1. Introduction
Herbivores require plants for survival and reproduction [
1] and for maintaining a stable population size [
2]. Plants have evolved systems for defense against herbivores, which hinder feeding, digestion, and metabolism, thus restricting food choices [
3]. These defense mechanisms include structural features, such as needles, thorns, and spines, to prevent feeding by herbivores. Alternatively, plant secondary metabolites (PSMs) can inhibit animal growth and development [
4].
The intestine is a critical site for nutrient digestion and absorption. Digestive enzymes break down the majority of the nutrients in food, and then intestinal microbes in the cecum convert undigested protein and undigested carbs to generate a wide variety of metabolites [
5]. In addition, the intestine is also an important immune organ that allows the required nutrients to enter the internal environment and acts as a barrier to prevent harmful substances from penetrating the intestinal lumen [
6]. The effects of PSMs on intestinal function in small mammals are incompletely understood.
Polyphenols are the most common complex secondary metabolites in plants [
7]. Tannins, the most extensively studied plant polyphenols, can be classified into condensed tannins or hydrolytic tannins according to their chemical properties. Tannic acid (TA), a hydrolytic tannin, possesses antioxidant, anti-inflammatory, and antibacterial properties [
8,
9]. However, TA is often considered cytotoxic and may be an anti-nutritional factor for many animals and alter the growth performance of animals owing to its ability to bind to proteins [
10]. Tannins have been shown to induce intestinal mucosal necrosis and villus damage in broilers [
11], but gallnut TA extract decreased intestinal lesions in pigs [
12]. Mbatha et al. [
13] reported that TA has a negative effect on the intestinal morphology of Boer goats. Mandal et al. [
14] found that TA could reduce the digestive enzyme activities in Indian major carps. Broilers fed high concentrations of TA show increased gut permeability [
15]. However, little is known about the effects of TA on the intestinal function and physiology of herbivores mediated via the intestinal flora.
Brandt’s vole (
Lasiopodomys brandtii) is a non-hibernating herbivore, largely inhabiting Inner Mongolia and other regions with degraded pastures [
16]. It feeds on grassland vegetation and is therefore extremely destructive to grasslands.
Leymus chinensis, the dominant plant in Inner Mongolia grassland, the preferred food of voles, contains TA [
17]. Accordingly, Brandt’s vole is a suitable subject to explore the effects of PSMs on the survival and reproduction of herbivores. TA can affect ovarian development in Brandt’s voles, thus affecting their reproductive capacity [
18]. However, it is not clear whether TA affects intestinal function, and therefore the survival of the species.
In the present study, we examined the impact of TA (at doses not exceeding the average content in the typical diet) on Brandt’s voles in terms of (1) growth performance, (2) intestinal morphology, (3) the activity of digestive enzymes and cecal fermentation, and (4) immune and microbial barrier functions of the intestine. These analyses allowed us to gauge the effects of TA on the intestinal function of Brandt’s voles, thereby potentially providing a new theoretical basis for the understanding of plant–herbivore interactions and the ecological role of PSMs.
4. Discussion
Previous research on tannins has focused more on their effects on the growth performance of poultry animals, and less on that of wildlife. Hydrolytic tannins are easily hydrolyzed into glucose and gallic acid and can be subdivided into two classes, gallotannins and ellagitannins [
28]. In this study, we investigated the effects of TA, a type of gallotannin, on the growth performance, intestinal morphology, digestive enzyme activity, cecal fermentation, intestinal barrier function, and gut microbiota composition of Brandt’s voles.
Previous studies have shown that tannins can affect the growth performance of animals. Tong et al. [
29] showed that plant tannins can improve the growth performance of broilers. By contrast, Choi et al. [
15] reported that the astringency and bitter taste of TA–saliva protein complexes reduced feed intake, and thus, reduce broilers growth performance. Consistent with the study of Choi et al., we found that TA reduced the BW and ADFI of voles compared with those of the control group, indicating that TA has an adverse effect on growth performance. The differences between studies might be owing to the differences in the source and amount of tannins, features of experimental animals, diet and living environment, duration of the experiments, and potential correlations between these factors.
