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Glycerol monolaurate (GML), a representative fatty acid glyceride, was used to promote growth. This study investigated the effects of GML in diets on growth, serum biochemical parameters, antioxidant capacity, liver morphology and lipid metabolism for juvenile pompano Trachinotus ovatus. Six groups of T. ovatus (mean weight = 14.00 ± 0.70 g) were fed with six diets, containing varying GML levels of 0.00% (G0), 0.05% (G5), 0.10% (G10), 0.15% (G15), 0.20% (G20), and 0.25% (G25) for 8 weeks. Fish were reared for in floating cages (length: 1 m; width: 1 m; and height: 2 m). The highest weight gain rate (WGR), specific growth rate (SGR), and protein deposit rate (PDR) were observed in the G15 group (P < 0.05). Hepatic glutathione peroxidase, total antioxidant capacity and superoxide dismutase activities were significantly increased in the G10 and G15 groups compared with the G0 group (P < 0.05), whereas hepatic malondialdehyde (MDA) was significantly reduced in the G10 and G15 groups (P < 0.05). The alkaline phosphatase activity was significantly higher in the G5, G10, G15, and G20 groups than in the G0 group (P < 0.05), and the highest AKP activity was observed in the G15 group. Alanine aminotransferase (ALT) activity was significantly lower in the G10, G15, G20, and G25 groups than in the G0 group (P < 0.05). The addition of GML to the feed significantly reduced triglyceride (TG) content (P < 0.05). In comparison with the G0 group, the G15, G20, and G25 groups had significantly lower total cholesterol content and significantly higher high-density lipoprotein content (P < 0.05). Low-density lipoprotein content was significantly lower in the G5 and G10 groups than in the G0 group (P < 0.05). Serious vacuolation occurred in the G0 group, but the cell boundaries in all added groups were obvious, and the rate of intracellular vacuolization decreased. As the GML level increased, carnitine palmitoyl-transferase Ι mRNA level was significantly up-regulated (P < 0.05). The expression of fatty acid synthesis in the G10, G15, G20, and G25 groups was significantly lower than that in the G0 group (P < 0.05). The expression of sterol regulatory element-binding protein-Ι was significantly lower in the G10, G15, G20, and G25 groups than in the G0 group (P < 0.05). Based on the broken-line model of WGR and PDR corresponding to GML levels, the optimum addition level of dietary GML was 0.14%-0.16% for juvenile T. ovatus. In conclusion, 0.15% GML significantly improved the growth performance, hepatic antioxidant ability, and lipid metabolism and protected the liver for juvenile pompano T. ovatus.

Little is known about the association of bile acids (BAs) with lipid deposition and lipid metabolism of fish in vitro. In the present study, we established the model of fatty-degenerated hepatocytes in pearl gentian grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀), and explored the effects and mechanism of BAs regulating on lipid metabolism in this model. The results showed that fatty-degenerated hepatocytes induced by lipid emulsion accumulated the intracellular triglyceride (TG), the enhanced expression of sterol responsive element binding protein 1 (SREBP1) protein, and the repressed expression of peroxisome proliferator-activated receptor alpha (PPARA), and phosphorylated PPARA (P-PPARA) proteins. BAs co-incubation reduced the content of TG, while increasing the expression of PPARA, farnesoid X receptor (FXR), and G protein-coupled bile acid receptor 1 (TGR5) proteins. Activation of FXR by INT-747 reduced the content of TG, while down-regulating the expression of SREBP1 and PPARA, and up-regulating the expression of P-PPARA, FXR, small heterodimer partner (SHP), and TGR5. Inhibition of FXR by guggulsterone increased the level of TG, while decreasing the expression of TGR5, increasing the expression of SHP. Activation of TGR5 by INT-777 reduced the content of TG, while down-regulating the expression of SREBP1 and SHP, up-regulated the expression of TGR5. Inhibition of TGR5 by SBI-115 elevated the level of TG, while reducing the expression of P-PPARA and TGR5. In conclusion, the FXR and/or TGR5 signaling pathways which were inhibited in fatty-degenerated hepatocytes from pearl gentian grouper, were activated after BAs co-incubation, then altering the lipid metabolism by repressing lipogenesis and enhancing lipolysis, and regulating transcriptional factors, thus reduces the lipid accumulation in vitro.

