Circadian effects of ionizing radiation on reproductive function and clock genes expression in male mouse.

BACKGROUND: Exposure to the ionizing radiation (IR) encountered outside the magnetic field of the Earth poses a persistent threat to the reproductive functions of astronauts. The potential effects of space IR on the circadian rhythms of male reproductive functions have not been well characterized so far. METHODS: Here, we investigated the circadian effects of IR exposure (3 Gy X-rays) on reproductive functional markers in mouse testicular tissue and epididymis at regular intervals over a 24-h day. For each animal, epididymis was tested for sperm motility, and the testis tissue was used for daily sperm production (DSP), testosterone levels, and activities of testicular enzymes (glucose-6-phosphate dehydrogenase (G6PDH), sorbitol dehydrogenase (SDH), lactic dehydrogenase (LDH), and acid phosphatase (ACP)), and the clock genes mRNA expression such as Clock, Bmal1, Ror-α, Ror-ß, or Ror-γ. RESULTS: Mice exposed to IR exhibited a disruption in circadian rhythms of reproductive markers, as indicated by decreased sperm motility, increased daily sperm production (DSP), and reduced activities of testis enzymes such as G6PDH, SDH, LDH, and ACP. Moreover, IR exposure also decreased mRNA expression of five clock genes (Clock, Bmal1, Ror-α, Ror-ß, or Ror-γ) in testis, with alteration in the rhythm parameters. CONCLUSION: These findings suggested potential health effects of IR exposure on reproductive functions of male astronauts, in terms of both the daily overall level as well as the circadian rhythmicity.

Irradiation of carotid baroreceptor with low-intensity pulsed ultrasound exerts different metabolic protection in perirenal, epididymal white adipose tissue and interscapular brown adipose tissue of obese rats.

This study was designed to clarify whether the irradiation of carotid baroreceptor (CB) with low-intensity pulsed ultrasound (LIPUS) protects against obesity by rebalancing the autonomic nervous system (ANS). Obesity was induced using a high-fat diet (HFD) for 8 weeks in Sprague-Dawley rats. Irradiation with LIPUS was daily (20 minutes a day) applied to the right CB. In our study, LIPUS significantly ameliorated metabolic disorders in obese rats. LIPUS partly restored norepinephrine (NE) and acetylcholine (ACH) levels in the perirenal white adipose tissue (PWAT), epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT), and plasma of obese rats. LIPUS partially rectified the dysregulated AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor (PPAR) α/É£ pathway in the PWAT, EWAT, and IBAT of obese rats. PPARγ and PPARγ target genes respond more sensitively to HFD and LIPUS in PWAT and EWAT than in IBAT. NE, ACH, uncoupling protein-1, phosphorylated AMPK, PPARα, and PPARα target genes respond more sensitively to HFD and LIPUS in IBAT than in PWAT and EWAT. Conclusion: LIPUS irradiation of CB exerts different metabolic protection in PWAT, EWAT, and IBAT by rebalancing the ANS and rectifying the AMPK/PPARα/É£ pathway in obese rats.

The effectiveness of traditional Japanese medicine Goshajinkigan in irradiation-induced aspermatogenesis in mice.

BACKGROUND: Infertility and gonadal dysfunction are well known side-effects by cancer treatment in males. In particularly, chemotherapy and radiotherapy induced testicular damage, resulting in prolonged azoospermia. However, information regarding therapeutics to treat spermatogenesis disturbance after cancer treatment is scarce. Recently, we demonstrated that Goshajinkigan, a traditional Japanese medicine, can completely rescue severe busulfan-induced aspermatogenesis in mice. In this study, we aimed to detect the effects of Goshajinkigan on aspermatogenesis after irradiation. METHODS: This is animal research about the effects of traditional Japanese medicine on infertility after cancer treatment. C57BL/6 J male mice received total body irradiation (TBI: a single dose of 6Gy) at 4 weeks of age and after 60 days were reared a Goshajinkigan (TJ107)-containing or TJ107-free control diet from day 60 to day 120. Then, two untreated females were mated with a single male from each experimental group. On day 60, 120 and 150, respectively, the sets of testes and epididymis of the mice in each group after deep anesthetization were removed for histological and cytological examinations. RESULTS: Histological and histopathological data showed that 6Gy TBI treatment decreased the fertility rate (4/10) in the control diet group; in contrast, in the TJ107-diet group, the fertility rate was 10/10 (p < 0.05 vs. 6Gy group). Supplementation with TJ107 was found to rescue the disrupted inter-Sertoli tight junctions via the normalization of claudin11, occludin, and ZO-1 expression and reduce serum anti-germ cell autoantibodies. CONCLUSIONS: These findings show the therapeutic effect on TBI-induced aspermatogenesis and the recovering disrupted gonadal functions by supplementation with TJ107.

