Chromatin structure in globozoospermia: a case report.

Sperm nuclear abnormalities in patients with globozoospermia have not been well characterized and may lead to the high rates of fertilization failure and embryo loss reported in patients with this form of teratozoospermia. This study used transmission electron microscopy (TEM), the sperm chromatin structure assay (SCSA), and single cell gel eletrophoresis assay (COMET) to assess if globozoospermia is associated with sperm chromatin structure abnormalities, DNA fragmentation, or both. The flow cytometric SCSA measures abnormal chromatin structure based on the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. COMET measures DNA fragmentation in individual sperm nuclei based upon gel electrophoretic patterns. Although sperm concentration (113 million/mL) and motility (66%) were normal in the patient, there was complete acrosome deficiency. TEM and SCSA data confirmed light microscopic examination that showed that sperm populations included a mixture of round and elongated sperm heads. Even though 100% of sperm had abnormal head morphology, only 13% demonstrated DNA denaturation (COMPalpha(t)), which is below our threshold of 15% COMPalpha(t), and consistent with high-fertility patients. Of interest, 13% of the sperm were also positive in the COMET assay, supporting our previous observations that SCSA-positive cells are also positive for DNA fragmentation. It was unexpected but of great interest that a human sperm population with 100% sperm morphology abnormalities had a chromatin integrity at the molecular level that is equivalent to sperm populations shown in previous studies to be highly fertile. These data are the first reported using SCSA and COMET assays to evaluate a patient with globozoospermia and support previous reports that intracytoplasmic sperm injection of globozoospermia may result in fertility/pregnancy. Lower success rates seen in some patients may be due to unrelated factors.

Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study.

Semen samples from a fertile patient presenting with influenza and a 1-day fever of 39.9 degrees C were obtained and analyzed at 18-66 days postfever (dpf) for sperm nuclear proteins, DNA stainability, free thiols (-SH), and susceptibility to DNA denaturation in situ. At 18 dpf, 36% of sperm demonstrated denatured DNA as measured by the sperm chromatin structure assay (SCSA), and decreased to 23% by 39 dpf. Samples at 33 and 39 dpf contained 49% and 30%, respectively, of cells with increased DNA stainability (HIGRN). A unique sperm nuclear protein band migrating between histones and protamines on acid-urea gels appeared at 33 and 39 dpf and nearly disappeared by 52 dpf. Amino acid sequencing of the first 8 N-terminal residues identified this protein as the precursor to protamine 2. The protamine P1 and P2 ratio remained normal, whereas the histone to protamine ratio increased slightly at 33 to 39 dpf. Flow cytometric measurements of nuclear -SH groups revealed the greatest reduction in free nuclear thiols at 33 dpf, and returned to normal by 45 dpf. The time of appearance of the unprocessed protamine 2 precursor and the relative increase in histone suggest a fever-related disruption of the synthesis of mRNA that codes for a P2 processing enzyme or enzymes. Increased DNA staining is likely due to the increased histone/protamine ratio. This case study demonstrates that fever/influenza can have latent effects on sperm chromatin structure and may result in transient release of abnormal sperm.

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques.

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.

Relationship between sperm nuclear protamine free -SH status and susceptibility to DNA denaturation.

Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples from 30 stallions were evaluated by the SCSA. Aliquots of the same samples were sonicated to liberate sperm nuclei, purified through a 60% sucrose gradient, stained with an -SH specific fluorochrome (CPM (7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin)), and the blue fluorescence of 5000 cells per sample was measured. If S=S bonds stabilize chromatin to inhibit DNA denaturation under the imposed low pH conditions, a low blue intensity would correlate with a low level of DNA denaturation. However, this study showed no correlation (r = -0.199, P = 0.31) of -SH stainability with the extent of DNA denaturation. Thus, other parameters, possibly DNA strand breaks, play a more significant role in susceptibility to DNA denaturation than the extent of S=S bonding within and between protamine molecules. These results also imply that rate of passage through the epididymis may not have significant effects on sperm fertility potential with regard to disulphide bonding status.

Density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged chromatin structure.

The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged chromatin structure was assessed by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in chromatin structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm chromatin structure as well as motility (%), forward progression (1-4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved chromatin integrity (SDalpha(t)) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.

Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic.

