Effects of season on boar semen parameters and antioxidant enzymes in the south subtropical region in Brazil

Although boar semen productivity is affected by seasonality, its effects are not equal among different regions which raise concerns regarding the profitability of boar stud farms. Therefore, the goals of this study were (i) to evaluate the seasonal effect on semen production in a commercial boar stud farm located in a subtropical climate region and (ii) to verify whether the activities of superoxide dismutase and glutathione peroxidase in spermatozoa and seminal plasma were associated with seminal traits of fresh and cooled semen. Nine boars were collected twice per season, and routine seminal parameter analyses were performed together with superoxide dismutase and glutathione peroxidase activities in seminal plasma and spermatozoa. Despite a reduction in sperm concentration in spring and summer, most seminal parameters were constant year‐round. Temperature–humidity index was higher in the summer compared to spring, autumn and winter (p < .05). Superoxide dismutase activity in spermatozoa was increased in summer compared to autumn and winter (p < .05). The activities of both enzymes in seminal plasma and spermatozoa glutathione peroxidase remained unaltered throughout the seasons. In conclusion, seasonality showed little influence in overall boar seminal parameters despite microclimatic differences among seasons, and spermatozoa collected during summer increased superoxide dismutase activity.     

Evaluation of the cryoprotective effect of seminal plasma on llama (Lama glama) spermatozoa

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated. A significant decrease in sperm motility, viability, membrane function and intact acrosomes was observed in thawed samples (0%, 10% and 50%) when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > 0.05), but a significant increase (p < 0.05) in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples compared to raw semen. Higher percentages of total and progressive sperm motility were observed when 0% and 10% of seminal plasma were used compared to 50%. However, no statistical differences were established for sperm viability, membrane function, morphology, acrosome status and DNA quality between thawed treatments. To conclude, neither of the percentages of seminal plasma used showed superiority nor cryoprotective effect on llama sperm survival.     

Use of post‐thaw semen quality parameters to predict fertility of water buffalo (Bubalus bubalis) bull during peak breeding season

This study was designed to predict the fertility of water buffalo bull using post‐thaw semen quality parameters during peak breeding season. Thirty ejaculates were collected from five bulls with artificial vagina and cryopreserved. At post‐thaw, semen was analysed for motility parameters, velocity distribution, kinematics, DNA integrity/fragmentation, viability, mitochondrial transmembrane potential, morphology, plasma membrane and acrosome integrity. Data of 514 inseminations were collected for estimation of in vivo fertility. Pearson's correlation coefficients showed that progressive motility (PM), rapid velocity, average path velocity, straight line velocity, straightness, supravital plasma membrane integrity, viable spermatozoon with intact acrosome or with high mitochondrial activity were correlated with in vivo fertility (r = .81, p < .01; r = .85, p < .01; r = .64, p < .05; r = .73, p < .05; r = .57, p < .05; r = .88, p < .01; r = .84, p < .01 and r = .81, p < .01 respectively). Step forward multiple regression analysis showed that the best single predictor of fertility was PM. However, combinations of semen quality parameters to predict fertility were better as compared to single parameter. In conclusion, fertility of buffalo bull can be predicted through some of the post‐thaw in vitro semen quality tests during peak breeding season.     

Seminal plasma vitamin B6 levels in men with asthenozoospermia and men with normal sperm motility,a measurement using liquid chromatography with tandem mass spectrometry

There is growing evidence that vitamin B₆ has a valuable contribution in maintaining normal sperm parameters; however, this contribution has not yet well-identified. Here, we aimed to measure the level of seminal plasma vitamin B₆ in men with asthenozoospermia compared to men with normal sperm motility. Ninety-seven human males with asthenozoospermia and eighty-eight human males with normal sperm motility (control) were recruited in this study. Collected semen samples were assessed for sperm motility, sperm count and semen volume. Liquid chromatography with tandem mass spectrometry was used to measure seminal plasma vitamin B₆ concentrations. A highly significant difference (p < .0001) in concentrations of seminal plasma vitamin B₆ was found between asthenozoospermic and control groups. Besides, no statistical correlations were found between seminal plasma vitamin B₆ level and sperm motility, sperm count, semen volume and men age in both tested groups. In conclusion, men with asthenozoospermia have lower seminal plasma vitamin B₆ level compared to men with normal sperm motility. Also, seminal plasma vitamin B₆ was found not to be correlated with sperm motility and count, semen volume and men age in both tested groups. These results may provide new contribution in the management of male infertility.     

