The ability of spermatozoa to withstand cryopreservation differs between stallions. While the underlying mechanisms of these differences are not fully understood, seminal plasma (SP) may play a crucial role in modulating sperm cryotolerance. Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs), nanometer-sized membrane particles that can transfer biomolecules to sperm modulating their function. This study aimed to isolate and characterize two sized-sEV subsets-small (S-) and large (L-)-from stallion SP, and to evaluate their involvement in sperm freezability. Seminal plasma samples (n = 10) were collected from 10 stallions, which were divided according to sperm freezability into: good (GFE, n = 5) and poor freezability ejaculates (PFE, n = 5). The sEV subsets were isolated using a size-exclusion-chromatography (SEC)-based procedure, and characterized according to MISEV2023, including morphology, concentration, particle size distribution, purity (albumin assessment) and presence of canonical EV-markers (CD9, CD63, and CD81, and HSP70). Results showed that stallion SP contains a heterogeneous population of sEVs, with S- and L-sEVs exhibiting distinct phenotypic and compositional profiles. The S-sEVs displayed a higher expression (P<0.01) of CD9 and HSP70 (mean±SD; 47.0%±10.5% and 51.0%±6.5%, respectively) than in L-sEVs (35.7%±7.5% and 39.0%±1.8%, respectively), suggesting functional differences between subsets. Interestingly, GFE contained higher (P<0.05) CFSE+ particles (intact sEVs) in S- and L-sEVs (66.4%±13.4% and 72.2%±5.0%, respectively) compared to PFE (45.1%±7.8% and 37.8%±9.5%, respectively). In conclusion, our study establishes an SEC-based procedure suitable for isolating two sized-sEV subsets in SP stallions that differ in the canonical EV-markers expression. The higher abundance of intact sEVs in GFE suggests their involvement in sperm freezability.

The follicular fluid (FF) is crucial for providing oocytes with an ideal environment that promotes their development and maturation. Not only does this fluid supply nutrients and hormones, but also other components that protect both follicular cells and the oocyte itself from potential harmful factors, such as those inducing oxidative stress (OS). The FF has also been suggested to have beneficial effects on sperm when they reach the oviduct. The objectives of this study were to describe the presence and activity levels of redox biomarkers in the preovulatory follicular fluid (PFF), and to analyze the impact of PFF on the motility, viability and reactive oxygen species (ROS) levels of donkey sperm. For this purpose, ten PFF samples obtained from Catalan jennies and nine ejaculates collected from Catalan donkeys were used. Redox biomarkers -including enzymatic and non-enzymatic antioxidants, and oxidative biomarkers- were analyzed in jennies' PFF. After collection, each semen sample was split into two aliquots of equal volume. The two aliquots were centrifuged to remove the seminal plasma; one pellet was resuspended in Tris Buffered Medium (TBM), and the other was resuspended in TBM supplemented with 20% PFF. Motility parameters (CASA) and other semen quality biomarkers (flow cytometry) were assessed after 0, 60 and 120 min of incubation at 38 °C. Exposure of donkey sperm to PFF reduced intracellular ROS levels and helped maintain sperm motility and viability. These findings suggest that, in the oviduct, the PFF components protect donkey sperm from oxidative stress. Furthermore, knowing better the composition of donkey PFF in terms of antioxidant biomarkers may be used to improve the formulation of media for oocyte maturation and fertilization in this and other species.

Background The oxytocin receptor (OR) is a G-protein-coupled receptor recently identified in human spermatozoa, whose origin and role in sperm physiology remain unknown.Objectives In this study, using the pig as a model, we examine the presence of the OR in ejaculated spermatozoa through immunofluorescence and immunoblotting, and investigate the receptor's origin in the male gamete via immunohistochemistry in testicular and epididymal tissues. Additionally, we assess the involvement of the OR in in vitro capacitation and the acrosome reaction by utilizing physiological concentrations of agonists (oxytocin and carbetocin) and an antagonist (L-371,257).Results The results indicate that, in addition to the expected presence in ejaculated spermatozoa, the OR is expressed during spermatogenesis. Besides, this receptor is found in Leydig and Sertoli cells, as well as in the principal, basal, and apical cells of the epididymis. Furthermore, our data suggest that, during epididymal maturation, the OR could be incorporated in spermatozoa via extracellular vesicles within the apical blebs. The OR is involved in sperm capacitation, as the combination of the antagonist (L-371,257) and the agonist (carbetocin) increases intracellular calcium levels and membrane lipid disorders, which are known as capacitation markers.Conclusions The presence of the OR in mammalian spermatozoa could originate from both spermatogenesis and epididymal maturation. Moreover, in the male gamete, this receptor regulates sperm capacitation by interacting with its ligand in the female reproductive tract.

