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Pulmonary arterial hypertension (PAH) is a disease that affects millions of people worldwide. Besides the effects on the lungs and heart, PAH can affect other organs, including the liver, kidneys, brain, glands, and testis. This study aimed to evaluate the impact of PAH and physical resistance training (RT), a complementary treatment for hypertension, on epididymis morphology and function and sperm parameters. Wistar rats were divided into four experimental groups (n = 8/ group): sedentary control, sedentary PAH, RT control, and RT + PAH. PAH was induced using monocrotaline injections on Day 1 and 7 of the experiment. Sixteen rats from RT groups underwent RT training for 30 days, while rats from sedentary groups did not exercise. The epididymis was processed and analyzed using microscopic, biochemical, and functional approaches. Sperm were harvested from the cauda epididymis and evaluated for morphology and motility. Our results showed that PAH compromised the epididymis antioxidant defense system and reduced NO levels, leading to an imbalance in the organ's mineral content. These alterations affected the epididymis morphology and reduced the sperm transit time in the proximal epididymis, resulting in an increase in abnormal sperm morphology in the cauda region. Unfortunately, RT was not a good therapy against the PAH effect on the epididymis. PAH negatively affected epididymis functions with consequences to male gametes. Dysfunctions in the post-testicular environment may lead to male infertility due to the disturbance of spermatozoa fecundity.
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Epidídimo , Condicionamiento Físico Animal , Ratas Wistar , Espermatozoides , Animales , Masculino , Epidídimo/metabolismo , Epidídimo/patología , Ratas , Condicionamiento Físico Animal/fisiología , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Motilidad Espermática/fisiología , Conducta Sedentaria , Estrés Oxidativo/fisiologíaRESUMEN
Background: Vitamin D (vitD) deficiency could affect male reproductive function. Our objective was to investigate the relationship between serum vitD concentrations and hormonal and seminal parameters in infertile patients and to compare the results with those in healthy controls. Materials and methods: Infertile patients (n = 29) and normozoospermic healthy donors (n = 27) were recruited for the study. Serum concentrations of vitD, total testosterone, estradiol, and sex hormone-binding globulin were determined using chemiluminescence assays, and free testosterone concentration was determined by radioimmunoassay. Semen analysis was performed as suggested by the World Health Organization. Statistical analysis was conducted using Student's t test, contingency tables, and linear regression studies. Results: VitD concentrations were lower in patients than in controls (p < 0.001). A significant association (p < 0.001) was observed between vitD concentrations <20ng/mL and infertility. In the control group, significant correlations were reported between vitD concentrations >30 ng/mL and the concentrations of testosterone (p < 0.05), free testosterone (p < 0.01), and estradiol (p < 0.05). A direct correlation was found between vitD concentration and percentage of sperm vitality (p = 0.01). VitD also positively correlated with the percentage of progressive sperm motility (p <0.05) and sex hormone-binding globulin concentrations (p < 0.01). Conclusions: VitD may affect male reproductive parameters, and its deficiency could be associated with infertility.
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PURPOSE: Ferroptosis is a type of iron-dependent regulated cell death characterized by increased bioavailability of redox-active iron, loss of GPX4 antioxidant capacity, and oxidation of polyunsaturated fatty acid-containing phospholipids mediated by reactive oxygen species (ROS). The aim of this study was to evaluate the effect of oxidative stress induced by arachidonic acid (AA) on ferroptotic cell death in human spermatozoa. MATERIALS AND METHODS: Spermatozoa from normozoospermic donors were exposed to AA (5, 25, and 50 µM) for 1 hour at 37 â, including an untreated control. Oxidative stress was confirmed by evaluation of cytosolic and mitochondrial ROS production, viability, mitochondrial membrane potential (ΔΨm) and motility. Subsequently, molecular markers of ferroptosis including iron content, levels of GPX4, SLC7A11, ACSL4, IREB2 and lipid peroxidation were evaluated. The analyses were carried out using either flow cytometry, a microplate reader or confocal laser microscopy. RESULTS: AA-induced oxidative stress showed increased cytosolic and mitochondrial ROS production accompanied by impairedΔΨm, viability and motility in human spermatozoa. These results were associated with biochemical and molecular markers related to ferroptotic cell death including an increase in iron content in the form of ferrous (Fe2+) ions, SLC7A11, ACSL4, IREB2, a decrease in the level of GPX4, and an increase in the level of lipid peroxidation compared to the untreated control. CONCLUSIONS: This study revealed that AA-induced oxidative stress induces cell death with biochemical characteristics of ferroptosis in human spermatozoa, demonstrating another mechanism of alteration of sperm function induced by oxidative stress and could establish new therapeutic objectives to prevent the decrease in sperm quality mediated by oxidative stress.
