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Aims The effect of increasing dietary cation–anion difference (DCAD) on rumen fermentation and ruminal microbial community in dairy cows under heat stress (HS) conditions were evaluated. Methods and Results This study was performed as a two‐period cross‐over design during the summer season, with eight lactating dairy cows randomly distributed to either a control DCAD diet (CON: 33·5 mEq/100 g DM) or high DCAD diet (HDCAD: 50·8 mEq/100 g DM). Throughout the present study, the temperature and humidity index (THI; 80·2 ± 4·29) was generally elevated above the threshold (THI = 72) that is reported to cause HS in lactating dairy cows. Rumen liquid samples were collected on 15 and 21 d during each 21 d‐period. The absolute concentration of ruminal total volatile fatty acid (TVFA) in HDCAD treatment was significantly (P < 0·05) higher than those in the control, whilst the ruminal pH, NH3‐N, and VFA molar percentages were unaffected through increasing DCAD. Furthermore, the copy numbers of the cellulolytic bacteria Ruminococcus albus and Ruminococcus flavefaciens in rumen fluid significantly (P < 0·05) rose along with the increment of DCAD. Although the Alpha diversity indexes and the bacterial microbiota structure were unaffected, increasing DCAD significantly (P < 0·05) enriched the phylum Fibrobacteres and genus Fibrobacter in the microflora of rumen fluid, whilst the genera Flexilinea and Dubosiella were the most differentially abundant taxa in the control. Conclusions Increasing DCAD under HS conditions resulted in a greater concentration of total VFA without affecting rumen bacteria diversity or structure, although the enrichment of some cellulolytic/hemicellulolytic bacteria was observed. Significance and Impact of the Study The present study provides information on the modulation of rumen fermentation and microbial community through the increment of DCAD in Holstein dairy cows under HS conditions.

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