Tributyrin Plays an Important Role in Regulating the Growth and Health Status of Juvenile Blunt Snout Bream (Megalobrama amblycephala), as Evidenced by Pathological Examination.

The present study aimed to assess the role of tributyrin (TB) in regulating the growth and health status of juvenile blunt snout bream (Megalobrama amblycephala) through an 8-week feeding experiment. Six groups were fed experimental diets with added TB percentages of 0% (control group), 0.03%, 0.06%, 0.09%, 0.12% and 0.15%. The present results showed that TB supplementation in feed had some positive impacts on FW, WG, FCR and SGR, and the best results were found in the 0.06% TB group (P<0.05). However, TB supplementation in feed had no significant effects on SR, CF, VSI or whole-body composition (P>0.05). TB supplementation in feed increased antioxidant capacity and immunological capacity and attenuated the inflammatory response by increasing the activity of T-SOD, GPx, CAT and the levels of anti-inflammatory cytokines (IL-10 and TGF-ß) and decreasing the levels of MDA and anti-inflammatory cytokines (TNF-α) (P<0.05). Furthermore, TB supplementation improved immunity by increasing the levels of immunoglobulins (IgM and IgG), C3 and IFN-γ (P<0.05). Surprisingly, 0.06%-0.12% TB supplementation significantly increased the content of IL-1ß (P<0.05). However, TB supplementation in feed had no significant effects on the plasma content of GSH, HSP70, IL-8 and the activity of T-AOC (P>0.05). The possible mechanism was that TB activated PI3K/Akt/Nrf2 and inhibits the NF-κB signaling pathway, further regulating the mRNA levels of key genes with antioxidant capacity and the inflammatory response; for example, it increased the mRNA levels of Nrf2, Cu/Zn-SOD, HO-1, CAT, Akt, PI3K, GPx, IL-10, and TGF-ß and decreased the mRNA levels of NF-κB and TNF-α (P<0.05). In addition, 0.06%-0.15% TB supplementation significantly increased the mRNA levels of IL-1ß (P<0.05). TB supplementation in feed had no significant effects on the mRNA levels of HSP70, Mn-SOD and IL-8 (P>0.05). Evidence was presented that TB supplementation decreased the mortality rate caused by Aeromonas hydrophila challenge. In pathological examination, TB supplementation prevented hepatic and intestinal damage. Generally, TB supplementation improved the growth performance of juvenile blunt snout bream. Furthermore, TB supplementation activated PI3K/Akt/Nrf2 and inhibited the NF-κB signaling pathway, regulating health status and preventing hepatic and intestinal damage.

Effects of dietary fenugreek seed extracts on growth performance, plasma biochemical parameters, lipid metabolism, Nrf2 antioxidant capacity and immune response of juvenile blunt snout bream (Megalobrama amblycephala).

Dietary administration of some plant-derived substances have been proved of great economic value in aquaculture. In order to investigate the effects of dietary fenugreek seed extracts (FSE) on juvenile blunt snout bream (Megalobrama amblycephala), a feeding trial was conducted for 8 weeks. The results showed that final weight (FW), weight gain (WG), feed conversion ratio (FCR) and specific growth rate (SGR) were not significantly affected by dietary FSE levels. The whole body lipid contents of fish fed with 0.04%, 0.08% and 0.16% FSE diets were significantly lowered compared to the control group. Dietary FSE diets significantly affected plasma complement component 3 (C3), immunoglobulin M (IgM), albumin (ALB) and total protein (TP). The relative expressions of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element binding protein-1 (SREBP1) mRNA in the liver of fish decreased significantly with increasing dietary FSE levels from 0% up to 0.04%. FSE supplementation diets lowered the liver pro-inflammatory genes expressions by regulating tumor necrosis factor-α (TNF-α) and interleukin 8 (IL-8) mRNA levels and increased anti-inflammatory genes expression by regulating transforming growth factor (TGF-ß) and interleukin 10 (IL-10). FSE diets increased growth factor-1 (IGF-1) and target of rapamycin (TOR) mRNA levels from 0% up to 0.04%, 0.04% FSE diets significantly increased growth factor-1 (IGF-1) mRNA levels and S6 kinase-polypeptide 1 (S6K1) mRNA levels compared to the control group. 0.04% FSE diets significantly increased superoxide dismutase (SOD) activities and 0.08% FSE diets significantly increased catalase (CAT) and glutathione peroxidase (GPx) activities, 0.16% FSE diets significantly increased total antioxidant capacity (T-AOC) activities compared to the control group. Additionally, compared to the control group, 0.04% dietary FSE significantly up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels and glutathione peroxidase-1 (GPx1) mRNA levels, at the same time, 0.02%, 0.04%, 0.08%, 0.16% FSE diets significantly down-regulated kelch-like ECH-associated protein 1 (Keap1) mRNA levels. However, no significant effects were observed on copper zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD). Our study indicated that dietary FSE could improve plasma biochemical parameters, regulate lipid metabolism related genes, promote Nrf2 antioxidant capacity and enhance immune response of juvenile blunt snout bream.

