Embryo development is impaired by sperm mitochondrial-derived ROS.

BACKGROUND: Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. RESULTS: We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. CONCLUSION: Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.

An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm.

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.

Assessment of sperm mitochondrial activity by flow cytometry and fluorescent microscopy: a comparative study of mitochondrial fluorescent probes in bovine spermatozoa.

CONTEXT: While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6 (3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. AIMS: The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. METHODS: Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6 (3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. KEY RESULTS: DiOC6 (3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6 (3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. CONCLUSIONS: JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6 (3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. IMPLICATIONS: Both DiOC6 (3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.

Sperm DNA damage compromises embryo development, but not oocyte fertilisation in pigs.

BACKGROUND: The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. RESULTS: The results showed that global DNA damage negatively correlates (P < 0.05) with normal sperm morphology (R = - 0.460) and progressive motility (R = - 0.419), and positively with the percentage of non-viable sperm (R = 0.507). Multiple regression analyses showed that non-viable sperm were related to SSB (ß = - 0.754). In addition, while fertilisation did not seem to be affected by sperm DNA integrity, global DNA damage, DSB and SSB were found to be correlated to embryo development outcomes. Specifically, whereas global DNA damage and DSB negatively affected (P < 0.05) the later preimplantation embryo stages (percentage of early blastocyst/blastocyst D6: for global DNA damage, R = - 0.458, and for DSB, R = - 0.551; and percentage of hatching/hatched blastocyst D6: for global DNA damage, R = - 0.505, and for DSB, R = - 0.447), global DNA damage and SSB had a negative impact (P < 0.05) on the developmental competency of fertilised embryos (R = - 0.532 and R = - 0.515, respectively). Remarkably, multiple regression analyses supported the associations found in correlation analyses. Finally, the present work also found that the inclusion of Comet assays to the conventional sperm quality tests improves the prediction of blastocyst formation (AUC = 0.9021, P < 0.05), but not fertilisation rates (P > 0.05). CONCLUSION: Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

The triple role of glutathione S-transferases in mammalian male fertility.

Male idiopathic infertility accounts for 15-25% of reproductive failure. One of the factors that has been linked to this condition is oxidative stress (OS), defined as the imbalance between antioxidants and reactive oxygen species. Amongst the different factors that protect the cell against OS, the members of the glutathione S-transferase (GST) superfamily play an important role. Interestingly, reduction or lack of some GSTs has been associated to infertility in men. Therefore, and to clarify the relationship between GSTs and male fertility, the aim of this work is to describe the role that GSTs play in the male reproductive tract and in sperm physiology. To that end, the present review provides a novel perspective on the triple role of GSTs (detoxification, regulation of cell signalling and fertilisation), and reports their localisation in sperm, seminal plasma and the male reproductive tract. Furthermore, we also tackle the existing correlation between some GST classes and male fertility. Due to the considerable impact of GSTs in human pathology and their tight relationship with fertility, future research should address the specific role of these proteins in male fertility, which could result in new approaches for the diagnosis and/or treatment of male infertility.

Parkinson Disease Protein 7 (PARK7) Is Related to the Ability of Mammalian Sperm to Undergo In Vitro Capacitation.

Parkinson disease protein 7 (PARK7) is a multifunctional protein known to be involved in the regulation of sperm motility, mitochondrial function, and oxidative stress response in mammalian sperm. While ROS generation is needed to activate the downstream signaling pathways required for sperm to undergo capacitation, oxidative stress has detrimental effects for sperm cells and a precise balance between ROS levels and antioxidant activity is needed. Considering the putative antioxidant role of PARK7, the present work sought to determine whether this protein is related to the sperm ability to withstand in vitro capacitation. To this end, and using the pig as a model, semen samples were incubated in capacitation medium for 300 min; the acrosomal exocytosis was triggered by the addition of progesterone after 240 min of incubation. At each relevant time point (0, 120, 240, 250, and 300 min), sperm motility, acrosome and plasma membrane integrity, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium and ROS were evaluated. In addition, localization and protein levels of PARK7 were also assessed through immunofluorescence and immunoblotting. Based on the relative content of PARK7, two groups of samples were set. As early as 120 min of incubation, sperm samples with larger PARK7 content showed higher percentages of viable and acrosome-intact sperm, lipid disorder and superoxide levels, and lower intracellular calcium levels when compared to sperm samples with lower PARK7. These data suggest that PARK7 could play a role in preventing sperm from undergoing premature capacitation, maintaining sperm viability and providing a better ability to keep ROS homeostasis, which is needed to elicit sperm capacitation. Further studies are required to elucidate the antioxidant properties of PARK7 during in vitro capacitation and acrosomal exocytosis of mammalian sperm, and the relationship between PARK7 and sperm motility.

