Swim-down separation of progressively motile sperm from poor quality human semen by the modified funnel procedure

The simultaneous swimming up and down from identical semen specimens suggested that the separation efficiency of progressively motile sperm by the swim-down method was superior to that of the swim-up method. The swimming down was performed in a small plastic funnel, the bottom of which was plugged with a 2.5 ml disposable syringe. In this funnel, 2.0 ml of 70% Percoll was poured and then the sperm resuspension was overlaid. After 1 h, progressively motile sperm penetrating into the lower part of Percoll layer (1.6 ml) was collected in the syringe. The technical strategies to yield higher density of progressively motile sperm were found to be centrifugal concentration of the sperm from whole ejaculate prior to the swimming down, and subsequent re-centrifugation of the separated sperm after the swimming down. Oligo-asthenozoospermic semen (22 +/- 4.4 x 10(6) ml1, 5.5 +/- 4.4% motility, n = 8) was processed by the present method, yielding sperm qualities of 32 +/- 19 x 10(6) ml1, 74 +/- 14% in the final preparations. Overall improvement in fertility index (sperm density ml1 x motility % x 10(8)) reached 27-folds on average.     

Secular and seasonal changes in semen quality among young Danish men: a statistical analysis of semen samples from 1927 donor candidates during 1977-1995

The objective of this study was to investigate whether semen quality has changed during the years 1977-1995 in a group of unselected semen donor candidates, and to determine whether semen quality is subject to seasonal variation, by analysis of time- and season-related changes in semen quality using multiple regression and ANOVA. The study was based on analysis of the first semen sample delivered by 1927 semen donor candidates in Copenhagen during the period 1977-1995, with determination of semen volume, sperm concentration, total sperm count, percentage motile spermatozoa, and a semiquantitative sperm motility score. Multiple linear regression analysis with year, sexual abstinence and season as covariates showed a significant increase in mean sperm concentration from 53.0 x 10(6)/mL in 1977 to 72.7 x 10(6)/mL in 1995 (p < 0.0001) and in mean total sperm count from 166.0 x 10(6) to 227.6 x 10(6) (p < 0.0001). Mean semen volume and percentage motile spermatozoa did not change. Sperm motility deteriorated, as the spermatozoa in 74.2% of the samples were of excellent motility in 1977-1980 compared to only 41.9% in 1993-1995 (chi 2 = 130.0, p < 0.0001). Analysis of variance showed significant variation between seasons regarding sperm concentration (p < 0.0001) and total sperm count (p < 0.0001). Highest sperm counts were found in spring, with a mean concentration (95% C.I.) of 77.6 x 10(6)/mL (71.9-83.7), and lowest in summer, with a mean of 57.5 x 10(6)/mL (50.1-65.4). No other semen parameter varied with season. It is concluded that sperm counts increased, whereas sperm motility decreased, in a group of Danish semen donor candidates, from 1977 to 1995. Due to the retrospective design and the anonymity of the donors, we were unable to control for variation in donor age, and we cannot exclude the possibility that some donor candidates were selected by being accepted as donors by other semen donor services in Copenhagen. With these limitations in mind, we suggest our results should be interpreted cautiously and regarded as a contribution to the ongoing dispute on whether or not there is a continuous decrease in sperm quality. The seasonal variations found in sperm concentration and total sperm count were pronounced and were not attributable to seasonal differences in the length of sexual abstinence. Additionally, the same seasonal pattern was observed in five successive year-intervals. These findings strongly indicate that human testicular function is influenced by season, a phenomenon well known in many lower mammals.     

