Transcriptomic changes in embryonic blood of subsequent generations following in ovo stimulation with nutriepigenetic factors

BACKGROUND: Epigenetic modifications regulate gene expression and are influenced by environmental factors, shaping phenotypic and clinical outcomes. These changes have the potential to persist across generations, although their stability may vary between tissues. This study aims to observe, using an in ovo model, the effects of single (F1) and repeated (F1-F3) prenatal stimulation with potential epigenetic factors on the transcriptome of embryonic blood in subsequent generations. METHOD: (PS) and choline were injected on day 12 of incubation. F1 embryos were assigned to control (0.9% NaCl), synbiotic (SYN, 2 mg PS), or synbiotic + choline (SYNCH, 2 mg PS + 0.25 mg choline) groups. In F2 and F3, SYN and SYNCH were split into two subgroups each: (A) injected only once in F1 embryos (SYNs and SYNCHs); and (B) repeatedly injected in every successive generation (SYNr and SYNCHr). Embryonic blood was collected from the dorsal aorta at HH stages 14-16, and RNA sequencing was performed on male samples. RESULTS: Transcriptomic changes in F3 embryonic blood were found in treatment groups vs. control after single (F1) and repeated (F1-F3) in ovo administration of synbiotic alone and synbiotic combined with choline. Gene expression differences detected in F3 embryonic blood in a single ancestral in ovo exposure lineages largely diminished in F4, suggesting that effects of these treatments may attenuate with generational distance. Repeated injections in SYNr and SYNCHr groups did not produce cumulative effects in F3 and F4 generations. Gene set enrichment analysis indicated that the most affected functional categories involved metabolism, detoxification, cytoskeletal organization, and protein regulation. CONCLUSIONS: These findings highlight that targeted prenatal interventions can induce transcriptomic modifications, which may be detectable in the subsequent generations (F3), though their persistence through the further generations may be limited. Embryonic blood may reflect a potential epigenetic footprint rather than a stably inherited signal. The tissue-specific and time-sensitive nature of the transcriptomic effects confirms the importance of analysis across different tissues, and across generations.