Ejaculate Samples Research Articles

Ejaculate Adjustment in Response to Sperm Competition Risk in Humans.

Previous research suggests that human males, like males of many mammalian and avian species, adjust their ejaculate quality in accordance with sperm competition risk. Men who spend less time with their regular female partner since the couple's last copulation produce ejaculates with more sperm at the couple's next copulation (Baker & Bellis, 1993). We conducted a conceptual replication of this research to investigate whether sperm competition risk predicts ejaculate adjustment in human males using additional measures of sperm competition risk (e.g., perceptions of partner infidelity, presence of potential sexual rivals) and updated laser-optic semen analysis technology. We collected data from 34 heterosexual couples (age range 18-32years) from a university population who completed self-report surveys on their relationship dynamics and provided six ejaculate samples (three copulatory and three masturbatory) across a 45-day period. Time spent together since the couple's last copulation was not significantly associated with ejaculate quality. However, sperm concentration for copulatory ejaculates was higher for men who perceived more potential sexual rivals. Discussion situates the current results within the literature on human sperm competition and suggests several directions for future research.

A semen-specific deoxyribonucleic acid methylation model for epigenetic age estimation and its robustness under environmental challenges.

In forensic investigations, semen samples are a common form of biological evidence, especially in cases involving sexual assault. Therefore, accurately estimating the age of an individual is crucial in criminal cases. This study presents a novel age estimation model based on semen-specific CpG methylation patterns. A multiplex panel was developed, consisting of 12 CpG sites (PARP14, C5orf25, cg23488376, MXRA5, PFKFB3, DLL1, NOX4, cg12837463, TTC7B, KCNA7, NKX2-1, and SYNE4), which exhibit strong correlations with age. Additionally, this study investigates the resilience of these methylation markers under simulated environmental challenges. We collected ejaculate samples from a diverse cohort of 115 male individuals, aged 20-71 years, who underwent deoxyribonucleic acid extraction and bisulfite conversion. Methylation levels of the selected CpG sites were assessed using a SNaPshot assay, which revealed significant correlations with chronological age. We developed and validated two robust age estimation models through stepwise and enter regression analyses, achieving reliable accuracy with mean absolute errors ranging from 3.81 to 4.1 years. Additionally, the study also investigated the robustness of semen stains under diverse environmental conditions, including fabric type, washing, hematin exposure, and UV-C light. The selected methylation markers demonstrated remarkable resilience despite the challenges posed by washing procedures and environmental exposure, confirming their potential for age estimation in forensic genetics. This research presents successful age estimation models, emphasizing the strong correlations between methylation levels and chronological age. The proposed methodology's accuracy is affirmed through model validation on an independent test set, while also highlighting the resilience of semen stains on fabrics under varying storage and washing conditions.

Microscopic ferning in human pre-ejaculate is highly correlated with the absence of sperm

ObjectiveWe characterize microscopic ferning in pre-ejaculate samples with and without sperm. Study designHealthy, male, withdrawal-experienced participants provided up to three paired pre-ejaculate and ejaculate samples. We centrifuged ejaculate samples to obtain a supernatant without sperm. After sperm analysis, we dried and evaluated pre-ejaculate, ejaculate, and supernatants for microscopic ferning. ResultsOf 57 pre-ejaculate samples (N = 24 men), seven (12.3%) contained sperm, none of which exhibited ferning. Sixty-six percent (33/50) of pre-ejaculate samples without sperm exhibited ferning. Neither ejaculate nor supernatant samples exhibited ferning. ConclusionFerning may distinguish clinical pre-ejaculate with and without sperm. Ferning exhibited 100% specificity for pre-ejaculate without sperm.

Analysis of the relationship between the frequency of erythrocytes with micronuclei, semen quality and reproductive indicators in artificial insemination of foxes

Relevance. Reproductive biotechnologies are widely used in various branches of animal husbandry. Their scientifically based application shows high results in obtaining offspring. In fur-bearing cellular animal husbandry, reproductive biotechnology methods also give certain results. At the same time, in order to increase and predict the success in reproduction of fur-bearing animals, it is necessary to control the selection and selection of parental individuals, in particular male sperm donors during artificial insemination.Methods. The studies were performed in February — May 2023. The object of the study were ejaculate samples, peripheral blood smears of 16 silver-black fox males, as well as the corresponding results of artificial insemination (AI). To evaluate ejaculates, the following were studied: sperm concentration — by photometry, their percentage distribution by type of movement — on the ISAS system, sperm morphology — by microscopy. In peripheral blood smears, the frequency of occurrence of erythrocytes with micronuclei was determined using a micronucleus test (MTA). The results of the IO were evaluated by the number and percentage of pregnant and missing females, the number of live and dead puppies born.Results. The results showed that with the frequency of occurrence of erythrocytes with micronuclei above 2%, the sperm content in ejaculates was from 45 to 54.5% with non-accessible movement (type c), and from 22.7 to 44.2% stationary (type d). Also, abnormal sperm forms prevailed in the ejaculates of these males (63–83%), while 66–100% of missing females were noted. At the same time, males with MINTS of less than 1.5% demonstrated good ejaculate quality and high efficiency of artificial insemination.

