Multi-Omics-Based Analysis Of Yeast Cultures For The Regulation Of Rumen And Intestinal Functions In Hu Sheep

With the development of the livestock industry,high grain diets are becoming more and more common in ruminant production in pursuit of faster growth rates.However,this feeding pattern can easily lead to abnormal rumen fermentation.Continuous low rumen p H may lead to subacute rumen acidosis(SARA),which induces metabolic disorders,hoof disease and inflammation in ruminants,posing a great threat to animal production and health.How to maintain high production performance while maintaining animal health is a current concern for nutritionists.Yeast culture(YC)is a mixture of yeast organisms and their metabolites obtained from the fermentation of Saccharomyces cerevisiae.It is widely used in husbandry as a safe and environmentally friendly green additive.YC has a positive effect in promoting ruminant production and health,but the effect is not stable and may be influenced by diet type,animal condition and product process.YC has shown good results in the regulation of rumen and intestinal health in dairy and beef cattle under high concentrate conditions,but related studies are lacking in sheep.Therefore,this paper takes Hu sheep as the research object,and uses multi-omics technology to deeply explore the regulatory effect of YC on the physiological functions of the gastrointestinal tract of sheep,aiming to provide a certain theoretical basis for the application of YC in sheep fattening production.Thirty lambs of similar weight at 3 months of age were selected and randomly divided into three groups: the basic diet group(CON),the 20 g/d YC-added group(LYC)and the 40 g/d YC-added group(HYC),with 10 lambs in each group,and a feeding experiment was conducted for 14 d of the pretest period and 60 d of the regular test period.At the end of the feeding experiment,blood was collected to detect immune indicators and abnormal metabolites.Ruminal epithelium and rumen contents samples for histomorphology,ruminal fermentation parameters,digestive enzyme activity,microbiome and metabolome determination.Intestinal tissues and contents for the analysis of intestinal villus morphology,immunoglobulins,cytokines,digestive enzyme activity and microbiota.The main results obtained from this study are as follows:1.Effects of YC on rumen fermentation,digestive enzyme activity and blood immunity of sheepThe results of production performance showed that compared with the CON group,HYC had an increase in final weight,DMI and ADG values,a decrease in daily feed intake,and an improvement in feed-to-meat ratio,but the difference was not significant(P>0.05);YC significantly increased the digestibility of ADF(P<0.05),and the apparent digestibility of CP and NDF tended to increase,but the difference was not significant(P=0.08).The results of rumen fermentation showed that YC could significantly increase the concentration of butyric acid and TVFA,and decrease the concentration of lactic acid,LPS and histamine(P<0.05).The results of digestive enzyme activities showed that YC significantly increased ruminal lipase and cellulase activities and decreased α-amylase activity(P<0.05).YC increased amylase and lipase activities in the jejunum(P<0.05),but had no significant effect on other intestinal enzyme activities(P>0.05).Blood indicators showed that YC significantly increased the level of Ig G and decreased lactate in the blood of the HYC group(P<0.05),and the level of LPS in the blood of HYC group decreased,but did not reach a significant level(P>0.05).2.Effects of YC on rumen epithelial development,microbiota and metabolitesThe results showed that YC improved rumen papilla development,significantly increased rumen papilla length and decreased cuticle thickness(P<0.05).The results of 16 S r DNA sequencing showed that the Shannon and Simpson indices were significantly higher in the HYC group than in the other two groups(P<0.05).PCo A analysis showed that the CON group and experimental groups were significantly separated in microbial composition(P<0.05).At the phylum level,YC increased the abundance of Tenericutes(P<0.05).At the genus level,YC decreased the relative abundance of ruminal Prevotella＿1 but increased the relative abundance of Ruminococcaceae＿UCG-005,Prevotellaceae＿UCG-001,and Ruminococcus＿1 etc.