Individual variation in the frequency of sperm aneuploidy in humans.

To examine interindividual differences in sperm chromosome aneuploidy, repeated semen specimens were obtained from a group of ten healthy men, aged 20-21 at the start of the study, and analyzed by multi-color fluorescence in situ hybridization (FISH) analysis to determine the frequencies of sperm aneuploidy for chromosomes X, Y, 8, 18 and 21 and of diploidy. Semen samples were obtained three times over a five-year period. Statistical analysis examining the stability of sperm aneuploidy over time by type and chromosome identified two men who consistently exhibited elevated frequencies of sperm aneuploidy (stable variants): one with elevated disomy 18 and one with elevated MII diploidy. Differences among frequencies of aneuploidy by chromosome were also seen. Overall, disomy frequencies were lower for chromosome X, 8 and 18 than for chromosomes 21 or Y and for XY aneuploidy. The frequency of chromosome Y disomy did not differ from XY sperm frequency. Also, the frequency of meiosis I (XY) and II (YY + XX) sex chromosome errors did not differ in haploid sperm, but the frequency of MII errors was lower than MI errors in diploid sperm. Frequencies of sperm aneuploidy were similar between the first sampling period and the second, two years later. However, the frequency of some types of aneuploidy (XY, disomy Y, disomy 8, total autosomal disomies, total diploidy, and subcategories of diploidy) increased significantly between the first sampling period and the last, five years later, while others remained unchanged (disomy X, 21 and 18). These findings confirm inter-chromosome differences in the frequencies of disomy and suggest that some apparently healthy men exhibit consistently elevated frequencies of specific sperm aneuplodies. Furthermore, time/age-related changes in sperm aneuploidy may be detected over as short a period as five years in a repeated-measures study.

Antiretroviral therapy effects on genetic and morphologic end points in lymphocytes and sperm of men with human immunodeficiency virus infection.

Many human immunodeficiency virus (HIV)-infected persons receive prolonged treatment with DNA-reactive antiretroviral drugs. A prospective study was conducted of 26 HIV-infected men who provided samples before treatment and at multiple times after beginning treatment, to investigate effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Several antiretroviral regimens, all including a nucleoside component, were used. Lymphocyte metaphase analysis and sperm fluorescence in situ hybridization were used for cytogenetic studies. Semen analyses included conventional parameters (volume, concentration, viability, motility, and morphology). No significant effects on cytogenetic parameters, semen volume, or sperm concentration were detected. However, there were significant improvements in sperm motility for men with study entry CD4 cell counts >200 cells/mm(3), sperm morphology for men with entry CD4 cell counts < or =200 cells/mm(3), and the percentage of viable sperm in both groups. These findings suggest that nucleoside-containing antiretrovirals administered via recommended protocols do not induce chromosomal changes in lymphocytes or sperm but may produce improvements in semen quality.

Factor analytic models of clustered multivariate data with informative censoring.

This article describes a general class of factor analytic models for the analysis of clustered multivariate data in the presence of informative missingness. We assume that there are distinct sets of cluster-level latent variables related to the primary outcomes and to the censoring process, and we account for dependency between these latent variables through a hierarchical model. A linear model is used to relate covariates and latent variables to the primary outcomes for each subunit. A generalized linear model accounts for covariate and latent variable effects on the probability of censoring for subunits within each cluster. The model accounts for correlation within clusters and within subunits through a flexible factor analytic framework that allows multiple latent variables and covariate effects on the latent variables. The structure of the model facilitates implementation of Markov chain Monte Carlo methods for posterior estimation. Data from a spermatotoxicity study are analyzed to illustrate the proposed approach.

Significance of incorporating measures of sperm production and function into rat toxicology studies.

The rat is the preferred species for reproductive toxicity testing. The inclusion of measures of rat sperm quality, such as motility and morphology, into reproductive test protocols often increases the sensitivity of the test to detect effects, and provides the toxicologist and risk assessor with valuable information about the nature of the reproductive toxicity of the test substance. Technical advances in computer-aided sperm analysis have made it possible to evaluate motion characteristics of rat spermatozoa. This technology can provide an objective means of classifying the motion of rat spermatozoa as progressive or non-progressive, as required in test protocols. More specific tests of rat sperm function are being applied for the purpose of evaluating modes and mechanisms of toxicant action. Computer-aided sperm analysis can be used to evaluate sperm motion during cultures that support sperm capacitation and to identify hyperactivated spermatozoa. Under the same culture conditions, acrosome-specific stains can be used to identify effects of toxicants on the acrosome reaction. These approaches, in combination with in vitro fertilization in rats, can pinpoint sperm functional deficits and thereby assist the toxicologist in addressing hypotheses regarding the cellular-molecular bases of toxicant-induced male infertility.

