Improved Flow Cytometric Sorting of X- and Y-Chromosome Bearing Sperm: Substantial Increase in Yield of Sexed Semen

The yield of flow cytometric sorted Xand Y-chromosome-bearing sperm in a given time period is an important factor in the strategies used for fertilization and the production of sex-preselected offspring. This yield is dependent on the efficiency with which the modified flow cytometer/cell sorter analyzes the DNA of spermatozoa. The efficiency is directly related to the number of sperm with the correct orientation during DNA analysis. Currently, the efficiency of flow cytometric sperm sorting is low since orientation of the sperm head to laser excitation is rate limiting. To overcome this problem, a new nozzle was designed to enhance sperm orientation and tested under flow cytometric sorting conditions. The degree of orientation improvement was determined with different sample rates using viable sperm and dead sperm of several different species. There was at minimum, a two-fold increase in the proportion of oriented sperm when comparing the new nozzle with the currently used modified flow cytometer/cell sorter employing a beveled needle. More than 60% of intact bull sperm and boar sperm were correctly oriented compared with 25% to 30% using the beveled needle system. A unique characteristic of the novel nozzle was that the proportion of oriented sperm was independent of sample rate and of sperm motility. The accuracy of DNA measurement together with high purity sorting was tested using the novel nozzle. The novel nozzle was unique in that accuracy of measurement and sorting performance were not diminished. Using the new nozzle, samples of 88% purity of sorted X-sperm and Y-sperm were obtained for viable bull and boar sperm. The yield of flow cytometric sorted Xand Y-chromosome-bearing sperm using the novel nozzle was, on average, twice that obtained by using the beveled needle system in conjunction with a standard equipment nozzle for orientation. Mol. Reprod. Dev. 52:50–56, 1999. Published 1999 Wiley-Liss, Inc.†