Comparative analysis of equine semen preservation techniques and DNA damage detection using Comet and SCSA assays

Maintaining the DNA integrity of equine spermatozoa during preservation is crucial for the success of assisted reproductive techniques. Sperm preservation generally relies on low temperatures to reduce or halt sperm metabolism to preserve functionality, but more recently methods of storing sperm above 15°C have been developed to reduce the effects of cold shock and cryodamage. The major damage that spermatozoa experience during preservation at any temperature is oxidative damage, which may lead to DNA damage. There are numerous DNA damage assays available, but their relative prognostic value in horses has yet to be evaluated. Therefore, the objective of the present study was to compare the sensitivity of two popular sperm DNA damage assays, the sperm chromatin structure assay (SCSA) and the single cell gel electrophoresis (neutral comet) assay in identifying sperm DNA damage in the horse. A total of 3 split ejaculates (diluted 2:1 with EquiPlus) from individual stallions were utilised. High-quality spermatozoa were isolated via single-layer colloidal centrifugation (EquiPure). Sperm pellets were resuspended in: L-EDTA cryodiluent with 20% egg yolk and 3% dimethylformamide; EquiPlus; and SpermSafe, after which they were either cryopreserved, chilled (5°C), or stored at 18°C respectively. The SCSA (DNA fragmentation index; DFI) and comet (tail intensity) analyses were performed at various relevant timepoints. While the SCSA revealed no significant increase in DNA damage at any timepoint, for any storage treatment, the comet assay was more sensitive, revealing a significant increase in DNA damage after 72h of storage in SpermSafe (0h: 21.1±11.4% vs 72h: 53.5±0.2%; p≤0.05), though this fell to non-significant levels by the 7-day timepoint (43.675±3.8%), and after cryopreservation (pre-freeze: 21.1±11.4% vs post-thaw: 67.2±3.5%; p≤0.05). There was no significant increase in DNA damage at any timepoint for the chilled, EquiPlus-stored samples. This study shows that the comet assay is more sensitive than the SCSA in detecting sperm DNA damage following various preservation methods. Notably, sperm stored in SpermSafe at 18°C exhibited unexpectedly high tail intensity, which contrasts with high pregnancy rates in commercial settings. We hypothesise that this may result from an artefact associated with capacitation-like changes and chromatin decondensation, due to the SpermSafe formulation mimicking oviduct fluid. Ongoing studies aim to investigate this further. Post-thaw, tail intensity (comet) more than tripled following cryopreservation, which could also be linked to capacitation-like changes or represent actual DNA damage that contributes to lower pregnancy rates seen with frozen-thawed sperm. A key limitation to this study is the small sample size, but additional replicates are being included in ongoing research. Despite this, our findings offer insights into sperm DNA integrity during preservation and suggest that the comet assay and SCSA may offer complementary prognostic value for equine assisted reproductive techniques.