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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]

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]

The aim of this study was to assess the quality and fertilizing potential of red deer epididymal spermatozoa stored in a liquid state for up to 11 days (D11). In Experiment 1, sperm quality was determined. In Experiment 2, the efficiency of in vitro fertilization (IVF) and artificial insemination (AI) of stored sperm were evaluated. An analysis of sperm quality on D5 of storage revealed a decrease (p < 0.05) in motility and morphology, and a higher proportion of apoptotic spermatozoa. On D1, D7 and D10, the total motility of sperm for IVF and AI was determined to be 82.6%, 71.0% and 64.8%, respectively. The results of IVF and AI demonstrated that the fertilizing potential of spermatozoa differs between days of storage. The percentage of blastocysts was higher when oocytes were fertilized on D1 (17.4 %) compared to D7 (8.5%) and D10 sperm (10.5%). Differences were noted in the pregnancy rates of inseminated hinds. The insemination with D1, D7 and D10 sperm led to live births (33% from D7 and D10). The results indicate that the quality of red deer epididymal spermatozoa remains satisfactory during ten days of storage in a liquid state, and that these spermatozoa maintain their fertility potential. [ABSTRACT FROM AUTHOR]

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]

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]

Sperm surface binding sites for non-zinc-binding proteins (nZnBPs) and zinc-binding proteins (ZnBPs) were studied by the fluorescence technique with biotin-labelled proteins. The nZnBPs binding pattern was unspecific, no characteristic sites on plasmalemma were found. ZnBPs were attached mainly to the acrosomal region of sperm head and to the sperm flagellum. ZnBPs added to the incubation mixture of the canine spermatozoa allowed the preservation of higher values for total motility, progressive motility, curvilinear line velocity, straight line velocity, and beat cross frequency (P < 0.05), both at time 0 and after 1 h incubation at 5ºC. The addition of nZnBPs to the incubation mixture caused only weak positive effects when compared with control sample (PBS). A higher percentage of canine-ejaculated spermatozoa with intact membranes were observed when ejaculate was incubated with ZnBPs in comparison to control sample stored with PBS (P < 0.05) or nZnBPs (P < 0.05). Spermatozoa diluted with ZnBPs and nZnBPs exhibited a higher percentage of cells with active mitochondria when compared with control, both at time 0 and after 1 h; however, no statistical differences were observed. Our results emphasise the role of seminal plasma protein in securing the correct quantity and availability of zinc ions as a component regulating the motility of canine spermatozoa. The protective effect of ZnBPs against the cooling effect may be due to their ability of preventing sperm membrane damage. [ABSTRACT FROM AUTHOR]

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]

The aim of the study was to assess the impact of solarium light therapy on selected biological and biochemical parameters of peripheral blood in recreational horses. The study involved 10 horses divided into two groups of young (aged 5 to 7 years) and old (aged 14 to 19 years) individuals. All animals participated in light therapy sessions every other day. Blood was sampled three times during the study: before the treatment, after five light sessions, and after ten light sessions. Morphological parameters, the activity of antioxidant enzymes, TAS values, and the levels of glutathione (GSH), vitamin D3, vitamin C, and malondialdehyde (MDA) were measured in the whole blood. Light therapy contributed to an increase in MCV, HDW, MCVr, CHr and MPV indices, and simultaneously a decrease in the basophil counts, MCHC, RDW and CHCMr indices in both groups of horses (p ≤ 0.05). At the same time reticulocytes fell in older whereas white blood cells and monocytes counts expanded in younger individuals. The treatment also increased the activity of glutathione reductase (GR) and glutathione peroxidase (GPx) in young but decreased the activity of mentioned enzymes in blood plasma of old horses. The total antioxidant status (TAS) of the blood plasma rose progressively, whereas GSH levels declined in all individuals. Moreover, vitamin D3 levels did not change, whereas vitamin C levels gradually decreased during the experiment. The therapy also helped to reduce levels of MDA in the blood plasma, especially of older horses (p ≤ 0.05). In turn, GPx and GR activities as well as MDA levels significantly declined, whereas GSH levels notably elevated in erythrocytes (p ≤ 0.05). Solarium light therapy appears to have a beneficial impact on the morphological parameters and antioxidant status of blood in recreational horses in the winter season. However, the observed results could in part be attributed to the natural physiological adaptation of each individual organism to the treatment. [ABSTRACT FROM AUTHOR]

