Flagellar Movement Research Articles

Enhancement mechanism of xanthan gum production in Xanthomonas campestris induced by atmospheric and room-temperature plasma (ARTP) mutagenesis.

Xanthan gum, produced by the aerobic fermentation of carbohydrates by Xanthomonas campestris, is a significant natural and industrial biopolymer known for its exceptional properties. Enhancing the yield of xanthan gum production remains a critical challenge. This study employed atmospheric and room temperature plasma (ARTP) technology to induce mutagenesis in X. campestris, resulting in a high-yielding strain, X20. The X20 mutant exhibited a substantial increase in xanthan gum yield, ranging from 13.3 % to 30.0 % over the starting strain across NaCl concentrations of 0, 6.0, and 8.0 g/L, along with improved viscosity and molecular weight. In the whole genome of X20 mutant, a total of 80 variant sites differing from the reference genome were identified, involving 76 mutated genes. Among these, 19 were missense mutations primarily associated with the two-component system. Transcriptome analysis highlighted their role in enhancing flagellar movement, biofilm formation, and metabolic synthesis, thereby elevating the capability of the mutant strain in xanthan gum production. This study demonstrates the potential of ARTP as an effective tool for microbial mutagenesis breeding, providing theoretical guidance for future studies on the synthesis regulation of xanthan gum and the engineering modification of X. campestris.

Continuous Inertial Alignment and Isolation of Spherical Microparticles and Nonspherical Flagellate Microalgae

Flagellate microalgae play an increasingly significant role in environmental management and biotechnology for valuable bioproducts, excellent photosynthetic capability, and autonomic movement. However, multiple flagellate microalgae practically live together in the ocean and lake areas, and they are susceptible to contamination as a result of improper operations. Enthused by these aspects, we develop a reliable inertial microfluidic method to overcome the influence of flagella movement and non-spherical shape on the alignment and isolation of target flagellate microalgae. Firstly, a computational model incorporating fluid-structure interaction was established to investigate influence of releasing position and shape parameters on the displacement and rotation of non-spherical microalgal cells and numerically studied the processes of shape- and size-based particle separation. Secondly, the movement of different-size particles under diverse flow rates in the channel was explored, and the capability of this method was validated by aligning and separating 10 μm and 20 μm polystyrene particles. Thirdly, this method was applied to align H. pluvialis and isolate Dunaliella salina from the mixed microalgal samples to explore the influence of flow rate on the alignment and isolation of flagellate microalgae. Fourthly, this method was engineered to select 20 μm polystyrene particles from three types of particles and isolate H. pluvialis from the mixture of multiple microalgae species. Finally, we leveraged this approach to realize separation of H. pluvialis and Synedra ulna to explore the performance of this method in shape-based cell separation, and we isolated Euglena from microalgal cell wastes, including dead cells, bacteria, and particles. This method has promising prospects to be a reliable tool to isolate target flagellate microalgae to address problematic issues in environmental monitoring, pharmaceutical synthesis, and chronic wound treatment for the advantage of good adaptability and reliability.

Enhancement of astaxanthin accumulation via energy reassignment by removing the flagella of Haematococcus pluvialis

Astaxanthin biosynthesis in Haematococcus pluvialis is driven by energy. However, the effect of the flagella-mediated energy-consuming movement process on astaxanthin accumulation has not been well studied. In this study, the profiles of astaxanthin and NADPH contents in combination with the photosynthetic parameters with or without flagella enabled by pH shock were characterized. The results demonstrated that there was no significant alteration in cell morphology, with the exception of the loss of flagella observed in the pH shock treatment group. In contrast, the astaxanthin content in the flagella removal groups was 62.9%, 62.8% and 91.1% higher than that of the control at 4, 8 and 12 h, respectively. Simultaneously, the increased Y(II) and decreased Y(NO) suggest that cells lacking the flagellar movement process may allocate more energy towards astaxanthin biosynthesis. This finding was verified by NADPH analysis, which revealed higher levels in flagella removal cells. These results provide preliminary insights into the underlying mechanism of astaxanthin accumulation enabled by energy reassignment in movement-lacking cells.

