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There has recently been an explosion of studies implicating the human microbiome in playing a critical role in many disease and wellness states. The etiology of abnormal semen analysis (SA) parameters is not identified in 30% of cases; investigations involving the semen microbiome may bridge this gap. Here, we explore the relationship between the semen microbiome and alterations of sperm parameters. We recruited men presenting for fertility evaluation or vasectomy consultation with proven biological paternity. SA and next generation sequencing was performed. Differential abundance testing using Analysis of composition of Microbiota with Bias Correction (ANCOM-BC) was performed along with canonical correlational analysis for microbial community profiling. Men with abnormal (N = 27) sperm motility showed a higher abundance of Lactobacillus iners compared to those with normal (N = 46) sperm motility (mean proportion 9.4% versus 2.6%, p = 0.046). This relationship persisted on canonical correlational analysis (r = 0.392, p = 0.011). Men with abnormal sperm concentration (N = 20) showed a higher abundance of Pseudomonas stutzeri (2.1% versus 1.0%, p = 0.024) and Pseudomonas fluorescens (0.9% versus 0.7%, p = 0.010), but a lower abundance of Pseudomonas putida (0.5% versus 0.8%, p = 0.020), compared to those with normal sperm concentration (N = 53). Major limitations are related to study design (cross-sectional, observational). Our results suggest that a small group of microorganisms may play a critical role in observed perturbations of SA parameters. Some of these microbes, most notably Lactobacillus iners, have been described extensively within other, fertility-related, contexts, whereas for others, this is the first report where they have potentially been implicated. Advances in our understanding of the semen microbiome may contribute to potentially new therapeutic avenues for correcting impairments in sperm parameters and improving male fertility.

Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.

Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

Pulmonary arterial hypertension (PAH) is a disease that affects millions of people worldwide. Besides the effects on the lungs and heart, PAH can affect other organs, including the liver, kidneys, brain, glands, and testis. This study aimed to evaluate the impact of PAH and physical resistance training (RT), a complementary treatment for hypertension, on epididymis morphology and function and sperm parameters. Wistar rats were divided into four experimental groups (n = 8/ group): sedentary control, sedentary PAH, RT control, and RT + PAH. PAH was induced using monocrotaline injections on Day 1 and 7 of the experiment. Sixteen rats from RT groups underwent RT training for 30 days, while rats from sedentary groups did not exercise. The epididymis was processed and analyzed using microscopic, biochemical, and functional approaches. Sperm were harvested from the cauda epididymis and evaluated for morphology and motility. Our results showed that PAH compromised the epididymis antioxidant defense system and reduced NO levels, leading to an imbalance in the organ's mineral content. These alterations affected the epididymis morphology and reduced the sperm transit time in the proximal epididymis, resulting in an increase in abnormal sperm morphology in the cauda region. Unfortunately, RT was not a good therapy against the PAH effect on the epididymis. PAH negatively affected epididymis functions with consequences to male gametes. Dysfunctions in the post-testicular environment may lead to male infertility due to the disturbance of spermatozoa fecundity. [ABSTRACT FROM AUTHOR]

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress. Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 106/mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes. The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity. • Olive fruit extract supplementation enhanced in vitro fertilization ability of buffalo sperm. • The addition of olive fruit extract improves buffalo sperm biological antioxidant potential. • The treatment with olive fruit extract increased the percentage of acrosome intact spermatozoa. [ABSTRACT FROM AUTHOR]

Five biological characteristics of honey bee drones were investigated under the influence of organic farming in comparison with non-organic ones. For this purpose, 10 honey bee colonies were established in each of the areas of 2.1 Hectare. The organic one was isolated from the other by 17.2 km. Each area was cultivated with Chamomile (Matricaria chamomilla), one under organic and the other under non-organic farming, for two seasons. As criteria for this study, amount of sealed drones brood produced, drone longevity, number of sperms in drones, percentage of drone maturity, and number of sperms in mated queens were chosen. The amount of sealed drone brood was higher in non-organic areas than organic ones, but a significant increase in the longevity of drones reared under organic farming was registered. Concerning reproductive futures, and although the number of sperms per million (mil.) was higher in newly emerged drones reared under non-organic farming, it began to decrease more significantly at 8 and 14 days in drones reared under non-organic than those reared under organic ones. Concerning the percentage of mature drones, the results showed a significant increase in drones reared under non-organic conditions at 14 days, but an insignificant increase at 8 days in comparison to drones reared under organic ones. For both groups, the percentage of mature drones of 14 days drones was significantly higher than those of 8 days. Lastly, the number of sperms in the spermatheca of queen mated with drones reared under organic farming was significantly higher than those reared under non-organic conditions. The results were discussed in the light of some biological aspects of drones. [ABSTRACT FROM AUTHOR]

In Bihar, an estimated 10 million are exposed to arsenic poisoning through drinking of water. The entire Gangetic plain region people are exposed to arsenic poisoning which is causing lot of health hazards including the problem of infertility. Usually, the infertility problem is associated with the hormonal imbalances and arsenic is a xenoestrogen toxicant which plays major role in causing the imbalance. Moreover, this reproductive disorder has become a major problem in the society causing social stigma in the exposed population. Therefore, there is indeed need for search of novel drug which may play major role in combating the present problem. The present study therefore aims to discover fertility booster against the arsenic induced male reproductive toxicity in animal models. The current study was conducted on Charles Foster Rats after approval by the Animal Ethics Committee of the Institution. A total of n=18 rats (12 weeks of age) with a mean weight of 160±20 g was used in this study. The study group consisted of n=6 controls and n=12 orally treated with sodium arsenite at a dose of 8mg/Kg b.w per day for 40 days. The n= 6 animals were dissected and rest n=6 was administered orally with Asparagus racemosus root ethanolic extract at the dose of 400mg/Kg b.w per day for 40 days. At the end of the entire experiment, all the animals were dissected out and their reproductive organs were taken out especially epididymis for sperm counts, sperm motility, sperm mortality, sperm morphology. The blood samples were taken for hormonal determination (testosterone and luteinizing hormone), as well as for haematological and biochemical analysis. The study showed a severe degeneration in the reproductive organs of the arsenictreated rats. There were degenerative changes in sperm count, sperm motility, sperm mortality, sperm morphology, in the hormonal parameters, as well as in the hematological and biochemical parameters in the arsenic treated rats. But, in the Asparagus racemosus root ethanolic extract treated group, there was significant normalization in all these parameters. Therefore, in the present study, the Asparagus racemosus root ethanolic extract at the dose of 400mg/Kg/body weight per day shows significant fertility boost effect on arsenic induced Charles Foster rats. [ABSTRACT FROM AUTHOR]

Signal peptide peptidase-like 2c (SPPL2c) is a testis-specific aspartyl intramembrane protease that contributes to male gamete function both by catalytic and non-proteolytic mechanisms. Here, we provide an unbiased characterisation of the in vivo interactome of SPPL2c identifying the ER chaperone calnexin as novel binding partner of this enzyme. Recruitment of calnexin specifically required the N-glycosylation within the N-terminal protease-associated domain of SPPL2c. Importantly, mutation of the single glycosylation site of SPPL2c or loss of calnexin expression completely prevented SPPL2c-mediated intramembrane proteolysis of all tested substrates. By contrast and despite rather promiscuous binding of calnexin to other SPP/SPPL proteases, expression of the chaperone was exclusively required for SPPL2c-mediated proteolysis. Despite some impact on the stability of SPPL2c most presumably due to assistance in folding of the luminal domain of the protease, calnexin appeared to be recruited rather constitutively to the protease thereby boosting its catalytic activity. In summary, we describe a novel, highly specific mode of intramembrane protease regulation, highlighting the need to systematically approach control mechanisms governing the proteolytic activity of other members of the aspartyl intramembrane protease family. [ABSTRACT FROM AUTHOR]

