1. Introduction
Combining different feedstuffs to formulate swine diets is essential for the supply of amino acids (AA), energy, and other nutrients necessary for optimal growth and nutrient utilization and production. While the main goal of diet formulation is meeting nutrient requirements, the impact different feedstuffs have on nutrient utilization (e.g., nitrogen retention) and intestinal physiology (e.g., gut health) of the pig also need to be considered. For instance, dietary components, such as dietary fibre (DF) and crude protein (CP), influence nutrient use and intestinal health. Mechanisms for these effects include reduced nutrient availability and microbial metabolite production [
1,
2] modulation of gut microbiome [
3], and changes in intestinal morphology [
4]. For example, DF can reduce nutrient digestibility [
5,
6] and increase endogenous AA losses [
7], resulting in greater threonine (Thr) requirement for protein deposition (PD) and growth [
2,
8,
9].
On the other hand, the inclusion of high-DF feedstuffs in swine diets can positively affect intestinal health, promoting the growth of beneficial bacteria and production of metabolites (e.g., short-chain fatty acids) [
10]. Feeding high-CP diets has been reported to negatively affect weanling pigs [
11] due to an increase in the amount of protein flow to the hind gut (i.e., fermentable protein (fCP)), promoting growth of pathogenic bacteria and production of harmful metabolites. Metabolites of protein fermentation (e.g., ammonia, amines, phenolic and indole compounds) have been associated with a negative impact on gut health and predisposition to post-weaning diarrhea following enterotoxigenic
E. coli challenge [
12]. Although feeding high DF increases endogenous Thr losses [
13] and increases dietary Thr requirement for growth and PD [
2,
9], the inclusion of DF in swine diets has been suggested as a potential mechanism to mitigate the negative effects of protein fermentation [
14] by providing gut microbes a preferential fermentation substrate. For example, inclusion of DF has been reported to reduce ammonia and putrescine levels in pigs fed a diet containing highly fermentable protein [
14]. The reduction in protein fermentation has been suggested to not only be dependent on the provision of alternative fermentable substrates, such as DF, but also the fibre type (e.g., soluble fibre vs. insoluble fibre) and site of fermentation [
15].
The objective of the present study was to investigate the interactive effects of DF and fCP on Thr requirement for PD and further elucidate how these factors affect markers of intestinal health. Based on the associated effects of fCP on the gut and impact on nutrient utilization, we hypothesized that feeding high DF and high fCP will interactively increase the Thr requirement for PD and to maintain intestinal health and barrier function.
4. Discussion
Understanding the effects of dietary components, such as DF and protein, on nutrient requirements and animal health is key to development of dietary strategies to optimize nutrient utilization, growth, and health of growing pigs. Therefore, in the present study we evaluated the independent and interactive effects of DF and fCP on the Thr requirement for protein deposition, largely because Thr has been shown to be important in maintaining gut health and barrier function in pigs [
23]. We further evaluated the impact of DF and fCP content on fermentation characteristics and gut health in the pig hindgut. Based largely on results of in vitro studies, protein fermentation products, such as ammonia and BCFA, have been shown to have detrimental effects on the intestinal epithelium largely, with fewer in vivo studies confirming this [
24]. As such, dietary strategies aimed at reducing protein fermentation metabolites are thought to improve intestinal health. For example, previous work in pigs showed that when DF is included in high protein diets, protein fermentation and production of harmful metabolites is reduced and there is increased production of beneficial metabolites, such as acetic acid, butyric acid, and propionic acid [
14]. Moreover, highly fermentable sources of DF, such as sugar beet pulp, can provide intestinal microbes an alternative, and potentially preferred, source of substrate for fermentation, allowing for the incorporation of N into the microbial biomass, further reducing the concentration of harmful fermentation metabolites (e.g., BCFA, ammonia) [
25,
26].
