Effect of Lactococcus lactis and Enterococcus faecium on growth performance,digestive enzymes and immune response of grouper Epinephelus coioides

The effect of dietary administration of Lactococcus lactis MM1 and Enterococcus faecium MM4, two strains of lactic acid bacteria isolated from the gut of juvenile grouper Epinephelus coioides, on the growth, feed utilization, digestive enzymes and immune response of juvenile E. coioides was evaluated. The fish were fed for 60 days with three different diets: control diet (without probiotic), diet P1 supplemented with 1.0 × 10⁸ CFU g^?1L. lactis or diet P2 with 1.0 × 10⁸ CFU g^?1E. faecium. Although growth and feed intake decreased slightly, significant improvement of feed utilization was observed in fish fed diet P1 and diet P2 for 60 days. The hepatopancreatic protease activities in the two probiotic treatments were significantly higher than that in the control. The phagocytic activity, serum superoxide dismutase activity and complement component 4 (C4) and IgM levels were not significantly affected by diet P1 and diet P2. Serum complement component 3 (C3) levels in the two treatments were significantly higher than that in the control. Significant increased serum lysozyme activity was observed in fish fed the diet P1, while significant decreased lysozyme activity was observed in fish fed the diet P2 compared with the fish fed the control diet. There was a significant interaction effect between diet and administration date for the complement C4. The study demonstrates potential for L. lactis MM1 and E. faecium MM4 to improve the feed utilization and modulate the immune function in E. coioides.     

Effects of dietary live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.     

Dietary probiotic supplementation in growth and health of live-bearing ornamental fishes

Bacillus subtilis, a probiont isolated from the intestine of Cirrhinus mrigala (Hamilton), was incorporated in fish feed at four different concentrations (5 × 10⁸, 5 × 10⁷, 5 × 10⁶ and 5 × 10⁵ cells g^?1) and fed to Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) for a period of 90 days. The growth parameters, proximate composition of whole body, digestive enzymes and gut microbiology were monitored at regular monthly intervals. The increments in length and weight and the survival were significantly higher (P < 0.05) and the values of food conversions were significantly lower (P < 0.05) in fishes fed the probiotic feeds. The administration of B. subtilis in diet resulted in an increase (P > 0.05) in body ash and protein content and in the specific activity of protease and amylase in the digestive tract of all the fishes. The counts of total heterotrophs, motile aeromonads, presumptive pseudomonads and total coliforms were significantly lower (P < 0.05) in the gut of the probiotic fed fishes. Bacillus subtilis persisted in the fish intestine and in the feed in high numbers during the feeding period. The high survival and low infectivity recorded in the probiotic feed fed fishes when challenged by immersion assay with the pathogenic Aeromonas hydrophila demonstrate that the probiotic strain confers disease resistance in live‐bearing ornamental fishes. A probiotic concentration of 10⁶ to 10⁸ cells g^?1 is adequate for improved growth performance and survival and for healthy gut microenvironment and use of higher concentration of the probiont did not always lead to significantly better results.     

Assessment of the effects of vegetative and lyophilized Pediococcus acidilactici on growth,feed utilization,intestinal colonization and health parameters of rainbow trout (Oncorhynchus mykiss Walbaum)

A study was conducted to assess the probiotic effect of different dietary forms of Pediococcus acidilactici on rainbow trout (Oncorhynchus mykiss Walbaum). Growth performance, feed utilization, intestinal colonization and basic health status were investigated after a 10 week feeding trial. Fish were fed either vegetative (Veg) or lyophilized (Lyo) cells incorporated into a basal diet at either 10⁷ (Lo) or 10⁸ (Hi) CFU g^?1. P. acidilactici temporarily colonized the digestive tract (as both epithelium associated and transient populations) in all probiotic groups during supplemented feeding. Scanning electron microscopy confirmed the presence of localized colonization of P. acidilactici‐like cells between intestinal folds of the probiotic fed fish. Compared to the control group, no significant improvements in growth performance, feed utilization or carcass composition were observed in the probiotic fed fish (P > 0.05). However, a significant reduction of condition factor (K) was evident in fish fed the lyophilized diets. Increased leucocyte levels were observed in fish fed the low level vegetative P. acidilactici supplemented diet yet leucocyte types were not affected. The study demonstrates some potential for the application of P. acidilactici with rainbow trout but further research is required to optimize applications.     

