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Title: SUBSTANTIAL INCREASE IN YIELD OF SEXED SEMEN BY IMPROVED FLOW CYTOMETRIC SPERM SEPARATION EFFICIENCY

Author
item RENS, W - USDA, ARS, LPSI, GGPL
item Welch, Glenn
item Johnson, Lawrence

Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: The USDA-Beltsville Sperm Sexing Technology has been developed for use by livestock producers, so that they might more efficiently produce offspring. The ability to use sex preselection in their particular production scheme increases their efficiency as well as the quality of the genetics. This manuscript details the use of a newly developed orienting nozzle for a flow wcytometer cell sorter which is used to sex sperm. In testing the nozzle i was found to increase the efficiency of the sperm sexing process by two to three fold for swine and cattle. It does this by orienting the sperm much closer to the laser beam than previously done when a beveled needle was the orienting device. Utilization of this advancement in conjunction with the standard sperm sexing procedures will give significant impetus for utilization of the sperm sexing technology on a widespread practical basis. It will be used by scientists to refine current protocols of sexing so as to optimize conditions of the sexing process and it will be used by the commercial sector as they develop the market for sexed semen.

Technical Abstract: The yield of flow cytometric sorted X- and 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. The efficiency of flow cytometric sperm sorting is low since orientation of the sperm 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 and with viable sperm and with dead sperm of several different species. There was at minimum, a twofold increase in the proportion of orientated sperm when comparing the new nozzle with the currently used modified flow cytometric/cell sorter using beveled needle. More than 50% of bull sperm and boar sperm were correctly orientated compared with 20% using the beveled needle system. A unique characteristic of the novel nozzle was that the proportion of orientated sperm were independent of motility of sperm and sample rate. In the second study, accuracy of DNA measurement together with high purity sorting were tested with 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 X- and Y-chromosome-bearing sperm using the novel nozzle was, on average, twice that obtained by using the beveled needle system for orientation.