** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** ARTIFICIAL INSEMINATION (AI) IS THE PREFERRED METHOD OF BREEDING IN THE SWINE AND DAIRY CATTLE INDUSTRIES. DESPITES DECADES OF TECHNOLOGICAL DEVELOPMENT, THE EFFICIENCY OF AI, MEASURED BY PREGNANCY RATES AND NUMBER OF OFFSPRING, IS SEVERELY REDUCED BY SHORTCOMINGS IN THE PROCESSING AND DISTRIBUTION OF SEMEN. IN PARTICULAR THIS IS DUE TO INADEQUATE, POTENTIALLY HARMFUL (TO SPERM CELLS) COMPOSITION OF SEMEN EXTENDERS, THE DILUENTS USED TO PREPARE MULTIPLE AI SEMEN DOSES FROM EACH SEMEN COLLECTION. DURING PREVIOUS FUNDING PERIOD OF THIS PROJECT, WE CONFIRMED THAT THE MAIN PROTEIN RECYCLING CELLULAR MACHINERY, THE UBIQUITIN-PROTEASOME SYSTEM (UPS), REGULATES THE SPERM CAPACITATION PROCESS. IF IT OCCURS INSIDE FEMALE REPRODUCTIVE TRACT AFTER AI, CAPACITATION MAKES THE SPERM CELLS COMPETENT TO FERTILIZE AN EGG, BUT IT KILLS THEM IF IT HAPPENS PREMATURELY DURING SEMEN PROCESSING AND DISTRIBUTION. WE DISCOVERED NEW FACTORS THAT AFFECT THE PROCESS OF SPERM CAPACITATION, INCLUDING OUR GROUNDBREAKING DISCOVERY OF ZINC REQUIREMENT FOR SPERM SURVIVAL AFTER SEMEN COLLECTION. CONSEQUENTLY, WE OBSERVED THAT SUPPLEMENTING THE AI DOSES WITH ZINC INCREASED THEIR LONGEVITY AND FERTILIZING ABILITY. WE TERMED THIS APPROACH ZINC RELOADING. BASED ON THESE ADVANCES, THE GOAL OF THIS RENEWED PROJECT IS TO HARNESS THE POWER OF UPS AND ZINC RELOADING TO DEVELOP SEMEN EXTENDERS THAT WILL YIELD HIGHER PREGNANCY RATES AFTER AI. OUR SPECIFIC OBJECTIVES FOR THE NEXT PROJECT PERIOD ARE TO: 1) DEVELOP A TECHNIQUE THE ASSESSMENT OF SPERM ZINC CONTENT AS A TOOL FOR EVALUATION OF SEMEN QUALITY AND MALE FERTILITY; 2) DEVELOP SEMEN EXTENDERS OPTIMIZED FOR SPERM ZINC CONTENT, TO MITIGATE PREMATURE SPERM CAPACITATION AND MAXIMIZE UPS FUNCTION; AND 3) IDENTIFY SPERM FUNCTIONS AND PROPERTIES THAT ARE REGULATED BY ZINC. WE EXPECT TO IMPROVE FARM ANIMAL REPRODUCTION BY MAXIMIZING SPERM SURVIVAL AND FERTILITY DURING SEMEN PROCESSING AND DISTRIBUTION TO FARMS FOR AI. NEW KNOWLEDGE FROM THIS PROJECT WILL INCREASED OUR UNDERSTANDING OF SPERM CAPITATION AND INFORM THE FUTURE METHODOLOGY FOR AI SEMEN PROCESSING IN SWINE AND CATTLE.