Comparison of two staining techniques on the manual and automated canine sperm morphology analysis.

Detailed and direct analysis of semen, including sperm morphology, enables a diagnosis of male fertility. This study aimed to describe an economical and verified protocol for canine spermiograms and compare the effectiveness of Sperm Stain® and Sperm Blue® (Microptic, Spain) in veterinary practice. Sperm assessment was conducted manually, using a standard optical microscope, and via computerized semen analysis using the SCA® CASA (Sperm Class Analyzer® CASA System-MICROPTIC, Spain). This study showed that Sperm Blue® is a better solution for computerized sperm quality analysis of healthy dogs. At the same time, Sperm Stain® turned out to be more helpful in identifying specific morphological defects of sperm. Automated canine sperm morphology analysis worked better with Sperm Blue stain, but Sperm Stain simplified manual evaluation of various organelles' defects. Standard, manual examination is more error-prone for an inexperienced andrology technician, but it seems to be still a gold standard technique for canine sperm assessment.

The Association between Clusterin Sialylation Degree and Levels of Oxidative-Antioxidant Balance Markers in Seminal Plasmas and Blood Sera of Male Partners with Abnormal Sperm Parameters.

Nearly 30% of infertility cases are caused by male factor. This study aimed at checking the associations between the sialylation degree of glycoprotein clusterin (CLU) and levels of oxidative-antioxidant balance markers in infertile men. Using lectin-ELISA with biotinylated lectins specific to α2,6-linked (Sambucus nigra agglutinin, SNA) and α2,3-linked (Maackia amurensis agglutinin, MAA) sialic acid (SA), the CLU sialylation in 132 seminal plasmas (SP) and 91 blood sera (BS) were analyzed. Oxidative-antioxidant status was measured by determining Sirtuin-3 (SIRT3), Sirtuin-5 (SIRT5), total antioxidant status (TAS), and ferric reducing antioxidant power (FRAP) levels. We indicate that multiple sperm disorders are associated with decreased expression of MAA-reactive SA in SP. Decreased SP SIRT3 concentrations may be associated with teratozoospermia and oligoasthenoteratozoospermia. ROC curve and cluster analysis revealed that SP relative reactivity of CLU glycans with MAA, the value of MAA/SNA ratio, and SIRT3 and SIRT5 concentrations may constitute an additional set of markers differentiating infertile oligoasthenoteratozoospermic patients (OAT) from normozoospermic (N), asthenoteratozoospermic (AT) and teratozoospermic (T). The multinomial logistic regression analysis confirmed the potential utility of SIRT3 determinations for differentiation between N and OAT groups as well as between N and T groups for SIRT3 and SIRT5. For BS, based on ROC curve and cluster analysis, relative reactivities of CLU glycans with SNA, MAA, SIRT3 and FRAP concentrations may be useful in the differentiation of normozoospermic patients from those with sperm disorders. The multinomial logistic regression analysis showed that the SNA relative reactivity with CLU glycans significantly differentiated the N group from AT, OAT and T groups, and FRAP concentrations significantly differed between N and AT groups, which additionally confirms the potential utility of these biomarkers in the differentiation of infertile patients with abnormal sperm parameters. The knowledge about associations between examined parameters may also influence future research aimed at seeking new male infertility therapies.

Does single-layer centrifugation with Bovicoll improve sperm quality of frozen-thawed semen in Fleckvieh bulls?

The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.

Transfer of orally administered ZnO:Eu nanoparticles through the blood-testis barrier: the effect on kinetic sperm parameters and apoptosis in mice testes.

Zinc-based nanoparticles are promising materials for various applications, including in biomedicine. The aim of our study was to determine the effect of fluorescent europium-doped zinc oxide nanoparticles (ZnO:Eu NPs) on sperm parameters, cell apoptosis and integrity of the blood-testis barrier (BTB) in mice. Nanostructures were orally administered to adult mice (n = 34). Animals were sacrificed after 3 h, 24 h, 7 d and 14 d following oral administration. Sperm was collected and analysed for viability and kinetic parameters. Collected testes were quantitatively analysed for accumulation of ZnO:Eu NPs. Microscopic evaluation based on immunofluorescence and histopathological studies were also conducted. Results showed that ZnO:Eu NPs were able to overcome the BTB with their subsequent accumulation in the testis. No toxic or pro-apoptotic effects of nanoparticles on the male reproductive system were observed. The results suggested that ZnO:Eu NPs were able to accumulate in the testis with no negative impact on sperm parameters, tissue architecture or the integrity of the BTB.

Changes in sperm function and structure after freezing in domestic cat spermatozoa.

