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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 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]

Abstract: For the majority of marine fish larvae a fully developed digestive tract, including gastric digestion, is acquired weeks to months (depending on species) after onset of exogenous feeding. Still, the processing capacity (capability to degrade and absorb dietary nutrients) of the larval gut is sufficient to support fast larval growth by digesting prey naturally available in the sea. However, the physiological constraints of the gut with respect to digestion of cultivated live prey and particularly formulated starter feeds still remain to be elucidated. This paper reviews some recent findings in the areas of control and efficiency of digestive function of marine fish larvae. For studies on the hormonal control, the peptide hormone cholecystokinin (CCK) has been targeted, since it is believed to play an important role in controlling digestion in vertebrates. Recent work on the processing capacity include studies of the digestibility and transfer kinetics of macronutrients from live prey and experimental work on absorption of protein, peptides and free amino acids from the lumen of the digestive tract into the tissues of larval fish and how this changes during ontogeny. The molecular and in vitro characterization of transporters is currently being integrated with ongoing in vivo studies. [Copyright &y& Elsevier]

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 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]

In studies of marine larvae, it is common to use days post-hatch as a developmental reference point. We show that age is a poor measure of morphological and physiological development in Atlantic cod. Therefore, we propose a set of five developmental stages of Atlantic cod from start-feeding until the juvenile stage, based on cranial ossification as previously done in Atlantic halibut. Cod follows a sequence of cranial ossification that is to a large extent preserved in most fish species examined. These stages are therefore tools to standardize sampling and to reduce growth dependent variation in the analysis of larvae during development. We show that several developmental stages are present in the same rearing unit at a given time. We also demonstrate that nutrition during early development is a vital foundation for robust skeletal development. Cod larvae supplied with copepods instead of rotifers followed by Artemia , develop less skeletal deformities at 10 cm standard length, despite given the same formulated feed from 1.8 cm standard length and onwards. Statement of relevance This paper provides developmental stages that are vital for best practice protocols in aquaculture. By relating farming practices to developmental stages and not age, the right treatment ect may be provided. This manuscript does also highlight the importance of nutrition during live feed stages on events that may occur late in the production cycle. [ABSTRACT FROM AUTHOR]

Abstract: In vitro methods have previously been utilised for the rapid and reliable evaluation of protein digestibility in fish. In this study we used in vitro methods to compare the digestibility of various live and artificial larval feeds and feed ingredients. Given previous suggestions that water-soluble dietary proteins are efficiently digested and utilised by stomachless fish larvae, we also analysed the content of water-soluble nitrogen in the feeds and feed ingredients and then measured the specific in vitro digestibility (simulated midgut conditions) of the water-soluble and insoluble fractions. The soluble nitrogen fractions were generally more digestible than the insoluble nitrogen fractions (P <0.05). A soluble reference protein (Na+-caseinate) was digested faster than the similar but insoluble reference protein (casein) although their final digestibility was the same (94%). Frozen live feeds (Artemia fransiscana and Calanus finmarchicus) contained high fractions of soluble nitrogen (54–67%) and also had high digestibility in vitro (84 and 87%, respectively). The in vitro digestibility of two formulated larval feeds tested was lower (53 and 70%) than the frozen live feeds. The digestibility of the ingredients of the protein-encapsulated feeds particles was reduced as a result of the production process (from 71 to 53%, respectively). Three meals of marine origin (fish meal, squid meal and fish roe meal) all had low contents of water-soluble nitrogen (11–17%) but showed different degrees of digestibility (77, 77 and 49%, respectively). The results also demonstrated that while pre-hydrolysis of a feed ingredient (fresh frozen cod fillet) almost doubled the water-soluble nitrogen fraction (from 34 to 65%) the positive effect of pre-hydrolysis on in vitro digestibility was much lower (from 80 to 86%). This demonstrates the complexity of assessment of bioavailability of dietary protein sources in larval fish; a number of factors such as leaching rates from feed particles, digestibility, digestion rates and absorption rates all need to be taken into account. [Copyright &y& Elsevier]