Molecular characterization and function of hif1a and fih1 in response to acute thermal stress in American shad (Alosa sapidissima).

In order to evaluate the function of hypoxia-inducible factor-1 alpha (hif1α) and factor inhibiting hif1α (fih1) in response to thermal stress, we first conducted a functional analysis of A. sapidissima hif1α and fih1, and determined hif1α and fih1 expressions in different tissues in response to thermal stress based on identified housekeeping genes (HKGs). The results showed that hif1α and fih1 were mainly located in the nucleus and cytoplasm. The full length cDNA sequence of hif1α and fih1 was 4073 bp and 2759 bp, respectively. The cDNA sequence of hif1α includes 15 exons encoding 750 amino acid residues, and the full length cDNA sequence of fih1 contains 9 exons encoding 354 amino acid residues. During the acute thermal stress transferring from 16 ± 0.5 °C (control) to 20 ± 0.5 °C, 25 ± 0.5 °C, and 30 ± 0.5 °C for 15 min, it was found that the expression trends of hif1α and fih1 showed an inhibitory regulation in the heart, while they consistently expressed in brain, intestine, muscle, gill, kidney and liver. In conclusion, this is the first study to identify the tissue-specific HKGs in A. sapidissima and found that ef1α and ß-actin are the most suitable HKGs. Hif1α and Fih1 are mainly the nuclear and cytoplasmic proteins, respectively, having high levels in the heart and brain. Alosa sapidissima countered a temperature increase from 16 to 25 ℃ by regulating the expressions of hif1α and fih1, but their physiological regulatory functions were unable to cope with acute thermal stress when the temperature difference was 14 ℃ (from 16 to 30 ℃).

Suitable Cottonseed Protein Concentrate Supplementation in Common Carp (Cyprinus carpio) Serves as an Effective Strategy for Fish Meal Sparing Based on Improvement in Intestinal Antioxidant Capacity, Barrier and Microbiota Composition.

The application of cottonseed protein concentrate (CPC) is an effective strategy to moderate the shortage of fish meal (FM) for the aquafeed industry. However, little attention has been paid to the effects of replacing fishmeal with CPC on cyprinid fish. This study used common carp (Cyprinus carpio) as the biological model and assessed the potential of applying CPC as a substitute for fishmeal in the diet of common carp. The proportion of fish meal substituted with CPC in the six diets was 0% (CPC0), 25% (CPC25), 50% (CPC50), 75% (CPC75), and 100% (CPC100). Each diet was fed to three replicate groups of common carp (4.17 ± 0.02 g) for 56 days. Results revealed that the CPC50 group significantly increased the growth indexes via up-regulating the genes of the GH/IGF axis and the TOR pathway. The intestinal digestive ability was also elevated in the CPC50 group via markedly increasing intestinal villus height, protease and lipase activities in the whole intestine, and the amylase activity of the foregut and midgut. The CPC50 group captured significantly higher activities and gene expressions of antioxidant enzymes and lower malonaldehyde contents via evoking the Nrf2/Keap1 signal pathway. The CPC50 group enhance the intestinal mechanical barrier via up-regulating the gene expressions of tight junction proteins and heighten the intestinal biological barrier by increasing the probiotics (Lactococcus) and decreasing the harmful bacteria (Enterococcus). But excessive substitution levels (75% and 100%) would compromise growth performance, intestinal antioxidant capacity, and immune function. The optimum substitution level was estimated to be 46.47%, 47.72%, and 46.43% using broken-line regression analyses based on mass gain rate, protein efficiency ratio, and feed conversion rate. Overall, the fishmeal in common carp feed could be substituted up to 50% by CPC without negative influence on growth, feed utilization, and or intestinal health.

Mulberry Leaf Polysaccharides Attenuate Oxidative Stress Injury in Peripheral Blood Leukocytes by Regulating Endoplasmic Reticulum Stress.

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H2O2)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 µM of H2O2 for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 µg/mL) for 3 h, and then stimulated with 100 µM of H2O2 for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs' antioxidant capacities. The 200 µg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H2O2-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (p < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca2+ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca2+ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p < 0.05), which rebounded to normal levels in the NC group (p > 0.05). These results indicated that MLP pre-treatment attenuated H2O2-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage.

Regulation of sirt1 and foxO1 in glucose metabolism of Megalobrama amblycephala.

