1. Introduction
As one of the dietary nutrients affecting intestinal function, dietary fiber has been widely recognized to prevent intestinal disorders, such as post-weaning diarrhea and inflammatory bowel disease in pigs [
1,
2,
3]. Hence, it can be considered a potential dietary constituent for alleviating stress-induced intestinal damage in pigs [
4]. The beneficial effect of dietary fiber on the intestine is mainly attributed to the alteration of hindgut bacterial composition and the short-chain fatty-acid (SCFA) acetate, propionate, and butyrate contents, both of which have a potent anti-inflammatory effect [
5]. Commensal bacteria and SCFA have been shown to modulate the intestinal barrier integrity. The messenger RNA (mRNA) expression levels of Occludin in CACO-2 cells have been found to be upregulated by
Lactobacillus spp. in vitro [
6].
Escherichia coli can disrupt tight junctions by dissociating the tight-junction proteins from epithelial cells [
7]. A study using ovine mammary epithelial cells as an in vitro model revealed that propionic acid could inhibit the internalization of
Staphylococcus aureus into the epithelial cells [
8], while studies on CACO-2, IPEC-J2, and LS174T human colorectal cells demonstrated that butyrate could maintain intestine barrier function by increasing the expression of mucin and tight-junction protein genes and enhancing transepithelial electrical resistance [
9,
10,
11]. These results suggest that bacteria and SCFA could affect the intestinal barrier integrity by regulating the expression levels of tight-junction proteins and mucin genes. Hence, dietary fiber may improve the function of the intestinal mucosa by modulating the intestinal bacteria and fermentation end-products in growing pigs.
Beet pulp has high levels of crude fiber (CF), nitrogen-free leachate, and crude protein (CP). Moreover, a large amount of
l-arabinose polymer exists in the CF of beet pulp [
12]. Pigs had a higher digestibility due to the fiber from beet pulp, mainly because of its high soluble fiber content [
13]. Short-term studies using piglets revealed that piglets fed a diet containing beet pulp or wheat bran had a higher weight of the large intestine and fewer
Enterococcus spp. in the feces [
14,
15]. The findings of these studies suggest that beet pulp has a positive effect on intestine microbiota composition. However, there is little available information on the systematic mechanism via which beet pulp promotes the intestinal health status. Consequently, we tested the hypothesis that dietary fiber obtained from beet pulp could affect gut health by regulating the intestinal morphology, nutrient digestion and absorption, and gut barrier function. In the present study, we modulated the dietary-fiber concentration by including beet pulp and systematically investigated the effects of a high-fiber diet on the gut health of growing pigs. Our findings will raise understanding about how dietary beet pulp inclusion beneficially influences the intestinal health in growing pigs.
4. Discussion
As a monogastric species, pigs have limited ability to utilize dietary fiber [
23]. Therefore, although pigs can tolerate a relatively high level of fiber, supplemental fiber in a pig’s diet is considered as an important factor affecting feed intake. Anguita et al. (2007) [
24] and Zhang et al. (2013) [
25] demonstrated that high fiber contents in diets can decrease the voluntary feed intake of the animals as a consequence of higher water retention capacity and gut fill, in agreement with the present study that a reduction in ADFI and ADG of pigs fed a beet pulp diet was found. However, the feed conversion rate of pigs was not affected by fiber levels, and an improved numerical value was even observed in beet pulp group, which indicated that feeding supplemental beet pulp possibly altered the intestinal digestive physiology and barrier function of pigs [
26].
