Diluent Containing Dimethylformamide Added With Sucrose Improves In Vitro Quality After Freezing/Thawing Stallion Sperm.

The research investigates the impacts of adding sucrose to the freezing extender used in the preservation of stallion sperm, revealing a positivity in semen quality, including enhanced motility and better membrane integrity, after thawing when different concentrations of sucrose were used.

Research Goal

• The main objective of this research was to evaluate the effects of the addition of sucrose to the freezing medium (extender) used for stallion sperm in terms of its effects on the quality of the sperm post-thawing.

Methodology

• Semen samples were acquired from six different stallions, totalled to 24 samples.
• These samples were diluted in a specifically formulated freezing medium comprising of skimmed milk, egg yolk, dimethylformamide with added sucrose. The sucrose concentrations used were in the degrees of 0 (Control), 25, 50, and 100 mM. A commercial freezing extender, BotuCrio, was also used for comparison.
• The diluted semen samples were then placed in straws and frozen, stored at -196°C.
• Upon thawing at 37°C, the samples were assessed for kinetics using Computer Assisted Sperm Analysis (CASA), and also checked for plasma and acrosomal membrane integrity, as well as mitochondrial membrane potential using flow cytometry.

Main Findings

• The study found that there were increased parameters of TM, PM, VCL, VSL, and VAP when 50 and 100mM amounts of sucrose were added to the freezing extender in comparison to the control group.
• The WOB parameter was higher when the concentration of sucrose was at 100 mM.
• Higher values of ALH and BCF were observed in the groups treated with sucrose (25, 50, and 100 mM), compared to the commercial extender, BotuCrio.
• The sperm samples that were frozen with 100 mM sucrose showed higher percentages of sperm with intact plasma and acrosomal membranes, as well as a high mitochondrial membrane potential compared to the other groups.

Conclusion

• The study concludes that adding sucrose to the equine semen freezing extender can enhance the sperm quality upon thawing. It was noticed to notably increase sperm motility at concentrations of 50 and 100 mM, and also preserve plasma, acrosomal membrane integrity, and maintain a high mitochondrial membrane potential at 100 mM concentration.