Multigenerational exposure to uranium changes morphometric parameters and global DNA methylation in rat sperm.

There is increasing evidence that environmental exposures early in fetal development influence phenotype and give rise to disease risk in the next generations. We previously found that lifelong exposure to uranium, an environmental contaminant, induced subtle testicular and hormonal defects; however, its impact on the reproductive system of multiple subsequent generations was unexplored. Herein, rats were exposed to a supra-environmental and non-nephrotoxic concentration of natural uranium (U, 40 mg·L-1 of drinking water) from postnatal life to adulthood (F0), during fetal life (F1), and only as the germ cells from the F1 generation (F2). General parameters (reproductive indices, epididymal weight) and sperm morphology were assessed in the three generations. In order to identify the epigenetic effects of U, we analyzed also the global DNA methylation profile and described for the first time the mRNA expression levels of markers involved in the (de)methylation system in rat epididymal spermatozoa. Our results showed that the F1 generation had a reduced pregnancy rate. Despite the sperm number being unmodified, sperm morphology was affected in the F0, F1 and F2 generations. Morphometric analysis for ten parameters was detailed for each generation. No common parameter was detected between the three generations, but the head and the middle-piece were always modified in the abnormal sperms. In the F1 U-exposed generation, the total number of abnormal sperm was significantly higher than in the F0 and F2 generations, suggesting that fetal exposure to uranium was more deleterious. This effect could be associated with the pregnancy rate to produce the F2 generation. Interestingly, global DNA methylation analysis showed also hypomethylation in the sperm DNA of the last F2 generation. In conclusion, our study demonstrates that uranium can induce morphological sperm defects and changes in the DNA methylation level after multigenerational exposure. The epigenetic transgenerational inheritance of U-induced reproductive defects should be assessed in further experiments.

Infrared photobiomodulation (PBM) therapy improves glucose metabolism and intracellular insulin pathway in adipose tissue of high-fat fed mice.

Obesity represents a continuously growing global epidemic and is associated with the development of type 2 diabetes mellitus. The etiology of type 2 diabetes is related to the resistance of insulin-sensitive tissues to its action leading to impaired blood glucose regulation. Photobiomodulation (PBM) therapy might be a non-pharmacological, non-invasive strategy to improve insulin resistance. It has been reported that PBM therapy in combination with physical exercise reduces insulin resistance. Therefore, the aim of this study was to investigate the effects of PBM therapy on insulin resistance in obese mice. Male Swiss albino mice received low-fat control diet (n = 16, LFC) or high-fat diet (n = 18, HFD) for 12 weeks. From 9th to 12th week, the mice received PBM therapy (LASER) or Sham (light off) treatment and were allocated into four groups: LFC Sham (n = 8), LFC PBM (n = 8), HFD Sham (n = 9), and HFD PBM (n = 9). The PBM therapy was applied in five locations: to the left and right quadriceps muscle, upper limbs and center of the abdomen, during 40 s at each point, once a day, 5 days a week, for 4 weeks (780 nm, 250 mW/cm2, 10 J/cm2, 0.4 J per site; 2 J total dose per day). Insulin signaling pathway was evaluated in the epididymal adipose tissue. PBM therapy improved glucose tolerance and phosphorylation of Akt (Ser473) and reversed the HFD-induced reduction of GLUT4 content and phosphorylation of AS160 (Ser588). Also, PBM therapy reversed the increased area of epididymal and mesenteric adipocytes. The results showed that chronic PBM therapy improved parameters related to obesity and insulin resistance in HFD-induced obesity in mice.

[Reaction of Reproductive System and Epididymal Spermatozoa .of Rats to Electromagnetic Radiation from Mobile Phone (1745 MHz) of Various Duration].