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ABSTRACT Purpose Varicocele is a condition known to cause damage to seminal parameters and sperm function. Furthermore, it has been hypothesized that the varicocele effect on fertility is time-dependent; however, little is known about the consequences of its establishment time on reproductive organs and/or sperm function. This study aimed to evaluate the effect of the duration of experimental varicocele on reproductive organs, sperm parameters, and sperm function. Materials and Methods Varicocele induction surgeries were performed in Wistar rats aged 40 or 100 days old. At 160-day-old, analyses were performed, including biometry of reproductive organs (prostate, seminal vesicles, epididymis, and testis), sperm parameters (vitality, morphology, and motility), and sperm function tests (nuclear DNA integrity, acrosome integrity, and mitochondrial activity). Results The analysis of the biometry of reproductive organs showed no differences between distinct ages in which varicocele was induced. The total abnormal sperm morphology was bigger in animals with varicocele induced to 100 days old than in animals with varicocele induced to 40 days old. Regarding nuclear DNA integrity, animals of varicocele induced to 100 days old showed worse results compared to animals of varicocele induced to 40 days old. Other parameters analyzed showed no differences between varicocele groups. Conclusion In this study conducted on rats, we conclude that varicocele adversely affects sperm, particularly its function. However, we did not observe a negative progressive effect on sperm.
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Background: Assisted reproduction techniques in birds have contributed to many species' conservation and sustainable use. One of these techniques is semen cryopreservation, which is possible following the discovery of suitable cryoprotectants. Aims: This study aimed to characterize the fresh and post-thaw ejaculates of different species of birds of prey. Methods: The following species were included in the study: red-tailed hawk (Buteo jamaicensis) n=3, golden eagle (Aquila chrysaetos) n=3, and Harris's hawk (Parabuteo unicinctus) n=3. Twenty-five ejaculates were obtained for each species. The percentage of spermatozoa motility, viability, and morphology were evaluated. Results: Evident differences were observed among the ejaculates of the three species, particularly in sperm length and between the fresh and post-thaw parameters of the same species in which the motility reduced to approximately 40% after thawing. It was demonstrated that sperm cryopreservation of the studied species was possible using the same freezing protocol. Conclusion: This study showed that sperm characteristics could influence the parameters obtained during their in vitro conservation, both in the fresh and post-thaw states.
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Sperm quality is essential to guarantee the success of assisted reproduction. However, selecting high-quality sperm and maintaining it during (cryo)preservation for high efficiency remains challenging in livestock reproduction. A comprehensive understanding of sperm biology allows for better assessment of sperm quality, which could replace conventional sperm analyses used today to predict fertility with low accuracy. Omics approaches have revealed numerous biomarkers associated with various sperm phenotypic traits such as quality, survival during storage, freezability, and fertility. At the same time, nanotechnology is emerging as a new biotechnology with high potential for use in preparing sperm intended to improve reproduction in livestock. The unique physicochemical properties of nanoparticles make them exciting tools for targeting (e.g., sperm damage and sexing) and non-targeting bioapplications. Recent advances in sperm biology have led to the discovery of numerous biomarkers, making it possible to target specific subpopulations of spermatozoa within the ejaculate. In this review, we explore potential biomarkers associated with sperm phenotypes and highlight the benefits of combining these biomarkers with nanoparticles to further improve sperm preparation and technology.
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Sperm morphology is considered the best indicator of male fertility. In Neotropical bats, important aspects of sperm morphology have been scantly studied. The aim of the present study was to characterize and compare the sperm morphology and morphometry of Artibeus planirostris and Sturnira erythromos. A total of 11 specimens were analyzed from the Colección de Mamíferos Lillo: five A. planirostris and six S. erythromos. The fixed epididymis were extracted and macerated in Farmer's solution, followed by the routine cytological procedure with different stains. To carry out the description and morphometric analysis, microphotographs were taken under an optical, epifluorescence and scanning electron microscope. A total of 50 sperm from each individual were measured for morphometric analysis. The percentage of normal/abnormal spermatozoa was estimated and the sperm abnormalities were classified. Both species showed morphologically simple spermatozoa with a spatulate head, a short neck, a helical midpiece and a tail that tapers at the final end, similar to other species of Phyllostomidae. The differences observed were: apex of the head was conical in A. planirostris and was oval in S. erythromos; longer head and midpiece in S. erythomos and longer sperm in A. planirostris. Both species showed a high percentage of sperm with normal appearance: 65% for A. planirostris and 72% for S. erythromos. The main sperm abnormalities were: scattered tails and heads, coiled tails, folded midpieces and presence of cytoplasmic droplets. The present work will improve the understanding of their reproductive biology. RESEARCH HIGHLIGHTS: Morphological descriptions and morphometric analyses of the sperm of Artibeus planirostris and Sturnira erythromos were carried out with optical, epifluorescence and scanning electron microscopy.
