Effects of red clover isoflavone on lactation performance, milk quality and nitrogen metabolism in dairy cows

Reducing nitrogen loss and improving nitrogen utilization efficiency in dairy cows can reduce economic costs and mitigate the environmental impact of nitrogen emissions. Red clover isoflavone, a natural compound derived from plant extract, has the advantage of biological safety. This study aimed to investigate the effects of red clover isoflavone on lactation performance and nitrogen metabolism in dairy cows. Sixty-eight Holstein cows (165 ± 21 d in milk, 710 ± 75 kg initial body weight, 2.30 ± 1.21 parities, 33.93 ± 3.81 kg/d milk yield) were chosen in a randomized complete block design. Cows were divided into 5 blocks by milk yield and randomly assigned to 4 treatments (17 cows per treatment). The experimental period lasted 84 d, during which cows were fed a basal diet supplemented with red clover extract (RCE) at 0, 2, 4, or 8 g/kg of total mixed rations (TMR) on a dry matter (DM) basis. These levels corresponded to red clover isoflavones of 0, 0.46 (0.31 g/kg formononetin and 0.15 g/kg biochanin A), 0.93 (0.67 g/kg formononetin and 0.26 g/kg biochanin A), and 1.83 g/kg (1.32 g/kg formononetin and 0.52 g/kg biochanin A), respectively. With increasing supplementation of RCE in TMR, milk yield increased quadratically by 4.65% to 9.64% (P = 0.020) and feed efficiency improved by 6.58% to 10.53% (P = 0.005), with the optimal dose identified as 2 g/kg. Additionally, RCE supplementation significantly increased the yield of milk components, including fat, protein, lactose, non-fat milk solid, and total milk solid (P < 0.05), with the highest in the 2 g/kg group. Regarding nitrogen metabolism, RCE supplementation linearly increased microbial nitrogen (MN) from 782.61 to 956.41 g/d (P = 0.003), which was accompanied by a linear increase in nitrogen utilization efficiency from 26.32% to 30.31% (P < 0.001). In contrast, urinary total nitrogen (P = 0.027) and milk urea nitrogen (P < 0.001) decreased quadratically with increasing doses of RCE, reaching their minimum at 4 g/kg. Following RCE supplementation, the apparent digestibility of DM, organic matter (OM), and neutral detergent fiber (NDF) exhibited a quadratic effect (P < 0.05), with significantly higher digestibility observed in the 2 g/kg group. Metabonomics results revealed that RCE supplementation significantly upregulated the top 12 differential metabolites. These findings suggest that RCE supplementation at levels ranging from 2 to 4 g/kg DM enhances lactation performance, milk quality, and nitrogen metabolism in dairy cows.