Effects of Dietary Aflatoxin B1 on Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) Growth, Intestinal Health, and Muscle Quality.

This study investigated the effects of varying doses of dietary aflatoxin B1 (AFB1) on the growth, intestinal health, and muscle quality of hybrid grouper. Four diets with varying AFB1 concentrations (0, 30, 445, and 2,230 µg kg-1) were used. Elevating AFB1 concentrations led to a decline in growth indexes, specifically the weight gain rate and the specific growth rate, although the survival rate remained unchanged. Morphological indicators showed a dose-dependent decline with AFB1 exposure. Intestinal MDA content and hindgut reactive oxygen species (ROS) levels increased, while antioxidant indexes and digestive enzymes decreased with higher AFB1 levels. AFB1 negatively influenced hindgut tight junction protein and antioxidant-related gene expression while promoting inflammation-related gene expression. The presence of AFB1 in the experiment led to a decrease in beneficial intestinal bacteria, such as Prevotella, and an increase in harmful intestinal bacteria, such as Prevotellaceae_NK3B31_group. Muscle lipid and unsaturated fatty acid content significantly decreased, while muscle protein and liver AFB1 content increased dramatically with higher AFB1 concentrations. AFB1 caused myofibrillar cleavage and myofilament damage, leading to increased spaces between muscle fibers. In conclusion, diets with AFB1 levels exceeding 30 µg kg-1 inhibited hybrid grouper growth, while levels surpassing 445 µg kg-1 resulted in hindgut ROS accumulation, inflammation, elevated intestinal permeability, reduced digestive enzyme activity, and compromised muscle quality.

Effects of α-Lipoic Acid Supplementation on Growth Performance, Liver Histology, Antioxidant and Related Genes Expression of Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂).

This study aimed to assess the impact of α-lipoic acid on the growth performance, antioxidant capacity and immunity in hybrid groupers (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatus) fed with a high-lipid diet. Groupers (8.97 ± 0.01 g) were fed six different diets, with α-lipoic acid content in diets being 0, 400, 800, 1200, 1600, and 2000 mg/kg, named S1, S2, S3, S4, S5, and S6, respectively. The results show that the addition of 2000 mg/kg α-lipoic acid in the diet inhibited the growth, weight gain rate (WGR), and specific growth rate (SGR), which were significantly lower than other groups. In serum, catalase (CAT) and superoxide dismutase (SOD) were significantly higher in the S5 group than in the S1 group. In the liver, CAT, SOD and total antioxidative capacity (T-AOC) levels were significantly increased in α-lipoic acid supplemented groups. α-lipoic acid significantly upregulated liver antioxidant genes sod and cat, anti-inflammatory factor interleukin 10 (il10) and transforming growth factor ß (tgfß) mRNA levels. Conclusion: the addition of 2000 mg/kg of α-lipoic acid inhibits the growth of hybrid groupers. In addition, 400-800 mg/kg α-lipoic acid contents improve the antioxidant capacity of groupers and have a protective effect against high-lipid-diet-induced liver oxidative damage.

Low-frequency Stimulation at the Subiculum Prevents Extensive Secondary Epileptogenesis in Temporal Lobe Epilepsy.

Secondary epileptogenesis is characterized by increased epileptic susceptibility and a tendency to generate epileptiform activities outside the primary focus. It is one of the major resultants of pharmacoresistance and failure of surgical outcomes in epilepsy, but still lacks effective treatments. Here, we aimed to test the effects of low-frequency stimulation (LFS) at the subiculum for secondary epileptogenesis in a mouse model. Here, secondary epileptogenesis was simulated at regions both contralateral and ipsilateral to the primary focus by applying successive kindling stimuli. Mice kindled at the right CA3 showed higher seizure susceptibilities at both the contralateral CA3 and the ipsilateral entorhinal cortex and had accelerated kindling processes compared with naive mice. LFS at the ipsilateral subiculum during the primary kindling progress at the right CA3 effectively prevented secondary epileptogenesis at both the contralateral CA3 and the ipsilateral entorhinal cortex, characterized by decreased seizure susceptibilities and a retarded kindling process at those secondary foci. Only application along with the primary epileptogenesis was effective. Notably, the effects of LFS on secondary epileptogenesis were associated with its inhibitory effect at the secondary focus through interfering with the enhancement of synaptic connections between the primary and secondary foci. These results imply that LFS at the subiculum is an effective preventive strategy for extensive secondary epileptogenesis in temporal lobe epilepsy and present the subiculum as a target with potential translational importance.