Effects of dietary tryptophan levels on antioxidant status and immunity for juvenile blunt snout bream (Megalobrama amblycephala) involved in Nrf2 and TOR signaling pathway.

Dietary administration of tryptophan has been proved improving growth performance of fish. An 8-week feeding trial was conducted to investigate the effects of dietary tryptophan level on antioxidant capacity and immune response through Nrf2 and TOR signaling pathway. The results showed that, 0.08% tryptophan level significantly increased plasma aspartate aminotransferase (AST), while immunoglobulin M (IgM) and alkaline phosphatase (ALP) were strikingly increased by 0.40% level. The level of plasma complement component 3 (C3), alanine aminotransferase (ALT) and albumin (ALB) were independent of tryptophan supplementation. Total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC) and glutathione (GSH) activity were increased with increasing dietary tryptophan level until 0.40% and then decreased, while the level of malondialdehyde (MDA) showed a reverse trend. 0.19% and 0.28% tryptophan level significantly improved the glutathione peroxidase 1 (GPx-1) activity. Compared with 0.08% dietary tryptophan level, 0.40% level significantly improved nuclear factor erythroid 2-related factor 2 (Nrf2), GPx, manganese superoxide dismutase (Mn-SOD), CAT and transforming growth factor-ß (TGF-ß) mRNA level, while Kelch-like ECH-associated protein 1 (Keap1) and interleukin 1ß (IL-1ß) mRNA level were significantly decreased. The relative expression of copper zinc superoxide dismutase (Cu/Zn-SOD), heme oxygenase-1 (HO-1), target of rapamycin (TOR), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase B (Akt) and interleukin 10 (IL-10) were significantly improved by 0.28% diet, while the mRNA level of tumor necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were increased by 0.08% diet. Interleukin 8 (IL-8) mRNA level was not significantly affected by dietary tryptophan. Based on MDA and T-SOD value, the optimal dietary tryptophan level of juvenile blunt snout bream was determined to be 0.33% (1.03% of dietary protein) and 0.36% (1.13% of dietary protein), respectively, using quadratic regression analysis.

Effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream.

