**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** OUR PROPOSED RESEARCH DEVELOPS METHODS AND PROCEDURES TO INVESTIGATE THE IMMUNE CELLS WITHIN THE RUMEN OF DAIRY COWS. CURRENTLY, WE DO NOT KNOW WHAT IMMUNE CELLS RESIDE IN THE RUMEN OR WHERE THEY RESIDE WITHIN THE RUMEN WALL. ALSO, WE DO NOT KNOW HOW DIET AFFECTS THESE IMMUNE CELL POPULATIONS. IMPROVING OUR UNDERSTANDING OF RUMINAL IMMUNE CELLS AND HOW THEY ARE AFFECTED BY DIET MAY HELP US DESIGN RATIONS OR FEED ADDITIVES THAT WILL IMPROVE HEALTH, WELFARE, AND EFFICIENCY OF LIVESTOCK. DOING SO WILL CONTINUE TO IMPROVE THE EFFICIENCY OF RUMINANT LIVESTOCK PRODUCTION AND SUSTAINABILITY.WE ARE GOING TO FEED DAIRY COWS DIETS THAT DIFFER IN THE AMOUNT OF GRAIN. THEN WE WILL COLLECT TISSUE SAMPLESFROM THE RUMEN AND USE FLOW CYTOMETRY AND IMMUNOHISTOCHEMISTRY TO INVESTIGATE THE IMMUNE CELLS IN THESE SAMPLES. FLOW CYTOMETRY AND IMMUNOHISTOCHEMISTRY ALLOW US TO QUANTIFY HOW MANY OF EACH SPECIFIC CELL TYPE ARE PRESENT AND DETERMINE WHERE THEY RESIDE WITHIN THE RUMEN EPITHELIUM. THIS DATA WILL THEN BE ANALYZED AND DISSEMINATED BY WRITING SCIENTIFIC ARTICLES, EXTENSION ARTICLES, AND THROUGH PRESENTATIONS AT SCIENTIFIC CONFERENCES.THE ULTIMATE GOAL OF THIS PROJECT IS TO ESTABLISH METHODS THAT CAN BE TO BETTER UNDERSTAND THE ROLE THAT IMMUNE CELLS PLAY IN THE RUMEN AND TO UNDERSTAND HOW FEEDING ADDITIONAL GRAIN TO CATTLE MAY AFFECT THE RUMEN TISSUE AND IMMUNE CELLS THAT MAY RESIDE THERE. FURTHERMORE, ESTABLISHING THESE METHODS WILL ALLOW FOR INVESTIGATION OF HOW HEAT STRESS, METHANE INHIBITING FEED ADDITIVES, PROBIOTICS,OR DIFFERENT MANAGEMENT STRATEGIES AFFECT HEALTH AND INFLAMMATION OF THE RUMEN. UNDERSTANDING HOW THE RUMEN IS AFFECTED UNDER EACH OF THESE SCENARIOS WILL YIELD INTERVENTIONS THAT IMPROVE THE HEALTH AND EFFICIENCY OF THE RUMEN WHICH WILL INCREASE ANIMAL WELFARE AND IMPROVE THE EFFICIENCY OF RUMINANT LIVESTOCK PRODUCTION.