Search Results (3,268)

Background: Antibiotic-free approaches to boar semen preservation are gaining importance to counter emerging antimicrobial resistance. Hypothermic storage at 5 °C, instead of the conventional 17 °C, is a promising strategy to eliminate antibiotics still commonly used in extenders. For practical adoption, the method must be simple and compatible with on-farm routines. Objective: To assess fertility when cooling was initiated on farm one day after delivery, and to evaluate the robustness of cold-stored semen to temporary warming and subsequent re-cooling, mimicking typical handling on insemination days. Methods: Individual ejaculates (n = 34) from six boars were extended in Androstar^® Premium either without antibiotics (5 °C) or with gentamicin (17 °C control). One day after collection, antibiotic-free doses were cooled on farm to 5 °C and used alongside controls in routine insemination of 270 sows. Sperm quality was evaluated by computer-assisted semen analysis and flow cytometry, and bacterial counts were monitored. In a separate test, cold-stored doses were exposed to 20 °C for 60 min and re-cooled to 5 °C. Results: Farrowing rates and litter sizes did not differ between groups (p > 0.05). In antibiotic-free samples after 120 h, bacterial counts were mostly not detectable or low (<10² CFU/mL). Sperm motility and plasma membrane integrity in cold-stored doses remained >80%, comparable to controls (p > 0.05). Temporary warming did not affect sperm quality or bacterial counts. Conclusions: Antibiotic-free semen storage at 5 °C is easy to implement in practice and maintains fertility under field conditions. Broader validation under routine conditions is encouraged in support of the One Health concept. Full article

Zeugodacus tau, a highly destructive agricultural quarantine pest causing severe economic losses to global fruit crops, urgently requires the development of male fertility-based control strategies. Here, we identified and characterized the testis-specific serine/threonine protein kinase 1 gene (ZtTSSK1) in Z. tau. The encoded protein of ZtTSSK1 is highly conserved among dipteran species. Spatiotemporal expression analysis revealed predominant expression in adult males, with specific localization to the testes. In situ hybridization further localized ZtTSSK1 transcripts to the transformation zone. Furthermore, functional characterization by RNA interference (RNAi) revealed that knockdown of ZtTSSK1 resulted in a significant 62% reduction in sperm counts. While egg numbers laid by females mated to dsZtTSSK1- versus dsGFP-injected males did not differ, hatching rates were significantly lower for eggs from dsZtTSSK1 matings. These findings establish ZtTSSK1 as a key regulator of spermatogenesis and male fertility in Z. tau, providing a theoretical foundation and candidate target for genetic-based sterile insect technique (gSIT) development. Full article

Cryptorchidism is one of the major reproductive diseases affecting testicular function in yaks. However, the mechanisms underlying its impact on testicular spermatogenesis remain unclear. In this study, high-throughput transcriptomics (RNA-seq) and proteomics technologies were employed to analyze the key signaling pathways involved in cryptorchidism-induced spermatogenic dysfunction in yak, and a mouse model was established for validation. The results indicate that differentially expressed genes (DEGs) in the testes of yak with cryptorchidism are primarily enriched in the hypoxia-inducible factor (HIF) signaling pathway and the PI3K-AKT signaling pathway. Experimental results from the hypoxic mouse model indicate that the hypoxic environment remarkably raised HIF-1α content in the blood of mice while activating the PI3K-AKT signaling pathway, accompanied by decreased testicular expression of the cell adhesion molecules (CAMs) claudin 2, claudin 3, E-cadherin, and N-cadherin. Cell culture experiments showed that cell adhesion molecule expression was significantly downregulated when HIF-1α and PI3K expression were inhibited among mouse Sertoli cells, indicating that the HIF-1α/PI3K-AKT signaling pathway regulated cell adhesion molecule expression among mouse testes. Decreased CAMs directly affect tight junctions and the adhesion of spermatogenic and Sertoli cells, thus affecting sperm production and potentially also testis development. This study provides data to support research on the regulatory mechanisms involved in reproductive function and hypoxia adaptation in male animals in a low-oxygen environment. Full article

