A NOVEL NOZZLE FOR MORE EFFICIENT SPERM ORIENTATION TO IMPROVE SORTING EFFICIENCY OF X-CHROMOSOME-BEARING AND Y-CHROMOSOME-BEARING SPERM

Efficient high-resolution detection of DNA for flow cytometric sorting of X and Y chromosome-bearing sperm is dependent on effectively orien tating the sperm head to the laser beam in orthogonally configured flo w systems. Normally, a beveled needle is required to enlarge the fract ion of properly orientated sperm (flat side facing the laser beam). In this report, a modification to a standard jet-in-air nozzle for impro ved sperm orientation is presented. Inside the modified nozzle (novel nozzle), orientation forces are applied lower in the nozzle than in th e current beveled injection needle system. The nozzle was tested with sperm heads from several species, This study shows that use of the noz zle to orientate cattle, swine, rabbit, mouse, and human sperm effecti vely improves the percentage of sperm that are properly oriented, The percentage of sperm heads oriented by use of the former system (bevele d needle) ranges around 30% for most species. With the newly designed nozzle, that percentage ranges around 60%. At least a twofold increase in analysis is achieved. It was found that, unlike results with the b eveled needle, the percentage of properly oriented sperm was independe nt of the sample rate. The introduced nozzle is a significant improvem ent over the beveled needle system for the analysis and sorting of spe rm on the basis of DNA content. In addition to the improvement in sort ed sperm production brought about by the novel nozzle when fitted to s tandard-speed cell sorters, it clearly also has significant potential for improving die efficiency of the Beltsville Sperm Sexing Technology for separating X and Y chromosome-bearing sperm when adapted to highs peed cell-sorting systems, (C) 1998 Wiley-Liss,Inc.(dagger)