Prebiotic Galacto-Oligosaccharide and Xylo-Oligosaccharide Feeds in Pig Production: Microbiota Manipulation, Pathogen Suppression, Gut Architecture and Immunomodulatory Effects

Abstract

Gastrointestinal health is critical to the productivity and welfare of pigs. The transition from milk to plant-based feeds represents an intestinal challenge at wean that can result in dysbiosis and pathogen susceptibility. Prebiotic galacto-oligosaccharides (GOS) and xylo-oligosaccharides (XOS) are non-digestible carbohydrates that can reach the hind gut to promote gut health, either by enhancing the abundance of beneficial members of the intestinal microbiota or via direct interaction with the gut epithelium. Amongst the changes in the intestinal microbiota, GOS and XOS promote populations of short-chain fatty acid (SCFA)-producing bacteria of the genera Lactobacillus, Bifidobacterium and Streptococcus. SCFAs benefit the host by providing nutritional support for the gut, enhance intestinal barrier function and regulate inflammatory responses. By modifying the indigenous microbiota, prebiotics offer a sustainable alternative to the use of antimicrobial growth promoters that have led to the dissemination of antimicrobial resistance and represent a growing threat to public health. This review examines microbial and cellular mechanisms whereby prebiotic feed supplements can support the development of a diverse and robust microbiota associated with a healthy and productive digestive system over the lifetime of the animal, and which is in sharp contrast to the development of dysbiosis often associated with existing antimicrobial treatments. The application of prebiotic feed supplements should be tailored to their modes of action and the developmental challenges in production, such as the provision of GOS to late gestational sows, GOS and XOS to pre-weaning piglets and GOS and XOS to growing/fattening pigs.

1. Introduction

Global animal protein production continues to increase but not without concerns relating to environmental impacts and the use of antimicrobial growth promoters (AGPs) in intensive agriculture [1,2,3]. Despite bans of AGPs in territories such as the EU, there has been an increase in the therapeutic use of antibiotics in animals and emergence of anti-microbial resistance (AMR) and in non-EU countries intensifying animal protein production, where AGPs are still used as growth promoters [4,5]. In this respect, it could be argued that bans of AGPs have led to higher therapeutic antibiotic use in relation to greater animal pathogenesis as a product of intensive production. Moreover, high doses of pharmaceutical zinc oxide have been used to suppress the gut microbiota to prevent diarrhoea and promote growth in weaning pigs [6]. However, this has led to environmental concerns and the promotion of methicillin-resistant Staphylococcus aureus (MRSA) in pigs [7,8], developments that have provoked legislation to restrict the use of zinc oxide after 2022 in the European Union (EU) [9]. Again, other jurisdictions may continue to use zinc-based AGPs without restriction. Nevertheless, there is a need for viable alternatives to AGPs in animal production globally, where pro- and prebiotics, although not necessarily replacing AGPs like for like, may have a beneficial role in the physiological, gut microbiota and immune health of animals. This is particularly true in pigs, considering that pork is the most consumed meat in the EU, which remains the second largest producer to China worldwide [10,11]. Overall, there is an absolute requirement for alternative strategies to improve pre- and post-weaning pig health and welfare in relation to non-infectious physiological failure to thrive and infectious causes of failure to thrive as well as possible zoonotic transmission of pathogens to human populations. In these respects, feeding with pro- and prebiotics may be useful in stimulating the proliferation of beneficial microbiota, enhancing gastrointestinal tract (GIT) microbial diversity, preventing pathogen colonization and thereby supporting gut health and mitigating dysbiosis. This imbalance in the gut microbiota is largely affected during weaning and is associated with reduced feed intake, growth retardation, gut inflammation and diarrhoea, which collectively are leading causes of morbidity and mortality in pigs with concomitant losses in productivity [12]. To this extent, this review considers the effects on the intestinal microbiota and health of production animals from using the prebiotic supplements galacto-oligosaccharides (GOS) in late gestational sows and pre-weaning piglets [13,14], and xylo-oligosaccharides (XOS) that are largely applied post-weaning [15]. These applications reflect the abrupt transition from a milk-based diet, where the prebiotics may be supplied as a creep feed supplement or formulated within a milk replacer, to a solid plant-based diet containing the prebiotic oligosaccharide supplements. The sources of the oligosaccharides are consistent with the dietary transition; porcine milk contains a diverse array of oligosaccharides that include GOS [16], while XOS is derived from xylan, which is ubiquitous in cereals that make up a large proportion of the post-weaning porcine diet [17]. The properties and effects of GOS and XOS are considered with respect to weaning and post-weaning productivity, which include fermentability and beneficial effects in vivo, including microbiome modulation and those upon gut architecture, immunomodulation, gut pathogen adhesion and neonatal viral suppression. Given there is a continuum whereby the early life of pre-weaning animals heavily influences the development and composition of the adult microbiota and intestinal innate immune functions [18,19,20], the effects of GOS and XOS supplementation throughout early and late stages of life are considered in response to the physiological and microbiological challenges in pig production. Thus, it is important to review and understand the challenges posed by endemic pathogens such as enterovirulent E. coli and rotaviruses, which can inform the potential for GOS and XOS prebiotic applications in animal production and welfare. Literature searches for prebiotic studies were conducted until February 2025 using the SCOPUS database for in vivo studies that included the search terms [“pig” or “piglet” or “porcine”] with either [“GOS” or “galacto-oligosaccharide” or “galacto-oligosaccharides”] or [“XOS” or “xylo-oligosaccharide” or “xylo-oligosaccharides”] and [“microbiome” or “microbiota”], and for in vitro studies [“GOS” or “galacto-oligosaccharide” or “galacto-oligosaccharides”] or [“XOS” or “xylo-oligosaccharide” or “xylo-oligosaccharides”] and [“vitro”].