Benzyl butyl phthalate (BBP), a plasticizer found in a wide range of consumer products including vinyl flooring, carpet backing, food packaging, personal care products, and children's toys, is an endocrine-disrupting chemical linked to impaired reproduction and development in humans. Despite evidence that BBP exposure perturbs the integrity of male and female gametes, its direct effect on early meiotic events is understudied. Here, using the nematode Caenorhabditis elegans, we show that BBP exposure elicits a non-monotonic dose response on the rate of X-chromosome nondisjunction measured using a high-throughput screening platform. From among the range of doses tested (1, 10, 100 and 500 μM BBP), we found that 10 μM BBP elicited the strongest effect on the germline, resulting in increased germ cell apoptosis and chromosome organization defects. Mass spectrometry analysis shows that C. elegans efficiently metabolizes BBP into its primary metabolites, monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP), and that the levels of BBP, MBP, and MBzP detected in the worm are within the range detected in human biological samples. Exposure to 10 μM BBP leads to germlines with enlarged mitotic nuclei, altered meiotic progression, activation of a p53/CEP-1-dependent DNA damage checkpoint, increased double-strand break levels throughout the germline, chromosome morphology defects in oocytes at diakinesis, and increased oxidative stress. RNA sequencing analysis indicates that BBP exposure results in the altered expression of genes involved in xenobiotic metabolic processes, extracellular matrix organization, oocyte morphogenesis, meiotic cell cycle, and oxidoreduction. Taken together, we propose that C. elegans exposure to BBP leads to increased oxidative stress and double-strand break formation, thereby compromising germline genomic integrity and chromosome segregation. Author summary: Endocrine disrupting chemicals (EDCs) have been linked to various health problems including effects on reproductive health. Benzyl butyl phthalate (BBP) is a commonly used plasticizer found in many consumer products that has been shown to act as an EDC and affect human reproduction and development. However, how it might impact the germline and affect the specialized cell division program of meiosis, which results in the formation of haploid gametes (i.e. eggs and sperm), remained unclear. Here, using the nematode C. elegans, we show that BBP exposure at levels within the range of what is detected in humans impairs accurate chromosome segregation, which can result in aneuploidy. Similar to mammals, BBP exposure in C. elegans results in a non-monotonic dose response, whereby lower doses result in the strongest effects, and BBP is efficiently metabolized, underscoring the use of this model system for gaining mechanistic insights into how this EDC impacts the germline. BBP results in increased levels of DNA double-strand breaks (DSBs), activation of a DNA damage checkpoint, and altered chromosome morphology in exposed germlines. We propose that BBP exposure alters gene expression and results in increased oxidative stress, resulting in increased DSBs, thereby compromising chromosome morphology and accurate segregation. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Materials and Methods Results Conclusions Hypogonadotropic hypogonadism (HH) is a treatable cause of nonobstructive azoospermic male infertility. Gonadotropin treatment can successfully induce spermatogenesis in most patients, although comprehensive quantitative summary data on spermatogenic outcomes like those required to induce pregnancy is lacking in the literature.Systematic review and meta‐analysis of outcomes related to male reproductive function following gonadotropin treatment.Our search strategy identified 41 studies encompassing 1673 patients with a mean age of 25 (± 5) years. Average sperm concentration achieved after a median of 18 months of gonadotropin treatment was 11.6 M/mL of ejaculate (95% CI 8.4–14.9). Sperm concentrations > 0, > 1, > 5, > 10 and > 20 M/mL were achieved by 78%, 55%, 36%, 24% and 15% of patients, respectively. Mean sperm output and the proportion of patients achieving all sperm thresholds were significantly greater following combined hCG/FSH treatment compared with hCG monotherapy. When compared by diagnosis, patients with congenital HH (CHH) had significantly lower mean sperm output compared with patients with hypopituitarism or mixed patient cohorts that did not differentiate between CHH and hypopituitarism. Treatment‐related increases in testosterone and testicular volume (TV) were not different between hCG and combined hCG/FSH treated patients, although increases in TV were lower in men with CHH compared with those with hypopituitarism.Gonadotropin treatment successfully induced spermatogenesis in most men with pathological gonadotropin deficiency. Sperm outputs more consistent with those typically needed to induce a natural pregnancy were less commonly achieved. Despite similar effects on serum testosterone and TV, combined hCG/FSH appeared more efficacious than hCG alone at inducing spermatogenesis. [ABSTRACT FROM AUTHOR]

Sperm capture techniques that immobilize sperm to halt their motility are essential for the long-term analysis of individual sperm. These techniques are beneficial in assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) by allowing selective screening of sperm. However, there is a notable lack of high-throughput and non-destructive sperm capture methods that allow the flagellum to beat freely, which is crucial for accurately reflecting the behavior of unfettered, freely swimming sperm. To bridge this gap, we introduce a novel microfluidic device specifically engineered to capture sperm without restricting flagellar motion. The design utilizes sperm's innate boundary-following behavior in both 3D and 2D environments to direct them into a capture zone. Once captured, the sperm head is restrained while the flagellum remains free to exhibit natural beating patterns. Utilizing this device, we explore the effects of hyperactivating agents, temperature, and their combined influence on the dynamics of bovine sperm flagella. The unrestricted flagellar motion offered by our device yields two prominent advantages: it mirrors the flagellar behavior of free-swimming sperm, ensuring research findings are consistent with natural sperm activity, and it prevents imaging overlap between the flagellum and the capture structures, simplifying the automation of flagellar tracking and analysis. This technological advancement facilitates the collection of waveform parameters along the entire flagellum, addressing inconsistencies that have arisen in previous research due to differing measurement sites, and enabling precise extraction of sperm behavioral properties. [ABSTRACT FROM AUTHOR]

Microfluidics provides unique opportunities for the high throughput selection of motile sperm with improved DNA integrity for assisted reproductive technologies (ARTs). Here, through a parametric study on dimensions and geometrical angles, a butterfly-shaped chip (BSC) is presented to isolate sperm with high progressive motility and intact DNA at a separation rate of 1125 sperm per minute. Using finite element simulations, the flow field and shear rates in the device were optimized to leverage the inherent motility characteristics of sperm for maximum selection throughput. The device incorporates a triple selection mechanism in series, initially activating sperm rheotaxis by rotation against the semen flow, penetrating the counter buffer flow and swimming against the direction of the buffer flow, leaving dead cells and debris behind, and subsequently leveraging boundary-following behavior to direct progressively motile sperm to swim along the walls and reach the device outlet. The device selects over 4.1 million sperm per mL within 20 minutes, with 29.2%, 68.2%, and 57.3% improvement in total motility, DNA integrity, and velocity parameter (VCL), as compared with the conventional swim-up method, respectively. Overall, the performance of the device to separate sperm with approximately 95.9% total motility, 97.8% viability, and 96.6% DNA integrity at high concentrations demonstrates its potential for enhancing the efficiency of conventional treatment methods. [ABSTRACT FROM AUTHOR]

