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A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (Paralichthys olivaceus) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 10 8 -10 9  CFU kg -1 of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the Lactobacillus abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In recent years, Bacillus spp. have garnered attention as probiotic supplements in aquafeed owing to the production of heat stable and low pH resistant spores. Herein, we isolated and characterized an autochthonous Bacillus licheniformis KCCM 43270 from the intestine of olive flounder (Paralichthys olivaceus) for supplementation in low-fishmeal extruded aquafeeds. The KCCM 43270 was screened based on amylase, protease, cellulase, and lipase as well as non-hemolytic activities. The isolate was able to grow in carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) when used as a single carbon source in the minimal nutrient M9 medium. The KCCM 43270 spores displayed complete survival in acid (pH 2.5) and bile (0.3%, w/v) for 3 hr, strong biofilm formation, and nearly 50% adhesion with intestinal mucus. The spores of the isolate also showed significant survival ability at 80, 90, 100°C for 60, 30, and 1 min, respectively. In addition, the spores in a blend of SBM complex carrier showed significant heat stability at 120°C for 5 min and under different drying conditions. Furthermore, the spores also survived the extrusion process during low-fishmeal aquafeed manufacturing, implying the potential application of B. licheniformis KCCM 43270 in aquafeed industry., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Bacillus sp. SJ-10 (BSJ-10) was identified from traditional Korean fermented fish, the previously recognized prebiotic β-glucooligosaccharides (BGO), and their combination as a synbiotic were prepared to evaluate their individual and synergistic effects in olive flounder (Paralichthys olivaceus). Four diets (one control and three treatments) were formulated containing neither BSJ-10 nor BGO (control), 1 × 10 8  CFU g -1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 10 8  CFU g -1 BSJ-10 + 0.1% BGO (BSJ-10 + BGO). Triplicates of 15 fish (weight 10 ± 0.25 g) were randomly allocated to the four diet groups and fed one of the diets for 8 weeks. At the end of the experiment, fish weight gain (WG), specific growth rate (SGR), feed conversion ratio, and protein efficiency ratio in BSJ-10, BGO and BSJ-10 + BGO diets were positively modulated (P < 0.05) compared with control. Specially, WG and SGR were significantly (P < 0.05) higher in BSJ-10 + BGO than that of BSJ-10 and BGO (individual component). The innate immune parameters such as respiratory burst, superoxide dismutase, and lysozyme activity (LSZ) of fish fed BSJ-10 and BSJ-10 + BGO (both groups) were significantly (P < 0.05) higher than the control. Moreover, myeloperoxidase activity (MPO) and LSZ of fish fed BSJ-10 + BGO were significantly higher compared with individual component. Compared with control, intestinal BSJ-10 content, expression of interleukin (IL)-1β in liver and kidney, and tumor necrosis factor (TNF)-α in liver were higher in both groups, but microvillus length was increased (P < 0.05) only in BSJ-10 + BGO. During in vivo challenge experiment with Streptococcus iniae (1 × 10 8  CFU ml -1 ), survival rate of fish was significantly higher in all treatment groups versus control. Moreover, in BSJ-10 + BGO, protection against S. iniae infection and transcription of TNF-α and IL-6 in gill were significantly (P < 0.05) higher than the individual component. Collectively, an improved WG, SGR, MPO, LSZ, transcription of IL-6 and TNF-α, and cumulative survival rate against streptococcosis clearly demonstrates a synergistic outcome of diet BSJ-10 + BGO as synbiotic in olive flounder., (Copyright © 2018. Published by Elsevier Ltd.)

Simple Summary: The marbled flounder (Pseudopleuronectes yokohamae) is a fish species with high commercial value in Korea and has been gaining attention as a new aquaculture target. To clarify the digestive physiology and feeding habits of the marbled flounder, this study conducted morphological, histological, and histochemical analyses of its digestive tract. In conclusion, the feeding habits of marbled flounder are close to those of carnivorous fish. In addition, we found that the marbled flounder has a stomach with a structure suitable for eating small amounts of hard food, and that most of its digestion takes place in the anterior intestine portion. Our study presents the histological and histochemical characteristics of the digestive tract of the marbled flounder, providing basic knowledge for both physiological and nutritional studies. This study investigated the morphological, histological, and histochemical characteristics of the digestive tract of the marbled flounder (Pseudopleuronectes yokohamae). The relative length of the gut of the marbled flounder digestive tract was 1.54 ± 0.10 (n = 20), and it had a simple stomach and 6–9 pyloric caeca. The mucosal folds of the marbled flounder digestive tract exhibited a general branched morphology. The thickness and mucosal fold length of the intestinal muscularis externa showed similar aspects in all areas. The thickness of the intestinal muscularis externa was the thickest in the posterior intestine portion, and the length of mucosal folds was the longest in the anterior intestine portion. It was indicated that food digested by gastric acid in the stomach moves to the anterior portion (including pyloric caeca) and mid portion of the intestine, ensuring effective stimulation of cholecystokinin (CCK)-producing cells. In addition, the distribution pattern of CCK-producing cells in the intestine was very similar to that of mucus-secreting goblet cells. The CCK-producing cells and goblet cells in the marbled flounder were well-adapted to promote optimal control of the digestive process. Based on the morphological and histochemical studies, it was concluded that the marbled flounder displays a digestive tract comparable to that of fish species with carnivorous habits. [ABSTRACT FROM AUTHOR]

