| After fed with broad bean for 120 days, common grass carp can become crisp grass carp that will gain appropriate hardness. At present crisp grass carp has become a characteristic industry in Zhongshan, Guangdong Province, China. The crisp grass carp market has gradually covered the entire country with Guangdong Province being the major crisp grass carp consuming market. However, insufficiently crisp grass carp and overly crisp grass carp often appear in crisp grass carp production, and this problem has puzzled many farmers. The physiological and biochemical changes during the transformation from grass carp to crisp grass carp have not been studied yet. Therefore, this article investigate the changes of microstructure and TPA value of muscle, intestinal microflora, serum enzyme, ultrastructure of head kidney and liver, immune gene(such as C-type lysozyme) expression of crisp grass carp in order to assess the physiological and biochemical changes during the transformation from grass carp to crisp grass carp. This study has an important scientific significance to the healthful aquaculture for crisp grass carp. The results were as follows:1. In order to ascertain the changes of muscle structure of crisp grass carp at the different crisping stages, microstructure and TPA value of muscle between crisp grass carp and grass carp were examined. The results showed that, on 120 d, diameter of muscle fiber of broad bean group decreased by 28.12% and density of muscle fiber of broad bean group increased by 99.14% compared with those of control group. On 120 d, hardness, spring, chewiness, cohesiveness and gumminess of muscle of broad bean group increased by 39.76% 、37.20% 、92.82% 、30.43% and 68.17% respectively compared with those of control group. The results demonstrated that, compared with those of grass carp, diameter of muscle fiber of crisp grass carp decreased gradually, density of muscle fiber of crisp grass carp increased gradually, hardness, spring, chewiness, cohesiveness and gumminess of muscle of crisp grass carp increased gradually.2. To obtain scientific data for the healthful aquaculture of crisp grass carp, intestinal microflora, serum enzyme and growth performance of the grass carp at different stages of feeding broad bean were examined to assess the physiological and biochemical changes during the transformation from grass carp to crisp grass carp. The results showed that, on 30 day there is no significant difference between the average weight in broad bean group and those in the control group, from 60 day to 150 day, the average weight of grass carp feeding broad bean group were lower than those of control group. On 30 and 60 day, the head kidney somatic index and hepatosomatic index of broad bean group were within normal levels compared with the control group, but from 90 day to 120 day, they were all lower than those of the control group, and on 150 day there is no significant difference between the hepatosomatic index in broad bean group and those in the control group. The activities of serum alkaline phosphatase, lysozyme and complement 3 of broad bean group were all lower than those of control group, but they were rising after taking artificial feed. Compared with the control group, the diversity of intestinal microflora of broad bean group decreased, the number of probiotics which belong to Cetobacterium, Bacillus and Eubacterium decreased and the number of opportunistic pathogens such as Aeromonas veronii, Aeromonas sobria and Vibrio anguillarum increased in broad bean group. Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria were the dominant intestinal microflora of broad bean group and control group. Our results demonstrated that, with the increase of crisping time, broad bean gradually changed the composition of intestinal microflora and maybe generate adverse effects to the intestine of grass carp. On the other hand, it inhibited the activity of serum enzyme and growth performance of grass carp, and these indexes of broad bean group were found to be a recovering trend after feeding artificial feed for 30 days.3. To investigate the changes of structure of immune organization of grass carp taking broad bean, this experiment observed the ultrastructural changes of head kidney, liver and intestine of crisp grass carp at different crisping stages. The ultrastructural observations showed that, vacuoles appeared in head kidney of broad bean group, damaged cells and necrotic areas observed in head kidney of broad bean group at later crisping stages. The hepatocytes of broad bean group became swelling gradually. In the broad bean group, mitochondria and endoplasmic reticulum became decreased and scattered disorderly in cytoplasm. Damages appeared in perisinusoidal space with the reduction of endothelial cell and microvilli on basal surface of hepatocytes. There were evident disorder and chaos in the shape of nucleus and structure of perisinusoidal space of broad bean group at later crisping stages. Electron microscopy of intestine epithelium showed an enteritis effect in broad bean group. Broad bean group displayed sparse and shorter microvilli. At later crisping stages, it displayed missing microvilli, exposure of tight junction and damaged enterocytes. The results demonstrated that, broad bean gradually changed the structure of of head kidney, liver and intestine of grass carp.With the increase of crisping time, more serious damage observed in the structure of immune organization.4. This experiment examined the changes of immune gene(C-type lysozyme, IL-1β, IFN-I, TNF-α, Ig M and MHC-I) expression in head kidney and liver of grass carp taking broad bean. The results showed that, the expression of C-type lysozyme, IFN-I, Ig M and MHC-I in head kidney of broad bean group were significantly decreased at the crisping stages. The expression of IFN-I, TNF-α, Ig M and MHC-I in liver of broad bean group were significantly decreased at the crisping stages. After feeding crisp grass carp with artificial diet for 30 d, the expression of aforementioned genes was found to be a recovering trend except the expression of C-type lysozyme in head kidney. The results demonstrated that, broad bean has an inhibitory effect on the expression of immune gene in head kidney and liver of grass carp. After feeding crisp grass carp with artificial diet for 30 d, the expression of inhibited genes was found to be a recovering trend.In conclusion, feeding crisp grass carp with artificial diet for 30 d didn’t affect the TPA value of muscle of crisp grass carp. After feeding broad bean, the diversity of intestinal microflora and the number of probiotics of grass carp decreased. On the other hand, feeding broad bean inhibited the activity of serum enzyme and the expression of immune gene, and damaged the ultrastructural changes of immune tissue. These indexes of broad bean group were found to be a recovering trend after feeding artificial feed for 30 days. This study has an important scientific significance to the healthful aquaculture for crisp grass carp. |
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