Seminal plasma proteins of adult boars and correlations with sperm parameters

The present study was conducted to identify the major seminal plasma protein profile of boars and its associations with semen criteria. Semen samples were collected from 12 adult boars and subjected to evaluation of sperm parameters (motility, morphology, vitality, and percent of cells with intact acrosome). Seminal plasma was obtained by centrifugation, analyzed by two-dimensional SDS-PAGE, and proteins identified by mass spectrometry (electrospray ionization quadrupole time-of-flight). We tested regression models using spot intensities related to the same proteins as independent variables and semen parameters as dependent variables (P ≤ 0.05). One hundred twelve spots were identified in the boar seminal plasma gels, equivalent to 39 different proteins. Spermadhesin porcine seminal protein (PSP)-I and PSP-II, as well as spermadhesins AQN-1, AQN-3 and AWN-1 represented 45.2 ± 8% of the total intensity of all spots. Other proteins expressed in the boar seminal plasma included albumin, complement proteins (complement factor H precursor, complement C3 precursor and adipsin/complement factor D), immunoglobulins (IgG heavy chain precursor, IgG delta heavy chain membrane bound form, IgG gamma-chain, Ig lambda chain V-C region PLC3, and CH4 and secreted domains of swine IgM), IgG-binding proteins, epididymal-specific lipocalin 5, epididymal secretory protein E1 precursor, epididymal secretory glutathione peroxidase precursor, transferrin, lactotransferrin and fibronectin type 1 (FN1). On the basis of the regression analysis, the percentage of sperm with midpiece defects was related to the amount of CH4 and secreted domains of swine IgM and FN1 (r² = 0.58, P = 0.006), IgG-binding protein (r² = 0.41, P = 0.024), complement factor H precursor (r² = 0.61, P = 0.014) and lactadherin (r² = 0.45, P = 0.033). The percentage of sperm with tail defects was also related to CH4 and secreted domains of swine IgM and FN1 (r² = 0.40, P = 0.034), IgG-binding protein (r² = 0.35, P = 0.043) and lactadherin (r² = 0.74, P = 0.001). Sperm motility, in turn, had association with the intensities of spots identified as lactadherin (r² = 0.48, P = 0.027). In conclusion, we presently describe the major proteome of boar seminal plasma and significant associations between specific seminal plasma proteins and semen parameters. Such relationships will serve as the basis for determination of molecular markers of sperm function in the swine species.     

Relationship between Lipids Levels of Serum and Seminal Plasma and Semen Parameters in 631 Chinese Subfertile Men

Objective

This prospective study was designed to investigate the relationship between lipids levels in both serum and seminal plasma and semen parameters.

Methods

631 subfertile men were enrolled. Their obesity-associated markers were measured, and semen parameters were analyzed. Also, seminal plasma and serum TC, TG, HDL and LDL and serum FFA, FSH, LH, total testosterone (TT), estradiol (E2) and SHBG levels were detected.

Results

Seminal plasma and serum TG, TC and LDL levels were positively related to age. Serum TC, TG and LDL were positively related to obesity-associated markers (P < 0.001), while only seminal plasma TG was positively related to them (P < 0.05). For lipids levels in serum and seminal plasma, only TG level had slightly positive correlation between them (r = 0.081, P = 0.042). There was no significant correlation between serum lipids levels and semen parameters. However, seminal plasma TG, TC, LDL and HDL levels were negatively related to one or several semen parameters, including semen volume (SV), sperm concentration (SC), total sperm count (TSC), sperm motility, progressive motility (PR) and total normal-progressively motile sperm counts (TNPMS). Moreover, seminal plasma TG, TC, LDL and HDL levels in patients with oligospermatism, asthenospermia and teratozoospermia were higher than those with normal sperm concentration, motility or morphology. After adjusting age and serum LH, FSH, TT, E2 and SHBG levels, linear regression analysis showed that SV was still significantly correlated with seminal plasma LDL (P = 0.012), both of SC and TSC with seminal plasma HDL (P = 0.028 and 0.002), and both of PR and sperm motility with seminal plasma TC (P = 0.012 and 0.051).

