Promotion of improved intestinal barrier health by soybean-derived bioactive peptides in Chinese mitten crab (Eriocheir sinensis) fed a low fishmeal diet.

To alleviate the growth inhibition, and intestinal damage of Chinese mitten crab (Eriocheir sinensis) induced by low fishmeal diets (LF), an 8-week feeding trial was conducted to evaluate the addition of dietary soybean-derived bioactive peptides (SBP) in LF diets on the regulation of growth, digestion and intestinal health. The crabs were fed isonitrogenous and isoenergetic conventional diet and LF diets (10 % fishmeal replaced by soybean meal, LF) supplemented with 0, 1 %, 2 %, 4 % and 6 % SBP, respectively. The results showed that LF diet inhibited growth while inclusion of SBP quadratically remitted the growth inhibition induced by LF. For digestive function, increasing addition level of SBP quadratically improved the α-amylase and trypsin activities. For antioxidant function, LF group significantly increased the malondialdehyde content, while SBP linearly decreased the malondialdehyde level and cubically increased the anti-superoxide anion activity and total antioxidant capacity level. For intestinal health, the peritrophic membrane (PM) almost completely separated from the inner wall of the intestinal lumen, the epithelial cells reduced, the muscularis became thinner and the apoptotic signals increased in LF group; with SBP addition, the intestinal morphology was improved, with the PM adhering to the inner wall of the intestinal lumen, an increase in the number of epithelial cells and an increase in the thickness of the muscularis. Additionally, there was a decrease in apoptotic signals. Dietary SBP also increased the expression of PT and Crustin1 quadratically and decreased the expression of ALF1 linearly, ALF3 and ILF2 quadratically.

Bile acid and short chain fatty acid metabolism of gut microbiota mediate high-fat diet induced intestinal barrier damage in Macrobrachium rosenbergii.

The limited tolerance of crustacean tissue physiology to a high-fat diet has captured the attention of researchers. Yet, investigations into the physiological response mechanisms of the crustacean intestinal barrier system to a high-fat diet are progressing slowly. Elucidating potential physiological mechanisms and determining the precise regulatory targets would be of great physiological and nutritional significance. This study established a high-fat diet-induced intestinal barrier damage model in Macrobrachium rosenbergii, and systematically investigated the functions of gut microbiota and its functional metabolites. The study achieved this by monitoring phenotypic indicators, conducting 16S rDNA sequencing, targeted metabolomics, and in vitro anaerobic fermentation of intestinal contents. Feeding prawns with control and high-fat diets for 8 weeks, the lipid level of 7 % in the CON diet and 12 % in the HF diet. Results showed that high-fat intake impaired the intestinal epithelial cells, intestinal barrier structure, and permeability of M. rosenbergii, activated the tight junction signaling pathway inhibiting factor NF-κB transcription factor Relish/myosin light chain kinase (MLCK), and suppressed the expression of downstream tight junction proteins zona occludens protein 1 (ZO-1) and Claudin. High-fat intake resulted in a significant increase in abundance of Aeromonas, Enterobacter, and Clostridium sensu stricto 3 genera, while Lactobacillus, Lactococcus, Bacteroides, and Ruminococcaceae UCG-010 genera were significantly decreased. Targeted metabolomics results of bile acids and short-chain fatty acids in intestinal contents and in vitro anaerobic fermentation products showed a marked rise in the abundance of DCA, 12-KetoLCA, 7,12-diketoLCA, and Isovaleric acid, and a significant reduction in the abundance of HDCA, CDCA, and Acetate in the HF group. Pearson correlation analysis revealed a substantial correlation between various genera (Clostridium sensu stricto 3, Lactobacillus, Bacteroides) and secondary metabolites (DCA, HDCA, 12-KetoLCA, Acetate), and the latter was significantly correlated with intestinal barrier function related genes (Relish, ZO-1, MLCK, vitamin D receptor, and ecdysone receptor). These findings indicate that gut microorganisms and their specific bile acids and short-chain fatty acid secondary metabolites play a crucial role in the process of high-fat-induced intestinal barrier damage of M. rosenbergii. Moreover, identifying and targeting these factors could facilitate precise regulation of high-fat nutrition for crustaceans.

Effects of high fat in the diet on growth, antioxidant, immunity and fat deposition of Macrobrachium rosenbergii post-larvae.