The attractiveness of crude feed ingredients to olive flounder and effects of including feed ingredients showing strong feeding attractiveness in extruded pellet (EP) on performance of fish were determined. Four types of experimental EP were prepared to evaluate the effects of feed ingredient manipulation on performance of fish. Five per cent anchovy meal component of the control diet was substituted with an equal amount of jack mackerel meal, sardine meal and hydrolysed fish meal to create the JM, SM and HFM diets respectively. The diets were prepared in commercial form as EP. Jack mackerel meal produced the strongest feeding attractant responses of olive flounder, followed by sardine meal and hydrolysed fish meal. The greatest weight gain, feed consumption and condition factor (CF) were observed in fish fed the JM diet, followed by fish fed the SM, HFM and control diets, in that order in the 8‐week feeding trial. The dietary manipulation of feed ingredients performed in this study showed that the inclusion of ingredients with strong feeding attractiveness improved feed consumption and eventually accelerated the growth performance and CF of the fish. [ABSTRACT FROM AUTHOR]

Prebiotics, probiotics, and synbiotics are considered natural functional food ingredients and an alternative feeding strategy for immunomodulation and antibiotic eradication. Olive flounder (Paralichthys olivaceus) is commercially cultured in China, Japan, and Korea. Antibiotics used in flounder aquaculture produce antibiotic-resistant pathogens and residual effects on human. Prebiotics are non-digestible oligosaccharides, fermented by intestinal microbiota to produce short-chain fatty acids that bind G protein receptors. Probiotics are usually incorporated into the diet at a certain concentration to alter the intestinal microbial population through colonization or implantation. Synbiotics are combinations of prebiotics and probiotics that produce synergistically better benefits than separately. Previous studies on olive flounder have revealed that various types of immunostimulants, prebiotics, probiotics, synbiotics, and potentially immunoreactive feed additives can increase growth and feed utilization; cellular and humoral immunity; immune gene expression; blood/serum biochemistry; and infectious-disease resistance. Additive concentrations, their activity, and viability after feed storage and in the intestine, and interactions with the intestinal microbial community are major limiting factors. This review summarizes and discusses changes in growth and feed utilization, innate immunity, biochemical parameters, gene transcription, and disease protection in olive flounder after diet supplementation with various types of additives. It also indicates areas needing greater attention for future research. [ABSTRACT FROM AUTHOR]

Abstract: This study was conducted to investigate the influence of dietary lipid level and supplemental soy lecithin on growth, feed utilization and body composition of juvenile flounder reared at two suboptimal water temperatures, 12 and 17 °C. Three isonitrogenous (CP 50%) diets containing 1% squid liver oil (S7), 7% squid liver oil (S14), and a mixture of 2% soy lecithin and 5% squid liver oil (SL14) were formulated to obtain 7% and 14% of crude lipid. Triplicate groups of fish (6.3±0.2 g) were fed to apparent satiation twice a day for 60 days. Weight gain, daily feed intake, feed efficiency and protein efficiency ratio were significantly higher for fish reared at 17 °C than at 12 °C. Daily feed intake was significantly decreased with the increase of dietary lipid level at the same water temperature, but weight gain was not affected by dietary lipid composition. At 12 °C, feed efficiency and protein efficiency ratio were significantly higher for fish fed the S14 and SL14 diets than those of fish fed the S7 diet, while at 17 °C feed efficiency and protein efficiency ratio of fish fed the S14 diet, which were not significantly different from those of fish fed the SL14 diet, were significantly higher than those of fish fed the S7 diet. There were no significant effects of dietary lipid composition or temperature range on the whole body contents of moisture, crude protein, lipid or ash. Dietary lipid composition and temperature affected the fatty acid content of the polar lipid from the liver; and the n-3HUFA contents such as 20:5n-3 and 20:6n-3 were higher at 12 °C than at 17 °C. The results of this study indicate that an increase of dietary lipid level from 7% to 14% has beneficial effects on feed utilization of flounder when reared in suboptimal water temperatures. The increase of water temperature from 12 to 17 °C improved growth and feed utilization, but 2% soy lecithin had no substantial effect on growth and feed efficiency of juvenile flounder. [Copyright &y& Elsevier]

