Application of computer-assisted sperm analysis system to elucidate lack of effects of cyclophosphamide on rat epididymal sperm motion.

A computer-assisted sperm analysis (CASA) system was used to examine the motion of epididymal spermatozoa derived from cyclophosphamide (CP)-treated male rats. Male rats were orally dosed daily for 1 week with 20 mg/kg of CP. Males were euthanized or were mated 3 times with untreated females at 1 day, 3 weeks, and 8 weeks after the final treatment. Significant decreases in testicular and epididymal weights and epididymal sperm counts of the treated animals were noted after 8-week recovery. Histopathological morphometry of the testis revealed minimal damage to spermatogonia at 1 day after the final treatment and to spermatocytes after 3-week recovery in the CP-treated group. On Caesarian section, increased post-implantation losses were found in females mated with CP-treated males in matings starting 1 day and 3 weeks after the final treatment. On the other hand, none of the sperm motion parameters of treated males derived from the CASA system exhibited significant changes at any time points, although the spermatozoa of treated males at 1 day and 3 weeks after the final treatment were damaged at the DNA level, and the spermatozoa of males after 8-week recovery had been the target cells of CP when they were spermatogonia in the testis. It was thus found that damaged spermatozoa could exhibit no changes on their motion when the damage was confined to the nuclei, and that the effect of CP on sperm nuclei was reversible.     

Effects of alpha-chlorohydrin on rat sperm motions in relation to male reproductive functions

alpha-chlorohydrin (ACH) is a known male reproductive toxicant and produces antifertility in rats. The present experiments were performed to determine the relationship between sperm motions and reproductive function, and to further examine the possible mechanism for antifertility. ACH was administered to male rats for 9 days at 1, 3 and 10 mg/kg/day. The males were mated with untreated females and their reproductive status was determined. All mated males failed to impregnate females at 10 mg/kg/day. Low pregnancy rate associated with a decreased implant number was seen at 3 mg/kg/day. When sperm motions were analyzed using the CellSoft computer-assisted sperm analyzer, percentage of motile sperm, curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were reduced at 10 mg/kg/day. At 3 mg/kg/day, VCL and ALH were reduced but the percentage of motile sperm was comparable to that of controls. In order to examine a possible mechanism for the effect of ACH on fertility, the number of sperm reaching the oviducts of mated females and the number of fertilized eggs was evaluated. Half of the females mated with ACH-treated males at 3 mg/kg/day had very low sperm numbers in the oviducts. At 10 mg/kg/day, all the mated females had a very low sperm number. The percent of fertilized eggs in the oviducts of mated females was decreased in a dose-dependent manner. These findings suggest that the effect of ACH on fertility was directly related to decreased VCL and ALH as well as percentage of motile sperm, and by the mechanism in which the sperm number reaching the oviducts after mating was reduced, so the reduction resulted in only a rare chance to fertilize.     

Reproductive Toxicology of Aluminum in Male Mice

The reproductive toxicology of aluminum was studied in mice.Adult male mice were treated intraperitoneally with aluminumnitrate at doses of 0, 50, 100, and 200 mg/kg/day for 4 weeksbefore mating with untreated females. Decreased body weightwas seen in all aluminum-treated groups. Decreased pregnancyrate was observed in the females mated with males previouslytreated with 100 or 200 mg/kg/day of aluminum nitrate. High-dosemale mice showed significantly decreased testicular and epididymalweights, as well as significant decreases in testicular andspermatid counts and epididymal sperm counts. Spermatid countswere also reduced at 100 mg/kg/day. However, the sperm motilitywas unaffected, and the percentages of morphological normalspermatozoa in all mice exposed to aluminum were comparableto the values in control mice. Histological changes, includingnecrosis of spermatocytes/spermatids, were observed in the testesof male mice treated with 100 and 200 mg/kg/day of aluminumnitrate, whereas the tubu lar diameters were unaffected by aluminumadministration. The current study demonstrates adverse effectsof parenteral aluminum exposure on the mouse male reproductivesystem. The "no observable adverse effect level" (NOAEL) was50 mg/kg/day.     