Lipids are essential nutrients for organisms, and high-fat feeds for shrimp may cause oxidative stress. This study evaluated the effects of feeding high fat in the diet on the growth, antioxidant, immunity, and liver fat accumulation of Macrobrachium rosenbergii post-larvae. Five groups with an initial body weight of 0.0084 ± 0.001 g were fed five isonitrogenous and isoenergetic diets (47.01% crude protein and 18.40 kJ/g gross energy) containing 8%, 10%, 12%, 14% and 16% (named L8, L10, L12, L14 and L16) lipid for 8 weeks, respectively. The results showed that the weight gain rate (WGR) and specific growth rate (SGR) of L8 group were significantly higher than those of L10, L12, L14 and L16 group (P < 0.05), and the feed coefficient (FCR) of L8 group was significantly lower than that of other groups (P < 0.05). With the increase of dietary fat level, the content of MDA and the activity of SOD increased significantly, and the activities of T-AOC and CAT decreased significantly (P < 0.05). H&E staining clearly revealed the occurrence of hepatocyte swelling, hepatocyte vacuolization and nucleus displacement to the peripheral cell vacuolization in the L16 group, and hepatic lipid accumulation was further observed in the L14 and L16 group by Oil red O staining. In addition, high-fat diet significantly upregulated the expression of Dorsal, Relish and IκBα mRNA, and also upregulated the expression of fat synthesis-related genes FAS, ACC, DGAT and fat transport-related gene FABP (P < 0.05), and significantly downregulated the expression of fat metabolism-related genes AMPK and CPT-1 (P < 0.05) compared to that of the L8 group. In conclusion, this study showed that feeding a high-fat diet could induce oxidative stress, inhibit growth performance, alter antioxidant capacity, cause hepatic fat deposition and affect the immune system of M. rosenbergii post-larvae.

Effects of dietary tea tree oil on the growth, physiological and non-specific immunity response in the giant freshwater prawn (Macrobrachium rosenbergii) under high ammonia stress.

This study aimed to investigate the effects of dietary tea tree oil (TTO) on the performance, intestinal antioxidant capacity, and non-specific immunity after ammonia nitrogen stress in Macrobrachium rosenbergii. Six experimental diets were formulated with 0, 25, 50, 100, 200, 400 mg/kg TTO, respectively. A total of 900 prawns (average initial weight, 0.39 ± 0.01 g) were randomly assigned to 6 groups in triplicate in 18 tanks. After an 8-week feeding trial, 20 prawns from each tank were changed with 20 mg/L ammonia stress for 24 h. The results showed that 100 mg/kg TTO significantly increased prawns performance and survival rate compared with the control group. Moreover, 100 and 200 mg/kg TTO significantly improved intestinal antioxidant capabilities by increasing SOD enzyme activities and decreasing MDA levels. In addition, the prawns fed with 100 mg/kg TTO diet showed the highest survival rate under ammonia stress. After ammonia stress, the group of 100 mg/kg TTO significantly improved antioxidant capacity by increasing hemolymph respiratory burst activity, as well as intestinal anti-superoxide anion activity and SOD. Coincidentally, 100 mg/kg TTO significantly upregulated the intestinal relative expression of antioxidant-related genes (peroxiredoxin-5). Further, it was found that 100 mg/kg TTO activated the toll-dorsal pathway in prawns, which performed the similar function as the classic NF-κB pathway by upregulating the TNF-α and IL-1. Finally, 100 mg/kg TTO increased the levels of iNOS activities and NO contents after ammonia stress and enhanced non-specific immunity. The results indicated that 100 mg/kg TTO could significantly improve the M. rosenbergii performance, antioxidant capacity and ammonia stress resistance. We suggested that the mechanisms may be attributed to that TTO enhanced the antioxidant capacity and non-specific immunity of M. rosenbergii via the NF-κB signal pathway.

Amelioration of ammonia-induced intestinal oxidative stress by dietary Clostridium butyricum in giant freshwater prawn (Macrobrachium rosenbergii).

