Semen Plasma Research Articles

Supplementation of glutamine in a short-term boar semen extender during 17°C holding time enhances post-thaw sperm quality for cryopreservation.

Glutamine is a nonessential amino acid and the most abundant amino acid found in the seminal plasma and sperm-rich fraction of boar semen. Glutamine plays an important role in enhancing glutathione (GSH) synthesis. It acts as an effective antioxidant in semen and provides intracellular defense to sperm against oxidative stress. This study aimed to improve the quality of frozen-thawed boar semen by using glutamine supplementation in a short-term semen extender during the holding time at 17°C before cryopreservation. The results indicate that the total motility, progressive motility, LIN, STR, and WOB were the highest in the 20 mM supplementation group at the 2h timepoint after thawing. Thus, the optimal concentration for glutamine supplementation in short-term boar semen extender during the holding time at 17°C was 20 mM. Interestingly, at all of the time points after thawing, 20 mM glutamine supplementation exhibited the highest level of sperm viability and membrane integrity when compared to the CONTROL (0 mM) and other experimental dilution groups. Moreover, the acrosome integrity, mitochondrial activity, and capacitation status (F pattern) were significantly greater in the 20 mM supplementation group than the other groups at the 2h timepoint after thawing. Supplementation of glutamine at a concentration of 20 mM in a short-term semen extender (Bio Pig®) during the 17°C holding time before cryopreservation, which had a standard freezing extender (9.0% glycerol and 1.9% Equex paste), could enhance the post-thaw sperm motility and quality parameters of cryopreservation.

Association between Exposure of Rare Earth Elements and Outcomes of In Vitro Fertilization-Embryo Transfer in Beijing

Objective The study aimed to investigate the impact of rare earth elements (REEs) exposure on pregnancy outcomes of in vitro fertilization-embryo transfer (IVF-ET) by analyzing samples from spouses.Methods A total of 141 couples were included. Blood and follicular fluid from the wives and semen plasma from the husbands, were analyzed for REEs using inductively coupled plasma mass spectrometry (ICP-MS). Spearman's correlation coefficients and the Mann–Whitney U test were used to assess correlations and compare REE concentrations among three types of samples, respectively. Logistic models were utilized to estimate the individual REE effect on IVF-ET outcomes, while BKMR and WQS models explored the mixture of REE interaction effects on IVF-ET outcomes.Results Higher La concentration in semen (median 0.089 ng/mL, P = 0.03) was associated with a lower fertilization rate. However, this effect was not observed after artificial selection intervention through intracytoplasmic sperm injection (ICSI) (P = 0.27). In semen, the REEs mixture did not exhibit any significant association with clinical pregnancy.Conclusion Our study revealed a potential association between high La exposure in semen and a decline in fertilization rate, but not clinical pregnancy rate. This is the first to report REEs concentrations in follicular fluid with La, Ce, Pr, and Nd found at significantly lower concentrations than in serum, suggesting that these four REEs may not accumulate in the female reproductive system. However, at the current exposure levels, mixed REEs exposure did not exhibit reproductive toxicity.

Evaluation of the effectiveness of the use of exosomes in the regulation of the mitochondrial membrane potential of frozen/thawed spermatozoa.

Numerous studies confirm the involvement of extracellular vesicles (EVs) in the regulation of physiological processes of mammalian sperm cells. It has been proven that they take part in the processes of capacitation, acrosonmal reaction, and anti-oxidation. Despite growing interest in the biomedical potential (including the search for new reproductive biomarkers) of EVs, the role of extracellular seminal vesicles in maintaining semen quality during cryopreservation has not yet been established. Therefore, the objective of this experiment was to evaluate the effectiveness of the use in the regulation of the mitochondrial membrane potential of bovine sperm and to explain the mechanisms of EV action during cell cryopreservation. Exosomes were isolated from bull semen plasma, measured, and used for extender supplementation. Semen samples were collected from Simmental bulls, diluted, and pre-evaluated. Then they were divided into equal fractions that did not contain EVs or were supplemented with 0.75; 1.5 and 2.25 mg/ml of EVs. The test samples were frozen/thawed and the mitochondrial membrane potential, DNA integrity, and viability were evaluated. EVs have been established to have a positive effect on cryopreserved sperm structures. The most favourable level of EVs was 1.5 mg / ml, which can be successfully to improve cell cryostability during freezing/thawing. In this study, exosomes isolated from the sperm plasma and supplemented with a concentrated dose in the extender for sperm freezing were shown to significantly improve cryostability of cells by supporting the potentials of the mitochondrial membrane and protecting the cytoplasmic membrane of spermatozoa.