Both silent information regulator 2 homolog 1 (sirt1) and forkhead box transcription factor 1 (foxO1) are crucial transcription factors involved in glucolipid metabolism and energy regulation. The presnt study aimed to understand their regulatory roles in glucose metabolism. Molecular cloning and sequencing of sirt1 gene of Megalobrama amblycephala (masirt1) was conducted and cellular localization of both the factors were analysed. Their effects and action patterns in the glucose metabolism of Megalobrama amblycephala (M. amblycephala) were investigated through acute and long-term glucose tolerance assays. The results revealed that the full-length masirt1 cDNA sequence was 2350 bp and closely related to Sinocyclocheilus rhinocerous. Sirt1 and foxO1 were found to be mutually dependent and localized in the nucleus. Acute glucose tolerance tests revealed that the expression levels of both factors in the liver of M. amblycephala showed an initial increase followed by a decrease. Plasma glucose levels in M. amblycephala significantly increased at 2 and 12 h (P < 0.05). In a long-term breeding experiment with high-sugar feeding, the expressions of the sirt1 and foxO1 genes in the kidney and intestine of M. amblycephala exhibited synergistic changes. The 51WS groups had significantly higher levels of sirt1 and foxO1 gene expression in the kidney and intestine compared to the 0WS and 17WS groups (P < 0.05). Overall, masirt1 is evolutionarily highly conserved, and the interaction site of sirt1 and foxO1 is located in the nucleus. In long-term hyperglycemic regulation, sirt1 and foxO1 exhibit synergistic regulatory effects in the kidney and intestine of M. amblycephala. This study provides insights into how sirt1 and foxO1 regulate glucose metabolism in M. amblycephala.

High-temperature stress response: Insights into the molecular regulation of American shad (Alosa sapidissima) using a multi-omics approach.

High temperature is an important abiotic stressor that limits the survival and growth of aquatic organisms. American shad (Alosa sapidissima), a migratory fish suitable for culturing at low temperatures, is known for its delicious taste and thus has high economic value. Studies concerning changes in A. sapidissima under high temperature are limited, especially at the gene expression and protein levels. High-temperature stress significantly reduced the survival rates and increased vacuolar degeneration and inflammatory infiltration in the gills and liver. High temperature increased the activities of SOD, CAT, and cortisol, with a trend of initial increase followed by decreases in MDA, ALP, and LDH, and irregular changes in T-AOC and Na-K-ATPase. Comprehensive analysis of the transcriptome, proteome, and metabolome of gills from fish treated with different culture temperatures (24, 27, and 30 °C) revealed that differentially expressed genes, proteins, and metabolites were highly enriched in pathways involved in protein digestion and absorption, protein processing in endoplasmic reticulum, metabolic pathways, and purine metabolism. Gene expression and protein profiles indicated that genes coding for antioxidants (i.e., cat and alpl) and members of the heat shock protein (i.e., HSP70, HSP90AA1, and HSP5) were significantly upregulated. Additionally, a conjoint analysis revealed that several key enzymes, including nucleoside diphosphate kinase 2, adenosine deaminase, and ectonucleoside triphosphate diphosphohydrolase 5/6 were altered, thereby affecting the metabolism of guanosine, guanine, and inosine. An interaction network further confirmed that levels of the essential amino acids DL-arginine and L-histidine were significantly reduced, and corticosterone levels were significantly increased, suggesting that A. sapidissima may be more dependent on amino acids for energy in vivo. Overall, this work suggests that living in a high-temperature environment leads to differential defense responses in fishes. The results provide novel perspectives for studying the molecular basis of adaptation to climate change in A. sapidissima and for genetic selection.

Ferrous Ion Alleviates Lipid Deposition and Inflammatory Responses Caused by a High Cottonseed Meal Diet by Modulating Hepatic Iron Transport Homeostasis and Controlling Ferroptosis in Juvenile Ctenopharyngodon idellus.

