This paper outlines a 25 yr research strategy to better understand how proteins in male and female reproductive fluids influence fertility. Extensive fertility data available from artificial insemination cooperatives were used to identify individual dairy bulls with phenotypes of above and below average fertility. We determined whether the protein composition of reproductive fluids from these bulls differed with fertility. Surgical alteration of bulls to equip them with indwelling vas deferens cannulae enabled recovery of accessory sex gland fluid and cauda epididymal fluid and sperm for in vitro studies. Extensive analyses of reproductive fluids from these unique individuals enabled identification of several proteins that were associated with fertility. Methods to cannulate the oviducts of cows were also used to evaluate the composition and function of oviductal fluid. In vitro testing of proteins purified from male and female reproductive fluids demonstrated that they facilitated sperm capacitation, fertilization and early embryo development. Collectively these studies pursued a reductionist approach that started with the phenotype of an animal, use of surgical techniques to acquire reproductive fluids, use of analytical tools to define the composition and their relationships to fertility, and ultimately the purified proteins were tested with gametes and embryos to assess function. Using this approach, hypotheses were tested to determine fertilityrelated functions of proteins in reproductive fluids. The results provide an understanding of how fertility-related proteins contribute to the fertility phenotype of an individual and provide insights into potential applications to improve the fertility of cattle