Role of pyridoxine and oxidative stress in asthenozoospermia

PurposeInfertility is a worldwide concern, and recent research indicates that vitamin B6 deficiency may play a role in male infertility, primarily by inducing hyperhomocysteinemia and oxidative stress. These processes can have a detrimental effect on semen quality, ultimately affecting male fertility. Here, we aim to evaluate the biochemical status of pyridoxine (vitamin B6) in relation to total glutathione and total antioxidant capacity. Materials and methodsA case control study samples were collected of asthenozoospermic (n = 63) and normospermic (n = 43) cases, with average men age 30.35 ± 7.03 years old. Semen plasma specimens representing both fertile and sub-fertile men visiting two different secondary care health institute in Irbid province, Jordan. All samples were assessed according to WHO guidelines (2021) and by using spectrophotometry to evaluate the semen plasma levels of vitamin B6, glutathione (GSH) and total antioxidant capacity (TAC). ResultsOur main finding is there is significant positive correlations between the seminal plasma concentration of GSH (p < 0.0001) and TAC (p < 0.0073) are significantly correlated with vitamin B6 deficiency in asthenozoospermia group in comparison to normozoospermia cases. A significant decrease (p < 0.0001) the levels of vitamin B6 in men with asthenozoospermia compared to normozoospermic men (control) with an approximate 80 % percent reduction in the mean levels between groups. ConclusionsThese findings suggest that pyridoxine deficiency may very well alter the GSH system, in so doing affecting the antioxidant defense mechanism against reactive oxygen species to sperm, impacting sperm development and maturation. leading to asthenozoospermia.

Ambient Ozone Exposure, Semen Plasma Metabolites, and Sperm Quality Decline among Adult Men in Wuhan China.

Ambient ozone (O3) exposure may be associated with a reduction of semen quality, yet the potential biological mechanisms remain unclear. We investigated the effects of certain seminal plasma metabolites on mediating the links between O3 exposure and the deterioration of semen quality. The untargeted metabolomics analysis was performed on semen samples of 200 Chinese adult men to determine candidate metabolites associated with characteristics of semen quality. Mediation analysis was adopted to examine whether these metabolites modulated the links between O3 exposure and semen quality. We found a significant reduction in sperm concentration by -28.1% (95% CI: -41.7%, -11.3%), and sperm count by -29.2% (95% CI: -43.7%, -11.0%) associated with each 10 μg/m3 increase in ambient O3 concentration during the period of sperm development. We delineated 7 metabolites in seminal fluid that substantially mediated the links between O3 exposure and declined semen quality, including myristoleic acid, aspartyl-isoleucine, phenylethyl primeveroside, ACar (18:2), ACar (18:1), FAHFA (22:6/22:3), and LPS (22:5). Among these, myristoleic acid exhibited the most pronounced mediation effects, with its indirect effect of which accounts for 46.4% of the overall association. Our findings suggested that exposure to ozone decreased sperm quality by disrupting fatty acid metabolism, particularly myristoleic acid.

