Pronounced melanin enrichment characterizes the Lueyang black-boned chicken, yet the transcriptional basis underlying this pigmentation phenotype remains incompletely understood. We established a standardized in vitro culture system for primary avian melanocytes to investigate CDKN2A-associated transcriptional changes related to melanocyte cellular states and pigmentation-related gene expression. Primary melanocytes were isolated and characterized by morphology, ferrous sulfate staining, and TRP2 immunofluorescence, with passages P2-P4 showing relatively stable cellular characteristics. Using CDKN2A overexpression and knockdown models combined with RNA-seq analysis, we identified transcriptional alterations associated with CDKN2A modulation. Notably, CDKN2A overexpression was associated with increased expression of cell cycle-related genes (e.g., CDKN1A) and melanogenesis-associated transcriptional markers, particularly MITF, suggesting an association between CDKN2A expression and pigment-related transcriptional programs. Conversely, CDKN2A knockdown produced opposite expression patterns. Gene set enrichment analysis (GSEA) further indicated that high CDKN2A expression was associated with enrichment of melanogenesis and MAPK signalling pathways, whereas CDKN2A knockdown was associated with enrichment of Wnt signalling pathways. By minimizing potential in vivo paracrine effects, this in vitro model suggested that CDKN2A-associated transcriptional changes might reflect alterations related to cell cycle status and melanocyte cellular states. Although this study provided transcript-level evidence, direct functional assays of melanin production were not performed; therefore, these findings should be interpreted as correlative rather than definitive evidence of CDKN2A-associated pigmentation regulation. These findings provide a transcriptomic resource for investigating the relationship between cell-state regulation and pigmentation-associated programs in avian melanocytes.

The precise targeted integration of large exogenous genes into chicken sex chromosomes is of particular interest for sex-linked trait manipulation, sex-control breeding, and the development of avian bioreactor models. However, efficient targeted integration at sex chromosome loci remains technically challenging, and optimized editing strategies for these loci are still lacking. To improve targeted genome editing at two previously identified chicken sex chromosome safe-harbor loci, EE0.6 and NC_006127.4, this study systematically evaluated and optimized key parameter affecting editing efficiency. First, we evaluated the effects of different sgRNA combinations on targeted knockout efficiency, establishing the advantage of a dual-sgRNA/Cas9 architecture, which achieved knockout efficiencies of 86.67% and 75.00% at the EE0.6 and NC_006127.4 loci, respectively. We next introduced the Cas9 nickase (Cas9n) system, which has previously been reported to exhibit improved editing specificity, and evaluated its performance at both target loci. Quantitative analysis showed that the dual-sgRNA/Cas9 targeting system successfully mediated the precise targeted integration of a 1.1-kb SV40-mCherry reporter cassette, reaching 100% (28/28) at the EE0.6 locus and 80.00% (20/25) at the NC_006127.4 locus. Based on this result, this study further investigated the effects of donor homology arm (HA) lengths (200 bp, 600 bp, and, 1000 bp) and vector topologies (circular and linearized) on targeted knock-in efficiency. The results revealed that in the circular donor system, the optimal HA lengths for the EE0.6 and NC_006127.4 loci were 200 bp (50.4% ± 4.4%) and 600 bp (30.1% ± 1.2%), respectively. However, upon the introduction of linearized donors with free ends, the knock-in efficiency of the exogenous target fragment was significantly enhanced, and its HA length preference underwent a significant reversal. The optimal HA length for EE0.6 was extended to 600 bp (78.9% ± 1.0%), whereas that for NC_006127.4 was shortened to 200 bp (49.9% ± 0.4%). In summary, this study established an efficient targeted integration strategy for chicken sex chromosome loci. The optimized system provides a foundation for future applications in sex-linked breeding and avian bioreactor development.

