Nanominerals have gained increasing attention in animal nutrition because of their potential to enhance feed utilization and nutrient digestibility, although their effects may vary depending on the type of nanomaterial and experimental conditions. This study investigated the effects of zinc oxide nanoparticles (ZnONPs) at different supplementation levels (0, 5, 10, 20, and 40 mg/kg dry matter (DM)) on the chemical composition, estimated organic matter digestibility (OMD), metabolizable energy (ME), and methane (CH4) percentage of dairy cattle concentrate feed under in vitro conditions. ZnONPs were synthesized using Prunus serrulata (Japanese Cherry Blossom) leaf extract. Dry matter content and CH4 percentage decreased in all supplemented groups compared with the control (P<0.05). Total carbohydrate (TC) and hemicellulose (HCL) contents decreased, whereas crude ash (CA) and acid detergent fiber (ADF) contents increased in all supplemented groups (P≤0.001). Ether extract (EE) content increased with ZnONP supplementation and reached its highest value at 40 mg/kg DM (P<0.001). Crude protein (CP) content remained unchanged at 5 and 10 mg/kg DM but decreased at 20 and 40 mg/kg DM (P<0.001). The lowest neutral detergent fiber (NDF) content was observed at 40 mg/kg DM (P<0.001). Estimated OMD increased at 10, 20, and 40 mg/kg DM compared with the control, with the highest value observed at 40 mg/kg DM (P<0.05). Metabolizable energy did not differ significantly among treatments (P>0.05). Methane values represent the percentage of total gas produced during the 24-hour in vitro incubation period.
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