O-119 Architecture and in situ arrangement of the CatSper-channel complex in the sperm flagellum

Abstract The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway. Here, we applied cryo-electron tomography to visualize the higher-order organization of the native CatSper complex in intact mammalian sperm. The repeating CatSper units form long zigzag-rows along mouse and human sperm flagella. Above each tetrameric channel pore, most of the extracellular domains form a canopy that interconnects to a zigzag-shaped roof. Murine CatSper contains an additional wing-structure connected to the tetrameric channel. The intracellular domains link two neighboring channels to a diagonal array, suggesting a dimer formation. Fitting of an atomic model of isolated monomeric CatSper to the in situ map reveals supramolecular interactions and assembly of the CatSper complex. Loss of EFCAB9-CATSPERζ alters the architecture and interactions of the channels, resulting in fragmentation and misalignment of the zigzag-rows and disruption of flagellar movement in Efcab9−/− sperm. This work offers unique insights into the structural basis for understanding CatSper regulation of sperm motility.

Exploring sperm cell motion dynamics: Insights from genetic algorithm-based analysis

Accurate analysis of sperm cell flagellar dynamics plays a crucial role in understanding sperm motility as flagella parameters determine cell behavior in the spatiotemporal domain. In this study, we introduce a novel approach by harnessing Genetic Algorithms (GA) to analyze sperm flagellar motion characteristics and compare the results with the traditional decomposition method based on Fourier analysis. Our analysis focuses on extracting key parameters of the equation approximating flagellar shape, including beating period time, bending amplitude, mean curvature, and wavelength. Additionally, we delve into the extraction of phase constants and initial swimming directions, vital for the comprehensive study of sperm cell pairs and bundling phenomena. One significant advantage of GA over Fourier analysis is its ability to integrate sperm cell motion data, enabling a more comprehensive analysis. In contrast, Fourier analysis neglects sperm cell motion by transitioning to a sperm-centered coordinate system (material system). In our comparative study, GA consistently outperform the Fourier analysis-based method, yielding a remarkable reduction in fitting error of up to 70% and on average by 45%. An in-depth exploration of the sperm cell motion becomes indispensable in a wide range of applications from complexities of reproductive biology and medicine, to developing soft flagellated microrobots.

Flagellar Motility is Mutagenic.

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but to also promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an E. coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation frequencies. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.

Enhancement of serrawettin W1-producing Serratia marcescens cell migration by resonant oscillation under alternating current electric field

Swarming migration is observed in flagellated bacteria on wet surfaces. As the secreted biosurfactant hydrates, friction between the cell and the wet surface is reduced, and weak flagellar movement appears to be a significant force for cell translocation. Developing an artificial swarming control method could greatly aid in establishing techniques for biofilm inhibition and detachment. In this study, the effect of forced cell vibration by an alternating current electric field (ACEF) on swarming motility was investigated using the serrawettin W1-producing Serratia marcescens strain. At frequencies close to the natural frequencies of microbial cells (12 MHz), swarming motion was effectively enhanced in biosurfactant-producing S. marcescens, but not in non-surfactant-producing Escherichia coli or non-flagellated Staphylococcus aureus. Electric field-assisted cell migration was significantly induced under swarming conditions with low friction resistance between the cell and gel surface. This finding suggests a direction for developing strategies to regulate biofilm formation and detachment using ACEF-assisted oscillation of cells attached to surfaces.

Homozygous variant in DRC3 (LRRC48) gene causes asthenozoospermia and male infertility.

Human infertility affects 10-15% of couples. Asthenozoospermia accounts for 18% of men with infertility and is a common male infertility phenotype. The nexin-dynein regulatory complex (N-DRC) is a large protein complex in the sperm flagellum that connects adjacent doublets of microtubules. Defects in the N-DRC can disrupt cilia/flagellum movement, resulting in primary ciliary dyskinesia and male infertility. Using whole-exome sequencing, we identified a pathological homozygous variant of the dynein regulatory complex subunit 3 (DRC3) gene, which expresses leucine-rich repeat-containing protein 48, a component of the N-DRC, in a patient with asthenozoospermia. The variant ENST00000313838.12: c.644dup (p. Glu216GlyfsTer36) causes premature translational arrest of DRC3, resulting in a dysfunctional DRC3 protein. The patient's semen count, color, and pH were normal according to the reference values of the World Health Organization guidelines; however, sperm motility and progressive motility were reduced. DRC3 protein was not detected in the patient's sperm and the ultrastructure of the patient's sperm flagella was destroyed. More importantly, the DRC3 variant reduced its interaction with other components of the N-DRC, including dynein regulatory complex subunits 1, 2, 4, 5, 7, and 8. Our data not only revealed the essential biological functions of DRC3 in sperm flagellum movement and structure but also provided a new basis for the clinical genetic diagnosis of male infertility.

