Mitochondrially active boar sperm are essential for fertilization but evade pelleting by centrifugation - implications for in-vitro sperm enrichment and antioxidant enzyme analysis.

Differential molecular mechanisms of heparin, BSA, and Ca2+-Induced boar sperm capacitation revealed by transcriptomics.

Non-thermal plasma enhances boar sperm quality via FOXO3-PINK1/Parkin-mediated mitophagy.

The targeted, substrate-specific degradation of paternal mitochondria inside the zygote, known as post-fertilization sperm mitophagy, is a crucial and evolutionarily conserved early embryonic event. It ensures the exclusive maternal inheritance of the mitochondrial genome. Post-fertilization sperm mitophagy was initially thought to only be achieved via the ubiquitin-proteasome system. Until pro-autophagic receptor proteins such as SQSTM1, GABARAP, as well as the proteasome-interacting ubiquitinated protein dislocase VCP, were identified as contributors to the degradation of the sperm mitochondria early after mammalian fertilization. This synergy of proteasomal and autophagic pathways ensures a timely degradation of sperm mitochondria shortly after fertilization. The discovery of these autophagic receptors lead researchers to believe there might be other autophagic receptors and determinants necessary for proper post-fertilization sperm mitophagy. Based on the established inventory of proteins from mass spectrometry trials of boar spermatozoa exposed to porcine oocyte extracts in an intra-specific porcine cell-free system (CFS), five candidate mitophagy determinants were further investigated in this study, namely LACTB, PRDX3, PSMA8, TOMM34, and FUNDC1. These proteins of interest were studied and validated by using in vitro fertilization (IVF) protocols, cell imaging of spermatids, spermatozoa, oocytes and zygotes, protein interactome analysis, and the porcine CFS. The proteins PSMA8 and TOMM34 behaved in accordance with our proteomic study predictions. The PSMA8 labeling increased after exposure to CFS; in agreement with the classification PSMA8 was given from the mass spectrometry findings. TOMM34 underwent a visible decrease in labeling after exposure to CFS, which also agreed with its proteomic classification; this labeling persisted in IVF zygotes. Except for LACTB, the examined proteins showed mutual interactions as well as interactions with previously identified sperm mitophagy factors in the STRING interactome analysis. Results from this study validate the novel porcine CFS as a valuable tool for the exploration of early fertilization events at a molecular level. Future phenotyping and functional studies using porcine CFS will advance the understanding of mitochondrial inheritance and zygotic development and potentially shed light on the origins of certain mitochondrial diseases arising from the failure of post-fertilization sperm mitophagy.

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.

Interest in identifying the type of endogenous compounds capable of initiating the formation of reactive oxygen species (ROS) in boar semen upon photoexcitation is driven by the use of photobiomodulation methods in artificial insemination of pigs and the unclear mechanisms underlying the stimulating effect of light on sperm function. One hypothesis regarding the mechanism of biological activity of light suggests the crucial role of ROS in mediating the effects of photobiomodulation. This study examined endogenous fluorophores in boar semen characterized by absorption in the UV-A and visible spectral regions and capable of generating ROS upon photoexcitation. For the first time, weak fluorescence of endogenous porphyrin sensitizers was detected in freshly collected boar ejaculate (native sperm) alongside intense fluorescence of NADH (NAD(P)H) coenzymes and flavins (flavin mononucleotide, FMN, and flavin adenine dinucleotide, FAD). Using steady-state and kinetic spectrofluorimetry, the types of porphyrins localized in the sperm were identified, and the presence of uroporphyrin III, coproporphyrin III, and their zinc complexes was established. Using a chemiluminescent assay for monitoring ROS levels and specific ROS quenchers (scavengers), it was shown that exposure of boar sperm to radiation of blue spectral region leads to the formation of singlet oxygen and hydrogen peroxide. It was concluded that light-induced generation of ROS (primarily singlet oxygen) by endogenous porphyrins and flavins can alter the redox state of sperm and, at low ROS concentrations, exert a mild regulatory effect on their functional activity.

