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Simple Summary: Flow cytometry (FC) is the recommended technique for assessing sperm quality. In comparison with fluorescence microscopy (FM), FC supports analyses of much larger sperm populations and generates more reliable results. However, FC is not always accessible, and sperm populations are often evaluated with the use of FM. In the present study, FC and FM were used to assess the functionality of various organelles in European red deer epididymal spermatozoa stored in liquid nitrogen. Spermatozoa were collected from the epididymides of hunter-harvested European red deer stags. The epididymides were stored in a refrigerator (2–4 °C) for 12 h before analysis. The study demonstrated that the refrigerated storage of the epididymides for 12 h had no significant effect on the sperm quality before cryopreservation, but it significantly influenced the percentage of early necrotic sperm after thawing. The results of FM and FC assays differed significantly, excluding in the assessment of the plasma membrane integrity. However, the results of both assays revealed significant correlations between the examined variables, except for mitochondrial activity. The study demonstrated that the spermatozoa from epididymides chill-stored for 12 h can be used for cryopreservation. Fluorescence microscopy and FC are equally reliable techniques, but FM was more useful for evaluating mitochondrial activity. 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. [ABSTRACT FROM AUTHOR]

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage. ● Sperm can be stored in the epididymides in the scrotum at 5 °C for up to 96 h. ● Motility parameters decline earliest during epididymal storage. ● Liquid storage is recommended for short-term preservation of epididymal sperm. ● Bovidyl® has a more positive effect on the progressive movement of sperm than Salomon's. [ABSTRACT FROM AUTHOR]

The objective of this study was a comparative analysis of the impact of the addition of lipoproteins from ostrich egg yolk (LPFo) and hen egg yolk (HEY) to a Tris-citric acid-fructose (TCF) extender on the quality of dog sperm preserved at 5°C for 7 days. The sperm-rich fraction of the ejaculate was manually collected and extended using the following extenders: control (TCF), TCF+ 5% addition of LPFo (TCF-LPFo) and TCF+ 20% of addition HEY (TCF-HEY). The analysis of quality included: sperm motility parameters (TMOT, PMOT, VAP, VSL, VCL, ALH, BCF, STR, LIN), normal apical ridge acrosome (NAR), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and ATP content. The addition of LPFo and HEY to the TCF extender had a beneficial effect on all quality parameters of the stored sperm compared with the TCF extender. However, the analysis of sperm motility parameters revealed differences (P=0.05) between TCF-LPFo and TCF-HEY extenders as affected by storage day. Semen extended in TCF-LPFo was characterized by lower values of TMOT and VAP on Day 5 and by lower values of TMOT and VCL on Day 7, compared to that extended in TCF-HEY. The analysis of PMOT, VSL, ALH, BCF, STR, LIN, PMI, MMP and ATP content showed no differences (P=0.05) between TCF-LPFo and TCF-HEY extenders. The results suggest that TCF extender supplementation with LPFo or HEY improves the quality of dog sperm stored at 5°C. Both LPFo and HEY protect motility, membrane integrity and can contribute to the improvement of the energy status of stored dog sperm. LPFo can be alternatively used instead of HEY for dog semen preservation in the liquid state. [ABSTRACT FROM AUTHOR]