Effects of natural mating, artificial insemination and intravaginal deposition of raw semen or seminal plasma on vaginal and uterine blood flow in German Holstein cows

AimThe present study was performed to characterize and compare the perfusion of vaginal and uterine arteries after challenging the reproductive tract of dairy cows via natural mating, artificial insemination (AI), or intravaginal deposition (vaginal fundus) of different biological fluids or a placebo.Materials and methodsIn a double-blind study, six German Holstein cows were administered PGF2α during dioestrus and 48 h later treated with GnRH. Intravaginal or intrauterine treatments were carried out 12 h after GnRH was administered. Animals served as their controls, using a cross-over design with an interval of 14 days between experiments. The experimental animals were allocated to receive the following treatments: natural mating (N), intrauterine artificial insemination (A), intravaginal deposition (vaginal fundus) of 6 mL raw semen (R) or 6 mL seminal plasma (S), and compared to their controls [control 1: 6 mL placebo (P: physiological saline); control 2: no treatment (C)). Corresponding time intervals were chosen for the untreated control oestrus. Blood flow volume (BFV) in the uterine (u) and vaginal (v) arteries ipsilateral to the ovary bearing the preovulatory follicle was determined using transrectal Doppler sonography.ResultsAll animals exhibited oestrus and ovulated between 30 and 36 h after GnRH. Transient increases (P < 0.05) in vaginal blood flow occurred between 3 and 12 h following mating as well as 3 to 9 h after deposition of raw semen and seminal plasma, respectively. The most distinct increases (199%) in vBFV occurred 6 h after mating compared to values immediately before mating (= time 0 h). Neither AI nor deposition of a placebo into the vagina affected vBFV (P > 0.05). Only mating and deposition of either raw semen, seminal plasma or AI increased uBFV (P < 0.003). The greatest rise in uBFV occurred after natural mating. Maximum uBFV values were detected 9 h after mating when values were 79% greater (P < 0.05) than at 0 h.ConclusionsThe natural mating, deposition of raw semen or seminal plasma and conventional AI affect vaginal and/or uterine blood flow to different degrees. The factors responsible for these alterations in blood flow and their effects on fertility remain to be clarified in future studies.

Various Saccharides Addition of Extender Ram Efficiency to Improve Cryopreservation in Semen Etawah Crossbreed Bucks with Seminal Plasma Replacement

Phospholipase in buck seminal plasma will trigger a coagulation incident resulting in the demise of the spermatozoa during liquid semen processing using milk or egg yolk substrates diluent. A plasma replacement process is performed to avoid this possibility. The cryopreservation process leads to sperm cell damage due to the freezing process, therefore cryoprotectant agents such as saccharides are required as protective agents. This study aimed to investigate the effect of various types of saccharides on the quality of frozen semen of etawah crossbreed (PE) bucks during cryopreservation and thawing with plasma replacement using Priangan ram semen plasma. Semen was collected using an artificial vagina once a week. Fresh PE bucks semen centrifuged at 3,000 RPM for 30 min. The supernatant (seminal plasma) was removed and replaced in equal volume with Priangan ram seminal plasma. Semen was divided into four treatments: goat semen with sheep semen plasma in Tris diluent (control); control with 0.6% dextrose added (monosaccharides); control with 0.6% sucrose added (disaccharides); control with 0.6% raffinose added (trisaccharides). Semen quality including percentages of motile spermatozoa, live spermatozoa, and intact plasma membrane (IPM) were evaluated after diluted, equilibrated, and thawing, respectively. Results of this study showed that viability and IPM of monosaccharides, disaccharides, and trisaccharides were significantly (P&lt;0.05) higher than control (54.40 and 51.40; 55.00 and 53.60; 55.60 and 52.20 vs 48.40 and 52.20, respectively). The motility of disaccharides was significantly (P&lt;0.05) higher compared to control and other diluent (47.00 vs 41.00, respectively). In conclusion, different types of saccharides were effective in maintaining the quality of etawah crossbreed buck frozen semen. The addition of disaccharides seems more effective compared to monosaccharides and trisaccharides in tris extender on the quality of etawah crossbreed buck frozen semen.

