Sperm Samples Research Articles

Tackling somatic DNA contamination in sperm epigenetic studies

IntroductionRecent interest in sperm epigenetics has stemmed from its implication in sperm DNA quality, sperm fertility, environmental toxicity, and transgenerational inheritance. Sperm epigenetic data may be significantly affected by somatic DNA contamination, resulting in misleading conclusions. However, detecting and dealing with somatic DNA contamination in semen samples can be a challenging task.MethodsIn the present study, we worked out a detailed and robust plan to deal with somatic cell DNA contamination in sperm epigenetic studies in order to draw error-free scientific conclusions. Apart from incorporating simple quality checks, such as microscopic examination and somatic cell lysis buffer (SCLB) treatment, we compared the Infinium Human Methylation 450K BeadChip data for sperm and blood samples to identify the CpG sites that were highly methylated in blood samples in comparison to sperm, but were unrelated to infertility.Results and discussionThe comparison of Infinium Human Methylation 450K BeadChip data for sperm and blood samples identified 9564 CpG sites that can be used as markers for analyzing somatic DNA contamination. We have put together a comprehensive plan including evaluation under a microscope, SCLB treatment, inclusion of CpG biomarkers for sample quality evaluation, and applying a 15% cut off at the time of data analysis to completely eliminate the influence of somatic DNA contamination in sperm epigenetic studies. We conclude that if this comprehensive plan is followed, the influence of somatic DNA contamination in sperm epigenetic studies can be completely eliminated.

Cryopreserved sperm among patients with Oligoasthenoteratozoospermia.

This study investigates sperm utilization and disposal patterns in Oligoasthenoteratozoospermia (OAT) patients undergoing long-term sperm storage. OAT is a major contributor to male infertility. Cryopreservation is a common practice as a "fertility insurance" in case of further deterioration until azoospermia. However, long-term storage practices and utilization rates remain unclear. In this retrospective study spanning from October 1993 to December 2021, we examined men diagnosed with OAT who underwent spermatozoa cryopreservation. Data from medical records included utilization and disposal of sperm samples, age and paternity status at initial cryopreservation. We analyzed the data over 10years using Kaplan-Meier curves, compared age and paternity status with the log-rank test. Among 238 patients undergoing semen cryopreservation due to the indication of OAT, 45 utilized sperm for fertility treatments, with a third using all frozen straws and others using only a portion. Sixteen patients used sperm due to azoospermia. Additionally, 54 requested the disposal of all cryopreserved straws. A Kaplan-Meier analysis revealed that after 10years, 25.9% of men used cryopreserved sperm, while 31.2% opted for disposal. Most participants did not use or dispose of preserved sperm. After 10years, no significant differences were observed in usage or disposal based on age or paternity status. Our analysis of sperm utilization in OAT patients undergoing cryopreservation revealed a key finding: nearly half exhibited a "wait-and-see" approach. Unlike other indication for sperm preservation, age and paternity did not significantly influence utilization or disposal decisions. This suggests unique factors drive decision-making in OAT patients, highlighting the need for tailored cryopreservation management strategies.

Screening semen samples for Zika virus infection: role for serologic and RT-PCR testing

IntroductionZika virus (ZIKV) is primarily transmitted through mosquito bites though may act as a sexually transmitted infection. Men can shed the virus in their semen for extended periods, with reports indicating shedding for up to six months after the initial infection. This poses a concern for couples planning pregnancy, whether through natural or assisted methods due to the risk of congenital Zika disease. Human reproductive clinics typically perform serologic tests to screen for Zika infection.MethodsIn this study, we evaluated semen samples stored in a human reproduction clinic in Salvador, Brazil, during the Zika virus outbreak, using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique.Results36 sperm samples from 23 donors were analyzed, including 5 with positive IgG serology for ZIKV and all negative for IgM. Despite these serologic findings, all analyzed samples were negative by RT-qPCR ZIKV.DiscussionDespite the limited number of samples, this study raises further concern that the use of serology may be an unreliable surrogate method to predict the presence of ZIKV in semen.