Aspirin decreases human sperm motility and vitality,chelates seminal calcium,but insignificantly reduces seminal nitric oxide production

Lately, there is a systematic research consensus that reveals adverse effects of aspirin on semen quality characteristics; however, such consensus is lacking further confirmation by human studies. Therefore, here, we asked whether sperm motility and vitality are affected in the presence of aspirin at 0.1 and 1 mM in the ejaculated semen, and whether such effect may be due to an alteration in seminal calcium ions or seminal nitric oxide production. Forty-three semen samples from different normozoospermic men were recruited in this study. Sperm motility was measured by Makler counting chamber, and sperm vitality was measured by Eosin test. Calcium chelating effect of aspirin and seminal nitric oxide production was measured spectrophotometrically. Aspirin at both tested concentrations significantly (p < .05) reduced progressive grade-a motility and vitality of spermatozoa. Additionally, aspirin was found to have significant ability (p < .05) to bind seminal calcium ions, but insignificantly reduced the amount of seminal nitric oxide. In conclusion, sperm motility and vitality were reduced in the presence of aspirin at 0.1 and 1 mM in semen. Such reduction may be attributable to the ability of aspirin to chelate seminal calcium ions, but not to an alteration in the amount of nitric oxide produced.     

The presence of seminal plasma,especially derived from stallion semen,helps preserve chilled Asian elephant (Elephas maximus) sperm motility

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris–glucose–egg yolk extender, supplemented with different SP types and preserved at 4°C for 48 hr. Experiment 1 (n = 31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p < .05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p < .05). Experiment 2 (n = 14) determined the effect of different SP from four stallions. All stallion SP gave higher (p < .05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage.     

The effect of cigarette smoking on human seminal parameters,sperm chromatin structure and condensation

Considerable debate still exists regarding the effects of cigarette smoking on male fertility. This work aimed to explore effects of cigarette smoking on semen parameters and DNA fragmentation on 95 infertile patients who were divided into infertile male nonsmokers (45) and infertile male smokers (50). Smokers were subdivided according to a number of cigarettes smoked per day into mild (≤10), moderate (11‐20) and heavy smokers (≥21). Semen analysis, sperm chromatin condensation integrity with aniline blue staining and sperm viability were compared between the study groups. A significant decrease has been shown in sperm count (p = .006), progressive motility (p = <.001), percentage of normal forms (p = <.001) and viability (p = .002) between infertile nonsmoker and infertile smokers. The percentage of abnormal sperm chromatin condensation was significantly higher in smokers compared to nonsmokers (p = <.001). A linear correlation was detected between the extent of cigarette smoking and the degree of worsening in progressive motility (p = .001), total motility (p < .001), viability (p < .001) and normal morphology (p < .001). These results indicate that cigarette smoking has detrimental effects on semen parameters. It negatively affected all conventional semen parameters in addition to sperm chromatin condensation and sperm viability. These abnormalities were also proportional to the number of cigarettes smoked per day and to the duration of smoking.     

Incubation of frozen-thawed llama sperm with seminal plasma

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C). A total of 20 ejaculates from five adult llama males (n = 5; r = 4) were evaluated. A significant decrease in sperm motility, membrane function and live sperm was observed in all thawed samples (0%, 10% and 50%) at 0 hr when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > .05), but a significant increase in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples versus raw semen. When evaluating thawed samples over time, a significant decrease of motility and membrane function was observed, while the percentages of total live sperm were preserved over the 3 hr of incubation in all final concentrations evaluated. To conclude, the addition of 10% or 50% of seminal plasma was incapable of preserving motility or membrane function of frozen-thawed llama sperm during 3 hr of incubation.     

