Stallion Sperm Cryopreservation Using Various Permeating Agents: Interplay Between Concentration and Cooling Rate

Area of Science:

• Reproductive biology
• Cryobiology
• Biotechnology

Background:

• Cryopreservation of stallion sperm is crucial for genetic preservation and artificial insemination.
• Understanding the effects of cryoprotective agents (CPAs) and cooling rates is essential for optimizing sperm survival.

Purpose of the Study:

• To investigate the interplay between cooling rate and CPA concentration for stallion sperm cryopreservation.
• To compare the efficacy of different CPAs, including glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO).

Main Methods:

• Modeling sperm dehydration during freezing using water permeability values.
• Experimental determination of sperm cryosurvival via flow cytometry (membrane intactness) and computer-assisted motility analysis.
• Testing CPAs at concentrations up to 1500 mM and cooling rates from 5°C to 55°C min⁻¹.

Main Results:

• Dimethylformamide (DMF) yielded the highest cryosurvival rates, followed by GLY and EG. PG and DMSO resulted in poor survival.
• Optimal CPA concentrations varied: DMF survival plateaued at 500 mM, while GLY and EG peaked between 250-500 mM.
• Cooling rate significantly affected survival only at low CPA concentrations; higher concentrations showed less sensitivity.
• DMF cryopreservation resulted in altered motility characteristics (hyperactivation), unlike GLY and EG.

Conclusions:

• DMF is a highly effective CPA for stallion sperm cryopreservation, demonstrating high survival across a range of concentrations and cooling rates.
• Optimal cryopreservation protocols require careful selection of CPA type, concentration, and cooling rate.
• Model predictions for optimal cooling rates did not align with experimental findings, highlighting the complexity of cryopreservation.