Fresh and frozen-thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765 mg/kg C57BL6/J or 600 mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P < 0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P < 0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P < 0.05).     

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.     

Testosterone production and spermatogenesis in free-ranging Eurasian lynx (Lynx lynx) throughout the year

Seasonal variation in reproduction is common in mammals as an adaptation to annual changes in the habitat. In lynx, male reproduction activity is of special interest because female lynxes are monoestric with an unusual narrow (about 1 month) breeding season. In Eurasian lynx, mating occurs between January and April depending on the latitude. To characterize the seasonal pattern of sperm and testosterone production in free-ranging Eurasian lynxes, long-term frozen-stored testis material obtained postmortem from 74 hunted or road-killed lynxes in Sweden was used to analyze annual changes in testis mass, testicular testosterone content, and spermatogenetic activity. Values of most gonadal parameters obtained in subadult lynxes were significantly different from the values observed in adult males. In adult lynxes, a moderate annual fluctuation of gonadal parameters was found which was most profound for testis weight and testicular testosterone concentration reaching highest values in March (median of 2.18 g and 2.67 μg/g tissue respectively). Grouping the data of pre-/breeding (January–April) and postbreeding season (May–September) revealed significant changes in testis weight and testosterone concentration. The relative spermatogenetic activity remained high in postbreeding testes. However, net sperm production decreased according to reduction of testis mass and a tendency to lower cauda epididymal sperm numbers in the postbreeding period was observed. Our results demonstrate that it is possible to analyze the gonadal activity of frozen testis/epididymis tissue postmortem and that male Eurasian lynxes show—opposite to the females—only moderate seasonal changes in their reproductive capacity.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

粗糙沼虾精巢发育的组织学

利用光镜技术,对粗糙沼虾精巢发育进行了研究,根据精子发生过程中每种生殖细胞所占的比例和发生的次序,并结合精巢的形态特征,把精巢发育过程分为五个时期,即精原细胞期,精母细胞期,精细胞期,成熟精子期及退化期,精原细胞期,精巢小,透明乳白色,生精小管内的生殖细胞以精原细胞为主;精母细胞期;精巢体积增大,半透明乳白色,主要由处于初级精母细胞的次级精母细胞阶段的生殖细胞组成;精细胞期,精巢体积继续增大,颜色加深,生精小管内的生殖细胞以精细胞为主;成熟精子期,精巢体积可达最大,紫红色,生精小管内充满着成熟的精子,退化期;精巢体积减小,半透明乳白色,生精小管内的成熟精子几乎排空。     

Another functional frame‐shift polymorphism of DEFB126 (rs11467497) associated with male infertility

DEFB126 rs140685149 mutation was shown to cause sperm dysfunction and subfertility. Indel rs11467497 is another 4‐nucleotide frame‐shift mutation (151bp upstream of rs140685149) that leads to the premature termination of translation and the expression of peptide truncated at the carboxyl terminus. In the present study, we performed a comprehensive association study to check the contribution of rs140685149 and rs11467497 to male infertility. Our results confirmed the previous findings that there was no association between rs140685149 and sperm motility. In contrast, we found a significant association of another indel rs11467497 with male infertility. Moreover, rs11467497 was shown to be associated with higher number of round cells in the infertile males with low sperm motility. Surprisingly, the two mutations commonly existed in the sperm donors (n = 672), suggesting a potential application of the two indels in the screening for eligible sperm donors. Western blotting assays showed the sperms with rs140685149 2‐nt deletion tended to have unstable DEFB126 protein in contrast of no DEFB126 protein expressed in the sperms with rs11467497 4‐nt deletion, suggesting a more severe consequence caused by rs11467497 mutation. In conclusion, our study presented a significant contribution of another functional frame‐shift polymorphism of DEFB126 (rs11467497) to male infertility.     