The effect of hydrolyzed fish protein powder (HFP) on the growth, intestinal development, gene mRNA expression, and enzyme activity in the intestine and liver of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ and Epinephelus lanceolatus ♂) was assessed after an 8-week feeding trial. Seven isonitrogenous (50%) and isolipidic (9%) diets were fed to hybrid grouper with 0% (CT), 1% (H1), 1.5% (H2), 2% (H3), 2.5% (H4), 3% (H5), and 4% (H6) HFP. No significant difference (p > 0.05) in weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), and survival rate (SR) was observed in all the groups. The crude protein content in the H6 group was significantly higher than in the other groups (p < 0.05). Intestinal lipase and trypsin activity were significantly higher in H3 and H5 groups (p < 0.05). In the serum, superoxide dismutase (SOD) activity was significantly higher in H5 and H6 groups, while malondialdehyde (MDA) activity was lower (p < 0.05) compared to other treatments. Insulin-like growth factor (IGF-I) and target of rapamycin (TOR) mRNA expression levels in the intestine and muscle were significantly higher in the H2 group and H1 group (p < 0.05), respectively. The most abundant intestinal bacteria found at the genus level are Acinetobacter, Vibrio, and Flavobacteriaceae. The villus was significantly longer in hybrid grouper fed with different levels of HFP compared to the control, and fish in the H2 group had thicker intestinal muscle compared to the other groups (p < 0.05). In conclusion, the addition of HFP to the low fishmeal (FM) diets of juvenile grouper improved the intestinal development and increased the levels of intestinal digestive enzymes.

This study evaluated the effects of lipid sources on the replacement of fishmeal with Clostridium autoethanogenum protein (CAP) in the diet of Pacific white shrimp (Litopenaeus vannamei). By using CAP to replace 40% fishmeal in the basal diet (contains 25% fishmeal), four diets (PC as the control diet, LSO as the low soybean oil level, MSO as the middle soybean oil level, and HSO as the high soybean oil level) were formulated by adding different proportions of fish oil and soybean oil. Each diet was assigned to four replicates (40 shrimps per replicate, initial weight = 1.79 ± 0.02 g). A 7-day Vibrio parahaemolyticus challenge test was conducted at the end of the 50-day feeding experiment. Weight gain was significantly higher and the cumulative mortality after challenge was lower in HSO treatment. The total antioxidant capacity (T-AOC) in hepatopancreas was significantly raised with increasing dietary soybean oil based on CAP substitution. After challenge, Pen3 mRNA expression was significantly higher in hepatopancreas and NF-κB pathway-related mRNA (dorsal, relish, and IKKβ) expression was higher in the intestine in HSO treatment. The height of mucosal folds and the thickness of the muscle layer were positively correlated with the level of dietary soybean oil. Transmission electron microscopy (TEM) analysis showed that the microvilli were damaged and the endoplasmic reticulum was swollen in shrimp fed the LSO diet. Diversity of intestinal microbiota was increased in shrimp fed the LSO diet, with a significant increase in beneficial bacteria such as Halocynthiibacter and Ruegeria as well as less harmful bacteria such as Vibrio. These results suggested that the replacement of fishmeal with CAP and the high proportion of soybean oil in the diet could improve the growth performance, disease resistance, and intestinal structure of Pacific white shrimp, and high fish oil in a low-FM diet raised the percentage of beneficial bacteria in the intestine of shrimp.

