Single-Cell Transcriptomic Analysis Revealed the Cell Population Changes and Cell-Cell Communication in the Liver of a Carnivorous Fish in Response to High-Carbohydrate Diet.

BACKGROUND: Carnivorous fish have a low carbohydrate utilization ability, and the physiologic and molecular basis of glucose intolerance has not been fully illustrated. OBJECTIVES: This study aimed to use largemouth bass as a model to investigate the possible mechanism of glucose intolerance in carnivorous fish with the help of single-nuclei RNA sequencing (snRNA-seq). METHODS: Two diets were formulated, a low-carbohydrate (LC) diet and a high-carbohydrate (HC) diet. The feeding trial lasted for 6 wk, and then, growth performance, biochemical parameters, liver histology, and snRNA-seq were performed. RESULTS: Growth performance of fish was not affected by the HC diet, while liver glucolipid metabolism disorder and liver injury were observed. A total of 13,247 and 12,848 cells from the liver derived from 2 groups were isolated and sequenced, and 7 major liver cell types were annotated by the marker genes. Hepatocytes and cholangiocytes were lower and hepatic stellate cells (HSCs) and immune cells were higher in the HC group than those in the LC group. Reclustering analysis identified 7 subtypes of hepatocytes and immune cells, respectively. The HSCs showed more cell communication with other cell types, and periportal hepatocytes showed more cell communication with other hepatocyte subtypes. Cell-cell communication mainly focused on cell junction-related signaling pathways. Uncovered by the pseudotime analysis, midzonal hepatocytes were differentiated into 2 major branches-biliary epithelial hepatocytes and hepatobiliary hybrid progenitor. Cell junction and liver fibrosis-related genes were highly expressed in the HC group. HC diet induced the activation of HSCs and, therefore, led to the liver fibrosis of largemouth bass. CONCLUSIONS: HC diet induces liver glucolipid metabolism disorder and liver injury of largemouth bass. The increase and activation of HSCs might be the main reason for the liver injury. In adaption to HC diet, midzonal hepatocytes differentiates into 2 major branches-biliary epithelial hepatocytes and hepatobiliary hybrid progenitors.

Implications on growth performance, glucose metabolism, PI3K/AKT pathway, intestinal flora induced by dietary taurine in a high-carbohydrate diet for grass carp (Ctenopharyngodon idella).

An 8-week experiment was performed to investigate the influence on growth performance, plasma biochemistry, glucose metabolism and the insulin pathway of supplementation of dietary taurine to a high-carbohydrate diet for grass carp. In this study, fish were fed diets at one of two carbohydrate levels, 31·49 % (positive control) or 38·61 % (T00). The high-carbohydrate basal diet (T00), without taurine, was supplemented with 0·05 % (T05), 0·10 % (T10), 0·15 % (T15) or 0·20 % (T20) taurine, resulting in six isonitrogenous (30·37 %) and isolipidic (2·37 %) experimental diets. The experimental results showed that optimal taurine level improved significantly weight gain, specific growth rate (SGR), feed utilisation, reduced plasma total cholesterol levels, TAG and promoted insulin-like growth factor level. Glucokinase, pyruvate kinase and phosphoenolpyruvate carboxykinase activities showed a quadratic function model with increasing dietary taurine level, while hexokinase, fatty acid synthetase activities exhibited a positive linear trend. Optimal taurine supplementation in high-carbohydrate diet upregulated insulin receptor (Ir), insulin receptor substrate (Irs1), phosphatidylinositol 3-kinase (pi3k), protein kinase B (akt1), glycogen synthase kinase 3 ß (gs3kß) mRNA level and downregulated insulin-like growth factor (igf-1), insulin-like growth factor 1 receptor (igf-1R) and Fork head transcription factor 1 (foxo1) mRNA level. The above results suggested that optimal taurine level could improve growth performance, hepatic capacity for glycolipid metabolism and insulin sensitivity, thus enhancing the utilisation of carbohydrates in grass carp. Based on SGR, dietary optimal tributyrin taurine supplementation in grass carp was estimated to be 0·08 %.

Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile pacific shrimp (L. Vannamei).

This study looked at the effects of adding butyric acid (BA) to the diets of juvenile Pacific shrimp and how it affected their response to survival, immunity, histopathological, and gene expression profiles under heat stress. The shrimp were divided into groups: a control group with no BA supplementation and groups with BA inclusion levels of 0.5 %, 1 %, 1.5 %, 2 %, and 2.5 %. Following the 8-week feeding trial period, the shrimp endured a heat stress test lasting 1 h at a temperature of 38 °C. The results showed that the control group had a lower survival rate than those given BA. Interestingly, no mortality was observed in the group receiving 1.5 % BA supplementation. Heat stress had a negative impact on the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the control group. Still, these activities were increased in shrimp fed the BA diet. Similar variations were observed in AST and ALT fluctuations among the different groups. The levels of triglycerides (TG) and cholesterol (CHO) increased with high temperatures but were reduced in shrimp-supplemented BA. The activity of an antioxidant enzyme superoxide dismutase (SOD) increased with higher BA levels (P < 0.05). Moreover, the groups supplemented with 1.5 % BA exhibited a significant reduction in malondialdehyde (MDA) content (P < 0.05), suggesting the potential antioxidant properties of BA. The histology of the shrimp's hepatopancreas showed improvements in the groups given BA. Conversely, the BA significantly down-regulated the HSPs and up-regulated MnSOD transcript level in response to heat stress. The measured parameters determine the essential dietary requirement of BA for shrimp. Based on the results, the optimal level of BA for survival, antioxidant function, and immunity for shrimp under heat stress is 1.5 %.

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.

Muscular lipidomics and transcriptomics reveal the effects of bile acids on lipid metabolism in high-fat diet-fed grouper.

Little information is available on how exogenous bile acids alter lipid metabolism in muscle of fish. In the present study, an 8-week feeding trial were used to investigate the impacts of bile acids on lipid deposition, lipid metabolism, lipidomics, and transcriptomics in muscle of pearl gentian grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) fed a high-fat diet (HD). The HD treatment significantly increased the crude lipid content, while bile acids diet (BD) treatment decreased it (p = 0.057). BD treatment significantly decreased triglycerides level and significantly increased phosphatidylcholines, phosphatidylethanolamines, and phosphatidylglycerol levels. The contents of TG (17:0/18:2/18:2), TG (17:1/18:2/22:6), PC (6:0/22:1), PC (9:0/26:1), PC (26:1/6:0), PC (17:2/18:2), PE (16:0/18:1), PE (18:0/17:1), PG (18:0/20:5), PG (18:3/20:5), PG (19:0/16:1), and PG (18:0/18:1) in muscle were well response to dietary lipid level and bile acids supplementation. HD and BD groups induced a variety of adaptive metabolic responses in transcriptomics. HD treatment increased the lipogenesis and decreased lipolysis, whereas BD treatment decreased the lipogenesis and increased lipolysis. Present study revealed the improvement of muscular lipid metabolism and lipid composition in response to bile acids administration in pearl gentian grouper.

The effects of sodium butyrate (NaB) combination with soy saponin dietary supplementation on the growth parameters, intestinal performance and immune-related genes expression of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

