Abstract Background Mastitis, one of the most prevalent inflammatory diseases in female mammals, causes significant economic losses in livestock farming. Notably, the natural flavonoid compound baicalin exhibits potent anti-inflammatory activity. However, its efficacy in alleviating mastitis severity and the underlying molecular mechanisms remain unexplored. Therefore, this study aims to investigate the protective effects of baicalin in alleviating mastitis and its key molecular mechanisms. Results This study demonstrated in vivo that baicalin effectively alleviates mastitis symptoms in dairy cows and mice, primarily manifested by reduced tissue pathological damage, decreased levels of pro-inflammatory cytokines, and maintain the integrity of the blood-milk barrier (BMB). Multi-omics sequencing analysis indicated that IL-17 and TNF signaling pathways play crucial roles in this process. Further studies demonstrated that IL-17RA −/− mice exhibited a phenotype similar to that observed with baicalin treatment, confirming the importance of this pathway. Notably, network pharmacology screening combined with molecular dynamics simulations revealed stable binding of baicalin to IL-17RA, suggesting that baicalin exerts its protective effect to alleviate mastitis by targeting IL-17RA. Mechanistically, both baicalin treatment and IL-17RA deletion block activation of key downstream pathways of the IL-17 signaling pathway, including MAPK, ERK and NF-κB, thereby suppressing excessive activation of the TNF signaling pathway, preventing exacerbation of the inflammatory response and barrier damage. Conclusions In conclusion, this study demonstrates that baicalin inhibits excessive activation of the IL-17/TNF signaling pathway by targeting IL-17RA, thereby reducing inflammatory responses and BMB damage within the mammary gland and alleviating mastitis severity.

As the global population continues to grow, the demand for protein is correspondingly increasing. Milk serves as a significant source of high-quality protein, with casein being its primary nutrient. Consequently, the cultivation of dairy animals with elevated casein expression has become a critical objective in the field of livestock breeding. Compared with traditional breeding methods, gene editing offers a more efficient approach to enhancing casein expression in dairy animals. However, progress in this area is constrained by the lack of key editing targets. Enhancers, which are core cis-acting elements enriched with transcription factor and cofactor binding sites, play a crucial role in regulating milk protein expression and represent ideal targets for gene editing. At present, comprehensive research on enhancers at the casein gene locus in livestock remains limited. This study identified 8 ultra-conserved regions through a cross-species comparison of casein gene loci in humans, mice, cattles, zebu cattle, goats, sheep, and camels. Following functional validation in silico, cross-species conserved enhancer sequences regulated by STAT5a were validated, with the CSN2 conserved enhancer (CSN-EN3) demonstrating the most potent regulatory effect. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation assays have demonstrated that STAT5a interacts with cofactors such as MED1, GR, ELF5, and NFIB, thereby synergistically regulating gene expression. The findings suggest that transcription factors and cis-acting elements associated with lactation exhibit high interspecies conservation, elucidating the pivotal role of STAT5a in lactation regulation. By altering the conserved enhancer CSN-EN3, its cis-regulatory control over STAT5a-dependent transcription was changed, leading to a 3.57-fold increase in casein expression. In conclusion, this study developed an enhancer identification system that integrates multi-species genome alignment with model animal epigenetic marker analysis, successfully identifying cross-species conserved enhancers at the mammalian casein locus. This research introduces a novel strategy to augment casein expression in ruminants, providing a significant theoretical foundation and technical support for the precision breeding of high-casein dairy cows and goats.

Dynamic changes in mammary cells are essential for sustaining lactation and maintaining epithelial homeostasis. However, the phenotypic transition process of mammary cells during lactation remains unclear. Here, single-nucleus RNA sequencing (snRNA-seq) of 64 199 cells and single-nucleus chromatin accessibility sequencing (snATAC-seq) of 78 984 cells were generated from the goat mammary gland of dry and lactation stages. A total of 18 cell types were annotated, and spatial transcriptomic analysis confirmed the localization of lactation-related cell types within the mammary tissue. Enrichment analysis of SNP within cell type-specific chromatin accessibility regions revealed strong associations between mammary epithelial cells (MECs) with milk production traits. To further explore the MECs functional diversification during lactation and their differences from the dry stage, four differentiation trajectories from luminal progenitor to luminal mature cells were reconstructed. Lineage-specific gene regulatory networks (GRNs) were constructed by integrating snRNA-seq and snATAC-seq data, and stage-specific signals were identified through cell-cell communications. Finally, to explore the evolutionary conservation and divergence of MECs, cross-species comparative analyses were conducted and revealed MEC differential evolutionary rates, conserved milk-producing subtypes, and lineage-specific populations driving species-specific differences in milk composition. Overall, these findings uncover the coordinated transcriptional and chromatin dynamics that drive mammary epithelial differentiation and functional maintenance during lactation.

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis in adult mammals. However, SSCs are highly heterogeneous, comprising a complex array of subpopulations whose identities and dynamic transitions remain greatly underappreciated. Through in silico analysis, we identified IL1R2 as a surface marker specific to the SSC subpopulation. IL1R2 enables the specific sorting of functionally active SSCs in both human and mouse. Il1r2 CreERT2/+ Rosa26 mTmG/+ mice allowed us to pulse-label and trace the lineage of Il1r2-expressing cells. We confirmed that IL1R2+ SSCs support spermatogenesis via both self-renewal and differentiation. Following spermatogenic disruption, IL1R2+ SSCs are reactivated for proliferation via the PI3K-AKT-mTORC1 pathway to replenish the SSC pool. Importantly, we demonstrated that PI3K-AKT-mTORC1 agonists can effectively enhance the recovery of spermatogenesis upon disruption. These findings highlight a promising therapeutic strategy to mitigate chemotherapy-induced infertility.

Bovine pluripotent stem cells (PSCs) hold significant potential for diverse applications in agriculture, reproductive biotechnology, and biomedical research. However, challenges persist in establishing stable bovine PSC lines and understanding the mechanisms underlying their pluripotency maintenance. Here, we derived bovine embryonic stem cells (bESCs) from Holstein cattle embryos. These cells exhibited robust differentiation capacity into three germ layers in vitro and in vivo. Transcriptome analysis revealed distinct molecular profiles compared to primed-state bESCs. Notably, bESC proliferation ceased on methanol-treated feeder cells, in contrast to mouse ESCs (mESCs), which proliferated normally. Pathway analysis identified key signaling events critical for bESC survival and proliferation, highlighting species-specific regulatory mechanisms. Furthermore, the derived bESCs demonstrated chimerism capacity in early bovine embryos, underscoring their functional pluripotency. This work provides a foundation for advancing bovine embryology research and stem cell-based biotechnologies in livestock.

Dairy goat milk possesses substantial nutritional value, and comprehending the regulatory mechanisms of lactation is crucial for enhancing the milk production performance of dairy goats. During lactation, the mammary gland of dairy goats exhibits marked alterations in the expression of numerous genes. While extensive research has clarified the mechanisms governing mammary gene expression at the transcriptional level, the regulation of these genes at the translational level remains largely unexplored. In this study, ribosome-sequencing and RNA-sequencing analyses were conducted on the mammary glands of dairy goats during both the nonlactation and lactation periods. The findings revealed that the lactation process significantly influences both the translation and transcription of genes, with a notably higher overall translation efficiency (TE) observed during lactation compared with the nonlactation period. Transcription and translation collaboratively regulate gene expression in mammary tissues, thereby constructing a complex regulatory network. We systematically identified small open reading frames (sORF) in mammary glands, demonstrating that upstream open reading frames suppress the translation of main open reading frames. The RNA-binding proteins (RBP) were found to significantly influence the gene TE. Notably, the sORF3917 located in the 5' untranslated region of the FASN gene was shown to regulate fatty acid synthesis-related gene expression, highlighting the role of sORF in lactation. This study presents novel insights into the regulatory mechanisms of lactation in dairy goats and provides valuable genetic resources for gene editing and breeding strategies aimed at enhancing dairy goat production.

