1. Introduction
Adding antibiotics to animal feed can improve livestock production. However, the use of antibiotics is controversial as it can lead to the emergence of antibiotic-resistant strains that pose a risk to both human and livestock health [
1]. In addition, the use of antibiotics is prohibited in some regions of the world, such as the European Union. Therefore, antibiotic alternatives need to be developed to improve livestock productivity. In this context, recent studies indicate that secondary metabolites such as tannins, saponins, flavonoids and essential oils have the potential to regulate digestive tract metabolism and improve animal production efficiency [
2,
3]. Compared with antibiotics, the use of secondary metabolites is advantageous as they are safe and effective, without hormonal consequences or other negative side effects [
4].
Saponins are widely distributed in ginseng, alfalfa, tea and other plants, and their roles can vary based on their sources [
5]. Tea saponins are triterpenoids widely distributed in the roots, stems, leaves and seeds of the genus
Camellia [
6]. Previous studies indicate that saponins have the potential to both increase feed digestibility, and reduce methane emissions in ruminants. For example, saponin supplementation in sheep appears to increase digestibility of organic matter, neutral detergent fibre and acid detergent fibre by 9.6%, 27.9% and 38%, respectively [
7]. Furthermore, in an in vitro fermentation study, addition to saponins led to a 32.5% reduction in methane production, coupled with significantly higher short chain fatty acid and metabolizable energy production [
8]. Overall, these studies support a potential role for tea saponins in regulating nutrient digestion.
Rumen is an important digestive organ in livestock, which houses a complex ecosystem composed of a large number and a variety of microbial species, primarily including bacteria, protozoa and fungi [
9]. These microbial species play a crucial role in the digestive physiology of ruminants, and therefore it is plausible that the effect of saponins on ruminant digestion is mediated by modulating rumen microbiota. In fact, recent studies already provide evidence that tea saponins improve rumen fermentation by killing protozoa [
10]. However, apart from protozoa, tea saponins may also modulate rumen metabolism by influencing ruminal bacteria and fungi [
10,
11]. Moreover, tea saponins may potentially cause toxicity when added to feed at high levels, and at least part of these toxic effects may be mediated via ruminal microbiota. To the best of our knowledge, previous microbiome-based studies have not characterized the effects of tea saponins on rumen bacteria and fungi. Therefore, this study aims to characterize the effect of feeding different levels of tea saponins on rumen microbiota and rumen function in Qinchuan cattle. The broader aim of this study is to provide a theoretical basis for the use of tea saponin in ruminants.
4. Discussion
Acetate, propionate and butyric acid constitute some of the main VFAs found in rumen, which are derived from the fermentation of carbohydrates in feed [
19]. In this study, increasing levels of tea saponin supplementation did not significantly alter the content of acetate, butyrate, iso-butyrate, valerate and iso-valerate fatty acids. However, feed supplementation with low levels of tea saponin significantly increased total VFA content and the proportion of propionate fatty acid. These changes are likely attributable to alterations in the relative abundance of microbial species involved in carbohydrate decomposition, observed in the LT treatment group, such as Actinobacteria, Saccharomyces and Aspergillus. Similar studies in the past have also reported increased proportion of propionate, in conjunction with alterations in the abundance of bacteria involved in fibre decomposition, such as Fibrobacter succinogenes and Ruminocus flavefaciens [
7,
20]. Overall, given that rumen fermentation is primarily influenced by rumen microbiota [
21], it is predicted that the rumen microbiota was altered in this study.
Alpha diversity indices, commonly used to assess microbial richness in community ecology, are known to be negatively correlated with feed conversion efficiency [
22]. In this study, Alpha diversity analyses indicated reduced bacterial diversity in the MT treatment group, which in turn suggests that cattle in this treatment group may have higher feed conversion efficiency.
Ruminal microbiota interact with hosts in a variety of ways to play a crucial role in ruminant physiology. Environmental factors, particularly dietary supplementation, can influence the composition of rumen microbial communities [
23], which in turn can modulate the digestion of food, thereby influencing metabolism in ruminants [
24]. Our study identified Bacteroidetes and Firmicutes as the dominant phyla represented in ruminal bacteria, which is consistent with previous studies on ruminants [
25,
26,
27,
28]. Bacteroidetes and Firmicutes are also the two most important bacteria involved in the degradation of plant polysaccharides and the production of VFAs by secreting a variety of metabolic enzymes, while other bacteria play a secondary role [
29]. This may explain the similarity in dominant bacterial communities observed in ruminants that feed mainly on plant-based diets. Apart from Bacteroidetes and Firmicutes, Actinobacteria represents another key phylum, which constitutes a relatively smaller proportion of gut microbiota, but it is crucial in maintaining intestinal health and homeostasis, biodegradation of resistant starch and modulation of host immune responses [
30,
31,
32]. Within Actinobacteria, Bifidobacteria constitute a main class of anaerobic Actinobacteria that use by glycosyl-hydrolases (GHS) to hydrolyze glycosidic bonds between two or more sugars, and cooperatively decomposes carbohydrate starch and polysaccharides, producing high concentrations of acetate that protect the host from infection of digestive tract pathogens [
33]. In this study, low levels of tea saponin supplementation resulted in a significant increase in the relative abundance of Actinobacteria, which indicates that saponins have potential value in influencing livestock productivity and health by modulating ruminal bacteria.
