Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14–16 Hamburger–Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Exploring how early-life nutritional interventions may impact future generations, this study examines the inter- and transgenerational effects of in ovo injection of bioactive compounds on gene expression in the cecal tonsils and cecal mucosa using a chicken model. Synbiotic PoultryStar^® (Biomin) and choline were injected in ovo on the 12th day of egg incubation. Three experimental groups were established in the generation F1: (1) a control group (C) receiving 0.9% physiological saline (NaCl), (2) a synbiotic group (SYN) receiving 2 mg/embryo, and (3) a combined synbiotic and choline group (SYNCH) receiving 2 mg synbiotic and 0.25 mg choline per embryo. For the generations F2 and F3, the SYN and SYNCH groups were each divided into two subgroups: (A) those injected solely in F1 (SYNs and SYNCHs) and (B) those injected in each generation (SYNr and SYNCHr). At 21 weeks posthatching, cecal tonsil and cecal mucosa samples were collected from F1, F2, and F3 birds for transcriptomic analysis. Gene expression profiling revealed distinct intergenerational and transgenerational patterns in both tissues. In cecal tonsils, a significant transgenerational impact on gene expression was noted in the generation F3, following a drop in F2. In contrast, cecal mucosa showed more gene expression changes in F2, indicating intergenerational effects. While some effects carried into F3, they were less pronounced, except in the SYNs group, which experienced an increase compared to F2. The study highlights that transgenerational effects of epigenetic modifications are dynamic and unpredictable, with effects potentially re-emerging in later generations under certain conditions or fading or intensifying over time. This study provides valuable insights into how epigenetic nutritional stimulation during embryonic development may regulate processes in the cecal tonsils and cecal mucosa across multiple generations. Our findings provide evidence supporting the phenomenon of epigenetic dynamics in a chicken model.

The exuberant development of targeted genome editing has revolutionized research on the chicken genome, generating chickens with beneficial parameters. The chicken model is a crucial experimental tool that can be utilized for drug manufacture, preclinical research, pathological observation, and other applications. In essence, tweaking the chicken’s genome has enabled the poultry industry to get more done with less, generating genetically modified chickens that lay eggs containing large amounts of lifesaving humanized drugs. The transition of gene editing from concept to practical application has been dramatically hastened by the development of programmable nucleases, bringing scientists closer than ever to the efficient producers of tomorrow’s medicines. Combining the developmental and physiological characteristics of the chicken with cutting-edge genome editing, the chicken furnishes a potent frontier that is foreseen to be actively pursued in the future. Herein we review the current and future prospects of gene editing in chickens and the contributions to the development of humanized pharmaceuticals.

Epigenetic mechanisms play a pivotal role in hereditary processes, shaping phenotypic outcomes across generations. This study investigates the transgenerational impacts of in ovo injection of bioactive substances on gene expression and DNA methylation in the male gonads using the Green-legged Partridgelike chickens as a model organism. Synbiotic PoultryStar^® (Biomin; PS) and choline were injected in ovo on the 12th day of egg incubation. In the F1 generation, three groups were established: (1) control (C, 0.9% physiological saline); (2) PS synbiotic (SYN, 2 mg/embryo); and (3) PS synbiotic combined with choline (SYNCH, 2 mg/embryo of synbiotic and 0.25 mg/embryo of choline). In subsequent F2 and F3 generations, groups SYN and SYNCH were further divided into two subgroups each: (A) only injected in F1 embryos (SYNs and SYNCHs); and (B) repeatedly injected in every generation (SYNr and SYNCHr). At 21 weeks post-hatching, gonadal tissues were sampled from F2 and F3 male chickens for transcriptomic and reduced representation bisulfite sequencing (RRBS). Synbiotic alone produced minimal and diminishing changes in gene expression across generations. In contrast, the single co-administration of synbiotic and choline in F1 embryos (SYNCHs) led to 1,897 differentially expressed genes (DEGs) and 786 differentially methylated regions (DMRs) in F3. Repeated administration across generations (SYNCHr) resulted in an even greater number of DEGs (2,804) and DMRs (2,880) in F3, reflecting a cumulative exposure effect. DEGs in SYNCH groups were mainly enriched in pathways related to cytoskeletal organization and extracellular matrix. In SYNCHs, methylation changes were associated with TGF-beta signaling, whereas SYNCHr showed additional enrichment in Wnt signaling, focal adhesion, and adipocytokine signaling pathways. Integrative analysis revealed coordinated changes in gene expression and DNA methylation, particularly in the F3 generation, identifying 37 genes (47 DMRs) in SYNCHs and 194 genes (306 DMRs) in SYNCHr. This study highlights the potential of prenatal epigenetic interventions to induce gene expression and DNA methylation changes across generations in reproductive tissues.