Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.

Association between ambient particulate matter exposure and semen quality in fertile men

Background Several studies have suggested adverse effects of particulate matter (PM) exposure on male reproductive health; few have investigated the association between PM exposure and semen quality in a large population of fertile men. Methods We evaluated 14 parameters of semen quality in 1554 fertile men in Nanjing from 2014 to 2016. Individual exposure to particular matter ≤10 μm in diameter (PM10) and ≤ 2.5 μm in diameter (PM2.5) during key periods of sperm development (0-90, 0-9, 10-14, 15-69, and 70-90 days before semen collection) were estimated by inverse distance weighting interpolation. Associations between PM exposure and semen quality were estimated using multivariable linear regression. Results Higher 90-days average PM2.5 was in association with decreased sperm motility (2.21% for total motility, 1.93% for progressive motility per 10 μg/m3 increase, P < 0.001) and four quantitative aspects of sperm motion (curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of lateral head displacement (ALH), P < 0.01). The association between PM2.5 exposure and semen quality were generally stronger for the earlier exposure window (70-90 days prior to ejaculation) than for recent exposure (0-9, 10-14, or 15-69 days). In the subgroup of men who had normal sperm parameters (n = 1019), similar results were obtained. Ninety-days PM10 exposure was associated only with decreased VCL and VAP and was not related to sperm concentration. Conclusions Exposure to PM2.5 adversely affects semen quality, specifically lower sperm motility, in fertile men. Graphical abstract

Characteristic of spermogram parameters in men with reproductive pathology in age-related aspect

Introduction. The prevalence of andrological diseases among adolescents and young adults resulting in lowered reproductive potential has been noted to progressively increase. At the same time, the number of couples starting to manage reproductive issues after 35–40 years of age highlighting the onset of male androgen deficiency continues to rise. Therefore, the analysis of spermogram as the key element in assessing male reproductive potential is better to conduct at different age periods of man's life.Aim: to compare spermogram parameters in different age groups of patients with reproductive pathology.Materials and Мethods. The analysis of spermograms in adolescents with left-sided grade II–III varicocele aged 17 years and in infertile males aged 22–48 years was performed. Semen analysis was conducted in accordance with the standards of the 5 th edition of the World Health Organization and included the following parameters: semen volume (ml), sperm concentration (million/ml), total sperm count (million), acidity, viscosity, progressive motility, total motility, viability, morphology, detected mucus, leukocytes, amyloid bodies, lecithin grains as well as sperm aggregation and agglutination. The stained preparations were used to assess the morphology of spermatozoa and spermatogenesis cells. According to the spermogram data obtained, the following conclusions were drawn: normozoospermia, oligozoospermia, asthenozoospermia, teratozoospermia. Statistical analysis was performed by using Statistica 10.0 software (StatSoft Inc., USA). The normality distribution was assessed using the χ2 test. Quantitative parameters were presented as arithmetic means and standard deviations (M ± SD). Assessing significance of differences was performed by using the Student's t-test, whereas inter-parameter correlation relations were analyzed by using the linear Pearson's correlation coefficient. A significance level between inter-group parameters was set at p < 0.05.Results. It was found that adolescents with varicocele vs. adult men had significantly decreased ejaculate volume. In particular, the average ejaculate volume in adolescents and adult men was 2.32 ± 1.22 ml and 3.50 ± 1.44 ml, respectively, so that the larger number of young patients were noted to have ejaculate volume below 1.5 ml. Compared to young subjects, aged patients had decreased sperm concentration (35.88 ± 25.74 versus 72.20 ± 49.32 million/ml) and total sperm count (120.58 ± 91.72 versus 173.07 ± 163.92 million). Young patients were found to have significantly superior data in all categories of sperm motility, whereas infertile men were diagnosed with impaired sperm motility. In particular, adolescents were featured with the average number of spermatozoa displaying fast and slow translational movement comprising 17.12 ± 11.04 % and 29.30 ± 12.29 %, respectively, the proportion of progressive motility spermatozoa was 46.20 ± 19.82 %. In contrast, similar parameters in adult men were 5.10 ± 6.36 %, 19.80 ± 9.61 %, and 24.95 ± 11.23 %, respectively. In infertile men prevalence of lacked spermatozoa with rapid forward movement was 46 (46.0 %), in adolescents – 8 (8.6%), whereas rate of immotile spermatozoa in infertile men, on average, accounted for 53.10 ± 14.56 %, in adolescents – 34.40 ± 21.83 %. In addition, adolescents with varicocele had significantly fewer spermatozoa with normal morphology – 14.14 ± 8.06 % (in adult men – 30.08 ± 17.94 %), there were more abundant defects in the sperm head – 58.01 ± 12.43 % (in men – 48.83 ± 18.95 %) and flagella – 17.24 ± 6.31 % (in men – 10.29 ± 6.21 %). The data obtained showed that adolescents were more often diagnosed with normozoospermia – in 49 (52.7 %) cases, in infertile men – in 12 (12.0 %) cases, whereas in aged men asthenozoospermia was detected in 82 (82.0 %) cases, in adolescents – 5 (5.4 %) cases.Conclusion. The abnormalities in the spermogram revealed in adolescents may be associated with unestablished spermatogenesis. Normozoospermia more common in adolescents with varicocele may evidence about preserved reproductive potential. Impaired sperm motility in aged patients seems to be related to the formation of oxidative stress and damage to spermatozoa by reactive oxygen species due to combined age-related changes, cumulation of the negative effects of environmental and lifestyle factors, as well as comorbidities.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

