Comparative Study On Sources Of Mixed Extracellular Vesicles In Dairy Rumen And Artificial Rumen

The structure and composition of rumen microbiome and its relationship in rumen ecosystem play an important role in the early development,health and physiology of ruminants.Since the discovery of rumen microorganisms,with the development of research methods and techniques,great progress has been made in the classification and function of rumen microorganisms and their interactions with each other and the host.However,effective means and tools have been lacking to explore the interactions between rumen microorganisms and the microbe and the host.This seriously affects our understanding of the rumen and its microbial function.There is increasing evidence that extracellular vesicles(EVs)from microbiome and host are key players in the transboundary cross talk in the gastrointestinal ecosystem,and they are promising to be new agents and means for resolving microbe-microbial and microbe-host interactions.At present,some researchers have preliminarily established a method for the isolation of rumen EVs and preliminarily characterized its basic characteristics,but the sources of rumen EVs have not been elaborated.The determination of the source and composition of rumen EVs is likely to provide a new means and approach for the in-depth understanding of rumen microecosystem and digestive physiology of ruminants,which is also a work with great workload and difficulty.In order to elucidate their origin,it is important to select systems with distinct differences in host cell and microbial composition.Sources of dairy rumen EVs(d EVs)include microorganisms and host cells,while sources of artificial rumen EVs(a EVs)basically only include microorganisms.By comparing rumen EVs from different microorganisms and host cells,the source composition of rumen EVs can be more accurately analyzed.Proteome,16 S sequencing is a common method to study the source of EVs.Therefore,this study intends to identify and analyze mixed EVs from artificial rumen and dairy rumen based on proteomics,16 S sequencing and ITS sequencing,so as to comprehensively understand the specific source composition of rumen mixed EVs.The research results are as follows:(1)Rumen EVs were isolated by differential centrifugation combined with density gradient centrifugation,and the lipid protein ratios of d EVs and a EVs were 0.29 and 0.77,respectively,indicating that rumen EVs isolated had high purity.Their particle size peaks were 81 nm and 79 nm,respectively,and obvious bilevels and cup-like structures were observed under transmission electron microscopy.Western blots failed to detect exosome marker proteins such as TSG101 and HSP70,suggesting that exosome related marker proteins may not be suitable for the detection of microbial origin extracellular vesicles.(2)Protein spectrum analysis was performed on d EVs and a EVs,and 1,964 and 2,558 proteins were identified,respectively.The sources of proteins included bacteria,fungi,archaea,viruses,protozoa,cattle,and plants.Bacteria-derived proteins make up the bulk.At the genus level,Bacteria from d EVs and a EVs mainly include Prevotella,Ruminococcus,Clostridium,Butyrivibrio,Lachnospira,Blautia,Coprococcus,Selenomonas,Muribaculaceae,Succiniclasticum,Atopobium and Acidaminococcus;At the species level,d EVs and a EVs source bacteria mainly include Lachnospiraceae bacterium,Butyrivibrio hungatei,Lachnospira pectinoschiza,uncultured Ruminococcus sp,Prevotella sp.and Prevotella bryantii.(3)Before d EVs and a EVs samples were sequenced,PCR amplification was performed on the full length of 16 S and ITS1 gene.The results showed that relevant bands were amplified,indicating that the samples contained nucleic acids of bacterial and fungal origin.16 S r RNA and ITS sequencing results showed that at the generic level,The bacteria genera from d EVs and a EVs mainly include Prevotella＿7,Prevotella＿1,Rikenellaceae＿RC9＿gut＿group,Pseudomonas,F082,Succiniclasticum,Lactobacillus,Olsenella,Candidatus＿Saccharimonas,Shuttleworthia,Christensenellaceae＿R-7＿group and Bifidobacterium.The fungi from d EVs and a EVs mainly include Cutaneotrichosporon,Pichia,Aspergillus and Alternaria.These results indicated that the rumen of dairy cows and the artificial rumen could secrete EVs with similar particle size distribution and morphological characteristics.The protein sources of rumen EVs included bacteria,fungi,archaea,viruses,protozoa,cattle and plants,and the bacterial protein accounted for the main part.The bacterial sources at the genus level of d EVs and a EVs mainly include Prevotella,Pseudomonas,Lactobacillus,Bifidobacterium,Ruminococcus,etc.Prevotella bryantii,Prevotella sp.,and uncultured Ruminococcus sp.are the main bacteria sources of d EVs and a EVs at the species level.