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PubMed2026

Non-invasive embryo assessment: Cell-free DNA-based genetic testing and amino acid metabolomics in relation to morphology: A case-control study.

BACKGROUND: Cell-free DNA (cfDNA) in spent culture medium (SCM) offers a non-invasive option for preimplantation genetic testing, but its low concentration and fragmentation reduce clinical reliability. Combining genetic assessment with metabolomic profiling may provide complementary information about embryo competence. OBJECTIVE: This study assessed pre-analytical cfDNA processing workflows and examined whether SCM amino acid metabolic patterns could act as practical markers of embryo quality. MATERIALS AND METHODS: In this case-control study (2021-2023), 90 embryos were evaluated using fluorescence in situ hybridization or array comparative genomic hybridization. SCM samples underwent rapid boiling, silica-based purification, or whole-genome amplification (WGA). Sex determination was performed using quantitative polymerase chain reaction (qPCR). For cfDNA quality control and aneuploidy screening, the multiplex IRFiling kit and quantitative fluorescent polymerase chain reaction (QF-PCR) were used. Amino acid profiles across embryonic developmental stages and quality grades were quantified via liquid chromatography-tandem mass spectrometry. RESULTS: Rapid boiling resulted in complete failure of DNA amplification. Conversely, silica-based purification yielded 70.0% concordance for qPCR-based sexing and 56.7% for QF-PCR. WGA achieved the highest efficacy (73.3% qPCR and 56.7% QF-PCR concordance), although quality control checks flagged occasional misclassifications. LC-MS/MS profiling revealed significantly elevated alanine and arginine levels in tripronuclear embryos. Furthermore, high-quality blastocysts exhibited elevated glutamic acid levels alongside a pronounced overall depletion of extracellular amino acids compared to low-quality counterparts and controls. CONCLUSION: WGA improves cfDNA detectability and qPCR accuracy compared with boiling or purification, but remains inadequate as a standalone screening approach. SCM amino acid profiling provides informative, complementary metabolic signatures of developmental competence, supporting a multimodal strategy for non-invasive embryo assessment.

Amino acid metabolism↗

PubMed1977

Cell surface carbohydrates of preimplantation embryos as assessed by lectin binding.

Preimplantation embryos were obtained from the uteri and oviducts of 2 strains of mice, Swiss CD-1 and B6CBA. After removal of the zona pellucida by treatment with pronase, FITC-lectins were bound to the embryonic cell surfaces at either 4 degrees C or 37 degrees C. Both morula and blastocyst stage embryos bound the following lectins, FITC-ConA, FITC-WGA, FITC-RCAII and FITC-RCAI. No difference in binding was observed between the morula stage and the blastocyst stage within each mouse strain for each specific lectin. However B6CBA embryos bound less FITC-ConA and FITC-WGA than the corresponding Swiss CD-1 embryos. The topographical arrangement of the lectin receptors was observed to differ between 4 degrees C and 37 degrees C for FITC-ConA, FITC-RCAII, and FITC-RCAI. While lectins bound at 4 degrees C showed a pattern of continuous labeling, the same lectin at 37 degrees C showed aggregation of lectin receptors into patches indicating lateral mobility of these receptors within the embryonic cell membranes. In contrast FITC-WGA bound at 4 degrees C and 37 degrees C demonstrated continuous labeling of embryos at both temperatures. FITC-fucose binding protein did not bind to Swiss CD-1 embryos. The invasiveness of trophoblastic cells of mouse blastocysts was studied by culturing isolated embryos without prior enzyme treatment on reconstituted collagen gels. After 4 days in BME containing only glutamine and bovine serum albumin as supplements, the embryos shed their zona pellucida and implanted into the collagen gel as indicated by zones of lysis in proximity to the embryonic cells when analyzed by scanning electron microscopy.