Soy saponins are generally known to have negative effects on growth and the intestines of aquatic animals, and appropriate levels of sodium butyrate (NaB) may provide some mitigating effects. We investigated the effects of low and high levels of soy saponin and the protective effects of NaB (based on high level of soy saponin) on growth, serum cytokines, distal intestinal histopathology, and inflammation in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). The experiment included four groups: fishmeal group (FM, 0.00% saponin and 0.00% NaB), low saponin group (SL, 0.30% saponin and 0.00% NaB), high saponin group (SH, 1.50% saponin and 0.00% NaB) and high saponin with NaB group (SH-NaB, 1.50% saponin and 0.13% NaB). The results showed compared to FM, the final body weight (FBW) and weight gain (WG) were significantly higher and lower in SL and SH, respectively (P < 0.05). Compared to SH, the FBW and WG were significant higher in SH-NaB (P < 0.05). In the serum, compared to FM, the interferon γ (IFN-γ) and interleukin-1ß (IL-1ß) levels in SH were significantly increased (P < 0.05). Compared to SH, the IFN-γ level was significantly decreased in SH-NaB (P < 0.05). In the distal intestine, based on Alcian Blue-Periodic Acid-Schiff (AB-PAS) observation, the goblet cell/µm was significantly increased and decreased in the SL and SH, respectively, compared to FM. The intestinal diameter/plica height ratio in the SH was significantly higher than those in the FM, SL and SH-NaB (P < 0.05). The NO and ONOO- levels in the SH were significantly higher than that in FM and SL (P < 0.05). At the transcriptional level in the distal intestine, compared to FM, the mRNA levels of tumor necrosis factor (tnfα), il1ß, interleukin-8 (il8) and ifnγ were significantly up-regulated in the SH (P < 0.05). Compared to the SH, tnfα, il8 and ifnγ were significantly down-regulated in the SH-NaB (P < 0.05). Compared to the FM, the mRNA levels of claudin3, claudin15, zo2 and zo3 were significantly up-regulated in the SL (P < 0.05). The mRNA levels of occludin, claudin3, claudin12, claudin15, zo1, zo2 and zo3 were significantly down-regulated in the SH compared to the FM (P < 0.05). Additionally, compared to the SH, the mRNA levels of occludin, claudin3, claudin12, claudin15, zo1, zo2 and zo3 were significantly up-regulated in the SH-NaB (P < 0.05). After the 7-day Vibrio parahaemolyticus challenge test, the survival was significantly higher and lower in the SL and SH, respectively, compared to FM (P < 0.05). Overall, low and high levels of soy saponins had positive and negative effects on growth, disease resistance, serum cytokines, and distal intestinal development and anti-inflammation, respectively, in hybrid grouper. NaB effectively increased disease resistance and improved distal intestinal inflammation in hybrid grouper, but the effects of NaB were mainly observed in improving distal intestinal tight junctions.

Growth, immunity and transcriptome response to different stocking densities in Litopenaeus vannamei.

The effects of different stocking densities on Litopenaeus vannamei were investigated from the aspects of growth performance, immune response and transcriptome in this experiment. L. vannamei (initial body weight: 0.30 ± 0.02 g) were reared for 8 weeks at three stocking densities of 100 (LSD), 200 (MSD) and 300 (HSD) shrimp/m³, respectively. The results showed that the survival rate (SR), final body weight (FBW), weight gain rate (WGR), specific growth ratio (SGR) and protein efficiency ratio (PER) of L. vannamei significantly decreased, while the feed factor (FCR) significantly increased with the increase of stocking density. After Vibrio parahemolyticus infection, the SR of L. vannamei in the HSD group was significantly lower than that in the LSD and MSD groups. Increasing stocking density significantly increased the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lysozyme (LYS) while significantly decreased the activities of catalase (CAT) and phenol oxidase (PO) in the serum of L. vannamei. Similar changes of the gene expression as the activities of immune enzymes were found in the hemocytes. Pairwise comparison between the LSD, MSD and HSD group in the transcriptome analysis identified that there were 304, 1376 and 2083 differentially expressed genes (DEGs) in LSD vs MSD, MSD vs HSD and LSD vs HSD, respectively. Among them, most of the immune-related DEGs were down-regulated and metabolism-related DEGs were up-regulated with the increasing stocking density. In addition, KEGG enrichment pathway analysis revealed that several immune and metabolic related pathways including PI3K-Akt signaling pathway and AMPK signaling pathway were significantly enriched. Of these, the PI3K-Akt signaling pathway had the most DEGs and was also the most significantly enriched pathway. Furthermore, 16 DEGs (such as FOXO, PCK2 and CTSC, etc.) and partial immune enzyme activity (such as AST, CAT and PO, etc.) changes were closely correlated with the increase of stocking density when partial immune-related DEGs and immune-related enzymes were analyzed jointly. All these results indicated that changes in stocking density had a significant effect on the growth performance, immunity and transcriptome of L. vannamei.

