Glucose prevents the acquisition of the capacitated state in pig spermatozoa.

BACKGROUND: 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. OBJECTIVES: To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process. MATERIAL AND METHODS: 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. RESULTS: 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. DISCUSSION AND CONCLUSIONS: 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.

Trophic Nutrition in ICU Patients Undergoing High-Flow Oxygen Therapy and/or Noninvasive Mechanical Ventilation: The Nutri-Trophic Study.

Enteral nutrition (EN) therapy in ICU patients requiring oxygen therapy with high-flow nasal cannula (HFNC) and/or noninvasive mechanical ventilation (NIMV) is controversial. A prospective, cohort, observational, and multicenter study was conducted in 10 ICUs in Spain to analyze the 90-day mortality, tolerance, side effects, and infectious complications of trophic EN in patients requiring HFNC therapy and/or NIVM. A total of 149 patients were enrolled. The mean age, severity scores, tracheobronchitis, bacteremia, and antimicrobial therapy were significantly higher in deceased than in living patients (p < 0.05), and the mortality rate was 14.8%. A total of 110 patients received oral trophic feedings, 36 patients received nasogastric tube feedings (NGFs), and 3 received mixed feedings. Trophic EN was discontinued in only ten (14.9%) patients because of feeding-related complications. The variables selected for the multivariate logistic regression on feeding discontinuation were SOFA upon admission (OR per unit = 1.461) and urea (OR per mg/dL = 1.029). There were no significant differences in the development of new infections according to the route of EN administration. Early trophic feeding administered to patients with acute respiratory failure requiring noninvasive ventilation is safe and feasible, and is associated with few dietary and infectious complications in a mortality, setting comparable to similar studies.

Quantitative Analysis of the Human Semen Phosphorometabolome by 31P-NMR.

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.

The compound YK 3-237 promotes pig sperm capacitation-related events.

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.

Bisphenol S Reduces Pig Spermatozoa Motility through Different Intracellular Pathways and Mechanisms than Its Analog Bisphenol A.

Bisphenol A (BPA: 2,3-bis (4-hydroxyphenyl) propane) is an environmental chemical widely used in the manufacturing of epoxy polymers and many thermoplastic consumer products. Serious concerns about its safety led to the development of analogs, such as BPS (4-hydroxyphenyl sulfone). Very limited studies about BPS's impact on reproduction, specifically in spermatozoa, exist in comparison with BPA. Therefore, this work aims to study the in vitro impact of BPS in pig spermatozoa in comparison with BPA, focusing on sperm motility, intracellular signaling pathways and functional sperm parameters. We have used porcine spermatozoa as an optimal and validated in vitro cell model to investigate sperm toxicity. Pig spermatozoa were exposed to 1 and 100 µM BPS or BPA for 3 and 20 h. Both bisphenol S and A (100 µM) significantly reduce pig sperm motility in a time-dependent manner, although BPS exerts a lower and slower effect than BPA. Moreover, BPS (100 µM, 20 h) causes a significant increase in the mitochondrial reactive species, whereas it does not affect sperm viability, mitochondrial membrane potential, cell reactive oxygen species, GSK3α/ß phosphorylation or phosphorylation of PKA substrates. However, BPA (100 µM, 20 h) leads to a decrease in sperm viability, mitochondrial membrane potential, GSK3ß phosphorylation and PKA phosphorylation, also causing an increase in cell reactive oxygen species and mitochondrial reactive species. These intracellular effects and signaling pathways inhibited might contribute to explaining the BPA-triggered reduction in pig sperm motility. However, the intracellular pathways and mechanisms triggered by BPS are different, and the BPS-caused reduction in motility can be only partially attributed to an increase in mitochondrial oxidant species.

