). Choline supplementation to low methionine diets thus may have beneficial effect on the metabolic health and lipid transport to extra-hepatic tissues. Taurine supplementation had no effect on phospholipids status, but increased taurine concentration in tissues and as such may affect oxidation status when methionine availability is low. [ABSTRACT FROM AUTHOR]

The current study aimed to investigate the effect of surplus dietary arginine on polyamine and amino acid metabolism and accretion of proteins and lipids, as previous studies have demonstrated a lipid-reducing effect following surplus arginine supplementation in viscera as well as increased muscle growth. Four plant protein-based diets were given increasing concentrations of L-arginine, from 21.1 to 36.1 g kg-1 DM, and fed to quadruplicate tanks of adult Atlantic salmon (1.1 kg) for 12 weeks. No effects on growth or deposition of fat or protein were observed. Free amino acid concentrations and related metabolites were unaffected in the liver, except for urea, while concentrations in muscle and plasma reflected production of arginine metabolites. Polyamine concentrations were unaffected in liver, muscle and white adipose tissue ( WAT), as were the abundance and activity of spermidine/spermine N1-acetyltransferase ( SSAT), the rate-limiting enzyme in polyamine turnover. Gene expression demonstrated differential regulation of ornithine decarboxylase ( ODC) in liver and WAT, although overall little effects were observed on gene expression. Liver S-adenosylmethionine ( SAM) concentrations decreased with arginine supplementation. We suggest that adult Atlantic salmon have adapted to surplus arginine, and the main long-term effect appears to be increased concentrations of arginine metabolites. [ABSTRACT FROM AUTHOR]

Cobia (8.4 ± 0.1 g body weight) were fed to satiation with three test diets of high plant protein-based ingredients and different lysine to arginine ratios, and one commercial diet (currently used for cobia rearing in Vietnam as a control for growth) for 6 weeks. The test diets contained 206 g marine ingredients kg−1, including fishmeal, krill meal and fish protein concentrate (in order of high to low inclusion), while the rest of the dietary protein was a blend of soya and pea protein concentrate, wheat protein and sunflower meal. Crystalline lysine and arginine were added in the test diets to produce either a balanced lysine to arginine ratio ( BL/ A; 1.1) and a high or low lysine to arginine ratio ( HL/ A; 1.8 and LL/ A; 0.8, respectively). There were no significant differences in final body weight, weight gain, feed conversion ratio or protein gain between cobia fed BL/ A- and commercial control diet ( CCT). Cobia fed BL/ A diet performed better than fish fed either HL/ A- or LL/ A diet. This was partly due to a higher feed intake and protein and lipid gain in cobia fed BL/ A diet as compared to HL/ A- and LL/ A diet. [ABSTRACT FROM AUTHOR]

The appetite-stimulating effect of krill hydrolysate ( KH), free amino acids and specific water-soluble low molecular N-compounds was explored in a short feeding trial with Atlantic salmon. A 100 g kg−1 fish meal positive ( MFM), a 30 g kg−1 fish meal negative control ( LFM) and six more LFM diets were produced added known attractants for fish- KH at two levels: low ( LAK) or high ( HAK), AMP, choline chloride ( CC); an amino acid mix resembling the free amino acid composition of KH ( AA) or a mix of AMP, CC and AA ( ACA). HAK, ACA and AA showed highest feed intake and significantly higher plasma phospholipids and cholesterol. The lower performing treatments ( LFM, AMP) showed higher liver lipids and hepatosomatic index. CC induced reduced liver lipids and increased plasma phospholipids and cholesterol. Appetite regulating neuropeptide gene expression analysis (q PCR) was performed in fish fed LFM, HAK and AA. Pyy showed the highest postprandial expression in LFM, whereas the expression of the anorexigenic neuropeptides cart, pomca1, pomca2 and pomca2s was low. These apparently contradictory results may be explained by initial appetite stimulation by HAK and AA, resulting in higher feed intake, followed by satiation and appetite downregulation. [ABSTRACT FROM AUTHOR]

