To explore the protective effects of selenium nanoparticles (SeNPs) against renal, hepatic, and reproductive toxicity induced by aflatoxin, male Sprague-Dawley rats (n = 36), 3 months old and weighing 162-233 g (198.17 ± 2.64 g), were equally divided into control, SeNPs, aflatoxin, and SeNPs-aflatoxin groups. Blood and tissue samples from the liver, kidneys, and testicles were collected. Luteinizing hormone (LH), testosterone, estradiol (E2), interleukin-β (IL-β), transforming growth factor-β (Tgf-β), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total antioxidants (TAC), nitric oxide (NO), AST, ALT, and creatinine were measured. DNA fragmentation and the expression of activity-regulated cytoskeleton-associated protein (Arc), Early growth response factor 1 (Egr1), and JunB Proto-Oncogene (JunB) were determined in the liver. SeNPs and SeNPs-aflatoxin increased testosterone (P < 0.001) and estradiol (P = 0.004) but lowered TGF-β (P = 0.013) compared with the aflatoxin group. Aflatoxin decreased SOD (P < 0.047) and CAT (P < 0.0001) activities and increased NO concentrations (P = 0.004). SeNPs, aflatoxin, and SeNPs-aflatoxin decreased AST (P < 0.01) and ALT (P < 0.0001) but increased creatinine (P < 0.001). Aflatoxin and SeNPs-aflatoxin increased DNA fragmentation% and hepatic Arc, Egr1, and JunB expression (P < 0.0001), whereas SeNPs decreased them. Aflatoxin caused severe degenerative alterations in the testis, liver, and kidneys, including vacuolization, necrosis, and structural disorganization. SeNPs mitigated the toxic effects of aflatoxin by improving tissue organization and minimizing cellular damage. Significant histological improvements were noted, although full restoration was not achieved. In conclusion, SeNPs could protect to some extent against the hepatic and testicular toxic effects of aflatoxin in male rats. Renal toxicity could not be ameliorated to normality due to the nephrotoxic effects of both SeNPs and aflatoxin.
The development of trophoblast organoids (TOs) has revolutionized placental research by providing proliferative, three-dimensional systems recapitulating the key aspects of placental architecture and function. These models have become invaluable tools for studying trophoblast differentiation and early placental development. Importantly, TOs also offer new opportunities for investigating mechanisms underlying pregnancy-related disorders such as preeclampsia or fetal growth restriction, and host-pathogen interactions, advancing our understanding of how infections impact early pregnancy. However, the relevance and translatability of experimental outcomes strongly depend on aligning the research question with the most appropriate model system. Despite their potential, current models still lack critical physiological components, including vascular and immune elements. This review discusses TOs application in translational science and highlights future directions toward integrating these systems into more complex microphysiological platforms to better model human placental function and disease. These advances not only deepen our understanding of placental biology but also pave the way for clinically relevant applications, notably to guide therapeutic interventions in pregnancy-related disorders.
Asthenozoospermia, a common cause of male infertility characterized by reduced sperm motility, has been linked to oxidative stress and ferroptosis. Long non-coding RNAs (lncRNAs) are emerging regulators of redox balance and cell death. This study aimed to investigate the expression profile of the lncRNA taurine upregulated gene 1 (TUG1) and its relationship with ferroptosis-related dysfunction in spermatozoa from men with asthenozoospermia. Semen samples were collected from Iranian men with asthenozoospermia and normozoospermic controls. A bioinformatic screen of ferroptosis-associated lncRNAs identified TUG1 as a candidate biomarker, with several isoforms predicted to be dysregulated in asthenozoospermic sperm. Quantitative real-time PCR was used to assess the expression of TUG1, the ferroptosis-suppressing antioxidant enzyme gene glutathione peroxidase 4 (GPX4), and the somatic-cell markers CD5 and CD34. Lipid peroxidation was quantified by measuring malondialdehyde (MDA) levels. Associations among gene expression, oxidative stress markers, body mass index (BMI), and semen parameters were analyzed. Asthenozoospermic men exhibited significant upregulation of TUG1 and downregulation of GPX4 compared with normozoospermic controls. Elevated MDA levels positively correlated with TUG1 expression and inversely with GPX4 mRNA level, suggesting increased oxidative stress and possible ferroptosis-related involvement. Reduced expression of CD5 and CD34 confirmed efficient removal of somatic-cell contamination. Higher BMI was negatively correlated with sperm motility indices. Altered expression of TUG1 and GPX4 in asthenozoospermic sperm is consistent with involvement of ferroptosis-related pathways in impaired motility. These findings provide mechanistic insight into oxidative-stress-mediated sperm dysfunction and may inform future diagnostic and therapeutic strategies for male infertility.
Understanding primate testicular development is essential for elucidating the evolution of male fertility. Here, we integrated publicly available single-cell RNA sequencing datasets from 23 human and 4 macaque testes spanning infant to adult stages. Integrated analysis identified 22 conserved germ cell subtypes, including spermatogonial stem cells, spermatocytes, and spermatids. Notably, a human-enriched late spermatid-like transcriptional state, Sperm.C2, was enriched for genes involved in chromatin condensation, acrosome formation, and fertilization readiness, but was not detected in the macaque dataset analyzed here. Comparative transcriptomic profiling revealed age-related transcriptional dynamics in SSCs, reflecting species-biased developmental strategies between humans and macaques. Moreover, candidate regulators such as CELF4 and CTCF were predicted to be associated with SSC maintenance and spermatid maturation. Together, these findings delineate conserved and species-biased molecular programs governing spermatogenesis and provide a comparative framework for understanding primate germline development.
Reliable prediction of semen production in breeding bulls requires an understanding of the interactions among developmental history, physiological maturity, and environmental conditions, yet these relationships remain poorly characterized in indigenous Bos indicus breeds. This study quantified the independent and interactive effects of developmental, physiological, and environmental factors on longitudinal semen production records of Kankrej breeding bulls using a Restricted Maximum Likelihood (REML)-based mixed-effects model. A total of 4012 ejaculates collected from 24 breeding bulls during 2011-2020 were analysed. Bull was included as a random effect to account for repeated observations, whereas season and period of birth, age at first semen collection, age at semen collection, season and period of semen collection, ejaculate number, and biologically relevant interaction terms were fitted as fixed effects. Mixed-model analysis revealed significant associations between semen production traits and several non-genetic factors, including biologically relevant interactions. Age at semen collection and ejaculate number consistently influenced semen production, while seasonal conditions modified these responses. The findings highlight the value of longitudinal mixed-effects modelling for evaluating non-genetic variation in semen production and may support evidence-based reproductive management, optimization of semen collection strategies, and improved utilization of elite indigenous breeding bulls in artificial insemination programmes.
In the conservation of endangered wildlife such as Eld's deer (Rucervus eldii), assisted reproductive technologies including artificial insemination play an important role in enhancing reproductive success and maintaining genetic diversity. However, semen cryopreservation induces molecular alterations that may impair post-thaw sperm function. This study aimed to characterize changes in the seminal plasma proteome of Eld's deer across fresh, pre-freeze, and post-thaw stages of cryopreservation, using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cryopreservation resulted in a consistent reduction of fertility-associated and immune-related proteins, including binder of sperm 1 (BSP1), spermadhesin-1 (SPADH1), polymeric immunoglobulin receptor (PIGR), and beta-defensin 1 (DEFB1). In contrast, proteins associated with oxidative stress defense and ubiquitin-mediated protein regulation, including glutathione S-transferase theta-4 (GSTT4), ubiquitin thioesterase OTUB1 (OTUB1), and ubiquitin carboxyl-terminal hydrolase 1 (USP1), were increased following cryopreservation. These findings indicate a functional shift in the seminal plasma proteome from fertility-related components toward stress-response and cellular maintenance pathways during cryopreservation. Notably, GSTT4, OTUB1, and USP1 were identified as candidate proteins associated with cryopreservation-related responses. Further validation is required to determine their potential utility as biomarkers of sperm cryotolerance and their relevance to improved semen preservation strategies for endangered Eld's deer.