Conhecimento dos profissionais da atenção primária à saude sobre segurança do paciente em Rondônia / Knowledge of primary health care professionals about patient safety in Rondônia / Conocimiento de los profesionales de atención primaria de salud sobre la seguridad del paciente en Rondônia

INTRODUÇÃO: O programa de Segurança do Paciente tem como principal objetivo a redução a um nível mínimo aceitável do risco de danos evitáveis associados à assistência em saúde. Visando qualificar esse cuidado na Atenção Primária à Saúde (APS), o Conselho Nacional de Secretários de Saúde tem desenvolvido a Planificação da Atenção à Saúde (PAS) com apoio de instituições de excelência e secretarias de saúde. OBJETIVO: Identificar o conhecimento dos profissionais da Atenção Primária à Saúde envolvidos na planificação acerca da segurança do paciente em Rondônia. MÉTODO: Estudo transversal conduzido no estado de Rondônia com profissionais e estudantes envolvidos na PAS que estiveram em um evento estadual nos dias 24 a 26 de agosto de 2022. A coleta de dados foi realizada por meio de formulário eletrônico em link e os dados foram analisados por meio do pacote estatístico Stata versão16. Este estudo faz parte do projeto matriz intitulado: Boas Práticas na Assistência ao Paciente, no Controle da Infecção e no Processamento de Produtos para a Saúde no Estado de Rondônia autorizado através do parecer número 3.771.377. RESULTADOS E DISCUSSÃO: Do total de 160 participantes do evento, 90 indivíduos aceitaram participar deste estudo. Entre os achados, constatou-se 80,7% de conhecimento geral sobre o tema, sendo que mais de 75% dos participantes apresentaram conhecimento sobre os conceitos de segurança do paciente e quase 68% avaliaram corretamente as situações de segurança do paciente na APS. O menor nível de conhecimento foi referente às situações específicas. CONCLUSÃO: Esse estudo contribui para um diagnóstico situacional do conhecimento dos trabalhadores da APS que participam do projeto de PAS no estado de Rondônia, podendo ser utilizado para o planejamento de outras ações educativas com a temática de segurança do paciente neste estado e em outros com o mesmo perfil dos trabalhadores e organização dos serviços de saúde.

INTRODUCTION: The main objective of the Patient Safety program is to reduce the risk of avoidable harm associated with health care to an acceptable minimum level. Aiming to qualify this care in Primary Health Care (PHC), the National Council of Health Secretaries has developed the Health Care Plan (PAS) with the support of institutions of excellence and health secretariats. OBJECTIVE: To identify the knowledge of Primary Health Care professionals involved in planning about patient safety in Rondônia. METHOD: Cross-sectional study conducted in the state of Rondônia with professionals and students involved in the PAS who attended a state event from August 24 to 26, 2022. Data collection was performed using an electronic form in the link and the data were analyzed using the statistical package Stata version16. This study is part of the matrix project entitled: Good Practices in Patient Care, Infection Control and Processing of Health Products in the State of Rondônia authorized through opinion number 3,771,377. RESULTS AND DISCUSSION: Of the total of 160 participants in the event, 90 individuals agreed to participate in this study. Among the findings, there was 80.7% of general knowledge on the subject, with more than 75% of the participants having knowledge about patient safety concepts and almost 68% correctly assessing patient safety situations in PHC. The lowest level of knowledge was related to specific situations. CONCLUSION: This study contributes to a situational diagnosis of the knowledge of PHC workers who participate in the PAS project in the state of Rondônia, and can be used for the planning of other educational activities with the subject of patient safety in this state and in others with the aim of same profile of workers and organization of health services.

INTRODUCCIÓN: El objetivo principal del programa de Seguridad del Paciente es reducir el riesgo de daño evitable asociado con la atención médica a un nivel mínimo aceptable. Con el objetivo de calificar este cuidado en la Atención Primaria de Salud (APS), el Consejo Nacional de Secretarías de Salud ha desarrollado el Plan de Atención a la Salud (PAS) con el apoyo de instituciones de excelencia y secretarías de salud. OBJETIVO: Identificar el conocimiento de los profesionales de la Atención Primaria de Salud involucrados en la planificación sobre la seguridad del paciente en Rondônia. MÉTODO: Estudio transversal realizado en el estado de Rondônia con profesionales y estudiantes involucrados en el PAS que asistieron a un evento estatal del 24 al 26 de agosto de 2022. La recolección de datos se realizó mediante un formulario electrónico en el enlace y los datos fueron analizados. utilizando el paquete estadístico Stata versión16. Este estudio forma parte del proyecto matriz titulado: Buenas Prácticas en Atención al Paciente, Control de Infecciones y Procesamiento de Productos de Salud en el Estado de Rondônia autorizado por dictamen número 3.771.377. RESULTADOS Y DISCUSIÓN: Del total de 160 participantes en el evento, 90 personas aceptaron participar en este estudio. Entre los hallazgos, se encontró un 80,7% de conocimientos generales sobre el tema, siendo más del 75% de los participantes con conocimientos sobre conceptos de seguridad del paciente y casi el 68% valorando correctamente situaciones de seguridad del paciente en la APS. El nivel más bajo de conocimiento estaba relacionado con situaciones específicas. CONCLUSIÓN: Este estudio contribuye para un diagnóstico situacional del conocimiento de los trabajadores de la APS que participan del proyecto PAS en el estado de Rondônia, y puede ser utilizado para la planificación de otras actividades educativas con el tema de seguridad del paciente en este estado y en otros con el objetivo de igualar el perfil de los trabajadores y la organización de los servicios de salud.

