Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 13, 1999
Publication Date: N/A
Interpretive Summary: For hundreds of years man has been interested in the possibility of sexing sperm in order to preselect the sex of his offspring or those of the livestock that he raises. For hundreds of years, there has been no way to preselect the sex of offspring nor has there been a way to identify the X and Y proportions within a sample of semen that has been prepared for use to produce sexed offspring. This paper describes the procedure of sort reanalysis for DNA content of sperm that have been sorted and are going to be used in practice for producing offspring. The necessity for laboratory determination of sperm sex ratio is essential prior to using sorted X or Y sperm in practical applications. To not know the outcome of using the semen in terms of sex, is very costly to the livestock producer. This technology has been applied for years to guide the development of the Beltsville Sperm Sexing Technology. It is an integral part of using any known or potential sexing technology.
Technical Abstract: Laboratory validation is essential in developing an effective methodology for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continu progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow-cytometric sperm sorting, is also well suited for validation in the laboratory by "sort reanalysis" of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consist of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen.