Immune responses and gut morphology in Senegalese sole (Solea senegalensis) fed dietary probiotic supplementation and following exposure to Photobacterium damselae subsp. piscicida

The aim of this study was to determine the effect of a dietary multi‐species probiotic on growth, gut morphology and immune parameters in Senegalese sole (Solea senegalensis). Fish were fed with two experimental diets, a control diet and the same diet supplemented with the probiotic for 72 days. A sub‐lethal bath challenge with Photobacterium damselae subsp. piscicida was performed after the growth trial. Intestine and blood samples were collected to study gut structure and plasmatic immune parameters. No significant differences were found in growth performance. The analysis of gut morphology showed a significant increase in intestinal villi height of Senegalese sole fed the probiotic. Regarding circulating leucocytes, dietary probiotic supplementation increased thrombocytes levels whereas a decrease in the proportion of lymphocytes was observed. No significant differences were observed in humoural immune parameters. Bath challenge differentially affected leucocyte counts and increased peroxidase activity. This study presents the possibility of using dietary probiotic supplementation to increase Senegalese sole welfare since gut morphology was positively affected. Although the immune response after a bacterial challenge was not modulated by dietary treatment, further studies would be instrumental to unravel eventual dietary benefits on immune mechanisms.     

Intestinal microbiota variation in Senegalese sole (Solea senegalensis) under different feeding regimes

The aim of this study was to determine the influence of the feeding regimes in Senegalese sole (Solea senegalensis) cultured under extensive, semi‐extensive and intensive production systems. A total of 254 bacterial isolates from guts of fish cultured under different production systems and feeding regimes were tested. Biochemical tests and genetic analyses based on the 16S rDNA sequence analysis were conduced to identify bacterial strains. Vibrio species were the most represented taxonomic group in the culturable microbiota of S. senegalensis guts tested. Particularly, Vibrio ichthyoenteri was the most frequently isolated Vibrio species. Comparison among diets showed a significant reduction (P<0.05) in vibrio percentages and a higher occurrence of Shewanella species in Senegalese soles fed polychaeta. In addition, a major influence of environmental temperature on microbiota composition was detected. Cold temperatures brought about a change in the percentages of Vibrio species and a higher representation of α‐Proteobacteria in both outdoor systems (extensive and semi‐extensive). The significant differences between intestinal bacterial composition in Senegalese soles fed commercial diets and natural preys (polychaeta) reveal the necessity to develop specific optimized diets for the intensive rearing of this fish species.     

Use of probiotic bacteria in the rearing of Senegalese sole (Solea senegalensis) larvae

Three candidate probiotics, which had shown antimicrobial activity in vitro against two fish pathogens, were used in the rearing of Senegalese sole larvae and postlarvae. These probiotics improved the survival of starved sole yolk‐sac larvae. A feeding experiment of sole larvae and postlarvae comprised three treatments: A, B and C. Cultures of a Vibrio sp. (2J18) were added to treatment A, whereas a gram‐positive (J84) and a Shewanella sp. strain (2J27), were added to treatment B, while in a control treatment C no bacteria were added. Addition of bacteria in treatment B increased survival of larvae in the first phase of the experiment [0–20 days after hatching (DAH) (P<0.05)] and decreased the numbers of colony‐forming units (CFU) in larval gut 5 DAH compared with the control treatment (P<0.05). No differences were observed in survival (25–47%) during the second phase of the experiment (20–60 DAH). Nevertheless, the total numbers of CFU in fish gut 40 DAH were significantly lower in treatment B (P<0.05). The colonization rates of the added bacteria were the highest 5 and 11 DAH, and the highest average values reached were 65%, 82% and 17% of the total CFU count for the strains 2J18, 2J27 and J84 respectively.     