Astaxanthin Improved the Quality of Hu Ram Semen by Increasing the Antioxidant Capacity and Mitochondrial Potential and Mitigating Free Radicals-Induced Oxidative Damage.

The objective of this research was to investigate the effect of astaxanthin supplementations of semen extender on the quality of Hu ram semen after up to five days of preservation at 4 °C. Semen samples were collected from five healthy Hu rams using an artificial vagina during breeding season (April to August 2023) and diluted with a basic extender supplemented with control (0), 1 µM, 2 µM, 3.5 µM, or 4.5 µM of AXT. Overall, 170 semen ejaculate samples (34 repetitions) from five healthy Hu rams were used in our research study. The results revealed that the addition of AXT (3.5 µM) significantly (p ≤ 0.05) increased the sperm kinematic indexes (T.M%, P.M%, MAD%, STR%, and LIN %), sperm viability, plasma membrane integrity, acrosome integrity, total antioxidant content (T-AOC), and mitochondrial membrane potential (MMP) of the Hu rams spermatozoa after up to five days of preservation at 4 °C. Contrary to that, the addition of the best concentration of AXT (3.5 µM) to the semen extender significantly (p ≤ 0.05) reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) concentration of Hu ram semen. In conclusion, the results of the current study indicate that the addition of a semen extender with AXT improves the quality of Hu ram spermatozoa by increasing the total antioxidant capacity (T-AOC) and mitochondrial membrane potential (MMP). On the other hand, reducing free radicals induced oxidative (ROS) and per oxidative (MDA) damage to Hu ram semen.

Метод Фурье ИК-спектроскопии в исследовании эякулята в норме и с олигозооспермией

The optical properties of the components (plasma, spermatozoa, protein and a solution after protein isolation from plasma) of a series of 24 ejaculate samples according to spermatograms in normal and with oligozoospermia were studied by Fourier IR spectroscopy. The analysis of the complex of spectral data obtained for a series of samples of the "norm" category showed in 50% of cases the presence of pronounced pathology in the optical properties of ejaculate, plasma and sperm. For the remaining 50% of the samples, the presence of spermatozoa of satisfactory quality in terms of material composition (proteins, lipids, carbohydrates) was noted with already well-marked signs of pathology in the ejaculate and plasma. A comparative analysis of their optical properties made it possible to note that the absence of clearly defined Amide-I and Amide-II bands in the spectra and the presence of peptide bands in the 1593–1572 cm-1 region is a stable sign of an existing or incipient pathology. Samples of the oligozoospermia category are quite well identified by the spectra of the cellular part of the ejaculate. The results of the study may be useful in modernizing clinical techniques and diagnostic approaches to the problem of male fertility.

DNA-fragmented sperm ability to bind to the zona pellucida

BACKGROUND: A high percentage of the infertile couples are classified as having unexplained infertility. At the same time, male partners may have both normozoospermia and high level of sperm DNA fragmentation. The investigation of the selectivity of the oocyte zona pellucida for spermatozoa with fragmented DNA is an extremely promising direction. The novel data could improve sperm selection methods, which in turn will increase the efficiency of IVF cycles.
 AIM: The aim of this study was to assess the ability of spermatozoa with fragmented DNA to bind to the zona pellucida.
 MATERIALS AND METHODS: The material for the study was ejaculate samples from patients with normozoospermia (n = 5) and sperm donors (n = 4), as well as the zona pellucida of immature oocytes (n = 25) from nine patients. Sperm preparation was carried out by centrifugation in a density gradient of silicone particles and the swim-up method. Sperm DNA fragmentation was assessed using the method of fluorescent labeling of single- and double-stranded DNA breaks (TUNEL assay). Manipulations with gametes and the zona pellucida were performed using micromanipulation equipment and a femtosecond laser.
 RESULTS: Spermatozoa with fragmented DNA can effectively bind to the oocyte zona pellucida. However, among the zona pellucida-bound spermatozoa, the proportion of cells with fragmented DNA was significantly lower than among the unbound ones. The data obtained suggest the key role of the oocyte zona pellucida in the selection of spermatozoa with intact DNA.
 CONCLUSIONS: Spermatozoa with fragmented DNA retain the ability to bind to the oocyte zona pellucida. The use of the selective binding of spermatozoa with intact DNA to the zona pellucida can serve as a method for selecting gametes for intracytoplasmic injection of spermatozoa into the oocyte. Data on the selective properties of the oocyte zona pellucida can serve to select the optimal strategy in cases of “hidden” male factor, in which men with normozoospermia experience impaired fertility, in particular, due to sperm DNA fragmentation.

