Diagnosis of sperm-bound anti-sperm antibodies by flow cytometry and their association with semen quality

Table 1. ASA-bound spermatozoa and ASA-positive samples in twenty Information on anti-sperm antibodies (ASAs) in stallion populations is scarce. The objectives were to investigate presence of sperm-bound ASAs in a group of stallions, the association of ASA with semen quality and other factors that may affect the diagnosis. It was hypothesized that ASA binding differed with breeding soundness classification and semen quality. Stallion age, breed, sexual rest and season were proposed as determinants. After a breeding soundness examination, twenty stallions (2 to 26 years old) were classified as satisfactory or non-satisfactory breeders. One ejaculate per stallion was analyzed for ASA binding. Spermatozoa were labeled with fluorescein isothiocyanate (FITC)-labeled anti-equine IgG or IgA, and propidium iodide. Isotype controls were included. Percentages of IgGand IgA-bound live spermatozoa were analyzed with flow cytometry. Dead spermatozoa were excluded from the analysis since dead cells uptake labeling antibodies nonspecifically. Regional specificity of antibody binding had previously been confirmed with confocal laser microscopy but was not evaluated in this study. The percentage of ASAbound spermatozoa was compared between satisfactory (n1⁄411) and non-satisfactory breeders (n1⁄49), semen collected during the breeding (n1⁄414) or non-breeding (n1⁄46) season, sexually (n1⁄417) or non-sexually rested stallions (n1⁄43), or stallions of Quarterhorse (n1⁄414) and Warmblood (n1⁄46) breeds using a Wilcoxon rank test. ASA binding was compared among age groups with a Friedman test. Reference intervals (RIs) for ASA binding were established using 95% of the distributionwithin the 0.975 fractile of satisfactory breeders. Semen samples with a percentage of ASA-bound spermatozoa above the upper limit of the RIs