Embryo biopsy is widely used in human assisted reproduction for preimplantation genetic testing, but its application in bovine in vitro fertilization (IVF) programs remains limited, largely due to the cost and complexity of laser-based systems. Here we describe a micromanipulation method for laser-free embryo biopsy in bovine embryos that relies on a mechanical separation technique to collect embryo samples while preserving embryo viability. This method provides a practical alternative for embryo biopsy in bovine IVF systems and may facilitate the incorporation of embryo genotyping into genetic improvement programs.
Endometrosis is a chronic degenerative condition of the endometrium in the mare, with fibrosis being the most prevalent. In the current study, the transcriptome signature of in vitro cultured fibroblasts derived from endometria with or without endometrosis was compared by RNA sequencing to elucidate alterations in gene expression that lead to dysregulated cellular function and the underlying molecular mechanisms driving fibrosis. A total of 191 genes were identified as altered, including 43 up-regulated and 148 down-regulated. In addition, the differentially expressed genes (DEGs) were annotated to nine KEGG pathways and almost 280 Gene Ontology (GO) terms. The DEGs classified as belonging to the biological process category were annotated with terms including extracellular matrix (ECM) organization, wound healing, and regulation of the innate immune response. The DEGs classified as 'molecular function' were annotated with terms including collagen binding, metaloendopeptidase activity, L-ascorbic acid binding, and fibroblast growth factor. Moreover, DEGs were assigned to KEGG pathways, including ECM-receptor interaction, transcriptional misregulation in cancer, focal adhesion, and others. The results showed that the transcriptomic profile of fibrotic fibroblasts was altered in comparison to non-fibrotic fibroblasts, which consequently could affect their function. The gene profile of enzymes responsible for ECM remodelling was downregulated, indicating that the excessive ECM deposition in the fibrotic process cannot be degraded. The expression of immune-related genes differed between fibrotic and non-fibrotic fibroblasts, suggesting that fibroblasts from fibrotic endometrium may be more active in modulating immune responses, which play a crucial role in the development of fibrosis.
Vitamin D3 (VD3) is a pleiotropic hormone with diverse physiological functions, but its role during early embryonic development in fish remains poorly understood. This study investigated the effects of aqueous VD3 exposure on embryonic survival, hatching, and the expression of the kiss1, kiss2, gnrh3, foxr1, and hsp70 genes in common carp (Cyprinus carpio L.). We also examined the hypothesis that common carp embryo is a site of VD3 non-genomic action and its metabolism, evaluated by measuring 25-hydroxyvitamin D3 (25OHD3) levels and confirmed by immunolocalization of VDR, PDIA3 as well as CYP24A1 and CYP27B1. The eggs (obtained from 6 females) were fertilized and incubated in water containing VD3 at concentrations: 0, 1, 10, 50, and 100 ng/mL. During early embryonic development (24, 48, 72 h post-fertilization - hpf), VD3 did not reveal developmental abnormalities and mortality in common carp but increased larvae hatching rate at the highest tested concentration. Furthermore, early life exposure to VD3 has shown no alteration in kiss1, kiss2, gnrh3, foxr1, hsp70 gene expression in newly hatched larvae. Crucially, VD3 exposure altered embryonic 25OHD3 concentrations. This finding, alongside the immunohistochemical localization of VDR, PDIA3, CYP27B1 and CYP24A1 in the tissue of common carp embryo, indicates the possible genomic and non-genomic action of VD3 as well as its active metabolism (synthesis and inactivation) at early life stages of this species. These findings encourage further studies to better understand the role of VD3 in reproduction and early life stages in cyprinids.