#253 : Administration of Umbilical Cord-Derived Mesenchymal Stem Cells Secretome to Sperm Preparation Medium: A Pilot Study

Background and Aims: As previously known, umbilical cord-derived mesenchymal stem cells (UC-MSCs) secretome has antioxidant potential that can reduce oxidative and nitrosative stress. This study is very important because understanding the effect of the UC-MSCs secretome on spermatozoa can assist in the development of sperm quality improvement techniques. This study aims to examine the sperm concentration and velocity before and after the addition of UC-MSCs secretome as a modification in sperm preparation with time duration and concentration variation. Method: A pilot study used a secretome produced at Dr. Cipto Mangunkusumo Hospital. Ejaculate samples were collected from married couples who already had children and the results of the semen analysis showed normozoospermia and DNA fragmentation index examination at a good level (15%). Sperm preparation was done using the Swim-up method. Samples were divided into 7 treatments with different ratios of secretions and ejaculate and washing medium (1:1 and 1:2) with incubation time durations (30, 45, and 60 minutes) to find optimal conditions to be used in further studies. The sperm concentration and velocity were observed before and after incubation. Results: Based on the results, it can be observed differences in sperm concentration and velocity from various incubation methods using UC-MSCs secretome. The 1:1 (v/v) secretome treatment had the highest spermatozoa concentration (68 million/mL), while the 1:2 (v/v) secretome treatment had the fastest spermatozoa velocity (30.51 μm/s), with the same incubation time of 45 minutes. The treatment group that was given UC-MSCs secretome 1:1 (60 minutes) and 1:2 (60 minutes) had slower spermatozoa velocity (22.91 μm/s and 21.45 μm/s, respectively) compared to the control group (26.39 μm/s). Conclusion: The administration of UC-MSCs secretome to the sperm preparation medium could increase the concentration and velocity of spermatozoa after incubation. However, this conclusion is provisional and needs further study to support this technique’s development.

Effect of Zinc Supplementation to Diluent Boar Semen on Sperm Characteristics and Activity of Antioxidant Enzymes

Background. Zinc is essential for male reproductive function as it is necessary for spermatozoa maturation, capacitation, acrosome reaction, and fertilization. It has been established that the use of chelates compounds of metals with amino acids, nucleotides, peptides, and carbohydrates is not only more effective but also economically justified. Objective. The aim of this study was to evaluate and compare the effect of different concentrations of Zn glutamic amino acid chelate (ZnGlu) on boar spermatozoa motility, viability parameters, and prooxidant-antioxidant homeostasis during in vitro incubation. Methods. Freshly ejaculated boar semen, after the addition of "Ecosperm" diluent, was divided into groups: a control group and three experimental groups. Zinc glutamate was supplemented to the experimental samples at concentrations of 1.0, 2.0, and 5.0 µg/ml, respectively. Semen samples were stored at 18 °C for 4 days. Every 24 hours, sperm motility and viability and the antioxidant status were assessed by the level of diene con­jugates (DC), concentration of malondialdehydere (MDA), and the activities of catalase and superoxide dismutase. Results. It was observed that boar sperm motility decreased with the extension of storage time, while the addition of 2.0 and 5.0 µg ZnGlu significantly improved sperm total motility and the percentage of vitality spermatozoa during 48–96 hours of incubation. Our studies also demonstrate that ZnGlu possess a protective effect in alleviating oxidative stress in boar sperm in vitro. The addition of ZnGlu significantly reduced the content of MDA and DC in ejaculate samples in all experimental groups during incubation, compared to the control group. Moreover, the activity of superoxide dismutase and catalase increased after adding ZnGlu to the boar semen, especially at concentrations of 2.0 and 5.0 mg/ml (P < 0.05). Conclusions. These data demonstrate that the supplementation of zinc glutamate enhances the antioxidant defence system of sperm and improves quality of boar semen in vitro.

Successful cryptozoospermia management with multiple semen specimen collection

To determine the prevalence of sperm suitable for intracytoplasmic sperm injection (ICSI) in fresh ejaculated semen samples provided by men scheduled for a microdissection testicular sperm extraction (mTESE) procedure. Secondary objectives included an evaluation of the effect of a short abstinence period on semen quality and ICSI outcomes for men with cryptozoospermia. Retrospective cohort study. Academic medical center. All men were scheduled to undergo a mTESE procedure by a single, high-volume surgeon at an academic center from September 1, 2015, to May 1,2021. Presence of sperm suitable for ICSI in the ejaculate on the day of scheduled mTESE. Prevalence of sperm suitable for ICSI in the ejaculate among previously diagnosed men with azoospermia. Secondary outcomes included changes in semen parameters, clinical pregnancy rate, and live birth rate. Of 727 planned mTESE procedures, 69 (9.5%) were canceled because sperm suitable for ICSI were identified in a fresh ejaculated sample produced on the day of scheduled surgery (typically one day before oocyte retrieval). Overall, 50 men (50/727, 6.9%) used these rare, ejaculated sperm for ICSI. Semen samples obtained with <24 hours of abstinence were more likely to have better motility than the sample initially provided on the day of the planned mTESE. The live birth rate per ICSI attempt using these rare, ejaculated sperm was 36% (19/53). Providing a fresh ejaculated semen sample on the day of mTESE allows nearly 10% of men with azoospermia to avoid surgery with satisfactory ICSI outcomes. Providing multiple ejaculated samples over a short period of time does not adversely affect sperm concentration and may enhance sperm motility in men with cryptozoospermia.