Background: The management of Non-Obstructive (NOA) Azoospermia or Obstructive Azoospermia (OA) patients relies on testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI). In NOA patients the sperm recovery is successful in only 50% of cases and therefore the ability to predict those patients with a high probability of achieving a successful sperm retrieval would be a great value in counselling the patient and his partner. Several studies tried to suggest predictors of a positive TESE (e.g. FSH concentration), but most concluded that diagnostic testicular biopsy (histology) is best. Methods: This is a retrospective analysis of 526 TESE patients. After the extraction of the testis, the resulting sample was immediately given to the embryologist, who examined the tubules for sperm cryopreservation. During the same procedure, a different specimen was destined to the histological analysis. The comparison between the two methodological approaches was carried out through a score. Results: Concordance between TESE and testicular histology outcomes was found in 70,7% of patients; discordance was found in 29,3% of patients. Among the discordance outcomes, in approximately 95% we found at least 1 sperm in the TESE retrieval, while the histology report did not find any spermatozoa or found not enough compared to our evaluation; in only 5% of cases we did not find any spermatozoa or found not enough compared to what was detected in the testicular histology. Conclusion: Based on our experience, to increase diagnostic accuracy, a larger biopsy should be sent to the histopathology laboratory; another option may be to use TESE cell suspension (the same embryologists employ for cryopreservation) for cytological evaluation of spermatogenesis. [ABSTRACT FROM AUTHOR]

As the global male infertility rate continues to rise, there is an urgent imperative to investigate the underlying causes of sustained deterioration in sperm quality. The gut microbiota emerges as a pivotal factor in host health regulation, with mounting evidence highlighting its dual influence on semen. This review underscores the interplay between the Testis-Gut microbiota axis and its consequential effects on sperm. Potential mechanisms driving the dual impact of gut microbiota on sperm encompass immune modulation, inflammatory responses mediated by endotoxins, oxidative stress, antioxidant defenses, gut microbiota-derived metabolites, epigenetic modifications, regulatory sex hormone signaling. Interventions such as probiotics, prebiotics, synbiotics, fecal microbiota transplantation, and Traditional natural herbal extracts are hypothesized to rectify dysbiosis, offering avenues to modulate gut microbiota and enhance Spermatogenesis and motility. Future investigations should delve into elucidating the mechanisms and foundational principles governing the interaction between gut microbiota and sperm within the Testis-Gut microbiota Axis. Understanding and modulating the Testis-Gut microbiota Axis may yield novel therapeutic strategies to enhance male fertility and combat the global decline in sperm quality. [ABSTRACT FROM AUTHOR]

Our research focuses on semen collection from 42 African penguin males, grouped by age, habituation levels, and reproductive season timing. We assess behavioral and physiological responses to dorso-abdominal massage, evaluating species-specific seminal traits using conventional and advanced methods. Positive behavioral responses corresponded with successful semen collection. Young and human-reared males exhibited more favorable behaviors, and samples containing spermatozoa were more likely collected during the reproductive season. Age did not influence sample collectability or spermatozoa presence, though mature males produced higher semen volumes. Young males exhibited more sperm morphological abnormalities, especially outside the breeding season. Sperm concentration and number per ejaculate showed no significant differences across age or seasonal groups. Young males had higher motile and progressive sperm percentages, while mature males had more static sperm. Additionally, percentages of live sperm and intact acrosomes were higher in mature males. Young males and samples from non-reproductive season presented more dead sperm with damaged acrosomes. Plasma membrane integrity positively correlated with age for live sperm and negatively for dying sperm. This research demonstrates the feasibility of semen collection from African penguins regardless of habituation level, fills the knowledge gap by describing sperm morphological abnormalities, and provides insights into using flow cytometry in Sphenisciformes. [ABSTRACT FROM AUTHOR]

Polyorchidism is an uncommon congenital condition often discovered incidentally, which may significantly impact male fertility. We present a case of a 34-year-old man with unilateral polyorchidism and associated severe asthenozoospermia and teratozoospermia. Despite normal hormonal levels and no detected genetic anomalies, the patient's sperm showed mitochondrial damage, and his fertility remained compromised after conservative management and assisted reproductive attempts. This case underscores the intricate relationship between polyorchidism and male infertility, highlighting the need for personalized management strategies and further research into its etiology and impact. [ABSTRACT FROM AUTHOR]

1. The present study assessed the effect of different antioxidants on the quality of chilled/frozen-thawed sperm of red-legged partridge.2. Sperm samples from 40 red-legged partridges were collected and extended 1:1 (v:v) with Lake and Ravie 84, supplemented with ascorbic acid or butylated hydroxytoluene (BHT) at 0, 0.2, 0.4, 0.8 mM and catalase (CAT) or superoxide dismutase (SOD) at 0, 100, 200 and 300 IU/ml. Ten sperm samples were used per concentration. Motility and viability were evaluated in fresh and after 6 h of chilling at 5°C or after freezing-thawing.3. For chilled sperm, the presence of ascorbic acid decreased viability and several motility variables; BHT 0.8 mM increased non-progressive motility (NPM, 26.7 ± 1.99 vs. 20.7 ± 2.12); CAT 200 IU/ml improved the rectilinear velocity (40.4 ± 4.63 μ/s vs. 29.9 ± 4.62 μ/s) and linear progression ratio (52.8 ± 3.11% vs. 45.4 ± 2.98%); SOD 100 IU/ml increased NPM (24.5 ± 1.21% vs. 19.3 ± 1.75%) and tended to improve total progressive motility (42.7 ± 3.33% vs. 33.2 ± 3.26%, p = 0.07). Using an extender supplemented with CAT 200 or SOD 100 did not improve the post-thawed sperm quality.4. The present work provides an advance in the optimisation of chilling and freezing protocols for red-legged partridge sperm. [ABSTRACT FROM AUTHOR]

Background: Recent advances in microbiome research have revealed the presence of diverse microbial communities in human tissues previously thought to be sterile. The present study delves into the emerging field of seminal plasma microbiomics, examining the relationship between semen microbes and semen parameters and post-freezing tolerance. Methods: The study involved a cohort of healthy fertility males and microbial genome analysis using 16S rRNA to characterize the microbial diversity of seminal plasma. Microbial diversity analysis identified unique amplicon sequence variants (ASVs) and genera dominant in seminal plasma. Spearman’s correlation coefficient was used to assess the relationship between flora and semen parameters. A paired t-test was used to compare the changes in microbiome expression in seminal plasma before and after cryo-resuscitation. Results: The relevant results show that the top five phyla in terms of abundance of seminal plasma microbiome were Firmicutes, Bacteroidota, Proteobacteria, Actinobacteriota, and Campylobacterota. Spearman correlation analysis highlighted the association between specific microbial species and semen parameters, between Porphyromonas_asaccharolytica and sperm concentration. Microbial changed significantly after cryo-resuscitation, affecting taxonomic units such as Campylobacter and Muribaculaceae, and KEGG enrichment analyses, suggesting that metabolic pathways are associated with sperm freezing. Eubacterium_coprostanoligenes and Eptoniphilus_duerdenii exhibited a potential impact, while Orynebacterium_tuberculostearicum demonstrated a positive correlation with the recovery rate of progressive motile sperm. Conclusion: The semen of normal fertile individuals contains a microflora component that is closely related to semen quality, including the sperm’s ability to withstand freezing. [ABSTRACT FROM AUTHOR]