The use of Clostridium butyricum in crustacean aquaculture for anti-abiotic stress is yet unknown. Feeds were formulated containing 0, 125, 250, 500, and 1000 mg/kg Clostridium butyricum (2 × 107 CFU/g), respectively. The giant freshwater prawns (Macrobrachium rosenbergii) were fed for 8 weeks in triplicate. The results showed that C. butyricum-supplemented groups improved growth performance significantly with the optimum level at 610 mg/kg. Ammonia stress reduced hemolymph glucose, total protein, total cholesterol, and triglyceride concentrations while dietary C. butyricum significantly increased hemolymph glucose and total protein levels after the ammonia challenge. Ammonia stress increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels, and the treatments supplemented with C. butyricum had considerably enhanced levels of iNOS and NO after stress. Treatment with C. butyricum increased the level of superoxide dismutase (SOD), and decreased the level of malondialdehyde (MDA) and superoxide anion, with the 125 mg/kg treated groups having the extreme value. Furthermore, C. butyricum-treated groups reduced the expression of HSPs after ammonia stress while the ammonia stress induced the expression of HSP60, HSP70, and HSP90. Dietary C. butyricum elevated the expression of peroxiredoxin-5 and toll in response to ammonia stress. The results indicate that dietary supplementation with 125-500 mg/kg of C. butyricum (2 × 107 CFU/g) improved biochemical and antioxidant features as well as intestinal immunity of M. rosenbergii under ammonia challenge by activating the toll signal pathway.

Effects of dietary threonine on growth and immune response of oriental river prawn (Macrobrachium nipponense).

A 70-day feeding trial was conducted to ascertain the effects of threonine on immune response of juvenile oriental river prawn (Macrobrachium nipponense). Six isonitrogen and isolipidic feeds were formulated according to levels of dietary threonine (0.35%, 0.79%, 1.18%, 1.67%, 2.08% and 2.48% respectively). The juvenile prawns were divided into six groups with four replicates, and stocked into 24 tanks with 50 prawns per tank (initial weight 0.20 ± 0.02 g). The results showed a significant increasing trend of final body weight, specific growth rate, protein efficiency ratio, and weight gain rate when threonine levels increased to 1.67% (P < 0.05). However, feed intake, feed conversion ratio, and whole-body lipid composition significantly decreased as threonine levels in the feed increased up to 1.67% (P < 0.05). Moreover, haemolymph N-urea content was significantly lowest at 1.67% threonine level (P < 0.05), whereas glucose was highest at 0.79% followed by 1.67% of threonine levels in the feeds. Aspartate aminotransferase (AST) enzyme activities were significantly decreased by an imbalance (except 1.67%) of threonine in the feed (P < 0.05). Activities of Alanine aminotransferase (ALT) and albumen (ALB) were not significantly affected by threonine in the feed (P > 0.05). Excessive dietary threonine level (2.48%) significantly activated haemolymph catalase (CAT) activity (P < 0.05), whereas malondialdehyde (MDA) content was significantly affected by deficient (0.35% and 0.79%) dietary threonine levels (P < 0.05). Inducible nitric oxide synthase (iNOS) activity and haemolymph complement component 4 (C4) content were significantly decreased by deficient levels of threonine in the feed (P < 0.05). Excess threonine concentration significantly down-regulated Toll, Dorsal, Relish, and heat shock protein 60 (Hsp60) gene expressions in the hepatopancreas of M. nipponense (P < 0.05), while all genes were significantly up-regulated by the optimal (1.67%) threonine level (P < 0.05). The threonine level at which maximum specific growth rate of M. nipponense occurred was estimated by second degree polynomial regression analysis as 1.65% of threonine level, equivalent to 4.44% dry weight bases of protein in the feed.

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1ß, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Cytotoxic effects, inflammatory response and apoptosis induction of cyclophosphamide in the peripheral blood leukocyte of blunt snout bream (Megalobrama amblycephala).

The present study was aimed to evaluate the effects of the cyclophosphamide (CY) exposure (Control, 0.032, 0.32, 1.0, 1.6 and 3.2 mg/mL) on the damage in the peripheral blood leukocytes of blunt snout bream for 24 h, which including cell viability, apoptosis, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (Δѱm), ROS, antioxidant enzyme activity and the relative mRNA levels of apoptosis. Results showed that cell viability and Δѱm effects of CY were greatly reduced, and occurred in a dose-dependent manner. CY exposure (0.32-3.2 mg/mL) significantly increased the LDH release and induced apoptosis accompanied by ΔΨm disruption and ROS generation compared to the control. The cellular ROS was significantly increased with increase of CY level from 0.032 mg/mL to 1 mg/mL and the plateau occurred at 0.32 mg/mL. Additionally CY exposure led to oxidative stress as evidenced by significantly the decrease of SOD and CAT and increase of MDA concentration after treating cells with 3.2 mg/mL of CY. Besides, the relative mRNA levels of caspase-3 in the dose of 0.032, 0.32 mg/mL CY, caspase-9 and interleukins-1ß (IL-1ß) in the dose of 0.32 mg/mL CY, tumor necrosis factor-alpha (TNF-α) in the dose of 0.032 mg/mL CY significantly higher than that of the control. In conclusion, 0.32-3.2 mg/mL CY could lead to cytotoxic effect, inflammatory response and induce the apoptosis of the peripheral blood leukocyte of Megalobrama amblycephala.

