Microbial recognition and signaling in intestinal epithelial cells

The intestinal epithelial cells (IEC) form a dynamic barrier between the host and the external environment (Peterson and Artis Reference Peterson and Artis2014). The composition and activity of the intestinal microbiota significantly influences IEC function through constant biochemical and molecular crosstalk (Kaur et al. Reference Kaur, Ali and Yan2022; Zhou et al. Reference Zhou, Yuan and Yang2022a). This complex interaction is shaped in part by the structural and biochemical diversity of intestinal bacteria, which differ in cell wall composition and the metabolites they produce (Yin et al. Reference Yin, Wang and Dai2023). Generally, gram-positive bacteria have a thick peptidoglycan (PGN) layer and lack an outer membrane (Masschalck et al. Reference Masschalck, Deckers and Michiels2002). In contrast, gram-negative bacteria contain a relatively thin PGN layer enclosed by an outer membrane containing lipopolysaccharides (LPS) that provide structural protection (Gauthier et al. Reference Gauthier, Rotjan and Kagan2022). These structural differences influence how bacteria interact with the intestinal environment, and both groups include commensal and potentially pathogenic members.

The PGN-derived microbial-associated molecular pattern (MAMP), γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), is more abundant in the PGN of gram-negative bacteria, but can be present in some gram-positive species (Wang et al. Reference Wang, Li and Han2023a). In contrast, muramyl dipeptide (MDP) is broadly present in both gram-positive and gram-negative bacteria (Cañas et al. Reference Cañas, Fábrega and Giménez2018). The IEC utilize pattern recognition receptors (PRRs) to detect and respond to these MAMPs (Chamaillard et al. Reference Chamaillard, Hashimoto and Horie2003; Watanabe et al. Reference Watanabe, Asano and Fichtner-Feigl2010). The PRRs recognize MAMP and endogenous alarm signals, damage-associated molecular patterns (DAMPs), allowing for rapid responses without the need for prior antigen exposure (Kawai and Akira Reference Kawai and Akira2010; Zhang and Liang Reference Zhang and Liang2016). Activation of PRRs initiates signaling cascades that regulate cytokine production, antimicrobial peptide secretion and oxidative responses (Menendez et al. Reference Menendez, Willing and Montero2013; van der Post et al. Reference van der Post, Birchenough and Held2021). Signaling by PRRs does not exclusively promote pro-inflammatory responses and can also drive the expression of anti-inflammatory mediators and immune tolerance, support epithelial integrity and prevent chronic inflammation (Hayashi et al. Reference Hayashi, Sato and Kamada2013; Latorre et al. Reference Latorre, Layunta and Grasa2018; Zhou et al. Reference Zhou, Cao and Fang2015). Among the PRRs of the intestine are Toll-like receptors (TLRs), located on the plasma membrane and within endosomal compartments (Yu et al. Reference Yu, Nie and Knowles2014) and nucleotide-binding oligomerization domain NOD-like receptors (NLRs), which reside in the cytoplasm (Chamaillard et al. Reference Chamaillard, Hashimoto and Horie2003). Some of the primary pro- and anti-inflammatory signaling pathways of the IEC are summarized in Figure 1.

Figure 1.

Signaling pathways of microbe-associated molecular patterns and metabolites in intestinal epithelial cells modulating immune responses and oxidative balance in small intestine of nursery pigs. Microbial components and metabolites interact with intestinal epithelial cells to shape pro-inflammatory (left cell) and anti-inflammatory (right cell) signaling and immune responses and influence epithelial barrier integrity and cellular redox homeostasis.

