Tea polyphenols protect against Flavobacterium columnare-induced gill injury via suppression of oxidative stress, inflammation, and apoptosis in grass carp.

Flavobacterium columnare (F. columnare) is one of the deadliest fish pathogens causing bacterial gill rot disease in various freshwater fish species globally. Tea polyphenols (TPs) are an inexpensive product extracted from tea that have received much attention as a feed additive in aquaculture. The current study was designed to investigate the underlying mechanisms and protective effects of dietary TPs against F. columnare-induced gill injury via suppression of oxidative stress, apoptosis, and inflammation in grass carp. TPs were not supplemented to the diet (control) and were supplemented at 40, 80, 120, 160 or 200 mg/kg diet. The feeding experiment was carried out for 60 days, followed by a 3-Day F. columnare challenge test. The results showed that 120 mg/kg TPs in the diet exerted the following five protective effects in fish gill: (1) control gill-rot disease and improved histopathology, (2) inhibit excessive apoptosis, (3) enhance the activity of antioxidant enzymes and upregulate related gene expression via the Nrf2/Keap1 pathway, (4) increase the activity of immune enzymes, And (5) mediate inflammatory cytokine gene expression via the JAK/STAT3 pathway. Taken together, dietary supplementation with TPs is a compelling approach to protect the gill function of fish against F. columnare.

A new insight on copper: Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella).

Copper (Cu) is a trace element, essential for fish growth. In the current study, in addition to growth performance, we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella). A total of 1080 fish (11.16 ± 0.01 g) were randomly divided into 6 treatments (3 replicates per treatment) to receive five doses of organic Cu, which were Cu citrate (CuCit) at 0.99 (basal diet), 2.19, 4.06, 6.15, and 8.07 mg/kg, and one dose of inorganic Cu (CuSO4·5H2O at 3.15 mg/kg), for 9 weeks. The results showed appropriate Cu level (4.06 mg/kg) enhanced growth performance, improved nutritional Cu status, and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas, intestine, and muscle of juvenile grass carp. Meanwhile, collagen content in fish muscle was increased after Cu intake, which was probably due to the following pathways: (1) activating CTGF/TGF-ß1/Smads signaling pathway to regulate collagen transcription; (2) upregulating of La ribonucleoprotein domain family 6 (LARP6) mRNA levels to regulate translation initiation; (3) increasing proline hydroxylase, lysine hydroxylase, and lysine oxidase activities to regulate posttranslational modifications. In addition, optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter >50 µm, which might be associated with upregulation of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen, myogenic determining factor (MyoD), myogenic factor 5, myogenin (MyoG), myogenic regulatory factor 4 and myosin heavy chain (MyHC) and downregulation of myostatin mRNA levels, increasing protein levels of MyoD, MyoG and MyHC in fish muscle. Finally, based on percentage weight gain (PWG), serum ceruloplasmin (Cp) activity and collagen content in fish muscle, Cu requirements were determined as 4.74, 4.37 and 4.62 mg/kg diet (CuCit as Cu source) of juvenile grass carp, respectively. Based on PWG and Cp activity, compared to CuSO4·5H2O, the efficacy of CuCit were 131.80% and 115.38%, respectively. Our findings provide new insights into Cu supplementation to promote muscle growth in fish, and help improve the overall productivity of aquaculture.

Dietary threonine improves muscle nutritional value and muscle hardness associated with collagen synthesis in grass carp (Ctenopharyngodon idella).

To further explain the improvement effect of threonine (Thr) on the fillet quality of fish, a 9-week feeding experiment was conducted. After feeding graded levels of Thr (2.38, 5.38, 8.38, 11.38, 14.38 and 17.38 g/kg), the compositions of fillet hydrolyzed amino acid and fatty acid, and the muscle hardness associated with collagen biosynthesis were mainly analyzed in grass carp (Ctenopharyngodon idella). The results showed that Thr increased the pH value, changed the amino acids and fatty acid composition of fillets, especially essential amino acid (EAA), C22:6n3 (DHA) and C20:5n3 (EPA). Furthermore, this study revealed for the first time that the improvement of muscle hardness by Thr was associated with collagen biosynthesis, and the TGF-ß1/Smads, LARP6a and Hsp47 regulate transcriptional processes, translation initiation and post-translational modifications in collagen biosynthesis, respectively. This study offered a basis for exploring the contribution of Thr in improving muscle quality in sub-adult grass carp.

Dietary cinnamaldehyde improves muscle protein content by promoting muscle fiber growth via PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling pathway in grass carp (Ctenopharyngodon idella).