This study evaluated functional and proteomic changes in ram sperm during 48 h of liquid storage at 5 °C, comparing samples collected at two breeding-season stages (July and November). Semen from six Assaf rams was diluted in INRA 96^® and stored at 15 °C for 6 h and at 5 °C for 24 and 48 h. Sperm functionality (motility and kinetics, viability, apoptotic-like changes, and mitochondrial reactive oxygen species content) and proteomic profiles were assessed at each storage protocol. Sperm collected in July showed a significantly faster decline in motility and kinetics, viability, and mitochondrial reactive oxygen species levels, along with increased apoptotic-like changes during storage (p < 0.05), resulting in lower values for several functional parameters after 48 h compared with November samples. Proteomic analyses revealed more pronounced changes in protein abundance during storage in July samples, including enrichment of metabolic pathways and reduction in proteins associated with membrane stability and ion regulation. In contrast, November samples showed fewer changes in protein abundance, mainly involving mitochondrial and regulatory proteins. These results indicate that ram sperm resistance to liquid storage varies across breeding-season stages and is associated with distinct proteomic profiles. The findings provide descriptive molecular insights that may support future studies aimed at improving semen preservation strategies in ovine reproduction. Full article

Oxidative stress (OS) and inflammation are common causes of male infertility; antioxidants in spermatozoa and especially in seminal plasma play a protective role. The study aims to evaluate whether OS lipid mediators (F₂-isoprostanes: F₂-IsoPs; Resolvin D1: RvD1) measured in seminal plasma, and Trolox equivalent antioxidant capacity (TEAC), measured in both seminal plasma and spermatozoa, are associated with male infertility. Semen samples (18 fertile and 62 infertile subjects) were examined following WHO guidelines and with a mathematically elaborated transmission electron microscopy analysis (fertility index-FI-, % sperm pathologies). F₂-Isops were measured by GC/NICI-MS/MS, RvD1 by ELISA, and TEAC by a commercially available antioxidant assay. F₂-Isops, RvD1, and sperm TEAC (positively interrelated with each other) correlated negatively with seminal parameters and FI and positively with sperm pathologies. F₂-Isops, RvD1, and sperm TEAC levels were significantly higher in infertile vs fertile subjects (p < 0.001). ROC curve analysis demonstrated that sperm TEAC (J index: 0.13 mM) and seminal RvD1 (J index: 38.26 pg/mL) discriminated between fertile and infertile subjects. Spermatozoa stimulate antioxidant capacity in the presence of an OS environment; this data suggests an association in which antioxidant defences may vary in relation to the surrounding seminal plasma. Moreover, sperm TEAC, and to a lesser extent seminal RvD1, emerge as potential markers for identifying infertile patients. Full article

Background/Objectives: Aberrant DNA methylation of imprinted genes and increased sperm DNA fragmentation (SDF) have been implicated in male infertility. However, their impact on assisted reproductive technology (ART) outcomes remains unclear. This pilot study aimed to investigate SDF and methylation status of H19, IGF2, and PEG1/MEST in relation to fertilisation and embryo development following intracytoplasmic sperm injection (ICSI). Methods: Twenty male partners of women undergoing ICSI were recruited and classified according to ART outcome into viable embryos (VEs, n = 7), non-viable embryos (NVEs, n = 7), and no fertilisation (NF, n = 6). Before sperm selection, an aliquot of each seminal sample was used for semen analysis according to WHO, 2021, SDF assessment (TUNEL assay), and sperm DNA methylation analysis of H19, IGF2, and PEG1/MEST (pyrosequencing). Results: Semen parameters were above the fifth percentile. SDF was significantly lower in the VE group compared with the other groups. H19 CpG1 methylation correlated positively with viable embryos (p = 0.016), while H19 CpG2 island showed a positive correlation with sperm concentration (p = 0.028). In male/couple infertility cases, total H19 methylation correlated negatively with SDF (p = 0.050). IGF2 CpG3 island methylation correlated positively with viable embryos (p = 0.027). Total PEG1/MEST methylation was positively correlated with fertilisation events (p = 0.002) and viable embryos (p = 0.011). PEG1/MEST CpG2 island also positively correlated with sperm motility (p = 0.034), while CpG3 and CpG4 showed significant correlations with fertilisation (p < 0.001; p = 0.004). Conclusions: This pilot study shows that SDF and sperm methylation levels of H19, IGF2, and PEG1/MEST are related to ICSI outcomes, supporting that sperm molecular and epigenetic features may influence fertilisation and embryo development. Full article