Small non-coding RNAs (sRNAs) act as post-transcriptional regulators and protectors of genomic integrity. Environmental stress and genomic variation can both alter the sRNA composition in sperm, leading to altered offspring phenotypes. Commercial pig breeds, which have undergone years of selective breeding, vary in purpose: some are optimized for meat production, others for high reproductive capacity - traits that are often antagonistic. This study aims to characterize and compare the sperm sRNA composition in four commercial pig breeds, two used for meat production (sire lines) and two for reproductive purposes (dam lines). We find that the two breeds propagated for reproduction traits (Landrace, Yorkshire) show high variability in PIWI-interacting RNA (piRNA) which target transposable elements. In contrast, the two meat production breeds (Duroc, Hampshire) were enriched in mitochondrial ribosomal RNA-derived sRNA (mt rsRNA). These trends may reflect an emphasis on genome protection among reproductive breeds, and on mitochondrial function and metabolic output in meat production breeds. Other notable differences included amongst the miRNAs, which were associated with numerous developmental and metabolic processes. Our study investigates the unique sRNA profiles in boar sperm associated with different breeding objectives, highlighting possible epigenetic mechanisms that could be leveraged in selective breeding programs to optimize fertility, growth and offspring health.

In brief: Successful fertilization in pigs depends on sperm that can navigate the complex geometry of the sow's reproductive tract, yet current sperm preparation methods select only on motility and can damage cells. This study demonstrates that a biomimetic microfluidic device selects boar sperm on thigmotactic competence, yielding functionally superior populations and revealing species-specific differences in angular navigation. Abstract: Efficient sperm selection is crucial for reproductive success in pigs, where artificial insemination is the main method of assisted reproduction. Traditional methods such as density gradient centrifugation (DGC) can enrich motile sperm but may cause mechanical and oxidative stress and do not replicate the natural filters of the sow's reproductive tract. This study assessed a biomimetic microfluidic (MF) device, previously validated in cattle, sheep, and dogs, for enrichment of high-quality spermatozoa from chilled boar ejaculates (n = 18). Unprocessed extended semen (RAW), DGC, and MF samples were evaluated for concentration, computer-assisted sperm analysis kinematics, viability, morphology, acrosome integrity, DNA fragmentation (sperm chromatin structure assay), and thigmotactic navigation. Microfluidic processing yielded a lower concentration (5.73 ± 2.22 × 106/ml) than RAW (65.20 ± 23.33 × 106/ml) and DGC (22.68 ± 10.95 × 106/ml; both p < 0.0001), an ∼11-fold reduction consistent with functional enrichment. Microfluidic sperm showed higher total motility (91.0% vs 77.4% DGC, 67.1% RAW), progressive motility (74.8% vs 59.6% and 33.1%; 2.2-fold over RAW), intact acrosomes (81.4% vs 71.9% and 62.6%), and lower DNA fragmentation (0.77% vs 1.68% and 4.38%; ∼5.7-fold reduction versus RAW; all p < 0.0001). Angular microchannels revealed graded thigmotaxis, with navigation success decreasing from 95.8% at 45° to 87.9% at 90° and 69.4% at 135°, comparable to bull and dog but lower than ram (89.1% at 135°). Biomimetic microfluidics isolates functionally superior boar sperm, offering a pump-free alternative to centrifugation with potential to enhance artificial insemination and in vitro embryo production.

ABSTRACTFlow cytometric sorting can be used to select sperm subpopulations with defined physiological or molecular traits; however, the impact of sorting on sperm function and subsequent embryo development in the porcine model remains unresolved. The objective of this study was to evaluate the effect of fluorescence‐activated cell sorting (FACS) on boar sperm motility and developmental competence following in vitro fertilization (IVF). Pooled semen from boars was divided into sorted and unsorted (control) treatments and analyzed for motility and kinematic parameters using computer‐aided sperm analysis (CASA) with both population‐level (conventional, population‐averaged) metrics and single‐cell track data collected before and after sorting. Oocytes were fertilized with either treatment, and cleavage (≥ 2‐cell stage) and blastocyst development were evaluated. Cleavage and blastocyst percentages were reduced in the sorted (48.8% ± 9.9% and 8.5% ± 4.2%) compared with control (92.2% ± 1.5% and 28.4% ± 2.8%, respectively; p < 0.001) group. Population‐level CASA revealed statistically significant but biologically modest changes in motility and kinematic parameters following sperm sorting. In contrast, single‐cell CASA coupled with k‐means clustering identified four distinct motility subpopulations and demonstrated that sorting redistributed existing motility states, characterized by depletion of fast, progressive sperm and enrichment of less energetically demanding populations. These findings suggest that FACS alters the composition of sperm motility subpopulations, alongside a marked reduction in post‐fertilization developmental competence in porcine IVF.