• Liquid-stored epididymal sperm of European red deer can survive up to 25 days. • Sperm motility exceeded 60% until storage day 11. • The integrity of sperm membranes exceeded 80% until storage day 11. • Sperm motility is more sensitive to storage than membrane integrity. • Mitochondrial membrane potential is positively correlated with membrane integrity. The aim of the study was to evaluate viability longevity and quality of liquid-stored epididymal sperm of European red deer (Cervus elaphus elaphus). Sperm samples were recovered from epididymides of 15 mature stags. Samples were diluted to 100 × 106 sperm⁄mL in modified Salomon's extender and stored at 5 °C for 25 days. Sperm were analyzed to determine total motility (TMOT), progressive motility (PMOT) and motion variables [CASA system], acrosomes with normal apical ridges (NAR), viability and acrosomal status (FITC-PNA/PI), plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) on the first day (D1) and every other day of storage (D3-D25). Data were analyzed using a one-way analysis of variance (ANOVA). Spermatozoa remained motile until D25, whereas TMOT exceeded 60%, NAR and PMI exceeded 80%, FITC-PNA/PI exceeded 75%, and MMP approximated 70% until D11. There was a lesser (P < 0.05) value for these variables on D5 (relative to D1). Furthermore, there was a decrease in values for motility variables as duration of storage increased, excluding amplitude of lateral head displacement, on D3. The results indicate that liquid-stored epididymal sperm of European red deer are characterized as having a long viability (25 days) with a retention of sperm quality for as long as 11 days in the liquid storage state. In vitro or in vivo studies, however, are required to confirm the findings in the present study. [ABSTRACT FROM AUTHOR]

Animal fertility is the result of a combination of genetic, physiological and environmental factors. Assessment of semen quality plays a key role in determining the reproductive performance of boars. The aim of the study was to determine the effect of two seasons of the year on the morphology, morphometry, cell membrane integrity and mitochondrial activity of sperm during storage of liquid boar semen at 17 °C. The study was carried out using 20 boars. Four ejaculates were collected from each boar in each of two seasons of the year. All tests were carried out five times: at 1, 24, 48, 96 and 168 h during storage. The results showed that sperm in ejaculates collected in summer are more sensitive to storage conditions than those from ejaculates obtained in winter. The percentage of sperm with morphological defects was shown to increase with the storage time of the diluted ejaculates, particularly between 96 and 168 h of preservation. In summer, the percentage of sperm with an intact cell membrane and the percentage with high mitochondrial membrane potential are much lower than in winter, at every hour of semen preservation. In the case of boars used for artificial insemination, it is worth taking into account the season when the semen is collected, especially if it is to be stored in liquid form. Assessment of sperm cell structures during storage of liquid semen should be implemented at insemination stations and should be carried out more often in summer. • The season affects the morphological structures of boar sperm. • Semen collected in summer is more sensitive to storage conditions than in winter. • The assessment of sperm cell structures during the storage of liquid boar semen should be implemented in insemination stations. [ABSTRACT FROM AUTHOR]

Zinc-binding proteins (ZnBPs) isolated from boar seminal plasma exert multiple effects on boar spermatozoa. The aim of this study was to determine whether the isolated zinc-binding proteins were capable of binding additional ligands. The affinity chromatography method was used for the first time to demonstrate that boar ZnBPs show affinity for both heparin (hep) and phosphorylcholine (pch). ZnBPs+hep accounted for approximately 20%, and ZnBPs+pch - for approximately 45% of total ZnBPs. Predominant polypeptides with molecular mass form 6.5 to 14 kDa were observed in both groups (ZnBPs+hep, ZnBPs+pch). The analyzed peptides' low molecular mass and their ability to bind zinc ions as well as heparin and phosphorylcholine suggest that they belong to the family of multifunctional boar spermadhesins which perform their functions in the fertilization process by binding more than one ligand. The results indicate that the ZnBPs of boar seminal plasma may be involved in the processes associated with oocyte fertilization not only by zinc ions binding but also through heparin and phosphorylcholine. [ABSTRACT FROM AUTHOR]

The aim of the study was to analyse sperm proteomes of ejaculates from Polish Large White (PLW) and Polish Landrace (PL) boars and to identify differences which putatively influence semen quality. Spermatozoa protein profiles were analysed by electrophoretic methods followed by selected techniques to evaluate semen quality on the following factors: sperm motility, lipid peroxidation levels (MDA production), ATP content, activities of superoxide dismutase (SOD) and catalase (CAT), total antioxidant status (TAS), and total oxidant status (TOS) of seminal plasma. A protein with an estimated molecular weight of 30 kDa was found in spermatozoa of selected ejaculates. Mass spectrometry demonstrated that this polypeptide is most similar to proacrosin binding protein (sp32). The presence of the protein was more frequently observed in sperm extracts obtained in spring-summer period. Ejaculates containing sp32-like protein demonstrated significantly higher spermatozoa motility, lower inhibition of MDA production by seminal plasma, and higher SOD activity in seminal plasma. Boar semen which included sp32-like protein also demonstrated lower ATP levels in spermatozoa as well as higher TAS and lower TOS of seminal plasma, though the differences were not statistically significant. Ejaculates from PLW boars, with sp32-like protein present in sperm, were characterised by significantly higher sperm motility, lower ATP content in spermatozoa, and higher TAS of seminal plasma. The diminished parameters of semen quality were observed in ejaculates from PL boars that also contained the discussed protein, but the differences were not statistically significant. These findings suggest that the presence of sp32-like protein in boar spermatozoa could influence semen quality [ABSTRACT FROM AUTHOR]