Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post-thaw sperm quality in stallions.

The research aims to explore the impact of replacing glycerol with alternative cryoprotectants, in various proportions, on the quality of stallion semen after it has been frozen and thawed. The findings suggest that reducing the concentration of glycerol in the extender used for freezing equine sperm can be accomplished effectively by adding urea as a cryoprotectant and increasing glucose concentration.

Research Objectives and Hypothesis

• The study was aimed at investigating the impact on the quality of frozen-thawed stallion semen after partial or complete replacement of glycerol in the freezing extender with alternative cryoprotectants.
• The hypothesis was replacement of glycerol, often found to have toxicity for equine sperm, with cryoprotectants found in cold-resistant frog, insect, or plant species, might result in equal or improved semen quality after freezing and thawing.

Methodology

• A commercial extender named Ghent was used with added ingredients like glucose and urea, trehalose and proline, or trehalose and betaine.
• The sperm was frozen either in Ghent control, Ghent glucose-urea extender, or a Ghent mixed extender in a computerized freezing machine, based on a series of preliminary tests.
• After freezing and thawing, the semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential, and chromatin condensation.

Findings

• No distinctions were observed between Ghent control and Ghent glucose-urea extender. However, all other endpoints minus DNA integrity were negatively affected in Ghent combined extender.
• It was concluded that successful reduction of glycerol concentration in commercial freezing extender for equine sperm could be achieved by increasing glucose concentration and adding cryoprotectant urea. However, complete replacement of glycerol with combination of betaine, proline and trehalose was less successful.