The present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream. Fish were fed six practical diets with graded arginine levels ranging from 0.87% to 2.70% for 8 weeks. The results showed that compared with the control group (0.87% dietary arginine level), significantly higher mRNA levels of adenosine monophosphate activated protein kinase (AMPK) and nitric oxide synthetase (NOS), activities of total nitric oxide synthetase (T-NOS) and nitric oxide synthetase (iNOS), and plasma nitric oxide (NO) contents were observed in fish fed with 1.62%-2.70% dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.22%-2.70%, the mRNA levels of iNOS were significantly improved. Dietary arginine also significantly influenced plasma interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) contents. Furthermore, dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase (GPx), the mRNA levels of pro-inflammatory factor including IL-8 and TNF-α and plasma malondialdehyde (MDA) content. However, total superoxide dismutase (T-SOD) activity, plasma complement component 3 (C3) content, plasma immunoglobulin M (IgM) content, plasma interleukin 1ß (IL-1ß) content and the mRNA levels of copperzinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD) and IL-1ß were not significantly affected by dietary arginine. After Aeromonas hydrophila challenge, the death rate was significantly lowered in fish fed with 1.62%-1.96% dietary arginine levels. Furthermore, the mRNA levels of AMPK, NOS and iNOS, plasma NO content and the activities of T-NOS and iNOS showed an upward trend with increasing dietary arginine levels. Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70% dietary arginine levels in enzyme-linked immune sorbent assay. At dietary arginine levels of 1.96%-2.31%, T-SOD activities were significantly improved. Significantly higher GPx activities were observed in fish fed with 1.22%-2.70% dietary arginine levels. At dietary arginine levels of 1.22%-2.31%, the plasma TNF-α and IL-8 contents were significantly decreased. Significantly lower plasma IL-1ß contents were observed in fish fed 1.62%-1.96% dietary arginine levels. Dietary arginine significantly influenced the mRNA levels of antioxidant and pro-inflammatory genes including Cu/Zn-SOD, Mn-SOD, GPx, IL-8, TNF-α and IL-1ß. Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%-1.96% arginine levels. Furthermore, plasma IgM contents were significantly improved at dietary arginine levels of 1.62%-2.31%. However, high dietary arginine group (2.70%) significantly improved the mRNA levels of pro-inflammatory genes including IL-8, TNF-α and IL-1ß and plasma MDA, IL-8, TNF-α and IL-1ß contents as compared with optimal dietary arginine levels (1.62% and 1.96%). The present results indicate that optimal arginine level (1.62% and 1.96%) could improve antioxidant capacity, immune response and weaken tissues inflammatory involved in arginine-AMPK-NO signaling pathway, while high arginine level resulted in excessive NO production, leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream.

Dietary leucine modulates growth performance, Nrf2 antioxidant signaling pathway and immune response of juvenile blunt snout bream (Megalobrama amblycephala).

The present study assessed the effects of dietary leucine on growth performance, antioxidant status and immunity in juvenile blunt snout bream. Fish were fed six practical diets of graded leucine levels ranging from 0.90% to 2.94% of dry basis for 8 weeks. Trail results showed that compared to control group (0.90%), 1.72% dietary leucine level significantly improved final weight (FW), weight gain rate (WG) and specific growth rate (SGR), and significantly lowered feed conversion ratio (FCR). Based on WG and SGR, the optimal dietary leucine level was obtained at 1.40% and 1.56%, respectively. Whole body crude lipid and protein contents were improved with increasing dietary leucine up to 2.14% and thereafter showed a downward trend, while whole body moisture content showed a converse trend. No significant change was found in whole body ash content. 1.72% dietary leucine level significantly improved the antioxidant capacity of fish by regulating the plasma superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, aspartate aminotransferase (AST) activities and malondialdehyde (MDA) content, furthermore, 1.72% dietary leucine level also significantly improved the antioxidant genes expressions of associated with Nrf2 signaling pathway by regulating heme oxygenase-1 (HO-1), GPx, copperezinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), 2.14% dietary leucine levels also significantly improved glutathione transferase (GST) mRNA level. Dietary leucine levels significantly affected plasma immunity parameters such as the contents of plasma complement component 3 (C3), immunoglobulin M (IgM) and lowered the hepatopancreas genes expressions of pro-inflammatory factor by regulating interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels. The present study indicated that optimal dietary leucine level plays an important role in improving growth, enhancing antioxidant and immune status to maintain the health in juvenile blunt snout bream.

Molecular cloning and expression analysis of Megalobrama amblycephala transferrin gene and effects of exposure to iron and infection with Aeromonas hydrophila.

Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5' and a 204 bp 3' untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl3-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl3-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.