Effects of Moringa oleifera leaf extract on growth performance, physiological and immune response, and related immune gene expression of Macrobrachium rosenbergii with Vibrio anguillarum and ammonia stress.

In order to study the effects of Moringa oleifera leaf extract on Macrobrachium rosenbergii under high ammonia exposure, freshwater prawns were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 0.25%, 0.5% and 1.0% M. oleifera leaf extract and 0.025% Enrofloxacin for 60 days, respectively. Then, freshwater prawns were exposed to high ammonia stress for 72 h and Vibro anguillarum infection. The growth, antioxidant capabilities, related immune genes as well as resistance to infection by V. anguillarum were determined. The results showed that compared with the control group, the weight gain, specific growth rate and protein efficiency rate, haemolymph catalase (CAT), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) increased while feed conversion ratio, haemolymph aspartate aminotransferase, alanine aminotransferase, nitrogen oxide (NO), hepatopancreas heat shock proteins (HSP70), immune deficiency (IMD) expression levels decreased in the group of 0.5% M. oleifera leaf extract before the stress. After ammonia stress, the group of 0.5% M. oleifera leaf extract also could improve the haemolymph SOD, glutathione peroxidase, NO, iNOS, hepatopancreas HSP70 expression levels and reduce haemolymph CAT, hepatopancreas peroxiredoxin 5 and NF kappa B inhibitor alpha expression level compared with the control group. The rate of mortality of the prawns challenged with V. anguillarum was lower in the supplemented groups in comparison with the control group with the lowest being in the group of 0.5% M. oleifera leaf extract. Antioxidant activities as well as biochemical parameters in the enrofloxacin group (0.025%E) were not significantly enhanced both pre and post challenge in comparison with the M. oleifera leaf extract groups, showing the superiority of the natural herb over the synthetic antibiotic. In summary, this study suggested that at an inclusion rate of 0.5%, M. oleifera leaf extract could increase the growth performance, even has positive effects on physiological and immune function and prevents high ammonia stress in the Freshwater prawn, M.rosenbergii.

The effects of crowding stress on the growth, physiological response, and gene expression of the Nrf2-Keap1 signaling pathway in blunt snout bream (Megalobrama amblycephala) reared under in-pond raceway conditions.

We evaluated the effects of high density stress on growth performance, antioxidant parameters, and Nrf2 pathway signaling molecules after different lengths of exposure (30, 60, or 90 days) of Megalobrama amblycephala to in-pond raceway aquaculture systems (IPRS). M. amblycephala (average initial weight 2.33 ±â€¯0.15 g) were reared at two different initial densities (low density group [LD] had 534 fish/m3 and high density group [HD] had 1073 fish/m3) for 90 days. The growth performance was adversely influenced by the high stocking density. The HD group had elevated white blood cell counts, hemoglobin content, and hematocrit on days 60 and 90. The mRNA levels of NOX2 on days 60 and 90, Nrf2 on days 30, 60, and 90, Keap1 on day 30, Bach1 on days 30 and 60, SOD on day 30, and CAT on day 30 were significantly higher in the HD group than in the LD group. Similarly, higher trends were observed in the enzymatic activities of SOD on day 60, CAT on days 60 and 90, and GPx on day 60 in the HD group, compared to the LD group. Furthermore, HD bream showed an increased MDA content on days 60 and 90 compared to that of the LD group. This study demonstrates that high density-induced antioxidant defenses were involved in modifications to the enzymatic and transcriptional regulation of Nrf2-Keap1 signaling molecules and that M. amblycephala growth was reduced in a crowded IPRS.

Comparative proteomic analysis of hepatic mechanisms of Megalobrama amblycephala infected by Aeromonas hydrophila.

