Search

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm‐associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild‐type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function. Summary: Analyzing the role of male reproductive tract‐specific proteins in male fertility using knockout models is an active area of investigation. Although a large number of knockout models for genes that are specific to the testis are generated and the reproductive function studied, there are very few studies that analyze the role of epididymal proteins that contribute to sperm function and fecundity. In this study, the effect of knockout of an epididymal‐specific gene, Spag11a, on male reproductive function, especially the fecundity, capacitation, acrosome reaction, and sperm proteome was analyzed. Our observations indicate subfertility and compromised sperm function associated with perturbations in sperm proteome. Using shRNA‐mediated Spag11a mRNA ablation and active immunization‐mediated SPAG11A protein ablation, we previously demonstrated the possible role of Spag11a in sperm function and fecundity. This study significantly differs from our previous studies because we used a higher level of gene ablation model, that is, the Spag11a knockout mice to provide further evidence. Furthermore, we also analyzed the molecular changes at the proteome level under conditions of Spag11a gene knockout. Thus, this study carried a lot of significance since it reiterates the observations made in our previous studies using an animal model that truly reflects the ablation of Spag11a expression. [ABSTRACT FROM AUTHOR]

β‐nerve growth factor (βNGF) plays a crucial role in reproductive physiology and sperm quality. Enzymatic activity of seminal plasma and vaginal fluids reduces available βNGF and it has been demonstrated that chitosan microspheres could protect rrβNGF from degradation. This study examined the effects of microencapsulated rrbNGF with chitosan on rabbit sperm viability, motility and capacitation status. Results showed that 0.5 and 1 μg/mL of microencapsulated rrβNGF, as well as free rrβNGF or empty microspheres, did not adversely affect sperm viability or total motility after 2 h of incubation. However, the highest progressivity kinetic parameters were observed with 1 μg/mL free rrβNGF, while the highest curvilinear velocity (VCL) occurred with 0.5 μg/mL microencapsulated rrβNGF. Empty chitosan microspheres did not induce acrosome reaction (AR), but both concentrations of free and rrβNGFch favoured AR during in vitro incubation. The study suggests that using chitosan spheres did not show any adverse effects on sperm traits, unlike free rβNGF and rrβNGFch promoted capacitation and AR. Further research is needed to explore the potential of rrβNGFch in modifying in vitro capacitation and inducing ovulation during artificial insemination. [ABSTRACT FROM AUTHOR]

Community health promotion offers a potential solution to persistent healthcare challenges, with community health workers playing a pivotal role. The Community Training Institute for Health Disparities (CTIHD) implemented a problem-solving curriculum in Community Health Promotion, integrating a competency-based learning model through two courses: Introduction to Community Health Promotion and Design of an Action Plan for the Promotion of Community Health. Each course comprised ten three-hour sessions, featuring pre/post-tests, evaluations, and a cognitive debriefing. Knowledge change was assessed using pre/post-test scores among 27 community leaders from southern Puerto Rico. Cohort 1 and Cohort 2 demonstrated an overall retention rate of 62.6% and 96.7%, respectively. Although differences in knowledge gained between cohorts and courses weren't statistically significant, a trend toward increased knowledge was noted. Cohort 1 experienced a 22% knowledge increase in Course 1 and a 24% increase in Course 2. Cohort 2 demonstrated a 41% knowledge increase in Course 1 and a 25% increase in Course 2. The CTIHD's Community Health Promotion Program has made significant strides in elevating awareness and knowledge, marking a positive step toward reducing health disparities and fostering healthier, empowered communities in southern Puerto Rico. [ABSTRACT FROM AUTHOR]

Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro , the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse. • Different treatments for equine sperm capacitation were evaluated in two media. • Combination of capacitating inducers boosts phosphorylation and acrosomal exocytosis. • Capacitating conditions improve sperm's zona pellucida binding capacity. [ABSTRACT FROM AUTHOR]

Backgroud: Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm's ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction. Results: In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa. Conclusions: The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize. [ABSTRACT FROM AUTHOR]

Background Objective Materials and methods Results Conclusion Recently, the metabolic pathways involved in energy production and the role of aquaglyceroporins in capacitation‐associated events have been studied in humans and mice. However, little is known about these in ram spermatozoa.The present study investigated bioenergetic and aquaglyceroporin 3 variations during in vitro capacitation of ram spermatozoa. In addition, differences in testosterone levels between males were examined to determine their influence on capacitation‐like changes.Spermatozoa obtained from nine rams (ejaculates = 36) were incubated for 180 min in three different media (control, capacitating, and aquaglyceroporin‐inhibitor media) at 38.5°C. At 0 and 180 min of incubation in each medium, sperm viability, kinetics, chlortetracycline patterns, adenosine triphosphate concentration, lactate excretion (final subproduct of glycolysis), and immunolocalization of aquaporin 3 were evaluated.The increment of the capacitated spermatozoa‐chlortetracycline pattern and the hyperactivated‐like movement characterized by the highest curvilinear velocity and amplitude of lateral head displacement and the lowest linearity was only recorded after 180 min in the capacitating medium. At this time and conditions, adenosine triphosphate content and lactate excretion decreased, whereas the aquaglyceroporin 3 location in the midpiece and principal piece increased compared to 0 min. Such changes were not observed in the control medium over time. Incubation in the aquaglyceroporin‐inhibitor medium for 180 min reduced drastically sperm motility and adenosine triphosphate content compared to the other media. Testosterone analysis revealed a significant individual variability, which was also present in all sperm parameters evaluated. Furthermore, testosterone was negatively correlated with adenosine triphosphate content but positively correlated with lactate excretion levels, sperm viability, motility, capacitated sperm‐chlortetracycline pattern, and aquaglyceroporin 3 immunolabeling in the midpiece and principal piece.Despite individual differences, capacitation of ram spermatozoa increases adenosine triphosphate consumption, energy metabolism, and aquaglyceroporin 3 location in the midpiece and principal piece, which seems to be related to the acquisition of hyperactivated‐like motility. Furthermore, testosterone levels may serve as a valuable tool to select those males with a greater sperm metabolism rate and fertilizing capacity. [ABSTRACT FROM AUTHOR]