The effects of differing nutritional levels and body condition score on scrotal circumference, motility, and morphology of bovine sperm

Bulls often experience various levels of nutrient availability throughout the year. Nutritional management is a critical factor on overall ejaculate composition and the ability to get females pregnant. We hypothesized that differing nutritional levels and body condition score (BCS) affects reproductive fertility parameters in bulls. Mature Angus bulls (n = 11) were individually housed and randomly assigned to one of two dietary regimens: 1) over-fed (n = 5) or 2) restricted (n = 6). Bulls were fed the same ration at different volumes to achieve desired effects resulting in 8 individual treatments: gain to an over-fed body condition score ([BCS]; GO), gain after nutrient restriction (GR), loss after an over-fed BCS (LO), loss from nutrient restriction (LR), maintenance at ideal adiposity (BCS = 6) after overfeeding (IMO), maintenance at ideal adiposity after nutrient restriction (IMR), maintenance at an over-fed BCS (BCS = 8; MO), and maintenance at a restricted BCS (BCS = 4; MR). Body weight (BW) and BCS were recorded every two weeks to monitor bull weight and BCS changes. Scrotal circumference was measured every 28 d. Body fat and sperm motility and morphology were evaluated every 84 d. Scrotal circumference, motility, and morphology were normalized to the initial value of each bull. Thus, allowing the individual bull to serve as a control. Statistical analyses were conducted with PROC GLIMMIX of SAS as a complete randomized design to determine if treatment influenced BW, BCS, scrotal circumference, motility, morphology, and adipose thickness. Scrotal circumference (P &lt; 0.001) had the least amount of deviation from initial during the LR (0.29 ± 0.44) treatment and the greatest during the MO (3.06 ± 0.44), LO (2.28 ± 0.44), MR (2.43 ± 0.44), GR (3.03 ± 0.44) and IMR (2.91 ± 0.44) treatments. Sperm motility was not affected by nutritional treatments (P = 0.55). Both head and total defects of sperm differed (P = 0.02) due to nutritional treatments. Increased head abnormalities occurred during the LO (37.60 ± 8.61) treatment, with no differences between the other treatments. Total defects increased during the LO (43.80 ± 9.55) treatment with similar increases in bulls during the GR (29.40 ± 9.55) and IMR (35.60 ± 9.55) treatments. In conclusion, male fertility was impacted when a deviation from a BCS of 6 occurred which could be detrimental to reproductive and beef production efficiency.