Proteomics and metabolomics analyses of mechanism underlying bovine sperm cryoinjury

BackgroundThe cryoinjury of semen during cryopreservation reduces sperm motility, constraining the application of artificial insemination (AI) in bovine reproduction. Some fertility markers, related to sperm motility before and after freezing have been identified. However, little is known about the biological mechanism through which freezing reduces sperm motility. This study investigated the selective effects of cryoinjury on high-motility sperm (HMS) and low-motility sperm (LMS) in frozen-thawed from the perspectives of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP levels. The molecular mechanism of decreased sperm motility caused by cryoinjury was explored through a joint analysis of 4D-label free quantitative proteomics and non-targeted metabolomics.ResultsThe results indicate that low levels of ROS and high degrees of MMP and ATP play a critical role in the survival of HMS during the freezing process. The sperm samples from the frozen-thawed HMS and LMS were analysed for proteomics and metabolomics, 2,465 proteins and 4,135 metabolites were detected in bovine sperm samples. In contrast to LMS, HMS have 106 proteins and 106 metabolites with high abundance expression, and 79 proteins and 223 metabolites with low abundance expression. Proteomics and metabolomics data exhibit that highly expressed antioxidant enzymes and metabolites in HMS can maintain sperm motility by regulating the ROS produced during freezing to prevent sperm from oxidative stress and apoptosis. Furthermore, the KEGG analysis of differential proteins and metabolites during the freezing process implies that the significant enrichment of glycolysis and cAMP in HMS can guarantee energy supply.ConclusionsThe results provided that during the process of bovine sperm freezing, highly expressed antioxidant enzymes can regulate the reactive oxygen species levels to avoid oxidative stress and the glycolysis signalling pathway ensures ATP production can sustain frozen-thawed sperm motility.

Effect of hydro-ethanol extract of Caesalpinia pulcherrima (L.) Sw. leaves in human and rat: In vitro approach of male contraceptive development.

The study focused the contraceptive efficacy of hydro-ethanolic (60:40) extract (HEE) of Caesalpinia pulcherrima leaves in human and rat sperm samples by in vitro study. Six young fertile adult males were selected for semen collection. Sperm samples were collected from six adult rat also by chopping the epididymis along with the collection of testicles, epididymis, and liver. The semen, sperm, and tissue samples were grouped into control, 1, 2, and 4 mg HEE exposed categories. Sensitive spermiological sensors, androgenic key enzymes, oxidative stress, and metabolic toxicity markers were assessed according to standard protocols. Human semen samples, rat sperm samples and metabolic tissue samples were divided into 16 test tubes in all of the above groups to find out the direct effect of the extract on such sensors in concentration and duration dependent manner. Spermiological sensors both in human and rat were decreased significantly (p<0.05) in concentration and duration dependent manner after in vitro exposure of HEE against the control group. Testicular ∆5,3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, superoxide dismutase, and catalase activities were significantly (p<0.05) decreased, and level of the end product of lipid oxidation-peroxidation was elevated (p<0.05) in rat after extract charging. No general toxicity imposition of the said extract indicated by the activities of hepatic transaminases. Phytochemical screening was done by qualitative and liquid chromatography-mass spectrometry analysis. Extract focused the promising male contraceptive potentiality at 2 mg/ml concentration. Mode of action will be unfolded from running in vivo study very shortly.