Microbiota-driven regulation of intestinal stem cell function

In addition to influencing the function of IIC, the intestinal microbiota and microbial metabolites also influence the activity of intestinal stem cells (ISC; Fig. 2). The absorptive cells of the small intestine undergo rapid turnover that is driven by ISC located at the base of the crypts (Gehart and Clevers Reference Gehart and Clevers2019). Although ISC are relatively protected from direct contact with the microbiota due to their location in the intestinal tissue as compared with IEC, they remain responsive to direct microbial interactions or from interactions between microbes and other host cells to exert downstream effects on ISC. When it comes to direct microbial interactions, the response of ISC is very similar to that of IEC, following the previously outlined pro- or anti-inflammatory signaling pathways. For example, activation of TLR4 on the surface of ISC can suppress cell turnover and proliferation (Yi et al. Reference Yi, Patel and Sodhi2012), whereas NOD2 signaling, enhances stem cell survival and supports intestinal regeneration (Nigro et al. Reference Nigro, Rossi and Commere2014).

Figure 2.

Regulation of intestinal stem cell signaling and renewal. Microbial components, microbial metabolites and host-derived signals interact with the intestinal stem cells within the crypt to influence intestinal stem cell activity, contributing to epithelial renewal and structural maintenance of the intestinal tissue.

In particular, ISC are highly responsive to SCFA (Luceri et al. Reference Luceri, Femia and Fazi2016; Uchiyama et al. Reference Uchiyama, Sakiyama and Hasebe2016; Wang et al. Reference Wang, Lee and Campbell2020a). Select commensal microbes produce SCFA that stimulate ISC proliferation and differentiation by increasing the expression of ligands of the wingless-related integration site (Wnt)/β-catenin signaling pathway (Lee et al. Reference Lee, Kim and Kim2018; Wu et al. Reference Wu, Xie and Miao2020; Xie et al. Reference Xie, Li and Dai2022). The ISC respond to the binding of Wnt ligands to their receptor to promote the expression of genes such as cellular myelocytomatosis oncogene (c-Myc), cyclin D1 and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), which drive ISC proliferation and epithelial renewal (Clevers and Nusse Reference Clevers and Nusse2012; Zhu et al. Reference Zhu, Wen and Sheng2018). Microbial metabolites and dietary nutrients also modulate the mammalian target of rapamycin (mTOR) pathway, a central signaling pathway related to nutrient sensing and growth (Park et al. Reference Park, Kim and Kang2015). Growth factors and pro-inflammatory cytokines further activate the mTOR signaling pathway, which can reinforce mechanisms of survival, differentiation and regenerative capacity, both in conditions of nutrient surplus and stress (Feng and Levine Reference Feng and Levine2010; Floyd et al. Reference Floyd, Favre and Lasorsa2007; Magagnin et al. Reference Magagnin, van den Beucken and Sergeant2008; Yang et al. Reference Yang, Xiong and Wang2016). Together, these findings highlight that the microbiota-derived metabolites and microbial recognition pathways influence the activity of ISC. Thus, the microbiota not only shapes immune and metabolic processes, but also serves as a key regulator of epithelial regeneration and homeostasis.

Protein and amino acids

Dietary protein is critical for growth, but diets for nursery pigs should prioritize highly digestible sources as protease production is inadequate at this age (Makkink et al. Reference Makkink, Berntsen and op den Kamp1994; Zhang and Piao Reference Zhang and Piao2022). Highly digestible protein sources, including enzyme-treated soybean meal and spray-dried porcine plasma, have been shown to enhance intestinal immune function and maintain epithelial integrity, thereby supporting optimal growth performance (Long et al. Reference Long, Ma and Piao2021; Tran et al. Reference Tran, Bundy and Li2014). In contrast, incomplete digestion of dietary protein allows undigested residues to accumulate in the intestinal lumen, where they become substrates for proteolytic bacteria, including members of Pseudomonadota (Heo et al. Reference Heo, Kim and Hansen2009; Li et al. Reference Li, Ding and Zhu2022; Pluske et al. Reference Pluske, Pethick and Hopwood2002). Many of the harmful metabolites produced by the fermentation of protein, including ammonia, putrescine and cadaverine, trigger inflammation and oxidative stress (Huang et al. Reference Huang, Mo and Jin2022; Li et al. Reference Li, Ding and Zhu2022; Wen et al. Reference Wen, Wang and Zheng2018). Even excessive provision of highly digestible protein sources can exceed the digestive capacity of the gastrointestinal tract. A previous study demonstrated that high levels of dietary crude protein derived from milk protein can negatively influence intestinal health by increasing inflammation, thereby damaging intestinal villi and limiting their absorptive capacity (Gao et al. Reference Gao, Yin and Xu2020). Fortunately, the use of crystalline amino acids, which are virtually 100% digestible, can reduce the crude protein level and still meet the amino acid requirement of the diet (Duarte et al. Reference Duarte, Parnsen and Zhang2024; Lee et al. Reference Lee, González-Vega and Htoo2022; Zhao et al. Reference Zhao, Weaver and Fellner2014).

