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The study aims to test whether predigested dietary protein enhances the utilization of formulated diets at weaning, and also whether it stimulates intestinal maturation. In this study, Atlantic cod [ Gadus morhua L.; 41 days posthatch (dph)] and Atlantic halibut [ Hippoglossus hippoglossus L.; 63 days postfirst feeding (dpff)] were weaned onto diets with graded levels of protein hydrolysate. By increasing the inclusion of dietary protein as hydrolysate from 0 to 400 g kg−1, cod increased the rate of survival from 7 ± 1% to 18 ± 2% (82 dph; regression, P = 4*10−7). In halibut, the survival rate decreased from 57 ± 9% to 22 ± 7% as the inclusion of protein in the form of hydrolysate increased from 0 to 450 g kg−1 (119 dpff; regression, P = 8*10−5). Growth was not affected in any of the species. Results in specific activities of the intestinal enzymes leucine aminopeptidase (LAP) and alkaline phospatase (AP) supported the results in survival in halibut and partly also in cod, by showing increased activities in groups with increased survival (anova, P < 0.05). The lower optimal level of hydrolysed protein in halibut than in cod is suggested mainly because of a slower feeding practice in halibut, which allows more extensive nutrient leaching before ingestion. [ABSTRACT FROM AUTHOR]

Atlantic halibut ( Hippoglossus hippoglossus) achieve a mature gastrointestinal tract approximately 2 months after first feeding (12 °C). The immature digestion may be the reason that compound diets fail to sustain growth and survival in first feeding halibut larvae and in larvae of other marine fish species. On the other hand, larvae fed with live feeds are capable of extraction of sufficient quantities of nutrients to sustain high growth rates. A lower availability of the protein in formulated diets compared with live prey is considered to be an important reason for the low performance of formulated diets. One approach to increase dietary protein availability is supplementation of pre-digested proteins. Experiments using tube fed individual larvae show that halibut larvae are able to utilize hydrolysed protein more efficiently than intact protein. However, Atlantic halibut in culture did not respond well to dietary supplementation of hydrolysed protein, in contrast to some other species. One reason may be extensive leaching of pre-hydrolysed proteins from the microparticulate feed. Atlantic halibut are slow feeders and may thus suffer more from nutrient leaching than species eating more rapidly. Feed formulation techniques affect dietary protein leaching, and in this paper, different techniques and their impact on feed properties are described. Microbound diets are most widely used in larval rearing, but show high rates of nutrient leaching. Lipid-based capsules seem to have the best potential to prevent leaching, however, they are not able to deliver a complete diet. The high need for improvements in larval feed formulation techniques are clearly stated, and some suggestions are given. Among these are production of complex particles, where small lipid-based capsules or liposomes containing the low molecular weight water-soluble nutrients are embedded. In such feed particles the water-soluble molecules are protected from leaching. Techniques for delivery of water-soluble nutrients that are needed in large quantities, i.e. free amino acids or hydrolysed and water-soluble protein, remain to be developed. [ABSTRACT FROM AUTHOR]

Atlantic halibut juveniles, which have been fed Artemia during larval development, frequently demonstrate malpigmentation and impaired eye migration. This is in contrast to the high percentage of normally developed larvae fed copepods, reared under similar conditions. Nutrition is therefore an important component influencing larval development. Analyses of the nutrient composition of Artemia and copepods show that Atlantic halibut larvae fed Artemia probably receive sufficient amounts of vitamin A by converting canthaxanthin, while iodine may be deficient, possibly leading to interrupted thyroid hormone synthesis. An unbalanced fatty acid composition, such as high levels of arachidonic acid and low levels of docosahexaenoic acid, can be another limiting factor in Artemia. Vitamin A, fatty acids and thyroid hormones have all been shown to affect pigmentation in flatfish. They are ligands to nuclear receptors, thyroid hormone receptors, retinoic acid receptors, retinoic X receptors and peroxisomal proliferator-activated receptors, which are members of the superfamily of steroid hormone receptors. The receptors interact with each other to promote gene expression that modulates proliferation and differentiation of cells. Our hypothesis is that these interactions are important for development during flatfish metamorphosis. Very little data exist on the topic of impaired eye migration. However, energy limitation, iodine deficiency and an unbalanced fatty acid composition have been proposed as possible explanations. Here, we review the literature on development of pigment cells and the possible mechanisms behind the effects of vitamin A, fatty acids and thyroid hormone on pigmentation and eye migration during development of Atlantic halibut larvae. [ABSTRACT FROM AUTHOR]