Germ cell mutagenicity of gamma-ethyl-gamma-phenyl-butyrolactone (EPBL) detected in the CF1 mouse-dominant lethal study

Female and male CFI mice weighing 25-30 g were given 0, 50, 100 or 200 mg/kg of gamma-ethyl-gamma-phenyl-butyrolactone (EPBL) for 5 days intraperitoneally. In the male-dominant lethal phase, males treated with EPBL were mated with untreated females following a 7-day mating schedule with three consecutive mating events. In the female-dominant lethal phase, females treated with EPBL were caged with untreated males. The above dosages and schedule treatments were used. The incidence of pregnancy of females mated on days 1-7 and 8-14 after males were given 200 mg/kg of EPBL and of females given 200 mg/kg when mated to untreated males was decreased. Upon examining surgically exposed uteri and ovaries of pregnant females during the first phase, on gestation days 13-15, an increased incidence of pre-implantation losses with 200 mg/kg of EPBL and an increased incidence of post-implantation losses with 100 and 200 mg/kg was observed. In addition, an increased frequency of pre- and post-implantation losses was seen in females treated with 200 mg/kg. These results support the conclusion that EPBL is a germ cell mutagen and its effects are more pronounced during the post-meiotic stage.     

Effects of valproic acid on fertility and reproductive organs in male rats

Crj:CD(SD)IGS rats were orally administered valproic acid at doses of 250, 500 or 1000 mg/kg/day for 4, 7 or 10 weeks. At each dose, one group of male rats was euthanized after 4-week dosage (4-week dose group) and the other two were mated with untreated females after 4 (7-week dose group) or 7 (10-week dose group) weeks of treatment with valproic acid and their fertility was evaluated. Females were euthanized on day 14-17 of gestation, and numbers of corpora lutea, implantations and live and dead fetuses were recorded. After 4, 7 or 10 weeks of treatment, males were euthanized, genital organs were weighed, the number of sperm in the cauda epididymis was counted, sperm motion analyzed, and histopathological examination of testes performed. The male rats of the 1000 mg/kg dose group died or were moribund 3 or 4 days after the start of treatment. No effects on fertility of male rats were observed up to the 500 mg/kg 10-week dose group. Treatment for 4 weeks at 500 mg/kg/day decreased epididymis weight. After 7 weeks at 500 mg/kg/day, the weights of epididymis, seminal vesicles and prostate were decreased, and the number of sperm heads per cauda epididymis and percentage of motile sperm were reduced. In the 500 mg/kg 10-week dose group, the weight of testis was decreased. On histopathological examination of the testis, degeneration of seminiferous tubules and loss or exfoliation of spermatids were observed, and the ratio of retention of step 19 spermatids in stage IX-XI was increased in the 500 mg/kg 4-, 7- and 10-week dose groups. These results suggest that analysis of sperm motion and histopathological evaluation of testes are sensitive methods for assessing toxicity of valproic acid on male reproductive organs.     

Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies

Adriamycin (ADR), an anticancer drug which induces testicular toxicity, was administrated to Slc:SD male rats at doses of 0.5, 1.0, and 2.0 mg/kg intravenously once a week for 4 weeks. The males treated with ADR were mated with untreated females, and sperm analyses (motion, count, and morphology) were performed. Sperm motion was analyzed by Hamilton-Thorne Sperm analyzer (HTM-IVOS) to investigate the useful parameters. Copulated females were necropsied at Day 13 of gestation, and reproductive status was evaluated. In ADR-treated groups, the testicular weight was dose-dependently decreased. Associated with this decrease was a depletion of the number of spermatogonia noted histopathologically at all dosage levels. Sperm morphological abnormalities, which were classified as tailless sperm and/or no-hook head sperm, were increased in both the 1.0 and 2.0 mg/kg groups. The males treated with ADR at 1.0 and 2.0 mg/kg had a decreased number of sperms per cauda epididymis. In sperm motion analysis, decreases in the percentage of motile sperm, percentage of progressive sperm, and sperm velocity (straight line velocity and curvilinear velocity) were noted at 2.0 mg/kg. Impaired fertility was noted at 2.0 mg/kg in the form of decreased numbers of implantations and live embryos, and an increased number of pre-implantation losses. In conclusion, ADR induced deterioration of sperm motion and sperm content, which were responsible for the adverse effect on male fertility. The most sensitive indicators to detect male reproductive toxicity induced by ADR were testicular weight and histopathological findings in the testis. Among the parameters generated by HTM-IVOS, the percentage of motile sperm, the percentage of progressive sperm, and sperm velocity are useful for assessing male fertility.     