New insights into the antibiofilm activity and mechanism of Mannosylerythritol Lipid-A against Listeria monocytogenes EGD-e

Listeria monocytogenes is one of the leading causative agents of foodborne disease outbreaks worldwide. Herein, the antibiofilm effect and mechanism of Mannosylerythritol Lipid-A against L. monocytogenes EGD-e is reported for the first time. MEL-A effectively attenuated biofilm formation while reducing the viability and motility of bacteria within the biofilm in the early stage, and influenced bacterial adhesion by affecting the secretion of extracellular polysaccharides and eDNA. RT-qPCR revealed that MEL-A significantly suppressed the expression of genes involved in flagellar movement and virulence. Untargeted LC-MS metabolomics indicated that MEL-A affected the fluidity and permeability of cell membranes by significantly upregulating unsaturated fatty acids, lipids and glycoside metabolites, and affected protein biosynthesis, nucleotide metabolism and DNA synthesis and repair by significantly downregulating amino acid metabolism and nucleic acid metabolism. These pathways may constitute the key targets of biofilm formation inhibition by MEL-A. Furthermore, MEL-A showed good removal effects on mature biofilms under different temperatures, different materials and milk. Our data indicated that MEL-A could be used as a novel antibiofilm agent to improve food safety. Our study provides new insights into the possible inhibitory mechanism of MEL-A and the response of L. monocytogenes EGD-e to MEL-A.

Dinoflagellate Proton-Pump Rhodopsin Genes in Long Island Sound: Diversity and Spatiotemporal Distribution.

Microbial proton-pump rhodopsin (PPR)-based phototrophy, a light-harvesting mechanism different from chlorophyll-based photosystems, may contribute significantly to solar energy entry into the marine ecosystem. PPR transforms solar energy into cellular energy that is used for various metabolic processes in the cells or flagellar movement. Although rhodopsins or their encoding genes have been documented in a wide phylogenetic range of cultured dinoflagellates, information is limited about how widespread and how spatiotemporally dynamical dinoflagellate PPR (DiPPR) are in natural marine ecosystems. In this study, we investigated DiPPR in Long Island Sound (LIS), a temperate estuary of the Atlantic Ocean between Connecticut and Long Island, New York, USA. We isolated six novel full-length dinoflagellate proton-pump rhodopsin cDNAs, expanding the DiPPR database that is crucial to PPR research. Based on these new sequences and existing sequences of DiPPR, we designed primers and conducted quantitative PCR and sequencing to determine the abundance and diversity of DiPPR genes spatially and temporally throughout a year in the water samples collected from LIS. DiPPR genes were found year-round and throughout LIS but with higher abundances in the eutrophic Western Sound and in April and July. The gene diversity data suggest that there are at least five distinct rhodopsin-harboring groups of dinoflagellates throughout the year. The abundance of DiPPR genes, measured as copy number per mL of seawater, appeared not to be influenced by water temperature or nitrogen nutrient concentration but exhibited weak negative correlations with orthophosphate concentration and salinity and a positive correlation with the abundance of DiPPR-harboring dinoflagellates. This first quantitative profiling of PPR in natural plankton reveals the prevalence and dynamics of this plastid-independent photoenergy harvesting mechanism in a temperate estuary and provides efficient DiPPR primers potentially useful for future research. Furthermore, this study shed light on the potential role of DiPPR in phosphor nutrition and dinoflagellate population, which warrants further studies.

