Exploring The Regulatory Mechanisms Of Inulin Supplementation On Subclinical Mastitis In Dairy Cows Based On The Profile Of Gastrointestinal Microbiota And Metabolites

Bovine mastitis is a persistent disease in the dairy farming industry,affecting the health of dairy cows,the quality of raw milk,and the efficiency and benefits of the industry.Especially Subclinical Mastitis（SM）,because of its inconspicuous clinical symptoms,strong concealment and long incubation period,the incidence rate in actual production is often higher than that of Clinical Mastitis（CM）,and SM dairy cows have a higher incidence rate.It is very easy to become a source of infection in the herd to infect other healthy cows.Because SM is an early stage of mastitis,there are few targeted prevention and control measures for SM in production,which can easily lead to further aggravation of inflammation.Recent studies have shown that the occurrence and development of mastitis is related to the structure of host gastrointestinal flora.Inulin is a recognized prebiotic product that has been shown in both human and animal studies to optimize gut microbiota structure and enhance host immunity.Therefore,in the early stage of mastitis（i.e.,SM）,whether the alleviation of mastitis can be achieved by means of nutritional regulation is a direction worthy of attention and research.In this study,the gastrointestinal flora and metabolite structure were targeted to explore the effect and mechanism of inulin supplementation on SM.First,by comparing the differences in the structure of microflora and metabolites in the milk and gastrointestinal tract（rumen and feces）of healthy and mastitis cows,the structural characteristics of the gastrointestinal flora and metabolites in the gastrointestinal tract of dairy cows with mastitis and their relationship with udder inflammation were initially revealed.and then to explore the effect of inulin supplementation on SM inflammatory symptoms and its regulatory mechanism.This study intends to initially reveal the characteristics of mastitis from the perspective of gastrointestinal flora structure,and try to provide feasible relief measures for the early stage of mastitis by means of nutritional regulation.Experiment 1 Differences in lactation performance and the profile of milk microbiota and metabolites among healthy,SM and CM dairy cowsThe profile of microflora and metabolite in the mammary gland are closely related to theincidence of mastitis.The current study aimed to investigate differences in microbial diversity and metabolites in milk from healthy（H）,SM,and CM cows.According to somatic cell counts（SCC）,differential somatic cell counts（DSCC）,California mastitis test（CMT）results and udder clinical symptoms,cows with H,SM and CM were selected（n=20 per group）.16S r RNA sequencing and non-targeted metabolomics technology were used to detect the microorganisms and metabolites in the above 3 types of milk samples.The results showed:1)With the aggravation of mastitis,milk yield（P=0.021）,milk fat（P=0.036）and milk lactose（P<0.01）were significantly decreased,while milk SCC was significantly increased（P<0.01）.Among milk fatty acids,C4（P=0.04）,C16:1（P=0.03）,C18:3n6（P=0.03）,and C18:1c9（P=0.02）,etc.,while C14:1（P=0.02）,C18:1t9（P=0.02）and C18:2t6（P<0.01）significantly increased.2)Firmicutes（P=0.038）were significantly enriched in CM group,Actinobacteria（P=0.002）,Bacteroidetes（P=0.002）and Spirochaetes（P=0.002）=0.029)were significantly enriched in the SM group.The correlation analysis of microbiome and metabolome showed that there was a positive correlation between Staphylococcus,Streptococcus and ceramide in milk of CM group.In SM group,Acinetobacter,Corynebacterium were positively correlated with testosterone glucuronic acid and 5-methyltetrahydrofolate.Several beneficial commensal bacteria（Dietzia,Aeromicrobium,Alistipes,Sphingobacterium,etc.）,and metabolites participated in