Reproductive toxicity evaluation of vanadium in male mice

The reproductive toxicity of vanadium was studied in mice. Male Swiss mice were exposed to sodium metavanadate at doses of 0, 20, 40, 60, and 80 mg/kg per day given in the drinking water for 64 days. To evaluate the fertility of the vanadium-treated animals, males were mated with untreated females for 4 days. A significant decrease in the pregnancy rate was observed at 60 and 80 mg/kg per day of sodium metavanadate. However, metavanadate did not reduce fertility in male mt 20 and 40 mg/kg per day. Reproductive toxicity was measured by sperm count, sperm motility, organ weights, and histologic evaluation of the testes. Decreased body and epididymis weight was only observed in the 80 mg/kg per day group, while testicular weights were not altered by the treatment with all doses used. Sperm coung was significantly decreased at 40, 60, and 80 mg/kg per day, but the sperm motility was unaffected. Histopathological examination revealed that the testes were normal and that the epididymis of treated male mice contained normal appearing sperm. The no observed adverse effect level (NOAEL) was 40 mg/kg per day. Consequently, vanadium would not cause any adverse effect on fertility or testicular function in male mice at the concentrations usually ingested by humansthrough the diet and drinking water.     

Effects of an agent inducing dominant lethals on rat sperm--examination with ethyl methanesulfonate

Ethyl methanesulfonate (EMS), an alkylating agent which induces dominant lethals, was administered in oral doses of 100 mg/kg to Crj:CD(SD)IGS male rats for 5 consecutive days. At the termination of treatment and after a 28-day withdrawal, mating with untreated females and sperm analysis (motion, number, and morphology) were performed. The copulated females were sacrificed at 20 days of gestation. At the termination of treatment, no clinical signs related to EMS were observed except for a decrease in body weight. Gross pathology and sperm analysis revealed no abnormalities in treated males. However, females mated at the termination of treatment had a clearly higher fetal mortality. Females mated after the 28-day withdrawal exhibited lower fetal mortality than females mated at the termination of treatment. On the other hand, females mated after the 28-day withdrawal exhibited a lower implantation rate that was not observed in females mated at the termination of treatment. For males after a 28-day withdrawal, sperm analysis revealed both a decrease in sperm motion and number and an increase in morphological change. These findings indicate that two types of male reproductive toxicity induced by EMS can be distinguished. One induces a low implantation rate that can be detected by sperm analysis, while the other induces fetal lethals that could not be detected by sperm analysis in this study.     

Atropine-induced inhibition of sperm and semen transport impairs fertility in male rats

Previous studies revealed that atropine reduced male fertility in rats without any effects on mating performance, sperm production and motility, and testicular morphology. The present study was conducted to investigate whether the impairment of male fertility induced by atropine was related to the inhibition of sperm and semen transports from the vas deferens and seminal vesicle to the urethra during the process of emission. Male rats were treated with atropine at 125 mg/kg/day for 10-17 days prior to mating with untreated females. After confirmation of mating, male rats were euthanized and sperm number in the vas deferens and weights of the seminal vesicle and copulatory plug were determined as indicators of inhibition of sperm and semen transports, respectively. Reproductive status of mated females was determined on gestation days 15-17. A low pregnancy rate associated with a decreased number of implants was observed in females that mated with the atropine-treated males. The average number of sperm in the vas deferens was increased in the atropine-treated males. The average seminal vesicle weight in the atropine-treated males was greater than that of controls. The copulatory plug weights were decreased in the atropine-treated males. These results suggest that inhibitions of sperm and semen transports from the vas deferens and seminal vesicle to the urethra during the process of emission result in reduced male fertility in rats.     

