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.     

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.     

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.     

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 fenvalerate on progesterone production in cultured rat granulosa cells

In this study, primary serum-free cultured rat granulosa cells (rGCs) were used as a cellular model to investigate the effects of fenvalerate on progesterone production. Various concentrations (0, 1, 5, 25, 125 and 625 microM) of fenvalerate were added to the cell cultures for 24 h. rGCs were stimulated by compounds such as follicle-stimulating hormone (FSH), 8-bromo-cAMP or 22(R)-hydroxycholesterol (22R-HC). Progesterone production and intracellular cAMP content were measured in control and treated groups. Expression of P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) were monitored by real-time PCR and Western blotting. Results showed that fenvalerate inhibited basal progesterone production in rGCs in the absence of stimulators. This inhibition was stronger in the presence of FSH and was not fully reversed by 8-bromo-cAMP or 22R-HC. The increase of cAMP content, stimulated by FSH, was inhibited by fenvalerate implicating that the intracellular cAMP-dependent signal pathway was involved. Fenvalerate reduced mRNA and protein expression of P450scc. These results suggested that multi-site inhibition of progesterone production by fenvalerate including a cAMP-dependent protein kinase pathway and reduction on P450scc gene expression and/or its enzymatic activity in rGCs.     

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.     

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.     

Inhibitory action of halothane on rat masculine sexual behavior and sperm motility

Adult male rats were exposed to inhale halothane in the following regime: 15 ppm/4 h/5 days/week/9 weeks. Sexual behavior observations and sperm motility test were made before halothane exposure (0 days) and at 15, 30, 45 and 60 days of exposure. Fifteen days after halothane exposure, this anesthetic inhibited the proportion of animals displaying ejaculation. In those animals ejaculating, halothane produced an inhibition of masculine sexual behavior reflected as an increase in the intromission latency, number of mounts and postejaculatory interval. At 30 days after exposure, only an increase in the intromission latency was observed. At 45 and 60 days, the inhibitory effect of halothane on sexual behavior disappeared. Similarly, at 15 and 30 days, but not at 45 or 60 days of halothane exposure, a reduced sperm motility was observed. Such transient effects of halothane suggest the development of tolerance to the inhibitory actions of this anesthetic on sexual behavior and sperm motility. These halothane effects are in line with an inhibition of masculine sexual behavior after stimulation of the GABAergic system.     

Preliminary studies on the in vitro and in vivo effect of salicylate on sperm motility.

We have studied the in vitro effect of different concentrations of salicylic acid on the motility of normal human spermatozoa. Sperm motility was evaluated over 48 h at 0, 50, 100, and 200 mg/L of salicylate concentrations. Results are reported as the mean % motility, where 100% motility at each time interval was taken to be the motility of the control (0 mg/L of salicylate). At 48 h, the mean % motility (n = 12) was 61, 43, and 33% at 50, 100, and 200 mg/L of salicylate. Twenty-four-hour values (n = 17) were 75, 65, and 48%. Eosin supravital staining demonstrated that the decrease in sperm motility was caused by direct inhibition of motility rather than by spermatozoal death. Four healthy males (17-51 years of age) gave a semen specimen prior to taking 650 mg of salicylate four times daily, followed by a second semen specimen 72 h later. Each individual, therefore, served as his own control. The mean % motility in the postdrug sample at 2, 24, and 48 h was 49, 53, and 47% of the motility found in the predrug sample (n = 4). We conclude that salicylate significantly decreases sperm motility in vitro and in vivo.     

Vitamin C protects against in vitro cytotoxicity of cypermethrin in rat hepatocytes.

The objective of this study was to investigate the effect of ascorbic acid (AA) on the in vitro cytotoxicity of cypermethrin (CM), and on glutathione (GSH) metabolism in rat hepatocytes. In vitro cell viability, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) leakage were measured, as indicators of hepatic damage, at 1, 15 and 30 min of exposure to CM. Glutathione and the activities of glutathione-S-transferase (GST) and gamma glutamyl transpeptidase (gamma-GT) were also measured. CM hepatotoxicity increased in a time and dose-dependent manner. In the presence of 30 microM CM, ALT and AST also increased 49 and 130% (P < 0.05), respectively, indicating metabolic hepatocyte damage. AA (1 mM) was capable to preserve 100% of cell integrity and modulated ALT and AST. Furthermore, CM induced a 27% reduction in the endogenous antioxidant GSH, and increased 203% GST and 283% gamma-GT (P < 0.05), indicating an oxidative insult. The presence of AA showed chemopreventive capacity against CM, recovering 60% of GSH and a 54% decrease in gamma-GT activity. These results suggest that AA in a 1:33 (CM:AA) ratio can modulate up to 90% of the damage caused to the cells by CM. It also demonstrates that AA can act as a primary antioxidant and hepatoprotector in rat hepatocytes.     

Pentoxifylline stimulates human sperm motility both in vitro and after oral therapy.

Pentoxifylline is a haemorrheologic agent often used in the treatment of peripheral vascular disorders. In this study, we measured sperm motility with a trans-membrane migration method and investigated the effect of this drug in the treatment of male infertility. We found that pentoxifylline increased motility of ejaculated spermatozoa in vitro from both normal and asthenozoospermic samples. After giving pentoxifylline to patients with asthenozoospermia for 3 months, sperm motility significantly increased, but sperm concentration did not increase. From the above results, it can be concluded that pentoxifylline is a useful drug in the treatment of normogonadotropic asthenozoospermia.     

Effects of chlordiazepoxide and FG 7142 on a rat model of diencephalic amnesia as measured by delayed-matching-to-sample performance

 The intralaminar thalamic nuclei (ILn) have been implicated as a critical site of pathology in amnesia. Lesions of the ILn have been found to produce behavioral effects comparable to benzodiazepine (BDZ) receptor agonists. We compared the effects of chlordiazepoxide (CDP), a BDZ agonist, and FG 7142, a partial inverse agonist at the BDZ receptor, in rats with thalamic lesions and in unlesioned controls. Delayed matching-to sample (DMS) performances were studied during treatment with ascending doses of CDP, counterbalanced trials with 2.5 mg/kg CDP and saline, ascending doses of FG 7142, and (for unlesioned controls only) counterbalanced trials with saline and higher doses of CDP. CDP had effects similar to the ILn lesion, decreasing response speed and percent correct responding in a delay-independent fashion. These effects were additive with the impairments associated with the ILn lesion. The effects of FG 7142 were more complex. At lower doses, it increased response speed without affecting response accuracy. At higher doses, it diminished both the speed and the accuracy of DMS responding. These results support the hypothesis that ILn lesions and BDZ agonists have similar effects on DMS performance. The biphasic effects observed for FG 7142 are consistent with other evidence that low doses of this drug enhance while higher doses impair memory performance. Although DMS accuracy was not improved, the enhancement observed for response speed provides evidence that partial inverse BDZ agonists have potential utility as treatments for cognitive impairments associated with amnesia. Received: 24 June 1998 / Final version: 1 October 1998     

Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas)

The steroid hormone progesterone (P4) is found at relatively high concentrations (～300 ng/L) in association with concentrated animal feeding operations (CAFOs). In an effort to better understand the potential endocrine disrupting effects of P4 in male fish, computer assisted sperm analysis (CASA) was used to evaluate the effects of this steroid on sperm motility in the fathead minnow (Pimephales promelas). The rationale for focusing on sperm motility is that certain progestins have been shown to bind to surface membrane receptors on fish spermatozoa and increase sperm swimming velocity. It was hypothesized, therefore, that sperm swimming velocity might be a useful indicator of progestin exposure in fish. Adult male fathead minnows (ages 6-12 months) were exposed to environmentally relevant doses of P4, both longer-term (1 week, in vivo exposure) and short-term (minutes, in vitro exposure). Sperm were then video recorded and analyzed by CASA. When fathead minnows were continuously exposed for 1 week to low levels of progesterone in vivo there was a significant dose-dependent reduction in sperm motility. There was no effect of short-term P4 exposure on fathead minnow sperm swimming characteristics. Additional research is required to elucidate the mechanism by which progesterone alters sperm swimming in the fathead minnow. With further validation, the fathead minnow sperm motility assay may be a useful tool to rapidly screen for endocrine disrupting chemicals in the aquatic environment.     