Detection and Quantification of Human Herpes Viruses Types-6 and Cytomegaly Viruses in Sperm Samples of Patients with Fertility Disorders

Background; Viral infections are common around the world and cause many health problems. Infertility in men is a widespread health problem that can affect lifestyle and psychological state. Aims of the study; Study of the effect of herpes virus type 6 and cytomegalovirus on infertility disorders in men. Methodology; A case control study was done for a 100 specimens collected from men. This study has been conducted in Al Sader Medical City / Infertility Center / Al-Najaf Al-Ashraf and privet Center Dr. Ali Al-Ibrahimi for Embryos and Infertility in najaf . During the period from 10/10/ 2023 to 10/4/2024. The current study consisted of a sample size of 100 males, which was subsequently separated into two groups. The control group comprised 40 fertile men, aged 25 to 45 years, who had experienced both primary and secondary fecundity for at least twelve months. The Patients Cases group, consisting of sixty infertile males aged between 22 and 46 years, was categorized into two subgroups based on the type of infertility. There were forty-six guys who were classified as main infertility. There were a total of fourteen men who experienced secondary infertility. Male participants who are reproductive age, normal seminal analysis which is healthy and have a child as a Control group (Fertile men). Name of patient, sample number, Age (years), Education, housing area, Type of work, height, weight, Smoking, chronic diseases, History of surgery, Primary infertility, Secondary infertility, Sperm count, Sperm motility and Sperm morphology. Detection of CMV in semen plasma by CMV ELISA kit and detection of HHV 6 in semen plasma by HHV 6 ELISA kit. Result; The results showed that there was no statistical significance in age between the two groups. They also showed that there was statistical significance in smoking, type of infertility, and duration of infertility. The results showed statistical significance in the results for herpes and cytomegalovirus. There is also no statistical significance regarding the number of cases of cytomegalovirus infection according to age. On the contrary, there was statistical significance regarding age in herpes. The results also indicated that there was statistical evidence in the sperm analysis between the two groups. Conclusions; The statistical evidence between the two groups in the number of infection cases for all groups or by age indicates the role of viral infections in causing infertility in men.

Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. N/A. This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Evaluation of telomere length, reactive oxygen species, and apoptosis in spermatozoa of patients with oligospermia.

50% of cases of infertility are caused by male factor, which acquired or congenital problems may bring on. Male infertility can be caused by oligospermia and asthenozoospermia, which are common. Since the same mutations that cause azoospermia in some people also cause oligozoospermia in others, oligozoospermia may be thought of as a less severe form of azoospermia. Studies have demonstrated telomere length, catalase activity, super oxide dismutase (SOD), and DNA fragmentation can be influential factors for male infertility. The amount of apoptosis, oxidative stress factors, telomere length, and DNA fragmentation were some aspects of healthy sperm that we chose to look into in this study and compare to oligospermia individuals. Oligospermia patients (n = 24) and fertile men (n = 27) semen samples were collected, and the apoptosis rate of sperms in both groups was analyzed (Flow cytometry). Also, gene expression of apoptotic and antiapoptotic markers and telomere length were examined (real-time polymerase chain reaction). The sperm DNA fragmentation kit was used to determine DNA fragmentation and to evaluate catalase and SOD activity; the specific kits and methods were utilized. Higher expression levels of caspase3 (p = .0042), caspase8 (p = .0145), caspase9 (p = .0275), and BAX (p = .0202) mRNA were observed in patients who had oligospermia. In contrast, lower mRNA expression of BCL-2 (p = .0009) was detected in this group. In addition, telomere length was decreased in the oligospermia group (p < .0001) compared to the health group. Moreover, the frequency of apoptosis is induced in patients (p = .0026). The catalase activity is low (p = .0008), but the SOD activity is high (p = .0015) in the patient group. As a result of our findings, we may list the sperm cell apoptosis rate, telomere length, the degree of sperm DNA fragmentation, and lastly, the measurement of significant and efficient oxidative stress markers like SOD and catalase in semen plasma among the principal diagnostic characteristics for oligospermia. Future studies will be better able to treat oligospermia by showing whether these indicators are rising or falling.