Abstract: Further improvement of growth performance in fish larviculture is closely linked to better understanding of the dietary amino acid (AA) requirements, and therefore of the processes involved in AA metabolism. In recent years, major advances in the understanding of fish larvae amino acid metabolism have been accomplished, in particular through the use of tracer studies. Modelling is a holistic approach to integrate knowledge on growth and metabolism and identify gaps in current understanding. A dynamic mechanistic model that simulates AA metabolism of fish larvae was developed. It aims to improve the understanding of larval digestion and absorption of dietary AA, and the postprandial AA metabolism and growth. The model also assists in the interpretation of results obtained from tracer studies. The model is driven by amino acid intake, with the absorbed dietary AA being used for energy production or for biosynthetic processes. The model is implemented for Senegalese sole (Solea senegalensis) larvae fed Artemia, and was parameterized using literature data. The model allows to integrate the results obtained after feeding a single meal with tracer AA, and following these tracer AA in the free AA and protein pools of larval gut and larval body at different time points after the meal. Model simulations suggest that there is a sharp dynamic change in the FAA pool after a meal while the protein pool is little affected. This suggests that the AA composition of the food has a major contribution to the FAA pool composition. This implies that sole larvae is highly sensitive to dietary AA imbalances, having high AA unavoidable losses unless the dietary AA profile is well balanced. The model also suggests that rates of protein synthesis and AA catabolism rapidly increases after the meal, with the peak for this postprandial metabolism occurring only 1h after the meal, and the rates returning to “basal” values 2h after the meal. This suggests a rapid processing of the Artemia protein by the larvae, and supports the need for feeding sole larvae at a high frequency in order to fully use its growth potential. Mechanistic modelling is useful and an important complement in evaluation of metabolism kinetics in nutrient flux studies. Moreover, due to its mechanistic nature, the present model can be used with different AA tracers, and also for other fish species. [Copyright &y& Elsevier]

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 <0.5 kDa hydrolysates). At 15 days after hatching (DAH), proteome expression changes were assessed in entire larvae by two-dimensional gel electrophoresis and the quality of larval skeleton was analysed at 28 DAH through double staining of cartilage and bone. Dietary PH fractions tested affected growth, the larvae fed diet L being significantly larger than those fed diet H, but it did not affect the incidence of deformed larvae, nor the number of deformities per fish. Two-dimensional analysis of larvae proteome allowed the detection and the comparative quantification of a total of 709 protein spots having a pI between 4 and 7, around half of which had an expression significantly affected by dietary treatment, the main difference being between proteome of Control larvae with those of both groups L and H. From these spots, 52 proteins involved in diverse processes such as cytoskeletal dynamics, energetic, lipoprotein, amino acid (AA), and nucleotide metabolisms, protein chaperoning and degradation, and signal transduction, were identified. This study revealed that the molecular weight of the dietary protein hydrolysate fraction had a minor impact on skeletal deformities in white seabream larvae, but affected growth performance and had a strong impact on larvae whole body proteome. [ABSTRACT FROM AUTHOR]

One of the main gastrointestinal hormones, cholecystokinin (CCK), was studied in order to advance understanding of the control of the digestive process in Atlantic cod Gadus morhua larvae after onset of first feeding. Larvae were fed either natural zooplankton or enriched rotifers in similar rearing systems and sampled from hatching to 22 days post-hatch (dph). CCK was visualized by immunohistochemistry and the first CCK-immunoreactive (IR) cells were detected at 8 dph corresponding to 6 days after first feeding. The CCK-IR cells were mostly found in the anterior midgut, and the number of CCK-IR cells was lower in the posterior midgut. They were also present in the hindgut of some of the larvae, but not in the foregut. No clear differences were found in the ontogenetic appearance and the distribution pattern of CCK-IR cells between the two dietary treatments. This indicates that the onset of CCK production in the gut as well as the spatial distribution of the CCK-IR cells is not differentially affected by these diets. To what extent the hormone production itself is influenced by dietary factors needs to be studied by more sensitive methods. [ABSTRACT FROM AUTHOR]

Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel ( Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3′-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na+ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na+-independent, H+-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process ( Km,app ∼ 1.5 mmol L−1) and that within the 0.1–10 mmol L−1 peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H+/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay. [ABSTRACT FROM AUTHOR]

The current best practice intensive culture of larval Atlantic cod includes feeding rotifers from onset of exogenous feeding until 25–30 days after hatching. These larvae grow considerably slower and develop higher frequencies of deformities than larvae reared in semi-extensive systems, using copepods as feed. The present study compares the micronutrient concentrations in rotifers with those of copepods, with the aim of identifying nutrients that may be limiting for normal growth and development of cod larvae. An additional criterion used is the nutrient requirements given for fish in general, by NRC (1993) , as nutrient requirements of cod remains to be determined. Rotifers were fed on four different diets, consisting of baker's yeast with cod liver oil (3.3 : 1 dry weight (DW)/v), baker's yeast with Algamac 2000TM (3.5 : 1 DW), baker's yeast with live algae Chlorella (4.1 : 1 DW), and Culture Selco 3000TM (CS). CS was a complete commercial diet for rotifers while the other diets are considered as based on raw ingredients. Compared with copepod nutrient levels, rotifers grown on yeast-based diets supplemented with either cod liver oil, Algamac 2000 or Chlorella were apparently sufficient for covering the requirements in cod larvae for all the B-vitamins, except thiamine. Rotifers cultured on the CS diet also had sufficient amounts of thiamine. Of the minerals, only calcium and magnesium were sufficient, using this criterion while iron was on the borderline. However, with reference to the requirements given for larger fish ( NRC 1993 ), only thiamine, vitamin A, manganese, selenium and perhaps copper, appear too low in the rotifers cultured without extra micronutrient supplementation. The other nutrients were present at levels intermediate between copepod and fish requirement levels. This study suggests that it is necessary to develop enrichment techniques to produce rotifers with sufficient amounts of all micronutrients. Such techniques will also be important tools for determining which nutrients are present at levels below the actual requirements in cod larvae. [ABSTRACT FROM AUTHOR]

