Search Results (78)

Sperm capacitation is essential for fertilization and involves coordinated changes in membrane organization, ion fluxes, and intracellular signaling. However, commonly used detection methods may reflect different biological events, which can be strongly influenced by experimental methodology. This study critically evaluated fluorescence-based approaches for assessing capacitation in boar spermatozoa, focusing on their specificity, interpretative limits, and methodological sensitivity. Ejaculated boar spermatozoa were incubated under in vitro capacitating conditions in TALP medium. Selected samples were subsequently treated with calcium ionophore to induce the acrosome reaction (AR). Phosphotyrosine (PTyr) immunofluorescence was assessed using five fixation and labeling protocols, acrosin redistribution was evaluated with the ACR.2 antibody, calcium ion redistribution was assessed using chlortetracycline (CTC) fluorescence, and acrosomal responsiveness was monitored by peanut agglutinin (PNA) lectin labeling. PTyr immunofluorescence was highly dependent on fixation protocol, indicating marked methodological sensitivity. Acrosin immunodetection revealed a clear capacitation-associated redistribution from weak or diffuse staining to a well-defined acrosomal pattern, whereas ionophore treatment caused a pronounced signal loss consistent with acrosomal exocytosis. PNA labeling confirmed that capacitation alone did not increase spontaneous acrosome loss, whereas ionophore treatment induced a robust AR. CTC staining showed a significant shift from whole-head pattern to acrosome in TALP-treated spermatozoa, indicating capacitation-associated Ca²⁺ redistribution. Together with CTC and Western blot data, these findings show that sperm capacitation status should be evaluated using multiple complementary markers rather than a single gold-standard assay. Full article

Capacitation is a key maturation process that enables spermatozoa to acquire fertilizing ability and can be induced in vitro using capacitation media. Because capacitation protocols differ markedly among laboratories, we compared three compositionally distinct Hepes-, Tris-, and TALP-based media. This study was performed in boar spermatozoa using 3–6 biological replicates of pooled ejaculates depending on the assay, with 46 ejaculate samples from 12 boars in total. The aim was to determine whether such non-standardized conditions differentially affect signaling pathways leading to capacitation and thereby influence the detection of commonly used capacitation markers. We found clear differences among the tested media. All three induced capacitation-associated events, but their functional and molecular effects were not equivalent. The Hepes-based medium supported sperm motility most effectively, increasing total and progressive motility to 60.0% and 48.7%, respectively, after 1 h of incubation and maintaining the highest motility throughout the incubation period. In contrast, the Tris-based medium maintained lower but relatively stable motility, whereas the TALP-based medium showed a rapid decline in total motility from 53.1% to 15.2% during the first hour. The TALP-based medium induced the highest and most sustained protein kinase A (PKA) activity, reaching 0.047 U/mL at 0 h and 0.040 U/mL after 3 h, whereas the Hepes- and Tris-based media showed lower and less sustained activity ranging from 0.003 to 0.030 U/mL during incubation. In addition, distinct patterns of protein tyrosine phosphorylation were observed depending on the medium used. In particular, the TALP-based medium containing bicarbonate and bovine serum albumin (BSA) and the Hepes-based medium with the highest BSA concentration were associated with the highest levels of total protein tyrosine phosphorylation. Phosphoproteomic analysis further revealed condition-specific phosphorylation events, indicating that sperm maturation is dynamically regulated by the surrounding molecular environment. In contrast, no significant differences were detected in oxidative phosphorylation or in electron transport system complexes among the tested media. These findings show that differences in capacitation media composition, particularly in bicarbonate and BSA content, can markedly alter signaling outcomes and the interpretation of capacitation markers, with important implications for reproductive technologies and experimental standardization. Full article

Sperm cryo-tolerance resulted in significant variations in post-thaw semen quality among breeds and individual boars. In the present study, semen samples from thirty-seven large white boars were cryopreserved to select individuals with strong and weak freezing tolerance according to their post-thaw sperm quality. Comparative TMT-based quantitative proteomic analysis between the two groups identified 22 significantly differentially expressed proteins. NT5C1B and ADA, the significantly downregulated proteins in the semen of the low cryo-tolerance group, were supplemented in the semen samples with lower cryo-tolerance. Supplementation with 1 µg/mL of NT5C1B dramatically (p < 0.05) improved kinematic parameters and structural integrity. In comparison with the control group, mitochondrial activity and antioxidant capacity were significantly enhanced in post-thaw sperm. In vitro fertilization assays revealed that the NT5C1B-treated group also has notably (p < 0.05) high sperm penetration and embryonic cleavage rates. ADA supplementation did not exhibit obvious freezing tolerance effects. NT5C1B can be a potential key functional protein to enhance the quality and cryo-tolerance during cryopreservation. Specifically, supplementation with 1 µg/mL NT5C1B significantly improved post-thaw motility, structural integrity, mitochondrial activity, and antioxidant capacity and ultimately enhanced the sperm penetration rate and embryonic cleavage rate in cryo-sensitive sperm, confirming its role as a functional protector during cryopreservation. Full article