Seminal plasma (SP), a fluid composed of secretions from the male genital tract, is rich in seminal extracellular vesicles (sEVs), nano-sized particles surrounded by a lipid bilayer membrane and loaded with functionally active molecules. Seminal EVs are secreted by functional cells of the male genital tract and play a key role in modulating reproductive processes, including sperm function and immune response in the female genital tract. The aim of this study was to isolate and characterize sEVs from donkey SP for the first time. Nine SP samples were collected from nine healthy and reproductive active donkeys. The SP samples were randomly pooled to create three pools (three SP samples per pool). The SP pools were subjected to differential centrifugation and size-exclusion chromatography to separately isolate two subsets of sEVs: small (S-) and large (L-). Orthogonal characterization of sEV samples was performed according to MISEV 2023 guidelines, including morphology (by cryogenic electron microscopy), concentration (by total protein concentration and total and CFSE positive particles by flow cytometry [FC]), particle size distribution (by dynamic light scattering), purity (by albumin assessment by FC), and specific EV protein markers (tetraspanins CD9, CD63, and CD81, and HSP70 by FC). The results showed that donkey SP is highly enriched in sEVs. Size differences were found between both sEV subsets, with S-sEVs being smaller (∼160 nm) and L-sEVs larger (∼290 nm). Both sEV subsets were positive for the four EV protein markers. However, the percentage of CD81-positive events was higher in S-sEV samples than in L-sEV samples (P < 0.05). This study is the first to isolate and characterize sEVs in donkey SP, demonstrating their heterogeneity and suggesting differences in biogenesis and function between S-sEVs and L-sEVs.

Semen contamination is one of the main issues of concern in livestock industry. While the presence of some bacteria in semen is considered as normal, high bacterial loads detrimentally affect sperm quality and fertilizing ability, thus having a negative repercussion on the efficiency of artificial insemination. For this reason, the present chapter focuses on the methods used for the assessment of semen contamination in farm animals. Routinely, bacterial culture is performed to assess the overall degree of contamination in sperm samples; in some cases, however, the identification of bacterial species is needed to determine the optimal antibiotic composition of semen preservation media and address whether those bacteria have developed antibiotic resistance. In addition, other techniques, such as scanning electron microscopy, can be used to evaluate sperm-bacteria interaction. This chapter also discusses the strategies to prevent bacteria contamination in semen. As the ejaculate is not a sterile fluid, media for preservation usually include, as aforementioned, antibiotics to avoid bacterial growth. Because of the mounting restriction in the usage of antibiotics, which is due to the increase of bacterial resistance, other approaches to prevent bacterial contamination of semen include preservation at low temperatures (5 °C) and the inclusion of alternative molecules such as antimicrobial peptides.

Background: Ion channels are essential for differentiation and maturation of germ cells, and even for fertilization in mammals. Different types of potassium channels have been identified, which are grouped into voltage-gated channels (Kv), ligand-gated channels (K-ligand), inwardly rectifying channels (K-ir), and tandem pore domain channels (K-2P). Material-Methods: The present review includes recent findings on the role of potassium channels in sperm physiology of mammals. Results-Discussion: While most studies conducted thus far have been focused on the physiological role of voltage- (Kv1, Kv3, and Kv7) and calcium-gated channels (SLO1 and SLO3) during sperm capacitation, especially in humans and rodents, little data about the types of potassium channels present in the plasma membrane of differentiating germ cells exist. In spite of this, recent evidence suggests that the content and regulation mechanisms of these channels vary throughout spermatogenesis. Potassium channels are also essential for the regulation of sperm cell volume during epididymal maturation and for preventing premature membrane hyperpolarization. It is important to highlight that the nature, biochemical properties, localization, and regulation mechanisms of potassium channels are species-specific. In effect, while SLO3 is the main potassium channel involved in the K+ current during sperm capacitation in rodents, different potassium channels are implicated in the K+ outflow and, thus, plasma membrane hyperpolarization during sperm capacitation in other mammalian species, such as humans and pigs. Conclusions: Potassium conductance is essential for male fertility, not only during sperm capacitation but throughout the spermiogenesis and epididymal maturation.

