| The scale of poultry farming in China is expanding,and the proposed and implementation of concepts such as welfare farming and green farming need theoretical and technical support.The small intestine is the most important digestive and absorption organ of broiler chickens,which is closely related to the nutrient metabolism of the body.Lipid uptake,transport and intracellular metabolism are important parts of the body’s nutritional and energy balance,and are also key links in the emergence of certain metabolic diseases in the body.Bacillus subtilis,as an excellent probiotic,can not only improve intestinal health,increase feed conversion rate and promote growth in production,but also plays an important role in improving lipid metabolism of the body.However,there are few relevant studies on the role and mechanism of Bacillus subtilis in the small intestinal lipid metabolism of broiler chickens at home and abroad.In particular,there is a lack of relevant studies on the effect of Bacillus subtilis on the level of DNA methylation in the small intestine.In this experiment,we investigated the effects of Bacillus subtilis on the uptake,intracellular transport and intracellular metabolism of lipids in various segments of the small intestine of broiler chickens at 40 d by adding different doses of Bacillus subtilis to the broiler feed,and explored the mechanisms involved in the regulation of DNA methylation level and the regulation of lipid metabolism by Bacillus subtilis.The broilers were randomly divided into 3 groups of 2000 birds each,and divided into Control(C)group,Low-Dose(L)group(1‰)and High-Dose(H)group(1.5‰)according to the dose of Bacillus subtilis(Viable count≥1.0×10~9CFU/g).Each group had 8 replicates.At40 days of age,one broiler was randomly selected from each replicate,and eight birds were collected from each group.DNA immunoprecipitation(Me DIP)to detect the level of DNA methylation Pparαand Cp G island methylation modification in the promoter region of Rxrα,a gene regulating lipid metabolism in jejunum.The results were as follows:1.Broiler intestinal index,villi morphology and blood lipid index:duodenal weight,jejunal weight,ileal weight and total small intestinal weight were highly significantly higher in groups L and H than in group C(P<0.01).duodenal index and small intestinal index were both highly significantly higher in groups L and H than in group C(P<0.01);ileal index was significantly higher in groups L and H(P<0.05).duodenal villi length was significantly higher in groups L and H(P<0.05);duodenal crypt depth was highly significantly increased in group L(P<0.01).jejunal crypt depth was highly significantly increased in group L(P<0.01);jejunal villus length/crypt depth ratio was highly significantly decreased in group L(P<0.01).ileal crypt depth was highly significantly increased in both group L and group H(P<0.01);ileal villus length/crypt depth ratio was highly significantly The triglyceride(TG)concentration in the serum of broiler chickens was significantly increased in group L(P<0.05),and TG was highly significantly increased in group H(P<0.01);lowdensitycholesterol(LDL-c)was highly significantly decreased in both group L and group H(P<0.01).2.Proteomic analysis of broiler small intestine and m RNA expression of lipid metabolism-related genes:the top 20 pathways in the jejunum and ileum differentially expressed genes KEGG enrichment analysis scores share lipid metabolism-related pathways:fatty acid degradation,and PPAR signaling pathway.Lipid metabolism-related gene m RNA expression.Fatty acid and cholesterol uptake related genes:Cd36 was highly significantly reduced in duodenum,jejunum and ileum of broilers in groups L and H(P<0.01),and Npc1l1was highly significantly reduced in duodenum and jejunum of broilers in groups L and H(P<0.01).Lipid intracellular transport-related genes:Slc27a4(Fabp4)was significantly reduced in the duodenum,jejunum and ileum of broilers in groups L and H(P<0.05);Fabp2 was highly significantly reduced in the duodenum,jejunum and ileum of broilers in groups L and H(P<0.01).Triglyceride synthesis-related genes:Dgat2 was significantly lower(P<0.05)in the duodenum and Mogat2 in the ileum(P<0.05)in groups L and H and broilers compared with group C.Lipoprotein assembly-related genes:duodenal and jejunal Acat2 and Apoa1were significantly lower(P<0.05),duodenal Mttp pole was significantly lower(P<0.01)and ileal Apoa4 was significantly higher(P<0.01)in broiler groups L and H.Lipolysis-related genes:duodenal and ileal Plin1 were highly significantly reduced in broilers in groups L and H(P<0.01),and jejunal and ileal Lpl were highly significantly reduced(P<0.01).Fatty acid metabolism-related genes:Fasn was significantly elevated in the jejunum and ileum of broilers in groups L and H(P<0.05);Fatp2,Hadh,Gcdh,and Ehhadh were extremely significantly reduced in the jejunum of broilers in groups L and H(P<0.01).3.The m RNA and protein expression of genes related to the regulation of lipid metabolism:the m RNA of duodenum and jejunum Pparαwas highly significant lower in broiler groups L and H(P<0.01),and Rxrαwas significantly lower in broiler groups L and H(P<0.05);however,there was no significant difference in protein expression;the m RNA and protein expression of jejunum Fxr were significantly higher in group L(P<0.05).The jejunum Dnmt3a was highly significant higher in group L(P<0.01)and Dnmt3b was significantly higher in group H(P<0.05).Me DIP results showed that the methylation level of Cp G islands in the Pparαpromoter region of jejunum was highly significant(P<0.01)in the L and H groups,and the methylation level of Cp G islands in the Rxrαpromoter region of jejunum was highly significant(P<0.01)in the H group.The above results showed that Bacillus subtilis increased small intestinal villus length and crypt depth,promoted intestinal digestion and absorption ability,reduced serum LDL-c concentration,and improved body fat metabolism of broiler chickens;promoted jejunal methyltransferase Dnmt3a and Dnmt3b expression,upregulated Cp G island methylation level in the promoter region of Pparαand Rxrα,inhibited the transcription of Pparαand Rxrα,which in turn acting on the PPAR signaling pathway,downregulated the expression of downstream lipid metabolism-related genes and reduce the overall intestinal lipid metabolism.Elevated jejunal FXR expression suggests that FXR is also involved in the regulation of small intestinal lipid metabolism... |
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