Bacterial Contamination of Boar Semen and its Relationship to Sperm Quality Preserved in Commercial Extender Containing Gentamicin Sulfate.

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°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 ×106 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.

Influence of chamber type integrated with computer-assisted semen analysis (CASA) system on the results of boar semen evaluation.

The objective of the study was to evaluate the effect of different types of chambers used in computer-assisted semen analysis (CASA) on boar sperm concentration and motility parameters. CASA measurements were performed on 45 ejaculates by comparing three commonly used chambers: Leja chamber (LJ), Makler chamber (MK) and microscopic slide-coverslip (SL). Concentration results obtained with CASA were verified by manual counting on a Bürker hemocytometer (BH). No significant differences were found between the concentrations determined with BH vs. LJ and SL, whereas higher (p<0.01) values of this parameter were obtained with MK. Compared to MK and SL, significantly higher values were recorded in LJ for velocity (VCL and VAP) as well as amplitude of the lateral head displacement (ALH) and beat cross frequency (BCF), which was associated with significantly higher percentages of motile, progressively motile and rapidly progressive motile spermatozoa. Higher values for the linearity (LIN) and straightness (STR) of sperm movement were obtained for the analysis performed in MK and SL. In both these chambers, the results of all the linearity and kinetic parameters of sperm were similar (p>0.05). The results obtained show that CASA assessment of boar semen should account for the effect of counting chamber on the results of sperm motility and concentration, which confirms the need for further study on standardizing the automatic analysis of boar semen.

Serum selenium concentration and glutathione peroxidase activity and selenium content in testes of Polish Konik horses from selenium--deficient area in North-Western Poland.

The aim of this study was to determine serum selenium concentrations in Polish Konik horses residing in the Odra Delta Nature Park (Poland) and to evaluate the activity of glutathione peroxidase and Se content in testes of this horse breed. In over 95% of cases, serum Se concentration was below the optimal range, and none of the horses examined was deficient in this trace element. The lack of Se deficiency in the animals examined suggests however, that the Polish Konik horses have a natural ability to the optimal use of nutrients available in their life area. Testicular content of Se and GSHPx activity in the colts was higher than those found in stallions, and a positive relationship between these antioxidants was demonstrated. The differences in Se contents and GSHPx activities in testes between colts and stallions suggest that selenoenzymes play important roles during the puberty of male horses.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

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.

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 µg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

Evaluation of sperm genomic integrity of normozoospermic men: a prospective study.

The objective of our study was to evaluate the incidence of spermatozoa with nuclear DNA strand breaks in patients with normal routine sperm parameters (26 subjects). Sperm DNA fragmentation was measured using TUNEL test assessed in flow cytometer. Variable percentages of sperm with damaged DNA (9.42 +/- 7.68%; range: 2-36) were found. Two categories of patients were distinguished: (1) patients (8 out of 26 subjects) with < or = 4% of TUNEL-positive sperm and (2) patients (18 out of 26 subjects) with > 4% of TUNEL-positive sperm. A significantly lower percentage of normal sperm forms was found in patients with > 4% of TUNEL-positive sperm than in patients with < or = 4% of TUNEL-positive sperm. Moreover, a significant negative correlation (r(s) = -0.50) was noted only between a proportion of normal sperm forms and a proportion of TUNEL-positive spermatozoa. In electron microscope, a large number of spermatozoa with immature chromatin was observed more frequently in subjects with > 4% of TUNEL-positive cells (11 out of 18 subjects). Our results suggest that in some patients with normal routine sperm parameters, DNA fragmentation may be associated with poor sperm morphology. The diminished sperm genomic integrity may result from molecular disturbances in nuclear remodeling process during spermiogenesis. TUNEL assay is a screening tool that may help to discriminate between fertile and infertile men and may help to predict successful in vitro fertilization.

Diagnostic evaluation of oxidoreductive capability of sperm mitochondria.

In the present paper, morphological and functional features of human sperm midpiece, contributing to the assessment of sperm fertility potential, have been described. The NADH-dependent NBT screening assay was used to identify and visualise: 1/ morphological defects of sperm midpiece, 2/ immature sperm forms with extensive cytoplasmic retention, reflecting developmental failure in spermatogenic remodelling process, 3/ cytoplasmic sperm conglomerates, related to apoptotic bodies and 4/ sperm NADH-dependent oxidoreductase system at the mitochondrial level, related to the reaction intensity. The used assay is an adequate marker of sperm mitochondrial activity and sperm maturity. It can also help discover sperm defects that result in asthenozoospermia and can be used as an additional indicator in the evaluation of the sperm midpiece, as well as in routine morphological examination of spermatozoa, having a considerable predictive value for in vivo and in vitro fertilization.