摘要Background While maternal proline(Pro)supplementation has demonstrated efficacy in enhancing placental angio-genesis and farrowing efficiency in swine,its regulatory role in fetal skeletal muscle ontogeny remains undefined.This study systematically evaluated the temporal-specific impacts of dietary Pro supplementation during critical phases of fetal myogenesis(encompassing primary myofiber formation and secondary myofiber hyperplasia)on offspring muscle devel-opment.A total of 120 sows with similar farrowing schedules were assigned to three groups:CON(basal diet),ST-Pro(0.5％Pro supplementation during secondary myofiber formation period,from d 60 gestation to farrowing),LT-Pro(0.5％Pro supplementation spanning primary and secondary myofiber formation period:from d 20 gestation to farrowing).Results LT-Pro group significantly increased the longissimus dorsi(LD)muscle mass per unit body weight in new-born piglets compared to CON group(P＜0.05),while no such effect was observed in the ST-Pro group.Metabolomic profiling revealed elevated Pro,lysine,and tryptophan levels in the LD muscle of LT-Pro group piglets,accompanied by reduced branched-chain amino acids(BCAAs;leucine,isoleucine,and valine)in both serum and muscle(P＜0.05).Histological analysis demonstrated a 45.74％increase in myofiber cross-sectional area in the LT-Pro group(P＜0.05).At the molecular level,LT-Pro group piglets exhibited upregulated mRNA expression levels of myogenic regulatory genes(MYOD1,MYF6)and the cell cycle accelerator CCND1(P＜0.05),coupled with activation of the STAT3 signal-ing pathway(phosphorylated STAT3 protein increased by 2.53-fold,P＜0.01).Furthermore,Pro supplementation enhanced oxidative metabolism,evidenced by elevated mitochondrial biogenesis markers(the mRNA expression levels of PPARGC1 A,OPA1,and SQSTM1)and a 61.58％increase in succinate dehydrogenase activity(P＜0.05).Notably,LT-Pro group piglets showed a selective shift toward slow-twitch oxidative fibers,with both MyHC1 mRNA and protein expression levels significantly upregulated(P＜0.05),while the mRNA expression levels of MyHCllb showed no signifi-cant change.Conclusions This study identified the primary fiber formation period as a critical window.Supplementation with Pro during G20-114 reprogrammed offspring skeletal muscle development through STAT3-CCND1-mediated myoblast proliferation,enhanced mitochondrial bioenergetics,and oxidative fiber specification.However,no such effects were observed during G60-114.These findings propose maternal Pro intervention as a novel strategy to enhance muscle yield and metabolic efficiency in swine production,with potential applications for improving meat quality traits linked to oxidative muscle phenotypes.