Changes in the weight of reproductive system organs, quantitative and qualitative indicators of the epididymal spermatozoa of rats on the Ist and 30th days after the end of an electromagnetic exposition from the mobile phone (1745 MHz, the 8 hour/day, power density 0.2-20 µW/cm²) of various duration (from 1 to 90 days) have been researched. A significant increase in the absolute and relative weight of the epididymis and seminal vesicles and the number of epididymal spermatozoa was revealed on the 1st day after electromagnetic expo- sure during 7 days. The increase in the exposure duration up to 14 days is accompanied by a fall of the above indicators, and after a longer exposure (30, 60 days) there is an increase of the absolute weight of the testes; in all other cases, significant deviations in terms of the weight of reproductive organs are not observed. Expo- sition of various duration at 1745 MHz, except for 7-day radiation exposure, had no significant effect on the amount of epididymal spermatozoa and DNA fragmentation in them; however, viability of mature male germ cells in irradiated animals decreases independently of the duration of exposure. The revealed disturbances of the researched indicators are mainly normalized in the remote period (on the 30th day) after electromagnetic radiation.

Effects of 2100 MHz radio frequency radiation on ductus epididymis tissue in rats.

PURPOSE: The use of mobile phones is widespread since the beginning of 1990s. A great debate exists about the possible damage that the Radio Frequency - RF radiation from mobile phones exerts on different organs. The objective of this study was to investigate the possible histopathological effects of 2100 MHz RF radiation on rat ductus epididymis tissue using a light microscopy and immunohistochemical method after one or two month exposure. MATERIALS AND METHODS: The study was performed on 36 adult Wistar albino rats. 2100 MHz RF radiation was used with a specific absorption rate (SAR) of 0.36 W/kg for 30 min/day, 6 days per week for one or two months. There were 3 groups (n = 6 for each group): one month RF exposed group, two months RF exposed group, and the control group. RESULTS: At the end of the study, the structural changes in ductus epididymis tissue were evaluated. In both 2100 MHz RF exposed groups, the rat ductus epididymis sperm were not observed in some channels, a reduction in sperm density in some of the channels drew an attention. The loss of connective tissue and edematous areas were observed in cross channel interstitial connective tissue. In addition, it was observed that vascularization was highly increased with respect to the control group in cross-channel interstitial connective tissue. CONCLUSION: 2100 MHz RF exposure resulted in some structural changes in the male genital ducts of rats (Tab. 1, Fig. 5, Ref 20).

[Effect of External Irradiation and Immobilization Stress on the Reproductive System of Male Rats].

We studied the state of the reproductive system of male rats after irradiation at a dose of 2.0 Gy, immobilization stress (6 hours/day for 7 days) and their combined effects. On the 30th day after the combined treatment (37 days after irradiation) a decrease in the testicular weight by almost 50% compared with the control and lesions connected with the process of spermatogenesis are observed. In the remote period--on the 60th day (67th after irradiation) the effect of irradiation and irradiation in combination with immobilization stress leads to a sharp drop in the number of epididymal sperm (up to 18% of the control), and a reduction of their viability. The reaction ofthe reproductive system to the immobilization stress is expressed in a certain increase in the mass of the testes and epididymis, moderate imbalances in the composition of spermatogenic cells in the testis tissue, and in the long term--in the increased number of epididymal sperm and the decrease in their viability. Changes of testosterone in the blood serum, especially significant for the combined effect, reflect impairments of the regulation of the reproductive system of males under these conditions. With regard to individual indicators of the reproductive system of male rats in some cases, the- combined effects of radiation and stress had a synergistic, or, on the contrary, antagonistic character.

Effects of prenatal exposure to a 900 MHz electromagnetic field on 60-day-old rat testis and epididymal sperm quality.

