Effects of reactive oxygen species on sperm function

Reactive oxygen species (ROS) formation and membrane lipid peroxidation have been recognized as problems for sperm survival and fertility. The precise roles and detection of superoxide (SO), hydrogen peroxide (HP), and membrane lipid peroxidation have been problematic, because of the low specificity and sensitivity of the established chemiluminescence assay technologies. We developed flow cytometric assays to measure SO, HP, membrane lipid peroxidation, and inner mitochondrial transmembrane potential in boar sperm. These methods were sufficiently sensitive to permit detection of early changes in ROS formation in sperm cells that were still viable. Basal ROS formation and membrane lipid peroxidation in the absence of ROS generators were low in viable sperm of both fresh and frozen-thawed boar semen, affecting less than 4% of the sperm cells on average. However, this is not the case in other species, as human, bovine, and poultry sperm have large increases in sperm ROS formation, lipid peroxidation, loss of motility, and death in vitro. Closer study of the effects of ROS formation on the relationship between sperm motility and ATP content in boar sperm was conducted using menadione (mitochondrial SO generator) and HP treatment. Menadione or HP caused an immediate disruption of motility with delayed or no decrease in sperm ATP content, respectively. Overall, the inhibitory effects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to a ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.     

Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 °C

Boar semen is occasionally transferred to different locations in liquid form at 15 °C for cryopreservation. However, the use of frozen boar semen is limited due to the high susceptibility of boar sperm to cold shock. The aim of this study was to help improve the quality of frozen boar semen by determining the changes in sperm membrane and ROS during the cryopreservation processes of 15 °C-stored boar semen. Semen was collected from ten Duroc boars and transferred to our laboratory in liquid form stored at 15 °C. After cooling to 5 °C and freezing-thawing, conventional sperm parameters (total motility, progressive motility, and normal morphology), plasma membrane integrity, acrosomal membrane status, and intracellular ROS were evaluated. Sperm function, as assessed by conventional parameters, was unaffected by cooling but was decreased by freezing-thawing (P<0.05). However, the cooling and freezing-thawing processes led to damages in the sperm plasma membrane, and the cooling process caused increase in mean PNA (peanut agglutinin)-fluorescence intensity in viable acrosome-intact sperm (P<0.05). In ROS evaluation, the cooling process decreased intracellular (·)O(2) and H(2)O(2) in viable sperm (P<0.05), while the freezing-thawing process increased intracellular H(2)O(2) (P<0.05) without change in intracellular (·)O(2) in viable sperm. Our results suggest that, in liquid boar semen stored at 15 °C, cooling may be primarily responsible for the destabilization of sperm membranes in viable sperm, while freezing-thawing may induce reductions in sperm function with increase in membrane damage and H(2)O(2).     

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.     

Evaluation of a cushioned method for centrifugation and processing for freezing boar semen

The purpose of this investigation was to evaluate the use of an iodixanol cushion during centrifugation on sperm recovery and yield after centrifugation (sperm recovery, sperm motility, viability, membrane lipid disorder, acrosome reaction and ROS generation); and to investigate how this procedure affects sperm function after freezing-thawing (sperm motility, membrane lipid disorder, acrosomal status and homologous in vitro penetration test). The sperm-rich fractions from fertile boars were centrifuged under two centrifugation régimes: 800xg for 10min (standard method) and 1000xg for 20min with an iodixanol (60% w/v) cushion at the bottom of the centrifuge tubes (Cushion method). The highest recovery was achieved using the cushion method (sperm loss for cushion method was 0.50%+/-0.18 versus 2.97%+/-0.43 for standard method, P<0.01) and sperm quality was not significantly affected by the centrifugation régime. The motion parameters (% progressive motility, % motility, VCL, VSL, VAP, ALH, BCF, P<0.05) of frozen-thawed samples showed higher values using the standard method. However, a higher number of viable spermatozoa with lower lipid disorders were found in spermatozoa processed with the cushion method. The in vitro penetration assay showed that the individual boar influenced the parameters studied but there were no differences between the two centrifugation régimes used. Our results support the hypothesis that the proportion of sperm loss in frozen-thawed semen was significantly influenced by the centrifugation régime. Therefore, the iodixanol cushion method is a suitable tool for cryopreservation of boar semen in order to reduce sperm loss without affecting sperm quality.     

Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.     

Effects of hypothermic storage on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and plasma membrane integrity in striped bass (Morone saxatilis) sperm

Experiments were conducted to determine the effect of hypothermic 24 h storage on striped bass sperm cell plasma membrane integrity, free intracellular Ca²⁺ ([Ca²⁺]_i), mitochondrial membrane potential (ΔΨ_m), and reactive oxygen species (ROS) formation (oxidation of hydroethidine to ethidium) as determined by flow cytometry; motion activation and ATP concentration as determined by Luciferin-Luciferase bioluminescence assay. Semen was stored for 1 or 24 h at 4 °C in an O₂ atmosphere undiluted or diluted (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma in the presence of various treatments. Viability (% cells excluding propidium iodide) approached 100% after 1 h storage in undiluted or diluted semen. After 1 h of storage the [Ca²⁺]_i marker, Fluo-3, was detected in only 3% of sperm cells in undiluted or diluted semen. In contrast to storage for 1 h, after 24 h the incidence Fluo-3 fluorescence intensity was increased (P < 0.05) in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM ethylene glycol tetraacetic acid (EGTA) blocked CaCl₂-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased (P < 0.05) to 30% after 24 h. However, motility activation failed in the presence of EGTA at 1 or 24 h. During storage ΔΨ_m was not affected by storage time or treatment. In contrast, sperm ATP was greater (P < 0.05) at 1 h than at 24 h and was greater in sperm stored in diluted than undiluted form. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation in the absence of menadione. The increased [Ca²⁺]_i found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.     

Reactive oxygen species-mediated loss of bovine sperm motility in egg yolk Tris extender: protection by pyruvate,metal chelators and bovine liver or oviductal fluid catalase

Improvement of bovine semen cryopreservation requires a better understanding of the properties of the currently used extenders. At present, about half of the spermatozoa die or become immotile following cryopreservation. The implication of an oxidative stress during or following the process of cryopreservation has been suspected to alter sperm functions. However, insufficient information is available on the effect of oxidative stress on sperm functions in their surrounding environment, the extender, such as the one based on egg yolk, Tris and glycerol. In this study, we investigated the effects of hydrogen peroxide (H2O2) and superoxide anion (O2*-) on bovine sperm motility in a widely used egg yolk Tris glycerol (EYTG) extender in comparison to a reference medium, the Tyrode's albumen lactate pyruvate (TALP). Bovine sperm were incubated for 6 h with or without concentrations of H2O2 ranging from 12.5 microM to 1.25 mM and with the hypoxanthine/xanthine oxidase system (X/XOD) that generates O2*-. Sperm motility was established by computer assisted semen analysis (CASA) in four similar experiments using the same frozen pool of semen. We have found that sperm motility was reduced significantly by H2O2 concentrations 20-fold lower in EYTG than in TALP medium. The differential resistance of the two media was explained by pyruvate present in TALP that acts as an antioxidant and metals ions, chelated by ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DETAPAC), found in egg yolk that might react with H2O2. Addition of only 5 U/ml of bovine liver catalase or oviductal fluid catalase (OFC) were sufficient to overcome the loss of sperm motility caused by 100 microM H2O2 in both EYTG and TALP. However, OFC was the most effective of the two catalases in EYTG. In addition to maintain sperm motility, catalase (5 U/ml) and pyruvate (5 mM) increased the intracellular sperm ATP level in comparison to sperm incubated alone for 6 h at 38.5 degrees C in EYTG. Moreover, EDTA, pyruvate and catalase prevented sperm ATP loss in presence of 100 mM of H2O2 in EYTG. These results indicated that EYTG has a very limited capacity to neutralize H2O2, and the addition of low amounts of catalase and millimolar concentrations of pyruvate greatly improved the antioxidant properties of a commonly used extender.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 °C or cryopreserved

To evaluate the influence of dietary supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) on storage of boar semen, three experiments were conducted: two involved long-term, fresh semen storage (Exp. 1 and Exp. 2), whereas the other involved cryopreservation (Exp. 3). Boars were allocated randomly to three dietary treatments (for 6-7 mo). In addition to a daily allowance of 2.5 kg of a basal diet, they received: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO), containing 18% docosahexanoic acid (DHA) and 15% eicosapentanoic acid (EPA); or 3) 60 g of tuna oil (TO), containing 33% DHA and 6.5% EPA. In Experiment 1 (n = 26) and Experiment 2 (n = 18), semen was cooled and stored in vitro for several days at 17 °C before assessment, whereas in Experiment 3 (n = 18), viability, motility, acrosomal integrity, susceptibility to peroxidation (LPO), and DNA fragmentation were determined in fresh and frozen-thawed sperm. In Experiment 1, sperm from boars fed TO had better resistance to fresh storage; even after 7 or 9 d of storage at 17 °C, there were more (P = 0.03) motile sperm in boars fed TO (>60%) than in those fed AF or MO. In Experiment 2, fish oil supplementation did not influence any aspect of sperm quality during semen storage (P > 0.10). In Experiment 3, cryopreservation decreased the proportion of motile and viable frozen-thawed sperm as well as acrosomal integrity and increased DNA fragmentation and LPO (P < 0.01) relative to fresh semen, although sperm quality was unaffected by treatments (P > 0.09). In conclusion, although adding fish oil to the diet failed to significantly improve the quality of cryopreserved boar sperm, inconsistent responses of long-term storage of cooled sperm to dietary n-3 PUFA supplementation warrant further investigation.     

Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3h (control non-dialysis) or dialyzed for 5h in semi-permeable dialysis bags of 12-14kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen-thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced (P<0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect (P>0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher (P<0.05) ATP content compared with the control after freezing-thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing-thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.     

Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing?

In this study a radioisotope method, which is based on the quantitative measurements of tritiated-labeled actinomycin D ((3)H-AMD) incorporation into the sperm nuclei ((3)H-AMD incorporation assay), was used to assess the chromatin status of frozen-thawed boar spermatozoa. This study also tested the hypothesis that frozen-thawed spermatozoa with altered chromatin were susceptible to DNA fragmentation measured with the neutral comet assay (NCA). Boar semen was diluted in lactose-hen egg yolk-glycerol extender (L-HEY) or lactose ostrich egg yolk lipoprotein fractions-glycerol extender (L-LPFo), packaged into aluminum tubes or plastic straws and frozen in a controlled programmable freezer. In Experiment 1, the chromatin status and DNA fragmentation were measured in fresh and frozen-thawed spermatozoa from the same ejaculates. There was a significant increase in sperm chromatin destabilization and DNA fragmentation in frozen-thawed semen as compared with fresh semen. The proportions of spermatozoa labeled with (3)H-AMD were concurrent with elevated levels of sperm DNA fragmentation in K-3 extender, without cryoprotective substances, compared with L-HEY or L-LPFo extender. Regression analysis revealed that the results of the (3)H-AMD incorporation assay and NCA for frozen-thawed spermatozoa were correlated. Boars differed significantly in terms of post-thaw sperm DNA damage. In Experiment 2, the susceptibility of sperm chromatin to decondensation was assessed using a low concentration of heparin. Treatment of frozen-thawed spermatozoa with heparin revealed enhanced (3)H-AMD binding, suggesting nuclear chromatin decondensation. The deterioration in post-thaw sperm viability, such as motility, mitochondrial function and plasma membrane integrity, was concurrent with increased chromatin instability and DNA fragmentation. This is the first report to show that freezing-thawing procedure facilitated destabilization in the chromatin structure of boar spermatozoa, resulting in an unstable DNA that was highly susceptible to fragmentation.     

Effect of Bcl-x(L) overexpression on reactive oxygen species,intracellular calcium,and mitochondrial membrane potential following injury in astrocytes