Phosphatidylserine Levels in Seminal Plasma of Iraqi Infertile Men Using Direct and Indirect Swim Up Sperm Preparation Techniques

Phosphatidylserine (PS), exposed on viable sperm, is required for fertilization as it is recognized by specific receptors located on the microvilli of the oocyte to promote sperm: egg fusion. The heads of viable and motile sperm exhibit PS. With (PS) exposure progressively increasing during sperm transit through the epididymis. PS asymmetry on the leaflets of plasma membrane is arranged in a complex way, via multiple proteins functioning in opposite directions; further, PS is also essential for many intracellular trafficking events. The aim of this study was to evaluate the phosphatidylserine PS level in seminal plasma of infertile men before and after activation using Direct Swim Up (DSU) and Indirect Swim Up (IDSU) activation method and make a comparison between them. Forty infertile men were included in this study during their attendance to the infertility clinic at High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain University. Normozoospermic or mild asthenozoospermic semen samples were used to choose the cases, according to WHO manual standards (1999 -2010). The collected semen samples obtained were divided into three parts: The first part prepared was as an in vitro sperm characterization before activation while the second and third parts were after activation using direct and indirect swim up techniques respectively. The level of seminal plasma phosphatidylserine was increased after the activation techniques from the baseline level before activation. Comparison of Seminal plasma phosphatidylserine levels between direct and indirect sperm activation technique were significantly higher in direct swim up activation technique than indirect one. Phosphatidylserine (PS) in semen plasma of infertile men was significantly increased after activation using Direct Swim Up DSU preparation method.

A metabolomic perspective on the mechanisms by which environmental pollutants and lifestyle lead to male infertility.

The incidence of male infertility (MI) is rising annually. According to epidemiological studies, environmental pollution (e.g., organic, inorganic, and air pollutants), occupational exposure (e.g., high temperature, organic solvents, and pesticides), and poor lifestyle (e.g., diet, sleep, smoking, alcohol consumption, and exercise) are important non-genetic causative factors of MI. Due to multiple and complex causative factors, the dose-effect relationship, and the uncertainty of pathogenicity, the pathogenesis of MI is far from fully clarified. Recent data show that the pathogenesis of MI can be monitored by the metabolites in serum, seminal plasma, urine, testicular tissue, sperm, and other biological samples. It is considered that these metabolites are closely related to MI phenotypes and can directly reflect the individual pathological and physiological conditions. Therefore, qualitative and quantitative analysis of the metabolome, the related metabolic pathways, and the identification of biomarkers will help to explore the MI-related metabolic problems and provide valuable insights into its pathogenic mechanisms. Here, we summarized new findings in MI metabolomics biomarkers research and their abnormal metabolic pathways triggered by the presented non-genetic risk factors, providing a metabolic landscape of semen and seminal plasma in general MI patients. Then, we compared the similarities and differences in semen and seminal plasma biomarkers between MI patients exposed to environmental and poor lifestyle factors and MI patients in general, and summarized some common biomarkers. We provide a better understanding of the biological underpinnings of MI pathogenesis, which might offer novel diagnostic, prognostic, and precise treatment approaches to MI.

Reduced expression of miR-221 is associated with the pro-apoptotic pathways in spermatozoa of oligospermia men

Oligospermia and asthenozoospermia, both frequent, can lead to male infertility. Oligospermia might be viewed as a milder form of azoospermia because the same mutations that produce azoospermia in some individuals also create oligospermia in other individuals. In this, we looked at different characteristics of oligospermia men, counting the level of apoptosis and a few related apoptotic and oxidative stress components, and compared them to solid controls. In this study, semen samples from healthy fertile men (n = 35) and oligospermia (n = 35) were collected, and sperm death rates in both groups were examined using flow cytometry. Also, gene expression of apoptotic and anti-apoptotic markers and miR-221 were investigated (Real-Time PCR). Moreover, for the evaluation of catalase and SOD activity and anti-inflammatory cytokines, including IL-10 and TGF-β, the specific ELISA kits and procedures were applied. As a result, higher gene and protein expression levels of PTEN, P27, and P57 were observed in patients with oligospermia. In contrast, lower mRNA expression of AKT and miR-221 was detected in this group. In addition, IL-10, TGF-β, and catalase activity were suppressed in the oligospermia group compared with healthy men samples. Moreover, the frequency of apoptosis of sperm cells is induced in patients. In conclusion, apoptosis-related markers, PTEN, and the measurement of significant and efficient oxidative stress markers like SOD and catalase in semen plasma could be considered as the critical diagnostic markers for oligospermia. Future studies will be better able to treat oligospermia by showing whether these indicators are rising or falling.