Glutamine improves growth and intestinal health in juvenile hybrid groupers fed high-dose glycinin.

Glutamine addition can improve immunity and intestinal development in fish. This study examined the protective roles of glutamine on growth suppression and enteritis induced by glycinin in juvenile hybrid groupers (female Epinephelus fuscoguttatus × male Epinephelus lanceolatus). The experiment set four isonitrogenous and isolipidic trial diets: a diet containing 10% glycinin (11S), 10% of 11S diet supplemented with 1% or 2% alanine-glutamine (1% or 2% Ala-Gln), and a diet containing neither 11S nor Ala-Gln (FM). A feeding trial was conducted in hybrid grouper for 8 weeks. Weight gain and specific growth rates in Groups 1% and 2% Ala-Gln were significantly higher than those of the 11S group but were similar to those of the FM group. The intestinal muscular layer thickness, plica height and width of the 2% Ala-Gln group were significantly higher than those of Group 11S. The enterocyte proliferation efficiency of the 11S group was significantly lower compared to other groups. Compared with the 11S group, Groups 1% and 2% Ala-Gln fish had increased intestinal lysozyme activities, complement 3 and immunoglobulin M as well as cathelicidin contents. The mRNA levels of tnf-α, il-1ß, ifn-α, and hsp70 genes were more downregulated in Groups 1% and 2% Ala-Gln than in Group 11S. Compared with FM group, fish from the 11S group had significantly lower mRNA levels of myd88, ikkß, and nf-κb p65 genes. These three values in the 2% Ala-Gln group were significantly lower than those in Group 11S but not significantly different from those of Group FM. The relative abundance of Vibrio in Group 11S was higher than that in Groups FM and 2% Ala-Gln. Intestinal glutamine, glutaminase, glutamic acid, α-ketoglutarate, malate dehydrogenase and ATP contents were higher in Groups 1% and 2% Ala-Gln than in Group 11S. These results suggest that glutamine is a useful feed additive to enhance growth and intestinal immunity, alleviate inflammation, and modulate gut microbiota in hybrid grouper fed high-dose glycinin.

The enhancement effect of low-dose dietary lipopolysaccharide on the growth and immunity of Litopenaeus vannamei, and transcriptome analysis.

In this study, the effects of dietary lipopolysaccharide (LPS) on Litopenaeus vannamei were investigated to determine whether LPS could play a role as a potential immunostimulant in shrimp. L. vannamei with an initial body weight of 0.30 ± 0.02 g were fed a diet containing LPS at doses of 0, 0.2, 1, 5, 25 or 125 mg kg-1 for eight weeks (groups LPS0, LPS0.2, LPS1, LPS5, LPS25 and LPS125, respectively). After eight weeks of feeding, the growth performance, immunity and transcriptome response of L. vannamei were analysed. Only dietary LPS at 0.2 and 1 mg kg-1 resulted in a significant increase in the growth of L. vannamei (P < 0.05). According to the weight gain rate (WGR) and specific growth rate (SGR), the optimum dietary LPS level was 2.462 and 2.455 mg kg-1, respectively. When compared with the control group, the survival rate (SR) of L. vannamei in the LPS0.2 group was significantly increased after white spot syndrome virus (WSSV) infection and the SR of L. vannamei in the LPS1 group was significantly increased after Vibrio parahaemolyticus infection (both P < 0.05). Compared with the LPS0 group, immune enzyme activity in the serum of L. vannamei could be significantly increased and the content of maleic dialdehyde (MDA) significantly decreased by dietary LPS. Transcriptome analysis of the haemocytes of L. vannamei identified 399 up-regulated differentially expressed genes (DEGs) and 5000 down-regulated DEGs in the LPS0.2 compared to the control group. Most of the DEGs were significantly enriched in the following pathways: phosphatidylinositol signalling, Wnt signalling, Jak-STAT signalling and inositol phosphate metabolism. In conclusion, this study revealed that diets supplemented with low-dose LPS had positive effects on the growth and immunity of L. vannamei.

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.

The Effect of Glycerol Monolaurate on Intestinal Health and Disease Resistance in Cage-Farmed Juvenile Pompano Trachinotus ovatus.