Many studies have demonstrated the protective effects of Bcl-x(L) against both apoptotic and necrotic cell death, but the mode of action of Bcl-x(L) remains unclear. This work analyzed effects of Bcl-x(L) overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca(2+)](i)), and mitochondrial membrane potential (DeltaPsi(m)) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H(2)O(2)). Upon exposure to GD or H(2)O(2), uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-x(L). Changes in DeltaPsi(m) in response to the two insults differed. H(2)O(2) induced a decrease in DeltaPsi(m) that was initially greater in Bcl-x(L) cells, but then held stable. DeltaPsi(m) in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in DeltaPsi(m,) which was not observed with Bcl-x(L) overexpression. Bcl-x(L) failed to inhibit the calcium dysregulation seen in control cells exposed to 400 microM H(2)O(2), but still improved cell survival. There was no increase in [Ca(2+)](i) with 5 h of GD. These data thus dissociate the effect of Bcl-x(L) on calcium homeostasis from effects on ROS, DeltaPsi(m,) and for H(2)O(2) exposure, cell survival.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage

The present study describes the effects of exposure of bovine sperm to mild and more intense ROS generating conditions. The membrane integrity of the incubated sperm was assessed and the incubated sperm were used for IVF after which the percentages of cleavage and blastocyst formation were determined for a period up to 9 days. The incubated sperm samples showed increased levels of molecular oxidation in the plasma membrane, the mitochondria, the cytosol and to a lesser extent in the sperm's DNA. The sperm membrane integrity as well as the first cleavage rates obtained with sperm from mild ROS generating conditions (100 microM H2O2) were not different from sperm incubated without pro-oxidants. However, exposure of sperm to more severe oxidative stress (500 mM H2O2 or a combination of 100 microM ascorbic acid, 20 microM FeSO4 and 500 microM H2O2) led to plasma membrane oxidation, reduced percentages of cleaved embryos and a reduction in the percentages of cleaved embryos that developed to the blastocyst stage. From these results, we conclude that the impact of oxidative stress to sperm becomes primarily manifest after the first cleavage of the formed zygote. Importantly, the level of lipid peroxidation in the sperm plasma membrane significantly correlates with blastocyst formation when the corresponding sperm is used for in vitro fertilization of oocytes.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa

Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 x 10(6)/ml) were incubated for 30 min at 38 C with neutrophils (0,0.5 x 10(6),1 x 10(6), 5 x 10(6) and 10 x 10(6)/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H2O2 was measured at T30 with the Amplex Red assay kit. At T30, there was a significant (P < 0.01) increase in H2O2 with the addition of 5 x 10 and 10 x 10(6) neutrophils/ml activated by FMLP (0.76 +/- 0.3 and 0.99 +/- 0.4 microM, respectively, versus 0.0024 +/- 0.002 microM in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 +/- 5.1 and 48 +/- 6.0%, respectively, versus 80 +/- 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H2O2 with the addition of 5 x 10(6) and 10 x 10(6) neutrophils/ml activated by PMA (1.88 +/- 0.2 and 2.07 +/- 0.3 microM, respectively, versus 0.0009 +/- 0.0006 microM in sperm alone). The results of this study demonstrate that 5 x 10(6) activated neutrophils/ml are sufficient to impair equine sperm motility in vitro.     

Freeze-thawing as the factor of spontaneous activation of spermatozoa motility in common carp (Cyprinus carpio L.)

In the present study, we investigated the possibility of spontaneous carp spermatozoa activation by freeze-thawing. To evaluate this, the parameters of spermatozoa motility percentage, velocity, ATP content level and fertility rate of sperm were used. The motility and velocity of spermatozoa activated by freeze-thawing were characterized by motile spermatozoa with a median value of 16% and a velocity of 98 μm/s. In addition, the motility and velocity of sperm from the thawed samples were significantly lower than in the control (median value of 100% for sperm motility and 175 μm/s for sperm velocity). Furthermore, a spontaneously activated spermatozoa motility terminated within five minutes post-thaw time. After freeze-thawing the ATP level significantly decreased with post-thaw time (46 nmol ATP/10⁹ and 10 nmol ATP/10⁹ at 25 s and 10 min after thawing, respectively). Fertility of spermatozoa was not significantly affected within 10 min post-thaw. On the other hand, the fertility of frozen-thawed sperm was significantly lower if compared to fresh sperm. We conclude that the freeze-thawing procedure spontaneously activated spermatozoa motility in common carp. However, this activation did not negatively affect the fertility of frozen-thawed sperm.     

Ubiquitination and its influence in boar sperm physiology and cryopreservation

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination (n=12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen-thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen-thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% (P<0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120min of induced capacitation (P<0.05). In conclusion, frozen-thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.