Fertilization success and sexual behavior in male medaka, Oryzias latipes, exposed to tributyltin

We evaluated the effects of tributyltin (TBT), polychlorinated biphenyls (PCBs), and a mixture of TBT and PCBs on reproduction, gonadal histology, and sexual behavior in Japanese medaka (Oryzias latipes). We administered TBT (1 microg g(-1) body weight [b.w.] daily), PCBs (1 microg g(-1) b.w. daily), or both to medaka for 3 weeks. We assessed reproductive success during week 3 and the sexual behavior of male medaka after the exposure period. Fertilization success was significantly decreased in groups that received TBT. The profiles of fertility developed a bimodal distribution in the TBT and TBT+PCBs groups, and the numbers of males that performed "following" and "dancing" were significantly decreased in the TBT+PCBs group. The frequency of "dancing" also decreased after treatment with TBT, whereas fish that received PCBs only showed no difference in sexual behavior. Testes that had oocytes showed normal spermatogenesis, even though two of the five males treated with TBT+PCBs had some indication of testis-ova, which did not occur in any other group. Our results indicate that TBT, but not PCBs, affects sexual behavior and reproduction in medaka.     

Reproduction of mallards following overwinter exposure to selenium

Forty pairs of mallards (Anas platyrhynchos) were fed 15 ppm selenium as selenomethionine for about 21 weeks during winter. Twenty pairs served as controls. At the end of 21 weeks, which coincided with the onset of the reproductive season, selenium treatment was ended. Four birds died while on selenium treatment. Treated females lost weight, and their egg-laying was delayed. Hatching success of some of the first eggs laid by selenium-treated females was lower than that of controls, and a few of these early eggs contained deformed embryos, but, after a period of about two weeks off the selenium-treated diet, reproductive success returned to a level comparable with that of controls. The return to normal reproductive success was the result of a corresponding decrease in selenium concentrations in eggs once selenium treatment ended.     

Dietary exposure to technical hexabromocyclododecane (HBCD) alters courtship, incubation and parental behaviors in American kestrels (Falco sparverius)

Hexabromocyclododecane (HBCD) is a high production volume brominated flame retardant that has been detected in the environment and wildlife at increasing concentrations. This study was designed to determine potential effects of dietary exposure to environmentally relevant levels of HBCD on behavior during reproduction in captive American kestrels. Twenty kestrel pairs were exposed to 0.51 μg technical HBCD g⁻¹ kestrel d⁻¹ from 4 weeks prior to pairing until chicks hatched (∼75 d). Ten pairs of controls received the safflower oil vehicle only and were used for comparison. During the courtship period the chitter-calls were reduced in both sexes (p = 0.038) and females performed fewer bonding displays (p = 0.053). Both sexes showed a propensity to be less active than controls during courtship. The reduction in male courtship behavior was correlated with reduced courtship behaviors of females (p ? 0.008) as well as reduced egg mass (p = 0.019). During incubation, nest temperatures of treatment pairs were lower at mid-incubation (p = 0.038). HBCD-exposed males performed fewer key parental behaviors when rearing nestlings, including entering the nest-box, pair-bonding displays and food-retrievals. HBCD-exposed females appeared to compensate for the reduced parental behavior of their mates by performing these same behaviors more frequently than controls (p = 0.004, p = 0.027, p = 0.025, respectively). This study demonstrates that HBCD affects breeding behavior in American kestrels throughout the reproductive season and behavioral alterations were linked to reproductive changes (egg size). This is the first study to report HBCD effects on reproductive behavior in any animal model.     

Biomarker responses and reproductive toxicity of the effluent from a Chinese large sewage treatment plant in Japanese medaka (Oryzias latipes)