Simple Summary: Capacitation confers competency to spermatozoa to fertilize the oocyte, yet its regulatory mechanisms are not fully understood. Thus, we aimed to investigate the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography–tandem mass spectrometry (LCMS/MS) combined with tandem mass tag (TMT) labeling strategy. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein–protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Together, our results provided the database for studying the differentially expressed proteins during ram sperm capacitation. Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography–tandem mass spectrometry combined with tandem mass tag labeling strategy. As a results, 2050 proteins were identified and quantified; 348 of them were differentially abundant, with 280 of the proteins upregulated and 68 of the proteins downregulated between the CAP and NC spermatozoa, respectively. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein–protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Our findings indicate that capacitation progress might be regulated through different pathways, providing insights into mechanisms involved in ram sperm capacitation and fertility. [ABSTRACT FROM AUTHOR]

Simple Summary: Research efforts over the past decade have uncovered new insights into functions of erythropoietin that point to new roles beyond erythropoiesis. Recent studies have demonstrated that erythropoietin is implicated in tissue protection, regeneration, and reproduction. In this context, the present study reveals for the first time that erythropoietin inhibits cell death by apoptosis and enhances the fertilizing capacity of bovine spermatozoa. Erythropoietin (EPO), a hormone secreted mainly by the kidney, exerts its biological function by binding to its cell-surface receptor (EpoR). The presence of EPO and EpoR in the male and female reproductive system has been verified. Therefore, some of the key properties of EPO, such as its antioxidant and antiapoptotic effects, could improve the fertilizing capacity of spermatozoa. In the present study, the effect of two different concentrations of EPO (10 mIU/μL and 100 mIU/μL) on bovine sperm-quality parameters was evaluated during a post-thawing 4-h incubation at 37 °C. EPO had a positive effect on sperm motility, viability, and total antioxidant capacity. Moreover, EPO inhibited apoptosis, as it reduced both BCL2-associated X apoptosis regulator (Bax)/B-cell lymphoma 2 (Bcl-2) ratio and cleaved cysteine-aspartic proteases (caspases) substrate levels in a dose-dependent manner. In addition, EPO induced sperm capacitation and acrosome reaction in spermatozoa incubated in capacitation conditioned medeia. These results establish a foundation for the physiological role of EPO in reproductive processes and hopefully will provide an incentive for further research in order to fully decipher the role of EPO in sperm physiology and reproduction. [ABSTRACT FROM AUTHOR]

Abstract Backgroud Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm’s ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction. Results In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P

Abstract Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

Abstract To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.

Mammalian spermatozoa rely on glycolysis and mitochondrial oxidative phosphorylation for energy leading up to fertilization. Sperm capacitation involves a series of well-regulated biochemical steps that are necessary to give spermatozoa the ability to fertilize the oocyte. Additionally, zinc ion (Zn2+) fluxes have recently been shown to occur during mammalian sperm capacitation. Semen from seven commercial boars was collected and analyzed using image-based flow cytometry before, after, and with the inclusion of 2 mM Zn2+ containing in vitro capacitation (IVC) media. Metabolites were extracted and analyzed via Gas Chromatography-Mass Spectrometry (GC-MS), identifying 175 metabolites, with 79 differentially abundant across treatments (p < 0.05). Non-capacitated samples showed high levels of respiration-associated metabolites including glucose, fructose, citric acid, and pyruvic acid. After 4 h IVC, these metabolites significantly decreased, while phosphate, lactic acid, and glucitol increased (p < 0.05). With zinc inclusion, we observed an increase in metabolites such as lactic acid, glucitol, glucose, fructose, myo-inositol, citric acid, and succinic acid, while saturated fatty acids including palmitic, dodecanoic, and myristic acid decreased compared to 4 h IVC, indicating regulatory shifts in metabolic pathways and fatty acid composition during capacitation. These findings underscore the importance of metabolic changes in improving artificial insemination and fertility treatments in livestock and humans. [ABSTRACT FROM AUTHOR]

Capacitation involves tyrosine phosphorylation (TP) as a key marker. Lifestyle-related factors, such as obesity and smoking, are recognized for their adverse effects on semen quality and male fertility, yet the underlying mechanisms, including their potential impact on TP, remain unclear. Moreover, the effect of sperm cryopreservation on TP at the human sperm population level is unexplored. Flow cytometry analysis of global TP was performed on pre-capacitated, post-capacitated and 1- and 3-hours' incubated fresh and frozen–thawed samples from sperm donors (n = 40). Neither being overweight nor smoking (or both) significantly affected the percentage of sperm showing TP. However, elevated BMI and smoking intensity correlated with heightened basal TP levels (r = 0.226, p = 0.003) and heightened increase in TP after 3 h of incubation (r = 0.185, p = 0.017), respectively. Cryopreservation resulted in increased global TP levels after capacitation but not immediately after thawing. Nonetheless, most donors' thawed samples showed increased TP levels before and after capacitation as well as after incubation. Additionally, phosphorylation patterns in fresh and frozen–thawed samples were similar, indicating consistent sample response to capacitation stimuli despite differences in TP levels. Overall, this study sheds light on the potential impacts of lifestyle factors and cryopreservation on the dynamics of global TP levels during capacitation. [ABSTRACT FROM AUTHOR]