Abstract Background While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated. Results Partial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation. Conclusions Inhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Background Despite their low abundance in sperm, mitochondria have diverse functions in this cell type, including energy production, signalling and calcium regulation. In humans, sperm mitochondrial DNA content (mtDNAc) has been reported to be negatively linked to sperm function and fertility. Yet, the association between mtDNAc and sperm function in livestock remains unexplored. For this reason, this study aimed to shed some light on the link between mtDNAc and sperm function and fertilising potential in pigs. A qPCR method for mtDNAc quantification was optimised for pig sperm, and the association of this parameter with sperm motility, kinematics, mitochondrial activity, and fertility was subsequently interrogated. Results First, the qPCR method was found to be sensitive and efficient for mtDNAc quantification in pig sperm. By using this technique, mtDNAc was observed to be associated to sperm motility, mitochondrial activity and in vivo, but not in vitro, fertility outcomes. Specifically, sperm with low mtDNAc were seen to exhibit greater motility but decreased mitochondrial activity and intracellular reactive oxygen species. Interestingly, samples with lower mtDNAc showed higher conception and farrowing rates, but similar in vitro fertilisation rates and embryo development, when compared to those with greater mtDNAc. Conclusions These findings enrich our comprehension of the association of mtDNAc with sperm biology, and lay the foundation for future research into employing this parameter as a molecular predictor for sperm function and fertility in livestock.

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.

Background: In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. Results: Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33-194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments .Conclusion: Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.

EDITORIAL article Front. Cell Dev. Biol., 22 February 2023Sec. Molecular and Cellular Reproduction Volume 11 - 2023 | https://doi.org/10.3389/fcell.2023.1152086

Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. Results This study determined chromatin protamination (Chromomycin A3 test, CMA 3 ), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions ( P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF ( P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed ( P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P < 0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs = 0.973; P < 0.001). Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction.

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20–28® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant (p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 staining and O2 consumption rate. Antimycin A, however, did not affect intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract the effects of light on motility, O2 consumption rate and CCO activity, but not to the same extent than that observed for antimycin A. Finally, the effects observed when sperm were co-incubated with antimycin A and PKCi were similar to those observed with antimycin A alone. In conclusion, red LED light acts on sperm function via a direct effect on mitochondrial electron chain. Additionally, light-activated PKC pathways have a supplementary effect to that observed in the electron chain, thereby modulating sperm parameters such as motility and CCO activity.

Over the last decades, selection in cattle has mainly been based on milk production rather than on reproductive efficiency. While, when applied, focus on reproduction has involved females, attention has barely been paid to males and, if so, it has only looked at classical sperm quality parameters. In effect, variables such as telomere length have been missed, despite the fact that longer telomeres have been suggested to be linked to male fertility in humans. For this reason, the present study aimed to determine the length of telomeres in bovine sperm and their relationship with a) sperm quality evaluated through the conventional spermiogram and flow cytometry, and b) bull reproductive performance. For this purpose, 29 bulls were involved in this study. Sperm telomere length was evaluated through quantitative Fluorescent In Situ Hybridization (qFISH), and sperm quality was determined at 0 h and 4 h post-thaw. Bull fertility was assessed as non-return to estrus rates after 90 days of artificial insemination. Although the mean telomere length in bovine sperm was 12.06 +/- 2.75 kb, the intra-individual variability in length led us to observe three different groups of telomeres in each sperm cell: short telomeres (7.14% +/- 5.79% of telomeres; 8.29 +/- 2.34 kb), medium telomeres (31.03% +/- 12.92% of telomeres; 16.00 +/- 2.72 kb) and long telomeres (61.93% +/- 18.11% of telomeres; 30.13 +/- 11.35 kb). Moreover, whereas reactive oxygen species (ROS) were found to be correlated to sperm telomere length (Rs = -0.492; P= 0.007), no correlation with other sperm quality parameters was found (P > 0.05). Reproductive performance after artificial insemination was not seen to be correlated to sperm telomere length (Rs = 0.123; P= 0.520). In conclusion, this study determined, for the first time, the mean telomere length in bovine sperm and also reported that there is a high variability within each sperm cell. Yet, while telomere length was found to be correlated to ROS generation, it was not related to bull reproductive performance. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).