Effect of Feeding Soybean and Sesame Seed Meal on Semen and Blood Plasma Traits in Awassi Rams

This study was done by using 15 Awassi male lambs, with an average body weight of 60.70 kg and age 10-12 months. Lambs were randomly distributed into three treatments, the first treatment was fed on a diet that contained (10%) soybean meal, the second treatment was fed on a diet that contained (11%) untreated sesame seed meal, and the third treatment was fed on a diet containing (11%) of formaldehyde-treated sesame seed meal. The results showed that daily feed intake decreased in T3 by 1.07 kg as compared with the T1 and T2 (1.34 and1.27 kg) respectively, semen characteristics did not differ as a result of treatments but dead sperm in the T3 was significantly decreased (p≤0.05) 11.31% as compared with the T2 15.57%. Blood glucose was higher (p≤0.05) in the T2 at 93.97 mg/dl as compared with the T1 and T3 (72.74, 73.26 mg/dl) respectively. also, cholesterol concentration increased significantly in T3164.57 mg/dl compared with T1 and T2 (134.27 and 140.05 mg/dl) respectively. Whereas, feeding sesame meal treated with formaldehyde led to a significant decrease (p≤0.05) in blood urea concentration of 42.88 mg/dl compared to 48.14 and 50.47 mg/dl, respectively, for the T1 and T2.

O-256 Evaluation of the fertility potential of semen using Proton NMR

Abstract Study question Is Proton NMR seminal plasma assessment a valid method for identifying biomarkers allowing to predict top-quality (TQ) embryo development? Summary answer Biomarkers important in the context of male fertility can be assessed using proton NMR What is known already Currently, the quality of sperm is assessed predominantly by examining its concentration, motility, and morphology. These parameters are insufficient in diagnosing male infertility and predicting treatment outcomes with assisted reproductive technology (ART). Metabolomics techniques have been shown to be potentially useful in identifying biomarkers related to infertility. It has been proven, using proton NMR, that there are a number of substances in the semen plasma that correlate with various physiological states of patients and may potentially indicate impaired functioning of various metabolic pathways. However, the relationship between biomarkers and embryo development has not yet been investigated Study design, size, duration This was a prospective case-control study and consisted of 30 semen samples from 13 patients who underwent IVF treatment between January – March 2022 at INVICTA Fertility Centre, Poland. Sperm motility, concentration, morphology, and DNA fragmentation of the samples were assessed. Before sperm preparation for fertilization, semen was frozen to perform 1H NMR analyses, which were preceded by centrifugation in order to separate the plasma. NMR analysis results were compared to the embryological results. Participants/materials, setting, methods It was possible to identify pre-selected biomarkers in semen plasma through the use of reference spectra and to quantify their concentrations using the internal standard addition method. Their relative concentrations (separately and in pairs) were collated with semen parameters, embryo development data and pregnancy outcomes. A Machine Learning approach was employed to identify the most important variables, which were then used for further modelling with the use of LGBM models. Main results and the role of chance NMR allows for the identification and measurement of biomarkers important in the assessment of the fertility potential of semen. The presence of amino acids, organic acids, sugar residues and fatty acid derivatives was confirmed by applying this technique. It was found that the concentration of these biomarkers is an individual feature - probably dependent on the physiological state of the organism. The results indicate that the ratios of biomarker concentrations to each other are more important than their individual concentrations. Machine learning modelling showed a strong correlation of several compounds with the chance of obtaining a TQ blastocyst. The amount of taurine, glutamic acid and choline together with its derivatives seem to be the most important factors in blastocyst development. The results indicate that if the amount of glutamic acid significantly surpasses glycerophosphocholine, choline exceeds taurine, the amount of taurine is higher than that of citrate, or the amount of choline is significantly higher than its phosphorus analogue the likelihood of obtaining a TQ blastocyst is higher. This would suggest that several metabolic pathways as the Krebs cycle, the one-carbon cycle or choline metabolism were modified signalling pathological conditions which the standard assessment of semen parameters cannot detect. Limitations, reasons for caution The NMR technique allows a fast determination of the content of many biomarkers, but results are in the mmol/l range. The results should be confirmed in a larger group of patients with different semen disorders Wider implications of the findings NMR allows for the rapid creation of unique metabolic profiles of patients, which can ensure the creation of personalized treatments for male infertility and the selection of sperm with the highest chance of obtaining a TQ blastocyst Trial registration number The studies were approved by the Ethics Committee at the Gdansk Regional Medical Board (No KB-3/22).

