**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 US DAIRY FARMERS INCLUDE FATTY ACID SUPPLEMENTS TO INCREASE MILK FAT YIELD AND MAXIMIZE RETURN ON INVESTMENT. HOWEVER, MANY FATTY ACIDSUPPLEMENTS WITH A WIDE VARIETYARE COMMERCIALLY AVAILABLE. FURTHERMORE, FATTY ACID SUPPLEMENTATION AFFECTS THE WHOLE ANIMAL METABOLISM. HENCE, OTHER PHYSIOLOGICAL FACTORS CONFOUND THE SPECIFIC ROLES OF FATTY ACIDS ON MAMMARY GLAND FAT SYNTHESIS.THE MOST WIDELY USED COMMERCIALLY AVAILABLE FATTY ACIDS ARE PALMITIC AND STEARIC. HOWEVER, THEIR SPECIFIC REGULATORY ROLES IN MILK FAT SYNTHESIS ARE UNCLEAR. WE HYPOTHESIZE FATTY ACIDS PALMITIC AND STEARIC ACID HAVE REGULATORY ROLES IN ENHANCING MILK FAT SYNTHESIS IN THE MAMMARY, EACH WITH UNIQUE MECHANISMS. THE STUDY'S FIRST OBJECTIVE IS TO DETERMINETHE SPECIFIC EFFECTS OF PALMITIC AND STEARICACIDS ON REGULATORS OF FATTY ACIDSYNTHESIS AND LIPOGENIC ENZYMES IN THE MAMMARY GLAND. WE WILL USE A MODIFIED CELL CULTURE MEDIUM WITH A MIXTURE OF FATTY ACIDS MIMICKING THE BLOOD PLASMA AS A BASAL MEDIA TO CHARACTERIZE THE IN-VIVO ENVIRONMENT BETTER. CELLS WILL BE TREATED WITH DIFFERENT DOSES OF PALMITIC AND STEARIC ACIDS, AND TOTAL RNA WILL BE EXTRACTED, SEQUENCED, AND THE WHOLE TRANSCRIPTOME ANALYZED. THE SECOND OBJECTIVE IS TO DETERMINETHE UPTAKE OF PALMITIC AND STEARICFATTY ACIDS THROUGH A MONOLAYER OFMAMMARY EPITHELIAL CELL LINESAND CHARACTERIZE THE PROTEINS THAT MEDIATE THE TRANSFER. WE EXPECT PALMITIC AND STEARIC FATTY ACIDS TO ENHANCE THE CELLULAR UPTAKE OF FATTY ACIDS AND TRIGLYCERIDE SYNTHESIS, EACH USING UNIQUE MECHANISMS OF ACTION AND AT DIFFERENT RATES. WE EXPECT STEARIC ACID TO HAVE A MORE PRONOUNCED INHIBITORY EFFECT ON DE NOVOBUT PALMITIC ACIDTO HAVE A MINIMAL INHIBITORY EFFECT ON DE NOVO LIPOGENESIS.