Use of negative staining technique and electron microscopy for the study of structural anomalies of outer dense fibres of human flagellum

Motility disorders due to tail defects are often seen in clinical andrology. Sperm motility should be assessed with regard to the morphology of the flagellum. Since suitable longitudinal sections are rarely obtained by routine transmission electron microscopy (TEM) and in view of the importance of dense fibres in modulating sperm motility and providing tensile strength, a detailed, study of human sperm flagellum by negative staining andTEM was attempted. The study was undertaken in two groups of men (I) fertile and (II) asthenozoospermic. The study revealed that outer dense fibres extend to 50–60% of the principal piece. Normal dense fibres were seen in 83% sperms and 23% sperms in groupsI andII respectively. The characteristics seen were variation in diameter, breakage or degradation with lacking or extended endpiece. The negative staining method provides an easy and useful analytical tool for identifying the defects of dense fibres and quantifying them.     

Supplementation of freezing media with alpha lipoic acid preserves the structural and functional characteristics of sperm against cryodamage in infertile men with asthenoteratozoospermia

The aim of this study was to examine the effects of alpha lipoic acid (ALA) supplementation during semen cryopreservation on the sperm quality, chromatin integrity, oxidative stress, and expression level of BAX, BCL2, HSP70 and iNOS genes in semen samples obtained from infertile men with asthenoteratozoospermia.MethodsTwenty freshly ejaculated semen samples were cryopreserved with sperm freezing medium supplemented with 0.00, 0.02, 0.05, 0.1, 0.5, and 1 mmol/mL of ALA. The samples were analyzed according to the WHO guidelines before and after freezing. Sperm ROS production level, DNA fragmentation and cryo-capacitation were assessed using flow cytometry, TUNEL assay and chlortetracycline (CTC) test, respectively. Expression level of stress protein (HSP70), pro-apoptotic Bax, anti-apoptotic Bcl-2, and iNOS genes was assessed by real-time PCR assay.ResultsThe effective concentrations of ALA (0.02 and 0.5 mM) significantly improved the motility, viability and morphology of the frozen-thawed sperms compared to the control group treated with 0.00 mM of ALA. During cryopreservation, treatment of semen with 0.02 mM of ALA, as the optimal concentration, significantly decreased DNA fragmentation and oxidative stress level (P < 0.05), protected the acrosome integrity, and led to insignificant reduction in BAX gene expression level and significant increase in expression level of BCL2, HSP70, and iNOS genes compared with control group.ConclusionOur findings revealed that the adding ALA to semen samples obtained from infertile men with asthenoteratozoospermia plays a significant protective role against cryodamage by preserving the sperm functional parameters.     

Oxidation of glyceraldehyde-3-phosphate dehydrogenase decreases sperm motility

The relation between the activity of the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) and the motility of sperms was investigated. It was found that the mean value of GAPDS activity in sperm samples with low motility is 2.5–3-fold lower than that in samples with high motility. Sperm motility was shown to diminish in the presence of superoxide anion, hydroxyl radical, and hydrogen peroxide. The decrease in sperm motility in the presence of hydrogen peroxide was proportional to the concentration of the oxidant and correlated with the decrease in GAPDS activity (r = 0.96). Based on the literature data on the importance of GAPDS for the motility of sperms together with the presented observations, it was concluded that the decrease in the sperm motility in the presence of reactive oxygen species is due to the oxidation of GAPDS and inhibition of glycolysis.     

A Single Genetic Determinant that Prevents Sex Reversal in C57BL-YPOS Congenic Mice

