Biochanin A (BCA), an isoflavone phytoestrogen with various functions (Wenjin et al., 2021), is a secondary metabolite produced mainly in leguminous plants such as red clover and chickpea (Křížová et al., 2019). The BCA content in red clover (Trifolium pratense L.) leaves during the flowering stage ranges from 4.57 to 6.86 mg/g dry matter (DM), with an average concentration of 5.76 mg/g DM, which accounts for approximately 40% of the total isoflavones (Lemeziene et al., 2015). Chickpea (Cicer arietinum L.) sprouts contain lower concentration of BCA than red clover, with a maximum concentration of 2.34 mg/g DM (Gao et al., 2015). Sonication of peanuts (Arachis hypogaea) in 80% aqueous ethanol yielded a BCA content of 0.964 mg/100 g DM (Chukwumah et al., 2009). Young plants of Astragalus glycyphyllos L. and Astragalus cicer L. had BCA contents of 8.81 and 11.4 mg/100 g DM, respectively (Butkute et al., 2018). Other natural sources of BCA include alfalfa (Kagan et al., 2015) and soybeans (Hu et al., 2021). Recent studies have demonstrated that BCA possesses numerous biological properties, such as anti-inflammatory, anti-microbial, and antioxidant activities (Jalaludeen et al., 2016; Sadri et al., 2017). Furthermore, BCA has been shown to be a potential drug for protection against osteoporosis (Lee and Choi, 2005), prevention and treatment of atherosclerotic cardiovascular disease (Yu et al., 2020), and alleviation of symptoms in postmenopausal women (Galal et al., 2018). Therefore, BCA is an important source of novel health foods and natural products.

Hyper-ammonia-producing bacteria (HAB) play a vital role in amino acid (AA) deamination in the rumen (Flythe and Kagan, 2010). In vitro, BCA was shown to possess antimicrobial activity against HAB isolated from the rumen fluid of bovines and caprines; however, it has to act synergistically with other heat-stable antibacterial compounds to inhibit HAB growth (Flythe and Kagan, 2010; Flythe et al., 2013). In addition, BCA exhibited selective antimicrobial activity against amylolytic and cellulolytic bacteria, and increased Lactobacilli, decreased gram-positive cocci, and directly inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8, and Ruminococcus albus 8 (Harlow et al., 2017b,2018). These results were attributed to the enhancement of the activities of native rumen antimicrobials (e.g., bacteriocins) by BCA (Flythe et al., 2013; Harlow et al., 2017b). Studies have also shown that BCA can inhibit lactic acid production and increase lactate-utilizing bacteria and lactic acid metabolism, thereby ameliorating pH decline, promoting rumen fermentation, and increasing the concentrations of acetate, propionate, and total volatile fatty acids (VFAs) (Harlow et al., 2017b,2018). Furthermore, BCA could be a substitute for antibiotics, such as monensin, for reducing rumen acidosis (Harlow et al., 2017c,2021). Supplementing steers fed dried distillers’ grains (DDG) with BCA improved crude protein (CP) digestibility of DDG and increased average daily gain, as the addition of BCA decreased ammonia production and improved the quality of absorbed protein by inhibiting HAB (Flythe et al., 2013; Harlow et al., 2017a). Similar results were observed with red clover whereby the average daily gain depended on the interaction between BCA and other isoflavones (Harlow et al., 2020).

In our lab, it was observed that BCA inhibited the activity of proteolytic and ureolytic bacteria and improved the efficiency of microbial protein synthesis (Liu et al., 2020). Therefore, BCA is hypothesized to be a potential novel urease inhibitor. However, to date, there is limited information on the in vivo effects of BCA on dairy livestock. Therefore, the objective of this study was to evaluate the effects of BCA on production performance, health, and rumen microbiome of dairy goats.

Currently, few studies have investigated the influence of BCA on the growth and production performance of dairy goats and cows. Supplementation of steers with BCA (6.3 g/d) exhibited an increase in the average daily gain. The reduction in ammonia concentration was attributed to BCA, which inhibited HAB fermentation of AA deamination (Harlow et al., 2017c). Steers supplemented with 0.91 kg of red clover hay (equivalent to 0.9–1.4 g BCA) and 1.51 kg DDG also exhibited daily gain (Harlow et al., 2020). When the grass silage was replaced with red and white clover silage, the DMI was increased by 1.2 and 1.3 kg, respectively, and the milk yield was increased by 1.5 and 2.2 kg/d, respectively (Steinshamn, 2010). In this study, milk yield and composition were increased when dairy goats were fed BCA. Similarly, supplementation with flavonoid-rich plant extracts improved the milk yield of dairy cows; however, flavonoids from different sources have different effects (Gessner et al., 2015; Zhan et al., 2017). Alfalfa flavonoid extract increased milk lactose synthesis and secretion by reducing the milk fat and protein content (Zhan et al., 2017). However, soy isoflavone supplementation reduced milk production (Kasparovska et al., 2016). These varying results could be caused by extracts from different sources containing different types and amounts of flavonoids. The expression of milk protein and lactose synthesis-related genes were inhibited by BCA and genistein, however, they were upregulated by formononetin and daidzein, and the metabolite of BCA, p-ethylphenol (Tsugami et al., 2017).