purine,vitamins and amino acids were significantly reduced.The results showed that potential pathogenic bacteria and inflammation-related metabolites were enriched in the milk of mastitis cows,while the abundance of commensal bacteria and compounds involved in maintaining normal physiological metabolism was decreased.Experiment 2 Differences in gastrointestinal microflora and metabolite among healthy SM and CM cowsThe invasion of pathogens from the external environment into the udder was previously thought to be the main cause of mastitis.However,recent studies reported that gastrointestinal microbes are vital in inflammation of distant extra-intestinal locations,including the mammary gland.Therefore,on the basis of experiment 1,the current study further compared the different characteristics of microbes and metabolites in gastrointestinal tract（rumen and feces）from H,SM and CM cows.The current experiment design was the same as experiment 1.The results showed that,with the increase of the degree of mastitis,the concentration of lactic acid（LA;P<0.01）,acetate,propionate,butyrate,valerate（P<0.001）and total volatile fatty acids（TVFA;P<0.01）in the rumen were decreased.In the rumen of CM cows,bacteria related to intestinal and oral inflammation were significantly increased,such as Lachnospiraceae（FDR-adjusted P=0.039）,Moraxella（FDR-adjusted P=0.011）,Neisseriaceae（FDR-adjusted P=0.036）,and the level of several pro-inflammatory metabolites,such as 12-oxo-20-dihydroxy-leukotriene B4(FDR-adjusted P=5.97×10^-9)and 10beta-Hydroxy-6beta-isobutyrylfuranoere mophilane(FDR-adjusted P=3.88×10^-10),were increased.In SM group,the increase in Ruminiclostridium＿9（FDR-adjusted P=0.042）and Enterorhabdus（FDR-adjusted P=0.043）were accompanied by methenamine(FDR-adjusted P=6.95×10^-6),5-hydroxymethyl-2-furfuraldehyde(FDR-adjusted P=2.02×10^-6)and 6-methoxy-bee-toxin(FDR-adjusted P=2.57×10^-5).The short-chain fatty acids producing bacteria and some probiotics,including Prevoterotoella＿1（FDR-adjusted P=0.045）,Bifidobacterium（FDR-adjusted P=0.035）,and 2-phenylbutyrate(FDR-adjusted P=4.37×10^-6),were significantly reduced.In the feces,compared with H group,the community diversity in SM and CM cows were decreased.Bifidobacterium（FDR-adjusted P=0.035）,Romboutsia（FDR-adjusted P=0.040）,less abundance of Lachnospiraceae＿NK3A20＿group（FDR-adjusted P=0.046）,Coprococcus（FDR-adjusted P=0.045）,Prevotellaceae＿UCG-003（FDR-adjusted P=0.046）and Alistipes（FDR-adjusted P=0.042）,and higher abundance of Proteobacteria（FDR-adjusted P=0.040）,Ruminococcus（FDR-adjusted P=0.040）0.041)and Escherichia-Shigellawas（FDR-adjusted P=0.043）in CM cows.Klebsiella（FDR-adjusted P=0.044）was significantly enriched in the feces of SM cows.Several similarities were observed in feces and serum metabolites,higher level of pro-inflammatory lipid products and less abundance of metabolites involved in secondary bile acids（SBAs）,energy and purine metabolism were identified both in SM and CM cows.In addition,serum IL-1β（P=0.038）,IL-6（P=0.022）,TNF-α（P=0.046）,triglyceride（TG;P=0.026）and total cholesterol（TC;P=0.039）concentrations from SM and CM dairy cows were increased.The level of high-density lipoprotein（HDL;P=0.041）was decreased.The current study suggested significant difference in the profile of microbiota and metabolites in gastrointestinal tract among H,SM and CM cows.Several potential pathogens and pro-inflammatory metabolites were enriched in the gastrointestinal tract of dairy cows with mastitis.Experiment 3 Effects of inulin supplementation on lactation performance,rumen and fecal fermentation parameters and expression of inflammatory factors in SM dairy cowsThe current study combined with in vitro and in vivo experiments.Eight inulin supplementation levels of 0.0（control）,0.2%,0.4%,0.6%,0.8%,1.0%,1.2%and 1.4%were set in vitro study.There were 9 culture flasks（3 replicates×3 incubation times）for each inulin supplementation