**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** METRITIS IS AN ACUTE INFLAMMATORY DISEASE OF MULTIPLE LAYERS OF THE UTERINE LINING WITH SYSTEMIC IMPLICATIONS THAT AFFECTS ~ 20% OF POSTPARTUM DAIRY COWS AND HAS MARKED WELFARE, HEALTH, PRODUCTION, REPRODUCTION, AND ECONOMIC CONSEQUENCES TO THE INDIVIDUAL ANIMAL AND THE HERD. THE DAIRY COW IS UNIQUE IN THE SENSE THAT VIRTUALLY ALL COWS ARE COLONIZED BY BACTERIA IN THE FIRST DAYS AFTER CALVING, AND FAILURE TO ELIMINATE PATHOGENIC BACTERIA LEADS TO THE ESTABLISHMENT OF DISEASE. OUR PREVIOUS WORK SHOWED THAT THE MICROBIOTA IS IDENTICAL BETWEEN COWS THAT DEVELOP METRITIS AND HEALTHY COWS UP UNTIL 2 DAYS POSTPARTUM, AFTER WHICH THE BACTERIAL NUMBER AND STRUCTURE DEVIATE IN FAVOR OF GREATER ABUNDANCE OF PATHOGENIC GRAM-NEGATIVE ANAEROBIC BACTERIA IN METRITIC COWS, PARTICULARLY FUSOBACTERIUM, BACTEROIDES AND PORPHYROMONAS. INTERESTINGLY, WE SAW THAT A SUB-COMMUNITY OF UTERINE PATHOGENS THAT INCLUDED FUSOBACTERIUM, BACTEROIDES AND PORPHYROMONAS WAS PRESENT IN BLOOD SHORTLY AFTER CALVING, WHICH INDICATE THAT A HEMATOGENOUS ROUTE OF UTERINE INFECTION WAS POSSIBLE IN COWS. THEREFORE, UTERINE PATHOGENS COULD BE TRANSFERRED TO THE UTERUS SHORTLY AFTER CALVING OR EVEN BEFORE CALVING. INDEED, A RECENT STUDY HAS IDENTIFIED A UTERINE MICROBIOME IN THE PLACENTA AND AMNIOTIC FLUID OF PREGNANT COWS, AND ANOTHER GROUP OF RESEARCHER WERE ABLE TO VISUALIZE IMPORTANT BACTERIA SUCH AS FUSOBACTERIUM NECROPHORUM AND PORPHYROMONAS LEVII WITHIN THE ENDOMETRIUM OF PREGNANT COWS USING FLUORESCENCE IN SITU HYBRIDIZATION (FISH). MEANWHILE, OUR GROUP HAS SHOWN THAT THETHE FIRST STOOL OF CALVES HAD A MICROBIOME, AND THAT THE ABUNDANCE OF BACTEROIDACEAE AND PREVOTELLACEAE, TWO KNOWN FAMILIES OF UTERINE PATHOGENS, IN CALVES' BLOOD WAS POSITIVELY CORRELATED WITH ABUNDANCE OF THESE BACTERIA IN COWS' BLOOD. THESE FINDINGS BEG THE QUESTION: IF BACTERIA CAN COLONIZE THE AMNIOTIC FLUID OF THE DAM AND THE GASTROINTESTINAL (GI) TRACT OF THE FETUS, AND CAN BE FOUND IN THE FETAL CIRCULATION, COULD IT ALSO COLONIZE THE UTERUS OF THE FETUS? IF THE ANSWER IS YES, WE WOULD LIKE TO STUDY POSSIBLE ROUTES OF BACTERIAL TRANSLOCATION FROM THE DAM TO THE FETAL UTERUS. IF THE ANSWER IS NO, THEN WE WOULD LIKE TO KNOW WHEN IN THE LIFE OF A CALF/HEIFER DOES THE UTERUS BECOME COLONIZED WITH