As interest in deep space travel grows exponentially, understanding human adaptation in becoming an interplanetary species is crucial. This includes the prospect of reproduction. This review summarizes recent updates and innovations in assisted reproductive technologies (ART) on Earth, while also discussing current challenges and areas for improvement in adapting ART studies to the space environment. We discuss the critical components of ART - gamete handling and preparation, fertilization, embryo culture, and cryopreservation - from the daily practice perspective of clinical embryologists and reproductive endocrinologists and lay out the complicated path ahead. In vitro embryo development in low Earth orbit and beyond remains questionable due to synergetic effects of microgravity and radiation-induced damage observed in simulated and actual in-space mammalian studies. Cryopreservation and long-term storage of frozen samples face substantial obstacles - temperature limitations, lack of trained personnel, and absence of adapted cosmic engineering options. We touch on recent innovations, which may offer potential solutions, such as microfluidic devices and automated systems. Lastly, we stress the necessity for intensive studies and the importance of an interdisciplinary approach to address numerous practical challenges in advancing reproductive medicine in space, with possible implications for both space exploration and terrestrial fertility treatments. [ABSTRACT FROM AUTHOR]

Background Objective Materials and methods Results Conclusions Infertility affects a significant percentage of couples worldwide, with male infertility contributing substantially in a considerable number of cases. Research indicates that oxidative stress is a critical factor impacting male fertility.To explore the relationship between semen static oxidation–reduction potential (sORP), sperm parameters, and validated biomarkers of oxidative stress in infertile men.This cross‐sectional study involved 202 men diagnosed with idiopathic male factor infertility and male partners from couples with unexplained infertility. Multivariable linear regression to query the associations between sORP, sperm parameters, and oxidative aggression biomarkers (lipid peroxidation, mitochondrial membrane potential, annexin V, and sperm DNA fragmentation).SORP has no linear association with any semen analysis parameter. Furthermore, its relationship with validated biomarkers of oxidative stress was inconsistent. sORP was inversely related to lipid peroxidation (multivariable linear regression coefficient: −0.64), positively associated with sperm DNA fragmentation (multivariable linear regression coefficient: 3.20), and unrelated to mitochondrial membrane potential or annexin V.There is no clear or consistent relationship between sORP and validated oxidative aggression biomarkers or sperm parameters. Our findings suggest that sORP is unlikely to be helpful in the evaluation of a male with idiopathic infertility. [ABSTRACT FROM AUTHOR]

Background: The deep-sea acorn worm Quatuoralisia malakhovi belongs to the phylum Hemichordata, class Enteropneusta, family Torquaratoridae, which was described in 2005. Owing to their epibenthic lifestyle and deep-sea habitat features, torquaratorids differ anatomically from shallow-water acorn worms; however, their morphology and fine structure are poorly studied. We have the opportunity to make three complete detailed series of histological sections of Q. malakhovi and to study the microscopic anatomy, histology and fine structure of the reproductive system of this acorn worm using scanning and transmission electron microscopy. Results: The sexes of Q. malakhovi are separate and indistinguishable externally. The lobed testes occupy the dorsal side of the genital wings and distinctly bulge into the peribranchial cavity by their mature lobes. The central part of the testis is always submerged into the genital wing and opens via a single gonad pore. The monociliary muscle cells stretch along the external wall of the testis and surround the gonad pore, probably taking part in the contraction of the testis lobes for spawning. The germinative epithelium of the testis contains spermatogenic cells at different stages of development and interstitial cells. Yolk cells are not found. Interstitial cells embrace the spermatogonia and spermatogenic columns, providing horizontal compartmentalization of the germinative epithelium, and contain numerous phagosomes with remnants of degenerating spermatogenic cells. The testis wall contains haemal lacunae, which are usually located on the side opposite the gonad pore. We describe the fine structure of spermatogonia, spermatocytes clustered in spermatogenic columns, spermatids, and spermatozoa. Spermatozoa are of the ectaquasperm type and consist of an acorn-shaped head and a flagellum 18–25 µm long. The sperm head includes a beak-shaped acrosomal part, a spherical nucleus and a midpiece containing a ring of 5 or rarely 6 mitochondria. Conclusions: The male reproductive system and sperm structure of Q. malakhovi, a representative of the family Torquaratoridae, have a number of differences from shallow-water acorn worms; however, the spermatogenesis and sperm structure of Q. malakhovi generally follow the pattern of the other three enteropneust families, and the phylogenetic significance of these deviations should be the subject of further research. [ABSTRACT FROM AUTHOR]

ABSTRACT Pollen development and germination are critical for successful generation of offspring in plants, yet they are highly susceptible to heat stress (HS). However, the molecular mechanism underlying this process has not been fully elucidated. In this study, we highlight the essential roles of two mRNA capping enzymes, named Arabidopsis mRNA capping phosphatase (ARCP) 1 and 2, in regulating male and female gamete development. The transmission efficiencies of gametes carrying arcp1 arcp2 from arcp1+/− arcp2−/− and arcp1−/− arcp2+/− mutants are 30% and zero, respectively. These mutants exhibited a significant increase in misshaped pollen, with germination rates approximately half of those in wild type. ARCP1/2 exhibit RNA triphosphatase and RNA guanylyltransferase activities, which are required for proper pollen development. Through RNA‐seq analysis, genes involved in pollen development/germination and HS response were identified as downregulated genes in pollen from arcp1+/− arcp2−/− mutant. Furthermore, ARCP2 protein is degraded under HS condition, and inducing the expression of ARCP2 can increase the pollen germination rate under elevated temperature. We propose that HS triggers the degradation of mRNA capping enzymes, which in turn disrupts the transcriptome that required for pollen development and pollen germination and ultimately leads to male sterility. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation in small ruminant is an efficient strategy to distribute spermatozoa for reproductive programmes, but this process reduces the fertility potential of frozen–thawed spermatozoa. The aim of the current research was to evaluate the impact of different concentrations of cysteamine (CYS) in soybean lecithin (SL)‐based medium on postthawed buck semen quality and fertility potential. Semen samples were collected from five bucks, twice a week, then diluted in the SL‐based extender containing different concentrations of CYS as follows: extender containing 0 mM (control, C0), 1 mM (C1), 2 mM (C2), 4 mM (C4) and 8 mM (C8) CYS. Motility characteristics, membrane integrity, abnormal morphology, mitochondrial activity, acrosome integrity, viability, apoptotic‐like changes, lipid peroxidation, DNA fragmentation, ROS concentration, pregnancy rate and kidding rate were evaluated after freeze–thaw process. In results, C1 resulted in greater (p ≤ 0.05) total motility, progressive motility, average path velocity, membrane integrity, mitochondrial activity, acrosome integrity, viability, pregnancy rate and kidding rate compared to the other groups. Furthermore, supplementation of freezing medium with 1 mM of CYS presented lower (p ≤ 0.05) apoptotic‐like changes, lipid peroxidation, DNA fragmentation and ROS concentration compared to the other groups. On the other hand, C8 presented the least (p ≤ 0.05) total motility, progressive motility, average path velocity, membrane integrity, mitochondrial activity, acrosome integrity and viability as well as the highest (p ≤ 0.05) apoptotic‐like changes, lipid peroxidation, DNA fragmentation and ROS concentration compared to the other groups. Therefore, supplementation of freezing medium with 1 mM CYS could be a helpful strategy to protect buck's spermatozoa quality and fertility potential during cryopreservation process. [ABSTRACT FROM AUTHOR]