Mastitis is a prevalent disease in dairy livestock, posing a significant threat to milk quality and the economic efficiency of the dairy industry. Transcriptomic analysis of mammary gland samples from healthy and mastitis-affected animals revealed that inflammatory responses and immune regulatory processes play central roles in the progression of the disease. Differentially expressed genes were significantly enriched in Gene Ontology (GO) categories related to cytokine-cytokine receptor interactions, cell adhesion molecules, and the JAK-STAT signaling pathway. Quantitative trait locus (QTL) association analysis indicated the importance of CXCR1/ CXCR2. Literature evidence further suggests that lactoperoxidase (LPO) protein can mitigate inflammation by reducing CXCL2, a ligand of CXCR1/CXCR2. In this study, siRNA-mediated knockdown of the LPO gene demonstrated that LPO silencing under bacterial lysate stimulation markedly increased the expression of pro-inflammatory cytokines (IL8, CXCL2, IL1 beta; p < 0.01). Moreover, weighted gene co-expression network analysis (WGCNA) highlighted a key role for ubiquitination in mastitis responses. Experimental results showed that inflammatory stimulation significantly upregulated both inflammatory mediators (NF kappa B, IL1 beta, Caspase1) and deubiquitinating enzymes (CYLD, A20; p < 0.01). Based on these findings, this study proposes that future mastitis intervention strategies may benefit from enhancing endogenous antimicrobial proteins while modulating ubiquitination processes to attenuate excessive inflammatory responses. In summary, this study systematically elucidates the key molecular mechanisms underlying mastitis and proposes therapeutic and breeding approaches, providing a foundation for more effective clinical interventions and the development of mastitis-resistant breeding lines, with potential implications for enhancing animal health, milk quality, and the sustainable development of the dairy industry.

Leydig cells (LCs) are the primary testosterone-producing cells. Their steroidogenic capacity is heavily influenced by the postnatal development of the LC lineage. The final developmental stage of LCs involves transition from immature LCs (ILCs) to adult LCs (ALCs) during puberty. However, the morphological and functional changes that occur during this transition in most mammals remain unclear. In this study, we conducted a comparative analysis of morphological and functional changes in developing LCs in dairy goats. The results revealed that during the transition from ILC to ALC, the ovoid LC transformed into an irregular round shape with a well-developed smooth endoplasmic reticulum (sER), numerous mitochondria, and small lipid droplets (LDs) distributed in the cytoplasm. Subsequently, the isolated primary ILCs were matured using chorionic gonadotropin (CG). CG stimulation increases the expression of steroidogenic genes and decreases the levels of testosterone-metabolizing enzymes. In vitro studies have shown that small LDs are the preferred source of cholesterol substrates in steroidogenesis, and that their interaction with mitochondria facilitates free cholesterol transport. Further analyses using confocal microscopy, ultrastructural analysis, and co-immunoprecipitation consistently demonstrated that vimentin filaments contributed to the interaction between LDs and mitochondria. Specifically, the knockdown of vimentin using siRNA substantially reduced the physical contact between LDs and mitochondria, resulting in inadequate cholesterol transport and ultimately impaired steroidogenesis. Overall, our findings indicate that significant morphological and functional changes occurred during the transition from ILCs to ALCs in goats. The cytoskeleton, primarily composed of vimentin filaments, is an important component of the interactions between LDs and mitochondria and contributes to cholesterol transport during testosterone synthesis.

Ruminants play a crucial role in dairy farming, pharmaceuticals, and embryonic stem cell research; thus, it is vital to prevent pregnancy loss and improve reproductive outcomes through a better understanding of placental development. Paternally-expressed gene 10 (PEG10) is a conserved gene essential for placental development in mammals, but its function in ruminants is not well understood. To develop insights into its role in placental development, this study investigated the gene structure and expression of PEG10 in cattle and goats. We found that PEG10's structure was conserved across species, and in the placenta, it retained the ability to bind to its own mRNA. Transcript analysis revealed differential expression patterns of PEG10 at early and late stages of placental development. We identified 70 proteins potentially interacting with PEG10 that were involved in biological processes like metabolism, signal transduction, cell proliferation, and immune responses. These proteins were grouped into seven clusters, associated with pathways such as amino acid degradation, the TCA cycle, longevity regulation, cardiomyopathy, proteasome function, and biosynthesis. Our findings suggest that PEG10 regulates placental development in ruminants by interacting with key proteins like CAST, ITGA6, and FTL, which are responsible for critical cellular processes in placental function.

Crossbreeding has emerged as a strategy to combine desirable traits from different sheep breeds, with the goal of enhancing productivity, disease resistance, and growth rates. This study compares the immune responses, rumen microbiomes, and serum metabolites of Hu sheep, East Friesian (EF) sheep, and crossbred Hu × EF (DH) sheep to explore the effects of crossbreeding on productivity and disease resistance. Hu sheep exhibited significantly higher lymphocyte counts (p < 0.05) and white blood cell (WBC) counts (p < 0.05) compared to EF and DH sheep, indicating stronger basal immune responses. DH sheep showed superior immune responses, with a higher cluster of differentiation 4+/cluster of differentiation 8+ (CD4+/CD8+) T cell ratio (p < 0.05) compared to EF sheep. Rumen microbiome analysis revealed distinct microbial profiles; DH sheep exhibited higher relative abundances of Prevotella (p < 0.05), which is associated with improved growth and disease resistance. Metabolomic analysis revealed significant differences in bile acid profiles: DH sheep exhibited higher levels of 6-keto lithocholic acid (6-ketoLCA), cholic acid and chenodeoxycholic acid (CDCA), and 3β-hyodeoxycholic acid (3β-HDCA) (p < 0.05), which is associated with improved immune function and gut health. These results indicate that crossbreeding improves immune resilience and metabolic efficiency, which has implications for breeding strategies designed to enhance livestock productivity and disease resistance.

