Origin, viability, and cell cycle status of seminal somatic cells for potential application in cattle cloning via nuclear transfer.

Cryopreservation of Drosophila melanogaster embryonic nuclei in a Dimethyl sulfoxide (ME2SO)-free solution.

Very low efficiency limits the application of interspecies somatic cell nuclear transfer (iSCNT) on preserving endangered species. Our study demonstrated when taxonomical distance increased from intra-species to inter-species to inter-subfamily, the blastocyst rates decreased gradually (27.8% to 12.4% to 0). Scriptaid treatment significantly improved blastocyst rate of Bearded pig iSCNT embryos, accelerated the division timing and enhanced the level of H3K9ac at earlier stages. Transcriptome analysis revealed that half of the comparison groups based on taxonomical relations or Scriptaid treatment, displayed high proportion of enriched GO terms related to mitochondrion structure and/or function. Inter-subfamily or inter-species comparison groups reflected in downregulated differentially expressed genes (DEGs) or upregulated DEGs, contrarily. Accordingly, all these comparison groups displayed KEGG pathway enrichment to oxidative phosphorylation under their top pathways. The key genes related to the GO terms were identified for taxonomical relations or Scriptaid treatment, which are mainly from the NADH-ubiquinone oxidoreductase (NDUF) or ND gene family, separately. These findings suggest that taxonomical distance or Scriptaid treatment is closely associated with development fate of iSCNT embryos in Suidae through regulating the expression of mitochondrion-related genes. Our distinct contribution is identifying specific dysregulation in the NDUF (nuclear-encoded) and ND (mitochondrially encoded) gene families as a potential molecular basis for the "taxonomic wall," specifically showing how Scriptaid partially rescues this uncoupling.

Transcriptional drug repositioning enhances the development of somatic cell nuclear transfer embryos by inhibiting CDK4/6 during the first cleavage of bovine pseudo-zygotes.

Somatic cell nuclear transfer (SCNT) is a valuable tool in regenerative medicine, yet its efficiency remains limited by epigenetic reprogramming barriers that have been partially corrected by global regulation of epigenetic enzymes. However, these approaches lack gene locus specificity and may disrupt normal gene regulation. Therefore, new strategies capable of broadly enhancing reprogramming fidelity are needed. Here, we demonstrate that overexpression of the pioneer transcription factor Nr5a2 in mouse SCNT embryos improves both zygotic genome activation and the morula-to-blastocyst transition, two major developmental barriers in SCNT, and enhances birth rates. Mechanistically, Nr5a2 recruits P300 to increase H3K27ac at genes with low expression, restoring transcriptional activity and promoting SCNT embryo development.

Pig somatic cell nuclear transfer (SCNT) has valuable applications in agriculture, biomedicine, and life sciences, yet low cloning efficiency remains a major constraint limiting its application. To systematically investigate factors related to the production efficiency of pig cloning, this study conducted a retrospective analysis of 367,701 SCNT embryos transferred into 2019 surrogate sows over five years, focusing on breeds of donor cells, the season of embryo transfers, and the number of embryos transferred per surrogate. Our data demonstrate that the genetic background of donor cells is a critical determinant. SCNT embryos generated by wild-type (WT) Pietrain and Duroc pigs yielded significantly higher cloning efficiencies compared to those from Large White and Yorkshire pigs. This breed-specific influence was also observed with genetically modified (GM) donor cells. Nevertheless, within the GM groups, GM-Duroc and GM-Yorkshire showed superior efficiency compared to GM-Large White and GM-Bama. Furthermore, Summer was identified as the least favorable season for embryo transfer, with significantly lower pregnancy rates, delivery rates, and cloning efficiency compared to the other seasons. Importantly, we established that transferring 100-150 embryos per recipient optimized cloning efficiency, significantly outperforming groups receiving higher embryo numbers without compromising pregnancy rates, delivery rates, or average litter sizes. Our findings provide valuable guidance for optimizing large-scale SCNT protocols in swine.

Inhibition of SUV39H1 and SUV39H2 promotes zygotic genome activation and improves the developmental competence of porcine somatic cell nuclear transfer embryos.