A rapid decline in wild animal biodiversity, as well as in the number of local livestock breeds of lower economical values, makes species and breeds protection and creation of genetic reserves a necessity and a challenge of the 21st century. For birds, semen freezing is still the best method of gene preservation ex situ in vitro. The aim of the presented study was to assess the susceptibility to freezing process and to establish the semen gene bank of twelve goose breeds covered by the Polish genetic resources conservation program. Sexually mature ganders were randomly selected from four northern local breeds: Kartuska, Pomorska, Rypinska, Suwalska, four southern local breeds: Garbonosa, Kielecka, Lubelska, Podkarpacka, and four foreign goose breeds: Kuban, Landes, Roman and Slovakian. Each breed was represented by 12 males, twelve repetitions were carried out. Semen samples were frozen in a computer-controlled cryogenic chamber using a method developed for White Koluda goose. The basic evaluation criterion was sperm morphology in fresh and freeze-thawed semen assessed in nigrosine-eosin stained histological smears. The percentage of live in total and live normal sperm in the fresh semen differed significantly (P<0.05) between breeds. Semen of Kartuska and Suwalska ganders (northern breeds), contained the highest percent of live sperm – 94.4 and 94.1 respectively, including 52.5% and 54.2% of live normal sperm, while the lowest number of these forms was observed for Slovakian goose – 86.4% and 29.2%, respectively. The freezing process resulted in a significant (P<0.05) reduction in the total number of live and live normal sperm and an increase in dead sperm in all groups. Cryopreservation process was the most effective in case of Suwalska goose semen (28.4% of live normal cells in thawed semen) and the less profitable for Landes (7.4% of live normal cells). The analyses showed significant differences between breeds in sperm morphology in fresh and thawed semen. Moreover, the high proportion of live spermatozoa in the fresh semen did not guarantee a high proportion of spermatozoa after thawing, indicating the differences in semen susceptibility to cryopreservation process depending on goose breed. [ABSTRACT FROM AUTHOR]

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm‐associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild‐type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function. Summary: Analyzing the role of male reproductive tract‐specific proteins in male fertility using knockout models is an active area of investigation. Although a large number of knockout models for genes that are specific to the testis are generated and the reproductive function studied, there are very few studies that analyze the role of epididymal proteins that contribute to sperm function and fecundity. In this study, the effect of knockout of an epididymal‐specific gene, Spag11a, on male reproductive function, especially the fecundity, capacitation, acrosome reaction, and sperm proteome was analyzed. Our observations indicate subfertility and compromised sperm function associated with perturbations in sperm proteome. Using shRNA‐mediated Spag11a mRNA ablation and active immunization‐mediated SPAG11A protein ablation, we previously demonstrated the possible role of Spag11a in sperm function and fecundity. This study significantly differs from our previous studies because we used a higher level of gene ablation model, that is, the Spag11a knockout mice to provide further evidence. Furthermore, we also analyzed the molecular changes at the proteome level under conditions of Spag11a gene knockout. Thus, this study carried a lot of significance since it reiterates the observations made in our previous studies using an animal model that truly reflects the ablation of Spag11a expression. [ABSTRACT FROM AUTHOR]

Sperm function is suggested to evolve by sexual selection but is also reduced by microbial damage. Here, we provide experimental evidence that male fertility can adapt to microbes. We found that in vivo, male fertility was reduced by one-fifth if sperm encountered microbes in the females that they had not previously been exposed to, compared to sperm from males that coevolved with these microbes. The female immune system activation reduced male fertility by an additional 13 percentage points. For noncoevolved males, fertility was larger if microbes were injected into females after they had stored away the sperm, indicating microbial protection as a previously unrecognized benefit of female sperm storage. Both medical and evolutionary research on reproductive health and fertility will benefit from considering our findings that the impact of microbes on sperm depends on their joint evolutionary history. Our results may assist in reconciling contradictory results of sexually transmitted disease effects on sperm and bring empirical realism to a recently proposed role of locally adapted reproductive microbiomes to speciation. [ABSTRACT FROM AUTHOR]

Objective: To study the effect of switching to a follicle-stimulating hormone (FSH) preparation other than that to which infertile male patients have not had an effective response. Patients and methods: Seventy-four normogonadotropinemic, non-obstructive, oligozoospermic patients who were poor responders to the administration of highly purified FSH (hpFSH) (Group 1 (n = 22) and Group 3 (n = 15)) or to recombinant human FSH (rhFSH) (Group 2 (n = 22) and Group 4 (n = 15)) were selected for this prospective study. After 3 months of washout from treatment with the first FSH preparation of choice, rhFSH was administered to patients in Groups 1 and 4 and hpFSH to those in Groups 2 and 3. Serum luteinizing hormone, FSH, total testosterone levels, conventional sperm parameters, testicular volume, and the number of pregnancies were evaluated at study entry and after the first and second treatment cycles. Results: Comparing treatment groups, the greatest improvement in sperm parameters was recorded in the groups of patients prescribed the switch in FSH preparation. Group 1 had the greatest benefit from therapy, with the highest pregnancy rate after the second treatment cycle. Indeed, eight couples achieved pregnancy (36.4%), compared to Groups 2 (n = 4; 18.2%), 3 (n = 1; 6.7%), and 4 (n = 2; 13.3%) (p = 0.04). Conclusions: The results of this study suggest that a therapeutic scheme involving the "switching" of the FSH preparation yields better results than a protocol using the same FSH preparation for six months. These findings, if confirmed by further studies, will help us better design a treatment strategy with FSH for infertile patients with oligozoospermia. [ABSTRACT FROM AUTHOR]

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility. [ABSTRACT FROM AUTHOR]

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor protein-coding gene, considered a master regulator of the cellular stress response. The genetic variations of the NRF2 could influence its transcriptional profile and, subsequently, the stress resilience in all cell types, including sperm cells. Therefore, the sperm-borne antioxidants abundance in association with the genetic variation of a GCC microsatellite located at the 5′ upstream region of the NRF2 gene was investigated in young (n = 8) and old (n = 8) Holstein bulls' sperm cells at different seasons. The sperm DNA was sequenced using Sanger sequencing, while- the sperm-borne mRNA analysis was carried out using the synthesized cDNA and qPCR. The data were statistically analyzed using GraphPad Prism 10.0.2 software. The results showed that two bulls had a heterozygous genotype of eight and nine GCC repeats, while biallelic of eight, nine, and fifteen repeats were identified in two, ten, and two bulls, respectively. The computational in silico analysis revealed that the NRF2 upstream sequence with 15, 9, and 8 GCC repeats bound with 725, 709, and 707 DNA-binding transcription factor proteins, respectively. Lower quality of sperm DNA was detected in the spring season compared to other seasons and in young bulls compared to old ones, particularly in the summer and autumn seasons. The mRNA expression analysis revealed that the PRDX1 gene was the abundant transcript among the studied sperm-borne antioxidants and was significantly determined in old bulls' spermatozoa. Moreover, two transcripts of the NRF2 gene and antioxidant (SOD1, CAT, GPX1, TXN1, NQO1) genes displayed differential expression patterns between the age groups across seasons in an antioxidant-dependent manner. The bulls with a heterozygous GCC sequence exhibited elevated sperm-borne mRNA levels of NRF2 and PRDX1 transcripts. Taken together, the findings suggest that the NRF2-GCC microsatellite may contribute to the transcription regulation of NRF2 transcripts and their subsequent downstream antioxidants in bovine sperm cells. [ABSTRACT FROM AUTHOR]

