Abstract
<title>Abstract</title> <p> Background Calves derived from <italic>in vitro</italic> embryo production (IVP) often have higher birth weights than those produced by artificial insemination or <italic>in vitro</italic> embryo transfer, and are frequently accompanied by malformations, mortality, or dystocia in dams. However, a subset of calves exhibits only increased body weight without adverse health outcomes. This study focused on the non‑pathological fetal overgrowth phenotype, aiming to refine the understanding of abnormal birth weight in IVP calves and provide new perspectives and a theoretical basis for the underlying regulatory mechanisms. Results In this study, whole‑transcriptome, metabolomic, physiological, biochemical, and hormonal analyses were integrated to compare nine IVP calves with the non‑pathological fetal overgrowth phenotype and nine IVP calves with normal birth weight. The results demonstrated that increased birth weight in IVP calves was accompanied by prolonged gestation length in recipient dams, and multiple physiological, biochemical, and hormonal parameters were significantly altered. Untargeted metabolomics identified 3,259 metabolites, among which 132 differential metabolites were screened, including 82 significantly up‑regulated and 50 significantly down‑regulated metabolites. Two core regulatory axes were identified in the ceRNA network: circ36279‑bta‑miR‑431 (targeting SPON2, TNN, INSIG1, and AEN) and circ35151‑bta‑miR‑154b (targeting SHD, TBX2, and IL20RA). Integrative analysis revealed 5 co‑enriched pathways, with arginine and proline metabolism as the core pathway, mediating multiple downstream branches including polyamine synthesis, ornithine metabolism, creatine synthesis, and proline metabolism through key enzymes such as AGMAT, ARG1, and CKM, collectively constituting a complex regulatory network. Conclusions Through multi‑omics integrative analysis, this study clarified that IVP‑associated non‑pathological fetal overgrowth is accompanied by systemic physiological and biochemical changes and identified two ceRNA regulatory networks and arginine and proline metabolism as a core pathway. These findings refine the molecular understanding of this phenotype and provide new perspectives for precise intervention in growth abnormalities of IVP calves. </p>