To investigate the mechanisms through which ferrous ion (Fe2+) addition improves the utilization of a cottonseed meal (CSM) diet, two experimental diets with equal nitrogen and energy content (low-cottonseed meal (LCM) and high-cottonseed meal (HCM) diets, respectively) containing 16.31% and 38.46% CSM were prepared. Additionally, the HCM diet was supplemented with graded levels of FeSO4·7H2O to establish two different Fe2+ supplementation groups (HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+). Juvenile Ctenopharyngodon idellus (grass carps) (5.0 ± 0.5 g) were fed one of these four diets (HCM, LCM, HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+ diets) for eight weeks. Our findings revealed that the HCM diet significantly increased lipid peroxide (LPO) concentration and the expression of lipogenic genes, e.g., sterol regulatory element binding transcription factor 1 (srebp1) and stearoyl-CoA desaturase (scd), leading to excessive lipid droplet deposition in the liver (p < 0.05). However, these effects were significantly reduced in the HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+ groups (p < 0.05). Plasma high-density lipoprotein (HDL) concentration was also significantly lower in the HCM and HCM + 0.2%Fe2+ groups compared to the LCM group (p < 0.05), whereas low-density lipoprotein (LDL) concentration was significantly higher in the HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+ groups than in the LCM group (p < 0.05). Furthermore, the plasma levels of liver functional indices, including alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (GLU), were significantly lower in the HCM + 0.4%Fe2+ group (p < 0.05). Regarding the expression of genes related to iron transport regulation, transferrin 2 (tfr2) expression in the HCM group and Fe2+ supplementation groups were significantly suppressed compared to the LCM group (p < 0.05). The addition of 0.4% Fe2+ in the HCM diet activated hepcidin expression and suppressed ferroportin-1 (fpn1) expression (p < 0.05). Compared to the LCM group, the expression of genes associated with ferroptosis and inflammation, including acyl-CoA synthetase long-chain family member 4b (acsl4b), lysophosphatidylcholine acyltransferase 3 (lpcat3), cyclooxygenase (cox), interleukin 1ß (il-1ß), and nuclear factor kappa b (nfκb), were significantly increased in the HCM group (p < 0.05), whereas Fe2+ supplementation in the HCM diet significantly inhibited their expression (p < 0.05) and significantly suppressed lipoxygenase (lox) expression (p < 0.05). Compared with the HCM group without Fe2+ supplementation, Fe2+ supplementation in the HCM diet significantly upregulated the expression of genes associated with ferroptosis, such as heat shock protein beta-associated protein1 (hspbap1), glutamate cysteine ligase (gcl), and glutathione peroxidase 4a (gpx4a) (p < 0.05), and significantly decreased the expression of the inflammation-related genes interleukin 15/10 (il-15/il-10) (p < 0.05). In conclusion, FeSO4·7H2O supplementation in the HCM diet maintained iron transport and homeostasis in the liver of juvenile grass carps, thus reducing the occurrence of ferroptosis and alleviating hepatic lipid deposition and inflammatory responses caused by high dietary CSM contents.

Momordica charantia saponins administration in low-protein-high-carbohydrate diet improves growth, blood biochemical, intestinal health and microflora composition of juvenile common carp (Cyprinus carpio).

An 8-week feeding trial was conducted to explore the feasibility of Momordica charantia saponins (MCS) administration to facilitate the protein-sparing action of high carbohydrate in diets for juvenile common carp (Cyprinus carpio) with initial mass of 5.41 ± 0.02 g. Based on our previous study, four diets with different the ratio of protein and carbohydrate (P/C ratio) were designed: 32%P/40%C, 30%P/43%C, 28%P/46%C, 28%P/46%C supplemented with 0.16% MCS (28%P/46%C + MCS). Each diet treatment was divided into 3 replicates. Results revealed that 30%P/43%C group increased growth performance and intestinal digestion, decreased intestinal inflammation, and optimized the intestinal microbiota compared to 32%P/40%C group, which presented the stronger protein-sparing action of high carbohydrate. But if the P/C ratio reduced to 28%P/46%C or less, the saving action would be restrained. However, compared to the 30%P/43%C and 28%P/46%C groups, 28%P/46%C + MCS group significantly elevated growth performance and activities of digestive enzymes and antioxidative enzymes, whilst the opposite trend occurred in the contents of glucose, triglyceride, total cholesterol, low density lipoprotein cholesterol, blood urea nitrogen, glutamic oxalacetic transaminase, glutamic-pyruvic transaminase and malondialdehyde. In addition, 28%P/46%C + MCS group markedly upregulated the expressions of GH/IGF axis genes, genes involved in protein synthesis, antioxidant genes and anti-inflammatory cytokine, whilst the opposite trend occurred in the expressions of pro-inflammatory cytokines. Moreover, 28%P/46%C + MCS group obtained the remarkably higher Enterococcus proportion and lower Lactococcus proportion compared to the 30%P/43%C and 28%P/46%C groups, whereas the opposite occurred in 30%P/43%C group, which indicated that there existed differences in the improvement mechanism on intestinal microflora composition between MCS and appropriate P/C ratio. Combined with the above mentioned changes in our research, we concluded that 0.16% MCS administration in a 28%P/46%C diet could facilitate the protein-sparing action of high carbohydrate in diets for common carp, which could decrease the 5% dosage of soybean meal and synchronously reduce the 4% crude protein of diets without affecting the growth and immune ability for common carp.

