Comparing DNA Damage Assays in Equine Sperm Preservation

DNA Damage Assays - cartoon horse with DNA machinesBackground & Purpose

Preserving sperm DNA integrity is critical for successful equine assisted reproduction. While low-temperature storage is traditional, above-15°C methods have emerged to reduce cold shock damage. Oxidative stress during any preservation method can cause DNA damage. This study compared the sensitivity of two DNA damage assays — the Sperm Chromatin Structure Assay (SCSA) and the neutral comet assay — in detecting sperm DNA damage across different preservation methods.

Study Design

  • 3 split ejaculates from individual stallions
  • High-quality sperm isolated via single-layer colloidal centrifugation
  • Three preservation treatments:
    • Cryopreservation (L-EDTA diluent + egg yolk + dimethylformamide)
    • Chilled at 5°C (EquiPlus)
    • Stored at 18°C (SpermSafe)
  • Both assays performed at multiple timepoints

Key Findings

  • SCSA detected no significant DNA damage increase under any condition or timepoint
  • Comet assay was more sensitive, detecting significant DNA damage in:
    • SpermSafe at 72h (tail intensity rose from ~21% to ~54%)
    • Post-cryopreservation (tail intensity more than tripled, ~21% to ~67%)
  • Chilled EquiPlus samples showed no significant DNA damage at any timepoint

Notable Observations

  • The high DNA damage signal in SpermSafe-stored sperm at 18°C was unexpected, given that commercial pregnancy rates with this product are reportedly high. The authors hypothesize this may be an artifact of capacitation-like changes, as SpermSafe mimics oviduct fluid and may cause chromatin decondensation rather than true DNA damage
  • Post-thaw damage after cryopreservation may reflect either the same capacitation-like artifact or genuine DNA damage contributing to the lower pregnancy rates typically seen with frozen-thawed equine sperm

Limitations & Conclusions

The small sample size (n=3) is acknowledged as a key limitation, with additional replicates underway. Despite this, the study concludes that the comet assay is more sensitive than SCSA and that the two assays likely offer complementary prognostic value for equine reproductive techniques. The unexpected SpermSafe findings highlight the importance of understanding how preservation media chemistry may influence assay results.

(Yaseen M, Swegen A, Gibb Z. 2025. Comparative analysis of equine semen preservation techniques and DNA damage detection using Comet and SCSA assays. JEVS 145:105335 – Presented in Association with and by Permission of the International Society for Equine Reproduction)