Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342.
OBJECTIVE
To develop methods for using a DNA-specific dye to discriminate between motile and nonmotile sperm and static particulate matter in fresh and diluted semen, using computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, TOX version (Hamilton-Thorne Research, Beverly, MA).


METHODS
Donor semen was divided for treatment as fixed stained sperm (Hoechst 33342 stain; Sigma Chemical Company, St. Louis, MO), fresh motile and nonmotile stained sperm, and unstained control sperm.


METHODS
Normal human volunteers in an academic research and medical environment.


METHODS
Selected healthy student volunteers.


METHODS
Delivered semen to the laboratory within 1 hour of collection.


METHODS
Semen quality measured by CASA.


RESULTS
Fixed or fresh human sperm stained with Hoechst 33342 dye should be diluted to < or = 50 x 10(6) sperm/mL to count sperm accurately. Motile and nonmotile sperm were stained suitably with 5 to 10 micrograms/mL of dye when diluted with a simple diluent, but the dye concentration should be increased to 40 micrograms/mL when egg yolk is in the diluent.


CONCLUSIONS
The DNA-specific dye, Hoechst 33342, can be used to discriminate between motile and nonmotile sperm and other particulate matter when evaluated by CASA with instrumentation equipped with suitable optics.
Farrell
P B
PB
Department of Animal Science, Cornell University, Ithaca, New York 14853-4801, USA.
Foote
R H
RH
Zinaman
M J
MJ
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007248:Infertility, Male / Q000175:diagnosis / Q000473:pathology; D008297:Male; D012661:Semen / Q000166:cytology / Q000502:physiology; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology*
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride