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Simple Summary: The choice of the optimal sperm preservation method is an important consideration in animal breeding. Stored semen can be used for reproductive purposes to introduce new genotypes and prevent inbreeding, which poses a considerable problem in cervid farms. The aim of this study was to evaluate and compare the effect of storage time and storage method (liquid state/epididymides) on the motility, morphology, and antioxidant status of European red deer sperm stored at 5 °C for up to six days (D0-D6). Sperm samples were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (based on malondialdehyde, MDA, content). Significant differences between storage variants were noted on D2 in sperm morphology; on D4 in the percentage of progressively motile sperm, MDA content, and SOD and GPx activity; and on D6 in the percentage of motile and viable spermatozoa. Sperm motility, viability, and antioxidant status are more effectively preserved during liquid storage than epididymal storage. Morphological and functional abnormalities of sperm were observed earlier during epididymal storage, which suggests that spermatozoa can be stored for shorter periods of time in the epididymides than in a liquid state. The aim of this study was to evaluate the motility, morphology, and antioxidant status of European red deer sperm stored in a liquid state (variant I) and in the epididymides (variant II). Spermatozoa were harvested post-mortem from the cauda epididymis. Sperm samples in both variants were stored for up to six days (D6) at 5 °C. Spermatozoa were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (malondialdehyde, MDA, content). Sperm samples were analyzed on storage days 0, 2, 4, and 6 (D0-D6). Storage time and storage method significantly (p ≤ 0.05) influenced the examined variables. On D2, a decrease in motility and acrosomal integrity was observed in both storage variants, whereas a decrease in viability and an increase in MDA content were noted in spermatozoa stored in the epididymides. On D4, higher values of SOD and GPx activity and MDA content were noted in variant I than in variant II. Catalase activity was very low. GPx is the key enzyme that participates in the reduction of hydrogen peroxide in sperm cells. Spermatozoa stored in a liquid state were characterized by higher motility and viability, improved morphology and antioxidant status than those stored in the epididymides; therefore, liquid storage is more recommended for short-term preservation of epididymal spermatozoa. [ABSTRACT FROM AUTHOR]

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]

However, liquid preservation is often the method of choice in many animal species due to its simplicity, low cost, and ability to preserve sperm fertilizing capacity. Henning et al. [[2]] reported that a high percentage of spermatozoa with morphological defects, such as cytoplasmic droplets in ejaculated boar semen (above 15%), can compromise sperm capacitation and decrease semen fertilizing capacity. The choice of the preferred sperm-preservation method (liquid or cryopreservation) is determined by the species. The sperm of various animal species differ in morphology, biological parameters, and susceptibility to cryopreservation [[1]]. [Extracted from the article]

Simple Summary: The use of commercial extenders for storing cervid epididymal spermatozoa in a liquid state can contribute to popularizing this method of semen preservation in cervid farms. The aim of this study was to assess the effect of various extenders (Bovidyl®, BoviFree®, and BioXcell®) on the quality of European red deer epididymal spermatozoa stored at 5 °C. The analyses demonstrated that the tested extenders and storage time significantly affected sperm motility and functionality of different sperm structures. The Bovidyl® extender containing chicken egg yolk exerted the most beneficial influence on the quality of stored sperm. Spermatozoa stored in the Bovidyl® extender were characterized by satisfactory motility (above 60% on storage day 9) and the functionality over a long period of time. In turn, extenders containing plant-based ingredients and glycerol (BioXcell® and BoviFree®) significantly decreased membrane integrity and mitochondrial activity of spermatozoa already on the first day of storage. These results indicate that European red deer epididymal spermatozoa can be stored in commercial extenders, but their viability and functionality are significantly influenced by the composition of the applied extenders. The extender containing chicken egg yolk without glycerol produced the most satisfactory results. The aim of this study was to evaluate the effect of different extenders on the quality of European red deer epididymal sperm stored at 5 °C. Epididymal spermatozoa were collected post mortem from 10 stags and diluted with three extenders (Bovidyl®, BoviFree®, and BioXcell®) and stored at 5 °C. Sperm motility (TMOT), motility parameters (system CASA), plasma membrane integrity (SYBR-14+/PI−), acrosomal membrane integrity (FITC-PNA−/PI−), mitochondrial activity (JC-1/PI), viability, and apoptotic-like changes (YOPRO/PI) were evaluated. The analyses were conducted on the first and successive days of storage (D1–D7). The applied extender, storage time, and the interactions between these factors significantly (p < 0.001) affected most of the analyzed parameters whose values were highest in sperm samples stored in Bovidyl®, regardless of storage time. In Bovidyl®, BoviFree®, and BioXcell® extenders, TMOT values were estimated at 83%, 63%, and 59%, respectively, on D3. The extenders significantly influenced DNA integrity on D7. The percentage of dead sperm increased from D3. The quality of stored sperm cells was significantly influenced by the extenders' biochemical composition. BoviFree® and BioXcell® contain glycerol which could contribute to deteriorating the quality of spermatozoa stored at 5 °C. Sperm cells stored in the egg yolk-based extender (Bovidyl®) were characterized by the highest viability and functionality. [ABSTRACT FROM AUTHOR]