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase (TYR) gene, resulting in melanin deficiency and severe visual impairments. Although mouse models provide insights into OCA1 pathogenesis, they exhibit significant anatomical and physiological differences from humans, particularly in ocular structure and function, thereby limiting their ability to recapitulate human OCA1 phenotypes. Therefore, in this study, we generated a porcine OCA1 model by selection-free genome editing via somatic cell nuclear transfer to characterize ophthalmological features and evaluate their translational relevance to human OCA1. Our approach utilized TYR-targeting CRISPR/Cas9 ribonucleoproteins without the need for single-cell-derived clonal expansion, thus streamlining the generation process. After somatic cell nuclear transfer with TYR knockout donor cells, the embryos demonstrated normal in vitro embryonic development comparable to the control, resulting in four healthy OCA1 piglets that exhibited characteristic OCA1 phenotypes with complete melanin loss in ocular and cutaneous tissues. Comprehensive ophthalmological analyses revealed significant structural abnormalities, including marked reduction in retinal layer thickness and elevated intraocular pressure. Remarkably, electroretinography revealed selective impairment of the rod bipolar pathway with reduced b-wave amplitudes and increased oscillatory potentials, indicating disturbances in synaptic processing. Overall, our study demonstrates the efficiency and reliability of selection-free genome editing for generating porcine OCA1 models. Moreover, the ophthalmological findings provide valuable insights for exploring retinal dysfunction and pigmentation mechanisms and advancing the preclinical evaluation of potential therapeutic interventions for human OCA1.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-28385-9.

Semen is a complex fluid that, in addition to spermatozoa, contains other cell populations, including immune cells, immature male germ cells, epithelial cells, and fibroblasts. These cells share the diploid condition, making them suitable candidates as nuclear donors for somatic cell nuclear transfer (SCNT) cloning. The generation of viable embryos and offspring has been demonstrated using these cells. Effective methods for isolating them from semen include centrifugation and osmotic gradient techniques; however, prolonged in vitro culture periods are necessary to establish primary cultures from these isolated cells. Furthermore, the samples that were obtained contained a high load of spermatozoa, which interferes with the establishment and maintenance of in vitro cultures. To date, primary cultures have only been successfully established from fresh semen samples, while attempts using cryopreserved semen have consistently failed. This limitation significantly restricts the potential to generate clones from cryopreserved semen samples. The present study proposes a simple microaspiration-based methodology for isolating somatic cells from cryopreserved equine semen. This approach allows for the rapid retrieval of a sufficient number of cells for immediate use in SCNT cloning procedures.

Regenerative medicine is one of the most innovative areas of modern medical science. It is engaged in the treatment of chronic diseases by restoring damaged tissues and organs both through the activation of their regenerative potential and through the introduction of stem cells as well as the implantation of tissue-engineered structures. This innovative field of medicine combines a large number of rapidly developing biomedical technologies. They are able to actively intervene in human nature, and therefore need theological thought from the point of view of Orthodox anthropology. Current areas of regenerative medicine are the development of cellular and tissue engineering products, as well as the creation of artificial organs using the 3D bioprinting method and the development of biomaterials with well-defined properties. Currently, a number of theological problems of regenerative medicine require close attention. First of all, these include the problem of finding an ethically acceptable source of stem cells from the position of Orthodox anthropology. Notable the theological assessment of new technologies for interfering with the cellular genome and implanting tissue-engineered structures in humans. The theological assessment of therapeutic cloning and 3D bioprinting methods is also important. The article is concerned with a review of the theological literature on the possibility of using key methods of regenerative medicine from the perspective of Orthodox anthropology.

Marmosets are a valuable model for studying human disorders, but developing specific genetic models requires efficient protocols for embryo genomic manipulation. Although marmoset embryos have been produced in vitro, oocyte retrieval traditionally involves extended recombinant human follicle-stimulating hormone (FSH) treatment (9-10 days), surgical laparotomy and ovarian exposure, limiting repeat procedures. Here we evaluated a simpler, cost-effective ovarian stimulation protocol using porcine pituitary FSH, combined with laparoscopic ovum pick-up (LOPU), for in vitro production of marmoset embryos via in vitro fertilization (IVF) and somatic cell nuclear transfer. A total of 3,922 oocytes were retrieved from 129 LOPU procedures, averaging 30.4 oocytes per LOPU, with 85.5% (26.0 per LOPU) graded as viable. The procedure was safe, with no adverse effects observed in ovaries or fimbria after up to nine LOPU sessions. Most collected oocytes were meiotically immature and matured in vitro before use. Following IVF, approximately 40% of oocytes cleaved, and 40% of cleaved embryos developed to the expanded blastocyst stage. LOPU-derived oocytes also supported somatic cell nuclear transfer, with 69.8% of embryos cleaving and 21.8% forming blastocysts. IVF-derived blastocyst quality was assessed by total cell count, immunodetection of SOX2 (inner cell mass) and CDX2 (trophectoderm), and cryotolerance. Embryo transfer to recipients resulted in successful live births. These findings demonstrate that a simplified pituitary FSH protocol followed by LOPU is an efficient, less invasive and safe method for retrieving developmentally competent marmoset oocytes, offering a promising approach for advancing marmoset-based research in disease modeling and reproductive biotechnology.

