In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane.

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C - PKC; phospholipases A2 and Cζ - PLA2, PLCζ; soluble adenylyl cyclase 10 - sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 - Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Epicatechin Prevents Cryocapacitation of Bovine Spermatozoa through Antioxidant Activity and Stabilization of Transmembrane Ion Channels.

Epicatechin (EPC) is a flavonoid belonging to the family of catechins; it has been described as a powerful scavenger of a wide spectrum of reactive oxygen species (ROS) and a modulator of ex vivo sperm vitality. In this study, we assessed the potential protective abilities of EPC on cryopreserved bovine spermatozoa. We focused on conventional quality parameters, as well as the oxidative profile of spermatozoa alongside capacitation patterns, and expression profiles of proteins involved in the process of capacitation. Semen samples were cryopreserved in the presence of 25, 50 or 100 µmol/L EPC and compared to native semen (negative control) as well as ejaculates frozen in the absence of EPC (positive control). A dose-dependent improvement of conventional sperm quality parameters was observed following EPC administration, particularly in case of the sperm motility, membrane, acrosome and DNA integrity in comparison to the positive control. Experimental groups exposed to all EPC doses presented with a significantly lower proportion of capacitated spermatozoa as opposed to the positive control. While no significant effects of EPC were observed in cases of superoxide production, a significant decrease in the levels of hydrogen peroxide and hydroxyl radical were recorded particularly in the experimental groups supplemented with 50 and 100 µmol/L EPC. Western blot analysis revealed that supplementation of particularly 100 µmol/L EPC to the semen extender prevented the loss of the cation channel of sperm (CatSper) isoforms 1 and 2, sodium bicarbonate cotransporter (NBC) and protein kinase A (PKA), which play important roles in the process of sperm capacitation. In summary, we may hypothesize that EPC is particularly effective in the stabilization of the sperm membrane during the freeze-thaw process through its ability to quench ROS involved in damage to the membrane lipids and to prevent the loss of membrane channels crucial to initiate the process of sperm capacitation. These attributes of EPC provide an additional layer of protection to spermatozoa exposed to low temperatures, which may be translated into a higher post-thaw structural integrity and functional activity of male gametes.

Effect of Environmental Stressors, Xenobiotics, and Oxidative Stress on Male Reproductive and Sexual Health.

This article examines the environmental factor-induced oxidative stress (OS) and their effects on male reproductive and sexual health. There are several factors that induce OS, i.e. radition, metal contamination, xenobiotic compounds, and cigarette smoke and lead to cause toxicity in the cells through metabolic or bioenergetic processes. These environmental factors may produce free radicals and enhance the reactive oxygen species (ROS). Free radicals are molecules that include oxygen and disbalance the amount of electrons that can create major chemical chains in the body and cause oxidation. Oxidative damage to cells may impair male fertility and lead to abnormal embryonic development. Moreover, it does not only cause a vast number of health issues such as ageing, cancer, atherosclerosis, insulin resistance, diabetes mellitus, cardiovascular diseases, ischemia-reperfusion injury, and neurodegenerative disorders but also decreases the motility of spermatozoa while increasing sperm DNA damage, impairing sperm mitochondrial membrane lipids and protein kinases. This chapter mainly focuses on the environmental stressors with further discussion on the mechanisms causing congenital impairments due to poor sexual health and transmitting altered signal transduction pathways in male gonadal tissues.

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

Exceptional Properties of Lepidium sativum L. Extract and Its Impact on Cell Viability, Ros Production, Steroidogenesis, and Intracellular Communication in Mice Leydig Cells In Vitro.

The prevalence of reproductive dysfunction in males has risen in the last few years, and alternative therapies are gradually gaining in popularity. Our in vitro study aimed to evaluate the potential impact of Lepidium sativum L. on mice TM3 Leydig cells, concerning basal parameters such as cell viability, cell membrane integrity, and lysosomal activity, after 24 h and 48 h exposure. Moreover, reactive oxygens species generation, sex-steroid hormone secretion, and intercellular communication were quantified. In the present study, the microgreen extract from Lepidium was rich in ferulic acid, 4-OH benzoic acid, and resveratrol, with a significant antioxidant activity. The results showed that lower experimental doses (62.5-250 µg/mL) could positively affect the observed parameters, with significant differences at 250 µg/mL after 24 h and 48 h, respectively. Potential risks could be associated with higher concentrations, starting at 500 µg/mL, 1000 µg/mL, and 2000 µg/mL of Lepidium. Nevertheless, biochemical quantification indicated a significant antioxidant potential and a rich content of biologically active molecules at the applied doses, and time determined the intracellular response of the cultured model.