This study aimed to determine the effect of sugarcane juice substitution in a citrate–egg yolk diluent (CE-Y) on the quality of frozen semen of landrace boars. The semen used in this study was collected from four landrace boars aged between 2–2.5 years using the glove gland method. The study employed a Completely Randomized Design with five treatments and four replications, namely T0 = 100% S-KT + 0% sugarcane juice, T1 = 90% S-KT + 10% sugarcane juice, T2 = 80% S-KT + 20% sugarcane juice, T3 = 70% S-KT + 30% sugarcane juice, and T4 = 60% S-KT + 40% sugarcane juice. All treatments were supplemented with 6% glycerol. Semen was evaluated macroscopically and microscopically. After evaluation, a holding time of 2 hours was applied in S-KT diluent with a ratio of 1:1 at a temperature of 24–26°C. The semen was then centrifuged at 3000 rpm for 15 minutes. The pellet was diluted using a base diluent and then distributed into tubes containing the treatment diluent. The semen was packaged in 0.5 mL straws and equilibrated for 2 hours at 3–5°C. After equilibration, the straws were frozen at a distance of 10 cm above the surface of liquid nitrogen for 10 minutes. The straws were then plunged into liquid nitrogen and stored in a liquid nitrogen container. Thawing was carried out by immersing the frozen semen straws in warm water at 37°C for 30 seconds. After dilution and equilibration, sperm quality was evaluated based on motility, viability, and abnormalities. Post-thaw evaluation included motility, viability, abnormalities, and recovery rate. All obtained data were analyzed using SPSS 27.0. The results showed that sugarcane juice substitution had no significant effect (P&gt;0.05) on motility, viability, abnormalities, and RR. It was concluded that the substitution of sugarcane juice with the addition of citrate–egg yolk diluent did not affect the quality of frozen spermatozoa of landrace boars at the stages of post-dilution, post-equilibration, and post-thawing.

This study investigated the effects of red LED light exposure on boar semen quality, sperm metabolism, and reproductive performance in sows. A total of 60 semen samples were collected from Duroc boars and randomly assigned to six light treatment procedures and a control. Among these, Procedure #1 (red light for 13 min - dark for 10 min - red light for 13 min) significantly improved sperm motility indicators, particularly reducing the amplitude of lateral head displacement (ALH) and enhancing beat-cross frequency (BCF) over time (P < 0.05). Flow cytometry analysis revealed that this procedure preserved mitochondrial membrane potential and acrosome integrity. A large-scale field trial involving 1,499 sows across four breeds demonstrated that insemination using red light-treated semen (Procedure #1) significantly increased pregnancy and delivery rates in Large White and Yorkshire sows, along with higher litter birth weight (LBW), total number born (TNB), and number born alive (NBA) (P < 0.05). Untargeted LC-MS/MS metabolomics identified 60 differential metabolites and revealed distinct clustering between treated and control groups. Enrichment and ROC analyses highlighted key altered pathways, including retinol metabolism, fatty acid degradation, sphingolipid metabolism, and arachidonic acid metabolism. Metabolites such as vitamin A, palmitoylcarnitine, psychosine, and prostaglandin E2 showed potential as biomarkers of red-light exposure. These findings suggest that optimized red LED exposure enhances boar semen quality and sow reproductive outcomes by modulating sperm motility and metabolic profiles, offering a novel strategy to improve reproductive efficiency in swine production.

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.

Seminal plasma extracellular vesicle-derived miR-296-3p targets PLA2G6 to enhance boar sperm motility by lowering Lys Phosphatidylcholine.