Integration of microRNA and mRNA analyses depicts the potential roles of Momordica charantia saponin administration in insulin resistance of juvenile common carp (Cyprinus carpio) fed with a high-starch diet.

Background: The regulation of target gene mRNA mediated by microRNA may play an important role in glucose metabolism in fish. Previous research findings of our research group revealed that Momordica charantia saponin (MS) administration in a high-starch diet could improve insulin resistance of common carp through renovating insulin signaling pathways, whose fundamental mechanisms have remained unknown by far. To reveal this potential mechanism, we aimed to investigate the difference in miRNA and mRNA expression profiles between common carp fed with high-starch diets containing MS (HS_MS1 and HS_MS2) and common carp fed with high-starch (HS) diets. Results: Through miRNA deep-sequencing, 10 significantly differentially expressed miRNAs in HC and HS_MS1, including one upregulated and nine downregulated miRNAs, were identified, whereas 10 significantly differentially expressed miRNAs in HC and HS_MS2, including four upregulated and six downregulated miRNAs, were identified. These miRNAs may not only be involved in the regulation of insulin signaling pathways and insulin resistance in common carp but also be the markers for liver insulin resistance in MS therapy for the remission of insulin resistance. This study identified 10 potential known miRNAs, namely, ccr-miR-10b, ccr-miR-122, ccr-miR-143, ccr-miR-146a, ccr-miR-155, ccr-miR-16c, ccr-miR-200a, ccr-miR-29a, ccr-miR-34, and ccr-miR-375, as candidates participating in modulating the liver insulin resistance. According to the biopathway enrichment analysis of the 252 target genes using the KEGG classical biopathway database, the relative expression levels of gsk3bb, pik3r1, and pik3r3b were analyzed using RNA-seq. Compared to the HC group, a significant decrease in the relative expression levels of pik3r1 and pik3r3b was observed in HS_MS1 and HS_MS2 groups (p < 0.05). This study raised a presumption of the presence of ccr-miR-29a targeting pik3r1 or ccr-miR-143 targeting pik3r3 playing likely roles in Momordica charantia saponins remitting the liver insulin resistance. Conclusion: The findings will further deepen the understanding of the carbohydrate metabolism of common carp and provide an important scientific basis for the application of Momordica saponins as functional nutrients to alleviate insulin resistance of fish in fish culture.

Effects of M. oleifera leaf extract on the growth, physiological response and related immune gene expression of crucian carp fingerlings under Aeromonas hydrophila infection.

We evaluated the effect of dietary supplementation with Moringa oleifera leaf extract on the resistance to Aeromonas hydrophila infection in crucian carp. The fish were randomly divided into five groups: the basal diet, the basal diet supplied with 0.25% (0.25 M), 0.5% (0.5 M), 0.75% (0.75 M) and 1.0% M. oleifera leaf extract (1.0 M) for 8 weeks. The growth, antioxidant capabilities, related immune genes as well as resistance to A. hydrophila infection were determined. The results showed that compared with the control group, the weight gain, specific growth rate in the group of 0.5% M. oleifera leaf extract, serum superoxide dismutase (SOD), albumin (ALB) and glutathione peroxidase (GSH-Px), the gene expression of hepatopancreas BTB and CNC homolog 1 (Bach1), NF-E2-related factor 2 (Nrf2), peroxidases (PRX) and NADPH oxidase (NOX) in the group of 0.5%-1.0% M. oleifera leaf extract increased, while feed conversion ratio, serum cortisol, red blood cell (RBC), alanine aminotransferase (ALT), malonaldehyde (MDA) decreased in the group of 0.5%-1.0% M. oleifera leaf extract before the stress. After the infection, the group of 0.5% or 0.75% M. oleifera leaf extract also could improve the serum ALB, hepatopancreas Kelch-like-ECH-associated protein 1 (Keap1), Bach1, Nrf2, TOR, PRX and NOX and reduce cortisol compared with the control group. In summary, this study suggested that 0.5% M. oleifera leaf extract inclusion increased the growth performance, even had positive effects on physiological and immune function, and enhanced resistance against pathogenic infections in crucian carp. The optimum level of M. oleifera leaf extract for crucian carp was estimated to be 0.35%-0.48% based on polynomial comparison with FCR and SGR.