Sperm cryopreservation allows for a long-term storage of genetic. However, changes due to factors as cold shock, osmotic and oxidative stress cause reduction in viability and fertilising ability of frozen/thawed spermatozoa. Therefore, evaluation of cryoinjury of cat spermatozoa is a key factor in achieving better cryopreservation results. This study analysed the changes in structural and functional after freezing in ejaculated domestic cats spermatozoa. Semen samples (n = 60) were analysed before and after freezing, progressive motility was determined with computer-assisted sperm analysis and viability, and acrosome intact spermatozoa, mitochondrial function and superoxide anion ( O 2 - ) were assessed by flow cytometry. The results demonstrated that cryopreservation induced changes in all sperm parameters (p < 0.05). Total sperm motility, viability, acrosome integrity and mitochondrial function of fresh samples were near to 80% and decrease near to 40% in frozen/thawed spermatozoa (p < 0.05); nevertheless, in contrast to all other sperm parameters, the sperm positive with O 2 - increased post/thawing (p < 0.05). In conclusion, changes in frozen/thawed spermatozoa could be related to the effect of oxidative stress due to the increase in the synthesis of O 2 - and a concomitant loss of functional competence. Therefore, the evaluation of these sperm parameters could contribute to complement the analysis of fresh or frozen semen used ART.

Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress.

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O2- localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.

Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients.

Our study compares the status of human seminal plasma immunoglobulin G (IgG) and IgA secretory component (SC) fucosylation between infertile leukocytospermic and normal, fertile normozoospermic patients. The seminal IgG and SC are decorated with AAL-reactive core fucose, and antennary UEA- and LTA-reactive fucose of Lewis(y) and Lewis(x) structures, respectively. However, a correlation between IgG core fucosylation and IgG concentration (r = -0.52; p < 0.0003) was observed. The IgG present in leukocytospermic samples is characterized by lower expression of core fucose than in the normal group (0.82 ± 0.3 AU and 1.2 ± 0.3 AU, respectively; p < 0.002). In seminal plasma the SC is present in two forms: 78-kDa and 63-kDa. The present study has also shown a higher AAL and LTA specific reactivity of glycans expressed in 63-kDa SC, in comparison to 78-kDa SC, in the normal group. In leukocytospermia, the values of specific lectin reactivity for core fucose, fucose α(1-2)- and α(1-3)- linked, were similar for both SC bands. Moreover, the present study has shown that in leukocytospermic samples the mean concentrations of IgG and S-IgA are twice as high (131.68 ± 102.6 mg/l and 36 ± 27 mg/l, respectively) as in the normal group (67.68 ± 29.2 mg/l; p < 0.02, and 19 ± 18 mg/l, p < 0.019, respectively). The analysis of IgG and SC fucosylation status and the determination of IgG and S-IgA concentrations in seminal plasma might constitute a valuable diagnosis tools for the evaluation of male infertility associated with leukocytospermia with accompanying inflammation.

Human seminal fibronectin fragmentation patterns and their domain immunoreactivities in leucocytospermic patients.

The aim of the work was to analyse fibronectin (FN) domain immunoreactivities and profiles of FN fragmentation in seminal plasmas of fertile normozoospermic and infertile leucocytospermic male patients. ELISA with domain-specific monoclonal antibodies and immunoblotting were used in these measurements. Immunoblotting of normal and leucocytospermic seminal plasmas revealed the presence of twelve FN bands of ~70-196kDa with nearly identical FN profiles under reducing and non-reducing conditions. The epitopes of the cell-, fibrin-, collagen-binding FN domains and the extra domain A (EDA) FN segment retained the ability to bind their specific monoclonal antibodies, whereas the fibrin-heparin domain (N-terminal end) and the area around the disulfide bridges (C-terminal end) of the FN polypeptide did not show any reactivities with their respective specific antibodies. The mean values of cell- (338.4±138.4 and 398.3±310mgL(-1)), fibrin- (79.1±38.5 and 145.2±188.8mgL(-1)) and collagen-binding (19±19.8 and 50.9±73.4mgL(-1)) FN domain immunoreactivities and the relative amount of (EDA)FN did not show any significant differences between the normal and leucocytospermic groups. The high values of standard deviations for the FN domain immunoreactivities in the leucocytospermic group probably results from different aetiology of leucocytospermia. The profile of FN fragmentation and alterations of FN domain immunoreactivities in seminal plasma may influence their engagement in the fertilisation process. The analysis of seminal FN molecular status would be helpful for selecting the highest quality spermatozoa for use in assisted reproduction techniques.

The Relationship between the Testicular Blood Flow and the Semen Parameters of Rams during the Selected Periods of the Breeding and Non-Breeding Seasons.

The study aimed to conduct advanced semen evaluation tests during routine ram examination periods in the breeding and non-breeding seasons and to investigate their correlation with the dynamics of testicular blood flow. Semen was collected from eighteen rams, and pulse wave Doppler examination before (BBS), during (BS), and after the breeding season (ABS). Routine and advanced semen analysis, including computer-assisted sperm analysis (CASA), sperm chromatin dispersion test (SCD), and motile sperm organelle morphology examination (MSOME), were conducted. In Doppler ultrasonography, the peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI), and pulsatility index (PI) were calculated. In BS period, high sperm concentration (p < 0.0001) and total sperm number/ejaculate (p = 0.008) were noted. During the BBS period, a low percentage of forwarding motility (p = 0.017) and high sperm abnormalities (p = 0.005) were found. Also during this period, both SCD and MSOME revealed high sperm DNA fragmentation (p < 0.0001) and signs of vacuolization (Grade II-IV, p < 0.05). The advanced features of higher sperm abnormalities (Grade IV of MSOME) correlated with an increase RI (ρє <0.60;0.61>) and PI (ρє <0.46;0.52>), whereas the basic percentage of sperm abnormalities correlated with the EDV (ρє <0.44;0.73>) value. One may conclude that the current preliminary study requires further research concerning the monthly examination of a ram to provide full yearly characteristics of the relation between advanced semen evaluation tests and the dynamics of testicular blood flow.

Are There Associations between Seminal Plasma Advanced Oxidation Protein Products and Selected Redox-Associated Biochemical Parameters in Infertile Male Patients? A Preliminary Report.

Oxidative stress (OS) is one of the reasons for male infertility. Seminal plasma contains a multitude of enzymes and ions which influence OS and thus may affect male fertility. The aim of the study was to check for associations between seminal plasma advanced oxidation protein products (AOPP) concentrations and levels of selected biochemical parameters (total protein, iron, uric acid, magnesium, calcium) in infertile men, and establish whether they are associated with sperm disorders. Seminal plasma AOPP, as well as total protein, iron, uric acid, calcium, and magnesium concentrations, were determined for the following patient groups: normozoospermic (N; n = 33), teratozoospermic (T; n = 30), asthenoteratozoospermic (AT; n = 18), and oligoasthenoteratozoospermic (OAT; n = 28). AOPP concentrations were significantly higher in N and T groups in comparison to AT and OAT groups. Total protein concentrations were significantly lower in the T group in comparison to the AT and OAT groups, whereas iron concentrations significantly decreased in the OAT group in comparison to the T and N patients. AOPP differentiates AT patients from men with other sperm disorders. Our results suggest that asthenozoospermia may be connected with total protein levels. Insufficient iron levels may reflect a decrease in sperm count.

The possible association of clusterin fucosylation changes with male fertility disorders.

In the seminal plasma (n = 118) and serum (n = 90) clusterin (CLU) the fucosylation and the expression of selected fucosyltransferases (FUTs) were analyzed. Samples from infertile men were divided into groups based on the results of the standard semen analysis: normozoospermic (N), teratozoospermic (T), asthenoteratozoospermic (AT) and oligoasthenoteratozoospermic (OAT). The CLU fucosylation was analyzed using lectin-ELISAs with biotinylated lectins specific to α1,3-, α1,2-linked antennary fucose, and α1,6-linked core fucose (LTA, UEA, and LCA, respectively). The concentrations of FUT3 and FUT4, reflecting the expression of Le oligosaccharide structures, were measured using ELISA tests. The differences in serum CLU and FUT4 concentrations, and in the expression of core fucose and antennary fucose α1,2-linked in CLU glycans between the N group and other groups examined suggest that the disturbances in sperm count, motility, and morphology are not the only cause of male infertility. Lack of similarities between levels of examined parameters in blood serum and seminal plasma may suggest the differences in mechanisms leading to glycoproteins glycosylation. It confirmed the observed differences in concentrations of seminal plasma CLU, FUT3, and FUT4 between the OAT group and N, T, AT groups, indicating that decreased sperm count may be related to these parameters expression. The serum CLU concentrations and expression of core fucose and fucose α1,2-linked in CLU, seem to be good markers differentiating normozoospermic men from those with abnormal sperm parameters, which was not observed for seminal plasma.

NT-proCNP as a new indicator of asthenozoospermia.