We investigated the effects of exposure in utero to a 900 megahertz (MHz) electromagnetic field (EMF) on 60-day-old rat testis and epididymis. Pregnant rats were divided into control (CG; no treatment) and EMF (EMFG) groups. The EMFG was exposed to 900 MHz EMF for 1 h each day during days 13 - 21 of pregnancy. Newborn rats were either newborn CG (NCG) or newborn EMF groups (NEMFG). On postnatal day 60, a testis and epididymis were removed from each animal. Epididymal semen quality, and lipid and DNA oxidation levels, apoptotic index and histopathological damage to the testis were compared. We found a higher apoptotic index, greater DNA oxidation levels and lower sperm motility and vitality in the NEMFG compared to controls. Immature germ cells in the seminiferous tubule lumen, and altered seminiferous tubule epithelium and seminiferous tubule structure also were observed in hematoxylin and eosin stained sections of NEMFG testis. Nuclear changes that indicated apoptosis were identified in TUNEL stained sections and large numbers of apoptotic cells were observed in most of the seminiferous tubule epithelium in the NEMFG. Sixty-day-old rat testes exposed to 900 MHz EMF exhibited altered sperm quality and biochemical characteristics.

Effects of microwaves (950 MHZ mobile phone) on morphometric and apoptotic changes of rabbit epididymis.

The effect of mobile phone radiation on human reproduction system is still a matter of debate. In this study, 18 male rabbits were randomly divided into two experimental groups and one control group. Experimental groups received simulated microwaves with the frequency of 950 MHz and the output power of 3 and 6 watts for 2 weeks, 2 h a day. After a week of rest, the microscopic slides from the quada of the excised epididymis were prepared. Then, the diameter of epididymis, the height of epithelium and the number of apoptotic cells in epithelium in study groups were determined. The data were compared using spss software and one-way anova test. The epithelial height and diameter of the epididymis in 3 watt and 6 watt groups had a significant decrease compared to the control group (P < 0.001), while the testosterone level only in 6 watt group was significantly decreased compared to control group. The rate of apoptosis in the epithelial cells of the epididymis had a significant increase only in 6 watt group compared to the control group (P < 0.001). This study showed that the microwaves with the frequency of 950 MHz can have negative impacts on morphometric and apoptotic changes of rabbit epididymis.

Long-term effects of 900 MHz radiofrequency radiation emitted from mobile phone on testicular tissue and epididymal semen quality.

The purpose of this study is to bridge this gap by investigating effects of long term 900 MHz mobile phone exposure on reproductive organs of male rats. The study was carried out on 14 adult Wistar Albino rats by dividing them randomly into two groups (n: 7) as sham group and exposure group. Rats were exposed to 900 MHz radiofrequency (RF) radiation emitted from a GSM signal generator. Point, 1 g and 10 g specific absorption rate (SAR) levels of testis and prostate were found as 0.0623 W/kg, 0.0445 W/kg and 0.0373 W/kg, respectively. The rats in the exposure group were subject to RF radiation 3 h per day (7 d a week) for one year. For the sham group, the same procedure was applied, except the generator was turned off. At the end of the study, epididymal sperm concentration, progressive sperm motility, abnormal sperm rate, all-genital organs weights and testis histopathology were evaluated. Any differences were not observed in sperm motility and concentration (p > 0.05). However, the morphologically normal spermatozoa rates were found higher in the exposure group (p < 0.05). Although histological examination showed similarity in the seminiferous tubules diameters in both groups, tunica albuginea thickness and the Johnsen testicular biopsy score were found lower in the exposure group (p < 0.05, p < 0.0001). In conclusion, we claim that long-term exposure of 900 MHz RF radiation alter some reproductive parameters. However, more supporting evidence and research is definitely needed on this topic.

Circadian alterations of reproductive functional markers in male rats exposed to 1800 MHz radiofrequency field.

In this study, we explored the circadian effects of daily radiofrequency field (RF) exposure on reproductive functional markers in adult male Sprague-Dawley rats. Animals in circadian rhythm (as indicated by melatonin measurements), were divided into several groups and exposed to 1800 MHz RF at 205 µw/cm(2) power density (specific absorption rate 0.0405 W/kg) for 2 h/day for 32 days at different zeitgeber time (ZT) points, namely, ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20. Sham-exposed animals were used as controls in the study. From each rat, testicular and epididymis tissues were collected and assessed for testosterone levels, daily sperm production and sperm motility, testis marker enzymes γ-GT and ACP, cytochrome P450 side-chain cleavage (p450cc) mRNA expression, and steroidogenic acute regulatory protein (StAR) mRNA expression. Via these measurements, we confirmed the existence of circadian rhythms in sham-exposed animals. However, rats exposed to RF exhibited a disruption of circadian rhythms, decreased testosterone levels, lower daily sperm production and sperm motility, down-regulated activity of γ-GT and ACP, as well as altered mRNA expression of cytochrome P450 and StAR. All of these observations were more pronounced when rats were exposed to RF at ZT0. Thus, our findings indicate potential adverse effects of RF exposure on male reproductive functional markers, in terms of both the daily overall levels as well as the circadian rhythmicity.