A Dose-Response Analysis of Methoxychlor-Induced Alterations of Reproductive Development and Function in the Rat

A Dose-Response Analysis of Methoxychlor-Induced Alterationsof Reproductive Development and Function in the Rat. GRAY, L.E., JR., OSTBY, J., FERRELL, J., REHNBERG, G., LINDER, R., COOPER,R., GOLDMAN, J., SLOTT, V., AND LASKEY, J. (1989). Fundam. Appl.Toxicol12, 92–108. In the present study rats were dosed fromweaning, through puberty and gestation, to Day 15 of lactationwith methoxychlor at 25, 50, 100, or 200 mg/kg/day. Morphologicallandmarks of puberty were measured, including the ages at vaginalopening, first estrus, and first estrous cycle in females andat preputial separation in males. In the female, estrous cyclicity,fertility, litter size, number of implantation sites, organweights, and ovarian and uterine histology were also measured.The viability of the offspring (F1) and their fertility wereevaluated using a continuous breeding protocol. Males were necropsiedafter breeding, the reproductive organs were weighed, and thecauda epididymal sperm counts were determined. One testis wasused for histopathology, while the other was used to quantifyinterstitial fluid (IF) content, IF testosterone concentration,and testicular sperm production. Testosterone and an drogen-bindingprotein were measured in the caput epididymis, and sperm motilityand morphology were evaluated from a caudal sample. The serumand pituitary were saved for hormonal determinations. Methoxychloraccelerated the age at vaginal opening and first estrus, andthe vaginal smears were cornified. Growth was retarded at 100and 200 mg/kg/day and fertility was reduced when the femaleswere bred with untreated or similarly treated males. In thehighest- dose group, the mated females went from constant estrusinto pseudopregnancy following mating, but they had no implants.In males, methoxychlor treatment markedly reduced growth, seminalvesicle weight, cauda epididymal weight, caudal sperm content,and pituitary weight. Puberty was delayed in the two highest-dosagegroups. Testicular sperm measures were much less affected thancaudal measures. Testis weight and histology were slightly affected,and testicular sperm production, sperm morphology, and motilitywere unaffected. Endocrine function of the testes and pituitarywas altered by methoxychlor administration. Leydig cell testosteroneproduction, in response to human chorionic gonadotropin challenge,was reduced and pituitary levels of prolactin, thyroid-stimulatinghormone (TSH), and follicle-stimulating hormone (FSH) were altered.In contrast, serum levels of prolactin, FSH, and luteinizinghormone were unaffected. Serum TSH was reduced by 50% of controlat 100 and 200 mg/kg/day, while pituitary levels were increased.Gonadotropin-releasing hormone concentration in the mediobasalhypothalamus was also elevated. In spite of the many reproductivealterations, the fertility of treated males was not reducedwhen they were mated with untreated females. Growth and viabilityof the offspring (F1) from the 50 mg/kg/day treatment groupwere normal, but in the females, vaginal opening was accelerated,estrous cyclicity was abnormal in the rats during middle age,and fecundity was reduced.     

Rat epididymal sperm motion changes induced by ethylene glycol monoethyl ether, sulfasalazine, and 2,5-hexandione