The Chinese rare minnow (Gobiocypris rarus), an important model fish in China, faces endangerment in the wild. Sperm cryopreservation facilitates the development of new strains and germplasm conservation, but the quality of its cryopreserved sperm remains low. This study evaluates the protective effects of different concentrations of antifreeze proteins (AFP I and AFP III) on the cryopreservation of Chinese rare minnow sperm. Cryopreserved sperm showed significant declines in progressive motility, curvilinear velocity (VCL), average path velocity (VAP), and lifespan compared to fresh sperm, except for straight-line velocity (VSL). The cryomedium containing 10 μg/mL AFP I improved these parameters to their highest levels. However, no significant difference was found in progressive motility and kinetic parameters between cryopreserved sperm with and without AFPs. Cryopreserved sperm with 10 μg/mL AFP I showed the highest plasma membrane integrity, mitochondrial activity, and DNA integrity, significantly better than without AFPs; importantly, the fertilization rate of cryopreserved sperm with 10 μg/mL AFP I was not significantly different from that of fresh sperm. These results indicate that the addition of 10 μg/mL AFP I to the cryomedium for Chinese rare minnow sperm does not improve kinetic parameters but significantly enhances sperm quality, aiding in its new strain development and germplasm conservation. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and ultrastructure. AgNPs were synthesized, purified and resuspended in the extender. Nine frozen–thawed donkey sperm samples were exposed to different concentrations of AgNPs (0, 1.25, 2.5, 5, 12.5, 25 and 50 μg/mL). Sperm parameters: total (TMOT, %) and progressive (PMOT, %) sperm motility, plasma (LIVE, %) and acrosomal membrane integrity (AIS, %), and sperm morphology (MORF, %) were evaluated immediately after AgNPs exposure (T0) and after 2 h of incubation (T2). The interaction beween AgNPs and spermatozoa was visualized by transmission electron microscopy (TEM). At T0, sperm motility and AIS were reduced (p <.05) when using concentrations ≥50 and ≥25 μg/mL, respectively. At T2, sperm motility and LIVE were significantly decreased (p <.05) in concentrations ≥25 and ≥50 μg/mL, respectively. TEM analysis revealed nanoparticle adhesion to the acrosomal region of the plasma membrane. In conclusion, AgNPs at concentrations ≥25 μg/mL impair motility, acrosome and plasma membrane integrity of donkey sperm, which may be mediated by adhesion to the acrosomal region of the sperm surface membrane. [ABSTRACT FROM AUTHOR]

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co‐incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization‐IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p >.05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p >.05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p >.05). In conclusion, in vitro exposure of boar sperm to OVGP1‐functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP. [ABSTRACT FROM AUTHOR]

In this study, we evaluated sheep sperm quality after using Tris‐citrate‐fructose‐based extender with and without egg yolk, a Tris‐citrate without fructose and with egg yolk and the commercial extender Biladyl®, preserving diluted semen at 15 and 23°C for different times (4, 24, 48 and 72 h). The results showed that the diluents with fructose and egg yolk gave the best results of seminal quality. Moreover, the production of ROS was higher for the temperature of 23°C compared to the temperature of 15°C (control). In addition, VCL and the percentage of spermatozoa with intact acrosome decreased with temperatures of 23°C. Finally, a drastic decrease in sperm quality was observed after 24 hours of preservation for most of the parameters evaluated. [ABSTRACT FROM AUTHOR]

The present study compares two protocols for the cryopreservation of chicken semen. Both protocols had an initial low cooling rate in the first step, followed by higher cooling rates around ice nucleation (Protocol 1) or following the dissipation of the latent heat of fusion (Protocol 2) in the second step. Semen ejaculates obtained from 12 roosters were diluted with Rootex with 6% dimethylformamide and frozen following either Protocol 1 (from +5°C to −10°C at 5°C/min and from −10°C to −130°C at 60°C/min) or Protocol 2 (from +5°C to −35°C at 7°C/min and from −35°C to −140°C at 60°C/min). Compared with fresh semen, following both protocols, cryopreservation resulted in reduced post‐thaw sperm quality (p <.001). Post‐thaw percentage of sperm with an intact plasma membrane was greater using Protocol 2 than Protocol 1 (p <.05). The results suggest that high cooling rates around the time of ice nucleation are not recommendable. [ABSTRACT FROM AUTHOR]

Germplasm banking is a fundamental tool for the preservation of autochthonous breeds. Semen cryopreservation is effective for this task, but protocols are adapted to commercial species, and post‐thawing sperm quality could be sensitive to environmental cues. We compared the post‐thawing sperm quality in doses from the CBA‐SERIDA bank in northern Spain for the Asturiana de la Montaña (AM) and Asturiana de los Valles (AV) autochthonous cattle breeds. Doses from 23 AM and 16 AV bulls (ejaculates from at least three different seasons) were assessed for motility (computer‐assisted sperm analysis), physiology and chromatin status (flow cytometry) after thawing and after 5 h at 38°C. Data were analysed using linear mixed‐effects and cosinor models for seasonal and breed effects and by correlations with the association of sperm quality with temperature‐humidity index (THI), considering the interval of spermatogenesis plus maturation. The breed affected sperm quality, with higher motility for AV and higher apoptotic ratio, mitochondrial activity, reactive oxygen species, DNA fragmentation and chromatin immaturity for AM. However, seasonality effects were minimal, and THI was not associated with sperm quality. In summary, the season seems to be a minor factor in the post‐thawing quality of the AM and AV autochthonous breeds, well‐adapted to their local environment. [ABSTRACT FROM AUTHOR]

Goat production is affected by reproductive seasonality. In vitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM‐199 with hormones, EGF and cysteamine; fertilized and cultured in BO‐IVF and BO‐IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO‐IVF, CAP) and non‐capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p <.05). After CAP, acrosome reaction and intracellular calcium were higher (p <.05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo in vitro capacitation. [ABSTRACT FROM AUTHOR]

A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT‐TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen–thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT‐TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT‐TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p <.001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p <.05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p <.001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF. [ABSTRACT FROM AUTHOR]

This study investigates the impact of bovine serum albumin (BSA) inclusion in the thawing extender on boar sperm quality. Thawing protocols in sperm cryopreservation are vital, yet underexplored. It has been determined that BSA can interact with membranes, stabilizing them and preventing damage during the freezing process, for this reason it could also have a beneficial effect during thawing. Our study explores modifications in the conventional Beltsville thawing solution, incorporating BSA at different concentrations (0.005, 0.01, 0.025 and 0.050 g/mL), and evaluating the sperm quality after up to 150 min post‐thawing incubation (37°C). All BSA concentrations preserved plasma membrane and acrosome integrity, relative to control (BSA absence). In addition, 0.025 g/mL BSA group preserves acrosome integrity over time without compromising motility or kinetic parameters. Our study suggests that BSA inclusion in thawing extenders should be considered as an additive for improving post‐thaw boar sperm quality. [ABSTRACT FROM AUTHOR]

The aim was to evaluate the influence of residual feed intake (RFI) on the semen characteristics of Dorper sheep after cryopreservation. Six Dorper sheep were used, classified from a population of 64 according to feed efficiency (groups of animals-positive RFI and negative RFI). The ejaculates were harvested three times a week, with a minimum interval of 48-h between collections, being seven collections per animal, totalling 42 ejaculates. Motility, plasma membrane integrity, acrosomal, mitochondrial activity and perivitelline membrane binding tests were performed. Negative RFI animals presented higher progressive motility (31.28%) than of RFI positive (25.37%). Negative and positive RFI animals present similar qualities in the assessment of plasma and acrosomal membrane integrity and mitochondrial activity, and negative RFI animals presented a higher number of membrane-bound spermatozoa than the positive RFI animals. Negative RFI animals displayed better rates of sperm motility after thawing, and are more efficient in the fertilisation in vitro. RFI did not influence the integrity of the plasma and acrosomal membrane, nor the mitochondrial activity of Dorper sheep spermatozoa after cryopreservation. [ABSTRACT FROM AUTHOR]

