The Ace and Chao indices are used to estimate the total num ber of microbial species. The Shannon and Simpson indices are used for estimating microbial diversity. Higher values of these latter indices indicate a higher level of community diversity. Flavonoids could generate an antimicrobial and antifungal effect by inhibiting bacterial nucleic acid synthesis and by destroying the bacterial cytomembrane [
4]. The microbial diversity of the rumen might therefore be reduced by AFE. The number of phyla and genera in the rumen of a dairy cow with a diet containing 20 mg/kg BW AFE was lower than that of a cow without AFE supplementation. The results of previous studies showed that flavonoids could inhibit
Escherichia coli,
Staphylococcus aureus, and
Enterococcus faecalis [
12,
13], whereas could not affect
Streptococcus bovis [
6]. Previous studies showed that not all bacteria could be inhibited by flavonoids. There is a range of microbes in the rumen, with relationships including mutualism, antagonism, competition, parasitism, and predation. Bacteriostatic action and flavonoid dose showed a positive correlation; a higher dose of flavonoids produced a stronger antibacterial effect [
14]. However, in the present study, the number of microbial species and microbial diversity in the groups receiving from 60 mg/kg BW to 100 mg/kg BW of AFE was increased. Possible explanations of these results are that: i) the flavonoids inhibited the growth of pathogenic bacteria and then promoted the growth of other microbes in competition with them; ii) flavonoids may be degraded by gut microbes. It has been reported that over a particular concentration range the degradation rate of flavonoids is increased with increasing dose of flavonoids [
15]. An increasing quantity of microbial flavonoid degradation products is therefore generated.
Previous studies have found that the principal phyla of micro bes in the rumen are
Firmicutes,
Bacteroidetes, and
Proteobacteria. The results of this study confirm previously reported proportions of these three phyla to be about 91% of the total [
16,
17].
Tenericutes, as a class of bacteria distinguished by the absence of a cell wall contains the class
Mollicutes [
18]. The primary genera includes the
Mycoplasma,
Spiroplasma,
Ureaplasma, and
Phytoplasma. Most of them have sterols that make the cell membrane somewhat more rigid.
Mollicutes are parasites of various animals and plants, living on or in the host’s cells. To maintain their parasitic mode of life, the
Mollicutes have developed rather sophisticated mechanisms to colonize their hosts and resist the host immune system.
Tenericutes bacteria are sensitive to tetracycline antibiotics. Flavonoids might not, therefore, be inhibitory to them; instead, flavonoids might inhibit the growth of bacterial competitors of the
Tenericutes, and thereby promote the growth of
Tenericutes class bacteria.
Euryarchaeota, as a major class of Archaea, contain
Methanogenus,
Halobacterium, and some hyperthermophilic anaerobic and aerobic bacteria. This study found that the abundance of
Methanomicrobia was increased by an increasing level of AFE. Therefore, the reason for increasing the abundance of
Euryarchaeota was that AFE may promote the growth of bacteria of the class
Methanomicrobia. It indicated that diet supplemented flavonoids extracted from alfalfa might promote methanogenesis. Ma et al [
19] found that mulberry leaf flavonoids decreased ruminal populations of methanogens. In addition, naringin and quercetin significantly suppressed the growth of methanogens [
7] and showed that flavonoids could inhibit methanogenesis. The result of this study was different and indicated that flavonoids from different sources have different effects. The phylum
Fusobacteria, commonly found in the oral cavity, is a phylum of anaerobic gram-negative bacilli with fusiform morphology [
20,
21]. The results of the present study showed that AFE could inhibit bacteria of the
Fusobacteria and the effect was positively correlated with dose. There are four characterized rumen
Prevotella spp.:
P. ruminicola,
P. bryantii,
P. albensis, and
P. brevis [
22], which degrade protein, starch, and fibre, thus playing a vital role in feed digestion. This study confirms the results of previous studies which showed that
Prevotella was the dominant bacterium in the rumen [
23]. Furthermore, the abundance of
Prevotella was reduced with increasing dose of AFE, indicating that AFE may inhibit the growth of
Prevotella. The genus
Pyramidobacter comprises strains that are anaerobic, non-motile, asaccharolytic bacilli that produce acetic and isovaleric acids and small quantities of propionic and isobutyric acids [
24].
Succinivibrio could ferment sugars and produce acetic and succinic acids as end-products of metabolism [
25]. In the present study, the abundance of
Pyramidobacter and
Succinivibrio was found to be reduced by AFE. The alfalfa flavonoids may decrease the content of acetic acid in the rumen. However, acetic acid could be transformed into methane by
Methanobacteria, or be used by the cow in the synthesis of milk fat [
26]. The results of this study showed that AFE may have the effect of reducing both methane and milk fat. Lee et al [
27] found that acetate stimulated particularly the growth of
Spirochaetes. The reduction in the abundance of
Spirochaeta in the present work may be because AFE reduced the generation of acetic acid.
Mogibacterium has been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals [
28].
Asteroleplasma are compulsory parasites/pathogens of vertebrates [
29]. In the present study, AFE had the effect of inhibiting
Mogibacterium and
Asteroleplasma. This indicated that AFE has the potential to protect animals from diseases caused by
Mogibacterium and
Asteroleplasma.
Suttonella is a gram-negative coccobacillus that can produce indole and ferment sucrose and glucose [
30]. Many studies have shown that indole plays a vital role as an intercellular signaling molecule in some enteric pathogens such as
Escherichia coli,
Edwardsiella tarda and
Vibrio cholera. The drug resistance and pathogenicity of pathogenic bacteria could be reduced by degrading or inhibiting the accumulation of indole signal molecules. AFE might therefore affect the release of indole and thereby influence competition within microbial consortia.