HP, as an isotropic physical stress, has been widely applied in cell biology and reproductive research to simulate the effects of environmental pressure on cellular functions. In this study, the elastic silicone membrane of a novel bionic insemination catheter was employed as the pressure medium, with semen perfused into a sealed silicone chamber. As the silicone membrane underwent controlled deformation, the liquid inside the chamber generated a nearly uniform isotropic pressure, thereby maintaining spermatozoa in a stable HP environment. Boar sperm are susceptible to physiological and functional damage under HP stress, which can impair fertilization capacity. This study aimed to investigate the effects of TMAO, BET, or their combination on the quality of semen from eight Landrace boars under HP during storage at 17 °C (experiment repeated three times). Semen was collected using the manual collection method and treated with different concentrations of TMAO or BET. Sperm motility parameters were assessed using a CASA system to determine the optimal concentrations. Subsequently, experimental groups were established: the fresh group, HP control group, T group (optimal TMAO), B group (optimal BET), and H group (optimal TMAO + BET). The results showed that the optimal concentrations were 8 mmol/L for TMAO and 20 mmol/L for BET. Compared with the HP control group, the T, B, and H groups showed significantly improved sperm viability, mitochondrial membrane potential (MMP), and plasma membrane integrity (p < 0.05), and significantly reduced DFI, ROS, MDA, and NO contents (p < 0.05), while acrosome integrity showed no significant differences (p > 0.05). Additionally, the B group showed significantly increased T-AOC (p < 0.05). Non-targeted lipidomic analysis revealed 49 differential lipids in the T group, 262 in the B group, and 269 in the H group compared with the HP control. These differential lipids were mainly associated with PC, AcCa, and sphingolipid signaling pathways, with key sphingolipid pathway lipids including Cer, SM, and DG. These findings indicate that BET and TMAO + BET improve HP-induced sperm damage by modulating the sphingolipid signaling pathway and maintaining PC and AcCa levels, whereas TMAO alone may exert protective effects through additional mechanisms. In conclusion, TMAO, BET, or their combination effectively mitigates the detrimental effects of HP on boar sperm. Full article

This research explored the effects of different concentrations of p-coumaric acid (PCA) on the quality of frozen-thawed boar semen. Boar sperm samples were pre-treated with different concentrations of PCA (0, 30, 60, 90, 120 μg/mL) prior to the freezing process. Subsequently, multiple parameters were analyzed post-freeze-thawing, including sperm morphological and kinetic characteristics, acrosome and membrane integrity, mitochondrial function, DNA integrity, antioxidant enzyme activities, the expression levels of the BCL-2, BAX, and Caspase-3 proteins, the in vitro fertilization rate of porcine oocytes, and the embryo cleavage rate. The findings indicated that, compared with the control group, the addition of 90 μg/mL PCA led to significant improvements in several key aspects. Sperm motility, average path velocity, straight-line velocity, curvilinear velocity, and beat cross frequency were all notably enhanced. Moreover, parameters related to sperm quality, such as acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity, also showed significant increases (all p < 0.05). In terms of antioxidant capacity, the 90 μg/mL PCA treatment significantly elevated the total antioxidant capacity, as well as the activities of superoxide dismutase, glutathione peroxidase, and catalase. Simultaneously, it caused a significant reduction in the contents of malondialdehyde and hydrogen peroxide (p < 0.05). Regarding protein expression, the addition of 90 μg/mL PCA significantly upregulated the expression level of the BCL-2 protein, while downregulating the relative expression levels of BAX and Caspase-3 (p < 0.05). Additionally, this concentration of PCA significantly improved the in vitro fertilization rate of porcine oocytes and the embryo cleavage rate (p < 0.05). In conclusion, incorporating PCA into the semen extender can potentially be advantageous for the cryopreservation of boar sperm, with 90 μg/mL being the optimal concentration. Full article

