Milk,caseinate and lactoferrin addition to equine semen cooling extenders

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk‐ and glucose‐based, C2) 0.6% caseinate, C3) C2 + Lf 200 μg ml^?1, C4) C2 + Lf 500 μg ml^?1 and C5) C2 + Lf 1000 μg ml^?1 extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 μm per ×10⁶ spermatozoa) compared with the fresh semen (8.6 ± 1.9 μm per ×10⁶ spermatozoa). In contrast, the H₂O₂ concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 μm per ×10⁶ spermatozoa) than in the milk extender (430.9 ± 199.8 μm per ×10⁶ spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 μm per ×10⁶ spermatozoa). Caseinate showed to be as efficient as milk to protect equine‐cooled spermatozoon.     

Lycopene and resveratrol improve post‐thaw bull sperm parameters: sperm motility,mitochondrial activity and DNA integrity

We focussed on evaluating the protective effect of lycopene and resveratrol on post‐thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10^?3 g ml^?1) and resveratrol (1 mm ), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post‐thawed computer‐assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.     

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk‐based extender with Trolox supplementation

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml^?1] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post‐thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml^?1 Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml^?1 groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml^?1 ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.     

Semen quality in adult male survivors 5 years after the 2008 Wenchuan earthquake

The influence of the Wenchuan earthquake on semen quality of adult male survivors is unclear. We investigated the semen quality included 673 male survivors from the worse‐affected counties in the earthquake between Aug 2008 and July 2013. Semen parameters including pH, volume, concentration, motility and morphology were measured according to the World Health Organization (WHO) criteria. Kruskal–Wallis analysis of variance was used to examine the statistical differences between years, and a logistic regression was used to analyse the impacts caused by earthquake on the changes of semen quality. We found the medians (5th and 95th) were 2.5 ml (0.6–5.5) for semen volume, 59.0 × 10⁶ ml^?1 [(13.0–133.0)] × 10⁶ ml^?1 for semen concentration, 46% (13–64%) for sperm progressive motility and 3.0% (0–17.5%) for normal morphology for adult male survivors. Semen concentration, the percentage of sperm progressive motility, total motility and sperm normal morphology were all decreased in the first 3 years, and the differences among years 1, 2 and 3 were significant except the percentage of sperm progressive motility (P < 0.05). The casualties and heavy housing damage caused by earthquake had a negative effect on semen quality. The main findings will provide further diagnosis and therapy basis of male fertility by data, for affected populations in the earthquake.     

Semen quality evaluation in a cohort of 28213 adult males from Sichuan area of south‐west China

The trends in semen quality are conflicting. Although many previous surveys on semen quality indicated a decline, the trends in semen quality in Sichuan area of south‐west China are not clear. We analysed the semen parameters in a cohort of 28 213 adult males close to general population in Sichuan between July 2007 and June 2012, and investigated the changes on semen quality. The semen parameters including pH, volume, concentration, motility, morphology were measured according to the World Health Organization (WHO) criteria. Kruskal–Wallis analysis of variance was used to examine the statistical differences of semen quality between groups. We found that the medians (5th and 95th percentiles) were 2.4 ml (1.0–5.0) for semen volume, 62.0 × 10⁶ ml^?1 (15.0–142.0) for semen concentration, 39% (18–60%) for sperm progressive motility and 10.5% (1.0–34.5%) for normal morphology. In these 5 years, sperm concentration and the percentage of sperm normal morphology were decreased from 66.0 × 10⁶ ml^?1 to 49.0 × 10⁶ ml^?1 and from 13.5% to 4.5%, respectively; among different reproductive history groups, sperm concentration and the percentage of sperm normal morphology were also decreased in these 5 years. And the incidence of azoospermia was increasing. These may imply that there is a decline in semen quality of adult males in Sichuan area.     