Effects of a dietary organic acids blend and oxytetracycline on the growth,nutrient utilization and total cultivable gut microbiota of the red hybrid tilapia,Oreochromis sp., and resistance to Streptococcus agalactiae

A 20‐week feeding trial was conducted to measure growth, nutrient utilization and faecal/gut bacterial counts in triplicate groups of red hybrid tilapia, Oreochromis sp., when fed diets supplemented with 0.5% organic acids blend (OAB), 1.0% OAB, 0.5% oxytetracycline (OTC) or a control diet (no additives). At the end of the feeding trial, tilapia were challenged with Streptococcus agalactiae for 22 days. Fish fed the OTC diet had significantly higher (P < 0.05) growth than the control treatment, while growth between fish fed the OTC or OAB diets was not significantly different (P > 0.05). Phosphorus, dry matter and ash digestibility were significantly higher in the 1.0% OAB diet than the control diet. Fish fed the OAB diets had significantly lower colony‐forming units of adherent gut bacteria compared to the control or OTC treatments while those fed the 1.0% OAB diet had the lowest total faecal bacterial counts. Tilapia fed the 0.5% OTC or OAB diet had significantly higher resistance to S. agalactiae than those fed the control diet. This study indicates that dietary organic acids can potentially replace OTC as a growth promoter and antimicrobial in tilapia feeds.     

The effect of dietary inulin on aerobic bacteria associated with hindgut of Arctic charr (Salvelinus alpinus L.)

The primary aim of the present study was to evaluate the population level of adherent (autochthonous) aerobic and facultative anaerobic bacteria in the hindgut of healthy Arctic charr (Salvelinus alpinus L.) fed dextrin or inulin. This was assessed by the dilution plate technique, and visualized using both transmission and scanning electron microscopy. A population level of 4.8 × 10⁵ adherent bacteria per gram wet mass was found in the hindgut of fish fed a casein‐based diet supplemented with 15% dextrin. However, substituting dextrin with 15% inulin reduced the bacterial population level in the hindgut (3.56 × 10⁴). A total of 217 bacterial isolates were identified by key phenotypical and biochemical characteristics. In addition, 22 strains were also identified by partial sequencing of the 16S rRNA gene. The composition of bacteria colonizing the hindgut of Arctic charr fed dextrin was dominated by the genera Staphylococcus, Pseudomonas, Micrococcus, Psychrobacter glacincola and Streptococcus. However, bacteria colonizing the hindgut of fish fed inulin were dominated by Gram‐positive bacteria of the genera Staphylococcus, Streptococcus, Carnobacterium and Bacillus. While Carnobacterium divergens‐like strains were isolated from charr fed dextrin, Carnobacterium maltaromicus‐like strains were isolated from the hindgut of fish fed inulin. Electron microscopical analysis of hindgut regions confirmed traditional culture‐based microbial analysis as fewer bacterial cells were observed between microvilli and associated with the surfaces of enterocytes of fish fed inulin rather than dextrin.     

Autochthonous probiotic bacteria modulate intestinal microbiota of Pirarucu,Arapaima gigas

This study aimed to investigate the ability of two probiotic strains (Lactococcus lactis subsp. lactis and Enterococcus faecium) to colonize Arapaima gigas intestines and their competence in modulating gut microbiota, morphology, and immunological status. A total of 135 A. gigas (58.86 ± 10.25 g) were divided into three treatments: (a) fish fed a diet containing L. lactis subsp. lactis at 1 × 10⁸ CFU^?1, (b) fish fed a diet containing E. faecium at 1 × 10⁸ CFU^?1, and (c) fish fed a diet nonsupplemented with probiotic. After 21 days, 12 fish per treatment were sampled. Blood and posterior intestine were sampled for further analysis. High‐throughput sequencing results demonstrated that the relative abundance of Cetobacterium genus was high in all treatments. The number of thrombocytes increased in both groups fed the probiotic, and eosinophils were higher in fish fed E. faecium. Serum antimicrobial activity of fish fed L. lactis subsp. lactis was higher when compared with the other two treatments. In conclusion, both strains could populate the intestinal mucosa of A. gigas and could be used as probiotics in pirarucu production as they can influence hematoimmunological parameters and decrease the abundance of pathogenic bacteria in the gut of pirarucu within 21 days of feeding.     

