The effects of two dietary synbiotics on growth performance, haematological parameters, and non-specific immune responses in Japanese eel (Anguilla japonica).

Feed additives have attracted increased attention in aquaculture due to their ability to modulate fish gut microbiota, resulting in improved fish growth and immunity. This study assessed two synbiotics' effects in Japanese eels: Bacillus subtilis with mannooligosaccharide (MOS) and Enterococcus faecium with fructooligosaccharide (FOS). Six diets, including a control (CON), oxytetracycline (OTC), and four synbiotic diets - B.subtilis at 1 × 106 and 107 CFU/g with 5 g/kg MOS (BS6MO and BS7MO) and E. faecium at 1 × 106 and 107 CFU/g with 5 g/kg FOS (EF6FO and EF7FO) - were fed to triplicate groups of 20 fish averaging 6.00 ± 0.07g for eight weeks. Fish fed the BSMOS diets showed significantly higher weight gain (WG), specific growth rate (SGR), and feed efficiency (FE) compared to the CON and OTC diets (P < 0.05), but not significantly different from those fed the EFFOS diets. Weight gain, SGR of fish fed EFFOS were not significantly different from those fed all other diets (P > 0.05). Fish fed the OTC diet showed a higher mean aspartate aminotransferase (AST) level, though the difference was not statistically significant. The myeloperoxidase (MPO) activity of fish fed the BS7MO diet was significantly higher than in all other diets, and the superoxide dismutase (SOD) activity of fish fed the BS7MO diet was also significantly higher than in the EF7FO diet. Overall, the BSMOS synbiotic diets were significantly more effective than the CON diet in enhancing fish survival against Vibrio anguillarum. Our findings suggest that synbiotics can be a preferable alternative to antibiotics in aquaculture.

Evaluation of Yeast Hydrolysate in a Low-Fishmeal Diet for Whiteleg Shrimp (Litopenaeus vannamei).

An eight-week feeding trial was performed to evaluate the effects of yeast hydrolysate (YH) supplementation in a low-fishmeal diet on the growth, immune responses, intestinal histology and disease resistance of whiteleg shrimp (Litopenaeus vannamei). Five experimental diets were produced by supplementing YH at 0 (CON), 0.5 (YH0.5), 1 (YH1), 2 (YH2) and 4 (YH4) % to a basal diet containing 10% fishmeal and compared with a positive control with 25% fishmeal (FM25). Shrimp with an initial average weight of 0.43 ± 0.005 g (mean ± SD) were stocked in 18 tanks and fed the experimental diets (38% protein and 8% lipid) four times a day. Results showed that shrimp fed the FM25 diet exhibited significantly higher final body weight, weight gain, specific growth rate and protein efficiency ratio than those fed CON, YH0.5, YH1 and YH2 diets (p < 0.05). However, there were no significant differences between shrimp fed the YH4 and FM25 diets (p > 0.05). In addition, there were no significant differences in whole-body proximate composition, hemolymph biochemical parameters and non-specific immune responses among treatments. Intestinal villi length and muscular layer thickness of shrimp fed the YH4 and FM25 diets were significantly higher than the other groups. At the end of the bacterial (Vibrio parahaemolyticus) challenge test, shrimp fed YH4 and FM25 diets showed a significantly higher survival rate than those of shrimp fed CON, YH0.5 and YH1 (p < 0.05). These results suggest that supplementing 4% YH in diet containing 10% fishmeal could beneficially influence growth, intestinal morphology and disease resistance of whiteleg shrimp.

Effect of Four Functional Feed Additives on Growth, Serum Biochemistry, Antioxidant Capacity, Gene Expressions, Histomorphology, Digestive Enzyme Activities and Disease Resistance in Juvenile Olive Flounder, Paralichthys olivaceus.