Hemorrhage syndrome is one of the most prevalent and epidemic diseases that is mainly caused by Aeromonas hydrophila invasion in Megalobrama amblycephala. Recent studies have uncovered a number of immune enzymes and transcripts that are differently expressed in this disease, but the molecular mechanism elicited still remain largely unknown. Here, we constructed an in vivo A. hydrophila infection to investigate the immune mechanism in M. amblycephala using comparative proteomic approach at the one day after infection. 30 altered protein spots were found to undergo differential expression against A. hydrophila infection in the hepatopancreas of M. amblycephala based on 2-DE and were all successfully identified using MALDI-TOF/TOF, representing 18 unique proteins. These proteins were functionally classified into metabolism, antioxidant, cofactors and vitamins, chaperone and signal transduction. Network interaction and Gene Ontology annotation indicated 13 unique proteins were closely related to immune response and directly regulated by each other. Compared with the control group, A. hydrophila infection significantly decreased the metabolism-related mRNA expressions of ENO3, APOA1, CAT and FASN, but increased the mRNA expressions of MDH, ALDOB and RSP12, which was consistent with the protein expression. Nevertheless, FAH was down-regulated at both levels but had no significant difference in mRNA level, ALDH8a1 was down-regulated at protein level but non-significantly up-regulated at the mRNA level. GSTm was up-regulated at protein level but down-regulated at the mRNA level. Consequently, these results revealed that A. hydrophila infection altered the related antioxidative proteins via complex regulatory mechanisms and reduced the immune ability of M. amblycephala at the one day after infection.

Oxidized fish oil injury stress in Megalobrama amblycephala: Evaluated by growth, intestinal physiology, and transcriptome-based PI3K-Akt/NF-κB/TCR inflammatory signaling.

Lipids are essential nutrients for animal. Oxidized lipid might induce injury stress for fish. Here we conducted a 12-week rearing experiment with diets containing 0, 2, 4, and 6% oxidized fish oil (6F, 4F2OF, 2F4OF, and 6OF) to describe the oxidative impairment mechanism on teleost fish blunt snout bream, Megalobrama amblycephala. Results were evaluated by growth performance, intestinal physiology, and transcriptome-based PI3K-Akt/NF-κB/TCR inflammatory signaling. From the results, 6OF reduced growth performance with increased FCR and reduced FBW, WGR and SGR compare with 6 F. Meanwhile, oxidized fish oil treatments also increased antioxidant enzyme activity, suggesting an impaired physiological condition. The plasmatic antioxidant enzyme activity of T-SOD, GSH-Px, ASAFR, concentration of MDA and cortisol were significantly increased in 6OF, while GSH concentration was decreased. Histological ultrastructure revealed the integrity of mid-intestinal cells and villus were destroyed in 6OF. Moreover, transcriptomic analysis revealed PI3K-Akt/NF-κB/TCR inflammatory signaling were active to oxidized fish oil stress. We verified the expression of twelve key genes related to this signaling by RT-PCR, which revealed TLR2, PI3K, Akt, NF-κB, MHCII-ß, TCR-α, TGF-ß, TNF-α, IL-6, IL-1ß, GPx1 and GSTm were all activated under 6OF stimulation. We found that oxidized fish oil may induce oxidative stress, destroy intestinal integrity, produce free radical, dysregulate lipid metabolism and oxidative balance, reversely affect the physiological adaptation, and eventually lead to growth inhibition. This study revealed the mechanism of PI3K-Akt/NF-κB/TCR inflammatory signaling in M. amblycephala under oxidized fish oil stress, which may help to understand the complex regulation involved in lipid oxidative stress resistance.

Effects of various feeding patterns of Bacillus coagulans on growth performance, antioxidant response and Nrf2-Keap1 signaling pathway in juvenile gibel carp (Carassius auratus gibelio).

The present study was conducted to evaluate the effects of various Bacillus coagulans feeding patterns on growth, antioxidant parameter and Nrf2 pathway in juvenile gibel carp. The similar size of gibel carp (initial weight: 14.33 ± 0.15 g) were subjected to three levels of B. coagulans supplementation (0, 500, and 1000 mg/kg) and two feeding modes (supplementing B. coagulans continuously or for two days of B. coagulans after 5 days of a basal diet) according to a 3 × 2 factorial design. The fish that were continuously fed 500 mg/kg B. coagulans (P2) and those fed the first basal diet for 5 days followed by 500 mg/kg or 1000 mg/kg B.coagulans for 2 days (P4 or P5) showed higher weight gain rate and specific growth rate than the other groups. Blood respiratory burst (RB), myeloperoxidase (MPO), and anti-superoxide anion free radical (AFASER) activities in the P4 group were higher than those of the control. White blood cell count (WBC), RB activity, MPO activity, and glutathione (GSH) content in the P5 group were also higher than those of the control. A similar higher trend was observed in the gene expressions of NADPH oxidase 2 (NOX2), NFE2-related factor (Nrf2), Kelch-like-ECH-associated protein(Keap1) in the P4 and NOX2, NRF2, CNC homolog 1 (Bach1), peroxiredoxin 2 (Prx2) in the P5 group compared with the control. Additionally, we observed a significantly lower level of plasma aspartate aminotransferase (AST), lower activity of alanine aminotransferase (ALT), a higher level of MPO, higher GPX activity, and increased NRF2 and Prx2 expression were all observed in the P2 treatment group compared with the control. Furthermore, the malondialdehyde (MDA) content in the P2, P3, and P4 groups was lower than that of the control. These results indicate that a diet supplemented with appropriate levels of B.coagulans could improve the growth, immune response, and antioxidant capability of gibel carp. We concluded that the pattern of two days of 500 or 1000 mg/kg B. coagulans after 5 days of a basal diet was recommended for gibel carp.