Cryopreservation of pufferfish sperm in small and large volumes: the effect on kinetic parameters, and fertilization and hatching rates

AbstractCryopreservation of fish gametes has many potential applications for ecological, scientific and aquaculture purposes, and cryobank management has been established for many freshwater and marine fish. Nevertheless, there are no studies for the long-term conservation of pufferfish (Takifugu alboplumbeus) sperm, thus the objectives of this work were (i) to develop a new cryopreservation protocol for pufferfish sperm using different sperm:extender ratios and vials; and (ii) to assess the fertilization capacity of cryopreserved sperm. Results showed that cryopreserved sperm samples showed excellent post-thawing motility results when they were frozen in straws, reaching values over 60% with both 1:20 and 1:50 sperm:extender dilution ratios. Samples frozen in cryotubes showed slightly lower motility results (around 50%) than those obtained in the straws. Due to the excellent results obtained in the cryopreservation trials, in vitro fertilization trials were undertaken using different sperm:egg ratios with both fresh and cryopreserved sperm. High fertilization (FR) and hatching (HR) rates (over 90%) were reached using high 1:105 and 1:106 ratios. However, when the spermatozoa amount was limited in the fertilization micro-environment (using lower egg:sperm ratios of 1:103 and 1:104), the cryopreserved sperm generated lower values of FR and HR than the fresh sperm. These results suggest that for achieving high FR and HR, it is essential to use 10 times more cryopreserved sperm than fresh sperm. We then recommend using egg:sperm ratios of 1:104 and 105 (for fresh and cryopreserved sperm, respectively) for "Takifugu" species in order to optimize the amount of gametes collected during aquaculture procedures. This study has laid the basis for the establishment of cryopreservation protocols in pufferfish, that will be helpful for further reproduction in captivity programs and genetic cryobanking.

Genome-Wide microRNA Expression Profiling in Human Spermatozoa and Its Relation to Sperm Quality.

Sperm samples are separated into bad and good quality samples in function of their phenotype, but this does not indicate their genetic quality. Here, we used GeneChip miRNA arrays to analyze microRNA expression in ten semen samples selected based on high-magnification morphology (score 6 vs. score 0) to identify miRNAs linked to sperm phenotype. We found 86 upregulated and 21 downregulated miRNAs in good-quality sperm (score 6) compared with bad-quality sperm samples (score 0) (fold change > 2 and p-value < 0.05). MiR-34 (FC × 30, p = 8.43 × 10-8), miR-30 (FC × 12, p = 3.75 × 10-6), miR-122 (FC × 8, p = 0.0031), miR-20 (FC × 5.6, p = 0.0223), miR-182 (FC × 4.83, p = 0.0008) and miR-191 (FC × 4, p = 1.61 × 10-6) were among these upregulated miRNAs. In silico prediction algorithms predicted that miRNAs upregulated in good-quality sperm targeted 910 genes involved in key biological functions of spermatozoa, such as cell death and survival, cellular movement, molecular transport, response to stimuli, metabolism, and the regulation of oxidative stress. Genes deregulated in bad-quality sperm were involved in cell growth and proliferation. This study reveals that miRNA profiling may provide potential biomarkers of sperm quality.

RNA Sequencing Data Analysis of Oligoasthenoteratozoospermia Patients' Sperms and Effects of Curcumin on the Expression of Some Genes during Sperm Cryopreservation and Freeze-Thawing Process.

The cause of oligoasthenoteratozoospermia (OAT) is unclear. In this original study, we examined OAT patients' sperm using RNA-sequencing and studied the impact of curcumin on gene expression during sperm cryopreservation and freeze-thawing. RNA-seq was performed using the Galaxy Europe server in IKHC (Imam Khomeini Hospital Complex), Tehran, Iran in 2023. Sperm samples were collected from 30 OAT patients and 30 healthy volunteers. Sperm parameters were analyzed, and samples were frozen with 20 μM curcumin at -196 °C for one week. Thawed samples were assessed for sperm parameters, and the expression levels of bax, bak, bcl-2, bclw, casp9, apaf-1, SOD, cat, and GPX4 genes were measured using RT-PCR. RNA-seq analysis showed increased expression of NANOS1, HSPA6, and ALOXE3 genes, while BHLHE41, Hey1, and PPM1D genes were down-regulated in OAT patients' sperm. Curcumin (20 μM) effectively preserves sperm parameters and motility during cryopreservation in healthy subjects and in OAT patients in particular. Cryopreserved sperm from both OAT patients and healthy individuals exhibited reduced expression of pro-apoptotic genes, increased expression of anti-apoptotic genes, and elevated levels of SOD and GPX4 genes. Altered expressions of NANOS1, HSPA6, ALOXE3, BHLHE41, Hey1, and PPM1D genes likely contribute to OAT development. Additionally, curcumin protects sperm parameters during cryopreservation for both healthy individuals and OAT patients.

