Evaluation of Sperm Retrieval Efficiency and Extender Impact in Cryopreserved Canine Epididymal Semen

(1) Background: Cryopreservation of epididymal spermatozoa in dogs is challenging due to their lower cryotolerance compared to ejaculated spermatozoa. Given the limited sperm volume obtained per individual, efficient recovery and preservation techniques are essential. (2) Methods: This study assessed sperm collection and cryopreservation methods from the cauda epididymis of ten dogs undergoing routine elective castration. After dissection and mincing, the cauda epididymidis tissue was incubated in 0.9% saline at 38 °C for either 10- or 30-min. Samples were analyzed for concentration and motility using AndroVision^® software (CASA; AndroVision™; Minitüb GmbH) (Tiefenbach, Germany). Additional evaluations included histological examination, hypoosmotic swelling test, live/dead staining, and morphological assessments. Three extenders, custom-made Tris-Fructose-Citrate (Tris), custom-made Uppsala, and commercial Optixcell^®, were used for cryopreservation and compared for post-thaw sperm quality. (3) Results: No significant differences were found between the 10- and 30-min incubation groups regarding sperm motility, viability, or histological integrity. The total sperm counts were 292 × 10⁶ ± 175 × 10⁶ for the 10 min group and 233 × 10⁶ ± 162 × 10⁶ for the 30 min group (<i>p</i> = 0.56). Histological sections revealed no significant difference in residual intraluminal spermatozoa between groups, indicating that 10 min of incubation is sufficient for effective sperm migration. Post-thaw sperm motility was significantly higher with Uppsala (17.2 ± 12.2%) and Optixcell^® (11.7 ± 6.5%) compared to Tris (4.7 ± 4.8%). Morphological abnormalities were lowest in samples preserved with Optixcell^® (37.5 ± 10.1%, <i>p</i> = 0.005). (4) Conclusion: A 10 min incubation period is adequate for efficient recovery of epididymal sperm in dogs. Among the tested extenders, Uppsala and Optixcell^® demonstrated superior cryoprotective effects, resulting in better post-thaw motility and reduced morphological abnormalities compared to Tris.