Effects of fenvalerate and cypermethrin on rat sperm motility patterns in vitro as measured by computer-assisted sperm analysis

Fenvalerate and cypermethrin were reported to impair male reproductive function, inducing significant reductions in epididymal sperm count. Further, fenvalerate was shown to reduce sperm motility. However, it is not clear whether fenvalerate and cypermethrin might impact sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens or their receptors. In this study, sperm suspensions were treated with fenvalerate and cypermethrin, respectively, at various concentrations (0, 1, 4, 16, or 64 micromol/L) for various times (1, 2, or 4 h). The motility parameters of sperm treated with these two insecticides were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of fenvalerate and cypermethrin on rat sperm motility patterns in vitro were also compared. Our study revealed that fenvalerate and cypermethrin reduced sperm motility in vitro in a concentration- and time-dependent manner. Cypermethrin exerted a greater effect on sperm motility in comparison to fenvalerate. These results provided evidence that fenvalerate and cypermethrin directly influence mature rat sperm motility.     

Effect of two insecticides and two herbicides on the porcine sperm motility patterns using computer-assisted semen analysis (CASA) in vitro

The recent decline in sperm concentration observed in men has developed over a short period of time, suggesting that it could be the result of environmental factors. The present study has evaluated the effects of insecticides Malathion and Diazinon, and herbicides Atrazine and Fenoxaprop-Ethyl on porcine sperm viability and motility patterns in vitro using the eosin-nigrosin staining and a computer-assisted semen analyzer (CASA), respectively. Malathion and Fenoxaprop-Ethyl exerted more deleterious effects than Diazinon and Atrazine. Progressive sperm motility was strongly affected whereas the effect on sperm viability was less pronounced. This suggests that a reduction of sperm motility is not necessarily the result of sperm death. Since sperm motility is dependent on energy metabolism the mechanism of action of these pesticides might be mediated at the level of the mitochondrion, producing a delay in motility and eventual cell death.     

Effects of nonylphenol on motility and subcellular elements of epididymal rat sperm

Nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical. The objective of these studies was to determine the effects of NP on epididymal rat sperm in vitro. Epididymal sperm samples from Sprague-Dawley rats were incubated in 1, 10, 100, 250, and 500 μg/ml NP for 1, 2, 3, or 4 h. Computer-assisted sperm analysis was used to determine motility. Epifluorescent microscopy was used to determine acrosomal status and flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity. Exposure of epididymal rat sperm to 250 or 500 μg/ml NP was highly detrimental to motility (P < 0.05), with complete loss of motility observed after exposure to 500 μg/ml NP (P < 0.05). The acrosomal integrity of sperm was significantly reduced with the lowest concentration (1 μg/ml) of NP, and higher concentrations resulted in a dose-dependent induction of the acrosomal reaction (P < 0.05). Similarly, the percentage of sperm with high MMP declined dramatically after exposure to 100, 250, and 500 μg/ml NP (P < 0.05). Duration of NP exposure did not have any effect on motility or MMP and NP did not appear to have detrimental effects on chromatin integrity (P > 0.05). These results indicate that major mechanism of action of NP on rat sperm is by adversely affecting their acrosomal integrity. However, NP-induced impaired sperm motility, decreased mitochondrial membrane potential also likely to play an important role in destruction of sperm function.     

Sources of variation in the computer-assisted motion analysis of rat epididymal sperm

Random and nonrandom factors associated with sample preparation and the automated analysis (CellSoft) of rat cauda epididymal sperm motion were studied. Random factors included inherent system variation at both the individual cell level and at the multiple cell level. Repeated analyses of identical tracks across grey level revealed a statistical interaction between grey settings and curvilinear velocity. However, in multiple track analyses, grey level was seen to be a factor only at higher settings. Nonrandom factors included time after sample preparation, dilution medium, and sample preparation procedures. Using a nicked preparation of the entire cauda epididymis from Long-Evans rats, the effects of time were studied on sperm suspended in 1) phosphate-buffered saline + 10 mg BSA/mL, 2) TEST yolk buffer, and 3) Medium 199. In PBS/BSA, the percent motile sperm estimate decreased (50% to 30%) over an hour, while the curvilinear velocity increased (127 to 142 microns/sec). Both sperm motion parameters were maintained in the TEST yolk buffer and in the Medium 199, although at lower values for the latter. Evaluation of the relative contribution of several factors, nested within sample, to the overall variance of three separate motion endpoints revealed that there was a large variation from field to field, negligible variation between overall CellSoft analyses of 200 cells or more, low variation at the preparation aliquot level, and moderate variation at the animal level. In planning experiments to test for effects on sperm motion endpoints, consideration of the relative contribution of the individual study factors to the overall variance of the parameter estimates will result in more sensitive experimental designs.     

Metabolism of 3-phenoxybenzoic acid and the enterohepatorenal disposition of its metabolites in the rat

The absorption, metabolism and disposition of 3-phenoxybenzoic acid (3PBA) and its 4'-hydroxylated derivative (4'HO3PBA) have been investigated in intact and bile duct-cannulated rats. Both acids are rapidly and extensively eliminated in the urine as the O-sulfate ester of 4'HO3PBA (4'HOSO2O3PBA). Biliary excretion was 45% of the dose when 3PBA was administered at 100 mg/kg, compared with 16% at 10 mg/kg. The major biliary metabolites, identified by mass spectrometry, were 3PBA glucuronide and the ether and ester glucuronic acid conjugates of 4'HO3PBA. These biliary metabolites were of minor importance in the urine. At a dose of 100 mg/kg, an apparent excretion balance figure approximately 120%, as determined from the extent of elimination of radioactivity in the urine (72%) of intact animals and bile of cannulated rats (45%) (only 7% appeared in the feces), suggests that the biliary glucuronides decompose and/or are enzymically cleaved in the gastrointestinal tract to the respective benzoic acids. The latter are subsequently reabsorbed and undergo further metabolism, principally to the sulfate ester which is excreted in the urine. This enterohepatorenal disposition of 3PBA has been confirmed by suppression of the gut microflora with antibiotics in vivo, which dramatically reduces the extent of deconjugation of biliary glucuronides and hence reabsorption of the free benzoic acids. The deconjugation by gut bacteria has also been demonstrated in vitro.     

Comparison of sperm motility test methods (except computer-assisted sperm analysis) in rats under the condition of alfa-chlorohydrin treatment--collaborative investigation

A comparison among rat sperm motility test methods including percent of motile sperm (% Motile), scoring method (Scoring), Ishii's method, Progressive Motility Test (PMT) and Sperm Quality Analyzer (SQA), was conducted using data gathered from eleven laboratories. As a unified study design, mature male rats were orally treated daily for approximately 1 week with alpha-chlorohydrin (ACH), which is known to affect the sperm motility at the epididymis, at dose levels of 2.5, 5 and 10 mg/kg, and then subjected to more than two test methods for sperm motility in each laboratory. Scoring (4 or 5 grades), Ishii's method, PMT and SQA showed high sensitivity for the detection of the effects of ACH, which were not considered to be inferior to a computer-assisted sperm analyzer (CASA). Longer incubation time before testing was considered to contribute to detecting the effects of ACH. In particular, we realized that Scoring was a favorable method even if the demerit of poor objectivity was allowed for. Percent Motile showed lower sensitivity than other test methods. The differences in sensitivity between % Motile and other methods were considered to be based on whether the defects of progressive motion could be detected. Although % Motile cannot clearly judge whether immotile sperm are dead or alive, the value is a great help for the interpretation of the result from other methods. Based on the characters for detectability, objectivity and efficiency, the most suitable method of sperm motility should be selected according to the purpose of the toxicity study.     

Acute toxicity of two pyrethroids,permethrin, and cypermethrin in neonatal and adult rats

The present study aims specifically at obtaining a comparison of the acute toxicity of cypermethrin (CY), a type I pyrethroid, and permethrin (PERM), a type II pyrethroid, administered orally as a single dose to neonatal and adult rats, and at assessing the importance of pyrethroid biotransformation in CY and PERM toxicity through use of drug metabolism inhibitors. Our experiments show that CY is more toxic than PERM to adult and neonatal rats. The sensitivity of neonatal rats both to CY and to PERM toxicity is higher, the younger the animals. CY is much more toxic than PERM in the neonatal rat, compared with the adult. In rats aged 8, 16, and 21 days, pretreatment with piperonil butoxide (PB), a monooxygenase inhibitor, or with tri-o-tolyl phosphate (TOTP), an esterase inhibitor, does not produce significant variations in the lethal effects of CY and PERM. Instead, in the adult rats, a significant increase in CY (X²=5.97;p<0.05) and PERM (X²=4.37;p<0.05) mortality occurred in rats pretreated with esterase inhibitors, whereas no increase in CY and PERM toxicity was found in adult animals pretreated with monooxygenase inhibitor. It was concluded that the higher level of sensitivity of the neonate rat to pyrethroid toxicity is probably due to incomplete development of the enzymes which catalyze the metabolism of pyrethroids in the liver of young animals. It is suggested that ester hydrolysis is an important pyrethroids detoxification reaction in the adult rat.     

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.     

Species variations in the renal and hepatic conjugation of 3-phenoxybenzoic acid with glycine

1. 3-Phenoxy[¹⁴C]benzoyl-CoA has been chemically synthesized, purified and characterized by field-desorption mass spectrometry. Biological activity of the purified thioester was > 92%.