2. The Development of Prebiotic Applications

2.1. The Prebiotic Concept

Prebiotics are dietary components that fortify autochthonous beneficial bacteria of the GIT microbiota, which, unlike probiotics, do not require a live ingested microorganism to survive intestinal transit [21]. The original definition of a prebiotic was as follows: “A prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon improving host health” [22]. This definition takes into account that food/feed should not be digested in the upper GIT but should be a selective substrate for beneficial bacteria to alter the colonic microbiota and induce effects beneficial to host health. Recently, this has been generalized to “a substrate that is selectively utilized by host microorganisms conferring a health benefit” [23]. At this time, the family of prebiotics was extended to galacto-oligosaccharides (GOS), human milk oligosaccharides, fructo-oligosaccharides (FOS), inulin and lactulose with candidate molecules including mannan-oligosaccharides (MOS), arabinoxylans, soya oligosaccharides and resistant starches [23,24], with XOS now recognized to exhibit prebiotic activities in multiple organisms [25]. The main prebiotic effects are considered as an improvement of gut microbiota composition, intestinal and barrier function, immune regulation and pathogen suppression [26].

2.2. Prebiotic Oligosaccharides

The development of prebiotic oligosaccharides was predicated on the beneficial effects of constituent oligosaccharides of the milk of placental mammals [27,28]. Human milk oligosaccharides have been the focus of research compared to recent investigations of animal milk oligosaccharides [28,29,30,31]. Although mammalian milk oligosaccharides have no direct nutritive value to the neonate through limited upper intestinal hydrolysis and absorption, their effects relate to stimulating the development of the intestinal microbiota in neonates and conferring a variety of health benefits, including innate and adaptive immune development [32,33]. In terms of shaping the neonatal GIT microbiome, milk oligosaccharides are digested by Bifidobacterium spp. to produce beneficial SCFAs such as acetate, propionate and butyrate and Bacteroides spp. to produce lactate and succinate, butyrate and propionate [34]. Acetate, butyrate and propionate are the main energy sources for intestinal enterocytes [35,36], while butyrate stimulates epithelial activator protein pathways that control cell proliferation and death, demonstrating the importance of SCFAs for epithelial regeneration and maintaining a healthy mucosa [37].

Milk oligosaccharides also exhibit a major protective effect by inhibiting bacterial and viral infection, either by binding to the pathogens in the GIT lumen and/or inhibiting their binding to cell surface glycan receptors, presumably acting as soluble cell surface decoys [32,38,39]. As immune modulators, milk oligosaccharides promote the maturation of small intestinal epithelial cells and enhance barrier function mainly through the upregulation of tight junction proteins, claudin-8 and claudin-5 [40,41]. Goblet cells (GCs) that line the entirety of the GIT are essential to maintaining barrier function, intestinal homeostasis, epithelial integrity and physical lubrication of luminal contents [42,43]. It is now recognized that GC intrinsic sensing of the GIT microbiota plays a critical role in regulating the exposure of the immune system to microbial challenges [44,45,46]. Further evidence suggests milk oligosaccharides can enhance mucosal barrier function through the direct modulation of GC function and upregulation of the genes for GC secretory products [47,48].

2.3. Porcine Oligosaccharides

Mammalian colostrum and milk contain, in addition to lactose, a variety of neutral and acidic oligosaccharides. These are typically composed of three to ten monosaccharide units, including glucose (Glc), galactose (Gal) and N-acetyl- glucosamine (GlcNAc) as well as fucose and sialic acids (N-acetylneuraminic and N-glycolylneuraminic acids). Porcine milk oligosaccharides (PMOs), like other mammalian milk oligosaccharides, contain bolt-on residues arranged across core polymerized structures, leading to complex and diverse sialylated and fucosylated structures (Figure 1A–C) [49,50]. The core moiety present at the reducing end of milk oligosaccharides is either lactose (Gal(β1–4)Glc) or N-acetyl-lactosamine (Gal(β1–4)GlcNAc) [29]. The dominant saccharide in mammalian milk or colostrum is lactose, which is synthesized from Glc and uridine diphosphate galactose (UDP-Gal) by β4galactosyltransferase in vivo in the mammary gland [29]. Most animal milk oligosaccharides are sialylated (sialic acid at glycoprotein terminal ends), containing N-acetylneuraminic acid (Neu5Ac) and/or N-glycolylneuraminic acid (Neu5Gc) [29,30]. Sialic acids are nine carbon atom sugars that are also present at the terminal ends of glycolipids and glycoproteins involved in cellular communication and survival (or non-survival) of pathogens [51]. Porcine milk oligosaccharides contain a range of structurally identified fucosylated, neutral core-structured, sialylated, fucosyl and sialylated oligosaccharides [50].

Figure 1. The structures of milk and commercial prebiotic oligosaccharides: (A) in general, milk oligosaccharides consist of five monosaccharide moieties. (B) The composition of the core oligosaccharide structure that can be elongated and/or variably decorated with fucose and/or sialic acid residues. (C) Examples of oligosaccharide structures found in porcine milk [49,50]. (D) Structures of commercially available galacto-oligosaccharide mixtures synthesized by the transgalactosylation of lactose using β-galactosidases, which are composed of between 1 and 7 β-(1→3), β-(1→4) and β-(1→6) linked galactose units with a terminal glucose [52]. (E) General structures of prebiotic xylo-oligosaccharides, where the degree of polymerization is between 2 and 10 repeating units of xylose joined by β-1,4 glycosidic bonds that are generated by the enzymatic hydrolysis of plant xylans using xylanase [53].

