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

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.

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.

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.

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.

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.