Sex-sorting sperm using flow cytometry/cell sorting.

The research studied a method for pre-determining the sex of mammalian offspring by sorting sperm with the help of flow cytometry. This technique allows scientists to separate and collect X-chromosome and Y-chromosome bearing sperm that have been identified with a DNA-specific stain, and has been used successfully in numerous species, including humans and farm animals.

Overview of the Research

• The goal of this research was to verify whether or not the sex of mammalian offspring could be predetermined by using flow cytometry/cell sorting to separate relatively pure populations of X- and Y-chromosome-bearing sperm.
• The team employed a specialized method which involved staining sperm DNA with the nucleic acid-specific fluorophore, Hoechst 33342, which is capable of differentiating between X-bearing and Y-bearing sperm due to differing DNA content.

The Methodology

• Once the sperm had been stained using the fluorescent compound Hoechst 33342, they were sorted using a high-speed cell sorter, the MoFlo(®) SX XDP.
• Following sorting, the sperm were transferred into a biologically supportive medium in preparation for reconcentration and cryopreservation.
• The sorting process was designed to gather sperm in quantities suitable for use in artificial insemination procedures for certain species, or for in vitro fertilization.

Results and Implications

• The researchers found that the flow cytometric sorting procedure could isolate X- or Y-bearing sperm in the 8,000 sperm/second range while maintaining an approximate purity of 90%.
• This level of success enabled the technique to be applied on a commercial basis for cattle, but it proved effective across various mammalian species including swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo and even humans.
• The ability to predetermine the sex of offspring using this method potentially allows for greater control in breeding programs, whether they be aimed at livestock farming, preservation of endangered species or human reproductive therapies.