Integrity of mitochondrial membrane potential reflects human sperm quality

The aim of this work was to evaluate intracellular reactive oxygen species (ROS) levels, phosphatidylserine (PS) externalisation and mitochondrial membrane potential integrity in the spermatozoa of healthy donors and outpatients who consulted for infertility and to correlate the results with the classic sperm parameters. For the evaluation of intracellular ROS levels, PS externalisation and mitochondrial membrane potential integrity, the fluorescent compounds dihydroethidium, annexin V-FITC and JC-1, respectively, were used and analysed by using flow cytometry. Conventional seminal analysis, including motility, viability, morphology, sperm count and volume, was performed according to the WHO criteria. The mitochondrial membrane potential and ROS results showed significant differences between the spermatozoa of individuals with a normal semen analysis and those of the group presenting abnormality in at least one of the sperm parameters. Mitochondrial membrane potential showed a significant and direct correlation with all the sperm parameters analysed. ROS were inversely correlated with motility, viability and morphology. PS externalisation, however, did not show any differences between the two groups, nor was it correlated with the sperm parameters examined. The evaluation of mitochondrial membrane potential integrity is a test that reflects sperm quality, which makes it highly recommendable to be applied as a complement to routine sperm analyses.     

Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa

The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment 3, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P <.005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by SOD, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment 3, cryopreservation of equine spermatozoa was associated with an increase (P <.01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.     

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μ m Fe²⁺/1 m m ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p  < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p  < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.     

Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential.

Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glycerol decreased the motility and viability of equine spermatozoa, the rapid removal of glycerol by dilution in isosmotic media resulted in an even greater decline in motility and viability compared with spermatozoa maintained under anisosmotic conditions. Likewise, the addition and rapid removal of 1.0 M glycerol, ethylene glycol, dimethylsulfoxide, or propylene glycol resulted in a significant decline in sperm motility and viability. Among these cryoprotectants, ethylene glycol had the least detrimental effect on either viability or motility of spermatozoa following the rapid addition and removal of these cryoprotectants. These data demonstrate that equine spermatozoa have a limited osmotic tolerance compared with published reports for mouse or human spermatozoa, and appear to be more similar to boar spermatozoa in their osmotic tolerance. Of the 4 cryoprotectants evaluated in equine spermatozoa, the addition and removal of glycerol resulted in a more marked osmotic stress as indicated by alterations in motility, viability, and acrosomal integrity. These data suggest that alternative cryoprotectants should be considered for cryopreservation of equine spermatozoa in order to reduce osmotic stress associated with the addition of these agents during semen freezing.     

冻存后大鼠精子活力变化与其结构的关系

目的　观察冷冻对大鼠精子活动能力的影响 ,并探讨其活动能力的变化与细胞膜完整性、DNA结构、线粒体鞘和顶体的关系。　方法　使用计算机辅助精子活动分析仪 (CASA)检测冻存前后大鼠精子活动力的变化 ;荧光素乙酰乙酸盐 (FDA)染色法检测大鼠精子细胞膜的完整性 ;双氢鲁丹明荧光反应观察精子线粒体鞘的变化 ;金霉素 (CTC)荧光检测法观察冻存对大鼠精子顶体的影响。　结果　复苏后精子的活动力下降 ,为冻存前的 39.7% ;FDA检测显示冻存后的大鼠精子细胞膜完整 ,未受到破坏 ;冻存后的大鼠精子线粒体鞘荧光强度下降 ,且荧光不连续、甚至消失 ;CTC荧光反应显示冻存后精子顶体反应 (AR)的类型发生改变 ,AR型精子比例明显下降 ,由冻存前的 6 8.6 %降至冻存后的 13.4 %。　结论　冻存后大鼠精子细胞膜的完整性未受到破坏 ;精子的线粒体鞘和顶体受到较明显的破坏。大鼠精子冻存复苏后活力下降与线粒体鞘和AR存在相关性。     

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.     