Proper functioning of the intestine depends on its morphological integrity. A change in intestinal morphology can affect the digestion, absorption, and transportation of nutrients [
30]. VH, CD, and the VH/CD ratio are the main indices used to evaluate the morphological structure of the small intestine [
31]. TA has been found to cause significant changes in these intestinal parameters. Mbatha et al. [
13] reported that TA had a negative effect on intestinal morphology by damaging the gut villi of Boer goats. Consistent with previous results, we did not detect significant differences in the length of intestinal segments when voles were fed TA at varying concentrations. However, TA feeding altered the intestinal morphology of voles by decreasing VH and the VH/CD ratio in the intestine. The current results demonstrated that TA damages the structure of the intestinal mucosa of Brandt’s voles.
In vitro digestive enzyme activity levels directly affect the absorption and utilization of nutrients, thus affecting the health status and growth rate of animals. The effects of TA on digestive enzyme activities have not been thoroughly studied. Studies have shown that TA can reduce the enzymatic activities of lipase, amylase, and protease in Indian major carps [
14]. In this study, TA decreased amylase, cellulase, and lipase activities in different intestinal segments. TA binds to proteins, thereby reducing their absorption. After protein binding, a sufficient hydrophobic layer is formed on the surface, which is affected by features of the protein (size, conformation, and charge), as well as the number and stereospecificity of binding sites on polyphenols and protein molecules [
32]. The effects of TA on digestive enzymes differ between segments of the small intestine, which may reflect functional differences among segments.
Most nutrients in food are digested and absorbed in the stomach and small intestine. Undigested protein and undigestible carbohydrates in the cecum are metabolized to generate a large number of metabolites by intestinal microorganisms [
33]. Brandt’s voles exhibit post-gut fermentation [
34]. The cecal pH, NH
3-N concentration, and SCFA content are important indices used to evaluate the level of cecal fermentation [
35]. It is generally accepted that SCFAs (mainly including acetate, propionate, and butyrate) play an important role as an energy source [
36]. Ammonia nitrogen is a major protein catabolite and an important substrate for bacterial protein synthesis by cecal microorganisms [
37]. Biagi et al. [
38] reported that TA significantly reduced the concentration of ammonia, isobutyric acid, and isovaleric acid in the cecum of weaned piglets. In line with previous findings, TA supplementation decreased the acetic acid and butyric acid contents in the cecum, indicating that fermentation decreased in TA-fed Brandt’s voles, compared with those in the control group voles.
The levels of endotoxins and zonulin in plasma are indicators of intestinal permeability [
21]. Increased intestinal permeability allows toxins to enter the blood and intestines, where they cause the production of several cellular inflammatory factors that result in intestinal inflammation [
39]. Choi et al. [
15] found that broilers fed high concentrations of TA displayed increased gut permeability. In this study, we found that TA increased plasma levels of zonulin compared with that in the control group, implying that TA could increase intestinal permeability.
The distribution and function of intestinal goblet cells play an essential role in maintaining an intact gut tissue morphology [
40]. The intestinal mucosal barrier formed by mucus secreted by goblet cells can prevent exogenous pathogenic substances from entering intestinal epithelial cells and protect the intestinal tissue morphology [
41]. In the present study, TA reduced the number of goblet cells in the ileum of voles, indicating that TA had adverse effects on the intestinal chemical barrier in the vole ileum. Additionally, the number of small intestinal goblet cells tended to increase from the duodenum to the ileum, consistent with the results of a previous study [
42]. Mast cells exhibit immune activity and secrete a variety of inflammatory mediators, such as TNF-α, in the intestine [
43]. In this study, a high concentration of TA reduced the number of mast cells in the duodenum and jejunum of voles.