Optimal dietary protein levels in juvenile Senegalese sole (Solea senegalensis)

A study was undertaken to determine the dietary protein level for optimal growth performance and body composition of juvenile Senegalese sole. Five experimental extruded diets were formulated to contain increasing levels of protein [430, 480, 530, 570 and 600 g kg^?1 dry matter (DM)] and a constant lipid level, ranging from 100 to 130 g kg^?1 DM. Triplicate groups of 35 sole (initial body weight: 11.9 ± 0.5 g) were grown over 84 days in 60‐L tanks supplied with recirculated seawater. Fish were fed by means of automatic feeders in eight meals per day. At the start and end of the trial, whole‐body samples were withdrawn for proximate composition analysis. At the end of 84 days of experimental feeding, daily weight gain and specific growth rate in fish fed diets P43 and P48 were significantly lower than those found in fish fed higher protein level diets (P53, P57 and P59). Similarly, feed efficiency was also significantly lower in fish fed diet P43 than in fish fed all other dietary treatments. Sole juveniles fed lower protein level diets (P43 and P48) showed a significantly lower protein content than fish fed the higher dietary protein level treatments (P53, P57 and P60). Changes within the tested dietary protein levels did not affect significantly protein productive value or total nitrogen (N) losses in fish. However, daily N gain was significantly higher (P < 0.05) in fish fed diets P53 and P60 than in fish fed the lowest protein level diet (P43). Data from the present study indicate that diets for juvenile Senegalese sole should include at least 53% crude protein to maintain a good overall growth performance. Based on a second‐order polynomial regression model, the daily crude protein requirement for maximum whole‐body N gain as estimated here for Senegalese sole juveniles was 6.43 g kg^?1 body weight day^?1 which corresponds to a value of 1.03 g N intake kg^?1 body weight day^?1. If the present data are expressed on a dietary crude protein concentration basis, the allowance for maximum protein accretion (N gain) would be met by a diet containing a crude protein level of 600 g kg^?1.     

Preliminary evaluation of growth performance and immune response of Nile tilapia Oreochromis niloticus supplemented with two putative probiotic bacteria

This study evaluated the effect of two bacteria, Bacillus amyloliquefaciens and the dairy yogurt (DY) Lactobacillus sp., on improving the growth performance, feed conversion ratio (FCR) and some immunological and haematological parameters. Nile tilapia (19.1 g) were stocked in 0.42 m³ tanks at 67 fish m^?3 and fed with two probiotic‐incorporated diets and a control diet for 99 days, followed by a normal diet for another 61 days. At the end of the probiotic feeding, the mean weight, FCR and production rate showed no significant difference among the treatments. However, after 61 days of feeding of the normal diet, the B. amyloliquefaciens fish showed significantly superior growth and better FCR than the control. The DY group had the lowest growth and the poorest FCR. Significantly higher serum lysozyme activity, head‐kidney superoxide dismutase, total immunoglobulin and serum bacterial agglutination titres were recorded in the probiotic groups than the control. Haematological parameters showed no difference between treatments. The B. amyloliquefaciens fed fish showed that the gut microflora was dominated by B. amyloliquefaciens even after the withdrawal of the probiotic. On the contrary, the probiotic bacterial species isolated from the dairy yoghurt did not persist longer in the gut. These results indicate the beneficial effect of administering the bacteria isolated from fish gut microflora, B. amyloliquefaciens, in improving growth, FCR and immunological parameters. The high persistence of B. amyloliquefaciens indicates that the probiotic colonization in the gut is essential for the best responses and economics.     

Senegalese sole larvae growth and protein utilization is depressed when co‐fed high levels of inert diet and Artemia since first feeding

A large effort has been dedicated in the past years to the development of nutritional balanced inert diets for marine fish larvae in order to suppress the nutritional deficiencies of live feed. In this study growth performance, Artemia intake, protein digestibility and protein retention were measured for Senegalese sole (Solea senegalensis Kaup), in order to provide insight into how protein utilization affects growth performance. Three feeding regimes were tested: ST – standard live feed; ArtRL – live feed and 20%Artemia replacement with inert diet (dry matter basis) from mouth opening; ArtRH – live feed and 58%Artemia replacement with inert diet from mouth opening. Artemia intake and protein metabolism were determined at 6, 15 and 21 days after hatching using ¹⁴C‐labelled Artemia protein and subsequent incubation in metabolic chambers. At the end of the experiment, sole fed exclusively with live feed were significantly larger than sole from Artemia replacement treatments. Protein digestibility decreased during sole ontogeny, and more sharply in ArtRH sole. Concomitantly retention efficiency increased during ontogeny but with a slight delay in ArtRH sole. Senegalese sole larvae growth and protein utilization is depressed when co‐fed high levels of inert diet and Artemia, mostly during metamorphosis climax.     

Effect of soybean meal,raffinose and stachyose on the growth,body composition,intestinal morphology and intestinal microflora of juvenile allogynogenetic silver crucian carp (Carassius auratus gibelio♀×Cyprinus carpio♂)

The study was conducted to investigate the effects of soybean meal (SBM), raffinose and stachyose on juvenile crucian carp (Carassius auratus gibelio♀ × Cyprinus carpio♂). The experimental diets consisted of one control diet based on fish meal (FM), one diet containing 300 g kg^?1 SBM and four FM‐based diets with the addition of either 6.7 g kg^?1 raffinose (Raf), 33.9 g kg^?1 stachyose (Sta), a combination of raffinose and stachyose (Raf?Sta) and finally a Raf?Sta diet supplemented with 2.5 g kg^?1 saponins (Raf?Sta?Sap). After 3 weeks of feeding, the relative gut lengths of SBM‐fed fish and the fish fed stachyose‐containing diets were shorter than those of the FM‐fed fish; further, more SBM‐fed fish showed fissures on the tips of the intestinal folds. After 8 weeks of feeding, the growth of SBM‐fed fish was significantly lower than that of FM‐fed fish (P<0.05). The fish fed Raf?Sta?Sap had a low relative gut length (P<0.05). In comparison with the other fish, the SBM‐fed fish had a higher number of large‐sized homogeneous vacuoles in the cytoplasm of epithelial cells and shorter microvilli. No significant difference was observed in body composition or intestinal microflora. The results indicated that raffinose and stachyose played no or only minor roles in the development of soybean‐induced growth reduction.     

Influence of dietary administration of a probiotic strain Shewanella putrefaciens on senegalese sole (Solea senegalensis,Kaup 1858) growth,body composition and resistance to Photobacterium damselae subsp piscicida

Probiotics are currently being supplemented to cultured fish due to their benefits for fish performance. Herein, we tested the health protection and nutritional effects of probiotic Shewanella putrefaciens (Pdp11 strain), both fresh and lyophilized cells, on Senegalese sole (Solea senegalensis) juveniles. Pdp11 was incorporated into fish feed at concentration of 10⁹ cells g^?1, and then provided to juvenile sole for 2 months. Growth rates were significantly higher in fish fed the fresh probiotic compared with fish fed the control diet. Growth of fish receiving lyophilized bacteria was not improved when compared with controls. Body compositions (protein, total lipids and fatty acids profile) were similar for each dietary treatment. At the end of the study, fish were challenged by intraperitoneal inoculation with a pathogenic strain of Photobacterium damselae subsp. piscicida. Relative percentages of survival (RPS) ranged between 25% and 43.8% for fish receiving both probiotic supplemented diets. Both fresh and lyophilized Pdp11 cells conferred protection against P. damselae subsp. piscicida. However, only fresh Pdp11 enhanced Senegal sole performance. These findings should be taken into account for industry purposes in which lyophilized Pdp11 might be easier to manage.     

The effect of dietary oxidized lipid levels on growth performance,antioxidant enzyme activities,intestinal lipid deposition and skeletogenesis in Senegalese sole (Solea senegalensis) larvae

Fish tissues, particularly rich in n‐3 PUFA, are prone to lipid peroxidation that can damage cellular membranes, cause severe lesions and subsequently incidences of disease and mortality. However, fish possess antioxidant defences, such as vitamin E (VE) and antioxidant enzymes, to protect them against oxidative damage. This study investigated the effects of an increasing gradient of oxidized dietary lipid on the survival, growth performance, skeletogenesis and antioxidant defensive processes occurring in Senegalese sole (Solea senegalensis) larvae. Four groups of fish were fed live prey enriched with experimental emulsions containing an increasing gradient of oxidized oil: non‐oxidized, NO+VE, 34.5 nmol MDA g^?1 w.w.; mildly oxidized, MO+VE, 43.1 nmol MDA g^?1 w.w.; highly oxidized, HO+VE, 63.3 nmol MDA g^?1 w.w. and highly oxidized without VE, HO‐VE, 78.8 nmol MDA g^?1 w.w. The oxidation levels increased in enriched rotifers following the oxidation gradient of the emulsions, but were not affected in enriched Artemia metanauplii. The oxidation status of Senegalese sole larvae increased during development, but this was not related to the dietary treatments. The increasing dietary oxidation levels did not affect the fatty acid profile, survival, growth performance and metamorphosis processes of sole larvae. Senegalese sole seem to activate antioxidant defence mechanisms in response to the increasing amounts of dietary peroxidized lipids, in a manner efficiently enough to prevent detection of any alterations of these physiological processes. Antioxidant systems and detoxification mechanisms appeared to occur through the consumption of dietary α‐tocopherol, the activation of the antioxidant enzymes (catalase, superoxide dismutase, glutathione S‐transferase, glutathione reductase) and the retention of oxidized fat in the intestinal enterocytes for detoxification prior to their utilization. However, fish fed the highest oxidized diet presented a reduction in bone mineralization, but lower incidence of deformities in the vertebral and caudal regions than fish fed the other diets. This study exemplifies the importance of rearing Senegalese sole larvae on non‐oxidized diets during the early larval development to avoid detrimental consequences in older fish, most notably in the process of skeletogenesis.     

