RAPID AND SIMULTANEOUS DETECTION OF CHROMOSOME-Y-BEARING AND CHROMOSOME-1-BEARING PORCINE SPERMATOZOA BY FLUORESCENCE IN-SITU HYBRIDIZATION

This study was carried out to develop a rapid and simultaneous detecti on system of chromosome Y- and 1-bearing porcine spermatozoa by fluore scence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Di g)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chrom osome and chromosome 1-specific DNA was applied to the preparation, im mediately denatured at 75 degrees C for 8 min, hybridized for 5 min at 37 degrees C and overall FISH steps were done within a few hours. Whe n double FISH with Dig-labeled chromosome Y-specific and biotin-labele d chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig- signal, 99.2% of the sperm nuclei had the biotin-signal and the averag e of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bea ring spermatozoa ranged from 49.8% to 52.8% among 5 boars and the aver age putative rate of Y-bearing spermatozoa was 51.0%. The results indi cated that a rapid and simultaneous FISH with chromosome Y- and 1-spec ific porcine DNA probes produced by PCR made possible more accurate as sessment of Y-bearing porcine spermatozoa, (C) 1996 Wiley-Liss, Inc.