Effect of probiotic on reproductive performance in female livebearing ornamental fish

A probiotic bacterial strain, Bacillus subtilis, isolated from the intestine of Cirrhinus mrigala (Hamilton), was incorporated in fish feed at four different concentrations (5 × 10⁸ cells g⁻², 5 × 10⁷ cells g⁻², 5 × 10⁶cells g⁻¹ and 5 × 10⁵ cells g⁻¹) and fed to four species of livebearing ornamental fish, Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) for one year duration to observe the effect of dietary probiotic supplementation on their reproductive performance. Sixty virgin females of each species were stocked separately in circular FRP tanks (350 L) and fed diets with varying levels of probiotic cells and control feed. Broodstock performance was evaluated based on gonadosomatic index (GSI), fecundity and fry production of female broodstock. The results showed that supplementation of feed with probiotics increased significantly (P<0.05) the GSI, fecundity and fry production of spawning females and length and weight of fry in all the four species of fish. The number of dead and deformed fry were also significantly lower (P<0.05) in fish fed with the probiotic feeds. The use of higher concentration of the probiont in diet did not always lead to significantly improved reproductive performance of the spawners. Collectively, this study showed that female livebearers benefit from inclusion of probiotics in diet during their reproductive stages.     

The viability of probiotic bacteria as a factor influencing the immune response in rainbow trout Oncorhynchus mykiss

The objective of the present study was to understand the immune response induced by different forms of the probiont Lactobacillus rhamnosus JCM 1136 in rainbow trout Oncorhynchus mykiss and to clarify the carry over effect after withdrawal of the probiotic diet. A prebiotic-based diet was formulated, which served as the control diet, and to it the selected lactic acid bacteria (LAB) was incorporated in one of the three forms: (1) heat-killed, (2) live-sprayed or (3) freeze-dried. A rearing trial was conducted in triplicate with juvenile rainbow trout (average 126 g), which were fed on the control and three other diets containing the respective forms of the probionts, two times daily until the satiation level for a period of 30 days, following which the test diets were withdrawn and control diet was fed up to 45 days. Sampling was done at 10, 20, 30 and 45 days for gut microflora composition and the non-specific humoral and cellular immune responses of the fish. The viable forms (live spray or freeze-dried) were found to induce better phagocytic activity and complement activity compared to that of the non-viable heat-killed form. The plasma immunoglobulin level showed an increasing trend in the fish groups that received the viable probiont, but the trend did not exist towards the end of the study. Upon withdrawal of the probiotic diets, the LAB disappeared from the intestine and the elevated immune parameters returned to the prefed level. This study elucidates that probiont viability could probably influence the immune responses they induce.     

Effects of dietary vitamin C on immunity, growth and survival of Indian major carp Labeo rohita, fingerlings

An 8‐week feeding trial was conducted to evaluate the efficacy of dietary l ‐ascorbic acid (AA) dosages on immunity, growth and survival of the fingerlings of Labeo rohita. AA was supplemented at 0, 100, 200 and 500 mg kg⁻¹ basal diets. Each diet was fed for 8 weeks to duplicate groups of fingerlings of rohu with initial body weight of 35 ± 5 g. Selected biochemical and haematological analyses were evaluated every second week. At the end of the feeding trial of 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens, Aeromonas hydrophila and Edwardsiella tarda. The mortality (%) and agglutinating antibody titre were recorded on 28th day postchallenge. Non‐specific immune parameters like superoxide anion production, lysozyme activity and complement activity increased independently of the levels of AA in the diet. Whereas phagocytic ratio, phagocytic indices, lymphokine production indices and serum bactericidal activity were better in the group of fish fed with 200 mg AA kg⁻¹ feed for 42 days. However, growth parameters such as specific growth rate, feed conversion ratio and survival were better in the group of fish fed with the highest level of AA through feed for 56 days. These data indicated that elevated levels of dietary AA could be recommended for optimum immunity, growth and survival of fingerlings of L. rohita.     