Ferroptosis is an iron-dependent form of regulated cell death characterized by labile iron accumulation, glutathione depletion, mitochondrial dysfunction, and lipid peroxidation. Although ferroptosis has been linked to impaired spermatogenesis and male infertility in vivo, its occurrence and functional relevance in ejaculated spermatozoa during liquid semen storage at 17 °C has not been established. This study evaluated how ferroptosis inducers and inhibitors modulate boar sperm function during liquid semen storage at 17 °C and determined whether ferroptosis contributes to spontaneous sperm deterioration over time. Semen samples from five Duroc boars were treated with ferroptosis inducers: Erastin (system Xc- antagonist) and RSL-3 (GPX4 inhibitor), inhibitors: Ferrostatin-1 and N-acetylcysteine (NAC), and vehicle/untreated controls. Sperm motility was assessed by computer-aided sperm analysis, and comprehensive ferroptosis-associated biomarkers were quantified using spectral flow cytometry with a 7-fluorophore panel including sperm identification (Hoechst 33342), labile ferrous iron (FerroOrange), glutathione (monochlorobimane), reactive oxygen species (DCFDA), lipid peroxidation (BODIPY 581/591 C11), mitochondrial membrane potential (MitoTracker Deep Red), and plasma membrane integrity (propidium iodide). RSL-3 induced declines in total and progressive motility accompanied by elevated Fe2+ accumulation, glutathione depletion, mitochondrial depolarization, and membrane disruption by day 3 (p < 0.05). Erastin produced similar but delayed effects, reaching maximum ferroptotic markers by day 7. Both Ferrostatin-1 and NAC mitigated oxidative injury and preserved motility and cellular integrity relative to inducers. Critically, untreated controls exhibited time-dependent increases in all ferroptosis-associated biomarkers over 7 days of liquid storage, indicating spontaneous ferroptotic activity independent of chemical induction. These findings establish ferroptosis as a mechanistically relevant contributor to sperm.

Capacitation is the process by which sperm undergoes physical, biochemical, and molecular changes to acquire the ability to fertilize an oocyte. The first stage occurs after ejaculation with the removal of cholesterol from the cell membrane, reorganization of the cell membrane, and permission of ion entry into the cell. Intracellular alkalization leads to signaling, resulting in the basic capacitation cascade (sAC-cAMP-PKA-PTP). Consequently, the process of hyperactivation and preparation for the acrosome reaction is completed. Significant interspecies differences are observed in farm animals, and identifying these differences will make important contributions to the development of reproductive technologies. Bull sperm is mostly dependent on heparin-like glycosaminoglycans; however, in ram, seminal plasma proteins are shown to play important roles during capacitation. In goat, the rapid and stress-sensitive capacitation mechanism and the tight zinc-dependent in vivo control of boar sperm are important. Furthermore, early and premature capacitation caused by cryopreservation is called cryocapacitation and directly affects fertilization success. Species-specific capacitation mechanisms have been investigated; however, understanding differences will be beneficial using omics approaches in fertilization prediction and in developing cryopreservation strategies. Moreover, this review compares the molecular mechanisms of sperm capacitation in bulls, rams, bucks, and boars. It highlights species-specific differences and their impact on reproductive technologies.

Spermatozoa strongly rely on their streamlined morphology to successfully fertilize an oocyte. A striking example of a morphological defect resulting in infertility is acephalic spermatozoa syndrome, a rare but severe condition leading to detachment of the sperm head and tail. Among the most common genetic causes for this syndrome are mutations in the linker of nucleo- and cytoskeleton (LINC) complex component SUN5. LINC complexes typically reside in the nuclear envelope, the double-membrane surrounding the nucleus, where they establish a physical bridge between nucleus and cytoplasm. This localization allows them to transduce mechanical signals from the cytoplasm to the nucleus and to regulate nuclear morphology. In sperm, LINC complexes are essential for a multitude of morphological changes during sperm development, including the reshaping of the nucleus and establishing a stable head-tail junction. Here, using superresolution fluorescence microscopy, we find that sperm-specific SUN5 localizes to the base of the head in human, mouse, and boar sperm. By applying in situ cryoelectron tomography, we find an extensive hexagonal lattice in the nuclear envelope in this region. This lattice appears to maintain a consistent close apposition between the inner and outer nuclear membranes (ONM). Further structural analysis supports a model in which LINC complexes form this lattice by laterally interacting at the ONM. Overall, this study sheds light on nuclear envelope organization in the highly streamlined sperm cell, providing a structural basis for uniform nuclear envelope spacing maintained by a LINC lattice and rationalizing the disruptive effects of SUN5 mutations.