Evaluation of aneuploidy and DNA damage in human spermatozoa: applications in field studies.

With the goal of incorporating measures of sperm nuclear integrity in an epidemiology study, semen samples from young Czech men were analysed for sperm aneuploidy and sperm chromatin structure in addition to routine measures of sperm production and quality. The exposure in question was to high seasonal air pollution containing reactive polyaromatic hydrocarbons potentially capable of affecting spermatogenesis and damaging sperm DNA. The sperm aneuploidy assay uses fluorescence in situ hybridization to label selected sperm chromosomes; as applied in this study, the sex chromosomes (X,Y) and chromosome 8 were targeted. The sperm chromatin structure assay detects sperm nuclei with increased susceptibility to denaturation, a feature that is associated with DNA damage. Logistically, these assays were relatively easy to incorporate into the study design. The aneuploidy assay provided information suggesting that exposure to high levels of air pollution may increase the risk of sperm aneuploidy and that it is important to control for exposure to cigarette smoke and/or alcohol in such studies. The sperm chromatin structure assay provided valuable baseline information about Czech semen donors and data suggestive of an adverse effect of smoking and air pollution on spermatozoa that merits further investigation.

Exposure to hazardous substances and male reproductive health: a research framework.

The discovery in the mid-1970s that occupational exposures to pesticides could diminish or destroy the fertility of workers sparked concern about the effects of hazardous substances on male reproductive health. More recently, there is evidence that sperm quantity and quality may have declined worldwide, that the incidence of testicular cancer has progressively increased in many countries, and that other disorders of the male reproductive tract such as hypospadias and cryptorchidism may have also increased. There is growing concern that occupational factors and environmental chemical exposures, including in utero and childhood exposures to compounds with estrogenic activity, may be correlated with these observed changes in male reproductive health and fertility. We review the evidence and methodologies that have contributed to our current understanding of environmental effects on male reproductive health and fertility and discuss the methodologic issues which confront investigators in this area. One of the greatest challenges confronting researchers in this area is assessing and comparing results from existing studies. We elaborate recommendations for future research. Researchers in the field of male reproductive health should continue working to prioritize hazardous substances; elucidate the magnitude of male reproductive health effects, particularly in the areas of testicular cancer, hypospadias, and cryptorchidism; develop biomarkers of exposure to reproductive toxins and of reproductive health effects for research and clinical use; foster collaborative interdisciplinary research; and recognize the importance of standardized laboratory methods and sample archiving.

Semen quality and reproductive health of young Czech men exposed to seasonal air pollution.

This study of male reproductive health in the Czech Republic resulted from community concern about potential adverse effects of air pollution. We compared young men (18 years of age) living in Teplice, a highly industrialized district with seasonally elevated levels of air pollution, to those from Prachatice, a rural district with relatively clean air. Surveys were scheduled for either late winter, after the season of higher air pollution, or at the end of summer, when pollution was low. Participation included a physical examination, donation of a semen sample, and completion of a questionnaire on health, personal habits, and exposure to solvents and metals through work or hobby. Analysis of data from 408 volunteers showed that the men from Teplice and Prachatice were similar in physical characteristics, personal habits, and work- or hobby-related exposures. Sixty-six percent (272) of these men donated a single semen sample for routine semen analysis, computer-aided sperm motion analysis, and sperm chromatin structure assay. The mean (median) sperm concentration and sperm count were 61. 2 (44.0) million/mL semen and 113.3 (81.5) million, respectively, and were not associated with district of residence or period of elevated air pollution. However, periods of elevated air pollution in Teplice were significantly associated with decrements in other semen measures including proportionately fewer motile sperm, proportionately fewer sperm with normal morphology or normal head shape, and proportionately more sperm with abnormal chromatin. These results suggest that young men may experience alterations in sperm quality after exposure to periods of elevated air pollution, without changes in sperm numbers.

Objective evaluation of hyperactivated motility in rat spermatozoa using computer-assisted sperm analysis.