Antioxidative effects of cysteamine,hyaluronan and fetuin on post‐thaw semen quality,DNA integrity and oxidative stress parameters in the Brown Swiss bull

The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm ), hyaluronan (0.25, 1 mg ml^?1) and fetuin (5, 10 mg ml^?1) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml^?1 of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml^?1 of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo‐osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml^?1 of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml^?1 of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze‐thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml^?1 of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml^?1 of hyaluronan and 7.5 mm of cysteamine after the freeze‐thawing process (P < 0.001).     

Anogenital distance and reproductive parameters in young men

The purpose of this study was to assess the relationship between anogenital distance (AGD) measures and semen quality and serum reproductive hormone levels in Caucasian young men from southern Spain. Two variants of AGD [from the anus to the posterior base of the scrotum (AGD_AS) and to the cephalad insertion of the penis (AGD_AP)] were assessed in 215 university students. Semen parameters (semen volume, sperm concentration, total sperm counts, motility and morphology) and serum reproductive hormones (follicle stimulating hormone, luteinising hormone, inhibin B, testosterone, calculated free testosterone, oestradiol and sex hormone‐binding globulin) were also determined. Associations between AGD measures and the semen quality and reproductive hormone levels were tested using multiple regression analyses. Overall, median sperm concentration was 44.0 × 10⁶ ml^?1 (5th–95th percentiles: 8.9–129 × 10⁶ ml^?1), median total sperm count was 121 × 10⁶ (18.0–400 × 10⁶), and mean (SD) testosterone level was 21.7 nmol/l (6.9). Mean (SD) AGD_AS and AGD_AP measures were 48.3 mm (11.6) and 128 mm (12.0) respectively. In the multivariable analysis, AGD measures were not associated with any semen parameters or any of the reproductive hormone levels, which is in contrast to results of studies of US young men or infertile men. Further research is warranted to confirm these findings.     

Saturated,omega‐6 and omega‐3 dietary fatty acid effects on the characteristics of fresh,frozen–thawed semen and blood parameters in rams

The aim of this study was to investigate the effects of several dietary fatty acids (FAs) on semen quality and blood parameters in rams. We gave diet‐supplemented treatments (35 g day^?1 ram^?1) by C16:0 (palm oil), C18:2 [sunflower oil (SO)] and an n‐3 source [fish oil (FO)] to 12 rams, who were fed for 15 weeks during their breeding season. Semen was collected once per week. Semen samples were extended with Tris‐based cryoprotective diluents, then cooled to 5 °C and stored in liquid nitrogen. Positive responses were seen with FO after 4 weeks. The mean prefreezing semen characteristics improved with the intake of FO (P < 0.05). Interestingly, maximum sperm output in FO was achieved 7.5 × 10⁹ when compared to palm oil 5.3 × 10⁹. Rams that received FO had the highest total testosterone concentrations (11.3 ng ml^?1 for FO, 10.8 ng ml^?1 for SO and 10.2 ng ml^?1 for palm oil) during the experiment (P < 0.05). FO also improved the rams' sperm characteristics after thawing (P < 0.05). Although C16:0 is a major saturated FA in ram sperm and all rams have been fed isoenergetic rations, the unique FAs of FO improved fresh semen quality and freezing ability compared to other oils.     

Consequences of bilateral cryptorchidism in adults

Bilateral cryptorchidism treatment results are often shadowed by the majority of unilateral cases. We report the long‐term follow‐up results of boys treated for bilateral cryptorchidism during childhood. Patients treated in two main paediatric surgery centres were selected from medical registries and invited for a clinical examination including scrotal ultrasound, salivary testosterone measurement and a semen sample. Thirty‐six men (38.3%) replied to the written invitation, and 21 agreed to be examined. The mean age at orchidopexy was 74 months (range 24–138). Sperm count was 0.42 × 10⁶ (SD ± 0.64 × 10⁶) ml^?1. The correlation between total testicular volume and total sperm count was statistically significant (r = 0.481; P = 0.032). These results show that surgical treatment of bilateral cryptorchidism after the age of 2 years does not prevent infertility. Sperm count and endocrine evaluation advocated after the treatment of bilateral cryptorchidism in all adult patients.     