Dietary ferulic acid supplementation improved cottonseed meal-based diet utilization by enhancing intestinal physical barrier function and liver antioxidant capacity in grass carp (Ctenopharyngodon Idellus).

The present study explored the effects of ferulic acid (FA) supplementation in cottonseed meal (CSM)-based diets on grass carp growth performance, feed utilization, liver antioxidation status, and intestinal physical barrier function. Here, four experimental diets supplemented with FA at graded levels (0, 50, 100 and 200 mg/kg) and CSM as the main protein source (384.6 g/kg feed) for an 8-week feeding trial. Our results indicated that 200 mg/kg FA supplementation in a CSM-based diet significantly improved growth performance [including final body weight (FBW), weight gain rate, and specific growth rate] and feed utilization [including feed conversion ratio and protein efficiency ratio] in grass carp (p < 0.05). The results of polynomial regression analysis based on FBW recommended that the optimal dose for FA supplementation was 204 mg/kg. Compared with that no FA supplementation, 200 mg/kg FA supplementation significantly reduced liver malondialdehyde levels and increased glutathione reductase activities (p < 0.05) and 100 mg/kg FA supplementation significantly increased liver total superoxide dismutase activities and reduced blood alanine transaminase levels (p < 0.05). Compared with the control group, 100 mg/kg FA supplementation also led to significantly increased mRNA expression of zo-1, zo-2, occludin, claudin-b, claudin-3, claudin-7a, and claudin-12, encoding intestinal tight junction proteins (p < 0.05). Notably, FA supplementation could reduce lipid deposition by regulating bile acid (BA) secretion. In this study, 100 and 200 mg/kg FA supplementation significantly increased blood and liver total BA levels, respectively (p < 0.05); 100 mg/kg FA also significantly activated mRNA expressions of fxr and cyp7a1 (p < 0.05). Furthermore, the whole-body composition results presented that FA treatment relieved lipid deposition, particularly 50 and 200 mg/kg FA supplementation (p < 0.05). Moreover, triglyceride and total cholesterol levels were significantly lower and high-density lipoprotein levels were significantly higher with 200 mg/kg FA supplementation than with no FA supplementation (p < 0.05). Taken together, the results indicated that FA may be a beneficial feed additive to boost fish growth performance and increase CSM utilization.

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.

Mulberry leaf meal: A potential feed supplement for juvenile Megalobrama amblycephala "Huahai No. 1".

This study was performed to evaluate the potential application of mulberry leaf meal (ML) and fermented mulberry leaf meal (FML) as feed supplements in aquatic animals for developing varieties of practical and economical feed ingredients. Juveniles Megalobrama amblycephala were fed a basal diet (35.7% crude protein, 10.4% crude lipid; control group) supplemented with 2.22% and 4.44% mulberry leaf meals (ML2, ML4) and fermented mulberry leaf meals (FML2, FML4) for 8 weeks. Generally, the two-way ANOVA showed the supplementation level exhibited a prominent effect on the growth performance and physiological status of fish. Furthermore, the two-way ANOVA showed the supplementary fermented mulberry leaf meal increased plasma complement 4 (C4) content (P < 0.05). The weight gain rate (WGR, 145.87%) and the specific growth rate (SGR, 1.63%) were significantly increased in FML2 group compared with the control group (P < 0.05). The muscle crude lipid content and hepatosomatic index (HSI) were higher in FML2 group than that in ML2 group (P < 0.05). The hepatic GSH content in ML4 group and CAT, T-SOD activities in FML4 group were significantly increased compared with the control group (P < 0.05). The hepatic MDA content in FML4 group was significantly decreased compared with the FML2 group (P < 0.05). Total cholesterol (TC) contents showed a significant decrease in ML4 and FML4 groups compared with the control group (P < 0.05). Regarding the gene expression, sirtiun 1 (Sirt1) gene expression was elevated in FML2 group compared with the ML2 group (P < 0.05). Compare to the control group, FML2 diet significantly increased the expression of i-kappa-B alpha (IKBα) gene in liver, and decreased the expression of forkhead box O1 α (FoxO1α), toll-like receptors 4 (TLR4) and nuclear factor-kappa B (NF-κB) genes (P < 0.05). In conclusion, 2.22% FML promoted the growth performance of M. amblycephala and enhanced the anti-inflammatory responses by inhibiting TLR4/NF-κB signaling pathway. On the other hand, 4.44% FML reduced plasma lipid content (hypolipedemic effect) and improved the hepatic antioxidant capacity of M. amblycephala.