上海地区正常生育力男性精液参考值初探

目的:回顾分析上海地区志愿捐精者与正常生育力男性精液分析各项主要参数的分布特征,比较两组男性精液质量的差别,探讨上海地区男性精液参数的正常参考值下限。方法:2010年10月至2011年7月上海市人类精子库招募正常生育力男性41例,健康捐精者100例,按《世界卫生组织人类精液检查与处理实验室手册》(第5版)进行精液常规检测,评估精液体积、精子浓度、前向运动(PR)精子百分率、精子总数和PR精子总数的均值,标准差,并进行t检验。同时统计正常生育力组上述各参数的分布,得出精液特征参数的正常参考值下限。结果:健康捐精组与正常生育力组精液常规各项主要参数(精液体积、精子浓度、PR精子百分率、精子总数、PR精子总数)间差异无统计学意义(P<0.05)。上海地区正常生育力男性精液参考值下限(P<0.05)为:浓度≥27.3×106/ml、PR≥8.1%、体积≥0.82 ml、精子总数≥44.73×106/1次射精、PR精子总数≥24.68×106/1次射精。结论:在评估男性生育力时,精子总数和PR精子总数可能是比精子浓度、精液体积和PR精子百分数更具参考价值的评价指标。     

Trends in semen parameters in the northeast of Scotland

The data on trends in semen quality are conflicting and sensitive to geographical variations. Although previous British surveys on semen quality indicate a decline, the northeast of Scotland has never been included in these surveys. This is an area with low out migration rates where andrology services for a population of 500 000 are centralized within a single laboratory, thus providing a unique opportunity to study population-based trends in semen quality over time. We investigated trends in semen parameters between 1994-2005, in a cohort of 4832 men attending for routine semen analysis at the Aberdeen Fertility Centre who had a sperm density of greater than 20 million per mL. The main outcome measures were trends in sperm density, sperm motility and motile density in the first semen sample. Linear regression and time series analysis were used to examine trends over time in the semen parameters. The mean and standard deviation (SD) age of all men (n=5204) in the study was 34(6) years. The median (inter quartile range) for sperm density and motile density for the study population were 61 (40-91) million/mL and 99 (47-181) million. The mean (SD) sperm motility was 49 (19)%. Among 4832 men (with sperm count >20 million per mL), data adjusted for age and period of abstinence showed a decreasing trend for sperm density over time, R2=0.45 (P=.017). There was no such trend in sperm motility and motile density. However, this trend has to be interpreted with caution due to fluctuations in semen parameters, population bias and the retrospective nature of the analysis.     

Effect of sample collection site on semen parameters of healthy young volunteers

The effect of sample collection site on semen parameters in ten men aged between 22 and 24 years was investigated. Sperm was collected at two sites: in a university hospital restroom for general use and in a one-person hospital room. Samples were collected from the same individual twice, with an interval of two weeks between collections. Semen parameters for the two sites were compared. Samples were collected after a minimum of three days and not longer than seven days of sexual abstinence. Sperm concentration did not differ significantly between the university hospital restroom location (86.8 +/- 25.4 x 10(6)/ml; mean +/- standard deviation) and the private hospital room (97.1 +/- 72.0 x 10(6)/ml). There was no difference in the total motile sperm count or daily sperm production between the collection sites. These results suggest that the collection site has little effect on semen parameters.     

Study of a group of 484 fertile men Part II: Relation between age (20–59) and semen characteristics

Commonly measured semen variables as well as post-thaw motility have been studied as a function of age in fertile men. The mean age was 34.6 (so = 6.6). No significant change with age was found for the sperm count, semen volume or total number of spermatozoa. Conversely, there were significant differences between age groups for the percentage of normal cells ( P < 0.01) and the percentage of motile forms ( P < 0.01) as well as for the after-thaw motility ( P < 0.001). These three variables rise, reach a maximum level at 30 to 35 years of age and then decrease. These changes are not explained by variations in the length of abstinence.     

