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Polymeric materials from renewable resources have been tested as additives for rubber blends and vulcanisates. Their influence on the vulcanisation process, tensile strength properties and dynamical‐mechanical properties has been studied and compared with silica. The corn starch, collagen hydrolysate, corn protein and various types of lignin have been added to natural and styrene butadiene rubber in order to find convenient fillers for rubber blends which are able to modify the properties of rubber products. Correlation between Wolf activity coefficient and mechanical properties of vulcanisates is discussed. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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 analysed 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., Competing Interests: Declaration of Competing Interest There is no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Background: Apricot kernels containing amygdalin (AMG) as the major cyanogenic glycoside are potentially useful as a complementary therapy for the management of several ailments including cancer. Nevertheless, little is known regarding the toxic and therapeutic doses of AMG, particularly in terms of male reproduction. Hence, this study evaluates selected qualitative characteristics of rabbit testicular tissue following in vivo administration of AMG or apricot kernels for 28 days., Methods: The rabbits were randomly divided into five groups (Control, P1, P2, P3, P4). The Control received no AMG/apricot kernels while the experimental groups P1 and P2 received a daily intramuscular injection of amygdalin at a dose of 0.6 and 3.0 mg/kg of body weight (b.w.) for 28 days, respectively. P3 and P4 received a daily dose of 60 and 300 mg/kg b.w. of crushed apricot kernels mixed with feed for 28 days, respectively. Changes to the testicular structure were quantified morphometrically, while tissue lysates were subjected to the evaluation of reactive oxygen species (ROS) production, total antioxidant capacity, activities of antioxidant enzymes, and glutathione concentration. The extent of damage to the proteins and lipids was quantified as well. Levels of selected cytokines were determined by the enzyme-linked immunosorbent assay while a luminometric approach was used to assess the activity of caspases., Results: Rabbits treated with 3.0 mg/kg b.w. AMG presented a significantly increased protein oxidation ( p = 0.0118) accompanied by a depletion of superoxide dismutase ( p = 0.0464), catalase ( p = 0.0317), and glutathione peroxidase ( p = 0.0002). Significantly increased levels of interleukin-1 beta ( p = 0.0012), tumor necrosis factors alpha ( p = 0.0159), caspase-3/7 ( p = 0.0014), and caspase-9 ( p = 0.0243) were also recorded in the experimental group P2 when compared to the Control. No effects were observed in the rabbits treated with apricot kernels at the oxidative, inflammatory, and histopathological levels., Conclusions: Apricot kernels did not induce toxicity in the testicular tissues of male rabbits, unlike pure AMG, which had a negative effect on male reproductive structures carried out through oxidative, inflammatory, and pro-apoptotic mechanisms., Competing Interests: Given her role as Guest Editor, Eva Tvrdá had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Amedeo Amedei. The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

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-Ctrl N ) as well as semen samples cryopreserved in the absence of KAE (positive control-Ctrl C ). 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 Ctrl C . Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa ( p < 0.001) when compared with Ctrl C . 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 Mg 2+ -ATPase ( p < 0.05) and Na + /K + -ATPase ( p < 0.0001) in comparison to Ctrl C . 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., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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.

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.

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.

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.

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.

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, β-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 β-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.

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.

Artificial insemination, as an essential pillar of the modern poultry industry, primarily depends on the quality of semen collected from stud roosters. Since the collection and storage of ejaculates is not a sterile process, antimicrobial agents have become essential supplements to semen extenders. While the use of traditional antibiotics has been challenged because of rising bacterial resistance, natural biomolecules represent an appealing alternative because of their antibacterial and antioxidant properties. As such, this study strived to compare the effects of 50 μmol/L curcumin (CUR) with 31.2 µg/mL kanamycin (KAN) as a conventional antibiotic on rooster sperm quality in the presence of Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa. Changes in sperm structural integrity and functional activity were monitored at 2 and 24 h of culture. Computer-assisted semen analysis revealed significant sperm motility preservation following treatment with KAN, particularly in the case of Salmonella enterica and Pseudomonas aeruginosa (p < 0.001) after 24 h. On the other hand, CUR was more effective in opposing ROS overproduction by all bacteria (p < 0.05), as determined by luminol-based luminometry, and maintained sperm mitochondrial activity (p < 0.001 in the case of Salmonella enterica; p < 0.05 with respect to Escherichia coli and Pseudomonas aeruginosa), as assessed by the fluorometric JC-1 assay. The TUNEL assay revealed that CUR readily preserved the DNA integrity of rooster sperm exposed to Salmonella enterica (p < 0.01) and Escherichia coli (p < 0.001). The bacteriological analysis showed higher efficiency of KAN in preventing the growth of all selected bacterial species (p < 0.0001) as opposed to CUR. In conclusion, CUR provided protection to rooster spermatozoa against alterations caused by uropathogens, most likely through its antioxidant activity. Hence, CUR supplementation to poultry semen extenders in combination with properly selected antibacterial substances may become an interesting strategy in the management of bacterial contamination during semen storage.