Addition of Bamboo Charcoal to Selenium (Se)-Rich Feed Improves Growth and Antioxidant Capacity of Blunt Snout Bream (Megalobrama amblycephala).

The ability of bamboo charcoal to reduce the negative effects of high dietary selenium (Se) concentrations was assessed by feeding juvenile blunt snout bream (Megalobrama amblycephala) one of five Se-rich diets (1.5 mg/kg Se; 36% protein, 8.7% lipid) containing graded levels (0-4 g/kg) of bamboo charcoal powder for eight weeks. There were four tanks (350 L) of fish (initial weight 16.0 ± 0.5 g) for each treatment, and the fish were fed to satiation four times each day. At the end of the feeding trial, all of the fish from each tank were weighed to calculate the growth performance. Blood samples were firstly obtained to collect plasma for the biochemical indexes determination. Liver tissues were then collected to determine the antioxidant enzyme activities and gene expression. Dorsal muscles were also collected to determine the nutrient composition. The results show that when the bamboo charcoal content in the Se-rich feed ranged between 0 and 3 g/kg, the weight growth rate (WGR) and specific growth rate (SGR) values increased with the higher dietary bamboo charcoal content, and the maximum WGR and SGR values were achieved when the bamboo charcoal content in the Se-rich feed was 2-3 g/kg (p < 0.05). The Se content in muscle tissues decreased significantly with the increased bamboo charcoal content (p < 0.05) in the Se-rich feed, which ranged from 0 to 4 g/kg. When the bamboo charcoal content in the Se-rich feed was 2-3 g/kg, the levels of glucose (GLU) and albumin (ALB) in plasma reached a maximum (p < 0.05), whereas the level of alkaline phosphatase (ALP) reached a minimum (p < 0.05). Additionally, the activities of catalase (CAT), total superoxide dismutase (T-SOD), total antioxidative capacity (T-AOC), and glutathione peroxidase (GSH-Px) were significantly enhanced (p < 0.05) when the bamboo charcoal content was 3 g/kg. In contrast, the malondialdehyde (MDA) level increased sharply when the bamboo charcoal content in the Se-rich feed was 1 g/kg, compared to the control group and the groups supplemented with 2-3 g/kg bamboo charcoal (p < 0.05). Regarding mRNA-level gene expression, the results show that dietary supplementation with 0 to 3 g/kg of bamboo charcoal increased the expression of keap1 and nrf2, whereas nfkb expression was inhibited (p < 0.05). The mRNA expression of the antioxidant enzymes cat, gpx, and mn-sod was consistently enhanced in the group fed with the 3 g/kg bamboo charcoal diet (p < 0.05). The expression of the pro-inflammatory cytokines tnfα and tgfß was inhibited in the groups supplemented with 2-3 g/kg bamboo charcoal, whereas the expression of anti-inflammatory cytokines (il10) increased in the bamboo charcoal supplementation groups compared to the control group (p < 0.05). Generally, supplementation with 2-3 g/kg of bamboo charcoal in Se-rich feed improved the growth performance, physiological status, and antioxidant enzyme activities of blunt snout bream. Moreover, bamboo charcoal supplementation in Se-rich diets stimulated the antioxidant system and inhibited the inflammatory response by activating Nrf2-Keap1 and suppressing NF-κB.

Appropriate leucine supplementation promotes glucose metabolism and enhances energy homeostasis in juvenile crucian carp (Carassius auratus gibelio var. CAS III).