Optimizing in vitro fertilization in four Caribbean coral species.

Larval propagation and seeding of scleractinian corals for restoration is a rapidly expanding field, with demonstrated applications to assist the recovery of declining populations on reefs. The process typically involves collecting coral reproductive material, facilitating in vitro fertilization (IVF), and settling and outplanting the resulting coral offspring. Optimizing IVF can reduce gamete wastage and increase larval yields for propagation, therefore improving the efficiency of this intervention. In this study we tested three IVF conditions in four Caribbean broadcast-spawning coral species (i.e., Diploria labyrinthiformis, Colpophyllia natans, Pseudodiploria strigosa, Orbicella faveolata) to determine sperm concentration, gamete age, and co-incubation time resulting in the highest fertilization success. For each species, we exposed eggs from a single dam to pooled sperm samples from three sires (1) at concentrations ranging from zero to 109 cell mL-1, (2) after letting gametes age for 2 to 6 h, and (3) for a period of 15 to 120 min. These experiments revealed a gamete longevity of at least 4 h and clear minimum sperm concentration thresholds (>105 to 106 cell mL-1) in all four species. Fertilization took place much faster than expected (≤15 min) in the three brain corals under study, whereas O. faveolata gametes required a co-incubation period of 60 to 120 min to achieve maximum IVF success. We present these results in the context of IVF data available for other hermaphroditic broadcast-spawning scleractinians. We then provide recommendations for coral breeding practitioners to maximize larval production from gamete collections, and finally, we discuss our findings' potential implications on fertilization dynamics during natural coral spawning events.

Carvacrol attenuates varicocele-induced infertility in rats.

Carvacrol is a common ingredient in the pharmaceutical, cosmetic, and perfume industries. It possesses various pharmaceutical properties including pain relief, anti-cell death, antioxidant, anti-cancer, and anti-inflammatory effects. We investigated the protective impact of carvacrol on infertility caused by varicocele in rats. The animals were assigned to nine groups randomly: control, sham-operated, carvacrol alone at 10, 20, and 40 mg/kg b.w./day, varicocele-induced control, and varicocele-induced rats treated with carvacrol. After thirty days of treatment, the serum was collected to evaluate testosterone levels, and left epididymal sperm samples were obtained to assess sperm quality. Additionally, the left testis was removed for biochemical and histopathological evaluation. The findings demonstrated that carvacrol administration (20 and 40 mg/kg) notably improved sperm quality in rats with varicocele. Furthermore, carvacrol treatment (20 and 40 mg/kg) increased antioxidant levels, reduced MDA levels, decreased AQP9 expression in testicular tissue, and improved testicular tissue structure. Hence, carvacrol (20 and 40 mg/kg) may serve as a therapeutic agent for male reproductive system disorders, particularly varicocele-related infertility, due to its antioxidant properties and protective effects on testicular function.

