Sex preselection: Laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA

Citation
Gr. Welch et La. Johnson, Sex preselection: Laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA, THERIOGENOL, 52(8), 1999, pp. 1343-1352
Citations number
23
Categorie Soggetti
Veterinary Medicine/Animal Health","da verificare
Journal title
THERIOGENOLOGY
ISSN journal
0093691X → ACNP
Volume
52
Issue
8
Year of publication
1999
Pages
1343 - 1352
Database
ISI
SICI code
0093-691X(199912)52:8<1343:SPLVOT>2.0.ZU;2-I
Abstract
Laboratory validation is essential in developing an effective method for se parating X and Y sperm to preselect sex. Utilizing sexed sperm from a parti cular experiment to test fertility and achieve the subsequent phenotypic se x without knowing the likely outcome at conception is too costly for most a pplications. Further, research advances need to be built on an ongoing asse ssment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, whi ch 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 l aboratory 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 333 42, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it als o can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists 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. Sepa rate analysis of each aliquot produces a histogram that is fitted to a doub le gaussian curve to determine proportions of X and Y populations. The rela tive breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is cons istently 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 to ol by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proport ions of X- and Y-bearing sperm based on DNA content is also an effective to ol for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis p rovides for a check on the potential sex ratio of any sample of semen. Publ ished by Elsevier Science Inc.