Stallion Sperm Lyophilisation: Effect of Source of Sperm and Reproductive Season

This study was conducted to study the quality and DNA integrity of freeze-dried stallion sperm, focusing on the influence of sperm origin (ejaculate vs. epididymis) and seasonality (breeding vs. non-breeding season). Ejaculates were collected from six healthy stallions and epididymal sperm were obtained from 12 stallions via cauda epididymis perfusion during the reproductive season and non-reproductive season in both cases. Sperm samples were diluted in an extender composed of 0.1 M trehalose and BSA 12.5% in PBS, freeze-dried, and stored at 4 °C for at least 3 months. After rehydration, sperm plasma membrane integrity and DNA integrity were analysed using eosin-nigrosin staining and a chromatin dispersion test (SCDt Halomax^®), respectively. Differences in DNA damage were found to be a function of sperm origin. The percentage of DNA fragmentation in ejaculated sperm was significantly higher than in epididymal sperm. However, when evaluating the effect of the season (breeding vs. non-breeding time), DNA integrity did not differ between seasonal groups. In relation to sperm plasma membrane integrity, no differences were found between the experimental groups. Changes in sperm DNA integrity were observed, independently of season, between epididymal and ejaculated stallion sperm after freeze-drying. This study identifies different factors that lead to improved sperm DNA quality after lyophilisation.