This experiment was conducted to assess the possibility of replacing fish meal (FM) with soy protein peptide (SPP) at different levels—0% (FM), 14.29% (S5), 28.57% (S10), 57.14% (S20), 71.42%(S25)—and its effects on growth, histology, gene expression related to liver lipid metabolism and intestinal immunity in juvenile pompano Trachinotus ovatus (initial mean weight = 39.88 ± 0.15 g). 600 healthy and uniformed-size fish were distributed to five groups of three replicates, each with 40 fish in each floating cage and fed twice daily for 8 weeks. Results showed that no significant difference in the growth was observed with SPP replacing FM (P > 0.05). Serum glutathione peroxidase activity in the S10 group was significantly higher than that in the FM group, and serum malondialdehyde content significantly decreased (P < 0.05). SPP significantly improved intestinal immunity by increasing alkaline phosphatase and lysozyme activities and up-regulating interleukin 10 and complement 4 mRNA levels while simultaneously decreasing triglyceride and total cholesterol content and down-regulating interleukin 1β mRNA expression. Villus length and muscle thickness in the S10 group were significantly higher than those in the FM group (P < 0.05). SPP significantly improved liver fat metabolism by increasing carnitine palmitoyl transferase I mRNA levels, and down-regulating fatty acid synthesis mRNA expression (P < 0.05). In summary, SPP substitution for FM promoted intestinal health, liver lipid metabolism and reduced liver fat accumulation for juvenile pompano T. ovatus, with no significant effect on growth performance. Based on the second-order polynomial analysis model of LYZ activity, the optimal replacement SPP level for juvenile pompano T. ovatus was 11.82%.

The supplementation of gossypol in excess is noted to cause detrimental effects such as the reduction of antioxidant enzymes and disruption of lipid metabolism in animals. Studies regarding the effects of different levels of gossypol are very rare; thus, this study was conducted to evaluate the effects of low and high dietary levels of gossypol and of supplementation with 0.13 % sodium butyrate (NaB) under high gossypol conditions on the growth performance and intestinal health of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂). Four treatments were used: Feed containing 40% fish meal was used as the control group [fishmeal (FM)], the FM diet plus 0.03% gossypol acetic acid (abbreviated as gossypol) as the low-level gossypol group (gL), FM + 0.15% gossypol was used as the high-level gossypol group (gH), and FM+0.15 % gossypol with 0.13 % NaB as the repair group (gHNaB). All diets were isonitrogenous and isolipidic. The results showed that the gL treatment significantly increased specific growth rate (SGR) and feed utilization; upregulated mRNA levels of distal intestinal transforming growth factor-β1 (tgfβ1), jam, occludin, claudin3, and zo1; and downregulated mRNA levels of il8, ifnγ, and akt. The gH treatment significantly reduced SGR and feed utilization; increased distal intestinal total nitric oxide synthase (NOS) activity and nitric oxide (NO) content; upregulated mRNA levels of distal intestinal tnfα, il1β, il6, ifnγ, caspase2, caspase9, and akt; and downregulated mRNA levels of tgfβ1, jam, and zo1. NaB supplementation significantly increased distal intestinal total NOS activity and NO content; downregulated distal intestinal tnfα, il1β, ifnγ, pi3k p85, and akt mRNA levels; and increased distal intestinal tgfβ1, jam, occludin, and zo1 mRNA levels. Above all, low- and high-level gossypol exhibited positive and negative effects on growth performance, distal intestinal anti-inflammatory capacity, and tight junctions, respectively, in hybrid groupers. NaB supplementation improved distal intestinal anti-inflammatory capacity and tight junctions in hybrid groupers to a certain extent.