The aim of this study was to use computer-assisted sperm analysis (CASA) to examine changes in motion parameters of rat spermatozoa incubated under culture conditions that support IVF. Rat cauda epididymal spermatozoa were evaluated in six replicate experiments, at 0 and 4h of incubation. CASA was conducted at 60 Hz on digital 1s tracks ( approximately 100 spermatozoa/rat). Mean values of CASA parameters that describe the vigour of spermatozoa [curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] increased, while those indicating progressiveness [straight line velocity (VSL), linearity (LIN) and straightness (STR)] decreased between 0 and 4 h. Visual inspection of sperm tracks after 4 h of incubation revealed classical hyperactivation patterns. Bivariate models were evaluated to objectively define the subpopulation of hyperactivated (HA) spermatozoa. Of all models considered, ALH and LIN, VCL and LIN, BCF and LIN, VCL and BCF, and VCL and ALH showed significant changes in the percentage of HA spermatozoa after the 4 h incubation period. The efficacy of detecting HA spermatozoa was evaluated using sperm tracks that were visually classified as HA or progressive. VCL and LIN provided the most accurate prediction of HA spermatozoa. It was concluded that analysis of CASA data using bivariate models could be used to detect and monitor hyperactivation in rat spermatozoa.

Workshop to identify critical windows of exposure for children's health: reproductive health in children and adolescents work group summary.

This work group report addresses the central question: What are the critical windows during development (preconception through puberty) when exposure to xenobiotics may have the greatest adverse impact on subsequent reproductive health? The reproductive system develops in stages, with sex-specific organogenesis occurring prenatally and further maturational events occurring in the perinatal period and at puberty. Complex endocrine signals as well as other regulatory factors (genetics, growth factors) are involved at all stages. Evidence from animal models and human studies indicates that many specific events can be perturbed by a variety of toxicants, with endocrine-mediated mechanisms being the more widely studied. Prioritized research needs include basic studies on the cellular-molecular and endocrine regulation of sexual differentiation and development; increased efforts regarding potential adverse effects on development in females, including breast development; expanded animal studies on different classes of chemicals, comparing responses during development (prenatal and postnatal) with responses in adults; and, more extensive explorations regarding the reproductive biology and toxicology of puberty in humans.

Evaluation of a container for collection and shipment of semen with potential uses in population-based, clinical, and occupational settings.

Large, population-based studies of semen quality are encumbered by the logistics and expense of obtaining semen samples from men who live in a variety of locations. A prototype semen collection and transportation kit, the TRANSEM100, can be distributed to study participants and then directly shipped to a central laboratory for analysis. This study was designed to evaluate the ability of male volunteers to correctly use the kit. Thirty volunteers aged 20 to 44 years with no history of diabetes, recent chemotherapy, fertility problems, or vasectomy were recruited through a newspaper advertisement, interviewed to obtain demographic information, and instructed on the use of the kit. Twenty-six of the initial subjects provided at least 1 semen specimen using the kit and returned the specimens by overnight delivery to the laboratory for analysis, 25 completed a follow-up interview on the use of the collection kit, and 20 submitted a second semen sample using the same method. The average volunteer was white, 27.8 years old, and held at least a college degree. Forty percent of the volunteers were married. In general, participants correctly followed the instructions for collecting, packaging, and shipping the semen samples. Volunteers were instructed to collect samples after at least 2, but no more than 7 days of abstinence. For the first and second samples submitted, participants collected semen samples after an average of 3.3 and 3.9 days of abstinence, respectively. Seventeen (65%) of the samples from the first sampling period and 16 (80%) of the samples from the second period were received in the laboratory the day after they had been collected. In summary, the TRANSEM100 may prove to be useful for collecting human semen in field studies. Further testing of this method is warranted to evaluate preservation of sample quality and use of the kit by men among diverse socioeconomic groups.

Summarizing the motion of self-propelled cells: applications to sperm motility.

Proper characterization of the motion of spermatozoa is an important prerequisite for interpreting differences in sperm motility that might arise from exposure to toxicants. Patterns of sperm movement can be extremely complex. On the basis of an exponential model that relates the discretely approximated curvilinear velocity to the tracking rate, we develop a statistic that indexes the predictability of the path for individual sperm. We summarize the path of each sperm using this and two other statistics: (1) the path displacement velocity and (2) linearity of movement. We apply the method to a set of rat sperm tracks representative of both normal and abnormal motion characteristics.

Fertility of rat epididymal sperm after chemically and surgically induced sympathectomy.

