4.2. Apparent Digestibility of NSP
The apparent colonic and total tract digestibility of total and soluble NSP in rye-fed pigs was higher compared to pigs receiving wheat-based diets. A higher ATTD of TDF and arabinose in rye-fed pigs was associated with an increased microbial fermentation in the large intestine [
16]. In rye compared to wheat a higher fermentative activity might be related to the higher solubility of rye AX, the higher content of β-glucans and the higher total amount of dietary fiber [
16,
37]. Therefore, the digestibility of NSP in the current study might have been caused by a higher uptake of fermentable fiber with the rye-based diets, including more sNSP and soluble AX. Moreover, wheat AX might be less degradable, because of cross-linkages to other polysaccharides such as cellulose [
37]. Cellulose is a major plant cell wall component, which is highly insoluble, poorly fermentable, and has a higher concentration in wheat than in rye [
18]. The increased concentrations of digesta SCFA and the higher relative abundance of major plant cell wall-degrading bacterial species in the jejunum indicate an enhanced bacterial fiber degradation and can further explain the higher digestibility of NSP in rye-fed pigs of the present study.
With respect to NSP digestibility in RSM-fed pigs, values for colonic and total tract digestibility of total and iNSP was lower than in SBM-fed pigs. Similarly, ATTD of neutral detergent fiber (NDF), acid detergent fiber (ADF), carbohydrates, and dietary fiber was lower in other studies investigating the feeding of RSM instead of SBM to pigs [
39,
40]. However, one of these studies did not show a different ATTD of NSP and cellulose between RSM and SBM [
39]. Microbial fiber fermentation and metabolic activity might be reduced as RSM is more lignified and insoluble than SBM [
18]. This is reflected in the greater amount of IDF and iNSP in RSM-based diets of the current study and the lower SCFA concentration in jejunum, colon, and feces of RSM-fed pigs compared to SBM.
Surprisingly, the present values for colonic digestibility of NSP were higher than ATTD of NSP. In contrast, other studies show an increasing digestibility of NSP and AX in caudal direction [
16,
38]. However, the values of NSP digestibility were numerically in the same range, and furthermore, the determination of digestibility in feces is susceptible to interference. Bacteria may act as “non-dietary interfering substance” in NSP analytical procedures resulting in overestimated values of NSP concentration that are higher than the true diet derived NSP concentration [
41]. Considering the higher number of bacteria in feces compared to colonic digesta and that in pigs and humans bacterial mass represents 40–50% of fecal DM [
42,
43], the interfering effect would be more pronounced with respect to ATTD of NSP compared to colonic digestibility. Additionally, some bacteria produce exopolysaccharides such as colonic acid (
Enterobacteria) or dextran (
Lactobacillales), which might have contributed to increased fecal concentration of NSP [
44].
4.3. Bacterial Metabolites and Composition of the Microbiome
In the current study, compared to wheat, rye-fed pigs had a higher concentration of SCFA in the jejunum and colon. As shown in a study comparing the feeding of an AX-rich diet (65% rye-flakes) with a diet based on wheat flour, complex cereal polysaccharides such as AX are not degraded enzymatically in the small intestine and might therefore promote the growth of SCFA-producing bacteria in the distal parts of the intestine [
38]. Another study focused on the feeding of wheat and rye breads to pigs, demonstrating that increased SCFA-production in the small intestine might be related to the more soluble fraction of dietary fiber, which is readily fermentable, whilst insoluble fractions would be degraded more distally [
37]. Moreover, rye might cause a higher bacterial production of butyrate due to the structure of AX and the lower content of cellulose [
45]. Therefore, the high concentration of TDF and SDF in the rye-based diets of this study may have provided more substrate for growth of SCFA-producing bacteria. Positive effects of SCFA derived from fermentation of dietary fiber on gut and animal health have been widely reviewed, including the use of acetate as an energy source and butyrate as the main fuel for colonocytes and its efficiency against possible pathogens [
7,
46]. In the present study, only data on digesta concentration of SCFA are available, but SCFA are absorbed rapidly via the gut wall [
47]. Rye-derived SCFA might be of systemic use as peripheral blood concentration of SCFA was increased by an AX-rich diet based on rye-flakes compared to a wheat flour-based diet [
48]. Despite the increased colonic concentration of SCFA and especially butyrate in the current study, no changes of the microbial community were determined in colonic digesta. This might be related to cross-feeding of AX-degrading bacteria, e.g., between
Bifidobacteria and butyrate-producing bacteria, and to an increased abundance of the phosphotransferase system (kl02060) regulating carbohydrate uptake into bacterial cells [
22]. Nevertheless, it is likely that rye compared to wheat enhanced the production of bacterial metabolites by providing a higher amount of easier fermentable substrate without a shift in the microbial community.
Compared to SBM-fed pigs, concentration of SCFA was lower in RSM-fed pigs in the jejunum and tendentially in the colon which indicates a lower metabolic activity of the resident microbiota. In contrast, previous studies showed an equal level of SCFA between RSM and SBM [
24,
25,
28]. However, compared to these studies, the inclusion level of RSM and consequently the content of lignin and IDF was higher in the present study. A high degree of lignification may hamper enzymatic as well as microbial fiber degradation in RSM-fed pigs [
39]. In combination with the lower digestibility of NSP and DM, this could explain the lower SCFA compared to SBM-fed pigs. Additionally, SCFA might have been absorbed rapidly or used by other microbiota in the sense of cross-feeding [
49].