This research studied the effects of glycerol monolaurate (GML) to diets on the digestive capacity, intestinal structure, intestinal microbiota, and disease resistance for juvenile pompano Trachinotus ovatus (mean weight = 14.00 ± 0.70 g). T. ovatus were, respectively, fed six diets containing 0.00, 0.05, 0.10, 0.15, 0.20, and 0.25% GML for 56 days. The highest weight gain rate was observed in the 0.15% GML group. In the intestine, amylase activities in the 0.10, 0.15, 0.20, and 0.25% GML groups were significantly increased, compared with 0.00% GML group (P < 0.05). Lipase activities in the 0.10 and 0.15% GML groups were significantly increased (P < 0.05). Similar significant elevations in the protease activities were also found in the 0.10, 0.15, and 0.20% GML groups (P < 0.05). Amylase activities were significantly higher in the 0.10, 0.15, 0.20, and 0.25% GML groups than that in the 0.00% GML group (P < 0.05). Villus lengths (VL) and muscle thicknesses (MT) of the 0.05, 0.10, 0.15, and 0.20% GML groups were significantly enhanced, and the villus widths (VW) in the 0.05, 0.10, and 0.15% groups were significantly increased (P < 0.05). Additionally, 0.15% GML significantly improved the intestinal immunity by upregulating interleukin 10 (il-10), increasing beneficial bacteria abundances (e.g., Vibrio, Pseudomonas, and Cetobacterium), downregulating nuclear factor kappa b (nf-κb) and interleukin 8 (il-8), and decreasing harmful bacteria abundances (e.g., Brevinema and Acinetobacter) (P < 0.05). After challenge test, GML significantly increased the survival rate (80%-96%) (P < 0.05). In addition, ACP and AKP activities in the GML-supplemented groups were significantly higher than those in the 0.00% GML group, and LZM activity was significantly higher in the 0.05, 0.10, 0.15, and 0.20% GML groups than that in the 0.00% GML group (P < 0.05). In summary, 0.15% GML significantly promoted the intestinal digestibility, improved the intestinal microflora, regulated intestinal immune-related genes, and increased resistance to V. parahaemolyticus of juvenile pompano T. ovatus.

Replacement of Dietary Fishmeal with Clostridium autoethanogenum Protein on Lipidomics and Lipid Metabolism in Muscle of Pearl Gentian Grouper.

Clostridium autoethanogenum protein (CAP) is an economical and alternative protein source. Here, three experimental diets were formulated with CAP replacing 0% (CAP-0), 30% (CAP-30), and 60% (CAP-60) of fishmeal to investigate the alterations of structure integrity, fatty acids profiles, and lipid metabolism in the muscle of pearl gentian grouper. With increasing levels of CAP substitution, the percentages of 16 : 0 or 18 : 0 were decreased in triglycerides (TG) and diacylglycerols (DG); 18 : 1 or 18 : 2 was increased at the sn-1 and sn-2 positions in phosphatidylethanolamines; 20 : 5n-3 was increased in TG and DG. The phosphatidylcholines (PC) (18 : 3/20 : 5), PC(22 : 6/17 : 1), and sphingomyelins (d19 : 0/24 : 4) were identified as potential lipid biomarkers between CAP treatments. The CAP-30 treatment enhanced lipolysis and lipogenesis, while the CAP-60 treatment inhibited lipogenesis. In conclusion, fishmeal replacement with CAP affected the lipid characteristics and lipid metabolism, whereas it did not affect the structural integrity and fatty acids profiles in the muscle of pearl gentian grouper.

Effects of Dietary Lysine Level on Growth Performance and Protein Metabolism in Juvenile Leopard Coral Grouper (Plectropomus leopardus).