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.     

Supplementation of heat‐inactivated Bacillus clausii DE5 in diets for grouper,Epinephelus coioides,improves feed utilization,intestinal and systemic immune responses and not growth performance

In recent years, more and more attentions have been paid to the development and application of probiotics in aquaculture, and viable probiotics have been extensively studied, while rare information was available about inactivated probiotics in aquaculture. Therefore, in this study, a feeding trial was designed to investigate the effect of dietary supplementation of heat‐inactivated probiotic Bacillus clausii DE5 on growth performance, immune response and key immune genes expression in head kidney and intestine in grouper Epinephelus coioides. Fish were fed for 60 days with control diet (C) and two experimental diets containing 1.0 × 10⁸ CFU/g live (T1) and heat‐inactivated (T2) B. clausii DE5, respectively. The probiotic treatments did not affect the final weight, weight gain (WG) and specific growth rate (SGR) of E. coioides at days 30 and 60 (p > .05), while both heat‐inactivated and live B. clausii DE5 significantly decreased the feed intake and feed conversion ratio (FCR) at day 60 (p < .05). Serum lysozyme activity and complement C3 level in the two probiotic treatments were significantly higher than those in the control (p < .05). The lysozyme activity and complement C3 level at day 60 were significantly higher than those at day 30 (p < .05), while no significant interaction effect between diet and administration date was observed. Moreover, the heat‐inactivated B. clausii DE5 significantly improved the expression of TLR5, pro‐inflammatory cytokines (IL‐8 and IL‐1β) and TGF‐β1 in head kidney and intestine (p < .05), while the live probiotic did not show any significant effect on the expression of these key immune‐related genes in head kidney and intestine. These results indicate that dietary supplementation of heat‐inactivated B. clausii DE5 effectively improved feed utilization and both the local and systemic immune responses of E. coioides.     

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.     

Dietary probiotic supplementation in juvenile rainbow trout (Oncorhynchus mykiss) reared under cage culture production: effects on growth,fish welfare,flesh quality and intestinal microbiota

This study was conducted to evaluate the effects of dietary probiotic supplementation on growth performance, fish welfare, sensory evaluation, freshness during ice storage and changes in intestinal microbiota of rainbow trout (Oncorhynchus mykiss). The 9‐week feeding trial was conducted in cages under commercial conditions during the winter season, when water temperature decreased from 14°C to 7°C. Fish were fed either a diet without probiotic supplementation (control) or with 0.2% (2 × 10⁹ CFU kg^?1 diet) of multi‐strain probiotic bacteria (Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.). Fish weight was recorded at 0, 2, 4, 6 and 9 weeks. Growth performance was not affected by dietary probiotic supplementation. Alternative complement activity (ACH50) was significantly higher in fish fed the diet with probiotic (58.4 ± 3.7 units mL^?1) than in the control group (46.9 ± 3.1 units mL^?1) (P = 0.03). Several biochemical markers were altered by the dietary treatment. 7‐ethoxyresorufin‐O‐deethylase (EROD) (P < 0.01), glutathione S‐transferase (GST) (P < 0.001), glutathione peroxidase (GPx) (P < 0.05) increased and lipid peroxidation (LP) (P < 0.01) decreased with dietary probiotic supplementation. Flesh quality and shelf‐life of fish stored on ice was not affected by dietary probiotic supplementation. Probiotic supplementation had a limited effect on the intestinal microbiota. Signal strength of the observed operational taxonomic units (OTUs) differed between the control and treatment samples and could indicate variations in the abundance of certain OTUs. Microbiota adhering to the intestinal mucus varied between sampling points, indicating that changes were associated with water temperature.     