The nitrogen intake of dairy goats was not significantly affected by BCA addition; however, urine secretion was increased with BCA addition. Urinary nitrogen excretion remained constant. BCA could delay the rate of urea hydrolysis in the rumen, thereby allowing for more urea to be used by rumen microorganisms for microbial protein synthesis. Furthermore, BCA reduced the degradation of proteins and AAs in the rumen by inhibiting the activity of proteolytic bacteria (Liu et al., 2020) and hyper-ammonia-producing bacteria (Harlow et al., 2017c). BCA supplementation increased milk yield, indicating that dietary N utilization efficiency was improved (Harlow et al., 2017c; Pereira et al., 2020). Fewer non-degradable rumen proteins increased protein utilization and produced more milk protein (Morales et al., 2010). In another study, the acetate, propionate, and total VFA contents increased, and pH was decreased by BCA supplementation, which affected cellulose fermentation by rumen cellulolytic bacteria (Harlow et al., 2018). Similar results were obtained when BCA was added to a corn diet fermented by amylolytic bacteria; BCA increased the activity of lactic acid-utilizing bacteria and reduced the starch degradation rate (Harlow et al., 2021). However, the VFA content remained unchanged when no dietary supplements were added to the artificial medium (Liu et al., 2020). In contrast to these studies, all the VFA contents were increased by BCA in this study, which may be caused by different fermentation substrates (Harlow et al., 2020, 2021). In addition, the total VFAs and propionate contents in the rumen were also increased by replacing corn silage with different proportions of red clover (Castro-Montoya et al., 2018). Thus, the increase in VFAs by BCA was dependent on the fermentation substrate.

In this study, rumen ammonia nitrogen concentration was linearly decreased with increasing BCA. The hydrolysis rate of urea was decreased, which ultimately resulted in the inhibition of AA degradation by BCA (Liu et al., 2020). The inhibition of AA degradation by BCA was mainly achieved by inhibiting the growth of hyper-ammonia-producing bacteria, which ferment AAs into ammonia in the rumen (Harlow et al., 2017c). Hyper-ammonia bacterial growth was reduced not inhibited by BCA through the enhancement, by rumen microorganisms, of antibacterial activity of bacteriocin, which reduced the ammonia nitrogen content rather than exerting an inhibitory effect, thereby indicating that BCA had synergistic antibacterial activity (Flythe et al., 2013). Crude red clover phenolic extracts also inhibited the growth of hyper-ammonia-producing bacteria (Clostridium sticklandii SR) (Kagan and Flythe, 2012), increased bypass protein, and promoted the by reducing protein nitrogen loss (Harlow et al., 2017c). Therefore, BCA promoted growth of steers rumen fermentation and increased microbial protein production. In addition, the SOD, GSH-Px, and catalase are the main antioxidant enzymes in animals. Serum GSH-Px activity reflected oxidative stress and contributed to oxidative defense in animals (Tuzun et al., 2002). The activities of SOD and GSH-Px were enhanced by BCA through inhibiting nicotinamide adenine dinucleotide phosphate and malondialdehyde production (Yu et al., 2019). In addition, BCA prevented oxidative stress by enhancing the total antioxidant status and the levels of SOD and catalase (Jalaludeen et al., 2016; Sadri et al., 2017). Isoflavone BCA is activating a novel nuclear factor erythroid 2-related factor 2-antioxidant response element activator and can protect against oxidative damage (Yu et al., 2019). Flavonoid extracts containing BCA had a similar effect on improving the antioxidant capacity of livestock and poultry. In chickens supplemented with soybean isoflavone, increased T-AOC and SOD levels in the plasma were reported (Jiang et al., 2007). Dietary supplementation with flavonoids from Scutellaria baicalensis Georgi enhanced the antioxidative ability of broilers (Liao et al., 2018). The activities of SOD and GSH-Px were also increased by feeding alfalfa flavone extract to dairy cows (Zhan et al., 2017). The GSH-Px activity was increased with an increase in BCA supplementation, indicating that BCA can improve the antioxidant performance of dairy goats by enhancing T-AOC and the activities of antioxidant enzymes.