Guanethidine, a chemical that selectively blocks sympathetic noradrenergic neurons, was used to investigate the role of sympathetic innervation in the fertility of rat epididymal sperm, using both natural mating and in utero insemination protocols. This animal model correlates, at least in part, with spinal cord injury (SCI) in men. Adult male rats were treated daily by i.p. injections, for 21 or 42 days, with 0 or 6.25 mg/kg guanethidine. To compare the effects of guanethidine-induced sympathectomy with those following surgically induced sympathectomy, the inferior mesenteric ganglion and the proximal hypogastric nerves were removed in another group of rats. Both chemically and surgically induced sympathectomy increased the weight of the epididymis and seminal vesicles/coagulating glands as well as the number and the transit time of cauda epididymal sperm. Neither serum testosterone levels nor LH was affected by treatment with guanethidine. Using natural mating, no litters were produced by guanethidine-treated rats. Chemically denervated rats failed to produce copulatory plugs or ejaculate into the uterus. However, distal cauda epididymal sperm from chemically or surgically denervated rats displayed normal fertilization ability (80%) using in utero inseminations. In addition, the sperm of denervated rats did not show abnormal sperm chromatin structure using an assay that detects DNA damage. We conclude that sympathectomy delays the transit of sperm through the cauda epididymidis and produces ejaculatory dysfunction but does not compromise sperm quality in the distal cauda epididymidis. Moreover, these data provide compelling evidence that there is no association between the prolonged transit time of sperm within the epididymis, i.e., pre-ejaculatory sperm aging, and the fertility of those sperm, which has important implications for artificial insemination using sperm from men with SCI.

Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites. III. Guidelines for field studies of male reproductive disorders.

Exposures to environmental toxicants can have detrimental effects on several aspects of human male reproduction: fertility, sexual function, hormone status, and pregnancy/birth outcomes. However, no simple prescreening methods are available for reliably identifying potential hazards; questionnaires alone are relatively imprecise and inefficient in the absence of field data. Multidisciplinary field studies are required that include detailed exposure information, health and reproductive histories, physical examinations, semen analyses, and possibly, hormone analyses. Semen analysis is a critical component of field studies for evaluating two aspects of male reproduction: 1) changes in sperm or seminal content, which may be indicative of adverse effects on the male reproductive system with possible implications for fertility potential; and 2) defects in sperm DNA or chromosomes, which may be associated with subsequent changes in viability during embryonic development and health risks to the offspring. Semen analyses may be tiered: 1) initially, each semen study may include conventional semen assays (concentration, motility, and morphology) as well as specific biomarkers indicated by the health effect of concern in the study cohort: and 2) archived samples (i.e., frozen, videotaped, or smeared) may be utilized in later second-tier analyses to further characterize specific findings. Before initiating any field study, it is cost effective to critically evaluate the suitability of the cohort by confirming exposure and determining that there are adequate numbers of male participants in each exposure category. Such evaluations must be based on the statistical sensitivities of the specific tissue biomarkers and health endpoints for detecting changes. This article summarizes the components of the ideal field study and identifies research needs for improving field studies of male effects and for understanding the mechanisms of male reproductive toxicity. Several promising semen methods currently under development are also discussed.

Sperm motion predicts fertility in male hamsters treated with alpha-chlorohydrin.

An understanding of the relationship between altered sperm motion and sperm function (fertility) is important when interpreting the biological significance of toxicant-induced changes in sperm velocity in rodent test species. Previous studies showed that a brief (4-day) exposure of male hamsters to the model chemical alpha-chlorohydrin (ACH) results in significant deficits in epididymal and uterine sperm velocity, which are associated with both a delay and a failure of fertilization in vivo. To characterize this effect in terms of fertility, similarly treated male hamsters were bred to untreated females and pups were counted the day before parturition. ACH treatment resulted in a dose-dependent decline in the percentage of sperm-positive females that were pregnant at the end of gestation (100, 78, 67, 22, and 0 where males were treated with 0, 33, 49, 66, and 83 mg ACH/kg/day, respectively). Cauda epididymal sperm from the same males were assayed for motion characteristics using computer-assisted sperm analysis (CASA), and for fertilizing ability in vitro. While the percentage of motile sperm was unaffected by ACH treatment, sperm velocity declined in a dose-dependent manner at all ACH treatment levels. Furthermore, the velocity of sperm from infertile males was shifted downward consistently across the entire velocity distribution. Since treated males tended to either be infertile (no pups) or have near normal litter size, the correlation between sperm velocity and litter size was nonlinear. Therefore, logistic regression models using velocity cut-off values were the most useful models for predicting fertility. These results support the contention that fertility relies on there being a sufficient number of sperm that exceed a velocity threshold. Sperm from treated males were also less likely to support in vitro fertilization (IVF), providing further evidence of impaired sperm function associated with acute exposure to ACH.