Cyclooxygenase 1 (COX1) as an indicator of sperm quality in humans

Sperm quality is important for in vitro fertilisation and embryo transfer and intracytoplasmic sperm injection in the treatment of human infertility. The purpose of this study was to screen for biomarkers that could evaluate sperm quality. We analysed semen samples in 172 fertile males; multivariate logistic regression analysis showed that the levels of COX1 (17.5 ng/ml) in seminal plasma may represent a useful biomarker for sperm quality (area under the curve: 0.745; sensitivity: 0.808; specificity: 0.722). Analysis indicated that the values of parameters related to sperm quality changed significantly (p < .05) between COX1 level ≥ 17.5 ng/ml group and COX1 level < 17.5 ng/ml group. Further analysis of two consecutive semen samples (1-hr interval) from 48 subjects revealed that the first semen samples (COX1 levels ≥ 17.5 ng/ml) had a higher sperm concentration and a larger proportion of spermatozoa showing progressive motility, a lower rate of sperm DNA fragmentation and a lower proportion of spermatozoa undergoing the acrosome reaction spontaneously (p < .05); identical results were observed for the second semen samples. These data indicated that COX1 could be used as an indicator for sperm status and may be helpful for identifying better quality spermatozoa for artificial insemination.     

Is addition or removal of seminal plasma able to compensate for the dilution effect of buffalo semen?

The present study aimed to compensate dilution effect using additional seminal plasma (SP) in conventional (80 million (M) spermatozoa/ml) dose and low spermatozoa/dose (8M spermatozoa/ml). We also attempted to confirm whether removal of SP before the extension of ejaculates affects post-thaw sperm quality of buffalo semen. For this, semen ejaculates (N = 15) were divided into four groups: control (CON), removal of SP by centrifugation (NSP), resuspension of the centrifuged semen pellet into SP (CEN) and extra supplementation of SP (ESP). All groups were diluted into two different semen doses to 20 and 2M spermatozoa/0.25 ml using tris egg yolk extender and subsequently cryopreserved. We found that neither addition nor removal of SP affected sperm motility, kinematics, longevity, mitochondrial superoxide production and high mitochondrial membrane potential (MMP). Further, the addition or removal of SP was not able to compensate dilution effect in 2M groups resulting in a significantly (p < .05) reduction in sperm motility, kinematics, sperm longevity, membrane integrity, MMP, and an increase production of mitochondrial superoxide. In conclusion, it appears that role of SP in the sperm cryopreservation process is insignificant.     

The changes in semen quality,arginase activity and nitric oxide level in dexamethasone-treated rams

This study was made to investigate the effects of intramuscular administrations of dexamethasone on seminal plasma nitric oxide levels and arginase activity, and some spermatological parameters in rams. Ten Akkaraman rams weighing 50–60 kg and 2 years old were used as material in this study. The study was performed during the breeding season (September–November) for rams. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th, 72nd and 96th hours for control group before dexamethasone administration. For treatment group, 0.25 mg/kg dexamethasone was administered and semen was collected at the time points described for control group. Spermatological characteristics of semen samples (semen volume, pH, sperm motility, density and abnormal sperm rate), seminal plasma arginase enzyme activities and nitric oxide levels were determined. It was determined that the administration of dexamethasone was detected to decrease seminal plasma arginase activity (p < .05 and .01) and nitric oxide level (p < .05), semen volume (p < .05 and .01), mass activity (p < .05 and .01), sperm density (p < .05) and sperm motility (p < .05 and .01), and to increase abnormal sperm rate (p < .05 and .01). In conclusion, dexamethasone is not recommended to be used during the breeding season as it damages the sperm quality of the rams.     

Age is a significant predictor of early and late improvement in semen parameters after microsurgical varicocele repair

Accumulating evidence indicates that varicocele repair improves sperm quality. However, longitudinal changes in sperm parameters and predictors of improved semen characteristics after surgery have not been fully investigated. We retrospectively reviewed data from 100 men who underwent microsurgical subinguinal varicocele repair at a single centre. Follow‐up semen examinations were carried out at 3, 6 and 12 months post‐operatively. Logistic regression was used to identify predictors of early (3 months) and late (≥6 months) improvement in semen parameters after varicocele repair. At 3 months post‐operatively, 76.1% of the patients had improved total motile sperm counts, which continued to improve significantly up to 12 months post‐operatively (p = .016). When comparing changes in semen parameters between younger (<37 years) and older (≥37 years) men, post‐operative improvements in sperm concentration and motility were greater among younger men. Multivariate analysis showed that younger age was associated with early (p = .043) and late (p = .010) post‐operative improvement in total motile sperm count. Our findings indicate that early varicocele repair improved semen parameters after surgery.     