Conclusion

The abnormal metabolism of lipids in male reproductive system may contribute to male factor infertility.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

A diet supplemented with l-carnitine improves the sperm quality of Piétrain but not of Duroc and Large White boars when photoperiod and temperature increase

It has been reported that a diet supplemented with l-carnitine can improve sperm quality in some mammalian species. Against this background, the current study seeks to determine the effects of feeding l-carnitine (625 mg·day⁻¹) on boar semen characteristics (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) in three different porcine breeds (Sus domesticus) (Piétrain, Duroc, and Large White) exposed to natural environmental changes in temperature and photoperiod over a 20-wk period (February to July 2007). One hundred twenty boars (40 per breed) were randomly separated into two groups (60 boars each): the first (20 boars per breed) was fed a control diet and the second (also 20 males per breed) the same diet supplemented with l-carnitine (625 mg·day⁻¹). Whereas the l-carnitine supplement did not affect ejaculate volume, concentration, motility, viability, or the osmotic resistance of spermatozoa, it did improve sperm morphology in Piétrain boars by reducing the percentage of immature spermatozoa when the temperature and the photoperiod increased. Conversely, no effect on sperm morphology from supplementing feed with l-carnitine was observed in both Duroc and Large White breeds. We can therefore conclude that the addition of l-carnitine to the diet of males may maintain the level of normal sperm morphology in Piétrain boars when a drop in sperm quality occurs (due to increases in photoperiod and temperature), without affecting the other sperm quality parameters.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi)

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential.     

Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality

To determine the effects of seminal plasma during and after cyopreservation on post-thaw sperm functions in semen from poor freezability boars, seminal plasma was removed immediately after collection, and sperm was subjected to cooling and freezing. Removal of seminal plasma did not significantly affect post-thaw sperm motility in good freezability boars; however, in boars with poor freezability, it increased post-thaw motility relative to control sperm cooled with seminal plasma (64.5+/-3.4% vs. 30.9+/-3.1%, P<0.01). Freezing sperm without seminal plasma increased both loss of the acrosome cap (37.5+/-1.6% vs. 18.4+/-2.8%, P<0.01) and expression of a 15 kDa tyrosine-phosphorylated protein (capacitation marker) in thawed sperm relative to controls; the addition of 10% (v/v) seminal plasma to the thawing solution significantly suppressed both changes and increased conception rate to AI (70% vs. 9% in the control group, P<0.05). In conclusion, our novel cryopreservation and thawing method increased the success of AI with frozen-thawed porcine semen, particularly from boars with poor post-thaw semen quality.     

Heat induced infertility of boars: The inter-relationship between depressed sperm output and fertility and an estimation of the critical air temperature above which sperm output is impaired

Eight groups of Large-White gilts were each inseminated with different numbers of normal motile sperm, in the range 0.28–7.0 × 10⁹. A significant (P < 0.05) relationship between conception rate and the number of motile sperm inseminated was shown. This relationship can be used to equate output of motile sperm with levels of fertility of boars. The optimal number of motile sperm for conception following intra-cervical insemination was near 5 × 10⁹ and the threshold number, below which animals did not conceive, was c. 4 × 10⁸.In a second experiment, three Large-White boars were subjected to graded thermal treatment (air temperature was increased by 1°C per day for 20 days, from a basal level of 20°C to a maximal level of 40°C) and responses of ejaculate and other physiological characteristics were monitored. Scrotal surface temperature, respiration rate and rectal temperature increased (P < 0.05) beyond basal levels at air temperatures of 30°C, 33°C and 35°C, respectively. Motility of sperm in ejaculates decreased when air temperature reached 30°C and this response was presumed to reflect hyperthermia in epididymal tissues, consistent with increasing scrotal surface temperature at this same air temperature. Motility fell below a pre-treatment level of about 93%, to 19% (P < 0.05), 3 weeks after heating. Volumes of seminal plasma and gel in ejaculates were also lower (P < 0.05) following heating. Changes in daily sperm production were minor and, as a result, daily motile sperm production levels paralleled changes in motility. Proportions of abnormal types of sperm increased (P < 0.05) to maximal levels in the last week of heating and all returned to pretreatment values 5 weeks later. High proportions of sperm with kinoplasmic droplets appeared in ejaculates collected after heating (P < 0.05), evidence that epididymal cell types in the boar are sensitive to heat.As a result of heat treatment, normal motile sperm production decreased from control levels (1.28 × 10¹⁰·day^?1) to 0.15 × 10¹⁰·day^?1, 3 weeks after heating ceased. However, the results suggest that normal sperm output by Large-White boars can be maintained at air temperatures as high as 29°C.By relating the results of both experiments, it is concluded that fertility of the boars in the second experiment (if mated once daily) would be depressed for about 5 weeks after heat treatment ceased. The findings support many field reports which indicate a contribution of boars to lower conception rates of sows during and immediately following summer and the results can be used in formulating strategies to circumvent this widespread problem.     

Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3±13.0 vs. 44.9±30.8%) and positive Spermac staining (51.9±14.5 vs. 7.5±14.4%), in addition to higher total volume (135.1±89.6 vs. 88.8±73.1 ml) and lower sperm concentration (473.0±511.2 vs. 1313.8±764.7×10⁶ cells ml⁻¹) compared to ejaculates exhibiting poor sperm motility (≤10%; P<0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ∼80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P<0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.     

Seminal prostaglandins in men with subnormal sperm motility and therapeutic treatment

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocelle and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE₂, PGI₂ and PGF_2α. There was a significant difference (p < 0, 025; F-test) between the PGI₂ concentrations in patients with impaired motility (5,6 ± 1,4 pg/mg protein) and normal men (8,8 ± 3,7 pg/mg protein). PGE₂ and PGF_2α were significantly different in patients with varicocele (p < 0,025, F-test). Wide ranges of prostaglandins occured in the Kallikrein-group with no significant differences. We conclude that: a) PGI₁ is an additional prostaglandin compound in seminal plasma. b)its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.     

Blood Serum and Seminal Plasma Selenium,Total Antioxidant Capacity and Coenzyme Q10 Levels in Relation to Semen Parameters in Men with Idiopathic Infertility

In this case–control study, we aimed to evaluate the serum and seminal plasma levels of Selenium (Se), total antioxidant capacity (TAC), and Coenzyme Q10 (CoQ-10) and determine their relationship with sperm concentration, motility, and morphology in men with idiopathic infertility. A total of 59 subjects were enrolled in the study. Forty four patients were diagnosed with idiopathic male infertility and had abnormal sperm parameters, and 15 subjects had normal sperm parameters with proven fertility. Serum Se, semen Se, and semen TAC levels were significantly different in the fertile and infertile groups (p?<?0.01, p?<?0.001, and p?<?0.001, respectively). However, serum TAC, serum, and seminal plasma CoQ-10 levels did not differ between fertile and infertile groups. When the levels of the measured parameters were compared in serum and seminal plasma, serum levels of Se were found to be correlated positively with the semen levels in all subjects included into the study (N?=?59) (r?=?0.46, p?<?0.01). A relationship was found between neither serum and semen levels of TAC nor between serum and semen levels of CoQ-10. Correlations among measured serum and semen parameters with sperm parameters demonstrated that both the serum and semen levels of Se were correlated positively with spermatozoa concentration, motility, and morphology. Additionally, seminal plasma levels of TAC correlated positively with all these sperm parameters. On the other hand, seminal plasma levels of CoQ-10 correlated only with sperm morphology but not with concentration or motility. No relationship was observed between serum levels of TAC or serum levels of CoQ-10 and sperm parameters. In conclusion, serum and seminal plasma Se deficiency may be a prominent determinant of abnormal sperm parameters and idiopathic male infertility. Measurement of serum Se levels may help determine nutritional status and antioxidant capacity in infertile patients, which may help distinguish those patients who will benefit from supplementation therapy.     

Colloidal centrifugation with Androcoll-E™ prolongs stallion sperm motility, viability and chromatin integrity

The objective was to investigate the changes in stallion sperm quality (sperm motility, viability, membrane integrity and chromatin integrity) occurring during cool storage, and to study the effect of sperm selection by single layer colloidal centrifugation on these parameters of sperm quality. Spermatozoa from 3 stallions (10 ejaculates, 3–4 per stallion) were selected by centrifugation through a single layer of colloid (SLC). The resulting sperm preparations and the control samples (extended but unselected semen samples) were stored at 5 °C for 48 h. Assessments of sperm quality, such as sperm motility, viability (SYBR-14/PI staining), membrane stability (Annexin-V/PI staining) and chromatin integrity, were performed on aliquots of the selected sperm preparations and unselected samples on the day of collection (3 h) and after 24 and 48 h of storage. In the SLC-selected sperm samples, sperm motility, sperm viability, proportions of spermatozoa with normal morphology and with intact chromatin were significantly better than in unselected samples (motility: 77 ± 4% vs. 64 ± 8% at 3 h; P < 0.001; viability: 79.5 ± 9% vs. 64.7 ± 9%, P < 0.001; normal morphology 89 ± 6% vs. 69 ± 9%; chromatin integrity DFI 11.3 ± 5% vs. 22.1 ± 10%). Membrane stability, however, was not different in the SLC-selected and unselected samples (74.6 ± 8% vs. 69.3 ± 8%). The deterioration seen in sperm quality in the unselected samples was prevented by SLC, so that sperm viability, membrane stability and chromatin integrity were unchanged in the selected samples by 48 h compared to 3 h (P < 0.001), whereas the unselected samples were significantly worse by 48 h (P < 0.001). Furthermore, it should be possible to send an aliquot of a normal insemination dose (i.e. unselected spermatozoa) overnight to a reference laboratory for analysis of both plasma membrane and chromatin integrity. In conclusion, centrifugation of stallion spermatozoa through a single layer of colloid is a useful technique for selecting the best spermatozoa from an ejaculate and, moreover, sperm quality is maintained during storage.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Prediction of porcine semen fertility by homologous in vitro penetration (hIVP) assay