Improving weaning strategies for Senegalese sole: effects of body weight and digestive capacity

To optimize Senegalese sole‐weaning strategies, three experiments were performed. The first trial tested four weaning strategies with a 10 mg sole. Artemia‐fed sole grew threefold less than fish fed an inert diet. Sudden weaning (abrupt change from Artemia to inert diet) and weaning with co‐feeding produced larger sole than did a late weaning treatment; delayed weaning negatively affected fish growth. In the second experiment, the digestive capacity of early‐weaned 1, 2 and 4 mg sole was investigated. The highest growth was observed in sole weaned at 4 mg. Digestive enzyme profiles suggest that sole have an adaptation period to inert diets, with reduced feed intake. This adaptation period is inversely proportional to post‐larvae weight. The third experiment examined weaning with co‐feeding at different weights (2, 5 and 11 mg). These studies demonstrate that sole of 5–10 mg can be weaned, with high survival rates. On the basis of the digestive enzyme profiles, the early introduction of inert diets in co‐feeding with Artemia seems to affect intestinal processes in smaller postlarvae. This study also suggests that trypsin and alkaline phosphatase may be used as indicators of nutritional status in sole of <5 mg.     

The ability of carnobacteria isolated from fish intestine to inhibit growth of fish pathogenic bacteria: a screening study

In the present study a microtitre plate assay was used to evaluate antagonistic activity of 157 intestinal bacteria belonging to Carnobacterium isolated from Arctic charr (Salvelinus alpinus L.), Atlantic salmon (Salmo salar L.) and wolf fish (Anarhichas lupus L.) against fish pathogenic bacteria. One hundred and forty‐nine strains isolated from Arctic charr fed; (a) different lipid levels and (b) different fatty acids were screened for their ability to inhibit growth of the fish pathogen Aeromonas salmonicida ssp. salmonicida strain AL 2020 (the causative agent of furunculosis). Carnobacterium maltaromaticum and Carnobacterium mobile isolated from fish fed a low‐lipid diet inhibited growth of the pathogen, while none of the Carnobacterium divergens isolated from fish fed the high‐lipid diet had this ability. When Arctic charr was fed different fatty acids, was the frequency of antibacterial ability of C. maltaromaticum highest in strains isolated from fish fed 4%α‐linolenic acid (18:3 n‐3) and lowest in strains isolated from fish fed 4% linoleic acid (18:2 n‐6). Extracellular growth inhibitory compounds harvested in exponential and stationary growth phase from eight carnobacteria strains isolated from three fish species were tested for their ability to inhibit growth of six fish pathogens [A. salmonicida, Vibrio splendidus strain VS11, Vibrio salmonicida strain LFI 315, Vibrio anguillarum strain LFI 317, Moritella (Vibrio) viscosa strain LFI 5000 and C. maltaromaticum (piscicola) CCUG 34645]. The highest antibacterial activity was found when cellular extracts of the producer isolate were harvested in stationary growth phase. Scanning electron microscopy (SEM) investigations of A. salmonicida showed that cell morphology was affected by the inhibitory substance produce by strain 8M851, a Carnobacterium inhibens‐like bacteria.     