The present study evaluated the effects of the dietary black soldier fly larvae meal (BSF) on growth performance, intestinal health, and susceptibility to Vibrio parahaemolyticus in the Pacific white shrimp Litopenaeus vannamei. The basal diet was formulated to contain 25% fish meal (FM), and then the FM was replaced with BSF for 10, 20, and 30% of the FM protein in the experimental diets, which are referred to as FM, BSF10, BSF20, and BSF30, respectively. Four hundred and eighty healthy and uniform-sized shrimp (~0.88 g) were distributed among four groups of three replicates, each with 40 shrimp in a 300-L tank and they were fed four times daily for 7 weeks. The results showed that the growth performance did not change significantly in shrimp fed with BSF10 and BSF20 diets, but significantly decreased in those fed with BSF30 diet compared to the ones fed with FM diet. After feeding trial and sampling, a V. parahaemolyticus infection challenge trial was conducted on shrimp. The results showed that the survival rate of shrimp fed with BSF10 was significantly higher than those fed with FM. The results of the midgut histology showed that the width and height of intestinal mucosal folds decreased significantly in shrimp fed with BSF20 and BSF30, and the early signs of apoptosis in the intestinal cells were found in shrimp fed with BSF30. The mRNA levels of non-specific immune-related genes dorsal and relish were downregulated in shrimp fed with BSF20 and BSF30 diets. The mRNA levels of antimicrobial peptides–related genes alf (anti-lipopolysaccharide factor) were upregulated in shrimp fed with BSF10 but downregulated in shrimp fed with BSF30. The mRNA level pen3 (penaeidins 3) was upregulated in shrimp fed with BSF10 and BSF20 diets. The intestinal bacterial communities on operational taxonomic unit levels among groups were not significantly differentiated according to the beta diversity analysis. At the genus levels, a decrease in Vibrio, Photobacterium, and Candidatus_Bacilloplasma, as well as the increase in Bacillus and Pseudoalteromonas abundance, indicated the improvement of intestinal microbiota in shrimp fed with dietary BSF. Therefore, the use of BSF in shrimp diet should be controlled at a dosage of 20% of the FM, which can improve the intestinal microbiota without causing any negative effects.

Oligosaccharides have recently received much attention from researchers owing to their multiple biological activities. This study was conducted to investigate the effects of a diet with reduced fish meal and chitosan oligosaccharide (COS) supplementation on a hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Seven isonitrogenous and isolipidic types of diet were formulated to feed the groupers for 56 days. To prepare the feed, a reference diet (FM group) containing 55% fish meal was used. Concentrated cottonseed protein (CPC) was used to replace 45% of the fish meal protein, and different COS supplementation levels (0, 0.2%, 0.4, 0.6, 0.8, and 1%) were added. After an 8-week breeding trial, Vibrio harveyi bacteria were injected into the groupers for a 7-day challenge test. The results showed that the FM and COS0.4 groups showed the best growth performance among the groups (p < 0.05); however, there was no significant difference in the survival rate (p > 0.05). Unlike in the FM group, adhesion and breakage of the intestinal plica occurred in the COS0 group. The height and width of the gut fold reached maximum values in the COS0.4 group (p < 0.05). Microbiome sequencing suggested that there was a stable microbiota core in the gut of the groupers. With increasing COS levels, the abundance of both beneficial bacteria and conditional pathogens increased; the activities of serum glucose oxidase, catalase, and total superoxide dismutase also increased (p < 0.05). In the gut tissue, the activities of glutathione peroxidase, glutathione reductase, and glutamine increased first but then decreased (p < 0.05); the contents of lysozyme, acid phosphatase, complements C3 and C4, and IgM showed upward trends (p < 0.05). Compared with that in the FM group, the expression of IL-1β and TNF-α in the COS0 group was upregulated. Gene expression levels of TLR22, TGF-β, and Nrf2 increased first but then decreased with COS supplementation levels (p < 0.05). COS supplementation reduced the cumulative mortality of the groupers in the challenge test (p < 0.05). In general, the results of this study demonstrated that dietary COS supplementation enhanced growth performance, intestinal health, and antioxidant and immune responses of groupers fed with a low-fish meal diet. The optimal and acceptable levels of COS supplement were 0.45 and 0.4–0.6%, respectively; these values can provide a reference for developing aquatic prebiotics.

