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The aim of study was to explore the beneficial effects of adding inositol to high-lipid diets on intestinal digestive enzymes, tissue morphology, and intestinal microbiota in juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu). Six isonitrogenous (CP 51 %) and isolipidic (CL 16 %) diets were formulated, each containing different levels of inositol: 0 % (J1, as the control group), 0.04 % (J2), 0.08 % (J3), 0.16 % (J4), 0.32 % (J5) and 0.64 % (J6) inositol. Grouper (initial body weight of 6.76 ± 0.34 g) were fed experimental diets continuously for 8 weeks. Results showed that inositol significantly increased the specific growth rate of J3, J4, J5, J6 groups compared to the control group (P > 0.05). Amylase, lipase, and trypsin activities generally increased with higher inositol levels. Amylase activities were significantly higher in J4, J5, and J6 groups compared to the control group (P < 0.05), while lipase and trypsin activities were significantly higher in all inositol-treated groups (P < 0.05). Inositol did not significantly affect intestinal plica height (P > 0.05), but increased plica width, muscularis thickness, and goblet cell number (P < 0.05). Analysis of intestinal microbiota revealed a higher number of OTUs in the J3 group compared to J1 and J6 groups (P < 0.05), with no significant differences in Ace and Chao1 indices (P > 0.05). The Simpson index was significantly higher in the J3 group compared to J1 group (P < 0.05), with no significant differences between J6 and J1, J3 groups (P > 0.05). The dominant intestinal phylum mainly consists of Proteobacteria and Cyanobacteria, while the dominant family mainly consists of Vibrionaceae and Burkholderiaceae. In summary, supplementing high-lipid diets with appropriate inositol can enhance digestive ability, improve the morphology of hybrid grouper intestines, increase the abundance of beneficial bacterial microbiota, maintain intestinal health, and promote growth.

Our previous study on plant oil substitution for fish oil found that corn oil induced inflammatory responses in grouper, while olive oil was effective in anti-inflammatory and immune enhancing. As a vital active ingredient in olive oil, squalene has been widely studied for its antioxidant and anti-inflammatory properties. Therefore, the present study was designed to use corn oil as the main lipid source to create a model of inflammation or low immunity and investigate the regulatory effect of squalene on the immunity of grouper, and to perform hepatic transcriptomic and metabolomic assays of grouper using omics technology, with the aim of providing basic data for the study of the mechanism of squalene. The results showed: squalene ingestion did not affect the growth performance of grouper (P > 0.05) but significantly improved immunity and the resistance of grouper to Vibrio harveyi (P

We previously found that squalene may influence immunity of grouper by affecting the Wnt/β-Catenin signaling pathway, but the exact mechanism is uncertain. We thus propose to interfere with the Wnt/β-Catenin signaling pathway and downstream pathways by intraperitoneal injection of PBS buffer (PBS), squalene (Sq), squalene + TWS119 (Wnt/β-Catenin signaling pathway activator, Sq + TWS119), and squalene + IWR-1 (Wnt/β-Catenin signaling pathway inhibitor, Sq + IWR-1) to initially elucidate the mechanism of squalene in hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu). The results showed: The injection test did not damage the liver structure of grouper, but Sq and Sq+TWS119 groups significantly increased the survival of Vibrio harveyi challenged and antioxidant capacity of grouper, and Sq+IWR-1 significantly decreased the survival of Vibrio harveyi challenged in grouper. The Sq and Sq+TWS119 groups activated the PI3K-Akt and Wnt/β-Catenin signaling pathway and upregulated the expression of anti-inflammatory factors in the NF-κB pathway, whereas Sq+IWR-1 inhibited the PI3K-Akt and Wnt/β-Catenin signaling pathway and upregulated the expression of pro-inflammatory factors in the NF-κB pathway. Conclusively, squalene can activate the PI3K-Akt-regulated Wnt/β-Catenin signaling pathway by binding to the interleukin 21 receptor, and the activation of Wnt/β-Catenin signaling pathway can inhibit NF-κB-mediated inflammatory response, consequently improving the immunity and disease resistance of grouper. It is expected that the regulation of NF-κB signaling pathway by Wnt/β-Catenin signaling pathway would contribute to the treatment the inflammation and inflammation-related diseases, and of course provide potential for the prevention of inflammation in aquaculture animals.