Semen analysis in non-human primates is widely used for colony management, reproductive studies, and experimental design. However, variability in sperm quality across repeated ejaculates and its implications for semen interpretation remain insufficiently characterized. In this study, repeated semen samples were collected from individual cynomolgus monkeys and evaluated using conventional semen parameters, computer-assisted sperm analysis (CASA)-derived kinematics, and functional sperm assessments, including viability, morphology, acrosome integrity, and acrosome reaction. Substantial variability was observed across repeated ejaculates. Progressive motility exhibited marked variability and was not consistently associated with sperm concentration at the ejaculate level. Kinematic parameters, such as amplitude of lateral head displacement, varied independently and did not uniformly correspond with motility percentages. Functional assessments further revealed heterogeneous patterns, with relatively stable viability and morphology but greater variability in acrosome-related parameters. Collectively, these findings indicate that sperm quality parameters do not vary in a concordant manner and reflect distinct biological properties. Sperm quality in cynomolgus monkeys therefore represents a multidimensional and ejaculate-dependent biological state rather than a fixed individual characteristic. Variability across repeated ejaculates may influence semen interpretation, indicating that single-ejaculate assessment may incompletely represent male reproductive status.
This study aimed to evaluate the influence of in vitro culture medium supplementation with zinc oxide nanocomposites (NCs) doped with magnesium (Mg²⁺), synthesized using magnesium chloride (MgCl₂) as the precursor (ZnO:0.5Mg-MgO) compared to MgCl2 supplementation at concentrations of 10, 20, and 30 µg/mL on the viability of bovine ovarian tissue fragments cultured in vitro. The following treatments were performed: Fresh Control, day 0 (FC D0); Control, day 7 (CONT D7); 10 µg/mL of MgCl2 (MG10); 20 µg/mL of MgCl2 (MG20); 30 µg/mL of MgCl2 (MG30); 10 µg/mL of ZnO:0.5Mg nanoparticles (NPs) (NPMG10); 20 µg/mL of ZnO:0.5Mg NPs (NPMG20); 30 µg/mL of ZnO:0.5Mg NPs (NPMG30). Cellular degeneration, reactive oxygen species (ROS) emission, cellular respiratory metabolism through nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) emission, and redox status were analyzed, in addition to follicular activation and viability. Statistical analyses were conducted using SigmaPlot software, applying analysis of variance followed by Fisher's LSD post hoc test for mean comparisons, with significance set at P < 0.05. The NPMG20 treatment showed lower cell degeneration rates, higher NAD and FAD autofluorescence, lower ROS generation, and higher follicular viability compared with the controls and MG20. These findings indicate that magnesium-doped ZnO proved effective, providing a favorable environment for cellular and follicular development. However, the potential role of ion interactions in mitigating toxicity remains to be further elucidated.
Pyroptosis, a form of inflammatory programmed cell death, has been implicated in the pathogenesis of non-obstructive azoospermia (NOA). However, pyroptosis-related gene function in NOA pathogenesis has yet to be fully characterized. Publicly available datasets were interrogated to pinpoint differentially expressed genes in patients with NOA relative to healthy controls. These genes were cross-referenced with curated pyroptosis-related gene sets to define pyroptosis-related differentially expressed genes (PRDEGs). Functional enrichment, Protein-protein interaction mapping and diagnostic evaluation were carried out. Immune infiltration was quantified by single-sample gene set enrichment analysis, with subsequent correlation assessment. A busulfan-induced azoospermia mouse model was established, and hub genes were validated by RT-qPCR, Western blotting, and immunofluorescence. Fifteen PRDEGs were identified and were significantly enriched in pathways related to proliferation, differentiation, and apoptosis. NFE2L2 and HMGB1 were identified as top hub genes, with strong diagnostic performance (AUC > 0.9) and markedly increased expression. Both genes were strongly correlated with most immune cell types (r > 0.5, p < 0.05). In the mouse model, Nfe2l2 and Hmgb1 mRNA levels and NFE2L2 and HMGB1 protein levels were increased in testicular tissues, with extensive extracellular localization of HMGB1. Our findings substantiate the involvement of NFE2L2 and HMGB1 in NOA pathogenesis and reveal strong associations between their expression and the organization of testicular immune infiltrates. Collectively, the results implicate NFE2L2 and HMGB1 in NOA pathogenesis, highlighting their dual utility as both diagnostic indicators and druggable targets.
