Fluorescence in situ hybridization with Y chromosome-specific probe in decondensed bovine spermatozoa

This study was carried out to demonstrate bovine Y chromosome-bearing sperm atozoa by rapid fluorescence in situ hybridization (FISH), using a digoxige nin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FIS H procedure, sperm heads were treated for decondensation with dithiothreito l (DTT) and glutathione (GSH) with or without heparin supplementation. Conc entrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used w ith 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially t hat of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), h owever, could induce reliable, repeatable detection of the Y chromosome sig nal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa. (C) 1999 by Elsevier Science Inc.