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The main aim of the study was to investigate the chemical composition, antioxidant, antimicrobial, and antibiofilm activity of Citrus aurantium essential oil (CAEO). The biofilm profile of Stenotrophonomonas maltophilia and Bacillus subtilis were assessed using the mass spectrometry MALDI-TOF MS Biotyper and the antibiofilm activity of Citrus aurantium (CAEO) was studied on wood and glass surfaces. A semi-quantitative composition using a modified version was applied for the CAEO characterization. The antioxidant activity of CAEO was determined using the DPPH method. The antimicrobial activity was analyzed by disc diffusion for two biofilm producing bacteria, while the vapor phase was used for three penicillia. The antibiofilm activity was observed with the agar microdilution method. The molecular differences of biofilm formation on different days were analyzed, and the genetic similarity was studied with dendrograms constructed from MSP spectra to illustrate the grouping profiles of S. maltophilia and B. subtilis . A differentiated branch was obtained for early growth variants of S. maltophilia for planktonic cells and all experimental groups. The time span can be reported for the grouping pattern of B. subtilis preferentially when comparing to the media matrix, but without clear differences among variants. Furthermore, the minimum inhibitory doses of the CAEO were investigated against microscopic fungi. The results showed that CAEO was most active against Penicillium crustosum , in the vapor phase, on bread and carrot in situ.

The aim of this study was to assess the chemical composition, antioxidant, antimicrobial and antibiofilm activity of the Coriandrum sativum essential oil. Changes in the biofilm profile of Stenotropomonas maltophilia and Bacillus subtilis were studied using MALDI-TOF MS Biotyper on glass and wooden surfaces. The molecular differences of biofilms in different days were observed as well. The major volatile compounds of the coriander essential oil in the present study were β -linalool 66.07%. Coriander essential oil radical scavenging activity was 51.05% of inhibition. Coriander essential oil expressed the strongest antibacterial activity against B. subtilis followed by S. maltophilia and Penicillium expansum. The strongest antibiofilm activity of the coriander essential oil was found against S. maltophilia. A clearly differentiated branch was obtained for early growth variants of S. maltophilia in case of planktonic cells and all experimental groups and time span can be reported for the grouping pattern of B. subtilis preferentially when comparing to the media matrix, but without clear differences among variants. The results indicate that coriander was effective against the tested Penicillium expansum in the vapor phase after 14 days with MID 50 367.19 and MID 90 445.92 µL/L of air.

Purpose: To evaluate the effect of sperm concentration adjustment in human ejaculates on the sperm DNA quality and longevity., Methods: Semen samples were obtained from 30 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended in PBS, and the sperm concentration adjusted to 200, 100, 50, 25, 12, and 6 × 10 6 /mL. Each set of samples was incubated at 37 °C for 24 h, and the sperm DNA damage was assessed using the chromatin-dispersion test following 0 h, 2 h, 6 h, and 24 h of incubation., Results: Sperm DNA fragmentation (SDF) did not differ between the selected experimental conditions at T0; however, Kaplan-Meier estimates for survival showed significant differences with respect to the dilution and time (all P values were smaller than .001). DNA fragmentation in semen samples adjusted to 200 × 10 6 /mL was approximately 3.3 times higher when compared to samples containing 25 × 10 6 /mL and 3.9 higher in comparison with samples adjusted to 12 × 10 6 /mL following 2 h of in vitro incubation. Although there was evidence of individual variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation., Conclusions: Incubation of spermatozoa for ART purposes should be done following a concentration adjustment below 25 × 10 6 /mL in order to avoid a higher susceptibility of the sperm DNA molecule towards fragmentation.