Animals↗

PubMed2026

Epigenetic reduction OF H3K9me3 and H3K27me3 by RK-701 and GSK 126 improves the developmental competence of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) failure has largely been attributed to incomplete epigenetic reprogramming, particularly the dysregulation of repressive histone modifications such as H3K9me3 and H3K27me3. Reducing these repressive marks has been shown to improve reprogramming efficiency in SCNT embryos. Although histone demethylase mRNA injection has been used for this purpose, it is labor-intensive, technically demanding, and time-consuming. In this study, we investigated a simplified approach that combined RK-701 and GSK-126 to reduce H3K9me3 and H3K27me3 levels, respectively, in bovine SCNT embryos. Three experimental groups were established: IVF embryos (control), SCNT-control (SCNT-C) embryos, and inhibitor-treated SCNT embryos (SCNT-T). The IVF group was used as a reference standard. Fused one-cell SCNT embryos were treated with 2 μM RK-701 and 0.2 μM GSK-126 from the one-cell stage to the 16-cell stage. Gene expression analysis at the 16-cell stage revealed a significant reduction in histone methyltransferase (HMT) expression (p < 0.05), and immunofluorescence analysis confirmed marked decreases in H3K9me3 and H3K27me3 levels. In addition, the expression of genes associated with zygotic genome activation (ZGA) and pluripotency was significantly higher in SCNT-T embryos than in SCNT-C embryos. Assessment of blastocyst quality revealed reduced reactive oxygen species (ROS) levels, decreased expression of apoptosis-related genes, and improved mitochondrial membrane potential in the treated group, as indicated by JC1 staining. Overall, this approach effectively reduced repressive histone marks, enhanced epigenetic reprogramming, and improved ZGA, thereby increasing the developmental rate and adhesion potential of bovine SCNT embryos. These findings suggest that combined treatment with RK-701 and GSK-126 may provide a simple and practical strategy for improving the efficiency of bovine cloning.

Bovine embryos↗

PubMed2026

ENTPD3 as a novel regulator of endometrial receptivity: suppressing EMT via the ATP-P2Y2 axis in patients with recurrent implantation failure.

BACKGROUND: Recurrent implantation failure (RIF) remains a major challenge in assisted reproductive technology and is primarily attributed to impaired endometrial receptivity. Despite its clinical significance, the precise mechanisms underlying RIF remain inadequately understood. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed on endometrial samples from patients with RIF and healthy controls during the secretory phase using the 10X Genomics Chromium platform. The expression and localization of ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) in the window of implantation (WOI) in the endometrium were examined using real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemistry (IHC). A mouse model with ENTPD3 overexpression was utilized to assess embryo implantation in vivo, and an in vitro blastocyst adhesion assay was performed to evaluate endometrial receptivity. Additionally, Ishikawa cells were transduced with an ENTPD3 recombinant adenovirus to explore the underlying molecular mechanisms. RESULTS: ENTPD3 expression was significantly upregulated in the endometria of patients with RIF during the WOI, and its apical surface localization in endometrial epithelial cells was confirmed by single-cell data and IHC. Functional studies demonstrated that ENTPD3 overexpression impaired endometrial receptivity by suppressing epithelial-mesenchymal transition (EMT). In vivo, ENTPD3 overexpression markedly reduced endometrial receptivity and inhibited embryo implantation in mice. Consistently, in vitro assays revealed that ENTPD3 overexpression diminished blastocyst adhesion to endometrial epithelial cells. Mechanistically, ENTPD3 hydrolyzes ATP, thereby suppressing EMT via the P2Y2 signaling pathway and ultimately disrupting endometrial receptivity. CONCLUSIONS: Dysregulated ENTPD3 expression contributes to RIF pathogenesis by impairing endometrial receptivity through ATP hydrolysis-mediated suppression of EMT via P2Y2 signaling. These findings highlight ENTPD3 as a potential therapeutic target for improving implantation success in affected patients.

Female↗

PubMed1977

Growth and mineral content of cultured chick embryos.