The digestion, absorption and post absorptive metabolism of a radiolabelled triacylglycerol (TAG; triolein) and a free fatty acid (FFA; oleic acid), delivered by tube feeding, was studied in herring Clupea harengus larvae, using metabolic chambers and video analysis. In general, a large amount of the delivered lipid was evacuated. Most of the evacuation occurred between 2 and 6 h after tube feeding although a group of larvae responded by rapidly evacuating the lipid (>50% before 2 h). The volume of the tube-fed lipid affected its utilization. A small volume of triolein (9·2 nl, representing c. 6% of gut filling capacity) resulted in a lower proportion of fast evacuating larvae and improved utilization (lower evacuation and higher absorption: body incorporation and catabolism) compared with 50·6 nl ( c. 17% of gut filling capacity). Increases in the volume of tube fed triolein enhanced only marginally label absorption and led to a steep rise in evacuation. At a comparable high volume (50·6 nl), oleic acid, which does not require digestion, was better absorbed and less evacuated than triolein. The video observation of the lipid digestive process revealed a considerable gut contractile activity that appeared effective in processing the tube fed lipid. Also, the gut wall seemed very sensitive to physical pressure. Signs of chemical degradation during lipid digestion were also noted. The metabolic studies, together with video image analysis, suggested that the limiting step for the utilization of high dietary lipid levels may have been the lipid absorption into the enterocytes and transport into the body, rather than lipid digestion. The results support the notion that the rate of lipid digestion and absorption in fishes is slower than that of mammals. [ABSTRACT FROM AUTHOR]

In order to assess the influence of dietary protein on digestive enzyme expression and cholecystokinin (CCK) content in sea bass larvae, four groups of larvae were fed experimental diets from mouth opening until day 42: three isonitrogenous diets with increasing protein hydrolysate levels (0%, 14% and 46% of crude matter) and one diet incorporating starch. The groups fed high starch or high protein hydrolysate level exhibited the lowest growth. The final weight in these groups was 9.5 and 5.6 mg, respectively, whereas it reached approximately 20.0 mg in the groups fed 0% or 14% protein hydrolysate level. The highest levels of trypsin secretion were observed in larvae fed the lowest protein hydrolysate level. Paradoxically, the groups fed diet containing starch also exhibited a high trypsin secretion level. There was a clear allometric relationship between larval CCK content and body mass, but there were also differences between dietary groups. On day 42, the CCK level in the group fed diet incorporating starch (40 fmol/mg dry weight of larvae) was more than twice as high as that found in the other groups. The lowest CCK level (13 fmol/mg) was found in the group fed the highest protein hydrolysate level. Our data suggested that dietary protein level and chain length combined with protein intraluminal proteolytic activity regulate the CCK level in fish larvae as in other vertebrates. The CCK concentration assayed in larvae fed diets with a low protein content or different protein hydrolysate levels is compatible with the existence of an indirect mechanism controlling CCK release and mediating pancreatic enzyme secretion. [Copyright &y& Elsevier]

In marine fish larvae, the sum of protein deposition, turnover and catabolism necessary for their rapid growth dictates a high amino acid (AA) requirement. Once the yolk is exhausted, the digestive tract becomes the vital organ that ensures a steady supply of dietary AA to the growing larval tissues. In this paper, we discuss the demand and availability of AA (free and polymerised pools) in relation to larval digestive capacity. The sources of AA from compound and live diets are described, and the early regulatory roles of cholecystokinin (CCK) and a retrograde peristaltic activity are highlighted. [Copyright &y& Elsevier]

The effect of acute changes of temperature and light on the rates of oxygen consumption and ammonia excretion of developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus ) reared at 15 °C were studied. Short-term temperature adjustments of ±2 °C gave a mean Q [sub 10] of 2.6 for the rate of oxygen consumption. Neither the presence nor the absence of light significantly influenced this response to acute temperature changes, despite light causing a significantly higher rate of oxygen consumption in the yolk-sac larvae between 9 and 12 days after fertilisation. This elevated metabolic rate in the presence of light occurred after pigmentation of the eyes and was probably due to activity associated with food-searching behaviour of this visual feeder. Similarly, for most development, the presence or absence of light and acute temperature adjustments did not cause any significant shift in the relative fraction of amino acids catabolised at 15 °C. However, on day 10 after fertilisation when peak rates of oxygen consumption occurred in the yolk-sac larvae exposed to light, the significantly elevated apparent nitrogen quotients indicated that greater amounts of amino acids were utilised for catabolic substrate oxidation. [ABSTRACT FROM AUTHOR]

