Inoculation Of Microbes Modified Rumen Microbial Colonization And Functions During Weaning Of Hu Lambs

Early life development and growth are critical in determining the lifelong growth performance and health of ruminants.Weaning poses the greatest challenge to young animals and induces the severe stress.As an important organ for solid feed digestion,the development of rumen itself and colonization of microorganisms are fundamental to the digestion of starter feed.Regulation of rumen microbial colonization process may promote digestion of starter and mitigate weaning stress.In this study,the effects of inoculation time,frequency and method of rumen microbial inoculation on the animal growth performance and gastrointestinal tracts development were evaluated.Moreover,the effects of lyophilized rumen contents,which can provide repeated stimulation on growth performance,health,metabolism and rumen microbial colonization of young ruminants before and during weaning were also determined.This study provides theoretical and experimental basis for early microbial regulation of young ruminants,which is a new perspective for efficient production of ruminants.1 Effects of inoculation with fresh rumen fluid before or during weaning on the microbiota composition and co-occurrence of the rumen and colon of lambsFifteen newborn Hu lambs(five triplets)were separated imediatelly after birth,colostrum and milk replacer were fed with bottle artificially.Lambs were allocated into three groups with one from each triplets,lambs were orally inoculated repeatedly on day 8,9,15,16 before weaning(IBW)or day 22,23 during gradual weaning(IDW)with the rumen fluid collected from adult sheep.Control lambs were inoculated with normal saline on each inoculation day.The oral inoculation did not significantly affect starter intake,growth performance,or ruminal fermentation.Based on sequencing analysis of 16S rRNA gene amplicons,however,the inoculation(both before and during weaning)affected the assemblage of the rumen microbiota,increasing or enabling some bacterial taxa to colonize the rumen.These included operational taxonomic units(OTUs)belonging to Moryella,Acetitomaculum,Tyzzerella 4,Succiniclasticum,Prevotella 1,Lachnospiraceae,Christensenellaceae R-7 group,Family XIII AD3011,and Bacteroidales S24-7 corresponding to inoculation before weaning;and OTUs belonging to Succiniclasticum,Prevotellaceae UCG-003,Erysipelotrichaceae UCG-004,Prevotella 1,Bacteroidales S24-7 gut group uncultured bacterium,and candidate Family ⅩⅢ AD3011 corresponding to inoculation during weaning.Compared to the inoculation during weaning,the inoculation before weaning resulted in more co-occurrences of OTUs that were exclusively predominant in the inoculum.However,inoculation during weaning appeared to have more impacts on the colonic microbiota than the inoculation before weaning.Considerable successions in the microbial colonization of the GI tracts accompanied the transition from liquid feed to solid feed during weaning.Repeated rumen fluid inoculation during early life can modulate the establishment of the microbiota in both the rumen and the colon and co-occurrence of some bacteria.Oral inoculation with rumen microbiota may be a useful approach to redirect the development of the microbiota in both the rumen and colon.2 Effects of repeated oral inoculation of artificially fed lambs with lyophilized rumen fluid on growth performance,rumen fermentation,and organ developmentIn this study,we aimed to investigate the effects of microbial intervention on the growth and development of young lambs and their rumen microbiota during early life because of the lower colonization resistance.To exclude the impact of chemicals,antigen and dead cells from rumen fluid,in the second study,lyophilized rumen fluid were determined for microbial colonization modulation.Forty-eight lambs were allocated to two groups,with one group being orally inoculated with lyophilized rumen fluid,and the other receiving same amount of skim milk served as the control during 5-21 days.Lambs had free acess to starter feed and fresh water from 5 days.Lambs were weaned in one week from the day when starter feed intake was greater than 200 g/d.Growth performance,rumen fermentation characteristics,and carcass measurements were determined weekly one week before and two weeks after weaning.Our results showed that the early life inoculation significantly increased the apparent digestibility of feed dry matter and crude protein before weaning,and post-weaning crude protein digestibility remained elevated in the inoculated lambs compared to the control lambs.Starch digestibility tended to be higher in the inoculated lambs than in the control lambs two weeks after weaning.Molar proportion of ruminal propionate and amylolytic enzyme activity were higher in the inoculated lambs than in the control lambs after weaning.The development of internal organs,including the lower gut and liver,was also stimulated by the inoculation.In conclusion,some rumen bacterial populations of young lambs were clearly affected by the inoculation with rumen microbiota,which could contribute to