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United States Department of Agriculture

Agricultural Research Service

Title: A Novel Nozzle for Faster Flow Cytometric Analysis of Sperm DNA Content.

Authors
item Rens, W - USDA, ARS, LPSI, GGPL
item Welch, Glenn
item Johnson, Lawrence

Submitted to: Cytometry Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 1, 1998
Publication Date: N/A

Interpretive Summary: The Beltsville Sperm Sexing Technology is being developed by commercial companies for providing the livestock industry with sexed semen. This gives the livestock producer greater options in terms of genetic progress and management flexibility. One of the drawbacks of the system is that it takes an hour to sort about 300,000 sperm. Too few for doing any significant type of artificial insemination. This study describes the development of a new nozzle that attaches to the cell sorter and allows the throughput of sperm to increase by two to three fold. The nozzle has been adapted to the Epics V and 750 series cell sorters and to the MoFlo cell sorter. The new nozzle provides the option of sorting sperm even with an older cell sorter at nearly one million sperm per hour. However by using a high speed sorter, the new nozzle allows sorting in the area of 4 million per hour. This newly developed system will enable sexed semen to be used in artificial insemination programs where prior to this it was only practical to use it for in vitro fertilization.

Technical Abstract: Efficient high resolution flow cytometric DNA analysis of X and Y chromosome bearing sperm is dependent on effectively orientating the sperm head to the laser beam. Normally a beveled needle is required to enlarge the fraction of properly orientated sperm. In this report a modification to a standard jet in air nozzle for improved sperm orientation is presented. Inside the modified nozzle (novel nozzle) orientation forces are applied lower in the nozzle (thus closer to the laser beam) then in the current beveled injection needle system. The novel nozzle was first tested with bull sperm heads with a low sample rate (<500/sec). The novel nozzle gave the same performance as the beveled needle system. Measurements of rabbit, boar, mouse and human sperm show that the novel nozzle is suitable for many species. Although mouse and human sperm have a hooked and angular shaped head respectively, about 70% were orientated by the novel nozzle. Higher sample rate experiments (2000/sec) with the novel nozzle demonstrated orientation performance superior to the beveled needle system. Generally a twofold increase in analysis rate is achieved with the novel nozzle. The use of the novel nozzle has no detrimental effect on the accuracy of fluorescence measurements. The novel nozzle design can be considered as an improvement on the beveled needle system for DNA analysis of sperm.

Last Modified: 10/24/2014
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