Fluorescence Microscopy and Flow-Cytometry Assessment of Substructures in European Red Deer Epididymal Spermatozoa after Cryopreservation

Abstract

Thawed spermatozoa, sampled post mortem from the fresh epididymides of European red deer and epididymides stored for up to 12 h at 2–4 °C, were evaluated by fluorescence microscopy (FM) and flow cytometry (FC). The sperm samples were extended and cryopreserved. The sperm motility (CASA), sperm viability (SYBR+/PI-), acrosome integrity, mitochondrial activity, apoptotic changes, and chromatin stability were assessed. Sperm were analyzed by FM before cryopreservation, and by FM and FC after thawing. Epididymal storage time (for 12 h) had no significant effect (p > 0.05) on the examined variables before cryopreservation. After thawing, the storage variants differed (p ˂ 0.05) in the percentage of apoptotic sperm (FM and FC) and DNA integrity (FC). The results of FM and FC differed (p ˂ 0.05) in all the analyzed parameters, excluding SYBR+/PI. Significant correlations (p ˂ 0.01) were observed between the sperm viability, acrosome integrity, and the percentage of non-apoptotic spermatozoa, regardless of the applied technique. In FM, the above parameters were also significantly correlated with mitochondrial activity. The study demonstrated that European red deer spermatozoa stored in the epididymides at 2–4 °C for 12 h can be used for cryopreservation. Both techniques were equally reliable, but FM was better suited for evaluating mitochondrial activity whereas FC was more useful in the evaluation of DNA fragmentation.