P-050 Using a second sample of ejaculate for IVF treatment

Abstract Study question Is it beneficial to use the second sample of ejaculate collected within 2 hours from the previous sample to fertilize the oocytes in IVF treatments? Summary answer A significantly higher clinical pregnancy rate was achieved using the second sample of ejaculate. What is known already The WHO recommends abstinence of 2 to 7 days prior to semen collection for standard evaluation. Although semen volume and sperm concentration may increase with prolonged abstinence, such abstinence may have a negative impact on sperm motility and viability. Previous studies have shown that a second ejaculate sample following a short period of abstinence exhibited improved sperm concentration, progressive motility, vitality, and decreased DNA fragmentation. It was suggested that a second ejaculate collected within 1 h might be preferable for ART procedures. To the best of our knowledge it has not yet been clinically implemented in routine IVF treatment cycles. Study design, size, duration A retrospective study of couples treated at our IVF unit between Dec 2020 and Dec 2022. All male partners were asked to give a second sample of ejaculate within 2-3 hours after the first. In all cases we used the second sample for fertilization whether it was better or worse than the first one. Participants/materials, setting, methods The study included 422 male partners of couples treated for IVF. Patients were divided into 2 groups. Group 1- 223 male partners that provided a second sample of ejaculate and Group 2- 199 patients unwilling to be detained that refused to provide an additional sample. Sperm count, motility percentile and sperm volume were recorded for each sample of ejaculate. Fertilization, cleavage, positive beta-hCG and clinical pregnancy rates were adjusted according to maternal and paternal age. Main results and the role of chance Of the 223 samples in Group 1 the second sample showed improved sperm quality in 123 samples, identical sperm parameters in 31samples and inferior sperm quality in 69 samples compared with the first sample. Regardless of sperm quality, only the second sample was used in all Group 1 participants. Demographic characteristics such as maternal age, cause of infertility, semen concentration and motility were similar in both groups. Paternal age was higher in Group 2 compared with Group 1 (37.2±7.0 and 35.2±6.3, p = 0.002 respectively). Clinical pregnancy rates were 36.9% in Group 1 and 21.8% in Group 2 (p &amp;lt; 0.003). Higher clinical pregnancy rates using the second sample remained after adjustment for maternal and paternal age OR = 1.92; 95% (1.15-3.19) and even when the second sperm sample was inferior in quality compared with the first. Limitations, reasons for caution The study limitation stems from the retrospective nature of the study and the fact that the groups might have a selection bias we were unaware of. Wider implications of the findings These findings suggest that it may be beneficial to routinely ask male partners, among couples undergoing IVF, for a second ejaculate 2-3 hours following the first. Further studies showing higher clinical pregnancy rates following the use of the second ejaculate for fertilization are needed to validate these findings. Trial registration number CMC-0017-22

P-068 Real-world data shows that frozen-thawed ejaculates used for ICSI with autologous oocytes have lower but clinically negligible cumulative live birth rates compared to fresh samples

Abstract Study question Does frozen-thawed sperm show an impact on success rates in ICSI with autologous oocyte cycles compared to fresh samples? Summary answer The slight decrease in cumulative live birth rates (CLBR) compared to using fresh samples should not overshadow the benefits offered by sperm freezing. What is known already Sperm freezing offers many advantages for cycle planning, logistics, sample safekeeping and patient comfort, becoming a reliable daily procedure in infertility clinics worldwide. Nevertheless, there is a lack of agreement amongst the published literature on the potential effect of using frozen-thawed sperm samples for ICSI cycles. The present study focused on outcomes of ICSI cycles using autologous oocytes and, in addition to expressing results as classical success rates, it assessed CLBR, which accounts for the contribution of all used oocytes and embryos from the same insemination procedure towards achieving a live birth. Study design, size, duration This is a retrospective multicenter observational cohort study involving 84,371 ICSI procedures with patients’ autologous oocytes (81,504 with fresh ejaculate semen samples and 2,856 with frozen ejaculate samples) from January 2008 to November 2021. The fresh sample group included real-world data from the clinical records of these patients and cycles from a cohort of 654,019 inseminated oocytes and 117,443 embryos transferred; whereas the frozen sample group considered 22,518 injected oocytes and 3,847 embryos replaced. Participants/materials, setting, methods Classical outcomes such as pregnancy rates and live birth rates (LBR) per embryo transfer (ET) were compared between the two study groups. Generalized linear models were used to obtain adjusted odds ratios (adjOR) and p-values for the comparisons. CLBR were expressed per ET, per embryo transferred and used oocyte, and were plotted as Kaplan-Meier curves. These were adjusted to the female patients’ age using Cox regression models to obtain adjusted hazard ratios and p-values. Main results and the role of chance There were statistically significant differences between both groups in terms of clinical and ongoing pregnancy rates per ET, and LBR per ET. However, after being adjusted to female and male patients’ age and BMI, total progressive motile sperm, semen capacitation, transfer before or at the blastocyst stage, and use of PGT, these differences were no longer statistically significant. Once three embryos were replaced, CLBR was 50.50% (49.91-51.08) in the fresh sample group, versus a 45.12% (41.77-48.28) in the frozen sample group. After five embryos transferred, these were 68.21% (67.27-69.13) and 64.62% (57.81-70.33) respectively (p &amp;gt; 0.001). The Cox regression resulted in an adjusted hazard ratio (adjHR) of 1.166 (1.076-1.264). The CLBR in the fresh sample group was 33.15% (32.69-33.61) after using 10 oocytes and 49.93% (49.31-50.54) after 14. In the frozen sample group, these were 28.73% (26.24-31.13) and 43.13% (39.78-46.30) (p &amp;gt; 0.001). The adjHR for this comparison was 1.220 (1.125-1.322). Limitations, reasons for caution The multivariate analyses aim to control to the best of our abilities the main limitation of the study, which is the potential biases that could be introduced due to the heterogeneity of the data and the retrospective nature of the present study. Wider implications of the findings Although frozen samples had slightly decreased CLBR per used oocyte and ET, there were no statistically significant differences in pregnancy and LBR per ET after the multivariate analysis. The extensive real-world data provided shows that slight declines in CLBR should not offset the many advantages that semen freezing offers. Trial registration number Not Applicable