This study investigated tea polyphenols (TP), α-lipoic acid (ALA) and their joint use on the antioxidant and lipid metabolic performance of hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu) took food with high-fat diets. Six high-lipid diets with isonitrogen (50% of dry matter) and isolipid (17% of dry value) were designed, in which a total content of 1,000 mg/kg additives were added to each group except for the control group (FL). The additives addition ratios in each group were ALA (AL), TP (PL), ALA : TP = 1 : 1 (EL), ALA : TP = 1 : 2 (OL), ALA : TP = 2 : 1 (TL). Each diet was divided into three repeat groups with 30 tails (6.84 ± 0.01 g) in each group and fed for 8 weeks. The consequences were as follows: (1) the highest weight gain rate, specific growth rate, as well as the lowest feed conversion ratio and ingestion rate were discovered in the OL team, which were opposite to the TL group. (2) The body fat content and muscle fat content in the fish oil group were the lowest (P

The study aimed to investigate the effects of oregano oil (OO) supplement in the diet on the growth performance, disease resistance, immunity, and intestinal health of hybrid grouper. Six isonitrogenous and isolipidic diets were fed to the grouper with an initial weight of 19.10 ± 0.12 g for 8 weeks, of which the dietary OO levels were 0 (Control), 0.1 (OO-0.1), 0.3 (OO-0.3), 0.6 (OO-0.6), 0.9 (OO-0.9), or 1.2 (OO-1.2) g kg-1. The results showed that there were no significant differences in growth performance among the six groups, while the OO supplement could improve the disease resistance ability of grouper. Our findings have demonstrated that the OO supplement, especially 0.3 g kg-1, could improve the intestinal morphology via increasing the villus number and muscle thickness and elevate digestive enzyme activities such as those of amylase (AMS) and lipase. The 0.3-g kg-1 OO supplement also can activate the antioxidant defense system, reduce nuclear factor erythroid 2-related factor 2-like 2 (Nrf2)/Kelch-like-ECH-associated protein 1 (Keap1) expression, and then enhance the immune ability via acid phosphatase (ACP), lysozyme (LYS), complement C3 (C3), and tight junction-associated signaling pathways in hybrid grouper. Meanwhile, the relative abundance of Firmicutes and Bacteroidetes in the OO-0.3 group was significantly higher than that of Control group (P< 0.05), while the relative abundance of Proteobacteria showed a basically opposite trend (P< 0.05). These findings suggested that dietary OO supplementation could significantly improve intestinal health and induce the antioxidant defense system and immunity, which means that OO could be used as a functional feed additive to protect the intestine, and the appropriate addition level of OO should be 0.3 g kg-1.

This study investigated the effect of lipid levels on growth, body composition, lipid metabolism enzymes activity and related genes expression in hybrid grouper (♀Epinephelus fuscointestinestatus × ♂E. lanceolatu). Three diets with different lipid levels of 6% (low-lipid feed, LF), 11% (medium-lipid feed, MF) and 16% (high-lipid feed, HF) were designed and each diet was fed to triplicate groups of 30 fish each (6.73 ± 0.00g) for eight weeks. The results revealed the following. 1) The maximum weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER) and the lowest feed conversion ratio (FCR) values were found in the HF group. There was no significant difference in the survival rate (SR) among the groups (P > 0.05). 2) Condition factor (CF), hepatosomatic index (HSI) and visceralsomatic index (VSI) increased with increasing dietary lipid. 3) Crude lipid content in the whole-body and muscle increased significantly as the dietary lipid level increased (P < 0.05). 4) Principal component analysis of fatty acids showed that sum n-3 polyunsaturated fatty acids (∑n-3PUFA)/sum n-6 polyunsaturated fatty acids (∑n-6PUFA) was significantly higher in the HF group than in the other two groups (P < 0.05). 5) Biochemical serum indexes showed higher triglycerides (TG), total cholesterol (TC), alkaline phosphatase (AKP) and low-density lipoprotein-cholesterol (LDL-C) levels but lower high-density lipoprotein-cholesterol (HDL-C) level in the HF group. 6) Liver lipid metabolism enzymes activity and biochemical indicators showed that fish fed the MF diet had higher lipoprotein lipase (LPL), hepatic lipase (HL) and triglyceride lipase (ATGL) activity but lower fatty acid synthase (FAS) activity and very-low density lipoprotein-cholesterol (VLDL-C) level. 7) The relative expression levels of lipoprotein lipase (lpl), adipocyte-type fatty acid-binding protein (a-fabp), liver-type fatty acid-binding protein (l-fabp), heart-type fatty acid-binding protein (h-fabp) and hormone-sensitive lipase (hsl) genes in the fish liver were higher in the MF group with increasing dietary lipid (P < 0.05), while the fatty acid synthase (fas) gene was expressed at lower relative level (P < 0.05). 8) Histological oil-red sections of the liver showed that fish in the HF group had larger and more hepatic lipid droplets than those fed the other diets. It was concluded that fish fed the HF diet had better growth performance but this diet was not conducive to the progress of lipid metabolism, resulting in lipid accumulation and affecting the quality of the fish. This study provides useful information for the formulation of environmentally friendly and cost-effective diets for hybrid grouper.

