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Microbial fermentation techniques are often used to improve their quality, where the keys are fermentation strains and fermentation time. This study studied the interaction between microbiota and environmental (or nutritional) factors and microbiota at different fermentation times to determine the most appropriate time, using lactic acid bacteria as fermentation strains. It can be concluded that fermentation improved the nutritional value of soybean meals. In the early stages of fermentation, debris in soybean meal highly proliferated and destabilized the microbial community, while pH and nutritional conditions played an important role in helping its stabilization. In addition, we must pay attention to the interspecific interactions of microorganisms, which makes it easy to understand how the microbial community maintains community stability. A 4-day fermentation of soybean meal with Lactobacillus is recommended.

To investigate the effects of dietary carbohydrate/lipid (CHO: L) ratios on non-specific immune responses, antioxidant capacity, and expression of TLR-MAPK/NF-κB signaling pathway-related genes of red swamp crayfish (Procambarus clarkii). Four isonitrogenous and isoenergetic diets containing different CHO: L ratios were formulated. The results showed that the group with a CHO: L ratio of 5.94 had better growth performance (P 0.05). The highest T-AOC, CAT, and SOD activities and the lowest MDA content in hemolymph and hepatopancreas were observed in the group with a CHO: L ratio of 5.94 (P 0.05). The lowest activities of ALT, AST, ACP, AKP, and ALB in the hemolymph were observed in CHO: L ratio 5.94 group (P 0.05), while the highest LZM activity, TP, and GLB content were observed in CHO: L 5.94 group (P 0.05). The highest mRNA expression levels of tlr3, myd88, and mapk3, and the lowest mRNA expression levels of nf-kb α, nf-kb β, nf-kb p105, and traf6 were observed in the CHO: L of 5.94 group (P 0.05). The highest mRNA expression levels of immune-related genes were observed in the CHO: L of 5.94 group (P 0.05). Overall, these results indicated that the optimum dietary CHO: L ratio is vital in promoting growth and enhancing antioxidants and immunity to maintain red swamp crayfish's intestinal and hepatopancreas health status. In conclusion, the diets with a CHO:L ratio of 5.94 (approximately 36.23% carbohydrate and 6.10% lipid) is optimal for juvenile red swamp crayfish's physiological condition and health status.

Ammonia is one of the major environmental pollutants in freshwater aquatic systems that affects the survival and growth of organisms. In the present study, a nine-week experiment was conducted to investigate the chronic toxicity of ammonia on survival and selected hematological parameters, antioxidant ability, and histological structure in juvenile Chinese blunt snout bream, Megalobrama amblycephala. Fish were exposed to one of five different ammonia concentrations (0, 5, 10, 15, and 20 mg L −1 total ammonia-N, pH = 7.86 ± 0.41, temperature = 28.23 ± 0.79 °C). Growth performance of fish in the high-ammonia treatment was lower than that of fish in the control group. The survival rate in the group exposed to the highest ammonia-N concentration (20 mg L−1) was significantly lower than that of the control group. The lowest level of white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, and plasma glucose and the highest level of plasma ammonia, cortisol and aspartate transaminase were observed in fish exposed to ammonia-N at 20 mg L −1 compared with the control group. The liver superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities in fish exposure to 20 mg L −1 ammonia-N group were significantly lower than those in the control group, while oxidative stress indices (levels of malondialdehyde, MDA) was significantly higher than those in the control group. Regression analysis found that growth performance, plasma parameters, hematological parameters, and antioxidant status of fish were significant linear correlated to ammonia-N concentration. Fish exposed to various concentrations of ammonia displayed histopathologic alterations in both gill and liver tissue. Furthermore, morphological examination by transmission electron microscopy revealed that gill tissues displayed vacuolation of the cytoplasm, damaged mitochondria, increased heterochromatin, and intense desquamation of pavement epithelial cells on the gill lamellae. Our findings suggest that the stress responses induced in juvenile blunt snout bream by chronic exposure to ammonia are not limited to simple growth inhibition, but are accompanied by changes in oxidative stress, plasma parameter effects, and alterations in the histology of the liver and gill. Blunt snout bream had a dose-dependent effect on ammonia-N. We preliminarily suggest that the level of plasma ammonia, MDA, CAT, AST, FER, and SGR can be used as an indicator for chronic ammonia-N toxicity, but further research and verification are needed in the future.