Effects of semen characteristics on IUI combined with mild ovarian stimulation

To determine the influence of sperm parameters inseminated on the outcome of intrauterine insemination (IUI) in patients undergoing ovarian stimulation with clomiphen citrat (CC) or human menopausal gonadotropin (HMG) therapy, a retrospective review was performed for 2 years on data from the IUI program. 190 couples underwent a total of 268 IUI cycles in which CC or HMG was used for ovulation induction. The initial sperm concentration (mil/ml), motility (percent), preprocessing total motile sperm (TMS) count (million), fast motile sperm (percent) and postprocessing sperm concentration (mil/ml), motility (percent), TMS count, fast motile sperm (percent), sperm morphology, hypoosmotic swelling (HOS) scores, semen leuocytes, and bacteria were analyzed. 268 inseminations were followed by a pregnancy rate of 12% and couple pregnancy rate of 17%. On multivariable logistic regression analysis, total motile sperm (TMS) count, percent motility, and percent of fast motile sperm were independent prognostic factors of fertility. The impact of the preprocessing and postprocessing sperm parameters on pregnancy outcome after IUI was evaluated. There was a trend toward an increasing percent of conception with increasing TMS count, motility, and percent of fast motile sperm. The TMS count, motility and percent of fast motile sperm independently predict success with IUI. Patients with original sperm motility > or = 30% had a higher cumulative pregnancy rate (74%) than patient with motility < 30% (p < 0.005). Pregnancy rate increased 4 times with motility of > or = 30%.     

Effect of age on quality of fresh and frozen－thawed semen in White Italian gandersin White Italian ganders

AIM: To comparatively evaluate the fresh semen quality of 1, 2 and 3-yr-old White Italian ganders (Anser anser L.) and the susceptibility of spermatozoa to freezing-thawing procedure. METHODS: Semen was collected by dorso-abdominal massage every 2 days~3 days from three groups of ganders: 1-yr-old (n=11), 2-yr-old (n=7) and 3-yr-old (n=9). In the pooled fresh semen samples, the following parameters were evaluated: the ejaculate volume, the blood or feca contamination and the motility, concentration and morphology of spermatozoa. Sperm motility and morphology were evaluated in the frozen-thawed semen. Semen diluted with EK extender was frozen in straws in a computerized freezing unit with 6 % dimethyl-formamide to -140 deg at a rate 60 deg/min and then transferred into the LN2 container. Straws with semen were thawed in a water bath at 60 deg. RESULTS: The ejaculate volume decreased with the age (0.21 mL for 1-yr-old, 0.18 mL for 2-yr-old and 0.14 mL for 3-yr-old ganders); the sperm concentration increased with the age (327 x 10(6) mL(-1) for 1-yr-old, 431 x 10(6) mL(-1) for 2-yr-old and 547x10(6) mL(-1) for 3-yr-old ganders); the number of live - normal sperm was significantly (P<0.01) lower in the 1-yr-old than that in the 2- and 3-yr-old ganders (26.61 %, 41.54 and 35.9 %, respectively). The percentage of normal cells survived the freezing-thawing process was 37.7 %, 43.3 % and 40.9 % for 1-, 2- and 3-yr-old ganders, respectively. CONCLUSION: Freezing and thawing processes more significantly (P<0.01) affected the motility, viability and morphology of spermatozoa in semen of 1-yr-old ganders in comparison with older males.     

Evaluation of reference values of standard semen parameters in fertile Egyptian men

The reference values of human semen, published in the WHO's latest edition in 2010, were lower than those previously reported. The objective of this study was to evaluate reference values of standard semen parameters in fertile Egyptian men. This cross‐sectional study included 240 fertile men. Men were considered fertile when their wives had recent spontaneous pregnancies with time to pregnancy (TTP) ≤12 months. The mean age of fertile men was 33.8 ± 0.5 years (range 20–55 years). The 5th percentiles (95% confidence interval) of macroscopic semen parameters were 1.5 ml for volume and 7.2 for pH. The 5th percentiles of microscopic parameters were 15 million/ml for sperm concentration, 30 million per ejaculate for total sperm count, 50% for total motility, 40% for progressive motility, 62% for vitality, 4% for normal sperm forms and 0.1 million/ml for seminal leucocyte counts. In conclusion , fertile Egyptian men had higher reference values of sperm total motility, progressive motility and vitality, and lower reference values for total sperm counts as compared to those determined by the latest edition of the WHO laboratory manual in 2010. Other semen parameters were identical to those defined by the WHO 2010 manual.     

Comparison of semen quality in older and younger men attending an andrology clinic

The effects of age on semen quality were analysed in patients referred to an andrology outpatient clinic in a retrospective study covering a period of 3 years. Semen analyses of older men (n = 66; > or = 50 years; median: 53) were compared with those of young patients (n = 134; 21-25 years; median 24). The duration of sexual abstinence was longer among older patients (median: 5.5 vs. 5.0 days; + 10%, P < 0.05). Even after adjustment for duration of sexual abstinence, progressive motility (- 27%, P < 0.01), percentage of morphologically normal spermatozoa (- 44%, P < 0.01) and semen volume (- 29%, P < 0.0005) were significantly lower in older than in younger men. Impaired sperm morphology was mainly attributed to abnormal staining of flagella (+ 50%, P < 0.05) and an increased portion of coiled or bent tails (+ 44%, P = 0.001) indicating epididymal dysfunction. Total sperm count and sperm concentration were unaffected by age. Age-dependent changes in accessory gland function, as indicated by fructose concentration (- 27%, P < 0.05), was correlated with lower semen volume. Moreover, serum testosterone levels were significantly reduced in the group of older men (median: 3.0 vs. 3.6 ng ml-1; -17%, P < 0.005).     

Poor semen quality from patients with malignancies does not rule out sperm banking

Cancer therapy can further impair the already poor semen quality in cancer patients. This study evaluated the prefreeze and postthaw semen quality before treatment of patients with malignancies to examine the rationale for sperm banking for these men. Records of nine patients with different malignant tumors, who had been referred for sperm cryopreservation between 1982 and 1997, were reviewed and the results were compared with those of 50 normal healthy donors. Patients did not differ from donors in age, ejaculate volume, or duration of sexual abstinence. The total motile sperm count (median and interquartile range) was significantly different between patients and donors for prefreeze specimens (P=0.026) and postthaw specimens (P=0.008). Also, the percent motility was significantly lower in the patients as compared with the donors in prefreeze (P=0.035) and postthaw specimens (P=0.005). The percentage change in motility after thawing was also larger for patient samples (−54% versus −47%,P=0.39). Other sperm motion characteristics did not significantly differ between the two groups except for postthaw curvilinear velocity (P=0.01). This study concludes that fresh and frozen–thawed semen from patients with malignant tumors is poor in quality but is still adequate for assisted reproductive techniques. As cancer therapy may further impair semen quality, patients should be offered the chance to bank sperm before undergoing cancer therapy. Received: 25 October 1999 / Accepted: 22 May 2000     

Semen quality changes among 2343 healthy Slovenian men included in an IVF-ET programme from 1983 to 1996

To determine whether semen quality in Slovenians has changed over 14 years (1983-96), we analysed retrospectively the semen of 2343 healthy men with a normal spermiogram, who were partners of women with tubal infertility included in the IVF-ET programme. Age at semen collection, duration of sexual abstinence, semen volume, sperm concentration, total sperm count, percentage of spermatozoa with progressive motility, and normal morphology were determined. Multiple regression analysis was used to assess the changes in sperm characteristics according to the year of semen collection, year of the man's birth and the duration of sexual abstinence. Semen volume, sperm concentration, sperm count and total sperm motility did not change between 1983 and 1996, whereas between 1988 and 1996 rapid progressive sperm motility decreased by 0.95% per year (p < 0.0001). Semen volume, sperm concentration, and sperm count increased with duration of sexual abstinence. After adjustment for the year of semen collection and duration of sexual abstinence, multiple regression analysis showed that sperm concentration decreased by 0.67% per each successive year of birth (p = 0.03). Thus the sperm concentration decreased from 87.6 x 10(6)/mL in men born in the 1940s to 77.3 x 10(6)/mL in those born between 1956 and 1960. After 1960, sperm concentration was found to increase. In 2343 healthy men, no decline in semen quality, except in rapid progressive motility, was observed in the study period. Lower sperm concentration was found among men born between 1950 and 1960. This could be related to worse socio-economic status, stress or negative environmental factors in this time period.     

Concentrating human sperm before cryopreservation

To improve the quality of cryo-preserved sperm, ejaculated semen was concentrated prior to freezing by means of continuous-step density gradient centrifugation. Freezing was simplified by employing liquid nitrogen vapor with KS-II cryo-medium. The original semen (n = 32, 41 +/- 13 x 10(6)/ml, 17 +/- 11% motility) was improved by the processing to be 68 +/- 29 x 10(6)/ml, 55 +/- 19% motility. Even after the specimens were thawed, sperm concentrations were similar to those of the original semen and 39 +/- 18% motility remained with a mean survival rate of 74 +/- 7.7%. The improved sperm motility and higher survival rate resulted in better post-thaw sperm quality than those of the original semen.     

Study of a group of 484 fertile men Part I: Distribution of semen characteristics

The semen characteristics studied were the sperm count, semen volume, morphology and pre-freeze and post-thaw motility. Two categories of fertile men were investigated: semen donor candidates for artificial insemination and pre-vasectomy subjects. Since mean values for each variable in the two series were similar, they could be considered as a single group of 484 fertile men. Only those subjects whose ejaculates were obtained after an abstinence of 5 days or less were retained. The distribution, mean and percentiles were determined for each variable. The 10th and 90th percentiles for sperm count, percentage of motile forms and percentage of normal cells were respectively 25 and 180 million per ml, 60% and 80% and 50% and 75% The three variables, sperm count, semen volume and total number of spermatozoa which were dependent on abstinence were analysed in the same manner for 3 days of abstinence. The group studied seemed to be as representative and as well defined as possible.     

Standardized methods for semen evaluation in a multicenter research study

Semen evaluation methodology is complex and difficult to standardize. Rigorously standardized laboratory protocols and strict quality control (QC) are essential for meaningful comparison of data from multiple sites. We describe the methods used for determination of semen volume, sperm concentration, and percent sperm motility in the Study for Future Families, a multicenter study of semen quality in the United States. Each of these 3 semen parameters was assessed using 2 techniques, which provided the opportunity to compare precision and assess suitability for multicenter studies. Detailed protocols were used, and technicians were centrally trained. A total of 509 semen evaluations were performed. Semen volume measured by weight was greater (P <.0001) than that determined by pipetting (3.7 +/- 1.6 mL vs 3.2 +/- 1.6 mL). Sperm concentration determined using hemacytometer chambers was consistently higher (P <.001) than that using disposable MicroCell chambers (81.0 x 10(6)/mL vs 65.9 x 10(6)/mL). Precision was slightly greater for the MicroCell chamber. The percentage of motile sperm was assessed by a simple counting technique as well as by the World Health Organization categorical method that assigns individual motile sperm to "a," "b," and "c" categories on the basis of progression. When these 3 categories were collapsed, the methods provided values that were not statistically different (P >.05), although the collapsed values tended to be higher (58.1% vs 51.6%) and less precise (CV 7.7% vs 4.1%) for the categorical method than for motility determined using the simple method. The data obtained in this study demonstrate the critical need for rigorous standardization of protocols and techniques for multicenter studies.     

Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing

Electroejaculate traits, testicular volume, and circulating FSH, LH, and testosterone concentrations were compared between two populations of domestic cats consistently producing either a high (greater than 60%, normospermic) or low (less than 40%, teratospermic) incidence of structurally normal spermatozoa/ejaculate. The effects of semen dilution in Biggers, Whitten and Whittingham (BWW) or modified Krebs Ringer bicarbonate (mKRB) medium and swim-up processing on sperm viability and duration of motility in vitro also were assessed. Ejaculate volume, percent sperm motility, sperm progressive motility, motile spermatozoa/ejaculate, testes volume, and mean serum FSH and LH concentrations were similar (P greater than 0.05) between normospermic and teratospermic cats. However, sperm concentration/ml of ejaculate was greater and circulating testosterone levels were lower in teratospermic males. Swim-up processing increased (P less than 0.05) percent sperm motility, progressive motility, and the number of structurally normal sperm cells recovered and also prolonged the duration of sperm motility in both cat populations. In teratospermic ejaculates, swim-up separation increased the proportion of morphologically normal spermatozoa recovered by more than two-fold. Diluting cat semen with either BWW or mKRB increased flagellar bending in both normospermic and teratospermic cats. The sperm motility characteristics of only the teratospermic ejaculates were influenced by medium type; mKRB increased percent sperm motility and progressive motility whereas BWW had no effect. Compared with undiluted raw ejaculates, the duration of sperm motility was improved 18- to 24-fold by diluting semen in either BWW or mKRB medium followed by swim-up processing. This study demonstrates that the electroejaculate characteristics of domestic cats vary markedly and that some males consistently produce high proportions of morphologically abnormal spermatozoa. Diminished serum testosterone concentrations and normal pituitary secretion of FSH and LH in teratospermic males suggest that there is an inverse relationship between gonadal androgen production and pleiomorphic spermatozoa in the domestic cat. The swim-up procedure is effective for recovering motile, structurally normal spermatozoa from teratospermic cats.     

The effect of chymotrypsin on the determination of total alpha-glucosidase activity in seminal plasma and the correlation between alpha-glucosidase level and semen parameters

To evaluate the effect of chymotrypsin on the examination of alpha-glucosidase activity in seminal plasma, thirty-nine samples of fresh liquefied semen with or without chymotrypsin and forty-eight samples of fresh un-liquefied semen with chymotrypsin were determined for the total alpha-glucosidase activity in seminal plasma. The total alpha-glucosidase level of each sample was assayed by the method of glucose oxidase. The correlations between alpha-glucosidase level and semen parameters, including semen volume, pH, sperm concentration, grade a and b motility and total motility, were analyzed with SPSS 11.0 software. The results showed that chymotrypsin had no effect on seminal alpha-glucosidase activity determination. Chymotrypsin could improve the liquefaction for un-liquefied semen, and there was no significant difference of alpha-glucosidase activity between liquefied and un-liquefied semen samples. There were significantly positive correlations between seminal alpha-glucosidase activity (U/ml) and sperm concentration (r = 0.338, p = 0.015) and between total alpha-glucosidase activity (U/ejaculate) and semen volume (r = 0.677, p = 0.000). However, there was no significant correlation between alpha-glucosidase level (U/ml) and semen volume, pH, sperm motility or grade a and b motility (r = -0.234 approximately 0.077, p = 0.099 approximately 0.993). The data indicated that chymotrypsin could be added into the un-liquefied semen samples for alpha-glucosidase activity determination, and there were different correlations between seminal alpha-glucosidase level and various semen parameters.     

Circannual variation in human semen parameters

The aim of the present study was to determine whether there were significant monthly variations in the semen parameters (i.e. volume, sperm count, total sperm count, motile and normal sperm count) of men living in a Mediterranean climate area. A total of 10 877 semen analysis results were included. Semen samples were obtained as a part of an initial screening of male partners from couples with infertility problems who were attending our laboratory from 1970 to 2000. Log transformation and cubic root transformation were used to test the sample distribution. Statistical significance was adjusted by year of examination, patient's age and sexual abstinence period by performing covariance analyses. Differences between months were assessed with the Bonferroni post-hoc test. There was an increase in March and a decrease in September in the adjusted mean sperm count (p < 0.0005), total sperm count (p < 0.0005), motile sperm count (p=0.01) and normal sperm count (p=0.002). There were no variations in semen volume in the study period. Monthly changes in semen quality are confirmed in this population.     