As an indigestible carbohydrate component of pig diet, fiber serves as the main fermentation substrate in the digestive tract, particularly for microorganisms in the hindgut, which maintains the normal physiological function of the digestive tract [
27]. Intestinal weight can be affected by dietary fiber. Short-term studies using piglets revealed a higher relative weight of the large intestine in piglets fed a diet containing beet pulp [
14,
15]. Similar results were observed in growing pigs fed a diet containing 23% beet pulp (8.1% CF). The weight of the stomach and whole intestine significantly increased in these pigs [
28]. In the present study, the beet pulp group had a higher weight of the whole intestine and small intestine and higher density of the large intestine than the control group, indicating that fiber could stimulate the growth of the intestinal mucosa and increase the mucosal weight [
29]. It is well known that IGF-1, EGF, GLP-2, and its receptor (GLP-2R) are main regulators of intestine length [
30,
31]. Therefore, we further investigated the influences of beet pulp on the mRNA expression levels of intestinal development-related genes in the intestine. As expected, the mRNA expression levels of EGF, GLP-2, and GLP-2R in the duodenum, EGF and GLP-2 in the jejunum, EGF in the ileum, and GLP-2 in the colon were significantly enhanced by dietary beet pulp supplementation. In addition, increased GLP-2 concentration was observed in the present study, which further suggested the regulating ability of intestine development brought by beet pulp.
The intestinal morphology is also affected by dietary fiber. A high-fiber diet containing 10% wheat straw increased the proliferation rate of jejunal and ileal epithelial cells and increased the villus height and crypt depth in the jejunum and ileum in growing pigs [
32]; these findings are consistent with those of the present study. Generally, the ratio of villus height to crypt depth represents the absorption capacity of the small intestine [
33]. In the present study, villus height and the ratio of villus height to crypt depth in the jejunum were increased relative to those in the control group, which suggests that beet pulp influences pig intestinal absorption capacity by regulating morphological structures. However, in another study, no difference was noted in the intestinal morphology on 15% soybean husk, 15% oatmeal husk, or 20% alfalfa meal supplementation [
34]. Chen et al. (2014) demonstrated that a diet supplemented with different sources of fiber (wheat fiber, pea fiber, soybean fiber, and corn fiber) caused variation in the structure of the small intestine in weaned piglets [
35]. It appears, therefore, that, in addition to fiber levels, fiber sources may have a certain impact on the intestinal morphology.
Several studies have shown that dietary fiber levels and physicochemical properties affect nutrient digestion and absorption in pigs. A high-fiber diet containing 15% soybean husk, 15% oatmeal husk, or 20% alfalfa meal decreased the apparent digestibility of energy, DM, and nitrogen in growing pigs [
34]. Moreover, a diet containing 20% beet pulp decreased the apparent digestibility of CP, crude fat, and organic matter, but increased the apparent digestibility of acidic detergent fiber and neutral detergent fiber in growing pigs [
13]. In the present study, the beet pulp group had lower ATTD of DM, CP, gross energy, and crude ash and higher ATTD of CF than the control group, which shows that digestion ability of dietary fiber could be increased by beet pulp. Moreover, we probed the influences of beet pulp supplementation on intestinal enzymatic activity. Our results showed that the activities of jejunal lactase and sucrase in beet pulp group were higher than those in the control group, specifically, that the development of disaccharidase activities can be induced by beet pulp supplementation. Similarly, pigs fed a high-fiber diet stimulated the activities of maltose and lactase in the brush border of the intestinal mucosa, and elevated the activities of trypsin, chymotrypsin, and total hydrolytic protease in the duodenal digesta [
15,
36]. As previously mentioned, it is essential for providing sufficient lactase, maltase, and sucrase to degrade disaccharides [
37]. In support of this notion, we speculated that beet pulp supplementation also can facilitate intestinal absorptive capacity through facilitating disaccharidase activities in growing pigs.
Fiber can affect the speed at which the digesta is passed through the intestine and reduce the retention time of the digesta in the small intestine, thus stimulating the compensatory secretion of endogenous digestive enzymes [
38]. This led to a decrease in the nutrient digestibility and an increase in the activities of lactase and sucrase in pigs supplemented with beet pulp in the present study. Nonetheless, an inconsistent study found that a diet supplemented with 5% wheat bran or beet pulp had no significant effect on the nutrient digestibility in growing pigs [
39]. Moreover, a diet supplemented with different sources of fiber had different digestibility both in weaned piglets and in growing pigs [
40]. Taken together, these different results may be due to the effect of dietary fiber levels, physicochemical properties, and sources. In addition, higher mRNA expression levels of intestinal GLUT-2, ZNT1, and DMT1 were observed in the beet pulp group in our present. These findings are sufficient to suggest that beet pulp has certain effect on the intestinal digestion and absorption function.