Abstract Folate mobilization from the yolk compartment during larval development was studied by analysing the folate concentration in whole body, embryo and yolk in a single batch of Atlantic halibut, Hippoglossus hippoglossus L., eggs and larvae that showed successful fertilization and development. There was a net loss of approx. 50% of folate from yolk during endogenous feeding. Further, only 23% of the decrease in yolk folate was retained in the larval body. The data suggest a need for folate for metabolic and growth purposes during embryogenesis of approximately 2 μ g g-1 weight gain. Relative to these data and published folate requirement for cold-water species, batches of egg from 16 Atlantic halibut brood fish contained variable and, for some batches, critically low levels of folate. This may constitute a potential problem for larval development until start feeding. [ABSTRACT FROM AUTHOR]

At 7 days after first feeding (DAFF), the peptide hormone cholecystokinin (CCK) content (fmol individual−1) and the tryptic activity [μmol arginine-methyl-coumarinyl-7-amide (MCA) min−1 individual−1] per individual gut of Atlantic halibut Hippoglossus hippoglossus larvae were low: 0·2±0·1 and 0·14±0·10, respectively. Thereafter, both parameters increased with the increase in gut mass and reached 19·67±5·58 and 2·71±0·64 at 26 DAFF, respectively. Due to the small sample size, the dry mass (MG, mg) of the individual gut could not be determined accurately at 7 DAFF. At 13 DAFFMG represented 5·5% of whole body dry mass (MW, mg) while at 26 DAFF it had increased to 23%. The mass specific tryptic activity [μmol MCA min−1 per mg dry mass (M)] in the gut increased from 2·74±1·98 at 13 DAFF to 5·00±0·78 at 26 DAFF. There was more individual variation in the mass specific CCK content (fmol M−1) but no significant differences were found, although the data indicated an increase (from 23·38±11·26 at 13 DAFF to 36·27±8·96 fmol M−1 at 26 DAFF). At 7 DAFF the CCK content of the gut represented c. 2% of the whole body CCK content while it increased to c. 62% of the whole body CCK content at 26 DAFF. This demonstrates that it is necessary to separate neural and gastrointestinal sources of CCK in order to determine its alimentary role in fish larvae. Trypsin activity was only found in the gut compartment. In larvae aged 45 DAFF dietary proteins delivery into the gut by tube-feeding appeared to stimulate post-prandial secretion of CCK from the gut as well as stimulate pancreatic trypsin secretion, suggesting that both factors contribute to protein digestion. Copyright 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. [Copyright &y& Elsevier]

Metamorphosis in flatfish (Pleuronectiformes) is a late post-embryonic developmental event that prepares the organism for the larval-to-juvenile transition. Thyroid hormones (THs) play a central role in flatfish metamorphosis and the basic elements that constitute the thyroid axis in vertebrates are all present at this stage. The advantage of using flatfish to study the larval-to-juvenile transition is the profound change in external morphology that accompanies metamorphosis making it easy to track progression to climax. This important lifecycle transition is underpinned by molecular, cellular, structural and functional modifications of organs and tissues that prepare larvae for a successful transition to the adult habitat and lifestyle. Understanding the role of THs in the maturation of organs and tissues with diverse functions during metamorphosis is a major challenge. The change in diet that accompanies the transition from a pelagic larvae to a benthic juvenile in flatfish is associated with structural and functional modifications in the gastrointestinal tract (GI-tract). The present review will focus on the maturation of the GI-tract during metamorphosis giving particular attention to organogenesis of the stomach a TH triggered event. Gene transcripts and biological processes that are associated with GI-tract maturation during Atlantic halibut metamorphosis are identified. Gene ontology analysis reveals core biological functions and putative TH-responsive genes that underpin TH-driven metamorphosis of the GI-tract in Atlantic halibut. Deciphering the specific role remains a challenge. Recent advances in characterizing the molecular, structural and functional modifications that accompany the appearance of a functional stomach in Atlantic halibut are considered and future research challenges identified. [ABSTRACT FROM AUTHOR]