The use of specific amino acids, including glutamine, glutamate, threonine and arginine, can be considered due to their role in regulating key metabolic pathways that improve intestinal health (Kim et al. Reference Kim, Mateo and Yin2007; Li et al. Reference Li, Sui and Gao2016; Zhang et al. Reference Zhang, Chen and Li2019a; Zheng et al. Reference Zheng, Song and Tian2018). Glutamine and glutamate are major energy substrates for enterocytes of nursery pigs (He and Wu Reference He and Wu2022). Supplementation with 1% L-glutamine in low-protein diets modulated the intestinal microbiota, increasing the abundance of beneficial bacteria such as Lactobacillus and reducing that of potentially pathogenic Streptococcus, in the feces of nursery pigs (Li et al. Reference Li, Bai and Yang2024). The downstream effects of these changes to the intestinal microbiota could be observed by a decrease in serum inflammatory markers, increased antioxidant capacity and improved growth performance. Threonine is an amino acid essential for mucin synthesis by intestinal goblet cells. When threonine is limited, the body prioritizes its use for mucin production over other protein synthesis processes, and therefore, supplementing threonine above the nutritional requirements could potentially benefit intestinal health (Munasinghe et al. Reference Munasinghe, Robinson and Harding2017). A study by Koo et al. (Reference Koo, Choi and Yang2020) investigated the effects of L-threonine supplemented at 115% of the nutritional requirement. Although the intestinal microbiota was not directly measured in this study, it could be inferred that the composition of the microbiota was positively influenced due to the reduced production of ammonia in the diets containing L-threonine above the requirement, in the digesta of nursery pigs fed corn-soybean meal-based diets (Koo et al. Reference Koo, Choi and Yang2020). The hypothesized changes to microbial composition and metabolite production are further supported by an observed decrease in the gene expression of inflammatory cytokines and positive changes to intestinal integrity, when L-threonine was supplemented at 115% of the requirement. Arginine plays a role in supporting the intestinal immune and antioxidant defense systems and promoting vascular development, which enhances nutrient absorption in the small intestine (Zhan et al. Reference Zhan, Ou and Piao2008). It is important to note that the benefits of arginine on intestinal health are primarily related to the direct role of arginine on the IEC and IIC, rather than on the microbiota. For example, supplementation of L-arginine at up to 1% of the diet was shown to improve the intestinal immune response in pigs challenged with LPS, as evidenced by increased numbers of intraepithelial lymphocytes, T cells and immunoglobulin production, and decreased Peyer’s patch apoptosis (Zhu et al. Reference Zhu, Liu and Xie2013). Similar outcomes were reported by Liu et al. (Reference Liu, Huang and Hou2008), who found that 1% arginine supplementation reduced the production of inflammatory cytokines in nursery pigs challenged with LPS, potentially through inhibition of downstream NF-κB signaling.