Flesh quality is evaluated according to nutritional value and sensory quality. Cinnamaldehyde (CIN) improves mammalian meat quality, but research relating this to aquaculture is scarce. In this study, five doses of CIN (0, 36, 72, 108, 144 mg/kg diet) were fed to grass carp (Ctenopharyngodon idella) for 60 days. The results show that CIN supplementation increased nutritional value by increasing crude protein content. CIN also improved the sensory quality by increasing the pH and collagen content, decreasing shear force, lactate, and cooking loss. These changes may be related to changes in muscle fiber growth by increasing myofiber diameter. The increased myofiber diameter induced by CIN is associated with TOR mRNA and protein levels, and down-regulated FOXO3a mRNA levels, which might be associated with PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling. Based on muscle crude protein content, optimal CIN supplementation dosage was 88.01 mg/kg.

Cellular antioxidant mechanism of mannan-oligosaccharides involving in enhancing flesh quality in grass carp (Ctenopharyngodon idella).

BACKGROUND: Deterioration of flesh quality has bad effects on consumer satisfaction. Therefore, effects of safe mannan-oligosaccharides (MOS) on flesh quality of grass carp (Ctenopharyngodon idella) muscle were studied. A total of 540 healthy fish (215.85 ± 0.30 g) were randomly divided into six groups and fed six separate diets with graded levels of MOS (0, 200, 400, 600, 800 and 1000 mg kg-1 ) for 60 days. This study aimed at investigating the benefits of dietary MOS on flesh quality (fatty acids, amino acids and physicochemical properties) and the protection mechanism regarding antioxidant status. RESULTS: Optimal MOS could improve tenderness (27.4%), pH (5.5%) while decreasing cooking loss (16.6%) to enhance flesh quality. Meanwhile, optimal MOS improved flavor inosine 5'-monophosphate (IMP) of 11.8%, sweetness and umami-associated amino acid, healthy unsaturated fatty acid (UFA) of 14.9% and n-3 polyunsaturated fatty acids (n-3 PUFAs) especially C20:5n-3 (15.8%) and C22:6n-3 (38.3%). Furthermore, the mechanism that MOS affected pH, tenderness and cooking loss could be partly explained by the reduced lactate, cathepsin and oxidation, respectively. The enhanced flesh quality was also associated with enhanced antioxidant ability concerning improving antioxidant enzymes activities and the corresponding transcriptional levels, which were regulated through NF-E2-related factor 2 (Nrf2) and target of rapamycin (TOR) signaling. Based on pH24h , cooking loss, shear force and DHA (docosahexaenoic acid, C22:6n-3), optimal MOS levels for grass carp were estimated to be 442.75, 539.53, 594.73 and 539.53 mg kg-1 , respectively. CONCLUSION: Dietary MOS is a promising alternative strategy to improve flesh quality of fish muscle. © 2022 Society of Chemical Industry.

Dietary mannan oligosaccharides strengthens intestinal immune barrier function via multipath cooperation during Aeromonas Hydrophila infection in grass carp (Ctenopharyngodon Idella).

In recent years, mannose oligosaccharide (MOS) as a functional additive is widely used in aquaculture, to enhance fish immunity. An evaluation of the effect of dietary MOS supplementation on the immune barrier function and related signaling molecules mechanism of grass carp (Ctenopharyngodon idella) was undertaken in the present study. Six diets with graded amounts of MOS supplementation (0, 200, 400, 600, 800, and 1000 mg/kg) were fed to 540 grass carp over 60 days. To examine the immune response and potential mechanisms of MOS supplementation on the intestine, a challenge test was conducted using injections of Aeromonas hydrophila for 14 days. Results of the study on the optimal supplementation with MOS were found as follows (1) MOS enhances immunity partly related to increasing antibacterial substances content and antimicrobial peptides expression; (2) MOS attenuates inflammatory response partly related to regulating the dynamic balance of intestinal inflammatory cytokines; (3) MOS regulates immune barrier function may partly be related to modulating TLRs/MyD88/NFκB and TOR/S6K1/4EBP signalling pathways. Finally, the current study concluded that MOS supplementation could improve fish intestinal immune barrier function under Aeromonas hydrophila infected conditions.

An emerging role of vitamin D3 in amino acid absorption in different intestinal segments of on-growing grass carp (Ctenopharyngodon idella).

Vitamin D3 (VD3), an essential nutrient for animals, has been demonstrated to stimulate the uptake of certain amino acids. However, the role of VD3 in the intestine, the primary site for digestion and absorption of nutrients, remains poorly characterized. Here, the grass carp (Ctenopharyngodon idella) was studied to assess the influence of different doses of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) on growth performance, intestinal morphology, digestive absorption, amino acid transport, and potential signaling molecule levels in a feeding experiment. As a result, dietary VD3 improved growth performance, intestinal structure, and digestive and brush border enzyme activities. Additionally, most intestinal free amino acids and their transporters were upregulated after VD3 intake, except for Ala, Lys, Asp, Leu, solute carrier (SLC) 7A7, SLC1A5, and SLC1A3 mRNA in different segments, Leu and SLC6A14 mRNA in the proximal intestine, and SLC7A5 mRNA in the mid and distal intestine. In the crucial target of rapamycin (TOR) signal pathway of amino acid transport, the gene and protein expression of TOR, S6 kinase 1, and activating transcription factor 4 were elevated, whereas 4E-binding protein 1 was decreased, further suggesting an advanced amino acid absorption capacity in the fish due to VD3 supplementation. Based on percentage weight gain, feed efficiency, and trypsin activity, the VD3 requirements of on-growing grass carp were estimated to be 968.33, 1,005.00, and 1,166.67 IU/kg, respectively. Our findings provide novel recommendations for VD3 supplementation to promote digestion and absorption capacities of fish, contributing to the overall productivity of aquaculture.