This paper reviews methodologies and recent findings in the study of the amino acid (AA) metabolism of fish larvae and post-larvae, in order to better understand the AA requirements. The larval indispensable AA (IAA) profile can be used as index of the IAA requirements. When turbot larvae and live food IAA profiles are compared, the profile of the latter seems to be deficient in some IAA. However, the larval IAA profile is only a rough indicator of AA requirements. A more precise estimate of the ideal dietary IAA profile implies the knowledge of the relative bioavailabilities of the individual AA, in particular, eventual differential rates of absorption and catabolism. Metabolic budgets (including unabsorbed AA, AA oxidation and AA retention) can be estimated using an in vivo method based on controlled tube-feeding of AA mixes containing a 14C-labelled AA. Results with fasted post-larval Senegal sole (Solea senegalensis) and fasted herring (Clupea harengus) larvae show a high retention of labelled doses of IAA (>60%) in the body, compared to catabolism as measured by liberated 14CO2 (<25%). In contrast, dispensable AA (DAA) show a higher catabolism (>40%) and a lower retention (<57%). So, from the onset of exogenous feeding, fish larvae have high catabolic losses of AA, but use DAA preferentially to IAA as energy substrates. A new method combining the use of 13C-labelled live food and 13C-NMR spectroscopy can be used to study simultaneously the relative bioavailability of several individual AA in fish larvae. In larval gilthead seabream (Sparus aurata) fed on rotifers, relative bioavailabilities (a combined measure of absorption efficiency and rate of catabolism) vary between AA being high for aspartate, glutamate and lysine and low for threonine. These estimates of relative biovailability of individual AA together with the IAA profiles of the larval seabream indicate that rotifers are deficient in threonine and leucine for larval seabream, threonine being the first limiting AA for protein synthesis. In order to define ideal IAA profiles for larval fish, further studies are needed on the factors affecting the relative bioavailability of IAA, such as species, age, developmental stage, temperature and the dietary nitrogen molecular form(s). Estimates of relative bioavailability of individual AA together with the IAA profile of the larval protein allow to determine the ideal dietary IAA profile for a given species. [Copyright &y& Elsevier]

Abstract The present study is focused to elucidate the main characteristics of the digestive function of this carnivorous fast-growing fish living at high temperatures. With this aim, we have examined the effects of an increased temperature from 30 to 34 °C on the daily pattern of gastrointestinal pH, enzymatic proteolytic digestive activity and the feed transit time in early juveniles of cobia (Rachycentron canadum), a species living in tropical and subtropical waters with an increasing aquaculture production. Fish were fed two meals a day. Gastric luminal pH was permanently acidic (mean pH values: 2.76–4.74) while the intestinal pH increased from neutral/slightly acidic to slightly alkaline when the digesta was present, with an increasing alkalinity from proximal to distal intestine (mean pH values: 6.05 to 7.69). The temperature did not affect the gastric pH but a slightly higher acidity was induced in the intestine at 34 °C. Pepsin activity showed a daily rhythm at 30 °C with maximum in the middle of the light period, while at 34 °C some hourly changes coinciding with feed adding without a clear daily trend during the 24-h period were observed. The trypsin activity exhibited a daily rhythm at both temperatures with an increase after morning feeding to reach a maximum several hours later. Average pepsin activity during the daily cycle was slightly higher at 34 °C (6.1 and 7.3 U mg−1 BW at 30 and 34 °C respectively), but values were significantly different only at 8 and 24 h after the morning meal. Similarly, the trypsin activity was significantly affected by the temperature only at 8 and 16 h after the morning meal, but daily activity averages were similar (1.20 and 1.29 U g−1 BW at 30 and 34 °C respectively). The partial transit rates of the first meal in the stomach for each period inter-samplings were higher during the first 4-h period and decreased progressively along the rest of the 24-h cycle at both temperatures, but no significant differences were detected at 30 °C. In addition, the transit was notably faster at 34 °C particularly during the first 8 h after feeding, with rates between 100 and 65% of total volume displaced (intake or released) during each 4-h period. In the intestine the transit rate was relatively constant and similar at both temperatures during 12 h after feeding. Then the rates remained very low during the following 12 h. Residence time of the first meal was longer at 30 than at 34 °C, particularly in the stomach (12 h:02 min vs 4 h:54 min respectively). In the intestine the difference was not so large (8 h:18 min vs 6 h:24 min respectively). In a parallel study under same conditions, cobia reared at 30 °C grew faster and showed a more favorable feed conversion ratio than those at elevated temperature (34 °C). The present results indicate that at 34 °C, a subtle increase of proteolytic activity cannot compensate for the faster gut transit rate. Therefore, 30 °C is more appropriate temperature for the early on-growing of cobia because at higher temperatures the digestion efficiency decrease being one of the causes for a lower growth. Highlights • Cobia exhibits a permanent gastric acidification. • Water temperature (30 and 34 °C) does not substantially affect the digestive proteolytic activities. • Both stomach and intestine are filled almost simultaneously. • Transit time was much faster and the residence time lower at 34 °C than at 30 °C. [ABSTRACT FROM AUTHOR]