In order to assess mineral uptake by chick embryos cultured outside of the shell, growth and mineral (Ca, Mg, K, Na) composition of control and cultured embryos and total egg contents (embryo plus residual egg contents) were measured from 8 through 21 days of incubation. At day nine and thereafter, cultured embryos contained significantly less Ca and Mg than did control embryos. At day 11 and thereafter, ash weight of cultured embryos was significantly less than that of controls. At 13 days and thereafter, wet weight, dry weight, toe length, K and Na content of cultured embryos were significantly less than in controls. To assess relative mineral uptake of cultured embryos, mineral (Ca, Mg, K, Na) content per gram embryo dry weight was plotted against incubation age. Values for the ratio of Mg, K and Na per gram dry weight were similar for control and cultured embryos. Control values for Ca per dry weight were consistently higher than those for cultured embryos during the period from 9 to 21 days. The data suggest that Ca may not be present in sufficient quantity, even on a relative growth basis, to perform its normal physiological functions in cultured embryos.

Animals↗

PubMed2026

Generation of germline-transmitting transgenic sheep by piggyBac-mediated transgenesis using pronuclear and cytoplasmic gene injection approaches.

Sheep represent an important large-animal model for biomedical research and biopharmaceutical production. Although the piggyBac transposon system offers efficient and stable genomic integration, the optimal gene delivery strategy for ovine embryos remains unclear. This study evaluated piggyBac-mediated transgenesis using pronuclear injection (PNI) in both in vivo- and in vitro-derived embryos and assessed cytoplasmic injection (CTI) as an alternative approach. In vivo-derived embryos were obtained from superovulated Kıvırcık ewes approximately 40 h after gonadotropin-releasing hormone administration, whereas in vitro-derived embryos were produced from slaughterhouse-derived oocytes. All embryos were injected with the hyperactive piggyBac transposase-based pmhyGENIE-3 construct (10 ng/µL). In vivo-derived embryos were transferred immediately after injection, whereas in vitro-derived embryos were cultured for 3 days and screened for EGFP expression prior to transfer. Among 65 in vitro-derived embryos injected by PNI, no transgenic offspring was obtained. In contrast, PNI of 19 in vivo-derived embryos resulted in one transgenic lamb (5.3%). CTI of 12 in vivo-derived embryos similarly produced one transgenic lamb (8.3%). Whole-genome sequencing of the healthy founder male produced by CTI identified a single detectable genomic integration locus on chromosome 10 within a non-coding RNA locus (LOC121820439). Germline transmission was confirmed by in vitro fertilization using sperm from the founder male, with EGFP expression detected in 10.1% (10/99) resulting embryos. These findings provide proof-of-concept evidence for piggyBac-mediated transgenesis in sheep and support the feasibility of cytoplasmic injection as an alternative gene-delivery approach under the conditions tested.

Animals↗

PubMed2026

From Static to Dynamic: Fluorescence Imaging Technology Advances Precise Embryo Evaluation.

Live-cell imaging technology has revolutionized our understanding of preimplantation embryonic development, shifting the field from static morphological descriptions to dynamic functional analyses. This has tremendously advanced the fields of in vitro fertilization (IVF) and embryonic development. At the heart of this transition lies the strategic application of fluorescent probes, which provide the requisite sensitivity and specificity to resolve complex biological events. This review provides a comprehensive overview of fluorescent probe-based strategies designed to address four cardinal questions in peri-implantation embryology: genomic stability, cell fate determination, tissue morphogenesis, and embryo-maternal interactions. We systematically evaluate the chemical design principles and imaging modalities of various probes, which range from small-molecule organic fluorophores to genetically encoded reporters and nanoparticle-based sensors. Furthermore, we discuss how these tools facilitate the real-time visualization of chromosomal aberrations, lineage segregation, biomechanical forces, and enzymatic activities within the delicate embryonic microenvironment. This review summarizes methodological strategies for selecting and developing optimal probes across diverse application contexts. By identifying current technical bottlenecks and proposing future directions, such as NIR-II imaging and noninvasive labeling, it aims to drive the translation of basic embryonic research into advanced reproductive medicine.

