Development of ovine hepatic organoids: a powerful

Background: The liver coordinates metabolic processes that determine growth, health, and production efficiency in livestock species. In ruminants, hepatic metabolism is adapted to support glucose homeostasis, lipid partitioning, and nitrogen utilization during ruminal fermentation. Therefore, predictive experimental models are essential for studying nutrient-mediated regulation of liver function. However, conventional two-dimensional hepatocyte cultures fail to reproduce the structural organization and functional complexity of native liver tissue, limiting their translational value. Methods: Hepatic organoids were developed from the progenitor cells of the liver of a male Assaf lamb, and were characterized by histological and immunofluorescence analyses, followed by transcriptomic profiling using RNA-seq and functional enrichment analysis. The metabolic response was assessed by adding DL-methionine and/or betaine and analyzing, by RT-qPCR, the expression of genes encoding key metabolic enzymes (e.g., Results: Hepatic organoids formed spherical epithelial structures with defined apical-basal polarity, intact tight junctions, albumin expression, and intracellular glycogen accumulation. Comparative transcriptomic analysis between organoids and native liver tissue supported the conservation of core cellular programs, although differential expression and functional enrichment analyses demonstrated that hepatic organoids exhibit a transcriptional profile biased toward cell proliferation, protein synthesis, and structural remodeling, while genes associated with mature liver metabolic functions, immune responses, and systemic homeostasis were relatively downregulated. Combined treatment with DL-methionine and betaine significantly increased the expression of Discussion: Ovine hepatic organoids reproduced structural and cellular features of liver tissue, supporting their relevance as an