Leucine has been identified to modulate feed intake and energy homeostasis in fish as in other vertebrates. Under allostatic conditions, energy expenditure may change, and adjustments to the processes that govern the energy homeostatic system may be necessary. We investigated the responsiveness of appetite-related neuropeptides involved in feed intake regulation in Atlantic salmon (Salmo salar) reared with high (35 g/kg leucine) or control (27.3 g/kg leucine) leucine-supplemented diets and/or under chronic stressor conditions (chasing) for eight weeks. We also analysed the response of amino acid transporters potentially involved in uptake of branched-chain amino acids (BCAA), including leucine, into areas of the brain where nutrient sensors may signal locally or to other areas involved in appetite control. At the end of the experiment, all fish were subjected to a novel-acute stressor (confinement). Our results show that fish fed with high leucine diet had a lower feed intake, growth, and hepatosomatic index (HSI) when compared to fish fed control leucine diet. In addition, increased mRNA expression of amino acid solute carrier (slc) genes in the diencephalon, and genes related to appetite control, such as proopiomelanocortin a1 (pomca1), in both the diencephalon and telencephalon, imply their involvement in leucine anorectic effect. Stress, as high leucine, reduced feed intake, growth and HSI of fish fed control or high leucine diet and antagonized the high leucine effect on the slc genes mRNA expression. An increase of neuropeptide y a1 (npya1) was observed both due to high dietary leucine and/or stress treatment which may represent a compensatory regulatory mechanism with the aim to reverse the decrease in feed intake. In summary, our results confirm an anorectic role of high dietary leucine via the activation of amino acid sensing mechanisms in the brain. Further, corticotropin-releasing hormone 1 b1 (crh1b1) and npya1 showed to play a role in the regulation of appetite in Atlantic salmon under stress conditions and/or high leucine levels. • High dietary leucine acts as anorectic factor in the control of energy balance in Atlantic salmon. • Both slc transporters and pomca1 neuropeptide are involved in the leucine anorexigenic control. • The same mechanisms do not seem to be responsible for such disruption under stressed conditions. • Instead, crf1b1 and npya1 play a role in the regulation of appetite under stressful conditions. [ABSTRACT FROM AUTHOR]

Sexual precocity in farmed European seabass is correlated to reduced growth and flesh quality, and is one of the major challenges for the industry. We evaluated the effects of one marine (M) and one plant (P) oil mix, supplemented at 2 dietary inclusion levels, low (LF) and high (HF), on sexual maturation of European sea bass males. The feeding experiment was conducted on maturing unsexed fish kept in indoor tanks at 10:14 LD photoperiod and reducing water temperatures from October to December. Fish performance of all the fish population, males and females, testis histology, blood hormone levels and gene expression of selected sexual maturation related hormones in the brain and testis of male fish were studied. European seabass males fed the marine oil diets had higher mRNA expressions levels of Cyp 11b, Cyp 19a and Kiss2 in both brain and testis tissues compared with male fish fed plant oil diets. Moreover, high expression of brain Kiss receptor gene (Kissr4) was found in males fed a plant oil diet suggesting a late recrudescence phase of maturation (stages III & IV) in these groups. Histological analyses of testis were in agreement with the hormone gene expression observations where male fish fed with plant oil diets were in early to mid-maturing phase (stages III-IV), while marine oil fed males were at mid to late maturing phase (stages IV-V). No significant effects were observed between the low or high dietary fat treatments. The present results suggest that supplementing diets with plant oils delays maturation in male European seabass. [ABSTRACT FROM AUTHOR]

Abstract: Following feeding of a single meal to Atlantic salmon, the temporal changes in the brain mRNA expression of neuropeptide y (npy), cocaine-amphetamine regulated transcript (cart), peptide yy (pyy), two isoforms of agouti-related protein (agrp), two isoforms of cholecystokinin (cck), and four isoforms of proopiomelanocortin (pomc) were assessed by q-PCR. In the course of 24h post-feeding (hpf), several of the brain neuropeptides displayed changes in mRNA expression compared to an unfed control group, indicating that food intake and processing affect the regulation of expression of these genes in Atlantic salmon. Expression of cart, cck-l, pomc-a1 and pomc-b all increased within 3h of feeding, while most of the feed was still in the stomach, suggesting that these neuropeptides play central anorexigenic roles similar to those described in higher vertebrates, including determining meal intervals. On the other hand, the npy and agrp isoforms which have been described as playing orexigenic roles in mammals, showed an opposite response in salmon and both were elevated in the first 3h after feeding. The different isoforms of cck, agrp and pomc had different mRNA expression patterns, which indicate specific roles related to feeding regulation. The minimal effect of feeding and digestion on pyy expression in the brain indicates that PYY plays a minor role in the central control of short-term food intake in Atlantic salmon. [Copyright &y& Elsevier]