Research question: Do the different fertilisation methods of conventional in vitro fertilisation (cIVF) or intracytoplasmic sperm injection (ICSI) affect abnormal cleavage (ABNCL) of early embryos? Design: A retrospective cohort study on 125,570 normally fertilised embryos from a large reproductive medicine centre. Normal cleavage (NC) and ABNCL embryos in the entire cohort were identified. Multivariable logistic regression with generalised estimating equations (GEE), stratified analysis and inverse probability weighting (IPW) were used to assess the incidence of ABNCL events in cIVF-and ICSI-derived embryos. Results: GEE analysis adjusted for multiple variables showed that the incidence of ABNCL was significantly higher in ICSI than cIVF (52.14 % vs 40.82 %, adjusted odds ratio [aOR] = 1.674, 95% confidence interval [CI]: 1.590-1.762, P < 0.001). Among the different female ages and ovarian stimulation regimens, higher incidences of ABNCL embryos were observed in ICSI compared with cIVF, and stratified analysis of sperm parameters showed that the incidence of ABNCL was significantly higher in ICSI than cIVF for the normozoospermia, mildly abnormal semen, and multiple abnormal semen groups (all P < 0.05). Conclusions: Embryos derived from ICSI were more prone to ABNCL than those derived from cIVF, and this association persists across female ages, different stimulation regimens and sperm parameter subgroups. Although semen parameters may modulate this risk, the consistent trend suggests that the ICSI procedure may be associated with an increased incidence of ABNCL.

Southwestern local chicken breeds in China have long been recognized for their strong adaptability and disease resistance, shaped by unique selective pressures during domestication. In this study, we combined Fst and π ratio analyses to detect selection signatures from RAD-seq data of four southwestern local breeds, Red Junglefowl, and 17 other Chinese local breeds. We identified 460 candidate genes under strong selection potentially associated with domestication, affecting behaviors, physiology, reproduction, immunity, and adaptability. Importantly, 34 genes showed strong and unique selection signals when comparing southwestern breeds with other local breeds. Among these, CCR6, CD40LG, and CSF3R represent candidate genes that have been previously associated with immune processes, while MAPK10, FGFR4, and STAT3 represent candidate genes potentially related to disease resistance. Furthermore, HTR1B, HSPB9, and RYR2 represent candidate genes potentially associated with environmental adaptability. Notably, ALX1, the only gene detected across all comparison groups, may reflect morphological adaptation related to feeding efficiency. Our findings provide valuable insights into the genetic basis of unique adaptations in southwestern local chicken breeds and highlight potential targets for breeding and conservation.

This study aimed to investigate how levels of serum follicle-stimulating hormone (FSH) on Day 7 of ovarian stimulation are related to the number of oocytes retrieved, and identify any cutoff or threshold point in protocols using gonadotropin-releasing hormone (GnRH) antagonists. We examined data from January 2017 to June 2024, covering 9,969 cycles from 7,981 patients using GnRH antagonists as part of in vitro fertilization processes. We found a complex link between serum FSH and oocyte collection. Lower serum FSH on Day 7 was associated with collection of fewer oocytes (β = −0.531, p < 0.0001), and different levels were associated with changes in the number of oocytes collected. The key point was 9.13mIU/mL. If serum FSH exceeded 9.13 mIU/mL on Day 7, each 1mIU/mL increase reduced the number of oocytes collected by 0.07 (effect β = −0.07, 95% confidence interval [CI] −0.10 to −0.05, P < 0.0001). When serum FSH was below 9.13 mIU/mL, each 1 mIU/mL rise increased the number of oocytes collected by 1.18 (effect β = 1.18, 95% CI 0.95 to 1.41, P < 0.0001). This association suggests that it may be possible to improve ovarian reaction by raising the FSH dose if serum FSH is under 9.13 mIU/mL. However, if serum FSH is already over 10 mIU/mL, it seems likely that other steps may be needed to increase ovarian reaction, although these hypotheses will need to be tested in future studies.

Sex determination is a crucial process in animal development, regulated by complex genetic networks. In avian species, Doublesex and mab-3 related transcription factor 1 (DMRT1) plays a vital role in gonadal development and sex determination. To unravel the function of DMRT1 in chicken sex determination, establishing an inducible DMRT1 knockout model is essential. In this study, we constructed an inducible DMRT1 knockout system and verified its efficiency and effects on related genes and physiological indicators. To achieve precise genomic ablation, we screened multiple sgRNAs targeting the DMRT1 locus and integrated the optimal sequence into a doxycycline-responsive (Tet-on) CRISPR/Cas9 architecture. For in vitro experiments, vectors were delivered via cell transfection and induced with 20 µg mL-1 doxycycline (DOX), achieving an 80% knockout efficiency. Following the administration of polyethylenimine (PEI)-encapsulated plasmids into chicken embryos, we successfully implemented the inducible system in vivo. Quantitative analysis confirmed a mosaic knockout of DMRT1 with an observed efficiency reaching 45%. Following targeted disruption, we evaluated sex-related gene and protein expression alterations via qRT-PCR and Western blot (WB). Furthermore, ELISA was performed to measure testosterone levels in male embryonic gonads across multiple developmental stages (E4.5 to E18.5). qRT-PCR analysis showed that after induction, female-related genes (CYP19A1, FOXL2, ESR1) were significantly upregulated, and male-related genes (DMRT1, SOX9, AMH) were significantly downregulated. WB results revealed increased protein expression levels of CYP19A1 and FOXL2, and decreased protein expression of SOX9 post-induction. ELISA confirmed that testosterone levels in the gonads of induced male embryos were significantly reduced compared to normal and non-induced males. The study successfully established an inducible DMRT1 knockout system in chickens. This system effectively regulates the expression of sex-related genes and reduces testosterone levels in male embryos, providing theoretical and technical support for breeding novel sex-controlled breeding materials.