The buffalo is an important species in many regions of the world, particularly in Asia, Africa and parts of Europe. Among the various species of buffalo, the water buffalo (Bubalus bubalis) is the most widespread, and it is well-known for its ability to thrive in wet and humid environments. In Europe, Italy is one of the leading producers of buffalo milk, and is particularly renowned for the production of Mozzarella di Bufala, a cheese made from the milk of water buffalo. Scientific knowledge on applied physiology, health, husbandry and reproduction in buffalo remains limited. This may be partly due to the fact that the species has historically been of greatest economic importance in regions where production systems are predominantly traditional and research investment has been limited. More recently, research has increasingly focused on production and feeding strategies and breeding practices. In addition, much of the research on the reproductive biology and management has concentered on the female buffalo, with particular emphasis on seasonality, oestruses detection and induction, whereas relatively little attention has been paid to male reproduction.This review discusses the reproductive physiology and behaviour of the male domestic buffalo in relation to differences in management and environmental conditions under which the species is reared, with a particular focus on the Italian Mediterranean buffalo breed (IMB).
Hyaluronic acid (HA)-based sperm selection has been proposed as a physiological strategy to enrich for mature spermatozoa with improved chromatin integrity during intracytoplasmic sperm injection (ICSI). However, whether these biological characteristics translate into improved embryo chromosomal competence remains uncertain. This prospective single-center sibling-oocyte randomized study evaluated the effect of HA-assisted ICSI on embryo euploidy in couples undergoing ICSI with preimplantation genetic testing for aneuploidy (PGT-A). Metaphase II oocytes retrieved within the same cycle were randomly allocated to HA-ICSI or conventional ICSI. The primary outcome was the proportion of euploid embryos among embryos with conclusive PGT-A results. Generalized estimating equations were used to account for embryo clustering within patients, and sperm DNA fragmentation was assessed using TUNEL assay with flow cytometry. Overall, 94 couples undergoing 95 cycles were included, yielding 258 embryos analyzed by PGT-A, including 119 in the HA-ICSI group and 139 in the conventional ICSI group. Euploidy rates were comparable between groups (52.1% vs. 54.7%; p = 0.773). In GEE analyses, sperm selection method was not associated with embryo euploidy (OR 1.20; 95% CI 0.68-2.13; p = 0.52). Fertilization outcomes were also comparable between groups. Most patients exhibited low sperm DNA fragmentation, with 81.1% showing values below 20%. These findings did not demonstrate a measurable advantage of HA-based sperm selection in embryo chromosomal competence in a population predominantly characterized by low sperm DNA fragmentation. However, smaller effects cannot be excluded.
This study aimed to characterize a long-term in vitro culture of feline oviduct epithelial spheroids (fOES) and to compare the effects of supplementation with fetal bovine serum (FBS) and bovine serum albumin (BSA) on fOES parameters. BSA improved structural preservation, reduced oxidative stress, and enhanced cell viability and motility. Despite morphological and metabolic changes, fOES preserved functional characteristics, supporting their use as a reliable in vitro model for reproductive studies in felids.
