ABSTRACT
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.[4] 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.[9] Hoechst 33342-stained sperm fluoresce bright blue when exposed to a laser beam of short wavelength light and the Xbearing 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).[10] 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]
FLOW SORTING SYSTEM
As the liquid stream containing sperm exits the sorter nozzle, it is vibrated at about 75,000 to 85,000 oscillations/sec to break the stream into individual droplets.[9] Although not all of these formed droplets contain sperm, those that do are electrically charged, either positive or negative, according to the DNA content information previously provided by detector. Droplets containing improperly oriented sperm, more than one sperm, or dead sperm, as determined by uptake of a vital dye, are disposed of as waste because no charge is applied to these droplets. As the droplets pass by an op-
positely charged plate they are deflected into a collection vessel according to the DNA content of the sperm. Those droplets containing Y-sperm are simultaneously directed to a separate collection vessel by applying an opposite charge to those drops to deflect them toward the opposing-charged plate. Opposing charges are applied to droplets containing X-sperm so that they are directed toward a separate vessel. Three streams of droplets containing X-sperm, Y-sperm, or no sperm as well as too many sperm are collected into separate vessels. Approximately 40% of the living sperm going through the sorter at a speed of approximately 75 km/hr can be accurately sexed and collected. Thus, at an event rate of 28,000 total sperm/sec, nearly 5000 live sperm/sec of each sex can be sorted simultaneously with 85-95% accura-cy.[8] The current system can produce approximately 10 to 15 × 106 live bovine sperm/hr of each sex-determining gamete. Considerable numbers of sperm are, however, lost in the centrifugation and other postsorting steps making actual yields somewhat lower.[1,2] Sperm of other species tend to sort at somewhat slower rates. Sorted sperm are dilute so they must be reconcentrated prior to being used for artificial insemination (AI) or in vitro fertilization (IVF).