(P<0.05).Metabolomic analysis(LC-MS)showed that a total of 61 differential metabolites were identified between the CON and HYC groups(FC>1.5,P<0.05).The annotated results showed that these differential metabolites were mainly involved in amino acid metabolism,lipid metabolism and vitamin metabolism pathways.The significantly enriched pathways were fat digestion and absorption,glycerol phospholipid metabolism and unsaturated fatty acid biosynthesis(P<0.05).Correlation analysis showed that Ruminococcaceae＿UCG-005,uncultured＿bacterium＿f＿Lachnospiraceae and Desulfovibrio were significantly negatively correlated with 4-Hydroxybenzaldehyde,Estrone and Phosphoric acid(P<0.05).However,they were significantly positively correlated with Chorismate,DMannonate,5-Methylthioribose,PC(16:1(9Z)/0:0)and Pimelic acid(P<0.05).3.Effects of YC on composition and function of microorganisms in different rumen nichesMetagenomics was used to further explore the effect of YC on the microbial composition and function of rumen solid-associated(SA)and liquid-associated(LA)niches.The results showed that the Simpson and Shannon indices of microorganisms in SA niche were significantly higher than those in LA niche(P<0.05).PCo A analysis showed that the microorganisms in the two niches were significantly separated(P<0.05).Further analysis of the effect of YC on microorganisms in different ecological niches showed that the effects of YC on microorganisms in SA ecological niches was greater(P<0.05).At the genus level,YC significantly increased the relative abundance of Dialister,Ruminococcus and Lactobacillus in SA system and decreased the relative abundance of Methanobrevibacter and Paraprevotella(P<0.05).YC significantly increased the relative abundance of Mitsuokella and Selenomonas in LA system(P<0.05).At the species level,PCo A analysis showed a significant effect of YC in SA system(P<0.05).Further analysis showed that YC significantly increased the relative abundance of some members of Prevotella,Dialister＿succinatiphilus and R.flavefaciens species,decreased the relative abundance of Prevotella sp.ne3005(P<0.05).LEf Se analysis showed that Dialister,Succinatimonas and Veillonellaceae were enriched in the HYC group,while Methanobrevibacter was the characteristic species in CON group.Analysis of lactic acid metabolism-related flora and methanogens showed that YC significantly altered the abundance of important lactic acid producing and utilizing bacteria in both systems,and YC significantly reduced the relative abundance of six methanogens including Methanobrevibacter and Methanosphaera in SA system.Functional analysis showed that microbial functions were significantly segregated by YC in SA system at the KEGG-KO level(P<0.05),but not in LA system(P>0.05).At the KEGG pathway-level 3 level,YC reduced microbial functions involving starch and sucrose metabolism,purine metabolism,antibiotic biosynthesis,and pyrimidine metabolism pathways(P<0.05).The results of the CAZymes annotation showed that YC significantly increased the relative abundance of GH43,GH78 and GH98 et al.members,decreased the relative abundance of GH130 family members(P<0.05).Correlation analysis showed that the concentration of LPS,lactate and amylase were positively correlated with the CON group and also with the relative abundance of Succinobacterium and Prevotella.However,cellulase activity was positively correlated with p H and also with the relative abundance of Dialister,Clostridium,Alistipes,Selenomonas and Treponema.4.Effects of YC on intestinal epithelial tissue morphology,immune function and microbiotaThe results showed that YC significantly improved intestinal villus length,crypt depth and V/C ratio(P<0.05).YC increased the content of mucosal cytokines such as IL-6,γIFN and TNFα(P<0.05),as a way to enhance intestinal barrier function and strengthen intestinal immunity.YC had a great effect on the composition of colon and cecum microbial community of sheep,mainly increasing the relative abundance of uncultured＿bacterium＿f＿Muribaculaceae,Christensenellaceae＿R-7＿group,and Coprococcus＿3 etc.(P<0.05).In conclusion,this study revealed the specific forms of YC action in the rumen and intestine by analyzing the effects of YC on sheep blood immunity,digestive tract enzyme activity,rumen and intestinal development and microorganisms.The study fills a gap in the study of the effect of YC on microorganisms in different ecological niches of the rumen,and systematically explores the regulatory role of YC in the small intestine and hindgut of ruminants.It provides a theoretical basis for the application of YC in ruminant production and provides new insights to resolve the mechanism of action of yeast cultures.