The Hydrogen Storage Nanomaterial MgH2 Improves Irradiation-induced Male Fertility Impairment by Suppressing Oxidative Stress

ObjectiveThis study aimed to reveal the protective effect of hydrogen storage nanomaterial MgH2 on radiation-induced male fertility impairment.MethodsThe characterization of MgH2 were analyzed by scanning electron microscopy (SEM) and particle size analyzer. The safety of MgH2 were evaluated in vivo and in vitro. The radioprotective effect of MgH2 on the reproductive system were analyzed in mice, including sperm quality, genetic effect, spermatogenesis, and hormone secretion. ESR, flow cytometry and western blotting assay were used to reveal the underlying mechanisms.ResultsMgH2 had an irregular spherical morphology and a particle size of approximately 463.2 nm, and the content of Mg reached 71.46%. MgH2 was safe and nontoxic in mice and cells. After irradiation, MgH2 treatment significantly protected testicular structure, increased sperm density, improved sperm motility, reduced deformity rates, and reduced the genetic toxicity. Particularly, the sperm motility were consistent with those in MH mice and human semen samples. Furthermore, MgH2 treatment could maintain hormone secretion and testicular spermatogenesis, especially the generation of Sertoli cells, spermatogonia and round sperm cells. In vitro, MgH2 eliminated the [·OH], suppressed the irradiation-induced increase in ROS production, and effectively alleviated the increase in MDA contents. Moreover, MgH2 significantly ameliorated apoptosis in testes and cells and reversed the G2/M phase cell cycle arrest induced by irradiation. In addition, MgH2 inhibited the activation of radiation-induced inflammation and pyroptosis.ConclusionMgH2 improved irradiation-induced male fertility impairment by eliminating hydroxyl free radicals.

Establishment of semen collection technique using electroejaculator and semen cryopreservation of Javan leopard (Panthera pardus melas Cuvier, 1809)

Background and Aim: The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to develop electroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen. Materials and Methods: The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl® (Minitüb, Tiefenbach, Germany)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a 0.25 mL MiniStraw® (Minitüb, Tiefenbach, Germany) then equilibrated at 4°C for 2 h. After equilibration, the straw was then frozen in liquid nitrogen vapor. Frozen semen was then stored in containers until further evaluation. Results: The results showed that ejaculation response occurred at all levels of stimulation, while erections did not always occur. The fastest ejaculation and erection occurred at the fourth voltage. The macroscopic evaluation showed that the semen volume was 0.80±0.26 mL, cloudy white, pH 7.44±0.14, and with watery semen consistency. The microscopic evaluation showed that the sperm motility was 66.98±0.39%, with sperm viability of 75.6±1.79%. Sperm concentration was 62.17±46.95×106 mL–1 with a total concentration of 42.14±23.51×106 cells. Normal sperm morphology is only 40.72±6.26%. Conclusion: This study concluded that the development of a semen collection technique using an EE preceded by imaging of the EE probe location using ultrasound was effective for the ejaculation of Javan leopards. The characteristics of the semen of the Javan leopard showed moderate semen volume, sperm motility, and viability. Javan leopard showed low sperm concentration and normal sperm morphology.

Localization and Distribution of Testicular Angiotensin I Converting Enzyme (ACE) in Neck and Mid-Piece of Spermatozoa from Infertile Men in Relation to Sperm Motility

Testicular angiotensin converting enzyme (ACE) is known to play an essential role in the male reproduction and fertility. Data about tACE in cases of male infertility are quite scarce, and in this respect we aimed to study localization and distribution of tACE protein in the neck and mid-piece of spermatozoa from pathological samples in relation to sperm motility. The enzyme expression during capacitation and acrosome reaction was quantitatively assessed. In human ejaculated spermatozoa tACE is localized on sperm plasma membrane of the head, the neck and mid-piece of the tail. The immunoreactivity becomes stronger in capacitated spermatozoa followed by a decrease in acrosome reacted sperm. In different cases of semen pathology (oligozoospermia, asthenozoospermia and teratozoospermia) fluorescent signals in the neck and mid-piece are in punctate manner whereas in normozoospermia they were uniformly distributed. The expression area of tACE the neck and mid-piece was decreased in ejaculated and capacitated sperm from pathological semen samples compared to normospermia. Significant positive correlation was established between tACE area and progressive sperm motility, whereas with immotile sperm the correlation was negative. Our data suggest that proper distribution of tACE in the neck and mid-piece is required for normal sperm motility that could be used as a novel biomarker for male infertility.