Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa

Abstract

Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75 °C and 37 °C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37 °C for 30 sec or at 75 °C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P = 0.84) between sperm motility after thawing at 37 °C and 75 °C. There was also no significant difference (P = 0.053) between the percentage of live spermatozoa using the calcein AM/ethidium homodimer stain after thawing at 37 °C and 75 °C. There was, however, a significant difference (P = 0.032) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37 °C compared with that at 75 °C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37 °C instead of 75 °C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa.