Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm.

This research investigated the replacement of egg yolk, commonly used in sperm preservation in horses, by a cyclodextrin-cholesterol complex, and the resulting impact on the semen’s quality. The goal was to offer better resistance to cell damage during the freezing process.

Research Aim

• The primary aim of the study was to experiment with completely eliminating egg yolk, usually used as a diluent in freezing horse sperm, and substitute it with a cyclodextrin-cholesterol complex.
• Simultaneously, the researchers endeavored to explore decreasing the glycerol content used for cryopreservation and establishing the ideal incubation time between semen and the freezing medium.

Methodology

• Horse ejaculates were frozen using four distinct extenders: A frozen reference medium with egg yolk and 2.5% glycerol (IF), and three media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin (HPβCD-C) cholesterol complex containing either 1% (G1), 2% (G2), or 3% glycerol (G3).
• Different incubation times at 4°C (90, 120, and 180 minutes) between the fresh semen and the specific media were tested before freezing.
• Post-thaw semen quality parameters like viability, progressive, and total motility were analyzed using computer-assisted semen analysis (CASA).

Findings

• Freezing media that included the HPβCD-C complex and either 1%, 2%, or 3% glycerol were found to significantly improve all three in vitro parameters of post-thaw semen quality compared to the reference medium IF.
• The greatest improvement was achieved using the G1 medium and the longest contact time.

Implications of the Study

• The study’s results suggest that the egg yolk typically used in extenders for freezing horse semen can be entirely replaced by the HPβCD-C complex.
• This adjustment reduces the protein load of the medium while maintaining the cholesterol supply to spermatozoa, improving its resistance to osmotic imbalance and increasing tolerance to glycerol toxicity.
• This development could have important implications for equine sperm preservation, as it could potentially improve post-thaw semen quality and thus the success rate of artificial insemination in horses.