Dietary tryptophan deficiency and its supplementation compromises inflammatory mechanisms and disease resistance in a teleost fish.

Tryptophan participates on several physiological mechanisms of the neuroendocrine-immune network and plays a critical role in macrophages and lymphocytes function. This study intended to evaluate the modulatory effects of dietary tryptophan on the European seabass (Dicentrarchus labrax) immune status, inflammatory response and disease resistance to Photobacterium damselae piscicida. A tryptophan deficient diet (NTRP); a control diet (CTRL); and two other diets supplemented with tryptophan at 0.13% (TRP13) and 0.17% (TRP17) of feed weight were formulated. Fish were sampled at 2 and 4 weeks of feeding and the remaining were i.p. injected with Phdp (3 × 106 cfu/fish) at 4 weeks and the inflammatory response (at 4, 24, 48 and 72 hours post-infection) as well as survival were evaluated. Results suggest that fish immune status was not altered in a tryptophan deficient scenario whereas in response to an inflammatory insult, plasma cortisol levels increased and the immune cell response was compromised, which translated in a lower disease resistance. When dietary tryptophan was offered 30% above its requirement level, plasma cortisol increased and, in response to bacterial infection, a decrease in lymphocytes, monocytes/macrophages and several immune-related genes was observed, also compromising at some degree fish disease resistance.

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.

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.

Metabolism of tryptophan, methionine and arginine in Diplodus sargus larvae fed rotifers: effect of amino acid supplementation.

Dietary amino acids imbalances have been described when fish larvae are fed rotifers, what may lead to a reduction in growth rate. The tube-feeding technique can be used to assess the effect of free amino acid short term supplementation. In this study supplementation of tryptophan, methionine and arginine were tested in Diplodus sargus. Single crystalline (14)C amino acids as well as a mix of (14)C amino acids were used as tracers to compare results of individual amino acids metabolism with the average of all amino acids. The results show low absorption efficiencies for tryptophan (70%) and arginine (80%) and similar absorption for methionine (90%) when compared with the average of all amino acids. Supplementation of these amino acids seems to be viable but it did not result in higher retention compared to the amino acid mix. This means that tryptophan, methionine and arginine are probably not the limiting amino acid when Diplodus sargus larvae are fed rotifers. However, supplementation in these IAA may be required for their roles as precursors of important molecules other than proteins, in order to improve larval quality and/or performance.

Stress response and changes in amino acid requirements in Senegalese sole (Solea senegalensis Kaup 1858).

Fish in aquaculture are often exposed to various stressors that may change their ability to survive or limit growth. Amino acids are used for processes other than growth, including stress response. This study intended to analyse how repeated acute handling stress can affect growth and amino acid requirements in fish. Senegalese sole juveniles were weekly held in the air during 3 min (Handling) for 9 weeks; Control groups were left undisturbed. Growth and plasma levels of stress indicators and of free amino acids were assessed at the end of the experiment. Plasma cortisol and osmolality levels showed that fish in the Handling treatment were stressed, but growth was unaffected. Plasma amino acid concentrations indicate that their requirements in stressed fish were altered, which probably reflects the synthesis of proteins or other specific compounds related to stress response.

Dietary neutral lipid level and source in Senegalese sole (Solea senegalensis) larvae: effect on growth, lipid metabolism and digestive capacity.

Contrary to larval essential fatty acid (EFA) requirements, the effect of dietary neutral lipid supply has been little investigated in marine fish larvae. The present work investigates the effect of feeding Senegalese sole larvae on Artemia enriched with higher or lower doses of lipid emulsion. Two lipid sources - soybean oil and fish oil - were compared. From 16 days after hatching (DAH) onwards, larvae were fed one of four experimental treatments: Artemia enriched on a high or low dose of soybean oil emulsion (HS and LS) or Artemia enriched on a high or low dose of fish oil emulsion (HF and LF). In terms of growth, the dietary lipid level did not have a significant effect while the soybean oil treatments induced a lower growth than the fish oil-enriched Artemia. The fatty acid (FA) composition of the larvae closely reflected the dietary quantitative and qualitative FA profile. Only slight dietary effects were noted in the activity of trypsin, lipase and alkaline phosphatase. A higher amount of lipid droplets was noticeable in the posterior intestine epithelia and in the hepatocytes of larvae fed Artemia enriched with higher lipid doses, while LS-Artemia induced the lower lipid accumulation on the basal zone of the enterocytes, in accordance with the lowest total lipid level measured in this treatment. These results suggest an important effect of dietary total lipid level on lipid accumulation in the enterocytes and on FA absorption. At 33 DAH a tube feeding trial was conducted with 14C-labelled oleic acid (OA) or triolein (TRI), showing that the lower accumulation of lipid droplets in the larvae fed LS was associated with a significantly higher absorption and retention in the gut and body tissues of the TRI label. For OA no significant differences between treatments were found. TRI label was considerably more evacuated than OA, indicating that sole larvae may have a lower capacity to incorporate a triacylglycerol, which needs to be digested. Finally, OA appears to be preferentially utilized for energy production, accumulating more in larval tissues when absorbed in higher amounts.

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.