Lysine supplement benefits the growth performance, protein synthesis, and muscle development of Megalobrama amblycephala fed diets with fish meal replaced by rice protein concentrate.

This study aimed to investigate the effects of lysine supplement on the growth performance of blunt snout bream Megalobrama amblycephala fed diets with fish meal (FM) replaced by rice protein concentrate (RPC) with the potential mechanisms characterized. Fish were fed three diets, including the FM diet (containing FM), the RPC diet (FM replaced by RPC), and the MRPC diet (the RPC diet supplemented with lysine) for 8 weeks. Weight gain, protein efficiency ratio, and nitrogen and energy utilization of fish fed the FM diet were all significantly higher than those of the RPC treatment, but they showed no statistical difference with those of the MRPC group. Fish fed the RPC diet showed shorter villi length of the distal intestine than that of the other treatments. No significance was found in whole-body composition and intestinal and hepatic cell proliferation among all the treatments. However, fish fed the RPC diet obtained relatively low transcriptions of growth hormone (GH), GH receptor, insulin-like growth factor-I (IGF-I), target of rapamycin (TOR), ribosomal protein S6 kinase 1, myoblast determination protein, myogenic factor 5, and myostatin a (MSTNa) but high levels of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) than those of the other groups. Furthermore, little difference was found in the transcriptions of 4E-BP2, myogenin, muscle-specific regulatory 4, and MSTNb in muscle. Overall, these results showed that dietary supplement of lysine benefits the growth performance of blunt snout bream fed FM-free diets through the mediation of the GH-IGF-I axis, TOR signaling pathway, myogenic regulatory factors, and MSTN.

Effects of partial replacement of fish meal by yeast hydrolysate on complement system and stress resistance in juvenile Jian carp (Cyprinus carpio var. Jian).

A 10-week feeding trial was carried out to investigate the effects of dietary fish meal replacement by yeast hydrolysate (YH) on growth performance, complement system and stress resistance of juvenile Jian carp (Cyprinus carpio var. Jian) (initial average weight 19.44 ± 0.06 g). In the study, there were five groups: one control group was fed with a basal diet (YH0), and four treatment groups were fed with dietary fish meal replaced by 1% YH (YH1), 3% (YH3), 5% (YH5) and 7% (YH7), respectively. Each group had four replicates. At the end of feeding trial, twelve fish from each group (three fish per replicate) were randomly selected for assessing the growth and immunity. Meanwhile, 20 fish per replicate were injected by Aeromonas hydrophila. The results showed that (1) Replacement levels of YH significantly affected the growth of the fish with the highest values of weight gain (WG) occurred in fish fed YH3 diet. However, no significant difference in feed conversion ratios (FCR) was observed among all groups. (2) Pre-stressed plasma lysozyme activity, total protein and albumin contents and complement component 3 (C3) and complement component 4 (C4) levels of fish fed YH3 diet were significantly higher than those of fish fed YH0 diet. However, post-stressed immune parameters of fish in all groups were significantly lower. (3) There was a trend that the expression levels of the complement-related genes (c1r/s-A, c4-1, c3-H1, c5-1, fb/c2-A, mbl-2 and masp) initially increased and then decreased except mbl-2 and masp, with the maximum values observed in fish fed YH3 diet. Before stress, the expression levels of the inflammation-related genes (alp, il-1ß and tnf-α) in the hepatopancreas and spleen of fish fed YH1 diet and YH7 diet were significant higher than that of fish fed YH0 diet. After stress, no significant difference in the expression levels of those genes was observed among all groups. These results indicated that FM replacement by YH could improve growth performance, enhance innate immunity, and activate complement via the alternative complement pathway (ACP) and the classical complement pathway (CCP).