A field trial was conducted to compare the fertility predicting capacity of different sperm assays applying classical semen analysis, sperm function and the homologous in vitro penetration test (hIVP) to 60 ejaculates from four boars collected over a period of 15 weeks. No differences were found between the groups of fertility (Low Fertility: <20%; Intermediate: 40–60% and High: >80%) for sperm-rich fraction volume collection, sperm concentration, total sperm number, cationic contents in seminal plasma and ATP concentration. Partial differences were found in the parameters of motility, normal morphology, normal apical ridge (NAR), viability with eosin–nigrosin stain, hypo-osmotic swelling test (HOS), osmotic resistance test (ORT) and functional membrane integrity (with carboxyfluorescein diacetate, DCF). These parameters would be useful for detecting sperm with poor fertility, but they are not precise enough to discriminate an ejaculate with higher fertility than the herd median. Only the penetration percentage (10.24±1.45 vs. 55.13±3.35 vs. 84.72±1.73) and sperm number per oocyte (1.29±0.07 vs. 11.29±1.79 vs. 25.86±1.43) in a hIVP system were parameters with a predictive capacity to discriminate between the three fertility groups. Consequently, hIVP was found to be the best seminal assay and it may improve the in vitro assessment of sperm fertilizing ability.     

Total reactive antioxidant potential and DNA fragmentation index as fertility sperm parameters

There is a growing evidence that oxidative stress play a major role in the etiology of defective sperm function including impaired morphology, motility, metabolism and fertility. The aim of the present study was to examine: 1/ total reactive antioxidant potential (TRAP) in seminal plasma; 2/ sperm DNA fragmentation index (DFI), 3/ sperm morphology and motility and 4/ cellular membrane integrity (hypoosmotic swelling test: HOS test) in patients attending in vitro fertilization/intracytoplasmatic sperm injection ( IVF/ICSI) program. According to the DFI value, the men were divided into: group 1 with DFI ≤15% (n=38) and group 2 with DFI ≥15% (n=37). Significant differences between the two groups were found in TRAP, sperm motility, morphology and concentration as well as HOS test scores. In group 1, DFI was negatively correlated with sperm motility and HOS test scores (p<0.05). The sperm morphology was positively correlated with sperm motility and HOS test scores in both groups. There was no correlation between TRAP and sperm chromatin fragmentation. Our results suggest that seminal plasma TRAP level may be a DFI independent parameter of sperm fertility.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Selenoprotein P in seminal fluid is a novel biomarker of sperm quality

Hepatically-derived selenoprotein P (SePP) transports selenium (Se) via blood to other tissues including the testes. Male Sepp-knockout mice are infertile. SePP-mediated Se transport to Sertoli cells is needed for supporting biosynthesis of the selenoenzyme glutathione peroxidase-4 (GPX4) in spermatozoa. GPX4 becomes a structural component of sperm midpiece during sperm maturation, and its expression correlates to semen quality. We tested whether SePP is also present in seminal plasma, potentially correlating to fertility parameters. Semen quality was assessed by sperm density, morphology and motility. SePP was measured by an immunoluminometric assay, and trace elements were determined by X-ray fluorescence spectroscopy. SePP levels were considerably lower in seminal plasma as compared to serum (0.4 ± 0.1 mg/l vs. 3.5 ± 1.0 mg/l); Se concentrations showed a similar but less pronounced difference (48.9 ± 20.7 μg/l vs. 106.7 ± 17.3 μg/l). Se and Zn correlated positively in seminal fluid but not in serum. Seminal plasma SePP concentrations were independent of serum SePP concentrations, but correlated positively to sperm density and fraction of vital sperm. SePP concentrations in seminal plasma of vasectomized men were similar to controls indicating that accessory sex glands are a testes-independent source of SePP. This notion was corroborated by histochemical analyses localizing SePP in epithelial cells of seminal vesicles. We conclude that SePP is not only involved in Se transport to testes supporting GPX4 biosynthesis but it also becomes secreted into seminal plasma, likely important to protect sperm during storage, genital tract passage and final journey.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Agreement between measures of total motility and membrane integrity in stallion sperm

Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and absolute difference) between TMOT and SMI was high (-0.7 and 5.6%, respectively), but decreased after 24 (10.8 and 15.1%, respectively) and 48 h (23.0 and 23.8%, respectively) of cooled storage as motility declined more rapidly than SMI. We concluded that TMOT and SMI measured separate aspects of sperm quality.