An 8-week feeding trial was executed to evaluate the efficacy of four functional feed additives in replacing antibiotics in juvenile olive flounder, Paralichthys olivaceus, fed with a low-fish-meal diet. A basal diet without feed additives was used as a control (CON); other diets were formulated by supplementing 0.50% taurine (TW), 0.30% peptide (PT), 0.23% mineral water (MW), 0.35% yeast-extracted nucleotides (GRO), 0.35% GRO + 0.50% taurine (GROTW), 0.35% GRO + 0.30% peptide (GROPT) and 0.35% GRO + 0.23% mineral water (GROMW) into the basal diet; in addition, one diet was supplemented with oxytetracycline (OTC) at 0.5% as a positive control. Triplicate groups of 25 fish with an average weight of 5.15 ± 0.06 g (mean ± SD) were fed one of the nine experimental diets. At the end of the feeding trial, the weight gain, specific growth rate and protein efficiency ratio of fish fed the GRO, GROMW, GROPT and GROTW diets were significantly higher than those of fish fed the CON diet (p < 0.05). The feed efficiency of fish fed the GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the TW and OTC diets. However, the survival, hepatosomatic index, viscerosomatic index and condition factor of fish, as well as their whole-body proximate composition, were not significantly affected by the experimental diets (p > 0.05). The serum glutamic pyruvic transaminase of fish fed the GROPT diet was significantly lower than that of fish fed the CON diet. However, glutamic oxaloacetic transaminase, glucose and total protein were not significantly affected by the experimental diets (p > 0.05). The serum superoxide dismutase activity of fish fed the PT, TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON diet. The lysozyme activity of fish fed the PT, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON and OTC diets. The myeloperoxidase activity of fish fed the TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON, PT and MW diets (p < 0.05). The flounder growth hormone gene expression of fish fed the TW, GRO, GROMW, GROPT, GROTW and OTC diets was significantly higher than that of fish fed the CON, PT and MW diets (p < 0.05). The interleukin 1ß and interleukin 10 gene expressions of fish fed the GRO, GROMW, GROPT and GROTW diets were significantly higher than those of fish fed the CON, PT, TW and MW diets (p < 0.05). Intestinal histology showed a significantly higher villi length for fish fed the GRO, GROMW, GROPT and GROTW diets compared to that of fish fed the CON diet (p < 0.05). Digestive enzyme activities such as trypsin activity were significantly higher in fish fed the GROMW, GROPT and GROTW diets than those in the rest of the diet groups (p < 0.05). Amylase activity in fish fed the MW, GRO, GROMW, GROPT, GROTW and OTC diets was significantly higher than that of fish fed the PT, TW and CON diets (p < 0.05). On the other hand, the lipase activity of fish fed the TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON, PT, MW and OTC diets (p < 0.05). The cumulative survival rate of fish fed the PT, GROTW, GROPT and GROMW diets was significantly higher than that of fish fed the CON, TW and MW diets after thirteen days of the challenge testing. Overall, the results demonstrate that the GRO, GROMW, GROPT and GROTW diets could be beneficial feed additives to replace antibiotics in juvenile olive flounder fed low-fish-meal diets.

Evaluation of dietary fermented tuna by-product meal as partial replacement for unprocessed tuna by-product meal in fishmeal-based diets for juvenile olive flounder Paralichthys olivaceus.

This study examined the effects of feeding fermented tuna by-product (FTBP) on the growth, non-specific immune response, liver and intestinal morphology, and disease resistance of olive flounder Paralichthys olivaceus. Olive flounders (n = 20; 2 g) were randomly assigned into four dietary groups in triplicates. Fish were fed four test diets (50% crude protein; 10% crude lipid) for 10 weeks in which unprocessed tuna by-product (TBP) meal was replaced (on a protein basis) with 0% (FTBP0 as the control diet), 15% (FTBP15), 30% (FTBP30), and 60% (FTBP60) of FTBP protein. Results showed that growth performance, blood parameters, and proximate composition were not influenced by FTBP inclusion. Non-specific immune parameters such as superoxide dismutase activity in the FTBP30 and FTBP60 groups were significantly higher than in the FTBP15 and the control groups, whereas lysozyme and myeloperoxidase activities were not different. Liver histopathology revealed normal architecture in groups fed FTBP0 and FTBP15 diets although mild alterations were noted in the FTBP30- and FTBP60-fed groups. Intestinal villi height and muscular thickness were not significantly altered with FTBP inclusion. Moreover, higher cumulative survival rate was observed in the FTBP60-fed group than fish fed with the other diets following the 13-day challenge with E. tarda. Together, these results demonstrate that fermented tuna by-product meal could be included at 60% in olive flounder diets.

Dietary Supplementation with γ-Aminobutyric Acid Improves Growth, Digestive Enzyme Activity, Non-Specific Immunity and Disease Resistance against Streptococcus iniae in Juvenile Olive Flounder, Paralichthys olivaceus.