Oxidative Stress in Aquatic Organisms.

Oxidative stress mainly refers to the imbalance between reactive oxygen species production and antioxidant defense systems in organisms [...].

Tea Tree Oil Improves Energy Metabolism, Non-Specific Immunity, and Microbiota Diversity via the Intestine-Hepatopancreas Axis in Macrobrachium rosenbergii under Low Fish Meal Diet Administration.

Tea tree oil (TTO) is an essential plant oil with diverse antibacterial and antioxidant properties; however, whether the role played by TTO in low fish meal (LF) diets induced the observed effects in the farmed crustaceans remains unclear. Therefore, this study used Macrobrachium rosenbergii as the model crustacean, and an 8-week feeding experiment with NF (normal fish meal), LF (soybean meal replacing 40% fish meal), and LFT (LF with 200 mg/kg TTO) diets was conducted to evaluate the positive effects of TTO under the LF diet. Compared to the NF diet, the LF diet reduced hemolymph antioxidant capacity and non-specific immunity, and induced hepatopancreas apoptosis and damage. However, in comparison with LF, LTF significantly ameliorated morphological impairment in the hepatopancreas, improved hepatopancreas energy metabolism by upregulating the Bcl-2/Bax and Akt/mTOR pathways, and enhanced antioxidant and non-specific immune capacity by activating the NF-κB/NO pathway. In addition, LFT repaired intestinal barrier injury and the imbalance of intestinal microbiota induced by the LF diet. Moreover, the Pearson correlation revealed the variations of the above indicators, which were related to the abundance changes of Klebsiella, Clostridium sensu stricto 12, Thermobifida, Bifidobacterium, and Alistipes, indicating that these microbes might serve as prospective targets for the intestine-hepatopancreas axis to affect hepatopancreas apoptosis, metabolism, and non-specific immunity. In summary, 200 mg/kg TTO supplementation mediated gut microbiota and positively improved energy metabolism and non-specific immunity, thereby alleviating hepatopancreas dysplasia and damage induced by the LF diet in M. rosenbergii.

High-Fat-Diet-Induced Oxidative Stress in Giant Freshwater Prawn (Macrobrachium rosenbergii) via NF-κB/NO Signal Pathway and the Amelioration of Vitamin E.

Lipids work as essential energy sources for organisms. However, prawns fed on high-fat diets suffer from oxidative stress, whose potential mechanisms are poorly understood. The present study aimed to explore the regulation mechanism of oxidative stress induced by high fat and the amelioration by vitamin E (VE) of oxidative stress. Macrobrachium rosenbergii were fed with two dietary fat levels (LF 9% and HF 13%) and two VE levels (200 mg/kg and 600 mg/kg) for 8 weeks. The results showed that the HF diet decreased the growth performance, survival rate and antioxidant capacity of M. rosenbergii, as well as inducing hypertrophied lipid droplets, lipophagy and apoptosis. A total of 600 mg/kg of VE in the HF diet alleviated the negative effects induced by HF. In addition, the HF diet suppressed the expression of toll-dorsal and imd-relish signal pathways. After the relish and dorsal pathways were knocked down, the downstream iNOS and NO levels decreased and the MDA level increased. The results indicated that M. rosenbergii fed with a high-fat diet could cause oxidative damage. Its molecular mechanism may be attributed to the fact that high fat suppresses the NF-κB/NO signaling pathway mediating pro-oxidant and antioxidant targets for regulation of oxidative stress. Dietary VE in an HF diet alleviated hepatopancreas oxidative stress and apoptosis.

Low fish meal diet supplemented with probiotics ameliorates intestinal barrier and immunological function of Macrobrachium rosenbergii via the targeted modulation of gut microbes and derived secondary metabolites.

