Search Results (264)

Assisted reproductive technologies (ART) use has increased over the past decades. However, reports concerning ART’s low efficiency continue to emerge, citing causes related to lower embryo quality and pregnancy rates compared to their in vivo counterparts. One of the setbacks of ART is oxidative stress, which can impair embryo developmental rates. Mitochondrial redox and energetic homeostasis determine both cell survival and death, so mitochondria are a key target for therapeutic intervention strategies. In the present work, our objective was to improve the quality of viable embryos by adding new mitochondria-targeted antioxidants in the embryo culture media to reduce oxidative stress. Two naturally derived antioxidants synthesized by our team, AntiOxBEN₂ and AntiOxCIN₄, based on hydroxybenzoic and hydroxycinnamic scaffolds, respectively, were studied in two different experimental protocols (here called experiments). The first experiment investigated the effects of the antioxidants on embryo development to determine their optimal concentrations. The first assay of the first experiment focused on the effects of AntiOxCIN₄ at concentrations of 1, 2.5, and 10 μM, while the second assay focused on the effects of AntiOxBEN₂ at the same concentrations. A control group without supplementation was run simultaneously. The second experiment aimed to compare the best concentrations of these antioxidant molecules in the embryo culture media and their effect on embryos’ resistance to vitrification/warming. In each experiment, the embryos were morphologically evaluated, and the total and viable cell numbers were examined. Reactive oxygen species (ROS) and mitochondrial polarization were also evaluated using specific fluorescent dyes. In experiment 1, an increased embryo quality was identified by using 2.5 μM AntiOxCIN₄ (p = 0.03) and 2.5 μM AntiOxBEN₂ (p = 0.001). Moreover, blastocysts supplemented with 2.5 μM AntiOxCIN₄ had higher viability (p = 0.008), while those supplemented with 2.5 μM AntiOxBEN₂ presented a greater total cell number (p = 0.01). An improvement in embryo cryosurvival following the supplementation during the culture process with either antioxidant was identified in experiment 2, with superior expansion scores after vitrification/warming and culture (2.5 μM AntiOxCIN₄, p = 0.056 and 2.5 μM AntiOxBEN₂, p = 0.059). In conclusion, both AntiOxCIN₄ and AntiOxBEN₂ had a beneficial effect on embryo development and cryosurvival, suggesting a potential intervention to reduce oxidative stress in assisted reproductive technologies. Full article

Three-dimensional (3D) cell culture models provide physiologically relevant systems that mimic the native endometrial environment better than 2D models and offer reliable platforms to study embryo implantation and maternal–embryo interactions. One widely used 3D culture model is the generation of spheroids. However, standardized and reproducible methods for generating uniform spheroids from trophoblast and endometrial stromal cells are limited. In this study, we established and validated a robust protocol for spheroid formation using human trophoblast (HTR8/SVneo, JEG3) and endometrial stromal (St-T1b, tHESC) cell lines. The protocol was further extended to generate spheroids from decidualized tHESC, representing a novel approach that closely reflects the receptive endometrial environment. Key parameters, including cell concentration and methyl cellulose supplementation, were optimized to produce compact and homogeneous spheroids. Spheroid formation was monitored at defined intervals (0, 8, 24, 32, and 48 h), and decidualized spheroids were assessed up to 72 h. Long-term cryopreservation over 11 months demonstrated high post-thaw viability across all spheroid types, as confirmed by Calcein-AM staining. This standardized workflow provides a reliable 3D model incorporating hormonally primed stromal cells and offers a practical platform to investigate the mechanisms underlying normal and trophoblast invasion in vitro. Full article

