**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THIS RESUBMITTED PROJECT RENEWAL APPLICATION BY PROJECT DIRECTOR PETER SUTOVSKY ADDRESSES THE PRIORITY AREA OF ANIMAL REPRODUCTION (A1211) WITHIN THE PROGRAM AREA OF ANIMAL HEALTH AND PRODUCTION AND ANIMAL PRODUCTS, WAS RANKED OUTSTANDING, #9 OVERALL (OUT OF 100 PROPOSALS REVIEWED IN FEBRUARY 2019). THUS, THE REVIEW PANEL RAISED ONLY MINOR CONCERNS AND THEREFORE, ONLY MINOR REVISIONS WERE MADE TO THIS RESUBMITTED PROPOSAL. AS IN THE PREVIOUS FUNDING PERIOD, OUR UMBRELLA HYPOTHESIS REMAINS THAT THE DEGRADATION OF PATERNAL MITOCHONDRIA AT FERTILIZATION (A.K.A. POST-FERTILIZATION SPERM MITOPHAGY) IS MEDIATED BY UBIQUITIN PROTEASOME SYSTEM (UPS) THROUGH AUTOPHAGY AND PROTEASOMAL PROTEOLYSIS. BASED ON OUR NEWEST DATA, WE NOW ALSO HYPOTHESIZE THAT THE CONTINGENT OF MITOPHAGY DETERMINANTS OF SPERMATOZOA IS REFLECTIVE OF SPERM MITOCHONDRIAL STRUCTURE AND FUNCTION, AND BY EXTENSION OF INDIVIDUAL MALES' SPERM QUALITY/FERTILITY. BUILDING ON EXTENSIVE PUBLISHED AND PRELIMINARY DATA FROM PREVIOUS FUNDING PERIOD (2013-2018), THE PROPOSAL CONTAINS TWO INDEPENDENT OBJECTIVES TIED TOGETHER BY OUR UNIQUE KNOWLEDGE OF UPS AND THE AUTOPHAGY/MITOPHAGY, TOGETHER PROMOTING MATERNAL INHERITANCE OF MITOCHONDRIA AND MITOCHONDRIAL GENES IN LIVESTOCK MAMMALS: OBJECTIVE #1: TO DECIPHER THE REGULATION OF POST FERTILIZATION SPERM MITOPHAGY IN PORCINE ZYGOTE AND RECAPITULATE IT IN A PORCINE CELL FREE SYSTEM. UNDER THIS OBJECTIVE, TWO EXPERIMENTS ARE DESIGNED TO DISCOVER AND VALIDATE KEY MOLECULES REGULATING SPERM MITOPHAGY IN THE IN VITRO FERTILIZED OOCYTES, USING OUR NOVEL PORCINE CELL FREE SYSTEM CELL FREE SYSTEM DATA AND TRANSGENIC MODEL CELLS FOR THE STUDY OF MITOPHAGY. OBJECTIVE #2: TO ESTABLISH THE RELATIONSHIP BETWEEN THE SPERM CONTINGENT OF MITOPHAGY DETERMINANTS AND MALE FERTILITY. UNDER THIS OBJECTIVE, WE WILL FOCUS ON AGGRESOMES, THE AUTOPHAGY-PRONE, UBIQUITIN-TAGGED PROTEIN AGGREGATES UNIQUELY FOUND IN THE MITOCHONDRIA OF FUNCTIONALLY AND MORPHOLOGICALLY NORMAL SPERMATOZOA, AND IN THE EXTRA-MITOCHONDRIAL AGGREGATES ASSOCIATED WITH ABERRANT SPERM PHENOTYPES. TWO EXPERIMENTS ARE DESIGNED TO CORRELATE THE AGGRESOME-BASED MORPHOMETRY OF SPERM TAIL MITOCHONDRIAL SHEATH AND EXTRAMITOCHONDRIAL AGGRESOMES WITH BOAR FERTILITY IN SINGLE SIRE AI SERVICE. FURTHERMORE, WE WILL MODULATE SPERM MITOCHONDRIAL METABOLISM DURING SEMEN STORAGE AND ASSESS THE INFLUENCE OF SPERM MITOPHAGY DETERMINANT CONTINGENT/AGGRESOME CONTENT ON INDIVIDUAL MALES' ABILITY TO RESPOND TO SUCH MITOCHONDRIAL BOOST/MODULATION. BY CONDUCTING THE PROPOSED, CAREFULLY THOUGHT-OUT, RESEARCH (SEE CHART, LEFT), WE ANTICIPATE ATTAINING BETTER UNDERSTANDING OF MECHANISMS PROMOTING CLONAL MITOCHONDRIAL INHERITANCE IN LIVESTOCK AND OTHER SPECIES, AND TO IMPROVE LIVESTOCK REPRODUCTIVE EFFICIENCY THROUGH BETTER MALE FERTILITY EVALUATION AND SEMEN PROCESSING.