Figure 1. The structures of milk and commercial prebiotic oligosaccharides: (A) in general, milk oligosaccharides consist of five monosaccharide moieties. (B) The composition of the core oligosaccharide structure that can be elongated and/or variably decorated with fucose and/or sialic acid residues. (C) Examples of oligosaccharide structures found in porcine milk [49,50]. (D) Structures of commercially available galacto-oligosaccharide mixtures synthesized by the transgalactosylation of lactose using β-galactosidases, which are composed of between 1 and 7 β-(1→3), β-(1→4) and β-(1→6) linked galactose units with a terminal glucose [52]. (E) General structures of prebiotic xylo-oligosaccharides, where the degree of polymerization is between 2 and 10 repeating units of xylose joined by β-1,4 glycosidic bonds that are generated by the enzymatic hydrolysis of plant xylans using xylanase [53].

Compared with other domestic animals, porcine milk contains the highest percentage of neutral oligosaccharides (20%), the most abundant variety of mono-sialylated and di-sialylated large oligosaccharides that are close to human milk oligosaccharide composition [28]. Between twenty-nine and sixty PMOs have been identified using a variety of high-performance liquid chromatography and mass spectrometry techniques [50,54,55,56,57,58]. However, the abundance and composition of PMOs changes throughout lactation. There is a lactation-stage-related decrease in the total number of PMOs by 36% and 24% in sow and gilt milk, respectively, over the course of lactation from colostrum to mature milk [50]. Even in the first week of lactation, the majority of PMOs decreased in abundance by 43%, with the concentration of acidic PMOs decreasing and that of neutral-fucosylated and neutral PMOs increasing [56,57]. Furthermore, significant decreases in sialylated PMOs correlate with significant increases in fucosylated PMOs at day fourteen post-partum [58], with an estimated decrease in sialylated PMOs from 80% content at farrowing to 60% in early lactation (days four to seven) to 40% in late lactation (day twenty-four) [54], indicating changes in functionality during lactation.

2.4. Commercially Available Galacto- and Xylo-Oligosaccharides

Commercially available GOS mixtures are typically composed of galactose units bound by various β(1-2), β(1-3), β(1-4) and β(1-6) linkages with a terminal glucose, where the degree of polymerization is two to eight residues (Figure 1D). These are generally synthesized by the transgalactosylation of lactose by β-galactosidases [52,59]. XOS are oligomers of xylose, generated by the hydrolysis of xylans derived from hemicellulosic components of lignocellulosic plant cell wall materials [60,61]. Once isolated from the lignocellulosic biomass, xylans are enzymatically hydrolysed, yielding mixtures of XOS with degrees of polymerization (DP) of 2 to 12 with the prospect of using diverse agro-industrial byproducts [25,62]. The chemical structure of XOS consists of xylose monomers linked by β(1-4) glycosidic bonds (Figure 1E), where the anomeric carbon of one xylose is linked to the hydroxyl group on the fourth carbon of the next xylose [63]. From a regulatory perspective, prebiotic GOS is included in the EU feed material register “https://www.feedmaterialsregister.eu/register (accessed on 28 February 2025)”, approved for use in human infant formula/foods in the USA, is generally regarded as safe (GRAS) “https://www.fda.gov/food/food-ingredients-packaging/generally-recognized-safe-gras (accessed on 28 February 2025)” and is considered a safe novel food by the European Food Safety Authority (EFSA) under the stipulated conditions of use [64]. Similarly, XOS holds GRAS and safe novel food status [65].

There are multiple reports of the use of mixed prebiotic supplements but fewer that are focused on commercial GOS or XOS in pigs that would normally be bound for production.

Table 1. Galacto-oligosaccharide effects on growth performance.

Growth Stage	BW, kg	Age, Days	Days Fed GOS	GOS Purity, %	In-Feed GOS, %	Effect of GOS, % Difference to Controls †			Reference
						BW	ADG	G:F
Post-wean	8.8	26	28	95	0.8	-	-	-	Alizadeh et al. [67]
Pre-wean	6.1	21	21	90	0.1	5.1	16.6		Tian et al. [68]
Post-wean	18.4	56	28	-	0.1	4.5	8.8	0.33	Xing et al. [69]
Pre-wean	5.9	28	7	90	0.001	-	-	-	Tian et al. [70]
Post-wean	6.3	28	7 + 7	90	2.0	-	-	-
Pre-wean *	4.0	21	21	90	1.0	-	-	-	Lee et al. [71]
Pre-wean	7.9	31	16	38	5	-	-	-	Boston et al. [66]
Post-wean	6.3	31	16 + 8	38	3.8	-	6.1	13.1

* Underperforming piglets. BW is body weight, ADG is average daily gain and G:F the gain-to-feed ratio ^† Percentage increases in the performance of animals on prebiotic oligosaccharide supplements are indicated compared to controls. - indicates metric measured but no significant difference compared to controls; blank cells denote no measurement.

shows pre- and post-wean studies using GOS, the majority of which do not show improvement in growth performance in pre-wean studies. However, two studies that provided GOS post-wean reported increases in average daily gain (ADG) and efficiency (gain-to-feed ratio; G:F). It is noteworthy that the recent study of Boston et al. provided GOS at higher inclusion rates of 5.0% in a gruel creep feed at farrowing and 3.8% of the nursery diet [66]. These rates may provoke more marked shifts in the hind gut microbiota and greater fermentation rates. The picture emerging from studies using XOS is clearer, with the majority of studies showing improvements in ADG (6/8), and half (4/8) also recording increases in body weight at the end of the period on the supplemented diet (

Table 2. Xylo-oligosaccharide effects on growth performance.