The effect of brown seaweed and polyphenol supplementation in male rabbits on the blood profile and antioxidant markers.

Currently, in animal nutrition, the replacement of synthetic substances with natural ones was expected to improve animal health. The aim of the present study was to evaluate the effects of a dietary brown seaweed and plant polyphenol mixture in adult male rabbits on the haematological profile and antioxidant markers. Twenty-four adult male rabbits were divided into three experimental groups receiving a control diet (C) or diets supplemented with 0.3% (T1) and 0.6% (T2) of a feed additive containing brown seaweed (Laminaria spp.) and plant extracts of seaweed origin. The trial lasted for 90 days. A lower potassium concentration was observed at 30 days in the T2 group, compared with the T1 and C groups. An increase in the antioxidant status was observed (P < 0.05) from day 60 of the trial in the rabbits fed diets with an algae-polyphenolic supplement (T1 and T2 groups). Concluding, the diet supplementation of brown seaweed and polyphenol stimulates the antioxidant status of the blood, however, it does not affect the haematological profile.

Inflammation, It's Regulation and Antiphlogistic Effect of the Cyanogenic Glycoside Amygdalin.

The inflammatory reaction accompanies in part or in full any disease process in the vascularized metazoan. This complicated reaction is controlled by regulatory mechanisms, some of which produce unpleasant symptomatic manifestations of inflammation. Therefore, there has been an effort to develop selective drugs aimed at removing pain, fever, or swelling. Gradually, however, serious adverse side effects of such inhibitors became apparent. Scientific research has therefore continued to explore new possibilities, including naturally available substances. Amygdalin is a cyanogenic glycoside present, e.g., in bitter almonds. This glycoside has already sparked many discussions among scientists, especially about its anticancer potential and related toxic cyanides. However, toxicity at different doses made it generally unacceptable. Although amygdalin given at the correct oral dose may not lead to poisoning, it has not yet been accurately quantified, as its action is often affected by different intestinal microbial consortia. Its pharmacological activities have been studied, but its effects on the body's inflammatory response are lacking. This review discusses the chemical structure, toxicity, and current knowledge of the molecular mechanism of amygdalin activity on immune functions, including the anti-inflammatory effect, but also discusses inflammation as such, its mediators with diverse functions, which are usually targeted by drugs.

In vivo effects of aflatoxin B1 and benzo[a]pyrene on the heart muscle of chicken embryos.

The purpose of the present study was to evaluate the effects of aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP) on the heart muscle of chicken embryos of the broiler line Ross 308. The benzo[a]pyrene in the organic oil solution was injected in ovo on the 6th day of the incubation in doses of: 0.1, 0.5, and 1 mg/kg weight of eggs; the aflatoxin B1 in the organic oil solution was injected in ovo on the 6th day of the incubation into the yolk in doses of 80, 120 and 240 ng/kg weight of eggs. Multiple biochemical and hepatic parameters have been observed, including sodium, potassium, chloride, cholesterol, uric acid, total proteins, aminotransferase aspartate, and aminotransferase alanine. A low dose of AFB1 and BaP administered in ovo during early embryonic development had a significant impact on chicken embryonic development, as demonstrated by alterations in biochemical, mineral, and hepatic parameters.

Resveratrol attenuates hydrogen peroxide-induced oxidative stress in TM3 Leydig cells in vitro.