Epididymal sperm was examined using the Hamilton-Thorne Sperm analyzer (HTM-IVOS, version 10.6) in male rats treated with known male reproductive toxicants that act by different mechanisms to detect effects on sperm motion. Three agents known to produce changes in sperm motion at high exposure levels were administered at lower levels. Ethylene glycol monoethyl ether (EGEE), sulfasalazine (SASP), and 2,5-hexandione (2,5-HD) were administered by oral gavage to adult male Sprague-Dawley rats at 250 or 500 mg/kg/day, at 300 or 600 mg/kg/day, or at 100 or 250 mg/kg/day, respectively. The males were treated with EGEE, SASP, and 2,5-HD for 35, 28, and 28 days, respectively. The males treated with EGEE and SASP were mated with untreated females to assess male fertility. All males were examined for body weight, testicular and epididymal weight, epididymal sperm count, and sperm motion. The sperm motion parameters included percentage of motile sperm, percentage of progressively motile sperm (progressive motility), curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness (STR). For the male rats treated with SASP, no treatment-related effects on percentages of motile sperm or sperm count were observed despite impaired male fertility. However, abnormal motion of epididymal sperm from the SASP treated males was detected by a significant reduction in mean progressive motility, VAP, and ALH, and an increase in BCF and STR. For the males treated with 2,5-HD for 4 weeks, most parameters generated by the HTM-IVOS indicated decreased sperm motion despite no remarkable changes in testicular weight, epididymal weight, or sperm count. In the EGEE-treated males at 250 mg/kg/day for 5 weeks, abnormal motion of epididymal sperm was detected by decreased progressive motility and increased BCF, although there were no treatment-related effects on testicular weight or male fertility. Progressive motility was decreased in all treated groups and the difference from the control value was of the greatest magnitude among the sperm motion parameters generated by the HTM-IVOS. Velocity parameters (VAP, VSL, VCL) responded sensitively to abnormal sperm motion in the SASP and 2,5-HD studies. In spite of decreased sperm motion, BCF values were significantly increased in all treated groups except the 7-week EGEE high-dose group, where there were no motile sperm to evaluate. ALH was significantly decreased in the treated groups in which remarkable effects on sperm motion were noted. There were no significant changes in ALH at the low-dose of EGEE at which only mild effects on sperm motion were observed. STR was increased for epididymal sperm from the males treated with SASP when compared with the controls. For the males treated with EGEE and 2,5-HD, however, STR was decreased when compared with the controls. There were no significant differences in LIN in any of the groups treated with SASP, in which remarkably reduced sperm motion was detected by the other parameters. In conclusion, among the parameters generated by the HTM-IVOS, progressive motility was significantly decreased in all treated groups and the most valuable for detecting slight changes in sperm motion induced by these three different target toxicants. Further investigation with a larger set of compounds is needed to evaluate which IVOS parameters are the most sensitive in detecting motion changes.     

Effects of Epichlorohydrin on Male and Female Reproduction in Long-Evans Rats

Male and female Long-Evans rats were treated with epichlorohydrin(ECH) by oral gavage (males: 12.5, 25, and 50 mg/kg/day; females:25, 50, and 100 mg/kg/day) for 21 and 14 days, respectively,prior to mating trials with untreated animals. Treated femaleswere further dosed until delivery. Fertility was assayed inthe high-dose males only and was found to be totally impaired.No measured parameters of female reproduction were changed relativeto controls. Treated males showed normal copulatory behavior.Sperm morphology and percentage motile sperm were not statisticallydifferent from control values in both ejaculated and cauda epididymalsamples from ECH-treated animals. The number of sperm in ejaculateswas normal while cauda epididymal sperm count was slightly decreasedin males at the 50 mg ECH/kg dose level. Mean curvilinear velocity,straight-line velocity, and amplitude of lateral head displacementof cauda epididymal sperm were significantly reduced by ECHat 12.5 mg/kg/day and above. Sperm track linearity was alsoreduced, but only at 50 mg/kg/day. Beat/cross frequency of spermwas significantly increased at 12.5 mg/kg/day and above. Allof the above sperm motion parameters showed dose-dependent trends.These effects are consistent with the spermatozoal metaboliclesions reported for -chlorohydrin, a metabolite of ECH.     

Reproductive toxicity studies in rats with rimexolone, a corticoid ophthalmic suspension.