The immune function of the intestinal mucosa is mediated by immune cells and cytokines [
44]. Immunoglobulins, especially IgG and IgA, play an important role in humoral immunity. An increase in the secretion of immunoglobulins indicates an improvement in immune function. SIgA is a major component of the intestinal mucosal defense system and a barrier to maintain the stability of the intestinal mucosal internal environment. Jafari et al. [
45] noted that oak acorn containing high levels of tannins can reduce immunoglobulin synthesis. In the present study, HT treatment reduced the levels of TNF-α, IL-6, lgG, and lgA in the serum and SlgA in the duodenum of voles, while LT treatment increased the level of SlgA in the ileum of voles. These findings suggest that TA had negative effects on immune function in voles.
In addition to immune barriers, commensal microorganisms in the gut form part of the intestinal epithelial barrier. These microbes interact with the host, forming a coevolutionary relationship [
46]. In this study, TA did not change the microbial alpha diversity; however, HT significantly changed the β-diversity of the colonic microbial community compared with that of the control group. Furthermore, an enrichment in biomarker OTUs was observed. These findings indicate that TA could alter the structure of the colonic microbiota. In the present study, the dominant bacterial phyla were Firmicutes and Bacteroidetes, consistent with previous results [
6]. Firmicutes can convert cellulose into volatile fatty acids to promote fiber degradation, growth, and weight gain [
47]. Bacteroidetes effectively break down carbohydrates, preventing obesity [
48]. In our study, we found a significantly lower relative abundance of Firmicutes and a lower Firmicutes/Bacteroidetes ratio, a marker of gut dysbiosis, in the HT group than in the control and LT groups. This result is consistent with the observed changes in BW, suggesting that weight loss is related to the HT treatment-induced reduction in Firmicutes.
Some bacteria, such as
Roseburia, Lachnospiraceae, and Butyricicoccaceae, produce SCFAs [
49].
Roseburia have been reported to produce SCFAs that break down indigestible carbohydrates. These compounds are usually involved in energy production and can protect the gut from pathogens [
50]. Furthermore,
Roseburia may play an important role in controlling inflammatory processes, especially intestinal inflammation. The dysregulation of
Roseburia may affect a variety of metabolic pathways [
51]. Lachnospiraceae, a potentially beneficial bacterium in the gut, produces acetic acid and butyrate for host energy and the maintenance of gut barrier integrity [
52]. Butyricicoccaceae can also produce butyrate and are related to ulcerative colitis [
53]. In the present study, the relative abundance of f_Butyricicoccaceae, g_
Roseburia, and g_
Lachnospiraceae was significantly lower in the HT group than in the control and LT groups. Furthermore, HT significantly reduced the butyrate content. Thus, we speculate that TA can decrease the abundance of these bacteria to reduce the production of SCFAs, thus damaging overall health.
Oxalobacter is a key oxalate-degrading bacterium in the mammalian gut. The degradation of oxalate in the intestine plays a key role in preventing nephrotoxicity in animals that feed on oxalate-rich plants [
54].
Sphingomonas can degrade macromolecular organic matter and is antagonistic to some pathogens. It can promote growth [
55].
Papillibacter are also thought to have probiotic effects [
56]. In our study, we found that HT treatment significantly reduced the relative abundance of o_Sphingomonadales, f_Oxalobacter, f_Sphingomonadaceae, g_
Papillibacter, and g_
Sphingomonas compared with the control and LT groups. Furthermore, the relative abundance of some pathogenic bacteria, such as f_Defluviitaleaceae and g_
[Eubacterium]_nodatum_group, was higher in the HT group than in the control group. Therefore, HT reduced the abundance of probiotics and increased the abundance of pathogenic bacteria in Brandt’s voles. KEGG function analysis revealed that TA induced a change in the metabolism, cellular processes, and organismal systems in the colonic bacteria and that the change was dose dependent. These findings provide a basis for further studies of the role of the gut microbiota in the health, environmental adaptation, and metabolism of Brandt’s voles.