Sex determination in the mammalian embryo begins with the activation of a gene on the Y chromosome which triggers a cascade of events that lead to male development. The mechanism by which this gene, designated SRY in humans and Sry in mice (sex determining region of the Y chromosome), is activated remains unknown. Likewise, the downstream target genes for Sry remain unidentified at present. C57BL mice carrying a Y chromosome from Mus musculus musculus or molossinus develop normally as males. In contrast, C57BL/6 mice with the Y chromosome from M. m. domesticus often show sex reversal, i.e., develop as XY females. It has been documented that C57BL mice with the Y chromosome from Poschiavinus (Y^POS), a domesticus subtype, always develop as females or hermaphrodites. This suggests that a C57BL gene either up- or downstream of Sry is ineffective in interacting with Sry, which then compromises the processes that lead to normal male sex development. Nonetheless, by selective breeding, we have been able to generate a sex reversal-resistant C57BL/6-congenic strain of mice in which the XY^POS individuals consistently develop as normal males with bilateral testes. Because the resistance to sex reversal was transferred from strain 129S1/Sv (nonalbino) by simple selection over 13 backcross generations, it is inferred that a single autosomal gene or chromosomal region confers resistance to the sex reversal that would otherwise result. XY^POS normal males generated in these crosses were compared to XY^POS abnormal individuals and to C57BL/6 controls for sexual phenotype, gonadal weight, serum testosterone, and major urinary protein (MUP) level. A clear correlation was found among phenotypic sex, MUP level, and testis weight in the males and in the incompletely masculinized XY^POS mice. The fully masculinized males of the congenic strain resemble C57BL/6 males in the tested parameters. DNA analysis confirmed that these males, in fact, carry the Y^POS Sry gene.     

Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Formation and Function in Assisted Fertilization

Spermatogenesis is a key developmental process allowing for a formation of a mature male gamete. During its final phase, spermiogenesis, haploid round spermatids undergo cellular differentiation into spermatozoa, which involves extensive restructuring of cell morphology, DNA, and epigenome. Using mouse models with abrogated Y chromosome gene complements and Y-derived transgene we identified Y chromosome encoded Zfy2 as the gene responsible for sperm formation and function. In the presence of a Zfy2 transgene, mice lacking the Y chromosome and transgenic for two other Y-derived genes, Sry driving sex determination and Eif2s3y initiating spermatogenesis, are capable of producing sperm which when injected into the oocytes yield live offspring. Therefore, only three Y chromosome genes, Sry, Eif2s3y and Zfy2, constitute the minimum Y chromosome complement compatible with successful intracytoplasmic sperm injection in the mouse.     

RAC1 controls progressive movement and competitiveness of mammalian spermatozoa

Mammalian spermatozoa employ calcium (Ca²⁺) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (t^w5/t^w32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells.     

Exploration of the Association between Obesity and Semen Quality in a 7630 Male Population

This study aimed to explore the association between body mass index (BMI), other anthropometric indexes and semen quality in a general male population in Taiwan. In this cross-sectional cohort study, the study cohort consisted of 7941 healthy male individuals aged 18 years or older who participated in a standard medical screening program run by a private firm from January 2008 to May 2013. Semen parameters including sperm concentration (SC), total sperm motility (TSM), progressive motility (PRM), and normal sperm morphology (NSM) were recorded. Anthropometric indexes including BMI, waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR) and body fat percentage were measured. A total of 7630 men were enrolled for the final analysis, of whom 68.5% had a normal weight distribution and 31.4% were overweight or obese. Total sperm motility, progressive motility, normal sperm morphology and sperm concentration showed a statistically linear decline with increasing age (p < 0.001, p < 0.001, p < 0.001 and p = 0.004). Sperm concentration showed a significantly negatively linear association with BMI (p = 0.005), and normal sperm morphology showed an inverse association with BMI and waist-to-height ratio (p < 0.001 and p = 0.004). The prevalence of abnormal total sperm motility, progressive motility, normal sperm morphology and sperm concentration increased with increasing age (p = 0.011, p < 0.001, p < 0.001 and p = 0.002). Lower normal sperm morphology and sperm concentration were associated with increasing body adiposity (p<0.05). No relationship between obesity and sperm motility was identified.     