Rimexolone is a potent anti-inflammatory corticosteroid with a lower potential for elevating intraocular pressure, relative to other ophthalmic steroids, and is indicated for postsurgical inflammation and uveitis. Fertility and peri/postnatal toxicities were evaluated at oral gavage doses of 50, 150 or 500 mg/kg, and developmental toxicity at 100, 500, or 1000 mg/kg. In the fertility study, male rats were treated daily beginning 4 weeks prior to mating and females were treated daily beginning 2 weeks prior to mating, and through gestation day 6. Females were necropsied on gestation day 15 and males were necropsied after 10 weeks of exposure. In males, dose-related reductions in mean body weights, body weight gains, and food consumption occurred in all groups. In the 500 mg/kg females, mean body weights were reduced during gestation, and there was an increase in early resorptions and concomitant decrease in viable fetuses at this level. There were no effects on copulation or fertility indices, or on the number of corpora lutea and implantation sites. The no-observed-effect level (NOEL) for fertility and reproductive effects was 150 mg/kg. In the developmental toxicity study, female rats were treated daily from gestation days 6 through 17, necropsied on gestation day 20 and fetuses were evaluated. Maternal toxicity occurred at 500 and 1000 mg/kg as indicated by reduced maternal body weights and body weight gains. However, there was no indication of a developmental effect on fetuses due to rimexolone. The NOEL was 1000 mg/kg for the developing fetuses. In the peri/postnatal toxicity study, female rats were treated daily from gestation day 6 through lactation day 20 and necropsied. F1 developmental and behavioral parameters were evaluated. Selected F1 animals were mated at 12 weeks, allowed to deliver, and necropsied on lactation day 21. At 500 mg/kg, F0 maternal body weights were reduced during gestation and lactation, and F1 pup weights were reduced during lactation and the growth phase. There were no effects on the F1 fertility or reproductive capabilities, or on F2 developmental parameters. The NOEL for the F0 females and F1 offspring was 150 mg/kg. Together, these studies indicate that, unlike some corticosteroids, rimexolone does not produce developmental or reproductive toxicity in rats.     

Investigation of cyclohexylamine sulfate for dominant lethal effects in the mouse

Cyclohexylamine sulfate was studied for mutagenic effects in the dominant lethal test on mice. 20 male NMRI mice were treated with 150 mg cyclohexylamine sulfate (approx. 102 mg cyclohexylamine) per kg body weight (0.6% solution in demineralized water) orally per day for 5 successive days. A parallel control group of 20 male mice received demineralized water only.After the last treatment each male was mated with 3 untreated females. In order to study successive germ cell stages of the males, each male was placed with 3 other untreated females every week for mating. The total mating period was 8 weeks. The uterus of the females was examined on about the 14th day of pregnancy, when pre- and post-implantative losses were determined as assessment criteria from the number of corpora lutea, implantations, and live and dead embryos.The treatment did not damage the males and did not impair their mating capacity and fertility.The treatment also had no influence on pre- and post-implantative losses: The frequency of pre-implantative and post-implantative deaths was practically identical in the treatment group and the untreated control. Thus, we found no indication of a mutagenic action of orally administered cyclohexylamine sulfate (5 doses of 150 mg/kg) in the dominant lethal test on mice.     

Assessment of reproductive and fertility effects of amitraz pesticide in male mice

Forty adult male Swiss mice were exposed to tap water containing 0, 40, 80, or 160 ppm amitraz for 12 weeks. Based on fluid consumption the mice received an average of 0, 5.42+/-0.47, 10.56+/-0.97, and 20.39+/-2.17 mg/kg/day amitraz, respectively. The average body weights gains and fluid consumption were significantly decreased in males exposed to amitraz pesticide. Fertility was significantly reduced in male mice ingesting 10.56+/-0.97 or 20.39+/-2.17 mg/kg/day amitraz in that the number of females impregnated by them was significantly reduced. The number of viable fetuses was significantly reduced in females mated with males that ingested 10.56+/-0.97 or 20.39+/-2.17 mg/kg/day amitraz. A significant increase in the total number of resorptions and the number of females with resorptions was observed in females impregnated with the exposed males. Absolute testis weight was significantly decreased at 10.56+/-0.97 mg/kg concentration. The weight of the epididymis was decreased in test males ingested 20.39+/-2.17 mg/kg amitraz. The seminal vesicles weights were significantly increased in male mice ingested 10.56+/-0.97 or 20.39+/-2.17 mg/kg/day amitraz. Similarly, the preputial gland weights were increased in males that ingested 5.42+/-0.47 or 10.56+/-0.97 mg/kg and decreased in males ingested 20.39+/-2.17 mg/kg amitraz. Testicular sperm counts and daily sperm production were significantly decreased in males that ingested 10.56+/-0.97 or 20.39+/-2.17 mg/kg/day amitraz. Epididymal sperm counts were significantly decreased in exposed male's at 10.56+/-0.97 or 20.39+/-2.17 mg/kg amitraz. These results strongly suggest that exposure to amitraz pesticide have an adverse effect on the fertility and reproductive system of male mice.     