Aim: The purpose of the study was to evaluate the impact of seminal plasma in human ejaculates on the sperm DNA quality and DNA longevity., Methods: Semen samples for this study were obtained from 20 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended either in the seminal plasma proceeding from its respective donor, or in an equal amount of PBS, adjusting the concentration to 50 × 10 6 /ml. Each set of samples was incubated at 37 °C for 24 h and the sperm DNA damage was assessed using the chromatin-dispersion test following 0, 2, 6, and 24 h of incubation., Results: Sperm DNA fragmentation (SDF) did not differ between the two experimental conditions at T0; however, Kaplan-Meier estimates to test for survival showed an statistical significant increase of SDF in the seminal plasma group when compared to the PBS group following all timeframes (p 0.000). With respect to sperm DNA longevity, the most dramatic loss of sperm DNA quality occurred during the first 2 h of incubation, with the rate of SDF (rSDF) in the seminal plasma being 2.1 more intense than in PBS. Interestingly, the rSDF was found to vary significantly between individuals, which was confirmed with significant correlations in all rSDF timeframes (rSDF T0-2, p 0.049; rSDF T2-6, p 0.000; rSDF T6-24: p 0.000)., Conclusions: Co-incubation of semen samples in seminal plasma after ejaculation increases iatrogenic sperm damage. This effect is statistically significant after 2 h of co-incubation. Subsequently, for ART purposes seminal plasma must be quickly removed after ejaculation-liquefaction, to avoid a higher susceptibility of sperm DNA towards fragmentation.

Nonylphenol is considered an endocrine disruptor and has been reported to affect male reproductive functions. In our in vitro study, we evaluated the effects of 4-nonylphenol (4-NP) on cholesterol levels, hormone formation and viability in cultured Leydig cells from adult ICR male mice. We also determined the potential impact of 4-NP on generation of reactive oxygen species (ROS) after 44 h of cultivation. The cells were cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg/mL of 4-NP in the present of 1 IU/mL human chorionic gonadotropin (hCG) and compared to the control. The quantity of cholesterol was determined from culture medium using photometry. Determination of hormone production was performed by enzyme-linked immunosorbent assay. Metabolic activity assay was used for quantification of cell viability. The chemiluminescence technique, which uses a luminometer to measure reactive oxygen species, was employed. Applied doses of 4-NP (0.04-5.0 μg/mL) slight increase cholesterol levels and decrease production of dehydroepiandrosterone after 44 h of cultivation, but not significantly. Incubation of 4-NP treated cells with hCG significantly (P < 0.001) inhibited androstenedione, but not testosterone, formation at the highest concentration (5.0 μg/mL). The viability was significantly (P < 0.05); (P < 0.001) increased at 1.0; 2.5 and 5.0 μg/mL of 4-NP after 44 h treatment. Furthermore, 44 h treatment of 4-NP (0.04-5.0 μg/mL) caused significant (P < 0.001) intracellular accumulation of ROS in exposed cells. Taken together, the results of our in vitro study reported herein is consistent with the conclusion that 4-nonylphenol is able to influence hormonal profile, cell viability and generate ROS., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Lycopene (LYC) is a natural carotenoid with powerful reactive oxygen species (ROS) scavenging activities. The aim of this study was to investigate if lycopene has the ability to reverse ROS-mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa subjected to ferrous ascorbate (FeAA). Spermatozoa were washed out of fresh bovine semen, suspended in 2.9 % sodium citrate and subjected to LYC treatment (0.25, 0.5, 1 or 2 mmol/L) in the presence or absence of FeAA (150 μmol/L FeSO4 and 750 μmol/L ascorbic acid) during a 6 h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry 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 culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA)., Results: FeAA treatment led to a reduced spermatozoa motility (P < 0.001), viability (P < 0.001) and a decline of the antioxidant capacity of spermatozoa (P < 0.001) but increased the ROS generation (P < 0.001), superoxide production (P < 0.001) and lipid peroxidation (P < 0.001). LYC administration resulted in a preservation of the spermatozoa motion parameters (P < 0.001), mitochondrial activity (P < 0.001) and antioxidant characteristics (P < 0.001 with respect to SOD; P < 0.01 in relation to CAT; P < 0.05 as for GPx and GSH) with a concentration range of 1 and 2 mmol/L LYC revealed to be the most effective., Conclusions: Our results suggest that LYC exhibits significant ROS-scavenging and antioxidant properties which may prevent spermatozoa alterations caused by oxidative stress, and preserve the functionality of male reproductive cells.