Feature-based 3D+t descriptors of hyperactivated human sperm beat patterns

The flagellar movement of the mammalian sperm plays a crucial role in fertilization. In the female reproductive tract, human spermatozoa undergo a process called capacitation which promotes changes in their motility. Only capacitated spermatozoa may be hyperactivated and only those that transition to hyperactivated motility are capable of fertilizing the egg. Hyperactivated motility is characterized by asymmetric flagellar bends of greater amplitude and lower frequency. Historically, clinical fertilization studies have used two-dimensional analysis to classify sperm motility, despite the inherently three-dimensional (3D) nature of sperm motion. Recent research has described several 3D beating features of sperm flagella. However, the 3D motility pattern of hyperactivated spermatozoa has not yet been characterized. One of the main challenges in classifying these patterns in 3D is the lack of a ground-truth reference, as it can be difficult to visually assess differences in flagellar beat patterns. Additionally, it is worth noting that only a relatively small proportion, approximately 10-20% of sperm incubated under capacitating conditions exhibit hyperactivated motility. In this work, we used a multifocal image acquisition system that can acquire, segment, and track sperm flagella in 3D+t. We developed a feature-based vector that describes the spatio-temporal flagellar sperm motility patterns by an envelope of ellipses. The classification results obtained using our 3D feature-based descriptors can serve as potential label for future work involving deep neural networks. By using the classification results as labels, it will be possible to train a deep neural network to automatically classify spermatozoa based on their 3D flagellar beating patterns. We demonstrated the effectiveness of the descriptors by applying them to a dataset of human sperm cells and showing that they can accurately differentiate between non-hyperactivated and hyperactivated 3D motility patterns of the sperm cells. This work contributes to the understanding of 3D flagellar hyperactive motility patterns and provides a framework for research in the fields of human and animal fertility.

Analysis of Ca2+-mediated sperm motility to evaluate the functional normality of the sperm-specific Ca2+ channel, CatSper.

Ca2+ is a key secondary messenger that modulates sperm motility by tuning flagellar movement in various species. The sperm-specific Ca2+ channel, CatSper, is a primary Ca2+ gate that is essential for male fertility in mammals. CatSper-mediated Ca2+ signaling enables sperm to develop hyperactivated motility and fertilize the eggs in the female tract. Therefore, altered CatSper function compromises the entry of Ca2+ into the sperm, followed by impairing hyperactivation and male fertility. However, methods to evaluate the function of the CatSper channel are limited to patch clamping and functional imaging using Ca2+ dye. Previous studies have revealed that various parameters for sperm motility are highly correlated with intracellular Ca2+ levels in mouse. Here, I cover a step-by-step protocol to analyze the change in Ca2+-mediated sperm motility by using computer-assisted semen analysis (CASA) to evaluate the functional normality of the CatSper channel in sperm. This approach analyzes sperm motility parameters during intracellular Ca2+ chelation followed by in vitro capacitation to recover intracellular Ca2+ via the activated CatSper channel. Thus, this Ca2+-handling method is handy and could be broadly applied in reproductive biology labs and clinics that have CASA equipment to examine the functional normality of the CatSper channel.

Microfluidic sperm trap array for single-cell flagellar analysis with unrestricted 2D flagellar movement.

Sperm capture techniques that immobilize sperm to halt their motility are essential for the long-term analysis of individual sperm. These techniques are beneficial in assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) by allowing selective screening of sperm. However, there is a notable lack of high-throughput and non-destructive sperm capture methods that allow the flagellum to beat freely, which is crucial for accurately reflecting the behavior of unfettered, freely swimming sperm. To bridge this gap, we introduce a novel microfluidic device specifically engineered to capture sperm without restricting flagellar motion. The design utilizes sperm's innate boundary-following behavior in both 3D and 2D environments to direct them into a capture zone. Once captured, the sperm head is restrained while the flagellum remains free to exhibit natural beating patterns. Utilizing this device, we explore the effects of hyperactivating agents, temperature, and their combined influence on the dynamics of bovine sperm flagella. The unrestricted flagellar motion offered by our device yields two prominent advantages: it mirrors the flagellar behavior of free-swimming sperm, ensuring research findings are consistent with natural sperm activity, and it prevents imaging overlap between the flagellum and the capture structures, simplifying the automation of flagellar tracking and analysis. This technological advancement facilitates the collection of waveform parameters along the entire flagellum, addressing inconsistencies that have arisen in previous research due to differing measurement sites, and enabling precise extraction of sperm behavioral properties.