Adropin, a peptide hormone first identified by microarray analysis of gene expression in mice’s liver, is expressed in multiple organs, including the brain, liver, and heart. It is a key regulator of carbohydrates and lipid metabolism. Moreover, it has anti-inflammatory and antioxidative effects. It enhances blood vessel dilation and is essential for the normal development and function of the cerebellum. It acts through binding to multiple receptors, primarily the orphan G protein-coupled receptor, which is expressed in various tissues, including the central nervous system, liver, heart, kidneys, and testis, suggesting a direct role of adropin in modulating the function of multiple organs. This review discusses recently identified testicular functions regulated by adropin. Some studies have demonstrated that adropin can stimulate testosterone synthesis in testicular Leydig cells by enhancing the expression of steroidogenic enzymes. Moreover, it increases germ cell proliferation and sperm formation by inhibiting apoptosis and oxidative stress. Current evidence remains limited, and further studies are required to clarify the underlying mechanisms of adropin in reproductive physiology. Moreover, its potential role in conditions associated with altered testosterone levels or impaired spermatogenesis remains speculative and requires validation in well-designed clinical studies. Full article

Infertility is a global health problem, with male factors contributing to nearly half of all cases. Up to 30% of male infertility is classified as idiopathic, in part because routine semen analysis does not assess sperm fertilizing competence. Capacitation is a complex process that endows spermatozoa with the competence to fertilize the oocyte, and it depends on oxidant-driven phosphorylation events. These events include increased PKA substrate and tyrosine phosphorylation, which promote hyperactivated motility and the acrosome reaction. These pathways are normally restrained by decapacitation factors that must be relieved in the female reproductive tract before capacitation can proceed. Protein CoAlation is an antioxidant modification of protein thiols through a disulfide bond with coenzyme A (CoASH). We previously detected protein CoAlation in human spermatozoa and observed that its levels decline during capacitation, but its function was unknown. We hypothesized that protein CoAlation functions as a decapacitation mechanism that prevents redox signalling, enabling oxidative activation of phosphorylation events during capacitation. Using spermatozoa from healthy human donors, we leveraged subcellular fractionation, immunocytochemistry, computer-assisted sperm analysis (CASA), and immunoblotting to determine the sperm protein CoAlation profile, assess CoASH biosynthetic enzymes, and test how pharmacological modulation of CoAlation levels influences capacitation. CoAlated proteins were distributed across intracellular sperm compartments, and spermatozoa possess the CoASH biosynthetic enzymes PANK2 and CoASY, indicating an intrinsic capacity for CoAlation. Inhibition of CoASH biosynthesis reduced CoAlation and enhanced PKA substrate phosphorylation, tyrosine phosphorylation, hyperactivated motility, and the progesterone-induced acrosome reaction under capacitating conditions. Pantothenic acid supplementation increased CoAlation and suppressed these processes without impairing viability or baseline motility. These findings indicate that high levels of protein CoAlation in several protein bands are a pre-existing feature of the non-capacitated state that restrains the redox-regulated events of capacitation and that its decline is required to permit sperm capacitation. CoAlation levels may emerge as a biomarker of sperm capacitation and fertilizing competence. Full article

Background: The cotton boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a major pest of cotton that causes significant yield losses and costly control measures. As a cotton-specialized species, its population dynamics are heavily influenced by environmental conditions and management practices during the off-season. This study examined the effects of off-season food sources on adult survival, reproductive capacity, and susceptibility to malathion in the last generation before the off-season and in the generation entering the next planting season. Methods: Females were fed diets representing off-season conditions (cotton terminals and pollen) and the standard diet (flower buds), and then evaluated for survival, copulation, sperm viability, and egg production. Concentration–mortality bioassays determined malathion susceptibility before and after the off-season. Results: Female survival was lowest in those fed cotton terminals, while pollen and flower buds supported higher survival rates. The likelihood of copulation and sperm viability remained similar among diets after 60 days, or up to 100 days in surviving females; however, females fed flower buds had more eggs in their oviducts. Females that mated early maintained viable sperm up to the end of the 100-day assessment period. Populations collected after the off-season were more susceptible to malathion than those collected at the end of the growing season. Conclusions: Food restrictions and reduced malathion exposure during the off-season may contribute to a reversion toward boll weevils’ susceptibility to malathion. Overall, the findings highlight the importance of the fallow period and proper management of residual off-season populations for effective long-term boll weevil control, confirming malathion’s continued viability as a management tool. Full article