The present study was conducted to assess the potential toxicity of the effluent from a large sewage treatment plant (GBD-STP) in Beijing. Japanese medakas (Oryzias latipes) at reproduction active period were exposed to a serial of graded concentrations of the effluent or 100 ng l-1 of 17-alpha-ethinylestradiol (EE2, positive control). Growth, gonadosomatic index (GSI), hepatosomatic index (HSI), reproductive success, induction potency of vitellogenin (VTG) in male fish and that of 7-ethoxyresorufin-o-deethylase activity (EROD) in male fish liver were used as test endpoints. The growth suppression of fish was observed in a dose-dependent manner, resulting in significant differences in both body length and body weight of medaka above 5% effluent. This effluent can inhibit the growth of gonad of medakas and are more sensitive to male than to female. At exposure concentration of 40% and higher, there was an unexpected decrease of HSI values, which may be resulted from sub-lethal toxicity of effluent to fish liver. VTG of plasma in males were induced in all exposure concentration levels, but not in a dose-dependent manner. The concentration of 5% effluent would be the lowest observed adverse effect level (LOAEL) affecting reproductive success when examining fertile individuals, fecundity and fertilization rate. The overt CYP1A response and higher reproductive toxicity may be indicative of low process efficiency of this STP.     

Effects of chlorpyrifos on reproductive performances of guppy (Poecilia reticulata)

Guppy (Poecilia reticulata) was selected to investigate the effects of chlorpyrifos on reproductive performances. Male and female guppy with proven fertility were selected from our own colony and the groups of fish (n=72/group) were exposed to pre-determined chlorpyrifos concentrations (0.002 microg/l, 2 microg/l) based on the 96-h LC50 for guppy. Mating behavior of males was recorded on the 2nd day of exposure. Offspring were counted and survival recorded on the 14th day. Gonopodial thrusts (8/15 min) in 0.002 microg/l and (4/15 min) in 2 microg/l were significantly different from the control group (11/15 min). Similarly, live birth reduced significantly to 8/female in 2 microg/l compared to 27/female in the control group. Survival of offspring after 14 days was reduced to 47% in the 2 microg/l group compared to 94% of survival in the control. Our findings demonstrate that low soluble concentrations of chlorpyrifos affect mating behavior, number of offspring and offspring survival of guppy.     

Reproductive toxicity of trenbolone acetate in embryonically exposed Japanese quail

This study was conducted to assess the effects of a one time embryonic exposure to trenbolone acetate on reproductive development and function in Japanese quail (Coturnix japonica). Embryos were exposed to either 0.05, 0.5, 5, or 50microg trenbolone or a sesame oil vehicle control at embryonic day 4. Onset of puberty, gonadal histopathology, sperm motility, cloacal gland size, and male copulatory behavior were assessed in adults. Trenbolone delayed onset of puberty in males, inhibited cloacal gland development, and reduced male reproductive behaviors. Industry laboratories have shown trenbolone acetate to be non-teratogenic in mammalian studies. Our study, however, shows that this one time in ovo exposure delayed onset of puberty in and suppressed adult copulatory behavior in quail males. These results suggest that this one time embryonic exposure to trenbolone may have disrupted development of either the central nervous system or the hypothalamic-pituitary-gonadal axis. This is the first study to demonstrate a demasculinizing effect on copulatory behavior in Japanese quail from embryonic exposure to a non-aromatizable androgenic chemical. More studies are needed to determine the mechanisms behind the observed effects.     

Effects of environmentally relevant concentrations of the xeno-androgen, methyldihydrotestosterone, on male and female mating behavior in Xenopus laevis

Endocrine disrupting compounds (EDCs) are well known to interfere with the hormone system of aquatic vertebrates and to affect their reproductive biology. 17α-Methyldihydrotestosterone (MDHT) is a widely used model compound for the assessment of androgenic EDCs, because it binds with high affinity to nuclear androgen receptors. It was previously shown to affect various aspects of reproductive biology in aquatic vertebrates, however, evidence for MDHT affecting mating behavior of aquatic vertebrate species is lacking. In order to test the assumption that MDHT affects reproductive behavior of aquatic vertebrates, we exposed male and female Xenopuslaevis to three environmentally relevant concentrations of MDHT (30.45 ng L⁻¹, 3.05 μg L⁻¹ and 30.45 μg L⁻¹). In males, MDHT at all concentrations led to enhanced levels of advertisement calling and decreased the relative proportions of rasping, a call type characterizing a sexually unaroused state of the male, indicating an increase in sexual arousal of MDHT exposed males. Temporal and spectral parameters of the advertisement call itself, however, were not affected by MDHT exposure. In females, MDHT (30.45 ng L⁻¹) did not have any effects, while MDHT at 3.05 μg L⁻¹ increased female receptivity, increased the duration of time females spent close to the speaker playing male advertisement calls and reduced their latency to respond. MDHT at 30.45 μg L⁻¹, on the other hand, decreased female receptivity and increased their latency to respond. In summary, this study illustrates that exposure to environmentally relevant concentrations of the androgenic EDC MDHT affects male and female mating behavior of X. laevis. Hence, we suggest that nonaromatizable androgens might play a direct and predominant role in the physiology and regulation of reproduction not only in male but also in female frogs.     