The unsuitable substitution ratio of fish meal by plant protein will reshape the intestinal microbial composition and intestine immunity. However, previous studies were mostly limited to investigating how different feed or probiotics characterized the microbial composition but ignored the biological interactions between bacteria and host physiology through secondary metabolites. Therefore, this study integrates the apparent indicators monitoring, 16S rDNA sequencing, and metabonomics to systematically investigate the effects of cottonseed protein concentrate (CPC) substitution of fish meal and Bacillus coagulans intervention on gut microbes, secondary metabolites, and intestinal immunity of Macrobrachium rosenbergii. Prawns were fed with three diets for 70 days: HF diets contained 25% fish meal, CPC in LF diets were replaced with 10% fish meal, and LF diets supplemented with 2 × 108 CFU/g diet B. coagulans were designated as BC diets. Results showed that CPC substitution induced a significant decrease in digestive enzyme activities (trypsin and lipase) and gut barrier protein PT-1 expression and a significant increase in γ-GT enzyme activity and inflammatory-related factors (Relish and Toll) expression. B. coagulans treatment mitigated the negative changes of the above indicators. Meanwhile, it significantly improved the expression levels of the barrier factor PT-1, the reparative cytokine IL-22, and Cu/Zn-SOD. CPC substitution resulted in a remarkable downregulated abundance of Firmicutes phyla, Flavobacterium spp., and Bacillus spp. B. coagulans treatment induced the callback of Firmicutes abundance and improved the relative abundance of Sphingomonas, Bacillus, and Ralstonia. Functional prediction indicated that CPC substitution resulted in elevated potential pathogenicity of microbial flora, and B. coagulans reduces the pathogenesis risk. Pearson's correlation analysis established a significant positive correlation between differential genera (Sphingomonas, Bacillus, and Ralstonia) and secondary metabolites (including sphingosine, dehydrophytosphingosine, amino acid metabolites, etc.). Meanwhile, the latter were significantly associated with intestinal immunoregulation-related genes (Cu/Zn-SOD, IL-22, PT-1, Toll, and Relish). This study indicated that B. coagulans could mediate specific gut microbes and the combined action of multiple functional secondary metabolites to affect intestinal barrier function, digestion, and inflammation. Our study revealed the decisive role of gut microbes and derived secondary metabolites in the model of dietary composition-induced intestinal injury and probiotic treatment from a new perspective.

Tea Tree Oil Mediates Antioxidant Factors Relish and Nrf2-Autophagy Axis Regulating the Lipid Metabolism of Macrobrachium rosenbergii.

Both oxidative stress and autophagy refer to regulating fat metabolism, and the former affects autophagy, but the role and mechanism of the antioxidant-autophagy axis in regulating lipid metabolism remains unclear. As an antioxidant, tea tree oil (TTO) has little research on the regulatory mechanism of lipid metabolism in crustaceans. This study investigated whether TTO could alter hepatopancreatic lipid metabolism by affecting the antioxidant-autophagy axis. Feed Macrobrachium rosenbergii with three different levels of TTO diets for 8 weeks: CT (0 mg/kg TTO), 100TTO (100 mg/kg TTO), and 1000TTO (1000 mg/kg TTO). The results showed that 100TTO treatment reduced the hemolymph lipids level and hepatopancreatic lipid deposition compared to CT. In contrast, 1000TTO treatment increased hepatopancreatic lipid deposition, damaging both morphology and function in the hepatopancreas. The 100TTO treatment promoted lipolysis and reduced liposynthesis at the transcriptional level compared to the CT group. Meanwhile, it improved the hepatopancreas antioxidant capacity and maintained mitochondrial structural and ROS homeostasis. In addition, it simultaneously activated the expression of transcription factors Keap1-Nrf2 and Imd-Relish. By contrast, the 1000TTO group significantly enhanced the ROS level, which considerably activated the Keap1-Nrf2 signaling expression but had no significant effects on the expression of Imd-Relish. The 100TTO group supplementation significantly enhanced lipid droplet breakdown and autophagy-related genes and protein expression. On the contrary, the 1000TTO group significantly inhibited the expression of genes and proteins related to autophagy. Pearson analysis revealed that Nrf2 has a positive correlation to lipid anabolism-related genes (Fasn, Srebp1, Pparγ) and autophagy regulators (mtor, akt, p62), and were negatively correlated with lipolysis-related genes (Cpt1, Hsl, Ampkα) and autophagy markers (Ulk1, Lc3). Relish was positively correlated with Atgl, Cpt1, Ampkα, Ulk1, and Lc3, and negatively correlated with Pparγ and p62. Moreover, Keap1 and Imd were negatively correlated with p62 and mtor, respectively. In sum, 100 mg/kg TTO enhanced antioxidant activity and increased autophagy intensity through the Relish-Imd pathway to enhance lipid droplet breakdown, while 1000 mg/kg TTO overexpressed Nrf2, thus inhibiting autophagy and ultimately causing excessive lipid deposition and peroxidation. Our study gives a fresh perspective for deciphering the bidirectional regulation mechanism of lipid metabolism by different doses of TTO based on the antioxidant-autophagy axis.