Growth Stage	BW, kg	Age, Days	Days Fed XOS	XOS Purity, %	In-Feed XOS, %	Effect of XOS, % Difference to Controls †			Reference
						BW	ADG	G:F
Weaning	8.8	28	28	95	0.01	-	-	-	Chen, et al. [72]
					0.05	4.7	9.6	7.1
					0.1	-	-	-
Weaning	6.3	21	28	50	0.02	-	16.6	14.3	Liu et al. [73]
Weaning	7.5	30	28	70	0.75	-	5.9	-	Pang et al. [74]
					1.5	8.6	15.5	-
					3	5.3	9.9	-
Nursing	19.94	ND	28	>35	0.04	10.6	33.1		Hou et al. [75]

BW is body weight, ADG is average daily gain and G:F the gain-to-feed ratio ^† Percentage increases in the performance of animals on prebiotic oligosaccharide supplements are indicated compared to controls. - indicates metric measured but no significant difference compared to controls; blank cells denote no measurement.

).

3. Biological Effects of Galacto- and Xylo-Oligosaccharides

3.1. Preventing Pathogen Adhesion

Diarrhoeal infections (scours) are estimated to account for 8.1 to 12.2% of piglet mortalities [76], with litters experiencing pre-weaning diarrhoea exhibiting reduced weight gain and a significantly increased risk of post-weaning diarrhoea (PWD) [77]. The withdrawal of sow’s milk and the protective immune factors that it contains, coupled with a susceptibility to enteric disease due to an immature mucosal immune system at weaning, induces stress to elevate the risk of infection [78,79]. Due to stress, dietary change and reduced feeding, a state of gut dysbiosis may be induced during weaning where disruption of the gut microbiota composition and intestinal inflammation lead to the expansion and domination of enteric pathogens [80]. Common pathogens playing an etiological role in infection are Escherichia coli, Clostridium perfringens, Lawsonia intracellularis, Salmonella enterica and Brachyspira (Serpulina) spp. [81]. The post-weaning growth check and the occurrence of PWD are major sources of economic loss, which can result in young pigs achieving less than 50% of expected performance [82,83,84]. Enterotoxigenic Escherichia coli (ETEC) are the main pathotypes in PWD in European pig farms post AGP bans [85], with total losses due to infection amounting to an estimated 17% in the EU [77]. The attachment of pathogens to intestinal epithelial cells, such as the use of fimbrial adhesins by the prominent pig pathotype ETEC, is a key step in the development of infection and disease [83,86]. However, GOS can prevent the cellular binding of ETEC or purified K88 fimbriae [87]. Studies have shown that GOS strongly inhibits the attachment of enterohepatic E. coli and Salmonella enterica serotype Typhimurium to human colon HT29 cells and adhesion of enteropathogenic E. coli (EPEC) to human GIT epithelial cells Hep-2 and Caco-2 lines by 65% and 75%, respectively [88,89]. The adherence inhibition of GOS outperforms that of other prebiotics, such as lactulose, inulin, raffinose and fructo-oligosaccharides, suggesting that the expression of complex mammalian milk oligosaccharides has evolved over time, not just to prime the microbiome but also to prevent infection through pathogen adherence inhibition [90]. Recent studies have shown that GOS significantly reduces adhesion of E. coli to HT-29 cells but also significantly reduced E. coli growth in vitro, demonstrating the capacity for GOS to react directly with pathogenic cells [91].

Rotavirus serogroups A to H affect pigs with rotavirus A (RVA), being the most widespread group causing acute dehydrating diarrhoea in veterinary and public health settings [92]. Rotavirus A reportedly accounts for 53% of pre-weaning and 44% of post-weaning diarrhoea in pigs [93], with the effects being significant mortality and morbidity in neonates, reduced performance in surviving growers and significant economic loss [92,94,95]. RVA infectivity of MA104 cells is reduced in the presence of GOS, with 3′-sialylated varieties proving most effective [96,97]. Human milk oligosaccharides have been demonstrated to shorten rotavirus-induced diarrhoea in piglets [98], and GOS has been shown to reduce the incidence and severity of rotavirus-associated diarrhoea in suckling rats [99].

Listeria monocytogenes can cause systemic bacterial disease in pigs at all stages of production, which is of concern for animal and public health [100]. Prebiotic GOS and XOS have been reported to prevent Listeria monocytogenes infection in guinea pigs [101]. However, XOS is reported to reduce the ability of Listeria monocytogenes to bind to human epithelial Caco-2 cells by reducing the expression of the adhesins internalin A (inlA) and Listeria adherence protein (lap), which are necessary for L. monocytogenes adhesion to intestinal cells [102]. Via a similar mode of action, XOS is reported to inhibit Salmonella enterica serovar Typhimurium adhesion to intestinal porcine enterocyte cells in vitro by reducing the expression of the adherence-associated genes bcfF, yejE and fljB [103].

