Search Results (54)

High semen quality is vital for reproductive success in the swine industry; however, seasonal fluctuations often compromise this quality. The molecular mechanism underlying these seasonal effects on semen quality remains largely unclear. This study employed untargeted metabolomic profiling of boar seminal plasma (SP) to identify metabolites and metabolic pathways associated with semen quality during the summer and winter months. Semen samples were collected from mature Duroc boars at a commercial boar stud and classified as Passed or Failed based on motility and morphology. SP from five samples per group was analyzed using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS). In total, 373 metabolites were detected in positive ion mode and 478 in negative ion mode. Several differentially expressed metabolites (DEMs) were identified, including ergothioneine, indole-3-methyl acetate, and avocadyne in the summer, as well as LysoPC, dopamine, and betaine in the winter. These metabolites are associated with key sperm functions, including energy metabolism, antioxidant defense, and capacitation. KEGG pathway analysis indicated enrichment in starch and sucrose metabolism, pyrimidine metabolism, and amino acid metabolism across the seasons. Overall, the results reveal that SP metabolomic profiles vary with the season, thereby influencing semen quality. The identified metabolites may serve as potential biomarkers for assessing semen quality and enhancing reproductive efficiency in swine production. Full article

Artificial insemination (AI) is a key breeding technique in the swine industry; however, the lack of reliable biomarkers for semen quality limits its effectiveness. Seminal plasma (SP) contains extracellular vesicles (EVs) that present a promising, non-invasive biomarker for semen quality. This study explores the biochemical profiles of boar SP to assess semen quality through near-infrared spectroscopy (NIRS) and proteomics of SP-EVs. Fresh semen from mature Duroc boars was evaluated based on sperm motility, classifying samples as Passed (≥70%) or Failed (<70%). NIRS analysis identified distinct variations in water structures at specific wavelengths (C1, C5, C12 nm), achieving high accuracy (92.2%), sensitivity (94.2%), and specificity (90.3%) through PCA-LDA. Proteomic analysis of SP-EVs revealed 218 proteins in Passed and 238 in Failed samples. Nexin-1 and seminal plasma protein pB1 were upregulated in Passed samples, while LGALS3BP was downregulated. The functional analysis highlighted pathways associated with single fertilization, filament organization, and glutathione metabolism in Passed samples. Integrating NIRS with SP-EV proteomics provides a robust approach to non-invasive assessment of semen quality. These findings suggest that SP-EVs could serve as effective biosensors for rapid semen quality assessment, enabling better boar semen selection and enhancing AI practices in swine breeding. Full article

In swine reproduction, immune-mediated mechanisms such as neutrophil extracellular trap (NET) formation can affect sperm function and reduce fertility outcomes. This study evaluated the capacity of antioxidant and reproductive compounds—butylated hydroxytoluene (BHT), prostaglandin E₂ (PGE₂), bovine serum albumin (BSA), and seminal plasma (SP)—to modulate NETosis in co-cultures of swine neutrophils and cryopreserved spermatozoa. NET formation was quantified by nuclear area expansion and validated by digital cytometry and immunofluorescence. BHT (0.5 mM) and PGE₂ (10 µM) produced the most significant inhibitory effects, reducing NETotic cell percentages from 34.5 ± 2.7% (sperm-exposed controls) to 12.2 ± 1.3% and 14.5 ± 2.1%, respectively (p < 0.01). SP at 20% decreased NETosis to 16.8 ± 1.8%, while BSA (0.5%) achieved a moderate reduction to 21.3 ± 2.5%. Flow cytometry revealed reduced peroxynitrite levels in sperm treated with SP and BSA. Two NET phenotypes (aggNETs and sprNETs) were identified. BTS medium enhanced NET formation, whereas DNase I degraded NETs effectively. These findings identify porcine NETosis as a redox-sensitive pathway modulated in vitro, suggesting an immunological role in enhancing sperm preservation for swine artificial insemination. Full article

(1) Background: The RoXsta^TM system has been developed as a rapid, effective means of profiling different types of antioxidant activity. The purpose of this study was to examine its performance utilizing a diverse array of biological fluids including semen, blood plasma, serum, urine, saliva, follicular fluid and plant extracts. (2) Methods: The RoXsta^TM system was used to assess the ability of different fluids to suppress free radical formation as well as scavenge a variety of toxic oxygen metabolites including free radicals and both hydrogen and organic peroxides. (3) Results: Human semen was shown to have significantly (p < 0.001) more peroxide scavenging power than any other fluid tested (10–14 mM vitamin C equivalent compared with 1–2 mM for blood serum or plasma), while urine was particularly effective in scavenging free radicals and preventing free radical formation (p < 0.001). The powerful antioxidant properties of human semen were shown to reside within the seminal plasma (SP) fraction, rather than the spermatozoa, and to be resistant to snap freezing in liquid nitrogen. Moreover, comparative studies demonstrated that human SP exhibited significantly (p < 0.001) higher levels of antioxidant potential than any other species examined (stallion, bull, dog) and that this intense activity reflected the relative vulnerability of human spermatozoa to peroxide attack. (4) Conclusions: The RoXsta^TM system provides valuable information on the antioxidant profile of complex biological fluids, supporting its diagnostic role in conditions associated with oxidative stress. Based on the results secured in this study, human semen is identified as a particularly rich source of antioxidants capable of scavenging both hydrogen and organic peroxides, in keeping with the high susceptibility of human spermatozoa to peroxide-mediated damage. Full article