This experiment was conducted to explore the effects of betaine on the DNA methylation level, expression level, and fat synthesis of VNN1 in goose hepatocytes by isolating the primary hepatocytes of goose at the cellular level and constructing a fatty degeneration model of goose hepatocytes. In the study, 23-day-old Taizhou goose embryos were used as the research object, and free fatty acid (PA:OA = 2:1) was used to induce steatosis of goose primary hepatocytes. The experiment was randomly divided into seven groups: control group, steatosis model group, and betaine (2, 10, 25, 50, 100mM) group. After 24 h of cell culture, cell viability, oil red O staining, and lipid metabolism-related indicators in cell supernatant were measured, and cells were collected to determine VNN1, FAS, ACC, SCD, SREBPQ gene expression and VNN1 promoter region DNA methylation level. (1) The addition of 0.5 mM fatty acids successfully determined the degeneration of goose hepatocytes. The levels of TG and LDL were significantly increased (p < 0.05), and the level of HDL was significantly decreased. (2) The addition of 100 mM betaine significantly reduced TG levels, and 10, 25, 50, and 100 mM betaine significantly reduced LDL levels. The addition of betaine had no significant effect on HDL level compared to the FFA group (p > 0.05), although a significant overall model effect was observed. Oil red O staining showed that the area of lipid droplets in cells with 50 mM betaine decreased most significantly. (3) The expression levels of VNN1, FAS, ACC, SCD, and SREBPQ genes in the fat model group were significantly higher compared to the control group (p < 0.05), and the DNA methylation level in the promoter region of the VNN1 gene decreased. (4) The addition of 2, 10, 25, 50, and 100 mM betaine significantly reduced the expression of VNN1. The expression of FAS in 2, 10, and 25 mM betaine groups significantly decreased. Betaine at 10 and 100 mM significantly reduced the expression of SREBPQ, but it showed no significant effect on ACC expression. Addition of 2, 50, and 100 mM betaine led to an increased DNA methylation status at the VNN1 gene promoter region. In summary, the addition of betaine can reduce the expression of fatty acid synthesizing genes such as FAS, SCD, and SREBPQ, down-regulate the expression level of the VNN1 gene, increase the DNA methylation level of the VNN1 promoter region, and reduce lipid deposition in goose liver steatosis cells. Full article

Short-term hypothermic storage at 4 °C represents a promising non-freezing alternative for transporting bovine embryos and synchronizing assisted reproductive procedures. However, chilling induces oxidative stress, mitochondrial dysfunction, and apoptosis, which markedly impair post-preservation embryonic viability. Glutathione (GSH), a key intracellular antioxidant, may mitigate these damaging effects, yet its protective mechanisms during bovine blastocyst hypothermic preservation remain unclear. Here, we investigated the impact of exogenous GSH supplementation on the survival, hatching ability, cellular integrity, mitochondrial function, and developmental potential of bovine blastocysts preserved at 4 °C for seven days. Optimization experiments revealed that 4 mM GSH provided the highest post-chilling survival and hatching rates. Using DCFH-DA, TUNEL, and γ-H2AX staining, we demonstrated that 4 °C preservation significantly increased intracellular reactive oxygen species (ROS), DNA fragmentation, and apoptosis. GSH supplementation markedly alleviated oxidative injury, reduced apoptotic cell ratio, and decreased DNA double-strand breaks. MitoTracker and JC-1 staining indicated severe chilling-induced mitochondrial suppression, including decreased mitochondrial activity and membrane potential (ΔΨm), which were largely restored by GSH. Gene expression analyses further revealed that chilling downregulated antioxidant genes (SOD2, GPX1, TFAM, NRF2), pluripotency markers (POU5F1, NANOG), and IFNT, while upregulating apoptotic genes (BAX, CASP3). GSH effectively reversed these alterations and normalized the BAX/BCL2 ratio. Moreover, SOX2/CDX2 immunostaining, total cell number, and ICM/TE ratio confirmed improved embryonic structural integrity and developmental competence. Collectively, our findings demonstrate that exogenous GSH protects bovine blastocysts from chilling injury by suppressing ROS accumulation, stabilizing mitochondrial function, reducing apoptosis, and restoring developmental potential. This study provides a mechanistic foundation for improving 4 °C embryo storage strategies in bovine reproductive biotechnology. Full article

Reactive oxygen species (ROS) are essential regulators of fertilization and early embryo development in mammals, including humans and various animal models, but they exert detrimental effects when produced in excess. In assisted reproductive technologies (ART), particularly in vitro fertilization (IVF), exposure to non-physiological conditions increases oxidative stress (OS), impairing gamete quality, embryo viability, and clinical outcomes. This review synthesizes experimental and clinical studies describing the endogenous and exogenous sources of ROS relevant to embryo development in IVF. Endogenous ROS arise from intrinsic metabolic pathways such as oxidative phosphorylation, NADPH oxidase, and xanthine oxidase. Exogenous sources include suboptimal laboratory conditions characterized by factors such as high oxygen tension, temperature shifts, pH instability, light exposure, media composition, osmolarity, and cryopreservation procedures. Elevated ROS disrupt oocyte fertilization, embryonic cleavage, compaction, blastocyst formation, and implantation by inducing DNA fragmentation, lipid peroxidation, mitochondrial dysfunction, and apoptosis. In addition, the review highlights how parental health factors establish the initial redox status of gametes, which influences subsequent embryo development in vitro. While antioxidant supplementation and optimized culture conditions can mitigate oxidative injury, the precise optimal redox environment remains a subject of ongoing research. This review emphasizes that future research should focus on defining specific redox thresholds and developing reliable, non-invasive indicators of embryo oxidative status to improve the success rates of ART. Full article