Recent research is increasingly shedding light on the important role that microbial metabolites such as γ-aminobutyric acid (GABA) play in the context of nutrition, cognition, immune function, and the modulation of the gut microbiome. Yet, very few trials were conducted to assess the effects of its supplementation on biomarkers of fish health. Therefore, an eight-week feeding trial was devised to evaluate GABA supplementation in juvenile olive flounder, (Paralichthys olivaceus). A total of 630 fish with an average weight of 4.90 ± 0.10 g (±SD) were randomly assigned to one of seven triplicate groups and fed a non-GABA supplemented diet (CON, with 92 mg/kg GABA content), a positive control with 4 g/kg oxytetracycline (OTC), and five other diets supplemented with 50, 100, 150, 200 and 250 mg/kg GABA (corresponding to a total GABA content of 154, 229, 282, 327 and 352 mg/kg, respectively). Growth, blood chemistry, nonspecific immunity, digestive enzyme activity and disease resistance were assessed. The results showed that 100 and 150 mg/kg GABA supplementation consistently yielded significant improvements (p < 0.05) in growth, intestinal amylase, serum lysozyme, and survival against infection with Streptococcus iniae. Based on polynomial analysis, the optimal supplementation level was determined to be 237 mg/kg. These results support GABA as an important functional feed additive in juvenile olive flounder.

Evaluation of Dietary Probiotic Bacteria and Processed Yeast (GroPro-Aqua) as the Alternative of Antibiotics in Juvenile Olive Flounder Paralichthys olivaceus.

We investigated the three probiotic bacteria and a processed yeast (GroPro-Aqua) as the replacers of antibiotics in juvenile olive flounder. A total of seven diets were used, that is, one basal or control (CON) diet; and six other diets, of which, three diets were prepared by supplementing probiotic bacteria such as Bacillus subtilis WB60 (BSWB60) at 1 × 108 CFU/g diet, Bacillus subtilis SJ10 (BSSJ10) at 1 × 108 CFU/g diet, and Enterococcus faecium SH30 (EFSH30) at 1 × 107 CFU/g diet; one diet with processed yeast (GRO) at 0.35% diet; and two other diets were supplemented with oxytetracycline (OTC) and amoxicillin (AMO) at 4 g/kg of each. Triplicate groups of fish (average 12.1 g) were fed one of the diets for eight weeks. At the end of the feeding trial, the fish that were fed the probiotic bacteria-supplemented diets had a significantly higher final weight, weight gain, and specific growth rate compared to the CON, OTC, and AMO diets. Fish that were fed the GRO diet had significantly higher feed efficiency and protein efficiency ratios than those of the fish that were fed the CON diet. Serum glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, glucose, and total protein were not affected by the diets. Lysozyme activity in fish that were fed the BSSJ10, BSWB60, and EFSH30 diets were significantly higher compared to the CON and OTC diets, whereas myeloperoxidase activity of fish fed the BSWB60 and EFSH30 diets were significantly higher than those of fish fed the CON and AMO diets. Flounder growth hormone gene expressions of fish that were fed BSWB60 and GRO diets were significantly higher compared to the CON, OTC, and AMO diets. The interleukin-1ß gene expression of fish that were fed the BSSJ10, BSWB60, EFSH30, OTC, and GRO diets was significantly higher than those of fish fed the CON diet. The interleukin-10 gene expression of fish that were fed the BSSJ10, EFSH30, and GRO diets was significantly higher than those of fish fed the CON and AMO diets. Posterior intestinal histology of fish showed significantly higher villi length in fish that were fed the BSSJ10, BSWB60, EFSH30, and GRO diets compared to the CON diet. After 15 days of challenge test with pathogenic bacteria Edwardsiella tarda, the cumulative survival rate of fish that were fed the BSSJ10, BSWB60, EFSH30, and GRO diets were significantly higher than those of fish that were fed the CON diet. Overall, the results indicate that dietary supplementation of B. subtilis (108 CFU/g diet), E. faecium (107 CFU/g diet), and processed yeast (GroPro-Aqua at 0.35% diet) could replace the antibiotics in terms of improving growth, immunity, gut health, and disease resistance in juvenile olive flounder.

Evaluation of Dietary Soluble Extract Hydrolysates with or without Supplementation of Inosine Monophosphate Based on Growth, Hematology, Non-Specific Immune Responses and Disease Resistance in Juvenile Nile Tilapia Oreochromis niloticus.