Effects of exposure to extremely low frequency magnetic fields on spermatogenesis in adult rats.

The constant exposure of modern society to extremely low frequency magnetic fields (ELF-MF) has raised considerable concerns about the potential risks to male reproduction. However, the epidemiological and experimental data remain contradictory and inconclusive. In the present study, we investigated the effects of 50 Hz ELF-MF of 500 µT applied 4 h/day, 7 days/week for 4 and 8 weeks on male reproduction, focusing on changes in spermatogenesis. Several biological endpoints related to testicular function and spermatogenesis were measured, including the following: body mass, masses of testes and epididymis, sperm count and abnormal sperm ratio in the caudal epididymis, serum testosterone level, testicular histology, frequency of 14 stages of the cycle of the seminiferous epithelium and of four stages of meiosis I, germ cell apoptosis and testicular oxidative status. No significant differences were found in the biological endpoints between the sham control and the exposed rats in either the 4- or 8-week exposure period. These negative results may result from the lack of change in serum testosterone. In conclusion, our study indicates that exposure to low intensity ELF-MF may have no adverse effects on spermatogenesis.

[The epididymal adipose tissue of mice after nanosecond pulse-periodic microwave irradiation].

The effect of pulse-periodic microwave radiation (PPMR) of the pulse repetition frequency of 8-25 pulseper second, the peak power density of 1500 W/cm2 on the epididymal adipose tissue of micewas investigated. The effect was assessed by the changes in the fat mass and size of the irradiated adipocytes. It was found that the fat mass and size distribution of adipocytes are affected by irradiation. The effects depend on the pulse repetition frequency and intensity of exposure.

Proteome analysis for profiling infertility markers in male mouse sperm after carbon ion radiation.

Ion radiation or radiotherapy is used to treat male patients with oligozoospermia, azoospermia, temporarily infertility, or even permanent infertility. The present study aims to investigate the potential infertility mechanism of sperm in mice after carbon ion radiation (CIR). The caudal epididymal sperm of male mice whole-body irradiated with carbon ion beam (0.5Gy and 4Gy) were used 7 days after irradiation. A two-dimensional gel electrophoresis approach was employed to investigate the changes in protein expression in the caudal edididymal sperm. Spot detection and matching were performed using the PDQuest 8.0 software. The criteria used to select spots for the analysis were more than a threefold difference in protein quantities (normalized spot volume), which allowed the detection of six differentially expressed proteins. Protein identification was performed using MALDI-TOF-TOF. Six specific proteins were identified by searching the NCBI protein sequence database. Among these proteins, HSP 70-2, PLC, GPX4, ß-tubulin, and GAPDHS were associated with sperm motility, which can affect fertility. ß-tubulin is important in axoneme migration flagellar movement and regulation, and GAPDHS is related to sperm energy supply. We analyzed their expressions using immunoblotting and immunofluorescence. The changes in sperm protein expression after CIR are mainly associated with motility. These proteins are potential markers for the mechanisms of infertility in space or radiotherapy.

The effects of simultaneous combined exposure to CDMA and WCDMA electromagnetic fields on rat testicular function.

Wireless mobile phones and other telecommunication devices are used extensively in daily life. We therefore examined the effects of combined exposure to radiofrequency electromagnetic fields (RF-EMF) on rat testicular function, specifically with respect to sensitive processes such as spermatogenesis. Male rats were exposed to single code division multiple access (CDMA) and wideband code division multiple access (WCDMA) RF signals for 12 weeks. The RF exposure schedule comprised 45 min/day, 5 days/week for a total of 12 weeks. The whole-body average specific absorption rate (SAR) of CDMA and WCDMA was 2.0 W/kg each or 4.0 W/kg in total. We then investigated the correlates of testicular function such as sperm count in the cauda epididymis, testosterone concentration in the blood serum, malondialdehyde concentrations in the testes and epididymis, frequency of spermatogenesis stages, and appearance of apoptotic cells in the testes. We also immunoblotted for p53, bcl2, GADD45, cyclin G, and HSP70 in the testes of sham- and combined RF-exposed animals. Based on the results, we concluded that simultaneous exposure to CDMA and WCDMA RF-EMFs at 4.0 W/kg SAR did not have any observable adverse effects on rat spermatogenesis.

