Effect of increased rearing temperature on digestive function in cobia early juvenile.

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.

Orchestrating change: The thyroid hormones and GI-tract development in flatfish metamorphosis.

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.

Transcriptome of the Atlantic halibut (Hippoglossus hippoglossus).

Although the Atlantic halibut (Hippoglossus hippoglossus) is an important commercial species, there is still a deficit with regard to the number of transcripts in the databases, which can be accessed and exploited for targeted candidate gene and pathway studies. In this study, the RNAs from head, skin and GI tract from different developmental stages were sequenced to generate 22,272 contigs of 500 base pairs or greater as a molecular resource for this species.

Postprandial effects on appetite-related neuropeptide expression in the brain of Atlantic salmon, Salmo salar.

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.

Pre-digestion of dietary lipids has only minor effects on absorption, retention and metabolism in larval stages of Atlantic cod (Gadus morhua).

The hypothesis of the present study was that cod larvae have a limitation in lipid digestion, and that absorption of lipids would increase by pre-hydrolysation. The diets used were designed to contain 15% lipid, of which 40% was phosphatidylcholine (PC) and 60 % was TAG. Cod larvae (40d post hatch (dph)) were fed a single meal where either PC or TAG was radioactively labelled, and the labelled PC or TAG was either intact or hydrolysed (pre-digested). The larvae were then incubated individually in chambers with collection of CO2 for 10 h. The following fractions were analysed for radioactivity: the incubation water (evacuated feed); the intestine; the body; the CO2 trap. The larvae ate a 16-29 µg diet, equivalent to 3·4-5·2 % of dry body weight. In the whole population, 0-16% of the lipid was evacuated. The larvae that had eaten less than 1·9-2·7 µg lipid absorbed close to 100% of the lipid, absorption being defined conservatively as the amount contained in the carcass and CO2, excluding the intestinal tissue. In these larvae, approximately 100 % of the absorbed lipid was also catabolised. In the larvae that ingested more than 1·9-2·7 µg lipid, there was a linear reduction in lipid absorption to a minimum of 55% at the highest lipid intakes parallel to an increasing retention of lipids in the carcass. There were only minor differences in digestion, absorption, retention and metabolism of lipids between the larvae fed the different diets, and the larvae tended to retain lipid classes as they were present in the feed. The study shows that 40-dph Atlantic cod larvae have an efficient utilisation of dietary lipids supplied as intact PC and TAG.

A reference growth curve for nutritional experiments in zebrafish (Danio rerio) and changes in whole body proteome during development.

Zebrafish is one of the most used vertebrate model organisms in molecular and developmental biology, recently gaining popularity also in medical research. However, very little work has been done to assess zebrafish as a model species in nutritional studies in aquaculture in order to utilize the methodological toolbox that this species represents. As a starting point to acquire some baseline data for further nutritional studies, growth of a population of zebrafish was followed for 15 weeks. Furthermore, whole body proteome was screened during development by means of bi-dimensional gel electrophoresis and mass spectrometry. Fish were reared under best practice laboratory conditions from hatching until 103 days post-fertilization (dpf) and regularly fed ad libitum with Artemia nauplii from 12 dpf. A growth burst occurred within 9-51 dpf, reaching a plateau after 65 dpf. Fork length and body weight were significantly lower in males than in females from 58 dpf onwards. Proteomics analysis showed 28 spot proteins differently expressed through development and according to sex. Of these proteins, 20 were successfully identified revealing proteins involved in energy production, muscle development, eye lens differentiation, and sexual maturation. In summary, zebrafish exhibited a rapid growth until approximately 50 dpf, when most individuals started to allocate part of the dietary energy intake for sexual maturation. However, proteomic analysis revealed that some individuals reached sexual maturity earlier and already from 30 dpf onwards. Thus, in order to design nutritional studies with zebrafish fed Artemia nauplii, it is recommended to select a period between 20 and 40 dpf, when fish allocate most of the ingested energy for non-reproductive growth purposes.

Distribution of cholecystokinin-immunoreactive cells in the gut of developing Atlantic cod Gadus morhua L. larvae fed zooplankton or rotifers.

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.

Expression of the oligopeptide transporter, PepT1, in larval Atlantic cod (Gadus morhua).

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.

Dietary TAG source and level affect performance and lipase expression in larval sea bass (Dicentrarchus labrax).

The influence of dietary TAG source (fish oil, triolein, and coconut oil) and level (7.5 and 15% of the diet) on growth, lipase activity, and mRNA level was studied in sea bass larvae, from mouth opening until day 24 and from day 37 to 52. Fish oil and triolein induced better growth in both experiments, this being significant at a higher dietary level. Coconut oil significantly decreased growth at the higher level, possibly as the result of an excessive supply of medium-chain TAG. Growth was not related to lipase specific activity, suggesting a production in excess to dietary needs. Body lipid content was positively related to dietary lipid level and was affected by lipid quality. In addition, larval FA composition generally reflected that of the diet. The source of dietary lipid, but not the quantity, was shown to affect lipase activity significantly. Coconut oil diets induced the highest lipase activity, whereas the effect of fish oil was age dependent-it was similar to coconut oil at day 24 but induced the lowest lipase activity in 52-d-old larvae. The differential lipase response was probably caused by differences in the FA composition of the diet, related to the specificity of lipase toward FA differing in chain length and degree of saturation. No significant differences were found in lipase/glyceraldehyde-3-phosphate dehydrogenase mRNA, which suggests the existence of a posttranscriptional regulation mechanism.

