An in vivo evaluation of induction of abnormal sperm morphology by some anthelmintic drugs in mice

Using the murine sperm-head abnormality test, the mutagenicity of pyrantel pamoate, levamisole, albendazole, mebendazole and niridazole was evaluated. Pyrantel pamoate and niridazole induced increases in sperm-head abnormalities statistically significant over the negative controls at all the dose levels that were considered; the induction was dose-dependent indicating that both drugs might be mutagenic. Levamisole, albendazole, mebendazole and thiabendazole, all were unable to induce statistically significant increases in sperm-head abnormalities over the negative controls at all the dose levels tested; there was no correlation between dose level of administered drugs and incidence of abnormal sperms, indicating that the drugs might not be mutagenic.     

In vivo evaluation of induction of abnormal sperm morphology in mice by an unsaturated aldehyde crotonaldehyde

Crotonaldehyde, a highly reactive unsaturated aldehyde is used for the manufacture of sorbic acid, synthesis of butyl alcohol, butylaldehyde, quinaldine, thiophenes, pyridines, dyes, pesticides, pharmaceuticals, rubber antioxidants, chemical warfare agents, etc. and also occurs naturally in meat, fish, in many fruits and vegetables, bread, cheese, milk, beer, wine and liquors. Human exposure to crotonaldehyde occurs from both man-made and natural sources. No human data was located describing carcinogenicity associated with crotonaldehyde exposure. In the present study we have evaluated whether or not exposure to crotonaldehyde results in a significant increase in the frequency of abnormal sperm heads in male Swiss albino mice. Adult male mice were treated with 8, 16 and 32 microl/kg b.w. of crotonaldehyde as a single intraperitoneal injection. The animals were killed 1, 3 and 5 weeks after treatment. Five animals were sacrificed per dose and time tested. Crotonaldehyde induced dose related increase in the percentage of abnormal sperm heads. Statistically significant increase in percentage of abnormal sperm heads was recoded at 16 and 32 microl/kg b.w. after 1 and 3 weeks of treatment and only at 32 microl/kg b.w. after 5 weeks of treatment.     

Radiation induction of mutations affecting sperm morphology in mice

To investigate the basis of the sperm abnormality assay, studies have been made of the frequency of sperm abnormalities in the genital tracts of the progeny of irradiated males. Male C57BL/6 mice were irradiated (75-600 rad X-rays to the testes) and were then bred in the pre-sterile period to untreated C57BL/6 females. The sperm of their male progeny were examined for the frequency of sperm abnormalities. Variant males with clearly elevated levels of sperm abnormalities were more frequently seen amongst the progeny of irradiated fathers than with the progeny of sham-irradiated controls (10 in 170 compared with 2 in 188; P less than 0.02). Although no clear dose-response relation could be discerned with the number of animals studied, similar differences were observed with irradiated male SWR, C3H/He in inbred crosses and with C57BL/6 in hybrid crosses with C3H/He females. In contrast, matings of males made at longer times following irradiation did not lead to a significant increase in the number of affected progeny for the number tested. Breeding experiments with the affected F1 males showed that the sperm morphology defect could be transmitted in 7 of 19 cases. Parallel cytogenetic studies showed that 3 of the initial affected progeny had detectable reciprocal translocations and that in 2 of these cases the translocation was transmitted with the sperm defect. The studies thus showed that radiation can induce mutations that affect the levels of sperm abnormalities and that these mutations can be associated with reciprocal translocations.     

Deficiency in fertilization by morphologically abnormal sperm produced by Azh mutant mice

Male mice homozygous for the azh mutation produce spermatozoa with abnormal head shapes and have significantly reduced fecundity, to between 5% and 10% that of wild-type or heterozygous mice. Several possible causes of this infertility were investigated. No gross endocrine disorders in azh/azh male mice were observed, and they exhibited apparently normal mating behavior. In addition, their sperm were motile, were capable of hyperactivated motility, and did not show premature acrosome reactions. However, quantitative analysis revealed slight but significant reductions in several motility parameters. Analysis of embryos following mating of azh/azh males with superovulated females indicated a reduction in the number of fertilized eggs compared to control matings. In vitro, spermatozoa from azh/azh mice failed to fertilize cumulus-intact/zona-intact and cumulus-free/zona-intact ova, although they successfully fertilized zonafree ova. These results indicate that the primary defect in fertility of azh/azh male mice is a result of sperm quality, likely, in sperm morphology, and is manifest at the level of interaction with the zona pellucida. © 1994 Wiley-Liss, Inc.     