3.2. Galacto- and Xylo-Oligosaccharides Are Substrates for Fermentation

Galacto-oligosaccharides are readily and completely fermented by pig faecal microbiota in vitro [104,105]. However, there are differences in the extent and duration of fermentation of GOS depending upon the structure of oligosaccharide molecules, be they inherently expressed PMOs or additive GOS. Acidic PMOs and GOS with degrees of polymerization between four and seven monomers were rapidly depleted within twelve hours of in vitro fermentation in contrast to more complicated molecules being fucosylated and phosphorylated PMOs, which were partially resistant to fermentation. GOS structures containing ß1-2 and β1-3 linkages were fermented in preference to GOS containing β1-4 and β1-6 linkages. This suggests that there are different physiological roles for different structures, with some readily lower-molecular-weight PMOs and GOS being preferentially fermented to SCFAs and others being less fermented but retaining sialylated, di-sialylated, fucosylated and large oligosaccharide structures implicated in preventing pathogen binding to piglet intestinal cells [28,105]. Using an in vitro model of the large intestine, inoculated with human or pig faeces, GOS fermentation and degradation was more pronounced with pig faecal inocula compared to human inocula. Prebiotic GOS significantly stimulated the growth of Lactobacillus and Bifidobacterium in both human and pig inocula, but with more complex communities that produced greater concentrations of SCFAs from pig faecal inoculates, indicating intrinsic differences between the microbiota [104]. GOS significantly increases SCFA production in vitro, largely producing acetate, propionate, butyrate, succinate and lactate [104,105,106]. These products are trophic for GIT epithelial cells, stimulate GIT cell proliferation, reduce the pH of the luminal contents, are antineoplastic (particularly butyrate) and favour the proliferation beneficial bacteria such as lactic acid bacteria [107,108]. There is also evidence that acetate and propionate production from GOS is responsible for inhibiting Salmonella enterica colonization in a pig in vitro fermentation model through a reduction in pH and modulation of the bacterial communities [106], as has been observed in broiler chicken intestine [109].

As with GOS, XOS is entirely fermented by piglet microbiotas [110,111]. In an in vitro fermentation model, caecal and colonic microbiota metabolized XOS more readily than ileal microbiota, with greater degradation of XOS by large intestinal communities after thirty hours of fermentation compared to ileal community members [111]. However, although caecal and colonic microbiota displayed similar fermentation profiles of short-, medium- and long-chained polymerized XOS, ileal microbiota metabolized short-chain XOS more readily than medium- and long-chained XOS [111]. The abundance of Bifidobacterium significantly increased in a colon model inoculated with human faeces and supplemented with XOS [112]. XOS DP 2 increased the abundance of total Bifidobacteria spp., while XOS DP 2–10 and 2–7 increased the abundance of Bifidobacterium lactis and total Bifidobacteria spp., indicating that there is a preference of B. lactis for greater polymerized XOS [112]. In a pig in vitro fermentation system, XOS was fermented by swine faecal microbiota to produce acetate, propionate and butyrate [113], with increased SCFA production, particularly acetate, observed when XOS was added to an in vitro batch culture system inoculated with porcine faecal microbiota compared to a control system lacking XOS [114].

3.3. Microbiota Mediated Beneficial Effects of Galacto- and Xylo-Oligosaccharides

Prebiotic GOS and PMOs are readily fermented in the GIT of pigs [56,67,88,115]. The majority of ingested GOS reaches the colon, where it is fermented by resident bacteria with very little absorbed systemically. At three and twenty-six days after feeding GOS to animals, only trace amounts could be found in the faecal and caecal digesta, indicating almost complete fermentation of GOS in vivo [115]. Even in nursing piglets receiving PMO-rich colostrum, no intact PMO structures in the faeces could be found in nursing piglets at one to two days, indicating intestinal fermentation of GOS at a very early age [56]. Similarly, no intact original molecular GOS structures could be found in piglet faeces at days three or twenty-six following GOS feeding in milk replacer [67]. Pigs fed 4% w/w GOS exhibited a lower proximal colonic pH than pigs fed a control diet [88], and significantly lower pH values were found in the caecal digesta of pigs fed GOS in milk replacer [67]. Lowering of the digesta pH is indicative of SCFA production in vivo, with increases in SCFAs in the proximal colon being almost entirely due to acetic acid, which is a major fermentation product of Bifidobacterium [88]. In contrast, GOS was reported to increase caecal butyrate digesta concentrations in pigs [67]. However, the pH of ileal digesta decreased in GOS-fed piglets with significantly increased concentrations of propionate, butyrate and valerate compared with controls [116]. The effects of GOS on pH and SCFA concentrations are not just limited to healthy pigs. In suckling piglets challenged with lipopolysaccharide (LPS) endotoxin derived from E. coli, the pH value of colonic digesta increased and the concentrations of acetate, butyrate and lactate significantly decreased. In contrast, animals fed GOS and challenged with LPS showed a significant decrease in the pH of the colonic digesta and a significant increase in acetate, butyrate, lactate and total SCFAs, demonstrating the ability of GOS to relieve colonic inflammation [117]. In 30 kg pigs receiving 2.5 g/kg GOS in their basal diet, Lactobacillus spp. were significantly increased in caecal and colonic GIT samples despite being challenged with 1 × 10⁸ cfu Salmonella enterica serovar Typhimurium [118], showing the ability of GOS to modulate the intestinal microbiota during infection. Prebiotic GOS also modulates the GIT microbiota in young pigs, with the addition of GOS to diets significantly increasing Lactobacillus and Bifidobacterium in the caecal contents and faeces [67,69,88]. After twenty-six days of feeding with GOS, significant reductions in E. coli and clostridial counts were observed [107]. Similar results were obtained by Xing et al. [69], who noted a significant decrease in the number of E. coli in a linear and dose-dependent manner from 500 mg/kg up to 2000 mg/kg GOS fed daily. The modulatory effects of GOS did not extend to the lower GIT or faeces. The ileal microbiota composition of the upper GIT was significantly enriched with Lactobacillus and reduced in Clostridium sensu stricto in twenty-one-day-old pigs following an initial ten-day GOS feeding period, demonstrating the ability of GOS to affect the microbiome over time [116]. GOS has also been shown to significantly increase the relative abundance of lactic-acid-producing bacteria, notably Lactobacillus and Bifidobacterium, throughout the GIT, from the duodenum to the colon, demonstrating benefits as a supplement to milk replacer in poorly performing piglets. Moreover, GOS significantly modulated GIT microbial communities, as demonstrated by β-diversity measures, with key effects on microbial community membership rather than structure, demonstrating that GOS promotes more diverse communities [71]. The comparative effects of dietary GOS on the intestinal microbiota of pigs are summarized in

Table 3. Galacto-oligosaccharide effects on the gut microbiota.