Bumblebees are key pollinators in agricultural and natural ecosystems; however, climate change is driving shifts in population size, body size, and diversity. Accelerating global warming affects bumblebee body size, thereby influencing the strength of sexual selection and overall population fitness. Thus, this study aimed to investigate the effects of body mass and temperature on sexual selection in Bombus terrestris reared under warm conditions, with mating conducted between equal sex ratios at both optimal (23 °C) and elevated (32 °C) temperatures. Mating success was lower at 32 °C than at 23 °C, regardless of body mass; however, larger queens and males consistently exhibited higher mating success at both temperatures than their smaller counterparts. Mate-choice patterns were similar across temperatures: large queens predominantly mated large males, whereas small ones mainly mated small males. However, small queens tended to mate large males at 32 °C than at 23 °C. Meanwhile, matings between small queens and small males occurred relatively later at both temperatures. Although small males exhibited longer mating durations than large ones, the amount of sperm transferred to the spermathecae of the queens was positively associated with the body size of both males and queens. Our findings indicate that, at equal sex ratios, mate choice in bumblebees is primarily determined by body mass at all temperatures. Thus, smaller bumblebees associated with higher temperatures had the lowest mating success. Full article

The highly compact chromatin and naturally fragmented RNA content of bovine sperm make it difficult to obtain sufficient total RNA for transcriptome sequencing. Bovine sperm studies have mostly adapted the somatic-cell protocol and used generic kits. To date, no commercial extraction kit is available for total RNA from sperm. These limitations prompted optimization of a sperm total RNA isolation protocol, tested using both fresh and cryopreserved sperm. Ejaculates were collected from two Girolando bulls (Bos taurus × Bos indicus) and were subsequently processed for cryopreservation. In total, eight RNA extraction protocols were tested, namely four TRIzol^®-based (GP) protocols and four spin-column (SC) methods. Across both fresh and cryopreserved sperm, Protocol D of SC protocols (SC-D) was the most suitable choice for total RNA sequencing. For fresh sperm, ~25–29 million filtered reads were obtained, and cryopreserved sperm yielded ~83–125 million filtered reads, indicating that cryopreserved sperm is also a good source of sperm RNA based on initial sequencing metrics. By pinpointing SC-D as the optimal balance in terms of yield and sequencing performance, and by identifying practical solutions for DNA carryover and storage-related duplication, the study’s results provide a streamlined protocol and quality control framework for total RNA sequencing of Girolando sperm. Full article

This study aimed to investigate the effects of supplementing with an essential oil blend and ascorbic acid on performance, semen characteristics, antioxidant status, gut microbiota, immunity, and gene expression in heat-stressed male rabbits. One hundred and forty male New Zealand White rabbits, aged 6 months, were randomly distributed into four dietary groups: the control group receiving basal feed (CON), the group receiving an essential oil blend (EOB, 200 mg/kg), the group receiving ascorbic acid (ASA, 1000 mg/kg), and the group receiving an essential oil blend and ascorbic acid (MAO). The experimental period lasted for eight weeks. The MAO mixture supported rabbits’ tolerance to heat stress by enhancing stress markers, as demonstrated by decreased glucose and HSP70 and increased triiodothyronine (T3). In addition, increased body weight, carcass weight, and nutrient digestibility, but reduced mortality rate, were observed in rabbits fed the MAO mixture. Additionally, semen density and volume, as well as sperm progressive motility and normality, were enhanced in rabbits fed the MAO mixture. Furthermore, MAO mixture supplementation decreases plasma cholesterol, triglycerides, AST, urea, and creatinine levels, while raising HDL and total protein levels. Adding MAO mixture contributed to an increase in plasma SOD and GPx levels, as well as seminal fluid TAC and GSH levels. Additionally, the incorporation of MAO increases IgA, IgG, and IL-10 levels while decreasing IL-6 and TNF-α levels. Adding the MAO mixture reduced C. perfringens and E. coli, as well as increased the expression of the MUC-2, CAT-1, and CLDN-1 genes. Combining an essential oil blend and ascorbic acid may contribute to improvements in performance, semen quality, immune response, antioxidant capacity, and gut health in heat-stressed male rabbits. Full article

