Methionine is an essential amino acid for fish. The present study was conducted to investigate the effects of dietary methionine on growth performance, digestive and absorptive ability, as well as antioxidant capacity in the intestine and hepatopancreas of sub-adult grass carp (Ctenopharyngodon idella). Dietary methionine deficiency significantly decreased percentage weight gain (PWG), feed intake, feed efficiency and protein efficiency ratio, as well as activities of hepatopancreatic glutamate-oxaloacetate transaminase and muscle glutamate-pyruvate transaminase in sub-adult grass carp (P < 0.05). Furthermore, methionine deficiency significantly reduced activities of trypsin, lipase and amylase in the intestine, Na+/K+-ATPase, alkaline phosphatase and γ-glutamyl transpeptidase in three intestinal segments, and creatine kinase (CK) in the proximal intestine (P < 0.05). However, an unexplained and significant increase in CK activity in the mid intestine was associated with dietary methionine deficiency. Malondialdehyde and protein carbonyl contents in the intestine and hepatopancreas were significantly increased by methionine deficiency (P < 0.05), whereas anti-hydroxyl radical capacity in the hepatopancreas and intestine, and anti-superoxide anion capacity in the intestine, were significantly decreased by methionine deficiency (P < 0.05). Moreover, methionine deficiency significantly decreased superoxide dismutase and glutathione reductase activities, glutathione contents in the hepatopancreas and intestine, as well as glutathione peroxidase activity in the intestine (P < 0.05), whereas it significantly increased activities of catalase in the hepatopancreas and glutathione-S-transferase in the hepatopancreas and intestine (P < 0.05). The present results demonstrated that dietary methionine deficiency induced poor growth, and decreased digestive and absorptive function and antioxidant capacity in the hepatopancreas and intestine of sub-adult grass carp. Methionine requirements for sub-adult grass carp (450-1, 170 g) based on PWG, intestinal trypsin, and hepatopancreatic anti-hydroxyl radical activities were estimated to be 6.12 g/kg diet (21.80 g/kg protein), 6.99 g/kg diet (24.90 g/kg protein) and 5.42 g/kg diet (19.31 g/kg protein), respectively, in the presence of 1.50 g cysteine/kg (5.35 g/kg protein).

Zearalenone (ZEA) is a prevalent mycotoxin with high toxicity in animals. In order to study its effect on juvenile grass carp (Ctenopharyngodon idella), six diets supplemented with different levels of ZEA (0, 535, 1041, 1548, 2002, and 2507 μg/kg diet) for 10 weeks were studied to assess its toxicity on intestinal structural integrity and potential mechanisms of action. Our report firstly proved that ZEA led to growth retardation and body deformity, and impaired the intestinal structural integrity of juvenile grass carp, as revealed by the following findings: (1) ZEA accumulated in the intestine and caused histopathological lesions; (2) ZEA resulted in oxidative injury, apoptosis, and breached tight junctions in the fish intestine, which were probably associated with Nuclear factor-erythroid 2-related factor 2 (Nrf2), p38 mitogen activated protein kinases (p38MAPK), and myosin light chain kinase (MLCK) signaling pathways, respectively. ZEA had no influence on the antioxidant gene levels of Kelch-like ECH associating protein 1 (Keap1)b (rather than Keap1a), glutathione-S-transferase (GST)P1, GSTP2 (not in the distal intestine (DI)), tight junctions occludin, claudin-c (not in the proximal intestine (PI)), or claudin-3c (not in the mid intestine (MI) or DI).

Fish is an important animal-source food for humans. However, the oxidative stress-induced by intensive aquaculture usually causes deterioration of fish meat quality. The nutritional way has been considered to be a useful method for improving fish flesh quality. This study using the same growth experiment as our previous study was conducted to investigate whether vitamin A could improve flesh quality by enhancing antioxidative ability via Nrf2/Keap1 signaling in fish muscle. Six diets with different levels of vitamin A were fed to grass carp (Ctenopharyngodon idella) (262.02 ± 0.45 g) for 10 weeks. Dietary vitamin A significantly improved flesh sensory appeal and nutritional value, as evident by higher pH_24h value, water-holding capacity, shear force, contents of protein, lipid, four indispensable amino acids (lysine, methionine, threonine, and arginine) and total polyunsaturated fatty acid in the muscle. Furthermore, dietary vitamin A reduced oxidative damage, as evident by decreased levels of muscle reactive oxygen species, malondialdehyde, and protein carbonyl, enhanced activities of antioxidative enzyme (catalase, copper/zinc superoxide dismutase (CuZnSOD), MnSOD, glutathione peroxidase, and glutathione reductase), as well as increased content of glutathione, which was probably in relation to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. These findings demonstrated that dietary vitamin A improved flesh quality probably by enhancing antioxidant ability through Nrf2/Keap 1a signaling in fish.

