Green synthesis, characterization, and in vitro evaluation of zinc oxide nanoparticles derived from pomegranate leaves for mitigating ruminal methane formation

Green-synthesized zinc oxide nanoparticles (nano-ZnO) may represent a sustainable alternative to chemical and antibiotic feed additives. This study aimed to synthesize and characterize nano-ZnO using pomegranate (Punica granatum) leaves and to evaluate its in vitro nutritional effects compared with normal ZnO and monensin. The gas production (GP) technique was used to evaluate the impact of different supplementation levels of nano-ZnO [0, 5, 50, 100 and 150 mg/kg dry matter (DM)] of diet compared to the normal-ZnO (300 mg/kg DM diet) and monensin (40 mg/ kg DM) on the rumen nutrient degradability, rumen protozoa, and methane (CH4) production. The nano-ZnO had sizes ranging from 15.74 to 18.36 nm, while the normal ZnO had high particle sizes of 134–234 nm. Total GP did not differ among treatments, whereas monensin produced the lowest (P < 0.05) true degraded neutral detergent fiber. A similar tendency for CH4 reduction (P = 0.06) was observed for monensin and nano-ZnO at 100 and 150 mg/kg DM compared to the control and normal-ZnO diets. Monensin tended to reduce total protozoa (P = 0.09) and Diplodinium spp. (P = 0.07), while nano-ZnO at 100 and 150 mg/kg DM tended to reduce Entodinium spp. (P = 0.09). Nano-ZnO at 150 mg/kg DM produced the lowest ammonia-N concentration (P = 0.03). Nano-ZnO at 100 mg/kg DM yielded the lowest acetate (P = 0.005) and highest propionate (P = 0.03) proportions versus the control. Nano-ZnO may serve as an alternative to ionophores at 100 mg/kg DM, reducing CH4 production without compromising nutrient degradability.