SPAIN IS ONE OF THE EUROPEAN COUNTRIES WITH THE LARGEST HORSE POPULATION. THE GENETIC QUALITY OF OUR BREEDS, INCLUDING THE SPANISH PURE BREED, IS INTERNATIONALLY RECOGNIZED. AT PRESENT, THERE IS AN INCREASING INTEREST IN THE DEVELOPMENT AND COMMERCIALIZATION OF ASSISTED REPRODUCTIVE TECHNIQUES IN THE HORSE. NOWADAYS THE EQUINE SEMEN INDUSTRY AND THE IN VIVO PRODUCTION AND TRANSFER OF EMBRYOS ARE WELL DEVELOPED TECHNIQUES, CONTRASTING WITH EQUINE IN VITRO EMBRYO PRODUCTION. IN VITRO FERTILIZATION (IVF) ENABLES THE PRODUCTION OF EMBRYOS IN A MORE COST-EFFECTIVE AND EFFICIENT WAY COMPARED TO THE IN VIVO SYSTEMS BUT, SO FAR, THERE ARE NOT REPEATABLE PROTOCOLS FOR CONVENTIONAL IVF IN THE HORSE YET. THE INCONSISTENCY OF EQUINE IVF AND THE FACT THAT IT HAS NEVER BEEN ESTABLISHED COMMERCIALLY IS DUE, AT LEAST IN PART, TO THE FACT THAT THE PHYSIOLOGY OF SPERM-OOCYTE INTERACTION AND SOME BASIC ASPECTS OF FERTILIZATION ARE STILL NOT WELL UNDERSTOOD IN THIS SPECIES. ON THE OTHER HAND, THE PHYSIOLOGY OF THE PHENOMENA PRECEDING FERTILIZATION LARGELY VARIES BETWEEN THE HORSE AND OTHER DOMESTIC SPECIES. A WIDE RANGE OF STUDIES HAVE BEEN PUBLISHED ON EQUINE IVF ALTHOUGH THE RESULTS ARE INCONSISTENT. EVEN WHEN EQUINE OOCYTES CAN BE MATURED IN THE LABORATORY AND SPERMATOZOA CAN BE CAPACITATED IN VITRO, GAMETE CO-INCUBATION DOES NOT RESULT IN CONSISTENT FERTILIZATION OR EMBRYO DEVELOPMENT. A PREVIOUS WORK DEMONSTRATED THAT EXPOSURE OF IN VITRO MATURED EQUINE OOCYTES TO HOMOLOGOUS OVIDUCTAL FLUID RESULTS IN ENHANCED FERTILIZATION RATES. IN OTHER SPECIES, IT HAS BEEN SHOWN THAT THE COMPOSITION OF PRE-OVULATORY AND POST-OVULATORY OVIDUCTAL FLUID IS DIFFERENT AND THAT THEIR EFFECT ON IVF OUTCOME ALSO VARIES. IN THE HORSE, THE VOLUME OF FLUID THAT CAN BE HARVESTED FROM A SINGLE OVIDUCT IS SCARCE AND IS OBTAINED FROM POST-MORTEM MARES, SO THE PROHIBITION OF HORSE SLAUGHTER IN OTHER COUNTRIES HAS RESULTED IN THE DISCONTINUATION OF THESE RESEARCH LINES. THEREFORE, OUR LONG-TERM OBJECTIVE IS TO DEVELOP A RELIABLE EQUINE IVF PROTOCOL THAT ALLOWS FOR THE OPTIMIZATION OF CURRENT FERTILIZATION RATES AND THE BIRTH OF VIABLE OFFSPRING. THE SPECIFIC OBJECTIVES ARE: 1 TO CHARACTERIZE THE COMPOSITION OF THE PRE- AND POST-OVULATORY EQUINE OVIDUCTAL FLUID; 2 TO DEVELOP TWO FERTILIZATION MEDIA FOR EQUINE IVF, BASED ON THE PRE- AND POST-OVULATORY COMPOSITION OF THE OVIDUCTAL FLUID; 3 TO OPTIMIZE EQUINE IVF PROTOCOLS BY COMPARING THE EFFECT OF NATURAL OVIDUCTAL FLUID AND THE TWO SYNTHETIC MEDIA ON EQUINE SPERM CAPACITATION; 4 TO OPTIMIZE THE EQUINE IVF PROTOCOL BY COMPARING THE EFFECT OF NATURAL OVIDUCTAL FLUID AND THE TWO SYNTHETIC MEDIA (PRE- AND POST-OVULATORY) ON THE IN VITRO MATURED EQUINE OOCYTE; 5 TO DEVELOP AN EFFICIENT EQUINE IVF PROTOCOL, USING PRE-AND POST-OVULATORY NATURAL OVIDUCTAL FLUID AND THE TWO SYNTHETIC MEDIA; 6 TO ASSESS THE PREGNANCY RATE OF THE BLASTOCYSTS PRODUCED AFTER TRANSCERVICAL TRANSFER TO RECIPIENT MARES (DAY 25 OF PREGNANCY). HENCE, OUR GOAL IS TO MAKE AVAILABLE TO THE SCIENTIFIC COMMUNITY AND THE EQUINE INDUSTRY AN EFFICIENT, AFFORDABLE AND REPEATABLE IVF PROTOCOL AS WELL AS TO IMPROVE THE PRODUCTIVE AND REPRODUCTIVE EFFICIENCY OF THIS SPECIES.