Donkey Semen Cryopreservation with Different Permeable Cryoprotectants

Donkey Semen Cryopreservation - cartoon donkey in lab with cryoprotectantsOptimizing cryoprotectant selection for donkey semen cryopreservation is critical, as excessive concentrations can negatively impact recipient mare fertility. This two-experiment study evaluated cryoprotectant type and concentration to identify the most effective preservation strategy for Pêga donkey semen.

Experiment 1 — Cryoprotectant Type (5 donkeys, 20 ejaculates):

  • Five cryoprotectants compared at 5% concentration: glycerol, ethylene glycol, methylformamide, dimethylformamide, and a standard glycerol/methylformamide control (1:4 ratio)
  • Dimethylformamide and the standard control achieved the highest total motility
  • Ethylene glycol produced the lowest total motility
  • Progressive motility was highest in the standard control versus glycerol and ethylene glycol
  • Dimethylformamide yielded the best rapid sperm percentage, comparable to the standard control
  • Dimethylformamide and the standard control best preserved plasma membrane integrity

Experiment 2 — Cryoprotectant Concentration (6 donkeys, 30 ejaculates):

  • Standard glycerol/methylformamide combination tested at 5%, 3%, and 2% concentrations
  • Total motility was similar across all concentrations
  • 5% yielded superior progressive motility and rapid sperm percentage versus 2%
  • 3% performed similarly to 5% for all parameters
  • Plasma membrane integrity was unchanged across concentrations

Conclusions:

  • Dimethylformamide is the superior single cryoprotectant for donkey semen cryopreservation
  • The glycerol/methylformamide combination at standard ratios remains a viable alternative
  • Reducing concentration to 3% is a promising strategy to minimize mare fertility risk without significantly compromising sperm quality

(Zanardi C, Carvalho A, Freitas-Dell’Aqua C, Alvarenga MA, Papa FO, Monteiro G, Segabinazzi L. 2025. Characterization of sperm traits of donkey semen cryopreserved with different permeable cryoprotectants. JEVS 145:105336 – Presented in Association with and by Permission of the International Society for Equine Reproduction)