Decoupled illumination detection in light sheet microscopy for 4D observation of spermatozoa at high-resolutions

We present the use of wavefront coding (WFC) combined with machine learning in a light sheet fluorescence microscopy (LSFM) system. We visualize the 3D dynamics of sperm flagellar motion at an imaging speed up to 80 volumes per second, which is faster than twice volumetric video rate. By using the WFC technique we achieve to extend the depth of field of the collection objective with high numerical aperture (NA=1) from 2.6 μm to 50 μm, i. e., more than one order of magnitude. To improve the quality of the final images, we applied a machine learning-based algorithm to the acquired sperm raw images and to the point spread function (PSF) of the generated cubic phase masks previous to the deconvolution process.

Comprehensive Genomic Characterization of Cronobacter sakazakii Isolates from Infant Formula Processing Facilities Using Whole-Genome Sequencing.

Cronobacter sakazakii is an opportunistic pathogen linked to outbreaks in powdered infant formula (PIF), primarily causing meningitis and necrotizing enterocolitis. Whole-genome sequencing (WGS) was used to characterize 18 C. sakazakii strains isolated from PIF (powdered infant formula) manufacturing plants (2011-2015). Sequence Type (ST) 1 was identified as the dominant sequence type, and all isolates carried virulence genes for chemotaxis, flagellar motion, and heat shock proteins. Multiple antibiotic resistance genes were detected, with all isolates exhibiting resistance to Cephalosporins and Tetracycline. A significant correlation existed between genotypic and phenotypic antibiotic resistance. The plasmid Col(pHAD28) was identified in the isolates recovered from the same PIF environment. All isolates harbored at least one intact phage. All the study isolates were compared with a collection of 96 publicly available C. sakazakii genomes to place these isolates within a global context. This comprehensive study, integrating phylogenetic, genomic, and epidemiological data, contributes to a deeper understanding of Cronobacter outbreaks. It provides valuable insights to enhance surveillance, prevention, and control strategies in food processing and public health contexts.

LRRC23 deficiency causes male infertility with idiopathic asthenozoospermia by disrupting the assembly of radial spokes.

Asthenozoospermia (AZS) is the primary cause of infertility in males. The radial spoke (RS) is an axonemal structure, connecting the peripheral doublet microtubules with the central pair of microtubules. This T-shaped multiprotein complex functions as a mechanochemical sensor to promote sperm motility. LRRC23 is a novel subunit of the RS complex that is necessary for flagellar assembly and movement in mice. However, the importance of LRRC23 in modulating RS formation in humans remains unclear. Here, we identified a homozygous nonsense mutation in LRRC23 (c.376C>T:p. Arg126X) in an infertile AZS patient whose parents were consanguineous. We verified the adversity of this novel mutation because of its ability to disrupt LRRC23 synthesis and impair RSs integrity. Furthermore, we demonstrated an interaction between LRRC23 and RSPH3 in vitro, indicating that LCCR23 is associated with RS in humans. Meanwhile, the LRRC23-mutant patient had a good prognosis following intracytoplasmic sperm injection. This study provides strong preliminary evidence that LRRC23 defects are potential causative factors of AZS in humans, which expands our knowledge for improved genetic counseling and better reproductive recommendations for patients with AZS.

Biointerfacial behavior of stallion spermatozoa adhered to hydrogel surfaces: Impact of the hydrogel chemical composition and the culture medium

Novel soft materials based on hydrogel are proposed to enhance the selection of high-quality stallion sperm based on their adhesion capacity. The hydrogel surfaces are derived from polyacrylamide (PAAm), which is copolymerized with neutral and ionic co-monomers to modify the interfacial properties. The hydrogels undergo characterization through FTIR spectroscopy, assessment of swelling capacity, and wettability under various experimental conditions. Sperm adhesion capacity on the hydrogels is examined through several parameters including the percentage of bound sperm (%Sp) to hydrogels, tail oscillation intensity and flagellar movement. The biointerfacial properties of sperm-hydrogel systems vary based on the chemical composition of hydrogel as well as the components present in the culture medium. High %Sp and excellent metabolic activity of the spermatozoa are observed on hydrogel surfaces that possess moderate hydrophilicity. Specifically, a cationic hydrogel in BGM3 culture medium and a neutral surface in BGM3 medium supplemented with BSA exhibit favorable outcomes. Scanning Electron Microscopy (SEM) reveals the normal morphology of the head and tail in spermatozoa adhered to the hydrogel. Therefore, these hydrogel surfaces are potential materials for selecting stallion sperm with high quality, and their application could be extended to the study of other mammalian reproductive cells.