This study was conducted to investigate the effects of nano-selenium (nano-Se) and glycyrrhiza extracts (GE) on reproductive performance, antioxidant capacity, and immune functions of primiparous sows, as well as growth performance of intrauterine growth retardation (IUGR) piglets. A total of 60 primiparous sows (Landrace × Yorkshire) were randomly divided into four groups (15 sows per group): 1) CON group, basal diet (0.30 mg·kg-1 Se, sodium selenite); 2) Nano-Se group, basal diet + 0.20 mg·kg-1 added Se (nano-Se); 3) GE group, basal diet + 500 mg·kg-1 added GE; 4) Combined group, basal diet + 0.20 mg·kg-1 added Se (nano-Se) and 500 mg·kg-1 added GE. The trial lasted for 46 days, from day 90 of gestation to weaning. In terms of growth performance, sole GE supplementation significantly improved the lactational litter weight gain and IUGR piglet weight gain (P < 0.05), and combined nano-Se and GE supplementation also exerted significant beneficial effects on these indicators compared with the CON group (P < 0.05), whereas individual nano-Se supplementation showed no significant effect on lactational litter weight gain (P = 0.335). For maternal antioxidant status, all dietary supplementation treatments increased serum glutathione peroxidase (GSH-Px) activity and decreased malondialdehyde (MDA) content in sows (P < 0.05), and the combined treatment further enhanced serum superoxide dismutase (SOD) activity (P < 0.05). Additionally, maternal nano-Se and GE supplementation significantly increased immunoglobulin G (IgG) and immunoglobulin A (IgA) levels in sow serum and colostrum (P < 0.05). In conclusion, perinatal dietary supplementation with nano‑Se and GE effectively enhances the antioxidant status and immune function of primiparous sows and improves postnatal growth performance in IUGR piglets.
We investigated the roles of bilateral intraovarian platelet-rich plasma (PRP) injection and ovarian mechanical damage during injection on the remaining functional ovarian reserve in rats with a premature ovarian insufficiency (POI) model. We analyzed serum anti-Mullerian hormone (AMH) levels, follicle-stimulating hormone (FSH) levels, follicle counts, fibrosis scores, histologic and immunohistochemical examinations, and related parameters. Twenty-eight adult female Wistar albino rats were randomly divided into four groups and treated with various combinations of cyclophosphamide, PRP, mechanical ovarian damage (MOD) and sodium chloride. In week eight, the rats were anesthetized, and their ovaries and blood samples were removed for examination. Although decreases in the numbers of primordial, secondary, and atretic follicles and increases in the numbers of primary and Graafian follicles were observed in the POI + MOD group compared with the POI + PRP + MOD group, these differences were not statistically significant. Serum FSH and AMH levels increased. In fibrosis scoring with Masson's trichrome staining, the H score also increased. Immunohistochemical staining with Ki-67 showed less proliferation, immunohistochemical staining with Caspase-3 showed more apoptosis but these were not statistically significant. This is an experimental animal investigation with a comparatively modest sample size and the first study, to our knowledge, to compare the effects of intraovarian PRP injection with those of mechanical ovarian injury occurring during PRP administration. Although the study attempted to discuss statistically significant and insignificant differences as much as possible, more studies and studies with larger samples are needed in this area.
Acetamiprid (Ace) is a widely used neonicotinoid insecticide whose intensive application in agriculture has raised concerns about its potential effects on the reproductive health of animals and humans. Successful fertilization depends on the integrity of the plasma membrane (PM) of the spermatozoa and the proper progression of the series of events involved in capacitation. The aim of this study was to evaluate the effects of Ace on the capacitation of boar spermatozoa and several associated functional parameters: membrane carbohydrate redistribution, membrane microdomains (MDM), cholesterol efflux, mitochondrial membrane potential (ΔΨm), DNA integrity, motility, and the capacity to achieve the acrosome reaction (AR). The median lethal concentration (LC50) of Ace was estimated by PROBIT analysis at 590 µM after testing 1/5 LC50, ½ LC50, and LC50. We found that Ace increased PM damage and was associated with reduced cholesterol efflux, capacitation, and AR, as well as altered distributions of N-acetylglucosamine and sialic acid residues and MDM. Ace significantly impaired membrane functionality during capacitation and increased both the number of spermatozoa with high ΔΨm and DNA damage. These results may be a consequence of damage to the PM function and the physiology of the spermatozoa, especially in processes related to capacitation and the AR. These effects have potential implications for the fertility of exposed populations.
