Feulgen-DNA content of individual bull spermatozoa

Abstract

D URING Feulgen-DNA measurements on individual bull spermatozoa with a view on establishing a contingent correlation between the spread of the values found within a sample and the fertilizing capacity of the semen we noted, first, that values obtained with our method showed a considerably lower spread than had been found previously and, secondly, that the results are compatible with the presence within the samples of two populations, differing some 3 per cent in DNA content. The present paper deals with an analysis of two sperm samples with respect to these observations. The cytophotometer used has been described previously [2]. It has been modified in that in the present apparatus the scanning is done in the image plane. It has been found empirically that this arrangement is less sensitive to focussing errors and allows of a slightly better resolution than that formerly employed. The standard deviation of repeated measurements on the same spermatozoon was found to amount to 1 per cent of its Feulgen-DNA content. This source of spread in measuring results will be referred to as the instrumental error. The standard Feulgen technique was employed but hydrolysis was performed in 5.5 N HCl at 2O’C ( f O.l’C). Under these circumstances, and in our material, the staining intensity is relatively independent of the hydrolysis time within the range 45-60 min. In the present investigation hydrolysis was terminated after 50 min. All baths were symmetrically agitated and every detail of the technique was rigorously standardized; nevertheless we were unable to obtain identical staining over the entire surface of 2 x 3 cm smears of semen. Within an area of a few square milimeters the spread of the values obtained on 50 spermatozoa was the same as that found in a similar area elsewhere on the same slide or on a slide treated simultaneously, but the mean of the values found in one area could differ from zero to as much as 4 per cent from that found in another area. In order to pool the measuring results obtained on a large number of spermatozoa we measured closely adjacent spermatozoa in groups of 40 or 50 and corrected for the apparently unavoidable difference in staining effectiveness between the groups by converting every group mean into 100 arbitrary units. The histograms for 760 spermatozoa from one ejaculate (P.C.), Fig. 1, and 450 spermatozoa from another bull (Joh. I.), Fig. 2, have been plotted in unit steps,