Search

Dietary lipid oxidation is a lasting concern to aquafeed manufacturers and aquaculture farmers. Here, we determined if there was an association between oxidized fish oil (OFO) and hepatopancreatic injury in Chinese mitten crab, and evaluated the alleviating effects of a combination of vitamins C and E on hepatopancreatic injury. There were significant reductions in the body weight gain, hepatopancreatic index, and feed efficiency in crabs that received OFO. Diets with OFO also resulted in reduced crude fat contents in the hepatopancreas and muscle tissues, and alternations in the hepatopancreatic fatty acid compositions, especially a reduction in polyunsaturated fatty acids. Moreover, dietary OFO exposure reduced the antioxidant defense and non‐specific immune response, caused hepatopancreatic injury, and even apparent whitening. An ultrastructural examination of the whitened hepatopancreas revealed that hepatopancreatic cells were injured at varying degrees following OFO exposure. The combination of vitamins C and E could efficiently maintain a constant fatty acid profile in the hepatopancreas exposed to OFO, thus alleviating OFO‐induced hepatopancreatic injury. In addition, a biochemical analysis indicated that a combination of vitamins C and E not only improved the antioxidant status by mitigating the harmful effects of oxidized oil, but also modulated non‐specific immune responses in the crab that received OFO. Overall, OFO‐induced hepatopancreatic injury may be alleviated by a dietary combination of vitamins C and E, and the feeding of rancid forage fish or diets that contain highly oxidized oil is likely a cause of hepatopancreatic necrosis that is frequently observed in farmed crabs.

Intestinal microbiota has increasingly been recognized as a major determinant of host health due to its critical role in the maintenance of immune homeostasis. An experimental fish model with disrupted intestinal microbiota allows us to identify potential strategies for restoring or modulating microbiota composition. The aim of this study was to develop a reliable grass carp model with disrupted intestinal microbiota by treatment with an antibiotic cocktail consisting of vancomycin, enrofloxacin, florfenicol, and metronidazole at different concentrations. To identify the appropriate concentration and exposure time of antibiotic treatment required to disrupt intestinal microbiota, five antibiotic regimens were compared in terms of their effects on intestinal microbiota composition, growth and health status, and histological changes in the intestine, hepatopancreas, and spleen. Antibiotic-induced changes in the expression of inflammation- and apoptosis-related genes in grass carp intestines were also examined. The results revealed that the highest antibiotic concentration over a 28 day period was required to disrupt both luminal and mucosal microbiota through significant expansion of Proteobacteria and suppression of Fusobacteria. Treatment with this regimen induced significant down-regulation of caspase-8, MLCK, ZO-1, IL-17N, and IL-23R, and up-regulation of IL-1β and IL-6 in the intestine, suggesting possible effects on intestinal epithelial tight junction and inflammatory conditions. This treatment did not produce substantial histopathological changes, and was not directly detrimental to growth and health. Therefore, this antibiotic regimen may provide a suitable option to disrupt intestinal microbiota in grass carp. In addition, our study also implies that fish may serve as a promising model for investigating growth-promoting effects of antibiotics, when treated with antibiotics in low doses.

The genomic and coding sequences of four members of grass carp interleukin (IL)-17 cytokine family, IL-17A/F2, IL-17A/F3, IL-17C and IL-17N, were isolated using rapid amplification of cDNA ends and conventional PCR techniques. A phylogenetic analysis showed that grass carp IL-17 family genes, including previously identified IL-17A/F1 and IL-17D, clustered with their counterparts from other fish species. IL-17D had two exons and one intron, while all the other members were composed of three exons and two introns. A multiple sequence alignment revealed that grass carp IL-17 family cytokines shared a cystine-knot motif that was highly conserved in mammals. A quantitative real-time PCR analysis revealed that these genes were expressed in the different examined tissues at varying levels. Moreover, they displayed different tissue expression patterns in fish at 24 h after intraperitoneal injection (IP) or anal intubation (AI) of Aeromonas hydrophila, implying roles in inflammatory responses. Next, we compared disease activity status and histopathological changes in intestinal tissues over a 7 day period after IP or AI, and then examine the potential differential effects of different infection routes on the expression profiles of IL-17 family genes in inflamed intestinal tissues. The intraperitoneal route resulted in more pronounced pathological symptoms than the anal route in terms of the disease activity index (DAI). Changes in the DAI highly coincided with the severity of intestinal inflammation. The two infection routes resulted in differential IL-17 family gene expression patterns over time in grass carp intestine, which suggests that IL-17 family members may play differential roles in mucosal and systemic immune responses and that IL-17N and IL-17A/F1 might possibly function as pro-inflammatory cytokines that provide defense against bacterial infection in grass carp intestine.

Interleukin-12 receptor β2 (IL-12Rβ2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rβ2 in teleost fish. In this work, we have cloned and characterized IL-12Rβ2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rβ2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rβ2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rβ2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rβ2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rβ2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rβ2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rβ2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rβ2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rβ2 is involved in the regulation of intestinal inflammation.

Two experiments were conducted to evaluate the feasibility of including high levels of rapeseed meal (RSM) and peanut meal (PNM) in juvenile crucian carp (Carassius auratus gibelio♀ × Cyprinus carpio♂) diets by investigating the growth performance, immunity, intestinal morphology, and microflora of these animals. Fish meal (FM)-based diets were used as controls in both experiments. In experiment 1, RSM and PNM replaced 250 (RSM25 and PNM25) and 500 (RSM50 and PNM50) g kg− 1 of FM in test diets. In experiment 2, RSM and PNM substituted 500 g kg− 1 of FM in test diets (RSM50′ and PNM50′, respectively). The diets in experiment 2 were isonitrogenous and isocaloric, and supplemental crystalline amino acids were added to compensate for the reduction in essential amino acids. More than 100 g kg− 1 of FM was maintained in the test diets in both experiments to ensure palatability, and fish were fed these diets for 8 weeks. No significant changes in feed intake were noticed among groups in both experiments. In experiment 1, the growth rate (GR) of fish fed test diets was significantly lower than in fish fed control diets; the feed conversion ratios (FCR) were higher in fish fed test diets than in fish fed control diets with the exception of fish fed the PNM25 diet. The lower GR observed in fish fed test diets was partly due to reduced dietary energy and protein levels. No significant differences were observed in the GR of fish in experiment 2; however, the FCR of fish fed the RSM50′ diet increased, whereas the protein efficiency ratio decreased in fish of both test groups, suggesting that growth retardation would have occurred over a longer period. No significant changes were observed in serum superoxide dismutase activity in both experiments. Compared with control groups, the lymphocyte proliferation index (LPI) decreased in fish fed the RSM50 diet but not in those fed the RSM50′ diet; the LPI increased in fish fed the PNM50 and PNM50′ diets. The secretion of mucus, which formed a peritrophic membrane-like structure, was enhanced when diets included high levels of RSM and PNM. Mild intestinal histological changes, but no significant inflammatory responses or intestinal microflora changes, were observed. From these results, we came to the general conclusion that the inclusion of high levels of RSM and PNM in the diet not only retarded growth but also produced health risks to the crucian carp.