Background: The apicomplexan parasite Toxoplasma gondii causes serious diseases in animals and humans. The in vitro efficacy of the antimicrobial peptide mixture tyrothricin, composed of tyrocidines and gramicidins, against T. gondii tachyzoites was investigated. Methods: Effects against T. gondii were determined by monitoring inhibition of tachyzoite proliferation and electron microscopy, host cell and splenocyte toxicity was measured by Alamar blue assay, and early embryo toxicity was assessed using zebrafish embryos. Differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was employed to identify potential molecular targets in T. gondii cell-free extracts. Results: Tyrothricin inhibited T. gondii proliferation at IC₅₀s < 100 nM, with tyrocidine A being the active and gramicidin A the inactive component. Tyrothricin also impaired fibroblast, T cell and zebrafish embryo viability at 1 µM. Electron microscopy carried out after 6 h of treatment revealed cytoplasmic vacuolization and structural alterations in the parasite mitochondrion, but these changes appeared only transiently, and tachyzoites recovered after 96 h. Tyrothricin also induced a reduction in the mitochondrial membrane potential. DAC-MS-proteomics identified 521 proteins binding only to tyrocidine A. No specific binding to gramicidin A was noted, and four proteins were common to both peptides. Among the proteins binding specifically to tyrocidine A were several SRS surface antigens and secretory proteins, mitochondrial inner and outer membrane proteins associated with the electron transfer chain and porin, and several calcium-binding proteins putatively involved in signaling. Discussion: These results suggest that tyrocidine A potentially affected multiple pathways important for parasite survival and development. Full article

Currently, in influenza vaccine production via the chicken embryo splitting method, embryo viability detection is a pivotal quality control step—non-viable embryos are prone to microbial contamination, directly endangering the vaccine batch quality. However, the predominant manual candling method suffers from unstable accuracy and occupational visual health risks. To address this challenge, we developed a novel real-time embryo viability detection system based on photoplethysmography (PPG) technology, comprising a hardware circuit for chicken embryo PPG signal collection and customized software for real-time signal filtering and time–frequency-domain analysis. Based on this system, we conducted three pivotal experiments: (1) impact of the source–detector spatial arrangement on PPG signal acquisition, (2) viable/non-viable embryo discrimination, and (3) embryo PPG signal detection performance for days 10–14. The experimental results show that within the sample size (15 viable, 5 non-viable embryos), the system achieved a 100% discrimination accuracy; meanwhile, it realized 100% successful multi-day (days 10–14) PPG signal capture for the 15 viable embryos, with consistent performance across the developmental stages. This PPG-based system overcomes limitations of traditional and existing automated methods, provides a non-invasive alternative for embryo viability detection, and presents significant implications for standardizing vaccine production quality control and advancing optical biosensing for biological viability detection. Full article

Scylla paramamosain is a commercially important crab species widely cultured in China. However, artificial breeding remains limited by the high mortality of ovigerous females and asynchronous embryo hatching. In vitro embryo hatching has emerged as a promising alternative, yet its practical feasibility and underlying molecular mechanisms have not been systematically investigated. In this study, we examined the developmental characteristics of S. paramamosain embryos under different temperature regimes and hatching modes, evaluated embryo viability following maternal death, and compared transcriptomic profiles of Zoea I larvae between in vitro and maternal hatching. Our results demonstrated that temperature had a pronounced effect on embryogenesis and survival, with 27–30 °C identified as the optimal range for development and hatching. Both low and high temperature extremes markedly reduced embryo survival. Developmental trajectories were largely comparable between in vitro and maternal hatching, confirming the reliability and feasibility of the in vitro approach. Embryos collected within 4 h after maternal death exhibited high hatching success, whereas those obtained after 8 h failed to hatch. Transcriptomic analysis revealed 3505 differentially expressed genes, including 1933 upregulated and 1572 downregulated, which were significantly enriched in pathways related to cell cycle regulation, energy metabolism, immune defense, and ion transport. These findings implied that in vitro embryos could maintain developmental competence by stabilizing genomic integrity, reallocating energy resources, and activating stress responsive mechanisms. This study provides the first comprehensive evidence supporting the feasibility of in vitro embryo hatching in S. paramamosain and offers practical insights for optimizing temperature regimes, improving the utilization of maternal resources, and advancing large scale seedstock production in crab aquaculture. Full article