Sperm Phosphoproteome: Unraveling Male Infertility.

Infertility affects approximately 15% of couples worldwide of childbearing age, and in many cases the etiology of male infertility is unknown. The current standard evaluation of semen is insufficient to establish an accurate diagnosis. Proteomics techniques, such as phosphoproteomics, applied in this field are a powerful tool to understand the mechanisms that regulate sperm functions such as motility, which is essential for successful fertilization. Among the post-translational modifications of sperm proteins, this review summarizes, from a proteomic perspective, the updated knowledge of protein phosphorylation, in human spermatozoa, as a relevant molecular mechanism involved in the regulation of sperm physiology. Specifically, the role of sperm protein phosphorylation in motility and, consequently, in sperm quality is highlighted. Additionally, through the analysis of published comparative phosphoproteomic studies, some candidate human sperm phosphoproteins associated with low sperm motility are proposed. Despite the remarkable advances in phosphoproteomics technologies, the relatively low number of studies performed in human spermatozoa suggests that phosphoproteomics has not been applied to its full potential in studying male infertility yet. Therefore, further studies will improve the application of this procedure and overcome the limitations, increasing the understanding of regulatory mechanisms underlying protein phosphorylation in sperm motility and, consequently, in male fertility.

Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro.

In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), ß-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14+/PI-), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA+/PI-) and reactive oxygen species (ROS) production (CellRox+/PI-), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation.

Patient Satisfaction Using a Home-Based Rehabilitation Protocol for the Non-Surgical Treatment of Proximal Humeral Fractures: A Prospective Longitudinal Cohort Study.

Supervised, center-based, daily physiotherapy presents limitations: transport, need for an accompanying person, or risk of infection. Home-based rehabilitation protocols (HBRP) can be effective alternatives. We use a HBRP for the non-surgically treated proximal humeral fractures (PHF) in older patients. OBJECTIVES: To assess patient satisfaction and preferences of using a booklet, videos, or an app to guide physiotherapy. PATIENTS AND METHODS: Prospective, single-center observational study of patients ≥55 years old who sustained a non-surgically treated PHF. The HBRP consisted of immediate mobilization, followed by 5 physiotherapist-guided, weekly sessions of rehabilitation and standard physiotherapy after 3 months, if needed. A booklet with images, videos, or a smartphone application were offered to guide the patients. RESULTS: Mean degree of satisfaction (1-5) was 4.66 ± .9: 84 patients (82.4%) were very satisfied, 11 patients (10.8%) were satisfied, and 5 patients (4.9%) were not satisfied at all. Mean Oxford Shoulder Score achieved was 40.5 ± 6.6. 59.8% patients preferred the booklet and 29.4% the videos. Exercise compliance was considered very high in 87.3% of patients, while 4% hardly never followed the HBRP. Only 17.7% patients needed center-based physiotherapy after the HBRP. DISCUSSION: Reasons for satisfaction were good final functional outcome, no need for transportation, being away from hospital, immediate rehabilitation availability and being capable of maintaining independence. Adherence is a major concern. Videos are more didactic explaining the exercises. CONCLUSION: If standard physiotherapy is not available, the HBRP can be a valid treatment option for PHF management in older patients, with a high degree of patient satisfaction. Older patients preferred the booklet to guide physiotherapy.

The Proteome of Equine Oviductal Fluid Varies Before and After Ovulation: A Comparative Study.

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro. Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle (n = 4) or a recent ovulation (n = 4); the samples were kept at -80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus. Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm-oviduct interaction, fertilization, and metabolism, among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses.

Impaired mammalian sperm function and lower phosphorylation signaling caused by the herbicide Roundup® Ultra Plus are due to its surfactant component.