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BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.
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Reacción Acrosómica , Acrosoma , Calcio , Plomo , Motilidad Espermática , Espermatozoides , Masculino , Espermatozoides/efectos de los fármacos , Calcio/metabolismo , Motilidad Espermática/efectos de los fármacos , Animales , Acrosoma/efectos de los fármacos , Plomo/toxicidad , Reacción Acrosómica/efectos de los fármacos , AMP Cíclico/metabolismo , Bovinos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Análisis de Semen , Daño del ADN/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Compuestos Organometálicos/farmacologíaRESUMEN
Bovine spermatozoa are highly susceptible to oxidative stress (OS), and it is known to affect their cellular functions. The main leukocyte producers of reactive oxygen species (ROS) in mammalian semen are polymorphonuclear neutrophils (PMN). PMN activation can result in the formation of neutrophil extracellular traps (NETs), which have been shown to affect the motility and function of spermatozoa. However, OS effects on bull spermatozoa derived from individual NETs components have not been investigated. The hypothesis of this study was that specific NETs components might generate OS on bull spermatozoa. Bovine sperm cells were incubated with five NETs-associated molecules, including 30 µg/mL histone 2A (H2A), neutrophil elastase (NE), 1 µg/mL myeloperoxidase (MPO), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), for a time course ranging from 15 to 240 min. Fluorescence microscopy was used to evaluate the coincubation of bovine PMN and sperm cells. Within 15 min, H2A, NE, and LL-37 caused membrane disruption, while MPO and Cat-G caused OS on bull spermatozoa after 1 h of coincubation. NET formation was observed within 15 min of coincubation in co-cultures of bovine PMN/sperm cells. This study is the first to report on the role of cytotoxic OS effects caused by NETs-derived components in bovine sperm in vitro.
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The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.
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Caseínas , Criopreservación , Inseminación Artificial , Análisis de Semen , Preservación de Semen , Motilidad Espermática , Animales , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Masculino , Femenino , Criopreservación/veterinaria , Criopreservación/métodos , Inseminación Artificial/veterinaria , Caseínas/farmacología , Análisis de Semen/veterinaria , Embarazo , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Crioprotectores/farmacología , Semen/efectos de los fármacos , Fertilidad/efectos de los fármacos , Ovinos , Oveja DomésticaRESUMEN
The evolving prevalence of anabolic androgenic steroids (AAS) abuse among nonathletes is alarming because of the known harm to an individual's health. Among the adverse effects of AAS abuse, male infertility and sexual dysfunction have been often reported in the literature, but little is known regarding its actual prevalence, possible underpinning mechanisms, and potential treatments either during or post-AAS usage. Thus, the current narrative review summarizes the state-of-art regarding the effects of AAS on male fertility and sexual function. Evidence was gathered from the latest reviews and recent original studies, specifically from prospective cohorts and clinical trials, ultimately resulting in five main topics of discussion. First, AAS usage is briefly characterized by its historical background, main physiological mechanisms, and the most frequently used AAS substances. Second, data on the prevalence of AAS-induced male infertility and sexual dysfunction are described. Third, some new insights on possible underpinning mechanisms of AAS-induced male infertility and sexual dysfunction are thoroughly discussed, with particular attention to histological data derived from animal models and the latest insights from prospective cohorts in humans. Fourth, the potential treatments during and after the AAS usage are presented, highlighting the odds of resolving male infertility and sexual dysfunction. Fifth, future directions on this topic are discussed, focusing on the methodological robustness of scientific studies.