Abstract Precise control of microscale object rotation is essential for numerous biomedical and microelectromechanical applications. For example, somatic cell nuclear transfer for aquatic biomedical models such as zebrafish faces significant technical challenges, particularly in egg trapping and alignment of an injection needle with the micropyle. In this study, we developed a 3D resin-printed microdevice to achieve frequency-selective electrorotation of dielectric microspheres using a quadrupole electrode configuration driven by phase-shifted alternating current (AC). Theoretical analysis based on the Clausius–Mossotti factor, which governs the polarization of a particle concerning its surrounding environment, highlights the critical role of its imaginary component in the induced dipole moment from the AC field that generates torque. Simulations conducted in COMSOL Multiphysics confirmed the formation of symmetric torque-driven rotation without significant micro-scale object translation. The frequency response of angular velocity exhibited a unimodal profile, with a peak near 4 MHz corresponding to maximum torque efficiency. Experimental validation using 700 µ m polystyrene microspheres in Dulbecco’s Phosphate Buffered Saline demonstrated consistent clockwise rotation, with a peak angular velocity of 8.1° s −1 observed at 900 kHz and 16 Vp–p. Although the experimental peak angular velocity occurred at a lower frequency than the theoretical maximum, the rotational trend followed the polarization relaxation behavior captured by Im[ K cm ]. Parameter studies further revealed that increasing microscale object permittivity amplified torque generation, while higher medium permittivity reduced it, underscoring the tunability of electrorotation via dielectric properties. This work demonstrates a robust and scalable platform for manipulating large microscale objects. It lays the foundation for future applications involving biologically relevant objects, such as eggs of biomedical research models.

CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.

Derivation of embryonic stem cells from cloned blastocysts using improved somatic cell nuclear transfer in common marmosets.

Artificial oocytes through haploidization of mouse endometrial stromal cells and bone marrow-derived mesenchymal stem cells.

To explore new ways to improve canine cloning efficiency, this study compared the effects of different concentrations and treatment times of glycolysis promoter PS48 and epigenetic modifiers (DNA methylase inhibitor RG108 and histone deacetylase inhibitor Scriptaid) on the developmental ability of canine-porcine interspecies somatic cell nuclear transfer (iSCNT) embryos. The results showed that (1) 5 μmol/L PS48 treatment on canine ear fibroblasts (cEFs) and canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) for 24 h significantly enhanced subsequent iSCNT embryo development. The cleavage rate, 4-cell stage rate and 8-cell stage rate of iSCNT embryos produced from PS48-treated cEFs were significantly higher than those of control iSCNT embryos (46.90±1.64% vs 13.30±1.61%, 32.30±1.55% vs 8.26±0.88%, and 10.62±1.68% vs 5.50±0.84%; P<0.05). The cleavage and 4-cell stage rates of iSCNT embryos generated from PS48-treated cAd-MSCs were significantly higher than those of control iSCNT embryos (49.51±3.00% vs 31.25±2.73%, 26.21±2.08% vs 15.18±1.58%; P<0.05). (2) Treatment of cEFs and cAd-MSCs with 20 μmol/L RG108 for 48 h had no significant effect on the developmental efficiency of iSCNT embryos. Treatment of cEFs and cAd-MSCs with 0 nmol/L, 400 nmol/L, 500 nmol/L and 600 nmol/L Scriptaid for 24 h had no significant effect on the developmental efficiency of iSCNT embryos. (3) Treatment of iSCNT embryos derived from two types of donor cells with 20 μmol/L RG108 significantly promoted their developmental competence (P<0.05). Treatment of iSCNT embryos derived from cEFs with 500 nmol/L Scriptaid for 16 h significantly increased their cleavage and 4-cell stage rates (23.08±2.94% vs 9.47±1.70%, 18.68±3.25% vs 6.32±1.07%; P<0.05). This study established some new methods that can significantly improve the developmental efficiency of canine-porcine iSCNT embryos, thereby contributing to the development and application of canine somatic cell cloning technology.

The success of cloning animal paves a new way to increase livestock production and conserve genetic resources. After the success of first cloned pig production, a large number of research reports related to cloned pig have been published. However, a few researches into reproductive ability of cloned pig were conducted. The evaluation of reproductive performance of cloning pig is of great importance in practical application for breeding and reproduction. The purpose of present study was to compare reproductive performance of cloned Large White boars with non-clones. First, cloned pigs were produced by somatic cell nuclear transfer (SCNT) technique. After confirmation of cloning success, two groups (clones versus non-clones) were created for a comparison. The semen quality and reproductive performance of pigs derived from SCNT were tested. Growth performance of their progeny was also compared. The results showed that there were no significant differences in semen quality and reproductive performance between clones and non-clones. The parameters related to growth performance such as weight and average daily gain were similar in both groups. To sum up, no remarkable differences were observed between clones and non-clones, indicating that SCNT technology could be applied not only to increase swine production but also to preserve and spread superior Large White pig species.