An 8-week feeding trial was conducted to evaluate the effects of dietary lysine level on growth performance and protein metabolism of juvenile leopard coral grouper (Plectropomus leopardus) and thereby obtained the optimal dietary lysine requirement of P. leopardus. Six isoproteic and isolipidic experimental diets were formulated to contain 1.10%, 1.69%, 2.30%, 3.08%, 3.56%, and 4.36% lysine of diets, respectively. Each diet was assigned at random to triplicate groups of 25 juveniles (initial mean weight is 10.57 g) per tank in a flow-through mariculture system maintained at 27-30°C. Dietary inclusion of 2.30-3.08% lysine improved the weight gain rate (WGR) and specific growth rate and decreased the feed conversion ratio (FCR) of juveniles (P < 0.05). The intestinal digestive enzyme (trypsin, amylase, and lipase) activities were overall enhanced by dietary inclusion of 3.08-3.56% lysine (P < 0.05). The mammalian target of rapamycin (mTOR) signaling pathway was activated in fish fed diets with 1.69-2.30% lysine by upregulating the relative expression levels of hepatic TOR and S6K1 (p70 ribosomal protein S6 kinase 1) but downregulating the relative expression level of hepatic 4E-BP2 (eIF4E-binding protein 2). Conversely, the amino acid response signaling pathway was inhibited in fish fed diet with 2.30% lysine by downregulating the relative expression levels of hepatic GCN2 (general control nondepressible 2), ATF3 (activating transcription factor 3), ATF4a (activating transcription factor 4a), and ATF4b (activating transcription factor 4b). Additionally, dietary 1.69-3.08% lysine enhanced the plasma total protein level and hepatic lysine α-ketoglutarate reductase activity but depressed the blood urea nitrogen level and hepatic adenosine monophosphate deaminase activity (P < 0.05). Moreover, dietary 3.08% lysine increased the contents of whole-body crude protein and total amino acids, while 1.69%-4.36% lysine depressed the whole-body lipid content (P < 0.05). These results indicated that optimal dietary lysine increased the digestive enzyme activities, promoted protein synthesis but depressed protein degradation, and thereby improved the growth performance of P. leopardus. Based on the second-order polynomial model, the optimal lysine requirement of juvenile P. leopardus for WGR, FCR, and lysine deposition was 2.60%-2.97% of diets (4.91%-5.60% of dietary protein).

Effects of Clostridium butyricum on Growth Performance, Intestinal Health, and Disease Resistance of Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) Fed with Cottonseed Protein Concentrate (CPC) Replacement of Fishmeal.

An 8-week feeding trial was conducted to investigate the effects of C. butyricum on the growth performance, microbiota, immunity response, and disease resistance in hybrid grouper fed with cottonseed protein concentrate (CPC) replacement of fishmeal. Six groups of isonitrogenous and isolipid diets were formulated including a positive control group (50% fishmeal, PC), a negative control group (CPC replaced 50% of fishmeal protein, NC), and Clostridium butyricum supplemented with 0.05% (C1, 5 × 108 CFU/kg), 0.2% (C2, 2 × 109 CFU/kg), 0.8% (C3, 8 × 109 CFU/kg), and 3.2% (C4, 3.2 × 1010 CFU/kg), respectively, to the NC group. The results showed that weight gain rate and specific growth rate were significantly higher in the C4 group than that in the NC group (P < 0.05). After supplementation with C. butyricum, the amylase, lipase, and trypsin activities were significantly higher than the NC group (P < 0.05; except group C1), and the same results were obtained for intestinal morphometry. The intestinal proinflammatory factors were significantly downregulated, and the anti-inflammatory factors were significantly upregulated in the C3 and C4 groups compared with the NC group after supplementation with 0.8%-3.2% C. butyricum (P < 0.05). At the phylum level, the PC, NC, and C4 groups were dominated by the Firmicutes and the Proteobacteria. At the genus level, the relative abundance of Bacillus in the NC group was lower than that in the PC and C4 groups. After supplementation with C. butyricum, grouper in the C4 group showed significantly higher resistance to V. harveyi than the NC group (P < 0.05). Above all, taking into account the effects of immunity and disease resistance, it was recommended to supplement 3.2% C. butyricum in the diet of grouper fed the replacement of 50% fishmeal protein by CPC.

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.

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 black soldier fly (Hermetia illucens) larvae meal on lipid and glucose metabolism of Pacific white shrimp Litopenaeus vannamei.