Intestinal inflammatory disease induced by excessive soy protein substitutions for fish meal (FM) protein is a common phenomenon. The pearl gentian grouper, Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂, a marine fish with important economical and nutritional values, exhibited a similar problem. As far as we know, there are no reports on the full-length transcriptome of the pearl gentian grouper. In the present study, seven isonitrogenous and isolipidic (10% lipid) diets were prepared and fed to fish for 10 weeks. The water volume in each barrel was about 1 m3, using natural light and temperature. The results showed that 40% dietary soy proteins significantly negatively affected the growth performance of the pearl gentian grouper. Compared to the FM control, the content of immunoglobulin M and the enzyme activities of glutathione reductase, glutathione peroxidase, and total superoxide dismutase in the intestine significantly increased; the content of malondialdehyde in the intestine significantly decreased; and the enzyme activities of alanine transaminase and aspartate transaminase in the liver significantly increased. A library composed of seven different treated distal intestine tissues, including the FM control group, 20% soybean meal substitute for FM (SBM20), SBM40, 20% soybean protein concentrate (SPC20), SPC40, 20% fermented soybean meal substitute for FM (FSBM20), and FSBM40, was constructed and sequenced using PacBio single-molecule real-time (SMRT) and the RNA-Seq technology. As a whole, this study obtained 420,006 full-length non-chimeric (FLNC) reads. After error correction, sequence clustering, and redundancy, 82,351 transcripts with high quality were obtained. In addition, a total of 77,815 transcripts were annotated in seven databases (non-redundant protein sequences, non-redundant nucleotide sequences, Protein family, Clusters of Orthologous Groups of proteins, Gene Ontology, Swiss-Prot, and KEGG Orthology). Also, 49,093 long non-coding RNAs (lncRNAs) and 141,702 simple sequence repeats were identified. Based on full-length transcriptome sequencing, the present study found that the Toll-like receptor/nuclear factor kappa-B signaling pathway plays an important role in the development of SBM- and FSBM-induced enteritis. SPC-induced enteritis is mainly accompanied by a general imbalance of the nutrition absorption-related signaling pathways, which only affects a small part of the immune-related signaling pathways. This study supplies new and valuable reference transcripts, which would better facilitate further research on the pearl gentian grouper.

Different doses of β-conglycinin produce different regulations on the intestinal health of aquatic animals, affecting the absorption of nutrients, indirectly changing water quality. Sodium butyrate (NaB) can effectively alleviate the negative effects caused by high-dose β-conglycinin. We investigated the positive response to low-dose (1.5%, bL) and negative response to high-dose (6.0%, bH) β-conglycinin and supplementation with NaB (6.0% β-conglycinin + 0.13% NaB, bHNaB) in terms of water pollutants, microbiota, transcriptome, and metabolome in hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂). The ammonia nitrogen, nitrite, total nitrogen, and total phosphorus contents were significantly higher in the water from bH than from FMb, bL, and bHNaB. Supplementing with NaB significantly reduced the ammonia nitrogen, nitrite, total nitrogen, and total phosphorus contents. Low-dose β-conglycinin increased the relative abundance of Pelagibacterium, Pediococcus, Staphylococcus, and Lactobacillus and promoted the “ribosome,” “peroxisome proliferator-activated receptor (PPAR) signaling” and “histidine metabolism.” High-dose β-conglycinin increased the relative abundance of pathogenic bacteria Ralstonia and Photobacterium and inhibited the “cell cycle” “PPAR signaling” and “starch and proline metabolism.” NaB supplementation at high-dose β-conglycinin reduced the Ralstonia and Photobacterium abundance and promoted the “cell cycle,” “linoleic acid metabolism,” and “ABC transporters.” Overall, these results reveal differences in the effects of high- and low-dose β-conglycinin, as well as NaB supplementation, on the utilization of proteins, carbohydrates, and lipids and on substance transport and signaling among distal intestinal cells of hybrid grouper. A total of 15 differential metabolite biomarkers were identified: FMb vs. bL contained 10-methylimidazole acetic acid, N-acetyl histamine, urocanic acid, creatinine, glutathione, taurine, nervonic acid, stearic acid, docosanoic acid, and D-serine; FMb vs. bH contained 4-L-fucose, sucrose, α,α-trehalose, and quercetin; and bH vs. bHNaB contained 4-N-acetyl histamine, urocanic acid, creatinine, and S-adenosylhomocysteine, respectively. Our study provides new insights into the regulation of intestinal health by β-conglycinin in aquatic animals and the protective mechanism of NaB.

