Sperm DNA integrity assessment can employ various techniques, each yielding distinct outputs. The sperm chromatin dispersion test (HALO) is often used for its simplicity, cost-effectiveness, and minimal specialised equipment requirements. The sperm chromatin structure assay is a high-throughput technique utilising flow cytometric analysis of the metachromatic shift of acridine orange (AO) fluorescence from green (double-stranded DNA) to red (single-stranded DNA). Both techniques present DNA integrity as a percentage of sperm with fragmented DNA within a population. While less common due to its resource-intensive nature, the alkaline single-cell gel electrophoresis (COMET) assay examines the degree of DNA fragmentation within individual sperm, making it useful for detecting subtle changes in DNA integrity. This study employed these three assays to investigate alterations in merino ram (n=9 biological replicates) sperm DNA integrity post-cryopreservation. Cryopreservation is essential for disseminating elite genetics in the sheep production industry; however, the cryopreservation process, including cold shock or the chemical components of the media, may negatively impact the integrity of critical intracellular components, including DNA.
The alkaline COMET assay revealed a statistically significant but minor decline in sperm DNA integrity after cryopreservation. Cryopreserved sperm exhibited higher mean tail DNA percent intensity (37.9%; p<0.01) compared to liquid-extended sperm (35.1%). Decreased DNA integrity of cryopreserved sperm was further confirmed by an increased mean tail moment, a parameter that considers both DNA fragment number and size by formulating comet tail DNA percent intensity and length for cryopreserved sperm (18.6; p<0.001) compared to liquid-extended sperm (16.6).
In contrast, neither AO nor HALO could distinguish the DNA integrity of cryopreserved sperm (3.98%; 2.16%) from liquid-stored sperm (4.66%; 2.65%), likely due to their population-based analysis methods. This research highlights the disparities among DNA integrity assays and their capacity to detect subtle changes following cryopreservation.