Dietary supplement of Enterococcus faecalis on digestive enzyme activities,short‐chain fatty acid production,immune system response and disease resistance of Javanese carp (Puntius gonionotus,Bleeker 1850)

This study investigated the effects of Enterococcus faecalis on digestive enzyme activities and short‐chain fatty acid production in fish intestine, resistance against Aeromonas hydrophila and humoral immunity response by 3 experiments on Javanes carp (Puntius gonionotus). The experiment 1 revealed that diet supplemented with E. faecalis significantly (P < 0.05) increased protease and lipase activities compared to control fed fish. Moreover, E. faecalis supplementation significantly enhanced the production of propionic and butyric acid in the intestine, while no significant difference (P > 0.05) in acetic acid production was observed. In the challenge study (experiment 2), fish were injected (intraperitoneal) with 10⁷ A. hydrophila per ml and survival was significantly improved when fish were fed diet supplemented with E. faecalis compared to control fish. In experiment 3, dietary E. faecalis affected immune system response as fish fed the probiont and exposed to 10⁶ A. hydrophila per ml displayed significantly elevated antibody levels compared to control fed fish. Fish fed diet supplemented with E. faecalis but not exposed to the pathogen revealed significantly higher antibody level than control fish (P < 0.05). Therefore, E. faecalis can be used as a probiotic in Javanese carp farming.     

Effects of dietary probiotic Lactobacillus plantarum and whey protein concentrate on the productive parameters,immunity response and susceptibility of Nile tilapia,Oreochromis niloticus (L.), to Aeromonas sobria infection

Immunostimulatory feed supplements have an increasingly interest in aquaculture management. Generally, an individual supplement was used in fish diets but it is expected that the use of multi‐supplements may show synergistic enhancements in fish performance, health, and immunity. Therefore, the present investigation was carried out to evaluate the use of dietary probiotic Lactobacillus plantarum and whey protein concentrate (WPC) in practical diets for Nile tilapia, Oreochromis niloticus. Hence, probiotic L. plantarum, WPC and their mixture were incorporated into a basal fish diet (300 g/kg crude protein) as follows: T1 = a basal control diet, T2 = a basal diet containing L. plantarum, T3 = a basal diet containing 1.0 g WCP/kg diet and T4, T5 or T6 = basal diets containing probiotic L. plantarum + 1.0, 2.0 or 3.0 g WCP/kg diet, respectively. Fish (15.2 ± 0.6 g) were fed on one of the tested diets up to apparent satiation twice a day for 60 days. After that, fish were intraperitoneally injected with pathogenic bacteria Aeromonas sobria and fish mortality was observed for 10 days postchallenge. Fish growth and feed intake were significantly improved by dietary probiotic L. plantarum (T2) and/or WPC (T3) over the control group (T1), and highest fish performance was observed in T5–T6 fish groups. Similarly, highest values of haematocrit, glucose, total proteins, albumin, and globulin were significantly observed in T5–T6 fish groups. Likewise, fish fed dietary probiotic L. plantarum (T2), WPC (T3), and their mixture (T4–T6) showed antioxidants and immune‐stimulating activities better than the control group. Fish fed the control diet were more susceptible to A. sobria infection showing highest fish mortality (75.0%). Meanwhile, dietary probiotic L. plantarum (T2), WPC (T3), and their mixture (T4–T6) enhanced significantly the fish resistance to A. sobria infection resulting in maximum values of relative percent of fish survival (73.3%–80.0%) in T5–T6 groups. The present investigation recommended the use of probiotic L. plantarum with 2.0 g WPC/kg diet to improve the growth, antioxidant, immunity responses and tolerance of Nile tilapia to A. sobria infection.     

Effects of an Enterococcus faecium‐based probiotic on growth performance and health of Pirarucu,Arapaima gigas

