The TUNEL assay underestimates the incidence of DNA damage in pig sperm due to chromatin condensation

Abstract

Inconsistencies in the relationship between sperm DNA fragmentation and reproductive outcomes as well as the low incidence in farm animals raise concerns on its actual value as a sperm quality parameter. Previous studies suggested that the different sensitivity of techniques evaluating DNA fragmentation could explain variations in the correlation with reproductive outcomes. While the TUNEL assay is one of the most standardized methods to detect DNA damage and cell death, the steric impediment for the terminal nucleotidyl transferase enzyme to access the highly condensed sperm nucleus may decrease the ability of this test to detect internal DNA breaks. In the present study, we sought to determine whether increasing chromatin decondensation makes the TUNEL assay more sensitive to detect DNA damage in pig sperm. We compared three chromatin decondensation treatments (2 mM DTT for 45 min; 5 mM DTT for 8 min and further 45 min; and 5 mM DTT+ 1 M NaCl for 8 min) through the Chromomycin A3 test (CMA3). While incubation with DTT increased the percentages of sperm with decondensed chromatin, regardless of concentration and time of incubation (P < 0.05), the extent of that decondensation was higher when 5 mM DTT was combined with 1 M NaCl. In addition, the TUNEL assay detected a higher number of DNA breaks in sperm with decondensed chromatin (1.89% ± 1.63% vs 8.74% ± 6.05%; P = 0.003). This study shows, for the first time, that previous chromatin decondensation increases the sensitivity of the TUNEL assay to detect DNA damage in pig sperm. These findings also support that larger chromatin decondensation is needed in order for DNA damage to be evaluated properly in species containing protamine P1 only.