Satellite glial cells in sensory ganglia play a wider role in chronic pain via multiple mechanisms.

Satellite glial cells are unique glial cells that surround the cell body of primary sensory neurons. An increasing body of evidence suggests that in the presence of inflammation and nerve damage, a significant number of satellite glial cells become activated, thus triggering a series of functional changes. This suggests that satellite glial cells are closely related to the occurrence of chronic pain. In this review, we first summarize the morphological structure, molecular markers, and physiological functions of satellite glial cells. Then, we clarify the multiple key roles of satellite glial cells in chronic pain, including gap junction hemichannel Cx43, membrane channel Pannexin1, K channel subunit 4.1, ATP, purinergic P2 receptors, and a series of additional factors and their receptors, including tumor necrosis factor, glutamate, endothelin, and bradykinin. Finally, we propose that future research should focus on the specific sorting of satellite glial cells, and identify genomic differences between physiological and pathological conditions. This review provides an important perspective for clarifying mechanisms underlying the peripheral regulation of chronic pain and will facilitate the formulation of new treatment plans for chronic pain.

Effects of High-Lipid Dietary Protein Ratio on Growth, Antioxidant Parameters, Histological Structure, and Expression of Antioxidant- and Immune-Related Genes of Hybrid Grouper.

The hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatus) is a new species of grouper crossed from giant grouper (E. lanceolatus) as the male parent and brown-marbled grouper (E. fuscoguttatus) as the female parent. We hypothesized that optimal levels of dietary protein may benefit liver function. High-lipid diets are energetic feeds that conserve protein and reduce costs, and are a hot topic in aquaculture today. Therefore, the objective of the research is to investigated the effects of dietary protein level in high-lipid diets on serum and liver biochemistry, liver histology, and liver immune and antioxidant indexes and gene mRNA expression of the juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatus). Six iso-lipidic (161 g/kg) diets were formulated containing graded levels of protein (510 as control, 480,450, 420, 390 and 360 g/kg). Each treatment consisted of three replicates and 30 fish (6.70 ± 0.02 g) in one replicate. After an 8-week feeding experiment, the results indicated the following: (1) With the decreasing of dietary protein level, the specific growth rate (SGR) of groupers increased gradually and then decreased; SGRs of the 390 and 360 g/kg groups were significantly lower than other groups (p < 0.05). (2) In terms of serum and liver, the activity of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD), and the total antioxidant capacity (T-AOC) content, and the activity of immune enzymes such as lysozyme (LYS) and immunoglobulin (IgM) was significantly increased under the appropriate protein level. (3) Based on liver histology, we know that high or low dietary protein levels cause liver damage. (4) Dietary protein levels can significantly affect the mRNA expression levels of an anti-inflammatory factor gene (tgfß), pro-inflammatory factor genes (il6, il8), heat shock proteins, and antioxidant and immune genes (hsp70 and hsp90, gpx, nrf2, keap1). It is concluded that the appropriate protein level can promote the growth performance of groupers, improve antioxidant activity and immune enzyme activity in serum and liver, and enhance the expression of immune genes.

Lycopene Alleviates the Adverse Effects of Feeding High-Lipid Diets to Hybrid Grouper (♀Epinephelus fuscoguttatus ×♂E. lanceolatus).