We performed an 8-week feeding trial to evaluate dietary soluble extract hydrolysates from fishery by-products, such as shrimp soluble extract (SSE) with or without inosine monophosphate (IMP), tilapia soluble extract (TSE) and squid soluble extract (SQSE), in juvenile Nile tilapia. A diet without feed additives was used as the control diet (CON); and five other experimental diets were formulated with 2% soluble extracts consisting of 100% SSE, 98% SSE + 2% IMP (SSEP2), 96% SSE + 4% IMP (SSEP4), 100% SQSE and 100% TSE. The diets were fed to 4.9 ± 0.07 g (mean ± SD) juvenile Nile tilapia in triplicate groups. The weight gain and specific growth rates of fish fed the SSE, SSEP2 and SSEP4 diets were significantly higher than those of fish fed the CON and SQSE diets. The superoxide dismutase activity levels of fish fed the SSE and SSEP4 diets were significantly higher than those of fish fed the CON, SSEP2, SQSE and TSE diets. Myeloperoxidase activity levels of fish fed the SSE and SSEP4 diets were significantly higher than those of fish fed the CON, SSEP2 and SQSE diets. Lysozyme activity levels of fish fed the SSEP4 and SQSE diets were significantly higher than those of fish fed the SSE and SSEP2 diets. Feed efficiency, protein efficiency ratio, survival rate, whole body proximate composition and hematological parameters were not significantly different among the groups. After ten days of challenge = against Aeromonas hydrophila, the cumulative survival rate of fish fed the SSE diet was significantly higher than those of fish fed the CON, SQSE and TSE diets. In conclusion, dietary shrimp soluble extract could improve the growth performance, non-specific immune responses and disease resistance in juvenile Nile tilapia, and inosine monophosphate did not add further benefits to this ingredient.

Evaluation of Potential Probiotics Bacillus subtilis WB60, Pediococcus pentosaceus, and Lactococcus lactis on Growth Performance, Immune Response, Gut Histology and Immune-Related Genes in Whiteleg Shrimp, Litopenaeus vannamei.

An eight-week feeding trial was conducted to evaluate the effects of different dietary probiotic supplements in juvenile whiteleg shrimp, Litopenaeus vannamei. A basal control diet without probiotics (CON), and five other diets by supplementing Bacillus subtilis at 107 CFU/g diet (BS7), B. subtilis (BS8), Pediococcus pentosaceus (PP8), and Lactococcus lactis (LL8) at 108 CFU/g diet, and oxytetracycline (OTC) at 4 g/kg diet were used. Whiteleg shrimp with initial body weights of 1.41 ± 0.05 g (mean ± SD) were fed with these diets. Growth of shrimp fed BS8 and LL8 diets was significantly higher than those of shrimp fed the CON diet (p < 0.05). Superoxide dismutase activity in shrimp fed PP8 and LL8 diets was significantly higher than that of shrimp fed the CON diet (p < 0.05). Lysozyme activity of shrimp fed probiotics and OTC diets significantly improved compared to those on the CON diet (p < 0.05). The intestinal histology showed healthier guts for shrimp fed the probiotic diets (p < 0.05). Immune-related gene expression in shrimp fed BS8, PP8 and LL8 diets was recorded as significantly higher than that of shrimp fed CON and OTC diets (p < 0.05). Also, results of the challenge test for 7 days and the digestive enzyme activity of shrimp fed BS8, PP8, and LL8 were significantly improved compared to those on the CON diet (p < 0.05). Therefore, these results indicated that L. lactis at 108 CFU/g could be an ideal probiotic for whiteleg shrimp, and also B. subtilis WB60 and P. pentosaceus at 108 CFU/g could improve the growth, immunity, histology, gene expression, digestive enzyme activity, and disease resistance, while replacing antibiotics.

Effects of Bacillus subtilis WB60 and Lactococcus lactis on Growth, Immune Responses, Histology and Gene Expression in Nile tilapia, Oreochromis niloticus.

An eight-week feeding trial was conducted to evaluate the effects of a basal control diet (CON), Bacillus subtilis at 107 (BS7) and at 108 CFU/g diet (BS8), Lactococcus lactis at 107 CFU/g (LL7) and at 108 CFU/g diet (LL8), and oxytetracycline (OTC) at 4 g/kg diet on Nile tilapia. Fish with initial body weight of 2.83 ± 0.05 g (mean ± SD) were fed two times a day. Weight gain, specific growth rate, feed efficiency, protein efficiency ratio and lysozyme activity of fish fed BS8, LL8 and LL7 diets were significantly higher than those of fish fed CON diet (p < 0.05). Superoxide dismutase and myeloperoxidase activity of fish fed BS8, LL8, BS7, LL7 and OTC diets were significantly higher than those of fish fed CON diet. Intestinal villi length and muscular layer thickness of fish fed BS8, LL8 and LL7 diets were significantly higher than those of fish fed CON and OTC diets. Also, heat shock protein 70 (HSP70), interleukin (IL-1ß), interferon-gamma (IFN-γ) and tumour necrosis factor (TNF-α) gene expression of fish fed BS8 and LL8 diets were significantly higher than those of fish fed CON diet. After 13 days of challenge test, cumulative survival rate of fish fed BS8 and LL8 diets were significantly higher than those of fish fed CON, BS7 and OTC diets. Based on these results, B. subtilis and L. lactis at 108 (CFU/g) could replace antibiotics, and have beneficial effects on growth, immunity, histology, gene expression, and disease resistance in Nile tilapia.