Effect of two extenders on the cryopreservation of Biyang donkey semen

The Biyang donkey is a medium-sized Chinese donkey. Very little research has been done on semen cryopreservation in this donkey breed. Dimethylformamide (DMF) and ethylene glycol (EG) are small molecular weight osmotic cryoprotectants that enter equine spermatozoa more readily and effectively than glycerol, and spermatozoa have reduced swelling mortality when equilibrated in amide-containing extenders. In recent years, EG and DMF have been used in extenders to improve the quality of cryopreserved donkey semen. It is, however, unknown whether their combination with glycerol is more effective than glycerol alone in protecting the semen of Biyang donkeys. In this experiment, we aimed to compare the effects of two different semen extenders on semen cryopreservation in Biyang donkeys. Both extenders were based on the CAU1 extender (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). The cryoprotectant 1 consisted of 2.5% egg yolk and 2.5% glycerol in CAU1 extender, cryoprotectant 2 consisted of 2.5% yolk, 1.5% DMF, 0.5% glycerin and 0.5% EG in CAU1 extender. In five donkeys, semen was collected three times to deplete epididymalsperm reserves, then one ejaculation of each jackass was collected for semen freezing was described (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). Briefly, after the semen was collected, an equal volume of CAU1 dilution was added and left to stand at room temperature (22-25°C) for 30 min, thereafter semen was divided into two equal portions and centrifuged to remove the semen plasma, one was diluted with cryoprotectant 1 and the other with cryoprotectant 2. Diluted semen was filled into 0.5 mL straws with a filling machine and left to stand at 4°C for 2-3 hours. Cryopreservation was carried out at a height of 3-5 cm above liquid nitrogen for 15 min, straws were then stored in liquid nitrogen. Two days later, semen was thawed in a water bath at 37°C for 40 seconds. Sperm motility was examined by CASA (TYCASA1.0, Beijing Tianyuan Aorui Biotechnology Co., Ltd). Motility of the raw semen (n=5) was 89.6±4.0%. The frozen-thawed motility was 56.0±4.18% and 51.6±4.3% in cryoprotectant 1 and cryoprotectant 2, respectively (not significant). In conclusion, the two cryo-extenders were equally suitable for cryopreservation of Biyang donkey semen.

Heat Shock Protein 70 Biomarkers in Sperm and Its Association on Oligoasthenozoospermia, and Asthenozoospermia in Male Infertility

Background: The heat shock protein (Hsp70) is a protein that helps cells survive in harsh conditions and is involved in a variety of cellular biological processes. Objectives: To study the relationship between the levels of (Hsp70) in semen plasma of infertile (oligoasthenozoospermia (OA), asthenozoospermia (AZ) and fertile men and the effect of an increase and decrease on fertility. Materials and Methods: The study was conducted on 119 semen samples, 31 men with (AZ), 28 men with (OA), whereas another 60 healthy controls were diagnosed, semen samples were collected from Iraqi patients who attended infertility clinics for infertility diagnosis and assisted reproductive technologies in Karbala for the period between November 2021 and May 2022. Results: The mean (Hsp70) in the patient group were significantly higher than in the control group (P &lt; 0.001). In comparison to (AZ) groups with (OA) found a significant difference in the mean level of (Hsp70) between (AZ) groups and (OA) groups (Hsp70) levels showed a negative significant correlation with the concentration, progressive cell and non-progressive cell, and a positive significant correlation with the sample volume, viscosity of semen, P &lt; 0.05. Conclusion: The total level of Hsp70 in the semen of men is higher in infertile men (AZ) and (OA), whereas we find lower levels in the semen of fertile men. A negative relationship was also found between the level of Hsp70 with semen concentrations and sperm motility and a positive relationship with the volume of the semen sample and its viscosity.

