RATIONALEMETHANE (CH4), A POTENT GREENHOUSE GAS, IS NATURALLY PRODUCED IN THE RETICULO-RUMEN BY METHANOGENIC ARCHAEA UTILIZING HYDROGEN (H2) RELEASED DURING FEED FERMENTATION BY BACTERIA. RESEARCH INDICATES THAT UNDERREPRESENTED METHANOL-REDUCING METHANOSPHAERA AND METHANOL- AND METHYLAMINE-REDUCING METHANOMASSILIICOCCALES MAY HAVE A GREATER SHARE IN OVERALL CH4 PRODUCTION COMPARED WITH CO2 REDUCING METHANOBREVIBACTER; HOWEVER, KNOWLEDGE ON WHO THE MAIN ARCHAEAL PLAYERS ARE AND THEIR PROPORTIONAL CONTRIBUTION TO TOTAL CH4 FORMATION IN THE RUMEN IS LACKING. FURTHERMORE, THE FATE OF H2 WHEN CH4 PRODUCTION IS INHIBITED REMAINS UNRESOLVED AS EXCESSIVE H2 ACCUMULATION CAN INHIBIT FEED FERMENTATION IN THE RUMEN. THE GOAL OF THIS PROJECT IS TO DETERMINE THE CONTRIBUTION OF INDIVIDUAL METHANOGENS TO TOTAL CH4 FORMATION AND DETERMINE THE IMPLICATIONS OF INHIBITING INDIVIDUAL METHANOGENS ON REGULATION OF H2 METABOLISM IN H2-PRODUCING BACTERIA IN THE RUMEN. WE PROPOSE TO USE MACROALGA ASPARAGOPSIS TAXIFORMIS (AT) AS A FEED SUPPLEMENT THAT CAN SERVE AS POTENT INHIBITOR OF METHANOGENESIS IN THE RUMENHYPOTHESISTHE OVERARCHING HYPOTHESIS IS THAT METHANOSPHAERA AND METHANOMASSILIICOCCALES MAY HAVE A GREATER SHARE IN TOTAL CH4 FORMATION THAN WAS ORIGINALLY THOUGHT AND THAT SUPPLEMENTING DAIRY COWS WITH THE MACROALGA ASPARAGOPSIS TAXIFORMIS (AT) MAY SELECTIVELY ELIMINATE METHANOSPHAERA IN THE RUMEN.SPECIFIC OBJECTIVESOBJECTIVE 1: DECIPHER AND QUANTIFY THE RELATIVE CONTRIBUTIONS OF METHANOBREVIBACTER, METHANOSPHAER,A AND METHANOMASSILIICOCCALES TO TOTAL CH4 FORMATION IN THE RUMEN OF DAIRY COWS.THE GOAL OF THIS OBJECTIVE IS TO DETERMINE THE FUNCTIONAL ROLE OF DIFFERENT METHANOGENS, QUANTIFY THEIR CONTRIBUTION TO TOTAL CH4 FORMATION, AND DETERMINE HOW AT SELECTIVELY INHIBITS METHANOSPHAERA. TO ACHIEVE THIS, WE WILL FIRST PERFORM IN VITRO EXPERIMENTS WITH PURE CULTURES OF INDIVIDUAL METHANOGENS AND DETERMINE THEIR SENSITIVITY TO AT. SECOND, WE WILL PERFORM IN VITRO RUMEN FERMENTATION ASSAYS WITH RUMEN INOCULUM OBTAINED FROM DAIRY COWS WITH HIGH AND LOW CH4-YIELD PHENOTYPES. METHANE EMISSIONS AND THE RELATIVE ABUNDANCE OF INDIVIDUAL METHANOGENS WILL BE ASSESSED AT DIFFERENT TIMES IN A 24H INCUBATION PERIOD WITH AND WITHOUT THE ADDITION OF AT. FINALLY, AN IN VIVO 4 X 4 LATIN SQUARE EXPERIMENT WILL BE CONDUCTED USING 20 COWS WITH 4 TREATMENTS (0, 0.25, 0.5, AND 0.75% AT DIETARY DRY MATTER BASIS). AT THE END OF EACH PERIOD, RUMEN SAMPLES WILL BE OBTAINED FROM THE COWS WHICH WILL BE SUBJECTED TO AMPLICON, METAGENOMIC, AND METATRANSCRIPTOMIC APPROACHES TO DETERMINE DIFFERENCES IN MICROBIAL COMPOSITION AS WELL AS THEIR GENES AND TRANSCRIPTS. IN ADDITION, WE WILL ALSO COLLECT DATA ON CH4 EMISSIONS, DISSOLVED AND FREE H2 AND FERMENTATION IN THE RUMEN, DIETARY DRY MATTER INTAKE, AND MILK YIELD AND COMPOSITION.OBJECTIVE 2: DETERMINE THE FATE OF H2 UNDER CH4 INHIBITION IN THE RUMEN OF DAIRY COWS WITH DIFFERENT CH4 YIELD PHENOTYPES WITH AND WITHOUT AT SUPPLEMENTATION.THE GOAL OF THIS OBJECTIVE IS TO UNDERSTAND THE MECHANISMS INVOLVED IN H2 PRODUCTION AND UTILIZATION IN THE RUMEN. THE HYPOTHESIS IS THAT