Response to cooling of pony stallion semen selected by glass wool filtration

The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Retained semen was considered the portion of cells that did not transpose glass wool barrier. Total motility from the control, filtered and retained groups after 24 h of cooling was 35.5%, 43.3% and 10% (p < .0001) respectively. Sperm membrane integrity percentage at the CFDA/PI test was 37.9%, 44.8% and 14.8% (p < .0001), on the control, filtered and retained groups respectively. The results confirmed that the passage of spermatozoa through glass wool increased the selection of spermatozoa from pony stallions with higher motility, mitochondrial viability and membrane integrity for cooling in milk extender up to 24 h. Moreover, it was not obtained higher sperm parameters to control after cooling 48 h under the conditions that the study was conducted.     

Caffeine increased progressive motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples and enhanced activity of seminal creatine kinase

Even though the effect of caffeine on humans' health has been revealed in various research studies, its effect on semen quality has yet to be well explained. Here, we measured the effect of caffeine at 1, 5, 10 and 20 mM on motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples, level of seminal nitric oxide, chelation of seminal calcium ions and activity of seminal creatine kinase. Fifty-one normozoospermic and ten asthenozoospermic semen samples were recruited in this study. Sperm motility was evaluated by Makler counter, and seminal nitric oxide, seminal-free calcium and activity of seminal creatine kinase were measured spectrophotometrically. Caffeine at 10 mM significantly (p < .05) increased progressive motility of spermatozoa in both tested groups. Also, caffeine significantly increased (p < .05) activity of creatine kinase and insignificantly (p > .05) altered nitric oxide and free calcium levels in seminal plasma. In conclusion, progressive motility of human spermatozoa was found to be higher in the presence of caffeine at 10 mM in normozoospermic and asthenozoospermic semen samples; this increase, albeit partially, could be due to increased activity of seminal creatine kinase, but not to increased production of nitric oxide or chelation of free calcium ions.     

Evaluation of the role of L-arginine on spermatological parameters,seminal plasma nitric oxide levels and arginase enzyme activities in rams

The purpose of this study is to investigate the effects of L-arginine on spermatological parameters, seminal plasma nitric oxide levels and arginase enzyme activities. Fertile rams that are 2–3 years old and weighing 50–60 kg were used as material. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th, 72nd, 96th and 120th hours for the control group before L-arginine administration. For treatment groups, L-arginine was administered intraperitoneally at a dose of 5 mg kg⁻¹ bw⁻¹ and semen was collected at the time point described for the control group. Spermatological characteristics of semen samples (semen volume, pH, sperm motility, concentration and abnormal sperm rate), seminal plasma nitric oxide levels and arginase enzyme activities were determined. Increased seminal plasma nitric oxide level (p < .01), seminal plasma arginase activity (p < .01), semen volume (p < .05), semen mass activity (p < .05), sperm motility (p < .05) and concentration (p < .01) and decreased abnormal sperm rate (p < .05 and p < .01) were observed by L-arginine administration. In conclusion, it may be concluded that L-arginine application in rams during the breeding season may have positive effects on rams' reproductive performance.     

Assessment of seminal mast cells in infertile men with varicocele after surgical repair

This study aimed to assess seminal mast cells in infertile men associated with varicocele (Vx) pre‐ and post‐surgical repair. Forty‐five infertile men associated with Vx were subjected to history taking and clinical examination. In addition, semen parameters and seminal mast cells stained with 1% toluidine blue were estimated pre‐varicocelectomy and three months post‐varicocelectomy. Vx surgical repair revealed a significant improvement in the mean sperm concentration, progressive sperm motility, total sperm motility and sperm abnormal morphology and a significant decrement in seminal mast cells (mean ± SD, 3.56 ± 2.23 cells per high‐power field (HPF) vs. 2.22 ± 1.06 cells per HPF, p = .01). The pre‐operative mean mast cell count demonstrated significant increases in cases with Vx grade III compared with other Vx grades and in cases with bilateral Vx compared with unilateral Vx cases. Seminal mast cells demonstrated a significant correlation with sperm concentration, progressive sperm motility and total sperm motility and a nonsignificant correlation with age and sperm abnormal morphology. It is concluded that seminal mast cells decrease significantly in infertile men with Vx after surgical repair showing a significant negative correlation with sperm concentration, progressive sperm motility and total sperm motility.     