Feed intake and growth performance of Senegalese sole (Solea senegalensis Kaup, 1858) fed diets with partial replacement of fish meal with plant proteins

To be able to study nutrient requirement and utilization in any species, a diet supporting normal feed intake and growth equally well as a traditional fish meal‐based diet is needed. Additionally the formulation of the diet should allow low levels of the nutrient under study. When studying the amino acid metabolism and requirements, one cannot rely on the fish meal‐based diets as fish meal are nicely balanced according to requirements. Therefore the current study aimed to develop a plant protein‐based diet (with low fish meal inclusion) to be used in the nutritional studies of Senegalese sole juveniles supporting feed intake and growth close to that obtained in a fish meal‐based control feed. Two experiments were conducted to evaluate whether Senegalese sole juveniles would accept and utilize diets containing high plant protein inclusion. For testing the acceptance of high plant protein inclusion, two diets were formulated: a reference diet that contained fish meal as the main protein source (450 g kg^?1 dry matter) whereas in the test diet, fish meal was substituted by a mixture of plant ingredients (soybean meal, corn and wheat gluten) with l ‐lysine supplementation. In order to improve the palatability, 50 g kg^?1 squid meal was added to both diets. The indispensable amino acids (IAA) profile of the test diet was made similar to the control diet by adding crystalline amino acids. Further, automatic feeders were used to improve the feed intake. Fish (24 g initial body weight) were fed the diets for a period of 4 weeks. As fish accepted both diets equally well, a second study was undertaken to test the growth performance. Fish (6 g initial BW) were fed the diets for a period of 12 weeks. The use of automatic feeders to deliver the feed and the addition of both squid and balancing the indispensable amino acids resulted in growth performance and accretion not differing from the fish meal fed control. It can be concluded that juvenile Senegalese sole are able to grow and utilize high plant‐protein diets when both diet composition and feeding regime are adequate for this species.     

Effects of Protein and Lipid Levels in Practical Diets on Growth and Body Composition of Tongue Sole,Cynoglossus semilaevis Gunther

A 9‐wk feeding experiment was conducted to estimate the optimal dietary protein and lipid levels for tongue sole, Cynoglossus semilaevis Gunther (initial average weight of 43.8 ± 0.18 g). Six practical test diets were formulated to contain three protein levels (45, 50, and 55%, respectively) at two lipid levels (12 and 16%, respectively) with P/E ratios ranging from 87.1 to 110.5 mg protein/kcal. Each diet was randomly fed to triplicate groups of 20 fish per tank (1000 L). The results showed that fish fed the diet with 55% protein and 12% lipid (P/E ratio of 110.5 mg protein/kcal) had the highest thermal‐unit growth coefficient (TGC), feed efficiency ratio, protein productive value, and energy retention. TGC was significantly increased with increasing dietary protein levels irrespective of dietary lipid levels (P < 0.05). However, fish fed the diet with 16% lipid showed significant lower growth than fish fed the diet with 12% lipid. These results suggest that the diet containing 55% protein and 12% lipid with P/E of 110.5 mg protein/kcal is optimal for tongue sole and the increase of dietary lipid level has no effective protein‐sparing effect.     

Soy protein concentrate as a protein source for Senegalese sole (Solea senegalensis Kaup 1858) diets: effects on growth and amino acid metabolism of postlarvae

The objective of this work was to evaluate the effect of a dietary amino acid imbalance, originating from the use of a soy protein concentrate (SPC) as the major protein source, on the growth performance and amino acid metabolism of Senegalese sole (Solea senegalensis) postlarvae. Senegalese sole (85.6±24.6 mg wet weight) were fed one of two experimental diets: one based on fish meal (FM) and another based on SPC. Diets were isonitrogenous (around 56% crude protein) and isoenergetic. Diet acceptability was very good and the growth rate was 6.9% day^?1 for sole eating the FM diet and 6.0% day^?1 for sole eating the SPC diet. Mass‐specific ammonia excretion and the activities of selected amino acid metabolic enzymes (ALAT, ASAT and GDH) did not present significant differences between treatments, although this may have been due to the high variability found for these parameters in the SPC treatment. This variability may suggest different capacities of individual fish to adapt to the possible methionine dietary deficiency. The utilization of amino acids as a substrate for lipogenesis does not seem to be affected by the dietary protein source, since NAPDH‐generating enzymes (G6PD and ME) had similar activities in both treatments. Amino acid metabolism in Senegalese sole postlarvae seems to be slightly affected by the dietary protein source. Nevertheless, the changes induced by the SPC diet do not seem to impair growth, at least at the high dietary protein level used in this experiment.     