Various physical stress factors on rat sperm motility, integrity of acrosome, and plasma membrane

The objective of this study was to determine the effects of various physical interventions such as centrifugation regimes, Percoll gradient separation, and repeated pipetting on various viability parameters of epididymal sperm of Fischer 344 (F-344) and Sprague-Dawley (SD) rat strains. Three experiments were conducted. In experiment 1, sperm motility and acrosomal and membrane integrity were compared after exposing sperm samples to 200, 400, 600, and 800 x g centrifugal forces for 5, 10, or 15 minutes. In experiment 2, sperm motility and acrosomal and membrane integrity were compared after passing them through a Percoll separation using centrifugal forces of 600, 800, 1000, and 1200 x g for either 15 or 30 minutes. In experiment 3, the effect of repeated pipetting (2, 4, 6, 8, and 10 times) on motility and membrane integrity of rat sperm was compared with that on mouse, ram, bull, and boar sperm. The results revealed that both F-344 and SD rat sperm motility and membrane integrity were significantly affected by centrifugation (P < .05). The acrosomal integrity of SD rat sperm was affected after using 800 x g centrifugation force for 10 or 15 minutes (P < .05), whereas F-344 rat sperm acrosomal integrity was not affected by any centrifugation regimes (P > .05). Sperm from SD rats also had higher motility and membrane integrity loss than did sperm from F-344 rats after centrifugation and pipetting (P < .05). Percoll gradient separation did not cause significant motility loss or acrosomal damage to either F-344 or SD sperm (P > .05). Repeated pipetting had a dramatic adverse effect on both rat and mouse sperm motility (P < .05) as compared with sperm from bull, boar, and ram, which were not affected at all (P > .05). These data suggest that rat sperm have unique properties that need to be considered during centrifugation, Percoll gradient separation, and pipetting procedures.     

Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.     

Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology

The accumulated data suggest that inflammation can increase the level of reactive oxygen species (ROS), which contribute to impaired sperm function and male infertility. Therefore, we propose that inflammation-mediated production of ROS in male and female reproductive tracts hinder sperm fertilisation. To test this hypothesis, phorbol myristate acetate (PMA) with polymorphonuclear leucocytes (PMNs) was applied to generate endogenous ROS. We evaluated the time-dependent effects of ROS on human sperm motility, viability and mitochondrial membrane potential (MMP). The results showed that after treatment with PMA and PMNs, the motility of human spermatozoa significantly decreased to 50% on Day 1 and 15% on Day 4 compared with that of the, respectively, negative controls (P = 0.012). The viability of human spermatozoa decreased on Day 4 of PMA + PMNs treatment (P = 0.028). The MMP of human spermatozoa significantly decreased from Day 2 to Day 4 in the PMA + PMN group compared with that of the controls (P = 0.019). Taken together, the 4-day cultivation approach provided an accurate evaluation of sperm quality, especially sperm motility and MMP. Our findings indicated that endogenous inflammation increased ROS levels, which might induce sperm oxidative damage. Additionally, sperm motility might be one of the earliest and most sensitive indicators of this damage.     

Antioxidant effects of the essential oil of Syzygium aromaticum on bovine epididymal spermatozoa

Focusing on its application in reproductive biotechnology, we evaluated the effects of the essential oil of Syzygium aromaticum (EOSA) on bovine epididymal sperm quality variables, including morphology, membrane functional integrity, membrane structural integrity, mitochondrial activity, metabolic activity, motility and oxidative stress by reactive oxygen species (ROS) levels. Bovine spermatozoa from eight males were incubated into the following groups: EOSA0 (without EOSA), EOSA10 (10 μg/ml of EOSA), EOSA15 (15 μg/ml of EOSA) and EOSA20 (20 μg/ml of EOSA); the incubation time with and without the EOSA was 1 or 6 hr. None of the sperm quality variables presented difference among the EOSA concentrations. However, the incubation time had a significant effect on the membrane functional integrity, membrane structural integrity, mitochondrial activity, progressive motility and some kinetic parameters. The effect of interaction among EOSA and incubation time was significant only on ROS levels. Spermatozoa incubated in the presence of 15 μg/ml of the EOSA for 1 hr had significantly reduced ROS levels compared with all other groups in the same time. In conclusion, the EOSA at a concentration of 15 µg/ml has antioxidant effects and protects bovine epididymal spermatozoa; hence, the EOSA may potentially be used in the field of reproductive biotechnology.     