Background Objectives Material and methods Results Discussion and conclusions Mammalian spermatozoa need to undergo a process named capacitation to be able to fertilize an oocyte. During their journey in the female tract, spermatozoa obtain energy while exposed to a changing environment containing a variety of metabolic substrates. The energy requirements for sperm capacitation are species‐specific. In addition, the available energy source can hinder the process of sperm capacitation and eventually the acrosome reaction.To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process.The effect of different metabolic substrates on sperm capacitation process was evaluated by analyzing phosphorylation in the p32 protein; the acrosome reaction and the ATP intracellular content.The presence of glucose in the in vitro capacitating medium diminishes, in a concentration‐dependent manner, parameters associated with the capacitated status: induced acrosome exocytosis, plasma membrane destabilization, and protein tyrosine phosphorylation. Conversely, sperm incubation with pyruvate or lactate, either individually or in combination, allows the attainment of the capacitated status. Unexpectedly, pig spermatozoa incubated without any extracellular energy substrates or with a non‐metabolizable substrate (l‐glucose) for 4 h displayed similar sperm viability to the control and exhibited a capacitated phenotype. The capacitation‐like phenotype observed in starved pig spermatozoa (absence of glucose, lactate, and pyruvate) was dependent on extracellular bicarbonate and calcium levels, and these spermatozoa exhibited lower intracellular ATP content compared to those not capacitated. Nevertheless, the intracellular content of calcium was not modified in comparison to the control.Our findings suggest that the metabolic substrates used to fuel pig sperm metabolism are important in achieving the capacitated status. The results of this work could be used to refine the capacitating medium employed in pig in vitro fertilization. [ABSTRACT FROM AUTHOR]

Context: The Rsa I polymorphism of the melatonin receptor MTNR1A gene affects seasonal reproduction in sheep, but its effect on ram spermatozoa and their response to melatonin is unknown. Aims: This study aims to evaluate whether Rsa I polymorphism of the MTNR1A gene influences the response of ram spermatozoa to in vitro added melatonin. Methods: Spermatozoa from rams carrying different Rsa I allelic variants were incubated with melatonin in a TALP medium or a capacitation-triggering medium during the reproductive and non-reproductive seasons. After incubation, sperm motility, membrane integrity, mitochondria activity, oxidative damage, apoptotic markers and capacitation status were assessed. Key results: In the reproductive season, the T/T genotype was related to some adverse effects of melatonin when spermatozoa were incubated in TALP medium, whereas the C/C genotype was linked with adverse effects when the hormone was added in a capacitation-triggering medium. The decapacitating effect of melatonin on spermatozoa was also different depending on genotype. Conclusions: The melatonin effect on spermatozoa from rams carrying different Rsa I genotypes differed depending on the season and the medium. Implications: The knowledge of the Rsa I allelic variant of the MTNR1A gene of rams could be helpful when carrying out in vitro reproductive techniques in the ovine species. Melatonin regulates seasonal reproduction in ovine species. This study explores the influence of the melatonin receptor gene and its variants on the response of ram spermatozoa to melatonin in different seasons and media. Results revealed that having specific variants of the gene affects how sperm cells react to melatonin. Understanding these genetic variations could aid in enhancing reproductive techniques in ovine species. Image by Victoria Peña-Delgado. This article belongs to the Collection Non-photoperiodic Actions of Melatonin. [ABSTRACT FROM AUTHOR]

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 μM of MnTBAP based on a Tris–egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 μM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 μM and 150 μM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 μM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 μM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 μM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze–thawing, improving the fertilization capacity, and leading to increased embryo development. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusion Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl‐alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa.Frozen–thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non‐capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl‐alcohol, bicarbonate and bovine serum albumin, polyvinyl‐alcohol and bovine serum albumin alone. Protein kinase A‐phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated.Incubation of frozen–thawed equine spermatozoa with bicarbonate and polyvinyl‐alcohol in atmospheric air for 45 min induced an increase in protein kinase A‐phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl‐alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl‐alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl‐alcohol = 120.9 ± 2.9 vs. NC = 76.91 ± 6.9 µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl‐alcohol = 1.15 ± 0.02 vs. NC = 0.77 ± 0.03 µm) (straightness: bicarbonate and polyvinyl‐alcohol = 0.76 ± 0.01 vs. NC = 0.87 ± 0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl‐alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre‐incubated in CAP condition for 45 min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05).These findings underscore the importance of bicarbonate and polyvinyl‐alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry. [ABSTRACT FROM AUTHOR]

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation--a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges--unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla. [ABSTRACT FROM AUTHOR]

Context: Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims: To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods: poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results: poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P < 0.05). Conclusions: poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm–oocyte interaction. Implications: The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm–oocyte interaction. We examined the tiny structures called extracellular vesicles that come from the fluid in pig uterus in order to understand how these vesicles change during the reproductive cycle of female pigs. Those from the late stages of the cycle seemed to have a strong connection with sperm and a possible key role in fertility. These vesicles regulate sperm function and interaction with oocytes, and they could potentially be used in fertility treatments to improve the chances of successful fertilisation. Photograph by SM Toledo-Guardiola. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

In most many countries worldwide, community leaders (CLs) are expected to play an important role in mobilising and soliciting contributions from others for the proactive implementation of climate change adaptation strategies. Due to a lack of capacity at the local leadership level, several local-level climate change mitigation and adaptation initiatives have not been effectively actioned with most of them frequently excluding local decision-makers, and key community stakeholders. Basing assessment on what is available in the literature, it is evident that there is urgent need for the scientific community to develop innovative strategies that facilitate the meaningful implementation of informed climate change policies. To successfully address the challenges of climate change, this paper posits that it is necessary for researchers, policy makers and planistrators must endeavour to explore and implement actionable local-level climate change mitigation and adaptation strategies. The take-home message from this paper is that the time to act is now and refrain from doing so until it is too late to do so for tomorrow may never come. The likely implications for the benefits of proactive and effective community leaders are considered against this backdrop and insights from literature-based multi-level governance, participatory learning, action-oriented transformative leadership initiatives that were sourced from the literature.

