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This study investigated the replacement of Leymus chinensis (LC) with whole-crop wheat hay (WCWH) in the diets of Holstein bulls in the fattening stage and examined the potential effects on blood parameters, fatty acids in rumen fluid and serum, and the rumen microbiomes. In this study, 12 Holstein bulls in the fattening period (body weight=485.0 ± 40.8 kg) were assigned to 1 of 4 dietary treatments using a 4 × 4 Latin square design. Each experimental period consisted of a 17-d adaptation period and a 5-d collection period. The dietary treatments consisted of 4 proportions of WCWH (0, 33, 67, and 100%) as a substitute for LC (designated as WCWH0, WCWH33, WCWH67, and WCWH100, respectively). On d 18 and 22 of each experiment period, blood and rumen content samples were collected for analysis, respectively. Real-time quantitative PCR was used to analyze the rumen microbiomes. The results from this study revealed no differences in the saturated, monounsaturated, and polyunsaturated long-chain fatty acid proportions of rumen liquid among the treatments. It was observed in the present trial that rumen microbiotal flora were not significantly different in the bulls fed LC compared with the bulls fed WCWH. Additionally, blood sample analysis demonstrated that the concentration of urea nitrogen in the WCWH100 group was higher than that observed in the other groups. Meanwhile, no differences were detected for other serum parameters. There were no differences in the proportions of serum saturated, monounsaturated, and polyunsaturated long-chain fatty acids among the treatments. In conclusion, our data revealed that LC can be replaced with WCWH in the diet of Holstein bulls in the fattening stage with no negative effects on the blood indicators, fatty acids, and microbiomes.