HVCN1 but Not Potassium Channels Are Related to Mammalian Sperm Cryotolerance.

Little data exist about the physiological role of ion channels during the freeze-thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O2-⁻ and H2O2 levels. General blockade of K+ channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O2-⁻ and H2O2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role.

HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa.

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

Effect of AQP Inhibition on Boar Sperm Cryotolerance Depends on the Intrinsic Freezability of the Ejaculate.

Aquaporins (AQPs) are transmembrane channels with permeability to water and small solutes that can be classified according to their structure and permeability into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs. In boar spermatozoa, AQPs are related to osmoregulation and play a critical role in maturation and motility activation. In addition, their levels differ between ejaculates with good and poor cryotolerance (GFE and PFE, respectively). The aim of this work was to elucidate whether the involvement of AQPs in the sperm response to cryopreservation relies on the intrinsic freezability of the ejaculate. With this purpose, two different molecules: phloretin (PHL) and 1,3-propanediol (PDO), were used to inhibit sperm AQPs in GFE and PFE. Boar sperm samples were treated with three different concentrations of each inhibitor prior to cryopreservation, and sperm quality and functionality parameters were evaluated in fresh samples and after 30 and 240 min of thawing. Ejaculates were classified as GFE or PFE, according to their post-thaw sperm viability and motility. While the presence of PHL caused a decrease in sperm quality and function compared to the control, samples treated with PDO exhibited better quality and function parameters than the control. In addition, the effects of both inhibitors were more apparent in GFE than in PFE. In conclusion, AQP inhibition has more notable consequences in GFE than in PFE, which can be related to the difference in relative levels of AQPs between these two groups of samples.

Integrating metabolomics into reproduction: Sperm metabolism and fertility enhancement in pigs.

The last decades of research have revealed that many other factors besides gamete genomes are able to determine the reproductive outcomes. Indeed, paternal factors have been observed to be capable of modulating multiple crucial features of the reproductive process, such as sperm physiology, the maternal environment and, even, the offspring health. These recent advances have been encompassed with the emergence of OMICS technologies, as they comprehensively characterise the molecular composition of biological systems. The present narrative review aimed to take a closer look at the potential of these technologies in the field of reproductive biology. This literature revision shows that most studies up to date have followed a non-targeted approach to screen mammalian seminal plasma (SP) and sperm metabolite composition through different metabolome platforms. These studies have proposed metabolites of multiple natures as potential in vivo fertility biomarkers. Yet, targeted approaches can be used to answer specific biological question, and their power is exemplified herein. For instance, metabolomic studies have uncovered not only that glycolysis is the main ATP energy source of pig sperm, but also that sperm metabolism can trigger DNA damage, hence compromise embryo development. In conclusion, this review shows the potential of both non-targeted and targeted metabolomics for the discovery of cell pathways that govern the reproductive process. Understanding these systems could help make progress in different areas, including livestock efficient breeding, the improvement of artificial reproductive technologies, and the development of biomarkers for infertility detection.

Sperm function, mitochondrial activity and in vivo fertility are associated to their mitochondrial DNA content in pigs.

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.

Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism.

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90ß). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.

An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration.

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Expression of miR-138 in cryopreserved bovine sperm is related to their fertility potential.