Methanogenic diversity and activity in municipal solid waste landfill leachates

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.     

Genotoxicity of leachates from a landfill using three bioassays.

In the city of Queretaro, around 500 tons of solid wastes are produced everyday and are deposited in a landfill. This is the result of social and economic activities of human beings or from their normal physiological functions. As a result of rain, leachates are produced, which, if not handled and treated correctly, may pollute the underground water. Among the bioassays developed for the detection of mutagenicity in environmental pollutants, plant systems have been proven to be sensitive, cheap, and effective. The purpose of this study was to determine the presence of genotoxic agents in the leachates of the landfill of the city using three bioassays: Tradescantia-micronucleus (Trad-MCN), Tradescantia stamen hair mutations (Trad-SHM) and Allium root anaphase aberrations (AL-RAA) and make a comparison of the results in the three assays. Leachates were sampled during both the dry and rainy seasons. Plant cuttings of Tradescantia or the roots of Allium were treated by submerging them in the leachates. Three replicates of each sample were analyzed in each of the three bioassays. As expected the samples of leachates collected during the dry season showed a higher genotoxicity than those collected during the rainy season. In conclusion, there are substances present in the leachates capable of inducing genotoxicity in the plant assays. On the other hand, the plant assays showed different degrees of sensitivity: the more sensitive was the Trad-MCN bioassay and the less sensitive the Trad-SHM assay. Therefore, when analyzing environmental pollutants it is recommended to use a battery of bioassays.     

Increases in chromosome aberrations and in abnormal sperm morphology in rubber factory workers

Subjects working at a rubber plant in a chemicals warehouse or in calandering and bambury units were analyzed for both sperm parameters and structural chromosome aberrations in peripheral blood lymphocytes. Sperm analysis war performed in a group of 24 workers for comparison with fertile (n = 24) and infertile (n = 24) control groups. The statistical analyses of semen volume, vitality and sperm count did not show significant differences between exposed and fertile groups but significant differences were found from the infertile group. A significantly lower proportion of normal sperm head shapes was found in exposed subjects when compared to the fertile group (40.1 vs. 57.8). Seven exposed workers were re-analyzed 1 year and their sperm parameters did not change. The cytogenetic analysis showed a significant increase (3.90%) in the percentage of cells with aberrations in bambury workers (n = 11). However, no differences were found between calandering workers (n = 8) and control subjects (n = 10). Workplace air samples taken on the day of tissue sampling did not show any increase above the Cuban maximal allowed concentration for benzo[a]pyrene or toluene.     

Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.     

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.     

The transport of morphologically abnormal sperm in the female reproductive tract of mice

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.     

Constructed wetlands for the treatment of landfill leachates: The Slovenian experience

Constructed wetlands are widely recognized as an economical, efficient and environmentally acceptable means to treat many different types of wastewater. Six systems have been constructed in Slovenia for the treatment of landfill leachates. This paper describes the early stages of two treatment systems operating from 1990 to 1993 that were used to treat leachates from municipal landfills. System S1 consisted of a sedimentation lagoon and a 600 m² reed bed. System S2 was designed with a sedimentation lagoon and two reed beds (total=450 m²). The subsurface flow in both was horizontal and fluctuated widely in S2, but was a constant 0.2 1 s^–1 in S1. Peat, soil, sand and gravel were in system S2, while gravel with a hydraulic conductivity of 5×10^–4 m s^–1, was in S1.The unanticipated fluctuations of hydraulic and organic loadings influenced their performance. The efficiency in reduction of organic matter, N, P, metals and fecal coliforms varied through the year. In 3 years of operation, the average removal efficiencies for COD, BOD₅ and TSS were 38%, 61%, and 81%, respectively, in S1, and 53%, 45%, and 47%, respectively, in 1.5 years of operation for S2. The reduction of ammonium did not reach the effluent standard of 10 mg l^–1 for either S1 or S2. As, Zn, Pb, and Cu accumulated in roots, and Cu, Fe, As, Ni and Pb accumulated in rhizomes after one year of operation in S2.     