The Quality of Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) Sperm Following Liquid-storage in Low Temperature

Indo-Pacific bottlenose dolphin (Tursiops aduncus) is a marine mammal that lives in relatively small populations. The geographic ranges of this species are susceptible to the effects of human activities, thereby necessitating conservation efforts to prevent extinction. Therefore, this study aimed to evaluate the daily quality of dolphin sperm after several days of refrigeration. The sperm of two male dolphins were stored at 4oC for 4 days, and the quality was observed daily to determine the motility, viability, membrane integrity, and sperm abnormalities. Sperm samples were divided into four groups, consisting of two centrifuged followed by the removal of seminal plasma, and two groups without centrifugation, containing 100x106 and 200x106 sperm/ml each. After liquid storage, the motility of sperm was 63-75% with no significant reduction in the first 3 days. Sperm viability following storage was 65-75% and the percentage with abnormal morphology ranged from 2-6%. Furthermore, there was no significant increase in abnormal morphology of sperm on any day of storage for 3 days. Sperm membrane integrity was 36-49%, with no significant reduction in the membrane integrity in the first 2 days. There was no significant difference in sperm quality, although centrifugation and removal of seminal plasm had a slight effect. The results of this study showed that Indo-Pacific bottlenose dolphin sperm could be stored for a short period as liquid storage while maintaining a quality that allows for future use.

Evaluation of carp sperm respiration: fluorometry with optochemical oxygen sensor versus polarography

The primary function of spermatozoa is to fertilize the oocyte, which depends on their motility and is directly associated with their metabolic state. The oxygen consumption rate (OCR) of spermatozoa reflects the respiratory capacity of sperm mitochondria under various physiological conditions and is an essential marker of sperm quality. We determined the OCR of common carp (Cyprinus carpio) sperm using two respirometry methods: the conventionally used polarographic method with a Clark-type electrode and fluorometric assay with an Oxo Dish optochemical oxygen sensor. The latter was used for the first time to evaluate spermatozoa oxygen consumption in various metabolic states (under different treatments) at different dilution rates. These two methods were compared using Bland–Altman analysis, and the applicability of the optochemical oxygen sensor for evaluating carp sperm oxygen consumption was discussed. Sperm motility and progressive velocity parameters were also assessed to evaluate the effect of sperm respiration under different metabolic states and dilution rates and preincubation period on the physiological status of spermatozoa. The comparison of these respirometry methods clearly shows that while the polarographic method allows immediate measurement of oxygen levels after adding a sperm sample, the optochemical oxygen sensor has a priority in the amount of data obtained due to simultaneous measurements of several samples (e.g., different males, different fish species, repetitions of the same sample or various experimental conditions), even at a later time after adding sperm to the measuring chamber. However, the compared methods are complementary, and the proposed methodology can be applied to other fish species.

The impact of COVID-19 on the male reproductive system.

The relevance of the study is determined by the deepening understanding of the global consequences of the coronavirus pandemic, which affect not only lung health but also a wide range of other body systems. In light of new data on the long-term effects of coronavirus infection, this study is highly significant. The purpose of this study is to investigate the impact of coronavirus infection on the male reproductive system and assess its potential influence on male fertility to refine the mechanisms of damage and provide recommendations for medical care. The study utilised a combination of methods, including a meta-analysis of medical organisation databases, analysis of clinical cases, representative sample method, and quantitative survey method. These approaches allowed for a comprehensive and multifaceted view of the problem. The samples of sperm showed a noticeable decrease in progressive motility, sperm concentration, and volume, especially in patients with moderate and severe symptoms of COVID-19, whereas patients with mild symptoms only experienced a decrease in progressive motility and overall sperm motility. The survey identified symptoms of male reproductive system dysfunction after recovering from COVID-19. Predominant symptoms included decreased libido (15%), impotence (13%), and infections of the genital organs (12%). Most surveyed men lacked sufficient awareness of other aspects of male reproductive health, including infections, genetic defects, chronic diseases, and available medical services. As a result of the study, it was concluded that coronavirus infection can have a negative impact on the male reproductive system. The practical value of this study lies in improving approaches to medical care for men who have recovered from COVID-19 and creating preventive programmes.

COVID-19 and Oncofertility: No SARS-CoV-2 in Semen but Inflammation Seems to Affect Sperm Parameters.

