Search Results (261)

Prime editing (PE) has emerged as a transformative genome editing technology, enabling precise base substitutions, insertions, and deletions without inducing double-strand DNA breaks (DSBs). However, its application in zebrafish remains limited by low efficiency. Here, we leveraged PE7, a state-of-the-art PE system, combined with La-accessible prime editing guide RNAs (pegRNAs), to enhance editing efficiency in zebrafish. By co-incubating PE7 protein with La-accessible pegRNAs to form ribonucleoprotein (RNP) complexes and microinjecting these complexes into zebrafish embryos, we achieved up to 15.99% editing efficiency at target loci—an improvement of 6.81- to 11.46-fold over PE2. Additionally, we observed 16.60% 6 bp insertions and 13.18% 10 bp deletions at the adgrf3b locus, representing a 3.13-fold increase over PE2. Finally, we used PE to introduce desired edits at the tyr locus, successfully generating zebrafish with the tyr P302L mutation that exhibited melanin reduction. These findings demonstrate that PE7 significantly enhances prime editing efficiency in fish, providing novel tools for functional gene studies and genetic breeding in aquatic species. Full article

Triphenyl phosphate (TPhP) is a widely used organophosphate flame retardant and plasticizer, raising concerns over its health impacts. This study examined the effects of embryonic TPhP exposure on axial skeletal development and metabolism in medaka (Oryzias latipes), a vertebrate fish model relevant to human bone biology. Medaka embryos were exposed to 1 µM TPhP and assessed through early larval stages. TPhP impaired vertebral ossification, causing shortened centra and reduced cartilage in the caudal complex, alongside disrupted distribution of osteoblast-lineage cells. Key osteogenic genes were significantly downregulated at 14 days post fertilization, and transcriptomic analysis revealed altered mitochondrial pathways linked to skeletal disorders. Functionally, TPhP-exposed larvae showed reduced caudal fin regeneration and decreased metabolic rate and oxygen consumption, consistent with mitochondrial dysfunction. These findings indicate that TPhP disrupts bone development and metabolism by affecting osteoblast differentiation and mitochondrial regulation, highlighting the value of small fish models for studying environmental toxicants and bone metabolic disease risk. Full article

In this study, we demonstrated that lrp6a, a co-receptor in the Wnt signaling pathway, is essential for proper median fin formation and somitogenesis in goldfish. We analyzed the gene’s sequence features and expression patterns in both wen-type and egg-type goldfish, uncovering distinct tissue-specific expression differences between the two varieties. To explore the functional role of lrp6a, we performed CRISPR/Cas9-mediated gene knockout using eight designed single-guide RNAs (sgRNAs), of which four showed effective targeting. Three high-efficiency sgRNAs were selected and co-injected into embryos to achieve complete gene disruption. Morphological assessments and X-ray microtomography (μCT) imaging of the resulting mutants revealed various abnormalities, including defects in the dorsal, caudal, and anal fins, as well as skeletal deformities near the caudal peduncle. These results confirm that lrp6a plays a key role in median fin development and axial patterning, offering new insights into the genetic regulation of fin formation in teleost fish. Full article

Collagen IV α1/α2 heterotrimers are the major constituents of all basement membranes (BM). Consistently, COL4A1/A2 mutations cause a complex multisystem disorder. While mouse models are invaluable, they alone cannot support the rapid functional validation needed for clinical translation. The FishCOLler project establishes zebrafish as a scalable in vivo platform to model COL4A1/A2 disease, employ rapid assays to monitor key disease traits, and enable mechanistic studies. Our first fish disease faithfully models patient symptoms, i.e., brain hemorrhage and ocular dysgenesis. The work supports FishCOLler as a platform for rapid variant interpretation, therapeutics testing, and highlights potential consequences of gene dosage modulation strategies. Full article