Ruminal fungi only account for ~8% of ruminal microbiota in terms of biomass, but they are known to secrete large amounts of cellulolytic enzymes that improve the degradation and utilization of plant feed [
34,
35]. In this study, Ascomycota, Neocallimastigomycota and Mucoromycota were identified as the most dominant phyla in cattle’s rumen, which accords with similar findings in previous studies on Yunnan yellow cattle, gayals, yak and Tibetan yellow cattle [
36]. However, a key point of difference with these other studies is that Mucoromycota was identified as a dominant phylum in this study, instead of Basidiomycota, which has been reported as the dominant phylum in other studies. Furthermore, in this study, low level supplementation with tea saponins resulted in a significant increase in the relative abundance of Ascomycota at the phylum level; and Saccharomyces and Aspergillus at the genus level. Functionally, Ascomycota are known to play a key role in degrading lignin and keratin [
37]; Neocallistigomycota mainly consume rumen degradable proteins [
38] and Mucoromycota are known to produce essential fatty acids and carotenoids [
39]. Saccharomyces and Aspergillus play a key role in fermenting carbohydrates that are conducive to the production of TVFAs [
40,
41]. In addition, Saccharomyces also provides vitamins and a variety of growth factors necessary for the growth of cellulolytic-hydrolyzing bacteria [
41]. Corresponding to these changes, the content of TVFAs in rumen in the low-level tea saponin treatment group was also found to be significantly increased relate to the control group in this study. Interestingly, there was no significant change in the content of TVFAs despite the significant decrease in Saccharomyces in the high-level tea saponin group, which may be related to the fact that Piromyces in the high tea saponin group compensated for the decrease in Saccharomyces. It has been previously reported that Piromyces are part of microbial consortia with high capacity of plant biomass degradation [
42]. Taken together, these findings suggest that cattle in the LT supplementation treatment group had a higher ability to digest forage. In fact, we also found that the apparent digestibility of crude fibre in LT treatment group was higher. These findings are consistent with findings of previous studies that have also reported digestibility to be significantly enhanced after addition of tea saponins to forage [
7].
Significant differences in microbial community structures of the four treatment groups were also identified via LEfSe analysis. More differentially abundant microbes were identified in the LT and MT treatment groups than in the CON and HT treatment groups. The LT treatment group in particular, was found to be enriched with Holdemania, Turicibacter, Propionibacterium, Akkermansia and Pseudomonas. Other researchers have reported that Propionibacterium can ferment lactate to propionate to enhance propionate production [
43]. Accordingly, we also found that low level tea saponin supplementation significantly increased the proportion of propionate in rumen fermentation parameters. Enhanced propionic acid can also potentially reduce methane production in rumen and improve energy utilization efficiency [
44]. Another genus that was found to be significantly enriched in the LT group was Akkermansia, which can decompose mucin and improve host health, thereby acting as a probiotics. Overall, these results indicate that tea saponins may modulate host physiology by altering the composition of ruminal microbiota.
Studies have shown that tea saponins are composed of sapogenins, glucosides and organic acids, among which glucosides include a variety of oligosaccharides [
6] that are beneficial to fibre-degrading microbes [
45]. Moreover, saponins can interact with cholesterol in protozoan cell membranes resulting in the destruction of protozoa [
46], which then also results in reduced protozoal predation of bacteria. This could explain the increased abundance of some microbes in the low level tea saponin treatment group compared with the control group. Interestingly, the relative abundance of several microbiota such as Actinobacteria, Ascomycota, Saccharomyces and Aspergillus, were found to be lower in the high level tea saponin treatment group, compared to low level of tea saponin supplementation. Previous studies have also shown that while low level saponins can stimulate the growth of some rumen bacteria such as cellulolytic bacteria, high level saponins can inhibit the growth of microbes [
47]. It has been reported that saponins can destroy the integrity of microbial cell membrane [
48]. Therefore, it is possible that at high levels, tea saponins destroy the cell membrane integrity of microorganisms, thereby inhibiting their growth.
The gut microbiota is a vital regulator of host metabolism. Therefore, the functional profile of the ruminal microbial community in cattle was predicted by PICRUSt. In our study, the majority of pathways identified to be associated with ruminal microbiota were associated with metabolism, e.g., Amino acid metabolism, Metabolism of cofactors and vitamins and Carbohydrate metabolism. These results were consistent with previous studies in yaks [
49] and Simmental bulls [
50]. Compared with the control group, the pathways belonging to the Xenobiotics biodegradation and metabolism increased significantly in LT treatment group, indicating that low levels of tea saponins promote the metabolism of xenobiotics in cattle. Similarly, the pathways belonging to the glycolysis III increased significantly in LT treatment group, indicating that low levels of tea saponins help promote glycolysis. Several previous studies have shown plant extracts can modulate functions associated with intestinal microbiota, thereby improving host growth performance [
51]. Thus, alterations in intestinal microbial function by tea saponin supplementation may lead to the performance improvements in cattle.