It was meaningful to evaluate the innovation introduction at the Council Chamber, through absorptive capacity of agents who have done courses sponsored by State Account Courthouse from Paraná. It was researched agents from Council Chamber of Paraná State, who have attended witness courses. Therefore, it was aimed to analyse if the public management courses may develop absorptive capacity on agents from public management to implement innovation in routines and process. It is a descriptive research, with quantitative and survey approach, they were applied to 1.863 agents from municipal public management, with variables which searched the absorptive capacity theory from Lane, Koka and Pathak (2006). Data were analysed through descriptive analysis, exploratory factorial and confirmatory analysis, and structures equation analysis. The respondent´s perception is that a percentage of innovation variability explained is around 62% while the percentage variability on absorptive capacity explained is around 50%. Finally, the public institutions may keep invest in capacitation, because there is confirmation that investment in external knowledge are valuable on good practical dimensions to routines and process, mainly the ones which allow the accounts authorizations and irregularities prevention. [ABSTRACT FROM AUTHOR]

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218 ± 23% and 245 ± 38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and β subunits (isoforms α1, α3, β1, β2, and β3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0 h control (33.0 ± 2.6% vs. 19.2 ± 2.6% capacitated sperm, respectively; p = 0.014) and altered the β2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N , N -dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3′-dipropylthiadicarbocyanine iodide and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare. [ABSTRACT FROM AUTHOR]

We aimed to investigate the impact of processing boar spermatozoa with phosphate-buffered saline (PBS) at 4 ˚C on acrosomal integrity and increase in 32 kDa tyrosine-phosphorylated protein (p32). Following cooled PBS washing, we observed a significant increase in p32 levels and in the proportion of dead spermatozoa with compromised acrosomal integrity compared to sperm washing using PBS at room temperature. Interestingly, this increase in p32 was effectively inhibited when cooled PBS was supplemented with 1 mM AEBSF, a serine protease inhibitor. Our findings suggest that the increase of p32 in response to cooled PBS washing in boar spermatozoa is associated with enhanced protease activity in dead spermatozoa. [ABSTRACT FROM AUTHOR]

Before fertilization of the oocyte, the spermatozoa must undergo through a series of biochemical changes in the female reproductive tract named sperm capacitation. Spermatozoa regulates its functions by post-translational modifications, being historically the most studied protein phosphorylation. In addition to phosphorylation, recently, protein acetylation has been described as an important molecular mechanism with regulatory roles in several reproductive processes. However, its role on the mammal's sperm capacitation process remains unraveled. Sirtuins are a deacetylase protein family with 7 members that regulate protein acetylation. Here, we investigated the possible role of SIRT1 on pig sperm capacitation-related events by using YK 3-237, a commercial SIRT1 activator drug. SIRT1 is localized in the midpiece of pig spermatozoa. Protein tyrosine phosphorylation (focused at p32) is an event associated to pig sperm capacitation that increases when spermatozoa are in vitro capacitated in presence of YK 3-237. Eventually, YK 3-237 induces acrosome reaction in capacitated spermatozoa: YK 3-237 treatment tripled (3.40 ± 0.40 fold increase) the percentage of acrosome-reacted spermatozoa compared to the control. In addition, YK 3-237 induces sperm intracellular pH alkalinization and raises the intracellular calcium levels through a CatSper independent mechanism. YK 3-237 was not able to bypass sAC inhibition by LRE1. In summary, YK 3-237 promotes pig sperm capacitation by a mechanism upstream of sAC activation and independent of CatSper calcium channel. [ABSTRACT FROM AUTHOR]

Background. Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. Methods. EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). Results. The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30&lowbar;200 nm) by NTA.CD9and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Conclusion. Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus. [ABSTRACT FROM AUTHOR]

Background: Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. Objective: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Materials and methods: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (10 mm) for 0.5 to 4 h. Response to calcium ionophore A23187 (5 μm) was used to evaluate capacitation status. Motility was evaluated using computer‐assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. Results: Incubation with lactate alone for 2 h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4 h induced a significant spontaneous increase in acrosome‐reacted, membrane‐intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome‐reacted spermatozoa. Sperm motility was significantly higher in pyruvate‐only medium than in glucose or lactate. The addition of pyruvate to lactate‐containing medium increased sperm motility but reduced the proportion of live acrosome‐reacted spermatozoa in a dose‐dependent fashion. Discussion: This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome‐reacted spermatozoa obtained is among the highest reported for equine spermatozoa. Conclusion: These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology. [ABSTRACT FROM AUTHOR]

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation—a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges—unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography–tandem mass spectrometry combined with tandem mass tag labeling strategy. As a results, 2050 proteins were identified and quantified; 348 of them were differentially abundant, with 280 of the proteins upregulated and 68 of the proteins downregulated between the CAP and NC spermatozoa, respectively. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein–protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Our findings indicate that capacitation progress might be regulated through different pathways, providing insights into mechanisms involved in ram sperm capacitation and fertility.

Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE‐sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility‐inhibitory factor of sheep I and II (MIFS‐I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS‐I and II, respectively. Specific activities of the partially purified MIFS‐I and II were 563 and 261 U/mg of protein, while the fold‐purification of the activity were 5119 and 2373, respectively. Both the proteins were heat‐labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS‐I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS‐I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross‐reactivity of MIFS‐I with polyclonal anti‐human SEMA3D antibody. It was concluded that the MIFS‐I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation. Significance statement: Oxidative damage caused by free radicals generated from metabolic activities in motile sperm leads to shorter survival and poor fertility of preserved ram semen. Hence, reversible blocking of sperm motility soon after semen dilution could be beneficial to minimize this damage. Sperm quiescent proteins of male reproductive tract fluid are known to inhibit sperm motility in a reversible manner; hence have great potential for use in semen preservation. Here we report partial purification of an 80 kDa sperm quiescent protein of ram cauda epididymal fluid and it is effect on sperm motility, viability, capacitation, and protein tyrosine phosphorylation during in vitro capacitation. [ABSTRACT FROM AUTHOR]

Context. Porcine gametes require energy for the physiological processes that allow fertilisation. Succinate dehydrogenase (SDH) plays a pivotal role in both, the tricarboxylic acid (TCA) cycle and the respiratory chain. Aims. The aimof this work was to study the participation of SDH in the in vitro oocytematuration, spermcapacitation and acrosome reaction in porcine species. Methods. Cumulus-oocyte complexes (COCs) from abattoir-derived porcine ovaries were collected by aspiration and were incubated in maturation media, with the addition of increasing concentrations (0, 1, 5 and 10 mM) of malonate (a specific inhibitor of SDH). Nuclear maturation and cytoplasmatic maturation were analysed. Semen samples were incubated for 2 h in capacitating medium with 40 mM sodium bicarbonate, as sperm capacitation inducer, and the addition of increasing concentrations of malonate (0, 1, 5 and 10 mM). Sperm capacitation state and true acrosomal reaction were evaluated. SDH activity was determined in sperm and oocyte extracts by the spectrophotometric method. Key results. The addition of 10 mM of malonate decreased both nuclear and cytoplasmic maturation rates (P < 0.05) without affecting COC viability (assessed using fluorescein diacetate). A lower level of capacitation (induced by bicarbonate) and acrosome reaction (induced by follicular fluid) was observed with the addition of 5 mM of malonate (P < 0.05) without affecting motility and viability of sperm at this concentration. The activity of SDH was 0.35 ± 0.1 × 10-5 and 2.37 ± 0.9 × 10-5 U/COC for immature and in vitro matured COC extracts (P < 0.05) respectively, and 0.44 ± 0.16 U/1010 sperm for boar sperm extracts. Conclusions. In conclusion, because it has been proposed that aerobic and anaerobic metabolic pathways of cells are changed depending on the oxygen availability and the composition of metabolic substrates in their environment, our results suggest that energy obtained through the mitochondrial respiration (TCA cycle and oxidative phosphorylation) is necessary to support oocyte maturation, sperm capacitation and acrosome reaction in the porcine species. Implications. The study of enzymatic activity in gametes is essential for understanding the mechanisms that control the energy production required to achieve successful fertilisation. This knowledge has significant implications for the development of assisted reproductive technologies. [ABSTRACT FROM AUTHOR]

In my research life of 35 years, growing with IETS as a researcher of in vitro maturation and fertilisation (IVM/IVF) of porcine and cattle oocytes, I suffered from hard times related to solving problems that prevented the progress of my research and conferment of my degrees. Many researchers may have similar problems. Thus, I would like to address a few examples of how I overcame these problems related to IVF and help young researchers with similar troubles. There were four main problems to be solved in my experiments. Problem 1: Establishment of IVF using only defined medium in pigs. Problem 2: Establishment of successful in vitro culture (IVC) of IVM/IVF bovine oocytes in defined medium. Problem 3: Low rate of male pronucleus formation in IVM porcine oocytes after IVF. Problem 4: Sedimentation of Ca2+ in the sperm capacitation solution for IVF in pigs. Problem 1 was solved by a lucky accident, in which a sperm suspension that would have otherwise been discarded happened to be successfully used for IVF in pigs. Problems 2, 3 and 4 were solved by communication with scientists whose fields were different from mine, where similar problems had been solved already. Young researchers are encouraged to transcend the boundaries of their research fields and solve problems by interacting with researchers in different fields. There are many good connections or answers around us that may be effective in resolving the problems that are hindering the progress of pending research. In vitro fertilisation (IVF) of livestock animals is socially important because it is useful for increasing livestock production, breeding improvement and gene conservation. However, there are problems that hinder the promotion of IVF, such as the necessity of co-culturing with other somatic cells for development of bovine IVF embryos. This paper presents examples of how other problems with IVF have previously been solved, in an attempt to promote joint research for the improvement of IVF. Image by Takashi Nagai. [ABSTRACT FROM AUTHOR]

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the in vivo situation, there is still a lack of standardization in vitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on in vitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2: Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC‐1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p <.01) but increased rAcro (p <.01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC‐1 and decreased TM (p <.01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p <.01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence in vitro sperm capacitation in this species. [ABSTRACT FROM AUTHOR]

Copyright of Revista Veterinaria is the property of Universidad Nacional del Nordeste and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Ritonavir (RTV) is an antiviral and a component of COVID-19 treatments. Moreover, RTV demonstrates anti-cancer effects by suppressing AKT. However, RTV has cytotoxicity and suppresses sperm functions by altering AKT activity. Although abnormal AKT activity is known for causing detrimental effects on sperm functions, how RTV alters AKT signaling in spermatozoa remains unknown. Therefore, this study aimed to investigate reproductive toxicity of RTV in spermatozoa through phosphoinositide 3-kinase/phosphoinositide-dependent protein kinase-1/protein kinase B (PI3K/PDK1/AKT) signaling. Duroc spermatozoa were treated with various concentrations of RTV, and capacitation was induced. Sperm functions (sperm motility, motion kinematics, capacitation status, and cell viability) and expression levels of tyrosine-phosphorylated proteins and PI3K/PDK1/AKT pathway-related proteins were evaluated. In the results, RTV significantly suppressed sperm motility, motion kinematics, capacitation, acrosome reactions, and cell viability. Additionally, RTV significantly increased levels of phospho-tyrosine proteins and PI3K/PDK1/AKT pathway-related proteins except for AKT and PI3K. The expression level of AKT was not significantly altered and that of PI3K was significantly decreased. These results suggest RTV may suppress sperm functions by induced alterations of PI3K/PDK1/AKT pathway through abnormally increased tyrosine phosphorylation. Therefore, we suggest people who use or prescribe RTV need to consider its male reproductive toxicity. [ABSTRACT FROM AUTHOR]

Infertility affects a significant percentage of couples worldwide, and male factors contribute significantly to this problem. Traditional assessments of male fertility rely primarily on parameters such as sperm motility, morphology, viability, and concentration. However, these metrics often do not provide a comprehensive understanding of sperm function, which is critical not only for fertilization but also for successful embryo development. Herein, we used porcine spermatozoa as a model to investigate the potential of sperm RNA markers in assessing various aspects of sperm function from motility to fertility. Using artificial insemination, we evaluated male fertility based on the litter size of sows inseminated with sperm from 20 boars. In addition, we measured parameters such as weaning rate, death births, live births, and mummy births. Sperm functional parameters, including motility and kinematics, were assessed before and after in vitro capacitation. Finally, correlations between various sperm functional parameters and sperm RNA markers were explored. Our results revealed interesting relationships between sperm functional parameters. While motility and kinematics were correlated, they were not correlated with sperm capacitation status. Surprisingly, no significant correlations were found between these parameters and male fertility. However, specific parameters of sperm capacitation status after in vitro capacitation were correlated with weaning rate and live births, highlighting their importance in predicting successful fertilization. Further analysis of sperm RNA markers identified genes related to male fertility, including IZUMO1, known for its role in sperm-egg fusion. These genes showed correlations with motility, capacitation, and fertilization parameters, shedding light on their potential roles in sperm function. In conclusion, our study demonstrates that sperm RNA markers hold promise for the diagnosis and prognosis of various aspects of sperm function, providing valuable insights into male infertility. These markers may serve as diagnostic tools to improve our understanding of male fertility problems, ultimately benefiting couples struggling with infertility. [ABSTRACT FROM AUTHOR]

To acquire the capacity to fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical reactions in the female reproductive tract, which are collectively called capacitation. The capacitated spermatozoa subsequently interact with the oocyte zona-pellucida and undergo the acrosome reaction, which enables the penetration of the oocyte and subsequent fertilization. However, the spontaneous acrosome reaction (sAR) can occur prematurely in the sperm before reaching the oocyte cumulus oophorus, thereby jeopardizing fertilization. One of the main processes in capacitation involves actin polymerization, and the resulting F-actin is subsequently dispersed prior to the acrosome reaction. Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR. In the present review, we describe the protective mechanisms that regulate sperm capacitation and prevent sAR. [ABSTRACT FROM AUTHOR]

Polyamines are polycationic molecules which contains two or more amino groups (-NH3+) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC. [ABSTRACT FROM AUTHOR]

Introduction: Lipidomics elucidates the roles of lipids in both physiological and pathological processes, intersecting with many diseases and cellular functions. The maintenance of lipid homeostasis, essential for cell health, significantly influences the survival, maturation, and functionality of sperm during fertilization. While capacitation and the acrosome reaction, key processes before fertilization, involve substantial lipidomic alterations, a comprehensive understanding of the changes in human spermatozoa's lipidomic profiles during these processes remains unknown. This study aims to explicate global lipidomic changes during capacitation and the acrosome reaction in human sperm, employing an untargeted lipidomic strategy using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Methods: Twelve semen specimens, exceeding the WHO reference values for semen parameters, were collected. After discontinuous density gradient separation, sperm concentration was adjusted to 2 x 106 cells/ml and divided into three groups: uncapacitated, capacitated, and acrosome-reacted. UPLC-MS analysis was performed after lipid extraction from these groups. Spectral peak alignment and statistical analysis, using unsupervised principal component analysis (PCA), bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) analysis, and supervised partial least-squares-latent structure discriminate analysis (PLS-DA), were employed to identify the most discriminative lipids. Results: The 1176 lipid peaks overlapped across the twelve individuals in the uncapacitated, capacitated, and acrosome-reacted groups: 1180 peaks between the uncapacitated and capacitated groups, 1184 peaks between the uncapacitated and acrosome-reacted groups, and 1178 peaks between the capacitated and acrosome-reacted groups. The count of overlapping peaks varied among individuals, ranging from 739 to 963 across sperm samples. Moreover, 137 lipids had VIP values > 1.0 and twenty-two lipids had VIP > 1.5, based on the O2PLS-DA model. Furthermore, the identified twelve lipids encompassed increases in PI 44:10, LPS 20:4, LPA 20:5, and LPE 20:4, and decreases in 16-phenyl-tetranor-PGE2, PC 40:6, PS 35:4, PA 29:1, 20-carboxy- LTB4, and 2-oxo-4-methylthio-butanoic acid. Discussion: This study has been the first time to investigate the lipidomics profiles associated with acrosome reaction and capacitation in human sperm, utilizing UPLC-MS in conjunction with multivariate data analysis. These findings corroborate earlier discoveries on lipids during the acrosome reaction and unveil new metabolites. Furthermore, this research highlights the effective utility of UPLC-MS-based lipidomics for exploring diverse physiological states in sperm. This study offers novel insights into lipidomic changes associated with capacitation and the acrosome reaction in human sperm, which are closely related to male reproduction. [ABSTRACT FROM AUTHOR]

