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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 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]

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: 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]