** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE BEEF AND DAIRY INDUSTRY ARE THE PRIMARY SUPPLIERS OF DIETARY PROTEIN SOURCES AIDING TO MEET THE GROWING GLOBAL DEMAND, WHICH RELIES HEAVILY ON ANIMAL REPRODUCTION AS A CENTRAL DETERMINANT OF PRODUCTION EFFICIENCY. UNFORTUNATELY, DAIRY CATTLE FERTILITY IS CONTINUALLY DECLINING WORLDWIDE, WHICH HAMPERS IMPLEMENTED EFFORTS TO ADVANCE ECONOMIC EFFICIENCY IN THE DAIRY SECTOR. EVEN THOUGH ASSISTED REPRODUCTIVE TECHNOLOGIES (ARTS) CURRENTLY ACCOUNT FOR A GROWING PORTION OF LIVESTOCK AND HUMAN POPULATIONS, ART-INDUCED OXIDATIVE STRESS CAN ALSO CONTRIBUTE TO IMPACTS ON EMBRYO DEVELOPMENT WITH LONG-TERM CONSEQUENCES ON FETAL GROWTH AND OFFSPRING HEALTH. AMONG THE SEVERAL MOLECULAR MECHANISMS ASSOCIATED WITH AN EMBRYO'S RESPONSE TO STRESS, WE HAVE PREVIOUSLY FOUND THE NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 - KELCH-LIKE ECH-ASSOCIATED PROTEIN 1 (NRF2-KEAP1) PATHWAY TO ACT AS A FIRST-LINE DEFENSE MECHANISM, WITH THE ABILITY OF THE EMBRYO TO ACTIVATE ITS NRF2-SIGNALING HIGHLY ASSOCIATED WITH ITS SURVIVAL AND VIABILITY UNDER OXIDATIVE STRESS CONDITIONS. THEREFORE, APPROACHES TO INDUCE NRF2 PROTEIN ACTIVITY IN BOVINE EMBRYOS WILL CONTRIBUTE TO IMPROVED SURVIVAL, VIABILITY, AND ENHANCED REPRODUCTIVE EFFICIENCIES FOLLOWING ARTS IN ANIMALS. MOREOVER, HERE WE AIM TO IMPLEMENT A DUAL-TARGETED APPROACH USING CRISPR-CAS9 MEDIATED KNOCKDOWN OF KEAP1 GENE (NRF2-INHIBITOR) IN SPECIFIC AIM 1 AND A PHARMACOLOGICAL STRATEGY USING THE ANTIOXIDANT QUERCETIN IN THE IN VITRO CULTURE SYSTEM IN SPECIFIC AIM 2, TO INCREASE THE RELATIVE ABUNDANCE OF LOCALIZED NRF2 PROTEIN IN BOVINE PREIMPLANTATION EMBRYOS TO PROMOTE THEIR SURVIVAL AND VIABILITY UNDER OXIDATIVE STRESS CONDITIONS. THE MODULATION OF THIS PATHWAY IN PREIMPLANTATION EMBRYOS IS EXPECTED TO REDUCE OXIDATIVE DAMAGE IN THE RESULTING BLASTOCYSTS TO ENHANCE THE SURVIVAL AND VIABILITY OF EMBRYOS UNDER SUBOPTIMAL CONDITIONS, PARTICULARLY WITH RESPECT TO CURRENTLY IMPLEMENTED ART PROCEDURES. MOREOVER, THE ACTIVATION OF NRF2 IN THE PREIMPLANTATION PERIOD HAS THE POTENTIAL TO MITIGATE STRESS-INDUCED ABNORMAL MOLECULAR ALTERATIONS, THEREBY ATTENUATING LONG-TERM NEGATIVE CONSEQUENCES OF STRESS ON FETAL GROWTH AND OFFSPRING HEALTH. THE PLANNED PROJECT WILL IDENTIFY GENES AND GENOME REGIONS, THAT CAN BE TARGETS FOR FUTURE INTERVENTION AGAINST PHYSIOLOGICAL AND ENVIRONMENTAL STRESS-INDUCED INFERTILITY PROBLEMS IN DAIRY AND BEEF CATTLE.