There is a trend towards using high-lipid diets in aquaculture, which can be optimized by using certain additives. This study investigated the effect of vitamin E (VE) supplementation of a high-lipid diet on the growth, serum and liver biochemical indexes, hepatic histology, and lipid metabolism of hybrid groupers (♀ Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus). Six groups of iso-protein (50.23%) and iso-lipidic high-lipid (15.36%) experimental diets were prepared by adding VE at concentrations of 49.6 (basic diet, controls), 100, 110, 163, 206, and 245 mg/kg. Each treatment consisted of three replicates and 30 fish (10.20 ± 0.02 g) in an 8-week feeding experiment. The results show that 1) compared with controls, growth performance was not affected by VE addition. However, the weight gain rate (WGR) and specific growth rate (SGR) were significantly lower in the high-VE group (245 mg/kg) than in the 163 and 206 mg/kg groups. 2) Compared with controls, VE significantly increased the contents of alpha-tocopherol in the liver and muscle. 3) Adding VE can reduce the fatty livers caused by high-lipid diets and significantly reduce total serum cholesterol, reduce the activity of lipid synthesis enzymes, and significantly increase the activity of lipolytic enzymes in the liver. 4) Compared with controls, appropriate amounts of VE significantly increased the expressions of the liver lipid-metabolism-related genes PPAR-α, PPAR-β, PPAR-γ, H-FABP , A-FABP, and L-FABP. Based on observations of lipid metabolism enzyme activity, histological sections, and lipid-metabolism-related gene expression, we conclude that the addition of 163–206 mg/kg VE to a high-lipid diet can promote lipid metabolism in groupers.

Long-term ingestion of high-lipid diets causes lipid metabolism disorder and excess lipid deposition. This investigation aimed to determine whether dietary tea polyphenols (TP) would alleviate the adverse effect of high-lipid diets in hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu). Six isonitrogenous (51 %) and isolipidic (16.7 %) diets were formulated: 0 %, 0.01 %, 0.02 %, 0.04 %, 0.08 % and 0.16 % dietary TP, and named TP1 (control), TP2, TP3, TP4, TP5, and TP6. After an 8-week feeding high-lipid diets trial, the results showed that weight gain rate significantly increased in TP-treated groups which had no difference in themselves. Protein efficiency ratio of TP4 group was significantly higher than that of the control group, while the other TP-treated groups had no significant difference from the control group. Hepatosomatic index dramatically decreased in TP-treated groups. TP markedly reduced lipid content and increased protein content of muscle and whole body. TP supplement significantly reduced contents of total cholesterol, low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol, but increased content of high-density lipoprotein cholesterol in the serum. Hepatic lipase, lipoprotein lipase, and fatty acid synthetase activities in the liver significantly increased in TP-treated groups. The histology of hepatic tissue indicated that lipid droplet reduced with the increase of dietary TP, and there was no obvious difference in lipid-lowering effect observed in TP4 and TP5. Hepatic expression of peroxisome proliferator-activated receptors (α, β and γ), hormone-sensitive lipase, adipose triglyceride lipase, carnitine palmitoyl transferase1, sterol regulatory element-binding protein-1, acetyl-CoA carboxylase and fatty acid synthetase were distinctly up-regulated by dietary TP (P

This investigation aimed at estimating the effects of two zinc (Zn) sources and dietary Zn levels on growth performance, serum biochemical and immune indexes and zinc content of tissues. There were two Zn sources, Zn sulphate heptahydrate (Zn-S) and Zn methionine (Zn-M) with five addition gradients (10, 20, 40, 60, 80 mg/kg), separately, added to the isonitrogenous and isolipidic basal diets. The group without additional Zn supplement served as the control group. In the eight-week experiment, Litopenaeus vannamei (0.34 g initial body weight) was fed 11 kinds of experimental diets respectively (shared control group) for 8 weeks. The results indicated that: 1) dietary Zn-S and Zn-M significantly improved weight gain rate (WGR), specific growth rate (SGR), protein efficiency rate (PER) of shrimp (P 0.05). WGR, SGR, PER and FCR were not influenced by Zn sources and IZSL (P > 0.05) while they were significantly influenced by Zn levels (P 0.05). In conclusion, dietary Zn-M and Zn-S both improved growth performance and immunity of shrimp. Based on WGR and Zn content of the whole body, the optimum requirements of Zn in Zn-S or Zn-M for shrimp were 93.36 ~ 112.28 mg/kg and 66.03 ~ 103.61 mg/kg. Additionally, Zn-M exhibited higher avail and immunity and was recommended in diets of Litopenaeus vannamei.