The use of cellular energy allocation (CEA) as a physiological energetic biomarker is useful for detecting the sublethal effects of environmental contaminants. The CEA assesses the health and energy status of organisms, serving as a reliable indicator for monitoring the health of aquatic ecosystems. This study aimed to evaluate the impact of emerging contaminants already listed as a priority for monitoring in freshwater ecosystems, namely sulfamethoxazole (0.156–2.50 mg/L), trimethoprim (25.0–400 mg/L), 4-chloroaniline (5.21–20.0 mg/L), and 3,4-dichloroaniline (0.38–4.00 mg/L), on the CEA of D. rerio embryos. A standard fish embryo toxicity test was conducted, and an adaptation of the allometric scaling approach was developed through the relationship between the size and the fresh weight of the embryos. All the compounds affected the fractions of the energy reserves (total carbohydrate, lipid, and protein contents) differently, with carbohydrates being the predominant energy fraction and the most responsive indicator. Although the energy consumed showed no significant changes, the CEA was notably altered after exposure to all the contaminants, indicating a direct connection to shifts in the available energy. The CEA alterations may indicate a reallocation of energy toward detoxification, combating the stress of contaminant exposure. Energy allocation biomarkers provide a comprehensive assessment of an organism’s physiological state, which is essential for evaluating emerging contaminants’ impacts, safeguarding aquatic ecosystems, and shaping effective environmental policies. Full article

The final recipient of nanoparticles, including various types of copper-based nanoparticles (Cu-based NPs), is the aquatic environment. Their increased production, especially as a component of antimicrobial agents, raises concerns about uncontrolled environmental release and subsequent ecological risks. The high reactivity of Cu-based NPs enables interactions with biotic and abiotic environmental components, leading to bioaccumulation and disorders in living organisms, such as fish in various life stages, especially in embryos or hatchlings. Increasing concentration of Cu-based NPs causes various toxic effects, mainly through the induction of oxidative stress. These effects include impairment of antioxidant mechanisms, as well as damage to genetic material, cells and tissues, growth retardation, metabolic disorders, increased mortality, or hatching inhibition. The aim of this review is to describe the release routes of Cu-based NPs and their adverse effects on fish, while emphasizing the need for further research on their toxicity and measures to control their release to the environment. Given the limited data on the toxicity of Cu-based NPs, especially concerning sensitive fish developmental stages, further studies are required. Full article

The presence of pesticides in surface waters has been widely reported worldwide and represents a significant problem that raises concerns on local, regional, national, and international scales. Among these, metolachlor is one of the most widely used herbicides to control annual grasses and broadleaf weeds in various crops. Despite the existing research, data on the effects of metolachlor on the nervous system of fishes, remain limited. The present study aims to investigate the impact of metolachlor during embryonic development on the formation of the nervous system and the subsequent inflammatory response in zebrafish (Danio rerio), focusing specifically on larvae at 24 h post-fertilization (hpf). To achieve this, transgenic zebrafish lines marking neuronal populations Tg(Hu:GFP), glial cells Tg(gfap:GFP), and circulating macrophages Tg(mpeg:GFP) were employed. Following exposure to sub-lethal doses of metolachlor, we observed a significant decrease in GPF-positive cells marking the neuronal population, accompanied by an increase in apoptotic cells within the brain region. Additionally, treated embryos exhibited a marked neuroinflammatory response, characterized by astrogliosis and the specific accumulation of microglia/macrophage-positive cells in the head region. In situ hybridization and real-time PCR analyses revealed a significant downregulation of the neurogenin-1 (ngn1) transcript and a noticeable upregulation of the pro-inflammatory cytokine interleukin-1 beta (il1b). Our findings contribute to the growing body of evidence suggesting that metolachlor, even at early developmental stages, can have detrimental effects on both the formation of the nervous system and the regulation of immune responses. Full article

This study investigated the effects of dietary supplementation with the probiotic Pediococcus acidilactici on the reproductive performance and offspring viability of male Atlantic salmon (Salmo salar). A total of 48 mature males were divided into three groups—Group A (control, no probiotic), Group B (60-day supplementation), and Group C (120-day supplementation). The fish were kept in 10 m³ tanks at a constant temperature of 9 °C under a natural photoperiod and evaluated for multiple reproductive and physiological parameters. The results demonstrate that Group C exhibited significantly higher gonad weight, gonadosomatic index (GSI), and sperm concentration than Groups A and B. The blood biochemistry revealed reduced bilirubin and alanine aminotransferase (ALT) levels in Group C; however, values were within normal reference ranges for the species in all groups. While sperm quality parameters such as membrane integrity and motility showed no significant differences, fertility and embryo viability were significantly higher in Group C across all three developmental stages (180, 300, and 380 accumulated thermal units, ATUs). Additionally, Group C showed lower rates of embryonic malformation and mortality at later stages of development. Statistical analyses confirmed a significant interaction between treatment duration and embryo viability, with the 120-day probiotic treatment (Group C) producing the best outcomes. These findings support the hypothesis that P. acidilactici supplementation positively influences male reproductive parameters and improves embryo quality and survival in Atlantic salmon. Full article