Non-starch polysaccharides

Because pigs lack endogenous enzymes to degrade non-starch polysaccharides (NSP), and their presence can reduce the digestibility of other nutrients, NSP inclusion should be limited, especially in diets of nursery pigs (Bach Knudsen and Hansen Reference Bach Knudsen and Hansen1991; Baker et al. Reference Baker, Deng and Gormley2025; Gutierrez et al. Reference Gutierrez, Serão and Kerr2014). The impact of dietary NSP on the microbiota and overall intestinal health is primarily determined by their physicochemical properties, and they can be generally classified as soluble or insoluble (Chen et al. Reference Chen, Chen and Tian2020b). Soluble NSP have an increased water holding capacity, forming gels that slow digesta passage, reduce nutrient digestibility, therefore increasing the opportunity for fermentation by intestinal bacteria (Choi et al. Reference Choi, Garavito-Duarte and Pasquali2024; McDonald et al. Reference McDonald, Pethick and Mullan2001). In contrast, insoluble NSP have a decreased water holding capacity compared to soluble NSP, and therefore, have an increased passage rate, decreasing bacterial fermentation (Choi and Kim Reference Choi and Kim2023; Huang et al. Reference Huang, Su and Li2015; Molist et al. Reference Molist, van Oostrum and Pérez2014).

Despite the potential negative effects of NSP on nutrient digestibility, the use of NSP may have some beneficial effects, particularly serving as a substrate for bacterial fermentation and the production of beneficial metabolites. A greater amount of NSP fermentation occurs in the large intestine, due to the increased retention time, compared to the small intestine (Metzler-Zebeli et al. Reference Metzler-Zebeli, Hooda and Zijlstra2010; Molist et al. Reference Molist, de Segura and Gasa2009). However, there is some fermentation of primarily soluble NSP that occurs in the small intestine (Baker et al. Reference Baker, Deng and Gormley2025; Ivarsson et al. Reference Ivarsson, Roos and Liu2014; Jha et al. Reference Jha, Rossnagel and Pieper2010). This fermentation could benefit intestinal health through the production of SCFA (Högberg et al. Reference Högberg, Lindberg and Leser2004; Owusu-Asiedu et al. Reference Owusu-Asiedu, Patience and Laarveld2006). As previously described, the presence of SCFA in the small intestine can provide beneficial effects such as reducing inflammatory signaling and maintaining TJ integrity, and these effects can be seen through with the combined use of soluble and insoluble NSP fractions (Chen et al. Reference Chen, Chen and Tian2020b; Jha et al. Reference Jha, Rossnagel and Pieper2010; Molist et al. Reference Molist, Hermes and de Segura2011). The microbial compositions can also adapt to the presence of different dietary NSP, with increased populations of NSP-degrading bacteria contributing to greater NSP utilization at the end of the digestive tract (Ellner et al. Reference Ellner, Wessels and Zentek2022; Niu et al. Reference Niu, Pu and Fan2022). Interestingly, although a faster passage rate may seem counterintuitive for nutrient utilization, insoluble NSP can promote pathogen clearance and reduce inflammation in pigs challenged with enteric pathogens (Molist et al. Reference Molist, Gómez de Segura and Pérez2010). Although less relevant in nursery diets compared to other life stages due to the significant influence of NSP on nutrient digestibility, the targeted use of NSP could benefit intestinal health of nursery pigs, primarily due to its influence on the intestinal microbiota, resulting in the production of beneficial metabolites.

Vitamins and minerals

Although vitamins and minerals are included to first fulfill nutritional requirements, the use of select vitamins and minerals beyond their nutritional requirement can influence the intestinal microbiota and have direct roles in maintaining intestinal health from a physiological standpoint. To date, vitamins have primarily been utilized for their direct influence on intestinal function and antioxidant capacity and less is known about their interactions with the intestinal microbiota, in the context of nursery pigs. Vitamin A has been shown to enhance transforming growth factor-beta (TGF-β) signaling, which promotes wound healing, in porcine ileum tissue (Yuen and Stratford Reference Yuen and Stratford2004). This is further supported by increased activity of small intestinal stem cells and improved structural development in weaned pigs supplemented with vitamin A (Wang et al. Reference Wang, Li and Wang2020). Vitamin D has context-dependent effects on the immune system, in some cases promoting tolerance, but supporting the production of immunoglobulins and antioxidative enzymes, thereby enhancing immune function and antioxidant capacity under stress conditions (Konowalchuk et al. Reference Konowalchuk, Rieger and Kiemele2013; Zhang et al. Reference Zhang, Liu and Piao2020). Uniquely, vitamin D has been shown to have a direct effect on the microbiota of weaned pigs, increasing the fecal abundance of beneficial Lachnospiraceae whereas reducing the abundance of harmful Streptococcaceae (Zhang et al. Reference Zhang, Yang and Piao2021). Research conducted in other species suggests that vitamins exert direct effects on the intestinal microbiota, however, this relationship remains poorly characterized in pigs.