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Objective: Seminal cryopreservation causes significant damage to the sperm; therefore, different methods of cryopreservation have been studied. The aim of the study was to compare the effects of density gradient processing and washing/centrifugation with seminal plasma removal for cryopreservation in semen parameters. Methods: Seminal samples of 26 normozoospermic patients were divided into 3 parts: with seminal plasma; after washing/centrifugation; and after selection through density gradient. The samples were cryopreserved for at least two weeks. Motility, sperm count, morphology and viability were evaluated before cryopreservation and after thawing. Results: Density gradient processing selected motile and viable sperm with normal morphology in fresh samples (p<0.05). Cryopreservation negatively affected all sperm parameters regardless of the processing performed, and even if the sperm recovery was lower in the density gradient after the thawing, progressive motility, total motility, viability and morphology remained higher (p<0.05). Conclusion: Cryopreservation significantly compromises sperm parameters (motility, morphology, viability). In normozoospermic patients, the density gradients select better quality spermatozoa compared to other processing methods; this benefit was kept after thawing.
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Criopreservación , Preservación de Semen , Adulto , Humanos , Masculino , Criopreservación/métodos , Semen , Análisis de Semen , Preservación de Semen/métodos , Motilidad Espermática , Factores de TiempoRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.
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PURPOSE: Varicocele is a condition known to cause damage to seminal parameters and sperm function. Furthermore, it has been hypothesized that the varicocele effect on fertility is time-dependent; however, little is known about the consequences of its establishment time on reproductive organs and/or sperm function. This study aimed to evaluate the effect of the duration of experimental varicocele on reproductive organs, sperm parameters, and sperm function. MATERIALS AND METHODS: Varicocele induction surgeries were performed in Wistar rats aged 40 or 100 days old. At 160-day-old, analyses were performed, including biometry of reproductive organs (prostate, seminal vesicles, epididymis, and testis), sperm parameters (vitality, morphology, and motility), and sperm function tests (nuclear DNA integrity, acrosome integrity, and mitochondrial activity). RESULTS: The analysis of the biometry of reproductive organs showed no differences between distinct ages in which varicocele was induced. The total abnormal sperm morphology was bigger in animals with varicocele induced to 100 days old than in animals with varicocele induced to 40 days old. Regarding nuclear DNA integrity, animals of varicocele induced to 100 days old showed worse results compared to animals of varicocele induced to 40 days old. Other parameters analyzed showed no differences between varicocele groups. CONCLUSION: In this study conducted on rats, we conclude that varicocele adversely affects sperm, particularly its function. However, we did not observe a negative progressive effect on sperm.
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Ratas Wistar , Análisis de Semen , Motilidad Espermática , Espermatozoides , Varicocele , Animales , Masculino , Varicocele/fisiopatología , Varicocele/patología , Espermatozoides/fisiología , Motilidad Espermática/fisiología , Factores de Tiempo , Modelos Animales de Enfermedad , Testículo/patología , Ratas , Factores de Edad , Epidídimo/patologíaRESUMEN
Resumen En el presente estudio se describe la histología del talón y del saco glandular anexo (SGA) del sistema reproductor de cinco especies de Megalobulimus (Gastropoda, Stylommatophora, Strophocheilidae). Para ello, se utilizó la técnica de coloración de Hematoxilina-Eosina. Se encontró que el talón es una estructura muscular compacta que alberga la bolsa de fertilización, una estructura tubular revestida por un epitelio cilíndrico ciliado replegado, en cuyo extremo proximal se conecta el conducto hermafrodita. Este conducto termina en la cámara de fertilización, desde donde parten los túbulos de la espermateca, los cuales se ramifican hasta su extremo ciego y almacenan espermatozoides exógenos. En los túbulos se observaron espermatozoides, en su mayoría con las cabezas fijadas en las células epiteliales ciliadas y las colas libres en el lumen. La pared del SGA presenta una capa externa de músculo liso y, recubriendo el lumen, un epitelio cilíndrico glandular no ciliado con distintos grados de plegamiento y células mucosas subepiteliales. Las secreciones de estas células desembocan en el conducto colector cerca de la base del talón. Lejos de la glándula de la albúmina se observó una sustancia similar junto con espermatozoides exógenos en los túbulos de la espermateca. Se identificaron diferencias entre las especies, destacándose como característica filogenéticamente relevante la diferenciación del talón, la cual fue más pronunciada en M. capillaceus en comparación con las otras cuatro especies estudiadas (M. popelairianus, M. carrikeri, M. leucostoma y M. maximus).