Abstract: The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract. [Copyright &y& Elsevier]

Considering that amino acids constitute an important energy fuel during early life of the Atlantic halibut (Hippoglossus hippoglossus L.), it is of interest to understand how the nitrogenous end products are handled. In this study we focused on the kinetics and fates of ammonia, urea and uric acid. The results showed that ammonia (TAmm: NH3+NH4+), and urea–N contents increased during final oocyte maturation. Urea–N excretion dominated the total nitrogenous end product formation in early embryos. Later, yolk TAmm levels increased in embryos and ammonia excretion was low. In the last part of the embryonic stage TAmm accumulation dominated, and was apparently due to yolk storage. Around hatching, the larval body tissues (larva with yolk-sac removed) accounted for 68% of whole animal urea–N accumulation, while TAmm levels increased predominately by yolk accumulation. Afterwards, ammonia excretion dominated and uric acid accumulation accounted for less than 1%. Urea, synthesised either through the ornithine–urea cycle, argininolysis or uricolysis, accounted for approximately 8% of total nitrogenous end product formation in yolk-sac larvae. The results suggested that a sequence occurred regarding which nitrogenous end products dominated and how they were handled. Urea excretion dominated in early embryos (<7 dPF), followed by yolk ammonia accumulation (7–12 dPF), and finally, ammonia excretion dominated in later embryonic and yolk-sac larval stages (>12 dPF). [Copyright &y& Elsevier]

Warm water treatment, i.e. exposure to sea water at a temperature of 28–34 °C for 20–30 s, has in recent years been widely used for delousing of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Norwegian aquaculture. High mortality and various lesions (e.g. injuries and/or bleedings in skin, fins, eyes, brain, and gills) have, however, been reported after industrial warm water treatments. The objective of this study was to reveal whether the thermal component of warm water treatment inflicts acute lesions on Atlantic salmon. The study was conducted by exposing individual, sedated Atlantic salmon post-smolts (w ¯ = 1117 ± 250 g) to sea water at a temperature of 34 °C (warm water treatment, n = 40) or 9 °C (control treatment, n = 20) for 30 s, and subsequently conducting welfare indicator scoring and histopathological examination of their skin, fins, eyes, snout, nasal pits/mucosa, palate, gills, thymus, pseudobranch, brain, heart, liver, kidney, pyloric caeca, pancreas, and spleen. The results showed that the prevalence and severity of acute lesions were not significantly different between the two treatment groups, except for higher prevalence of injuries on the caudal (p = 0.002), dorsal (p = 0.002), and right pelvic fins (p = 0.014) in the warm water treatment group. The main cause of these fin injuries may have been a strong behavioural reaction displayed by the fish when exposed to warm water. Possible consequences of fin injuries, the use of anaesthetic, and statistical limitations were discussed. It was concluded that exposure of Atlantic salmon to sea water at a temperature of 34 °C for 30 s did not lead to any statistically significant change in the prevalence of acute lesions except an increase in minor, possibly behaviour-related, fin injuries. Detection of a lower lesion prevalence than was possible in this study, but which may concern many individuals in an industrial setting, requires examination of a larger number of fish. • Warm water treatment has in recent years been widely used for delousing of Atlantic salmon and rainbow trout in Norwegian aquaculture. • The objective of this study was to reveal whether the thermal component of warm water treatment inflicts acute lesions on Atlantic salmon. • Exposure of sedated Atlantic salmon post-smolts to sea water at a temperature of 34 °C for 30 did not lead to any statistically significant change in the prevalence of acute lesions except an increase in minor fin injuries. • The main cause of these fin injuries may have been a strong behavioural reaction displayed by the fish when exposed to warm water. [ABSTRACT FROM AUTHOR]