Diabetes leads to testicular damage and infertility. Mesenchymal stem cells and their secretory trophic factors have shown potential as regenerative therapies for diabetes and its associated complications. This study examined the effects of conditioned medium derived from Wharton's jelly mesenchymal stem cells (WJMSCs‐CM) on sperm parameters, reproductive hormones, biochemical parameters, and histological changes in the testes of diabetic rats. Fifty‐six male Sprague–Dawley rats (250–300 g) were assigned to eight groups: control, diabetes, and six diabetic groups receiving early or late treatments with WJMSCs‐CM (D‐CME, D‐CML), insulin (D‐INSE, D‐INSL), or DMEM (D‐DME, D‐DML). In the early treatment groups, insulin (3 U/day, subcutaneously) and WJMSCs‐CM (10 mg/week, intraperitoneally) were administered immediately after diabetes induction; in the late treatment groups, these interventions began 30 days postinduction. Blood glucose and insulin levels, along with sperm parameters, were assessed. Sex hormones, testicular antioxidant enzyme activity, malondialdehyde (MDA), and glutathione (GSH) concentrations were measured using colorimetric methods. Real‐time PCR detected Bax, Bcl‐2, and tumor necrosis factor‐alpha (TNF‐α) gene expression. Our results showed that diabetes increased blood glucose levels, decreased insulin and sex hormone levels, induced testicular oxidative stress and apoptosis, and reduced sperm parameters compared to the control. WJMSCs‐CM significantly ameliorated hyperglycemia, increased insulin and sex hormone levels, and improved sperm quality. In WJMSCs‐CM‐treated diabetic rats, MDA levels were reduced, while GSH and antioxidant enzyme activity increased. Furthermore, WJMSCs‐CM decreased the testicular Bax/Bcl‐2 ratio and TNF‐α expression, as well as enhanced spermatogenic, Sertoli, and Leydig cells. In conclusion, WJMSC‐CM administration effectively mitigated diabetes‐induced testicular damage by reducing oxidative stress, inflammation, and apoptosis. Early treatment with WJMSCs‐CM was more effective than late treatment for diabetes‐induced reproductive dysfunction. [ABSTRACT FROM AUTHOR]

Background: Antioxidants of natural origin can be used to improve cryopreservation of semen. Objective: To evaluate the effect of dietary supplementation with Erythrina americana on the seminal characteristics of rams. Methods: Ten rams were randomly distributed in two treatments: l) Group supplemented with E. americana (20% dry matter basis; AEA, n=5), and 2) Control group not supplemented (SEA, n=5). Both diets were iso-proteic and iso-energetic. The feeding period was eleven weeks. Ejaculates were obtained through an artificial vagina and were evaluated fresh (37 °C) and refrigerated (5 °C). Semen volume (VOL) was assessed by visual examination in a calibrated (mm) tube; sperm concentration (CON x106) with a photometer; and mass motility (MM, 1-5), individual motility (IM), normality (N), and live sperm (L) were assessed by microscopic observation. Results: Feeding E. americana did not affect (p>0.05) VOL, CON, MM, and N in fresh semen, but decreased (p<0.05) IM and L. The L and N variables did not differ (p>0.05) among treatment groups in the refrigerated semen; however, IM improved in the AEA group (p<0.05) compared to the SEA group. Conclusion: Dietary supplementation of rams with E. americana reduces sperm quality in fresh semen but is beneficial for the cooling process of refrigerated semen. [ABSTRACT FROM AUTHOR]

Aim: To assess the efficacy of intratesticular injection of autologous platelet‐rich plasma (PRP) in men with non‐obstructive azoospermia (NOA) and a history of failed microdissection‐testicular sperm extraction (mTESE) procedures. Methods: A prospective case series of a cohort study was conducted involving couples diagnosed with NOA. Patients with at least one failed mTESE procedure were included. Intratesticular PRP injection was performed using a standardized protocol. Follow‐up assessments included sperm analysis, hormonal evaluation, and in vitro fertilization (IVF) outcomes. Results: Data from 177 men with NOA were analyzed, with 135 patients meeting eligibility criteria. PRP treatment resulted in positive sperm retrieval rates of 27.5% in patients with one prior failed mTESE procedure and 16.4% in patients with two or more failed attempts. IVF outcomes showed fertilization rates of 86.4% and 100.0% in respective groups, with pregnancy rates of 36.8% and 22.2% per embryo transfer. Histopathological examination post‐mTESE revealed varied patterns, including Sertoli cell‐only syndrome and maturation arrest. Conclusions: Intratesticular PRP injection shows promise as a potential therapeutic approach for NOA patients with prior failed mTESE procedures, demonstrating improved sperm retrieval rates and favorable IVF outcomes. Further randomized controlled trials are warranted to validate these findings and refine the technique's efficacy in male infertility management to answer the question of whether PRP could significantly improve the second attempt retrieval rate. [ABSTRACT FROM AUTHOR]

1. This study investigated the potential use of glycerol as a cryoprotectant for quail sperm cells. Its role in maintaining sperm fertilising ability in vivo and in vitro quality parameters, such as motility, was assessed. 2. The data showed that the presence of glycerol in semen samples was associated with infertility, which suggested that removal prior to insemination is mandatory. Removal through serial dilution centrifugation was associated with fewer than 5% of motile sperm cells and resulted in no fertility. 3. In conclusion, glycerol alone is not suitable for quail semen cryopreservation, and other approaches need to be investigated to develop cryobanking programmes for this species. [ABSTRACT FROM AUTHOR]

Background: Currently, approximately half of all pregnancies worldwide are unintended. Contraceptive use significantly reduces the risk of unintended pregnancy; however, options for men are particularly limited. Consequently, efforts are underway to develop novel, safe, and effective male contraceptives. Results: This review discusses research into emerging male contraceptive methods that either inhibit sperm production or impair sperm function. It focuses on those in the preclinical or early clinical stages of development. [ABSTRACT FROM AUTHOR]

Environmental exposure is associated with increased incidence of respiratory and cardiovascular diseases and reduced fertility. Exposure to air pollution can influence gene expression through epigenetic mechanisms. In this study, we analysed gene-specific CpG methylation in spermatozoa of city policemen occupationally exposed to air pollution in two Czech cities differing by sources and composition of the air pollution. In Prague, the pollution is mainly formed by NO2 from heavy traffic. Ostrava is a hotspot of industrial air pollution with high concentrations of particular matter (PM) and benzo[a]pyrene (B[a]P). We performed genome-wide methylation sequencing using the SureSelectXT Human Methyl-Seq system (Agilent Technologies) and next-generation sequencing to reveal differentially methylated CpG sites and regions. We identified differential methylation in the region chr5:662169 – 663376 annotated to genes CEP72 and TPPP. The region was then analysed in sperm DNA from 117 policemen using targeted methylation sequencing, which proved its hypermethylation in sperm of Ostrava policemen. [ABSTRACT FROM AUTHOR]