The equine reproductive microbiota has emerged as an important factor influencing reproductive health and fertility in both mares and stallions. Traditionally, the equine uterus was considered sterile, and microbial presence was interpreted primarily in the context of infection. However, sequencing-based studies have demonstrated that the reproductive tract, including the uterus and semen, contains detectable microbial communities or microbial DNA signatures, challenging this traditional paradigm. In mares, the vaginal microbiota is consistently dominated by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, whereas the uterine environment represents a low-biomass niche in which interpretation is complicated by contamination risk and the inability of sequencing-based methods to distinguish viable from nonviable microorganisms. Culture-based studies consistently identify opportunistic pathogens such as Streptococcus equi subsp. zooepidemicus, Escherichia coli, and Pseudomonas aeruginosa in association with endometritis and persistent breeding-induced endometritis, while sequencing-based studies suggest broader community-level dysbiosis rather than simple pathogen presence. In stallions, semen is not sterile and commonly contains taxa such as Porphyromonas, Corynebacterium, Peptoniphilus, and other opportunistic bacteria that may influence sperm quality and microbial transmission to mares during breeding. However, most reported associations remain correlative, and direct longitudinal evidence for persistent stallion-to-mare microbial transmission is limited. This review synthesizes current evidence on microbial composition, hormonal influences, dysbiosis, and reproductive implications of the equine reproductive microbiota, integrating culture-based and sequencing-based findings while emphasizing methodological limitations associated with low-biomass samples. Improved understanding of these microbial ecosystems may support more evidence-based reproductive diagnostics and microbiome-informed fertility management in horses. Full article

Background: Cryopreservation induces the production of excessive reactive oxygen species (ROS), which decreases sperm physiological functions. Phytochemicals with antioxidant properties, such as fisetin, have shown promising results in reducing oxidative stress (OS). Aim: We aimed to evaluate whether fisetin can counteract the OS exerted on sperm. Methodology: Fisetin (15 and 30 µM) was tested on normozoospermic semen samples that were either frozen in liquid nitrogen or treated with H₂O₂ to induce OS. Sperm motility, sperm viability, mitochondrial membrane potential, metabolic activity, ROS content, lipid peroxidation, reduced glutathione, ATP contents, and apoptosis were tested and compared to controls. Results: The protective effect of fisetin on human sperm was observed against OS-induced stress. Fisetin significantly improved sperm motility, viability, mitochondrial and metabolic activity, and ATP content by reducing OS and lipid peroxidation. Fisetin reduced necrotic cell death and improved sperm survival under H₂O₂-OS. Conclusions: Fisetin protects human sperm from OS, with 30 µM showing greater effectiveness, supporting its potential use in sperm preservation and OS conditions. Further studies are needed to optimize its concentration, elucidate its mechanism of action, and confirm its putative use as an additive in sperm cryoprotective media. Full article

Background and Objectives: The microbiome plays a pivotal role in male infertility, with distinct microbial species exerting both beneficial and deleterious effects on reproductive function. Sexually transmitted bacteria and several viruses, including human papillomavirus (HPV), have been identified in semen. This cross-sectional study aimed to examine the prevalence of single and co-infections of sexually transmitted bacteria (STB)—such as Chlamydia trachomatis, Mycoplasma spp., and Ureaplasma spp.—with various HPV subtypes in Greek male partners of infertile couples and to evaluate their potential impact on sperm parameters. In addition, the possible effect of cryopreservation on the maintenance of these pathogens was assessed. Materials and Methods: Eighty-two semen samples were initially collected from 82 individuals undergoing routine sperm analysis. In total, 80/82 (97.6%) participants proceeded to further analysis, as 2/82 (2.4%) were excluded due to poor DNA quality. Results: A total of 18/80 (22.5%) sperm samples tested positive for STB, with Ureaplasma spp. representing the most frequently detected pathogen. Co-infection of Ureaplasma spp. and Mycoplasma hominis was observed in 4/80 (5%) samples. Twelve samples (12/80, 15%) were positive for HPV, including low-risk (LR) and high-risk (HR) types, and HPV 16 was the predominant HR genotype. Notably, a co-infection of STB and HPV was not found in our specimens. STB-positive samples demonstrated significantly higher sperm concentration and improved progressive motility compared with STB-negative samples. HPV-positive samples exhibited lower sperm volume and concentration and increased non-progressive motility compared with HPV-negative samples. Following three months of cryopreservation, LR HPV and STB were no longer detectable, whereas HR HPV types remained detectable. Conclusions: These preliminary findings are interesting, as they could be useful for routine screening of HPV and STB in sperm samples preserved in sperm banks and highlight the need for future research. Full article