Growth Stage	In-Feed GOS, %	Bacterial Source	Effect of GOS on 16S rRNA Community Taxa †	Reference
Post-wean	0.8	Faecal	Bifidobacterium ↑, Lactobacillus ↑	Alizadeh et al. [67]
Pre-wean	age variable	Colon	Prevotella ↑, Barnesiella ↑, Parabacteroides ↑, Porphyromonada ↑, Dorea ↓	Wang et al. [119]
Pre-wean	0.001	Colon	Ruminococcaceae UCG-014 ↑, Faecalibacterium ↑	Tian et al. [70]
			Clostridium sensus strico 1 ↑, Terrisporobacter ↑
Post-wean	2		Dorea ↑ Phascolarctobacterium ↑
Pre-wean *	1.0	Caecum	Lactobacillus ↑, Bifidobacterium ↑, Leuconostoc ↑, Streptococcus ↓	Lee et al. [71]
Pre-wean	0.4	Faecal	Anaerostipes ↑, Mitsuokella ↑, Prevotella ↑, Clostridium IV ↑, Bulleidia ↑, Bilophila ↓, Clostridium XIVb ↓, Enterococcus ↓	Eudy et al. [120]
Pre-wean	5	Caecum	No significant effect	Boston et al. [66]
			Fusicatenibacter ↑, Collinsella ↑, Agathobacter ↓,
Post-wean	3.8		Ruminococcaceae ↑, Frisingicoccus ↓, Campylobacter ↓

* Underperforming piglets. ^† Alizadeh et al. used qPCR calibrated against pure cultures to measure faecal concentrations of the target bacteria; all others determined the relative abundance of taxa from sequencing 16S rRNA gene sequences amplified from DNA extracted from intestinal and/or faecal contents. Effects of oligosaccharide supplements compared to control animals where ↑ denotes a significant increase in the taxa abundance and ↓ a significant decrease. Lactic acid bacteria are indicated in bold.

.

In a rotavirus A rat infection model, GOS significantly reduced the incidence, duration and severity of diarrhoea, with a second RVA challenge failing to provoke significant disease in GOS-treated groups [99]. Similar studies have shown that GOS/FOS mixtures prevent infection and gut dysbiosis caused by rotavirus [121]. Including prebiotic oligosaccharides in human infant formulas containing mixtures of short-chain GOS and long-chain FOS at a 9:1 ratio significantly reduced the incidence of all types of infection in infants [122]. GOS/FOS mixtures also have modulatory effects in pigs infected with RV. These oligosaccharides increased luminal pH, lowered the dry matter content of the colon, enhanced numbers of butyrate-producing bacteria and reduced the duration of RV-induced diarrhoea in piglets [39]. Stressful weaning results in increased malondialdehyde (MDA) [123], a biomarker of oxidative stress and cellular damage by reactive oxygen species (ROS) [124]. Piglets rely on antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) to combat ROS-induced damage [124]. Studies have shown that GOS decreases the concentration of MDA and increases the concentration of GSH-Px and SOD in the serum and intestinal mucosa of suckling piglets [70,117].

Like other non-digestible carbohydrates, XOS is naturally fermented by GIT bacterial community members in the pig gut [125]. Bacteroidetes, for example, produce plant-cell-wall-degrading enzymes to digest lignocellulosic material in the large intestine [126]. Studies have demonstrated that dietary supplementation with XOS have similar effects to GOS supplementation; lowering the pH of pig digesta by increasing the fermentation activity of the resident microbiota, leading to increased production of SCFAs. In an in vitro fermentation system, XOS significantly increased levels of acetate, butyrate and propionate, resulting in a reduction in pH after 48 h [127]. Additionally, weaning pigs fed a diet containing 1% XOS exhibited greater concentrations of ileal SCFAs compared to pigs fed a control diet during a 28-day period [128]. XOS supplementation also impacts SCFA concentrations in the porcine large intestine, with increased caecal propionate and butyrate [129] and faecal acetate, propionate and butyrate concentrations following 28 days of supplementation in weaning piglets [74,130]. This demonstrates the ability of XOS to modulate microbial fermentation activity throughout the whole digestive tract of the weaning pig. As with GOS, weaning pigs receiving a diet containing 0.02% w/w XOS and challenged with E. coli LPS experienced a significant increase in caecal acetate, propionate, isobutyrate, valerate and isovalerate concentrations compared to pigs receiving a basal diet containing no XOS [131]. This demonstrates the ability of XOS to relieve inflammation in the large intestine. Lactic acid bacteria (LAB) of the genera Lactobacillus, Lactococcus, Bifidobacterium and Streptococcus contribute to SCFA synthesis, which subsequently lowers the pH of the gut. In weaning pigs receiving 1% XOS in their diet, Lactobacillus and Bifiodbacterium spp. were significantly increased in ileal digesta [128], while weaning pigs receiving 0.05% in-feed XOS experienced an increase in ileal and caecal Lactobacillus spp. abundances and a decrease in the ileal Escherichia-Shigella molecular taxa [129], demonstrating the ability of XOS to modulate microbiota throughout the GIT and to increase the abundance of beneficial LAB as well as to decrease the abundance of enteropathogens. The exception to this occurred when caecal increases in the abundance of the Escherichia-Shigella taxa were reported in conjunction with E. coli LPS challenge experiments [131]. However, XOS supplementation is reported to alleviate the diarrhoeal effects of high protein plant diets by reducing the colonic abundances of enteric pathogens and promoting barrier function [132]. Overall, XOS supplementation in pig diets improves the diversity of microbial communities, as demonstrated by increased alpha diversity measures [15,128], with the main effects related to community membership rather than structure, similarly to what was observed for GOS [71]. A summary of the comparative effects of dietary XOS supplementation on the intestinal microbiota of pigs is summarized in Table 4.