Plasma biochemical parameters reflected the metabolism of nutrients and health conditions of the animal body (Wang et al., 2011). No differences were observed in the total protein, albumin, globulin, uric acid, creatinine, and β-hydroxybutyric acid contents, suggesting that BCA had no effect on the health of dairy goats. Alanine and aspartate aminotransferase are vital indices of liver function, reflecting the permeability and metabolic function of liver cells (Li et al., 2017). Increases in alanine and aspartate aminotransferase activities were caused by damage to the integrity of the hepatic membrane architecture (Kotyzova et al., 2013). In this study, although there is no significant difference, aspartate aminotransferase activity had a decreased trend in the BH group, indicating that a high dose of BCA may have a hepatoprotective effect in dairy goats, and this trend was also observed in a previous study in rats (Breikaa et al., 2013). PUN and NH₃-N were the final protein metabolites that reflected AA metabolism (Li et al., 2017). PUN concentrations reached a peak approximately 2 h after feeding, then gradually decreased, reverting to normal levels at 6 h (Sinclair et al., 2012). A similar trend was observed in the NH₃-N concentrations, which may have resulted from the decreased hepatic clearance of NH₃-N (Ludden et al., 2000). Soybean isoflavone, an isoflavone phytoestrogen, is a physiological regulator of the reproductive and nutritional processes of the body through the neuroendocrine system to improve the production performance of animals (Jiang et al., 2007). Isoflavone promoted the development of mammary glands, increased milk yield, and accelerated animal growth by inducing changes in endogenous hormone levels in blood, such as prolactin, growth hormone, and IGF-1 (Tsugami et al., 2017). In this study, BCA treatment significantly increased the levels of 17β-estradiol, growth hormone, and IGF-1. Similarly, the levels of 17β-estradiol, growth hormone, and prolactin in the plasma of mid-lactation dairy cows were also increased by soybean isoflavone daidzein (Tsugami et al., 2017). These hormones can regulate the lactation of dairy goats and the structural development of mammary gland, and estradiol and IGF-1 can regulate mammary epithelial cells by binding to estrogen receptor and maintaining its expression (Tsugami et al., 2017). However, there are limited reports on the effects of BCA on the endocrine system of animals; therefore, further studies are required to explore the underlying mechanisms.

In this study, the flora structure of the rumen microbiome was modulated by BCA supplementation, which promoted nitrogen metabolic efficiency in the rumen. Similarly, in another study, the abundance of Bacteroidetes was increased by isoflavone-enriched feed, resulting in a high nitrogen metabolic efficiency in the rumen (Kasparovska et al., 2016). Rumen microbial diversity was not significantly affected, whereas the abundance of rumen microbiota was significantly affected by red clover isoflavones (Melchior et al., 2018). These results indicated that the nitrogen metabolic efficiency in the rumen was improved by BCA treatment by modulating the structure of the rumen microbiome. In this study, the flora of nitrogen metabolism and cellulolytic bacteria in rumen was regulated by BCA, and these rumen microbiota can promote nitrogen metabolic efficiency of dairy goats, resulting in enhancing feed conversion and increasing VFA production. As we know, acetate and butyrate are the precursors of milk fat, and propionate is the precursor of lactose in milk. Therefore, the increased VFA led to the improvement in milk production. In addition, reduced ammonia can be used by rumen microbes, and the increase of microbial protein can increase the milk protein, also leading to the increased feed conversion and VFA production. Additionally, in health dairy goats, the improvement in antioxidative capacity can enhance their resistance of pathogen and diseases, which is beneficial to the health of goats. Moreover, unchanged plasma biochemical parameters indicated that BCA had no side effects on the immune and health of dairy goats. The altered endocrine indexes indicated BCA can increased the production performance of dairy goat through hormone to some extent, including 17β-estradiol, GH, and IGF-1.

Milk production performance, nitrogen metabolism, and the feed conversion ratio of dairy goats were improved by BCA supplementation. Rumen AA degradation were inhibited, whereas VFAs were increased with BCA supplementation, indicating that rumen fermentation was promoted. Antioxidant performance (GSH-Px activity and T-AOC) and endocrine hormones levels (prolactin, growth hormone, and IGF-1) were also increased by BCA supplementation. The relative abundance of rumen microbiota were also affected by BCA supplementation, such as decreased Verrucomicrobiota and increased nitrogen metabolism and cellulolytic bacteria, including Prevotella sp., Treponema sp., Ruminococcus flavefaciens, and Ruminobacter amylophilus. Thus, dairy goat supplemented with BCA at a concentration of 6 g/day per goat can improve its performance and health status.

The original contributions presented in this study are publicly available. This data can be found here: https://nmdc.cn/resource/genomics/project/detail/NMDC10018266.

This animal study was reviewed and approved by Animal Care and Use Committee of the Institute of Animal Science of the Chinese Academy of Agricultural Sciences (approval no. IAS2020-97).

YL, JW, and SZ conceived and designed the experiments. QX and YL wrote and prepared the original draft. QX, WD, NZ, JW, and SZ edited the manuscript. QX, JW, and SZ critically reviewed the manuscript. All authors reviewed and approved the final manuscript.