It has been found that high-lipid diets (HLDs) disrupt lipid metabolism in fish, leading to an excessive accumulation of lipids in various tissues of the fish body. The objective of this study was to investigate if the inclusion of lycopene (LCP) in an HLD may mitigate the adverse consequences of excessive dietary lipid intake in hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatus). The experimental design incorporated a control group (L0), which was administered a diet consisting of 42% protein and 16% lipid. The diets for groups L1, L2, and L3 were developed by augmenting the control diet with 100, 200, and 400 mg/kg LCP, respectively. The duration of the trial spanned a period of 42 days. The results of the study showed that the weight gain rate (WGR) and protein efficiency ratio (PER) of the three LCP treatment groups (L1, L2, and L3) tended to increase and then decrease, with a significant increase in WGR and PER in L2 (P < 0.05). Visceral somatic index and hepatic somatic index tended to decrease and then increase in all treatment groups, with a significant decrease in L2 (P < 0.05). In serum dietary LCP significantly reduced triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) content and significantly increased high-density lipoprotein (HDL) content (P < 0.05). In the liver, dietary LCP reduced TC, TG, and very LDL levels and improved lipoprotein lipase, hepatic lipase, fatty acid (FA) synthetase, and acetyl-CoA carboxylase activities. The number and area of hepatic lipid droplets decreased significantly with increasing LCP content. In the liver, the addition of appropriate levels of LCP significantly upregulated lipoprotein lipase (lpl) and peroxisome proliferator-activated receptor α (pparα). In summary, dietary LCP improves growth and reduces lipid deposition in the liver of hybrid grouper by increasing lipolytic metabolism and decreasing FA synthesis. Under the experimental conditions, the fitted curve analysis showed that the recommended LCP additions to the high lipid diet for juvenile hybrid grouper were 200-300 mg/kg.

Effect of Tea Polyphenols, α-Lipoic Acid and Their Joint Use on the Antioxidant and Lipid Metabolism Performance of Hybrid Grouper (♀Epinephelus fuscoguttatus × â™‚E. lanceolatu) Fed with High-Lipid Diets.

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 × â™‚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 < 0.05), while those of the TL group were the highest. (3) Serum catalase, glutathione peroxidase, total antioxidant capacity, and superoxide dismutase activities were the highest, and the content of reactive oxygen species was the lowest in the OL group. (4) The OL group has the highest hepatic lipase activity and the lowest very low-density lipoprotein content of the liver. In contrast, the TL group had the highest fatty acid synthetase (FAS) activity (P < 0.05). (5) The oil-red aspects of liver tissue displayed lipid particles in other groups were reduced to different degrees compared with FL group, and the OL group showed the best lipid-lowering effect. (6) Compared with the FL group, the relative expressions of FAS, acetyl-CoA carboxylase (acc), and apolipoprotein b-100 (apoB100) genes in the liver were decreased. The relative expressions of lipoprotein lipase (lpl) and peroxisome proliferators-activated receptors-α (pparα) genes related to lipid catabolism were increased, among which the OL group had the most significant change (P < 0.05). (7) According to the 7-day challenge test of Vibrio alginolyticus, the OL group had the highest survival rate. To sum up, both ALA and TP have positive effects on relieving the lipid metabolism disorder of hybrid grouper. If they are jointly used, adding ALA : TP in a ratio of 1 : 2 (OL) may have the best effect, and an addition ratio of 2 : 1 (TL) may inhibit the hybrid grouper growth and increase the feeding cost.

Integrated analysis of microbiome and host transcriptome reveals the damage/protective mechanism of corn oil and olive oil on the gut health of grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu).

As digestive and immune organs of animals, the gut was frequently used to evaluate the health status of aquatic animals. In previous oil source alternatives study, corn oil (CO) had been found to induce gut inflammation, while olive oil (OO) had been found to be effective in protecting intestinal health. Three diets with different oil sources (fish oil, CO, OO) were formulated for an 8-week culture experiment, and it was proposed to combine 16S sequencing and transcriptome sequencing analysis to preliminarily elucidate the damage/protection mechanism of CO and OO on the gut health of grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu). We found that CO indeed damaged to gut health and destroyed the gut structure, while OO had a positive outcome in protecting the gut structure, promoting digestibility and relieving enteritis. Photobacterium, Romboutsia and Epulopiscium were significantly enriched in OO group and Staphylococcus were significantly enriched in CO group. Transcriptome sequencing further revealed CO could activated Complement and coagulation cascades, Staphylococcus aureus infection, Systemic lupus erythematosus, and Tuberculosis pathways; conversely, OO activated B-cell signaling receptors, promoted B-cell proliferation and apoptosis, and thus activated B-cell signaling pathways to enhance immunity, whereas OO can regulate IL17 signaling pathway and TNF signaling pathway to inhibit NF-κB signaling pathway to reduce pro-inflammatory response. By integrating the microbiome and transcriptome, further identified all differential microorganisms were directly and significantly correlated with differential genes, and Clostridium_sensu_stricto_1, Romboutsia, Staphylococcus might as the core regulates the expression of differential gene in the organism. These results reveal that different oil sources alter gut gene expression mainly by modulating the composition and abundance of gut microbiota, further regulating the health status of the gut. Gut microbiota could be used as biomarkers to provide reference and solutions for the mitigation of inflammation in aquatic animals.

