Developing a dynamic test to identify stallions with reduced sperm DNA stability

Abstract

Sperm DNA integrity is usually evaluated using a ‘static’ test. However, recent studies suggest that a ‘dynamic’ test to examine how rapidly spermDNA degenerates at 37 C is a more sensitive index of (sub)-fertility. This study validated a ‘DNA stress’ test by examining whether sperm DNA from different stallions degraded repeatedly at different rates during incubation at 37 C. After daily semen collection for 1 week to reach daily sperm output, semen was collected on three consecutive days from each of eight 3-year old Warmblood stallions, i.e. three ejaculates per stallion. All ejaculates had >60% morphologically normal and >60% motile sperm. The semen was diluted to 50x106 sperm/mL in an egg yolk-skimmed milk extender (Spervital EVD ; Spervital) and divided into two portions; one portion was centrifuged (800xg, 20min) and re-suspended. Centrifuged and non-centrifuged samples were split again and incubated at 37 C and 5 C. Semen was assessed during 37 C incubation after 4, 8, 24 and 48h; during 5 C incubation after 48, 96, 144 and 192h. At each time-point, sperm motility was evaluated using a computerized system (SpermVision ; Minitub), viability by SYBR14/PI staining assessed by fluorescence microscopy, and DNA integrity by SCSA . Differences in percentage of motile, viable or DNAintact sperm were compared between incubation temperatures, times and stallions using analysis of variance. Percentages of motile and viable sperm at 5 C were higher (p<0.05) for centrifuged than non-centrifuged semen, and there were no between-stallion differences in the rate of loss of motility or viability. Centrifugation did not reduce the rate at which the percentage of motile or viable sperm decreased during 37 C incubation. The rate of change of sperm DNA degradation, as reflected by the alpha t value, did not differ between these 8 stallions when semen was