Primordial germ cells (PGCs) are the progenitor cells of sperm and eggs. Xenotransplantation of chicken PGCs can achieve germline transmission. However, there are still challenges in obtaining many PGCs from endangered birds in vitro . In this study, at first, by incorporating 2i factors, the embryonic stem cells (ESCs) culture conditions were optimized, successfully yielding and validating pluripotent ESCs clones. Then, during induction ESCs, bFGF, activin A, and 1% KSR were added to Epiblast-like cells (EpiLCs). Quantitative real-time polymerase chain reaction (qRT-PCR) showed Pax6, Eomes, and Vimentin expression patterns similar to primary epiblast, indicating successful EpiLCs induction. During EpiLCs to Primordial germ cell-like cells (PGCLCs) transformation, we evaluated BMP4, BMP8b, EGF, LIF, and SCF combinations' impact on induction efficiency. Flow cytometry, qRT-PCR, and immunofluorescence showed high expression of Cvh, C-kit, Dazl, CVH, and DAZL in PGCLCs, suggesting successful EpiLCs differentiation. Induced PGCLCs injected into 2.5-day chick embryos migrated to gonads by day 7–7.5, demonstrating migration and colonization. This study optimized a two-step protocol for in vitro differentiation of chicken ESCs into PGCLCs. This research's results not only provide a reference for obtaining many PGCLCs in vitro but also open up a new approach for the development and application of genetic resource preservation technology in domestic chickens.

The Tibetan chicken (Gallus gallus domesticus), a native breed inhabiting the Qinghai-Tibet Plateau, has developed remarkable tolerance to chronic hypoxia. However, the molecular and epigenetic mechanisms underlying its high-altitude adaptation remain unclear. In this study, we integrated genome, transcriptome, and DNA methylome data from Tibetan chickens (TC) and three low-altitude breeds. Principal component analysis revealed clear genetic, epigenetic, and transcriptional divergence between TC and lowland chickens. Cardiac enzyme assays showed significantly higher activities of LDH, SDH, SOD, CAT, and GSH-Px in TC (p < 0.05), indicating enhanced oxidative metabolism and antioxidant defense under hypoxia. Transcriptomic analysis identified 2,532 common differentially expressed genes (co-DEGs), with upregulated genes enriched in oxidative phosphorylation, fatty acid metabolism, and hypoxia response pathways. Integration with methylome data demonstrated a significant negative correlation between promoter methylation and gene expression. Among 144 genes showing promoter hypomethylation coupled with transcriptional activation, five key genes—PDK4, BNIP3L, ATG3, SLC7A5, and OMA1—were identified as central regulators of hypoxia adaptation, participating in metabolic reprogramming, mitochondrial homeostasis, and autophagy. Our findings reveal that promoter hypomethylation acts as a major epigenetic mechanism mediating transcriptional activation of hypoxia-responsive genes in Tibetan chickens. The coordinated regulation of energy metabolism, antioxidant defense, and mitochondrial quality control contributes to their physiological resilience in high-altitude environments. This study provides novel insights into the molecular and epigenetic basis of high-altitude adaptation in avian species and offers valuable references for hypoxia-resistance breeding in poultry.

