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This study analyzed the expression of growth‐related genes of olive flounder, Paralichthys olivaceus, fed a low‐fish meal (FM) diet to investigate the replacement of the FM diet in the field. The expression of growth hormone (GH)/insulin‐like growth factor (IGF) axis genes in the brain/pituitary/liver and plasma hormone concentrations were measured. A basal experimental diet was formulated using 63% FM and 12% soybean meal as the primary protein sources, and then two other diets were made by replacing 20% and 30% of the FM with soy protein concentrate, tankage meal, and poultry by‐products meal. Each diet was fed to duplicate groups of juvenile flounder (150 ± 3.0 g) twice a day. After 20 weeks, the plasma growth‐related hormone concentrations were similar between the control and FM20 groups. Moreover, GH/IGF axis gene mRNA expression in the brain/pituitary/liver was similar between the control and FM20 groups. Immunoblotting of muscle and liver showed the same pattern. This study showed the utility of replacing 20% of the FM diet and supports the possibility of field application. [ABSTRACT FROM AUTHOR]

Simple Summary: The demand for fishmeal is increasing due to aquaculture development, but the supply is unstable. This indicates the need to reduce the fishmeal content in the feed and develop an optimal fish-feed formulation through substitutes. However, most studies on reducing fishmeal content in feed were conducted at the laboratory level. In this study, the application of a low-fishmeal diet as feed to olive flounder was evaluated in terms of growth-related factors, digestive physiology, and microbiota raised for five months in a fish farm using four feed formulations- FM70 [control (CON), 70% fishmeal], FM45 (45% fishmeal), FM35A (35% fishmeal), and FM35B (35% fishmeal + insect meal). There was no difference in growth-related factors, digestive physiology, and gut microbiota diversity compared with the CON-fed fish. Therefore, reducing the fishmeal content of the feed by up to 35% does not adversely affect growth and physiological characteristics under farm conditions. In olive flounder (Paralichthys olivaceus), growth performance, expression of growth-related factors, digestive physiology, and gut microbiota were assessed under farm conditions in the fish fed diets with low levels of fishmeal. Four experimental diets were prepared, FM70 [control (CON), 70% fishmeal], FM45 (45% fishmeal), FM35A (35% fishmeal), and FM35B (35% fishmeal + insect meal), and fed to the fish for five months. The CON-fed fish had the highest plasma GH, but IGF-1 and hepatic IGF-1 mRNA expression of the olive flounder fed diets with low-fishmeal levels did not significantly differ among diets. The intestinal villus length, muscular thickness, and the number of goblet cells were statistically similar, and ocular examination of hepatopancreas showed no discernable difference in all experimental diets. The chymotrypsin content of FM35B-fed fish is significantly lower, but trypsin and lipase contents are similar. The diversity of gut microbiota did not differ among groups, although the FM35B group had a higher composition of Firmicutes. Thus, a diet with reduced fishmeal content and several alternative protein sources can be used as feed ingredients in feed formulation for olive flounder reared under typical aquaculture farm conditions. [ABSTRACT FROM AUTHOR]

A 20‐week feeding trial was performed to evaluate the optimum level of animal and plant protein sources as substitutes of fishmeal (FM) in the diets of sub‐adult olive flounder (Paralichthys olivaceus) under farmed conditions. Groups of ninety fish averaging 478 ± 4.7 g (mean ± SD) were randomly distributed into four treatments in triplicates. An FM‐based diet was used as control (CON) and three other diets replaced 20% (APP20), 30% (APP30) and 40% (APP40) of FM with animal (tankage meal and poultry by‐product meal) and plant (soy protein concentrate) protein sources. Weight gain, specific growth rate and protein efficiency ratio were not significantly different among fish fed all the experimental diets (p > 0.05). The feed efficiency of fish fed the APP40 diet was significantly lower than those of fish fed the other experimental diets (p < 0.05). Non‐specific immune responses such as superoxide dismutase activity and lysozyme activity of fish fed all the experimental diets were not significantly different (p > 0.05). Intestinal villi length of fish fed the CON diet was significantly higher than those of fish fed the other diets (p < 0.05). Based on the results of growth performances and the non‐specific immune responses, the dietary combination of animal and plant protein sources could replace up to 40% of FM in sub‐adult olive flounder. [ABSTRACT FROM AUTHOR]