Kinetics and fates of ammonia, urea, and uric acid during oocyte maturation and ontogeny of the Atlantic halibut (Hippoglossus hippoglossus L.).

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 (T(Amm): NH(3)+NH(4)(+)), and urea-N contents increased during final oocyte maturation. Urea-N excretion dominated the total nitrogenous end product formation in early embryos. Later, yolk T(Amm) levels increased in embryos and ammonia excretion was low. In the last part of the embryonic stage T(Amm) 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 T(Amm) 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).

Assimilation and catabolism of dispensable and indispensable free amino acids in post-larval Senegal sole (Solea senegalensis).

The postprandial metabolism of dietary free amino acids (AA) was studied in post-larval Senegal sole, Solea senegalensis, aged 32 days after hatching (DAH). The diet was administered as a single pulse (36 nl, 43.1 mmol/l) using a micro tube-feeding technique and a dissolved mixture of crystalline AA. In four separate treatments the diet contained L [U-(14)C] tracer for two indispensable AA (IAA), lysine and arginine or two dispensable amino acids (DAA), glutamate and alanine. The post-larva absorbed all tested AA from the diet with similar efficiency (97.5%). A small fraction of the IAA was catabolised (11.5+/-1.1 and 15.1+/-3.3%, for lysine and arginine, respectively) and a high proportion was retained in the body (86.7+/-1.3 and 81.6+/-4.1%). For the DAA more were catabolised (64.9+/-5.3% and 41.4+/-7.2% for glutamate and alanine, respectively) and less were retained (32.9+/-5.1% and 56.3+/-7.2%). On this basis, it appears that post-larval Senegal sole use DAA in preference to IAA as energy substrates while the retention (assimilation efficiency) is better for the IAA. These results support other recent studies that early stages of fish have a better capacity to regulate AA catabolism than previously believed and that indispensable AA are saved for body growth.

Ontogeny of cholecystokinin-immunoreactive cells in the digestive tract of Atlantic halibut, Hippoglossus hippoglossus, larvae.

The appearance and distribution of cholecystokinin (CCK)-producing cells were investigated in the digestive tract of developing larvae of cultured Atlantic halibut, Hippoglossus hippoglossus. The CCK-producing cells were detected immunohistochemically, by use of a primary antiserum against CCK cloned for the Japanese flounder, Paralichthys olivaceus. No CCK-immunoreactive (IR) cells were detected in first-feeding larvae (33 days after hatching, DAH). Forty-five DAH or 12 days after first feeding, there were a few scattered CCK-IR cells in the epithelium of the anterior midgut in about 30% of the examined larvae. All larvae older than 52 DAH had CCK-IR cells in the anterior midgut, particularly frequent in the most anterior region adjacent to the pyloric caeca. No CCK-IR cells were detected in the foregut, the hindgut, or the midgut posterior to the first curvature. The CCK-IR cells spanned the intestinal epithelium from the basal lamina to the lumen and were triangular in shape, with the nucleus in the basal part and a thin apex toward the lumen. The mechanisms controlling release of bile, pancreatic enzymes, and peristalsis during the 12 days between first feeding and the first detection of CCK-IR cells remain to be clarified.

Pathways for urea production during early life of an air-breathing teleost, the African catfish Clarias gariepinus Burchell.

Embryos and larvae of the African catfish Clarias gariepinus excrete significant quantities of urea. The present study focused on the potential urea-generating pathways during early development of this teleost; uricolysis, argininolysis and the ornithine-urea cycle (OUC). Uricase, allantoinase, allantoicase and ureidoglycollate lyase of the uricolytic pathway were expressed in all early life stages and in adult liver of C. gariepinus. Uricase activity increased in starved larvae compared with yolk-sac larvae. The key regulatory enzyme of the teleost OUC, carbamoyl phosphate synthetase III (CPSase III), was expressed predominantly in muscle of developing C. gariepinus larvae and showed negligible activity in the absence of its allosteric effector N-acetyl-L-glutamate. CPSase III and ornithine carbamoyl transferase activities increased in fed larvae compared with starved larvae. In contrast to the early developmental stages, adult C. gariepinus expressed only low and variable levels of CPSase III, suggesting that, under the experimental conditions employed, OUC expression is influenced by developmental stage in this species. The data indicate that early C. gariepinus life stages express the enzymes necessary for urea production by uricolysis, argininolysis and the OUC, and this may explain why urea tissue levels and urea excretion rates are substantial during the early development of this air-breathing teleost.

Detection and basic properties of carbamoyl phosphate synthetase III during teleost ontogeny: a case study in the Atlantic halibut (Hippoglossus hippoglossus L.).

