Reshaping Gastrointestinal Microbiota Of Cows With Microbiota Transplantation And It’s Ecological Mechanism

Gastrointestinal microbiota plays multiple important roles in intestinal and physiological homeostasis as well as the feed digestion of host,especially for ruminants.Previous studies demonstrated using microbiota transplantation(MT)to reprogram gastrointestinal microbiota be promise for redressing intestinal and physiological disorders.However,the efficiency of MT for ruminants as well as the response difference in different gastrointestinal sections was remained largely unknown,and the underlying mechanism of MT was still unclear.To address these questions,with High throughput sequencing technology,we firstly assess the bacterial profiles in rumen digesta as well as liquid and solid fractions to explore whether it is suitable to use rumen fluid to perform MT;Then with rumen fluid transferred from healthy cows to antibiotics treated cows,we explored the efficience of MT to reshape the disturbed gastrointestinal microbiota in both rumen and rectum of cows;Finally,we tested the alterations in MT efficiency with different gut preparation procedures and explored the underlying mechanism of MT with mice model,which model has demonstrated having merits of well background and was widely accepted in MT study.In experiment 1,we compared bacterial profiles in rumen digesta,as well as liquid and solid fraction of rumen digesta.Samples of rumen digesta from 6 cows fed either a high fiber diet(HFD)or a high energy diet(HED)were collected via rumen fistulas.Rumen digesta was then squeezed through four layers of cheesecloth to separate liquid and solid fractions.The bacterial profiles of rumen original digesta,liquid and solid fractions were analyzed with High-throughput sequencing technique.Rumen bacterial diversity was mainly affected by diet and individual cow(P>0.05)rather than rumen fraction.Bias distributed bacteria were observed in solid and liquid fractions of rumen content using Venn diagram and LEfSe analysis.Fifteen out of 16 detected biomarkers(using LEfSe analysis)were found in liquid fraction,and these 15 biomarkers contributed the most to the bacterial differences among rumen content fractions.These results demonstrated the ecological similarity of microbiota in different rumen fractions,and similar results were found when using samples of original rumen digesta,rumen liquid or solid fractions to assess diversity of rumen bacteria;However,bacterial abundance difference of particular bacterial taxa in different ruminal fractions was also observed.These results summarily demonstrated that rumen fluid harbored almost all the rumen bacteria,and it was applicable to use rumen fluid to perform the MT.In experiment 2,we assessed the efficiency of microbiota transplantation on gastrointestinal microbiota reshaping of cows.Here,we used 15 cows with rumen fistula,assigned 5 cows as donor cows and the other cows received antibiotics treatment lasting 14 days,after antibiotics treatment,the 10 cows were randomly allocated into two groups with one group cows received rumen fuild microbiota transplantation(MT group)and the other group cows received saline solution infusion(CON group),we investigated the microbiota shift in rumen and rectum of lactating cows after antibiotics exposure as well as after antibiotics withdrawal with(MT group)or without(CON group)microbiota transplantation.We were able to demonstrate that microbiota in both foregut and hindgut significantly changed after 3 or 14 days of antibiotics exposure,and the changes persisted over long period of time(>18 days)after withdrawing the antibiotics.We further observed a faster restoration of microbiota in both foregut and hindgut of MT group than CON group,microbiota in foregut was mainly benefited from microbiota transplantation by restoring the alpha-diversity as well as within-group similarity,while microbiota in hindgut was primarily benefited from microbiota transplantation by reestablishing the co-occurrence network(nodes number,edges number,density,modularity as well as closeness centrality).These results together demonstrated that MT was efficient to reshape the rumen and gut microbiota of cows;However,the response in different gastrointestinal sections to MT might be differed,thus target gastrointestinal section should be under consideration when performing the MT.In experiment 3,we tested the hypothesis that gut microbiota reprogramming efficiency would be differentiated by different gut preparation and thus to explore the underlying mechanisms of MT.We prepared the guts of mice for MT by administering one of the three most-clinically-used pretreatments(antibiotics,bowel cleansing solution,or no pretreatment),30 mice were randomly assigned to 3 groups with each group mice receiving one of the above gut preparation treatment,after gut preparation for 3 days,each group mice revieved MT from human gut microbita,digesta and gut mucus sample from jejunum and colon was collected after gut preparation as well as 7 days after MT.We found that preparing the gut with antibiotics induced a more efficient modification of the gut bacterial community than was induced by either of the other two pretreatment types.The increased efficiency of antibiotic treatment appeared to occur via increasing the xenomicrobiota colonization.Further analysis demonstrated that antibiotic treatment of mice induced intestinal microbiota disruption,mostly by expelling antibiotic-sensitive bacteria,while the indigenous microbiota was maintained after treatment with a bowel cleansing solution or in the absence of pretreatment.The amount of antibiotic-resistant bacteria increased shortly after antibiotics usage but subsequently decreased after MT administration.Together,these results suggested that MT relied on the available niches in the intestinal mucosa and that preparing the gut with antibiotics facilitated xenomicrobiota colonization in the intestinal mucosa,which thus enhanced the overall gut microbiota reprogramming efficiency.In summary,we firstly assess the bacterial profiles in rumen digesta as well as liquid and solid fractions,which demonstrated that different fractions of rumen harbored similar bacterial ecology;Then with rumen liquid transfer from healthy cows to antibiotics treated cows,we demonstrated that MT was efficient to reshape the disturbed gastrointestinal microbiota in both rumen and rectum of ruminants;Finally,we tested the alterations in MT efficiency caused by different gut preparation procedures with mice model and demonstrated that MT efficiency was regulable and xenomicrobiota colonization in gut relied on the available niches.