Production and characterization of a turkey sperm mitochondrial monoclonal antibody and its usefulness for assessment of sperm integrity

Abstract

A monoclonal antibody (4C7/2E4) that binds to an antigen located on the midpiece of turkey spermatozoa has been produced and characterized. The antibody was also reactive to antigens on the midpiece of chicken, Japanese quail, and bobwhite quail spermatozoa but was unreactive to spermatozoa from guinea fowl, human, horse, boar, and bull. Immunogold transmission electron microscopy of turkey spermatozoa showed that the antibody was associated with the outer mitochondrial membrane. Titratable antibody binding was observed for extracts of turkey spermatozoa and isolated mitochondria. The antibody did not recognize proteins separated by SDS-PAGE, but on Western blots of proteins separated by native gradient-PAGE, the antibody recognized a broad band greater than 660 kDa. No effects on fertilizing capacity, embryonic mortality, or hatchability of eggs were observed when the antibody was added to turkey semen prior to insemination. When semen stored for 0, 3, 6, 24, and 48 h was immunolabeled with 4C7/2E4, the percentage of spermatozoa with unlabeled midpieces was significantly increased by 6 h of storage. The number of free mitochondria and damaged midpieces also significantly increased with storage.