To achieve our objective, diets were formulated to vary in DF and fCP content in addition to graded levels of Thr to allow for determination of the effects of diet components on Thr requirement for PD. In the current study, dietary inclusion of fibre and fibre type as well as protein content were based on previous studies examining the impact of DF and fCP on gut health and animal performance [
2,
9,
14]. To achieve the HF diet, we included sugar beet pulp and wheat bran at the expense of corn, a low fibre ingredient. This contrasts with Pieper et al. [
14] who added fibre at the expense of wheat, a high fibre ingredient, which resulted in increased insoluble DF content but decreased soluble DF. Our strategy to replace corn in the HF diet with sugar beet pulp and wheat bran ensured that the HF diets had greater levels of both soluble and insoluble DF, which we have reported previously [
2,
9]. Likewise, while dietary inclusion of soybean meal and dietary protein content was based on Pieper et al. [
14], adjustments were made to target values to ensure that the LfCP diet would meet all essential AA requirements and would not limit growth due to AA deficiencies. In the current study, adjustments in ingredient content were made to achieve treatment objectives in DF and fCP content while maintaining similar nutrient content across diets that met or exceeded nutrient requirements, such as energy, amino acids, and protein according to NRC [
16]. Inevitably, the choice to use typical feed ingredients, rather than purified diets, to achieve the DF and fCP targets resulted in some unavoidable differences in nutrient content. This is a common feature in nutrition studies, and especially in those examining different DF and fCP content [
2,
9,
14,
27]. Moreover, differences in calculated vs. analyzed nutrient content are not unexpected and are largely due to variation in ingredient nutrient content, diet preparation, and laboratory analysis. Results of the current study should be interpreted given these limitations in diet formulation and maintaining consistent nutrient content across diets when varying DF and fCP.
In the current study, the inclusion of high fCP in the diet increased ammonia concentration, whereas the inclusion of DF reduced ammonia concentration in the cecal and colonic digesta. This observation agrees with Bikker et al. [
28], who reported that ileal and colonic ammonia concentration was higher in the digesta of pigs fed a high protein diet, but when DF (i.e., sugar beet pulp and wheat middlings) was added, ammonia concentration in the digesta was reduced. Other studies have reported similar effects of high protein diets on digesta ammonia and other biogenic amine (putrescine, spermidine, spermine etc.) concentrations in the gut and the counteracting effect of the addition of a high fibre source [
1,
25]. Digesta SCFA and BCFA are considered key indicators of DF and protein fermentation in the gut and are generally considered to have positive and negative impacts on gut health, respectively. In the present study, we observed that high DF did not exert any significant independent effects on the concentration of fibre fermentation metabolites (e.g., acetic acid, butyric acid, and propionic acid) in the gut; however, the effects of fibre were evident in reducing the concentrations of BCFA (e.g., isobutyrate and isovalerate). The HfCP diet increased the concentration of BCFA in the cecal digesta while the inclusion of high DF generally reduced digesta BCFA concentration. The observations in the current study largely confirm a previous study indicating the role of DF as an alternate substrate for fermentation in high protein diets [
26].
Under normal health conditions, organisms are protected against oxidation of tissues through antioxidant systems [
28]. In situations of antioxidant deficiency, concentrations of oxidants are high, and tissues undergo oxidative stress [
28]. Fermentation metabolites from DF and protein substrates (e.g., ammonia and SCFA) can increase oxidative stress in pigs and can have negative consequences on intestinal health and function [
29]. The increase in serum antioxidant capacity with increasing dietary Thr observed in the current study is consistent with Azzam et al. [
30], who demonstrated that increasing dietary Thr increased serum antioxidant capacity in laying hens. This indicates that, while antioxidant defense systems may not be impacted by DF and fCP, dietary Thr may be a limiting factor for optimal antioxidant capacity. Gene expression analysis showed significant treatment effects on target genes only in the colon. In the colonic tissue, feeding the HfCP diet reduced the expression of MUC2 gene, with no effect on MUC5ac, while feeding high DF increased the expression of both MUC2 and MUC5ac genes, which agrees with previous reports [
14,
31,
32]. Expression of MUC2 was previously shown to be correlated with mucin secretion [
31] which increases Thr requirements for protein deposition [
2]. Therefore, this observation provides further support for the increase and decrease in Thr requirement for PD observed in the current study with high DF and high fCP diets, respectively. Reduced mucin secretion, and the associated barrier function, in high fCP diets may indicate a potential mechanism for greater incidence of pathogen-associated diarrhea in weanling pigs. High protein diets have been reported previously to negatively affect tight junction protein assembly and epithelial transport in pigs [
33]. We observed in the present study that, feeding HF diet increased tight junction protein ZO-1expression in the LfCP but not the HfCP diet, which is indicative of the effects of high protein diets on tight junction assembly. Again, with a significant fibre × Thr interaction, we observed that ZO-1 expression increased as Thr increased in the LF, but not the HF fed pigs, indicating that the impact of Thr on ZO-1 expression is dependent on fibre level in the diet. On the other hand, the fCP × Thr interaction observed suggests that increasing Thr increased ZO-1 expression in the HfCP fed pigs but not in the LfCP fed pigs. These observations further prove that Thr utilization could be conserved and prioritized for maintaining intestinal integrity and function [
22,
34].