The COVID-19 pandemic, driven by SARS-CoV-2, led authorities to recommend halting assisted reproductive technology programs, focusing instead on fertility preservation, for cancer patients. The presence of SARS-CoV-2 in semen remains controversial. This multicentric prospective cohort study, conducted across 12 university medical centers, aimed to determine if SARS-CoV-2 is present in spermatozoa/seminal plasma in cancer patients by RT-PCR and to assess its impact on standard semen parameters. The levels of cytokines and TNF-α were measured in seminal fluid by ELISA. We enrolled 129 men who underwent sperm cryopreservation between July 7, 2020, and June 30, 2021. The 63 were included and tested for COVID-19 in nasal swab samples by RT-PCR and/or by serology. All patients were asymptomatic on the day of semen collection: 50 were uninfected, 8 had a positive nasal swab (PCR+) and 5 were seropositive. SARS-CoV-2 RNA was not detected in the seminal fluid or spermatozoa. Ejaculate volume was significantly lower in the PCR+ group compared to the uninfected group (median [IQR]: 2.6 mL [1.6-3.4] vs. 4.6 mL [2.6-5.2] p < 0.05). Total and progressive motility were lower in the PCR+ group compared to the seropositive group (32.5% [25.0-45.0] vs. 50% [49.0-55.0] p < 0.05, and 22.5% [10.0; 32.5] vs. 44.5% [40-49] p < 0.05). Higher TNF-α level was observed in the PCR+ group (1.9 pg/mL [0-3.9]) compared to the uninfected group (0 pg/mL [0-0.4]) p < 0.05. Although SARS-CoV-2 was not detected in the sperm samples of cancer patients who were PCR+, the infection appears to impact sperm parameters, likely due to inflammation.

DNA breakpoints and free DNA fragments as potential predictors of infertility

The study investigated the impact of varicocele on male infertility by analyzing sperm samples from 42 varicocele patients and 22 healthy individuals. Basic sperm parameters such as concentration and vitality were assessed, along with the DNA Fragmentation Index (DFI) using the SCSA method. Additionally, the mean number of sperm DNA breaks (MDB) and free DNA fragments amount in seminal plasma (fDFA) were analyzed using the TDT-Strand Displacement Probe Technique. Results showed higher levels of DFI, MDB, and fDFA in varicocele-related infertility cases. Combining MDB and fDFA proved more effective in predicting fertility compared to individual assessments or the DFI alone. While these findings suggest a potential role for MDB and fDFA as predictors of male fertility, further rigorous studies are needed to validate their application in varicocele-related male infertility.

Evaluation of circANKLE2 & circL3MBTL4 -RNAs Expression in Fertile and Infertile Men.

There are many factors that affect male fertility such as chronic health problems, psychological factors, and illnesses. Male infertility can be caused abnormal sperm function, low sperm production or even blockages that prevent the delivery of sperm. The aim of the work is to determine the expression pattern of the circularANKLE2 and circularL3MBTL4 RNA in spermatozoa from fertile and infertile males, as well as the relationship between these circRNA transcripts and sperm quality. The study involved two groups: a control group comprising 40 healthy, fertile men and an experimental group of 90 infertile males. Semen samples were collected and processed for analysis using computer-assisted semen analysis. Following RNA extraction from sperm samples, reverse transcription and real-time PCR were performed to assess the levels of circular ANKLE2 and circular L3MBTL4 RNA. There was a significant up-regulation of circularANKLE2 RNA expression (p < 0.05), and a significant down-regulation of circularL3MBTL4 RNA expression (p < 0.05) in asthenozoospermia, astheno-teratozoospermia, and oligo-astheno-teratozoospermia groups, as well as, in immature spermatozoa separated from normozoospermic samples. Moreover, the altered expression of both circular L3MBTL4 and circular ANKLE2 RNA showed significant correlations with the associated sperm parameters. In conclusion, the expression of circular ANKLE2 RNA and circular L3MBTL4 RNA may play a significant role in male fertility and could serve as potential biomarkers of sperm quality, warranting further investigation for their application in infertility diagnostics.