Host gut-derived Bacillus probiotics supplementation improves growth performance, serum and liver immunity, gut health, and resistive capacity against Vibrio harveyi infection in hybrid grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus).

Several reports have revealed the vital role that probiotics play in fish growth and health. However, few works are available for host gut-derived probiotics on the growth, immunity, and gut microbiota of fish, especially in hybrid grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus) due to their isolation difficulty and functional verification. This study aimed at assessing 3 host gut-derived Bacillus species' effects on the growth, immune and antioxidant-biochemical responses, haematological parameters, intestinal morphology, immune-related gene expression, gut microbiota, and disease resistance against Vibrio harveyi in hybrid grouper. A total of 480 hybrid grouper (initial weight = 9.03 ± 0.02 g) were randomly allotted into 4 groups, namely, the group fed a basal diet without probiotic inclusion (control, B0), the group fed the basal diet with Bacillus velezensis GPSAK4 (BV), the group fed the basal diet with Bacillus subtilis GPSAK9 (BS), and the group fed the basal diet with Bacillus tequilensis GPSAK2 (BT) strains at 1.0 × 109 CFU/g. After a 6-week feeding trial, the results revealed significant improvements (P < 0.05) in the growth performance, whole fish-body proximate composition, blood haematological parameters, serum, liver, and intestinal biochemical indexes, intestinal morphology, and protection against V. harveyi pathogen in the probiotic-treated groups compared with the untreated. Additionally, the expressions of intestinal tight junction genes (occludin and ZO1), pro- and anti-inflammatory genes, including IL1ß, IL6, IL8, TNFα, MyD88, IL10, and TGFß, were upregulated (P < 0.05) after Bacillus species administration. Host gut-derived Bacillus supplementation shaped the gut microbiota by significantly increasing (P < 0.05) the relative abundance of Proteobacteria, Bacteroidetes, Actinobacteria (except the BS group), Acidobacteria (except the BT group), Cyanobacteria (except the BV and BT groups), and Verrucomicrobia phyla, as well as known beneficial genera (Romboutsia, Turicibacter, Epulopiscium, Clostridium_sensu_stricto 1 and 13, Lactobacillus, and Bacillus), but significantly decreased (P < 0.05) the abundance of Firmicutes, Chloroflexi, and Fusobacteria phyla, and purported pathogenic genera (Staphylococcus and Photobacterium) compared with the control group. Collectively, the results suggest that B. velezensis GPSAK4, B. subtilis GPSAK9 (especially this strain), B. tequilensis GPSAK2 dietary supplementation at 1.0 × 109 CFU/g has positive effects on the intestinal health of hybrid grouper via microbial composition modulation, thus enhancing the assimilation and absorption of nutrients to boost fish growth, immunity, and disease resistance.

Effects of choline supplementation on growth performance, liver histology, nonspecific immunity and related genes expression of hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu) fed with high-lipid diets.

This study was conducted to evaluate the effect of dietary choline levels on growth performance, liver histology, nonspecific immunity and related gene expression of hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatus) fed with high-lipid diets. The fish (initial body weight 6.86 ± 0.01 g) were fed diets containing different choline levels (0, 5, 10, 15, and 20 g/kg, named D1, D2, D3, D4, and D5, respectively) for 8 weeks. The results showed that:(1) dietary choline levels had no significant effect on final body weight (FBW), feed conversion rate (FCR), visceral somatic index(VSI) and condition factor (CF) compared with the control group (P > 0.05). However, the hepato somatic index (HSI) in the D2 group was significantly lower than that in the control group and the survival rate (SR) in the D5 group was significantly lower (P < 0.05). (2) with dietary choline level increasing, alkaline phosphatase (AKP) and superoxide dismutase (SOD) of serum showed a tendency to increase and then decrease, and the maximum values were obtained in the D3 group, but the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased significantly (P < 0.05). (3) Immunoglobulin M (IgM), lysozyme (LYZ), catalase (CAT), total antioxidative capacity (T-AOC), and SOD in the liver all showed a trend of first increase and then decrease with the dietary choline level increased, and all of them achieved the maximum value at D4 group (P < 0.05), while reactive oxygen species (ROS) and malondialdehyde (MDA) in the liver decreased significantly (P < 0.05). (4) results from liver sections suggest that appropriate levels of choline can improve cell structure, compared with the control group, the damaged histological morphology of the liver was relieved and even returned to normal in D3 group. (5) in the D3 group, choline significantly upregulated the expression of hepatic sod and cat mRNA, whereas the expression of cat in the D5 group was significantly lower than that in the control group (P < 0.05); And the supply of choline stimulated a significant down-regulation of interleukin 6 (il6), myeloid differentiation factor 8 (myd88), toll-like receptor 22 (tlr22) mRNA expression levels in liver, while the expression of cellular tumor antigen p53 (p53) and interleukin 10 (il10) showed an upward and then downward trend (P < 0.05). In general, choline can improve the immunity of hybrid grouper by regulating non-specific immune-related enzyme activity and gene expression and reducing oxidative stress induced by high-lipid diet.