Mastitis poses a huge economic burden, with antibiotics treatment leading to drug residues and the emergence of bacterial resistance. Therefore, there is an urgent need to develop alternatives to antibiotics for the treatment of mastitis. Anthocyanins (ACN) have excellent anti-inflammatory properties. However, the key pathways of mastitis disease progression and the therapeutic effects of ACN on mastitis remain poorly understood. In this study, using cows, goats, and mice as animal models, the efficacy of ACN extracts from crabapple fruit is investigated in alleviating the severity of mastitis and blood milk barrier (BMB) damage. A monomeric component, cyanidin 3-O-galactoside (C3Gal) is identified, that exhibited considerable effect on mastitis. Mechanistically, C3Gal regulated mastitis progression by inhibiting PANoptosis activation, and suppressing gasdermin D N-terminal activity to regulate BMB damage during mastitis. These findings establish PANoptosis as a critical pathway in the rapid progression of mastitis and highlight the potential of ACN extracts as effective alternatives to antibiotic for mastitis treatment. This study provides a promising strategy for the discovery and application of drugs for the treatment of inflammatory diseases, expands the application of ACN in inflammatory diseases, and elucidates the molecular mechanisms underlying its anti-inflammatory effect.

Ujimqin sheep, known for its distinctive multi-vertebrae phenotypes (T13L7, T14L6, and T14L7) and economic value, has garnered significant attention. However, conventional phenotypic detection methods suffer from low efficiency and high costs. In this study, based on a key SNP locus (ABCD4 gene, Chr7:89393414, C > T) identified through a genome-wide association study (GWAS), a TaqMan-MGB (minor groove binder) genotyping system was developed. the objective was to establish a high-throughput and efficient molecular marker-assisted selection (MAS) tool. Specific primers and dual fluorescent probes were designed to optimize the reaction system. Standard plasmids were adopted to validate genotyping accuracy. A total of 152 Ujimqin sheep were subjected to TaqMan-MGB genotyping, digital radiography (DR) imaging, and Sanger sequencing. the results showed complete concordance between TaqMan-MGB and Sanger sequencing, with an overall agreement rate of 83.6% with DR imaging. For individuals with T/T genotypes (127/139), the detection accuracy reached 91.4%. This method demonstrated high specificity, simplicity, and cost-efficiency, significantly reducing the time and financial burden associated with traditional imaging-based approaches. the findings indicate that the TaqMan-MGB technique can accurately identify the T/T genotype at the SNP site and its strong association with the multi-vertebrae phenotypes, offering an effective and reliable tool for molecular breeding of Ujimqin sheep.

The mammary gland is a unique organ in mammals and serves as the core tissue for lactation. Its development directly impacts lactation capacity and the quality of dairy products. Each developmental stage (embryonic, pubertal, pregnancy, lactation, and involution) is precisely regulated by various hormones, transcription factors, and signaling pathways. For large dairy animals, lactation not only provides essential nutrition for offspring but also serves as a critical source of high-quality raw materials for dairy product production for humans. Therefore, this review aims to elucidate the morphological changes, hormonal regulation, and related molecular regulation at different stages of mammary gland development in dairy animals. By summarizing these endogenous regulatory mechanisms, this review provides a theoretical reference for further research aimed at enhancing dairy animal production performance. Furthermore, the review presents a multidimensional perspective on the factors influencing mammary gland development and highlights emerging research directions, particularly in the application of advanced molecular technologies and multi-omics approaches to broaden the current understanding of mammary gland development.

