DECIPHERING THE CROSSTALK BETWEEN BACTERIA-ARCHAEA INTERACTIONS IN THE RUMEN AND METHANE-YIELD PHENOTYPE OF DAIRY COWS.PRINCIPAL INVESTIGATOR: DIPTI PITTA, UNIVERSITY OF PENNSYLVANIACO-PRINCIPAL INVESTIGATOR: ALEX HRISTOV, THE PENNSYLVANIA STATE UNIVERSITYRATIONALEMETHANE (CH4) ACCOUNTS FOR 14% OF TOTAL GLOBAL GREENHOUSE GAS (GHG) EMISSIONS AND IS 28 TIMES MORE POTENT THAN CO2. IN RUMINANTS, METHANE IS NATURALLY PRODUCED IN THE RETICULO-RUMEN BY METHANOGENIC ARCHAEA UTILIZING HYDROGEN RELEASED BY OTHER MICROBES DURING FEED FERMENTATION. ACCORDING TO USEPA, ENTERIC CH4 EMISSIONS FROM FARMED RUMINANTS ACCOUNTS FOR 25% OF THE TOTAL METHANE EMISSIONS IN THE UNITED STATES, POSING AN ENVIRONMENTAL THREAT. FOR THE RUMINANT HOST, CH4 PRODUCTION IS AN ENERGY INEFFICIENT PROCESS AS UP TO 12% OF GROSS FEED ENERGY IS LOST IN CH4 FORMATION. SEVERAL MITIGATION STRATEGIES HAVE BEEN PROPOSED, SOME HAVE AN ADVERSE EFFECT ON ANIMAL PRODUCTIVITY AND HEALTH; THE EFFECTIVENESS OF ONLY A FEW HAS BEEN DEMONSTRATED IN LIVE ANIMAL EXPERIMENTS. INCONSISTENT RESPONSE TO MITIGATION EFFORTS IS IN PART DUE TO A LACK OF UNDERSTANDING OF THE MECHANISTIC BASIS OF METHANOGENESIS AND THE IMPLICATIONS OF MITIGATION STRATEGIES ON RUMEN MICROBIOTA AND CONSEQUENTLY FERMENTATION. WE PROPOSE THAT UNDERSTANDING THE ROLE OF METHANOGENS IN METHANE PRODUCTION AND INVESTIGATING THE BACTERIA-ARCHAEA INTERACTIONS AND THEIR IMPACT ON RUMEN FERMENTATION IS NEEDED TO REDUCE METHANE FORMATION AND AS A CONSEQUENCE IMPROVE NUTRIENT UTILIZATION AND ULTIMATELY INCREASE PRODUCTIVITY OF DAIRY COWS.HYPOTHESISTHE LONG-TERM GOAL OF THIS PROJECT IS TO IDENTIFY ARCHAEA AND THEIR FUNCTIONAL GENE REPERTOIRES THAT ARE RESPONSIBLE FOR LOW-METHANE EMISSION PATHWAYS IN THE RUMEN AND DETERMINE THE SYNERGISTIC INTERACTIONS BETWEEN BACTERIA-ARCHAEA COHORTS NEEDED TO PARTIALLY DIVERT H2 TO ALTERNATE PATHWAYS TO SUSTAIN EFFICIENT RUMEN FERMENTATION AND ANIMAL PRODUCTIVITY. THE OVERARCHING HYPOTHESIS IS THAT SPECIFIC COHORTS OF BACTERIA AND ARCHAEA ARE INTERCONNECTED TO PARTIALLY SPARE HYDROGEN FROM FORMING METHANE IN THE RUMEN.SPECIFIC OBJECTIVESOBJECTIVE 1: IDENTIFY DIFFERENCES IN METHANOGENIC PATHWAYS AND DETERMINE THE RELATIONSHIP BETWEEN BACTERIA-ARCHAEA, HYDROGEN CONCENTRATION, AND FERMENTATION PATTERN IN THE RUMEN OF DAIRY COWS WITH CONTRASTING METHANE-YIELD PHENOTYPES.OUR GOAL IS TO UNDERSTAND THE ROLE OF BACTERIA-ARCHAEA INTERACTIONS IN COWS DIFFERING IN METHANE YIELD PHENOTYPE. PREVIOUS WORK INDICATES VOLUME OF METHANE FORMED IS HERITABLE AND REPEATABLE, REVEALING A NATURAL VARIATION IN METHANE PHENOTYPE OF DAIRY COWS. AS METHANE IS A SEQUEL OF MICROBIAL ACTIVITY, WE HYPOTHESIZE THAT DAIRY COWS DIFFERING IN METHANE YIELD WILL HAVE DIFFERENT MICROBIAL COMPOSITION. TO TEST THIS, WE WILL SCREEN DAIRY COWS FOR THEIR METHANE EMITTING POTENTIAL AND DIFFERENTIATE THEM AS HIGH AND LOW METHANE EMITTERS. FROM THESE COHORTS, WE WILL SELECT HIGH AND LOW METHANE EMITTING COWS FOR RUMEN CANNULATION TO SAMPLE THEIR RUMEN CONTENTS. THESE RUMEN SAMPLES WILL BE SUBJECTED TO AMPLICON, METAGENOMIC AND METATRANSCRIPTOMIC APPROACHES TO DETERMINE DIFFERENCES IN MICROBIAL COMPOSITION, THEIR GENES AND TRANSCRIPTS. IN ADDITION, WE WILL ALSO COLLECT DATA ON METHANE EMISSIONS, DISSOLVED AND FREE HYDROGEN AND FERMENTATION IN THE RUMEN, DIETARY DRY MATTER INTAKE AND MILK YIELD AND COMPOSITION.OBJECTIVE 2: DETERMINE THE EXTENT TO WHICH METHANE INHIBITORS CAN AFFECT RUMEN MICROBIOTA, RUMEN FERMENTATION, AND DAIRY COW PRODUCTIVITY.OUR GOAL IS TO INHIBIT METHANOGENESIS BY SUPPLEMENTING DAIRY COWS WITH METHANE INHIBITORS AND INVESTIGATE THE IMPACT ON MICROBIOTA AND THEIR GENE EXPRESSION, METHANE EMISSIONS AND PRODUCTIVITY. IN ADDITION TO KNOWN MITIGATION AGENTS, WE PROPOSE TO USE SEAWEED VARIETIES AS A NOVEL STRATEGY TO DECREASE ENTERIC METHANE EMISSIONS FROM DAIRY COWS. AS AN INITIAL STEP, WE WILL SCREEN SEAWEED VARIETIES AT DIFFERENT INCLUSION RATES FOR THEIR METHANE EMITTING POTENTIAL USING ESTABLISHED IN VITRO ASSAYS. SEVERAL VARIETIES WILL BE FURTHER EVALUATED IN VIVO IN LACTATING DAIRY COWS. MEASUREMENTS ON RUMEN MICROBIAL AND ANIMAL PERFORMANCE DATA WILL BE SIMILAR TO OBJECTIVE 1.OBJECTIVE 3: DETERMINE IF NON-INVASIVE METHODS SUCH AS ORAL SWABS, BOLUS, RUMEN FLUID USING GASTRIC LAVAGE AND FECAL SAMPLES CAN SERVE AS PROXIES FOR MICROBIAL COMPOSITION OF RUMEN CONTENTS TO IDENTIFY MICROBIAL MARKERS AND CONSEQUENTLY LOW METHANE-YIELD PHENOTYPE IN DAIRY COWS.EXPECTED RESULTSWE EXPECT THAT THE RUMINAL MICROBIOTA WILL BE DIFFERENT BETWEEN HIGH AND LOW METHANE-YIELD PHENOTYPE DAIRY COWS. FURTHERMORE, WE PREDICT THAT SPECIFIC COHORTS OF BACTERIA AND ARCHAEA ARE SELECTIVELY INHIBITED IN LOW-METHANE YIELD PHENOTYPE DAIRY COWS AND THAT THE USE OF MITIGATION AGENTS WILL FURTHER INHIBIT THESE RUMEN MICROBES, THUS GREATLY REDUCING ENTERIC METHANE EMISSIONS. WE ANTICIPATE THIS PROJECT WILL ENHANCE OUR UNDERSTANDING OF THE FUNCTIONAL ROLE OF MICROBES IN METHANOGENESIS BY USING THE COMBINED APPROACH OF METAGENOMICSAND METATRANSCRIPTOMICS. FURTHER, WE EXPECT TO GAIN NOVEL INFORMATION THROUGH DELINEATING THE INTERACTION BETWEEN METHANE PRODUCTION, HYDROGEN UTILIZATION, FERMENTATION VARIABLES AND THE MICROBIOTA TO SHED LIGHT ON METABOLIC ACTIVITIES OF BACTERIA AND ARCHAEA IN METHANE PRODUCTION AND NUTRIENT UTILIZATION.ANTICIPATED IMPACTBY IDENTIFYING DIFFERENCES IN MICROBIAL GENE EXPRESSION ASSOCIATED WITH METHANOGENESIS IN HIGH AND LOW METHANE-YIELD PHENOTYPE DAIRY COWS, THIS PROJECT WILL IDENTIFY POTENTIAL MICROBIAL MARKERS AND/OR TARGETS FOR METHANE MITIGATION. WE ALSO PREDICT THIS PROJECT WILL LAY THE FOUNDATION FOR SELECTION OF MICROBIAL MARKERS THAT CAN BE USED IN CONJUNCTION WITH HOST GENES TO IDENTIFY SELECTION CRITERIA FOR IMPROVING SELECTION OF DAIRY CATTLE.