To study the effects of acute ammonia stress on the poisoning reaction of the hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu), 300 healthy grouper juveniles with an initial body weight of 51.4 ± 2.57 g were selected for an acute ammonia stress experiment using a half-lethal concentration of ammonia for 24, 48, 72, and 96 h with triplicate. The results show that (1) The half-lethal concentrations of ammonia for a hybrid grouper were 39.5 mg/L for 24 h, 27.3 mg/L for 48 h, 26.5 mg/L for 72 h, and 25.0 mg/L for 96 h, and the safe concentration was 2.50 mg/L. (2) The biochemical indices of the serum and livers of the groupers fluctuated under acute ammonia stress (P < 0.05), and the contents of serum cortisol and lactate reached the maximum value in 72 h (P < 0.05). (3) 24 h ammonia stress increased the activities of alkaline phosphatase, acid phosphatase, catalase, glutathione peroxidase, superoxide dismutase, lysozyme, and the content of immunoglobulin M in the liver (P < 0.05). Meanwhile, all ammonia-stressed fish groups had an increased amount of malondialdehyde in the liver (P < 0.05). (4) The ammonia poisoning caused significantly up-regulation of antioxidant genes, inflammatory cytokines, and apoptosis genes (P < 0.05), and the expression of inflammatory cytokines and apoptosis genes were the highest in 24 h ammonia stress group. (5) The ammonia content in the water changed the abundance and evenness of intestinal microbes in grouper, mainly in that ammonia stress significantly increased the relative abundance of fusobacteria, but significantly decreased the relative abundance of gemmatimonadetes (P < 0.05). It was speculated that apoptosis induced by ammonia poisoning in grouper may be related to oxidative stress and the activation of inflammatory factors, and excessive inflammatory stress may be one of the causes of ammonia poisoning in the hybrid grouper.

Lysine is known to be a functional nutrient from yeast to human beings. However, the mechanisms for lysine to regulate body growth and metabolism in fish species are far from fully illuminated. In order to elucidate the molecular effects of lysine on growth performance and metabolism, largemouth bass (Micropterus salmoides), which is extremely sensitive to protein composition, was chosen as the experimental model. Juvenile largemouth bass (4.16 ± 0.02 g) were fed with the lysine control diet (LC diet), the lysine supplementation diet (LS diet), and the low lysine diet (LL diet) for 8 weeks. The results showed that the LS diet group significantly increased fish growth, feed efficiency, and nutrient retention compared with those of the LL diet group in largemouth bass. Moreover, compared to the LL diet, lysine supplementation also elevated the plasma total protein, total EAA, total AA, and some individual AA concentrations. Mechanistically, lysine supplementation had a significant effect to decrease the mRNA expression levels of peptide and AA transporters induced by lysine restriction. Lysine supplementation also had an important impact on regulating the target of rapamycin (TOR) signaling pathway. More importantly, the key regulators in the amino acid response (AAR) signaling pathway were also down-regulated by lysine supplementation. Our results provide a clear elucidation of how dietary lysine affected growth performance, physiological and biochemical responses, and signaling responses and represent a sound foundation for using lysine to improve the nutrient utilization of poultry by-product meal in largemouth bass.