| In this study, we successfully prepared oxidation konjac glucomannan (OKGM) and its acidolysis products (A-OKGM) by oxidizing and acidolysis, and their characteristics were analyzed. Firstly,450 fish were fed a basal diet or basal diet plus 4.0,8.0,16.0,32.0 g kg-1 OKGM for 60 days, and the intestinal morphology and intestinal microflora were examined. Then, the diets with seven levels (0,8.0,16.0, 32.0 g kg-1 of OKGM and 8.0,16.0,32.0 g kg-1 of A-OKGM) were fed to Schizothorax prenanti. After 60 days feeding trial, the effect of OKGM and A-OKGM on growth performance, immune function of Schizothorax prenanti were studied. The challenge experiment with Aeromonas hydrophila was conducted to determine the impact of OKGM and A-OKGM on the disease resistance of Schizothorax prenanti. We studied the effect of OKGM and A-OKGM on the expression of TNF-α, IL-1β and TLR22 of Schizothorax prenanti, moreover, the action of OKGM and A-OKGM on TNF-α, IL-1β and TLR22 from Schizothorax prenanti during the early stages of infection with Aeromonas hydrophila was determined. Furthermore, we cloned the fragments of LPL and FAS, and then investigated the effects of OKGM and A-OKGM on the expression of the two genes. The results are as follows:1. The intrinsic viscosity of OKGM is 822.82 cm3/g, and A-OKGM is 46.55 cm3/g, which is roughly 17 times lower than that of OKGM. Moreover, the Mη of OKGM is about 4.7×105 Da, and A-OKGM is about 9.2×103 Da. SEM examination indicated that KGM had scale-like surfaces, and the surface of OKGM was smoother than KGM, but A-OKGM showed erosion surfaces in our study. DSC analysis showed that the thermal stability of A-OKGM was far below that of KGM and OKGM. Moreover, when compared to KGM, no obvious changes in OKGM and A-OKGM were observed in infrared spectra.2. The mucosa fold height of fish fed with the OKGM diets were significantly increased (P<0.05). In anterior and mid intestine, the mucosal epithelial height of fish fed the 8.0 g kg-1 and 16.0 g kg-1 OKGM diet were significantly higher (P<0.05) than that of fish in other three groups. In posterior intestine, mucosal epithelial height was significantly increased by OKGM diets (P<0.05). In anterior and mid intestine, the submucosa height of fish fed the OKGM diets were significantly higher (P<0.05) than those of fish fed the basal diet, however, in posterior intestine, no significant difference in the submucosa height was observed among each groups except the 4.0 g kg-1 OKGM group (P<0.05). Fish fed the 4.0 g kg-1 OKGM diets exhibited the highest longitudinal muscularis thickness (P<0.05). In anterior intestine, circular muscularis thickness of fish fed the OKGM diets were significantly higher (P<0.05) than that of fish fed the basal diet, and the similar results also be observed in mid intestine (P<0.05), but the difference between the basal diet group and 32.0 g kg-1 OKGM group was not significant. In posterior intestine, fish fed 16.0 g kg-1 OKGM diet showed the highest circular muscularis thickness (P<0.05), which was significantly superior to the other groups. In anterior intestine, the number of goblet cell was significantly lower in the 32.0 g kg-1 OKGM group (P<0.05), however, no significant difference was found in mid intestine and posterior intestine when compared to the control group (P>0.05). The intestine flora of fish fed the 4.0 g kg-1 and 8.0 g kg-1 OKGM diet were classified into the same cluster and showed high similarity, then formed a clade with the 16.0 g kg-1 OKGM group. There was a low similarity between the OKGM diets groups and the control group. The Shannon Weaver Diversity Index of control group was significantly lower than that of other groups (P<0.05).3. Compared with the control group, the weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of fish fed 8.0 g kg-1 A-OKGM diet was significantly (P<0.05) increased, and the feed coefficient was significantly reduced (P<0.05). There was no significant (P>0.05) difference in head kidney index, mesonephros index, spleen index, intestinal length index and intestinal weight index between each groups. The lysozyme activity of fish fed the 8.0 g kg-1 and 