The objective of present study was to investigate in vitro protective potential of resveratrol in TM3 Leydig cells with induced oxidative stress using hydrogen peroxide (H2O2). Leydig cells experiencing oxidative stress exhibit reduced activities in androgens production, and become hypofunctional with age, which is also related to growing oxidative stress, while resveratrol has received growing attention as a cytoprotective agent. TM3 mouse Leydig cells were cultivated during 24 h in the presence of resveratrol (5, 10, 25, 50 and 100 µM) alone, or in combination with H2O2 (300/600 µM) to induce oxidative stress. Mitochondrial activity was evaluated using MTT test, triple assay was used in order to assess cell viability parameters, intracellular generation of superoxide was determined by the nitroblue-tetrazolium assay, and quantification of steroid hormones was performed by the enzyme- linked immunosorbent assay. Resveratrol alone treatment led to the most significantly improved values of all tested parameters in the cells of experimental group with addition of 10 µM of resveratrol in comparison to the control group. In the case of cells with induced oxidative stress (300 µM H2O2) resveratrol administration resulted in significantly increased (P < 0.05) metabolic activity, as well as cell membrane integrity at concentration 10 µM. Significantly improved (P < 0.001) lysosomal activity showed cells treated with 5 and 10 µM of resveratrol, and the level of both measured hormones was significantly higher (P < 0.05) in cells supplemented with 10 µM of resveratrol. Significant decline of superoxide radical production was observed in all experimental groups in comparison to the control exposed to H2O2 alone. With respect to cells exposed to higher concentration of H2O2 (600 µM), results showed positive effect of resveratrol only in biosynthesis of both androgens with significant increased values in experimental group treated with 5 µM (P < 0.05) and 10 µM (P < 0.01) of resveratrol, in addition, in the case of testosterone we recorded significant higher (P < 0.05) values in cells with addition of 25 and 50 µM resveratrol when compared to H2O2 control. More specific and systematic research focused especially on androgen biosynthesis is necessary related to the biological activity of resveratrol in male reproductive system due to inconsistent results of studies.

Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro.

Copper is an environmental risk factor, which has various effects on reproductive endocrinology. In this study human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of copper sulfate (CuSO4.5H2O) on steroidogenesis and cytotoxicity. The cell cultures were exposed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of CuSO4.5H2O and compared to control group (medium without CuSO4.5H2O). Cell viability was measured by the metabolic activity assay. Quantification of sexual steroid production directly from the medium was performed by ELISA assay. Following 48 h culture of NCI-H295R cell line in the presence of CuSO4.5H2O a dose-dependent depletion of progesterone release was observed even at the lower concentrations of CuSO4.5H2O. The lowest levels of progesterone were detected in groups with the higher doses (≥ 250 µM) of CuSO4.5H2O, which elicited significant cytotoxic action. Testosterone production decreased significantly, and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at 1000 µM of CuSO4.5H2O. The cytotoxic effect of CuSO4.5H2O was evident at all concentrations used in the study. The presented data suggest that copper has detrimental effects on sexual steroid hormones and consecutively on reproductive physiology.

Exposure to non-ionizing electromagnetic radiation of public risk prevention instruments threatens the quality of spermatozoids.

The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation-based examinations. For the determination of effect of non-ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand-held metal detector in various exposition times (0, 10 s, 15, 30 and 60 min-groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10 s, 1 and 5 min (insemination doses radiated before cryoconservation-CB, CB10, CB1, CB5; samples radiated after freezing-CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post-thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short-term spermatozoa stimulation induced by NIR.

Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus Faecalis in Rabbit Semen.

Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

Identification of in vitro effect of 4-octylphenol on the basal and human chorionic gonadotropin (hCG) stimulated secretion of androgens and superoxide radicals in mouse Leydig cells.

The aim of our in vitro study was to assess the potential effect of 4-octylphenol (4-OP) on the basal and human chorionic gonadotropin (hCG)-stimulated cholesterol levels and biosynthesis of steroid hormones in cultured mouse Leydig cells. In addition, we evaluated the intracellular superoxide production following 4-OP treatment. Isolated mouse Leydig cells were cultured in the presence or absence of 1 IU/mL (hCG) with the addition of 0.04; 0.2; 1.0; 2.5 and 5.0 µg/mL 4-OP during 44 h. The level of cholesterol was determined from the culture medium using photometry. Quantification of steroid hormones was performed by the enzyme linked immunosorbent assay and intracellular generation of superoxide radicals was assessed by the nitroblue-tetrazolium assay. Administered concentrations of 4-OP (0.04-5.0 µg/mL) did not affect basal and hCG-stimulated cholesterol level significantly. However, basal DHEA secretion was increased significantly (P < 0.001) in the highest experimental dose (5 µg/mL) of 4-OP. By hCG-stimulated DHEA secretion, a significant (P < 0.001) decrease was recorded at 5.0 µg/mL 4-OP in comparison to the control group. The stimulatory effect of 4-OP was also confirmed in androstenedione secretion, when 2.5 and 5.0 µg/mL increased hormone secretion significantly (P˂0.05; P˂0.001). Exposure to experimental concentrations (0.04 to 5.0 µg/mL) of tested chemical reduced hCG-stimulated androstenedione formation, but not significantly. Measurements of basal testosterone production have shown significant (P˂0.01; P˂0.001) increase at 1.0; 2.5 and 5.0 µg/mL of 4-OP. Furthermore, 44 h treatment by 4-OP (1.0-5.0 µg/mL) caused significant (P˂0.01; P˂0.001) intracellular accumulation of superoxide radicals in exposed cells. A considerably more detailed and systematic research is required for a better understanding of risks associated with male reproductive system in humans and wildlife.

