** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** DISCOVERY OF THE ROLE OF INTERFERON-TAU (IFNT) IN PREGNANCY RECOGNITION IS CONSIDERED A SEMINAL FINDING IN RUMINANT REPRODUCTION. ALL TYPE I INTERFERONS, INCLUDING IFNT, ACTIVATE THE TYPE I INTERFERON RECEPTOR, IFNAR, WHICH IS A HETERODIMER MADE UP OF TWO SUBUNITS: IFNAR1 AND IFNAR2. WE HAVE PRODUCED GENETICALLY MODIFIED SHEEP WITH IFNAR2 INACTIVATED ON EITHER ONE OR BOTH CHROMOSOMES. EXPERIMENTS CONDUCTED OVER THE PAST YEAR HAVE ESTABLISHED THAT BIALLELIC IFNAR2 KNOCKOUT EWES THAT DO NOT HAVE A FUNCTIONAL TYPE I INTERFERON RECEPTOR CAN BECOME PREGNANT AND HAVE A COMPLETELY NORMAL PREGNANCY. THIS UNEXPECTED AND NOVEL DISCOVERY IMPLIES THAT THE ACCEPTED PARADIGM FOR PREGNANCY RECOGNITION IN RUMINANTS NEEDS TO BE REVISED. THIS PROJECT IS BASED ON THE GENERAL HYPOTHESIS THAT SINCE IFNT SIGNALING VIA IFNAR IS NOT REQUIRED FOR PREGNANCY IN SHEEP, THERE MUST BE A REDUNDANT OR ALTERNATIVE MECHANISM OF PREGNANCY RECOGNITION IN RUMINANTS. THE SPECIFIC AIMS FOR THE PROJECT ARE TO: (1) DETERMINE DIFFERENCES IN TROPHOBLAST, ENDOMETRIAL EPITHELIAL CELL, AND CORPUS LUTEUM GENE EXPRESSION IN WILDTYPE VERSUS BIALLELIC IFNAR2 KNOCKOUT PREGNANCIES WHERE NEITHER THE DAM NOR CONCEPTUS EXPRESSES IFNAR; AND (2) IDENTIFY CANDIDATE ANTILUTEOLYTIC OR LUTEOTROPIC AGENTS PRESENT IN UTERINE LUMINAL FLUID AND SECRETED BY CULTURED TROPHOBLAST CELLS. FOR AIM ONE, GENE EXPRESSION WILL BE CHARACTERIZED BY SINGLE-CELL RNA-SEQ. FOR AIM TWO, LC-MS/MS WILL BE USED TO CHARACTERIZE UTERINE LUMINAL FLUID AND TROPHOBLAST CELL SUPERNATANTS. THIS PROPOSAL DIRECTLY ADDRESSES TWO ANIMAL REPRODUCTION PROGRAM PRIORITY AREAS, EMBRYONIC AND FETAL DEVELOPMENT,AND GONADAL FUNCTION.