Flatfish metamorphosis is initiated by the actions of thyroid hormones (TH) and iodine is an essential part of these hormones. Hence, an iodine deficiency may lead to insufficient levels of TH and incomplete metamorphosis. In this study, different iodine sources for enrichment of Artemia were evaluated and the levels of iodine obtained in Artemia were within the range of 60–350 μg g−1 found in copepods. Larval Atlantic halibut was fed Artemia enriched with either normal DC-DHA Selco or DC-DHA Selco (commercial enrichments) supplemented with iodine from days 9 to 60 postfirst feeding. There was no significant difference in growth, mortality or metamorphic development between the groups. The analyses showed that we were able to enrich Artemia with iodine. Further, the larvae-fed iodine-enriched Artemia had higher whole body iodine concentration compared to larvae-fed Artemia without iodine enrichment. [ABSTRACT FROM AUTHOR]

Atlantic halibut ( Hippoglossus hippoglossus L.) larvae were fed enriched Artemia or zooplankton in duplicate tanks from 0 to 60 days after first-feeding. Both diets and the larvae were analysed for vitamin A (VA) in order to confirm earlier findings, in which Artemia fed larvae had lower levels of VA compared with larvae fed zooplankton. Furthermore, we wanted to investigate the composition of the retinoids in the larvae. The results showed that Artemia and zooplankton contains low levels of VA, probably too low to sustain the assumed requirement. Nevertheless, larvae fed Artemia had the same level of retinal and retinol as larvae fed zooplankton. We found a significant lower level of retinyl esters in larvae fed Artemia. The total VA level was lower in larvae fed Artemia only at the end of the feeding trial after the onset of metamorphosis. Our conclusion is that feeding Artemia to Atlantic halibut larvae is not likely to cause VA deficiency. [ABSTRACT FROM AUTHOR]

Atlantic halibut larvae were fed docosohexanoic acid- (DHA) selco enriched Artemia (RH-cysts) or wild zooplankton in duplicate tanks from first-feeding and 60 days onward. The zooplankton were collected from a fertilized sea water pond and consisted mainly of different stages of Eurytemora affinis and Centropages hamatus . There were no differences in survival, or in growth during the first 45 days of feeding, between larvae fed the two prey items, but the larvae fed Artemia showed much higher incidence of malpigmentation and impaired eye migration than larvae fed zooplankton. The prey organisms contained similar amounts of dry matter and protein, but Artemia was higher in lipid and glycogen than the zooplankton. Larvae fed Artemia were higher in both glycogen and lipid than the zooplankton-fed larvae towards the end of the feeding period. There were large differences between the prey organisms in the concentrations of essential fatty acids (% of total fatty acids) which was reflected in the fatty acid composition of the larval body. It is concluded that the macronutrient composition of Artemia in the present study was probably within the optimal range for promotion of growth and survival in young Atlantic halibut. The concentration of n-3 HUFA, and especially DHA, is however, very much lower in enriched Artemia than in copepods, and may be one of the factors triggering developmental errors in Atlantic halibut. [ABSTRACT FROM AUTHOR]