Chicken embryos and stem cells require precisely regulated levels of reactive oxygen species (ROS) to maintain self-renewal and pluripotency. However, excessive ROS induces oxidative stress, leading to DNA damage, chromosomal aberrations, loss of mitochondrial membrane potential, and ultimately, abnormal differentiation or cell death. This oxidative imbalance is a significant barrier to successful embryonic development and the in vitro culture of stem cells. Innate antioxidant defense systems exist within chicken and mammalian embryos to scavenge excess ROS, a finding that has prompted the strategic addition of antioxidants to culture media. The present review has two primary foci. Firstly, it seeks to expand the understanding of the antioxidant defense mechanisms of the chicken embryo. Secondly, it explores the role of exogenous antioxidant supplementation in the culture of various stem cell types, including embryonic stem cells (ESCs), primordial germ cells (PGCs), spermatogonial stem cells (SSCs), and induced pluripotent stem cells (iPSCs). Enhancing in vitro stem cell survival and directed differentiation through antioxidant supplementation holds significant promise for advancing fields such as tissue regeneration, organ transplantation, and the development of transgenic chicken models for improved production traits, vaccine development, and recombinant protein production.

BackgroundsAbnormal cleavage (ABNCL) in early human embryos is a frequent phenomenon associated with impaired developmental competence and reduced reproductive potential. However, the clinical determinants of ABNCL and its subtypes remain insufficiently defined. This study aimed to identify patient- and treatment-related factors associated with ABNCL in embryos generated through in vitro fertilization.MethodsThis retrospective cohort included 138,178 normally fertilized embryos cultured in a time-lapse imaging (TLI) system, comprising 77,260 normal cleavage (NC) and 60,918 ABNCL embryos. Univariate, stratified, and multivariable logistic regression with generalized estimating equation (GEE) analyses were performed to evaluate the associations between clinical characteristics—including demographics, controlled ovarian stimulation (COS) parameters, and insemination methods—and ABNCL occurrence and its subtypes. ABNCL was classified into direct cleavage (DC; including DC1 and DC2), rapid cleavage (RC), chaotic cleavage (CC), and other atypical patterns according to established morphokinetic criteria.ResultsAmong the 138,178 embryos analyzed, 55.9% exhibited NC and 44.1% demonstrated ABNCL. Stratified analyses revealed significant variability in ABNCL rates across baseline characteristics and stimulation-related factors. In GEE models, endometriosis, unexplained infertility, higher oocyte yield, intracytoplasmic sperm injection (ICSI), and rescue ICSI were independently associated with an increased risk of ABNCL. Conversely, the use of a mild stimulation protocol and higher estradiol (E2) levels on the hCG trigger day were linked to a reduced risk. Subtype analyses showed distinct factor-specific patterns: higher total gonadotropin (Gn) doses increased the likelihood of DC and “other” ABNCL types, while elevated initial Gn doses were associated with higher risks of RC and CC. Additionally, the gonadotropin-releasing hormone antagonist protocol specifically increased the risks of RC and “other” ABNCL patterns.ConclusionsABNCL is a common event during early embryo development and is significantly influenced by both patient characteristics and treatment strategies. Endometriosis, unexplained infertility, higher oocyte yield, ICSI, and rescue ICSI were associated with increased ABNCL risk, whereas mild stimulation and higher E2 levels on the trigger day appeared protective. These findings underscore the importance of individualized COS and fertilization strategies to promote optimal early embryo development in assisted reproductive technology; however, their implications are limited to cleavage-stage outcomes and should not be extrapolated to later developmental or clinical endpoints.

The sex-linked barring of chicken feather pattern is a fascinating trait, and it has great application value in chicken breeding by enabling autosexing. In this study, the transcriptome changes in skin follicle tissues from the back (SKs) and crown of the head (SKHs) were explored by RNA-seq in Wenshang Barred chickens during their feather pattern formation. The key genes and signaling pathways regulating sex-linked barring formation in chickens were analyzed. As a result, a total of 2291 and 4496 differentially expressed genes (DEGs) were found in SKs and SKHs, respectively, during barred feather formation. The expression pattern clusters of Profile 6 and Profile 7 were the important and mainstream expression trends of the DEGs. Melanogenesis KEGG pathway and GO terms of melanocyte differentiation, melanosome organization and melanin biosynthetic biological processes were significantly enriched in the DEGs, including the well-known TYR, TYRP1, EDNRB and PMEL genes, clustered in Profile 6 expression pattern. A series of DEGs, including CDKN2A, SFN, CDKN2B and CDK1, were significantly enriched in the cell cycle pathway and were mostly clustered in Profile 7. Protein‒protein interaction (PPI) network analysis revealed TYR, TYRP1, CDKN2A and SFN were the hub genes. It was speculated that CDKN2A may target SFN to regulate the melanocyte cycle arrest, causing the formation of white bands of sex-linked barring. TYR, TYRP1, EDNRB and PMEL genes may play important regulatory roles by melanogenesis pathway in the early growth of black feathers, as well as in the later formation of black stripes. The different expression patterns of SLC45A2, KIT, MC1R, ASIP, DCT and SOX10 in SKs and SKHs may contribute to the different formation processes of barred feather patterns on the head and the back. These results provide new insights into the regulation of sex-linked barred feather patterns in Chinese native chicken and provide a valuable theoretical foundation for future chicken breeding and related efforts.

