| High-grain diet is often formulated in production to meet the higher energy needs of high-yielding ruminants,but it can easily cause abnormal rumen metabolism and cause metabolic diseases such as subacute rumen acidosis（SARA）.SARA has a high incidence,no obvious symptoms and a long course of illness,which reduces feed utilization efficiency and production performance,causes premature elimination of animals.Under the same high-grain diet,the risk of SARA in dairy animals is different.However,there is a lack of systematic research on the occurrence of SARA and the regulatory mechanism of the difference in susceptibility.Rumen degradable starch（RDS）is one of the key factors to measure the fermentation characteristics and health of the rumen.Therefore,this study builds a SARA model of dairy goat by increasing the RDS content of the diet.Then combined with the dynamic monitoring of the rumen microbial flora and the transplantation of the rumen flora into the intestinal test of mice pretreated with mixed antibiotics,to clarify the regulatory role of rumen microorganisms in the occurrence of rumen inflammation caused by SARA.On this basis,to screen individuals with differences in SARA susceptibility by gradually increasing the RDS level,using rumen microbiota transplantation technology and multi-omics combined analysis technology and other means to reveal the occurrence of SARA and the host-microbial interaction mechanism of susceptibility differences.Experiment 1 Construction of SARA model of dairy goat and dynamic monitoring of environment and microbiota in rumenThis experiment studied the dynamic effects of high RDS diets on the fermentation parameters and microbiota structure of the rumen of dairy goats by increasing the RDS content of the diet.In the experiment,12 healthy dairy goats of similar weight and age（54±2.4 kg,3-4 years old）were selected,permanent rumen fistulas were installed,and randomly divided into two groups.They were fed a low RDS diet（Health group,forage to concentrate ratio=3:7）and a high RDS diet（SARA group,forage to concentrate ratio=7:3）.The trial period included a diet adaptation period of 18 d and a sample collection period of 7d.The results showed that:compared with the Health group,the rumen p H value of dairy goats in the SARA group was significantly reduced（P<0.05）,the rumen fermentation type and microbiota were significantly changed（P<0.05）,the rumen LPS concentration was significantly increased（P<0.05）,and the proportion of rumen acetate and acetate to propionate ratio was significantly decreased within 1-6 h after morning feeding（P<0.05）.In1 h and 6 h after morning feeding,the proportion of propionate was significantly increased（P<0.05）.Within 2-6 h after morning feeding,the proportion of butyrate was significantly increased（P<0.05）.The 16S r RNA gene sequencing results showed that the diversity and richness of the rumen microbiota in the SARA group decreased significantly within 1-6 h after morning feeding（P<0.05）,and there were significant differences in the structure of the microbiota between the two groups（P<0.05）.The SARA group showed the continuous increase of starch-degrading bacteria such as Rumenococcus and the continuous decrease of fiber-degrading bacteria such as Prevotella,which led to significant differences in the function of the rumen microbiota.DNF00809,Candidatus Saccharimonas,Lachnospiraceae NK3A20 group,Erysipelotrichaceae unclassified,Corynebacterium 1,Ruminococcus 2 and Clostridiales unclassified were significantly increased in the SARA group,which may be the main bacteria that cause SARA,and it was significantly negatively correlated with rumen p H and positively correlated with LPS concentration.This experiment shows that with the increase of RDS content in the diet,SARA occurs in dairy goats,which is manifested as a decrease in rumen p H,an increase in LPS concentration,a significant change in VFAs concentration and proportion,and the fiber-degrading bacteria showed a decreasing trend over time,and the starch-degrading bacteria increased significantly.Experiment 2 Effects of rumen microbial transplantation on intestinal flora and inflammatory response in miceExperiment 1 showed that the occurrence of SARA was accompanied by significant changes in the structure of the rumen microbiota.In this experiment,the rumen microbiota transplantation of two groups of dairy goats in experiment 1 was transplanted into the intestines of mice pretreated with mixed antibiotics to further explore the important regulatory role of the rumen microbiota in the occurrence of SARA.63 male KM mice weighing 18-20 g were selected,and the mice were randomly divided into 5 treatment groups,S group（N=6）,Health-S group（n=3）,Anti-S group（n=6）,Anti-Health-S group（n=24）and Anti-SARA-S group（n=24）.