Abstract
Although sperm chromatin damage, understood as damage to DNA or affectations in sperm protamination, has been proposed as a biomarker for sperm quality in both humans and livestock, the low incidence found in some animals raises concerns about its potential value. In this context, as separate methods measure different facets of chromatin damage, their comparison is of vital importance. This work aims at analyzing eight techniques assessing chromatin damage in pig sperm. With this purpose, cryopreserved sperm samples from 16 boars were evaluated through the following assays: TUNEL, TUNEL with decondensation, SCSA, alkaline and neutral sperm chromatin dispersion (SCD) tests, alkaline and neutral Comet assays, and chromomycin A3 test (CMA3). In all cases, the extent of chromatin damage and the percentage of sperm with fragmented DNA were determined. The degree of chromatin damage and the percentage of sperm with fragmented DNA were significantly correlated (p < 0.05) in direct methods (TUNEL, TUNEL with decondensation, and alkaline and neutral Comet) and CMA3, but not in the indirect ones (SCD and SCSA). Percentages of sperm with fragmented DNA determined by alkaline Comet were significantly (p < 0.05) correlated with TUNEL following decondensation and CMA3; those determined by neutral Comet were correlated with the percentage of High DNA Stainability (SCSA); those determined by SCSA were correlated with neutral and alkaline SCD; and those determined by neutral SCD were correlated with alkaline SCD. While, in pigs, percentages of sperm with fragmented DNA are directly related to the extent of chromatin damage when direct methods are used, this is not the case for indirect techniques. Thus, the results obtained herein differ from those reported for humans in which TUNEL, SCSA, alkaline SCD, and alkaline Comet were found to be correlated. These findings may shed some light on the interpretation of these tests and provide some clues for the standardization of chromatin damage methods.
Highlights
The research of biomarkers that predict sperm fertilizing ability has gained much interest in the last years and has led to the discovery of factors affecting reproductive outcomes in both humans and production animals [1,2,3,4]
We comprehensively described the correlations among eight methodological variants assessing different facets of chromatin damage, namely, DNA breaks and poor protamination
Our results showed that the intensity of DNA damage, given by the amount of DNA breaks, is correlated with the percentages of sperm with fragmented DNA in direct (TUNEL and Comet), but not in indirect methods (SCSA and sperm chromatin dispersion (SCD) assays)
Summary
The research of biomarkers that predict sperm fertilizing ability has gained much interest in the last years and has led to the discovery of factors affecting reproductive outcomes in both humans and production animals [1,2,3,4]. Artificial insemination is the most used method for breeding, and the quality of liquid-stored and cryopreserved sperm is evaluated to determine the potential fertility of males [8, 9] In this context, finding sperm quality biomarkers that allow the selection of the most suitable boars is crucial to increase reproductive efficiency in farms [10,11,12]
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