Prenatal and postnatal exposure to diazinon and its effect on spermatogram and pituitary gonadal hormones in male offspring of rats at puberty and adulthood

The objective of this research is to study the possible reproductive adverse effects of diazinon on rat offspring exposed in utero and during lactation. Twenty-four Sprague–Dawley female rats (10–12 week old) were randomly assigned to four groups, each consisting of six rats. Group 1 served as the control and these rats were given normal saline orally. Rats in groups 2, 3, and 4 were administered diazinon, dissolved in saline at 10, 15, 30 mg/ kg^?1 body weight, per oral, once daily, during mating, pregnancy and lactation. The male offsprings were examined at puberty and adulthood for body weight, testis weight, epididymis weight, sperm count, motility and morphology, pituitary-gonadal hormone levels. At 30 mg kg^?1 dose, the male offsprings showed a decrease in testicular weight, sperm count, motility, with an increase in abnormal sperm percentage and a decline in pituitary-gonadal hormones, at puberty. Upon attaining adulthood, there was a decrease in testicular weight, sperm count and motility with an increase in abnormal sperm percentage and a decrease in pituitary hormone level. There was evidence of some adverse reproductive effects on the male offspring at the 15 mg/ kg^?1 dose. Most of the adverse effects were irreversible and were evident at both puberty and adulthood in the offsprings, although a few parameters reverted to the normal growth pattern. Diazinon is a reproductive toxicant for male offsprings if exposed during prenatal and postnatal phases.     

Effect of 17beta-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Estrogenic compounds such as 17beta-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis-ova. Males treated with E2 concentrations = 55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis-ova in male medaka exposed to environmentally relevant concentrations of E2.     

Effects of tributyltin maternal and/or waterborne exposure on the embryonic development of the Manila clam, Ruditapes philippinarum

We examined the effect of tributyltin (TBT) on embryonic development of the Manila clam, Ruditapes philippinarum. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, 0.310, or 0.350 microg/l at 20-22 degrees C for 3 weeks, and the embryo developmental success (the ratio of normal D-larvae to all larvae) was measured. There was a significant negative correlation between embryo developmental success and TBT concentration in the female Manila clams (p < 0.001). These results indicated that TBT accumulated in the female clam decreased embryo developmental success. In a waterborne exposure test, fertilized eggs (4 h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.062, 0.140, 0.320, or 0.640 microg/l for 23 h. Embryo developmental success was also significantly decreased in all TBT treatment groups compared with that in the control group. TBT accumulated in female adults and waterborne TBT clearly inhibit reproductive success of the clam.     

Suppression of aromatase activity in populations of bream (Abramis brama) from the river Elbe, Germany

Aromatase activity was determined in brain and gonads of wild bream collected along the river Elbe, Germany, and correlated with other endocrine and reproductive endpoints such as plasma sex steroid concentrations, secondary sex characteristics (STI), plasma vitellogenin, gonad size (GSI), and maturation stages of germ cells (MS) that were reported for the same fish in a previous study. Furthermore, regional patterns of aromatase activity were correlated to a number of environmental factors such as exposure to environmental contaminants and parasitism. While aromatase activity was not detectable in the gonads of male and female fish with the assay used, fish of both genders revealed relatively great brain enzyme activities. As for most of the endocrine and reproductive parameters, with the exception of plasma testosterone (T), aromatase activities were significantly less in fish from a river stretch characterized by elevated exposures to organic contaminants and metals. Brain aromatase activity was positively and significantly correlated with plasma estradiol (E2) and MS in females, and showed a similar trend with plasma 11-ketotestosterone (11KT) and STI in males. No comparable trend occurred for T. This decrease of the reproductively relevant hormones 11KT and E2 may be indicative of a disruption of the last step in sex hormone synthesis, a hypothesis that was supported for E2 by the strong (R2=0.78, p<0.05) linear regression between aromatase activity and E2 in female bream. It is also hypothesized that the effects on brain aromatase activity were likely to be related to the disruption of other reproductive parameters including sexual maturity and expression of secondary sex characteristics. Although a number of factors such as exposure to pollutants and prevalence of the tapeworm Ligula intestinalis correlated with the suppression of aromatase activity, the exact causes for the regional decrease in brain aromatase activity remain unclear due to inconsistencies of these correlations between sampling events or gender.     