In poultry, primordial germ cells (PGCs) play a crucial role in preserving and manipulating genetic resources for animal production. PGCs face challenges from oxidative stress during in vitro culture and manipulation processes. While the NRF2 pathway is known to centralize cellular homeostasis and stress responses in various cell types, its role in in vitro cultured PGCs remains poorly understood. This study investigates the impact of NRF2 inhibition on PGC proliferation, cellular basal characteristics, and potential underlying mechanisms. All experiments were performed with at least three biological replicates. The results showed that treatment with 24 μM ML385 significantly reduced PGC numbers after 3 d of culture compared to controls (2.83 ± 0.04 vs. 1.77 ± 0.06, × 105 cells), accompanied by a decrease in EdU-positive cells (28.85 ± 1.92% vs. 23.41 ± 1.49%) and a significant increase in apoptotic cells (3.50 ± 0.06% vs. 6.34 ± 0.13%). Transcriptomic sequencing analysis revealed 377 differentially expressed genes (234 up-regulated, 143 down-regulated) in ML385-treated PGCs, with significant enrichment of apoptosis-related pathways and downregulation of cell cycle pathways. NRF2 inhibition induced oxidative stress, with a 2.2-fold increase in reactive oxygen species levels and a 3.3-fold decrease in the GSH/GSSG ratio. Mitochondrial damage, including decreased mitochondrial number (27.86 ± 2.74 vs. 17.57 ± 1.55), vacuolization (9.54 ± 1.64% vs. 41.27 ± 11.18%) and hyperpolarization of mitochondrial membrane potential, was observed, along with increased markers of ferroptosis, including a 2.7-fold increase in MDA and a 2.4-fold increase in Fe²⁺ levels, and enhanced autophagy, as evidenced by increased LC3-II expression and upregulation of MAP1LC3B, ATG5, and BECN1. Notably, while in vivo migration efficiency of PGCs to recipient gonads was unaffected, the number of PGCs colonizing the gonads was significantly reduced (182.93 ± 23.48 vs. 120.66 ± 18.50) in the ML385 group. These findings demonstrate that NRF2 is essential for maintaining chicken PGC proliferation and survival under culture conditions, and its deficiency leads to oxidative stress, mitochondrial dysfunction, apoptosis, and increased susceptibility to ferroptosis.

In the original publication [...].

Background/Objective: It has been established that HINTW plays a pivotal role in the female differentiation of chickens; nevertheless, the underlying molecular mechanism remains to be fully elucidated. Method: To investigate the role of HINTW in avian sex determination, a prokaryotic expression vector containing its key structural domain was constructed, and its in vitro expression was achieved. Pull-down assays were performed to capture interacting proteins from male and female gonadal tissues, followed by a silver staining analysis to compare interaction profiles between ovaries and testes. Mass spectrometry was utilized to identify differentially bound proteins. Additionally, functional characterization and co-immunoprecipitation (Co-IP) assays were conducted to validate the interaction between HINTW and its candidate binding partner. Result: A total of 1590 differentially bound proteins were identified between ovarian and testicular tissues. Functional analysis and Co-IP assays confirmed a specific interaction between HINTW and UBE2I in the ovary, suggesting that HINTW may facilitate female differentiation in chicken embryos through its interaction with UBE2I. Conclusions: This study provides novel insights into the molecular mechanisms underlying HINTW-mediated female differentiation in chickens and contributes to a deeper understanding of avian sex determination.