Abstract Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g−1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1β, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 108 CFU mL−1), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis. Highlights • Heat killed Bacillus sp. SJ-10 (HKBSJ-10) positively improved growth and feed utilization in olive flounder. • HKBSJ-10 enhanced humoral innate immunity and pro-inflammatory cytokine transcriptions. • Administration of HKBSJ-10 could increase resistance against streptococcosis. [ABSTRACT FROM AUTHOR]

The effects of Bacillus sp. supplementation of different types of cells, such as spores and vegetative cells, on fish growth performance, intestinal microbial diversity alteration and immunity in juvenile olive flounder (Paralichthys olivaceus) were investigated. The fish were divided into three groups and provided with either a basal diet (control), Bacillus sp. vegetative cell (VCS) or spore (SS) supplemented diet for 8 weeks. At the end of the experiment, significant differences (P <.05) were found in terms of weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio between the VCS and control groups. Respiratory burst, superoxide dismutase activity, and lysozyme activity, which are innate immune indicators, were observed with positive alterations (P <.05) in SS compared to controls. Intestinal microbial diversity analysis showed that the SS group had an increased richness estimate (Chao 1, ACE, and Jackknife) compared with the control group. Diversity estimates (Shannon and reverse Simpson) revealed that the VCS group was increased compared with the control group. Analyzing the similarity between groups through beta-diversity, Venn diagram, and heatmap analysis, the control and VCS groups were similar, and the SS group was different. IL-1β and TNF-α expression levels in the liver and spleen were increased in the SS group compared with the control group. In growth-related gene expression analysis, only growth hormone increased in the VCS group compared with the control group. In an in vivo challenge experiment with Streptococcus iniae (1 × 108 CFU/mL), the survival rates of the VCS and SS groups were 14.29% and 28.57%, respectively, when the control mortality reached 100%. Therefore, we concluded that supplementation of Bacillus sp. with different physiological processes affects the growth performance and immune activity of flounder. In particular, VCS can induce growth performance and growth-related gene expression, and SS can alter nonspecific immune and immune-related gene expressions. • The effect of dietary supplementation is affected by the Bacillus physiological process. • Dietary Bacillus vegetative cell supplementation improves growth performance. • Bacillus spore supplementation affects the intestinal microbial composition. • Nonspecific immunity is altered by Bacillus spore supplementation. • Pro-inflammatory cytokines are upregulated by dietary spore supplementation. [ABSTRACT FROM AUTHOR]

The overall performance and well-being of juvenile olive flounder were evaluated in response to three isonitrogenous and isolipidic extruded feed pellets containing either wheat flour (WF), potato (PS) or corn starch (CS), as a dietary carbohydrate source. Following the 2-week acclimation period, triplicate groups of fish (16.6 ± 0.01 g) were assigned to each experimental diet and fed to apparent satiation for 8 weeks. The results of the extruded feed pellet's physical quality tests revealed that dietary inclusion of PS could significantly enhance the water stability of the extruded feed pellets. In addition, PS and CS diets had significantly higher bulk density than WF diet. Feeding juvenile flounders with these experimental diets, however, did not induce any significant changes in their overall performance. Significantly higher serum lysozyme activity was recorded in fish fed the PS and CS diets than those fed the WF diet. Fish fed the WF and CS diets had significantly higher superoxide dismutase activity than PS group. Expression of the immune-related genes, including SOD2, EPX-L, TNF-L, IL-6, and TIPRL, was notably up-regulated in liver of fish fed the PS and CS diets compared to those fed the WF diet. Interestingly, however, an inverse expression pattern was found for the same genes in the spleen where lower expression levels were detected in the PS and CS groups than the WF group. Overall, our findings revealed that while dietary utilization of PS produced an extruded feed pellet with markedly higher physical quality than either WF or CS, neither of these starch sources were able to significantly impact the flounder's performance. Further studies are needed to elucidate the potential long-term consequences of the invoked immune response in the PS and CS groups. • Overall performance and well-being of juvenile olive flounders were evaluated in response to the extruded feed pellets containing either wheat flour, potato starch or corn starch as dietary starch sources. • Diets containing potato starch showed significantly higher water stability than the other two experimental diets. • Dietary inclusion of either potato or corn starch in the extruded diet significantly affected immune-related genes expression in the liver and spleen of flounders. • The expression patterns of the target genes involved in the immune response were opposite in the liver and spleen after feeding with different starch sources. [ABSTRACT FROM AUTHOR]

