Effect Of Bacillus Subtilis On The Enzyme Activity,Tissue Structure,Intestinal Microflora And Immune Gene Expressions Of Juvenile Paralichthys Olivaceus

In this experiment,Bacillus subtilis were added to the formula feed of juvenile Paralichthys olivaceus by direct spraying.Continuous and discontinuous feeding methods were adopted to study the effects of Bacillus subtilis on the enzymes activity,histological structure,immune-related genes expressions and intestinal microflora in juvenile Paralichthys olivaceus to provide references for efficient and rational use of Bacillus subtilis.The main results were as follows:1.Effects of Bacillus subtilis on liver enzyme activity of juvenile Paralichthys olivaceus.Bacillus subtilis could improve the enzymes activity in the liver.Both continuous feeding method E1 and discontinuous feeding method E2 increased the activities of AKP,LZM,ALT,SOD,GSH and LPS in the liver of juvenile fish,but failed to improve the activities of AMS and MDA;E2 was better than E1 in the enhancement of LZM,SOD and GSH activities;E1was better than E2 in the enhancement of LPS,AKP and ALT activities.2.Effects of Bacillus subtilis on histological structure of stomach,intestine and liver of juvenile Paralichthys olivaceus.The liver structure was normal,and no significant differences were observed in the liver structure among groups.In the experimental group,the intestinal mucosal layer was thicker and intestinal villi was more richer and longer with more mucous cells than the control group;the gastric folds in the experimental group was richer,especially in the E2 group;the type II,III and IV mucous cells were observed in the gastric mucosa,and only type III mucous cell was observed in the intestinal mucosa;After feeding with Bacillus subtilis,the number of mucous cells in intestinal mucosa and gastric mucosa increased and reached the peak at 15 days after feeding,and there was no significant difference between the E1 and E2 groups;the number of type II and type IV mucous cells in the gastric mucosa increased,showing the mucus composition has changed from neutral mucopolysaccharide to acid mucopolysaccharide.3.Effects of Bacillus subtilis on immune gene expression in spleen of juvenile Paralichthys olivaceus.Bacillus subtilis could activate the up-regulated expression of immune-related genes in the spleen of flounder,and the SOD,LZM and IL-1 genes up-regulated expressions were obvious;the E2 was better at up-regulating the SOD and IL-1 genes in the spleen of flounder than that of E1;No significant difference was observed in the up-regulation of LZM gene between E1 and E2.4.Effects of Bacillus subtilis on intestinal microflora structure of juvenile Paralichthys olivaceus.The average OTU numbers of E0,E1 and E2 in the intestinal flora were 657,1120 and988,respectively.The shannon,simpson,Chao1 and Ace indices in the intestinal flora of E1 were the largest,therefore the flora diversity was the highest with evenly species distribution.The relative abundance of Firmicutes in the E1 and E2 increased,reaching 14.20% and 19.68%,respectively,compared with 7.24% in the control group.The relative abundance of Bacillus in the E1 and E2 groups increased significantly,reaching 1.09% and 1.06%,respectively,while the control group was only 0.07%,indicating that the Bacillus subtilis has successfully colonized the intestinal tract,and no significant difference in colonization rates was seen between E1 and E2.There were 15 figures,14 tables and 121 references in this thesis.