O-015 Spontaneous alterations in semen parameters are associated with age, accessory gland function and the FSHB c.-211G&amp;gt;T variant

Abstract Study question Which parameters are associated with spontaneous alterations in semen analysis in infertile men? Summary answer Changes in semen parameters are modulated by abstinence time, accessory gland function and fluctuations in FSH serum concentration, which is bound to FSHB c.-211G&amp;gt;T variant. What is known already There is a strong within-subject alteration of semen parameters in men with infertility. The WHO therefore recommends two ejaculate analyses. However, it remains unknown in which subgroup variations are likely to occur and which semen parameters are affected. We assume that in some men with idiopathic infertility, ejaculate parameters may spontaneously change without interventional medication apart from regression to the mean effects. The study aims to identify factors in conventional semen analysis based on the patients’ clinical data, including reproductive hormones like follicle stimulating hormone (FSH), and factors impacting endocrine activity, like the FSHB-211 promoter gene. Study design, size, duration In this retrospective longitudinal study we selected 3456 deeply phenotyped men with idiopathic infertility who had visited our centre for fertility workup from 2010-2020. The patients were selected from our well curated database Androbase© for analysis of repeated ejaculate samples. Participants/materials, setting, methods Men with idiopathic infertility and at least two ejaculate analyses (70-90 days distance) were included. Exclusion criteria comprised: sperm concentration &amp;lt;1 million/ml, abnormal FSH or low testosterone, bitesticular volume (bTV) &amp;lt;10ml, orchiectomy, cryptorchidism, varicocele testis, oncological disease, genetic findings (i.e. abnormal karyotype, AZF deletions), medication affecting spermatogenesis. Grouped linear 2-level nested mixed-effect models were applied. Analyzed parameters included: somatic, hormonal (including FSH and FSHB c.-211 variants), and semen parameters (including abstinence time, accessory gland markers). Main results and the role of chance Groups were built according to sperm concentration: A (n = 397): ≥1.0 to &amp;lt; 5.0 mill/ml, B (n = 708): ≥5.0 to &amp;lt; 15.0 mill/ml, C (n = 2351): ≥15.0 mill/ml. A, B and C: changes in ejaculate volume were associated with alterations in total sperm count and motility (p &amp;lt; 0.003). Changes were, controlled for abstinence time (p &amp;lt; 0.001), related to alpha-glucosidase, fructose or zinc (p = 0.005-0.02). A+B: fluctuations in FSH concentration influenced sperm concentration/count (p = 0.004-0.02), albeit only in men with FSHB c.-211 GG (p = 0.007-0.02). T-allele carriers did not show changes in FSH concentration (p &amp;gt; 0.1). B: age &amp;lt;50 years (p = 0.007-0.01) and normal bTV (p = 0.008-0.02) were associated with spontaneous increases of sperm concentration, count and motility. Semen parameters exhibit intra-individual alterations associated with organic, hormonal and genetic variables. Changes are pronounced in younger men with normal bTV and oligo- to almost normozoospermia. The effect is modulated by abstinence time, accessory gland function and fluctuations in FSH concentrations which, in turn, are bound to the FSHB-211 promotor gene polymorphism. Limitations, reasons for caution A limitation of our approach is the retrospective nature of the study, which cannot exclude the possibility of bias within the data set. Wider implications of the findings Judgement of semen analysis should be based on two semen samples, with abstinence time between 4-5 days. In future clinical trials, abstinence time should be kept constant. It might be investigated if younger men with normal bTV and FSHB c.-211 GT/TT benefit from improving accessory gland function and increasing FSH. Trial registration number not applicable