This study was carried out to investigate the effect of dietary differences on intestinal microbial population alteration and its relationship to immune-related gene expression in juvenile olive flounder (Paralichthys olivaceus). Initially, flounder were divided into three groups and supplemented with fishmeal diet (FM), fishmeal replaced by plant protein (termed low-fishmeal (LFM)), and Bacillus sp. SJ-10 probiotic-inoculated LFM (Pro) for 4 weeks. At the start (Initial group) and after completing the feeding trial, intestines were subjected to microbiota analysis and immune-related gene expression. The results of the intestinal microbiota analysis demonstrated that the FM and LFM groups' microbial richness (Chao1, ACE, and Jackknife) and diversity (Shannon and Simpson) did not differ significantly (P >.05) compared to the Initial group. However, the Pro group exhibited increased (P <.05) richness compared to all other groups, but its diversity was lower than that of the Initial group. A similar alteration phenomenon was also found in community composition between these two groups. At the phylum level, the most abundant phylum was Proteobacteria for all groups, but the second most abundant phylum in the Initial and Pro groups was Bacteroidetes, whereas in the FM and LFM groups, it was Firmicutes. An additional analysis of genus-level community composition and prediction of functional composition analysis showed differences among the groups. In the intestine, the transcription of interleukin (IL)-10 and IL-1β was increased more in the LFM and Pro groups compared to the FM group. IL-6 was increased only in the Pro group, and tumor necrosis factor-α (TNF-α) gene expression did not differ significantly among the groups. Therefore, we concluded that dietary changes affect intestinal microbiota composition and immune-related gene expression; in particular, diets containing probiotics can lead to greater change during the same feeding period. • Dietary changes affect intestinal microbiota composition and immune-related gene expression of olive flounder. • Probiotic have a greater impact on intestinal microbial diversity and composition than changes in fishmeal content. • Feed composition changes and probiotic supplements can manipulate the intestinal microbiota of olive flounder. [ABSTRACT FROM AUTHOR]

In an eight-week feeding trial, encapsulated sodium butyrate (ESB), gamma-aminobutyric acid (GAB), selenium-yeast (SeY), yeast extract nucleotides (YEN), yucca meal (YUM), song-gang stone (SGS) and protease (PRT) were evaluated as functional feed additives in a low fishmeal (FM) diet for olive flounder Paralichthys olivaceus. Eight experimental diets were fed to 5.26 ± 0.17 g (mean ± SD) juvenile olive flounder in triplicates. A low-FM basal diet without feed additive was considered as control (CON); seven other diets were prepared by adding 4 g ESB, 158 mg GAB, 2 g SeY, 4 g YEN, 1.5 g YUM, 4 g SGS, and 175 mg PRT per kg of basal diet. Weight gain, specific growth rate, feed efficiency, and protein efficiency ratio of fish fed diets YEN, GAB, and SGS were significantly higher than those of fish fed CON diet (P <.05). Superoxide dismutase activity of fish fed YEN and myeloperoxidase (MPO) and nitroblue tetrazolium (NBT) of fish fed YEN, GAB, SGS, ESB, and YUM were significantly higher than those of fish fed CON diet. Whereas, there were no significant differences in non-specific immune responses among fish fed YEN, GAB, SGS, ESB, and YUM and diets (P >.05). Heat shock protein 70 gene expression of fish fed YEN and SeY was significantly higher than those of fish fed the CON diet. Intestinal villi length of fish fed YEN, GAB, SGS, and ESB was significantly higher than those of fish fed CON. Also, intestinal trypsin activity of fish fed YEN, GAB, SGS, ESB, and PRT was significantly higher than those of fish fed CON diet (P <.05). After seven days of challenge test, fish fed diets YEN, GAB, SGS, PRT, and YUM had higher cumulative survival than those of fish fed CON diet. In conclusion, YEN, GAB, and SGS could have beneficial effects on growth and feed utilization performance, MPO and NBT activity, intestinal villi length, intestinal trypsin activity, and cumulative survival of olive flounder fed low-FM diets. • Functional feed additives such as YEN, GAB, and SGS can enhance growth performance of olive flounder • YEN, GAB, and SGS improved intestinal villi length, trypsin activity, non-specific immune responses, and disease resistance • Selective functional feed additives (YEN, GAB, and SGS) can be considered cost-effective solutions in low fish meal diets [ABSTRACT FROM AUTHOR]