Chronic effects of Cd on reproduction of Polypedilum nubifer (Chironomidae) through water and food

Chronic effects of Cd on reproduction of Polypedilum nubifer (Chironomidae) were investigated by Cd-exposure from the egg stage using a flow-through aquarium. No significant effect of Cd on reproduction was observed in the midge larvae which had been exposed to 10 or 20 microg Cd litre(-1). Emergence of the Cd-exposed larvae peaked several days before that of the control, although growth was impaired in 1st and/or 2nd instars. The percentage emergence success decreased to about 46% of the control at 40 microg Cd litre(-1). However, other reproductive processes (adult sex ratio, oviposition success and egg hatchability) were not impaired. The emergence success decreased to less than 3% of the control at 80 microg Cd litre(-1). The emergence success of midge larvae which had been fed Cd-contaminated food with 220 or 1800 microg Cd g(-1) decreased to nearly 60% of the control. However, other reproductive processes were not impaired. Emergence success of larvae exposed to food containing 22 microg Cd g(-1) was unaffected.     

Reproduction, larval growth, and reproductive development in African clawed frogs (Xenopus laevis) exposed to atrazine

Reproductive success and development of F2 offspring from F1 adult African clawed frogs (Xenopus laevis) exposed to atrazine throughout larval development and as sexually mature adults was examined. Larval X. laevis were exposed to one of four nominal concentrations of atrazine (0, 1, 10, 25 microg atrazine/l) beginning 96 hr after fertilization and continuing through two years post-metamorphosis. Clutch size and survival of offspring were used as measurement endpoints to gauge reproductive success of the F1 frogs. Larval survivorship and time to metamorphosis were used to gauge developmental success of the F2 offspring from atrazine-exposed frogs. Testes in F1 and F2 frogs were examined for incidence of anomalies, such as testicular ovarian follicles, and sex ratios in F2 offspring were investigated to determine if exposure to atrazine caused trans-generational effects (effects on F2 individuals due to exposure of F1 individuals). There were no effects of any of the studied concentrations of atrazine on clutch size of F1 frogs. There were also no effects on hatching success or time to metamorphosis. Sex ratios did not differ between F2 offspring among treatments. There was no evidence to suggest a transgenerational effect of atrazine on spawning success or reproductive development of X. laevis. This is consistent with the presence of robust populations of X. laevis in areas where they are exposed to atrazine that has been used for several decades for weed control in production of corn. Our observations also are consistent with the results of most other studies of frogs where no effects were found to be associated with exposure to atrazine. Our data do not support the hypothesis that atrazine significantly affects reproductive fitness and development of frogs.     

Evaluation of reproductive safety of beta-sitosterol on the American mink (Neovison vison)

beta-Sitosterol is a weakly estrogenic phytosterol used extensively in functional foods to lower elevated serum cholesterol concentrations due to its inhibitory action on intestinal cholesterol absorption. It caused previously decreased sex steroid concentrations in fish and lowered sperm counts in rats. In the American mink (Neovison vison), litter size increased slightly due to dietary beta-sitosterol supplement. The aim of the present experiment was to conduct a dose-response study on the effects of beta-sitosterol on the reproduction of the American mink. Juvenile male and female mink (n=480) were exposed to 0, 5, 10 or 50 mg of peroral beta-sitosterol kg(-1)d(-1) for 10 months. After 3 months of exposure in November, 15 males per group were sacrificed and general biochemical variables reflecting overall health were determined. The beta-sitosterol-treated male mink had increased absolute and relative masses of intraabdominal fat and higher blood hemoglobin and serum high-density lipoprotein cholesterol concentrations. In spring, the top-rated male mink were mated with multiple females within each study group and reproductive success was assessed. No differences in the reproductive performance of the males (10-11 per group) or females (47-50 per group) could be detected in the exposed groups and the kits of all groups developed in a similar manner. The results suggest that dietary beta-sitosterol presents no significant risk to mammalian fertility.     