Humans↗

PubMed1979

Replication of turkey herpesvirus and Marek's disease virus in chick embryo skin organ culture.

Turkey herpesvirus (HVT) and an attenuated Marek's disease virus (MDV) replicated in organ cultures of chick embryo skin as assessed by immunofluorescence and/or electron microscopy. HVT-specific immunofluorescent antigen was detected in the feather follicle epithelium (FFE) and in the surface layer of the skin epidermis. Electron microscopy of infected explants revealed herpes-type cytopathology. Immature particles of both viruses appeared first in the nucleus. Oval or horseshoe-shaped non-enveloped particles of HVT and enveloped virions of MDV were seen in the cytoplasm of some transitional cells. The difference in the ability of HVT and MDV to form an envelope was believed to account for the difference in their transmissibility in chickens. The results indicated that HVT replicated in the FFE and in the epidermis of the skin. However, attempts to localise the site(s) of MDV replication by electron microscopy were unsuccessful.

Animals↗

PubMed1978

Gastrulation in the mouse: assessment of cell populations in the epiblast of tw18/tw18 embryos.

Homozygous tW18 embryos die prior to organogenesis. They develop gross abnormalities shortly after primitive streak formation. Anatomically, the lesion appears to be confined to the mesoderm with that tissue showing ultrastructural deficiencies and abnormal migration (Spiegelman & Bennett, 1974), and failing to develop in teratomas produced from mutant embryos (Artzt & Bennett, 1972). Analysis of growth rate by determining cell number increase, and by mapping mitotic activity and planes of cleavage in the epiblast shows that the mutant embryos are small but paradoxically show overall a very high mitotic activity, approximately double that of their normal litter mates. They also show a marked disorientation of the planes of cleavage in most of the epiblast. In pre-primitive streak embryos, before gross abnormality is detectable, two types of embryo can be found. One group constitutes the small embryos which also show the mitotic disturbances characteristic of the later stage mutants. The second group, larger embryos, do not show mitotic abnormalities. The tW18 allele thus seems to act several hours before primitive streak formation. Since there is no difference in the amount of cell between mutant and normal embryos until 6.75 days p.c. it seems that arrest in division is the cause of the elevated mitotic index in mutants. Significantly a small region of the epiblast in mutant embryos is free of the mitotic abnormalities characteristic of the tissue as a whole. This region is the so-called proliferative zone (Snow, 1977) and the data suggest that it may be from this region that some of the ectoderm of the later embryos is produced.

Animals↗

PubMed1976

Effects of stimulation on embryonic activity in the chick.

Effects of stimulation on the stereotyped and coordinated movements in the chick embryo were investigated using the type of stimulation (regular loud clicks) that is known to accelerate development and the time of hatching. The number of coordinated movements, the amount and approximate size of all activity, heart rates and, in 20-day embryos, respiration rates were assessed during a 15-min control period, a 15-min period of stimulation, and a 15-min period of no stimulation. The results showed an increase in the number of coordinated movements, but not their size, and some changes in the heart and respiration rates after the onset of stimulation. These effects occurred in most cases only after several minutes of delay. It is suggested that this delay may be connected with the reciprocal relationship between the coordinated movements and random motility in that the former may be triggered more readily at times when the latter are suppressed.

Acoustic Stimulation↗

PubMed1977

Basal lamina of embryonic salivary epithelia. Production by the epithelium and role in maintaining lobular morphology.