[Effect of anabolic preparation fenobolin on blood, reproductive system and hormone levels in blood serum of fractionally irradiated male rats].

Influence of anabolic drug (phenobolin) injection at a dose of 2.5 mg/kg on some indexes of blood, reproductive system, the level of thyroid hormones, corticosterone and testosterone in blood serum of the male rats exposed to a fractional irradiation at the total dose of 2.0 Gy have been studied. A significant decrease in the quantity of leucocytes and lymphocytes after a fractional irradiation without and with phenobolin introduction was determined. Introduction of anabolic drug resulted in a significant drop of relative mass of testes and, particularly, epididymes, and also in a sharp decrease of the number of mature germ cells isolated from epididymes. A combined action of phenobolin and fractional irradiation (2.0 Gy) led to a significant reduction (3-5 times) in the quantity of mature germ cells in an epididymis. In some cases phenobolin influenced the level of testosterone in blood serum of the intact and irradiated animals. The anabolic steroid drug affects negatively the state of reproductive system of male rats.

Sperm abnormalities in high- and low-dose-rate Gamma-irradiated Korean dark-striped field mice, Apodemus agrarius coreae.

To evaluate the in vivo effects of low-dose-rate (0.7 mGy h(-1)) gamma radiation, abnormal shapes of sperm in the caudal epidydimus of Apodemus agrarius coreae (A. a. coreae) were used. The six categories of abnormal forms (amorphous heads, blunt hooks, excessive hooks, two heads and tails, folded tails and short tails) of sperm were observed eight days after gamma irradiation (0, 0.5, 1 and 2 Gy) with a high dose rate (0.8 Gy min(-1)) and a low dose rate. The frequency of total abnormal sperm gradually increased starting from 0.5 Gy after high-dose-rate radiation. Blunt hooks and short tails shaped sperm, in particular, were gradually increasing in the high-dose-rate irradiated mice. Dose rate reduction effects for the frequency of abnormal sperms in low-dose-rate irradiated mice to high-dose-rate irradiated mice were 1 at 0.5 Gy, 0.7 at 1 Gy and 0.5 at 2 Gy. Our results indicate that low-dose-rate radiation is not detrimental to spermatogenic cells.

The lack of histological changes of CDMA cellular phone-based radio frequency on rat testis.

We examined the histological changes by radiofrequency (RF) fields on rat testis, specifically with respect to sensitive processes such as spermatogenesis. Male rats were exposed to 848.5 MHz RF for 12 weeks. The RF exposure schedule consisted of two 45-min RF exposure periods, separated by a 15-min interval. The whole-body average specific absorption rate (SAR) of RF was 2.0 W/kg. We then investigated correlates of testicular function such as sperm counts in the cauda epididymis, malondialdehyde concentrations in the testes and epididymis, frequency of spermatogenesis stages, germ cell counts, and appearance of apoptotic cells in the testes. We also performed p53, bcl-2, caspase 3, p21, and PARP immunoblotting of the testes in sham- and RF-exposed animals. Based on these results, we concluded that subchronic exposure to 848.5 MHz with 2.0 W/kg SAR RF did not have any observable adverse effects on rat spermatogenesis.

Effect of long-term exposure to a randomly varied 50 Hz power frequency magnetic field on the fertility of the mouse.

A double blind study on BALB/c mice was conducted to examine the effects of extremely low-frequency electromagnetic fields on fertility. The mice were continuously exposed or sham-exposed from conception for two generations to magnetic fields varying between 0.5 and 77 muT. Biological parameters related to fertility were evaluated. Serum testosterone levels and mass of testes and adrenals were determined. No significant difference was found between the sham-exposed and exposed groups for all the biological endpoints, except for sperm motility. A significant difference between the two groups was found prior to the swim-up test with quantitative analysis of sperm motility as well as after the swim-up test for quantitative and qualitative analysis of sperm motility. ELF-EMFs significantly decreased the number of living sperm and the quality of movement of sperm, although these adversities did not impact on the outcome of the other parameters investigated.