PURPOSE: C-type natriuretic peptide (CNP) is a proinflammatory peptide. The highest concentration of CNP is found in male reproductive organs. This study aims to analyze the role of N-terminal C-type natriuretic propeptide (NT-proCNP) as a new indicator of asthenozoospermia. MATERIAL/METHODS: Semen was collected after 3-5 days of sexual abstinence from 86 men. The participants were between 25 and 38 years old, 51 of which had asthenozoospermia and 35 of which had normal sperm motility. Semen was analyzed for the concentrations of NT-proCNP, spermatozoa, percentages of live sperm, and sperm exhibiting a specific type of movement. The laboratory tests and analyses were performed using accepted methods and under appropriate conditions. RESULTS: A significant difference in the concentration and motility of spermatozoa was observed between the focus and control groups. The concentration of spermatozoa in the focus group was significantly lower than in the control group (median: 38.5 vs. 69.8mln/ml [p=0.016] respectively). The progressive motility of spermatozoa demonstrated a significantly lower performance in the focus group than in the control group (median: 10.4% vs. 45% respectively). The concentration of NT-proCNP was significantly higher in the focus group (median: 29.1 vs. 17.9pmol/l; p<0.001). The Area under the Receiver Operating Characteristic (AUROC) curve for the concentration of NT-proCNP - as an indicator of asthenozoospermia - was 0.733. Participants with a concentration of NT-proCNP higher than 28.8pmol/l had asthenozoospermia with 52.9% sensitivity and 94.3% specificity. CONCLUSIONS: NT-proCNP - an indicator of inflammatory reaction - should be evaluated as an indicator of asthenozoospermia.

[Sperm evaluation in testicular cancer patients before and after chemotherapy]. / Ocena parametrów nasienia u chorych na raka jadra przed i po chemioterapii.

OBJECTIVES: Over the last 20 years treatment results of testicular cancer have improved. At present, up to 90% of patients are cured. DESIGN: Semen analysis has been performed in 50 patients before and 3, 6, 12, 12, 18, 24, 30 months after i.t. MATERIALS: 50 Patients with testicular cancer underwent chemotherapy based on cisplatin. Before and after treatment the semen analysis has been performed on each patients. The results of the patient groups have been compared to healthy group of men. RESULTS: The quality of the semen is much worse in the group with cancer compared to healthy controls. The deepest impairment of the spermatogenesis has been found 3 mounts after chemotherapy. CONCLUSIONS: Orchidectomy has no influence on sperm parameters. Semen analysis revealed normospermia in 30% of the patients. 30 months after chemotherapy.

Fucose and sialic acid expressions in human seminal fibronectin and α{1} -acid glycoprotein associated with leukocytospermia of infertile men.

INTRODUCTION: The aim of this study was to compare fucose and sialic acid residue expression on fibronectin and α{1}-acid glycoprotein in the seminal plasma of men suspected of infertility and suffering from leukocytospermia. SUBJECTS AND METHODS: Seminal ejaculates were collected from 27 leukocytospermic and 18 healthy, normozoospermic men. The relative degree of fucosylation and sialylation of fibronectin and α{1}-acid glycoprotein was estimated by ELISA using fucose and sialic acid specific lectins from Aleuria aurantia, Lotus tetragonolobus, and Ulex europaeus as well as Maackia amurensis and Sambucus nigra, respectively. RESULTS: Leukocytospermic seminal fibronectin, in comparison with fibronectin of normal fertile group, showed lower relative reactivity with AAL, LTA and UEA, and higher reactivity with MAA and SNA, while the AGP of the leukocytospermic group was less reactive with AAL, and the relative reactivity with LTA and MAA was significantly higher. Fibronectin and α{1}-acid glycoprotein reactivity with UEA and MAA showed high positive correlations. DISCUSSION: Leukocytospermia was associated with the alterations of terminal monosaccharide expression in human seminal fibronectin and α{1}-acid glycoprotein. The increase of sialyl-Lewis{ x} antigen in α{1}-acid glycoprotein can be used as a marker of genital tract inflammation manifested by leukocytospermia.

Lower expression of the alpha2,3-sialylated fibronectin glycoform and appearance of the asialo-fibronectin glycoform are associated with high concentrations of fibronectin in human seminal plasma with abnormal semen parameters.

BACKGROUND: Sialic acid isoforms expressed on glycoconjugates are known to be involved in different biological functions. The aim of this study was to compare alpha2,3- and/or alpha2,6-linked sialic acid expression on fibronectin in the seminal plasma of male partners from infertile couples. The data obtained were analyzed in relation to normal and abnormal semen parameters, as well as to fibronectin concentrations. METHODS: Fibronectin concentrations were determined by ELISA using a specific monoclonal antibody. The relative degree of sialic acid linked to fibronectin glycans either by alpha2,3 or alpha2,6 isomeric linkage was estimated by lectin-fibronectin ELISA utilizing lectins from Maackia amurensis and Sambucus nigra, respectively. RESULTS: High seminal fibronectin concentration was more frequently associated with abnormal semen parameters. The glycans of seminal plasma fibronectin, in contrast to blood plasma fibronectin, contained sialic acid more frequently attached by alpha2,3 than by alpha2,6 isomeric linkage. The relative amounts of both alpha2,3- and alpha2,6-linked sialic acid fibronectin isoforms were lower in the seminal plasma of men suspected of infertility. CONCLUSIONS: Seminal fibronectin showed an oncofetal type of sialylation and the distribution of hypo- and asialylated fibronectin glycoforms were associated with abnormal semen parameters and with high concentrations of fibronectin.