BACTERIA. WE WOULD ALSO LIKE TO DETERMINE THE PROGRESSION OF THE UTERINE MICROBIOME FROM FETAL LIFE TO THE PREGNANT STATE. THIS KNOWLEDGE WOULD GIVE US A BETTER UNDERSTANDING OF THE UTERINE MICROBIOTA AND UTERINE DISEASE ETIOLOGY AND COULD COMPLETELY CHANGE THE WAY WE APPROACH UTERINE INFECTION IN COWS, FROM PREVENTION OF UTERINE INFECTION TO PREVENTION OF UTERINE MICROBIOME DYSBIOSIS. OUR LONG-TERM GOAL IS TO DEVELOP SOLUTIONS FOR IMPROVING ANIMAL HEALTH AND WELFARE. HEREIN, WE WILL INVESTIGATE THE UTERINE MICROBIOME FROM FETAL LIFE UNTIL PREGNANCY ESTABLISHMENT WITH THE OVERALL GOAL OF DETERMINING WHEN AND HOW A UTERINE MICROBIOME IS ESTABLISHED AND DETERMINING THE PROGRESSION OF THE UTERINE MICROBIOME UNTIL THE FIRST PREGNANCY. ADDITIONALLY, WE WILL INVESTIGATE THE ESTABLISHMENT AND PROGRESSION OF A MICROBIOME IN OTHER MAJOR ORGANS SUCH AS THE GI TRACT, LUNGS AND THE MAMMARY GLAND. OUR CENTRAL HYPOTHESIS IS THAT BACTERIA FROM THE DAM COLONIZE THE GI TRACT OF THE FETUS AND THEN TRANSLOCATE TO THE UTERUS OF THE FETUS OR THAT BACTERIA FROM THE DAM TRANSLOCATE TO THE BLOOD CIRCULATION OF THE CALF AND THEN COLONIZE THE UTERUS OF THE FETUS. ALTERNATIVELY, A CALF WOULD BE BORN WITHOUT A UTERINE MICROBIOME BUT WOULD ACQUIRE A MICROBIOME BEFORE THE ESTABLISHMENT OF ITS FIRST PREGNANCY. OUR RATIONALE FOR THIS PROPOSAL IS THAT DEVELOPING AN UNDERSTANDING OF THE ORIGIN AND PROGRESSION OF THE UTERINE MICROBIOME WILL PROVIDE INSIGHT ON UTERINE DISEASE ETIOLOGY AND ALLOW FOR THE DEVELOPMENT UTERINE MICROBIOME MODULATION STRATEGIES. ALTHOUGH OUR PROJECT IS FOCUSED ON THE UTERINE MICROBIOME, WE WILL ALSO DEVELOP AN UNDERSTANDING OF THE MICROBIOME OF MAJOR ORGANS SUCH AS THE GI TRACT, LUNGS AND THE MAMMARY GLAND FROM FETAL LIFE UNTIL ADULTHOOD, WHICH WILL PROVIDE INSIGHT INTO THE ETIOLOGY OF GI DISEASE, PNEUMONIA AND MASTITIS, THE MAJOR DISEASES OF DAIRY CALVES AND COWS. THIS IS EXPECTED TO IMPROVE ANIMAL HEALTH, WELFARE, AND FERTILITY WHICH IS EXPECTED TO ENHANCE FARM PROFITABILITY AND SUSTAINABILITY. FURTHERMORE, REDUCING DISEASE BURDEN WILL DECREASE ANTIBIOTIC USE, WHICH IS EXPECTED TO DELAY THE DEVELOPMENT OF BACTERIAL RESISTANCE TO ANTIBIOTICS USED IN HUMAN MEDICINE, WHICH WILL IMPROVE PUBLIC SENTIMENT TOWARDS ANIMAL AGRICULTURE WHICH WILL ULTIMATELY IMPROVE THE SUSTAINABILITY OF THE DAIRY INDUSTRY.