Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage.

The research examines the benefits of using dimethylformamide in enhancing the quantity and quality of thawed stallion sperm by reducing cellular damage.

Study Design and Methodology

• The study was conducted with a total of 42 ejaculates from seven Pure Spanish stallions, each providing six ejaculates.
• The ejaculates were separated and frozen in freezing media with diverse concentrations and mixes of cryoprotectants: Cáceres (a milk-based extender) containing glycerol, and Cáceres with a combination of glycerol and dimethylformamide in varying ratios.
• The samples were stored in liquid nitrogen for at least 4 weeks before being thawed and analyzed using computer-assisted sperm analysis and flow cytometry.
• The tests were designed to evaluate membrane lipid architecture, integrity, sublethal damage, and mitochondrial membrane potential.

Findings

• The study revealed that samples frozen in a freezing media containing 4% dimethylformamide or a combination of 1.5% glycerol and 2.5% dimethylformamide yielded more promising results.
• Total motility, a measure of sperm movement, increased by 16% in the 4% dimethylformamide group compared to those frozen in 2.5% glycerol.
• An increase in the percentage of progressive motile sperm was observed in the 1.5% glycerol and 2.5% dimethylformamide group. The percentage of such sperm was nearly double that in the 2.5% glycerol group.
• Samples frozen with 4% dimethylformamide contained a larger proportion of intact sperm (YoPro-1 negative) post-thawing, compared to the ones frozen in 2.5% glycerol.
• Interestingly, the membrane lipid architecture of sperm remained unaffected by any cryoprotectants used in the experiment.
• However, sperm frozen in 4% dimethylformamide presented a smaller proportion of mitochondria with lower membrane potential.

Conclusion

• The research concluded that dimethylformamide enhances the effectiveness of cryopreservation of stallion sperm, primarily by reducing sublethal cryodamage, thus improving sperm quality upon thawing.