Nile tilapia (Oreochromis niloticus) is the most widely farmed tilapia species globally, making it one of the most important aquaculture species. To meet increasing demand, hatcheries occasionally use artificial breeding techniques such as hormonal induction to synchronize breeding. Despite the common use of human chorionic gonadotropin (hCG) in fish breeding, no detailed protocol has been established specifically for Nile tilapia. The objective of this study is to establish an effective hCG-induced artificial breeding protocol for gene editing and aquaculture production, optimizing fertilization, hatching, and survival rates. We employed a single intramuscular injection of 2 IU/g hCG to induce ovulation. The protocol achieved an average fertilization rate of 88.3% and a larval survival rate of 90.5%, demonstrating its potential for obtaining high-quality embryos for functional studies and enhancing reproductive performance on a commercial scale. Full article

With the escalating frequency and intensity of global wildfires driven by climate change, fire retardants (FRs) have become essential tools in wildfire management. Despite their widespread use, the environmental safety of newer FR formulations—particularly in relation to aquatic ecosystems and developmental toxicity—remains insufficiently understood. In particular, their effects on fish embryos, which represent a sensitive and ecologically important life stage, are poorly characterized. This study investigated the acute toxicity of three commercially available FRs—N-Borate, N-Phosphate+, and N-Phosphate-—on early life stages of zebrafish (Danio rerio), based on an OECD 236 Fish Embryo Toxicity (FET) test. Notably, N-Phosphate- FR exhibited significant toxicity with a 96 h LC50 of 60 mg/L (0.0055%), while N-Borate (>432 mg/L, >0.032%) and N-Phosphate+ (>1181 mg/L, >0.08%) showed substantially lower toxicity. Sublethal effects, including reduced yolk sac absorption and yolk sac darkening, were observed across all FRs, highlighting potential developmental disruptions. The elevated toxicity of N-Phosphate- FR likely stems from its surfactant content. These findings reveal variations in the acute toxicity of different FR formulations, emphasizing the need for ecotoxicological assessments to guide the selection of safer FRs for wildfire management and to protect aquatic biodiversity. The results highlight the importance of incorporating developmental endpoints in FR risk assessments and provide foundational data for regulatory decisions regarding FR application near aquatic habitats. Further research is necessary to elucidate the mechanisms underlying observed effects and to evaluate cross-species toxicity at environmentally relevant concentrations. Full article

Oil spills pose a significant threat to marine ecosystems, with potentially adverse impacts on fish in early-life stages. Despite numerous studies reporting the developmental toxicity of oil exposure, knowledge about the recovery capacity of fish after oil exposure remains limited. Therefore, this study investigated the effects of water-accommodated fractions (WAFs) of Oman crude oil on the development and oxidative stress of marine medaka (Oryzias melastigma) embryos during a 7-day acute exposure period followed by a 14-day recovery period in clean seawater. Results revealed that WAF exposure caused concentration-dependent developmental toxicity gradually becoming apparent during the recovery period, including reduced survival and hatching rates, and increased morphological abnormalities. During the exposure period, low WAF concentrations triggered antioxidant responses (elevated SOD and CAT activities, and GSH content), while higher concentrations caused a concentration-dependent increase in lipid peroxidation (elevated MDA content). Differently, during the recovery period, all groups showed impaired antioxidant capacity (decreased SOD, CAT, GSH) and immune function (reduced AKP activity). Principal component analysis revealed strong correlations between survival, oxidative stress markers, and developmental toxicity. These findings could provide valuable insights into the recovery capacity of fish exposed to crude oil and give references for assessing the recovery potential of marine ecosystems after oil spills. Full article

In the field of fish genetic breeding, accurately determining the DNA content and ploidy of fish is of great significance. This article introduces the use of flow cytometry (FCM) to measure the DNA content and conduct ploidy analysis by sampling different tissues of freshwater fish species. It describes the FCM detection methods and their effectiveness for different individual tissues. These tissues include embryos and fry, as well as the blood, caudal fins, and sperm of adult live fish, and also specific tissues such as testes, ovaries, gills, spleens, and livers under anatomical conditions. Moreover, the application of ploidy detection to different tissues or individuals in different stages in the practice of fish genetic breeding is analyzed. This research covers samples from different growth stages and a variety of tissue types. The results show that this method exhibits high stability and reliability in the detection of different tissue samples, providing solid data support for subsequent research. It holds significant application value in fish genetic breeding. Full article