Stable transgene expression in the mammary gland is crucial for recombinant protein production in livestock, yet it is frequently hampered by transgene silencing and random integration. To address this, we profile chromatin accessibility in goat mammary epithelial cells (GMECs) using ATAC-seq and identify 15 highly accessible genomic regions. Three of these regions are confirmed to support stable transgene expression. Notably, we identify a goat-derived ubiquitous chromatin opening element (UCOE) in the SF3B1-COQ10B intergenic region, with a high GC content (65%) and CpG island enrichment. This UCOE improves hfCas12Max-mediated integration of large DNA fragments and maintains high-level expression of human lactoferrin (hLTF) in GMECs. Subsequently, we precisely integrate the UCOE-hLTF cassette into the highly accessible loci and generate a transgenic goat via somatic cell nuclear transfer, without detectable off-target effects. Our pipeline, which integrates chromatin accessibility profiling, UCOE discovery, and precision editing, demonstrates the role of CpG island-containing UCOEs in preventing transgene silencing. The study provides valuable tools for enhancing recombinant protein production, supports the breeding of dairy goats for milk with high lactoferrin content, and advances the understanding of the interactions between chromatin, regulatory elements, and transgenes in molecular breeding.

Background Ruminants possess a rich repository of natural antimicrobial peptides(AMPs) within their bodies, surpassing those found in humans and mice. These peptides, including Defensin, Cathelicidin, and Lysozyme, are integral to the body's innate and adaptive immune responses and represent promising alternatives to antibiotics with significant application potential. Results In the present study, we conducted a systematic analysis of 40 Defensins, 38 Cathelicidins, and 61 Lysozymes in cattle and sheep. Our findings revealed that these peptides have retained functional integrity through the evolutionary history of these species. However, they exhibit unique gene duplication and expansion events when compared to humans and mice, indicating their potent roles in cattle and sheep. Notably, the Cathelicidin gene family experienced the most substantial expansion in these ruminants. The newly expanded genes were highly expressed in tissues and organs such as the tongue surface, intestine, mammary gland, and others, exhibiting tissue-specific preferences. This expression pattern is associated with the unique behaviors and high lactation capacity of ruminants. An in vitro bacterial inhibition assay demonstrated that EBD, LALBA, LYSB, and CATHL4 exhibited significant broad-spectrum antibacterial activity. Additionally, loci dB1, dB5, cB2, cB3, and yB1 were pinpointed as key co-regulatory elements in the antimicrobial peptide motifs within cattle mammary epithelial cells. Conclusions This research illuminates the structure-function relationship and antimicrobial potency of natural AMP genes in cattle and sheep, providing a theoretical foundation for the development of novel veterinary drugs to treat common bacterial diseases in ruminants and for enhancing animal health care. The identified transcriptional regulatory sites offer a new perspective on the molecular regulation of AMP genes expression, which can be leveraged to improve the disease resistance of domestic animals. This work contributes to a broader understanding of the evolution and regulation of AMP genes, with potential applications for animal health and breeding programs.

Improving the reproductive ability and fertility of male ruminants is a central concern in animal husbandry. Phytogenic feed additives, known for their anti-inflammatory, antioxidant, and immunomodulatory properties, are commonly used in animal feed. Icariin (ICA), the primary flavonoid glucoside derived from Epimedium, is a traditional tonic in Chinese herbal medicine. However, its potential to enhance the reproductive performance of male ruminants remains unclear. In this study, twelve healthy adult male dairy goats were divided into two groups. The goats received oral administration of ICA at doses of 0 (control) and 50 mg/kg body weight daily for a consecutive period of 80 days during the breeding season. The effects of ICA on the reproductive performance was analyzed by histological examinations, semen quality analysis, and ELISA experiments. ELISA results showed a progressive increase in serum levels of GnIH, LH, and testosterone with the prolonged ICA treatment (p < 0.05). However, the serum concentration of GnRH in the ICA group initially increased, followed by a subsequent decrease (p < 0.05). The hypothalamic concentrations of dopamine (DA) and 5-hydroxytryptamine (5-HT) were significantly higher in the ICA group compared to the control group (p < 0.01). The CASA system analyzed sperm kinematics and revealed that ICA increased ejaculate volume, with both total motile and progressive motile sperm gradually increasing over time (p < 0.05). ICA did not affect the body weight of the goats but significantly increased the organ coefficient of the testes (p < 0.01). Additionally, there was an upregulation of hormone receptor expression in testicular tissue and an improvement in the antioxidant capacity of the testes after ICA treatment (p < 0.01). Furthermore, ICA was implicated in testosterone synthesis by modulating the expression of key enzymes associated with steroidogenesis and promoting the differentiation of spermatogonial stem cell to enhance spermatogenesis. In conclusion, our results indicate that icariin, as a phytogenic feed additive incorporated into the diet of ruminants, offers potential benefits in improving the reproductive performance of male dairy goats.

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3′ terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals.