Analysis Of Rumen Microbial Diversity In Ketosis Cows And Effect Of Bacillus Paralicheniformis On Rumen Fermentation Characteristics In Vitro

In recent years,the incidence of ketosis in dairy cows in China has been increasing year by year,and it can cause other perinatal diseases,causing serious economic losses to domestic dairy farming industry.Ketosis is a kind of energy metabolic disorder.The principle of its occurrence is that postpartum feed intake of cows cannot meet the nutritional requirements for high milk yield,which leads to a large amount of fat mobilization in the body and the production of a large number of ketone bodies into the blood,thus causing ketosis.The purpose of this study is to study and identify the diversity and difference analysis of rumen microorganisms among clinical ketosis,subclinical ketosis and healthy dairy cows,and to study the effect of Bacillus paralicheniformis on Rumen in vitro fermentation characteristics of dairy cows through in vitro fermentation simulation test,so as to provide theoretical basis for its scientific application in production practice.The first part of this study was divided into three groups:clinical ketosis group,subclinical ketosis group and healthy group,with 9 cows in each group.Rumen fluid samples were collected and detected by 16S rRNA high-throughput sequencing technology.The results showed that a total of 19 p Hyla were identified in rumen fluid samples,and the dominant groups whose abundance was more than1%were Bacteroidetes,Firmicutes,Proteobacteria,Fibrobacteria,Spiricobacteria and Tricobacteria.At the genus level,142 genera were detected in rumen fluid samples.There were significant differences in species richness and evenness between clinical ketosis group（CR）,subclinical ketosis group（SR）and healthy group（NR）（P<0.05）.The richness and species evenness of Cr and Sr samples were higher than those of NR samples.Through Beta diversity analysis and cluster analysis,it was found that there were significant differences between clinical ketosis group and healthy group in rumen microbiota（P<0.05）.The bacteria in Cr,Sr and NR samples showed significant differences at the level of p Hylum,class,order,family,genus and species.At the p Hylum level,there were a small number of unclassified bacteria in CR,SR and NR groups respectively,and there was no significant difference between them（P>0.05）.Compared with the rumen fluid samples from the healthy group,the number of Firmicutes,Proteobacteria,Trichobacteria,Bacillus verrucoides and Bacillus fabularis in the clinical ketosis group was significantly higher（P<0.05）,while the relative abundance of the other p Hyla was not significantly different between the two groups.At the genus level,the abundance of Cr,Sr and NR groups also differed.The relative abundance of unclassified genera in the samples（p>0.05）was40.9%,and then the genera with a proportion greater than or equal to 0.1%in the rumen fluid samples were analyzed.The number of Sucinivibrionaceae（P=0.044）,RF32（P=0.022）and Sucinivibrio（P=0.028）in Proteobacteria were significantly decreased in CR group compared with NR group.In CR group,Rumenococcaceae（P=0.009）,Christensenellaceae（P=0.005）and Mogibacteriaceae（P=0.006）were classified into Firmicutes.BS11（P=0.012）and RF16（P=0.000）in Bacteroidetes and Anaeroplasma（P=0.005）in Trichoderma were significantly increased compared with NR group.In addition,we observed that in the CR and NR groups,Clostridiales（P=0.078）,Coprococcus（P=0.071）,Shuttleworthia（P=0.093）,Pseudobutyrivibrio（P=0.058）,Clostridiaceae（P=0.067）,Anaerostipes（P=0.086）and RFP12（P=0.057）in the PHylum Formicutes)but showed a trend.Conclusion:there are significant differences between the clinical ketosis cows and the healthy cows,which mainly reflected in the relative abundance of some special bacteria,but they were not found in the subclinical ketosis cows.In the second part,the rumen fermentation in vitro was simulated by single factor design.The experiment was divided into four groups:group I,group II,group III and group IV.the fermentation substrate was total mixed ration（TMR）for dairy cows,and each group was fermented for 72 hours.Ⅰas control group,not adding bacillus licheniformis,Ⅱ,Ⅲ,Ⅳas experimental group,respectively,to join the 0.02 ml and 0.2 ml,2 ml of bacillus licheniformis bacteria liquid,the number of living bacterium 10⁸,10⁹,10¹⁰,respectively.Each set had 3replicates,with 3 nylon bags in each bottle and 2g of fermentation substrate in each bag.The p H value,NH₃-N concentration,BCP concentration,VFA concentration and DM degradation rate of the fermentation broth were determined in a fermentation flask at 6,12,24,36,48,60 and 72 hours respectively to study the effects of Bacillus paralicheniformis on the rumen fermentation characteristics in vitro.Results show that the fermentation test process,test group each group p H change trend is consistent,in each period were extremely significant difference compared with control group（p<0.01）,the experimental groupⅡand experimentalⅣat 6 h and 24 h compared with significant difference（p<0.05）,while patients withⅢⅡ,Ⅲcompared with p HⅣdifferences were not significant;Group each group of NH₃-N concentration change trend is the same,the experimental group at various time points were higher than control group and the difference was significant（p<0.01）,while groupⅡ,Ⅲ,NH₃-N concentration differences betweenⅣtends to significantly（p<0.1）;Experimental groups at various time points BCP concentrations were significantly higher than that of control group（p<0.01）,the experimental groupⅡ,Ⅲ,Ⅳbetween each time differences are not significant（p>0.05）;Group each group total volatile fatty acid concentration（TVFA）at various time points compared with the control group were extremely significant difference（p<0.01）,12 hours in fermentation,ⅣⅡtrial group and test group compared with significant difference（p<0.05）,in 36 hours,fermentationⅡ,ⅢⅣtrial group and control group and test group compared with the differences were significant（p<0.01）,other time period,patientsⅡ,Ⅲ,Ⅳbetween difference was not significant;In vitro fermentation between 24 hours to 48 hours,the experimental group DM degradation rate were not significant difference compared with control group,other time extremely significant difference（p<0.01）,in vitro fermentation after 72 hours,experimental groupⅢextremely significant difference compared with control group（p<0.01）,ⅣⅡ,test group and test group and control group the difference was not significant（p>0.05）.Conclusion:addition of Bacillus paralicheniformis can improve rumen fermentation in vitro.