Over the past decades, there has been an emphasis on assessment of the use of natural compounds in the prevention or repair of oxidative injury to spermatozoa. Curcumin (CUR) is a natural phenol with powerful antioxidant properties. The aim of the present study was to examine if CUR could reverse reactive oxygen species (ROS)-mediated alterations to the motility, viability and intracellular antioxidant profile of bull spermatozoa subjected to a prooxidant (i.e., ferrous ascorbate - FeAA). Spermatozoa were washed from recently collected semen samples, suspended in 2.9% sodium citrate and subjected to CUR treatment (5, 10, 25 and 50μmol/L) in the presence or absence of FeAA (150μmol/L FeSO4 and 750μmol/L ascorbic acid) during a 6h in vitro culture. Spermatozoa motility characteristics were assessed using the SpermVision computer-aided spermatozoa analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified using luminometry and the nitroblue-tetrazolium (NBT) test was used to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the culture to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). Treatment with FeAA led to a reduced spermatozoa motility (P<0.001), viability (P<0.001) and decreased the antioxidant characteristics of the samples (P<0.001) but increased the ROS generation (P<0.001), superoxide production (P<0.001) and lipid peroxidation (P<0.001). The CUR treatment led to a preservation of spermatozoa motion (P<0.001), mitochondrial activity (P<0.001) and antioxidant characteristics (P<0.05 with SOD and GSH; P<0.01 with CAT and GPx), revealing the concentration range of 25-50μmol/L CUR to be the most effective for sustaining spermatozoa viability. Data from the present study suggest that CUR exhibits significant protective and ROS-scavenging characteristics which may prevent oxidative insults to spermatozoa and thus preserve the functional activity of male gametes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This in vitro study was designed to assess the impact of divalent (Fe(2+)) or trivalent (Fe(3+)) iron on the activity and oxidative balance of bovine spermatozoa at specific time intervals (0, 2, 8, 16, and 24 h) during an in vitro culture. Forty-five semen samples were collected from adult breeding bulls and diluted in physiological saline solution supplemented with different concentrations (0, 1, 5, 10, 50, 100, 200, 500, 1000 μmol/L) of FeCl2 or FeCl3. Spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Both divalent and trivalent iron exhibited a dose- and time-dependent impact on the spermatozoa physiology and oxidative balance. Concentrations ≥50 μmol/L FeCl2 and ≥100 μmol/L FeCl3 led to a significant decrease of spermatozoa motility (P < 0.05) and mitochondrial activity (P < 0.001 with respect to 200-1000 μmol/L FeCl2/FeCl3; P < 0.01 in case of 100 μmol/L FeCl2/FeCl3), accompanied by a significant superoxide overproduction (P < 0.001 in terms of 200-1000 μmol/L FeCl2 and 500-1000 μmol/L FeCl3; P < 0.01 with respect to 100 μmol/L FeCl2 and 100-200 μmol/L FeCl3). On the other hand, concentrations below 10 μmol/L FeCl2 and 50 μmol/L FeCl3 proved to stimulate the spermatozoa activity, as shown by a significant preservation of the motility and viability characteristics (P < 0.001 in case of the motility parameters; P < 0.01 with respect to the spermatozoa viability), alongside a significant decline of the superoxide generation (P < 0.05). In a direct comparison, divalent iron has been shown to be more toxic than trivalent iron. Results from this in vitro study show that high concentrations of both forms of iron are toxic, while their low concentrations may have spermatozoa activity-promoting properties. In vitro concentrations of divalent or trivalent iron that could be regarded as critical are 50 μmol/L FeCl2 and 100 μmol/L FeCl3 when iron ceases to be an essential micronutrient in order to become a toxic risk factor.

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.