Transcriptomic Analysis of Liver in Silver sillago, Sillago sihama Fed with High-Level Low-Gossypol Cottonseed Meal in Replacement of Fishmeal Diet.

Understanding the molecular mechanisms involved in adaptation to alternate diets has become a significant concern, as increasing amounts of fishmeal (FM) protein in aquafeeds are being substituted with plant protein. Thus, the goal of this study was to assess growth performance, quality, and liver function of juvenile Sillago sihama (S. sihama) through growth indices, whole-body composition, histology of the liver, and RNA-sequencing (RNA-seq), after they were fed a formulated diet with 64% low-gossypol cottonseed meal (LCSM) for 56 days, compared to those fed a traditional FM-based diet. Indicators of growth, including final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and condition factor (CF), were considerably lower in the 64% LCSM (R64) group than in the FM diet group. In the R64 diet, the whole crude lipid was significantly lower than in the FM diet. The hematoxylin-eosin section showed that dietary high levels of LCSM resulted in diffuse lipid vacuolation in the liver of S. sihama. According to a liver transcriptome analysis, high LCSM intake in the diet significantly impacted lipid synthesis and catabolism, elevated pathways for cholesterol synthesis, blocked several amino acid metabolic pathways, and adversely affected hepatic gluconeogenesis and glycolysis. The findings of this study indicate that feeding high levels of LCSM in S. sihama is harmful to the growth of the organism and can harm the liver's structural integrity, as well as obstruct the normal metabolism of amino acids, lipids, and carbohydrates. Therefore, it is not recommended to substitute LCSM for high levels of FM in the diet of S. sihama.

Substituting fish meal with castor meal in diets of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂): Effects on growth performance, immune response, antioxidant and digestive enzyme activities, gut morphology, and inflammatory-related gene expression.

The present study was conducted to investigate the effects of replacing fishmeal (FM) with castormeal (CM) on the growth performance, immune response, antioxidant and digestive enzyme activities, intestinal morphology, and expression of inflammatory-related genes in juvenile hybrid grouper (Epinephelus fuscoguttatus♀ ×E. lanceolatus♂). Six iso-nitrogenous (50% crude protein) and iso-lipidic (10% crude lipid) diets were formulated; namely, a reference diet (FM) containing 50% FM and five experimental diets (4% (CM4), 8% (CM8), 12% (CM12), 16% (CM16), and 20% (CM20)) in which FM protein was substituted with CM at varying levels to feed fish (initial weight: 9.12 ± 0.01 g) for 8 weeks. The results showed that the final weight, weight gain rate, and specific growth rate were highest in the FM, CM4, and CM8 groups, whereas the feed conversion ratio, hepatosomatic and viscerosomatic indexes were significantly enhanced in the CM4 group in comparison to the others. The CM4 and CM12 groups were observed to show the highest intestinal length index values compared to the other groups, with the CM20 revealing the worst growth performance. The serum total protein content first increased (P < 0.05) in the CM4 group and decreased (P < 0.05) afterward. Nonetheless, a decreasing significant (P < 0.05) cholesterol and triglyceride contents were witnessed with the increasing replacement of FM with CM. Compared to the control group, a significant increase (P < 0.05) in the activities of serum and liver immunoglobulin-M, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and complement-3 (except serum activity for CM12 group); liver lysozyme; intestinal amylase, and lipase, was witnessed in the CM groups. However, the serum lysozyme activity was highest (P < 0.05) in the CM4 group and lowest in the CM20 group. While the least serum malondialdehyde contents were observed in the CM4 group, that of the liver malondialdehyde was least witnessed in the FM, CM4, CM8, CM12, and CM16 groups as compared to the CM20. The intestinal histological examination revealed a significantly decreasing trend for villi height and villi width with increasing replacement levels. However, the muscle thickness, crypt depth, and type II mucus cells first increased upto 4% replacement level and later decreased. The increasing of dietary replacement levels significantly up-regulated pro-inflammatory (il-1ß, tnf-α, myd88, ifn-γ, tlr-22, and il-12p40) and down-regulated anti-inflammatory (il-10, tgf-ß, mhc-iiß) and anti-bacterial peptide (epinecidin and hepcidin) mRNA levels in the intestine. The mRNA levels of il-6 was up-regulated firstly upto 4 and 8% replacement levels, and later down-regulated with increasing replacement. These results suggested that, although higher dietary CM replacement enhances the immune, antioxidant and digestive enzymes, it aggravates intestinal inflammation. Replacing 4 and 8% of FM with CM could enhance the growth performance of fish.