The role of the basal lamina in maintaining the normal morphology of mouse embryo submandibular epithelia was assessed by examining its production as well as the cellular and organ culture changes associated with its removal and replacement. The lamina was removed from epithelia isolated free of mesenchyme by brief treatment with testicular hyaluronidase in the absence of calcium. The treatment causes rounding-up of the cells, loss of cellular cohesion, appearance of microvilli, and changes in the organization of cytoskeletal structures. The lamina is not removed and the cellular alterations do not occur in the absence of hyaluronidase in calcium-free medium or when both enzyme and calcium are present, possibly because digestion of chondroitin sulfate, a component of the lamina, is inhibited by calcium. Within 2 h after treatment, in the absence of mesenchyme or biological substrata, the epithelia deposits a new lamina, which is identical by several criteria to the preexisting lamina, and reverses the cellular alterations. Epithelia treated with hyaluronidase lose lobular morphology during culture with mesenchyme. Delaying culture with mesenchyme, to allow restoration of the lamina and of normal cellular architecture, prevents the loss of lobular morphology. The results indicate that the basal lamina imposes morphologic stability on the epithelium, while the mesenchyme apparently affects processes involved in changes in morphology, possibly by selective degradation of the basal lamina.

Animals↗

PubMed1977

Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials.

The ultrastructural organization and the composition of newly synthesized glycosaminoglycan (GAG) in the epithelial basal lamina of mouse embryo submandibular glands were assessed. The labeled GAG accumulating in the lamina is distinct from that in its tissue of origin, the epithelium, or from that in the surrounding mesenchyme. In the lamina, hyaluronic acid accounts for approximately 50% of the labeled GAG, chondroitin-4-sulfate is twice the chondroitin-6-sulfate, and there is a low proportion of chondroitin. This composition is constant regardless of whether the lamina is labeled by whole glands or, in the absence of mesenchyme, by isolated epithelia retaining a lamina and by isolated epithelia generating a lamina de novo. The results andicate that the labeled GAG are bona fide components of the lamina, and suggest that laminar GAG is deposited in units of constant composition. Ultrastructural observations following ruthenium red staining or tannic acid fixation extablish that the lamina is a highly ordered specialization of the basal cell surface. Discrete structures in macroperiodic arrays apparently attached to the plasmalemma are visualized. This organization is seen in intact glands and in the laminae produced by epithelia in the absence of mesenchyme or biological substrate. The data are interpreted as indicating that the basal lamina contains supramolecular complexes of hyaluronic acid and proteoglycan which are organized into an extracellular scaffolding which imposes structural form on the epithelium.

Animals↗

PubMed1979

Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations.

Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei are both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brings estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells.

Animals↗

PubMed1978

Conversion of androgen to estrogen by the human fetal ovary.

The conversion of radiolabeled androgen to estrone and 17 beta-estradiol was assessed in tissues of human embryos that varied from phenotypically indifferent stages (1-3 cm crownrump length) to midgestation (15. 1-20 cm crownrump length). Significant rates of estrogen synthesis were demonstrated only in ovaries, liver, and brain. Estrogen synthesis was undetectable in gonads from 1-3 cm fetuses, but by the 3.1-5-cm stage it had reached an average rate of 1.9 pmol . h-1 . mg protein -1 in ovaries and remained at this level of activity through the latest stages examined. Estrogen formation was undetectable in testes at all stages examined, but the time of appearance of the capacity to form estrogens in the fetal ovary is similar to the onset of the capacity of the fetal testis to synthesize testosterone. The capacity of the fetal ovary to form estrogen develops before histological differentiation of the tissue.

Androgens↗

PubMed1977

Estrogen formation by the ovary of the rabbit embryo.

The conversion of [1,2,6,7-3H]- testosterone to radioactive estradiol was assessed in tissue slices of 18 different tissues from rabbit embryos that varied in age from 16 to 29 days gestation. Significant rates of estradiol synthesis were demonstrated only in ovaries [4.2 +/- 0.7 (mean +/- SEM) pmol/h/mg) protein], placenta (0.7 +/- 0.2 pmol/h/mg protein) and brain (0.3 +/- 0.1 pmol/h/mg protein). Estradiol formation was undetectable in day 16 gonads of both sexes and in tests at all ages examined, but by day 18 it was demonstrable in ovaries and rose rapidly to reach a level of 6 pmol/h/mg protein by day 19. The time of appearance of the enzymatic capacity to convert testosterone to estradiol in the ovary is similar to the onset of the enzymatic capacity to form testosterone by the fetal testis, suggesting that the acquisition of the enzymatic activies that allow specific endocrine function by these two tissues may be regulated by the same or similar factors during embryonic development.