The use of worldwide glyphosate-based herbicide Roundup® is growing and to date its effects on mammalian spermatozoa are controversial. This study aims to investigate the functional impact of in vitro exposure of pig spermatozoa to low concentrations of Roundup® Ultra Plus (RUP), similar to those present as environment contaminants, to its active ingredient glyphosate, and to the non-active component, surfactant polyoxyethyleneamine (POEA). Pig spermatozoa were incubated in Tyrode's basal medium (TBM) or Tyrode's complete medium (TCM) (1 h at 38.5 °C) with several RUP dilutions or equivalent concentrations of glyphosate or POEA. RUP treatment causes a significant dilution-dependent decrease in sperm motility, a significant increase in plasma membrane disorganization and reduction in GSK3ß phosphorylation (TBM) and in two PKA substrates (TBM and TCM), whereas does not affect sperm viability or mitochondrial membrane potential (MMP). Equivalent glyphosate concentrations do not affect any functional sperm parameters. However, POEA concentrations equivalent to RUP dilutions mimic all RUP sperm effects: decrease sperm motility in a concentration-dependent manner, increase sperm plasma membrane lipid disorder and significantly inhibit GSK3ß phosphorylation (TBM) and two PKA substrates without affecting sperm viability or MMP. In summary, low concentrations RUP herbicide cause sperm motility impairment without affecting sperm viability. This adverse effect could be likely due to a detrimental effect in the plasma membrane lipid organization and to inhibition of phosphorylation of both, GSK3ß and specific PKA substrates. Importantly, our results indicate that negative effects of low RUP concentrations in pig spermatozoa function are likely caused by the surfactant included in its formulation and no by its active ingredient glyphosate.

Boar spermatozoa proteomic profile varies in sperm collected during the summer and winter.

Boar sperm quality is less during the summer as a result of the different photoperiod or ambient temperatures as compared with the winter. The present study was conducted to elucidate possible variations in proteomic profiles of boar spermatozoa collected during the summer and winter. Effects of season on sperm viability, total motility, progressive motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were also analyzed. Only sperm viability and mitochondrial membrane potential were less during the summer (P <  0.05). Spermatozoa were processed and evaluated using the nano LC-MS/MS QTof procedures. A total of 1028 characterized proteins were identified in sperm collected during both seasons of the year (False Discovery Rate < 0.01) and, among the total, 85 proteins differed in sperm collected in the winter and summer, with there being a lesser abundance of these proteins when there were ejaculate collections during the summer (q-value ≤ 0.05). The results from enrichment assessments for these protein networks utilizing UniProtKB procedures for determining reproductive processes indicates there were 23 proteins that were less abundant in the summer than winter. These proteins have essential functions in spermatogenesis, sperm motility, acrosome reaction and fertilization. These results are the first where there was ascertaining of proteomic differences in boar spermatozoa collected in the summer and winter. These results might help to explain the decreased sperm quality and prolificity when semen of boars is used for artificial insemination that is collected during the season of the year when ambient temperatures are relatively greater.

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the major metabolites present in equine preovulatory follicular fluid (FF) obtained from postmortem mares using proton nuclear magnetic resonance spectroscopy (1H-NMR). The results were contrasted against the composition of the most commonly used media for equine oocyte IVM: tissue culture medium 199 (TCM-199) and Dulbecco's modified eagle medium/nutrient mixture F-12 Ham (DMEM/F-12). Twenty-two metabolites were identified in equine FF; among these, nine of them are not included in the composition of DMEM/F-12 or TCM-199 media, including (mean ± SEM): acetylcarnitine (0.37 ± 0.2 mM), carnitine (0.09 ± 0.01 mM), citrate (0.4 ± 0.04 mM), creatine (0.36 ± 0.14 mM), creatine phosphate (0.36 ± 0.05 mM), fumarate (0.05 ± 0.007 mM), glucose-1-phosphate (6.9 ± 0.4 mM), histamine (0.25 ± 0.01 mM), or lactate (27.3 ± 2.2 mM). Besides, the mean concentration of core metabolites such as glucose varied (4.3 mM in FF vs. 5.55 mM in TCM-199 vs. 17.5 mM in DMEM/F-12). Hence, our data suggest that the currently used media for equine oocyte IVM can be further improved.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

Stage-specific metabolomic changes in equine oviductal fluid: New insights into the equine fertilization environment.