Accurate assessment of sperm morphology is essential for artificial insemination using liquid-preserved boar semen. This study compared nine commonly used staining techniques, eosin, eosin–nigrosin, Diff-Quick^®, Hemacolor^®, Sangodiff-G^®, Spermac^®, Formol–Citrate–Rose Bengal stain, Testsimplets^®, and Methyl Violet, based on morphological assessment, cost, time efficiency, and storage stability. Each staining method was applied to 36 slides, totaling 324 samples, and evaluated four times each (1296 evaluations). Slides were analyzed four times: immediately after staining and after 1 day, 1 week, and 3 months of storage. The results indicated that Eosin was the fastest and most cost-effective method, providing strong contrast, though it increased detection of structural alterations. Eosin–nigrosin offered detailed morphology but formed colored crystals over time. Diff-Quick^® and Hemacolor^® showed good initial performance, but Hemacolor^® lost pigment clarity after 3 months (p = 0.0273). Sangodiff-G^® had poor contrast and reduced detection of abnormalities (p = 0.00229). Spermac^® delivered high contrast but was time-consuming. Formol–Citrate–Rose Bengal stain required extensive preparation and showed significant post-storage changes (p < 0.0001). Testsimplets^®, despite their high cost, suffered from declining interpretability (p < 0.0001). Methyl Violet lacked sufficient resolution and was highly unstable over time (p < 0.0001). In conclusion, Eosin emerged as the most practical and economical staining method for routine morphological evaluation of liquid-preserved boar semen. While eosin–nigrosin was also effective, its storage instability limits broader application. Other methods showed specific weaknesses, emphasizing the need to tailor stain selection to laboratory goals and constraints. Full article

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen–thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing. Full article

This study aims to analyse the effect of selected variation factors on the ejaculate characteristics of boars and to characterise changes in ejaculate characteristics in Landrace, Large White, Duroc, and Pietrain boars during their use for artificial insemination. The original value of this work lies in the estimation of the percentage share of individual components of variability in shaping the traits of boar ejaculate. A total of 943 ejaculates collected from 77 boars used for artificial insemination were analysed. This study began when the boars were at 8–9 months old. Ejaculates were collected in nine consecutive months from the start of the boars’ use. Immediately after collection, they were analysed for ejaculate volume, sperm concentration, percentage of sperm with progressive motility, total number of spermatozoa, and number of insemination doses per ejaculate. The results were analysed according to three criteria: breed of boar (Landrace, Large White, Duroc, and Pietrain), age of boar (up to 10 months, 11–13 months, 14–17 months, and more than 17 months), and season (spring, summer, autumn, and winter). The analysis of the variation in ejaculate characteristics took into account the share of each factor (boar breed, boar age, and season) in the variation, as well as the interactions between factors. The effects of the three factors and interactions between them were calculated using an ANOVA (analysis of variance). The variation was shown to depend mainly on the breed and age. These two factors and the interaction between them determine about 80% of the variation in ejaculate characteristics. The season also has an effect, but its share in the influence of variation on ejaculate characteristics is relatively small. Ejaculates from Landrace boars are the most favourable for insemination, with a large volume, a relatively high sperm concentration, and the highest number of sperm. The highest number of insemination doses can be prepared from Landrace ejaculates—on average, 2.7–6.7 more doses than from the other breeds. Duroc boar ejaculates are most distinctive, with a very low volume but a very high sperm concentration and the highest sperm motility. The ejaculates of Pietrain boars showed the opposite pattern, with the largest volume but the lowest sperm concentration. The sexual development of young boars, expressed as an increase in ejaculation performance, progresses during their first year of insemination use. Full article

The physiological functions of mammalian sperm, such as motility, hyperactivation, and capacitation, require substantial energy. This study investigates the effects of two classic cryopreservation extenders—TCG (tris-citrate-glucose) and LEY (lactose-egg yolk)—on the energy metabolism of frozen–thawed boar semen. By comparing the quality indicators, key metabolite levels, and the activities of critical enzymes involved in glycolysis and the tricarboxylic acid cycle, we aim to understand how these different semen extenders influence the spermatozoa vitality of frozen–thawed boar semen. Following thawing, the LEY-cryopreserved sperm demonstrated significantly elevated motility parameters (viability, VCL, VSL, and VAP) and enhanced plasma membrane and acrosomal integrity compared with the TCG group (p < 0.05), though both cryopreserved groups exhibited significantly reduced performance relative to fresh semen controls. Cryopreservation markedly reduced intracellular adenosine triphosphate (ATP), pyruvate, and acetyl coenzyme A (A-CoA) levels (fresh > LEY > TCG; p < 0.05). The LEY-preserved spermatozoa retained higher activities of glycolysis-related enzymes (phosphofructokinase, PFK; pyruvate kinase, PK) compared with the TCG group, which, in turn, showed elevated lactate dehydrogenase (LDH) activity. Critically, TCG-suppressed pyruvate dehydrogenase (PDH) activity (p < 0.05) coincided with diminished A-CoA, indicating impaired mitochondrial oxidative phosphorylation. These results demonstrate LEY’s superior preservation of motility and membrane stability but highlight cryodamage-induced energy metabolism dysregulation, particularly TCG’s disruption of the glycolysis–TCA cycle coordination essential for spermatozoa function. In conclusion, the choice of semen extender has a significant impact on the energy metabolism and overall quality of frozen–thawed semen, highlighting the importance of optimizing cryopreservation protocols for improved spermatozoa viability and functionality. Full article

Artificial insemination (AI) is essential in intensive pig production, which significantly depends on semen quality from boars selected for health, genetics, and fertility. While AI aims to improve productivity, larger litters often result in smaller and less resistant piglets. Beyond fertility and genetic traits, boars also influence offspring health. This study investigated the relationship between sperm parameters of highly fertile boars and both reproductive outcomes and piglet physiological indicators. Multivariate analysis revealed significant paternal effects on blood markers reflecting organ function, including those of the pancreas, liver, and kidneys, as well as on glucose homeostasis, lipid metabolism, oxidative stress, protein and carbohydrate metabolism, muscle contraction, and neural signaling. Notably, sperm velocity was correlated with mitochondrial function, which is crucial for sperm motility, capacitation, DNA integrity, and embryo development—factors likely linked to healthier, more resilient offspring. Boars transmitting superior sperm velocity, erythropoiesis efficiency, and oxygen transport capacities produced piglets with better glucose regulation, growth, and resistance to neonatal hypoglycemia. These findings underscore the broader impact of sperm quality on offspring vitality and suggest that advanced sperm analysis could improve boar selection and enable more effective, health-oriented breeding strategies. Full article

This study investigated the impact of an increased arginine (ARG) level in a boar diet on semen production, sperm quality, and seminal plasma proteome. Adult Nebraska Index Line boars were assigned to two groups, one receiving a control diet with 0.77% arginine (n = 4) and the other a high-arginine diet with 1.77% arginine (n = 5). Semen was collected twice a week over the whole experiment, including one week before, six weeks during, and six weeks after the supplementation. Parameters such as semen volume and concentration were assessed immediately after collection, alongside sperm motility and morphology. Centrifugation of raw semen samples yielded seminal plasma for a gel-based proteome analysis. The seminal plasma proteins were extracted, quantified, and separated via 2D gel electrophoresis, allowing protein identification through mass spectrometry. Data analysis involved two-way ANOVA for comparisons (p < 0.05). Results showed that arginine supplementation improved semen volume and total sperm counts, with averages of 21 ± 3 doses in the control group versus 24 ± 2 in the ARG group (p = 0.05). Although sperm motility and morphology remained unaffected (p > 0.05), dietary arginine upregulated ten proteins and downregulated two. In summary, increased dietary arginine did not significantly alter key parameters of semen output or sperm quality but significantly impacted seminal plasma proteome, warranting further research on sperm viability. Full article

Spermatozoa are highly specialized male cells that are characterized by a unique ability to move, which is a critical factor for successful fertilization. The relative simplicity of motility assessment, especially in livestock, has made it a widely used parameter for evaluating ejaculate quality or cryopreserved semen in the clinical field, and an advanced tool in reproductive physiology and toxicology research. Technological advances in image analysis and computational methods have substantially increased its accuracy through the use of computer-assisted sperm analysis (CASA) to minimize subjective bias in motility assessments. Nevertheless, this more objective method still presents some significant challenges, including variability in the sample preparation, imaging conditions, and analytical parameters. These issues contribute to inconsistency and impair the reproducibility and comparability of data between laboratories. The implementation of standardized protocols, combined with comprehensive training and rigorous evaluation, can serve to mitigate some of the emerging inconsistencies. In addition, the in vitro conditions under which CASA analyses are performed often differ significantly from the natural environment of the female reproductive tract in vivo. This review discusses the methodologies, critical issues, and limitations of sperm motility analyses using CASA, with a particular focus on the boar as an important agricultural and biomedical model species in which this system is widely used. Full article