This study aims to analyze the influence of hormone-treatment duration in &amp;lt;i&amp;gt;in vitro &amp;lt;/i&amp;gt;maturation of porcine immature oocytes on the pig cloning efficiency. Although there were no significant differences in the maturation rate of immature oocytes and cleavage rate of somatic cell nuclear transfer embryos according to the hormone-treatment period (22, 27, 32, 37 and 42 h), the blastocyst rates of somatic cell nuclear transfer embryos derived from oocytes cultivated for 22, 27 and 32 h (19.0%, 21.5% and 22.4%, respectively) were significantly (&amp;lt;I&amp;gt;P&amp;lt;/I&amp;gt;&amp;lt;0.05) higher than those for 37 and 42 h (13.2% and 12.4%, respectively). In particular, there were significant (&amp;lt;I&amp;gt;P&amp;lt;/I&amp;gt;&amp;lt;0.05) increase in the hatching blastocyst rates of 27 and 32 h cultivated groups (13.7% and 16.5%, respectively) compared to 22, 37, 42h cultivated groups (8.2%, 5.6% and 6.7%, respectively). Moreover, the cloning efficiency and the delivery rates of normal lives of 27 (1.4% and 1.2%, respectively) and 32h (1.5% and 1.3%, respectively) cultivated groups were significantly (&amp;lt;I&amp;gt;P&amp;lt;/I&amp;gt;&amp;lt;0.05) higher than those of 22h cultivated group (0.9% and 0.6%, respectively). To sum up, this study shows that the increase of hormone-treatment duration in&amp;lt;i&amp;gt; in vitro&amp;lt;/i&amp;gt; maturation of porcine immature oocytes to a certain extent has a positive impact on the cloning efficiency.

The intensification of global climate warming exacerbates the issue of heat stress in dairy cows, making the SLICK mutation in the prolactin receptor (PRLR) gene a critical target for enhancing heat tolerance in these animals. This study aims to investigate the effects of CRISPR/Cas9-mediated editing of the PRLR gene on the biological characteristics of bovine fibroblasts and early embryonic development following somatic cell nuclear transfer (SCNT). Using the CRISPR/Cas9 system, we targeted and edited a 20 bp-150 bp region within exon nine of the PRLR gene. After conducting off-target predictions and activity screenings, we identified optimal guide RNA (sgRNA) sequences and established stable transgenic cell lines. Transcriptome sequencing was performed on edited cells to identify key genes and validate their expression profiles. Edited cells were utilized as donor cells for SCNT, during which we assessed oocyte levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial function to analyze embryonic developmental performance. We constructed a cellular stress resistance network aimed at mitigating damage transmission while maintaining embryonic developmental homeostasis. This research provides technical support and theoretical reference for genetic editing breeding programs aimed at improving heat tolerance in dairy cattle.

Placental enlargement in somatic cell nuclear transfer (SCNT)-derived mice is attributed to biallelic expression of noncanonical (H3K27me3-dependent) imprinted genes owing to loss of imprinting (LOI). Here, we investigated whether a similar mechanism underlies placental enlargement in intersubspecific hybrids between BDF1 (Mus musculus domesticus) and HMI (M. m. castaneus) mice. Quantitative and allelic expression analyses revealed gene-specific LOI in (BDF1 × HMI)F1 placentas: Jade1 (Phf17) and Slc38a4 showed LOI in all placentas regardless of expression levels, whereas Gab1 and Sfmbt2 exhibited LOI only when expression levels were elevated. Notably, Jade1 and Slc38a4 also showed biallelic expression at lower levels in normal-sized (BDF1 × JF1 [M. m. molossinus])F1 placentas. Maternal knockout of Jade1, Slc38a4, Sfmbt2, or the Sfmbt2 miRNA cluster restored monoallelic expression and significantly reduced the weight of (BDF1 × HMI)F1 placentas, indicating that these genes were collectively responsible for placental enlargement in intersubspecific hybrid placentas. Transcriptomic analysis revealed that LOI of noncanonical imprinted genes occurred after implantation. These findings suggest that placental enlargement in (BDF1 × HMI)F1 hybrids is driven by overexpression of multiple noncanonical imprinted genes, resulting from LOI after implantation and additional hybrid-specific, yet unidentified, upregulation mechanisms.