Evaluation of Porcine Sperm Activity Using an Electrochemical Device

[Introduction] In recent years, in vitro fertilization has become an important method in the field of infertility treatment. Research is progressing in the livestock industry to improve fertility and pregnancy rates. The motility of sperm can be evaluated through time-lapse microscopy. This method is cost-effective and requires little space for activity evaluation. In addition, methods for selecting motile sperm include those that utilize centrifugation, such as density gradient centrifugation. However, there is a concern that centrifugation may physically damage sperm due to the large gravitational force it receives. We previously measured the respiratory activity levels of sperm and somatic cells in raw cow milk using amperometry; however, we did not evaluate sperm motility [1]. Therefore, in this study, we developed an electrochemical device that collects motile spermatozoa using moving gravity sedimentation without adding centrifugation, and we reported the examined changes in the oxygen reduction current before and after sperm movement. [Experiment] Porcine (Duroc) sperm (approximately 108 sperm/mL) was purchased from the Miyagi Prefecture Livestock Experiment Station for use. The device was filled with 5.7 mL of 11.4 mM glucose-containing phosphate buffer solution (pH = 7.4), and a three-electrode system was constructed. A platinum electrode (diameter = 1.6 mm) was installed in the middle tube and a platinum electrode was installed in the counter electrode (Fig. 1). The applied voltage was maintained at -0.5 V vs. Ag/AgCl, and the sampling time was 0.1 s. After the start of measurement, 300 µL of the sperm sample was dropped onto the bottom of the outer tube, and amperometry was performed for approximately 60 minutes. [Results and discussion] Fig. 2 shows the amperogram from 40 to 55 minutes after dropping the sperm sample. After approximately 42 minutes, the oxygen reduction current started to decrease. After approximately 10 minutes, the current became constant, and a decrease of approximately 100 nA was observed. This result suggested that the pig sperm potentially consumed oxygen during movement from the bottom of the outer ring to the center of the well. The number of sperm that swam upward and moved to the center was approximately 4.4 × 106 sperm/mL in 60 minutes, and the recovery rate was approximately 15.0%. Compared to the literature value for human sperm (approximately 13.8%), the experimental results were considered reasonable, suggesting the possibility of capturing the real-time respiratory activity of moving sperm [2]. In this paper, we thoroughly discussed the relationship between the initiation of porcine sperm migration and the oxygen reduction current. [References] [1]S.Kasai, A.Prasad, R.Kumagai, K.Takanohashi, Biology,2022,11,549.https://doi.org/10.3390/biology11040549.[2] K.Tatsumi, T.Tatsumi, T.Uchida, K.Saito, H.Saito, F&S Reports, 2020,1(2), 106-112. Figure 1

Association of NOX5 Expression with Sperm Activity and Motility in Pathospermic Infertile Men.

The newest NOX isoform, NOX5, has been found in mammalian spermatozoa. Many physiological and pathological situations in spermatozoa are mediated by reactive oxygen species (ROS). NOX5 is the main source of ROS in spermatozoa. Our purpose was to investigate the changes in NOX5 expression and the effect of NOX5 expression on sperm motility, chromatin integrity, and oxidative status in oligoasthenozoospermic compared to normozoospermic men. Semen samples were collected from 30 normozoospermic (NS) and 30 oligoasthenozoospermic (OAS) men. NOX5 protein expression in sperm samples was evaluated by immunohistochemistry and western blot. Oxidative stress status was evaluated by total antioxidant capacity (TAC), total oxidant capacity (TOC), and oxidative stress index (OSI) parameters. Chromatin integrity in spermatozoa was evaluated by toluidine blue staining. NOX5 expression levels were significantly higher in OAS group than in NS group (p<0.001). In addition, chromatin integrity was significantly higher in the OAS group in comparison to NS group (p<0.001). TAC levels were higher in the NS group, but OSI and TOC levels were significantly higher in OAS group (p<0.001). It was found that NOX5 protein expression was positively correlated with oxidative stress and chromatin integrity and negatively correlated with motility (p<0.01). These results suggest that overexpression of NOX5 may be the source of excessive ROS production and oxidative stress injuries in oligoasthenozoospermic men. Considering that NOX5 expression is positively correlated with oxidative stress and chromatin integrity but negatively correlated with motility, it can be considered a biomarker to be used in assisted reproductive procedures.