The presence of carbamoyl phosphate synthetase III (CPSase III), catalyzing the first step of the urea cycle in fish, in Atlantic halibut (Hippoglossus hippoglossus L.) yolk-sac larvae and adult white muscle has been established using gel filtration chromatography to separate the CPSase III from the pyrimidine-pathway related CPSase II. The results are consistent with the hypothesis that teleostean fish express urea cycle enzymes during early development and with recent observations of low levels of CPSase III in muscle tissue. The presence of CPSase III in crude extracts could not be established using sensitive assay conditions to discriminate between CPSase III and CPSase II. However, kinetic characterization after chromatographic separation identified each as typical CPSase II and CPSase III activities, respectively. The CPSase III was less sensitive to activation by N-acetyl-L-glutamate and had a higher Km for ammonia than CPSase III found in other species. These results suggest that precise quantitation of low levels of CPSase III in the presence of CPSase II by assaying crude extracts may be difficult unless the enzymes are first separated and the kinetic properties of CPSase III are determined; the results indicate that assaying larval extracts of Atlantic halibut in the presence of uridine triphosphate results in CPSase activity that reflects mostly CPSase III and can, therefore, be used to measure changes in CPSase III activity.

Free amino acids are absorbed faster and assimilated more efficiently than protein in postlarval Senegal sole (Solea senegalensis).

To improve the formulation of diets for the early stages of marine fish, assimilation rates of free amino acids (FAA) and protein in postlarval Senegal sole (Solea senegalensis) were determined. Fish (2.45 +/- 0.87 mg dry weight) were tube fed 36 nL of a diet of FAA containing L-[(35)S] methionine (FAA diet) or bovine serum albumin, containing L-[methylated-(14)C]bovine serum albumin (Prot-diet), both at a concentration of 4.08 g/L. A time series was performed, and the amounts of label in incubation water, liver, gut and body carcass were quantified. The FAA diet was absorbed with a 3.5-times-higher transfer rate (P < 0.001) from the gut into the larval body tissues compared with the Prot-diet. The FAA diet also was assimilated with greater efficiency than the Prot-diet (80% versus 58%, P: = 0.001). If we assume that the label present in the gut represents amino acids incorporated into the intestinal tissue, the assimilation efficiencies for the two diets were 89 and 64%. Therefore, FAA seems to be superior to protein as a dietary source of amino acids in Senegal sole postlarvae. However, because the absorption dynamics of protein and FAA differ, care should be taken when using the sources together to avoid amino acid imbalance.

Bronchial response to breathing dry gas at 3.7 MPa ambient pressure.

In diving, pulmonary mechanical function is limited by the increased density of the gas breathed. Breathing cold and dry gas may cause an additional increase in airways resistance. We have measured forced vital capacity, forced expired volume in 1 s (FEV1) and forced midexpiratory flow rate (FEF25%-75%) before and after breathing dry or humid gas at 29-32 degrees C during a standardized exercise intensity on a cycle ergometer at an ambient pressure of 3.7 MPa. The atmosphere was a helium and oxygen mixture with a density of 6.8 kg.m-3. Six professional saturation divers aged 26-37 years participated in the study. There were no significant differences in convective respiratory heat loss between the exposures. The mean evaporative heat loss was 67 W (range 59-89) breathing dry gas and 37 W (range 32-43) breathing humid gas, corresponding to water losses of 1.7 g.min-1 (range 1.5-2.2) and 0.9 g.min-1 (range 0.8-1.1), respectively. There was a significant reduction in FEV1 of 4.6 (SD 3.6)% (P < 0.05), and in FEF25%-75% of 5.8 (SD 4.7)% (P < 0.05) after breathing dry gas. There were no changes after breathing humid gas. By warming and humidifying the gas breathed in deep saturation diving bronchoconstriction may be prevented.

Respiratory effects of warm and dry air at increased ambient pressure.

We have measured in 7 divers forced vital capacity (FVC), forced expired volume in 1 s (FEV1), and forced midexpiratory flow rate (FEF25-75%) before and after exposure to dry or humid breathing gas of 35.3 degrees-36.8 degrees C (air) when diving to pressures of 117-600 kPa. The response was compared with the subjects' reactivity to pharmacologic bronchoprovocation with methacholine. Baseline FEV1 and FEF25-75% decreased in accordance with increasing gas density. Relative to baseline, there was a significant reduction after the dives in FEV1 of 4.0 +/- 6.1% (P less than 0.05) and in FEF25-75% of 8.6 +/- 9.7% (P less than 0.01) with exposure to dry breathing gas. By analysis of variance the reduction in the lung function variables below baseline were related to the breathing gas characteristic (dry/humid) (P less than 0.01), bronchial hyperreactivity (P less than 0.02), and ambient pressure (P less than 0.02) independently of each other. There was no significant change in FVC after the exposures. Humid breathing gas was considered more comfortable than dry breathing gas, and the upper comfort limit for breathing gas temperature was higher with humid breathing gas. Convective respiratory heat loss was negligible in these experiments, indicating that dry gas itself had a significant bronchoconstrictive effect. Bronchial hyperreactivity may cause increased risk of development of bronchial obstruction and air trapping during diving.