Bu Ying, Zhang Xiaoyin, Xiong Zhanbo, Li Kexin, Zhang Shiqi, Lin Miao, Zhao Guoqi, Zheng Nan, Wang Jiaqi, Zhao Shengguo
State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
Yangzhou University, Yangzhou, 225000, China.
Appl Microbiol Biotechnol. 2025 Apr 30;109(1):107. doi: 10.1007/s00253-025-13497-z.
Red clover isoflavones, particularly biochanin A and formononetin, are known for their benefits in enhancing feed efficiency and nitrogen utilization in ruminants. However, their specific effects on rumen fermentation and microbial diversity remain insufficiently explored. This study investigated the impacts of red clover isoflavones on rumen function and bacterial diversity in dairy cows, utilizing both in vivo and in vitro methodologies. In the in vivo study, 40 Holstein dairy cows were allocated to four groups, each receiving red clover isoflavones at doses of 0, 0.4, 0.8, and 1.6 g/kg. Rumen fluid was collected for analysis of fermentation parameters, enzyme activity, and microbial composition through shotgun metagenomic sequencing. Concurrently, an in vitro rumen fermentation trial was conducted to evaluate the effects of biochanin A and formononetin on urea hydrolysis. Results from the in vivo experiments showed that red clover isoflavones significantly decreased ammonia nitrogen (NH₃-N) concentrations and urease activity in the rumen (P < 0.05). Species level metagenomic analysis indicated a reduced abundance of proteolytic and ureolytic bacteria, such as Prevotella sp002317355 and Treponema_D bryantii_C, with a corresponding increase in cellulolytic bacteria, including Ruminococcus_D sp900319075 and Ruminococcus_C sp000433635 (P < 0.05). The in vitro trial further demonstrated that biochanin A and formononetin significantly reduced urea decomposition rates (P < 0.05), with biochanin A exerting a more pronounced effect. These findings align with the observed reduction in ureolytic and proteolytic bacteria, along with an increase in cellulolytic bacteria across both trials. In conclusion, biochanin A emerged as the primary active component of red clover isoflavones, modulating urea nitrogen hydrolysis and rumen fermentation. This study substantiates previous findings and highlights the potential of red clover isoflavones for enhancing rumen microbial fermentation, offering a promising strategy for future dairy industry applications. KEY POINTS: • Red clover isoflavones inhibit urease activity to decrease the abundance of urealytic bacteria. • Biochanin A reduces ammonia nitrogen and urease activity, promoting protein efficiency. • Red clover isoflavones may improve dairy cow rumen health and nitrogen utilization.
红三叶草异黄酮，尤其是鹰嘴豆芽素A和芒柄花黄素，因其在提高反刍动物饲料效率和氮利用率方面的益处而闻名。然而，它们对瘤胃发酵和微生物多样性的具体影响仍未得到充分研究。本研究利用体内和体外方法，调查了红三叶草异黄酮对奶牛瘤胃功能和细菌多样性的影响。在体内研究中，40头荷斯坦奶牛被分为四组，每组分别接受剂量为0、0.4、0.8和1.6 g/kg的红三叶草异黄酮。通过鸟枪法宏基因组测序收集瘤胃液，用于分析发酵参数、酶活性和微生物组成。同时，进行了一项体外瘤胃发酵试验，以评估鹰嘴豆芽素A和芒柄花黄素对尿素水解的影响。体内实验结果表明，红三叶草异黄酮显著降低了瘤胃中氨氮（NH₃-N）浓度和脲酶活性（P < 0.05）。物种水平的宏基因组分析表明，蛋白水解和尿素分解细菌的丰度降低，如普氏菌属sp002317355和密螺旋体_D布氏_C，而纤维素分解细菌的丰度相应增加，包括瘤胃球菌_D sp900319075和瘤胃球菌_C sp000433635（P < 0.05）。体外试验进一步表明，鹰嘴豆芽素A和芒柄花黄素显著降低了尿素分解率（P < 0.05），其中鹰嘴豆芽素A的作用更为明显。这些发现与两项试验中观察到的尿素分解和蛋白水解细菌减少以及纤维素分解细菌增加一致。总之，鹰嘴豆芽素A是红三叶草异黄酮的主要活性成分，可调节尿素氮水解和瘤胃发酵。本研究证实了先前的发现，并突出了红三叶草异黄酮在增强瘤胃微生物发酵方面的潜力，为未来乳制品行业应用提供了一个有前景的策略。要点：• 红三叶草异黄酮抑制脲酶活性，以降低尿素分解细菌的丰度。• 鹰嘴豆芽素A降低氨氮和脲酶活性，提高蛋白质效率。• 红三叶草异黄酮可能改善奶牛瘤胃健康和氮利用率。