Interplay between bacterial 5'-NAD-RNA decapping hydrolase NudC and DEAD-box RNA helicase CsdA in stress responses.

Both prokaryotic and eukaryotic RNAs can be 5'-capped by the metabolite nicotinamide adenine dinucleotide (NAD). Nudix hydrolases, such as bacterial NudC, specifically remove NAD-caps; however, the molecular and cellular functions of these epitranscriptomic modulators remain elusive. Here, we discuss the roles of NudC under stress conditions and the effects of extracellular cues on the NAD epitranscriptome. Our proteome-wide analysis detected the proteins associated with the RNA degradosome as well as ribosomes and stress-responsive proteins in a NudC interactome. Moreover, we confirmed the physical association of NudC with the cold shock DEAD-box RNA helicase CsdA and the RNA chaperone Hfq. Interestingly, knocking out csdA similar to ∆nudC leads to an increased number of identified 5'-NAD-RNA species compared to wild type, exposing CsdA as a new player in a potentially unexplored layer of NAD-epitranscriptomic landscape. Mass spectrometry analysis also revealed the drastic up-regulation of 5'-NAD-RNA in response to cold. Furthermore, the inactivation of NudC in bacteria changes the levels of sRNA and protein-coding transcripts associated with bacterial chemotaxis and flagellar assembly pathways. We experimentally demonstrate that the decapping hydrolase NudC suppresses the flagellar movement, while CsdA stimulates it. Thus, the interplay between NudC and CsdA regulates bacterial mobility and coordinates stress-avoidance behavior. IMPORTANCE Non-canonical 5'-caps removing RNA hydrolase NudC, along with stress-responsive RNA helicase CsdA, is crucial for 5'-NAD-RNA decapping and bacterial movement.

Analyzing Flagellum Structure of Trypanosoma Brucei Using Electron Tomography Datasets from Electron Microscopy Data Bank

African trypanosomiasis is a vector-borne parasitic disease. The main parasite that causes the disease is Trypanosoma brucei, also known as T. brucei, which infects its hosts through the tsetse fly. Trypanosoma brucei has an important structure, the flagella. The parasite relies on the flagella for survival and movement, so studying the flagella structure in detail is essential to understanding its function. Therefore, we use electron microscopes for multi-angle observation and photography. We identified some important structures of flagella which aligned with previous literature including the 92+2 axoneme, and the paraflagellar rod (PFR), and we even captured some novel features including densities connecting the axoneme and PFR, densities that interconnect the microtubule, and densities that bridges the different zones of PFR. We also did literature reviews and proposed hypotheses about the functions of these structures. Our structural study could shed light on the inhibition of the flagella movement, thereby treating trypanosomiasis.

Pharmacological Evidence Suggests That Slo3 Channel Is the Principal K+ Channel in Boar Spermatozoa.

Sperm ion channels are associated with the quality and type of flagellar movement, and their differential regulation is crucial for sperm function during specific phases. The principal potassium ion channel is responsible for the majority of K+ ion flux, resulting in membrane hyperpolarization, and is essential for sperm capacitation-related signaling pathways. The molecular identity of the principal K+ channel varies greatly between different species, and there is a lack of information about boar K+ channels. We aimed to determine the channel identity of boar sperm contributing to the primary K+ current using pharmacological dissection. A series of Slo1 and Slo3 channel modulators were used for treatment. Sperm motility and related kinematic parameters were monitored using a computer-assisted sperm analysis system under non-capacitated conditions. Time-lapse flow cytometry with fluorochromes was used to measure changes in different intracellular ionic concentrations, and conventional flow cytometry was used to determine the acrosome reaction. Membrane depolarization, reduction in acrosome reaction, and motility parameters were observed upon the inhibition of the Slo3 channel, suggesting that the Slo3 gene encodes the main K+ channel in boar spermatozoa. The Slo3 channel was localized on the sperm flagellum, and the inhibition of Slo3 did not reduce sperm viability. These results may aid potential animal-model-based extrapolations and help to ameliorate motility and related parameters, leading to improved assisted reproductive methods in industrial livestock production.