Utilisation of sperm‐binding assay combined with computer‐assisted sperm analysis to evaluate frozen‐thawed bull semen

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm² of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r² = 0.91 and CASA: r² = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.     

Comparison and evaluation of capacitation and acrosomal reaction in freeze‐thawed human ejaculated spermatozoa treated with L‐carnitine and pentoxifylline

Cryopreservation is used to preserve the spermatozoa; however, it leads to a reduction in sperm quality. L‐carnitine (LC) influences sperm motility and preserves the sperm membrane and DNA integrity. The objectives of this study were to evaluate the protective effects of LC on the membrane integrity of normal human spermatozoa and compare it with pentoxifylline (PT) during cryopreservation. Thirty normal semen samples, prepared by swim‐up procedure, were divided into three aliquots: a control without any treatment and two experimental aliquots that were incubated in PT or LC for 30 min. All aliquots were cryopreserved and thawed after 48 hr. To evaluate the percentages of intact, acrosomal‐reacted and capacitated spermatozoa, lectin histochemistry and flow cytometry were performed by wheat germ agglutinin, peanut agglutinin and Con A. Statistical analyses were performed using ANOVA. LC supplementation elevated the percentage of noncapacitated spermatozoa compared with control and PT‐treated samples and the percentages of acrosomal intact spermatozoa compared with PT‐treated samples. PT pre‐treatment improved the motility but not membrane integrity. LC supplementation reduced the percentages of acrosomal‐reacted spermatozoa compared with the control and PT‐treated samples. Although LC did not improve motility, it protected the plasma membrane and acrosomal integrity. Therefore, LC may be the superior choice compared to PT for maintaining the sperm integrity.     

Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa

Motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa after different stages of cryopreservation (ie, dilution, cooling to 4 degrees C, equilibration at 4 degrees C, and freezing and thawing) were examined. Semen ejaculates from 4 buffalo bulls were pooled (n = 5), diluted in tris-citric acid extender, cooled to 4 degrees C over 2 hours, equilibrated at 4 degrees C for 4 hours, dispensed into 0.5-mL straws, and frozen in a programmable cell freezer before plunging into liquid nitrogen. Frozen semen was thawed at 37 degrees C for 15 seconds. After completion of each stage, sperm motion characteristics, plasma membrane integrity, and acrosomal morphology were determined using computer-assisted semen analysis, hypo-osmotic swelling assay, and phase-contrast microscopy, respectively. Data were presented as mean +/- standard error of the mean. Visual and computerized motility did not differ due to dilution, cooling, or equilibration (77.3% +/- 2.3% and 90.5% +/- 1.2%, respectively), but was reduced (P < .05) after freezing and thawing (53.0% +/- 4.6% and 48.6% +/- 6.5%, respectively). Linear motility of spermatozoa was lower (P < .05) after dilution or equilibration (56.2% +/- 2.4%) than after cooling or freezing and thawing (79.6% +/- 1.4%). Sperm curvilinear velocity was reduced (P < .05) from 112.4 +/- 5.3 microm/sec after dilution to 96.0 +/- 5.8 microm/s after cooling, and from 87.6 +/- 4.1 microm/s after equilibration to 69.4 +/- 2.0 microm/s after freezing and thawing. Sperm lateral head displacement differed (P < .05) after each stage (ie, dilution, 3.9 +/- 0.2 microm; cooling, 2.3 +/- 0.2 microm; equilibration, 3.1 +/- 0.3 microm; and freezing and thawing, 1.7 +/- 0.2 microm). Spermatozoa with intact plasma membranes were 80.2% +/- 3.9% after dilution, reduced (P < .05) to 60.4% +/- 5.6% after equilibration, and then to 32.6% +/- 3.8% after freezing and thawing. The percentage of spermatozoa with normal acrosomes remained higher after dilution, cooling, or equilibration (73.2% +/- 2.4%) than after freezing and thawing (61.8% +/- 2.4%; P < .05). In conclusion, the maximal damage to the motility apparatus, plasma membrane, and acrosomal cap of buffalo spermatozoa occurs during freezing and thawing followed by equilibration.     

