| Biofloc technology(BFT)is a culture technology with the advantages of maintaining aquaculture water quality,reducing ammonia nitrogen and nitrite nitrogen,improving feed utilization rate,digestion and metabolism,immunity and survival rate of farmed animals.In this study,biofloc technology was combined with the culture of Hyriopsis cumingii to explore whether H.cumingii can feed on bioflocs,the changes of digestion,metabolism,immune index and intestinal flora of H.cumingii after feeding,as well as the changes of water quality of cultured water.The results show that:1.Construction and characteristic analysis of biofloc system in this study,the biological flocculation system was constructed by ectopic culture,and Bacillus subtilis containing fluorescent plasmid(GFP-pMD,Kan+)was used as flocculant.Biofloc were cultured by mixing aerated tap water and pond water at 1:1 and aerated for 24 h.The growth status of flocs was observed at 17:00 every day and the culture period was 21 days.The morphology and growth of biofloc were observed by light microscope on day 7,14,21 of culture.The results showed that the flocs matured gradually in the process of culture,and the concentration of flocs in the reactor reached 200 mg/L at day 21,and the particle size of flovs reached 100 μm.The microbial structure analysis of mature biofloc showed that Proteobacteria,Verrucomicrobia,Bacteroidetes and Firmicutes were the dominant populations at the phylum level,while Rhodobacterales,Spartobacteria,Bacilli and Xanthomonadales were the dominant populations at the phylum level.At the genus level,Paracoccus,Gemmobacter,Bacillus,Spartobacteria_genera_incertae_sedis and Stenotrophomonas are the dominant populations.Therefore,in this study,the content of probiotics in biofloc cultured by B.subtilis is higher than that of pathogens,which lays a foundation for the follow-up culture experiment of H.cumingii.2.Effects of biofloc on physiological and biochemical indexes of H.cumingii in this study,Chlorella vulgaris was used as the basic feed for H.cumingii culture experiment.The final concentration of Chlorella was 4.25×105 cells/L water.The experiment set up 1 control group and 3 treatment groups,adding biofloc of 0%(control group C),25%(T1 group),50%(T2 group)and 100%(T3 group)of Chlorella mass respectively,3 parallel in each group,and the culture period was 28 d.After 1,7,14,21 and 28 days of culture,the hepatopancreas,intestinal tract,Gill and blood of H.cumingii were taken to determine the activities of digestive enzymes,metabolic enzymes and immune enzymes.The main results are as follows:(1)the effect of biofloc on digestive enzymes of H.cumingii.In the liver,the amylase activity in T3 group was significantly higher than that in T2 and C groups on the 14th day(P>0.05),which was 1.47 times higher than that in C group,and reached the maximum on the 28th day,which was significantly higher than that in the control group(P>0.05).On the 7th day,amylase activity in group T2 was significantly higher than that in group C on the 7th day,and amylase activity in group T3 was also significantly higher than that in group C on the 28th day,and reached 8.62mg/min/mgprot,which was the highest in all groups.The lipase activity in liver and intestine in treatment group was significantly higher than that in group C at each monitoring time point(P>0.05),but there was no significant difference between treatment groups(P≤0.05).The cellulase activity in liver and intestine increased gradually with the increase of culture time.The cellulase activity of T2 group was significantly higher than that of other groups on the 7th day.On the 28th day,the cellulase activity of T3 group reached 1.06U/mg,which was 3.25 times higher than that of C group.The results showed that feeding biofloc could significantly improve the digestibility of H.cumingii,and the increase of digestive enzyme activity was positively correlated with the addition of biofloc.(2)the effect of biofloc on metabolic enzymes of H.cumingii.Compared with the control group,the activity of glutamic oxaloacetic transaminase in hepatopancreas and Gill in the treatment group decreased.On the 28th day,the activity of aspartate aminotransferase in hepatopancreas in group T3 was significantly lower than that in group C(P>0.05),but there was no significant difference between treatment groups(P≤0.05).The Gill was the lowest on the 28th day,and it was significantly higher in T3 group than that in T1,T2 and C groups(P>0.05).In the liver,the glutamic pyruvic transaminase activity in group T2 reached the lowest on the 28th day,and the activity was only 64%of that in group C,which was significantly lower than that in group T1(P>0.05).On the 28th day,the Gill glutamic pyruvic transaminase activity in T2 and T3 groups was significantly lower than that in T1 and C groups(P>0.05).The results showed that biofloc could effectively reduce the activity of transaminase in hepatopancreas and Gills and protect the organs of H.cumingii,and moderate addition(T2 group)could play a better protective effect.In terms of glucose metabolism,the activities of hexokinase and pyruvate kinase in the liver and Gill of H.cumingii were significantly higher than those in the control group with the extension of culture time,and showed better effect in the low dose group.The results showed that biofloc could improve the ability of sugar metabolism and feed utilization of H.cumingii.