| In ruminant,rumen is the main digestive and metabolic place of nutrients,and the rumen health was directly related to the health of animal and the production performance.This thesis aimed to study the effects of subacute ruminal acidosis(SARA)induced by high-concentrae diets on rumen health and lactation performance,and the research included the following four parts.1.Effects of SARA on the strucutre and composition of rumen bacterial community in dairy cowsThe objective of this study was to evaluate the changes in bacterial populations in the rumen of dairy cows fed high-concentrate diet using pyrosequencing.Four cannulated primiparous Holstein dairy cows were assigned to either a 40%(Control diet,CON group)or 70%(High-concentrate diet,SARA group)concentrate feeds in a 2 × 2 crossover experimental design.Rumen fluid samples for pyrosequencig were collected at 0 h following the morning feeding on days 12,17 and 21 of each experimental period.As expected,the duration for which ruminal pH was less than 5.8 was more than 5 h after the first feed in the SARA group,indicated that the SARA model was constructed successfully.As compared with CON group,the concentration of ruminal lactate,propionate,butyrate,total VFA and free LPS were significantly higher(P<0.05)in the SARA group.Pyrosequencing results showed that at a high taxonomic level,the percentage of Proteobacteria and Bacteroidetes were reduced(P<0.05)by SARA,whereas Firmicutes and Actinobacteria were more abundant(P<0.05)in the SARA group than in the CON group.At the genus level,as compared with the CON group,the abundance of Prevotella,Treponema,Anaeroplasma,Papillibacter,Acinetobacter and unclassified populations including unclassified Lentisphaerae,and unclassified bacteria were lower(P<0.05),while the percentages of Ruminococcus,Atopobium,unclassified Clostridiales and Bifidobacteriu were increased(P<0.05)in the SARA group.SARA reduced the diversity of the rumen microbial community and the abundance of Gram-negative bacteria,and there was a linear negative correlation(r=-0.446,P=0.029)between the level of LPS and the gene copies of Bacteroidetes.Taken together,our findings provide a comprehensive picture of current knowledge of the community structure of the rumen bacterial ecosystem during SARA.Furthermore,our results suggest that these genera significantly affected in percentage by SARA may have an important role in preventing the pathophysiological changes or stimulating the development of SARA.2.Effects of SARA on rumen metabolism in dairy cowsAs we known that rumen microbiota are closely related to alterations in rumen metabolism,hence this study aimed to uncover the effects of SARA on rumen health via ruminal fluid metabolome.Rumen fluid samples for metabolome analysis based on GC-MS were collected at Oh following the morning feed on days 12,17 and 21 of each experimental period.After rigorous quality control and identification,we detected 233different metabolites,mainly organic acids,fatty acids,sugars,amino acids,purines,amines,sugar alcohols.Results showed that SARA significantly increased(P<0.05)the concentration of bacterial degradation products such as xanthine,hypoxanthine,uracil,etc.The level of some harmful or proinflammatory compounds such as LPS,biogenic amines,ethanolamine and glutaric acid in rumen were also elevated(P<0.05)in the SARA group compared with those in the CON group.Results also showed that 15 amino acids(included alanine,leucine,glycine,glutamic acid,isoleucine etc.)exhibited higher(P<0.05)content in SARA group compared with CON group.The enrichment analysis of differentially expressed metabolites indicated that Aminoacyl-tRNA biosynthesis,Phenylalanine,tyrosine and tryptophan biosynthesis,and Valine,leucine and isoleucine biosynthesis theses three pathways were significantly enriched(P<0.05)after diet treatments.These findings suggested that SARA can cause metabolic disorders in rumen,and hence may be detrimental to rumen health.3.Effects of SARA on gene expression profiles in rumen epithelium of dairy cowsThe objective of this study was to characterize the mRNA expression profile related to The objective of this study was to reveal the effects of SARA on gene expression profiles in rumen epithelium using microarray,and then to characterize the mRNA expression profile related to rumen epithelial inflammation combined the in vitro experiment.Results showed that 245 differentially expressed genes(DEGs)were detected in the cows in SARA group relative to the CON group.The DEGs were first annotated,and results revealed that the expression of inflammation-related genes,including IL-1?,IL-2,IL-22,CCL19,CCL8,CX3CR1,CXCL6,INHBE,LEPR,PRL,and TNFRSF9 found in the cytokine-cytokine receptor pathway were up-regulated in SARA group,indicating local inflammation in the rumen epithelium was initiated.The expression of IL-1?,IL-2,and IL-6 was further validated by qRT-PCR.To demonstrate whether there were relationships between cytokine mRNA expression and ruminal factors(pH and LPS),the isolated ruminal epithelial cells were cultured in vitro.Results showed that the mRNA expression of IL-1?,IL-2,IL-6,and IL-8 increased(P<0.05)after the LPS treatment,while low-pH treatment elevated(P<0.05)the mRNA expression of TNF-?,suggesting that low-pH coupled with higher levels of LPS in rumen of cows fed the high-concentrate diet may be mainly responsible for the initiated local ruminal inflammation.In general,our results indicate that ruminal local inflammation response might be initiated in SARA group,and these findings also enhance the knowledge of rumen epithelial adaptation to high-concentrate diet at the molecular level.4.Effects of SARA on lactating performance and milk bacterial community in dairy cowsThe objective of this study was to evaluate the impact of SARA on lactating performance and milk bacterial community in dairy cows.Milk samples were collected on day 17,18,and 19 of each experimental period.And blood samples were taken from the coccygeal vein at 0 and 4 h following the morning feeding on d 12,17,and 21 of each experimental period.Results showed that dry matter intake(DMI),milk yield and milk composition were not affected by different diets(P>0.05).Compared with CON group,white blood cells,lymphocytes and the concentration of albumin in SARA group increased significantly(P<0.05),while concentrations of serum total protein,globulin,cholesterol and low density lipoprotein in the SARA group decreased sifnificantly(P<0.05).Results of 454 pyrosequencing showed that the bacterial community of milk was dominated by Actinobacteria,Firmicutes,Proteobacteria,and Bacteroidetes.Compared with CON group,there were a higher proportion of some mastitis-causing pathogen bacteria in SARA group,such as Stenotrophomonas maltophilia,Streptococcus parauberis,and Brevundimonas diminuta,as well as of Psychrotrophic bacteria,such as Pseudomonas,Brevundimonas,Sphingobacterium,Alcaligenes,Enterobacter,and Lactobacillus.However,the diversity of the ruminal bacterial microbiota(Ace,Chao,and Shannon,index)was not affected by SARA.These findings indicate that SARA may increase the risk of dairy cows suffering mastitis,decrease the organoleptic quality of raw milk and dairy products,and limit the shelf life of processed fluid milk.In summary,using a combination of muti-omics approaches,our data provide comprehensive profiles of alterations in the community structure of the rumen bacterial microbiota,metabolomic composition,gene profiles in the rumen epithelium,and the bacterial community of raw milk during high-concentrate diets induced SARA.These findings contributed to reveal the internal interactions of rumen health and host health,and to provide the theoretical basis for the healthy feeding of dairy cows. |
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