Study of the immunoglobulin and oxidized protein content of semen under infertility

Among the causes of male infertility, enough attention is paid to oxidative stress, which in turn is a pathogenetic link in the inflammatory process. However, there is practically no information on the content of oxidized modified proteins in the semen, which makes it difficult to study the pathogenesis of diseases of the male reproductive system. In part, protein oxidation may be due to the production of reactive oxygen species by microorganisms, both directly and indirectly through the activation of immune system cells. The aim of the research was to study the level of oxidized modified proteins and changes in immunoglobulin concentrations in the semen under bacteriospermia. A study was made of the ejaculate of 48 men who applied to the clinic for infertility in marriage. The comparison group consisted of 32 practically healthy men who had no growth of microorganisms in the ejaculate samples. When conducting bacteriological analysis, the studied samples were diluted 10 times and used the generally accepted method. The concentration of albumin, immunoglobulins A, M, G, E was determined in the spermatic fluid. The oxidative modification of proteins was evaluated in the reaction with 2,4-dinitrophenylhydrazine. The concentration of oxidized proteins was expressed in nmol/mg of the total protein of the studied biological fluid. The biuret method was used to determine the protein concentration. Statistical analysis of the results was performed using descriptive statistics and Student's t-test for paired data. The concentration of protein in the seminal fluid did not differ significantly among the studied groups. The albumin concentration (16.96±1.28 mg/mL) was statistically significantly lower in the absence of microorganism growth than in bacteriospermia. With bacteriospermia, a decrease in the concentration of IgM and IgA and an increase in the level of IgG were noted. The degree of protein oxidation is maximum when enterobacteria are isolated from seminal fluid. Thus, during the studies it was found that, despite the absence of a clinic, with asymptomatic bacteriospermia, the secretion of immunoglobulins G into the semen is observed. The accumulation of oxidized proteins in the seminal fluid in bacteriospermia has been shown.

Effect of Poria cocos Mushroom Polysaccharides (PCPs) on the Quality and DNA Methylation of Cryopreserved Shanghai White Pig Spermatozoa.

In this study, we explore the effects of Poria cocos mushroom polysaccharides (PCPs) on the quality and DNA methylation of the cryopreserved spermatozoa of Shanghai white pigs. A total of 24 ejaculates (three ejaculate samples per boar) from eight Shanghai white pigs were manually collected. The pooled semen was diluted with a based extender supplemented with different concentrations of PCPs (0, 300, 600, 900, 1200, and 1500 μg/mL). Once thawed, the quality of the spermatozoa and their antioxidant function were assessed. In the meantime, the effect of spermatozoa DNA methylation was also analyzed. The results show that compared with the control group, 600 μg/mL of PCPs significantly improves the spermatozoa viability (p < 0.05). The motility and plasma membrane integrity of the frozen-thawed spermatozoa are significantly higher after treatment with 600, 900, and 1200 μg/mL of PCPs compared with the control group (p < 0.05). In comparison with the control group, the percentages of acrosome integrity and mitochondrial activity are significantly enhanced after the application of 600 and 900 μg/mL PCPs (p < 0.05). The reactive oxygen species (ROS), the malondialdehyde (MDA) levels, and the glutathione peroxidase (GSH-Px) activity, in comparison with the control group, are significantly decreased in all groups with PCPs (all p < 0.05). The enzymatic activity of superoxide dismutase (SOD) in spermatozoa is significantly higher in the treatment with 600 μg/mL of PCPs than in the other groups (p < 0.05). As compared with the control group, a significant increase in the catalase (CAT) level is found in the groups with PCPs at 300, 600, 900, and 1200 μg/mL (all p < 0.05). In comparison with the control group, the 5-methylcytosine (5-mC) levels are significantly decreased in all groups with PCPs (all p < 0.05). As a result of these findings, a certain amount of PCPs (600-900 μg/mL) added to the cryodiluent can significantly improve the quality of Shanghai white pig spermatozoa and can also reduce the methylation of spermatozoa DNA caused by cryopreservation. This treatment strategy may establish a foundation for the cryopreservation of semen from pigs.