level,and 3 incubation times（24,48 and 72 h）.Four healthy mid-lactating Holstein cows were selected as rumen fluid donors.The results showed that gas production（P=0.03）was significantly increased in the 0.8%inulin group,propionic acid（P=0.03）,butyric acid（P=0.04）,and total volatile fatty acids（P=0.02）were significantly increased at 0.8%and 1.0%The%inulin group increased significantly,while the NH₃-N concentration（P=0.02）and the ratio of acetate to propionic acid（A/P;P=0.04）were lowest in the 0.8%and 1.0%inulin groups.Microprotein concentrations（P=0.03）were significantly increased in the 0.8%,1.0%and 1.2%inulin groups.The calculation results of single-factor correlation effect index and multi-factor correlation effect index suggested that the 0.8%inulin supplementation showed the better responds.According to the results of in vitro test,40 SM cows with similar body condition were selected for the in vivo test,and the inulin supplementation levels were 0（control）,100,200,300 and 400 g/d/head,respectively.The experiment was lasted for 8 weeks.The results showed that:1)compared with the control group,the milk yield（P=0.031）,milk protein（P=0.034）and milk lactose rate（P=0.027）were increased,and the milk SCC was significantly decreased（P<0.01）in cows fed 300 g/d inulin.In addition,saturated fatty acids in milk,C4（P=0.032）,C11（P=0.044）,C13（P=0.041）,C15（P=0.022）,antiisoc15:0（P=0.041）,antiisoc17:0（P=0.041）and iso C17:0（P=0.035）ratios both increased in the 300 g/d inulin group,while the unsaturated fatty acid ratio（P=0.062）showed a downward trend with C20:1（P=0.087）.The proportions of C18:2n6c（P=0.093）,C18:3n3（P=0.081）,and all long-chain fatty acids（P=0.063）decreased with inulin supplementation.2)In the rumen and feces,inulin supplementation increased the concentrations of propionate（P=0.021;P=0.041）,butyrate（P=0.034;P=0.047）and total volatile acid（P=0.041;P=0.058）,and decreased the A/P（P=0.041;P=0.061）.The p H value（P=0.083;P=0.086）showed a downward trend.3)Inulin supplementation increased the concentration of IL-4（P=0.042）in the serum of SM cows and decreased Ig G（P=0.013）,IL-6（P=0.035）,IL-8（P=0.034）,TNF-α（P=0.042）,TC（P=0.042）,TG（P=0.037）and low-density lipoprotein（LDL）（P=0.020）concentrations.In addition,inulin supplementation decreased the LPS concentrations in the rumen（P=0.040）and serum（P=0.045）,with no significant changes in feces.The results of this experiment showed that inulin supplementation improved the lactation performance,increased the concentrations of propionate and butyrate in the rumen and feces,and decreased the milk SCC and expression levels of pro-inflammatory factors in SM dairy cows.In addition,300g/d inulin addition level showed better responds on most detected indexs.Experiment 4 The mechanism of inulin supplementation regulating SM inflammatory response in dairy cowsThe current study was performed based on experiment 3 to investigated the effects ofinulin supplementation on the profile of microflora and metabolites in the gastrointestinal tract and milk as well as the levels of serum metabolites and blood lipids in SM cows.To further explored the regulatory mechanism of inulin on the inflammatory response of SM.The results showed:1)In the rumen and feces,300g/d inulin supplementation increased the abundance of Bacteroidota（FDR-adjusted P=0.033;FDR-adjusted P=0.048）and decreased Firmicutes（FDR-adjusted P=0.037;FDR-adjusted P=0.047）.At the genus level,the relative abundance of propionate-and butyrate-producing bacteria,such as Bifidobacterium（FDR adjusted P=0.043;FDR adjusted P=0.032）and Lachnospiraceae＿NK3A20＿group（FDR adjusted P=0.046;FDR adjusted P=0.034）and Alistipes（FDR adjusted P=0.034;FDR-adjusted P=0.046）in the rumen and feces,and unclassified＿o＿＿Bacteroidales（FDR adjusted P=0.040）)in the rumen,were increased.Some pro-inflammatory microorganisms such as Clostridia＿UCG-014（FDR-adjusted P=0.035）,Streptococcus（FDR-adjusted