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.     

The micronutrient supplements,zinc sulphate and folic acid,did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty‐three OAT men participated in a 16‐week intervention randomised, double‐blind clinical trial with daily treatment of folic acid (5 mg day^?1) and zinc sulphate (220 mg day^?1), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A₃ staining; and semen and blood folate, zinc, B₁₂, total antioxidant capacity ( TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10⁶ ml^?1) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.     

A study on the association between serum amyloid A and sperm concentration

Our aim was to compare peripheral blood and seminal fluid serum amyloid A (SAA) protein levels in men classified on the basis of sperm concentration and investigate whether SAA protein is an important marker of male infertility. A total of 74 first‐attempt IVF male partners of infertile couples classified as azoospermic (n = 25), oligozoospermic (n = 25) and normozoospermic group (n = 24) were recruited for this cross‐sectional study. There was no difference with respect to age, BMI, infertility period and smoking ratio. No difference in haematologic parameters including white blood cell count, neutrophil ratio, lymphocyte ratio, neutrophil‐to‐lymphocyte ratio and blood SAA level was found between the groups. Seminal fluid SAA level was 17.85 ± 2.21 ng ml^?1 in azoospermics, 16.13 ± 3.58 ng ml^?1 in oligozoospermics and 15.67 ± 4.77 ng ml^?1 in normozoospermics, showing no significant difference. Seminal SAA level was found to be not correlated with blood SAA levels. Therefore, we could not find any associations between these parameters at all. However, further studies with more participants are needed to address the exact action of SAA on spermatogenesis.     

Porcine sperm vitrification I: cryoloops method

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 10⁶ spermatozoa ml^?1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra‐rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6‐carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo‐osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.     

Cryoprotective effect of l‐carnitine on motility,vitality and DNA oxidation of human spermatozoa

Successful cryopreservation for human spermatozoa markedly influences the reproductive outcomes of assisted reproductive technologies. But in spite of its usefulness, cryopreservation significantly decreases sperm quality. l ‐carnitine has been found to improve the quality of spermatozoa in selected cases with male infertility. Here, we examined the efficacy of l ‐carnitine in improving sperm motility and vitality and reducing sperm DNA oxidation during cryopreservation. Semen samples from infertile patients (n = 22) were collected and analysed. Cryopreservation medium supplemented with l ‐carnitine was mixed with the semen at a ratio of 1 : 1 (v/v). The final l ‐carnitine concentration in each cryovial was 0.5 mg ml^?1 per 5 × 10⁶ cell ml^?1. Controls were cryopreserved without addition of l ‐carnitine. After 24 h of cryopreservation, thawed sperm samples were analysed for motility, vitality and DNA oxidation. Sperm vitality was assessed by the eosin–nigrosin test, while sperm DNA oxidation was measured by flow cytometry. Addition of l ‐carnitine significantly improved sperm motility and vitality (P < 0.05) compared with the control. The flow cytometry experiment showed no statistical difference (P > 0.05) in the levels of DNA oxidation between samples and controls. In conclusion, l ‐carnitine improves human sperm motility and vitality, but has no effect on sperm DNA oxidation after cryopreservation.     