The mycobiota of landfill leachates in the pretreatment process in a sequencing batch reactor

The paper discusses the dynamics of the accumulation of microscopic fungi, depending on the sludge load (Bx), in activated sludge used for landfill leachate pretreatment. The propagule washout from the sludge into pretreated leachates is determined, including genera and species that may threaten environmental health. An increased accumulation of microscopic fungi in sludge flocs occurred at Bx=0.23−0.45 mg chemical oxygen demand (COD) mg⁻¹ d⁻¹. Microscopic fungi were eluted at the maximal Bx value tested of 1.64 mg COD mg⁻¹ d⁻¹. Both the activated sludge and the leachate runoff from the sequencing batch reactor (SBR) pose health risks to the environment due to the occurrence of fungi such as Aspergillus fumigatus, Purpureocillium lilacinum, Cyberlindnera jadinii (C. utilis), Geotrichum candidum and G. fragrans. Their count is sufficient to cause multi-organ infections in homeothermal animals and in humans.     

Effect of method and clinician on stallion sperm morphology evaluation

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.     

Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice

Sperm morphology and spermatocyte chromosomes were examined in mice maintained on a Torula yeast diet for 5 weeks. In the selenium-deficient group, the proportion of abnormal sperm was high, ranging from 6.8% to 49.6%, while in the control group it ranged from only 4.0% to 15.0%. The most frequently occurring abnormalities in sperm shape were in the sperm head. There was also a tendency for abnormalities in other regions (neck, midpiece and tail) to be increased. However, in metaphase-I spermatocytes, the frequencies of various types of abnormal chromosomes (univalent chromosomes, translocations and structural anomalies) did not differ between the selenium-deficient and control groups. These findings indicate that selenium may be an essential constituent for spermatogenesis in mice.     

Effects of vinyl acetate and acetaldehyde on sperm morphology and meiotic micronuclei in mice

The testicular genotoxic effects of vinylacetate (VA) and its hydrolysis product, acetaldehyde (AA), were studied in mice by analyzing the induction of morphologically abnormal sperm and meiotic micronuclei. VA significantly increased the frequency of sperm abnormalities at 500 mg/kg/day while lower doses were ineffective. AA did not induce abnormal sperm. Neither of the compounds influenced the frequency of meiotic micronuclei. VA, but not AA, caused a dose-dependent decrease in sperm production and a reduction of testicular weight at 500 and 125 mg/kg/day.     

Sperm competition does not influence sperm hook morphology in selection lines of house mice

Sperm show a remarkable degree of variation in size, shape and complexity. Murine rodents exhibit a sperm head morphology that differs greatly from the ovoid shape that is characteristic of most mammals. These rodents have sperm that bear one or more apical hooks, the function of which is currently surrounded by much controversy. It has been suggested that the hook serves to facilitate the formation of sperm groups, which in some species exhibit relatively faster velocities than single cells and thus, may provide an advantage when ejaculates are competing for fertilisations. In support of this hypothesis, a comparative study reported a positive association between the strength of sperm competition (estimated from testes size) and the curvature of the sperm hook amongst 37 murine species. Here, we assessed whether sperm competition influences sperm hookedness at the intra-specific level. Following 16 generations of selection, we used geometric morphometry (GM) to describe sperm head morphology in selection lines of house mice evolving with (polygamous) and without (monogamous) sperm competition. Although the GM analysis returned two relative warps that described variation in the curvature of the sperm hook, we found no evidence of divergence between the selection lines. Thus, we can conclude that sperm competition does not influence the degree of sperm hookedness in house mice.     

An assessment of sperm motility estimation for evaluation in rams

There is disagreement about the value of estimating spermatozoal motility as a predictor of fertility in rams. We tested the hypothesis that sperm motility can help one to predict lambing rate by evaluating sperm motility in rams prior to breeding and by subsequently breeding the rams regardless of their sperm motility results. A total of 943 rams (Merino, n=630 and Romney Marsh, n=313) was used during two breeding seasons. For fertility evaluation only 804 rams that bred at least ten ewes each were included in the study. Rams were grouped into 4 groups according to sperm motility scores, ranging from the lowest, which included rams with azoospermia, oligospermia, non-motile and single-motile sperm, through rams with 10 to 30%, 30 to 60%, to the highest at 60 to 100% motile sperm. The corresponding fertility rates found in our study in Merino and Romney breeds were 58.3 and 47.4%, 75.1 and 59.1%, 79.4 and 64.1% and 81.5 and 64.0%, respectively. We concluded that there was a statistically confirmed difference in fertility between the lowest and all the other motility categories. The differences in fertility among rams that had motility categories above 10% were not statistically significant. Evaluation of breeding soundness in large scale surveys of rams, based on sperm motility estimation, may be misleading. Clinical examination of genital organs seems to offer a reliable source of information about rams, and may be considered adequate in sheep breeding.