Study On Regulation And Its Mechanism Of Rumen Microbiota Transplantation On Gastrointestinal Microflora In Lambs

The microorganisms habituated in rumen are essential for nutrient digestion and host physiological health.This study first evaluated the effects of rumen microbiota from different sources on fermentation parameters and microbial community structure through an in vitro fermentation experiment,then selected rumen mixed phase,solid phase and liquid phase to produce three kinds of rumen lyophilized powder according to the results,and studied the regulatory effect and mechanism of rumen lyophilized powder on growth performance and gastrointestinal microflora of lambs,providing technical support for enriching the theory of ruminant microbial regulation and promoting efficient production.Our main results were as follows:Ⅰ.Study on the role and mechanism of rumen microbiota in regulating diet fermentation efficiency in VitroRumen microorganisms are closely related to feed utilization efficiency,but there is little attention paid to the differences in microbial composition and function between the solid and liquid phases of the rumen.In this study,the fresh fluid（FF）and fresh solid phase（FS）were collected by filtering rumen contents and FS was washed for fresh solid washing solution（FW）.The fractions of FF,FS and FW were autoclaved to obtain autoclaved fluid（AF）,solid（AS）and washing solution（AW）.Then,formed 8treatments based on cross design of two factors.After 24 hours fermentation,the gas production in AFFS,FFFS and FFAS were significantly higher than other groups.AFAS and AFAW had significantly lower alpha diversity than that of other groups.The solid phase was enriched with fiber-degrading bacteria including Treponema,Succinivibrio and Ruminococcus;the fluid phase was dominant by Christensenellaceae R-7 group,Prevotella,Rikenellaceae RC9 gut group;and the washing solution significantly enriched Ruminobacter,Pseudobutyrivibrio and Fibrobacter.Moreover,double-autoclaved phase increased abundances of harmful bacteria as AFAS and AFAW had higher Streptococcus and Prevotellaceae YAB2003 group.Network analysis found that the signature microbiota in AFAS and AFAW were negatively associated with the keystone microbiota in the other groups.PICRUSt2 revealed that the key microbiota in the rumen fluid phase and solid phase recombination played different functions.In addition,the correlation between characteristic microbial communities and fermentation parameters was also determined.In summary,AFFS had the best in vitro fermentation result.FFAS and FFFS also had good fermentation effects,providing a theoretical references for the next step of using rumen solid phase,liquid phase,and mixed phase for RMT.Ⅱ.Study on the regulation and mechanism of rumen lyophilized powder on the microflora of lamb gastrointestinal tractProbiotic intervention is considered an effective method for reshaping the balance of the microbiota.This experiment first collected the rumen contents of healthy adult sheep and prepared mixed phase,solid phase,and liquid phase lyophilized powder（BMIX,BSOL,BLIQ in sequence）,and then selected 68 healthy 12-week-old lambs of the same breed with similar weight and were randomly divided into control group and three treatment groups,each lamb of treatment groups were fed 10 g of corresponding lyophilized powder per day,while the control group（CON）was not fed with lyophilized powder.Daily feed intake of lambs was recorded.Body weight was measured,and rumen fluid samples and rectal fecal samples were collected every two weeks to determine rumen fermentation parameters and 16S r RNA gene sequencing.Blood samples were collected at the end of the experiment to determine serum biochemical indicators and serum metabolites.Results showed that SOL group and LIQ group had a tendency to affect the weight of lambs（0.05<P<0.1）and SOL group had a tendency to affect ADG of lambs in the second stage.As for rumen fermentation,concentration of NH₃-N,MCP,formate,total VFA,the proportion of acetate,propionate,isobutyrate and the ratio of acetate to propionate were significantly affected by treatment factor（P<0.05）.Specifically,MIX group decreased the total VFA concentration at weeks 4 and 6 of the experiment,while the LIQ group increased the total VFA concentration at week 4.By studying the rumen microbiota of lambs,it was found that MIX group has the highest species diversity（P<0.05）,and the clustering of microbial species and functions was obvious clustering separation from other groups（P<0.05）.Only Euryarchaeota was significantly affected by treatment factor（P<0.05）.Succinivibrio,Succiniclasticum,Roseburia,Erysipelotrichaceae UCG-002,Prevotella,Methanobrevibacter,Succinivibrionaceae UCG-001,Christensenellaceae R-7 group,NK4A214 group,Lachnospiraceae NK3A20 group were found to be key hubs for the structural transformation of rumen microbiota.The key OTUs affecting the concentration of total VFA,acetate,propionate and the ratio of acetate to propionate were predicted by random forest analysis.Treatment factor made an increasing difference in the microbial structure of rectal feces（P<0.05）.The key nodes affecting the interaction of intestinal microbiota were found,such as Lachnospiraceae UCG-008,Subdoligranulum,Parabacteroides,UCG-009,Faecalibacterium,Akkermansia,Blautia.The effects of treatment factor on the relative abundance and function of rumen and rectal microbiota were generally different in the three periods,and the functional differences were the largest in the second period.By tracing the characteristic microbiota and analyzing the correlation with phenotypic parameters,it was found that ADG was affected by the characteristic microbiota with p H,acetate and isobutyrate as important nodes（R>0.35&P<0.05）,and the concentration of acetate and propionate showed a significant negative correlation（R<-0.85&P<0.05）.The effect of lyophilized powder on rumen microbiota was greater than that of rectal intestinal microbiota.The key rumen microbiota affected by liquid lyophilized powder were the most,and the key rectal fecal microbiota affected by lyophilized powder were the characteristic bacteria of BMIX.In the analysis of serum parameters of 18-week-old lambs,IGF-1 was significantly reduced by treatment factor（P<0.05）.GSH-Px concentration of MIX group was significantly decreased（P<0.05）.Treatment factor reduced the concentration of most co-differential metabolites in the serum of 18-week-old lambs（P<0.05）.And combined with the weight gain results,it was suggested that the lamb at the end of the experiment has partial adaptability to lyophilized powder.In summary,the effect of continuous transplantation of lyophilized powder for four weeks was best with BSOL,and the effect of BLIQ transplantation was the best throughout the entire period.In conclusion,this study systematically elaborated the role and regulation mechanism of rumen microbiota transplantation on gastrointestinal microflora of lambs,screened potential functional strains conducive to rumen fermentation,and provided a theoretical basis for early artificial intervention strategies of young ruminants.