Differential distribution of 5-formylcytosine and 5-carboxylcytosine in human spermatogenic cells and spermatozoa

Background. The epigenome of gametes is formed under the control of the developmental programme and the influence of environmental factors. How cytosine oxidation patterns are formed and altered in human spermatogenesis remains obscure so far.&#x0D; The aim of the study was to assess 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) patterns in human spermatogenic cells and spermatozoa.&#x0D; Materials and Methods. The study was performed on testicular biopsy samples of 10 azoospermic patients and ejaculate samples of 5 sperm donors and 8 patients from infertile couples. The microscope slides were prepared for further indirect immunofluorescence to detect 5fC and 5caC and FISH to determine spermatogenic cell ploidy. &#x0D; Results. 5fC and 5caC were undetectable in mitotic and meiotic chromosomes of spermatogenic cells, and was present exclusively in some spermatogonia and spermatid interphase nuclei as well as in some ejaculated spermatozoa. The frequency of spermatozoa with 5fC and 5caC varied in a wide range and was higher in patients than in sperm donors (p=0,007, p=0,028). The increase in frequency of spermatozoa with 5fC and 5caC was accompanied with the decrease in frequency of morphologically normal and progressively motile spermatozoa.&#x0D; Conclusions. 5fC and 5caC are differentially distributed in human spermatogenic cells and spermatozoa. The immunocytochemically detected increase of 5fC and 5caC in individual spermatozoa is most likely induced by oxidative stress caused by effects of internal and external factors rather than developmental programme. The evaluation of 5fC and 5caC in spermatozoa can be potentially used as an additional criterion of ejaculate quality.

154 Growth, Libido, Semen Quality, and Reproductive Organ Weights of Hair Sheep Rams Rendered Short-Scrotum at Birth or Weaning

Abstract Rendering male lambs short-scrotum interferes with testicular development and thermoregulation, and has been proposed as a management tool to control male reproduction. This study evaluated at the effect of timing of the procedure (birth or weaning) on growth rate, testis size, libido, ejaculate characteristics and carcass traits in male landrace hair sheep lambs. April- (n = 31) and August-born (n = 23) Barbados Blackbelly (BB) and St. Croix (SC) male hair sheep lambs, balanced by breed and birth type, were rendered short-scrotum (SS) using elastrator bands at birth (B) or weaning (W), or left intact (I). Lambs were born on pasture, weaned at 9 weeks, transitioned in pens for two months post weaning, and then rotationally grazed as a single group to a target weight of 40 kg. At 6-mo of age (peri-pubertal), and again at target weight (post-pubertal), mating behavior was recorded (day) and ejaculate samples collected (day 2). Mating behavior was recorded in a0-min test with males individually exposed to estrus-induced ewes pen. An initial attempt was made to collect semen using an estrus-induced teaser ewe, and if not successful, the semen sample was collected by electro-ejaculation. At target weight lambs were harvested, scrotal content collected, and carcass characteristics (April cohort) determined. Data were analyzed in a model with sex class, breed and cohort as main effects. Pen and pasture ADG (142 and 129 g/d, respectively) and age at target weight (287 d) was not affected (P &amp;gt; 0.1) by SS treatment. However, April-born lambs grew 19 g/d less (P &amp;lt; 0.01) and reached target weight 40 d later (P &amp;lt; 0.001) than August-born males. As for breed, STX had greater pasture ADG (139 vs 124 g/d; P &amp;lt; 0.05) and reached target age 19 days earlier (P&amp;lt; 0.05) than BB males. Libido (number of mounts and services) was not affected by SS treatment, breed or cohort. The frequency of ejaculates containing sperm was less (P &amp;lt; 0.001) in SS (26 to 33%) than intact males (97%) and increased with age in SS but not I males. Sperm concentration and motile sperm in ejaculates were greater (P&amp;lt; 0.05) in I than SS males. Testis and epididymis were larger (P &amp;lt; 0.001) in I (300 and 44.3 g) than in B (134 and 20.0 g) and W (154 and 23.8 g), which were not different. Cohort and breed had no effect (P &amp;gt; 0.1) on mating behavior, ejaculate characteristics, and organ weights. Results suggest that SS and its timing had no effect on growth rate or on mating behavior. Though the procedure depressed the presence of sperm in the ejaculate and decreased testis weight, the suppression was not complete, and caution is warranted in utilizing the technique to facilitate co-grazing ewe lambs.

Morphological and morphometric characteristics of fresh and cryopreserved spermatozoa of saanen bucks

Background. Cryopreservation of spermatozoa with the best morphofunctional characteristics may allow their use for assisted reproductive technologies in goat breeding throughout the year to effectively increase livestock. Aim. To evaluate the influence of seasons on morphological and morphometric characteristics of fresh and cryopreserved goat sperm. Methods. The ejaculate samples of three sexually mature bucks of the Saanen breed were obtained during the breeding and non-breeding seasons. To assess the morphological characteristics, the smears of sperm were stained and then visualized under a light microscope with a magnification of ×1,000. For morphometric measurements, sperm micrographs were taken and analyzed using the ImageJ software. The cryopreservation of sperm was performed in a medium with 10 % glycerol and 20 % egg yolk. The samples were thawed in a water bath, then the morphological and morphometric characteristics of the cells were evaluated. Results. After the cryopreservation, the number of sperm with abnormal morphological structures was significantly higher compared to fresh sperm, both in the breeding and non-breeding seasons (p ≤ 0.05). After the cryopreservation, the length and width of the head decreased significantly (p ≤ 0.05) compared to the corresponding sperm sizes before the cryopreservation. Analyzing the morphological char- acteristics of the tail part, we revealed the fact of its reduction after the cryopreservation, probably due to twisting, loop formation, and detachment of its part. Conclusions. It was found that in the non-breeding season, the number of spermatozoa with damaged morphological structures increases, compared to the breeding season, which must be taken into account when using reproductive technologies. The morphological and morphometric characteristics of Saanen buck spermatozoa change after the cryopreservation, regardless of the season, which indicates the need to choose the fertilization tactics that will ensure the selection of spermatozoa without any morphological abnormalities.

