Rapid determination of sperm cell concentration in bovine semen by flow cytometry

A flow cytometric technique is described for determining sperm concentration in fresh or extended semen with improved accuracy, precision, repeatability, ease of conduct, and rapidity. The technique is designed to measure the ratio of a known number of fluorescent beads admixed with sperm stained with either acridine orange or propidium iodide. A significant advantage of the technique is the distinct resolution between sperm and other particles (e.g., somatic cells, fat droplets, and bacteria in the semen or extender) that interfere in other counting protocols. Field testing of this protocol over the past 3 yr has demonstrated its superiority over the Coulter counter, hemacytometer, and spectrophotometer for accuracy in counting sperm in extended semen and the accuracy of counting sperm in straws based on preextension spectrophotometric determination of sperm concentration. Sperm chromatin quality can be determined simultaneously with this sperm counting procedure. This approach to counting sperm provides an excellent procedure for quality control of sperm numbers in processed semen.