The effect of cysteine and glutamine on human sperm functional parameters during vitrification

Assuming the adverse effects of reactive oxygen species (ROS) on sperm function, this study was conducted to assess the effects of cysteine and glutamine as effective antioxidants on human sperm parameters under vitrification. Twenty normozoospermic samples were used. The samples were subjected to a vitrification process and cysteine (5 and 10 mM) and glutamine (10 and 15 mM). The sperm motility parameters, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), DNA damage and intracellular ROS damage were assessed for each sample. Statistical analyses showed that motility, mitochondrial membrane potential and DNA damage decreased in the vitrified groups with cysteine 5, 10 mM and glutamine 10, 15 mM separately. Also intracellular ROS increased significantly compared to the fresh group (p < .05). No significant differences were observed for PMI compared with the fresh group (p > .05). Supplementation of cysteine and glutamine in both concentrations separately decreased intracellular ROS and DNA damage of spermatozoa with significant increase in PMI, MMP and progressive motility compared to vitrified control group (p < .05). The results showed no significant effect of a specific concentration in cysteine and glutamine on sperm parameters compared to other concentrations. Both amino acids have the potential to improve the harmful effects of freezing on sperm parameters.     

Goat semen cryopreservation with rainbow trout seminal plasma supplemented lecithin-based extenders

The objective of this study was to determine the optimum concentrations of rainbow trout seminal plasma (RTS) supplemented extenders for goat semen quality at post-thaw and after incubation. Five sexually mature Saanen goat (Capra aegagrus hircus) were used for semen collection. Pooled semen was diluted with soybean lecithin-based extender without RTS (control) or supplemented with different concentrations of RTS (1%, 2%, 4% or 8%), at a final concentration of 150 × 10⁶ spermatozoon/ml. Sperm motility, plasma membrane functional integrity (HOST), damaged acrosome (PSA-FITC), mitochondrial activity (rhodamine123) and DNA integrity (TUNEL) were evaluated. Spermatological parameters were evaluated at post-thaw and after 6 hr incubation. RTS8 group preserved sperm motility, acrosomal integrity, plasma membrane functional integrity and mitochondrial function better than the control group (p < .05). The study demonstrated that RTS supplemented lecithin-based extenders have useful effects on goat spermatozoa. In addition, the results of the current study represented the positive effect of using 8% RTS supplemented extender.     

In vitro effects of nonylphenol on motility,mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml^?1 for 1, 2, 3 or 4 h. Computer‐assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml^?1 was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml^?1) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml^?1 NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose‐dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml^?1 NP for boar spermatozoa and 10 μg ml^?1 NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.     

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa

Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable-capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live-capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat-cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten's medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short-term incubation.     

The effect of sildenafil on human sperm motion and function from normal and infertile men

The aim of this report was to study the effect of sildenafil, a specific type-5 phosphodiesterase inhibitor, on human sperm motility, viability, membrane integrity and sperm penetration assay. Spermatozoa were obtained from normal donors (n = 6) and infertile men (n = 6) were washed using a single Percoll (80%) gradient, suspended in Ham's F-10 medium, and incubated with various doses of sildenafil (125, 250 and 750 ng/ml); pentoxifylline (3 mM) was used as a positive control, and Ham's F-10 was used as a reagent control. Sperm motility, grade, viability, membrane integrity (by hypo-osmotic swelling test), and motion evaluation were carried out at various time intervals. Hamster ova sperm penetration assay (SPA) was used to evaluate overall sperm function. Sildenafil did not affect sperm motility, viability or membrane integrity under these conditions as compared to our Ham's control (P> 0.05). Incubation with pentoxifylline significantly enhanced sperm motility (P < 0.05) and viability without affecting membrane integrity (P < 0.05). Sperm incubated with sildenafil and pentoxifylline from both normal donors and infertile patients demonstrated no significant change in sperm penetration assay from respective controls. In conclusion, sildenafil, at the doses evaluated, did not significantly alter the motility, viability, membrane integrity or sperm penetration characteristics of human spermatozoa from normal donors and infertile patients.     

Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes.

Mammalian spermatozoa are sensitive to oxygen-induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll-separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS-immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that .O2- and .OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.