SUPPLEMENTING AT TO LOW CH4-YIELD COWS MAY BE EFFECTIVE IN NOT ONLY REDUCING CH4 BUT ALSO IN DIVERTING H2 TO NON-METHANOGENIC BACTERIA AND MAY IMPROVE THE ENERGY EFFICIENCY OF RUMEN FERMENTATION. FURTHER, BY DETERMINING THE EFFECT OF AT ON INDIVIDUAL METHANOGENS, AND TRACKING HYDROGENASES AND H2 UPTAKE BY NON-METHANOGENIC SINKS, THIS STUDY WILL BE ABLE TO ACCOUNT FOR H2 THAT IS SPARED WHEN CH4 IS INHIBITED AND WILL ALSO BE ABLE TO PREDICT FERMENTATION PATHWAYS. WE WILL USE BOTH METAGENOMICS AND METATRANSCRIPTOMICS TO TRACK HYDROGENASES AND H2 UPTAKE BY NON-METHANOGENIC SINKS IN THE RUMEN OF HIGH AND LOW CH4 EMITTERS SUPPLEMENTED WITH AT TO BETTER UNDERSTAND H2 METABOLISM IN THE RUMEN. EIGHT TO 10 COWS EACH FOR HIGH AND LOW CH4-YIELD PHENOTYPES WILL BE IDENTIFIED WITH HALF THE COWS IN EACH GROUP RECEIVING AT (0.5% DIETARY DRY MATTER) AND THE REMAINING COWS SERVING AS CONTROL. EACH EXPERIMENTAL PERIOD WILL BE 28 D, WITH 21 D FOR ADAPTATION TO THE DIET AND 7 D FOR SAMPLING AND DATA COLLECTION. DATA FOR CH4, CO2, AND H2 EMISSIONS, TOTAL TRACT DIGESTIBILITY, URINARY NITROGEN LOSSES, RUMEN FERMENTATION (INCLUDING DISSOLVED H2), RUMEN MICROBIOTA, MILK FATTY ACID PROFILE, AND PRODUCTION VARIABLES (DMI, MILK YIELD AND COMPOSITION) WILL BE ANALYZED.OBJECTIVE 3: DETERMINE THE MECHANISMS BY WHICH BUTYRATE PRODUCTION IS INCREASED IN H2-PRODUCING BACTERIA IN THE RUMEN WITH INHIBITED METHANOGENESIS USING LABELED PRECURSORS.THE GOAL OF THIS OBJECTIVE IS TO INVESTIGATE THE MECHANISMS THROUGH WHICH BUTYRATE-PRODUCING BACTERIA SENSE H2 UNDER INHIBITED METHANOGENESIS BY AT. WE WILL USE LABELED GLUCOSE AS A VFA PRECURSOR TO UNDERSTAND HOW BUTYRATE PRODUCTION IS ENHANCED UNDER CH4 INHIBITION WITH AT AND TO WHAT EXTENT THE LABELED GLUCOSE IS DIRECTED TO ACETATE, PROPIONATE, AND BUTYRATE USING IN VITRO EXPERIMENTS. TOTAL GAS PRODUCTION, GAS COMPOSITION, FERMENTATIONVARIABLES, FIBER DEGRADABILITY, AND MICROBIAL PROTEIN SYNTHESIS AND MICROBIOME COMPOSITION WILL BE MONITORED.EXPECTED RESULTSWE EXPECT TO QUANTIFY THE ROLE OF INDIVIDUAL METHANOGENS IN METHANOGENESIS AND TOTAL CH4 FORMATION IN THE RUMEN. FURTHER, WE ALSO ANTICIPATE UNDERSTANDING THE MECHANISM OF ACTION OF AT IN INHIBITION OF METHANOGENESIS. BY SUPPLEMENTING AT TO LOW CH4-YIELD DAIRY COWS, WE ANTICIPATE NOT ONLY INHIBITING METHANOGENESIS BUT ALSO DIVERTING H2 AWAY FROM CH4 FORMATION TO OTHER SINKS THAT HELP INCREASE ENERGY EFFICIENCY IN THE RUMEN. FROM THIS PROJECT, WE ANTICIPATE ENHANCING KNOWLEDGE ON MECHANISMS BY WHICH BUTYRATE-PRODUCING BACTERIA SENSE CHANGES IN H2 CONCENTRATIONS IN RESPONSE TO CH4 INHIBITION. OVERALL, THIS PROJECT MAY PROVIDE NEW INSIGHTS ON ENHANCING ENERGY EFFICIENCY WHILE MITIGATING CH4 EMISSIONS FROM DAIRY COWS.ANTICIPATED IMPACTBY UNDERSTANDING THE ROLE OF INDIVIDUAL METHANOGENS AND ALSO SELECTIVELY INHIBITING INDIVIDUAL METHANOGENS, THIS PROJECT WILL SIGNIFICANTLY REDUCE CH4 EMISSIONS FROM DAIRY COWS WITHOUT ADVERSELY AFFECTING INTAKE AND PRODUCTION PERFORMANCE. THE PROJECT WILL ALSO SHED LIGHT ON ALTERNATE PATHWAYS OF FERMENTATION IN THE RUMEN THAT HAVE THE POTENTIAL TO IMPROVE PRODUCTION EFFICIENCY IN DAIRY COWS.