Intensive fish farming has resulted in an increased concern for disease outbreaks. Probiotic use is one of the strategies being developed to improve fish health and productivity. Measures of probiotic colonization, growth performance, haematological characteristics and parasite load were used to evaluate the effect of diets supplemented with Enterococcus faecium on growth and health of Arapaima gigas juveniles. A completely randomized design with four treatments (diet with E. faecium at 1 × 10⁶ CFU/g and 1 × 10⁸ CFU/g, control diet and diet with the culture medium MRS) and three replicates was used. Ninety‐six Arapaima juveniles were distributed in 12 cages fed with the specified diet for 68 days. Colonization of the intestinal tract by lactic acid bacteria reduced the total number of heterotrophic bacteria in fish fed with probiotics compared to controls. Fish fed a supplemented diet containing 1 × 10⁸ CFU/g presented higher values of weight gain, survival and fish growth uniformity, and lower values of feed conversion ratio. The prevalence of Trichodina sp. could have affected the survival of fish in the control group. Reduction in parasite load and an increase in haematocrit, the number of erythrocytes, thrombocytes, neutrophils and monocytes were also observed in fish fed the diet containing 1 × 10⁸ CFU/g. Enterococcus faecium presented a probiotic effect in A. gigas juveniles and can be recommended for use at a concentration of 1 × 10⁸ CFU/g to modify the gut microbiota, improve growth performance and haematology and reduce parasitic load.     

Autochthonous probiotic Lactobacillus sp. in the diet of bullfrog tadpoles Lithobates catesbeianus improves weight gain,feed conversion and gut microbiota

Dietary supplementation with probiotics in animal production is an alternative to antibiotics. In frog culture, studies involving native strains of probiotic bacteria and their effects on the performance and intestinal histology of farmed animals are scarce. Therefore, this study aimed to evaluate a diet supplemented with Lactobacillus sp. in tadpoles of Lithobates catesbeianus. This randomized test was performed with two dietary treatments: non‐supplemented control diet and diet supplemented with Lactobacillus sp., with nine replications. The growth performance of Lactobacillus sp., including its bacterial enzymatic activity and stability in feed, as well as colonization and histology of the intestinal tract, was evaluated after 42 days of experimentation. Animals fed with a supplemented diet showed higher weight gain and concentration of lactic acid bacteria in the gut and lower feed conversion. No significant difference was observed in survival, total heterotrophic bacterial count or histological change in the gut between the two treatments. The Lactobacillus sp. strain was able to colonize the intestinal tract and feed and remain at a high concentration of 10⁷ and 10⁶ CFU g^?1, respectively. It produced several enzymes, which might have contributed to the greater weight gain and lower feed conversion in the supplemented animals, thus demonstrating its probiotic potential for use as a dietary supplement in bullfrog tadpoles.     

Haematologic and immunologic parameters of bullfrogs,Lithobates catesbeianus,fed probiotics

The effects of two probiotics (P₁–Lactobacillus acidophilus, Bifidobacterium bifidum and Enterococcus faecium and P₂–Bacillus subtilis) supplemented to commercial feed (40% crude protein) on the haematological and immunological parameters of the bullfrog Lithobates catesbeianus were studied. Two doses of each probiotic (5 and 10 g kg^?1 of food) were added to the diets and fed to frogs, totalling five treatments over 112 days. Haematological analyses consisted of total and differential leucocyte counts, erythrocyte and thrombocyte counts, haematocrit, haemoglobin levels and RBC indices (mean corpuscular volume, mean corpuscular haemoglobin – and mean corpuscular haemoglobin concentration) and the immunological parameters included phagocytic capacity and phagocytic index of peritoneal phagocytes. The results showed that the probiotics did not significantly influence any of the haematological parameters measured. However, immunological assays showed that the probiotics had an immunostimulating effect. The greatest effects were seen with probiotic P₁ fed at a dose of 10 g kg^?1 of diet and probiotic P₂ fed at 5 g kg^?1 of diet.     

Isolation and characterization of Bacillus spp. M001 for potential application in turbot (Scophthalmus maximus L.) against Vibrio anguillarum