Effects of 4-nonylphenol on the steroidogenesis of human adrenocarcinoma cell line (NCI-H295R).

In this study, the human H295R adrenocarcinoma cell line was exposed to different concentrations (0.04, 0.2, 1.0, 2.5 or 5 µg/mL) of nonylphenol (NP) to investigate its impact on the inhibition or induction of the steroid hormones production during 48 h of in vitro culture. The hormone production was measured using ELISA kits. Results of this in vitro study suggest various effect of nonylphenol in relatively low concentrations on the selected steroid hormones production by the human H295R adrenocarcinoma cell line. The inhibiting impact on progesterone and androstenedione production was observed. The amount of progesterone was significantly decreased at 1.0, 2.5 and 5 µg/mL NP. Equally, the androstenedione production significantly decreased at 5 µg/mL NP. On the other hand, the amount of testosterone and 17ß-estradiol was induced after nonylphenol exposition. The significant increase of testosterone level was found out at treatment with 5 µg/mL NP. 17ß-estradiol production significantly increased at the doses of 2.5 and 5 µg/mL NP.

Resveratrol offers protection to oxidative stress induced by ferrous ascorbate in bovine spermatozoa.

Resveratrol (RES) is a natural polyphenol and phytoestrogen exhibiting cardioprotective, anticancer, antibacterial and vasorelaxing properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. This study was designed to determine the efficiency of RES to reverse the ROS-mediated impairment of the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to RES treatment (5, 10, 25 and 50 µmol L(-1)) in the presence or absence of a pro-oxidant, i.e., ferrous ascorbate (FeAA; 150 µmol L(-1) FeSO4 and 750 µmol L(-1) ascorbic acid) during a 6-h in vitro culture. Spermatozoa motion parameters were assessed using the SpermVision computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro experiments in order to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (P < 0.001) and viability (P < 0.001), decreased the antioxidant parameters of the samples (P < 0.001 in case of SOD; P < 0.01 with respect to CAT; P < 0.05 in relation to GSH) but increased the superoxide production (P < 0.001) and lipid peroxidation (P < 0.001). RES supplementation resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (P < 0.001 in case of SOD; P < 0.01 with respect to 25-50 µmol L(-1) RES and P < 0.05 in relation to 10 µmol L(-1) RES; P < 0.05 in case of GSH), with 50 µmol L(-1) RES proving to be the most effective RES concentration. Our results suggest that RES possesses significant antioxidant properties that may prevent the deleterious effects caused by ROS to spermatozoa, and preserve the fertilization potential of male reproductive cells.

Endocrine disruptive effects of cadmium on steroidogenesis: human adrenocortical carcinoma cell line NCI-H295R as a cellular model for reproductive toxicity testing.

Cadmium (Cd) is a known endocrine disruptor with the ability to affect the production of hormones involved in the regulation of reproductive processes. In this study human adrenocortical carcinoma cell line NCI-H295R was used as an in vitro biological model to study the effect of cadmium (CdCl2) on steroidogenesis. The cell cultures were exposed to different concentrations of CdCl2 (1.90, 3.90, 7.80, 15.60, 31.20 and 62.50 µM) and compared to control (medium without CdCl2). Cell viability was measured by the metabolic activity (MTT) assay for estimation of mitochondria structural integrity. Quantification of sexual steroid production directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Following 48 h culture of the cells in the presence of CdCl2 a concentration-dependent depletion in progesterone production was observed at the lower concentrations of CdCl2. The lowest amount of progesterone was significantly detected in groups with the higher doses (≥ 31.20 µM) of CdCl2, which elicited significant (P < 0.01) cytotoxic action, too. Cadmium decreased testosterone release in the whole applied range even at the lower concentration of CdCl2. The release of 17ß-estradiol decreased as well, but the decline was less pronounced compared to decrease of progesterone and testosterone. The cytotoxic effect was significantly (P < 0.01) detected at all concentrations of CdCl2 (1.90-62.50 µM) used in the study. However, the cell viability remained relatively high (>75%) up to 7.80 µM of CdCl2 and significantly (P < 0.01) decreased at 15.60 µM and higher concentrations of CdCl2. These results suggest that cadmium has endocrine disruptive effects on sexual steroid synthesis even at very low concentrations.

