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This study was carried out to evaluate the use of tannin extract from Acacia mearnsii as a strategy to reduce methane (CH 4 ) in two distinct cattle genotypes and its effect on intake, digestibility, microbial efficiency and ruminal fermentation. Four Nellore (Bos indicus) and four Holstein (Bos taurus) dry cows fitted with rumen cannula were assigned to two 4 × 4 Latin square design, in a 2 × 4 factorial arrangement, where each genotype represented a square receiving four tannin levels (commercial extract of A. mearnsii) in the diet (0%, 0.5%, 1.0% and 1.5% of dry matter). Tannin levels used did not cause a reduction in feed intake or rumen passage rate for both genotypes (p > 0.05), although there was a linear reduction in the degradation rate and ruminal disappearance of diet (p < 0.05). The increase in tannin levels reduced the amount of entodiniomorph protozoa in the Nellore cattle (p < 0.05). There was no change in N retention or microbial efficiency (p > 0.05), despite the linear reduction of nutrient digestibility and the synthesis of microbial nitrogen (p < 0.05). The ruminal CH 4 production was reduced (p < 0.05) without reducing the short-chain fatty acid production. The threshold of 0.72% of tannin in the diet was estimated as the starting point for the reduction of ruminal CH 4 production with long-term efficacy. Therefore, the use of low levels of tannin extract from A. mearnsii is a potential option to manipulate rumen fermentation in Nellore and Holstein cattle and needs to be further investigated., (© 2022 Wiley-VCH GmbH.)