Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene

Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to improve the survival rate and nutritive efficacy when using plant-based diets.     

Patterns of gastric evacuation, digesta characteristics and pH changes along the gastrointestinal tract of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

A comparative study of gastric evacuation rates (GERs) and digesta content, moisture and pH values along the gastrointestinal tract was performed between gilthead sea bream and European sea bass. In order to distinguish species-specific differences from diet-elicited effects, all parameters were determined under either a fishmeal diet or a carob seed germ meal diet that contained high levels of total and soluble non-starch polysaccharides. GERs were significantly different between species and they were not affected by diet. Similarly, species-specific patterns were revealed in the distribution of digesta and water content along the gastrointestinal tract. In sea bream, stomach digesta and water content decreased with time, whereas in sea bass stomach retained the highest digesta and water content throughout the sampling period. The anterior and distal intestine exhibited the lowest accommodating capacities of digesta and water in either species. Overall, sea bream performed stomach digestion at lower hydration levels and higher pH compared with sea bass. Diet affected stomach moisture in both species and pH of stomach digesta in sea bass and of all intestinal sections in sea bream. The results obtained indicated that water and inorganic ion exchanges through the gut may differentiate between the species and warrant further investigation.     

Diversity of microbial communities of the intestinal mucosa and intestinal contents of fish from Lake Chany (Western Siberia)

The diversity of microbial communities associated with the intestinal mucosa and the intestinal contents of fish with different nutritional profiles have been investigated. Differences in the intestinal microbiota of fish with different dietary preferences have been revealed using group-specific primers for the main large taxa of bacteria. Representatives of the phyla Planctomycetes, Vernicomicrobia, and Cyanobacteria have been detected in the intestinal contents of all investigated nonpredatory (crucian carp, roach, and dace) and predatory (pikeperch, pike, and perch) fish. Representatives of the phylum Firmicutes were detected in the intestinal mucosa and intestinal contents of predatory fish species, but not in those of the nonpredatory fish.     

Nutritional assessment of somatolactin function in gilthead sea bream (Sparus aurata): concurrent changes in somatotropic axis and pancreatic hormones

The role of somatolactin (SL) in the regulation of energy homeostasis in gilthead sea bream (Sparus aurata) has been analysed. First, a down-regulation of plasma SL levels in response to gross shifts in dietary amino acid profile and the graded replacement of fish meal by plant protein sources (50%, 75% and 100%) has been observed. Thus, the impaired growth performance with changes in dietary amino acid profile and dietary protein source was accompanied by a decrease in plasma SL levels, which also decreased over the course of the post-prandial period irrespective of dietary nitrogen source. Secondly, we examined the effect of SL and growth hormone (GH) administration on voluntary feed intake. A single intraperitoneal injection of recombinant gilthead sea bream SL (0.1 microg/g fish) evoked a short-term inhibition of feed intake, whereas the same dose of GH exerted a marked enhancement of feed intake that still persisted 1 week later. Further, we addressed the effect of arginine (Arg) injection upon SL and related metabolic hormones (GH, insulin-like growth factor-I (IGF-I), insulin and glucagon) in fish fed diets with different nitrogen sources. A consistent effect of Arg injection (6.6 micromol/g fish) on plasma GH and IGF-I levels was not found regardless of dietary treatment. In contrast, the insulinotropic effect of Arg was found irrespective of dietary treatment, although the up-regulation of plasma glucagon and glucose levels was more persistent in fish fed a fish meal based diet (diet FM) than in those fed a plant protein diet with a 75% replacement (diet PP75). In the same way, a persistent and two-fold increase in plasma SL levels was observed in fish fed diet FM, whereas no effect was found in fish fed diet PP75. Taken together, these findings provide additional evidence for a role of SL as a marker of energy status, which may be perceived by fish as a daily and seasonal signal of abundant energy at a precise calendar time.     

Rearing temperature enhances hepatic glucokinase but not glucose-6-phosphatase activities in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) juveniles fed with the same level of glucose

The aim of this work was to elucidate if the previous results observed in hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities in European sea bass and gilthead sea bream are due to temperature per se or to differences in feed intake at different water temperatures. For that purpose triplicate groups of fish (30 g initial body weight) were kept at 18 degrees C or 25 degrees C during two weeks and fed a fixed daily ration of a glucose-free or 20% glucose diet. At the end of the experimental period, plasma glucose levels in both species were not influenced by water temperature but were higher in fish fed the glucose diet. Higher hepatic GK activity was observed in the two fish species fed the glucose diet than the glucose-free diet. In the glucose fed groups, GK activity was higher at 25 degrees C than at 18 degrees C. Glucose-6-phosphatase activities in both species were not influenced by water temperature. In European sea bass and in contrast to gilthead sea bream it was observed an effect of dietary composition on G6Pase activities with surprising higher activities recorded in fish fed the glucose diet than in fish fed the glucose-free diet. Overall, our data strongly suggest that European sea bass and gilthead sea bream are apparently capable to strongly regulate glucose uptake by the liver but not glucose synthesis, which is even enhanced by dietary glucose in European sea bass. Within limits, increasing water temperature enhances liver GK but not G6Pase activities, suggesting that both species are more able to use dietary carbohydrates at higher rearing temperatures.     

Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg^?1), B. subtilis (10⁷ cfu g^?1), or B. subtilis + inulin (10⁷ cfu g^?1 + 10 g kg^?1) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.     

The IMTA-cultivated Chlorophyta Ulva spp. as a sustainable ingredient in Nile tilapia (Oreochromis niloticus) diets

Increasing levels of a mixture of Ulva spp. produced in an integrated multi-trophic aquaculture (IMTA) system were evaluated in Nile tilapia juveniles for partial replacement of dietary fish meal. A control diet (CTRL) was compared with three experimental diets containing 10 % (U10), 15 % (U15), and 20 % (U20) of Ulva spp. meal. Triplicate groups of fish (13 g initial body weight) were fed each diet for 63 days at 26 °C. Nutrient apparent digestibility coefficients and nitrogen retention efficiency did not vary significantly among diets. By the end of the trial, all groups of fish more than tripled their initial body weight. Specific growth rate and final body weight of U10 diet were similar to CTRL and significantly higher than U15 and U20 diets. Increasing Ulva dietary incorporation levels significantly increased feed conversion ratio (FCR), from 1.0 (CTRL) to 1.4 (U20). Fish fed with U10 diet had the highest protein efficiency ratio and nitrogen retention efficiency allowing this fish to growth and reach a final body weight similar to the CTRL group. Protein content was highest in fish fed with the CTRL diet, whereas the highest lipid content was observed in fish fed with U20 diet. The results show that the incorporation of IMTA-produced Ulva meal in Nile tilapia diets is possible up to 10 % without compromising growth performance, protein utilization, and protein retention of juveniles. The high capacity of Nile tilapia to digest all experimental diets suggests that Ulva meal is a practical partial replacement for fish meal in Nile tilapia diets.     

Short-term cell culture technique for obtaining chromosomes in marine and freshwater fish

In this paper we describe a simple and reliable short-term fish cell culture technique, for obtaining chromosome spreads with minimum stress involving the specimens sampled. Fibroblasts were derived from primary cultures from the caudal fin of some economically important freshwater species: northern pike, Esox lucius L. , goldfish, Carassius auratus L., and marine species: gilthead sea bream, Sparus aurata L. and cardinal fish, Apogon imberbis L.     

Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions

The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota.     

Evaluation of Hydrodictyon reticulatum as protein source in feeds for Oreochromis (Tilapia) niloticus and Tilapia zillii

The green filamentous alga Hydrodictyon reticulatum was evaluated as a protein source in fish-meal substituted diets for Oreochromis niloticus and Tilapia zillii fingerlings. The fingerlings were fed in duplicate groups each of six different diets for 50 days. Five of the diets contained 30% crude protein supplied by varying proportions of fish meal and H. reticulatum meal. The five diets were formulated to supply fishmeal protein: H. reticulatum meal protein ratios of 30:0 (diet 1), 15:5 (diet 2), 20:10 (diet 3); 15:15 (diet 4), 10:20 (diet 5) respectively. A sixth diet containing only 25% crude protein supplied entirely by H. reticulatum meal was also fed. The best growth and protein utilization was obtained at lower levels of H. reticulatum substitution for both species of fish. Carcass analysis revealed a decrease in lipid contents of the fishes with increasing levels of the alga in the diet.     

Pea protein concentrate in diets for sharpsnout sea bream (Diplodus puntazzo): effects on growth and health status

Four diets for sharpsnout sea bream juveniles (14 g body weight) with four levels of air-processed pea protein concentrate (PPC) (0, 160, 320 and 487 g/kg diet) were tested in triplicate. The experimental diets were isonitrogenous (43% crude protein) and isolipidic (19% ether extract) and the fish were fed to satiation twice a day. After 125 d, fish growth was diminished by the inclusion of PPC. Feed conversion did not show significant differences in any treatment. Neither the body analyses nor the protein and individual essential amino acid retention efficiencies were affected by the inclusion of PPC in the diet. However, histological gut examinations revealed noticeable differences. Fish fed the diet with the highest inclusion level of PPC presented the longest villous length and the most goblet cells, and the width of the lamina propria increased in the anterior intestine. Although no negative changes in nutritive parameters were detected, these alterations might affect nutrient transport, with negative consequences for fish growth. It was concluded that the PPC in the amounts tested here is an inappropriate substitute for fishmeal in diets for sharpsnout sea bream juveniles.     

