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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 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]

The purpose of this study was to characterize the bacteria isolated from rockfish intestines and to investigate the effects of feed supplementation in rockfish aquaculture. Bacillus sp. KRF-7 isolated from the intestine of rockfish (Sebastes schlegelii) was demonstrated to be safe based on in vitro tests confirming the absence of hemolysis, cytotoxicity, and genes with toxigenic potential. In a feeding trial, providing a supplemental diet of 1 × 108 CFU g−1 Bacillus sp. KRF-7 was observed to positively alter the weight gain, specific growth rate, feed conversion ratio, and protein efficiency ratio of juvenile rockfish. KRF-7 supplementation showed positive regulation of nonspecific immune parameters, such as superoxide dismutase, lysozyme activity, and myeloperoxidase activity. This analysis also revealed a change in the composition of the intestinal microbiota at the phylum level from Proteobacteria to Firmicutes. In both the kidney and spleen, the expression levels of IL-10, NF-κB, and B cell activating factors in the KRF-7-supplemented group were significantly increased compared to those in the control group. Therefore, this study verified the safety of KRF-7 isolated from the intestine of rockfish and suggests that dietary supplementation with KRF-7 enhances the growth performance of rockfish and has beneficial effects on the regulation of the intestinal microbiota and immune response. • The probiotic properties of Bacillus sp. KRF-7 were investigated. • Dietary supplementation of KRF-7 upregulated the growth performance of rockfish. • Supplementation with KRF-7 modulated gut microbial community of rockfish. [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]