Impacts of tea polyphenols on growth, antioxidant capacity and immunity in juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) fed high-lipid diets.

The experiment aimed to investigate the alteration of tea polyphenols (TP) in growth and immunity for hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) fed high-lipid diets. Six concentrations of TP (0, 0.01, 0.02, 0.04, 0.08, 0.16%, named TP1 (basic diet control), TP2, TP3, TP4, TP5, TP6) were supplied in isonitrogenous (51%) and isolipidic (16.7%) experimental diets. These diets were fed to the juvenile grouper (8.68 ± 0.22 g) for 8 weeks. The results showed that dietary TP significantly increased the weight gain rate and specific growth rate (P < 0.05), compared with the control group. The protein efficiency ratio in TP4 group was significantly higher than that of the control group (P < 0.05). TP supplement in high-lipid diets increased antioxidant capacity in the serum (CAT, GSH-Px, T-AOC) and liver (SOD, CAT, GSH-Px, T-AOC). Additionally, dietary TP decreased oxidative stress (ROS, MDA) and improved immunity (ACP, AKP, LYS, IgM) in the liver. The histology of hepatic tissue indicated that dietary TP alleviated pathological symptoms caused by high-lipid diets. Compared with the control group, appropriate dietary TP significantly up-regulated expression of sod, cat, gsh-px, nrf2, keap1, hsp70, hsp90, myd88, tnfα and down-regulated expression of tlr22, il8, il1ß, il10 in the liver (P < 0.05). In the head kidney, expression of myd88, il1ß, tnfα and il6 were significantly up-regulated and expression of tlr22 and il10 were significantly down-regulated by dietary TP (P < 0.05). After the challenge of Vibrio harveyi, survival rate in higher doses of TP group (TP4 âˆ¼ TP6) was evidently higher, compared with the control group. In conclusion, TP supplement in high-lipid diets improved antioxidant capacity and enhanced immunity of grouper. We speculate that TP may play the role of an immunostimulant, enhancing immunity and disease resistance by cytokine-medicated immune responses. Based on the second-order regression, 0.092-0.106% tea polyphenols were recommended in juvenile grouper high-lipid diets.

Ultra-Performance Liquid Chromatography-Mass Spectrometry-Based Untargeted Metabolomics Reveals the Key Potential Biomarkers for Castor Meal-Induced Enteritis in Juvenile Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