The aim of this study was to screen Bacillus strains from the guts of Scophthalmus maximus, Paralichthys olivacues, Epinephelus coioides and Clupanodon punctatus, for use as probiotics in aquaculture. Eight Bacillus strains were screened, and strain M001 was selected for probiotic study based on its antagonistic activity against multiple aquatic bacterial pathogens including Vibrio anguillarum, V. campbellii, V. vulnificus, V. parahamolyticus, Streptococcus sp. and Edwardsiella tarda. M001 was identified as B. amyloliquefaciens based on the biochemical tests and 16S rRNA gene analysis. In vitro experiments revealed that M001 was able to grow at a wider range of temperature, pH and salinity and was capable to use turbot mucus as nutrient for growth. Additionally, M001 isolate greatly inhibited the growth of V. anguillarum by producing antibacterial substances and was acid tolerance, non‐antibiotic resistance and non‐harmful. Thereafter, the potential probiotic effect of M001 was tested in turbot by dietary administration of M001 at a dose of 10⁸ CFU g^?1 diet for 42 days. No significant differences of weight gain, specific growth rate and feed ratio were found in the M001‐diet group of fish compared with control fish, but which increased, respectively, by 5.5%, 4.7% and 7.0% after 42 days of feeding. Several digestive enzyme activities were found to increase significantly in the M001‐diet group, including protease and amylase activities in hepatopancreas, protease activity in intestine and lipase activity in stomach (P < 0.05). Sera superoxide dismutase activity and total protein content (P < 0.05) were also increased significantly (P < 0.05) in the M001‐diet group. The challenge experiment showed that the M001‐diet group of fish showed a relative per cent of survival of 62.7% against V. anguillarim infection. The Bacillus M001 identified from this study has good potential to provide vibriosis control as probiotic feed additive for turbot aquaculture.     

Aqualase®, a yeast‐based in‐feed probiotic,modulates intestinal microbiota,immunity and growth of rainbow trout Oncorhynchus mykiss

Yeast probiotics have great promise, yet they received little attention in fish. This study investigated the influence of Aqualase^®, a yeast‐based commercial probiotic composed of Saccharomyces cerevisiae and Saccharomyces elipsoedas, on health and performance of rainbow trout (Oncorhynchus mykiss). Probiotics were incorporated in the diets at three different inclusion levels (1%, 1.5% and 2%) and administered to the fish for a period of 8 weeks. After the feeding trial, intestinal total viable aerobic bacterial count was significantly higher in fish group that received 2% in‐feed probiotics. In addition, a significant increase in at least 11% in intestinal lactic acid bacteria population was observed in all probiotic‐fed groups. Total protein level and lysozyme activity in skin mucus were significantly elevated following probiotic feeding. Inhibitory potential of skin mucus against fish pathogens was significantly enhanced by at least 50% in probiotic‐fed groups. Humoral and cellular immune parameters were influenced by probiotic feeding and the effects were dependent on inclusion level. Digestive physiology was affected by in‐feed probiotics through improvement of intestinal enzyme activities. All growth performance parameters were significantly improved following probiotic administration specifically at inclusion rate 1.5% and above. Taken together, the results revealed that Aqualase^® is a promising yeast‐based probiotic for rainbow trout with the capability of modulating the intestinal microbiota, immunity and growth.     

Culture-independent characterization of the autochthonous gut microbiota of grouper Epinephelus coioides following the administration of probiotic Enterococcus faecium

Probiotics have been applied extensively in aquaculture and demonstrated a range of benefits, but the mechanisms mediating such responses remain largely unknown. A clear illustration of the effect of probiotics on the autochthonous gut microbiota is the first step in understanding the underlying mechanisms. In this study, the autochthonous microbiota in the foregut, midgut and hindgut of juvenile grouper Epinephelus coioides following the dietary administration of probiotic Enterococcus faecium MM4 for 60?days were assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) with subsequent sequencing analysis. Generally similar DGGE patterns, with 15 common bands, were observed in the foregut, midgut and hindgut of fish fed the probiotic diet or the control diet, while more visible bands were present in fish fed the probiotic diet. The similarity dendrogram showed that the foregut samples formed an independent cluster distinctly different from the midgut and hindgut samples, but a high similarity (81.4%) was observed among the three gut sections. Significantly increased species richness and Shannon index were observed in the midgut and hindgut samples in the probiotic group compared with the control group, which suggested that probiotic E. faecium could elevate the autochthonous microbial diversity in the midgut and hindgut. Sequences analysis showed that probiotic E. faecium stimulated the growth of some unidentified bacteria and potentially beneficial bacteria, while exerted no apparent effect on potentially harmful Staphylococcus saprophyticus.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.