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This study compared the efficacy of the egg yolk (EY) and soy lecithin (SL) semen extender during 72-h storage at 4 °C in the presence/absence of streptomycin, penicillin, lincomycin, and spectinomycin. Bull semen samples (n = 12) were obtained and stored in either ANDROMED® (SL extender) or TRILADYL CSS® (EY extender). After 24, 48, and 72 h of storage at 4 °C, bacterial colonies (CFU - colony forming units) were grown and counted on tryptic soy and blood agar, and the bacterial isolates were identified using the MALDI-TOF MS Biotyper. Sperm motility, mitochondrial membrane potential (MMP), membrane and acrosome integrity, sperm DNA fragmentation, and reactive oxygen species (ROS) production were analysed. The results showed that the type of semen diluent could be crucial for the efficacy of used antibiotics, since significantly decreased bacterial occurrence following 48 h or no bacterial growth after 72 h was recorded in the SL-based groups. Even the SL medium itself did not favour bacterial growth, as significantly decreased bacterial load was observed after 48 h (P < 0.01) and 72 h (P

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [CtrlC]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (CtrlN). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity (p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to CtrlC. All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa (p < 0.001) than CtrlC. Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR (p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 (p < 0.01), while all TBR doses stabilized the levels of PKC (p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

This study examined the effects of three selected doses of kaempferol (KAE; 12.5, 25 or 50 μM) on bovine sperm motility and oxidative profile directly related to cold storage. We also elucidated the effect of KAE on the expression profiles of heat shock proteins (HSPs) 70 and 90 as well as the pro-apoptotic BCL2-associated X (BAX) protein and the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein. Frozen samples supplemented with KAE were compared with a native control (fresh spermatozoa) and a cryopreserved control, frozen in the absence of KAE. Our results demonstrate that the administration of all KAE doses led to a higher degree of sperm motility (p < 0.05) when compared with the cryopreserved control. The highest levels of protection of sperm DNA (p < 0.05), lipids (p < 0.05) and proteins (p < 0.05) were detected in samples exposed to 25 μM KAE when compared with samples frozen without KAE. Administration of 25 μM KAE led to a significant increase in HSP70 and HSP90 (p < 0.05) when compared with the unsupplemented frozen control. No significant differences were observed in the expression patterns of BAX; however, a significant up-regulation of Bcl-2 protein was observed in the frozen samples enriched with 25 μM KAE when compared with the cryopreserved control (p < 0.05). In summary, we may consider KAE as an effective agent in stabilizing the sperm membranes by preventing reactive oxygen species (ROS) overproduction in the mitochondria and subsequent oxidative damage to molecules critical for a proper sperm architecture and function. These protective properties of KAE may lead to higher post-thaw sperm activity and viability.

Semen quality plays a crucial role in poultry production; however, it may be impaired by the presence of numerous bacterial species. This study researched the impact of bacterial contamination of Lohmann brown rooster semen on the biochemical parameters of seminal plasma to evaluate its potential consequences on the sperm progressive motility. Semen was collected from 27 stud roosters, and the sperm concentration and progressive motility were measured using computer-assisted semen analysis (CASA). Seminal plasma was separated, and selected biochemical parameters were measured using commercially available assays. An aliquot of each semen sample was cultured, the colonies were counted and the MALDI Biotyper was used for bacterial identification. The samples were divided into three categories based on their sperm progressive motility and the data were compared and statistically evaluated. Moreover, Pearson’s correlation analysis was performed. The results showed that the lower the sperm progressive motility, the higher the level of colony-forming units. Moreover, sperm concentration was significantly higher (p < 0.05) in the group with the highest bacterial occurrence and the lowest proportion of progressively motile spermatozoa. Calcium, magnesium, creatinine, uric acid, alkaline phosphatase, and total proteins significantly changed in semen samples with the lowest proportion of progressive motility. In conclusion, seminal plasma biochemistry may mirror changes occurring in semen as a result of bacterial presence in the reproductive tract of poultry.

