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.

Seasonal assessment of selected trace elements in grass carp (Ctenopharyngodon idella) blood and their effects on the biochemistry and oxidative stress markers.

Environmental pollution by anthropogenic activity is still a highly relevant global problem. Aquatic animals are a specifically endangered group of organisms due to their continuous direct contact with the contaminated environment. Concentrations of selected trace elements in the grass carp (Ctenopharyngodon idella) (n = 36) blood serum/clot were monitored. Possible effects of the elements on selected biochemical and oxidative markers were evaluated. The concentrations of trace elements (Al, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Ga, Mn, Mo, Ni, Pb, Sr, Tl, and Zn) were analysed in the fish blood serum and blood clot by inductively coupled plasma optical emission spectrometry (ICP OES). A general scheme of decreasing concentrations of trace elements in the blood serum samples was: Zn ˃ Fe ˃ Sr ˃ Ba ˃ Ni ˃ Al ˃ Cu ˃ Be ˃ Co; < LOQ (below limit of quantification): Bi, Cd, Cr, Ga, Mn, Mo, Pb, Tl; and in the case of the blood clot, the scheme was as follows: Fe ˃ Zn ˃ Sr ˃ Al ˃ Ni ˃ Ba ˃ Cu ˃ Be ˃ Co ˃ Mn; < LOQ (below limit of quantification): Bi, Cd, Cr, Ga, Mo, Pb, Tl. Significant differences among the seasons were detected. The Spearman R correlation coefficients and linear or non-linear regression were used to evaluate direct relationships between trace elements and selected blood biomarkers. The correlation analysis between biochemical parameters (Na, K, P, Mg, AST, ALT, ALP, GGT, TAG, TP, urea, glucose) and trace elements (Al, Ba, Be, Cu, Fe, Ni, Sr, and Zn) concentrations confirmed statistically significant interactions in both seasons (summer and autumn). The regression analysis between oxidative stress markers (ROS, GPx, creatinine, uric acid, and bilirubin) and elements (Al, Ba, Co, Cu, Fe, Ni, and Sr) content confirmed statistically significant interactions. The results point to numerous connections between the observed elements and the physiological parameters of freshwater fish.

Signaling Roleplay between Ion Channels during Mammalian Sperm Capacitation.

In order to accomplish their primary goal, mammalian spermatozoa must undergo a series of physiological, biochemical, and functional changes crucial for the acquisition of fertilization ability. Spermatozoa are highly polarized cells, which must swiftly respond to ionic changes on their passage through the female reproductive tract, and which are necessary for male gametes to acquire their functional competence. This review summarizes the current knowledge about specific ion channels and transporters located in the mammalian sperm plasma membrane, which are intricately involved in the initiation of changes within the ionic milieu of the sperm cell, leading to variations in the sperm membrane potential, membrane depolarization and hyperpolarization, changes in sperm motility and capacitation to further lead to the acrosome reaction and sperm-egg fusion. We also discuss the functionality of selected ion channels in male reproductive health and/or disease since these may become promising targets for clinical management of infertility in the future.

Alpha-linolenic acid alleviates the detrimental effects of lipopolysaccharide during in vitro ovine oocyte development.

During the transition period and early lactation of ruminants with higher production, the reproductive organs are exposed to various stressors, like inflammation stimulators such as lipopolysaccharides (LPS), as a consequence of high concentrate consumption. In this study, we aimed to determine the probable potential of α-linolenic acid (ALA) in alleviating LPS-induced effects in ovine oocytes in vitro as well as the underlying controlling mechanisms. Different concentrations of LPS (0, 0.01, 0.1, 1, and 10 µg/mL) were added to the oocyte maturation medium to evaluate its effect on oocyte developmental competence. Likewise, different concentrations of ALA (0, 10, 50, 100, and 200 µM/mL) were added to the maturation medium to define its effects on oocyte developmental competence. Accordingly, a combination of ALA and LPS in a dose-dependent manner was added to the maturation medium to elucidate their effect on oocyte developmental competence and uncover any possible potential of ALA to alleviate the detrimental effect induced by the presence of LPS. The expressions of candidate genes were measured in mature oocytes treated either with ALA, LPS, or ALA plus LPS. Adding LPS to the maturation medium decreased the cleavage rate of the treated oocytes, and those oocytes reached the blastocyst stage at a lower rate. Adding ALA to the maturation medium in the presence of LPS alleviated the detrimental effects of LPS in a dose-dependent manner, which ultimately led to higher cleavage and blastocyst formation. A higher expression of Trim26, GRHPR, NDUFA, PGC-1α, SOD, CS, SDH, p53, and CAT was observed in LPS-treated oocytes compared with the ALA and control groups. Additionally, CS and CAT transcripts were down-regulated in oocytes in LPS plus ALA-treated group compared to that of the LPS-treated group. These findings revealed that ALA has the potential to alleviate the detrimental effects induced by LPS on in ovine oocytes during maturation in vitro. Thus, LPS-detrimental effect and ALA-preventing mechanisms seem to be regulated through the expression of genes involved in mitochondrial biogenesis and function, oxidative stress, and antioxidant systems.

