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

The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL progressive motile spermatozoa (PMS) in addition with the cryoprotectant (3 per cent G, 3 per cent EG, 6 per cent EG, 9 per cent EG). Sperm motility was assessed both by microscopy (in raw and frozen-thawed semen immediately after thawing) and with an HTM-IVOS analyzer (Hamilton-Thorne Research, MA, USA), at 0, 1, 4, 6, and 12 h after thawing and storage at 21 degrees C. Raw and frozen-thawed (0 h) semen samples for G and EG at 3 per cent were also submitted to the HOS test with a 100 mOsm sucrose solution and were evaluated to detect the presence of swollen tails. The higher EG concentrations (i.e. 6 per cent EG and 9 per cent EG)significantly reduced the percentage of motile and PMS, immediately after thawing. At the same concentration, i.e. 3 per cent, G resulted in a higher percentage of PMS than EG (36.2 vs. 30 per cent, P 0.05), but at 12 h after thawing and storage at 21 degrees C, no significant differences were detected between G and EG at 3 per cent. The correlations between progressive motility (assessed by direct microscope observation or measured through the HTM analyzer) and the HOS test results for 3 per cent G and EG were r