Androstenedione↗

PubMed2026

Clinical applications of digital twin technology in In Vitro Fertilisation.

BACKGROUND: Digital twin technology, originating from aerospace and manufacturing industries, has emerged as a transformative tool in healthcare. In vitro fertilisation (IVF) faces persistent challenges including suboptimal embryo selection, unpredictable treatment outcomes, and limited personalisation of protocols. Despite advances in assisted reproductive technology, existing literature exhibits fragmentation: artificial intelligence applications in embryo selection, ovarian stimulation, and endometrial assessment have been developed independently without systematic integration into comprehensive treatment frameworks. Digital twin technology offers unprecedented opportunities to create virtual replicas of biological systems, enabling real-time monitoring, predictive modelling, and personalised treatment strategies. AIM: This narrative review aims to critically examine the current applications of digital twin technology in IVF, evaluate its potential benefits and limitations, synthesize existing evidence into an integrative conceptual model, and identify future directions for implementation in reproductive medicine. METHOD: A comprehensive narrative review was conducted using PubMed, Scopus, Web of Science, and IEEE Xplore databases. A narrative review approach was selected over systematic review to accommodate the heterogeneity of evidence types in this emerging field, including theoretical frameworks, simulation studies, and proof-of-concept implementations that would be excluded from systematic reviews. Search terms included "digital twin," "IVF," "in vitro fertilisation," "assisted reproductive technology," "embryo selection," and "predictive modelling." Studies published between 2015 and 2025 were included, focusing on original research articles, systematic reviews, and proof-of-concept studies describing digital twin applications in reproductive medicine. RESULTS: Digital twin technology in IVF demonstrates significant potential across multiple domains including embryo development simulation, ovarian response prediction, endometrial receptivity modelling, and personalised stimulation protocols. Current applications integrate artificial intelligence, machine learning algorithms, time-lapse imaging, and omics data to create comprehensive virtual models. Early evidence suggests improvements in embryo selection accuracy, ovarian response prediction, and treatment protocol optimization, though large-scale randomized controlled trials remain limited. Implementation challenges include data integration complexity, computational requirements, regulatory considerations, and validation requirements. CONCLUSION: Digital twin technology represents a paradigm shift in IVF practice, offering personalised, predictive, and precision medicine approaches. This review synthesizes existing evidence to propose an integrative conceptual model for digital twin implementation across the IVF treatment spectrum, identifies critical knowledge gaps, and establishes research priorities to advance clinical translation. Despite current limitations, continued advancement promises improved success rates and patient outcomes.

Humans↗

PubMed1977

[Effect of some organosilicon compounds (silatranes) on the biosynthesis of collagen in cartilagenous tissue of chick embryo].

Effect of some organosilicon compounds from the class of silatranes on the biosynthesis of collagen in cartilagenous tissue of chick embryos in vitro was studied. The criteria for assessing the intensity of collagen biosynthesis was the formation of peptide bond 14C-hydroxyproline from 14C-proline of incubation medium. All the compounds studied (methylsilatrane, ethoxysilatrane and chloromethylsilatrane) stimulated total protein biosynthesis and collagen biosynthesis in cartilagenous tissue of chick embryos. The most pronounced stimulation of the biosynthesis of collagen was observed at 3-10(-3) M concentration of silatranes within 180 min. of incubation (14C-proline was added to the incubation medium 60 min after the beginning of the incubation). The activity of partly purified collagen prolyl-hydroxylase in the presence of silatranes was also studied.

Animals↗

PubMed2026

Evaluation of germline transmission of electroporation-mediated double gene-edited cattle lines.

Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.

Animals↗