Synergistic effects of dietary supplementation of Bacillus subtilis WB60 and mannanoligosaccharide (MOS) on growth performance, immunity and disease resistance in Japanese eel, Anguilla japonica.

This study evaluated the synergistic effects of dietary Bacillus subtilis WB60 and mannanoligosaccharide (MOS) in juvenile Japanese eel, Anguilla japonica. Seven treatment diets were formulated to contain three different levels of B. subtilis (0.0, 0.5, and 1.0 × 107 CFU/g diet denoted as BS0, BS0.5, and BS1, respectively) with two MOS levels (0 and 5 g/kg diet denoted as M0 and M5, respectively), and one diet with oxytetracycline (OTC) at 5 g/kg diet. Each diet (BS0M0 (CON), BS0M5, BS0.5M0, BS0.5M5, BS1M0, BS1M5, and OTC) was fed to triplicate groups of 20 fish averaging 9.00 ±â€¯0.11 g (mean ±â€¯SD) for eight weeks. Average weight gain, feed efficiency, specific growth rate and protein efficiency ratio of fish fed the BS0.5M5 and BS1M5 diets were significantly higher than those of fish fed CON, BS0.5M0 and OTC diets (P < 0.05). Significant increases in the nonspecific enzymatic activities (e.g., lysozyme and myeloperoxidase) were detected from fish fed the BS0.5M5, BS1M5, and OTC diets compared to the CON, BS0.5M0, and BS0M5 diets (P < 0.05). Whereas, immunoglobulin M expressions were recorded significantly higher for fish fed the BS0.5M5 and BS1M5 diets compared to those of fish fed the other diets (P < 0.05). Also, heat shock protein 70 mRNA levels of fish fed BS0.5M5 and BS1M5 diets were significantly higher than those of fish fed the CON diet (P < 0.05). Histological observations of the intestinal morphology showed healthier gut for fish fed BS0.5M5 and BS1M5 diets than those fed CON, BS0M5, and OTC diets. Additionally, resistance to bacterial challenge with Vibrio anguillarum was recorded significantly lower for fish fed the CON diet than those fed other diets (P > 0.05). Therefore, the results for growth performance, non-specific immune responses, intestinal morphology, and disease resistance demonstrated that supplementation of B. subtilis at 0.5 × 107 CFU/g diet and mannanoligosaccharide at 5 g/kg diet could have beneficial synergistic effects in Japanese eel. The isolated probiotic from eel and the selected prebiotic could lead to the development of a specific and potential synbiotic in Japanese eel aquaculture.

Comparative evaluation of dietary probiotics Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 on the growth performance, immunological parameters, gut morphology and disease resistance in Japanese eel, Anguilla japonica.

The current experiment was conducted to evaluate and compare the efficacy of two different probiotics Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 in diet of Japanese eel, Anguilla japonica. Seven experimental diets were formulated to contain no probiotics (CON), three graded levels of B. subtilis at 106 (BS1), 107 (BS2), 108 (BS3) and L. plantarum at 106 (LP1), 107 (LP2), 108 (LP3) CFU/g diet. Twenty fish averaging 8.29 ± 0.06 g were distributed in to 21 aquaria and were randomly assigned to one of the experimental diets in triplicate groups. Average weight gain (WG), feed efficiency (FE), and protein efficiency ratio (PER) of fish fed B. subtilis at 107 (BS2) and 108 (BS3) CFU/g diet were significantly higher than those of fish fed other experimental diets (P < 0.05). Nonspecific enzymatic activities including lysozyme, superoxide dismutase (SOD), myeloperoxidase (MPO) from fish fed B. subtilis at 107 (BS2) and 108 (BS3) CFU/g diet were detected to be significantly higher than that from fish fed CON diet (P < 0.05). Whereas, level of intestine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 70, 90 (HSP70, 90) and immunoglobulin (IgM) from fish fed B. subtilis at 107 and 108 CFU/g diet were recorded to be significantly higher than those of fish fed other experimental diets (P < 0.05). Parameters for intestinal morphology and histology suggested a healthier gut for the fish fed108 (BS3) CFU/g diet as compared to other treatment groups. Whereas, results from the disease challenge test with bacteria Vibrio angulillarum showed significantly lower survival rate for fish fed CON diet than those of fish fed other experimental diets. Therefore, these results indicated that oral supplement of B. subtilis at 108 (BS3) CFU/g diet could be a more effective source of probiotic compared to L. plantarum in Japanese eel.