A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (1H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF samples were lactate, myoinositol, creatine, alanine and carnitine. Only fumarate and glycine showed significant differences in their concentrations between PRE and PST OF samples, with higher concentrations in the PST samples. In a preliminary study, stallion spermatozoa (n = 3 ejaculates) were incubated with different concentrations of PST OF from one mare (0, 0.0625, 0.125, 0.25, 0.5 or 1%; v:v). After 4 h of sperm incubation, protein tyrosine phosphorylation (PY) by western blotting, sperm motility, and acrosomal status were evaluated. An increase of PY was observed in sperm from two stallions when treated with 0.0625% and 0.125% of OF; however no change in PY was noted in the other stallion. There were no effects of OF on spermatozoa motility or acrosome status. These results provide the first information on the metabolomics of equine OF at different stages of the estrus cycle, and present the possibility that OF may affect PY in stallion spermatozoa.

Effect of boar semen supplementation with recombinant heat shock proteins during summer.

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 µg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.

Antioxidants and Male Fertility: from Molecular Studies to Clinical Evidence.

Spermatozoa are physiologically exposed to reactive oxygen species (ROS) that play a pivotal role on several sperm functions through activation of different intracellular mechanisms involved in physiological functions such as sperm capacitation associated-events. However, ROS overproduction depletes sperm antioxidant system, which leads to a condition of oxidative stress (OS). Subfertile and infertile men are known to present higher amount of ROS in the reproductive tract which causes sperm DNA damage and results in lower fertility and pregnancy rates. Thus, there is a growing number of couples seeking fertility treatment and assisted reproductive technologies (ART) due to OS-related problems in the male partner. Interestingly, although ART can be successfully used, it is also related with an increase in ROS production. This has led to a debate if antioxidants should be proposed as part of a fertility treatment in an attempt to decrease non-physiological elevated levels of ROS. However, the rationale behind oral antioxidants intake and positive effects on male reproduction outcome is only supported by few studies. In addition, it is unclear whether negative effects may arise from oral antioxidants intake. Although there are some contrasting reports, oral consumption of compounds with antioxidant activity appears to improve sperm parameters, such as motility and concentration, and decrease DNA damage, but there is not sufficient evidence that fertility rates and live birth really improve after antioxidants intake. Moreover, it depends on the type of antioxidants, treatment duration, and even the diagnostics of the man's fertility, among other factors. Literature also suggests that the main advantage of antioxidant therapy is to extend sperm preservation to be used during ART. Herein, we discuss ROS production and its relevance in male fertility and antioxidant therapy with focus on molecular mechanisms and clinical evidence.

Supplementation of freezing/thawing media with GSK3 inhibitor alsterpaullone does not bypass the harmful effect of cryopreservation on boar spermatozoa.

Frozen-thawed boar sperm have less motility and fertility capacity in comparison to fresh sperm. Glycogen Synthase Kinase 3 (GSK3) contributes to sperm motility in fresh semen. In addition, GSK3 inhibition in boar spermatozoa in fresh semen improves motility variables. The role of GSK3 on boar cryopreserved sperm, however, is still unknown. The hypothesis in the present study was that GSK3 pathway inhibition by alsterpaullone (AST) could result in enhancement of the quality of sperm afer cryopreservation. Two different strategies were evaluated: i) AST supplementation to the freezing medium (AST + Cryo); ii) AST supplementation after sperm thawing (AST + Thaw). Sperm motility was evaluated using the CASA system and different sperm quality variables were evaluated using flow cytometry, as well as amount of GSK3 phosphorylation of thawed spermatozoa after 30 and 90 min incubation at 38.5 °C. Results indicate that AST supplementation had detrimental effects on sperm viability (live spermatozoa) and mitochondrial membrane potential when it was added after thawing (P < 0.05) The AST supplementation after thawing, however, had a protective effect on plasma membrane lipid disorganization (P < 0.05). The percentage of motile spermatozoa was not modified by AST supplementation. Nonetheless, after 30 min post-thawing, STR and LIN variables (related to straightness of the movement) as well as the percentage of rapid lineal spermatozoa were increased with both AST supplementation protocols. The GSK3α phosphorylation was not modified through the incubation time in boar thawed sperm. In summary, results do not support the idea of adding AST to the cryopreservation/thawing medium to improve boar sperm quality after cryopreservation.

Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions.

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 µM CZ, 200 µM W-7, or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.