Effect Of Rumen Microbiota In The Susceptibility Of Subacute Ruminal Acidosis In Dairy Cows

Subacute ruminal acidosis（SARA）is a relatively common nutritional and metabolic disease in high-producing dairy cows.The disease causes a decrease in milk production and productivity,which in turn lead to a series of diseases such as diarrhea,mastitis and laminitis,increase the mortality and elimination rate of dairy cows,and bring huge economic losses to the dairy farming industry.It was found that the probability of SARA risk varied among individual dairy cows despite identical feeding management conditions and that there were differences in SARA susceptibility among individual cows.Therefore,it is of scientific importance to analyze the mechanism of individual SARA susceptibility in dairy cows for clinical control of the disease and dairy cattle breeding screening.As the most important digestive organ of ruminants,rumen is the main site of microbial fermentation and feed digestion.The fermentation efficiency of rumen contents,nutrient absorption rate and animal immunity,which are important life activities,are closely related to the composition of rumen microbiota.There is growing evidence that rumen microbes play a critical role in dairy cow health and disease development.Some experts have proposed the idea of "healthy rumen,healthy cows".Recent studies have reported that the occurrence of SARA has an important association with rumen microorganisms.During the occurrence of SARA,rumen flora diversity and abundance were significantly reduced and rumen flora were disturbed.Accordingly,we hypothesized that SARA susceptibility was related to the rumen microbial community and that differences in rumen flora diversity might be the main reason for the differences in SARA susceptibility of individual dairy cows.To this end,this project firstly clarified the problem of SARA susceptibility differences among individual dairy cows by feeding a high-concentration diet to induce SARA disease model,and then detected the diversity of rumen bacterial and fungal flora in dairy cows by 16 S rRNA and ITSI-IF sequencing,and analyzed the differences in the initial flora of SARA-prone and SARA-resistant cows in order to elucidate the mechanism of SARA susceptibility differences in dairy cows.Firstly,20 Holstein cows of similar age,lactation days and body weight were selected and all were fed a low-concentration diet（ratio of fine to coarse: 4:6）for 15 days to allow cows to acclimatize to the environment.Then,a high-concentration diet（ratio of fine to coarse: 7:3）was selected to feed the cows continuously for 6 weeks to establish the SARA model.After 6 weeks,the p H of rumen fluid was analyzed.The results showed that after 6 weeks of continuous high-concentration diet,12 of the cows successfully established the SARA model,while we found that the remaining 8 cows did not meet the criteria for SARA disease.The p H of rumen fluid in SARA-positive cows was 5.77 ± 0.10,while that of SARA-negative cows was 6.32 ±0.09.Cows were further divided into SARA-resistant（SARA negative cows after 6 weeks on highconcentration diet）and SARA-prone（SARA positive cows after 6 weeks on highconcentration diet）groups according to their morbidity,pending subsequent testing and analysis of rumen microbiota in different groups.We found that although the conditions such as feeding management of the cows were the same,the cows still showed differences in the probability of SARA.These results suggest that there are differences in SARA susceptibility among individual cows.Secondly,the initial rumen bacterial microbiota of SARA-resistant and SARAprone cows was analyzed by 16 S rRNA sequencing.Ruminal fluid was collected from SARA-resistant and SARA-prone cows,and the association of rumen flora with SARA susceptibility was analyzed by 16 S rRNA sequencing to identify differential genera.16 S rRNA sequencing analysis showed that there were differences in the composition of rumen bacterial community between SARA-resistant and SARA-prone cows.Compared with SARA-resistant cows,the abundance of unidentified＿Spirochaetaceae,Anaeroplasma and Prevotella＿ruminicola in the rumen of SARA-prone cows was relatively higher.The abundance of Tyzzerella＿sp＿Marseille＿p3062 was higher in SARA-resistant cows.The above results suggest that changes in the abundance of unidentified＿Spirochaetaceae,Anaeroplasma,Prevotella＿ruminicola and Tyzzerella＿sp＿Marseille＿P3062 in the rumen fluid of dairy cows may be associated with susceptibility to SARA.Finally,fungi play an irreplaceable role in digesting lignocellulosic biomass in the rumen of ruminants.They coexist and interact with other microorganisms in the rumen.Based on the differences of rumen bacterial microbiota in the above experiments,we believe that fungi will also have relative differences.The initial rumen fungal microbiota of SARA-resistant and SARA-prone cows was analyzed by ITS1-IF sequencing technology.The ITS1-IF assay showed that the abundance of Sarocladium and Fusarium＿oxysporum was higher in the rumen of SARA-prone cows compared to SARA-resistant cows.In contrast,the abundance of Aspergillus and Papiliotrema＿laurentii was higher in SARA-resistant cows.The above results suggest that changes in the abundance of Sarocladium,Fusarium＿oxysporum,Aspergillus and Papiliotrema＿laurentii may be associated with SARA susceptibility.In conclusion,this study found that there were differences in SARA susceptibility among dairy cows.At the same time,through 16 S rRNA and ITSI-IF sequencing analysis,it was found that there were significant differences in rumen bacterial and fungal microbiota between SARA-resistant cows and SARA-prone cows.In particular,unidentified＿Spirochaetaceae,Anaeroplasma,Prevotella＿ruminicola,Sarocladium,Fusarium＿oxysporum,Tyzzerella＿sp＿Marseille＿P3062,Aspergillus and Papiliotrema＿laurentii may be related to differences in the susceptibility of cows to SARA.However,the role of these bacteria in SARA needs to be further investigated.This study provides preliminary experimental data to resolve the clinical differences in SARA susceptibility among individual dairy cows,and provides new directions and targets for the clinical control of SARA in dairy cows by targeting rumen flora.