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The Beltsville Sperm Sexing Technology: High-Speed Sperm Sorting Gives Improved Sperm Output for In Vitro Fertilization and AI

USDA, ARS, Germplasm and Gamete Physiology Laboratory, Beltsville, MD 20705

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.

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