This study attempted to evaluate the possible impact and mechanism of leucine (Leu) on fish intestinal barrier function. One hundred and five hybrid Pelteobagrus vachelli ♀ × Leiocassis longirostris ♂ catfish were fed with six diets in graded levels of Leu 10.0 (control group), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g/kg diet for 56 days. Results showed that the intestinal activities of LZM, ACP, and AKP and contents of C3, C4, and IgM had positive linear and/or quadratic responses to dietary Leu levels. The mRNA expressions of itnl1, itnl2, c-LZM, g-LZM, and β-defensin increased linearly and/or quadratically (p < 0.05). The ROS, PC, and MDA contents had a negative linear and/or quadratic response, but GSH content and ASA, AHR, T-SOD, and GR activities had positive quadratic responses to dietary Leu levels (p < 0.05). No significant differences on the CAT and GPX activities were detected among treatments (p > 0.05). Increasing dietary Leu level linearly and/or quadratically increased the mRNA expressions of CuZnSOD, CAT, and GPX1α. The GST mRNA expression decreased linearly while the GCLC and Nrf2 mRNA expressions were not significantly affected by different dietary Leu levels. The Nrf2 protein level quadratically increased, whereas the Keap1 mRNA expression and protein level decreased quadratically (p < 0.05). The translational levels of ZO-1 and occludin increased linearly. No significant differences were indicated in Claudin-2 mRNA expression and protein level. The transcriptional levels of Beclin1, ULK1b, ATG5, ATG7, ATG9a, ATG4b, LC3b, and P62 and translational levels of ULK1, LC3Ⅱ/Ⅰ, and P62 linearly and quadratically decreased. The Beclin1 protein level was quadratically decreased with increasing dietary Leu levels. These results suggested that dietary Leu could improve fish intestinal barrier function by increasing humoral immunity, antioxidative capacities, and tight junction protein levels.

Ochratoxin A (OTA) contamination widely occurs in various feed ingredients and food crops, potentially posing a serious health threat to animals. In this research, 1260 juvenile grass carp were separately fed with seven distinct experimental diets (0, 406, 795, 1209, 1612, 2003 and 2406 µg of OTA/kg of diet) for 60 consecutive days to evaluate OTA’s toxic effect on the intestinal apical junctional complex (including the tight junction (TJ) and the adherents junction (AJ)) and the underlying action mechanisms. Our experiment firstly confirmed that OTA caused fish growth retardation and disrupted the intestinal structural integrity. The detailed results show that OTA (1) depressed the feed efficiency, percentage weight gain and specific growth rate; (2) accumulated in the intestine; (3) caused oxidative damage and increased intestinal permeability; and (4) induced the RhoA/ROCK signaling pathway, destroying intestinal apical junctional complexes. Notably, OTA intervention did not result in changes in the gene expression of claudin-3c (in the proximal intestine (PI)), claudin-b and ZO-2b (in the mid intestine (MI) and distal intestine (DI)) in the fish intestine.

Mannan oligosaccharides (MOS) are a type of functional oligosaccharide which have received increased attention because of their beneficial effects on fish intestinal health. However, intestinal structural integrity is a necessary prerequisite for intestinal health. This study focused on exploring the protective effects of dietary MOS supplementation on the grass carp’s (Ctenopharyngodon idella) intestinal structural integrity (including tight junction (TJ) and adherent junction (AJ)) and its related signalling molecule mechanism. A total of 540 grass carp (215.85 ± 0.30 g) were fed six diets containing graded levels of dietary MOS supplementation (0, 200, 400, 600, 800 and 1000 mg/kg) for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila for 14 days. We used ELISA, spectrophotometry, transmission electron microscope, immunohistochemistry, qRT-PCR and Western blotting to determine the effect of dietary MOS supplementation on intestinal structural integrity and antioxidant capacity. The results revealed that dietary MOS supplementation protected the microvillus of the intestine; reduced serum diamine oxidase and d-lactate levels (p < 0.05); enhanced intestinal total antioxidant capacity (p < 0.01); up-regulated most intestinal TJ and AJ mRNA levels; and decreased GTP-RhoA protein levels (p < 0.01). In addition, we also found several interesting results suggesting that MOS supplementation has no effects on ZO-2 and Claudin-15b. Overall, these findings suggested that dietary MOS supplementation could protect intestinal ultrastructure, reduce intestinal mucosal permeability and maintain intestinal structural integrity via inhibiting MLCK and RhoA/ROCK signalling pathways.