Dose- and time-dependent effects of bisphenol A on bovine spermatozoa in vitro.

The present study was to evaluate the effect of bisphenol A (BPA) at the doses 1, 10, 100 and 200 µg mL(-1) on the bovine spermatozoa motility, viability and production of superoxide radical. The CASA system was used to determine the spermatozoa motility. The initial motility showed the significant differences (P < 0.001) between the groups higher than 100 µg BPA mL(-1) and the control group. Evaluation of the spermatozoa motility after 6 h of cultivation at the doses > 10 µg BPA mL(-1) was found to decrease motility significantly. After 24 h it was observed that the doses < 10 µg BPA mL(-1) statistically increased motility, while the doses > 100 µg BPA mL(-1) significantly decreased motility in comparison to control. The viability of spermatozoa as detected by the MTT assay decreased in all experimental groups, but significant differences were noted only at the highest doses of BPA after 24 h of in vitro cultivation. The intracellular superoxide production was observed by the NBT test after 24 h of BPA exposure. The results indicated that in all experimental groups the amount of superoxide increased as compared to the control group; significant changes were observed at the doses > 100 µg BPA mL(-1). In conclusion, the results from our experiments suggest the negative effects of BPA at the highest doses used on motility and viability of bovine spermatozoa and production of superoxide radical.

Effect of mercury on porcine ovarian granulosa cells in vitro.

The objective of this in vitro study was to examine dose-dependent changes in the secretion activity [progesterone (P4) and insulin-like growth factor-I (IGF-I)] of porcine ovarian granulosa cells after experimental mercury (Hg) administration, including its apoptotic potential so as to ascertain the possible involvement of Hg in steroidogenesis. Ovarian granulosa cells were incubated with mercuric chloride [mercury (II) chloride or HgCl2] at the doses 50-250 µg mL(-1) for 18 h and compared with control group without Hg addition. Release of P4 and IGF-I by ovarian granulosa cells was assessed by RIA and apoptosis by TUNEL assay. Observations show that P4 release by granulosa cells was significantly (P < 0.05) inhibited at all the doses, while IGF-I release was not affected at any of the doses used, although a decreasing trend in the release of IGF-I was noted in comparison to control. An increasing trend of apoptosis of granulosa cells was noted, the difference being significant (P < 0.05) only at the dose 130 µg mL(-1) HgCl2, in comparison to control. Obtained data suggest a direct effect of Hg on the release of steroid hormone progesterone but not growth factor IGF-I, and a dose-dependent effect on apoptosis of porcine ovarian granulosa cells. Results indicate the interference of Hg in the pathways of steroidogenesis and apoptosis of porcine ovarian granulosa cells.

Blood concentration of copper, cadmium, zinc and lead in horses and its relation to hematological and biochemical parameters.

Environmental pollution results in serious health hazards to animals and blood analysis serves as a good alternative for health status assessment. The target of this study was to analyze the concentration of selected metals in equine blood, to analyze the blood parameters and to find possible correlations. Blood samples were collected from the vena jugularis of healthy adult horses. The highest concentration of all elements was found in whole blood (Cu 3.84 ± 0.90 mg L(-1); Cd = 0.81 ± 0.90 mg L(-1); Zn 26.67 ± 14.12 mg L(-1); Pb 9.33 ± 5.76 mg L(-1)). Higher concentrations of copper, cadmium, zinc and lead were detected in blood clots compared to blood sera (44.04%). A similar tendency was found for cadmium (50%), zinc (13.08%) and lead (46.02%), which showed generally higher concentrations in blood clots (cells). Correlation analysis proved some relations between analyzed elements. In blood clots there is a strong positive correlation between Cd - Pb (r = 0.93) and Zn - Pb (r = 0.71) was detected. For biochemical and hematological parameters mainly medium correlations were detected. Obtained results prove different correlations of analyzed elements in blood components as well as the effect on parameters of blood biochemical and hematological profiles.