Testicular toxicity and infertility in male rats treated with 1,3-dinitrobenzene

Weanling male Sprague-Dawley rats were gavaged 5 d/wk with 1,3-dinitrobenzene (m-DNB) at dosages of 0, 0.75, 1.5, 3.0, and 6.0 mg/kg X d. Males were bred to untreated females during treatment wk 10 and were killed during treatment wk 12. Although males dosed with 3 mg/kg X d inseminated the females and evidence of mating was observed in males dosed with 6 mg/kg X d, none of the males in these groups sired litters. Diminished sperm production (reduced testicular sperm head counts), decreased cauda epididymal sperm reserves, nonmotile spermatozoa, atypical sperm morphology, decreased weights of the testes and epididymides, seminiferous tubular atrophy, and incomplete spermatogenesis were also observed in these groups. Sperm production was also decreased in males dosed with 1.5 mg/kg X d. Changes in the spleen included increased weight at dosages of 1.5 mg/kg X d or higher and splenic hemosiderosis, which ranged from slight in rats treated with 0.75 mg/kg X d to moderately severe in those dosed with 6 mg/kg X d. The data indicate that m-DNB is a potent testicular toxicant in the male rat, capable of producing extensive damage to reproductive tissues and reproductive failure. Limited data on four rats that received 6 mg/kg X d and were allowed a 5-mo posttreatment recovery period suggested that the testicular effects are at least partially reversible.     

Impairment of male fertility induced by muscarinic receptor antagonists in rats

Male rats were treated with a muscarinic receptor antagonist at 3, 10, and 100 mg/kg/day for 4 weeks prior to mating with untreated females and their reproductive status was determined on gestation days (GD) 15–17. Treatment-related decreases in the pregnancy rate were observed at 100 mg/kg/day without any effects on mating performance. Impairment of male fertility by this compound was also observed after treatment for 1 week, but there were no effects after a 1-week withdrawal period suggesting reversibility of the effect. There were no treatment-related effects on sperm production or motility, or testicular histopathology in any group. In order to determine whether the reduced fertility was a class effect of muscarinic receptor antagonists, atropine was examined. Males received atropine for 1 week at 62.5 and 125 mg/kg/day and were mated with untreated females. A low pregnancy rate associated with a decrease in the number of implantations was observed at 125 mg/kg/day. The effect on implantation was also observed at 62.5 mg/kg/day. These findings suggest that the impairment of fertility in male rats induced by muscarinic receptor antagonists is a class effect, and has a relatively short onset of effect and is quickly reversible.     

Effects of three male reproductive toxicants on rat cauda epididymal sperm motion

The sensitivity of the CellSoft™ computer-assisted sperm analysis (CASA) system to detect changes in rat sperm motion was evaluated. CASA motion endpoints were measured in cauda epididymal sperm from Long-Evan rats treated with each of three known male reproductive toxicants reported to affect the epididymis and epididymal sperm motility: -chlorohydrin, ornidazole, and trimethylphosphate. Significant changes in endpoints describing sperm swimming vigor (curvilinear velocity and straight-line velocity) and pattern (linearity and amplitude of lateral head displacement) were observed for rats dosed with each agent when evaluations included mean values and other statistical parameters (i.e., percentiles and distributional shape). -Chlorohydrin (ACH) treatment (10 mg/kg/day; 8 days) resulted in reductions in the mean percentage of motile sperm, curvilinear velocity (VCL), straight-line velocity (VSL), lateral head displacement (ALH), and linearity (LIN). Treatment with ornidazole (ONZ) (200mg/kg/day/14 days) reduced the percentage of motile sperm. Mean VCL, VSL, and ALH were reduced by 400 mg ONZ/kg/day treatment. Trimethylphosphate (TMP) treatment led to (a) a reduction in the 75th and 90th percentiles for ALH (100 mg TMP/kg/day; 5 days) (P≤0.04), (b) a reduction in VCL, VSL, and ALH (250 mg TMP/kg/day), (c) a reduction in the percentage of motile cells and in the 10th and 25th percentiles for VSL (600 mg TMP/kg/day), and (d) increases in the 90th percentile for VSL, in the mean, 75th, and 90th percentiles for VCL, and in the 75th and 90th percentiles for ALH (600 mg TMP/kg/day). The general utility of these analytic approaches in reproductive toxicology studies was demonstrated in the observations of effects at or below dose levels previously reported.     