In teleosts, two PepT1-type (Slc15a1) transporters, i.e., PepT1a and PepT1b, are expressed at the intestinal level. They translocate charged di/tripeptides with different efficiency, which depends on the position of the charged amino acid in the peptide and the external pH. The relation between the position of the charged amino acid and the capability of transporting the dipeptide was investigated in the zebrafish and Atlantic salmon PepT1-type transporters. Using selected charged (at physiological pH) dipeptides: i.e., the negatively charged Asp-Gly and Gly-Asp, and the positively charged Lys-Gly and Gly-Lys and Lys-Met and Met-Lys, transport currents and kinetic parameters were collected. The neutral dipeptide Gly-Gln was used as a reference substrate. Atlantic salmon PepT1a and PepT1b transport currents were similar in the presence of Asp-Gly and Gly-Asp, while zebrafish PepT1a elicited currents strongly dependent on the position of Asp in the dipeptide and zebrafish PepT1b elicited small transport currents. For Lys- and Met-containing dipeptides smaller currents compared to Gly-Gln were observed in PepT1a-type transporters. In general, for zebrafish PepT1a the currents elicited by all tested substrates slightly increased with membrane potential and pH. For Atlantic salmon PepT1a, the transport current increased with negative potential but only in the presence of Met-containing dipeptides and in a pH-dependent way. Conversely, large currents were shown for PepT1b for all tested substrates but Gly-Lys in Atlantic salmon. This shows that in Atlantic salmon PepT1b for Lys-containing substrates the position of the charged dipeptides carrying the Lys residue defines the current amplitudes, with larger currents observed for Lys in the N-terminal position. Our results add information on the ability of PepT1 to transport charged amino acids and show species-specificity in the kinetic behavior of PepT1-type proteins. They also suggest the importance of the proximity of the substrate binding site of residues such as Lys PepT1a /Gln PepT1b for recognition and specificity of the charged dipeptide and point out the role of the comparative approach that exploits the natural protein variants to understand the structure and functions of membrane transporters., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vacca, Gomes, De Gennaro, Rønnestad, Bossi and Verri.)

Taurine is a sulphur-containing amino acid with important physiological roles and a key compound for the synthesis of bile salts, which are essential for the emulsion and absorption of dietary lipids. This study aimed to evaluate the effects of taurine supplementation to low-fishmeal diets on the metabolism of taurine, bile acids, and lipids of Senegalese sole. A fishmeal (FM) and a plant-protein-based (PP0) diet were formulated, and the latter was supplemented with taurine at 0.5 and 1.5% (diets PP0.5 and PP1.5). Diets were assigned to triplicate tanks containing 35 fish (initial weight ~14 g) for 6 weeks. Fish from the PP0 treatment presented lower taurine and bile-acid concentrations compared with the FM treatment, and a downregulation of cyp7a1 and abcb11 was observed. Triolein catabolism decreased in PP0-fed fish, resulting in increased hepatic fat content and plasma triglycerides, while no effects on plasma cholesterol were observed. Taurine supplementation to plant-based diets resulted in a higher taurine accumulation in fish tissues, increased bile-acid concentration, and upregulation of cyp7a1 and abcb11 . Hepatic fat content and plasma triglycerides decreased with increasing dietary taurine supplementation. Taurine supplementation mitigated part of the negative effects of plant-based diets, leading to better lipid utilisation.

During the first feeding period, fish will adapt to exogenous feeding as their endogenous source of nutrients is depleted. This requires the development of a functional physiological system to control active search for food, appetite, and food intake. The Atlantic salmon ( Salmo salar ) melanocortin system, a key player in appetite control, includes neuronal circuits expressing neuropeptide y ( npya ), agouti-related peptide ( agrp1 ), cocaine- and amphetamine-regulated transcript ( cart ), and proopiomelanocortin ( pomca ). Little is known about the ontogeny and function of the melanocortin system during early developmental stages. Atlantic salmon [0-730 day degrees (dd)] were reared under three different light conditions (DD, continuous darkness; LD, 14:10 Light: Dark; LL, continuous light) before the light was switched to LD and the fish fed twice a day. We examined the effects of different light conditions (DD LD , LD LD , and LL LD ) on salmon growth, yolk utilization, and periprandial responses of the neuropeptides npya1, npya2, agrp1, cart2a, cart2b, cart4, pomca1 , and pomca2 . Fish were collected 1 week (alevins, 830 dd, still containing yolk sac) and 3 weeks (fry, 991 dd, yolk sac fully consumed) into the first feeding period and sampled before (-1 h) and after (0.5, 1.5, 3, and 6 h) the first meal of the day. Atlantic salmon reared under DD LD , LD LD , and LL LD had similar standard lengths and myotome heights at the onset of first feeding. However, salmon kept under a constant light condition during endogenous feeding (DD LD and LL LD ) had less yolk at first feeding. At 830 dd none of the neuropeptides analyzed displayed a periprandial response. But 2 weeks later, and with no yolk remaining, significant periprandial changes were observed for npya1, pomca1 , and pomca2 , but only in the LD LD fish. This suggests that these key neuropeptides serve an important role in controlling feeding once Atlantic salmon need to rely entirely on active search and ingestion of exogenous food. Moreover, light conditions during early development did not affect the size of salmon at first feeding but did affect the mRNA levels of npya1, pomca1 , and pomca2 in the brain indicating that mimicking natural light conditions (LD LD ) better stimulates appetite control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Norland, Gomes, Rønnestad, Helvik and Eilertsen.)