| Summary: | The objective of this study was to evaluate the effects of feeding sugarcane-derived polyphenolic supplement (Polygain, The Product Makers Australia, Keysborough, VIC, Australia) on enteric methane (CH<sub>4</sub>) emission, rumen microbiota, and performance of second-cross lambs. For this purpose, 24 Poll Dorset × (Border Leicester × Merino) lambs were allocated to 3 different treatments: Control (C), 0.25% Polygain (0.25 PG), and 1% Polygain (1 PG) diets with a uniform basal feed (25% cracked wheat grain, 25% cracked barley grain, 25% oaten chaff, 25% lucerne chaff). Both doses of Polygain reduced the total CH<sub>4</sub> production (g/day; <i>p</i> = 0.006), CH<sub>4</sub> yield (CH<sub>4</sub>, g/kg of dry matter intake; <i>p</i> = 0.003) and CH<sub>4</sub> intensity (CH<sub>4</sub>, g/kg of BW; <i>p</i> = 0.003). Dry matter intake tended to be greater (<i>p</i> = 0.08) in sheep fed 1 PG compared to the C group, with the 0.25 PG group being intermediate. The average daily gain of the lambs was improved (<i>p</i> = 0.03) with 1% Polygain supplementation. The relative abundance of genera <i>Methanobrevibacter_</i>unidentified, <i>Methanomethylophilaceae_</i>uncultured, <i>Methanogenic archaeon</i> mixed culture ISO4-G1, <i>Methanosphaera</i> uncultured rumen methanogen, <i>Methanogenic archaeon</i> ISO4-H5, and <i>Methanobrevibacter boviskoreani</i> JH1 were reduced with Polygain supplementation. In conclusion, feeding Polygain reduced lambs’ enteric CH<sub>4</sub> emissions, altered the rumen microbiome, and improved the growth performance of lambs.
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