Effective replacement of protein‐bound amino acids by crystalline amino acids in Senegalese sole (Solea senegalensis) juveniles

The objective of this study was to evaluate the potential of free amino acids to replace protein‐bound amino acids in diets for Solea senegalensis juveniles. For this purpose, fish with 4.26 g mean initial weight were fed during 55 days with diets containing 500 g kg^?1 protein and 100 g kg^?1 lipid, based on fish meal and fish oil as main protein and lipid sources (control diet), or diets where 20%, 35% and 50% of fish meal were replaced by crystalline amino acids, using L‐lysine HCl as lysine source. To access the efficiency of the crystalline lysine source, another diet was formulated with 35% fish meal replacement but using L‐lysine sulphate instead of L‐lysine HCl as lysine source. Overall, growth performance, feed utilization, nutrient retention, whole‐body and amino acid composition did not show significant differences among groups. In conclusion, up to 50% protein‐bound amino acids can be replaced by crystalline amino acids in diets for Senegalese sole juveniles without negative effects in fish performance. Additionally, it was shown that L‐lysine sulphate and L‐lysine‐HCl may be used interchangeably by Senegalese sole juveniles, with the potential economic benefits involved.     

Microbiota of yellow grouper (Epinephelus awoora Temminck & Schlegel, 1842) fed two different diets

The rapidly growing yellow grouper industry has experienced relatively severe bacterial disease problems in China. The proliferation of pathogens in fish can be suppressed by commensal microbiota. In this background, we used nested polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) and sequence analysis to investigate microbiota in the skin, gills and intestines, including adherent bacteria and non‐adherent bacteria in yellow grouper fed with natural diet and complete feed. A total of 21 bacterial species were identified using phylogenetic analysis. The γ‐Proteobacteria group (81.0%, 17 species) dominated the bacterial communities in yellow grouper completely. Others belonged to Firmicutes (9.5%, two species), Actinobacteria (4.75%, one species) and Verrucomicrobia (4.75%, one species). The higher similarities (above 91%) of the DGGE band patterns in skin, gill and intestinal‐non‐adherent bacteria between two groups of fish indicated that existed more stable microbial communities existed in these specifically ecological niches in yellow grouper. However, considerable differences existed between two intestinal‐adherent bacteria (IAB) samples; that is, compared with natural diet fed yellow grouper, higher bacterial apparent species richness and possibly less abundance existed in IAB in fish fed with complete diets, probably indicating that the community structures in IAB were affected easily and significantly by diet.     

Effects of Bacillus subtilis Strains on Growth,Immune Parameters,and Streptococcus iniae Susceptibility in Nile Tilapia,Oreochromis niloticus

This study was conducted to evaluate the effects of probiotic‐amended diets fed to juvenile Nile tilapia, Oreochromis niloticus, on growth and susceptibility to Streptococcus iniae infection. Fish (average weight 16.5 ± 0.2 g) were fed five diets formulated with Bacillus subtilis strains SB3086, SB3295, SB3615, or AP193 either individually or in combination of strains SB3086 and SB3615 at a targeted concentration of approximately 4 × 10⁷ colony‐forming units (CFU)/g of feed or with a basal control diet with no additives for 21 d. After the 21‐d growth trial, no significant difference in growth performance was observed with any probiotic‐amended diet. Results from serum bactericidal activity showed a significant difference between treatments and the control (P = 0.0002), except for the SB3295‐amended diet (P = 0.9020). Lysozyme activity was also significantly different in fish fed probiotic diets from those fed control diet (P = 0.0001). After 21 d of feeding, fish were challenged with S. iniae by intraperitoneal injection at a dosage of 8 × 10⁶ CFU per fish. Results from the challenge also showed a significant difference between treatments and control (P = 0.0001). Overall, fish fed with strain SB3615 showed the lowest percent mortality (44.0 ± 7.2%) and those fed the control diet showed the highest mortality (77.3 ± 7.0%). The combined feeding with strains SB3086 and SB3615 did not result in any significant difference in reducing mortality because of S. iniae infection in juvenile Nile tilapia when compared with the individual probiotic treatments.