Evaluation of ascorbate and urate antioxidant capacity in human semen

Summary. The ascorbate/urate ratio in the seminal plasma was studied in 76 randomly chosen infertile men. The levels of ascorbate and urate were found to vary widely (range: 93–954 μmol 1^?1 and 127–670 μmol 1^?1, respectively), while the ascorbate/urate ratio was 1.03 ± 0.63 (mean ± SD), indicating almost equimolar concentrations of both compounds in more than 60% of the subjects investigated. No relationship of ascorbate with any biochemical marker of accessory sex gland secretions was observed, whereas an inverse correlation of urate with some prostatic markers, acid phosphatase (?0.37; P<0.001), zinc (?0.35; P<0.002) and citric acid (?0.33; P<0.003), was found. In vitro experiments were conducted on an artificial suspension containing ascorbate and urate at physiological levels and activated polymorphonuclear leukocytes in the normal range (as proposed by WHO) to determine the extent to which the presence of superoxide anion-generating leukocytes contribute to the depletion of ascorbate and urate. The ascorbate level did not change in the presence of 0.2 × 10⁶ leukocytes ml^?1 while higher amounts of activated polymorphonuclear leukocytes initiated ascorbate oxidation, the intensity of which was in correlation with the extent of leukocyte contamination. After incubation (37 °C, 30 min) in the presence of 0.4, 1.0 and 1.5 × 10⁶ cells ml^?1, the average decline from the initial ascorbic acid level was 24, 43 and 49%, respectively. However, exposure of whole semen, instead of buffer, to oxidants released from the same amount of activated polymorphonuclears led to only 2.6, 11 and 22% decrease of the ascorbic acid, most probably due to the action of other superoxide anion scavenger compounds present in semen. Excessive superoxide anion production due to the presence of activated leukocytes had no influence on urate level either in the artificial suspension or in semen. The ability of ascorbate to afford protection against leukocyte-associated superoxide anions in not hampered in the semen of infertile men, provided that leukocyte contamination does not exceed 1 × 10⁶ cells ml^?1. The possible role of urate in stabilizing the ascorbate antioxidant activity in seminal plasma should be further investigated.     

Effect of Coenzyme Q10 supplementation on antioxidant enzymes activity and oxidative stress of seminal plasma: a double‐blind randomised clinical trial

Low seminal plasma concentrations of coenzyme Q10 (CoQ10) have been correlated with impaired sperm parameters, but the exact mechanism remains of dominating interest. This randomised, placebo‐controlled study examined the effect of CoQ10 on catalase, superoxide dismutase (SOD) and F₂‐isoprostanes in seminal plasma in infertile men and their relation with CoQ10 concentration. Sixty infertile men with idiopathic oligoasthenoteratozoospermia (OAT) were randomised to receive 200 mg d^?1 of CoQ10 or placebo for 3 months. 47 persons of them completed the study. Semen analysis, anthropometric measurements, diet and physical activity assessment were performed for subjects before and after treatment. Independent and paired t‐test, chi‐square test and ancova were compared outcomes of supplementation between two groups. CoQ10 levels increased from 44.74 ± 36.47 to 68.17 ± 42.41 ng ml^?1 following supplementation in CoQ10 (P < 0.001). CoQ10 group had higher catalase and SOD activity than the placebo group. There was a significant positive correlation between CoQ10 concentration and normal sperm morphology (P = 0.037), catalase (P = 0.041) and SOD (P < 0.001). Significant difference was shown between the mean of changes in seminal plasma 8‐isoprostane in two groups (P = 0.003) after supplementation. Three‐month supplementation with CoQ10 in OAT infertile men can attenuate oxidative stress in seminal plasma and improve semen parameters and antioxidant enzymes activity.     

Seminal level of clusterin in infertile men as a significant biomarker reflecting spermatogenesis

The objective of this study was to assess the impact of seminal clusterin level on spermatogenesis in infertile men. This study included 89 men who visited our clinic due to infertility, consisting of 28, 33, and 28 diagnosed with normospermia, oligozoospermia and nonobstructive azoospermia (NOA) respectively. The seminal clusterin concentrations measured by enzyme‐linked immunosorbent assay were 47.9, 28.2 and 18.4 ng ml^?1 in men with normospermia, oligozoospermia and NOA, respectively, with significant differences among these three groups (P < 0.01). Microdissection testicular sperm extraction (MD‐TESE) was performed in the 28 men with NOA, and spermatozoon was successfully retrieved from 9. There was a significant correlation between seminal clusterin level and testicular clusterin protein expression evaluated by immunohistochemical staining in these men with NOA (P = 0.026). Of several parameters available before MD‐TESE, the univariate analysis identified serum follicle‐stimulating hormone (FSH) level <10 IU ml^?1 and seminal clusterin level ≥18 ng ml^?1 as significant predictors of sperm retrieval, and of these, only serum FSH level <10 IU ml^?1 was shown to be independently associated with sperm retrieval in the multivariate analysis. Accordingly, it might be worthy to further evaluate the significance of seminal clusterin level as a biomarker for the assessment of spermatogenic status in infertile men.     

Participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa

The aim of this work was to study the participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml^?1) or forskolin (1–75 μm ), a well‐known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2′,5′‐dideoxyadenosine (6–25 μm ). Spermatozoa capacitated with forskolin (25 μm ) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25‐μm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2′,5′‐dideoxyadenosine prevented forskolin‐induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25‐μm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.     

Effects of smoking on fatty acid composition of phospholipid sperm membrane and the malondialdehyde levels in human seminal plasma

The aim of this study was to investigate fatty acids composition of sperm phospholipids, level of lipoperoxidation represented by malondialdehyde and to examine differences between recent smokers and nonsmokers. The levels of malondialdehyde were in the group of all patients 1.51 ± 0.56 μmol l^?1, in smokers 1.36 ± 0.59 μmol l^?1 and in nonsmokers 1.53 ± 0.55 μmol l^?1. Total sperm membrane phospholipid fatty acids were profiled into several groups, saturated acids (in smokers 61.86 ± 9.02%, in nonsmokers 61.20 ± 11.66%), polyunsaturated acids n‐3 (in smokers 12.62 ± 8.18%, in nonsmokers 14.28 ± 13.65%), polyunsaturated acids n‐6 (in smokers 9.13 ± 4.37%, in nonsmokers 10.10 ± 3.79%) and other acids (in smokers 14.36 ± 3.94%, in nonsmokers 13.88 ± 2.31%). Significant correlations were found between the level of malondialdehyde (MDA) and total sperm motility in all patients (r = ?0.358, P = 0.013), between both the level of MDA and progressive motility (r = ?0.465, P = 0.001) and between the level of MDA and total motility (r = ?0.382, P = 0.037) in nonsmokers. There were no statistically significant differences between composition of sperm phospholipid important fatty acids in smokers and nonsmokers. Significant correlations between selected sperm fatty acids and sperm motility and morphology in smokers and nonsmokers were not observed.     

Differential leucocyte detection by flow cytometry improves the diagnosis of genital tract inflammation and identifies macrophages as proinflammatory cytokine‐producing cells in human semen

Genital tract inflammation is considered as a major cause of male infertility with leucocytospermia as widely used diagnostic marker. However, threshold of 10⁶ leucocytes ml^?1 recommended by the WHO is a matter of debate. Moreover, leucocyte subpopulations and their impact cannot be identified by the routine peroxidase method (POM). Ejaculates of subfertile men (n = 47) were analysed by flow cytometry (FACS) using a bead‐based method. Leucocytes were identified by CD18 and further divided into macrophages (HLA‐Dr+/CD66abce‐) and neutrophils (HLA‐Dr‐/CD66abce+). IL‐1β, TNF‐α and IL‐6 production was investigated in these subpopulations. It was found that CD18‐positive cells correlated significantly with POM. However, only in samples with POM below 10⁶ per millilitre, FACS detected significantly higher leucocyte numbers. Moreover, in 31% of these samples, FACS leucocyte detection reached threshold values greater than 1 × 10⁶ ml^?1, fulfilling the criteria for diagnosis of leucocytospermia. Neutrophils were the predominating leucocyte population. Nevertheless, in 24% of samples, macrophages encountered more than 50% of leucocytes. Most interestingly, only macrophages produced significant amounts of IL‐1β, TNF‐α and IL‐6. It is concluded that FACS improves detection and functional differentiation of seminal leucocytes as one of the diagnostic hallmarks of male genital tract inflammation.