Development of a sandwich enzyme-linked immunosorbant assay for the quantification of vitellogeinin in Bullfrog (Rana catesbeiana)

A sandwich enzyme-linked immunosorbant assay (ELISA) was developed to quantitatively detect Bullfrog (Rana catesbeiana) vitellogenin (VTG) levels. This procedure involved a sandwich ELISA using monoclonal antibodies of BVmA1 and BVmA2 against Bullfrog-VTG, and BVmA1 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 9.4 ng/ml and 1200 ng/ml and the recovery of the VTG added to Bullfrog control male serum was 92.0-108.8%. Male Bullfrog was induced by injection 17beta-estradiol (E2) for four weeks and Bullfrog-VTG levels were measured each week. Histological analysis was performed for investigating the correlation of the effect to male reproduction and Bullfrog-VTG level variation depending on E2 dose. After two weeks of E2 exposure, the induced Bullfrog-VTG level was significantly higher than Bullfrog control female (p<0.05). After four weeks of E2 exposure, the rupture and fusion of seminiferous tubule in the testes of male Bullfrog were shown and provided direct evidence that the reproduction of male Bullfrog was affected by estrogenic compounds. Bullfrog-VTG bioassay, using the sandwich ELISA, could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.     

Tributyltin causes abnormal development in embryos of medaka, Oryzias latipes

We examined the effects of tributyltin (TBT) on embryonic development, hatching success and sexual differentiation in Japanese medaka (Oryzias latipes). Embryos (within 8h after fertilization) were exposed to TBT in ovo via nanoinjection at concentrations of 0 (control), 0.16, 0.80, 3.96, 19.2 and 82.1 ng/egg. Embryonic survival, development and hatching were observed. Hatched fry were reared until 60 days when they sexually matured, and sexual differentiation was also examined by accordance of genetic and phenotypic sex, based on existence of DMY (a male determining gene in medaka) and secondary sex characteristics. As results, TBT caused a concentration-dependent mortality and impaired the embryonic development. However, no masculinization was detected at 60 dph medaka adults. Lowest observed effective concentration for inducing abnormal embryonic development was estimated to 0.16 ng/egg (ca. 160 ng/g egg).     

Influence of a diet of fluoride-fed cockerels on reproductive performance of captive American kestrels

In 1982, twenty-four pairs of captive American kestrels (Falco sparverius) were forced to renest by removal of their first clutches 6 days after their completion. Immediately following, each of three groups of eight pairs was randomly assigned to one of three daily dietary regimes for 10 days: (1) three 1-day old cockerels with background levels of F(-) (62.4+/-51ppm, mean+/-SD) in their femurae, (2) two 10-day old cockerels with 4512+/-810ppm of F(-) in their femurae, (3) two 10-day old cockerels with 7690+/-417ppm of F(-) in their femurae. Fluoride levels in femurae of treated kestrels were significantly (P<0.0025) higher than those of control birds. Clutch sizes tended to be smaller as more fluoride was added to the diet, but not significantly so, due to an increase of the variance in the treatment group. Per cent fertility and per cent hatchability were not significantly affected by treatment. The fluoride content in eggshells in the fluoride-treated groups differed significantly from those of the control group (P<0.001).     

Effects of in utero di-butyl phthalate and butyl benzyl phthalate exposure on offspring development and male reproduction of rat

The study was conducted to assess the effects of in utero di-butyl phthalate (DBP) and butyl benzyl phthalate (BBP) exposure during late gestation on offspring’s development and reproductive system of male rats. Pregnant rats were treated orally with DBP (2, 10, 50 mg/kg), BBP (4, 20, 100 mg/kg), and diethylstilbestrol (DES) 6 μg/kg (positive control) from GD14 to parturition. A significant reduction in dams’ body weight on GD21 in DBP-, BBP-, and DES-treated groups was observed. The gestation length was considerably elevated in the treated groups. Decline in male pups’ body weight was significant at PND75 in DBP- (50 mg/kg), BBP- (20,100 mg/kg), and DES-treated groups. The weight of most of the reproductive organs and sperm quality parameters was impaired significantly in DBP- (50 mg/kg) and BBP- (100 mg/kg) treated groups. Further, a non-significant decline in testicular spermatid count and daily sperm production was also monitored in treated groups. A significant reduction in serum testosterone level in BBP (100 mg/kg), whereas the testicular activity of 17β-HSD was declined non-significantly in the treated groups with respect to control. The data suggests that DBP and BBP exposure during late gestation period might have adverse effects on offspring’s development, spermatogenesis, and steroidogenesis in adult rats.     