“S”means that mice are fed high-starch（5%cellulose）feed,“Anti”means that mice are irrigated with mixed antibiotics,and“Health”and“SARA”indicate that the mice are irrigated with rumen microorganisms from dairy goat donors in the experiment 1.The results showed that the bacterial richness of the small intestine and colon of mice after mixed antibiotics treatment decreased significantly（P<0.05）,and the structure of the microbiota changed significantly（P<0.05）,which shows that mixed antibiotics can deprive the original microorganisms in the intestines of mice to a certain extent.Compared with non-transplanted mice,theαdiversity of the small intestine and colon of mice after RMT increased significantly（P<0.05）,the structure of the microbiota also changed significantly（P<0.05）,and the microbiota structure and fermentation mode of the colon of mice after RMT are more similar to that of the donor.After transplanting the rumen flora of the SARA group in mice,the m RNA expression of IL-1βand IFN-γin colon tissue was significantly increased（P<0.05）,and the m RNA expression of Claudin-7 was significantly reduced（P<0.05）,indicating that colitis mediated by the rumen microorganisms of the SARA group occurred in mice.In view of the relieving effect of a high-fiber diet on SARA in dairy goats,we further study the effects of a high-fiber diet on colitis in mice.“F”means that mice are fed a high-fiber diet（50%cellulose）,and the rest of the abbreviations are the same as above.The results showed that compared with the Anti-F group,the gene expression of inflammatory factors in the Anti-SARA-F group did not change significantly（P>0.05）,and the gene expression associated with the intestinal barrier in the colon of mice was increased（P<0.05）.It has been shown that a high-fiber diet can reduce colitis in mice mediated by rumen microorganisms in SARA dairy goats and enhance the function of the colon barrier.In summary,this experiment successfully constructed a mouse model treated with mixed antibiotics,which verified the important regulatory role of rumen microorganisms in the occurrence of SARA,and the colitis of mice mediated by rumen microorganisms in SARA dairy goats can be alleviated to a certain extent by feeding a high-fiber diet.Experiment 3 The ruminal metabolic characteristics of the SARA susceptible dairy goats and the molecular mechanism of the inflammatory response of the rumen epithelium47 health dairy goats of similar weight and age were selected,permanent rumen fistulas were installed,and the RDS content of the diet was gradually increased by increasing the proportion of food concentrate first,and then using crushed corn instead of whole corn.Through continuous monitoring of the rumen p H,SARA susceptible and tolerant dairy goats were selected.After successful screening,they were divided into the control group of low-grain whole corn（LGW-CON）,the SARA susceptibility group of high-grain whole corn（HGW-SARA）,the SARA tolerance group of high-grain whole corn（HGW-Health）,the SARA susceptibility group of high-grain crushed corn（HGC-SARA）and the SARA tolerance group of high-grain crushed corn（HGC-Health）.Each group randomly selected 5dairy goats for slaughter sampling.In this experiment,the LGW-CON,HGW-SARA and HGC-SARA groups were selected to explore the mechanism of SARA occurrence and the cause of susceptibility in dairy goats.The results showed that compared with LGW-CON,the rumen p H of the two SARA susceptible groups was significantly reduced（P<0.05）,significantly increased the concentration of rumen NH3-N,acetate,propionate,isobutyrate and total acid（P<0.05）,the HGC-SARA group significantly increased the concentration of rumen butyrate and lactate（P<0.05）.The HGC-SARA group significantly increased the thickness of the rumen stratum corneum（P<0.05）.It shows that the environment in the rumen of SARA-susceptible individuals has changed significantly.Metagenomic results showed that there were significant differences in rumen microbiota between LGW-CON and the two SARA susceptible groups（P<0.05）.Compared with the LGW-CON group,the SARA susceptibility group significantly reduced the