Purpose: Optimal sperm DNA integrity is essential for fertilization and embryo health. Research indicates that testicular sperm (TS), obtained via TESA or TESE, typically show lower sperm DNA fragmentation (SDF) than ejaculated sperm after standard abstinence. Shortening abstinence to less than 2 days might reduce SDF, offering a less invasive and more cost-effective alternative to surgical sperm retrieval. Yet, no studies have directly compared the efficacy of shorter abstinence against TS extraction for lowering SDF. Our meta-analysis aims to address this gap by comparing SDF levels in TS to those in ejaculated sperm after a short abstinence period. Methods: Meta-analysis of 16 randomized controlled and prospective observational studies included 4 on TS and 12 on short abstinence ejaculation. The meta-analysis followed MOOSE guidelines, scrutinizing databases including Cochrane Library, Web of Science, Embase, MEDLINE(R), and PUMBED up to November 16, 2023. The analysis was conducted using RevMan. The observational studies' methodological quality was assessed using the Newcastle–Ottawa Scale, and the overall evidence quality was evaluated following the GRADE criteria. To compare short ejaculation duration and TS (are not directly compared in the literature) for SDF levels, we analyzed relevant data from studies of each method. We adjusted the participant numbers in the TS group by 1/3 and included each TS study three times, to perform a comparison against the short duration studies which were in a ratio of 1:3. This approach maintained an unaltered cumulative subject count for the meta-analysis of TS studies. Results: A total of 641 patients were included, comprising 120 and 521 patients with SDF measurements following TS and ejaculation after a short abstinence period, respectively. The studies had varied inclusion criteria, with not all patients having an initial elevated SDF. Some studies had incomplete details on age and other demographics. However, the mean ± SD age of 93 TS patients was 38.15 ± 5.48 years vs. 37.7 ± 6.0 years of 444 short abstinence patients, demonstrating no significant difference (P = 0.544). Short abstinence durations ranged from 1 to 48 h. Diverse DNA fragmentation tests were used: TUNEL assay in three testicular sperm studies, SCD assay in one, and in the short abstinence group, four used TUNEL and six used SCD assays, along with one each using SCSA and Halosperm. The mean ± SD SDF was lower in the TS group than in the short abstinence group (mean difference − 9.48, 95%CI − 12.45 to − 6.52, P < 0.001, I2 = 85%). Sensitivity analysis revealed that no single study significantly influenced the results. Employing the GRADE criteria, the initial assessment categorized the overall quality of evidence as low due to the observational nature of the acquired data. All studies were of medium to high quality. Conclusion: This study suggests testicular sperm may be better than ejaculated sperm for improving SDF in infertility cases. Direct comparisons are needed, before deeming short abstinence less effective. Future research should directly compare reproductive outcomes using both methods. [ABSTRACT FROM AUTHOR]

Little is known about the effects of low gravity on human gametes. The aim of this study was to analyze if fresh human sperm samples after fast gravity load changes suffered any detrimental effect in comparison to the splits maintained in Earth's gravity. Fifteen fresh samples from normozoospermic donors were analyzed. Statistically significant differences in vitality (69.7 ± 9.9 % vs 72.4 ± 9.7 %, [95 % CI: 0.002,0.07]); motile sperm concentration (23.7 ± 15.3 M/ml vs 31.5 ± 25.1 M/ml, [95 % CI: 1.03,14.65]); grade "a" sperm concentration (8.7 ± 6.5 M/ml vs 11.7 ± 9.9 M/ml, [95 % CI: 0.71,5.28]); percentage of progressive motility sperm (30 ± 12.9 % vs 36 ± 14.3 %, [95 % CI: 0.10,0.37]) and curvilinear velocity VCL: 45.7 ± 12.8 μm/s vs 47.7 ± 13.3 μm/s, [95 % CI: 0.79,3.22]) were observed. No statistical differences were observed in other sperm kinematic parameters, morphology, DNA fragmentation, apoptosis, and oxidative stress. In conclusion, even though it did not result in a total loss, heavy gravity load changes including microgravity causes a significant decrease in sperm vitality and motility suggesting that negative consequences would be even higher if the exposure were longer. The results obtained indicate that further research is really needed before Assisted Reproduction will be considered for the future human reproduction outside the Earth. • Motility and Vitality of human sperm are significantly decreased after parabolic flight including short periods of microgravity. • Curvilinear Velocity of human male gametes was particularly decreased. • No changes were found in other sperm kinematic parameters, morphology, DNA fragmentation, apoptosis and oxidative stress. • Results were found after short exposure; suggesting more negative consequences regarding human fertility in a longer time exposure. • More studies needed before Assisted Reproduction Techniques can be considered for the likelihood of Human Reproduction in Space. [ABSTRACT FROM AUTHOR]

Embryo development is stimulated by calcium (Ca2+) signals that are generated in the egg cytoplasm by the fertilizing sperm. Eggs are formed via oogenesis. They go through a cell division known as meiosis, during which their diploid chromosome number is halved and new genetic combinations are created by crossing over. During formation the eggs also acquire cellular components that are necessary to produce the Ca2+ signal and also, to support development of the newly formed embryo. Ionized calcium is a universal second messenger used by cells in a plethora of biological processes and the eggs develop a "toolkit", a set of molecules needed for signaling. Meiosis stops twice and these arrests are controlled by a complex interaction of regulatory proteins. The first meiotic arrest lasts until after puberty, when a luteinizing hormone surge stimulates meiotic resumption. The cell cycle proceeds to stop again in the middle of the second meiotic division, right before ovulation. The union of the female and male gametes takes place in the oviduct. Following gamete fusion, the sperm triggers the release of Ca2+ from the egg's intracellular stores which in mammals is followed by repetitive Ca2+ spikes known as Ca2+ oscillations in the cytosol that last for several hours. Downstream sensor proteins help decoding the signal and stimulate other molecules whose actions are required for proper development including those that help to prevent the fusion of additional sperm cells to the egg and those that assist in the release from the second meiotic arrest, completion of meiosis and entering the first mitotic cell division. Here I review the major steps of egg formation, discuss the signaling toolkit that is essential to generate the Ca2+ signal and describe the steps of the signal transduction mechanism that activates the egg's developmental program and turns it into an embryo. [ABSTRACT FROM AUTHOR]

Introduction: Since boar spermatozoa show a marked deterioration in sperm quality when cooled, insemination doses are usually stored at 16-18 °C. However, maintaining this temperature during transport of semen doses is challenging, particularly during the summer months. An alternative could be to store the doses at 4 °C if cold-shock to the sperm could be prevented. The objective of this study was to evaluate boar sperm quality and fertility in in vitro fertilization after storage in AndroStar Premium at 4 °C for 1 week. Methods: Insemination doses (n =9) in AndroStar Premium from a commercial boar semen collection station were transported to the laboratory at approximately 20 °C. At the laboratory, sperm quality evaluation and was preformed and each dose was split; half of each ejaculate was stored in a climate-controlled box at 16-18 °C, the other was slowly cooled to 4 °C. Both samples were stored for 1 week before further sperm quality evaluation and in vitro fertilization (IVF) were performed. Mean values were tested using generalized linear regression, with treatment and boar as fixed factors; p ≤ 0.05 was considered significant. Results: Sperm membrane integrity (mean±sem: 91±0.05 and 83±0.09% for 16 and 4 °C, respectively) and superoxide production (6.79±2.37 and 13.54±6.23% for 16 and 4 °C, respectively), were different between treatments. The DNA fragmentation index was lower in cold-stored samples than in conventionally stored samples (3.74±2.25 and 7.40±3.36% for 4 and 16 °C, respectively). The numbers of oocytes developing to blastocyst on Day 6 (mean±sd: 9.0±8.0 and 6.0±5.0%, for storage at 16 and 4 °C, respectively) were not different between treatments. Discussion: Therefore, storage of boar semen doses in AndroStar Premium at 4 °C for up to 7days would be a viable alternative to current praxis. [ABSTRACT FROM AUTHOR]