The effect of Tribulus terrestris extract on motility and viability of human sperms after cryopreservation

BackgroundSemen cryopreservation produces significant amounts of reactive oxygen species (ROS), which may lead to impairment of sperm morphology, function, and ultimately, male fertility. Since Tribulus terrestris has antioxidant and free-radical-scavenging properties, this study aims to reveal the effect of the Tribulus terrestris extract on motility and vitality of human sperms after cryopreservation.Materials and methodsSemen specimens from 80 healthy volunteers were divided into eight groups: fresh control (group I), freeze control (group II), groups III, IV, and V, which had 20, 40, and 50 μg/mL doses of Tribulus terrestris extract added before cryopreservation, and groups VI, VII, and VIII, which were supplemented by these extract doses after the freeze-thaw process. To evaluate the effects of the Tribulus terrestris extract, the semen samples were incubated with the extract and evaluated with a light microscope for motility and viability.ResultsAfter cryopreservation, a significant improvement in spermatozoa viability was observed in group VII. In groups VII and VIII, motility, according to World Health Organization (WHO) criteria, increased considerably (p < 0.001). There was no significant difference among groups III, IV, and V.ConclusionThe present study demonstrated that the protective effects of Tribulus terrestris, which improves human sperm motility and viability, may be due to its antioxidant properties. On the basis of the results, the researchers concluded that Tribulus terrestris can be used as a safe therapeutic alternative to current modalities for the management of motility dysfunction in males.     

Generation of fertile offspring from Kitw/Kitwv mice through differentiation of gene corrected nuclear transfer embryonic stem cells

Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit^w/Kit^wv mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kit^w/Kit^wv somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kit^w/Kit^wv infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.     

Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution

The purpose of the present study was to investigate the impact of carcinogenic polycyclic aromatic hydrocarbons and volatile organic compounds on sperm quality in a group of city policemen in Prague during a period of increased concentrations of ambient air-pollutants (winter season) compared to a period of low exposure (spring). Polymorphisms in metabolic genes (CYP1A1, EPHX1, GSTM1, GSTP1, GSTT1), folic acid metabolism genes (MTR, MTHFR) and DNA repair genes (XRCC1, XPD6, XPD23, hOGG1) were evaluated in these men as potential modifiers of associations between air pollution exposure and changes in sperm quality. The study population was a group of 47 policemen working in the center of the city. Seasonal differences in exposure were verified by ambient and personal monitoring. Markers of sperm injury included semen volume, sperm concentration, sperm morphology, sperm motility, and sperm DNA damage measured with the sperm chromatin structure assay The sperm chromatin structure assay (SCSA) includes a measure of DNA damage called DNA Fragmentation Index (DFI). The % of cells with detectable DFI (detDFI) by this assay includes sperm with either medium or high DNA damage; the term hDFI is used to define the % of sperm with only high DNA damage. The assay also detects immature sperm defined by high density staining (HDS).No significant differences were found in any of the standard semen parameters between the sampling periods except for vitality of sperms. Both DFI and HDS were significantly higher in winter than in spring samples for all men and for non-smokers. At the bivariate level, significant associations between hDFI or detDFI and polymorphisms of the repair genes XRCC1, XPD6 and XPD23 were observed. In multivariate models, polymorphisms of the genes XPD6, XPD23 and CYP1A1MspI were associated with hDFI and HDS. Moreover, HDS was significantly associated with polymorphisms in GSTM1 gene.     

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions.     

Generation of reactive oxygen species in sperms of rats as an earlier marker for evaluating the toxicity of endocrine-disrupting chemicals

Abstract

Bisphenol A (BPA) and diethylstilbestrol (DES) have been reported to cause sperm toxicity. To identify an earlier marker of toxicity of environmental substances or food additives, this study determined whether the levels of reactive oxygen species (ROS) in sperms could serve as indices for the prediction of sperm toxicity and quality. Male Wistar rats were given drinking water containing various doses of BPA or DES for 8 weeks. Some rats were treated with 0.45% N-acetyl cysteine (NAC) for 2 days prior to the administration of DES or BPA. Administration of BPA or DES to rats for 1 week dose-dependently increased the production of ROS, even at doses and time points which had no effect on sperm motility. 4-Hydroxy-2-nonenal modified proteins increased in sperms 8 weeks after BPA or DES treatment. NAC reversed oxidative stress and prevented the loss of sperm function in the DES or BPA-treated group. During observation, changes in the sperm motility, sperm count and morphology were not correlated to the increase in ROS levels. These results suggest that ROS levels may be used as an early indicator of sperm count and quality decreases which result from chronic toxicity.