Context: Several assisted reproduction techniques have been proposed for collared peccaries due to the interest in its conservation. However, there is little information about the requirements for sperm capacitation, an initial step in in vitro fertilisation. Aims: We aimed to determine the optimal conditions for collared peccary sperm capacitation by comparing Tyrode's albumin lactate pyruvate (TALP) or the same media plus heparin, caffeine, or their combination at different exposure times (1, 3, and 6 h). Methods: The samples were evaluated for kinetic parameters, membrane functionality and integrity, mitochondrial activity, morphology, DNA integrity, reactive oxygen species (ROS) expression levels, capacitation, and sperm binding ability using swine oocytes. Key results: Samples incubated with caffeine or heparin had a higher percentage of capacitated spermatozoa. The maximum percentage of capacitation was achieved after 3 h of incubation with either agent. Moreover, spermatozoa subjected to heparin capacitation showed better motility than when subjected to caffeine, and lower ROS expression levels after 1 h. No differences were observed among incubation times for the binding ability. Conclusions: In summary, collared peccary spermatozoa can be capacitated with caffeine or heparin; however, heparin better maintains sperm motility and ROS expression levels. The co-incubation of gametes in a medium with heparin for 3 h could be efficient for in vitro fertilisation in collared peccaries. Implications: This result will contribute to the development of assisted reproduction techniques for conservation and productivity of collared peccaries. In vitro fertilisation can contribute to the conservation and knowledge of the reproductive characteristics of collared peccary. There is little information about the requirements for sperm capacitation in the collared peccary, an initial step in in vitro fertilisation. We compared different capacitating media and incubation times and we observed that heparin improved sperm motility and promoted the capacitation of collared peccary spermatozoa. This result will contribute to the development of assisted reproduction techniques for conservation and productivity of collared peccaries. [ABSTRACT FROM AUTHOR]

This study was designed to analyze changes in the spermatozoa of three species of Phodopus hamsters incubated under different conditions. Cauda epididymal sperm were incubated for 4 h in modified Tyrode's medium containing albumin, lactate, pyruvate, and Hepes (mTALP-H), in the same medium with the addition of bicarbonate (mTALP-BH), or with bicarbonate and 20 ng/mL of progesterone (mTALP-BH+P4). Media with bicarbonate are believed to promote capacitation in rodent species. Sperm motility, viability, capacitation patterns, and kinematics were assessed at different times. Capacitation in live cells was quantified after staining with Hoechst 33258 and chlortetracycline. Patterns believed to correspond to non-capacitated cells (F pattern), capacitated, acrosome-intact cells (B pattern), and acrosome-reacted cells (AR pattern) were recognized. Kinematics were examined via computer-assisted sperm analysis (CASA). The results showed a decrease in total motility in all three species in different media, with a sharp decrease in progressive motility in bicarbonate-containing media (without or with progesterone), suggesting hyperactivated motion. However, none of the other signs of hyperactivation described in rodents (i.e., decrease in STR or LIN, together with an increase in ALH) were observed. F pattern cells diminished with time in all media and were generally lower in P. roborovskii and higher in P. campbelli. B pattern cells increased in mTALP-BH media in all species. Progesterone did not enhance the percentage of B pattern cells. Finally, AR pattern cells increased in all species incubated in different media, showing the highest percentage in P. roborovskii and the lowest in P. campbelli. Comparisons between media revealed that there were higher percentages of F pattern cells and lower percentages of B pattern cells over time in medium without bicarbonate (mTALP-H) in comparison to media containing bicarbonate (mTALP-BH; mTALP-BH+P4). Overall, changes consistent with the acquisition of capacitation and development of hyperactivated motility were found; however, further studies are required to better characterize media necessary to support the pathways involved in these processes in Phodopus species. [ABSTRACT FROM AUTHOR]

It is postulated that alterations in membranous lipids construction, surface characteristics, fluidity, Ca2+ permeability, and cholesterol levels collectively contribute to the process of capacitation induction. The change in sperm membrane calcium permeability following capacitation is the main signal for the acrosome reaction. The acrosome response is commonly induced in vitro using calcium ionophore. The study's objectives are to determine how the calcium ionophore (ionomycin) and the solvent dimethyl sulfoxide (DMSO) affect the motility, hyperactivity, and velocity of bovine frozen–thawed spermatozoa at varying concentrations and incubation times. The capacitation medium consisted of Bracket–Oliphant medium. Four capacitation media were prepared; two treated groups were supplemented with 25 nM and 50 nM of ionomycin, one group was without ionomycin, and the control group was supplemented with DMSO. Sperm motility decreased substantially with increasing time period in all four groups, according to the findings. The result found that there was some variance in the sperm parameters between the groups and treatment times. Overall, the group exposed to 25 nM had better semen and sperm parameters than the group exposed to 50 nM. In conclusion, the role of calcium ionophore had better semen and sperm parameters in the group exposed with a lower concentration than a higher concentration on sperm.

Bisphenol A (BPA) is an endocrine-disrupting compound, used as the key monomer of polycarbonate plastics and epoxy resins. BPA has been detected in both humans and farm animals and has been correlated with decreased sperm counts and motility. BPS and BPF are structural analogs of BPA and are increasingly being used in manufacturing as BPA substitutes. In this study, we aim to assess the direct outcomes of BPA, bisphenol S (BPS), and bisphenol F (BPF) exposure on bovine sperm parameters in vitro to elucidate how they affect sperm quality and fertilization potential, and to assess whether BPS and/or BPF are less harmful than BPA. Sperm from three or more bulls was obtained from either fresh samples or cryopreserved straws and exposed to 0.05 mg/mL of BPA, BPS, and BPF in vitro. After 4 h incubation, motility, capacitation, apoptosis/necrosis, and mitochondrial membrane potential levels were measured by computer-assisted sperm analysis or computational flow cytometry. Results showed that BPA exposure significantly reduced both fresh and cryopreserved sperm motility, capacitation, viability and mitochondrial membrane potential levels. Furthermore, BPF significantly decreased motility, capacitation and mitochondrial membrane potential in cryopreserved sperm only. BPS did not have any significant effects on any of the parameters measured. Our results suggest that BPA is the most harmful to sperm, while BPF is toxic under certain conditions, and BPS seems to be the least detrimental. Overall, this study provides an understanding of how the ubiquitous environmental chemicals, bisphenols, may impact male fertility even after ejaculation.