Compared to vitamins, the direct effect of minerals on the intestinal microbiota is more well defined. Zinc oxide has long been used at pharmacological levels to prevent post-weaning diarrhea in nursery pigs, although concerns about antimicrobial resistance and the environment have caused the greater pig industry to seek suitable alternatives (Bonetti et al. Reference Bonetti, Tugnoli and Piva2021). Zinc oxide has strong antibacterial and bactericidal function and therefore proposed alternatives should also either reduce the proliferation of harmful bacterial populations or prevent their growth through enhancing beneficial populations for competitive exclusion (Mendes et al. Reference Mendes, Dilarri and Forsan2022; Ng’ang’a et al. Reference Ng’ang’a, Tous and Ballester2025). One potential replacement could be zinc chelates, which have greater bioavailability than zinc oxide and may allow for reduced dietary zinc inclusion, thereby decreasing environmental excretion and maintain intestinal zinc concentrations associated with beneficial effects (Jang et al. Reference Jang, Moita and Martinez2023; Mazzoni et al. Reference Mazzoni, Merialdi and Sarli2010). When under challenge with an enteric pathogen, 400 mg/kg of zinc glycinate, an organic form of zinc, was able to reduce populations of pathogenic bacteria, potentially due to the increased availability of zinc ions for use in the formation of antibacterial proteins (Jang et al. Reference Jang, Moita and Martinez2023). Copper is another mineral that can be used due to its antimicrobial effects and its role in redox responses. Copper ions act as an antimicrobial by stealing electrons from the bacterial cell wall and disrupting their function (Højberg et al. Reference Højberg, Canibe and Poulsen2005; Salah et al. Reference Salah, Parkin and Allan2021). Copper also exhibits beneficial effects on the intestinal function by serving as co-factor for antioxidant enzymes and improving the overall redox balance in the intestine (Jiao et al. Reference Jiao, Zhang and Wu2018). The direct effects of copper on the intestinal microbiota have been observed in previous studies, where the supplemental use of copper markedly reduced the total bacteria in feces (Højberg et al. Reference Højberg, Canibe and Poulsen2005; Miller et al. Reference Miller, James and Smith1986; Villagómez-Estrada et al. Reference Villagómez-Estrada, Pérez and Darwich2020). Although copper has been shown to reduce total bacteria in feces, its overall effects on microbial diversity remain unclear. Thus, copper supplementation should be approached cautiously due to concerns about microbial resistance, environmental accumulation and the loss of beneficial microbes from its broad antibacterial activity (Agga et al. Reference Agga, Scott and Amachawadi2014; Dai et al. Reference Dai, Yang and Yuan2020; Zhang et al. Reference Zhang, Zhou and Dong2019b). Selenium is yet another trace mineral that can be supplemented to beneficially modulate the intestinal microbiota (Conway et al. Reference Conway, Sweeney and Dowley2022). More importantly, selenium is required for the synthesis of selenoproteins, which play a crucial role in maintaining intestinal redox homeostasis (Kasaikina et al. Reference Kasaikina, Kravtsova and Lee2011). Selenium is often supplemented in combination with vitamin E or other functional additives to further support the antioxidant defense system, thereby contributing to mucosal redox homeostasis and assisting in maintenance of the beneficial members of the intestinal microbiota (Liu et al. Reference Liu, Cottrell and Furness2016; Wang et al. Reference Wang, Zhang and Chen2022).

