Effect of pre-freeze sperm concentration, freezing extender, and epididymal flushing technique on post-thaw quality of cryopreserved epididymal stallion sperm

Cryopreservation of stallion epididymal sperm has become a common clinical procedure after routine castration, euthanasia, or acute death. Unique features of epididymal sperm compared to ejaculated sperm include the requirement to remove sperm from the cauda epididymis, lack of exposure to seminal plasma, and potential recovery of large sperm numbers. In this study, the effect of the flushing technique (Extender (INRA) vs. AIR), freezing extender (LE, CMLE, MFR5, CFR5, or BOTU), and the concentration at which sperm are cryopreserved (200, 400, or 800 × 10⁶ sperm/mL) on post-thaw epididymal sperm quality were studied. More total sperm were recovered when the cauda epididymides were flushed with INRA (14 × 10⁹) compared to AIR (10 × 10⁹). Epididymal sperm cryopreserved in BOTU and CMLE yielded higher post-thaw total and progressive motility than LE, MFR5, and CFR5 (P < 0.05); BOTU, MFR5, and CFR5 yielded higher post-thaw viable/acrosome-intact sperm than CMLE or LE (P < 0.05); CMLE, CFR5, and BOTU yielded higher post-thaw curvilinear velocity than LE or MFR5 (P < 0.05); an effect of extender on sperm DNA damage (COMP_αt) was not observed (P > 0.05). Overall, similar post-thaw epididymal sperm quality parameters were observed among different cryopreserved sperm concentrations (P < 0.05). This study provides evidence of some factors that can affect the post-thaw quality of epididymal sperm from stallions.