Abstract In this study Atlantic halibut (Hippoglossus hippoglossus L.) larvae (0.12 ± 0.04 g) were, from day 40 post first feeding, offered six diets in which 10% or 30% of the dietary protein was hydrolysed with (a) pepsin (P), (b) pepsin + trypsin (PT) or (c) pepsin + trypsin + chymotrypsin (PTC). In addition, a diet without hydrolysed protein was offered, and enriched Artemia was fed as control. The amount of soluble protein increased progressively with the enzyme treatments P, PT and PTC and with higher inclusion levels of hydrolysed protein. Survival was highest among the larvae offered Artemia (83 ± 0%) or the diet 10P (10% pepsin hydrolysed protein; 67 ± 4%). The diet 10P supported survival significantly better than the more hydrolysed diets 10PTC, 30P, 30PT and 30PTC, but not significantly better than the non-hydrolysed diet and 10PT. Specific growth rate (SGR) was 1.76 ± 0.20 in average for all groups of larvae and was not significantly affected by the diets. Still, the larvae offered pepsin hydrolysed diets tended to have better growth (2.10 ± 0.05 SGR; P < 0.06) than the larvae offered the other hydrolysed diets. The larvae offered the formulated diets did not differ in chemical composition. [ABSTRACT FROM AUTHOR]

Abstract Small raceways were used in a weaning experiment with Atlantic halibut (Hippoglossus hippoglossus L.) larvae. The size of the tanks was 1.0 × 0.4 m with a 1- to 2-cm water level. Duplicate larval groups were transferred to the raceways from circular first feeding tanks at 0.07, 0.10 and 0.16 g wet weight, while recommended weaning size of this species is 0.2–0.3 g. During the first 7 days of weaning, Artemia was used as a food supplement in combination with the formulated dry feed. Thereafter only dry feed was used. The dry feed used in this experiment was produced by a special heat technique. The 0.07, 0.10 and 0.16 g larval groups were evaluated after 31, 25 and 17 days respectively (same date) The corresponding average survival was 81.4%, 78.0% and 96.6% and the specific daily growth rate was 3.18%, 3.17% and 2.38% respectively. In the Artemia control group, a survival rate of 96.0% and a growth rate of 5.28% was achieved. To evaluate the weaning success, the groups were followed in a 22-day post-weaning period on a commercial dry diet. Higher growth rates, 5.8–6.9%, were then obtained in all experimental groups, except control. The survival here averaged approximately 80% in the three experimental groups, but showed some differences between replicates. One hundred per cent survival was achieved during weaning in the former Artemia group. From the start of weaning to the end of the post-weaning period, the survival rates averaged 64% for the 0.07 and 0.10 g groups, approximately 80% for the 0.16 g group and 96% in the Artemia control group. Higher variance (CV) through the experiment and highest growth of the 75% quartiles of the fish groups compared with the 50% and 25%, indicated suppressed growth of the smaller fish. The successful weaning at these small sizes considerably reduced the live food period. Based on the present knowledge of the energetic demands of this species, it is calculated that weaning at 0.07 g compared with 0.25 g will reduce the amount of Artemia needed by at least 60%. No differences in pigmentation or degree of completed eye migration were detected between groups, indicating that this is determined at earlier developmental stages. [ABSTRACT FROM AUTHOR]

Abstract: To investigate if digestion is limiting for absorption of dietary neutral lipids in Atlantic halibut (Hippoglossus hippoglossus, L.) larvae, absorption of triacylglycerol (TAG), diacylglycerol, (DAG), and monoacylglycerol, (MAG) as well as phosphatidylcholine (PC) was studied by tube-feeding larvae with radio-labelled lipids and tracing the radioactivity. PC was included to evaluate any difference in absorption of neutral versus polar lipids. The labelled lipids where deposited by tube-feeding before larvae were incubated individually for 18 h with collection of expired CO2. At sampling, the gut was separated from the body carcass and radioactivity traced in four different compartments, namely body carcass, gut, incubation water and CO2 expired from larvae. The relative (%) isotope distribution of each compartment was calculated and the dose size effect analysed by regression analysis. The results show that there is an increasing amount of evacuated unabsorbed neutral lipids as the complexity of the lipid increases (TAG>DAG>MAG). The larval faecal evacuation ranged from 66±20% of TAG to 9±6% of MAG. DAG was intermediate with 52±21% evacuated. Of the labelled PC, 37±16% was absorbed, but this can not be directly compared to the neutral lipids due to the different digestive enzymes that specifically hydrolyse neutral and polar lipids. Increasing the administered amount of lipids only slightly increased the total amount of labelled TAG and DAG that were actually absorbed, while there was a linear correlation between fed and absorbed MAG. The absorption of PC was also linearly related to the administered amount. The difference in net absorption of labelled TAG, DAG and MAG diets indicates that digestion is a limiting factor for absorption of neutral lipids in Atlantic halibut larvae. [Copyright &y& Elsevier]