Applying non-thermal plasma (NTP) to seeds prior to sowing is recognized for its ability to enhance germination and promote plant growth. This study investigated the effects of NTP seed treatment on alfalfa seed surface characterization, germination, growth, and biochemical traits under varying water conditions. NTP modified seed surface properties by decreasing water contact angle, roughening the coat, and reducing O–H/N–H and C–H band intensities, while major functional groups remained intact. Short plasma exposures (<2 min) enhanced germination, whereas prolonged treatment (10 min) reduced viability, indicating embryo sensitivity. In pot experiments, both 1 and 5 min treatments improved fresh and dry weight, stem and root elongation, pigment accumulation, and protein content, particularly under normal and moderate water stress, while extended exposure (10 min) offered limited benefits and could be detrimental under severe drought. Root growth was most responsive, suggesting enhanced water and nutrient uptake. Plasma had modest effects on polyphenols and flavonoids but influenced early physiological responses and antioxidant activity. These findings highlight NTP as a promising seed priming tool to improve alfalfa performance, though further studies are needed to clarify the mechanisms and specific contributions of plasma components. Full article

Background: Artificial Intelligence (AI) has emerged as a transformative tool in in vitro fertilization (IVF) as it has done in other sectors. In IVF, AI offers advancements in embryo selection, treatment personalization, and outcome prediction. It does so by leveraging deep learning and computer vision, as well as AI-driven platforms such as ERICA, iDAScore, and IVY where the goal is to address the limitations of traditional embryo assessment. Key amongst them are the issues of subjectivity, labor intensity, and limited predictive power. Despite rapid technological progress, the integration of AI into routine IVF practice faces key challenges. These are issues related to clinical validation, ethical dilemmas, and workflow adaptation. Rationale/Objectives: This review synthesizes current evidence to evaluate the role of AI in IVF, focusing on six critical dimensions: (1) the evolution of AI from traditional embryology to algorithmic assessment, (2) clinical validation and regulatory considerations, (3) limitations and ethical challenges, (4) pathways for clinical integration, (5) real-world applications and outcomes, and (6) future directions and policy recommendations. The objective is to provide a comprehensive roadmap for the responsible adoption of AI in reproductive medicine. Outcomes: AI demonstrates significant potential to improve the precision and efficiency of IVF. Studies report that AI models can achieve 10 to 25% higher accuracy in predicting embryo viability and implantation potential compared to traditional morphological assessment by embryologists. This enhanced predictive power supports more consistent embryo ranking, facilitates elective single-embryo transfer (eSET) strategies, and is associated with 30 to 50% reductions in embryologist workload per embryo cohort. Early adopters report promising trends. However, large-scale randomized controlled trials have yet to conclusively demonstrate a statistically significant increase in live birth rates per transfer compared to expert embryologist selection. The most immediate and evidenced value of AI lies in hybrid decision-making models. This is where it augments embryologists by providing data-driven, objective support, thereby standardizing workflows and reducing subjectivity. Wider Implications: The sustainable integration of AI into IVF banks on three key aspects: robust evidence generation, interdisciplinary collaboration, and global standardization. To foster these, policymakers ought to establish regulatory frameworks for transparency and bias mitigation. On their part, clinicians need training to interpret AI outputs critically. Ethically, safeguarding patient trust and equity is non-negotiable. Future innovations, mainly AI-enhanced genomics and real-time monitoring, could further personalize care. However, their success depends on addressing current limitations. By balancing innovation with ethical vigilance, AI holds the potential to revolutionize IVF while upholding the highest standards of patient care. Full article

This literature review systematizes current data on the development of triploid seedless hybrids in the Cucurbitaceae Juss family. The absence of seeds simplifies the consumption and industrial preparation of products from cucurbits. In addition, triploids showed larger plant habitus, field resistance to infections, extended shelf life, and higher fruit quality. Phenotypic differences in polyploids can stem from altered chromatin organization and gene regulation, as the nucleus must accommodate a doubled chromosome set. The triploid watermelon cultivation method developed in 1951 failed to gain traction among other crops in the gourd (Cucurbitaceae) family. The challenges of triploid seed production and use include the need for the development of tetraploid and diploid parental lines, as well as bypassing the problem of the low viability of tetraploid parent pollen and the issue of thick seed coats and underdeveloped embryos in triploids. The research findings presented in this review can be applied to the development of triploid seedless hybrids for other members of the Cucurbitaceae family. Full article

