Comparison of the Effects of Four Freezing Methods on Motility Characteristics, Morphology, and Viability of Postthaw Stallion Epididymal Sperm

Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freeze-thaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period.