Intrauterine insemination with double ejaculate compared with single ejaculate in male factor infertility: a pilot study

The aim of this study was to compare the efficiency of using a double ejaculate with the efficiency of using a single ejaculate for intrauterine insemination in male subfertility. Eligibility for the study was a total motile sperm count between 1 x 10(6) and 5 x 10(6) on postwash sperm analysis. Thirty-nine couples were randomized to the study group, while another 50 couples were randomized to serve as the control group. Males in the study group were asked to produce a second semen sample within 2 hours of the first sample on the day of insemination. In the study group, the mean total motile sperm count in the first sample was 3.83 x 10(6) (SD +/- 0.85 x 10(6); range 1.1-4.9) and 3.99 x 10(6) (SD +/- 0.72 x 10(6); range 0.9-4.4) in the control group. The mean total motile sperm count in the second sample was 3.52 x 10(6) (SD +/- 1.46 x 10(6); range 0.9-3.7) in the study group. The mean total motile sperm count in the final inseminate was 7.35 x 10(6) (SD +/- 1.90 x 10(6); range 2.9-10.6) in the study group. The difference in total motile sperm counts between the study and the control group was statistically significant (P < .001). There were 6 pregnancies in the study group, providing a pregnancy rate of 15.3%, whereas there were 5 pregnancies in the control group, representing a pregnancy rate of 10% (P = .44). We concluded that although it does not increase the pregnancy rate significantly, obtaining a second semen sample when the motile sperm yield of the first semen sample is 1 million to 5 million significantly increases the total motile sperm count in the final inseminate.     

Characterizing the reproductive physiology of the male southern black howler monkey, Alouatta caraya

Limited reproductive data are available for any species of howler monkey, including those listed as threatened (Alouatta pigra) and endangered (A. palliata) by the Convention on International Trade in Endangered Species Status (CITES) report. The Southern black howler monkey (A. caraya) is being considered as a model species to develop assisted reproductive technology (ART) for vulnerable howler species. Specific objectives of this study were to evaluate the effect of 1) time of year on ejaculate quality and testosterone concentration, 2) age of male on ejaculate quality, and 3) seminal plasma on sperm longevity in vitro. Three adult (4.5 to 5 years) and 3 subadult (1.5 to 2.5 years) males were evaluated for a 1.5-year period. Semen samples were obtained by electroejaculation, and testosterone levels were monitored by fecal steroid metabolite radioimmunoassay. Males produced coagulum-free ejaculates throughout the year. Likewise, most (4/6) males exhibited constant testosterone levels (3.66 +/- 0.45 ng/g) during the year. Testosterone levels for the remaining 2 males, housed as a bachelor troop, were elevated (43 ng/g) during the months of May and June. Seminal characteristics were similar (P > .05) between age groups. Average semen volume was higher during the summer months (P < .05). Sperm concentrations were highly variable through the year and ranged from 7.0 x 10(6) sperm/mL to 583.0 x 10(6) sperm/mL. Percentages of motile sperm (73% +/- 2.3%) and forward progressive sperm motility (3.3 +/- 0.1), however, were consistent (P > .05) throughout the year. The average pH (8.9 +/- 0.1) and osmolality (356.7 +/- 26.1 mmol/kg) of raw semen also did not vary (P > .05) throughout the year. Ejaculates from subadult males, however, contained more (P < .05) morphologically abnormal spermatozoa than adult ejaculates. In addition, in vitro sperm longevity was poor (<2 hours) for subadult male samples, regardless of the presence or absence of seminal plasma (P > .05). For adult males, seminal plasma was detrimental to sperm longevity; however, spermatozoa survived more than 5 hours in vitro when seminal plasma was removed. Although subadult males produce semen, these ejaculates would not be ideal for further characterization of seminal traits or development of ART for other howler monkey species.