The objective was to investigate the improvement of the flesh quality and nutritional value of hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu) by Lactobacillus pentosus (BL-15). The two diets were prepared with 9% oxidized fish oil (OFO negative control group) or 9% fresh fish oil (FFO positive control group) as the major lipid source, and the OFO diet were sprayed with L. pentosus at 1.0×107, 1.0×108, 1.0×109 CFU/g, respectively. Five kinds of diets were fed to hybrid grouper with initial weight of 21.36 ± 0.03 g for 60 days. The results showed that (1) consumption of OFO diet significantly increased muscle crude lipid, total cholesterol, and total monounsaturated fatty acids contents and significantly decreased muscle C20:5n3, C22:6n3, ∑PUFA levels of grouper (p < 0.05), whereas the addition of L. pentosus to OFO diet significantly reduced muscle crude lipid, malondialdehyde, reactive oxygen radicals, total cholesterol concentration, increased C20:5n3, C22:6n3, ∑PUFA levels, increased muscle crude protein content, and enhanced muscle hardness, gumminess, and chewiness compared with FFO or OFO group (p < 0.05). (2) In addition, ingestion of OFO diet significantly increased muscle phenylalanine content and significantly decreased aspartic acid content (p < 0.05), while the addition of L. pentosus restored aspartic acid to the level not significantly different from that of FFO group (p > 0.05). The addition of L. pentosus also increased tyrosine, lysine, leucine, phenylalanine, arginine, and alanine content compared to FFO group at the same time (p < 0.05). (3) Furthermore, the intake of OFO diet would lead to deterioration in muscle flavor quality and increase in noxious odors in grouper, and the addition of L. pentosus can mitigate or rectify these detrimental consequences. In further analysis, consumption of OFO group significantly downregulated the mRNA expression of muscle growth factors and caused serious damage to the muscle antioxidant system (p < 0.05), which was protected against by the potent antioxidant properties of L. pentosus. We recommend the supplementation level of L. pentosus to be 1.0×109 CFU/g under the highly oxidized conditions of the present trial fish oil.

The aim of the study was to investigate the effects of fresh fish oil (FFO) and oxidized fish oil (OFO) diets on the muscle quality of hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu). Hybrid grouper were fed with diets containing 9% FFO or OFO for 60 days. Muscle sample were collected at 0, 30, and 60 days and the selected indexes of muscle were measured. Malondialdehyde (MDA), reactive oxygen species (ROS), total cholesterol (TC) and triglycerides (TG) in grouper muscle accumulated gradually with prolonged ingestion time, especially OFO group. Total saturated fatty acids (ΣSAFA) was significantly reduced and total polyunsaturated fatty acids (ΣPUFA) was significantly increased of muscle in FFO group; meanwhile, the muscle ΣSAFA and monounsaturated fatty acids (ΣMUFA) contents in the OFO group were significantly higher than those in the FFO group and the ΣPUFA (especially C22:5n3, C22:6n3) contents was significantly lower than that in the FFO group at 60 days. Consumption of OFO diet for 60 days reduced the diversity of volatile compounds, significantly reduced the content of total esters and increased the content of total aldehydes and total aromatics in grouper muscle. Furthermore, ingestion of OFO diet significantly reduced the mRNA expression of fraction growth factors and antioxidant genes in the muscle of grouper. In conclusion, the increasing MDA content in FO and the oxidative rancidity of PUFA can cause the deterioration of grouper quality and flavor due to oxidative muscle damage.

The spleen is an important organ in the immune function of fish, and it is also important for hematogenesis and antibody and granulocyte production. However, the effect of oxidized fish oil on the spleen of hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus) is unknown. In this study, hybrid groupers were fed with oxidized fish oil and the spleen index, antioxidant ability, histology and transcriptome were investigated. Oxidized fish oil did not affect the spleen index. Levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in the spleen were significantly increased as the amount of oxidized fish oil in the diet increased, but the vitamin E concentration was significantly decreased. The morphological organization of the spleen was damaged with increased oxidative stress. And the spleen reacted to oxidative stress by platelet activation, FOXO and notch signaling pathways, which involved amyloid beta precursor protein binding family B member 1 interacting protein (APBB1IP) gene, glucose-6-phosphatase (G6PC) gene, histone acetyltransferase p300 (EP300) gene, insulin gene and notch 2 gene. In conclusion, the oxidized fish oil caused oxidative stress and damaged its structure. Additionally, oxidized fish oil changed the transcription profile of the spleen.