Oxidative stress status and sperm DNA fragmentation in fertile and infertile men

Evidence suggests that disturbing the balance between reactive oxygen species levels and antioxidant contents in seminal plasma leads to oxidative stress resulting in male infertility. This study was carried out to identifying clinical significance of seminal oxidative stress and sperm DNA fragmentation in treatment strategies of male infertility in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in patients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma were significantly (p < .001) lower in infertile men. Significant negative correlations were observed between MDA levels and sperm motility and normal morphology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men and significantly correlated with MDA levels and SOD and GPx activities. MDA of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut‐off limits to discriminate infertile patients from fertile men. The results show the leading role of oxidative stress in aetiology of male infertility in southwest Iran and indicate that evaluation of seminal antioxidant status and DNA integrity can be helpful in men attending infertility clinics during fertility assessment.     

Quality assessment of frozen bull semen with the precursor A-kinase anchor protein 4 biomarker

In this study, the quality of frozen bull semen was evaluated with the proAKAP4 level test. Sixty straws of frozen bull semen from various batches (n = 30) belonging to six bulls were used in the current study. The frozen bull semen samples were analysed in terms of proAKAP4 levels, sperm morphology and sperm movement parameters at hour 0 and hour 3 after thawing. The semen samples were divided into three groups according to the proAKAP4 levels: low concentration (<25 ng/10x10⁶ spermatozoa), moderate concentration (25 to 39 ng/10x10⁶ spermatozoa) and high concentration (≥40 ng/10x10⁶ spermatozoa). A positive correlation was found between the proAKAP4 level and total motility (TM₃), progressive motility (PM₃), VSL₃ and VCL₃ values obtained after the third-hour thermoresistance test (p < .05). There was a negative correlation between the percentage of sperm abnormal tail and the proAKAP4 level (p < .01). In addition, it was observed that the semen samples with proAKAP4 concentrations of 25 ng/10⁶ spermatozoa and higher preserved the TM₃ and PM₃ motility characteristics. In conclusion, the proAKAP4 has the potential to become a biomarker protein to evaluate in the quality analysis of frozen-thawed semen.     

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5 mM caffeine-treated groups). Compared to that in the control, the addition of 5 mM caffeine induced an increase in sperm motility (38.9 ± 2.8 versus 32.6 ± 3.4%), and a decrease in nitrite concentration (11.4 ± 2.1 versus 12.8 ± 2.9 µM/µg protein, p < .05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.     

Decreased prostate-specific membrane antigen levels in the seminal plasma of oligoasthenoteratozoospermic men

Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein with glutamate carboxypeptidase activity. However, its precise function in the prostate, prostasomes and seminal plasma with regard to male fertility remains unknown. This study was conducted to investigate the seminal plasma PSMA levels in fertile men and patients with oligoasthenoteratozoospermia (OAT) and to analyse its association with sperm parameters. Twenty fertile men and twenty patients admitted at the urology clinic of our institution with the diagnosis of OAT were included in the study. Following semen analysis, seminal plasma was isolated from semen ejaculates. PSMA concentrations in the seminal plasma were determined by ELISA. The correlations between seminal PSMA concentrations and semen parameters were statistically analysed. Seminal plasma PSMA concentration was significantly lower in OAT patients compared to fertile controls (p < .01). In fertile men, PSMA concentration was significantly correlated with the sperm concentration (r = −.481, p < .05), whereas in the patient group no statistically significant correlation was found between the sperm parameters and seminal PSMA level. This is the first study in the literature to investigate PSMA levels in the seminal plasma from infertile men. Decreased levels of seminal plasma PSMA might suggest a role for compromised prostasome function in the pathogenesis of OAT syndrome.