16.0 g kg-1 A-OKGM diet was significantly higher (P<0.05) than that of fish fed the basal diet, and there was no significant difference (P>0.05) among other groups. The Complement C3 activity of fish fed the 8.0 g kg-1 OKGM,8.0 g kg-1 A-OKGM and 16.0 g kg-1 A-OKGM diet was significantly higher (P<0.05) than that of fish fed the basal diet. Compared to the control group, the IgM activity of fish fed the A-OKGM diet was significantly increased (P<0.01). Moreover, the IgM activity of fish fed the 8.0 g kg-1 OKGM was higher than that of fish fed the basal diet, but the difference was not significant (P>0.05). The serum SOD activity of fish fed 32.0 g kg-1 OKGM diet and 8.0 g kg-1 A-OKGM diet was extremely significantly(P<0.01) higher than the control group, and fish fed 8.0 g kg-1 diet,16.0 g kg-1 OKGM diet and 16.0 g kg-1 A-OKGM diet showed significant(P<0.05) higher serum SOD activity than the control group. Serum MDA content of each experimental group was significantly (P <0.01) lower than that in the control group. Two days after the injection of A. hydrophila, the difference of the survival rate of each group was not significant (P>0.05). Six day later, fish fed 8.0 g kg-1 A-OKGM showed significantly higher (P<0.05) survival rate than control group. At the end of the challenge test,8.0 g kg-1 A-OKGM group also exhibited the highest survival rate among groups (P<0.05).4. The partial cDNA fragment of IL-1β, TNF-α and TLR22 had been cloned, and the length of the three fragments was 120 bp,116 bp and 174 bp. In the spleen, IL-1β was significantly up-regulated of all A-OKGM diet and 32.0 g kg-1 OKGM diet fed fish when compared to fish fed the basal diet (P<0.05). In the head kidney, the expression of IL-1β was affected by dietary OKGM and A-OKGM. IL-1β mRNA expression level of fish fed the 8.0 g kg-1 A-OKGM was significantly higher (P<0.05) than the fish of control group, but the difference between other groups was not significant (P>0.05). In the mesonephros, no significant difference was seen between the groups (P>0.05). In gut, IL-1β expression was significantly increased in fish fed the 8.0 g kg-1 OKGM and 16.0 g kg-1 A-OKGM diet (P<0.05) when compared to the control group. TNF-a mRNA expression in spleen was significantly increased (P<0.05) in fish fed with 16.0 g kg-1 and 32.0 g kg-1 A-OKGM diet when compared to the control group. In the head kidney, TNF-α mRNA expression level of fish fed the 32.0 g kg-1 OKGM diet and 8.0 g kg-1 A-OKGM diet was significantly higher (P<0.05) than that of fish fed the basal diet. In mesonephros, the expression of TNF-α in fish fed the 8.0 g kg-1 OKGM diet was significantly higher (P<0.05) than the fish of control group, and no significant difference (P>0.05) was seen between other groups. In the gut, a significant increase (P<0.05) of TNF-α expression was observed in fish of 32.0 g kg-1 A-OKGM group when compared to the control group. In the spleen, TLR22 expression in fish of 16.0 g kg-1 A-OKGM group was significantly higher than the control group (Fig.4). The expression of TLR22 in head kidney was significantly higher (P<0.05) in fish fed the 8.0 g kg-1 A-OKGM diet than that of fish fed basal diet, and no significant difference (P>0.05) was seen between other groups. In mesonephros, TLR22 mRNA expression level in fish fed the 16.0 g kg-1 A-OKGM diet was significantly higher (P<0.05) than that in fish fed the basal diet. As for gut, a higher expression of TLR22 was detected in fish fed the 8.0 g kg-1 OKGM diet, and the expression of TLR22 was lowest in fish fed the 32.0 g kg-1 OKGM diet.5. The expression of TLR22, TNF-a and IL-1(3 was induced in head kidney, spleen, mesonephros and gut at 6 h post-injection. In the head kidney, IL-1β mRNA expression level of fish fed OKGM and A-OKGM diet was significantly increased (P<0.05) after injection of Aeromonas hydrophila. In the mesonephros, after challenged with Aeromonas hydrophila, IL-1β mRNA expression level of fish fed 8.0 g kg-1,32.0 g kg-1 OKGM diet, and 8.0 g kg-1,32.0 g kg-1 A-OKGM diet was significantly up-regulated (P<0.05). After treatment with Aeromonas hydrophila, IL-1β expression of fish fed 32.0 g kg-1 