the digestion of the nonstructural carbohydrates available in the starter feed.Improved digestion of starter feed may enhance the energy and nutrient harvest and utilization in young ruminants during the critical weaning phase.3 Effects of repeated oral inoculation of artificially fed lambs with lyophilized rumen fluid on blood measurements,gluconeogenesis-related genes expression and rumen microbial compositionEight lambs from each group were euthanized before weaning,one week after weaning,and two weeks after weaning for rumen content,liver and blood samples.In control lambs,results showed blood NEFA decreased significantly after weaning and blood glucose had decreasing trend after weaning,while BHBA increased significantly after weaning.These measurements were stable in Inoc lambs after weaning.Blood lactate,insulin and glucagon were not affected by inoculation.Gene expression of PCK1,PCK2,PC,MCT1 and MCM were not affected by inoculation.Expression of G6PC was increased after inoculation,which may increase the production of glucose in liver and mitigate the energy deficiency.Different temporal fluctuation patterns of microbial taxa in the rumen of the inoculated lambs compared to the control lambs after weaning.After weaning,Mitsukella,Olsenella and Succiniclasticum were more abundant in Inoc lambs,they were also positively correlated to propionate and blood glucose.Module network analysis indicating treatment improved the propionate producing microbial modules associated with family Prevotellaceae,Succinivibrionaceae and Erysipelotrichaceae could be responsible for converting lactic acid to propionate in rumen.In conclusion,oral administrate of lyophilized mature rumen bacteria successfully introducing lactate producing bacteria in to rumen of weaning lamb and finally facilitate the propionate production in rumen and the subsequent hepatic glucose production for better energy homeostasis during weaning.4 Effects of repeated oral inoculation of artificially fed lambs with lyophilized rumen fluid on rumen microbial functional gene abundanceIn BW1,the abundant of 1,047 differential abundance genes in the Inoc group are significantly higher than that of the control group;98 differential abundant genes are significantly lower than the control group.Among the top 25 most abundant DAGss SusC,GHs and other carbohydrate utilization-related gene abundances were significantly higher in Inoc lambs than those in the control lambs,indicating that the Inoc lambs had a stronger ability to digest feed carbohydrates before weaning than the control lambs.At AW1,there were a total of 829 DAGs in the rumen of the Inoc lambs had significantly higher abundance than the control lambs,another 180 DAGs had significantly lower abundance than the control group.Among them,SusC,CBM and other gene abundances were maintained at a high level in the rumen of the Inoc lambs after weaning.At the same time,the functional gene abundance of peptidase and pectate lyase was also higher in the Inoc lambs than the control lambs,which may indicate higher potential of abilities to utilize protein and hemicellulose of Inoc lambs after weaning.At AW2,the abundance variance of functional genes between the two groups decreased,inoculation of lyophilized rumen fluid accelerated the colonization of rumen microbes and development of rumen microbial functions.Among the carbohydrate-related functional genes,the abundance of GH5,GH13,GH43,GH141,GH142 and other families in the Inoc lambs before weaning was significantly higher than that of the control lambs.However,after weaning,the functional genes between the two groups were no longer significant.The gene abundances of GH5,GH13,GH43 and GH142 in the rumen of the control lambs altered significantly after weaning,while it remained relatively stable in the Inoc lambs.Consistent with the previous results of this study,Inoc accelerated the colonization process of rumen microorganisms,maintain the relative stability of rumen microorganism functional genes during weaning.The KEGG pathway enrichment results showed that the functional genes of rumen microbes before weaning were significantly enriched in starch and sucrose utilization pathways,which further demonstrated that Inoc can increase the potential of rumen to use feed ingredient and play an important role in the weaning process.In conclusion,repeated manipulation of rumen microorganisms can promote the colonization of mature rumen microorganisms such as Mitsukella,Olsenella,Succiniclastcum and other microorganisms in the rumen,promote the development and maturation of rumen microorganisms,and enhance the ability of the lamb to digest the food before weaning.On the one hand,the application of inoculation in practice can improve the growth performance of juvenile ruminants,alleviate the negative energy balance during weaning stress,and promote the healthy development of juvenile ruminants;on the other hand,rumen microorganisms have strong plasticity during early life of ruminants.Manipulation of the rumen microbes during early life may change the fate of colonization of rumen microbes and achieve long-term promotions.