Separation of sperm according to the sex chromosome is based on the DNA content of the sperm. Mammalian semen can be sexed because the X-chromosome-bearing sperm that produce females contain about 4% more DNA than do the Y-chromosome-bearing sperm that produce males. The procedure is 85-95% accurate for the selected sex.[1-3] Sorting of live mammalian sperm according to their DNA content first was developed the USDA Beltsville Agricultural Research Center. This flow cytometric sorting procedure for sperm[5-8] is patented and exclusively licensed worldwide for non-human mammals to XY, Inc., a private company. For the sorting procedure, freshly collected sperm are stained with a DNA-specific bisbenzimidazole dye, Hoechst 33342, for approximately 1 hr prior to sorting. Hoechst 33342-stained sperm fluoresce bright blue when exposed to a laser beam of short wavelength light and the X-bearing sperm are differentiated from the Y-bearing sperm because they fluoresce brighter due to their greater DNA content. The fluorescence of each stained sperm is measured in a stream of fluid as it passes in front of a photomultiplier tube (PMT). The resultant data are integrated using a powerful computer. Only the DNA content of properly oriented sperm can be measured because the flat surface of each sperm head must be properly oriented relative to the PMT.[1,2]
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