XOS supplementation leads to a decreased abundance of pathogenic microbiota in the pig gut by promoting the growth of SCFA-producing bacteria. This shift in microbial composition reduces the incidence of diarrhoea in weaning pigs [74,128], thereby demonstrating XOS’s efficacy in reducing PWD and its associated economic losses. However, increasing in-feed XOS to 3% w/w showed a trend toward a return to control diet levels of diarrhoea incidence, indicating that excessive XOS supplementation may be detrimental to the animal [74]. Furthermore, increased luminal LAB and SCFA concentrations have been shown to downregulate Salmonella pathogenicity islands, which could reduce the incidence of PWD in pigs [133]. It has been demonstrated that supplementing pig diets with XOS increases the concentrations of SOD, CAT and GSH-Px in porcine serum [72,74,75] and intestinal mucosa [128,134], indicating that XOS supplementation plays a role in alleviating oxidative stress in pigs.

Table 4. Xylo-oligosaccharide effects on the gut microbiota.

Growth Stage	In-Feed XOS, %	Bacterial Source	Effect of XOS on 16S rRNA Community Taxa †	Reference
Weaning	0.025	Ileum	Acinetobacter ↑, Herbaspirillum ↑, Xanthobacter ↑	Ding et al. [135]
		Colon	Bifidobacterium ↑, Sharpea ↑, Slackia ↑, Veillonella ↑
Weaning	0.1	Ileum	Streptococcus ↑, Bifidobacterium ↑, Ruminococcus ↓	Gao et al. [134]
Weaning	1	Ileum	Lactobacillus ↑, Bifidobacterium ↑	Sun et al. [128]
Weaning	0.05	Ileum	Lactobacillus ↑, Escherichia-Shigella ↓, Clostridium sensu stricto 1 ↓	Chen et al. [129]
		Caecum	Lactobacillus ↑, Clostridium sensus strico 1 ↓, Terrisporobacter ↓
Weaning	0.02	Caecum	Escherichia-Shigella ↑, Streptococcus ↑	Wang et al. [131]
Weaning	0.05	Caecum	Lactobacillus ↑, Intestinibacter ↓, Anaerotruncus ↓, Ruminiclostridium 9 ↓, Clostridium sensu stricto 1 ↓, Turicibacter ↓	Tang et al. [136]
Weaning	0.01	Intestinal contents	Streptococcus ↑, Turicibacter ↑, Lactobacillus ↓	Yin et al. [15]
Growing/fattening	0.01	Intestinal contents	Lactobacillus ↑, Citrobacter ↓	Pan et al. [137]
Weaning	0.02	Faecal	Lactobacillus ↑, Escherichia coli ↓	Liu et al. [73]
Weaning	1.5	Faecal	Lactobacillus ↑, Bifidobacterium↑, Fusicatenibacter ↑, Ruminococcus ↓, Eubacterium coprostanoligenes ↓, Clostridia UCG-014 ↓	Pang et al. [74]
Weaning	0.01/0.025 /0.05	Faecal	Lactobacillus ↑, Bifidobacterium ↑	Su et al. [138]
Growing	0.02	Faecal	Prevotellaceae_NK3B1 ↑, Muribaculaceae ↑	Sutton et al. [139]
Pre-weaning	5	Faecal	Cloacibacillus porcorum ↑, Clostridium sensus strico 1 ↓, Parabacteroides goldsteinii ↓	Bai et al. [140]

^† The relative abundances of the taxa were determined by sequencing 16S rRNA gene sequences amplified from DNA extracted from intestinal and/or faecal contents. Effects of oligosaccharide supplements were compared to control animals, where ↑ denotes a significant increase in the taxa abundance and ↓ a significant decrease. Lactic acid bacteria are indicated in bold.

Table 4. Xylo-oligosaccharide effects on the gut microbiota.

Growth Stage	In-Feed XOS, %	Bacterial Source	Effect of XOS on 16S rRNA Community Taxa †	Reference
Weaning	0.025	Ileum	Acinetobacter ↑, Herbaspirillum ↑, Xanthobacter ↑	Ding et al. [135]
		Colon	Bifidobacterium ↑, Sharpea ↑, Slackia ↑, Veillonella ↑
Weaning	0.1	Ileum	Streptococcus ↑, Bifidobacterium ↑, Ruminococcus ↓	Gao et al. [134]
Weaning	1	Ileum	Lactobacillus ↑, Bifidobacterium ↑	Sun et al. [128]
Weaning	0.05	Ileum	Lactobacillus ↑, Escherichia-Shigella ↓, Clostridium sensu stricto 1 ↓	Chen et al. [129]
		Caecum	Lactobacillus ↑, Clostridium sensus strico 1 ↓, Terrisporobacter ↓
Weaning	0.02	Caecum	Escherichia-Shigella ↑, Streptococcus ↑	Wang et al. [131]
Weaning	0.05	Caecum	Lactobacillus ↑, Intestinibacter ↓, Anaerotruncus ↓, Ruminiclostridium 9 ↓, Clostridium sensu stricto 1 ↓, Turicibacter ↓	Tang et al. [136]
Weaning	0.01	Intestinal contents	Streptococcus ↑, Turicibacter ↑, Lactobacillus ↓	Yin et al. [15]
Growing/fattening	0.01	Intestinal contents	Lactobacillus ↑, Citrobacter ↓	Pan et al. [137]
Weaning	0.02	Faecal	Lactobacillus ↑, Escherichia coli ↓	Liu et al. [73]
Weaning	1.5	Faecal	Lactobacillus ↑, Bifidobacterium↑, Fusicatenibacter ↑, Ruminococcus ↓, Eubacterium coprostanoligenes ↓, Clostridia UCG-014 ↓	Pang et al. [74]
Weaning	0.01/0.025 /0.05	Faecal	Lactobacillus ↑, Bifidobacterium ↑	Su et al. [138]
Growing	0.02	Faecal	Prevotellaceae_NK3B1 ↑, Muribaculaceae ↑	Sutton et al. [139]
Pre-weaning	5	Faecal	Cloacibacillus porcorum ↑, Clostridium sensus strico 1 ↓, Parabacteroides goldsteinii ↓	Bai et al. [140]