The intensification of aquaculture to help kerb global food security issues has led to the quest for more economical new protein-rich ingredients for the feed-based aquaculture since fishmeal (FM, the ingredient with the finest protein and lipid profile) is losing its acceptability due to high cost and demand. Although very high in protein, castor meal (CM), a by-product after oil-extraction, is disposed-off due to the high presence of toxins. Concurrently, the agro-industrial wastes' consistent production and disposal are of utmost concern; however, having better nutritional profiles of these wastes can lead to their adoption. This study was conducted to identify potential biomarkers of CM-induced enteritis in juvenile hybrid-grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) alongside their growth and distal intestinal (DI) health evaluation. A total of 360 fish (initial weight = 9.13 ± 0.01g) were randomly assigned into three groups, namely, fish-meal (FM) (control), 4% CM (CM4), and 20% CM (CM20). After the 56-days feeding-trial, the DI tissues of FM, CM4, and CM20 groups were collected for metabolomics analysis. Principal components analysis and partial least-squares discriminant-analysis (PLS-DA, used to differentiate the CM20 and CM4, from the FM group with satisfactory explanation and predictive ability) were used to analyze the UPLC-MS data. The results revealed a significant improvement in the growth, DI immune responses and digestive enzyme activities, and DI histological examinations in the CM4 group than the others. Nonetheless, CM20 replacement caused DI physiological damage and enteritis in grouper as shown by AB-PAS staining and scanning electron microscopy examinations, respectively. The most influential metabolites in DI contents identified as the potential biomarkers in the positive and negative modes using the metabolomics UPLC-MS profiles were 28 which included five organoheterocyclic compounds, seven lipids, and lipid-like molecules, seven organic oxygen compounds, two benzenoids, five organic acids and derivatives, one phenylpropanoids and polyketides, and one from nucleosides, nucleotides, and analogues superclass. The present study identified a broad array of DI tissue metabolites that differed between FM and CM diets, which provides a valuable reference for further managing fish intestinal health issues. A replacement level of 4% is recommended based on the growth and immunity of fish.

The positive effects of dietary inositol on juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu) fed high-lipid diets: Growthperformance, antioxidant capacity and immunity.

The objective of the present research was to assess the influence of inositol supplementation on growth performance, histological morphology of liver, immunity and expression of immune-related genes in juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu). Hybrid grouper (initial weight 6.76 ± 0.34 g) were fed isonitrogenous and isolipidic diets (16%) with various inositol levels of 0.17 g/kg (J1, the control group), 0.62 g/kg (J2), 1.03 g/kg (J3), 1.78 g/kg (J4), 3.43 g/kg (J5), 6.59 g/kg (J6), respectively. The growth experiment lasted for 8 weeks. The results indicated that dietary inositol had a significant promoting effect on final mean body weight of the J5 and J6 groups and specific growth rate (SGR) of the J3, J4, J5 and J6 groups (P < 0.05). In the serum, superoxide dismutase (SOD) of the J4 group became significantly active compared with that of the control group (P < 0.05), while aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (AKP) activities in the inositol-treated groups showed distinctly decreased compared with those of the control group (P < 0.05). In the liver, dietary inositol could significantly increase the activities of SOD, catalase (CAT), lysozyme (LYZ) and the contents of total antioxidative capacity (T-AOC) and immunoglobulin M (IgM) (P < 0.05), and distinctly reduce the content of malondialdehyde (MDA) as well as reactive oxygen species (ROS) (P < 0.05). Compared with the control group, the damaged histological morphology of the liver was relieved and even returned to normal after an inositol increase (0.4-3.2 g/kg). In the liver, the remarkable up-regulation of SOD, CAT, glutathione peroxidase (GPX), heat shock protein70 (HSP70) and heat shock protein90 (HSP90) expression levels were stimulated by supply of inositol, while interleukin 6 (IL6), interleukin 8 (IL8) and transforming growth factor ß (TGF-ß) expression levels were down-regulated by supply of inositol. In head kidney, the mRNA of toll-like receptor 22 (TLR22), myeloid differentiation factor 88 (MyD88) and interleukin 1ß (IL1ß) expression levels were significantly down-regulated (P < 0.05), which could further lead to remarkable down-regulation of IL6 and tumor necrosis factor α (TNF-α) expression (P < 0.05). These results indicated that high-lipid diets with supply of inositol promoted growth, increased the antioxidant capacity, and suppressed the inflammation of the liver and head kidney by inhibiting the expression of pro-inflammation factors (IL6, IL8, TGF-ß and TNF-α). In conclusion, these results indicated that dietary inositol promoted growth, improved antioxidant capacity and immunity of hybrid grouper fed high-lipid diets. Based on SGR, broken-line regression analysis showed that 1.66 g/kg inositol supply was recommended in high-lipid diets of juvenile grouper.

Vitamin E amelioration of oxidative stress and low immunity induced by high-lipid diets in hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu).