A 12‐week feeding trial was designed to assess the probiotic potential of indigenous Bacillus amyloliquefaciens and/or Bacillus subtilis singly or in combination with Bacillus licheniformis in an extruded feed for olive flounder (Paralichthys olivaceus) juveniles. A high fish meal (FM) diet (control) and a low‐FM diet containing an alternative protein blend (30% FM replacement, FM30) were formulated. Three other experimental diets were prepared by inclusion of B. amyloliquefaciens (BA), B. subtilis (BS), or a mixture of B. amyloliquefaciens, B. subtilis, and B. licheniformis (BASL) into FM30 diet, with a final concentration of 106 CFU/g diet. Results indicated that the FM30 diet was well tolerated by flounder, and the overall performance was not affected by dietary treatments. Lysozyme activity and total immunoglobulin level were significantly reduced in flounders when fed with the FM30 diet compared with the BASL and BA diets, respectively. The Bacillus additives neither enriched the relative abundance of the corresponding Bacillus spp. in the relevant gut microbiota of olive flounder nor modulated the presumptive gene functions of the gut microbiome. Despite the absence of growth‐promoting effect, the tested probiotics could still be economically viable for use as immunostimulants in commercial flounder diets with partial FM replacement. [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]

Simple Summary: Increasing demand for an efficient and economic fishmeal feed for sustainable aquaculture has urged the aquafeed sector to seek an optimum fish-feed formulation. This study investigated the physiological response in olive flounder fed various fishmeal diets in a typical fish farm. The fish were farmed for 20 weeks, using the following experimental feeds: a control feed (CON), a replacement by 20% (F20), and 30% (F30) of the fish meal content of the CON. All groups showed no significant difference in growth and survival rates. However, due to investigating hormone expression associated with maturation, high expression of PSS-I and low expression of FSH-β, ER-α, and ER-β in FM30 compared to other experimental groups were observed. Therefore, up to 30% fishmeal replacement does not affect growth, but it appears to have a slight effect on the sexual development of olive flounder. Olive flounder (Paralichthys olivaceus) is a commercially important and valuable species for aquaculture in Korea. Due to the unstable supply of fishmeal for farmed fish, an optimum fish-feed formulation should be researched to ensure the sustainability of P. olivaceus aquaculture. This study investigated the effect of three experimental diets: Con (basal diet); FM20 (20% fishmeal replacement of CON); and FM30 (30% fishmeal replacement of CON) on P. olivaceus over 20 weeks at a typical farm by monitoring the growth and factors relating to sexual maturation. The results showed that no differences in growth were observed between the CON and diet-replacement groups. Gonadal oocyte development was similar between the CON and diet-replacement groups. Moreover, sbGnRH and GH expression did not differ between the CON and diet-replacement groups. The levels of Erβ and Vtg expression were significantly higher in the FM20 group than in the CON and FM30 groups after the experimental period. The expression of PSS-I was significantly higher in the FM30 group than in the CON and FM20 groups. Therefore, although growth occurred when 30% of the fishmeal was replaced, such high dietary protein replacement may be ill-advised during the maturation of olive flounder at the commercial fish farm. [ABSTRACT FROM AUTHOR]

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]

Abstract 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 × 108 CFU g −1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 108 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 × 108 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. Highlights • Bacillus sp. SJ-10 (BSJ-10) is identified as a potential probiotic in olive flounder. • BSJ-10+β-glucooligosaccharides produce synergistic effects on growth and innate immunity. • Transcription levels of pro-inflammatory cytokines are upregulated by dietary synbiotic. • Dietary synbiotic is able to increase microvillus length in the intestine. [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]

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]