P=0.048）,Staphylococcus（FDR-adjusted P=0.045）,Neisseriaceae（FDR-adjusted P=0.046）and Escherichia-Shigella（FDR-adjusted P=0.045）in the rumen were decreased.Paeniclostridium（FDR-adjusted P=0.034）,unclassified＿c＿＿Clostridia（FDR-adjustedP=0.034）,and unclassified＿f＿＿Peptostreptococcaceae（FDR-adjusted P=0.040）were decreased in the feces.Meanwhile,metabolites involved in energy and amino acids uch as melibiose（FDR-adjusted P=0.006;FDR-adjusted P=0.003）and L-glutamate（FDR-adjusted P=0.017;FDR-adjusted P=0.046）were decreased both in the rumen and feces.Elevated SBAs（deoxycholic acid（FDR-adjusted P=0.006）,taurodeoxycholic acid（FDR-adjusted P=0.040）,and faeostanoic acid（FDR-adjusted P=0.032））were observed in feces and serum.However,lipid pro-inflammatory metabolites（ceramide（d18:0/13:0）（FDR-adjusted P=0.010）and 12-oxo-20-dihydroxyleukotriene B4（FDR-adjusted P=0.046）were decreased in the rumen,feces and serum.2）In the milk,300g/d inulin supplementation increased the relative abundance of probiotics（Lactobacillus（FDR-adjusted P=0.044）,Bifidobacterium（FDR-adjusted P=0.026））in SM cow,and decreased mastitis-causing and potentially pro-inflammatory bacteria such as Escherichia-Shigella（FDR-adjusted P=0.045）,Pseudomonas（FDR-adjusted P=0.038）,Rhodococcus（FDR-adjusted P=0.031）,and Burkholderia-Caballeronia-Paraburkholderia（FDR-adjusted P=0.031）etc.).Meanwhile,arachidonic acid pro-inflammatory mediators(leukotriene E3(FDR-adjusted P=4.52×10^-5),20-carboxy-leukotriene B4(FDR-adjusted P=5.59×10^-5),12-Oxo-c-leukotriene B3（FDR-adjusted P=0.001）)and phospholipid metabolite levels were reduced.Whereas,the level of compound involved in energy metabolism（citric acid（FDR-adjusted P=0.002）,L-carnitine（FDR-adjusted P=0.001）,etc.）were increased.The results of this experiment showed that inulin supplementation promoted the proliferation of propionate-and butyrate-producing bacteria and commential bacteria in the gastrointestinal tract and milk of SM cows,increased the levels of compounds involved in energy,purine,amino acid and SBAs metabolism,inhibited theproliferation of potential of pathogens and abundance of lipid pro-inflammatory metabolites.In this experiment,the microbiota and metabolite regulated by inulin were partially similar to the differential flora and metabolite species identified in experiments 1 and 2,indicating that inulin can target SM-specific flora and metabolites.In conclusion,the present study showed significant differents in the profile of microflora and metabolites in gastrointestinal tract（rumen and feces）and milk among healthy,SM and CM cows.The potential pathogenic bacteria and pro-inflammatory metabolites were riched,while the commensal bacteria and compounds involved in maintaining normal physiological metabolism and short-chain fatty acids were decreased in the rumen,feces and milk of mastitic cows.During mastitis,abnormal blood lipid levels are mainly manifested by elevated TC and TG.Supplemention with inulin to SM cows increased the concentration of propionate and butyrate-producing bacteria and some probiotics,and the concentration of propionate and butyrate in the rumen and feces,whereas inhibit several potential pro-inflammatory and pathogens.Inulin supplementation elevated the levels of metabolites involved in the energy,amino acids and SBAs metabolism in the gastrointestinal tract,and decreased the levels of pro-inflammatory lipid metabolites.Inulin intake also inhibited the expression of inflammatory cytokines and the concentration of TG and TC in serum,which may be related to increased propionate and SBAs metabolites.The improvements of gastrointestinal tract environment further influence the mammary gland environment.Increased beneficial symbiotic bacteria and less mastitis-causing bacteria,pro-inflammatory metabolites and milk SCC were found in milk of SM cow after inulin intake.Meanwhile,the milk yield,milk protein and lactose rate were improved.