Background: Chicken Primordial Germ Cells (PGCs) are one of the few germ cells that can be cultured for a long time in vitro, but challenges remain such as low culture efficiency and unclear roles of nutrient factors and signaling pathways. Method: In this study, protein kinase B (AKT) pathway activator insulin-like growth factor 1 (IGF-1) was screened for its ability to promote cell proliferation by transcriptome results using various inhibitors of pathway activation. The effects of IGF-1 on PGCs were evaluated through EdU assays, qRT-PCR, flow cytometry, and migration experiments. Results: This study systematically examined the effects of insulin and IGF-1 on the proliferation, cell cycle, ferroptosis, migration capacity, and establishment efficiency of PGCs. The findings demonstrated that IGF-1 exhibited comparable effects to insulin and could effectively replace insulin in PGC culture systems. Conclusions: The research results are expected to provide a solid theoretical basis for optimizing the chicken PGC cultivation system and promoting its practical application.

The chicken iPSCs have been successfully established and exhibit significant potential for the recovery and conservation of endangered avian species. However, the efficiency of induction remains quite limited. To enhance the induction system, a six-factor reprogramming approach incorporating OCT4, SOX2, NANOG, LIN28, KLF4, and C-MYC (OSNLKM) was employed via lentiviral infection, in combination with the small-molecule compound OAC2 and mouse embryonic fibroblast (MEF) feeder layers. The results demonstrated that the six-factor system significantly accelerated the reprogramming process and improved induction efficiency compared to the conventional four-factor system (OCT4, SOX2, NANOG, and LIN28; OSNL). Additionally, the MEF feeder layers were found to be essential for the successful reprogramming of chicken somatic cells. The inclusion of Oct4-activating compound 2 (OAC2) further optimized the reprogramming conditions, enabling the successful induction of iPSCs from chicken embryonic fibroblasts (CEFs) and Sertoli cells. Collectively, this study establishes an optimized reprogramming system for chicken somatic cells, enhancing iPSCs induction efficiency and expanding the potential applications of iPSCs technology in avian conservation and genetic modification.

The Luhua chicken is an outstanding local breed in China that has been placed under conservation due to the impact of specialized breeding and the widespread adoption of commercial varieties. As such, this study analyzed reproductive traits across three consecutive generations and utilized whole-genome resequencing data from 60 Luhua chickens to assess conservation efficacy through genetic diversity, run of homozygosity (ROH) distribution, kinship, and population structure so as to better conserve the breed. The results show that, across generations, the body weight at first egg increased, the age at first egg was delayed, and the egg weight at first laying increased. No significant variations were found in the body weight at 300 d or the total egg number. The key genetic parameters of the polymorphism information content (PIC), expected heterozygosity (HE), observed heterozygosity (HO), and mean identical-by-state (IBS) distance were 0.234, 0.351, 0.277, and 0.782, respectively. The majority of ROHs ranged from 0.5 to 1 Mb, and the inbreeding coefficient based on ROHs was calculated at 0.021. The findings reveal that these traits remained unchanged across the three generations. Our research suggests that optimizing the mating plan of Luhua chickens is essential to minimize inbreeding risk. Furthermore, the methodology applied in this study provides a valuable reference for the conservation monitoring of other indigenous chicken breeds.

Primordial germ cells (PGCs) are the progenitor cells of sperm and eggs. Xenotransplantation of chicken PGCs can achieve germline transmission. However, there are still challenges in obtaining many PGCs from endangered birds in vitro. In this study, at first, by incorporating 2i factors, the embryonic stem cells (ESCs) culture conditions were optimized, successfully yielding and validating pluripotent ESCs clones. Then, during induction ESCs, bFGF, activin A, and 1% KSR were added to Epiblast-like cells (EpiLCs). Quantitative real-time polymerase chain reaction (qRT-PCR) showed Pax6, Eomes, and Vimentin expression patterns similar to primary epiblast, indicating successful EpiLCs induction. During EpiLCs to Primordial germ cell-like cells (PGCLCs) transformation, we evaluated BMP4, BMP8b, EGF, LIF, and SCF combinations' impact on induction efficiency. Flow cytometry, qRT-PCR, and immunofluorescence showed high expression of Cvh, C-kit, Dazl, CVH, and DAZL in PGCLCs, suggesting successful EpiLCs differentiation. Induced PGCLCs injected into 2.5-day chick embryos migrated to gonads by day 7-7.5, demonstrating migration and colonization. This study optimized a two-step protocol for in vitro differentiation of chicken ESCs into PGCLCs. This research's results not only provide a reference for obtaining many PGCLCs in vitro but also open up a new approach for the development and application of genetic resource preservation technology in domestic chickens.