Background: Seminal plasma is an important component of semen and has a significant effect on sperm function. However, the relationship between seminal plasma and sperm freezing capacity has not been fully studied. Purpose: Exploring metabolites and proteins related to the boar sperm freezing capacity in seminal plasma, by metabolomic and proteomic approaches, and directly verifying the protective effect of seminal plasma on the cryopreservation of boar sperm using high and low freezability seminal plasma as base freezing extender. Methods: Semen samples were collected from 30 different boars, 11 high and 11 low freezing-resistant boars were selected after freezing 2~4 times, and seminal plasma was selected at the same time. Sperm motility and movement parameters were analyzed using a CASA system. Reproductive hormones (Testosterone, progesterone, estradiol, prolactin, prostaglandin F2α, luteinoid hormone) in seminal plasma were detected by ELISA. Analysis of proteins and metabolites in high and low freezing-resistant seminal plasma by proteomics and metabolomics techniques. Results: The six reproductive hormones tested were not significantly associated with sperm freezing resistance. A total of 13 differentially expressed metabolites (DEMs) and 38 differentially expressed proteins (DEPs) were identified, while a total of 348 metabolites and 1000 proteins were identified. These DEMs were related to energy metabolism, drugs, or environmental pollutants, while the DEPs were mainly involved in the cytoskeletal dynamics and cell adhesion processes. There were 33 metabolites and 70 proteins significantly associated with mean progress motility (PM) at 10 min and 2 h after thawing. The 70 related proteins were associated with cell division and cycle regulation in gene ontology (GO) terms, as well as KEGG pathways, thermogeneration, and pyruvate metabolism. Using highly freezable boar SP as a base freezing extender made no difference from using lowly freezable boar SP, and both were not as good as the commercial control. Conclusion: There were significant differences in seminal plasma with different freezability, but the similarity was much greater than the difference. The protection effect of seminal plasma is not remarkable, and it does not exhibit superior cryoprotective properties compared to commercial semen cryoelongators. Significance: This study provides a deeper understanding of how seminal plasma composition affects sperm freezabilty. It provides potential biomarkers and targets for improving sperm cryopreservation techniques. Full article

Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen–thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14⁺/PI⁻) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen–thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP. Full article

Seminal plasma (SP) accounts for more than 90% of semen volume. It induces inflammation, regulates immune tolerance, and facilitates embryonic development and implantation in the female reproductive tract. In the physiological state, SP promotes endometrial decidualization and causes changes in immune cells such as macrophages, natural killer cells, regulatory T cells, and dendritic cells. This leads to the secretion of cytokines and chemokines and also results in the alteration of miRNA profiles and the expression of genes related to endometrial tolerance and angiogenesis. Together, these changes modulate the endometrial immune microenvironment and contribute to implantation and pregnancy. However, in pathological situations, abnormal alterations in SP due to advanced age or poor diet in men can interfere with a woman’s immune adaptation to pregnancy, negatively affecting embryo implantation and even the health of the offspring. Uterine pathologies such as endometriosis and endometritis can cause the endometrium to respond negatively to SP, which can further contribute to pathological progress and interfere with conception. The research on the mechanism of SP in the endometrium is conducive to the development of new targets for intervention to improve reproductive outcomes and may also provide new ideas for semen-assisted treatment of clinical infertility. Full article

Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll^® gradient densities (PG 45–90% and PG 35–70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45–90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35–70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45–90% was used for fertilization. PG 45–90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2–26.3%) and morula (8.1–10.5%) rates did not differ between the groups. Therefore, PG 45–90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes. Full article

Infertility is a growing concerning health problem affecting around 15% of couples worldwide. Conventional semen parameters have limited accuracy for male infertility potential determination. Current advances in the understanding of male infertility indicate that environmental and occupational exposure to chemical contaminants are important etiological factors leading to infertility problems. In this context, some heavy metals (HMs) can be considered as endocrine-disrupting compounds (EDCs), thus altering the seminal quality. This systematic review aims to summarize the key points to detect and quantify HMs in human seminal plasma (SP) and the involved analytical tools. Our results showed that that for HM quantification, atomic absorption spectroscopy (AAS) and inductively coupled plasma (ICP) were the most employed techniques while Zn, Cd, Pb, and Cr were the analytes most often detected. Fast, reliable, and sensitive quantification of EDCs in SP could be important for the development of accurate diagnostic and preventive strategies to address male infertility towards providing personalized therapy. Full article

Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30–400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility. Full article

This study aimed at evaluating the relationship between biomarkers of oxidative stress (OS) in seminal plasma and sperm motility in bulls before and after cryopreservation. Three ejaculates per bull were collected from 20 young bulls. Each ejaculate was analyzed for motility before and after cryopreservation (by CASA), and the SP concentration of Advanced Oxidation Protein Products (AOPP), thiols, and carbonyl groups (CT) were examined. Then, based on their motility, the ejaculates were grouped into: high motility fresh (HMF), low motility fresh (LMF), high motility thawed (HMT), and low motility thawed (LMT) groups. Higher AOPP and thiol concentrations on SP were related (p < 0.05) to the higher LIN and BCF and lower ALH of fresh semen. In addition, AOPP and thiols were significantly higher in HMF than LMF. As a confirmation of this, the Receiver Operating Characteristic (ROC) curve analysis showed that AOPP and thiol concentrations in SP were able to discriminate between HMF and LMF ejaculates (Area Under the Curve of 71.67% and 72.04%, respectively). These observations give an alternative perspective on the relationship between sperm motility and the OS parameters of SP, which need further investigations. Full article

Seminal plasma (SP) mirrors the local pathophysiology of the male reproductive system and represents a non-invasive fluid for the study of infertility. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) provides a high-throughput platform to rapidly extrapolate the diagnostic profiles of information-rich patterns. In this study, dispersive solid phase extraction (d-SPE) combined with MALDI-TOF-MS was applied for the first time to the human SP, with the aim of revealing a diagnostic signature for male infertility. Commercially available octadecyl (C₁₈)-, octyl (C₈)-bonded silica sorbents and hexagonal mesoporous silica (HMS) were tested and the robustness of MALDI-TOF peptide profiling was evaluated. Best performances were obtained for C₁₈-bonded silica with the highest detection of peaks and the lowest variation of spectral features. To assess the diagnostic potential of the method, C₁₈-bonded silica d-SPE and MALDI-TOF-MS were used to generate enriched endogenous peptide profiles of SP from 15 fertile and 15 non-fertile donors. Principal component analysis (PCA) successfully separated fertile from non-fertile men into two different clusters. An array of seven semenogelin-derived peptides was found to distinguish the two groups, with high statistical significance. These findings, while providing a rapid and convenient route to selectively enrich native components of SP peptidome, strongly reinforce the prominent role of semenogelins in male infertility. Full article

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. Full article

This study monitored the chemical and biochemical composition of bovine seminal plasma (SP). Freshly ejaculated semen (n = 20) was aliquoted into two parts. The first aliquot was immediately assessed to determine the sperm motion parameters. Another motility measurement was performed following an hour-long co-incubation of spermatozoa with SP at 6 °C. The other aliquot was processed to obtain the SP. Seminal plasma underwent the analyses of chemical composition and quantification of selected proteins, lipids and RedOx markers. Determined concentrations of observed parameters served as input data to correlation analyses where associations between micro and macro elements and RedOx markers were observed. Significant correlations of total oxidant status were found with the content of Cu and Mg. Further significant correlations of glutathione peroxidase were detected in relation to Fe and Hg. Furthermore, associations of chemical elements and RedOx markers and spermatozoa quality parameters were monitored. The most notable correlations indicate beneficial effects of seminal Fe on motility and Mg on velocity and viability of spermatozoa. On the contrary, negative correlations were registered between Zn and sperm velocity and seminal cholesterol content and motility. Our findings imply that seminal plasma has a prospective to be developed as the potential biomarker of bull reproductive health. Full article

Seminal plasma (SP) plays a crucial role in reproduction and contains a large number of proteins, many of which may potentially modify sperm functionality. To evaluate the effects of SP identity and its protein composition on human sperm function, we treated the sperm of several males with either their own or multiple foreign SPs in all possible sperm–SP combinations (full-factorial design). Then we recorded sperm motility and viability in these combinations and investigated whether the sperm performance is dependent on sperm and SP identity (or their interaction). Finally, we studied whether the above-mentioned sperm traits are affected by the abundance of three SP proteins, dipeptidyl peptidase-4 (DPP4), neutral endopeptidase (NEP), and aminopeptidase N (APN). The identity of the SP donor affected sperm swimming velocity, viability, and the proportion of hyperactivated sperm, but males’ own SP was not consistently more beneficial for sperm than foreign SPs. Furthermore, we show that sperm performance is also partly affected by the interaction between sperm and SP donor. Finally, we found that DPP4 and NEP levels in SP were positively associated with sperm swimming velocity and hyperactivation. Taken together, our results highlight the importance of seminal plasma as a potential source of biomarkers for diagnostics and therapeutic interventions for male-derived infertility. Full article