The experiment was conducted to investigate the effects of vitamin E (VE) on growth, oxidative stress and immunity for hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu) fed high-lipid diet. Six groups of iso-protein (50.23%) and iso-lipidic high-lipid (15.36%) experimental diets were prepared by adding 0 (basic diet control), 0.01%, 0.02%, 0.03%, 0.04%, 0.05% α-tocopherol respectively in basic diet. Each treatment consisted of 3 replicates and 30 fish (10.20 ± 0.02 g) in each replicate for 8 weeks. The results showed that: 1) compared with the control group, the growth performance of grouper was not affected by the addition of VE in high-lipid diet, but the specific growth rate (SGR) in high VE dose (0.6%) group were significantly decreased compared with 0.02% and 0.03% groups. 2) Adding VE to high-lipid diet can alleviate the hepatic oxidative damage caused by high-lipid diet, and significantly improve the serum and liver antioxidant enzyme activity. 3) Compared with the control group, appropriate VE significantly increased the expression of liver anti-inflammatory factors TGF-ß and IL10, and significantly decreased the expression of proinflammatory factors IL8 and IL6. In conclusion, adding appropriate amount of VE into high-lipid diet can improve antioxidant capacity and immunity of grouper, we speculated that VE may alleviate lipid peroxidation by improving antioxidant capacity to reduce the inflammatory response. In combination with the results of the current study, we recommend an additional dose of 0.02%-0.03% of α-tocopherol in this experiment under high-lipid conditions.

Degradation of Muscle Quality in Hybrid Grouper (♀ Epinephelus fuscoguttatus × â™‚ Epinephelus lanceolatu) Due to Oxidative Damage Caused by Ingestion of Oxidized 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 × â™‚ 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.

In vitro Assessment of the Safety and Potential Probiotic Characteristics of Three Bacillus Strains Isolated From the Intestine of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂).

Probiotics serving as an alternative to the criticized antibiotics mainly focus on improving animal's growth and health. After realizing the dangers posed by diseases that have led to lots of economic losses, aquaculture scientists have sought the usage of probiotics. However, most probiotics are ineffective in eliciting aquatic animals' preferred effects, since they are from non-fish sources. Again, there are even a few marine aquatic probiotics. Given this, a study was conducted to investigate the probiotic potential of the bacteria species isolated from the digestive tract of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). Based on the morphological, biochemical, 16S rRNA sequencing analysis and evolutionary relationships, the isolated species were identified as Bacillus tequilensis GPSAK2 (MW548630), Bacillus velezensis GPSAK4 (MW548635), and Bacillus subtilis GPSAK9 (MW548634), which were designated as GPSAK2, GPSAK4, and GPSAK9 strains, respectively. Their probiotic potentials including their ability to tolerate high bile salt concentration, low pH, high temperatures, adhesion ability (auto-aggregation and cell-surface hydrophobicity), antimicrobial activity and biosafety test, compatibility test, hemolytic activity, and antibiotic susceptibility test were evaluated. While GPSAK2 and GPSAK9 strains were γ-hemolytic, that of GPSAK4 was α-hemolytic. All the isolates were resistant to low pH (1) and higher bile salt concentration (0.5%), showed higher viability ability after higher temperature exposure (80, 90, and 100°C), as well as higher cell-surface percentage hydrophobicity and auto-aggregation. All isolates exhibited positive compatibility with each other, signifying their ability to be used as multispecies. The three strains were susceptible to ampicillin (except GPSAK9, which was resistant), penicillin, kanamycin, ceftriaxone, chloramphenicol, erythromycin, clindamycin, furazolidone (except GPSAK2 and GPSAK9, which were moderately susceptible and resistant, respectively), polymyxin B, vancomycin (except GPSAK9, which was resistant), sulfamethoxazole (except GPSAK9, which was moderately susceptible), amikacin, minocycline, ofloxacin, norfloxacin, doxycycline, neomycin, gentamicin, tetracycline, carbenicillin, midecamycin (except GPSAK9, which was moderately susceptible), ciprofloxacin, piperacillin, and cefoperazone. All isolates demonstrated good antimicrobial activity against four pathogens, viz. Streptococcus agalactiae, Streptococcus iniae, Vibrio harveyi, and Vibrio alginolyticus. The results collectively suggest that Bacillus strains GPSAK2, GPSAK4, and GPSAK9 could serve as potential probiotic candidates that can be used to improve the growth and health status of aquatic animals, especially grouper.