The hypothesis of the present study was that increased growth in spring, stimulated by increasing temperature and daylength, leads to oxidative stress in Atlantic salmon with accumulation of oxidation products in the tissues and increased utilization of antioxidants. The drop in fillet pigmentation and astaxanthin, often observed in spring by the industry, could be explained by oxidative stress. Furthermore, oxidative stress may cause production related diseases such as development of cataracts and melanin spots in the fillet. We sampled Atlantic salmon from two cages in a commercial scale experiment in Northern Norway (67°N), every month from April until August and then every second month until December (510 ± 160–3060 ± 510 g, mean weight ± std). The specific growth rate (SGR) increased with increasing temperature until midsummer and decreased thereafter. We found that vitamin E in the fillet and vitamin C in the liver were depleted in the spring and were restored in the autumn, even though the dietary concentrations were stable. Astaxanthin concentration in the muscle was constant during the spring and summer and increased in the autumn, concomitant with an increase in astaxanthin supplementation. Cataract increased from zero in May until July, when 90% of the fish were affected. The glutathione based redox-potential in the lenses became more reduced from June, indicating a protective mechanism against oxidative stress and cataract. The number of fish with melanin spots was high in June and decreased in August and October, but the size and intensity of the remaining spots increased in the same period. The change in vitamin C and E concentrations, cataract and glutathione metabolism during spring and early summer, indicate that the fish became oxidized in this period, while malon-di-aldehyde (MDA) and astaxanthin concentrations did not support the hypothesis. There are too few data to draw conclusions on possible effects of oxidative stress on melanin spots.

This study investigated the effects of dietary lipid sources on juvenile blunt snout bream, Megalobrama amblycephala. Juvenile blunt snout bream (14.40 ± 0.01 g) were fed diets containing 75 g/kg of fish oil (FO), linseed oil (LO), soybean oil, coconut oil (CO), rapeseed oil or beef tallow (BT) for 9 weeks. Survival rates, final weight, weight gain, feed efficiency ratio and feed intake remained unchanged. Viscerosomatic ratio, hepatosomatic index and condition factor were highest in fish fed the CO diet (p

Nitrite (NO2−) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ± 0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

Nitrite (NO2−) contamination of water can severely impact the health of aquatic life and is a major concern for commercial aquaculture. In order to study the effect of nitrite on Aristichthys nobilis, we investigated the oxygen-carrying capacity, NF-κB/HIF-1α pathway, and the gill tissue structure under nitrite stress. In the current study, bighead carp (initial weight 180.05 ± 0.092 g) were exposed to nitrite (48.634 mg/L) for 96 h and then for 96 h recovery test. After nitrite exposure for 6 h, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) mRNA expression increased significantly in the gill of bighead carp (P

A nine-week feeding trial was performed to determine the dietary linolenic acid (LNA; 18:3n–3) requirements of juvenile blunt snout bream. Six iso-nitrogenous, semi-purified diets were prepared with different concentrations of LNA (0–25 g/kg). Dietary LNA had no significant effects on survival rate. However, final fish weight, weight gain (WG), specific growth rate (SGR) and feed efficiency ratio (FER) increased with increasing dietary LNA concentrations up to 20 g/kg. Dietary LNA increased muscle LNA and total n-3 polyunsaturated fatty acid (PUFA) contents, but decreased total saturated fatty acid content. Fish fed 20 g/kg LNA had the highest plasma alkaline phosphatase activity, total protein, albumin and white blood cell count levels. Additionally, fish fed 20 g/kg LNA had higher triglyceride levels than control fish. Plasma glucose increased with increasing dietary LNA concentrations. Superoxide dismutase and glutathione peroxidase activities significantly increased with increasing dietary LNA concentrations up to 15 g/kg. Based on SGR and FER, the optimal dietary LNA requirements of juvenile blunt snout bream were 17.5 and 15.6 g/kg respectively.

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 ± 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.

Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this study, we isolated the caspase-8 gene from Megalobrama amblycephala, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The 2034 bp full-length M. amblycephala caspase-8 cDNA sequence contained an ORF of 1467 bp encoding a polypeptide of 489 amino acid residues, a 5'-UTR of 102 bp and a 3'-UTR of 462 bp. The caspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10, caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. Phylogenetic analysis revealed that M. amblycephala caspase-8 were clustered with the caspase-8 from other vertebrate. Real-time quantitative PCR analysis revealed that caspase-8 transcripts were detected in liver after exposure to ammonia. Meanwhile using Western blot analysis, caspase-8 cleaved fragment was detected and significant alteration of procaspase-8 level was found with the same ammonia treatment condition. Furthermore, the result of immunohistochemical detection showed that remarkable changes of immunopositive staining were observed after ammonia treatment. Accordingly, the results signify that caspase-8 of fish may play an essential role in ammonia induced apoptosis.

Objective Nitrite exposure induces growth inhibition, metabolic disturbance, oxidative stress, organic damage, and infection-mediated mortality of aquatic organism. This study aimed to investigate the mechanism in responses to acute nitrite toxicity in bighead carp (Aristichthys nobilis, A. nobilis) by RNA-seq analysis. Methods Bighead carps were exposed to water with high nitrite content (48.63 mg/L) for 72 h, and fish livers and gills were separated for RNA-seq analysis. De novo assembly was performed, and differentially expressed genes (DEGs) between control and nitrite-exposed fishes were identified. Furthermore, enrichment analysis was performed for DEGs to annotate the molecular functions. Results A total of 406,135 transcripts and 352,730 unigenes were tagged after de novo assembly. Accordingly, 4108 and 928 DEGs were respectively identified in gill and liver in responses to nitrite exposure. Most of these DEGs were up-regulated DEGs. Enrichment analysis showed these DEGs were mainly associated with immune responses and nitrogen metabolism. Conclusions We suggested that the nitrite toxicity-induced DEGs were probably related to dysregulation of nitrogen metabolism and immune responses in A. nobilis, particularly in gill.

A 9-week feeding trial was conducted to investigate taurine distribution by supplementation of methionine in juvenile Megalobrama amblycephala at a constant dietary cystine level. Six semi-purified diets were formulated to contain graded dietary methionine levels from 0.39-1.54% in about 0.25% increments. At the end of feeding trial, plasma methionine content significantly increased with increasing dietary methionine level from 0.39 to 1.0% (P < 0.05) and thereafter reached a plateau. The taurine content of muscle, intestine, liver, and eye in the fish fed dietary methionine level ranged from 1.24-1.54% and was significantly higher than that of the control group (P < 0.05). Taurine content of the brain in fish fed 1.54% methionine diet was higher than the fish fed 0.39% (P < 0.05). Cysteine sulfinic acid decarboxylase content significantly increased with increasing dietary methionine level from 0.39-0.85% (P < 0.05) and thereafter stayed at a similar level. Supplementation with methionine significantly increased (P < 0.05) plasma total protein (TP) content, complement 3 (C3) content and superoxide dismutase (SOD) activity (0.85% methionine diet groups), aspartate transaminase (AST) activity (1.24% methionine diet groups) and albumin content (1.0% methionine diet groups). Supplemented groups had significantly decreased plasma urea content (0.85% methionine diet groups) (P < 0.05). In conclusion, these results indicate that crystalline methionine can be utilized efficiently, juvenile blunt snout bream were able to convert methionine to taurine directly, and appropriate dietary methionine supplementation improves non-specific immune responses in blunt snout bream.

Adaptation to hypoxia is a complex process involving various pathways and regulation mechanisms. A better understanding of the genetic influence on these mechanisms could permit selection for hypoxia-sensitive fish. To aid this understanding, an integrated analysis of miRNA and mRNA expression was performed in Megalobrama amblycephala under four acute hypoxia and reoxygenation stages. A number of significantly differentially-expressed miRNAs and genes associated with oxidative stress were identified, and their functional characteristics were revealed by GO function and KEGG pathway analysis. They were found to be involved in HIF-1 pathways known to affect energy metabolism and apoptosis. MiRNA-mRNA interaction pairs were detected from comparison of expression between the four different stages. The function annotation results also showed that many miRNA-mRNA interaction pairs were likely to be involved in regulating hypoxia stress. As a unique resource for gene expression and regulation during hypoxia and reoxygenation, this study could provide a starting point for further studies to better understand the genetic background of hypoxia stress.

