The main point in the results reported in this manuscript is that inclusion of XOS plus AXRF improved growth efficiency of broiler chickens associated with a decrease in gut epithelial cell maturation and migration. The maintenance of a healthy gut epithelia accounts for roughly 20% of the energy and protein synthesis requirements [25, 35–39]. Several studies have reported the impact of dietary XOS supplementation on improving growth performance in chickens [14, 17, 40–44]. This positive effect has been associated with their capacity to modulate the gut microbiota, regulate the immune function, and enhance gut health [16, 19, 45, 46]. Intestinal tissue weights and protein synthesis were significantly increased by the presence of gut microbiota [47]. This is consistent with our findings showing that the supplementation of XOS/AXRF improved feed efficiency by 5% associated with reduced intestinal cell turnover suggesting a decreased energy requirement for the maintenance of the gut by about 15.7 kcal/chick/d.
Proteome analysis of the jejunum in XOS/AXRF supplemented chickens showed that DAP were related to reduced activity in biological pathways involved in cell metabolism, epithelial cell differentiation, and actin activity relevant to cell migration along the intestinal villus. These results are consistent with previous reports where dietary XOS shown a favourable impact on intestinal morphology and enhancement of the intestinal epithelial barrier function [48, 49]. The intestine has a high rate of cell renewal (including differentiation, maturation and migration of cells), which requires a large amount of energy (in the form of ATP) mainly obtained from glycolysis and mitochondrial oxidative phosphorylation [50, 51]. In the cell, the TCA cycle takes place in the mitochondria where pyruvate is oxidised leading to the production of electron donors and reducing factors utilised by the electron transport chain, hence driving ATP synthesis. An increase in mitochondrial ATP production has been associated with the increased energy demand of incremental cell migration and the promotion of wound repair [52, 53]. In contrast, our results indicate that XOS/AXRF supplementation of broiler diets reduced the ATP demand in jejunal epithelial cells, with lower abundance of critical proteins (e.g., pyruvate kinase) participating in the central metabolic pathways. In brief, it appears that XOS/AXRF fed birds have a lower need for ATP to maintain epithelial integrity compared to controls. Regarding cell differentiation, there was a reduced abundance of proteins like Annexin A4 (ANXA4), a calcium/phospholipid-binding protein that has been described to be upregulated upon cell differentiation and pathologic events in the intestine [54].
Cell migration is an active process critical for adequate cell turnover in the intestine, where cells move together and actin protrusions are directed towards the tip of the villus [55]. This process involves forces created by cell adhesions controlled by the assembly of actin to the cell membrane and regulated by the Rho family of small GTPases [56]. It is known that CDC42 and Rac1 proteins from the Rho proteins influence cell motility/migration due to its interaction with the cytoskeleton and the formation of protrusive structures [57, 58]. Healing requires migration of cells, which in turn requires the organization and enhanced activity of a number of cellular components including the cytoskeleton, a process known as intestinal restitution [59–61]. Cytoskeletal reorganisation is necessary for intestinal epithelial cell mobilisation involving the hydrolysis of ATP, which has been reported to account for almost 20% of the energy expenditure of the intestine [62, 63]. Based on this, XOS/AXRF fed chickens would utilise around 12.3 kcal/d in cytoskeleton dynamics compared to 12.8 kcal/d in non-supplemented chickens. Reorganization of the cytoskeleton involves the polymerisation and depolymerisation of actin in a reversible process known as treadmilling, where globular actin (G-actin) is added to the barbed-end of a filamentous actin (F-actin) and disassembled from the pointed-end of the filament [64]. In this study, several proteins involved in the formation and activity of microfilamentous structures, including gelsolin, an actin depolymerizing protein, and ARPC4, a subunit of the Arp2/3 complex that has an important role in polymerization of F-actin and reorganization of the cytoskeleton, showed lower abundance in XOS/AXRF fed birds [65–68]. The protein actin is the major constituent of the cytoskeleton, a crucial component regulating movement of epithelial cells [50]. The results of the study indicate that actin cytoskeletal reorganisation was reduced in XOS/AXRF chickens, while proteins like villin and non-muscular Myosin II, both participating in epithelial cell migration upon injury, were downregulated [61, 69–72]. In addition, this study showed decrease dynamics of actin in jejunal cells, including changes in polymerization, depolymerization, and turnover of the protein. This, in turn, indicates a reduction of cell migration along the villous-crypt axis in chickens that received XOS/AXRF in the diet (Fig. 3). To the best of our knowledge, this is the first time that dietary XOS supplementation has been associated with mechanisms relevant to cell mobilization.
A potential mechanism explaining the positive effect of XOS in gut health might be related to the production of SCFA [73]). It has been extensively described in the literature that XOS influence the microbiome and favours the production of SCFA [16, 48, 74, 75]. Studies in humans indicate that SCFA are involved in proliferation and differentiation of epithelial cells and have been described to influence epithelial cell migration relevant for cell restitution after injury [76–78]. According to Park [79], gram-positive bacteria are involved in the turnover of cells in the intestinal epithelium, an activity that is believed to be facilitated by SCFA. However, it is unclear that XOS-associated SCFA production in the chicken occurs in functional amounts in the jejunum. Thus, changes in XOS-associated microbial SCFA production and their possible effect relevant to intestinal epithelial cell turnover warrants further investigation.
Overall, XOS/AXRF supplementation leads to a phenotype which is consistent with an improved gut integrity and gut health requiring lower cell differentiation and migration. This improvement in intestinal health results in reduced energy requirements for cell differentiation and turnover, ultimately leading to more efficient growth.