In order to characterize the molecular mechanisms by which leucine regulates carbohydrate metabolism and energy homeostasis, juvenile crucian carps (Carassius auratus gibelio var. CAS III) fed with a high carbohydrate diet were supplemented with different levels of dietary leucine: 0% (Leu0), 0.4% (Leu4), 0.8% (Leu8), 1.2% (Leu12), 1.6% (Leu16), 2.0% (Leu20), and 5.0% (Leu50). After 8 weeks, RNA sequencing was performed on samples collected from the Leu0, Leu8, Leu12 and Leu50 groups. Differentially expressed genes were then detected and analyzed. The results showed a total of 91.6 Gb of clean bases were generated. Moreover, a total of 1131, 5254, and 1539 DEGs were detected in Leu8, Leu12, and Leu50 compared with Leu0, respectively, encompassing 161 common DEGs. STEM analysis elucidated four significant clusters of DEGs that were associated with "glycerophospholipid metabolism," "glycerolipid metabolism," "PPAR signaling pathway," and "adipocytokine signaling pathway." Moreover, the mRNA expression levels of acyl-CoA synthetase long chain family member 5 (ACSL5), choline kinase beta (CHKB), cryptochrome-1 (CRY1), lon protease homolog 2, peroxisomal isoform X2 (LONP2), lipin 1 (LPIN1), membrane bound O-acyltransferase domain containing 2 (MBOAT2), phosphoenolpyruvate carboxykinase 1 (PEPCK), and uridine-cytidine kinase 2b (UCK2b) were then further investigated in all leucine treatment groups at starvation times of 0 h, 24 h, and 48 h. The results revealed that the expression levels of UCK2b and MBOAT2 were negatively correlated with the addition of leucine, whereas CHKB, LONP2, CRY1, PEPCK, and LPIN1 were positively correlated. In conclusion, dietary leucine supplementation below 1.2% enhanced carbohydrate metabolism in juvenile crucian carp fed with a high-carbohydrate diet, whereas concentrations above 2.0% is a better choice for energy homeostasis under starvation.

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.

Molecular cloning and functional characterization of the hypoxia-inducible factor-1α in bighead carp (Aristichthys nobilis).

HIF-l is the earliest documented and most widely studied hypoxia-inducible factor (HIF) and plays a key role in the cell hypoxia signal transduction pathway. Particularly, the HIF-1α protein is sensitive to oxygen and plays a critical role in hypoxia regulation. This study is the first to report on the molecular cloning and characterization of HIF-1α in bighead carp (Aristichthys nobilis; anHIF-1α). The full-length cDNA of anHIF-1α was 2361 bp, and encodes an estimated 674 amino acids with a predicted molecular mass of 76.10 kDa and a theoretical isoelectric point of 7.72. Moreover, the conserved basic Helix-Loop-Helix domain along with two Per-ARNT-Sim domains (A/B), and C-TAD were identified in this protein. Interestingly, the tertiary structure of the anHIF-1α protein was found to be extremely similar to that of mice. Multiple comparison and phylogenetic tree results demonstrated that anHIF-1α was highly conserved. Under normoxic conditions, anHIF-1α mRNA transcripts could be detected in all tissues examined with the highest expression level in the heart. With gradually decreasing oxygen concentrations, anHIF-1α mRNA level was upregulated significantly in the gill, liver, kidney, spleen, intestine, brain, and muscle tissues (P < 0.05). Similarly, anHIF-1α was expressed in all examined bighead carp tissues, and the results suggested that the upregulation of anHIF-1α at the transcriptional level may be an important stress response adaptation to hypoxia in bighead carp. Finally, based on the tertiary structure comparative analyses between anHIF-1α with mouse HIF-1α, we think the physiological function, and protein structure of HIF-1α could be compared between fish and mammal in the future.

Molecular characterization and expression of sirtuin 2, sirtuin 3, and sirtuin 5 in the Wuchang bream (Megalobrama amblycephala) in response to acute temperature and ammonia nitrogen stress.