BACKGROUND: MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of 22-24 nucleotides that regulate gene expression. In the last decade, miRNAs have been described in sperm of several mammals, including cattle. It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans; however, little is known about the content, expression, and function of sperm-borne miRNAs in early bovine embryo. In this study, total sperm RNA was isolated from 29 cryopreserved sperm samples (each coming from a separate bull) using a RNeasy kit and treatment with DNase I. RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer. The expression of 10 candidate miRNAs in bovine sperm (bta-miR-10a, bta-miR-10b, bta-miR-138, bta-miR-146b, bta-miR-19b, bta-miR-26a, bta-miR-34a, bta-miR-449a, bta-miR-495 and bta-miR-7), previously identified in testis and/or epididymis, was evaluated with RT-qPCR. The cel-miR-39-3p was used as a spike-in exogenous control. Nonparametric Mann-Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility [HF; non-return rates (NRR) ranging from 39.5 to 43.5] and those with subfertility (SF; NRR ranging from 33.3 to 39.3). Several sperm functionality parameters (e.g., viability, membrane stability or oxygen consumption, among others) were measured by multiplexing flow cytometry and oxygen sensing technologies. RESULTS: RNA concentration and purity (260/280 nm ratio) (mean ± SD) from the 29 samples were 99.3 ± 84.6 ng/µL and 1.97 ± 0.72, respectively. Bioanalyzer results confirmed the lack of RNA from somatic cells. In terms of the presence or absence of miRNAs, and after applying the Livak method, 8 out of 10 miRNAs (bta-miR-10b, -138, -146b, -19b, -26a, -449a, -495, -7) were consistently detected in bovine sperm, whereas the other two (bta-miR-10a, and -34a) were absent. Interestingly, the relative expression of one miRNA (bta-miR-138) in sperm was significantly lower in the SF than in the HF group (P = 0.038). In addition to being associated to fertility potential, the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption. The expression of three other miRNAs (bta-miR-19b, bta-miR-26a and bta-miR-7) was also correlated with sperm function variables. CONCLUSIONS: In conclusion, although functional validation studies are required to confirm these results, this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond, and supports its use as a fertility biomarker in cattle.

Sperm physiology and in vitro fertilising ability rely on basal metabolic activity: insights from the pig model.

Whether basal metabolic activity in sperm has any influence on their fertilising capacity has not been explored. Using the pig as a model, the present study investigated the relationship of energetic metabolism with sperm quality and function (assessed through computer-assisted sperm analysis and flow cytometry), and fertility (in vitro fertilisation (IVF) outcomes). In semen samples from 16 boars, levels of metabolites related to glycolysis, ketogenesis and Krebs cycle were determined through a targeted metabolomics approach using liquid chromatography-tandem mass spectrometry. High-quality sperm are associated to greater levels of glycolysis-derived metabolites, and oocyte fertilisation and embryo development are conditioned by the sperm metabolic status. Interestingly, glycolysis appears to be the preferred catabolic pathway of the sperm giving rise to greater percentages of embryos at day 6. In conclusion, this study shows that the basal metabolic activity of sperm influences their function, even beyond fertilisation.

Comparison of the metabolite profile of donkey and horse seminal plasma and its relationship with sperm viability and motility.

Previous research revealed that several seminal plasma (SP) metabolites are related to sperm functionality, fertility, and preservation. While it is understood that variations between species exist, whether the SP metabolome differs between donkeys and horses has not been previously investigated. The aim of this work, therefore, was to characterize and compare donkey and horse SP metabolites using nuclear magnetic resonance (NMR) spectroscopy, and relate them to sperm viability and motility. For this purpose, ejaculates from 18 different donkeys and 18 different horses were collected and separated into two aliquots: one for harvesting the SP by centrifugation and obtaining the metabolic profile through NMR, and the other for evaluating sperm viability and motility. Based on total motility and sperm viability, samples were classified as with good (GQ) or poor (PQ) quality. The metabolomic profile of donkey and horse SP revealed the presence of 28 metabolites, which coincided in the two species. Yet, differences between horses and donkeys were observed in the concentration of 18 of these 28 metabolites, as well as between ejaculates classified as GQ or PQ and in the relationship of metabolites with sperm motility and viability. These findings suggest that sperm from donkeys and horses differ in their metabolism and energetic requirements, and that the concentration of specific SP metabolites may be related to sperm functionality. Further research should shed light on the metabolic needs of donkey and horse sperm, and evaluate how the knowledge collected from the contribution of these metabolites can help improve semen preservation in the two species.

Seminal plasma, and not sperm, induces time and concentration-dependent neutrophil extracellular trap release in donkeys.

