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This study was designed to test the hypothesis that sperm-bound IgG and IgA decrease binding of bull spermatozoa to oviductal epithelial cells in vitro. Three ejaculates were cryopreserved from each of four antisperm antibody (ASA)-negative satisfactory breeder bulls. Bulls were then immunized with autologous spermatozoa, and three ASA-positive ejaculates were cryopreserved from each bull post-immunization. First, microscopy methods were compared to select the most appropriate assay for evaluation of oviductal binding index (BI). The BI did not differ when the evaluation was performed under fluorescence microscopy (131.1 sperm/mm2; 62.5–251.1 sperm/mm2), phase-contrast microscopy (160.5 sperm/mm2; 56.8–397.4 mm2) or their combination (116.4 sperm/mm2; 56.8–249.6 sperm/mm2) (Median; IQR). The combination of microscopy methods was selected as it allowed better visualization of cells. Then, BI was compared between ASA-negative and ASA-positive ejaculates, and the association between BI and ASA binding was evaluated. The BI was less in ASA-positive (114.9; 0 to 201.8 sperm/0.1 mm2) than ASA-negative samples (218.9; 24.7 to 276.8 sperm/0.1 mm2) (P = 0.0002). This reduction in BI was significant in three of the four bulls. Regression analysis identified a negative association between BI and the percentage of IgG-bound (p = 0.013) but not IgA-bound spermatozoa. In conclusion, sperm-bound IgG decreased the ability of bovine spermatozoa to bind to oviductal epithelial cells in vitro.