Assessing chronic toxicity of bisphenol A to larvae of the African clawed frog (Xenopus laevis) in a flow-through exposure system

A number of currently used industrial chemicals are estrogenic, and therefore have potential to disrupt sexual differentiation in vertebrate wildlife during critical developmental windows. We assessed the effect of larval exposure to bisphenol A (BPA) on growth, development and sexual differentiation of the gonad in the African Clawed frog, Xenopus laevis. Larvae were maintained in flow-through conditions at 22 +/- 1 degrees C and exposed to BPA at mean measured concentrations of 0.83, 2.1, 9.5, 23.8, 100, and 497 microg/l, from developmental stages 43/45-66 (completion of metamorphosis). Each test concentration, plus dilution water control (DWC) and positive control (17beta-estradiol (E2), 2.7 microg/l) employed four replicate test vessels with 40 larvae per tank. Individual froglets were removed from test vessels upon reaching stage 66, and the study was terminated at 90 days. Froglets were dissected and sex was determined by inspection of gross gonadal morphology. Test concentrations of BPA had no effect on survival, growth, developmental stage distributions at exposure days 32 and 62, or mean time to completion of metamorphosis, compared to DWC. Analysis of post-metamorphic sex ratio, determined by gross gonadal morphology, indicated no significant deviations from expected (50:50) sex ratio, in DWC or any BPA test concentration. In contrast, exposure of larvae to (E2) resulted in feminisation, with sex ratio deviating significantly (31% male, replicates pooled). Exposure to BPA in the concentration range 0.83-497 microg/l in flow-through conditions had no observable effect on larval growth, development or sexual differentiation (as determined by gross gonadal morphology) in this study.     

Detection of lead-induced oxidative stress in the rat epididymis by chemiluminescence

It has been shown that lead (Pb) is able to induce lipid peroxidation, one of the main manifestations of oxidative stress. In this study we examined the relationship between chronic Pb exposure and level of reactive oxygen species (ROS) in reproductive system tissues of sexually mature male Wistar rats. One group of animals (control, K) was allowed to drink distilled water, the second group (Pb) was allowed to drink freely 1% aqueous solution of lead acetate. Another groups had a following supplements: rats were allowed to drink distilled water containing vitamin C (vit C) at concentration of 500 mg/l or Trolox (a vitamin E analog) at concentration of 48 mg/l or vit C (500 mg/l) + Trolox (48 mg/l). The similar groups among Pb-treated animals were examined after treatment with the same vitamins and using the same vitamin doses, dissolved in 1% aqueous solution of lead acetate. In all cases the time of drinking was 6 months. It was found that lead content in samples of tissues from testis, epididymis and in a whole blood in Pb- and Pb with antioxidants treated rats was significantly elevated. Chemiluminescence (CL) emitted by the Pb-treated tissues was significantly higher when compared to the light emission by tissues isolated from the animals of control group. The increase in the CL caused by lead occurs in the following increasing order within the studied tissues: cauda of epididymis < testis < caput of epididymis (19%, 39% and 51%, respectively). Dietary vit C supplementation to the Pb-treated rats for 6 months period decreased the CL from caput of epididymis, cauda of epididymis and testis (by 43%, 24%, 39%, respectively) more effectively in comparison to the control group (35%, 17%, 33%, respectively). Also stronger quenching effect on the light emission from the above mentioned tissues after Trolox supplementation was observed in the Pb-treated group (42%, 21%, 35%, respectively) than in the control group (23%, 13%, 13% respectively). The combination of both antioxidants treatments (vit C and Trolox) did not give a higher significant quenching effect compared to the treatment with the vitamins separately. No ultrastructural changes were found in the seminiferous epithelium of Pb-treated animals. However, we found abnormalities in ultrastructure of epididymal epithelial cells and epididymal spermatozoa in rats of Pb-treated groups. These findings provide ex vivo evidence that Pb causes oxidative cellular damage in reproductive system tissues of adult male rats, which may be closely associated with the ROS production.