gene abundance of coding（half）cellulosic degradase in the rumen（P<0.05）,significantly increased the gene abundance of lysozyme（P<0.05）,reduces the fiber degradation capacity of the rumen.The differentially expressed genes（DEGs）of the rumen epithelium were significantly enriched to inflammation-related pathways（P<0.05）.Compared with the LGW-CON group,the gene which was enriched to inflammation-related pathways was significantly upregulated in HGW-SARA,and significantly downregulated in HGC-SARA（P<0.05）,which indicates that no inflammation of the rumen epithelium occurred in the HGC-SARA group.The single-cell nuclear transcriptome of the rumen epithelium showed that compared with the LGW-CON and HGC-SARA groups,Th17 cells and macrophages were significantly increased in the HGW-SARA group,and the genes that were significantly upregulated in the HGW-SARA group were significantly enriched into the Th17 cell differentiation pathway.In order to explore the reasons for the differences in Th17 cell proliferation and differentiation,further analysis of the microorganisms and metabolites of HGW-SARA and HGC-SARA found that Bifidobacteria,Mitsuokella and Selenomonas（LDA>2）were significantly enriched in HGC-SARA,and the different metabolites of the two groups were significantly enriched in the tryptophan metabolic pathway（P<0.05）.Changes in these key microorganisms and metabolites may affect the differential differentiation of Th17 cells in the rumen epithelium.This experiment showed that the environment in the rumen of SARA-susceptible individuals was disturbed,and the the fiber degradation capacity decreased,and the microbial flora and function changed.This experiment found that there was no inflammation of the rumen epithelium in the SARA state for the first time.The occurrence of inflammation of the rumen epithelium in SARA individuals may be related to the proliferation and differentiation of Th17 cells mediated by key bacteria and molecules such as Bifidobacteria and tryptophan metabolites,and its mechanism needs to be further clarified.Experiment 4 The ruminal metabolic characteristics of SARA-tolerant dairy goats and its molecular mechanism for maintaining the homeostasis of the environment in the rumenThe LGW-CON,HGW-SARA,HGW-Health and HGC-Health groups were selected in this experiment to explore the host-microbial interaction mechanism of the differences in SARA susceptibility of dairy goats fed on a high RDS diet.The results showed that the p H of the rumen in the HGW-SARA group was significantly lower than that of the other three groups（P<0.05）,and the concentration of VFAs and NH3-N in the rumen was significantly increased（P<0.05）,and there was no significant difference between tolerance groups and the control group.Metagenomic results showed that compared with the susceptible group,the structure of the rumen flora in the tolerant group was closer to that of the control group,and the abundance of genes encoding xylanase andβ-glucanase in the tolerant dairy goats was significantly increased.It shows that the environment and flora structure of the rumen in the tolerant group are similar to that of the healthy group fed with low RDS,and both have higher rumen p H and fiber degradation capabilities.The DEGs of the rumen epithelium were significantly enriched to the metabolic pathways associated with inflammation and the epithelial barrier（P<0.05）.Compared with the HGW-SARA group,the inflammatory pathway-related genes were significantly downregulated（P<0.05）,the epithelial barrier pathway-related genes were significantly upregulated in the tolerance group（P<0.05）.It shows that there is no inflammatory response in the rumen epithelium of the tolerant group and it has a complete epithelial barrier function.The single-cell nuclear transcriptome of the rumen epithelium showed that compared with the HGC-SARA group,the number of Th17cells in the HGW-Health and HGC-Health groups was significantly reduced,and the number of epithelial cells was significantly increased.The epithelial cells were subdivided into subgroups,and compared with the HGC-SARA group,the number of basal and spinous cells which were responsible for the absorption of VFAs were increased in the other three groups.Among the epithelial DEGs of individuals with different SARA susceptibility,the solute carrier family-related genes increased more in the tolerant individuals.The VFAs concentration in blood of 6 h after morning feeding in the