Over the past few decades, demand for sea urchin roe has risen, while wild sea urchin populations have declined. This trend has increased interest in aquaculture techniques and the development of formulated feeds to support ecological restoration and research. Here, we examined the effects of a high-protein formulated feed on gonad development in Paracentrotus lividus, compared to fresh feeds (maize and spinach), across three replicated tanks. We assessed gonad maturation, gamete viability, and larval development, and developed a new histopathological index applicable to both sexes. Formulated feed significantly enhanced gonad maturation, increased gamete production, and led to heavier gonads with higher gonadosomatic indices compared to fresh feeds, which were insufficient to promote maturation within four weeks. Notably, no histological alterations were observed in the gonads. Fertilization trials showed that embryos were produced, but none reached the pluteus stage, indicating decreased embryo viability. Although the formulated feed improved gonad development, it adversely affected water quality, increasing nutrient concentrations and lowering pH. Overall, these findings suggest that high-protein formulated feeds could potentially improve aquaculture production by enhancing gonad maturation and gamete output, but additional measures may be needed to support complete larval development. Full article

Selecting high-quality seeds is an effective approach for increasing wheat yields. Phenotype-based seed selection has emerged in recent years as a simple and convenient method. However, due to the irregular shape of seeds, accurately measuring shape parameters via imaging for seed viability assessment poses certain challenges. This study statistically analyzed the embryo morphology of wheat variety Bainong 207, identifying three predominant phenotypes. The morphological parameters of seeds and embryos were measured for these three phenotypes. Germination tests were conducted on these three categories of wheat in accordance with Chinese national standards. The seeds with Phenotype Ⅰ exhibited the highest germination force (89.33%) and the highest germination percentage (96.00%), representing a statistically significant difference from Phenotype Ⅱ and Ⅲ. Morphological parameters related to seed vigor, including germinative force, germination percentage, seedling height, and root length, were measured. By exploring the relationship between embryo phenotypes and wheat seed viability and yield potential, principles and considerations for wheat seed selection based on embryo phenotypes were discussed. The YOLOv8 model was employed to classify wheat seeds with different embryo phenotypes. Under the global labeling of seeds, the classification accuracy for the three categories reached 99.9%. Classification results from various labeling methods were compared, validating the feasibility of machine vision for seed selection and providing technical support for large-scale wheat seed improvement. Full article

Chickpea seed quality is highly susceptible to mechanical damage during handling and to rapid deterioration under postharvest storage. Atmospheric pressure Non-Thermal Plasma (NTP) has shown positive effects on seed quality in several species, but its long-term impact on chickpea remains poorly understood. This study evaluated the effect of NTP on the physiological germination process and postharvest deterioration of Cicer arietinum L. (Fabaceae) ’Felipe UNC-INTA’ seeds. Seeds were treated for three minutes with dielectric barrier discharge using O₂ and N₂ as carrier gases. Results showed that NTP optimized the triphasic germination response in embryo, especially in phases II and III, where radicle protrusion occurred earlier in treated (27 and 30 h) than in control (33 h) seeds, accompanied with a partition ratio < 1, indicating the roots’ preferential assimilate allocation. Fungal incidence decreased notably, e.g., Aspergillus decreased from 31% (control) to 11% (N₂) and 10% (O₂). O₂-treated seeds exhibited higher germination (94%) than the control (90%) and an 11% reduction in individual electrical conductivity, indicating enhanced membrane integrity. After six months of storage, both treatments delayed aging, maintaining higher vigor than untreated seeds. Overall, NTP emerges as a promising postharvest technology to enhance and preserve seed vigor and viability in C. arietinum. Full article

Gene editing technologies are playing an increasingly important role in the study of insect gene functions. Appropriate incubation humidity helps improve the hatching rate of insect eggs. However, to date, no study has evaluated the impact of humidity on the hatching rate of gene-edited eggs or the subsequent development of the larvae. Using the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) as a model, we investigated the weight and hatching rate of microinjected eggs under different humidity conditions. We determined the larval development time and pupal weight and calculated the larval gene editing efficiency. The results showed that post-microinjection incubation under high humidity (RH = 95%) had a significant influence on CRISPR/Cas9 gene-edited eggs. The key research results indicate that, compared with the incubation environment with RH ≤ 80%, after 48 h of incubation under 95% RH, the hatching rate of H. armigera eggs increased by more than 27.5%, with higher egg saturation and greater egg weight; meanwhile, the larval developmental duration was shorter and the pupal weight was also greater. These results establish a humidity-regulated developmental recovery protocol for gene-edited insects, which provides theoretical and practical support for optimizing post-microinjection recovery processes in gene-edited insects. Full article