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.

The aim of this study was to evaluate the impact of diabetes mellitus type 2 (DM2) on the male endocrine system of Zucker Diabetic Fatty (ZDF) rats. Sexually mature ZDF rats were divided to a lean (control) and obese group, and had diabetes confirmed by blood tests. For the in vivo experiment, fasting blood was collected to obtain blood plasma. In case of the in vitro experiments, testicular fragments were cultured for 24 h, and the culture medium was collected. The concentrations of testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA-S), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were quantified in the blood plasma and the medium by the ELISA method, while cholesterol (CHOL) was assessed spectrophotometrically. A significant decline of T (36.31 %), A4 (25.11 %) and FSH (26.99 %) as well as a significant increase of CHOL and E2 (36.17 %) was observed in the blood plasma of obese ZDF rats in comparison to the control. Under in vitro conditions, a significant decrease of FSH (23.35 %) accompanied by an increase of E2 was observed in the obese group compared to the control. In the case of CHOL, LH, T, DHEA and A4 no significant differences were observed. Our results suggest that except for FSH and E2 all steroid biomolecules were synthetized normally by the testicular tissue, however a dramatic endocrine disturbance was observed at the system level. We may conclude that DM2 has negative effects on systemic hormone secretion and these alterations are more pronounced in combination with obesity.

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.

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., Competing Interests: Conflict of interest: Authors state no conflict of interest., (© 2022 Eva Tvrdá et al., published by De Gruyter.)

In this study, we evaluated the in vitro effects of 1-50 μM zearalenone (ZEA), deoxynivalenol (DON) and T-2 toxin (T-2) on rabbit spermatozoa for as much as 8 h of in vitro exposure. Our results indicate that all sperm quality parameters were negatively affected by these fusariotoxins in a time- and dose-dependent manner. The most prominent structure affected by ZEA was the plasma membrane, exhibiting alterations consistent with the onset of apoptosis and reactive oxygen species (ROS) overproduction. This correlated with the most prominent decline of the sperm motility among all selected fusariotoxins. Significant necrotic changes and mitochondrial dysfunction were primarily responsible for the sperm damage in the presence of T-2. Finally, exposure of spermatozoa to DON led to a significant decrease in the DNA integrity. This study may provide new information on the specific mechanisms of action involved in the in vitro toxic behavior of fusariotoxins on male gametes.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Conflict of interest: Authors state no conflict of interest., (© 2022 Eva Tvrdá et al., published by De Gruyter.)

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen ( n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

Although bacterial contamination of ejaculates may cause difficulties in cattle reproduction, standard protocols for a routine microbiological analysis of bovine semen are still missing. Understanding of the mechanisms of bacterial damage to spermatozoa may contribute to the prevention and management of bacteriospermia in the future. Therefore, this study was designed to investigate bacterial profiles of fresh bovine ejaculates (n = 30), while at the same time we focused on assessing the relationships between bacteriospermia and selected sperm quality parameters as well as an array of oxidative stress and inflammatory markers. The samples were divided into three quality groups according to the sperm motility: Excellent (EX) - over 90% > Good (GO) - between 89% and 80% > Moderate (MO) - under 80%. The results showed a significant increase in reactive oxygen species (ROS) generation in the GO group when compared to the EX group. In the MO group, a deterioration of almost all quality parameters was observed when compared to the EX group. In particular, sperm motility, mitochondrial membrane potential, ROS production and IL-6 concentration exhibited a significant decline. Pearson correlation analysis revealed positive associations among bacterial load and the presence of leukocytes in semen (r = 0.965), malondialdehyde concentration (r = 0.816) and DNA fragmentation (r = 0.784). MALDI-TOF MS Biotyper analysis showed a prevalence of the Staphylococcus genus. The quantification of bacterial colonies revealed a significantly increased (P < 0.01) bacterial load in the MO group when compared with the EX as well as the GO group. Overall, our results suggest that sperm quality may be affected by both, bacterial composition, and bacterial load. It appears that an increased presence of bacterial species triggers the immune response, causes oxidative stress, and thereby contributes to sperm structural alterations while diminishing their fertilization ability. Abbreviations: EX: Excellent; GO: Good; MO: Moderate; MOT: Motility; ROS: Reactive Oxygen Species; MMP: Mitochondrial Membrane Potential; IL-1: Interleukin 1; IL-6: Interleukin 6; IL-8: Interleukin 8; IL-12: Interleukin 12; CRP: C-reactive protein; DNA: Deoxyribonucleic acid; MALDI-TOF MS: Matrix-assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry; LPO: Lipid peroxidation; CFU: Colony-forming units MDA: Malondialdehyde; CASA: Computer-assisted Sperm Analysis; WS: Working solution; RIPA: Radio-immunoprecipitation assay; TBARS: Thiobarbituric Acid Reactive Substances; BHB: D-β-hydroxybutyrate.

