Long-Term Liquid Nitrogen Storage Effects on Frozen Stallion Semen Quality and Messenger RNA Abundance

Storage Effects on Frozen Stallion Semen - nitrogen tank and shipper being filled showing vapor in the air in a labCryopreserved semen stored in liquid nitrogen is widely assumed to maintain fertility indefinitely, yet emerging reports suggest possible long-term quality decline. This study examined whether extended storage duration affects conventional sperm quality parameters and fertility-associated messenger RNA abundance in commercially cryopreserved stallion semen.

Study Design:

  • 18 stallions (Warmblood, Thoroughbred, Lipizzaner); semen cryopreserved between 1998 and 2024; storage duration 0–26 years
  • Post-thaw assessment: concentration, motility, membrane integrity, mitochondrial membrane potential, and chromatin condensation
  • Messenger RNA extracted from 12 semen batches; abundance of 10 fertility-associated genes measured by quantitative reverse transcription polymerase chain reaction
  • Spearman correlation analysis performed between all endpoints and storage duration

Key Results:

  • No significant correlations between storage duration and any conventional sperm quality parameter
  • No significant correlations between storage duration and messenger RNA quality metrics
  • Four genes showed significant negative correlations with storage duration:
    • Protamine 1 (r=–0.338)
    • Protamine 3 (r=–0.373)
    • Peroxiredoxin 6 (r=–0.336)
    • Cysteine-rich secretory protein 3 (r=–0.507)

Conclusions:

  • Conventional sperm quality is not meaningfully affected by liquid nitrogen storage for up to 26 years
  • Fertility-associated messenger RNA abundance does decline with extended storage duration for select candidate genes
  • Major sperm quality changes occur at cryopreservation processing, not during subsequent storage
  • Larger sample sizes are needed to confirm messenger RNA findings

(Vigolo V, Ertl R, Aurich C. 2025. Long-term storage does not result in considerable changes of frozen stallion semen characteristics but affects semen mRNA abundance. Journal of Equine Veterinary Science 145:105332. https://doi.org/10.1016/j.jevs.2024.105332)