Abstract: To determine the status of development of the intestine, the activities of intestinal leucine–alanine peptidase (Leu–ala), brush border membrane (bbm)-bound alkaline phosphatase (AP) and bbm-bound leucine aminopeptidase N (LAP) were analysed from first feeding to passed weaning in Atlantic halibut (Hippoglossus hippoglossus; 1–78 days post first feeding; dpff) and Atlantic cod (Gadus morhua; 0–72 dpff). Before conducting the ontogenetic studies, the enzyme assays used were optimised in relation to temperature and pH (only AP and LAP), and the Michaelis constant (K M; All enzymes) determined, in order to ensure valid data and to maximise the activity potentials. Leu–ala was used as a marker of intracellular digestion which was expected to decrease during the experimental periods. AP and LAP were used as markers of development of the bbm and achievement of an adult mode of digestion. In halibut, Leu–ala increased 11-folds (P =0.0002) in specific activity (mU (mg protein)−1) from first feeding to 34 dpff and thereafter a small decrease (0.65-fold; P =0.006) in activity was observed. In cod, the specific activity of Leu–ala varied randomly during the experiment. Distinct increases in specific activities of AP and LAP were observed from 29 to 52 dpff in halibut (fivefold; P <0.002) and from 29 to 37 dpff in cod (two–threefold; P <0.02). The bbm-bound activities of AP and LAP in percent of total intestinal activity of the respective enzyme increased from approximately 2 to 15% between 34 and 43 dpff in halibut (P <0.0003), confirming the results of specific activities. In cod, a similar result with AP was found between 29 and 51 dpff (P =0.0003). The increase in percentage of bbm-bound activity of LAP was later, increasing exponentially from constituting 5 to 31% of total individual activity between 37 and 72 dpff (P =0.0003). The experiments indicated that the digestion in halibut had advanced towards the adult mode at 40 to 50 dpff, while time of reaching the same maturational level in cod was less certain due to the different activity profiles of AP and LAP. As AP is well recognised as a bbm differentiation marker, it can be assumed that the intestine in cod matured when AP activity had reached an elevated level at approximately 40 to 50 dpff, whereas LAP possibly was induced later. Ceasing of the larval mode of digestion, expected to be indicated by reduced activity of Leu–ala, was not detected. [Copyright &y& Elsevier]

To determine developmental stages independent of eye migration, a highly plastic feature in Atlantic halibut, 180 sibling halibut larvae reared at an average temperature of 11.9 °C from first feeding and for a further 46 days were examined, cleared and stained for ossification and the cranial development was recorded. Morphological development and cranial ossification generally coincided. The order of ossification of cranial structures was: jaw structures, hyoid arch, opercular bones and structures of the neurocranium. The Frontale exhibited torsion correlated with eye migration, but calcification began earlier and full calcification was independent of ocular displacement. The appearance of ossified elements was used to group larvae into Stages 5–9, comprising premetamorphosis to climax metamorphosis, with significant morphometric differences between stages. The trajectory of juvenile development appears fixed by Stage 8. There was a linear relationship between stage and myotome height (R2=0.86) and stage and standard length (R2=0.80). The stage definitions were validated on two groups (n=23, n=101) of commercially produced larvae. Because metamorphosis is protracted in halibut, use of these stages and especially myotome height should help standardize sampling and analysis between experiments and between producers. [Copyright &y& Elsevier]