2. The two enzymic steps involved in the conjugation of 3-phenoxybenzoic acid (3PBA) with glycine have been investigated in hepatic and renal tissues from various mammalian species.

3. A 10- to 300-fold excess of acyl-CoA: glycine N-acyltransferase activity as compared with acyl-CoA synthetase activity was found in most tissue preparations, while the rate of the activating step matched that of the overall process. This suggests that formation of the acyl-CoA thioester (3PBA-CoA) is the rate-limiting step in the conjugation of 3PBA with glycine.

4. In most of the species tested, renal activities were higher than those of corresponding liver preparations.

5. The gerbil and ferret, which excrete 3-phenoxybenzoylglycine as the principal urinary metabolite of 3PBA, gave the highest 3PBA-CoA synthetase and glycine N-acyltransferase activities in vitro. By contrast, the hamster, which excretes only small amounts of the glycine conjugate of 3BPA, had the lowest enzymic activities in vitro.

6. In the mouse and rat there were differences between the patterns of metabolism found in vivo and in vitro, and possible reasons for this are discussed.     

Species differences in the metabolism of 3-phenoxybenzoic acid

The metabolism of 3-phenoxybenzoic acid (3PBA) (10 mg/kg, ip) has been studied in ten mammalian and one avian species in comparison with that of benzoic acid. 3PBA exhibits wide species diversity in its metabolism, unlike benzoic acid, of which benzoylglycine (hippuric acid) is the major urinary metabolite in all species studied. With 3PBA, glycine conjugation is the major route of metabolism in three species (sheep, cat, and gerbil), whereas in the mouse the taurine conjugate is the principal metabolite. The ferret eliminates similar amounts of each of these metabolites, whereas the glycylvaline dipeptide conjugate is the major metabolite isolated from the excreta of the mallard duck. Conversely, glucuronic acid conjugates of 3PBA and its 4'-hydroxy derivative (4'HO3PBA) are the major urinary metabolites in the marmoset, rabbit, guinea pig, and hamster; the rat appears unique in eliminating the O-sulfate conjugate of 4'HO3PBA as the principal urinary metabolite. In most cases, where amino acid conjugates are the major excretory products, the proportions of hydroxylated metabolites present are minimal. The pattern of metabolism of 3PBA does not significantly vary with dose (0.1-100 mg/kg) or route (ip and po) in the sheep, gerbil, or mouse. When administered 4'HO3PBA, the gerbil and mouse eliminate principally glucuronide and sulfate conjugates rather than amino acid conjugates, which are only minor components (less than 10% of the dose) in each case. This implies that hydroxylation is a primary metabolic event in determining the eventual fate of 3PBA in many species.     

O-dephenylation and conjugation with benzoylornithine. New metabolic pathways for 3-phenoxybenzoic acid in chickens

Metabolism of [benzyl-14C]fluvalinate by chickens produces 3-phenoxybenzoic acid, which is further degraded by two new pathways. The first pathway involves O-dephenylation, not reported previously for related pyrethroids in birds or mammals. O-Dephenylation is a major metabolic route (12% of the applied 14C). In the second pathway, 2% of the applied dose is converted into four conjugates of benzoylornithine (two with 3-hydroxybenzoic and two with 3-phenoxybenzoic acids). The predominant conjugate with benzoylornithine is N2-(3-hydroxybenzoyl)-N5-benzoylornithine.     

Formation of an N-acetylornithine conjugate of 3-phenoxybenzoic acid in the chicken

The principal metabolite detected in the excreta of chickens treated orally with 3-phenoxybenzoic acid is alpha-N-acetyl-delta-N-(3-phenoxybenzoyl)-L-ornithine. This conjugate has been isolated and purified by preparative HPLC and its structure authenticated by mass and NMR spectrometry. Although diacyl ornithine conjugates of aromatic carboxylic acids are formed in some bird species, monosubstituted derivatives are rare. Moreover, this is the first example of the occurrence of an N-acetylornithine conjugate in the biotransformation of a xenobiotic in animals. The conjugation of such nonnutritive acids in avian species is discussed.     