Reproductive and Developmental Toxicity Studies of a Linear Alkylbenzene Mixture in Rats

Alkylate 215, a mixture of linear decyl- to tridecylbenzenes,is an intermediate in the manufacture of detergent sulfonates.A two-generation reproduction study and a developmental toxicitystudy were conducted using single daily doses given by gastricintubation in a corn oil vehicle. In the reproduction study,groups of 30 rats/sex/group were given doses of 0, 5, 50, or500 mg/kg/day. F₀ animals received a 10-week premating treatmentperiod and were then mated to produce a single litter; F₁ adultswere selected from the F₁ litters. F₁ animals were dosed for11 weeks before mating to produce a single litter. Adults andweaned pups received a gross postmortem examination. Histopathologystudies were conducted on reproductive tissues, tissues withgross lesions, and the pituitary gland taken from each adultin the control and high dose groups. In the developmental toxicitystudy, groups of 24 mated female rats were given 0, 125, 500,or 2000 mg/kg/day on Days 6 through 15 of gestation. Dams wereterminated on gestation Day 20 and fetuses were examined forexternal, soft tissue, and skeletal defects. Results of thereproduction study were as follows. At 50 mg/kg/day, pup weightswere decreased at Day 7 in the F₁ litter. At 500 mg/kg/day,decreases were found in the F₁ females in premating and earlylactation weight gains; in both generations in premating weightgains in males and in weight gains during gestation in females;and in litter size, pup viability at birth, Day 0–4 sur vival, and pup weights on Days 14 and 21. The NOAEL for reproductiveeffects was 5 mg/kg/day. The developmental toxicity study foundeffects on several parameters. The only effect noted at 125mg/kg/day was a slight decrease in maternal weight gain. Maternalweight gains were depressed to a greater extent at 500 and 2000mg/kg/day. Ossification variations and delayed ossificationwere increased significantly at 2000 mg/kg/day and were abovecontrol levels at 500 mg/kg/day. The NOAEL for developmentaltoxicity was 125 mg/kg/day. Alkylate 215 did not have any unusualor selective reproductive or developmental toxicity.     

Effects of corticosterone on reproduction in male sprague-dawley rats

Corticosterone, the predominant circulating adrenal corticosteroid in rodents, was investigated for its effects on reproduction in male Sprague-Dawley rats. Male rats (in groups of 50, 25, and 50) were administered corticosterone at doses of 0,10, and 25 mg/kg/d, respectively, by subcutaneous injection once daily for 6 weeks; the highest dose was decreased to 20 mg/kg/d after 15 d. During the last 2 weeks of the 6-week treatment period, 25 males per group were paired with untreated females. The remaining 25 males from the 0 and 25/20 mg/kg/d groups were allowed a 6-week recovery period and, during the last 2 weeks of this period, these males were also paired with untreated females. At the end of the treatment period, the males had markedly elevated plasma corticosterone concentrations and decreased weight gain. They also produced fewer copulatory plugs than controls, which may have been secondary to observed adverse effects on the accessory sex organs (decreased weights and microscopic changes in prostate and seminal vesicles). However, no adverse effects on sperm motility, sperm count, or microscopic features of the testes were observed. Serum testosterone concentration of the high-dose males was elevated, but luteinizing hormone was unaffected. The numbers of embryonic implantation sites and live fetuses in females mated to these males were reduced. All of these effects except decreased prostate weights were reversible upon cessation of corticosterone administration. Thus, exogenous administration of corticosterone to male rats produced reversible effects on implant count and litter size of female rats mated to these males. These effects on male rat reproduction may have been secondary to reduced accessory sex organ function, which resulted in diminished secretions and fewer copulatory plugs.