Evaluation of cytokine profile of ejaculate in adolescents with varicocele

Varicocele is a vascular disease characterized by abnormal tortuosity and dilation of the veins in pampiniform plexus that drains the testis. Due to difficult outflow of blood via the altered veins, the intratesticular blood flow becomes impaired, leading to pathological changes in the testicular tissue caused by hyperthermia, ischemia, hypoxia and development of inflammatory reaction. Seminal plasma contains numerous proteins, molecules, a wide range of cytokines, chemokines, growth factors. Their properties and levels largely determine the stages of post-testicular maturation of spermatozoa. At the same time, cytokines are an integral part of the inflammatory effect and are synthesized by various immunocompetent cells present in the male reproductive tract. Increased cytokine levels in ejaculate may act as a marker of local inflammatory process, being a significant factor of male infertility. The purpose of our study was to assess cytokine profile of ejaculate in adolescents with varicocele.The level of cytokines IL-1β, IL-6, IL-8, TNFα, IL-4, IL-10, VEGF was determined in ejaculate of adolescents aged 17 years. The main group consisted of 100 adolescents with II-III degree varicocele; the comparison group included 30 adolescents without varicocele.Adolescents with varicocele have statistically higher levels of all studied pro-inflammatory cytokines, except of IL-8 and IL-10, which may suggest presence of a local inflammatory process. We did not find significant differences in the levels of cytokines between groups with grade II and III varicocele. However, the levels of pro-inflammatory IL-1β, IL-6 cytokines in ejaculate proved to be increased in the patients with grade III varicocele. When comparing the results between both groups, depending on the period after varicocelectomy and in the comparison group, statistically higher levels of IL-1β, IL-6, TNFα were detected in patients with more recent surgical correction and IL-10 levels in both subgroups with varicocele. This finding, given the progressive course of this disease, may be considered an unfavorable factor, since the pro-inflammatory status of testicular tissue returned, at later terms after surgery.In adolescentswith varicocele, increased levels of pro-inflammatory cytokineswere revealed in the ejaculate. There were no statistically significant differences in the level of cytokines in ejaculate of the patients with II and III degrees of varicocele. At the longer postsurgical period, an increased level of pro-inflammatory cytokines was revealed in the ejaculate samples.

Spontaneous alterations in semen parameters are associated with age, accessory gland function and the FSHB c.-211G>T variant.

There is a strong within-subject alteration of semen parameters in men with infertility. However, it remains unknown in which subgroup variations are likely to occur and which semen parameters are affected. To evaluate parameters associated with spontaneous alterations in semen analysis. We retrospectively selected 3456 men with infertility without known causes affecting spermatogenesis or sperm output for analysis of repeated ejaculate samples. Exclusion criteria comprised sperm concentration <1 million/mL, abnormal follicle-stimulating hormone or low testosterone, and low bitesticular volume (<10mL). Grouped linear two-level nested mixed-effect models were applied. The analyzed parameters included abstinence time, bitesticular volume, age, accessory gland markers, follicle-stimulating hormone, and FSHB c.-211 variants. Groups include A (n=397): ≥1.0 to <5.0 million/mL, B (n=708): ≥5.0 to <15.0 million/mL, and C (n=2351): ≥15.0 million/mL. Groups A, B, and C: changes in ejaculate volume were associated with alterations in total sperm count and motility (p<0.003). Changes were, controlled for abstinence time (p<0.001), related to α-glucosidase, fructose, or zinc (p=0.005-0.02). Group A+B: fluctuations in follicle-stimulating hormone level influenced sperm concentration/count (p=0.004-0.02), albeit only in men with FSHB c.-211 GG (p=0.007-0.02). T-allele carriers did not show changes in follicle-stimulating hormone levels (p>0.1). Group B: age <50 years (p=0.007-0.01) and normal bitesticular volume (p=0.008-0.02) were associated with spontaneous increases in sperm concentration, count, and motility. Semen parameters exhibit intra-individual alterations associated with organic, hormonal, and genetic variables. Changes are pronounced in younger men with normal bitesticular volume and oligozoospermia to almost normozoospermia. The effect is modulated by abstinence time, accessory gland function, and fluctuations in follicle-stimulating hormone level, which is bound to FSHB c.-211G>T variant. Judgment of semen analysis should be based on two semen samples, with abstinence times between 4 and 5 days. As a future perspective, it might be investigated whether younger men with normal bitesticular volume who are unable to elicit increases in serum follicle-stimulating hormone (FSHB c.-211 genotype of GT/TT) benefit from improving accessory gland function and increasing follicle-stimulating hormone.