Feed additives

Feed additives are recognized as tools to support the intestinal microbiota, intestinal health and growth performance of nursery pigs (Zheng et al. Reference Zheng, Duarte and Sevarolli Loftus2021). With the global trend to reduce antibiotic growth promoters and pharmacological levels of zinc oxide, functional feed additives have become increasingly relevant (McAlpine et al. Reference McAlpine, O’Shea and Varley2012; Satessa et al. Reference Satessa, Kjeldsen and Mansouryar2020). These feed additives are not included to satisfy nutritional requirements, but rather to influence the intestinal microbiota to confer beneficial effects on overall intestinal health (Yin et al. Reference Yin, Wang and Dai2023).

Phytobiotics are feed additives made up of plant-derived compounds with diverse bioactive properties. Their biological effects depend on their chemical composition and structure and they may exert antimicrobial, antioxidant, or anti-inflammatory effects within the intestine as reviewed by Garavito-Duarte et al. (Reference Garavito-Duarte, Deng and Kim2025a). The antimicrobial properties of phytobiotics are primarily attributed to their ability to disrupt the cell membrane and induce oxidative stress within bacteria, inhibiting their proliferation (Lopez-Romero et al. Reference Lopez-Romero, González-Ríos and Borges2015; Pei et al. Reference Pei, Zhou and Ji2009). Previous studies have demonstrated that phytobiotics can be strategically utilized to support the microbiota during times of stress, such as at weaning or under disease pressure. For example, herb- and essential oil-based phytobiotics reduced harmful bacteria in the jejunal mucosa of Escherichia coli challenged nursery pigs, thereby supporting redox balance and epithelial regeneration to maintain intestinal integrity (Garavito-Duarte et al. Reference Garavito-Duarte, Duarte and Kim2025b). Additional studies support these findings, showing that phytobiotics can promote the growth of lactic acid bacteria, which competitively exclude opportunistic pathogens and enhance intestinal health of pigs post-weaning (Duarte and Kim Reference Duarte and Kim2022b; Juhász et al. Reference Juhász, Molnár-Nagy and Bata2023; Moita et al. Reference Moita, Duarte and da Silva2021). This positive modulation of the intestinal microbiota reduces downstream activation of inflammatory pathways, as evidenced by reduced production of inflammatory cytokines and oxidative damage products, and by maintenance of intestinal morphology in pigs fed diets supplemented with phytobiotics (Duarte and Kim Reference Duarte and Kim2022b; Moita et al. Reference Moita, Duarte and da Silva2021).

Organic acids including lactic, benzoic and butyric acids have been widely used in pig diets to decrease intestinal pH, inhibit pathogen proliferation by disrupting cellular metabolism, and improve nutrient digestibility and redox balance (Choi et al. Reference Choi, Chen and Longo2023; Long et al. Reference Long, Xu and Pan2018; Park et al. Reference Park, Sun and Wongchanla2025). Consistent with these functions, mixed organic acids containing formic, acetic and propionic acids have been shown to reduce the prevalence of pathogenic E. coli and diarrhea incidence in nursery pigs (Long et al. Reference Long, Xu and Pan2018). Similarly, in pigs challenged with E. coli, formic and propionic acid attenuated inflammatory immune response and this effect can potentially be attributed to decreased proliferation of pathogenic bacteria (Ren et al. Reference Ren, Zhou and Guan2019). Additionally, dietary supplementation with sodium butyrate combined with benzoic acid enhanced the intestinal microbiota by increasing proliferation of beneficial bacteria and reducing that of potentially harmful bacteria (Wei et al. Reference Wei, Bottoms and Stein2021). Aside from direct antimicrobial effects, a blend of several organic acids improved the intestinal antioxidant capacity and intestinal barrier integrity, yielding similar results to antibiotics when it came to managing weaning stress (Cai et al. Reference Cai, Zhao and Chen2024). Together, these effects highlight the role of organic acids in improving intestinal health through targeted reduction of pathogenic bacterial populations and direct influence on the intestinal epithelium.