Previous studies have reported a reduced efficiency of utilizing dietary Thr for PD with increasing DF content, thereby increasing the dietary Thr requirement growth [
2,
8,
9]. This effect is largely attributed to increased endogenous protein/AA losses [
7,
13,
32]. In the present study, we observed that the HF diet reduced ATTD of N, which is consistent with reports from previous studies [
2,
8], but no significant effect of fCP was observed. Again, HF diet increased fecal N output and reduced urinary N output, which agrees with previous studies demonstrating a shift in N output from urine to feces which may be due to increased incorporation of N into microbial biomass [
35]. The interactive association between DF and fCP on PD indicates that HF increased PD in the LfCP-fed but not the HfCP fed pigs. This observation is quite novel, as previous work has shown that feeding high DF will reduce growth performance when dietary Thr level is lower than requirement [
8,
9]. The negative effects of feeding high DF may be more evident when there is high fCP in the diet, as observed in the present study.
Within the LfCP fed pigs, HF increased PD compared to the LF fed pigs (165.4 vs. 154.7 g/d); however, in the HfCP group, HF did not increase PD compared to LF (173 vs. 176 g/d). This observation may suggest a possible negative physiological impact of combining HF and HfCP on the gut. Furthermore, when estimating the Thr requirement for maximum PD, we observed that, within the LfCP diet, feeding HF increased the Thr requirement for PD (0.73%, SID) compared to the LF diet (0.68%, SID). The same effect of DF was observed in HfCP-fed pigs, where inclusion of HF increased (0.64% SID) Thr requirement estimate for PD, compared to the estimate of 0.60% SID when fed an LF diet. However, while feeding high DF consistently increased the Thr requirement for PD, feeding a high fCP diet reduced the Thr requirement for PD. This observation agrees with previous studies which demonstrated an increased Thr requirement for PD [
2], and growth performance [
8,
9] with the inclusion of high DF in the diet; however, the reduced Thr requirement in HfCP-fed pigs was unexpected. Previous studies have demonstrated the negative effects of high fCP on the intestinal epithelium and the subsequent effect on growth and nutrient metabolism [
15,
36]. It was our expectation that the negative effects of HfCP, as indicated by the parameters measured in the current study, would increase Thr requirements for PD; however, this was not the case. It is possible that the HfCP diet contained more available Thr than the LfCP diet; however, analysis of the diets showed no differences in total or SID dietary Thr across diets. Another possible reason for this observation could be the ages of pigs (>25 kg) used in the present study, which deviate from previous studies examining the impact of high protein diets. In the majority of these studies, these effects are studied in the immediate post-weaning period, during which pigs may be more susceptible to the negative effects of high protein diets on gut health (e.g., increased post-weaning diarrhea) [
11,
12]. Finally, it is also possible that the primary effect of fCP is not on dietary Thr and instead on another dietary factor, such as another AA, and, therefore, dietary Thr was not first limiting in the HfCP diets used in the current study. Overall, the HfCP diet showed negative effects on the gut; however, it did not result in increased dietary Thr requirement for PD, and the reason for this remains unclear.