** 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 PROPOSAL ADDRESSES THE UNMET NEED FOR DEVELOPING HIGH THROUGHPUT PHENOTYPING FOR COLLECTING COMPREHENSIVE PHENOTYPES OF PRODUCTION, REPRODUCTION AND FITNESS TRAITS, A MAJOR EMPHASIS AREA OF THE USDA BLUEPRINT FOR ANIMAL GENOME RESEARCH 2022-2027. WE IDENTIFIED A LARGE SET OF DELETERIOUS GENE VARIANTS ASSOCIATED WITH SPECIFIC SPERM DEFECTS IN THE GENOMES OF 85 GENETICALLY VALUABLE BULLS WITH SUBOPTIMAL ARTIFICIAL INSEMINATION (AI) FERTILITY. WE NOW PROPOSE TO DEVELOP A SECOND GENERATION, LABEL-FREE SPERM PHENOTYPING PIPELINE, ELIMINATING VARIANCE AND COSTS ASSOCIATED WITH THE CONVENTIONAL, PROBE-BASED APPROACHES, BASED ON THE SPECIFIC GENOMIC DETERMINANTS OF ABERRANT SPERM QUALITY RESULTING IN UNIQUE MORPHOMETRIC AND OPTICAL PROPERTIES OF SPERMATOZOA, CAPTURED BY THE NEXT-GENERATION, BRIGHTFIELD IMAGE-BASED FLOW CYTOMETRY (IBFC) AND TRANSLATABLE TO HIGH-PRECISION PHENOTYPING OF HERITABLE PRODUCTION TRAITS. OBJECTIVE #1 IS TO PHENOTYPE AND VALIDATE RARE DELETERIOUS POLYMORPHISMS ASSOCIATED WITH ABERRANT SPERM HEAD AND TAIL MORPHOLOGIES WILL GROUP THE 85 SEQUENCED BULLS GROUPED BASED ON PREVAILING SPERM DEFECT PHENOTYPE, TO BE PHENOTYPED BY USING A MULTIPLEX PROTEOMIC APPROACH VALIDATED BY OUR PRELIMINARY DATA, OBJECTIVE #2 WILL DEVELOP A LABEL FREE SPERM PHENOTYPING PIPELINE FOR BULL FERTILITY TESTING, TRANSLATABLE TO SOMATIC CELL PHENOTYPING FOR OTHER HERITABLE TRAITS BY USING IBFC'S DEDICATED FEATUREFINDER SOFTWARE INTEGRATED WITH OUR MACHINE LEARNING PIPELINE, FOLLOWED BY VALIDATION ASSESSING SPERM STRUCTURAL INTEGRITY, GENTLE LIVE CELL SORTING AND METABOLOMICS. SUCH HIGHLY INNOVATIVE GENOME-TO-PHENOME PROJECT WILL ALIGN ANIMAL GENOMIC SELECTION WITH STATE-OF-THE-ART ANIMAL ANDROLOGY, VETERINARY DIAGNOSTICS AND PRECISION BREEDING, SUPPORTING THE IMPROVEMENT OF LIVESTOCK INDUSTRY EFFICIENCY THROUGH GLOBAL APPROACHES INTEGRATING PRODUCTION, REPRODUCTION, NUTRITION, HEALTH, AND WELFARE.