A healthy and stable microbiota prevents the development of intestinal diseases and results in improved gut performance. Dietary fiber has been shown to selectively regulate gut bacteria, including stimulating the proliferation of probiotics and inhibiting the proliferation of potential pathogens [
41,
42,
43,
44]. Fiber depravation significantly altered the profile of the intestinal microbiota, greatly increased the number of mucus-degrading bacteria, and sharply decreased the number of fiber-decomposing bacteria in mice, thereby reducing the thickness of their mucus layer and increasing their susceptibility to pathogenic bacteria [
45]. As a new dietary fiber, resistant starch can also promote the growth of beneficial bacteria, inhibit the number of harmful bacteria, promote the production of propionic acid and butyric acid in the colon [
46], and stimulate the secretion of microbial enzymes [
47]. Moreover, beet pulp can regulate the composition and quantity of intestinal microbiota. Previous studies demonstrated a lower number of
Enterococcus in the feces of piglets fed a diet containing beet pulp [
14,
15]. Similarly, a diet containing 23% beet pulp significantly increased the numbers of
Lactobacillus spp. and
Bifidobacterium spp. and decreased the numbers of
Enterobacteriaceae spp. and
Enterococcus spp. in the hindgut of growing pigs [
28]; this finding is consistent with the finding of the present study. With an increase in the
Bifidobacterium spp. populations in the cecum of pigs fed the beet pulp diet, the establishment of
Escherichia coli was inhibited possibly by a phenomenon known as colonization resistance [
48]. Furthermore, our present study discovered that a diet containing 25% beet pulp increased the SCFA contents and SCFA receptor expression in the colon of pigs. It is well known that SCFAs are closely related to intestinal growth and barrier function. Hence, fiber may improve the intestinal health by modulating the microbiota composition and further changing the profile of SCFA [
9,
49]. In view of the previously mentioned reasoning, we summarized that the increased populations of
Bifidobacterium spp. and the decreased populations of
Escherichia coli in the intestine improved the gut health in response to dietary beet pulp supplementation, which may be associated with the changes in the intestinal barrier integrity.
The intestinal barrier integrity is mainly maintained by the tight junction, including the peripheral membrane protein ZO family and the transmembrane protein Occludin and Claudin families, which participate in the formation of the intestinal mucosal barrier and prevent antigens from invading the body [
50]. In a study, measurement with Ussing-type chambers revealed that dietary fiber improved the intestinal function by alleviating the increase in intestinal cell permeability induced by 51Cr-EDTA [
51]. An in vitro study revealed that the adherence of
Eschericha coli to intestinal epithelial monolayers can give rise to the degradation of Occludin and ZO-1 and can downregulate the mRNA expression levels of both the genes, thereby diminishing the barrier function [
52]. In our study, pigs in beet pulp group had lower counts of
Eschericha coli and higher mRNA levels of ZO-1 and Occludin in intestine. These results suggest that the changed intestinal flora can influence the integrity of intestinal barrier by regulating the expression level of tight junction protein genes with beet pulp provided. As an important cell in the intestinal epithelium, goblet cells are found along the crypt–villus axis of the intestine and secreted mucins and peptide trifoliate factor, which contribute to the mucus layer in the intestine, providing an intestinal chemical barrier function [
53]. A diet containing 23% beet pulp significantly increased the count of goblet cells in the jejunum of growing pigs [
28]. Fiber intake increased the number of goblet cells in the intestinal mucosa compared to the fiber-free group, which is consistent with the results of the beet pulp group in this study [
54]. In the current study, more goblet cells in the ileum and colon were noticed after beet pulp addition, accompanied by an upregulated MUC2 and REG-Ⅲγ transcriptional level in the intestine, indicating that fiber obtained from beet pulp also improved the intestinal chemical barrier function in growing pigs. Therefore, it can be speculated that fiber obtained from beet pulp can upregulate the expression of tight-junction proteins and antimicrobial proteins, thereby maintaining the intestinal barrier.