| The purpose of this study was to investigate the effect of Lactobacillus johnsonii BS15 supplementation on lipid metabolism and intestinal microflora in broiler chickens with subclinical necrotizing enteritis.A total of 180 healthy male 1-day-old Cobb 500chickens were randomly divided into 3 groups,respectively as blank control group(NC group),modeling group(SNE group)and BS15 prevention group(BS15 group).Broilers were raised for 28 days.In the BS15 group,the whole bacteria solution of BS15 was added with live bacteria of 1.0 x 10~9 cfu/m L during the experiment.At 15 days of age,chicks were gavaged with 20,000 Eimeria acervulina oocysts and 5000 Eimeria maxima oocysts per chick except for the NC group.Unchallenged control chicks received the same volume of sterile phosphate buffered saline(PBS)instead of Eimeria oocysts.All Eimeria oocysts gavaged chicks were tolerated orally with 1 m L of C.perfringens(2.2×10~8 cfu/ml)on days 18-22.Meanwhile,unchallenged chicks accepted 1 ml of sterile fluid thioglycollate broth.On the 28th day,broiler samples were taken for detection.The test results were as follows:1.Compared with the NC group,the average daily weight gain of the SNE group was significantly lower(P<0.05),and the feed weight ratio was significantly higher(P<0.05).Compared with the SNE group,the average daily weight gain of broilers in the BS15 group was significantly increased(P<0.05).2.Serum biochemical tests showed that,compared with the NC group,serum concentrations of alanine transaminase and aspartate transaminase in broilers in the SNE group were significantly higher(P<0.05),serum total cholesterol was significantly higher(P<0.05),and serum high-density lipoprotein cholesterol was significantly lower(P<0.05).Compared with the SNE group,the serum concentration of alanine aminotransferase in broilers in the BS15 group was significantly decreased(P<0.05),and the serum total cholesterol was significantly decreased(P<0.05).3.Compared with the NC group,the abdominal fat rate of broilers in the SNE group was significantly higher(P<0.05),while that in the BS15 group was significantly lower(P<0.05).Real-time fluorescent quantitative PCR(q RT-PCR)technology to detect chicken belly fat tissue lipid metabolism related enzyme gene expression is found,SNE group of peroxidase in chicken belly fat body growth activated receptorγ(PPARγ)and triglyceride lipase(ATGL)gene expression quantity compared with NC group significantly(P<0.05),and abdominal fat of lipoprotein lipase(LPL)gene expression quantity compared with NC group significantly(P<0.05).Compared with the SNE group,the expressions of PPARγand ATGL genes in the abdominal fat of broiler chickens in the BS15 group were significantly increased(P<0.05).4.High throughput transcriptome sequencing(RNA-seq)was used to detect the expression of lipid metabolism related genes in the liver of broiler chickens in each group.It was found that a total of 407846912 clean reads were obtained after filtration in this study.The average number of clean reads in each sample was 45316324.Compared with the NC group,385 genes were significantly expressed in the liver of broilers in the SNE group(P<0.05),among which 268 genes were up-regulated and 117 genes were down-regulated.Through the GO analysis of differentially expressed genes,it was found that the number of pathway entries enriched by differentially expressed genes was 658,among which the biological processes involved in the regulation of lipid metabolism included lipid transport,cholesterol biosynthesis,steroid metabolism,phospholipid synthesis and triglyceride decomposition.Through KEGG analysis of differentially expressed genes,a total of 61 signaling pathways were enriched,including 12 pathways related to lipid metabolism,including steroid biosynthesis,insulin signaling pathway,adipocytokine signaling pathway,and PPAR signaling pathway.Compared with the SNE group,126 genes were significantly expressed in the liver of broiler chickens in the BS15group(P<0.05),including 60 up-regulated genes and 66 down-regulated genes.GO analysis and KEGG analysis of differentially expressed genes detected and enriched 442pathways and 56 pathways respectively,and 11 pathways related to lipid metabolism were found,including arachidonic acid metabolism,PPAR signaling pathway,glycerophospholipid metabolism,adipocytokine signaling pathway,and glyceride metabolism.5.Intestinal development and digestive tests showed that,compared with the NC group,the protein levels of insulin-like growth factor-1(IGF-1)and epidermal growth factor(EGF)in the jejunum of broilers in the SNE group were significantly decreased(P<0.05),the ileum of IGF-1 protein level significantly decreased(P<0.05),while compared with the SNE group,the protein levels of IGF-1 and EGF in the jejunum and ileum of broilers in the BS15 group were significantly increased(P<0.05).Compared with the NC group,the jejunal amylase and trypsin activities in the SNE group were significantly decreased(P<0.05),and the ileal lipase activities were significantly decreased(P<0.05).Compared with the SNE group,the jejunal amylase and trypsin activity in the BS15 group increased significantly(P<0.05),and the ileal lipase activity increased significantly(P<0.05).6.Using qRT-PCR technique to detect bacteria in the jejunal and ileal of chickens found that the abundance of Streptococcus spp.in jejunum and ileum,and Clostridium cluster I in jejunum,as well as Enterobacteriaceae in ileum were remarkably enhanced(P<0.05).The content of Bacteroidetes,Lactobacillus spp.and Clostridium cluster XIVa in jejunum and ileum,and Clostridium cluster IV in ileum were significantly reduced(P<0.05)upon treatment with SNE infection as compared with the normal chickens.In BS15treatment,the content of Lactobacillus spp.and Clostridium cluster XIVa in jejunum,and Bacteroidetes,Lactobacillus spp.and Clostridium cluster IV in ileum remained at higher level,whereas that of Streptococcus spp.in jejunum and ileum were markedly lower than those in the SNE group(P<0.05).In conclusion,the data presented here highlights the preventative mechanism of Lactobacillus johnsonii BS15 that may contribute to lipid metabolism,mainly by regulation of the PPAR signaling pathway and the arachidonic acid metabolism.And the mechanistic basis of Lactobacillus johnsonii BS15 that may promote the growth performance and lower fat deposit in subclinical necrotic enteritis broilers,which may contribute to the improved lipid metabolism in liver and adipose tissue as well as better intestinal development,enhanced digestive ability and balanced microflora in small intestines. |
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