Short-Term Storage of Rooster Ejaculates: Sperm Quality and Bacterial Profile Differences in Selected Commercial Extenders.

Bacterial contamination of semen has become an important contributor to the reduced shelf life of insemination doses in the poultry industry, which is why antibiotics (ATBs) are an important component of semen extenders. Due to a global rise in antimicrobial resistance, the aim of this study was to assess the efficiency of selected commercially available semen extenders to prevent possible bacterial contamination of rooster ejaculates. Two selected extenders free from or containing 31.2 µg/mL kanamycin (KAN) were used to process semen samples from 63 healthy Lohmann Brown roosters. Phosphate-buffered saline without ATBs was used as a control. The extended samples were stored at 4 °C for 24 h. Sperm motility, viability, mitochondrial activity, DNA integrity and the oxidative profile of each extended sample were assessed following 2 h and 24 h of storage. Furthermore, selective media were used to quantify the bacterial load and specific bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicate that semen extenders enriched with KAN ensured a significantly higher preservation of sperm quality in comparison to their KAN-free counterparts. Bacterial load was significantly decreased in diluents supplemented with ATBs (p ≤ 0.001); however, KAN alone was not effective enough to eradicate all bacteria since several Escherichia coli, Enterococcus faecalis, Enterococcus faecium and Micrococcus luteus were retrieved from samples extended in KAN-supplemented commercial extenders. As such, we may suggest that more focus should be devoted to the selection of an optimal combination and dose of antibiotics for poultry extenders, which should be accompanied by a more frequent bacteriological screening of native as well as extended poultry semen.

Strategies for Bacterial Eradication from Human and Animal Semen Samples: Current Options and Future Alternatives.

The primary role of semen processing and preservation is to maintain a high proportion of structurally and functionally competent and mature spermatozoa, that may be used for the purposes of artificial reproduction when needed, whilst minimizing any potential causes of sperm deterioration during ex vivo semen handling. Out of a multitude of variables determining the success of sperm preservation, bacterial contamination has been acknowledged with an increased interest because of its often unpredictable and complex effects on semen quality. Whilst antibiotics are usually the most straight-forward option to prevent the bacterial contamination of semen, antimicrobial resistance has become a serious threat requiring widespread attention. As such, besides discussing the consequences of bacteriospermia on the sperm vitality and the risks of antibiotic overuse in andrology, this paper summarizes the currently available evidence on alternative strategies to prevent bacterial contamination of semen prior to, during, and following sperm processing, selection, and preservation. Alternative antibacterial supplements are reviewed, and emphasis is given to modern methods of sperm selection that may be combined by the physical removal of bacteria prior to sperm preservation or by use in assisted reproductive technologies.

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.

Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability.

For decades now, sperm cryopreservation has been a pillar of assisted reproduction in animals as well as humans. Nevertheless, the success of cryopreservation varies across species, seasons, and latitudes and even within the same individual. With the dawn of progressive analytical techniques in the field of genomics, proteomics, and metabolomics, new options for a more accurate semen quality assessment have become available. This review summarizes currently available information on specific molecular characteristics of spermatozoa that could predict their cryotolerance before the freezing process. Understanding the changes in sperm biology as a result of their exposure to low temperatures may contribute to the development and implementation of appropriate measures to assure high post-thaw sperm quality. Furthermore, an early prediction of cryotolerance or cryosensitivity may lead to the establishment of customized protocols interconnecting adequate sperm processing procedures, freezing techniques, and cryosupplements that are most feasible for the individual needs of the ejaculate.

