Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization.
This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately 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. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled 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 average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa.
Kawarasaki
T
T
Shizuoka Swine and Poultry Experiment Station, Japan.
Sone
M
M
Yoshida
M
M
Bamba
K
K
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008969:Molecular Sequence Data; D013094:Spermatozoa / Q000502:physiology*; D013552:Swine; D014998:Y Chromosome
D017931:DNA Primers