Bacterial contamination of semen is an important factor connected to the health status of bulls that may significantly affect semen quality for artificial insemination. Moreover, some important bovine diseases may be transmitted through semen. Up to now, only a very limited number of complex studies describing the semen microbiome of bulls have been published, as many bacteria are hard to cultivate using traditional techniques. The 16S rRNA high-throughput sequencing strategy allows for the reliable identification of bacterial profiles of bovine semen together with the detection of noncultivable bacterial species. Fresh samples from Holstein Friesian breeding bulls ( n = 55) were examined for the natural variability in the present bacteria. Semen doses were selected randomly from Slovak Biological Services in Nitra, Slovak Republic. The most predominant phyla within the whole dataset were Firmicutes (31%), Proteobacteria (22%), Fusobacteria (18%), Actinobacteria (13%) and Bacteroidetes (12%). Samples of semen were divided into two separate clusters according to their microbiome compositions using a cording partition around a medoids analysis. Microbiomes of the first cluster (CL1) of samples ( n = 20) were based on Actinobacteria (CL1 average = 25%; CL = 28%) and Firmicutes (CL1 = 38%; CL2 = 27%), while the second cluster (CL2; n = 35) contained samples characterized by a high prevalence of Fusobacteria (CL1 = 4%; CL2 = 26%). Some important indicator microbial groups were differentially distributed between the clusters.

Bacteriospermia has become a serious factor affecting sperm quality in swine breeding, this is why antibiotics (ATBs) are a critical component of semen extenders. Due to ever-increasing antimicrobial resistance, the aim of this study was to assess the efficiency of selected commercially available semen extenders to prevent a possible bacterial contamination of boar ejaculates. Three Androstar Plus extenders containing different combinations of antibiotics were used to process ejaculates from 30 healthy Duroc breeding boars. Androstar Plus without antibiotics was used as a control. The extended samples were stored at 17 °C for 72 h. Sperm motility, viability, mitochondrial activity, DNA integrity and oxidative profile of each extended sample were assessed following 24 h, 48 h and 72 h. 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 ATBs ensured a significantly higher preservation of the sperm quality in comparison to the ATB-free control. The total bacterial count was significantly decreased in the extenders supplemented with ATBs ( p < 0.001), however gentamycin alone was not effective enough against Gram-positive bacteria, while a few colonies of Enterococcus hirae , Bacillus subtilis and Corynebacterium spp. were present in the samples extended in the presence of a triple combination of ATBs. In conclusion, we may suggest that semen extenders enriched in antibiotics were not able to fully eliminate the bacteria present in the studied samples. Furthermore, selection of suitable antibiotics for semen extension should be accompanied by adequate hygiene standards during the collection and handling of boar ejaculates.

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus -induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll ® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 10 8 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus , S. warneri , S. kloosii , and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

This study focused on the identification of naturally occurring bacteria in the reproductive fluid and impact on the quality of ejaculates obtained from the turkey breed British United Turkeys (BUT) Big 6 (n = 60). We determined possible relationships between the bacterial load and advanced sperm quality parameters that are important for effective artificial insemination and high fertility, as well as the concentration of selected antimicrobial proteins and pro-inflammatory markers of turkey semen. Sperm motility was assessed with computer-assisted sperm analysis (CASA), while the membrane and acrosome integrity were examined with smearing and staining methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL assay, and the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cell lysates were prepared to investigate the extent of lipid and protein oxidation. Furthermore, levels of interleukins 1 and 6 (IL-1, IL-6), C-reactive protein, cathelicidin, and β-defensin were quantified in the seminal plasma using the ELISA method. The most dominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was Escherichia coli , Proteus mirabilis , Staphylococcus lentus, and Citrobacter braakii . The bacterial load had a negative effect on the sperm motility ( p < 0.001), as well as membrane ( p < 0.05) and acrosome integrity ( p < 0.01). A strong positive relationship between the bacterial load and DNA fragmentation ( p < 0.001) was detected as well. Positive associations were recorded between the increasing presence of bacteria, ROS overgeneration ( p < 0.001), and a subsequent oxidative damage to the proteins ( p < 0.001) and lipids ( p < 0.01). It was revealed that the antimicrobial peptides β-defensin ( p < 0.001) and cathelicidin ( p < 0.001) had a positive relationship with the motility. In contrast, pro-inflammatory markers, such as IL-1 ( p < 0.001) and IL-6 ( p < 0.001), had a negative impact on the motion behavior of turkey spermatozoa. Our results suggest that the semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia is associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for a failed artificial insemination in turkey breeding.

In this study, we evaluated the impact of 5-50 μM quercetin (QUE) and naringenin (NAR) on extended boar spermatozoa in the BTS (Beltsville Thawing Solution) medium for 72 h. Spermatozoa motion, membrane, acrosome, and DNA integrity were investigated immediately after sample dilution (0 h) as well as after 24 h, 48 h, and 72 h of semen storage. Furthermore, reactive oxygen species (ROS) and superoxide production, as well as the extent of oxidative damage to the sperm proteins and lipids, were assessed to determine the potential of QUE and NAR to prevent a potential loss of sperm vitality due to oxidative stress development. Our results indicate that the most notable parameter influenced by QUE was the mitochondrial activity, which remained significantly higher throughout the experiment ( p < 0.001 and p < 0.0001; 10 μM), and which correlated with the most prominent maintenance of sperm motility ( p < 0.01, 48 h; p < 0.05, 72 h). A significant membrane stabilization ( p < 0.01, 24 h and 48 h; p < 0.0001, 72 h) and prevention of lipid peroxidation ( p < 0.05, 24 h and 48 h; p < 0.01, 72 h) was primarily observed following administration of 10 and 25 μM NAR; respectively. Administration of 10 μM QUE led to a significant decrease of superoxide ( p < 0.0001, 48 h and 72 h) while the most notable decline of ROS generation was recorded in the case of 10 and 25 μM NAR ( p < 0.001). This study may provide new information on the specific mechanisms of action involved in the favorable effects of natural biomolecules on spermatozoa.

Schisandra chinensis is a woody vine native to China, Korea, and Russia, which has been used as a traditional herbal remedy to treat male infertility. As very little information is available concerning its effects on ejaculated spermatozoa, the aim of this study was to investigate the chemical, antioxidant, and antibacterial properties of the S. chinensis berry ( Omija ) extract followed by an assessment of its in vitro effects on bovine sperm function and oxidative balance. Phytochemical components of the Omija extract were determined by high performance liquid chromatography. The content of polyphenols, flavonoids, and carotenoids was assessed by spectrophotometric protocols. Antioxidant characteristics of the Omija extract were determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and molybdenum-reducing antioxidant power (MRAP) assays. The disc diffusion method and determination of the minimal inhibitory concentration were applied to study the antibacterial properties of Schisandra . Thirty semen samples were exposed to different concentrations of Omija (1, 5, 10, 25, 50, and 75  µ g/mL) for 0, 2, and 24 h. Sperm motility, mitochondrial activity, and superoxide and reactive oxygen species production, as well as total antioxidant capacity and oxidative damage to proteins and lipids were determined. Our data reveal that the Omija extract, particularly at a concentration range within 5-50  µ g/mL, exhibited dose-dependent motion-promoting and metabolism-enhancing properties, accompanied by significant antioxidant effects. We may conclude that the biomolecules present in the Omija extract such as schisandrins and phenolic molecules offer protection to critical sperm structures against oxidative insults and/or possible bacterial contamination, leading to a higher preservation of mammalian sperm viability and functional activity., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Eva Tvrdá et al.)

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.