(3)the effect of biofloc on the immunity of H.cumingii.After adding biofloc to the diet,the activities of alkaline phosphatase,acid phosphatase and lysozyme in serum of each treatment group showed an upward trend,which was significantly higher than that of the control group on the 7th day(P>0.05),and reached the maximum on the 28th day.However,there was no significant difference among the treatment groups,and it showed a strong immune enhancement effect at low dose.The results showed that biofloc could effectively improve the non-specific immunity of H.cumingii.3.Effects of biofloc on intestinal health and nitrogen elements in water of H.cumingii.On day 28,the gills,hepatopancreas and intestines of H.cumingii in group C and T3 were taken for histological observation,and another mesentery was taken for microbial classification and sequencing.Water samples were taken and preserved before each change of water during the culture period,which were used to determine the contents of ammonia nitrogen,nitrate nitrogen and nitrite nitrogen in the water.Tissue section observation showed that,compared with the control group,the gill filaments of the T3 control group were neatly arranged,and the gap was only 56%of that of the control group;the thickness of epithelial tissue was 1.7 times that of the control group,and the number of nuclei was significantly higher than that of the control group(P>0.05).The digestive lumen of hepatopancreas was significantly reduced in T3 group(P>0.05).The length of intestinal villi in T3 group was significantly higher than that in control group(P>0.05),and the number of nuclei in the same region of intestinal epithelial cells in T3 group was 1.8 times higher than that in control group.The results showed that the addition of biofloc to the diet could improve the structure of gill filament and intestine,and improve the feeding metabolism and intestinal digestion of H.cumingii.The results of microbial classification and sequencing of intestinal flora showed that Bacillus subtilis was colonized in the intestinal tract after feeding biofloc.At the gate level,Proteus and Bacteroides were still the most dominant bacteria in group C and T3.In group T3,Proteus decreased from 90.88%to 60.64%,and Bacteroides increased from 7.11%to 17.14%.At the genus level,the proportion of Bacillus increased from 0.11%to 2.75%.The potential probiotic oligotrophomonas in T3 group increased from 6.36%to 9.07%.The proportion of total pathogenic bacteria decreased from 72.41%to 49.85%in T3 group.The correlation between bioflocus flora and the composition of intestinal flora in T3 group was significantly higher than that in the control group,and the proportion of potential probiotics with high relative abundance increased in T3 group.The results showed that the addition of biofloc could improve the intestinal microbial composition of H.cumingii to some extent,so as to improve the digestion,metabolism and immunity of H.cumingii.During the experiment,the concentration of three-state nitrogen in the culture water of each treatment group was significantly lower than that of the control group.The concentration of ammonia nitrogen in each group reached the peak on the 4th day,and then decreased,and the concentration of ammonia nitrogen in the treatment group was significantly lower than that in the control group.The concentration of nitrite nitrogen increased greatly in the early stage and decreased slowly in the later stage.The concentration of nitrite nitrogen in the control group was the highest and accumulated obviously,while that in T2 and T3 groups was the lowest.After 18 days,the concentration of nitrite nitrogen in each treatment group was significantly lower than that in the control group.The concentration of nitrate nitrogen in culture water also increased at first and then decreased,but the concentration of nitrate nitrogen in each treatment group was significantly lower than that in the control group.The results showed that biofloc could regulate the nitrogen elements in culture water,and the concentrations of three-state nitrogen in the treatment group were significantly lower than those in the control group,indicating that biofloc could improve the absorption and utilization of nitrogen elements in culture water.To sum up,the addition of biofloc to the diet can improve the digestion,metabolism and immunity of H.cumingii,and B.subtilis cultured with biofloc can be colonized in the intestinal tract of H.cumingii,and can also improve the intestinal flora structure of H.cumingii.Biofloc can also improve the water environment of H.cumingii culture,which provides a new idea for healthy culture of H.cumingii. |
PDF Full Text Request