Glutathione oxidation is associated with altered microtubule function and disrupted fertilization in mature hamster oocytes.

We hypothesized that depletion of glutathione (GSH) with diamide, a relatively specific GSH oxidant, may alter the meiotic spindle apparatus in mature hamster oocytes. Immunofluorescent analysis of oocytes exposed to diamide for 1.5 or 3 h revealed time- and concentration-dependent disruption of spindle morphology accompanied by chromosome clumping. In oocytes first cultured in diamide for 1.5 h and then in diamide-free medium for 1.5 or 3 h, microtubules appeared to repolymerize, but normal spindle structure was not regained. HPLC confirmed that diamide oxidized oocyte GSH under conditions identical to those associated with spindle-related abnormalities. Exposure of oocytes to 25 or 50 microM diamide before in vitro fertilization did not affect their ability to undergo fertilization. A significant proportion of the fertilized oocytes that had been exposed to 50 microM diamide before insemination exhibited abnormal multiple female pronuclei with an apparently normal male pronucleus. These observations indicate that mature hamster oocytes are susceptible to oxidative stress during the critical period that precedes fertilization and provide further evidence that GSH plays important roles in oocyte spindle function and pronucleus development.

Comparison of rat epididymal sperm counts by IVOS HTM-IDENT and hemacytometer.

Epididymal sperm counts, a common measurement in male reproductive toxicity studies, are routinely determined using a hemacytometer. Recently, computer assisted methods for automated sperm counts have been developed. In the present study we evaluated an automated system, the TOX IVOS (Hamilton Thorne Research, Beverly, MA) HTM-IDENT option, that utilizes a DNA-specific stain and fluorescence illumination to identify sperm for enumeration. Cauda and caput epididymal sperm counts were determined in 48 adult male Sprague-Dawley rats, using both the hemacytometer and HTM-IDENT. The mean hemacytometer and HTM-IDENT counts (+/- SD) were 250 +/- 43 and 254 +/- 52 million, respectively, for cauda sperm, and 123 +/- 13 and 127 +/- 18 million, respectively, for caput sperm. The average coefficient of variation using the hemacytometer was 13.8% as compared to 17.3% for the HTM-IDENT. Comparison of the machine count and a visual count from the Display Statics screen of the HTM-IDENT indicated that when two or more sperm heads touched or overlapped, the machine counted them as one. Manual (visual) and machine counts when compared over a range of nine concentrations from 3.7 to 47.8 million/mL differed by 4 to 12% at the lowest to highest concentration. The concentration of epididymal sperm samples used in comparing the two counting methods ranged from 5.8 to 17.7 million/mL. Therefore, the HTM-IDENT undercounting error attributable to sperm heads touching was less than 6%. Overall the data indicate good agreement between the HTM-IDENT and the hemacytometer counts. Furthermore, both counting time and technician fatigue were markedly reduced. Thus the HTM-IDENT option improves the efficiency of epididymal sperm counting without loss of precision.

Teplice program--the impact of air pollution on human health.

The aim of the Teplice Program is to investigate and assess the impact of air pollution on the health of the population in the district of Teplice, Czech Republic. Characterization of the air pollutants demonstrated unusually high concentrations during winter inversions of fine particles dominated by acidic sulfates, genotoxic organic compounds, and toxic trace elements. The major source of airborne fine particles is the burning of coal for heating and power. Human exposure and biomarker studies demonstrated large seasonal variations in air pollution within the Teplice District and higher seasonal average pollution levels than the comparative district, Prachatice. Personal exposures to fine particles and organic carcinogens [e.g., polycyclic aromatic hydrocarbons (PAH)] were correlated with excretion of PAH metabolites in urine, several trace metals in blood, and DNA adducts in white blood cells. Respiratory and neurobehavioral studies of school children were conducted using questionnaires and clinical measures. A significantly higher prevalence of adverse respiratory symptoms and decreased lung function were found in the Teplice district than in Prachatice. The neurobehavioral studies indicated significantly higher teacher referrals for clinical assessment in Teplice, but the majority of objective performance measures did not differ. Reproductive studies were conducted in both males and females. A study of the effects of exposure on pregnancy and birth found an excess prevalence of low birth weight and premature births in Teplice; these adverse effects were more common in infants conceived in the winter and whose mothers were smokers. Based on questionnaires and medical examination, the reproductive development of young men was not different between districts and seasons, however, measures of semen quality suggest that exposure to high levels of air pollution are associated with transient decrements in semen quality.