Protein Identification of Seminal Plasma in Bali Bull (Bos javanicus).

The purpose of this study was to identify seminal plasma proteins in Bali bull and their potential as biomarkers of fertility. Semen was collected from 10 bulls aged 5-10 years using an artificial vagina. Fresh semen was then centrifuged (3000× g for 30 min). The supernatant was put into straws and stored in liquid nitrogen. The semen plasma protein concentration was determined using the Bradford method, and the protein was characterized using 1D-SDS-PAGE. Coomassie Brilliant Blue (CBB) was used to color the gel, and the molecular weight of the protein was determined using PM2700. A total of 94 proteins were identified in the seminal plasma of Bali bulls analyzed using LC-MS/MS (liquid chromatography-mass spectrometry). Proteins spermadhesin 1 (SPADH1), C-type natriuretic peptide (NPPC), clusterin (CLU), apoliprotein A-II (APOA2), inositol-3-phosphate synthase 1 (ISYNA1), and sulfhydryl oxidase 1 (QSOX1) were identified as important for fertility in Bos javanicus. These proteins may prove to be important biomarkers of fertility in Bali bulls. These proteins are important for reproductive function, which includes spermatozoa motility, capacitation, and acrosome reactions. This study provides new information about the protein content in seminal plasma in Bali bulls. The LC-MS/MS-based proteome approach that we applied in this study obtained 94 proteins. The identification of these seminal plasma proteins of Bali bulls and their potential as fertility biomarkers may have an impact on the success of future artificial insemination (AI).

Characterization of proteases in the seminal plasma and spermatozoa of llama

Camelids' semen has peculiar characteristics that differentiate it from other species, including the highly viscous aspect of seminal plasma that greatly difficult sperm manipulation and the development of techniques such as cryopreservation, artificial insemination, and/or in vitro fertilization. The presence of proteases in the seminal plasma is responsible for semen liquefaction, and sperm functionality to achieve fertilization. The enzymatic and molecular composition of the semen of llama remains unknown. Therefore, the goal of the study was to characterize the protease activity and composition of the seminal plasma and sperm of llama semen. The proteolytic activity was performed using gelatine zymography and the composition by mass-spectrometry.Metallo-proteases were the major source of gelatinolytic activity in seminal plasma, while serine-peptidases were the main enzymes of sperm cells. Matrix Metalloproteinase 2 (MMP2) was the most prominent metallo-protease of llama seminal plasma characterized under the exposure of different inhibitors (EDTA and benzamidine) and by a specific immunodetection. Moreover, the prostate and epididymis were identified as potential sites of its synthesis and secretion.Outstandingly, this metalloproteinase was undetectable in llama sperm. Regarding, the molecular composition of semen by mass-spectrometry, 4 metallo-, 9 serine-, 8 threonine-, and 1 aspartic-peptidases were identified alongside 15 regulators in the sperm cell; where 24 were directly or indirectly interacting. Whereas 6 metallo-, 12 serine-, 3 cysteine-, and 1 aspartic-peptidases were identified, besides 7 inhibitors and 5 regulators in llama seminal plasma where 30 of them were directly or indirectly interconnected. This is the first study describing a partial degradome of llama seminal plasma and spermatozoa suggesting significant differences especially the absence of MMP2 in spermatozoa in contrast to data observed in other species. The characterization of proteases in llama semen will provide a better understanding of the molecular mechanisms involved in the in vivo or in vitro fertilization process in this species.