Dialysis as a new method for addition of glycerol to stallion semen before freezing

The main purpose of this study was to analyse the effect of dialysis, as a method for the addition of glycerol, on the quality of frozen / thawed stallion spermatozoa. Three fertility-proven Lipizzaner stallions and 20 ejaculates were included. Each ejaculate was divided into two equal portions in order to use a split ejaculate technique. Glycerol was added either by pipetting, where the semen pellet was diluted after centrifugation with an extender containing 5% glycerol, or using dialysis in which the semen pellet was first diluted with the extender containing no glycerol, and then glycerol was added with the use of a dialysis system. All the samples were gradually cooled to 5ºC, frozen in nitrogen vapor, and kept in liquid nitrogen until analysed. The semen was thawed in a water bath at 37ºC and the samples were analysed after 10 minutes and 3 hours of incubation at 37ºC, for motility, progressive motility, and concentration, using the CASA system (Computer Assisted Sperm Analysis). The quality of the thawed spermatozoa was further analysed using the hypoosmotic swelling (HOS) test and morphological analysis. The addition of glycerol by means of dialysis significantly increased total motility (P<0.001), progressive motility (P<0.001) 10 minutes after thawing, the percentage of HOS positive spermatozoa after 10 minutes and 3 hours of incubation (P<0.001, P<0.05, respectively), and the percentage of morphologically normal spermatozoa immediately after thawing (P<0.05). The results of this study confirmed the hypothesis that the negative effect, caused by the fast addition of glycerol to semen by pipetting, can be reduced by the gradual addition of glycerol by dialysis.