Ejaculatory Abstinence Affects the Sperm Quality in Normozoospermic Men-How Does the Seminal Bacteriome Respond?

This study was designed to describe bacterial profiles of ejaculates collected following a long and short ejaculatory abstinence set in the context of changes in the conventional, oxidative, and immunological characteristics of semen. Two specimens were collected in succession from normozoospermic men (n = 51) following 2 days and 2 h, respectively. Semen samples were processed and analyzed according to the World Health Organization (WHO) 2021 guidelines. Afterwards, sperm DNA fragmentation, mitochondrial function, levels of reactive oxygen species (ROS), total antioxidant capacity, and oxidative damage to sperm lipids and proteins were evaluated in each specimen. Selected cytokine levels were quantified using the ELISA method. Bacterial identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that samples collected following two days of abstinence presented with a higher bacterial load and diversity, and a greater prevalence of potentially uropathogenic bacteria including Escherichia coli, Staphylococcus aureus and Enterococcus faecalis. Only staphylococci and Escherichia coli remained present in specimens obtained after 2 h of abstinence. Whilst all samples accomplished the criteria set by WHO, a significantly higher motility (p < 0.05), membrane integrity (p < 0.05), mitochondrial membrane potential (p < 0.05), and DNA integrity (p < 0.0001) were detected following 2 h of ejaculatory abstinence. On the other hand, significantly higher ROS levels (p < 0.001), protein oxidation (p < 0.001), and lipid peroxidation (p < 0.01) accompanied by significantly higher concentrations of tumor necrosis factor alpha (p < 0.05), interleukin-6 (p < 0.01), and interferon gamma (p < 0.05) were observed in specimens collected after two days of abstinence. It may be summarized that shorter ejaculatory abstinence does not compromise sperm quality in normozoospermic men, while it contributes to a decreased occurrence of bacteria in semen which is accompanied by a lower probability of damage to spermatozoa by ROS or pro-inflammatory cytokines.

Seminal Bacterioflora of Two Rooster Lines: Characterization, Antibiotic Resistance Patterns and Possible Impact on Semen Quality.

This study aimed to characterize the bacterial profiles and their association with selected semen quality traits among two chicken breeds. Thirty Lohmann Brown and thirty ROSS 308 roosters were selected for semen quality estimation, including sperm motility, membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. The oxidative profile of the semen, including the production of reactive oxygen species (ROS), antioxidant capacity, protein, and lipid oxidation, were assessed as well. Moreover, the levels of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukins 1 and 6 (IL-1, IL-6) and C-reactive protein, as well as the concentrations of selected antibacterial proteins (cathelicidin, ß-defensin and lysozyme) in the seminal plasma were evaluated with the enzyme-linked immunosorbent assay. The prevailing bacterial genera identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were Citrobacter spp., Enterococcus spp., Escherichia spp. and Staphylococcus spp. While the bacterial load was significantly higher in the ROSS 308 line (p < 0.05), a higher number of potentially uropathogenic bacteria was found in the Lohmann Brown roosters. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains, particularly to ampicillin, tetracycline, chloramphenicol, and tobramycin. Furthermore, Lohmann Brown ejaculates containing an increased proportion of Escherichia coli presented with significantly (p < 0.05) elevated levels of TNF-α and IL-6, as well as ROS overproduction and lipid peroxidation. Inversely, significantly (p < 0.05) higher levels of ß-defensin and lysozyme were found in the semen collected from the ROSS 308 roosters, which was characterized by a higher quality in comparison to the Lohmann Brown roosters. In conclusion, we emphasize the criticality of bacteriospermia in the poultry industry and highlight the need to include a more complex microbiological screening of semen samples designated for artificial insemination.

Bacterial DNase activity as a putative inductor of sperm DNA fragmentation in infected bull frozen-thawed semen samples.