Acute exposure of female hamsters to carbendazim (MBC) during meiosis results in aneuploid oocytes with subsequent arrest of embryonic cleavage and implantation.

A single oral dose of the fungicide and microtubule poison, MBC, administered to female hamsters at proestrus, results in infertility and early pregnancy loss (1). To characterize the site and mode of action of this effect, direct assessments of oocyte chromosomes, fertilization, and preimplantation embryo development were made. Female hamsters were given a single dose of MBC (1,000 mg/kg) on the afternoon of proestrus (to coincide with meiotic maturation of the oocytes) and either killed shortly after ovulation (day 1) to recover oocytes, or bred and killed on gestation day (gd) 1 to 5 of pregnancy to assess fertilization and preimplantation embryo development and enumerate early implantation sites. Chromosome analysis in unfertilized oocytes revealed an MBC-induced increase in aneuploidy (37 vs. 14% in controls). When animals were bred after dosing, MBC had no effect on the number of oocytes recovered or fertilized. However, significant increases were found in the proportion of embryos that failed to reach the expected stage of development, namely, the eight-cell stage on the afternoon of gd 3, the morula stage by the morning of gd 4, and the blastocyst stage by the afternoon of gd 4 (a time when some embryos have implanted). The mean number of implantation sites, revealed by Evans Blue staining, was also significantly lower in treated females on the afternoon of gd 4 and the morning of gd 5. These simple direct assessments elucidated a mechanism of MBC-induced early pregnancy loss, induction of aneuploidy in oocytes. They also ruled out an effect on fertilization, but demonstrated a subsequent arrest of preimplantation embryonic development accompained by a decrease in the likelihood of implantation.

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report. ILSI Risk Science Institute Expert Working Group on Sperm Evaluation.

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Dibromoacetic acid affects reproductive competence and sperm quality in the male rat.

Recently, we demonstrated with short-duration tests that dibromoacetic acid (DBAA), a commonly occurring by-product of water disinfection, alters sperm morphology and motility in the male rat. These results suggested that the effects of DBAA on sperm quality were likely to compromise reproductive competence of the male rat early in subchronic exposure. The present studies were undertaken to investigate the dose response and time course of alterations in fertility and sperm quality. Proven breeder male rats were gavaged daily with 0, 2, 10, 50, or 250 mg DBAA/kg for up to 79 days; interim and terminal measurements of sperm quality and reproductive outcome were made. Because of the known neurotoxicity of the analogue, dichloroacetic acid, both natural breeding and artificial inseminations were evaluated in untreated females to distinguish between possible behavioral and spermatogenic effects. DBAA compromised male fertility during the second treatment week in naturally bred rats dosed with 250 mg/kg. The early antifertility effect appeared to be the result of behavioral changes since females artificially inseminated with sperm collected on Day 9 successfully produced offspring. However, sperm morphology and motility also were rapidly affected by DBAA treatment so that no offspring via natural insemination and only one litter via artificial insemination were produced subsequent to Day 15. Through 31 days, substantial effects on sperm motility, sperm morphology, and epididymal sperm numbers were observed, but there was no demonstrable effect on serum testosterone or sperm production. Because severe toxicity developed in the group given 250 mg/kg, exposure of these animals was prematurely terminated after 42 doses and their recovery was monitored through a 6-month posttreatment period; decreased testis weights and only limited recovery of reproductive performance were observed. Exposure to 50 mg/kg resulted in moderate changes in sperm morphology and motility and moderate decreases in epididymal sperm counts in rats dosed for 31 or 79 days. However, these males remained fertile, litter size was unaffected, and no paternally mediated developmental defects were noted in their offspring. No effects on sperm quality were detected at dosages of 2 or 10 mg/kg. However, compared to controls, naturally bred DBAA-treated rats tended to have fewer inseminations, fewer copulatory plugs, and fewer multiple litters, suggesting that DBAA may have altered mating behavior at dosages as low as 10 mg/kg.