In accordance with a recent meta-analysis that identified
Firmicutes,
Proteobacteria, and
Bacteroidetes as the most abundant phyla of the core microbiome in pigs [
50],
Firmicutes and
Bacteroidetes were the predominant phyla of the pigs in the current study.
In rye-fed piglets of this study, composition of the microbial community was only different to wheat-fed pigs in the jejunum. This was unexpected, because according to other studies, the major impact of rye on the microbial community is expected in the proximal parts of the large intestine [
16,
23,
38]. A higher relative abundance of
Firmicutes in the small intestine might be explained by the higher content of sNSP in the rye-based diets, since more insoluble substrate would be degraded further distally [
7]. The phylum
Firmicutes contains many plant cell wall-degrading species including
Clostridium sensu stricto 1 and
Terrisporobacter. These two genera are known to ferment complex indigestible plant polysaccharides such as hemicellulose and cellulose [
51]. Therefore, the fiber provided by the rye-based diets might have served as a substrate and stimulated
Firmicutes’ proliferation.
Firmicutes contains many butyrate-producers [
52], therefore an increased abundance could be considered as a positive effect of the feeding of rye. However, SCFA are absorbed primarily in the caecum and proximal colon, and might not utilized by the host in the same extent in the jejunum [
47].
Proteobacteria had a lower jejunal relative abundance in rye-fed pigs of the current study. Many putative pathogens such as
E. coli and
Salmonella belong to
Proteobacteria and a lower presence of
Proteobacteria was associated with an increased intestinal barrier function and a higher anti-inflammatory capacity of the local immune system [
53].
In contrast to an increased concentration of SCFA in jejunal digesta, the analysis of microbial diversity in rye-fed pigs showed a lower richness compared to wheat. It is possible that the increased production of metabolites might be driven by only a few selected genera of Firmicutes which were more abundant in the rye-fed pigs.
With respect to effects of RSM on the relative abundance of microbiota compared to SBM, the present study resulted in a reduced abundance of
Firmicutes at the three sampling sites. As mentioned above,
Firmicutes contains many fiber-degrading species and is known to produce SCFA. The lower NSP and DM digestibility together in RSM-fed pigs compared to SBM indicate that the fiber provided by the RSM-based diets was not used as a suitable substrate for fermentation by microbiota, especially fiber-degrading
Firmicutes. Consequently, the metabolic activity and growth of microbiota was lower as illustrated by the lower concentration of SCFA and the lower relative abundance of
Firmicutes. In line with this, compared to alfalfa meal, wheat bran and pure cellulose containing a higher amount of IDF also reduced the relative abundance of
Firmicutes in large intestinal mucosa of suckling pigs [
54]. However, another study investigating the feeding of RSM instead of SBM showed an increase of
Firmicutes, although the lower inclusion level and lower content of IDF might have prevented an inverse result [
55]. A higher ratio of
Firmicutes:Bacteroidetes was associated with a reduction of the incidence of diarrhea and infections [
55]. Nonetheless, previously published results of the present study showed that the fecal score of the pigs was in a physiological range throughout the trial [
14]. Within
Firmicutes, the predominant genera
Clostridium sensu stricto 1 and
Terrisporobacter were also decreased by RSM in the current study. This might explain the reduced concentration of SCFA in RSM-fed pigs because another study showed a strong correlation between the relative abundance of these genera with the production of large amounts of metabolites from plant fiber [
51].
In line with another study investigating the feeding of RSM [
25], RSM increased the relative abundance of
Actinobacteria along the gastro-intestinal tract in the current study. In contrast, another study did not show differing values of abundance of
Actinobacteria in RSM- or SBM-fed pigs [
55].
Actinobacteria efficiently use hemicellulose and cellulose [
52], both mainly insoluble fiber fractions. Despite RSM and SBM containing similar amounts of cellulose [
18] the higher inclusion of RSM compared to SBM and the higher content of IDF in RSM-diets might have promoted growth of
Actinobacteria in RSM-fed pigs. In the jejunum,
Bifidobacterium, a genus belonging to
Actinobacteria, was also increased by the feeding of RSM.
Bifidobacterium is considered to improve gut health [
7,
56] and was increased by the feeding of cellulose in another study [
57].
As mentioned above, an increased relative abundance of
Proteobacteria may be a risk for animal health. Nevertheless, the pigs of both RSM and SBM groups were in a good condition throughout the trial.
Proteobacteria contains many proteolytic genera [
58]. RSM may lead to a lower AID of protein and amino acids than SBM [
59] which also was demonstrated in this study [
14]. However, there was no correlation detected between AID of crude protein and total amino acids and the jejunal relative abundance of
Proteobacteria. It is likely that the protein was not available as a substrate for growth of
Proteobacteria, because IDF in RSM caused an encapsulation in the rigid cell wall and an increased digesta passage rate [
39].
In RSM-fed pigs, the increased relative abundance of
Bacteroidetes is most likely related to the higher content of IDF than in SBM-based diets.
Bacteroidetes as the second most abundant phylum of the gut microbiota in pigs [
50] was also increased by resistant starch in humans [
60] and by corn bran in pigs [
61], both sources of IDF. In contrast, a study investigating the feeding of RSM instead of SBM to pigs showed a lower abundance of
Bacteroidetes which was related to the high pectin content [
25]. An increased abundance of
Bacteroidetes is associated with weight loss in humans, mice [
62], and pigs [
63]. The negative correlation between abundance of
Bacteroidetes and ADG in the last six days of the trial might suggest that the shift towards more colonic
Bacteroidetes was connected to the reduced weight gain in RSM-fed pigs.