The Effect Of Dietary Fiber/Starch On Growing Rabbits’ Growth, Immune Response, Intestinal Bacterial Community Composition And Intestinal Proteomics Study

Enteric diseases frequently occur in rabbit restricted the farming development. Due to the extensive use of antibiotics in breeding can lead to bacterial resistance and food safety issues, and consumers as well as breeders require alternative breeding strategies including diet that improve the health of animals without using in-feed antibiotics.Two hundred weaner rabbits were randomly assigned to4groups(Ⅰ、Ⅱ、 Ⅲ、Ⅳ) with50replicates in each group, in which they were fed each diet with NDF/starch2.3,1.9,1.4,1.0, respectively. Around the trial rabbit production performance, cecal fermentation, gastrointestinal tract development, intestinal membrane shape, non-specific immunity, intestinal microbial diversity and abundance etc, which were determined to evaluate the effect of dietary fiber/starch on rabbit body environment. Proteomics was to explore intestinal protein expression differences between the health and diarrhea group trial rabbits. The results are summarized as follows:1. The effect of differnt NDF/starch on rabbit production, cecal fermentation, gastrointestinal developmentTen82-day old trial rabbit per treatment group were slaughtered to determined the relevant datas. Dietary NDF/starch had significantly influences on average daily gain (ADG) and feed/gain (F/G)(P<0.05),ADG and F/G deteriorated with decreasing NDF and increasing starch, F/R was the highest(4.51:1) in Diet I (P<0.05); Diarrhea index were affected by dietary NDF/starch (P<0.01), the diarrhea index was the lowest (0.026-0.03) in Diet Ⅰ and Ⅱ and largest in Group Ⅳ(3.27). The stomach weight (g/kg SW), small intestine weight (g/kg SIW) was influenced significantly among all treatment groups (P<0.05), The stomach weight (SW/LW, g/kg), small intestine weight (SIW/LW,g/kg) in Diet Ⅱ all were largest compared to Diet Ⅲ>Ⅳ treatment groups(P<0.05), and no difference with Diet Ⅰ. The NH3-N concentration increased with the descended of dietary NDF/starch, but every treatment had no significant influence (P>0.05); SIW/LW and SW was significant difference among four dietary treatments (P<0.05), while SIL SIL/LW, CW, CW/LW was not significant. Acetic acid was affected significantly by dietary NDF. Dietary NDF/starch had significant influenced illus height of the duodenum (p<0.05) and was the largest in Group Ⅱ (851μm). In conclusion, the appropriate fiber/starch in the diet of weaner to82-day-old growing rabbit is1.9.2. The effect of different NDF/starch on rabbits’ Non-specific immuneUsing ELISA, flow cytometry, immunohistochemistry method, the following parameters were assessed:the number of membranous epithelial (M) cells in the appendix, total SIgA titers in the gut, total IgG levels in the serum and the CD4+/CD8+T cell ratio in the peripheral blood; as these measures were taken to determine the effect of diet with age, samples were obtained at52,62,72and82days of age. Monitoring the trail rabbits’non-specific immune changes at52,62,72,82days of age, respectively. The effect of Diet×age on trail rabbits’M cells number had significantly different (P<0.001). The number of M cells was the highest in Diet I at52and62days of age (P<0.00\), while was the lowest in Diet Ⅳ at52days (P<0.001). However, at72and82days of age, there was no difference (P>0.05) in M cell number among the four diets treatments. Using immunohistochemistry, the high fiber/low starch diet (Diet I) resulted in an increase in M cell size and number when compared with the other diets. Diet×age effected on SIgA levels in the ileum and duodenum at52and62days (P<0.05), which was no difference at72-82days of age. The SIgA level change of duodenal mainly concentrated in the72days of age and the diet IV group had the lowest (P<0.05). Serum IgG titers were only affected (P=0.05) at82days of age. The ratio of CD4+/CD8+T cells in the peripheral blood was not affected. The study revealed that increased levels of fiber might improve mucosal functionality, but only during the early stage of growth of rabbits. The effect of dietary fiber/starch levels on intestinal immunity was greater than their effect on humoral immunity in the peripheral blood. Combined with clinical diarrhea index,dietary NDF/starch1.9rabbits can maintain intestinal health.3. The effect of different NDF/starch on trail rabbit intestinal flora abundance and diversityIn this work, we used a pyrosequencing-based analysis of16S rRNA genes to compare the cecal microbiota of rabbits fed diets containing different dietary fiber/starch ratio to determine the how the overall microbiome structure varies according to age (from52to82days of age) and diet. The different categories of bacterial phyla and species relative abundance in rabbits’cecum can be changed in early period through regulating NDF/starch. Research results show that high-fiber diets (Diet I) increased the abundance of Bacteroidetes phyla and decreased the abundance of Firmicutes during early peirod, While the abuandance of Firmicutes phyla bacteria from high-starch diet (diet IV) group was relatively stable in trial period. In the species level, including high-fiber diet (Diet Ⅰ,Ⅱ) were found Escherichia and Bifidobacteria in rabbits’ cecum,While found Desulfovibrionales for pathogenic bacteria from Diet Ⅰ, Ⅲ group.the cellulose-decomposing bacteria belonging to the genus Ruminococcus which are not affected by the composition of the diet. A weighted UniFrac analysis revealed that abundance of the gut microbiome was similar at72and82days old. A hierarchical clustering heatmap analysis based on the bacterial community profiles at the family level revealed that the major samples at72and82days of age grouped together. By Venn diagram, all samples have more OTUs in common at72and82days of age. DGGE profiles cluster analysis results show that the individual flora difference among trial rabbit was found. However, flora from all diets treatments gradually gathered a branch with aging. Conclusions:Our data suggest that the bacterial community in the rabbit cecum is able to change and adapt gradually to reach a new equilibrium in response to a nutritional disturbance. More reasonable distribution of intestinal flora was established when the NDF/starch1.9. 4. Intestinal proteomics research between Non-pathogenic diarrhea rabbit and health rabbitUnder the application of two-dimensional gel electrophoresis and mass ctrometry experiments,we finally obtained organization different proteins of14shared proteins of health, including1up-regulated protein and13proteins down in health rabbits. With the application of two-dimensional LC-MS analysis, we could obtain55specific protein of diarrhea group. In order to confirm the accuracy of our experiment, we selected two proteins (GRP94, a-Smooth muscle actin) among health and diarrhea rabbits’ tissues by Dimensional gel electrophoresis and mass spectrometry experiments respectively and verified the accuracy of two-dimensional gel electrophoresis by immunity signature experiment. We verified the accuracy of Two-dimensional LC-MS experiment by immunity signature.