OKGM diet and A-OKGM diet was significantly enhanced in the spleen (P<0.05). As for gut, IL-1β mRNA expression level of fish fed OKGM diet and 16.0 g kg-1 A-OKGM diet was significantly up-regulated after the treatment with Aeromonas hydrophila (P<0.05). After injection of Aeromonas hydrophila, the TNF-a mRNA expression level of 32.0 g kg-1 OKGM group,16.0 g kg-1 and 32.0 g kg-1 A-OKGM group was significantly increased in the head kidney at 6 h (P<0.05). In the mesonephros, the significant up-regulation of TNFa was observed in all test groups (P<0.05) except the 16.0 g kg-1 OKGM group (P>0.05) after the injection. In the spleen, following Aeromonas hydrophila treatment, the TNF-α gene expression of the fish fed 8.0 g kg-1 A-OKGM diet was significantly enhanced (P<0.05). In the gut, after bacteria treatment, the expression of TNF-α of the fish fed 8.0 g kg-1 and 16.0 g kg-1 OKGM diet,8.0 g kg-1 and 16.0 g kg-1 A-OKGM diet was increased sharply at 6 h (P<0.05). Following Aeromonas hydrophila treatment, TLR22 expression of fish fed 32.0 g kg-1 OKGM diet,8.0 g kg-1 and 32.0 g kg-1 A-OKGM diet was significantly increased in head kidney (P<0.05). In the mesonephros, TLR22 mRNA expression of fish fed the basal diet and the A-OKGM diet was significantly up-regulated (P<0.05). After challenged with Aeromonas hydrophila, TLR22 mRNA expression of fish fed OKGM diet and 8.0 g kg-1 A-OKGM diet significantly increased in the spleen. In the gut, TLR22 expression of fish fed the basal diet,8.0 g kg-1 and 16.0 g kg-1 OKGM diet,8.0 g kg-1 and 16.0 g kg-1 A-OKGM diet was significantly enhanced by the injection of Aeromonas hydrophila (P<0.05). The serum lysozyme activity and complement C3 content of fish fed 8.0 g kg-1 A-OKGM was significantly higher than that of fish fed the control diet.6. Using the design of LPL and FAS primers, two cDNA fragments had been amplified:512bp of LPL cDNA fragment (KF738266), encoding 170 amino acids, and 524bp of FAS cDNA fragment, encoding 174 amino acids. Fish fed 8.0 g kg-1 and 32.0 g kg-1 A-OKGM group diet showed significant (P<0.05) higher crude fat content in back muscle than the control group, and the abdominal muscle fat content of 16.0 g kg-1 OKGM group was significantly lower than (P<0.05) the control group. In liver, the expression of LPL of 8.0 g kg-1 A-OKGM group was significantly higher (P<0.05) than the control group, however, there was no significant (P>0.05) difference between each groups in muscle. The difference of FAS mRNA expression in the liver and muscle was not significant among seven groups (P>0.05).Therefore, we can draw the following conclusions:1. OKGM was degraded by hydrochloric acid, and its backbone structure did not change. Moreover, the acidolysis products of OKGM showed lower intrinsic viscosity and molecular weight, so the method is feasible.2. OKGM supplementation could improve gut morphology, and influence the intestinal microbiota in terms of microbial abundance and species richness of Schizothorax prenanti. The optimum dose of OKGM in the basal diets is 16.0 g kg-13. OKGM or A-OKGM diets could improve the growth performance, enhance the immune function and antioxidant activity of Schizothorax prenanti, and improve the survival rate of fish injected Aeromonas hydrophila, and the 8.0 g kg-1 A-OKGM diet showed the best effect.4. OKGM or A-OKGM diets induced the expression of IL-1β, TNF-α and TLR22 gene expression of Schizothorax prenanti, and the 8.0 g kg-1 and 16.0 g kg-1 A-OKGM group showed stronger effect.5. OKGM and A-OKGM diets up-regulated the expression of IL-1β, TNF-α and TLR22 of fish after injected Aeromonas hydrophila. However, the expression of each gene was different in different tissues.8.0 g kg-1 A-OKGM diet enhanced the serum lysozyme activity and improved the content of serum complement C3.6. The fat content of Schizothorax prenanti was affected by OKGM and A-OKGM diets. In liver, the expression of LPL of fish fed 8.0 g kg-1 A-OKGM diet was significantly higher than that of fish fed the control diet, and the difference of FAS mRNA expression in the liver and muscle was not significant among seven groups. |
PDF Full Text Request