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
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.