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine semen straws infected with bacteria showed clear evidence of DNase activity when compared to plasmid incubated in similarly prepared non-infected bovine diluent supernatant (Experiment 1). This DNase activity was subsequently found to be time dependent (0-60 min) and its activity prevented in the presence of EDTA (10 and 20 mM; Experiment 2). Semen straws infected (n = 10) and not infected (n = 10) with bacteria where incubated at 37 °C for up to 48h post-thaw. Semen infected with bacteria showed an exponential increase in bacterial growth and a corresponding increase in sperm DNA fragmentation. Non-infected semen samples showed no change in the incidence of sperm DNA fragmentation over the same period of incubation (Experiment 3). Our experiments reinforce the idea that exogenous DNases present in the semen should be considered as one of the primary contributing causes of sperm DNA fragmentation post ejaculation. In the case of the bull, post-thaw incubation of commercial straws contaminated with bacteria, resulted in increased levels of sperm DNA fragmentation, most likely associated with DNase activity (potentially restriction endonucleases) derived from the bacteria. Such adverse changes in sperm DNA fragmentation, as described here in vitro, may be also operative after insemination in the female reproductive tract (in vivo) and highlight the importance of implementing high levels of hygiene practice during semen processing, especially in light of future trends of bacterial resistance to the common antibiotics used in semen diluents.

Quercetin Ameliorates Testicular Damage in Zucker Diabetic Fatty Rats through Its Antioxidant, Anti-Inflammatory and Anti-Apoptotic Properties.

The aim of this study was to investigate the effects of quercetin (QUE) on the testicular architecture as well as markers of oxidative, inflammatory, and apoptotic profile of male gonads in Zucker diabetic fatty (ZDF) rats suffering from Type 2 diabetes mellitus in the absence or presence of obesity. QUE was administered orally at a dose of 20 mg/kg/day for 6 weeks. Morphometric analysis revealed that QUE treatment led to an improvement in testicular appearance, particularly in the case of Obese ZDF rats. Furthermore, a significant stabilization of the antioxidant capacity (p < 0.05), superoxide dismutase and catalase activity (p < 0.01), with a concomitant decrease in lipid peroxidation (p < 0.05) were observed in Obese ZDF animals exposed to QUE. Our data also indicate a significant decline in the levels of interleukin (IL)-1 (p < 0.05), IL-6 (p < 0.01) and tumor necrosis factor alpha (p < 0.001) following QUE supplementation to Obese ZDF rats in comparison with their respective control. Finally, a significant down-regulation of the pro-apoptotic BAX protein (p < 0.0001) was observed in Obese ZDF rats administered with QUE, while a significant Bcl-2 protein overexpression (p < 0.0001) was recorded in Lean ZDF animals when compared to their untreated control. As such, our results suggest that QUE is a potentially beneficial agent to reduce testicular damage in ZDF rats with Type 2 diabetes mellitus by decreasing oxidative stress, chronic inflammation, and excessive cell loss through apoptosis.

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.

Characterization of the structural, oxidative, and immunological features of testis tissue from Zucker diabetic fatty rats.

The purpose of this study was to characterize the testicular profile of Zucker diabetic fatty (ZDF) rats presenting with type 2 diabetes mellitus (DM2) in the absence or presence of obesity. To achieve this, testes were collected from 270-day-old male Wistar (n = 15), ZDF nonobese (n = 15), and ZDF obese rats (n = 16). Changes to the testicular structure were quantified morphometrically, while immunocytochemistry was employed to assess caspase-3 activity. Reactive oxygen species (ROS) production, fluctuations of major antioxidant molecules, and the extent of damage to the proteins and lipids were assessed in tissue lysates. Levels of selected interleukins (ILs) were determined by enzyme-linked immunosorbent assay. The results reveal significant alterations to the testicular structure accompanied by caspase-3 overexpression, particularly in ZDF obese rats. The most notable disruption of the oxidative balance, characterized by ROS overproduction, antioxidant deficiency, protein, and lipid deterioration was recorded in ZDF rats suffering from both DM2 and obesity. Accordingly, the highest concentrations of pro-inflammatory IL-1, IL-6, and IL-18 accompanied by reduced levels of the anti-inflammatory IL-10 were found in testicular tissue collected from ZDF obese rats. This study highlights the vulnerability of male gonads to pathophysiological changes caused by hyperglycemia, which are further exacerbated by excessive adipose tissue.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences.