The study aimed to evaluate the effect of the addition of aloe vera raw extract on the capacitation of bovine spermatozoa during the in vitro fertilization process. Hyper activated motility (HAM) and acrosome reaction (AR) of sperm cells, as well as in vitro fertilization and cleavage rates are the main parameters used to estimate the aloe vera’s effect on bovine spermatozoa’s fertilizing ability. In the current study, two different concentrations of aloe vera (5 and 10 µg/ml) were used. Frozen thawed semen spermatozoa were subjected to a swim-up technique to separate the motile spermatozoa and capacitated in modified Sperm Tyrode’s albumin lactate pyruvate (S-TALP) medium supplemented with heparin only without aloe vera treatment (positive control), heparin+5µg/ml aloe vera, heparin+10µg/ml aloe vera, and finally the negative control tube, which was supplemented with 5µg/ml aloe vera without adding heparin. Sperm cells were incubated for 90 minutes at 39C0 in a 5% CO2 incubator and evaluated every 30 minutes at intervals. Cumulus oophorus complexes (COCs) were matured in a 5% CO2 incubator at 39C0 and inseminated in vitro with frozen-thawed bull sperm at the above concentrations. The inseminated oocytes were incubated at 39C0 in a 5% CO2 incubator for 24 hours and then examined for evidence of fertilization. The current study revealed that the aloe vera concentration of 10µg/ml at incubation time (60 minutes) had the best effect on sperm capacitation and acrosome reactions. The overall percentage of spermatozoa with progressive motility (PM) had declined across all groups as time progressed, with a significantly lower value found at the above concentration, as well as the effect of sperm treated with aloe vera on fertilization rate. The results showed that the proportion of fertilized oocytes was significantly increased at the aloe vera concentration of 10µg/ml compared to the other concentrations (5µg/ml), positive control, and negative control, respectively. In conclusion, treatment of bull spermatozoa with 10 µg of aloe vera extract/ml semen was considered the most efficient concentration for enhancement of sperm capacitation as a high IVF rate was obtained in Holstein Bull.

Progesterone (P) is a well-known enhancer of hyperactivation which is associated with the success of in vitro fertilization (IVF). In this study, we examined whether P-enhanced hyperactivation affected IVF success in rats. When rat spermatozoa were exposed to 10, 20, and 40 ng/ml P, 20 ng/ml P enhanced hyperactivation via the membrane progesterone receptor. In addition, the enhancement of hyperactivation by 20 ng/ml P was regulated by phospholipase C, transmembrane adenylate cyclase, and protein kinase A. However, 20 ng/ml P did not affect IVF success. These results suggest that 20 ng/ml P enhances rat spermatozoal hyperactivation through non-genomic pathways. Because the concentration of P changes during the estrous cycle, it seems that rat spermatozoa are hyperactivated in response to the oviductal environment. However, the effect of 20 ng/ml P does not seem to fully capacitate spermatozoa.

Erythropoietin (EPO), a hormone secreted mainly by the kidney, exerts its biological function by binding to its cell-surface receptor (EpoR). The presence of EPO and EpoR in the male and female reproductive system has been verified. Therefore, some of the key properties of EPO, such as its antioxidant and antiapoptotic effects, could improve the fertilizing capacity of spermatozoa. In the present study, the effect of two different concentrations of EPO (10 mIU/μL and 100 mIU/μL) on bovine sperm-quality parameters was evaluated during a post-thawing 4-h incubation at 37 °C. EPO had a positive effect on sperm motility, viability, and total antioxidant capacity. Moreover, EPO inhibited apoptosis, as it reduced both BCL2-associated X apoptosis regulator (Bax)/B-cell lymphoma 2 (Bcl-2) ratio and cleaved cysteine-aspartic proteases (caspases) substrate levels in a dose-dependent manner. In addition, EPO induced sperm capacitation and acrosome reaction in spermatozoa incubated in capacitation conditioned medeia. These results establish a foundation for the physiological role of EPO in reproductive processes and hopefully will provide an incentive for further research in order to fully decipher the role of EPO in sperm physiology and reproduction.

Mammalian spermatozoa rely on glycolysis and mitochondrial oxidative phosphorylation for energy leading up to fertilization. Sperm capacitation involves a series of well-regulated biochemical steps that are necessary to give spermatozoa the ability to fertilize the oocyte. Additionally, zinc ion (Zn2+) fluxes have recently been shown to occur during mammalian sperm capacitation. Semen from seven commercial boars was collected and analyzed using image-based flow cytometry before, after, and with the inclusion of 2 mM Zn2+ containing in vitro capacitation (IVC) media. Metabolites were extracted and analyzed via Gas Chromatography-Mass Spectrometry (GC-MS), identifying 175 metabolites, with 79 differentially abundant across treatments (p < 0.05). Non-capacitated samples showed high levels of respiration-associated metabolites including glucose, fructose, citric acid, and pyruvic acid. After 4 h IVC, these metabolites significantly decreased, while phosphate, lactic acid, and glucitol increased (p < 0.05). With zinc inclusion, we observed an increase in metabolites such as lactic acid, glucitol, glucose, fructose, myo-inositol, citric acid, and succinic acid, while saturated fatty acids including palmitic, dodecanoic, and myristic acid decreased compared to 4 h IVC, indicating regulatory shifts in metabolic pathways and fatty acid composition during capacitation. These findings underscore the importance of metabolic changes in improving artificial insemination and fertility treatments in livestock and humans.

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 μM of MnTBAP based on a Tris–egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 μM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 μM and 150 μM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 μM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 μM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 μM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze–thawing, improving the fertilization capacity, and leading to increased embryo development.