Abstract In this study, the histology of the talon and the annex glandular sac (AGS) of the reproductive system from five species of Megalobulimus (Gastropoda, Stylommatophora, Strophocheilidae) is described. Hematoxylin-Eosin staining technique was used. It was found that the talon is a compact muscular structure housing the fertilization pouch, a tubular structure lined by folded ciliated cylindrical epithelium, to whose proximal end the hermaphroditic duct connects. This duct terminates in the fertilization chamber, from which the spermathecal tubules originate. These tubules branch out to their blind ends and store exogenous sperm. In the tubules, spermatozoa were observed, mostly with their heads attached to the ciliated epithelial cells and their tails free in the lumen. The wall of the AGS has an outer layer of smooth muscle and, lining the lumen, a non-ciliated glandular columnar epithelium with various degrees of folding and subepithelial mucous cells. The secretions from these cells flow into the collecting duct near the base of the talon. Far from the albumen gland, a similar substance was observed along with exogenous sperm in the spermathecal tubules. Differences between species were identified, with the most significant and phylogenetically relevant feature being the differentiation of the talon, which was more pronounced in M. capillaceus compared to the other four species studied (M. popelairianus, M. carrikeri, M. leucostoma, and M. maximus).
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BACKGROUND: Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. OBJECTIVE: To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction-infected men up to 3 months after hospital discharge. MATERIALS AND METHODS: This cross-sectional study included 13 confirmed moderate-to-severe COVID-19 patients enrolled 30-90 days after the diagnosis. Semen samples were obtained and examined with real-time polymerase chain reaction for RNA detection and by transmission electron microscopy. RESULTS: In moderate-to-severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA-based extracellular traps were reported in all studied specimens. DISCUSSION AND CONCLUSION: Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA-based extracellular traps, probably in a cell-free DNA-dependent manner, similar to those previously described in the systemic inflammatory response to COVID-19. In moderate-to-severe cases, the blood-testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post-testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single-stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell-free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA-based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa.
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l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through ß-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1-10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.
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Carnitina , Membrana Celular , Criopreservación , Crioprotectores , Preservación de Semen , Motilidad Espermática , Espermatozoides , Animales , Masculino , Caballos , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Criopreservación/métodos , Criopreservación/veterinaria , Espermatozoides/efectos de los fármacos , Carnitina/farmacología , Crioprotectores/farmacología , Motilidad Espermática/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Congelación , Fenómenos Biomecánicos/efectos de los fármacosRESUMEN
Objective: This review provides a comprehensive overview of the existing research on the seminal microbiome and its association with male infertility, while also highlighting areas that warrant further investigation. Methods: A narrative review was conducted, encompassing all relevant studies published between 1980-2023 on the male reproductive tract microbiome in humans. This review considered studies utilizing culture-based, polymerase chain reaction (PCR)-based, and next-generation sequencing (NGS)-based methodologies to analyze the microbiome. Data extraction encompassed sample types (semen or testicular tissue), study designs, participant characteristics, employed techniques, and critical findings. Results: We included 37 studies comprising 9,310 participants. Among these, 16 studies used culture-based methods, 16 utilized NGS, and five employed a combination of methods for microorganism identification. Notably, none of the studies assessed fungi or viruses. All NGS-based studies identified the presence of bacteria in all semen samples. Two notable characteristics of the seminal microbiome were observed: substantial variability in species composition among individuals and the formation of microbial communities with a dominant species. Studies examining the testicular microbiome revealed that the testicular compartment is not sterile. Interestingly, sexually active couples shared 56% of predominant genera, and among couples with positive cultures in both partners, 61% of them shared at least one genital pathogen. In couples with infertility of known causes, there was an overlap in bacterial composition between the seminal and vaginal microbiomes, featuring an increased prevalence of Staphylococcus and Streptococcus genera. Furthermore, the seminal microbiome had discernible effects on reproductive outcomes. However, bacteria in IVF culture media did not seem to impact pregnancy rates. Conclusion: Existing literature underscores that various genera of bacteria colonize the male reproductive tract. These organisms do not exist independently; instead, they play a pivotal role in regulating functions and maintaining hemostasis. Future research should prioritize longitudinal and prospective studies and investigations into the influence of infertility causes and commonly prescribed medication to enhance our understanding of the seminal microbiota's role in reproductive health.
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Infertilidad Masculina , Microbiota , Femenino , Embarazo , Humanos , Masculino , Semen , Estudios Prospectivos , Espermatozoides , Bacterias/genéticaRESUMEN
Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant.
RESUMEN
This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris-citric acid-based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris-citric acid-glucose plus 20% EY (TCG-EY) and tris-citric acid-fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing-thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing-thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.