Effects of cypermethrin, propetamphos, and combination involving cypermethrin and propetamphos on lipid peroxidation in mice

Insecticides are the chemicals widely used in agriculture, environmental health, human-and animal-health fields. Exposure to insecticides has been associated with many hazardous effects, including antioxidative metabolism. In the current study, the effect of cypermethrin (CYP), propetamphos (PRO) and their mixtures on oxidative stress in mice to understand the possible health effects to animals and human beings was investigated. In the present study, 245 male Albino mice weighing 35-40 g were used. The mice were divided into seven groups. The first group served as the control group. The second and third groups were administered CYP at doses of 5 mg/kg/bw and 10 mg/kg/bw, respectively, and the fourth and fifth groups were given PRO at doses of 2.5 mg/kg/bw and 5.0 mg/kg/bw, respectively. The sixth and seventh groups received combination regimens containing 5 mg/kg/bw CYP plus 2.5 mg/kg/bw PRO and 10 mg/kg/bw CYP plus 5 mg/kg/bw PRO, respectively, in feed for 60 days. Blood samples were collected by cardiac puncture on the 15th, 45th and 60th days. Serum nitric oxide (NO) and plasma malondialdehyde (MDA) levels and erythrocyte superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were measured. In conclusion, the alterations observed in the MDA and NO levels and SOD, CAT, and GSH-Px activities of the trial groups, demonstrate the administration of certain doses of CYP and PRO, either alone or combined, to mice for a period of 60 days to produce oxidative stress. The degree of oxidative stress was found to be related to the dose administered, the duration of exposure and the administration of the indicated compounds either alone or as a combination.     

The toxic effect of opioid analgesics on human sperm motility in vitro

Opioid analgesics are the most common therapeutic analgesic for acute pain. In this study, the toxicological and pharmacological features of a group of opioid analgesics were characterized by the motility of human sperm. Aliquots of sperm were incubated with various concentrations of opioid analgesics in vitro. Computer-assisted sperm analysis was used to assess sperm motility at 15 minutes, 2 hours, and 4 hours after drug addition to the medium. Butorphanol and dezocine showed marked reduction of motility after incubation with sperm for 15 minutes. Butorphanol was more effective than dezocine in immobilizing sperm. Other opioids studied, such as fentanyl, alfentanil, and sufentanil, showed only partial inhibitory activity. Based on the data reported herein, we have found that butorphanol and dezocine exert a sperm-immobilizing effect. However, fentanyl, alfentanil, and sufentanil exhibit only partial inhibition of sperm motility. Given the increasing use of opioids and their potential effect on sperm motility, these findings are greatly relevant to male reproductive health.     

In vitro effects of arecoline on sperm motility and cyclooxygenase-2 expression

Semen samples were obtained from 30 volunteers who had never consumed betel quid. Swim-up spermatozoa from the 30 seminal samples of non-betel quid chewers and also non-smokers, usually not exposed to passive smoking, were treated in vitro with arecoline at different concentrations to evaluate the action of these drugs on sperm motility. Highly motile sperms were collected and divided into 5 equal fractions. Four fractions were supplemented with various concentrations of arecoline and one as control. The study was carried out at time 0 and +1, +2, +3 and +4 hr of incubation. Sperm cells were also extracted and blotted with COX-2 antibody after arecoline treatment after 4 hr incubation. The sperm motility parameters, i.e., motility, average path velocity, curvilinear velocity, straight-line velocity and linearity, were significantly decreased after arecoline treatment. In vitro, arecoline induces the COX-2 expression of sperm cells in a dose-dependent manner. This is the first report to demonstrate that arecoline may mediate COX-2 expression in human sperms, resulting in inflammation response. This situation may act on the structure responsible for the flagellar motion and cause the reduction of sperm motility.     

Effects of metronidazole,ipronidazole, and dibromochloropropane on rabbit and human sperm motility and fertility

Treatment of protozoal pathogens in the reproductive system with chemical agents exposes flagellated sperm cells to potential toxicants. A widely used antiprotozoal agent is metronidazole. Its effect on rabbit and human sperm was compared with a more soluble 5-nitroazole compound, ipronidazole, and with a systemic environmental toxicant, dibromochloropropane (DBCP). The percentages of motile rabbit and human sperm incubated with the compounds, the velocity of sperm, migration of sperm in polyacrylamide gel, young born in rabbits, and penetration of hamster oocytes by treated human sperm were measured in seven experiments. Up to 10 mg/ml metronidazole and 1 mg/ml DBCP had little effect on most sperm characteristics. However, 10 mg/ml metronidazole and 5 mg/ml of ipronidazole increased attachment of human sperm to hamster oocytes, but oocyte penetration was unaffected. Rabbit sperm exposed to 5 mg/ml ipronidazole were infertile. No oocytes were penetrated by DBCP-treated human sperm.     

Effects of lithium and haloperidol on human sperm motility in-vitro.

Two psychotropic drugs, lithium and haloperidol, were evaluated for their in-vitro effects on sperm motility using a transmembrane migration method. Sperm motility was measured either immediately after semen had been mixed with the drug or after a 2 h incubation period at 37 degrees C. Lithium inhibited human sperm motility in a dose-dependent manner with an EC50 of 10 mM when the semen-lithium mixture had been incubated. Sperm motility was increased to 127% of control when semen had been incubated with 0.027 microM haloperidol; this concentration was within the therapeutic range.