Bacterial infestation of the male reproductive system with subsequent effects of bacteria on the structural integrity and functional activity of male gametes has become a significant factor in the etiology of male reproductive dysfunction. Bacteria may affect male fertility either by directly interacting with structures critical for sperm survival or indirectly by triggering a local immune response, leukocytospermia or reactive oxygen species (ROS) overproduction followed by oxidative stress development. This review aims to provide an overview of the currently available knowledge on bacteriospermia-associated sperm damage with a special emphasis on oxidative mechanisms underlying sperm deterioration caused by bacterial action. At the same time, we strive to summarize readily available alternatives to prevent or counteract alterations to spermatozoa caused by bacterial colonization of semen or by oxidative stress as an accompanying phenomenon of bacteriospermia.

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.

In the reproductive tissues, free radicals are formed as a result of exogenous and endogenous effects. Their excessive production leads to a state of imbalance with the body's antioxidant system, leading to oxidative stress. Oxidative stress is an important factor that affects the condition of male fertility. Preservation and handling of reproductive structures in vitro are accompanied by oxidative damage. Antioxidants are substances that alleviate the effects of oxidative stress and are responsible for slowing the oxidation of biomolecules. The flavonoid quercetin is a bioactive substance exhibiting free radical-quenching activities. This antioxidant is present in a wide variety of fruits and vegetables. In this study we analyzed the effect of various concentrations of quercetin (1 μmol/L; 10 μmol/L; 100 μmol/L) on the oxidative properties of testicular tissue in rats. Testes from adult Wistar rats were used. Quercetin-enriched medium was added to the testicular fragments, except for the control group, and the samples were cultured at 37 °C (5% CO2) for 24 hours. Lysates were obtained from the samples and used to determine the production of reactive oxygen species (ROS), total antioxidant capacity (TAC), protein carbonyl production, malondialdehyde production (MDA). In the groups supplemented with quercetin, a significant decrease in ROS (P

Cryopreservation is a technique, commonly used for a long-term storage of male gametes as an effective way to preserve their longevity and vitality. During cryopreservation and following the thawing process, sperm cells are exposed to thermal stress, which may lead into disruptions of their morphological structures and a subsequent functional dysfunction. The objective of our study was to monitor HSP90 (heat shock protein 90) gene expression in cryopreserved bovine spermatozoa. HSP90 is one of the main heat-related cellular proteins, which belongs into chaperone protein family. It is involved in the formation of protein complexes, which stabilize proteins against heat stress and aid in the protein degradation. For the experimental purposes were used semen samples from 12 sexually mature Holstein-Friesian bulls. The samples were divided into two identical fractions, one fraction was cryopreserved and kept in liquid nitrogen at -196°C, while the other fraction was processed as non-cryopreserved fresh semen. The sperm motility rate was evaluated using CASA (Computer Assisted Semen Analysis). Quantification of isolated RNA was performed by real-time PCR and the expression of the HSP90 gene was assessed with the Western blot technique. For a proper analysis, we compared cryopreserved sperm samples with fresh ones. The data from our study indicate a reduction in the sperm motility (P

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.

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

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.

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.

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.

Emerging evidence from in vivo as well as in vitro studies indicates that natural biomolecules may play important roles in the prevention or management of a wide array of chronic diseases. Furthermore, the use of natural compounds in the treatment of male sub- or infertility has been proposed as a potential alternative to conventional therapeutic options. As such, we aimed to evaluate the effects of selected natural biomolecules on the sperm production, structural integrity, and functional activity. At the same time, we reviewed their possible beneficial or adverse effects on male reproductive health. Using relevant keywords, a literature search was performed to collect currently available information regarding molecular mechanisms by which selected natural biomolecules exhibit their biological effects in the context of male reproductive dysfunction. Evidence gathered from clinical trials, in vitro experiments and in vivo studies suggest that the selected natural compounds affect key targets related to sperm mitochondrial metabolism and motion behavior, oxidative stress, inflammation, DNA integrity and cell death. The majority of reports emphasize on ameliorative, stimulating and protective effects of natural biomolecules on the sperm function. Nevertheless, possible adverse and toxic behavior of natural compounds has been indicated as well, pointing out to a possible dose-dependent impact of natural biomolecules on the sperm survival and functionality. As such, further research leading to a deeper understanding of the beneficial or adverse roles of natural compounds is necessary before these can be employed for the management of male reproductive dysfunction.

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.