New Insight on the Immune Modulation and Physical Barrier Protection Caused by Vitamin A in Fish Gills Infected With Flavobacterium columnare.

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp (Ctenopharyngodon idella) infected with Flavobacterium columnare. The fish were fed different concentrations of vitamin A diets for 10 weeks and then infected with F. columnare by immersion. We observed that optimal vitamin A significantly prevented gill rot morbidity in fish infected with F. columnare. Further investigations revealed that vitamin A boosted the gill immunity by increasing the contents of complements (C3 and C4), activities of acid phosphatase (ACP) and lysozyme, mRNAs of ß-defensin-1, liver-expressed antimicrobial peptide 2A and 2B (LEAP-2A and LEAP-2B), hepcidin, and anti-inflammatory cytokines like transforming growth factor ß1 (TGF-ß1), TGF-ß2, interleukin-10 (IL-10), and IL-11. It also enhanced the levels of various related signaling molecules including inhibitor protein κBα (IκBα), target of rapamycin (TOR), and ribosome protein S6 kinase 1 (S6K1) but downregulated the expression of pro-inflammatory cytokines including IL-1ß, IL-8, tumor necrosis factor α (TNF-α), and interferon γ2 (IFN-γ2) and related signaling molecules including nuclear factor κB p65 (NF-κB p65) (rather than NF-κB p52), IκB kinase ß (IKKß), IKKγ (rather than IKKα), eIF4E-binding protein 1 (4E-BP1), and 4E-BP2 mRNA levels in fish gills. In addition, dietary vitamin A markedly lowered the concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC), increased both the activities and mRNAs of copper/zinc superoxide dismutase (Cu/ZnSOD), MnSOD, glutathione transferases (GSTs), glutathione peroxidase (GPx), and glutathione reductase (GR) associated with upregulation of NF-E2-related factor 2 (Nrf2) mRNAs and downregulation of Kelch-like-ECH-associated protein (Keap1a) and Keap1b mRNAs. Moreover, vitamin A decreased the mRNAs of different apoptotic mediators [caspases 8, 9, 3 (rather than 7)] associated with downregulation of signaling molecule p38 mitogen-activated protein kinase (p38MAPK) mRNAs in fish gills. Besides, vitamin A promoted tight junction (TJ) complex mRNAs [including claudin-b, -c, -3, -7, -12, occludin, and zonula occludens-1 (ZO-1)] that have been linked to the downregulation of myosin light chain kinase (MLCK) signaling. Taken together, the current study demonstrated for the first time that vitamin A markedly enhanced gill health associated with immune modulation and physical barrier protection. Based on protecting fish against gill rot morbidity, ACP activity, and against lipid peroxidation, optimum vitamin A concentrations in on-growing grass carp (262-997 g) were found to be 1,991, 2,188, and 2,934 IU/kg diet, respectively.

[Clinical efficacy observation on abdominal acupuncture for cyclomastopathy].

OBJECTIVE: To verify clinical efficacy of abdominal acupuncture for cyclomastopathy. METHODS: One hundred and twenty-one cases of cyclomastopathy were randomly divided into an abdominal acupuncture group (64 cases) and a routine treatment group (57 cases). In the abdominal acupuncture, basic treatment (including psychological counseling and Chinese patent medicine Rupixiao) and abdominal acupuncture at Zhongwan (CV 12), Xiawan (CV 10), Qihai (CV 6), Guanyuan (CV 4) and Huaroumen (ST 24) were applied at the same time. In the routine treatment group, only basic treatment was applied. Before and after the treatment, visual analogue scale (VAS) of main symptoms and WHO Quality of Life-100BREF score were observed, also clinical efficacy of both groups was compared. RESULTS: The scores of VAS and WHO Quality of Life-100BREF in both groups had statistical significances before and after the treatment (both P < 0.05), indicating two treatments could both effectively relieve pain and improve life quality. The total effective rate was 84.4% (54/64) in the abdominal acupuncture group, which was superior to 68.4% (39/57) in the routine treatment group (P < 0.01). The abdominal acupuncture had best effect for moderate pain (P < 0.05). CONCLUSION: The abdominal acupuncture could improve clinical symptoms and life quality of patients with cyclomastopathy.