Screening Of Probiotic Lactic Acid Bacteria From Yak Vagina And Its Effect On Endometritis

Endometritis in cattle is mainly caused by pathogenic microbial infections during or after delivery,which can lead to destruction of uterine microecology,disruption of hormone secretion,decreased milk production,and even infertility.Traditionally,antibiotics are mainly used for treatment,but the problems they cause,such as drug residues,microbiota dysbiosis and pathogenic bacteria resistance,cannot be ignored.Studies have shown that probiotics have the ability to compete with pathogens for adhesion sites,antagonize pathogens,regulate the innate immune system,and improve host microecological balance,and can positively influence host physiological and metabolic functions.Due to the long-term evolution of probiotics,most of them are beneficial to host health only in specific natural ecological niches,thus vaginal-derived probiotics have the potential to prevent or treat endometritis in cattle.Yaks live in high altitude and alpine regions and have a unique micro-ecosystem.However,there is still a gap in the research on yak vaginal lactic acid bacteria（LAB）.Therefore,the following studies were conducted in this study.1.Isolation and identification of LAB from yak vaginaIn this experiment,25 samples were collected from the vagina of healthy yaks in Linxia City,Gansu Province,and the LAB were isolated and purified by Ca CO₃-MRS medium,then identified phenotypic and molecular biological methods.Five strains of LAB were finally identified as Leuconostoc mesenteroides,Lactiplantibacillus plantarum,Enterococcus hirae,Lacticaseibacillus camelliae,Limosilactobacillus mucosae.2.In vitro evaluation of the probiotic properties of LABIn this experiment,the probiotic properties of five LAB were evaluated by growth curve,acid production curve and H₂O₂ production test,acid and bile salt tolerance test,hydrophobicity determination,self-agglutination and coagglutination assay,bacterial inhibition test and adhesion test to bovine endometrial epithelial cells（BEECs）.The results showed that all strains had certain acid and bile salt tolerance properties,aggregation ability,effective inhibition against E.coli,S.aureus and Salmo.typhimurium,and could adhere to BEECs effectively.3.In vitro safety evaluation of LABTo evaluate the in vitro safety of five LAB,drug sensitivity test,virulence factor test,hemolytic test,gelatinase test and compatibility test were performed.The results showed that the five strains were sensitive to most antibiotics,did not carry virulence factors,did not have hemolytic and gelatinase activities,and were compatible between strains.4.In vivo safety evaluation of LABIn this experiment,YD6,YD9 and YD25,which showed good performance in in vitro probiotic tests and had potential as a mixture for the control of bovine endometritis,were selected to evaluate the in vivo safety of three LAB by the acute oral toxicity test and the 28-d oral toxicity test in mice.The acute oral toxicity test indicated that three LAB had no significant affect on activity,feeding and body weight of mice compared with the control group,and there were no significant organ lesions and no significant differences between organ index groups.The 28-day oral toxicity test indicated that three LAB had no effect on activity,feeding and body weight of mice,no significant organ lesion,no differences in organ indices between groups,and no displacement of bacteria.There was no significant effect of LAB on blood count,blood glucose,blood lipids,liver function and kidney function of mice.In conclusion,three LAB had no toxic side effects on mice and were safe in vivo.5.Effect of L.plantarum on LPS-induced inflammation in BEECsIn pre-experiments,L.plantarum was able to stimulate BEECs to produce a small amount of inflammatory factors,which was considered to have an immunostimulatory effect and was therefore used for further studies.In this experiment,an in vitro inflammation model of BEECs was firstly established with LPS,and then the effect of L.plantarum on the LPS-induced inflammation model of BEECs was detected by q RT-PCR and WB.The results showed that LPS significantly increased the expression of TLR4-mediated My D88-NF-κB signaling pathway-related proteins and inflammation-related factors（TNF-α,IL-1β,IL-6 and IL-8）in BEECs,while L.plantarum pretreatment of BEECs was able to decrease the expression of NF-κB signaling pathway-related proteins and inflammation-related factors in the LPS-induced inflammation model in BEECs.In conclusion,five LAB with in vitro probiotic properties and in vitro safety were isolated from yak vagina in this experiment.The toxicity test in mice showed that three LAB were safe in vivo.Cellular assays showed that L.plantarum may reduce the expression of LPS-induced inflammation-related factors through the TLR4signaling pathway.In summary,this experiment provide strain support and a basis for further development of microecological agents for the prevention and treatment of bovine endometritis in experimental studies.