ABSTRACT The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. It is an effective means of producing progeny of predetermined sex in cattle, swine, sheep, and laboratory animals. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for surgical intratubal or intrauterine insemination, deep-uterine insemination, regular artificial insemination in some cases, in vitro fertilization to produce sexed embryos for transfer, and intracytoplasmic sperm injection of ova. Skewed sex ratios of 85 to 95% of one sex or the other have been repeatably achieved in most species. The method has been used worldwide to produce several hundred morphologically normal animal offspring of the predicted sex. It has also been validated in the laboratory using DNA reanalysis of the sorted sperm populations and by fluorescence in situ hybridization and PCR of individual sperm. We developed a new orienting nozzle that we have fitted to both conventional and high-speed cell sorters that have been modified for sperm sorting. Recently we completed the adaptation of the new orienting nozzle to a Cytomation MoFlo high-speed cell sorter modified for sperm. This adaptation of the nozzle has increased the overall production rate of sorted X and Y sperm from about .35 million/h to 5 or 6 million sperm/h (each population). Calves have been born from cows artificially inseminated using conventional technique and sexed sperm. In addition, numerous litters of pigs have been born after transfer of embryos produced from X or Y sorted sperm.
Implications Sex preselection by flow-cytometric cell sorting is currently the only proven method that skews the sex ratio of mammals. The Beltsville technology will produce 5 to 6 x 106 sperm/h at 85 to 95% purity for X (female producing) or Y (male producing) sperm in most livestock species. One limitation of cell-sorting technology is that the process must be carried out one cell at a time. This makes the systems inherently slow, because millions (for cattle) and billions of sperm (for swine) are needed for conventional AI. Other than DNA, no other difference is known to exist between X and Y sperm that would allow their separation while maintaining viability. Improvements in the sperm-sorting technology have increased the throughput of sperm by 10 to 15 times. This high-speed technology using a special orienting nozzle opens the way for a much broader use of the method in terms of AI. Further improvements encompassing a dedicated sperm-sorting instrument would likely simplify the process and make it even more applicable. Continued progress in the development of greater efficiency in sorting sperm into X and Y sperm populations offers great encouragement that practical application of the sperm-sexing technology will occur within a few years.
Key Words: Spermatozoa, Flow Cytometry, Cells, Artificial Insemination
© 1999, by the American Society of Animal Science and the American Dairy Science Association. All rights reserved.
AM Symp. 213-220