The present study investigated the effect of black soldier fly (Hermetia illucens) larvae meal (BSF) on haemolymph biochemical indicators, muscle metabolites as well as the lipid and glucose metabolism of Pacific white shrimp Litopenaeus vannamei. Four diets were formulated in which the control diet contained 25 % of fishmeal (FM) and 10 % (BSF10), 20 % (BSF20), and 30 % (BSF30) of FM protein were replaced with BSF. Four hundred and eighty shrimp (0·88 ± 0·00 g) were distributed to four groups of three replicates and fed for 7 weeks. Results showed that growth performance of shrimp fed BSF30 significantly decreased compared with those fed FM, but there was no significant difference in survival among groups. The whole shrimp crude lipid content, haemolymph TAG and total cholesterol were decreased with the increasing BSF inclusion. The results of metabolomics showed that the metabolite patterns of shrimp fed different diets were altered, with significant changes in metabolites related to lipid metabolism, glucose metabolism as well as TCA cycle. The mRNA expressions of hk, pfk, pk, pepck, ampk, mcd, cpt-1 and scd1 in hepatopancreas were downregulated in shrimp fed BSF30, but mRNA expression of acc1 was upregulated. Unlike BSF30, the mRNA expressions of fas, cpt-1, fbp and 6pgd in hepatopancreas were upregulated in shrimp fed BSF20. This study indicates that BSF20 diet promoted lipid synthesis and lipolysis, while BSF30 diet weakened ß-oxidation and glycolysis as well as affected the unsaturated fatty acids synthesis, which may affect the growth performance and body composition of shrimp.

Catalytic and regulatory subunits of casein kinase 2 in Penaeus vannamei: Cloning, identification, expression profiles and functional analysis.

As a highly conserved serine/threonine kinase with catalytic and regulatory subunits distributed ubiquitously in eukaryotic organisms, casein kinase 2 (CK2) is involved in multiple cellular functions, including immune regulation. In this study, two variants of the catalytic subunit (designated PvCK2α-1 and PvCK2α-2) and the regulatory subunit homologs (designated PvCK2ß-1 and PvCK2ß-2) in Penaeus vannamei were cloned and characterised. PvCK2α-1 and PvCK2α-2 shared the same genomic sequence consisting of six exons and five introns and encoded the same protein of 350 amino acids with an S_TKc domain, although there was a sequence deletion in 3'-UTR in PvCK2α-2 when compared with PvCK2α-1. Because of the sequence deletion in the ORF, PvCK2ß-1 and PvCK2ß-2 encoded different proteins with a CK_II_beta domain. The gene structures of PvCK2ß-1 and PvCK2ß-2 were identical and consisted of four exons and three introns. Semi-quantitative RT-PCR analyses revealed that PvCK2α and PvCK2ß were constitutively expressed in all P. vannamei tissues tested, with higher levels detected in the immune-related tissues including hemocytes, hepatopancreas, gills and intestine. In these four tissue types, all variants of PvCK2α and PvCK2ß were induced upon challenge with white spot syndrome virus (WSSV), Vibrio parahaemolyticus and Staphyloccocus aureus. The inhibition of PvCK2α, PvCK2ß-1 and PvCK2ßComb (the amount of PvCK2ß-1 and PvCK2ß-2) significantly reduced the survival rates of P. vannamei after WSSV infection and significantly increased the WSSV viral loads. Knockdown of PvCK2 by RNAi could distinctly decrease the expression of NF-κB related genes. All of these results suggest that PvCK2 plays an important role in the innate immune response to pathogen challenges in P. vannamei, with a positive role in anti-WSSV response which may be mediated through regulating the expression of NF-κB drived antimicrobial peptide genes.

Effects of dietary chenodeoxycholic acid supplementation in a low fishmeal diet on growth performance, lipid metabolism, autophagy and intestinal health of Pacific white shrimp, Litopenaeus vannamei.

