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This study was designed to determine the degree and type of bacterial contamination in boar semen (79 ejaculates from Large White and Landrace boars) and its consequences for sperm quality during storage (27 extended semen samples, 16 o C for five days) under practical conditions of artificial insemination (AI). The results revealed the presence of aerobic bacteria in 99% of the ejaculates (from 80 to 370 ×10 6 colony-forming units/mL). Most of the ejaculates contained two or three bacterial contaminants, while the Staphylococcus, Streptococcus, and Pseudomonas bacterial genera were most frequently isolated. Also detected were Enterobacter spp., Bacillus spp., Proteus spp., Escherichia coli, P. fluorescens, and P. aeruginosa. In general, the growth of certain bacterial types isolated prior to semen processing (Enterobacter spp., E. coli, P. fluorescens, and P. aeruginosa) was not discovered on different days of storage, but fluctuations (with a tendency towards increases) were found in the frequencies of Bacillus spp., Pseudomonas spp., and Staphylococcus spp. isolates up to the end of storage. Semen preserved for five days exhibited decreases in sperm motility and increases in the average number of total aerobic bacteria; this was associated with sperm agglutination, plasma membrane disruption, and acrosome damage. We inferred that, due to the different degrees and types of bacterial contaminants in the boar ejaculates, the inhibitory activity of some antimicrobial agents used in swine extenders (such as gentamicin sulfate) may be limited. Because such agents can contribute to the overgrowth of certain aerobic bacteria and a reduction in the quality of stored semen, procedures with high standards of hygiene and microbiological control should be used when processing boar semen.

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Abnormal standard semen characteristics and reduced sperm chromatin maturity can appear with increasing male age. However, the influence of paternal age on semen parameters is still controversial. Therefore, this study was designed to estimate the influence of paternal age not only on conventional semen characteristics but also on sperm DNA integrity. This research was carried out on ejaculated sperm cells obtained from men (n = 1124) aged ≥40 y and <40 y. Our data revealed a decreased semen volume and an increased percentage of DFI (sperm DNA fragmentation index) in older men compared to younger men in the entire study cohort, in men with normozoospermia and in men with abnormal semen parameters. Moreover, there was a higher incidence of sperm DNA damage (>10% DFI, low fertility potential) in the groups of men aged ≥40 y than in the groups of men aged <40 y. Older men had over twice the odds ratio for high sperm DNA damage as younger men. Our findings suggest a detrimental effect of advanced paternal age on sperm chromatin integrity. The data show that the evaluation of sperm DNA has greater clinical utility than standard semen analysis in case of male fertility potential assessment.

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The aim of this study was to analyze changes in sperm plasma membrane integrity, mitochondrial activity, and extracellular environment during storage of boar semen at 5 °C, 16 °C, and 25 °C for 10 days. Progressive sperm motility, plasma membrane integrity (SYBR-14/PI-test and HOS test), aspartate aminotransferase (AAT), and mitochondrial activity (JC-1-test and NADH-dependent NBT assay), as well as pH and osmolality were assessed in nine ejaculates of Polish Landrace boars. The plasma membrane integrity of the semen stored at 5 °C was similar to that of the semen stored at 16 °C and 25 °C; however, an increase in AAT activity of the semen stored at 5 °C revealed sperm membrane disorders as early as day 2. Significant differences in the progressive motility of the semen stored at 5 °C and 16 °C were observed at each of the evaluation times. This reduced motility was consistent with decreased sperm mitochondrial transmembrane potential and oxidoreductive capability. We inferred that the cooling of boar semen to 5 °C increases the permeability of the sperm plasma membrane, which may not be revealed by SYBR-14/PI staining or HOS testing. The loss of boar sperm motility that occurs during hypothermic liquid preservation may be connected with alterations in the plasma membrane stability and disorders of mitochondrial transmembrane potential and oxidoreductive capability.

The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.