Enrichment of Bovine X-and Y-Chromosome-Bearing Sperm with Monoclonal H-Y Antibody-Fluorescence-Activated Cell Sorter

Bovine spermatozoa were assessed indirectly for the presence of a Y chromosome by monitoring expression of the H-Y antigen. Spermatozoa labeled with a monoclonal H-Y antibody (MoAb) and fluorescein-conjugated goat antibody to mouse F(ab)2 were counted with both a fluorescent microscope and a fluorescence-activated cell sorter (FACS). Of ejaculated spermatozoa, 40% to 60% fluoresced by this procedure compared to 1% to 15% of sperm reacted with nonimmune serum. Semen from three bulls was exposed to nonimmune serum (control) or MoAb, sorted by FACS, and analyzed for DNA content with a scanning microdensitometer. Control samples showed two distinct peaks with a mean difference in DNA content of 3.95%; these peaks were assumed to represent Y- and X-chromosome-bearing spermatozoa populations, respectively. DNA analyses of the MoAb-treated sperm of three bulls that sorted positively for H-Y antigen (fluorescent sperm) yielded ratios of Y- to X-chromosome-bearing spermatozoa of 76 : 24, 88 : 12, and 77 : 23, and those sorted negatively for H-Y antigen (nonfluorescent sperm) yielded ratios of 26 : 74, 35 : 65, and 23 : 77. The proportions of Y- and X-chromosome-bearing spermatozoa in nonsorted samples were not different from 50 : 50. Suitable MoAbs can be used in conjunction with FACS to enrich the proportion of Y- or X-chromosome-bearing spermatozoa in bovine semen.