Effect of paraoxon on erythrocyte metabolism as measured by oxygen uptake in vitro

1. Oxygen consumption in vitro and persistence in the general circulation of rabbit erythrocytes treated with the cholinesterase inhibitor paraoxon were determined.2. Paraoxon in vitro reduced oxygen consumption below a measureable level within 2 hours. By contrast, the metabolic inhibitor N-ethylmaleimide (NEM) produced complete inhibition within 15 minutes.3. Erythrocytes from rabbits orally dosed with parathion also exhibited marked depression of oxygen consumption.4. Glutathione (GSH) restored oxygen uptake to pretreatment levels within 15 min in erythrocytes previously inhibited with NEM or paraoxon.5. Erythrocytes treated with NEM were rapidly removed from the general circulation while paraoxon treated cells were removed at a rate comparable to untreated cells.     

alpha-chlorohydrin induced changes in sperm fertilizing ability in the rat: association with diminished sperm ATP levels and motility

In the present study, alpha-chlorohydrin (ACH) (5, 10, 25, 50 and 75 mg/kg, po) was administered to rats and the effects on sperm ATP levels, sperm motility, and the ability of sperm to bind and penetrate rat oocytes were determined. Groups of rats were killed 5 days and 3 h following treatment. At both time points, sperm from ACH-treated rats (>/=10 mg/kg) had significantly lower levels of ATP when diluted in media containing glucose. No diminution of ATP was seen in sperm diluted in phosphate-buffered saline (PBS). Computer analysis of sperm motility indicated that straight-line velocity (VSL) was the most sensitive parameter to ACH treatment and was significantly decreased in rat sperm three hours after ACH exposure (25 mg/kg). A clear drop in percent penetration (35% vs. 85% in control) of zona-free rat oocytes by rat sperm of both ACH groups was observed at 10 mg/kg. Higher dose levels produced no significant further decrease in percent penetration. Overall, the fertilizing ability of sperm was highly sensitive to ACH doses that caused minor but significant changes in sperm ATP levels and no significant changes in motility. These data are consistent with the spermatozoan's need for an uncompromised energy supply to maintain its ability to bind and penetrate the oocyte.     

Effects of caffeine on alertness as measured by infrared reflectance oculography

RATIONALE: Caffeine is a well-known stimulant that can be used to increase alertness and performance especially in low arousal situations such as monotonous highway driving or after sleep deprivation. The effects of caffeine in rested, alert, participants are less clear, and this may be attributable to difficulties in objectively assessing small changes in alertness. OBJECTIVES: The present study examined the effects of caffeine in non-sleep-deprived participants with methods that have previously been shown to be sensitive to changes in alertness. In order to avoid confounding results, low, or non-users of caffeine, were sought as participants. MATERIALS AND METHODS: Twelve subjects participated in a within-subjects double-blind placebo-controlled design study and were administered either a capsule containing 200 mg of caffeine or placebo on two separate days. Ten-minute long tests of vigilance were performed at baseline and then at 30, 60, 120, 180, and 240 min after swallowing the capsule. During vigilance tests, eye blink variables were measured using infrared reflectance oculography and converted into a drowsiness score, Johns Drowsiness Scale (JDS). RESULTS: Caffeine significantly reduced JDS scores (drowsiness) and reaction times, and these changes persisted for 3 to 4 h. Self reports of sleepiness were not as sensitive, with Karolinska Sleepiness Scale scores only being significantly lower in the caffeine compared to placebo condition at 30 min post capsule administration. CONCLUSIONS: The results demonstrated that despite being well rested, administration of caffeine significantly increased alertness and enhanced performance, and these changes were able to be detected with the JDS.     

Inhibition of metabolic cooperation in vitro and enhancement of enzyme altered foci incidence in rat liver by the pyrethroid insecticide fenvalerate

The synthetic pyrethroids cypermethrin, deltamethrin, fenvalerate, permethrin, and the fenvalerate metabolite p-chlorophenylisovaleric acid were investigated for inhibition of gap-junctional intercellular communication in vitro in the Chinese hamster lung fibroblast (V79) metabolic cooperation assay. Fenvalerate was furthermore studied for enhancement of gamma-glutamyl transpeptidase-positive enzyme altered foci incidence in partially hepatectomized, nitrosodiethylamine-initiated male Sprague Dawley rats. The in vitro studies showed that fenvalerate and p-chlorophenylisovaleric acid were inhibitors of intercellular communication at non-cytotoxic concentrations while cypermethrin, deltamethrin, and permethrin were inactive. In the in vivo study in rat liver, fenvalerate administered p.o. (75 mg/kg/day) 5 days a week for 10 weeks induced significantly more foci per cm³ and a larger percentage of liver tissue occupied by foci tissue compared to a vehicle control group. Analysis of size distributions of foci in fenvalerate- and vehicle-treated rats showed elevated foci incidences in fenvalerate-treated rats at all foci sizes. Fenvalerate induced no hepatotoxic effects as judged by plasma transaminase activities and histopathology. The results of this study suggest fenvalerate to be a potential tumour promoter.     

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.