Polystyrene nanoplastics are known to exert impact on the mammalian reproductive system, primarily via oxidative damage; however, the specific mechanisms underlying these effects have yet to be determined. Here, the mechanisms responsible for the oxidative damage experienced by mammalian sperm when exposed to polystyrene nanoplastics were investigated, along with how these effects could be mitigated by supplementation with antioxidants. The influence of Astragalus polysaccharides on the toxic effects of polystyrene nanoplastics on boar sperm was investigated by evaluating sperm quality, antioxidant functionality, and protein tyrosine phosphorylation. Exposure of capacitated boar sperm to polystyrene nanoplastics at doses of 1 and 2 μg/mL significantly reduced the proportion of progressive sperm, antioxidant capacity, and protein tyrosine phosphorylation in the sperm flagellum. Sperm were then exposed to different doses of Astragalus polysaccharides (0.25, 0.5, 0.75, and 1 mg/mL); analysis revealed that the supplementation of 1 mg/mL of Astragalus polysaccharides in the presence of 1 μg/mL of polystyrene nanoplastics increased the proportion of progressive sperm, antioxidant capacity, and protein tyrosine phosphorylation in the sperm flagellum. Collectively, Astragalus polysaccharides was confirmed to improve the quality, antioxidant capacity, and protein tyrosine phosphorylation of sperm exposed to polystyrene nanoplastics by increasing total antioxidative capacity activity and reducing total reactive oxygen species. The in vitro exposure of boar sperm to polystyrene nanoplastics was not affected by other metabolic factors and directly reflects the relationship between Astragalus polysaccharides and polystyrene nanoplastics exposed sperm. Cite this article as: Hu, Q., Li, Y., &amp; Gao, Z. (2026). Astragalus polysaccharides ameliorate the toxic effects of polystyrene nanoplastics on boar sperm. Acta Veterinaria Eurasia, 51, 0153, doi:10.5152/actavet.2026.25153.

Hydrogen sulfide in boar reproduction: Cystathionine β-synthase and cystathionine γ-lyase localization in reproductive tissues and sperm across maturation stages.

Identification and functional prediction of ceRNA networks regulating ATP levels in boar spermatozoa treated with non-thermal plasma.

Cryopreserved boar sperm yields lower conception rates and litter sizes than liquid-stored semen due to oxidative damage from elevated reactive oxygen species. Our prior study demonstrated that mitochondrial regulators, SIRT5 and IDH2, were significantly downregulated during cryopreservation; however, how this dysregulation alters sperm metabolism remains unknown. Here, SIRT5 and IDH2 were knocked down in boar sperm via siRNA electro-transfection, and differential metabolites were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results showed that SIRT5/IDH2 knockdown dysregulated antioxidant pathways and significantly downregulated L-methionine (p < 0.05). Supplementation with 80 µM L-methionine significantly increased total sperm motility (fresh: ∼+6% at 48 hr; cryopreserved: ∼+10%), progressive motility, ATP content, and mitochondrial function, while maintaining acrosomal and plasma membrane integrity. Notably, L-methionine reduced reactive oxygen species levels by 9% (p < 0.05) in fresh sperm at 48 hr and 13% in post-thawed sperm. This study elucidates a mechanism-driven strategy wherein the SIRT5/IDH2 axis maintains sperm antioxidant capacity by regulating L-methionine and associated metabolites. These findings enhance our understanding of energy metabolism in sperm and offer a targeted approach to improve cryopreserved semen quality, potentially increasing artificial insemination success rates.