An 8-week feeding trial was conducted to evaluate the effects of chenodeoxycholic acid (CDCA) on growth performance, body composition, lipid metabolism, and intestinal health of juvenile white shrimp, Litopenaeus vannamei fed a low fishmeal diet. Four practical diets were formulated: HFM (25% fishmeal), LFM (15% fishmeal), LB1 (LFM + 0.04% CDCA), LB2 (LFM + 0.08% CDCA). Each diet was assigned to four tanks with forty shrimp (initial weight 0.33 ± 0.03 g) per tank. The results indicated that the growth performance of shrimp were similar between the four groups; the crude lipid content of shrimp fed the LB2 diet was significantly lower than those fed the HFM diet (P < 0.05). The lipase activity content in hepatopancreatic were significantly higher in the two CDCA supplemented groups than that in LFM group; the contents of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol in hemolymph were significantly lower in LFM group, LB1 group and LB2 group than that in HFM group (P < 0.05). The shrimp fed LB1 diet was significantly decreased the intestinal expression levels of tube than those fed in HFM diet; the intestinal gene expression of imd and toll were significantly lower in LB2 group than those in HFM group (P < 0.05). The results of hepatopancreas gene expression suggest that shrimp fed the LFM diet showed significantly upregulated expression levels of sterol regulatory element-binding protein (srebp), acetyl-CoA carboxylase (acc), and carnitine palmitoyltransferase 1 (cpt-1) than those fed the HFM diet; shrimp fed the LB1 diet showed significantly upregulated expression levels of srebp, acc, and AMP-activated protein kinase (ampk) than those fed the HFM diet; shrimp fed the LB2 diet had higher expression levels of srebp, acc, and cpt-1 than those fed the HFM diet (P < 0.05). In the hepatopancreas, the shrimp fed the LFM diet shown significantly up-regulated the expression levels of beclin1 compared to those fed HFM diet; the expression levels of autophagy-related protein13 (atg3), autophagy-related protein 12 (atg12) of in shrimp fed the LB1 diet were significantly higher than those fed the HFM diet; and the expression levels of autophagy-related protein13 (atg13), beclin1, atg3, atg12, autophagy-related protein 9 (atg9) of shrimp fed LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). The atg3 in intestine of shrimp fed the LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). Intestinal mucous fold were damaged, hepatic tubules were disorganized and B cells appeared to be swollen in LFM group. The fold height and width of shrimp fed the diets supplemented with CDCA increased significantly than those fed the LFM diet (P < 0.05), the hepatic tubules were neatly arranged, and R cells increased. In conclusion, supplementary CDCA in a low fishmeal diet promoted lipid metabolism, enhanced autophagy of shrimp, also improved the health of the intestine and hepatopancreas.

Effects of fishmeal replacement by black soldier fly on growth performance, digestive enzyme activity, intestine morphology, intestinal flora and immune response of pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

An 8-week feeding trial was conducted to investigate the influence of partial replacement of fishmeal (FM) by black soldier fly (BSF) (Hermetia illucens) on the growth, distal intestine morphology, intestinal flora, and intestinal immune response of pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Four diets were formulated, 0% (0 g kg-1), 10% (50 g kg-1), 20% (100 g kg-1) and 30% (150 g kg-1) fishmeal were replaced with BSF, named as FM, BSF10, BSF20, BSF30, severally. The study found that, with the increasing dietary BSF levels, growth and feed conversion ratio of fish decreased significantly (P < 0.05). Chitinase and trypsin activities were significantly increased with increasing dietary BSF levels (P < 0.05). With the increasing dietary BSF levels, distal intestinal muscularis thickness and mucosal fold length decreased significantly (P < 0.05), as well as total abundance of intestinal flora. The relative abundance of four phyla and six genera among the top 20 genera were significantly affected by dietary BSF levels (P < 0.05). With the increasing dietary BSF levels, the mRNA levels of nf-κbem1, r-cel and il-10 up-regulated significantly (P < 0.05). For fish fed BSF30 diet, the mRNA levels of myd88 and tlr22 were significantly higher than fish fed FM diet (P < 0.05). In conclusion, replacement fishmeal with BSF increased activity of digestive enzymes, but negatively affected growth performance and intestinal health of pearl gentian grouper.