This study was conducted to investigate the effects of oral administration of a high concentration of glucose on the respiratory burst, antioxidant status, and hepatic gene expression of heme oxygenase-1 (ho1) and PI3K/Akt/Nrf2-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala). Blunt snout bream juveniles with an initial body weight of 19.94 ± 0.58 g were orally fed with a high concentration of glucose (3 g/kg body weight). The results indicated that plasma glucose exhibited a biphasic response. Acute and persistent hyperglycemia due to the oral glucose administration significantly reduced (P < 0.05) the white blood cell count, red blood cell count, and hemoglobin content and caused oxidative stress (significantly increased alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and glucose levels) and early apoptosis of hepatocytes in the fish. Hepatic superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities increased rapidly (P < 0.05) as protection from oxidative stress and were downregulated (P < 0.05) because of persistent hyperglycemia. Blood respiratory burst was significantly reduced (P < 0.05) because of hyperglycemia and showed a trend that was opposite to that of plasma glucose. Slight upregulation of nrf2 mRNA and antioxidants acts as a compensative protection mechanism, and the downregulated PI3K/Akt pathway blocked this function of Nrf2. In conclusion, the PI3K/Akt pathway and Nrf2 mediated the antioxidative mechanism independently in the blunt snout bream juveniles subjected to the oral administration of a high glucose concentration.

De novo transcriptome sequencing is a robust method for microRNA (miRNA) target gene prediction, especially for organisms without reference genomes. Following exposure of Megalobrama amblycephala to ammonia (0.1 or 20 mg L−1 ), two cDNA libraries were constructed from the fish gills and sequenced using Illumina HiSeq 2000. Over 90 million reads were generated and de novo assembled into 46, 615 unigenes, which were then extensively annotated by comparing to different protein databases, followed by biochemical pathway prediction. The expression of 2666 unigenes significantly differed; 1961 were up-regulated, while 975 were down-regulated. Among these, 250 unigenes were identified as the targets for 10 conserved and 4 putative novel miRNA families by miRNA target computational prediction. We examined expression of ssa-miRNA-21 and its target genes by real-time quantitative PCR and found agreement with the sequencing data. This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for Megalobrama amblycephala and will be useful for gene expression profile analysis and miRNA functional annotation., Highlights • Several differentially expressed genes and miRNA were identified. • Target prediction indicated that a number of miRNAs involved in immunity. • The study provides an understanding of molecular mechanisms of ammonia-induced toxicology in fish.

The present study was aimed at evaluating the effects of the feed additive curcumin on the growth and non-specific immune responses in juvenile Wuchang bream (Megalobrama amblycephala). Six experimental diets were formulated to contain graded curcumin levels (0, 15, 30, 60, 120 and 240 mg/kg of diet, respectively). After 60 days feeding trial, fish fed the 60 mg/kg curcumin diet had significantly higher weight gain rate (WGR) and specific growth rate (SGR), and significantly lower feed conversion ratio (FCR) than the control group. The number of leucocytes (WBC), interleukin-1 beta (IL-1β), tumor necrosis factor-α (TNF-α), alternative pathway of complement (ACH50), and respiratory burst activity increased with increasing dietary curcumin levels up to 60 mg/kg, and thereafter declined. Significantly lower alanine transaminase (ALT) and aspartate transaminase (AST) activities were observed in fish fed the 60 mg/kg curcumin diet compared to those in the control group. Fish fed with the diets containing 60 and 120 mg/kg curcumin had significantly higher TNF-α and ACH50 activities than those in the control, and the fish fed diets supplemented with 60 and 240 mg/kg curcumin had significantly higher IL-1β activities than those in the control. The results suggest that appropriate dietary curcumin supplementation (60 mg/kg curcumin of diet) significantly improved growth and non-specific immune responses in juvenile M. amblycephala.