The objective of this study was to evaluate the effects of dietary supplementation of malate (MAL) and active dried yeast (ADY) on feed intake, rumen fermentation parameters, rumen microbial populations, selected blood metabolites, and milk production during a sub-acute ruminal acidosis (SARA) challenge in primiparous lactating dairy cows. Six rumen-fistulated Holstein dairy cows (body weight: 630 ± 55 kg, 110 ± 25 days in milk, mean ± SD) were assigned to the following treatments in a 3 × 3 Latin square design: (1) control TMR (CON); (2) a TMR supplemented with 80 g of sodium–calcium malate/head per day (MAL); and (3) a TMR supplemented with 10 g of active dried yeast providing 20 × 109 CFU of Saccharomyces cerevisiae/head per day (ADY). Each experimental period consisted of 14 days of adaptation to the experimental treatments, 4 days of SARA challenge, and 10 days of rest. Dry matter intake (18.4 vs. 19.8 kg/day), and milk yield (29.3 vs. 30.4 kg/day) were depressed during SARA compared with adaptation. Malate and ADY had no effect on DMI and milk yield during the adaptation and SARA phases. Malate and ADY had no effect on ruminal pH characteristics during adaptation. During SARA, maximum and mean ruminal pH was not affected by supplementation, but minimum ruminal pH tended to be higher for ADY compared to CON and MAL. Time spent with ruminal pH <5.6 and < 5.8 tended be lower for ADY compared to CON. During adaptation, ADY tended to decrease rumen NH3-N concentration compared to CON. Malate and ADY had no effect on selected blood metabolites, but glucose and insulin concentration increased during SARA compared to adaptation. The population of Fibrobacter succinogenes tended to increase with MAL and ADY during adaptation, but its population was decreased during SARA compare with adaptation. During SARA, the abundance of Megasphaera elsdenii tended to be higher for ADY than for MAL. The abundance of Streptococcus bovis increased during SARA, but was not affected by MAL or ADY supplementation. The relative abundances of protozoa in the rumen decreased during SARA. In conclusion, ADY supplementation to dairy cows with SARA can potentially improve rumen function, as indicated by a tendency for an improved ruminal pH and greater abundance of M. elsdenii within the rumen. However, supplementation of MAL provided no benefit to dairy cows under SARA condition, at least with the inclusion rates used in this study

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