BACKGROUND: In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. OBJECTIVES: To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in response to frozen-thawed semen. STUDY DESIGN: In vitro experiments. METHODS: Jenny PMNs were exposed to jackass fresh or frozen-thawed semen, isolated sperm or seminal plasma (SP), over the course of three experiments. NET formation in response to different treatments was assessed through manual quantification of stained slides. A one-way analysis of variance (ANOVA), followed by a post hoc Sidak test, was carried out to determine statistical significance. RESULTS: NET release occurred in a semen concentration- and incubation-time-dependent manner. Surprisingly, frozen-thawed donkey sperm did not increase NETosis rate in comparison with the control (23 ± 2.5% vs. 31 ± 3.7%; P > .05), whereas fresh semen exposure did (78 ± 5.7% vs. 26 ± 3.2%, P < .01). NETosis increased in the presence of SP, regardless of the presence or absence of sperm, in comparison with the control in both fresh (84 ± 5.2% and 77 ± 5.0% vs. 12 ± 2.7%, respectively; P < .01) and frozen (95 ± 2.2% and 94 ± 2.9% vs. 14 ± 3.8%, respectively; P < .01) samples. Moreover, exposure of PMN to viable and motile sperm, in the absence of SP, did not increase NETosis rates (P > .05). CONCLUSIONS: Donkey SP, and not sperm-intrinsic factors, is able to trigger NETosis in both time- and semen concentration-dependent manner. The physiological relevance of such response against semen in the donkey remains to be elucidated.

Telomere length in bovine sperm is related to the production of reactive oxygen species, but not to reproductive performance.

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.

Metabolic profiling of preovulatory follicular fluid in jennies.

Follicular fluid is formed from the transudation of theca and granulosa cells in the growing follicular antrum. Its main function is to provide an optimal intrafollicular microenvironment to modulate oocyte maturation. The aim of this study was to determine the metabolomic profile of preovulatory follicular fluid (PFF) in jennies. For this purpose, PFF was collected from 10 follicles of five jennies in heat. Then, PFF samples were analysed by nuclear magnetic resonance (NMR) and heteronuclear single quantum correlation (2D 1H/13C HSQC). Our study revealed the presence of at least 27 metabolites in the PFF of jennies (including common amino acids, carboxylic acids, amino acid derivatives, alcohols, saccharides, fatty acids, and lactams): 3-hydroxybutyrate, acetate, alanine, betaine, citrate, creatine, creatine phosphate, creatinine, ethanol, formate, glucose, glutamine, glycerol, glycine, hippurate, isoleucine, lactate, leucine, lysine, methanol, phenylalanine, proline, pyruvate, threonine, tyrosine, valine, and τ-methylhistidine. The metabolites found here have an important role in the oocyte development and maturation, since the PFF surrounds the follicle and provides it with the needed nutrients. Our results indicate a unique metabolic profile of the jennies PFF, as it differs from those previously observed in the PFF of the mare, a phylogenetically close species that is taken as a reference for establishing reproductive biotechnology techniques in donkeys. The metabolites found here also differ from those described in the TCM-199 medium enriched with fetal bovine serum (FBS), which is the most used medium for in vitro oocyte maturation in equids. These differences would suggest that the established conditions for in vitro maturation used so far may not be suitable for donkeys. By providing the metabolic composition of jenny PFF, this study could help understand the physiology of oocyte maturation as a first step to establish in vitro reproductive techniques in this species.

Seminal extracellular vesicles subsets modulate gene expression in cumulus cells of porcine in vitro matured oocytes.

Seminal plasma (SP), a fluid composed mainly by secretions from accessory sex glands, contains a heterogenous population of extracellular vesicles (EVs), involved in several reproductive physiological processes. Seminal plasma has been found to modulate ovary function, in terms of hormone secretion and immune regulation. This study evaluated the potential effect of SP-EV-subsets on the modulation of cumulus-oocyte-complex (COCs) physiology during in vitro maturation (IVM). Two SP-EV-subsets, small-EVs (S-EVs) and large-EVs (L-EVs), were isolated from pig SP by size-exclusion-chromatography. Next, COCs were IVM in the absence (control) or presence of each SP-EV-subset to evaluate their uptake by COCs (PKH67-EVs labelling) and their effect on oocyte and cumulus cells (CCs) (gene expression, and progesterone and estradiol-17ß levels). S-EVs and L-EVs were able to bind CCs but not oocytes. Supplementation with L-EVs induced changes (P ≤ 0.05) in the transcript levels of oocyte maturation- (HAS2) and steroidogenesis-related genes (CYP11A1 and HSD3B1) in CCs. No effect on nuclear oocyte maturation and progesterone and estradiol-17ß levels was observed when COCs were IVM with any of the two SP-EV-subsets. In conclusion, while SP-EV-subsets can be integrated by CCs during IVM, they do not affect oocyte maturation and only L-EVs are able to modulate CCs function, mainly modifying the expression of steroidogenesis-related genes.