Transgenic rabbits are excellent animal models for human diseases and suitable bioreactors for the production of recombinant proteins on an experimental and commercial scale. The aim of this study was to compare the structure of the mWAP-hFVIII transgenic and non-transgenic rabbit ovarian and testicular tissue. Ovarian and testicular tissue samples were taken from transgenic and non-transgenic New Zealand White rabbits, examined by optical microscopy and analyzed morphometrically. An increase of the relative volume of primary follicles and a decrease of the relative volume of antral follicles was detected in the transgenic ovarian structure (P < 0.05), but other developmental follicular stages and follicular diameters were not affected (P > 0.05). In the testes a significant decrease (P < 0.05) of the epithelial height was detected in the transgenic testicular structure, but the relative volume of all basic structures (germinal epithelium, interstitium and lumen) was unaltered (P > 0.05). Generally, this study demonstrates a weak negative effect of mWAP-hFVIII transgenesis on rabbit gonadal structure.

Mutual relationships between selected chemical elements (Na, K, Fe, Cu, Mg, and Zn), basic motility characteristics (motility and progressive motility), and markers of the oxidative balance (superoxide dismutase, catalase, glutathione, albumin, and malondialdehyde) were investigated in bovine seminal plasma and spermatozoa. Computer assisted sperm analysis was used to assess the motility parameters; mineral concentrations were determined by the voltammetric method and flame absorption spectrophotometry; antioxidants and malondialdehyde were evaluated by UV/VIS spectrophotometry. Concentrations of chemical elements in both seminal fractions were in the following descending order: Na > K > Zn > Mg > Fe > Cu. Higher amounts of all minerals and nonenzymatic antioxidants were detected in the seminal plasma (P < 0.01; P < 0.001), while higher MDA concentration and activity of enzymatic antioxidants were recorded in the cell lysates (P < 0.01; P < 0.001). Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant parameters (P < 0.05; P < 0.01; P < 0.001). Inversely, K exhibited the positive associations with malondialdehyde (P < 0.05). This study demonstrates that most chemical elements are integral components of bovine semen and are needed for the protection against oxidative stress development.

The aim of this study was to investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility-MOT, progressive motility-PROG) as well as selected markers of the prooxidant-antioxidant balance (catalase-CAT, glutathione-GSH, malondialdehyde-MDA) in bovine seminal plasma and spermatozoa. Twenty five semen samples were collected from breeding bulls and used in the study. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. The samples were centrifuged, fractions of seminal plasma and spermatozoa were separated, lysates were prepared from the sperm cell fractions. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidants and MDA were analyzed by UV/Vis spectrophotometry. The analysis showed that the average concentration of Pb in the seminal plasma was 0.23 ± 0.02 μg/mL, while its amount in the sperm cells was significantly higher (0.41 ± 0.07 μg/mL; P < 0.05). The average Cd content in bovine seminal fractions was similar and non-significant: 0.09 ± 0.01 μg/mL in the seminal plasma and 0.11 ± 0.01 μg/mL in spermatozoa (P > 0.05). The correlation analysis revealed that both heavy metals were significantly negatively correlated with MOT and PROG (P < 0.05; P < 0.01; P < 0.001), CAT (P < 0.05; P < 0.01) as well as GSH (P < 0.05; P < 0.01) but significantly positively associated with MDA as the marker of lipid peroxidation (P < 0.05; P < 0.01). Moreover the samples were categorized in three quality groups (Excellent, Good, Moderate) according to their motility values. The lowest Pb, Cd and MDA concentrations but the best antioxidant characteristics were found in samples of the best quality, moderate quality samples exhibited the highest Pb, Cd and MDA content together with the worst antioxidant capacity. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of seminal oxidative stress development and a subsequent decrease of male fertility.

Oxidative stress is a state related to increased cellular damage caused by oxygen and oxygen-derived free radicals known as reactive oxygen species (ROS). It is a serious condition, as ROS and their metabolites attack DNA, lipids and proteins, alter enzymatic systems and cell signalling pathways, producing irreparable alterations, cell death and necrosis. While small amounts of ROS have been shown to be required for several functions of spermatozoa, their excessive levels can negatively impact the quality of spermatozoa and impair their overall fertilising capacity. These questions have recently attracted the attention of the scientific community; however, research aimed at exploring the role of oxidative stress and antioxidants associated with male fertility is still at its initial stages. This review summarises the current facts available in this field and intends to stimulate interest in basic and clinical research, especially in the development of effective methods for the diagnosis and therapy of semen damage caused by oxidative stress.