HGW-Health group was higher than that of HGW-SARA group（P<0.05）.The concentration of organic acids in different metabolites and the total VFAs concentration of rumen were significantly reduced in the tolerance group（P<0.05）,which proves that the enhanced absorption capacity of organic acids by the rumen epithelium may be the main reason for maintaining the homeostasis of the rumen in the tolerance group.In summary,this experiment showed that the environment and microbial flora of tolerant individuals in the rumen are similar to that of healthy sheep fed with low RDS,and there is no inflammatory response in the rumen epithelium,the ability of the rumen to absorb organic acids is enhanced,Maintain the stability of the environment in the rumen and ensure the health of the rumen of dairy goats.Experiment 5 The regulatory effects of rumen microorganisms and their metabolites on SARA susceptibilityThis experiment was based on the individuals with different susceptibility screened in experiment 3.The HGW-SARA and HGC-Health groups exchanged the contents of the rumen to observe the occurrence of SARA in receptor dairy goats,and explore the regulatory effects of microorganisms and their metabolites on the occurrence of differences in SARA susceptibility.The results showed that after the rumen microflora of HGC-Health group was transplanted to HGC-SARA group,rumen p H of recipient HGW-SARA group was significantly increased（P<0.05）,which could alleviate SARA symptoms,and the original microflora of HGW-SARA group was significantly changed（P<0.05）,which was more similar to that of donor HGC-Health group.And the HGW-SARA group of rumen microflora after transplantation to HGC-Health group,receptor HGC-Health groups have not been SARA,and microflora back to a steady state of tolerance group before transplantation.The abundance of fiber degrading bacteria was significantly increased in both recipient groups compared with the donor group（LDA>3）,indicating that the recipient group showed the microbial characteristics of tolerance group after RMT.Rumen fermentation parameters showed that the concentration of isobutyrate,valerate,lactate and LPS in the receptor group was significantly lower than that of the susceptible donor group（P<0.05）.The data of the metabolome of rumen,differential metabolites between the receptor and the donor were significantly enriched into the tryptophan metabolic pathway,and the N-acetyl serotonin and xanthine acid in this pathway were significantly increased in the receptor group.The rumen epithelial transcriptome showed that the DEGs between the receptor group and the susceptible donor group were significantly enriched to multiple immune system-related pathways.Among the DEGs that were significantly enriched to the IL-17 signaling pathway,was significantly downregulated in the receptor group compared to the susceptible donor group,which shows that no inflammatory response of the rumen epithelium occurred in the receptor group.This experiment showed that after the exchange of rumen flora,the tolerant host could reconstruct the microflora from the susceptible group with its strong absorption capacity of organic acids in the rumen epithelium,and the stable rumen microbial structure of the tolerant individuals could regulate the rumen fermentation mode of the susceptible individuals,so as to improve the rumen p H value,alleviate SARA occurrence,and maintain the homeostasis of the rumen environment.The above results show that The occurrence of SARA in dairy goats is accompanied by the changes of rumen microflora,and the microbial changes are involved in the occurrence of rumen epithelial inflammation.Under the same high RDS diet,SARA susceptible individuals have reduced rumen p H and fiber degradation ability,and inflammation of the rumen epithelium mediated by the proliferation and differentiation of Th17 cells occurs,but key bacteria and molecules such as Bifidobacteria and tryptophan metabolites can mediate the proliferation and differentiation of Th17 cells,affecting the occurrence of inflammation of the rumen epithelium.The proportion of basal layer cells and spinous cells responsible for VFAs absorption in the rumen epithelium of SARA-tolerant individuals has increased,and the number of Th17 cells has decreased,which may be the key mechanism for maintaining the homeostasis of the environment in the rumen and reducing inflammation. |
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