The sperm chromatin structure assay (SCSA) was used to measure over 500 human semen samples from two independent studies: Study I, 402 samples from 165 presumably fertile couples wishing to achieve pregnancy over 12 menstrual cycles; Study II, samples from 115 patients seeking fertility counselling. The SCSA measures susceptibility to DNA denaturation in situ in spermatozoa exposed to acid for 30 s, followed by acridine orange staining. SCSA data from the male partners of 73 couples (group 1) achieving pregnancy during months 1-3 of Study I were used as the standard of 'sperm chromatin compatible with high fertility' and were significantly different from those of 40 couples (group 3) achieving pregnancy in months 4-12 (P < 0.01) and those of male partners of 31 couples (group 4) not achieving pregnancy (P < 0.001). Group 2 contained couples who had a miscarriage. SCSA values for Study II were almost twice that of the Study I fertility standards. Within-couple repeatability tended to be less for group 3 than for groups 1, 2 or 4. Based on logistic regression, spermatozoa with denatured DNA (cells outside the main population, COMP alpha t) were the best predictor for whether a couple would not achieve pregnancy. Some 84% of males in group 1 had COMP alpha t < 15%, while no couples achieved pregnancy in group 1 with > or = 30% COMP alpha t, a threshold level considered not compatible with good fertility. Using selected cut-off values for chromatin integrity, the SCSA data predicted seven of 18 miscarriages (39%).

Spermatogenesis in early and chronic phases of experimental spinal cord injury in the rodent model.

A rodent model was used to study the degree and dynamics of potential spermatogenic alterations during both acute and chronic phases after experimental spinal cord injury (SCI). Sexually mature Sprague-Dawley rats underwent controlled impact SCI by exposure of the thoracic spine, T-10 laminectomy, and intraoperative somatosensory-evoked potential latency and amplitude. A 50 gm-cm SCI was produced in 35 experimental subjects. Sham surgery was performed on 16 control subjects through exposure of the dura without weight drop. SCI was verified by obliteration of the somatosensory-evoked potential following injury and subsequent neurologic assessment (modified hindlimb Tarlov scale) 4 weeks after injury. Flow cytometry with acridine orange as the DNA probe was used to measure potential spermatogenic alterations in testicular cell development and integrity of epididymal sperm chromatin structure between 2 and 20 weeks following SCI. Flow cytometric analysis revealed that nine of the 35 SCI animals demonstrated altered spermatogenesis; it is not clear whether these effects are specific or nonspecific stress related. These responder animals contributed to dramatic differences in relative percent testicular haploid cells (spermatids) and concurrent differences in percent diploid cells at 2, 4, 8, 12, and 16 weeks. Percentages within the three spermatid populations (round, elongating, and elongated) also differed at these time points. The sperm chromatin structure assay (SCSA) revealed significant epididymal sperm nuclear structure differences at 2, 4, and 12 weeks (P < 0.001). These findings are in concordance with our clinical observations of spermatogenesis in spinal cord injured men and suggest that significant spermatogenic deficit may occur, even in the early phase of injury.

Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis.

Susceptibility of mammalian sperm DNA to low pH- or heat-induced denaturation in situ has shown very strong dose-response relationships with animal and human exposure to chemical and physical toxicants and also fertility potential. In this study, 23 human semen samples representing a wide range in percentage (7-86%) of sperm exhibiting abnormally high susceptibility of DNA in situ to denaturation were studied for the integrity of their DNA using alkaline comet assay (single-cell microgel electrophoresis, pH 10.0). The percentage of comets observed for these samples ranged from 5 to 95%; these data correlated strongly with the percentage of sperm with increased DNA denaturability (r = 0.973; P < 0.001). Labeling of 3' ends of nicked DNA sites with 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP) followed by tagging with FITC-BrdUTP monoclonal antibody and flow cytometry also indicated significantly strong correlations of BrdUTP incorporation with both abnormal susceptibility of DNA to denaturation (r = 0.859, P < 0.001) and comet assay (r = 0.812, P < 0.001). The relationship among susceptibility of sperm chromatin to acid denaturation in situ, BrdUTP incorporation, and formation of comets suggests that DNA fragmentation monitored by these assays may have important physiological relevance in terms of sperm quality and fertility potential.

Effects of heat stress on mouse testicular cells and sperm chromatin structure.

Scrotal regions of mice were exposed to a 38.0, 40.0, or 42.0 degrees C (+/-0.1) H2O bath for 60 minutes to determine the effects of elevated temperatures on testicular cells and sperm chromatin structure. Mice were killed on various days after exposure, and ratios of acridine orange-stained testicular cell populations were determined by flow cytometry. Testicular weights of mice exposed to 42.0 degrees C decreased significantly day 1 (P < 0.01) through 35 (P < 0.001). Also, a significant relative decrease in testicular haploid cells was seen on days 3-35 (P < 0.001) with a corresponding increase in the diploid population (P < 0.001). Testicular analyses of mice exposed to 38.0 degrees C were not significantly different from control values. Testis weights of mice exposed to 40.0 degrees C were not affected, but a relative decrease in percent haploid cells occurred on days 11 and 14 (P < 0.001). The sperm chromatin structure assay (SCSA) was used to measure the susceptibility of cauda epididymal sperm DNA to in situ denaturation at low pH. Caudal epididymides of mice exposed to 42.0 degrees C had no sperm. Caudal epididymal sperm from mice exposed to 40.0 degrees C were most susceptible to acid-induced DNA denaturation on days 3 (P < 0.05), 7, 11, and 14 (all P < 0.001). The 38.0 degrees C exposed mice showed some minor sperm chromatin abnormalities at later time points (days 11-35). When compared to sperm head morphology measurements, SCSA parameters were more sensitive indicators of heat-induced sperm abnormalities. These results show that mouse spermatogenesis is disrupted by scrotal exposure to environmental temperatures several degrees over normal physiological temperature and, of more biological interest, that some thermal ranges above normal allowed production of sperm with compromised nuclear chromatin structure.

Bull sperm head morphometry related to abnormal chromatin structure and fertility.

This study investigated the relationship between morphologically abnormal sperm, sperm chromatin structure, and fertility. Semen samples were obtained from 13 bulls. The sperm chromatin structure assay (SCSA), a sensitive measure of denaturability of sperm nuclear DNA in situ following acid treatment, was performed on each sample to quantitate abnormal chromatin structure. Feulgenstained sperm head morphology was measured by computerized image analysis (ONCOR-Image): Sixteen parameters were measured for each of 200 nuclei per sample. Fertility estimates were available for nine of the bulls. The SCSA variable SD alpha t was correlated with fertility ranking (r = 0.617, P < 0.01). No correlations were seen between means of the imaging variables and SCSA variables % COMP alpha t or SD alpha t. Significant correlations (P < 0.05) were seen between SD alpha t and the variation of imaging variables eccentricity, width, and light blobs. Significant correlations (P < 0.05) were seen between % COMP alpha t and the variation of imaging variables area, perimeter, p2a, bending energy, nmac, sphericity, eccentricity, length, and width. A regression model for fertility rankings incorporating the standard deviation of the imaging variables area, bending energy, nmac, eccentricity, condensity, light blobs, and dark blobs was highly significant (r2 = 0.999, P < 0.05). These results indicate that variation of morphometry measurements is likely a sensitive biomarker related to fertility potential and abnormal chromatin structure.

Effects of X-irradiation on mouse testicular cells and sperm chromatin structure.

The testicular regions of male mice were exposed to x-ray doses ranging from 0 to 400 rads. Forty days after exposure the mice were killed and the testes and cauda epididymal sperm removed surgically. Flow cytometric measurements of acridine orange stained testicular samples indicated a repopulation of testicular cell types following x-ray killing of stem cells. Cauda epididymal sperm were analyzed by the sperm chromatin structure assay (SCSA), a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation. The SCSA detected increased susceptibility to DNA denaturation in situ after 12.5 rads of x-ray exposure, with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until the testes were exposed to 60 rads of x-rays. These data suggest that the SCSA is currently the most sensitive, non-invasive method of detecting x-ray damage to testicular stem spermatogonia.

Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay.

Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P < 0.001), bulls (r = 0.78, P < 0.001), and stallions (r = 0.65, P < 0.001). No significant differences were seen when using fresh or frozen samples for either assay. These results suggest that sperm cells that are more susceptible to in situ DNA denaturation may have a greater number of accessible endogenous DNA strand breaks. We hypothesize that the DNA strand breaks produced in the normal transition from a somatic cell histone complex to a protamine complex are not ligated properly, resulting in residual DNA strand breaks and altered chromatin structure. Alternatively, altered chromatin structure could lead to the accessibility of the endogenous DNA strand breaks.

Studies on sperm chromatin structure alterations and cytogenetic damage of mouse sperm following in vitro incubation. Studies on in vitro-incubated mouse sperm.

Mouse epididymal sperm incubated in Tyrode's T6 fertilization media were analyzed over time for chromosome damage by two methods. First, cytogenetic analysis was done on paternal pronuclei metaphase chromosomes. After 6 hours incubation 11% of the cells demonstrated chromosome structural abnormalities. Secondly, sperm nuclei were measured by the sperm chromatin structure assay, which is a measure of the susceptibility of sperm DNA to the nuclei demonstrated an increased susceptibility to DNA denaturation, reaching near 100% by 48 hours. Changes in chromatin structure at the molecular level may lead to chromosome breaks seen in pronuclear chromosomes.

Glutathione depletion potentiates ethyl methanesulfonate-induced damage to sperm chromatin structure.

Male rats were treated with phorone at dosages previously shown to reduce glutathione in rodent reproductive tracts, followed by a single challenge with ethyl methanesulfonate, a known mutagenic and clastogenic agent. Epididymal sperm collected 8 and 15 days after exposure from phorone pretreated animals had a significantly greater alteration of sperm chromatin structure, defined as an increased susceptibility to DNA denaturation in situ, relative to sperm obtained from animals injected with saline alone or saline+EMS (50, 100, 150, or 200 mg/kg bw). These data support the hypothesis that ethyl methanesulfonate-induced alkylation of developing sperm chromatin protamines causes a significant stress on chromatin structure leading to increased DNA damage. This is the first report showing that glutathione depletion potentiates EMS-induced chromatin structural alterations that are likely related to dominant lethal mutations.

Zinc-silicon interactions influencing sperm chromatin integrity and testicular cell development in the rat as measured by flow cytometry.

Flow-cytometric procedures were used to determine effects of dietary Zn and Si variations on rat testicular cell development, including integrity of caudal epididymal sperm chromatin structure defined as the susceptibility of DNA to denaturation in situ. Concentrations of 4 (deficient), 12 (adequate), and 500 (excessive) mg of Zn/kg of diet were used with Si concentrations of 0 (low), 540 (medium), and 2,700 (high) mg/kg of diet in a 3 x 3 factorial arrangement. Three-week-old Sprague-Dawley male rats were fed the experimental diets for 8 wk. Rats fed the Zn-deficient/Si-low diet demonstrated significant deviations in the ratio of testicular cell types present, including a reduction of S phase and total haploid cells. Furthermore, approximately 50% of epididymal sperm had a significant decrease in resistance to DNA denaturation in situ. In the Zn-deficient/Si-medium treatment, the effects of Si on animal and testicular growth, distribution of testicular cell types, and sperm chromatin structure integrity were quite similar to the effects of the Zn-adequate diets. A toxic effect of Zn on sperm chromatin structure integrity observed in the Zn-excess/Si-medium treatment seemed to be counteracted by Si in the Zn-excess/Si-high treatment. Silicon at medium and high levels seems to affect Zn metabolism through potentiation and antagonistic reactions, respectively. Zinc deficiency likely disrupts the normal sperm chromatin quaternary structure in which Zn plays a role by providing stability and resistance to DNA denaturation in situ.

Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure.

The effects of hydroxyurea (HU) on testicular cell kinetics and sperm chromatin differentiation were investigated in mice. Whole testis, minced testicular cell suspensions and caudal epididymal sperm cells were obtained at 8 and 29 days after i.p. injections containing 0, 25, 50, 100, 200, 400 and 500 mg/kg HU x 5 days. Testis weights were unaffected by 25 mg/kg HU while 500 mg/kg caused up to a 50% loss of testicular weight by 29 days. Flow cytometrically measured acridine-orange (AO) stained testicular cells revealed altered population ratios at the highest dosages at 8 days and for all dosages except 25 mg/kg HU at 29 days. At 8 days, 400-500 mg/kg HU caused a near depletion of tetraploid cells. Flow cytometry of AO stained sperm, previously treated with acid to potentially induce DNA denaturation, was used to follow the shift from normal chromatin structure to an abnormal form with increased sensitivity to DNA denaturation in situ. The extent of DNA denaturation was quantitated for each cell by the computer-derived value alpha t, alpha t = [red/(red+green) fluorescence]. The flow cytometry measures, standard deviation of alpha t (SD alpha t), mean of alpha t (X alpha t) and cells outside the main peak of alpha t (COMP alpha t), gave similar dose response curves to the sperm head morphology assay. SD alpha t was more sensitive than the X alpha t as a measure of HU-induced alteration of chromatin structure. The major conclusions reached are that HU inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase, causing maturation depletion of pachytene spermatocytes and, subsequently, depletion of meiotic daughter cells and differentiated cell types leading to mature sperm. This inhibition of DNA synthesis is related to an alteration of sperm chromatin structure and abnormal sperm head morphology.

Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics.

Effects of methyl methanesulfonate (MMS) on mouse testicular cell kinetics and sperm chromatin structure were determined flow cytometrically. Mice were exposed to a single ip injection of saline containing 0 or 150 mg/kg MMS. Relative ratios of 1N, 2N and 4N testicular cells were not affected until 22 days postexposure. Ratios of 1N cell types were altered from 13 to 22 days and were near normal by 25 days. This study revealed an MMS induced alteration of chromatin structure in testicular, elongated spermatids by the sperm chromatin structure assay (SCSA), a flow cytometric measure of the susceptibility of acridine orange stained sperm DNA to denaturation in situ. The SCSA also detected alterations in cauda sperm chromatin structure at 3 days, which was 8 days prior to alterations in sperm head morphology, indicating the increased sensitivity of the SCSA. SCSA data were practically similar whether measuring either fresh or frozen/thawed sperm, or whether measured by two different types of flow cytometers: a) laser driven, orthogonal optical axis; or b) low cost mercury arc lamp system with epiillumination. The data support the model of Sega and Owens [Mutat Res 111:227-244:1983] that MMS alkylates cysteine-SH groups in sperm protamines, thereby destabilizing sperm chromatin structure and leading to broken chromosomes and mutations.

Rapid determination of sperm cell concentration in bovine semen by flow cytometry.

A flow cytometric technique is described for determining sperm concentration in fresh or extended semen with improved accuracy, precision, repeatability, ease of conduct, and rapidity. The technique is designed to measure the ratio of a known number of fluorescent beads admixed with sperm stained with either acridine orange or propidium iodide. A significant advantage of the technique is the distinct resolution between sperm and other particles (e.g., somatic cells, fat droplets, and bacteria in the semen or extender) that interfere in other counting protocols. Field testing of this protocol over the past 3 yr has demonstrated its superiority over the Coulter counter, hemacytometer, and spectrophotometer for accuracy in counting sperm in extended semen and the accuracy of counting sperm in straws based on preextension spectrophotometric determination of sperm concentration. Sperm chromatin quality can be determined simultaneously with this sperm counting procedure. This approach to counting sperm provides an excellent procedure for quality control of sperm numbers in processed semen.

Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay.

Eight monthly semen samples from 45 men not known to be exposed to industrial toxicants were measured by the flow cytometric sperm chromatin structure assay (SCSA). This assay determines susceptibility of sperm DNA to in situ, acid-induced denaturation and is quantitated by the metachromatic shift of acridine orange fluorescence from green (native DNA) to red (denatured DNA). The observed green versus red fluorescence scattergram (cytogram) patterns were generally unique between donors and homogeneous within a donor over time. Within a donor, the cytogram patterns were the same whether intact sperm cells or detached nuclei were measured. For some individuals the cytogram patterns differed for some months and then returned to the original pattern. Intraclass correlations for mean and standard deviation of alpha t [alpha t = red/(red + green) fluorescence] were higher (.67 to .90) than any classically measured semen variables, suggesting that SCSA results within an individual were more consistent than other measures. Furthermore, average within-donor CV of alpha t parameters expressed as a percent of any given individual's means was around 10%, which is significantly lower than those derived from common semen measures. The SCSA is an objective, technically sound, biologically stable, sensitive, and feasible measure of semen quality.

Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.

Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.