Evaluation of the Detrimental Impact of Low-Intensity Laser Radiation on the Characteristics of Sperm Movement, Motion, and DNA Damage

It is generally accepted that low-level laser therapy promotes cellular energy production and increases the synthesis of ATP by triggering the mitochondrial electron transport chain's photosensitive cytochrome c oxidase complex. Nevertheless, this investigation aimed to examine the possible adverse impact on sperm function and DNA integrity of low-intensity laser radiation with a wavelength of 980 nm. Following standard analysis, forty semen samples were collected and categorized as either asthenospermic, oligospermic, or normospermic. The remaining semen was subjected to standard semen analysis, and the aliquots were divided into treatment and control groups. A continuous-wave 980 nm laser with an output power of 100 mW and an energy density of 10 J/cm2 was applied to the treated samples for 30 s. Divided samples underwent incubation at 37oC, and semen analysis was used to assess aliquots at 30 s and 2 h intervals. Following the incubation time, each sample was frozen in 250 mL at -80oC until DNA fragmentation was examined using flow cytometry. Thirty minutes after treatment, there was a noticeable increase in sperm movement; the rise was largest in oligospermic and asthenospermic samples (88%) while samples of normal sperm displayed the least increment (7.5%). There was no discernible increase in DNA damage in the treatment samples compared to the control samples. The treatment samples had 21.8% and the control samples had 25.3% of the DNA fragmentation index. The dynamics of sperm motion were noticeably altered. Short-term exposure to low-level laser radiation appeared to improve the motion and movement of treated sperm, and after 2 h, there was no increase in DNA damage. Asthenospermia and mitochondrial dysfunction may be related in some circumstances. To fully understand the ramifications of these results for possible medicinal applications, more research is needed. Our research's conclusions suggest that low-level laser stimulation is a useful and safe technique for identifying viable immotile spermatozoa in a particular sample. It may also cause these spermatozoa to move about, which could have positive therapeutic effects.

Detection of Androgen Receptors in Spermatozoa of Small Ruminants: A Putative Modulation Pathway for Cryoresistance Through AQP3.

This work was aimed to identify androgen receptors (AR) in the spermatozoa of wild and domestic ruminants and to assess the effect of testosterone on sperm localization of aquaporin-3 (AQP3) and cryopreservation process. Sperm samples from wild species were incubated with testosterone (T group), 1,3-propanediol (PDO group), phloretin (PHL group), PDO+T group, PHL+T group. Western blot identified the presence of AR as a single band of about 48 KDa. Immunolabelling of AR was located in the equatorial segment of the sperm head. In mouflons, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T than in Control and T groups. In ibexes, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T, PHL, and T group than in the Control group; the cryoresistance ratios for sperm kinematic variables were lower (p < 0.05) in PDO+T than in Control. No changes were found among treatments in the proportion of spermatozoa showing AQP3 in the different membrane domains after incubation and thawing in both mouflon and ibex. In conclusion, testosterone negatively affected sperm cryoresistance expressed as acrosome integrity, enhancing the effects of the AQP blocker PHL. Our findings provide a sound knowledge of the molecular mechanisms that explain the seasonal variation in sperm freezability from ruminants.