| Ageratina adenophora is a toxic invasive exotic plant that can cause multi-organ damage when ingested by animals,with its toxic effects mainly in the liver,spleen and kidney.Studies on the effects of A.adenophora on the intestinal tract of animals have focused on the intestinal structure and immune barrier,but studies on intestinal flora and intestinal metabolites are still lacking.In order to investigate the effects of A.adenophora on physiological indexes,intestinal flora and intestinal metabolites,in this study,12 5-6-month old,weight 20 kg-25 kg male Chengdu Hemp Sheep were randomly divided into control and experimental groups(fed 0% and 40% A.adenophora grass powder diets,respectively),serum and cecum contents were collected after 90 days of feeding to detect the changes of physiological indexes and serum liver function indexes.The changes of intestinal flora diversity,abundance and structure were analyzed by 16 sr DNA highthroughput sequencing,and the changes of intestinal metabolites were detected and analyzed by non-targeted metabolomics(LC-MS/MS).The main results of this study were as follows:1.The effect of feeding goats 40% A.adenophora grass powder diet on the physiological indexes of Chengdu Hemp sheep.The sheep showed clinical symptoms of poisoning such as depression,unstable standing,and lack of light in the coat at the late stage of the experiment.The body temperature of Chengdu Hemp sheep in the test group started to drop at 15 d and was significantly lower than that of the control group at 90 d(P<0.01).The body weight of the test group started to decrease continuously at 30 d and showed significant difference when compare to control group,control group(P<0.05),and was significantly lower than that of the control group at 60 d,75 d and 90 d(P<0.01).The levels of AST、ALP、 ALT、 GGT and TC in the serum were significantly increased by A.adenophora at the end of the experiment.The results indicated that the feeding of 40%A.adenophora can intoxicate Chengdu Hemp sheep and lead to stunted growth and liver damage.2.Based on 16 sr DNA high-throughput sequencing,we investigated the changes of cecum flora of Chengdu Hemp sheep intoxicated by A.adenophora.The results of Alpha diversity showed that there were significant differences in the diversity and homogeneity of the intestinal flora between the test and control groups.Abundance of Firmicutes were significantly increased,abundance of Bacteroides and Proteobacteria were significantly lower.A total of 64 different genera were identified at the genus level.9 biomarkers were indicated by LEf Se analysis.3.LC-MS/MS non-targeted metabolomics analysis was applied to analyze effect of A.adenophora on intestinal metabolites in Chengdu sheep.The results of multivariate statistical analysis showed significant group differences in metabolites in the intestine of test group compared to the control group.A total of 495 significantly different metabolites screened by t-test,178 metabolites were up-regulated and 317 metabolites were downregulated.KEGG enriched 4 significant different metabolic pathways(P<0.05): steroid hormone biosynthesis,folate biosynthesis,iron death,ovarian steroidogenesis;and bile acid metabolism which was enriched to a higher number of metabolites.Pearson correlation analysis of significantly different genera and significantly different metabolites revealed that Ruminococcaceae was positively correlated with β-estradiol(P<0.05),hydrocortisone was positively correlated with Parabacteroides(P<0.05),and Prevotella was positively correlated with dihydrofolate(P<0.05)and negatively correlated with folic acid(P<0.05).Eubacterium＿nodatum＿group,Christensenellaceae＿R-7＿group,Eubacterium＿siraeum＿group,Family＿XIII＿UCG-001 and some other clostridia were negatively correlated with taurocholic acid(P<0.05).Lithocholic acid was positively correlated with Eubacterium＿nodatum＿group,Christensenellaceae＿R-7＿group,UCG-007,and Negativibacillus.Conclusion:Liver damage was evident in Chengdu sheep fed with A.adenophora for 90 d.16 sr DNA sequencing showed that A.adenophora reduced the diversity of intestinal flora in Chengdu sheep and showed significant differences in flora abundance at the phylum and genus levels between test group and the control group.Non-targeted metabolomics identified 495 significantly different metabolites and KEGG enriched in four significantly different metabolic pathways.Correlation analysis revealed that the cecum flora was associated with cecum metabolites. |
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