Feulgen-DNA Cytophotometry of Bovine Spermatozoa

Abstract

Summary Two methods have been compared for quantitating the Feulgen-DNA in the same 240 mature bovine spermatozoa, using a fixed light beam cytophotometer. The two-wave-length method measured absorbance of single stained nuclei in a circular light beam having a diameter slightly larger than the long axis of the sperm head and eliminated the need for nuclear-size determinations. An over-all Feulgen-DNA coefficient of variability of 20.4% and a within sample coefficient of 14.6% were found. The other method measured nuclear absorbance of a monochromatic light beam with a slightly smaller diameter than the nuclear width and required an accurate nuclear volume determination related to the volume traversed by the light beam. Nuclear volume was estimated from linear measurements using three different formulae. All yielded Feulgen-DNA values with over-all coefficients of variability ranging from 12.3 to 12.9% and within sample coefficients of 6.5 to 7.3%. Two formulae gave falsely precise Feulgen-DNA values due to systematic errors in area calculation resulting from false assumptions of sperm nuclear shape. Nuclear area of each cell calculated by the van Duijn formula multiplied by its extinction value for a light beam of known area gave the best estimate of sperm nuclear Feulgen-DNA.