Effect of water temperature and dietary starch on growth and metabolic utilization of diets in gilthead sea bream (Sparus aurata) juveniles

We evaluated the effect of dietary starch level on growth performance, feed utilization, whole-body composition and activity of selected key enzymes of intermediary metabolism in gilthead sea bream juveniles reared at 18 and 25 degrees C. A diet was formulated to contain 48% crude protein, 12% lipids and 30% gelatinized maize starch (diet 30GS). Two other diets were formulated to include the same level of ingredients as diet 30GS except for the gelatinized starch, which was included at 20% (diet 20GS) or 10% (diet 10GS). No adjustment to diet composition was otherwise made. Each diet was fed to triplicate groups of gilthead sea bream (30 g initial mass) for 8 weeks, on a pair-feeding scheme. The higher temperature improved growth performance but the opposite was true for feed efficiency and protein efficiency ratio. Independently of temperature, growth performance, feed efficiency and protein efficiency ratio were lower in fish fed diet 30GS. No effect of temperature or dietary starch level on whole-body composition was noticed. Hepatosomatic index and liver glycogen were higher at 18 degrees C and, within each temperature, in fish fed diet 30GS. Glycemia was not affected by temperature, but was lower in fish fed diet 10GS. Data on enzyme activities showed that increasing water temperature enhances liver glucokinase (GK) and pyruvate kinase (PK) activities, suggesting that gilthead sea bream is more apt to use dietary starch at higher temperatures. No effect of temperature was noticed on hexokinase (HK), fructose-1,6-bisphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) activities. Dietary starch enhanced PK and FBPase activities while depressed GDH activity, suggesting a lack of significant regulation of hepatic glucose utilization and production in this species. HK, GK and G6PD activities were unaffected by dietary composition. Irrespectively of water temperature, gelatinized starch may be included up to 20% in diets for gilthead sea bream juveniles; at higher dietary levels, growth and efficiency of feed utilization are depressed.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.     

Aquacultured Rainbow Trout (Oncorhynchus mykiss) Possess a Large Core Intestinal Microbiota That Is Resistant to Variation in Diet and Rearing Density

As global aquaculture fish production continues to expand, an improved understanding of how environmental factors interact in fish health and production is needed. Significant advances have been made toward economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and toward defining the effect of rearing density on fish health and production. Little research, however, has examined the effects of fishmeal- and grain-based diets in combination with alterations in rearing density. Moreover, it is unknown whether interactions between rearing density and diet impact the composition of the fish intestinal microbiota, which might in turn impact fish health and production. We fed aquacultured adult rainbow trout (Oncorhynchus mykiss) fishmeal- or grain-based diets, reared them under high- or low-density conditions for 10 months in a single aquaculture facility, and evaluated individual fish growth, production, fin indices, and intestinal microbiota composition using 16S rRNA gene sequencing. We found that the intestinal microbiotas were dominated by a shared core microbiota consisting of 52 bacterial lineages observed across all individuals, diets, and rearing densities. Variations in diet and rearing density resulted in only minor changes in intestinal microbiota composition despite significant effects of these variables on fish growth, performance, fillet quality, and welfare. Significant interactions between diet and rearing density were observed only in evaluations of fin indices and the relative abundance of the bacterial genus Staphylococcus. These results demonstrate that aquacultured rainbow trout can achieve remarkable consistency in intestinal microbiota composition and suggest the possibility of developing novel aquaculture strategies without overtly altering intestinal microbiota composition.     

Phytase can reduce theneedfor monocalcium phosphate supplementation in soybean and rapeseed meal‐based diets of black sea bream,Acanthopagrus schlegelii (Bleeker, 1854)

A feeding trial was conducted to study the effect of partial replacement of dietary monocalcium phosphate (MCP) with phytase on growth performance, feed utilization and phosphorus discharge in black sea bream, Acanthopagrus schlegelii. In the feeding trial, the control diet (designated as P1.5) was prepared with 1.5% MCP but without phytase, and the three other diets (designated as PP1.0, PP0.5 and PP0, respectively) were supplemented with 1.0%, 0.5% and 0% MCP, respectively, along with 200 mg (400 U) phytase/kg diet in each. Each diet was tested in triplicate tanks and fish were fed twice daily to satiation. After an 8‐week feeding trial in indoor flow‐through cylindrical fibreglass tanks (25 fish per tank, initial body weight: 11.5 ± 0.12 g), fish fed with PP1.0 and PP0.5 had no significant change in weight gain (WG), specific growth rate (SGR), protein efficiency rate (PER) or feed conversion ratio (FCR) compared to the control (p > .05), whereas fish fed with PP0 showed a significantly lower growth performance in the above parameters (p < .05). The addition of phytase did not affect the body composition or muscle composition. The apparent digestibility coefficients (ADCs) of crude protein and phosphorus increased when fish were fed diets in which MCP was replaced by phytase. Phosphorus discharge was also significantly reduced in fish fed diets in which MCP was replaced by phytase (10.2 ± 0.50 to 8.01 ± 0.47 g/kg weight gain). The present study suggests that dietary MCP can be reduced when phytase is added to the black sea bream diet, with a maximum MCP reduction level of up to 1% when phytase is supplemented at 200 mg (400 U)/kg diet. Thus, phytase in the diet of black sea bream is economically and ecologically beneficial.     

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss)

AIMS: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. METHODS AND RESULTS: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. CONCLUSIONS: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.