This study investigates the impact of nutritional supplementation on semen quality, epigenetic-related gene expression, and oxidative status in boars. Thirty boars were divided into a control group and a treatment group receiving Espermaplus (a supplement containing various vitamins, amino acids, omega-3 fatty acids, and trace elements with antioxidant properties). The experiment was performed for 12 weeks. Semen samples were collected at four moments: before starting the supplementation and after 3 weeks, 8 weeks, and 12 weeks. Spermatozoa concentration, motility, and kinematics were assessed using the CASA system. The measured parameters included curvilinear velocity—VCL; straight-line velocity—VSL; average path velocity—VAP; curvilinear distance—DCL; straight line distance—DSL; distance of average path—DAP; amplitude of lateral head displacement—ALH; beat-cross frequency—BCF; and head activity—HAC. Moreover, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity in seminal plasma, as well as the concentration of thiobarbituric acid reactive substances (TBARS), were measured to assess oxidative stress levels in boar’s seminal plasma. The expression of epigenetic-related genes such as Protamine 1 (Prm1), Protamine 2 (Prm2), and DNA-methyltransferase 3 alpha (Dnmt3a) were evaluated using real-time PCR. The treated group showed a significant increase in spermatozoa concentration (p = 0.003), total motility (p = 0.001), and progressive motility (p = 0.002) after 12 weeks compared to the control group. Kinematic parameters such as VCL, VSL, and VAP were also significantly higher (p < 0.001; p = 0.028; p < 0.001, respectively) in the treated group by the end of the experiment. SOD and GPx activities were consistently higher (p < 0.01; p < 0.001, respectively) in the treated group, indicating enhanced antioxidative capacity. TBARS levels as an indicator of lipid peroxidation and oxidative damage were significantly lower (p < 0.01) in the treated group by the end of the study. Significant changes were observed in the expression of epigenetic-related genes. The supplementation of boar diets with Espermaplus significantly improved semen quality, reduced oxidative stress, and had an impact on the expression levels of certain epigenetic-related genes, suggesting that dietary antioxidants and bioactive compounds can enhance boar semen. Full article

Mammalian spermatozoa rely on glycolysis and mitochondrial oxidative phosphorylation for energy leading up to fertilization. Sperm capacitation involves a series of well-regulated biochemical steps that are necessary to give spermatozoa the ability to fertilize the oocyte. Additionally, zinc ion (Zn²⁺) fluxes have recently been shown to occur during mammalian sperm capacitation. Semen from seven commercial boars was collected and analyzed using image-based flow cytometry before, after, and with the inclusion of 2 mM Zn²⁺ containing in vitro capacitation (IVC) media. Metabolites were extracted and analyzed via Gas Chromatography-Mass Spectrometry (GC-MS), identifying 175 metabolites, with 79 differentially abundant across treatments (p < 0.05). Non-capacitated samples showed high levels of respiration-associated metabolites including glucose, fructose, citric acid, and pyruvic acid. After 4 h IVC, these metabolites significantly decreased, while phosphate, lactic acid, and glucitol increased (p < 0.05). With zinc inclusion, we observed an increase in metabolites such as lactic acid, glucitol, glucose, fructose, myo-inositol, citric acid, and succinic acid, while saturated fatty acids including palmitic, dodecanoic, and myristic acid decreased compared to 4 h IVC, indicating regulatory shifts in metabolic pathways and fatty acid composition during capacitation. These findings underscore the importance of metabolic changes in improving artificial insemination and fertility treatments in livestock and humans. Full article

To address the safety problems posed by the transportation of boar semen using LN, this study was conducted on the short-term storage of frozen boar semen in dry ice (−79 °C). Boar semen frozen in LN was transferred to dry ice, kept for 1 day, 3 days, 5 days, 7 days, or 8 days, and then moved back to LN. The quality of frozen semen stored in LN or dry ice was determined to evaluate the feasibility of short-distance transportation with dry ice. The results showed that 60 °C for 8 s was the best condition for thawing frozen semen stored in dry ice. No significant differences in spermatozoa motility, plasma membrane integrity, or acrosome integrity were observed in semen after short-term storage in dry ice compared to LN (p > 0.05). There were no significant changes in antioxidant properties between storage groups either (p > 0.05). In conclusion, dry ice could be used as a cold source for the short-term transportation of frozen boar semen for at least 7 days, without affecting sperm motility, morphological integrity, or antioxidant indices. Full article