Comparison between glycerol and ethylene glycol for the cryopreservation of equine spermatozoa: semen quality assessment with standard analyses and with the hypoosmotic swelling test.

This study compared the effectiveness of glycerol and ethylene glycol for preserving stallion semen in a skimmed milk extender, finding that ethylene glycol could potentially replace glycerol when used at the same or lower concentrations.

Overview and Purpose of the Study

• This study aimed to compare two cryoprotectants – glycerol (G) and ethylene glycol (EG) – for the preservation of stallion semen. The materials were compared under standard conditions and concentrations.
• The researchers aimed to assess the quality of semen using a range of EG concentrations and comparing manual and computerized analysis methods with the hypoosmotic swelling (HOS) test. This test is used to evaluate the functional integrity of the sperm cell membrane and has become a common method to evaluate sperm quality.

Research Methodology

• Ejaculates were collected from two stallions over three weeks, resulting in a total of six ejaculates per stallion. These were diluted, divided into four fractions, and mixed with the cryoprotectant (3% G, 3% EG, 6% EG, or 9% EG).
• Sperm motility was assessed immediately after thawing using microscopy and a specialized machinery known as an HTM-IVOS analyzer. This was carried out at varying intervals (0, 1, 4, 6, and 12 hours) after thawing and storing at 21 degrees Celsius.
• The researchers performed the HOS with a 100 mOsm sucrose solution on raw and 0-hour-freeze-thawed semen samples for G and EG at a 3% concentration and evaluated them to detect the presence of swollen tails.

Study Findings

• The study found that higher concentrations of EG (6% and 9%) significantly reduced the percentage of motile and progressive motile spermatozoa (PMS) right after thawing.
• When cryoprotectant concentration was 3%, glycerol resulted in a higher percentage of PMS than EG (36.2% vs 30%). However, after 12 hours of thawing and storage at 21 degrees Celsius, glycerol and ethylene glycol at 3% did not differ significantly in their effect on sperm motility.
• The study confirmed the HOS test’s usefulness as a complementary method of analyzing stallion semen. It found correlations between progressive motility (assessed by direct microscope observation or measured through the HTM analyzer) and the HOS test results for 3%G and EG were r = 0.61 and r = 0.35, respectively.
• The major conclusion from this research was that EG, if used at similar or lower concentrations, could replace G as a cryoprotectant for stallion semen when using a skimmed milk extender.