Exogenous enzymes, such as phytase, xylanase and mannanase, modulate the intestinal microbiota primarily by altering substrate availability for microbial fermentation. By degrading otherwise indigestible NSP, these enzymes reduce the availability of fermentable substrates that support the proliferation of opportunistic and pathogenic bacteria, thereby shifting microbial populations towards a more favorable composition (Chen et al. Reference Chen, Zhang and Kim2020a; Jo et al. Reference Jo, Ingale and Kim2012). For example, phytase supplementation decreased the relative abundance of Prevotella, a diverse genus of gram-negative bacteria that are associated with both positive and negative health outcomes, but that could potentially compete with beneficial microbes for mucosal binding sites in the jejunum of nursery pigs (Duarte and Kim Reference Duarte and Kim2022a; Moita and Kim Reference Moita and Kim2023). Because many gram-negative bacteria can activate inflammatory signaling pathways, reductions in their relative abundance are often accompanied by reductions in the concentration of TNF-α and MDA in the small intestine, as was observed in this study (Moita and Kim Reference Moita and Kim2023). Similarly, supplementation with xylanase and β-glucanase limited the growth of pathogenic bacteria, such as Helicobacter rappini by reducing digesta viscosity and increasing passage rate (Choi et al. Reference Choi, Garavito-Duarte and Pasquali2024). In contrast, the reduction in substrate availability can also limit the growth of beneficial bacterial populations. A previous study utilizing a multi-enzyme xylanase and carbohydrase blend in weaned pigs fed diets high in NSP found decreased populations of Lactobacillus in the colon compared to diets without exogenous enzymes (Li et al. Reference Li, Schmitz-Esser and Loving2019). This result is likely due to substrate degradation earlier in the gastrointestinal tract, indicating that diet composition, enzyme characteristics and desired physiological outcome should be considered prior to utilizing exogenous enzymes in a feeding program.

Prebiotics, probiotics and postbiotics are commonly used to support the intestinal microbiota to exert specific effects on microbial composition and intestinal health. Prebiotics are indigestible compounds that are selectively utilized by beneficial bacteria to enhance their growth and activity (Hill et al. Reference Hill, Guarner and Reid2014). Of note, galacto-oligosaccharides, like those found in sow milk, have been shown to support the development of a functional microbiota and intestinal structure in young pigs (Alizadeh et al. Reference Alizadeh, Akbari and Difilippo2015). Fructo-oligosaccharides have a similar effect, as they increased the proliferation of beneficial bacteria, which positively influenced small intestinal structure and barrier integrity by increasing the expression of TJ proteins and overall antioxidant activity (Zhang et al. Reference Zhang, Zhang and Zhang2022b). In contrast, probiotics consist of live microorganisms, commonly Lactobacillus, Bacillus, or Enterococcus species, that directly modulate microbial composition through competitive exclusion of pathogens and establishment of beneficial populations (Hill et al. Reference Hill, Guarner and Reid2014; Pan et al. Reference Pan, Zhao and Ma2017). Supplementation with Bacillus licheniformis increased populations of beneficial bacteria such as Lactobacillus, which was accompanied by improvements in small intestinal morphology, including increased villus height (Sun et al. Reference Sun, Chen and Meng2023). The use of probiotics has also been shown to mitigate the negative impacts of enteric pathogens such as E. coli on the intestinal microbiota, further demonstrating their role in stabilizing the microbiota (Sun et al. Reference Sun, Duarte and Kim2021; Zhang et al. Reference Zhang, Xu and Liu2010). Postbiotics, more recently defined than prebiotics and probiotics, include inanimate microbial cells, cell components or metabolites derived from yeast, bacteria or fungi that exert beneficial effects on the host (Salminen et al. Reference Salminen, Collado and Endo2021). Emerging research indicates that postbiotics can favorably modulate the intestinal microbiota by reducing harmful bacterial populations and supporting the redox balance, as evidenced by reductions in the production of oxidative damage products (Gormley et al. Reference Gormley, Duarte and Deng2024; Kim and Duarte Reference Kim and Duarte2024). Additionally, inclusion of Saccharomyces yeast in nursery pig diets has been shown to enhance the gene expression of mTOR and increase the cell proliferation in the crypts, thereby promoting intestinal turnover and accelerating intestinal repair following injury (Duarte and Kim Reference Duarte and Kim2024).