Low temperatures during cryopreservation activate a cascade of changes, which may lead into irreversible damage and reduction of the fertilization potential, including the process of premature capacitation. The aim of our study was to evaluate the range of cell damage following the cryopreservation process and possible activation of cryocapacitation in bovine spermatozoa. For the experiments semen samples were obtained from 30 sexually mature Holstein bulls. Within the analysed parameters, we focused on the functional activity, structural integrity, capacitation status and oxidative profile. The samples were divided into three experimental groups, control (CTRL), in vitro capacitated (CAP) and cryopreserved (CRYO). Based on the collected data, there was a significant decrease in the sperm motility, mitochondrial membrane potential and concentration of cyclic adenosine monophosphate in the CRYO group when compared to CAP and CTRL (P<0.0001). A significant decrease (P<0.01; P<0.0001) in the membrane and acrosome integrity as well as DNA fragmentation index and a significant increase (P<0.0001) of necrotic cells were observed in the CRYO group. Following capacitation, a significant increase (P<0.01; P<0.0001) was recorded in the number of cells which underwent the acrosome reaction in the CRYO group against CAP and CTRL. Changes in the oxidative profile of the CRYO group indicates an increase (P<0.0001) in the reactive oxygen species generation, except for the superoxide radical, which was significantly higher (P<0.0001; P<0.001) in the CAP group in comparison with CRYO and CTRL. In summary, premature capacitation may be considered a consequence of cryopreservation and the assessed parameters could serve as physical markers of cryogenic damage to bovine spermatozoa in the future.

Bacteriospermia - A formidable player in male subfertility.

Bacterial colonization of male reproductive tissues, cells, and fluids, and the subsequent impact of bacteria on the sperm architecture, activity, and fertilizing potential, has recently gained increased attention from the medical and scientific community. Current evidence strongly emphasizes the fact that the presence of bacteria in semen may have dire consequences on the resulting male fertility. Nevertheless, the molecular basis underlying bacteriospermia-associated suboptimal semen quality is sophisticated, multifactorial, and still needs further understanding. Bacterial adhesion and subsequent sperm agglutination and immobilization represent the most direct pathway of sperm-bacterial interactions. Furthermore, the release of bacterial toxins and leukocytic infiltration, associated with a massive outburst of reactive oxygen species, have been repeatedly associated with sperm dysfunction in bacteria-infested semen. This review serves as a summary of the present knowledge on bacteriospermia-associated male subfertility. Furthermore, we strived to outline the currently available methods for assessing bacterial profiles in semen and to outline the most promising strategies for the prevention and/or management of bacteriospermia in practice.

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men.

This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis, Staphylococcus capitis and Micrococcus luteus. The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.

Chemical and Biological Characterization of Melaleuca alternifolia Essential Oil.

The essential oil of Melaleuca alternifolia, commonly known as tea tree oil, has many beneficial properties due to its bioactive compounds. The aim of this research was to characterize the tea tree essential oil (TTEO) from Slovakia and its biological properties, which are specific to the chemical composition of essential oil. Gas chromatography/mass spectroscopy revealed that terpinen-4-ol was dominant with a content of 40.3%. γ-Terpinene, 1,8-cineole, and p-cymene were identified in contents of 11.7%, 7.0%, and 6.2%, respectively. Antioxidant activity was determined at 41.6% radical inhibition, which was equivalent to 447 µg Trolox to 1 mL sample. Antimicrobial activity was observed by the disk diffusion method against Gram-positive (G+), Gram-negative (G-) bacteria and against yeasts, where the best antimicrobial activity was against Enterococcus faecalis and Candida albicans with an inhibition zone of 10.67 mm. The minimum inhibitory concentration showed better susceptibility by G+ and G- planktonic cells, while yeast species and biofilm-forming bacteria strains were more resistant. Antibiofilm activity was observed against Pseudomonas fluorescens and Salmonella enterica by MALDI-TOF, where degradation of the protein spectra after the addition of essential oil was obtained. Good biological properties of tea tree essential oil allow its use in the food industry or in medicine as an antioxidant and antimicrobial agent.

Aflatoxin B1 impairs in vitro early developmental competence of ovine oocytes.

Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 µM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 µM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 µM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 µM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 µM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.