Background/Objectives: Male infertility is becoming a serious problem affecting about 7% of all men worldwide and is a major or contributory factor in 50% of infertile couples overall. Men with abnormal semen parameters have a significantly increased risk of aneuploidy, presenting a serious concern in programmes of assisted reproductive technologies. Recently, the introduction of preimplantation genetic testing for aneuploidies (PGT-A) has increased the pregnancy rate and live births. We investigated the effect of PGT-A on the success of IVF treatment in couples with the male factor of infertility. Methods: Two experimental groups and one control group were studied: Group A (110 couples)—male partners with abnormal semen parameters, with PGT-A; Group B (110 couples)—male partners with abnormal semen parameters, without PGT-A; and Group C (105 couples)—control, male partners with normal spermograms, with PGT-A. A Day 3 blastomere biopsy was followed by FISH-based PGT-A. A total of 880 embryos from Group A and 890 embryos from Group C was analysed. Results: In patients with abnormal semen parameters, embryonic aneuploidy was twice as common compared to the control (13.6% vs. 5.8%, p < 0.001). Group B had the lowest clinical pregnancy rate (28.2%), with two out of three pregnancies ending in a miscarriage. Only 10% of IVF cycles in this group resulted in live birth compared with 35.5% for Group A and 49.5% for Group C. Conclusions: Our data demonstrate that PGT-A screening as part of IVF treatment drastically increases the clinical pregnancy rate and chances of live birth in couples where male partners have semen abnormality. Full article

To address the growing consumer demands for improved fish meat quality, desirable morphological traits, and sustainable production practices, researchers have intensified efforts in the selective breeding and genetic improvement of carp (Cyprinus carpio) varieties. However, traditional breeding methods are often time-consuming and inefficient, which poses challenges to the sustainable development of the carp aquaculture industry. The establishment of germ stem cell lines offers a crucial tool for the study of germ cells, genetic improvement, and species conservation. In this study, we successfully established a spermatogonial stem cell line (YRSSCs) from Yellow River carp (Cyprinus carpio haematopterus) that can be cultured in vitro for the long term. We optimized the culture conditions to maintain their self-renewal and differentiation capabilities. The results demonstrated that YRSSCs have a diploid karyotype and can stably proliferate for over a year in L-15 medium supplemented with 5 mmol/L HEPES, 50 μmol/L β-mercaptoethanol, 15% FBS, 2 ng/mL bFGF, 2 ng/mL LIF, 1% carp serum, 800 IU/mL penicillin, 0.8 mg/mL streptomycin, 2 μg/mL amphotericin B, 1% zebrafish embryo extract, and 1% glutamine at 30 °C in the absence of CO₂. The cells exhibited a typical germ stem cell gene expression profile, with strong expression of the vasa, plzf-a, and Oct4-a genes. Additionally, this study found that YRSSCs possess the ability to differentiate in vitro and functionally colonize in vivo within recipient bodies. This research explored the establishment of YRSSCs and their differentiation potential both in vitro and in vivo, providing a novel strategy for the genetic improvement of aquaculture fish species through germ stem cell-based gene editing and transplantation technologies. Full article

Herein, we demonstrate that soluble factors extracted from the distinct phases of the development of zebrafish embryos (ZFEs) exhibit a specific miRNA profile. We removed proteins and concentrated miRNAs in different phase-related samples, which we investigated further. We observed that ZFEs modulate miRNA expression in both normal and cancerous breast cells, significantly inhibiting the invasiveness and motility of triple-negative breast cancer cells. Namely, ZFEs reactivate the synthesis of miR-218-5p in cancerous cells, leading to the downregulation of PI3K, which consequently alters the distribution of phosphoinositides (such as PIP2/PIP3). Moreover, the silencing of miR-218-5p abolished the ZFE effects. Restoring a proper PIP2/PIP3 ratio is crucial for promoting the regression of the malignant phenotype. Phenotypic reversion follows the extensive cytoskeleton rearrangement and the re-emergence of E-cadherin/β-catenin complexes. In addition, ZFEs antagonize the Epithelial Mesenchymal Transition (EMT) by modulating several pathways, including the TCTP-p53 axis. Overall, these results show that embryo extracts enriched with fish miRNAs reactivate endogenous miR-218-5p in cancerous cells, which in turn downregulates critical pathways involved in tumor progression and metastasis. Full article