This study sought to characterize sirtuin 2 (sirt2), sirtuin 3 (sirt3), and sirtuin 5 (sirt5) in Megalobrama amblycephala (M. amblycephala) by cloning the open reading frame (ORF) of sirt2, sirt3, and sirt5. The full-lengths of the resulting M. amblycephala sirt2, sirt3, and sirt5 cDNA sequences were 1845, 1534, and 1920 bp, respectively, with 92%, 98%, and 91% similarities to Danio rerio sequences. Based on our bioinformatic analyses and predictions, the sirt2 and sirt3 genes of M. amblycephala were classified within the Sir2 I family, whereas sirt5 belonged to the Sir2 III family. Furthermore, sirt2, sirt3, and sirt5 were widely distributed in different M. amblycephala tissues. Particularly, sirt2 and sirt5 were highly expressed in gills, intestines, and liver (P < 0.05), whereas sirt3 was highly expressed in gills, kidney, liver, and spleen (P < 0.05). A 2 × 2 factorial experiment was also conducted to analyze sirt2, sirt3, and sirt5 expression patterns in response to acute temperature (25 and 32 °C) and ammonia nitrogen (0 and 20 mg/L) stress. Notably, these two stressors were found to interactively affect sirt2, sirt3, and sirt5 expression patterns in M. amblycephala liver. At the higher water temperature (32 °C) and ammonia nitrogen concentration (20 mg/L) tested herein, sirt2, sirt3, and sirt5 had similar expression levels and exhibited a down-regulation trend at 6 and 48 h post-stress but became up-regulated thereafter to counteract the stressors at 96 h post-stress.

Molecular and functional characterization of sirt4 and sirt6 in Megalobrama amblycephala under high glucose metabolism.

We analyzed the sequences of sirt4 and sirt6 and their changes in expression after oral glucose administration in blunt snout bream (Megalobrama amblycephala). We cloned sirt4 and sirt6 and found that their full-length cDNA sequences were 1530 bp and 1723 bp, respectively; their amino acid sequences were 93% and 92% identical to Danio rerio. Megalobrama amblycephala were fed a high glucose solution (3 g/kg). Normally, sirt4 expression is higher in spleen, intestine, and gill (P < .05), and sirt6 expression is higher in intestine and gill (P < .05). After oral glucose administration, sirt4 and sirt6 expression increased in liver and gill, and sirt4 expression increased in intestine at 0.5 h (P < .05). In contrast, sirt4 in kidney and sirt6 in head kidney were downregulated at 1 h (P < .05). Expression of sirt4 was upregulated in brain, head kidney, spleen, muscle, and liver 2 h, 4 h, 4 h/24 h, 8 h, and 24 h, respectively (P < .05). Expression of sirt4 was downregulated in kidney at 8 h-48 h (P < .05). Expression of sirt6 was upregulated in intestine, liver, muscle, kidney, and spleen at 4 h/24 h, 8 h/24 h, 12 h, 12 h, and 24 h, respectively (P < .05). We report that sirt4 and sirt6 are highly conserved in evolution and exhibit tissue-specific expression profiles. We demonstrate that the expression of sirt4 and sirt6 are tissue-specific, and depend upon tissue-specific responses to glucose metabolism.

High glucose affected respiratory burst activity of peripheral leukocyte via G6PD and NOX inhibition in Megalobrama amblycephala.

High glucose levels are known to impair growth and immune function in fish. Here we investigated the role of glucose-6-phosphate dehydrogenase (G6PD) and NADPH oxidase (NOX) in high glucose-associated impairment of leukocyte respiratory burst activity in Megalobrama amblycephala. We cultured peripheral leukocytes isolated from M. amblycephala with media containing no glucose (non-glucose group), 11.1 mmol/L d-glucose (physiologic glucose group), 22.2 mmol/L d-glucose (high-glucose group), or 11.1 mmol/L d-glucose + 100 µmol/L dehydroepiandrosterone (DHEA) (DHEA-treated group). After 24 h, we assayed production of reactive oxygen species (ROS) as a measure of respiratory burst function as well as activity of G6PD and NOX. The high-glucose group and DHEA-treated group showed significantly reduced respiratory burst function, reduced production of ROS, and reduced G6PD and NOX activity at 24 h, compared to the non-glucose and physiologic glucose groups (P < 0.05). The degree of impairment was similar between high-glucose and DHEA-treated groups (P > 0.05). These findings suggest that reduced NADPH availability likely underlies the suppression of respiratory burst function in M. amblycephala leukocytes exposed to high glucose levels.

In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala.

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.