^† The relative abundances of the taxa were determined by sequencing 16S rRNA gene sequences amplified from DNA extracted from intestinal and/or faecal contents. Effects of oligosaccharide supplements were compared to control animals, where ↑ denotes a significant increase in the taxa abundance and ↓ a significant decrease. Lactic acid bacteria are indicated in bold.

3.4. The Effects of Galacto- and Xylo-Oligosaccharides on the Gut Architecture

A healthy, well-differentiated intestinal mucosa has long, regular villi and high villus-to-crypt ratios [141]. Moreover, the healthy maturation of the jejunum is beneficial for maintaining high growth rates in neonates [142] and is considered the main organ for nutrient absorption in pigs [68]. Although some studies have shown little effect of GOS on the intestinal architecture [68], others report differences in histomorphology (as summarized in

Table 5. Galacto-oligosaccharide effects on gut architecture.

Growth Stage	GOS Purity, % (w/w)	In-Feed GOS, %	Effect of GOS, % Difference to Control †				Reference
			Jejunum		Ileum
			VH	VCR	VH	VCR
Post-wean	59	0.8	21	50	16.3	-	Alizadeh et al. [67] Tian et al. [68]
Weaning	90	0.1	-	-
Pre-weang *	90	1.0	16–64	14–76	10–13	10–22	Lee et al. [71]
Pre-wean	38	5	-	-			Boston et al. [66]
Post-wean		3.8	-	-

* Underperforming piglets. ^† Percentage increases in the histomorphometric measurement of the intestinal section of animals on prebiotic oligosaccharide supplements are indicated compared to controls, where VH is villus height and VCR villus-to-crypt ratio. - indicates metric measured but no significant difference compared to controls; blank cells denote no measurement; denotes a significant increase but the metric was not reported.

).

Alizadeh et al. reported increases in villus height after three days on GOS feed, and after twenty-six days, the jejunal villi were thicker and larger, with a significant increase in villus height, villus breadth top and villus breadth base in GOS-fed pigs compared with controls [67]. In LPS-challenged pigs, GOS has a protective effect upon the GIT mucosa and alleviates inflammation. Histomorphological differences were observed in the jejunum of LPS plus GOS pigs who had significantly higher villus height and VCR compared to the LPS control pigs who were not fed GOS [117,131]. Higher villus heights were also seen in the duodenum of LPS GOS-fed pigs [117,143]. In an extensive study of the gut architecture of poorly performing piglets receiving GOS in milk replacer, jejunal and ileal villus height and VCR were significantly increased in those animals receiving GOS. In addition, GC density per mm² tissues significantly increased throughout the GIT of GOS-fed piglets compared with non-GOS-fed piglets [71]. This is an important finding since GCs are known to be essential to barrier function and immune regulation in animals [42,43,45]. GOS directly modulates the expression of GC secretory products that contribute to the production of barrier-enhancing mucins via cell surface receptors [47]. However, this may be indirectly modulated by the microbiota, particularly the lactic-acid-producing bacteria Bifidobacterium and Lactobacillus spp., as these are significantly increased by GOS [71], which supports intestinal cell regeneration and the proliferation of intestinal stem, Paneth cells and GCs through the action of lactic acid [144].

Reports of the impact of XOS supplementation on gut architecture and histomorphology are variable, with some studies observing XOS to have no effect on the duodenum of weaning pigs [73], while others indicate XOS can promote histological changes in the GIT [72]. A summary of the comparative effects of dietary XOS on the intestinal histomorphology of pigs is summarized in Table 6. For instance, XOS impacts gut architecture in the jejunum, with one study reporting a 7.5% increase in the villus-to-crypt ratio in 28-day-old pigs fed 0.05% w/w XOS in their diet for 28 days compared to control pigs not receiving XOS [129]. Furthermore, 21-day-old pigs fed 0.05% w/w XOS in their diet for 56 days displayed an increase of 14.6% in their villus-to-crypt ratio compared to control animals receiving no XOS [138], indicating that the beneficial effects of XOS on gut architecture are more pronounced with prolonged supplementation. XOS supplementation exerts its most significant histomorphological benefits in the ileum, where most beneficial effects on the gut architecture are observed. After 28 days of XOS supplementation, pigs exhibited longer ileal villi and a greater villus-to-crypt ratio compared to control animals [129]. Additionally, supplementation of XOS in 21-day-old weaning pigs resulted in approximately 20%-longer ileal villi [131], demonstrating the potent impact of XOS on gut architecture in young pigs. Longer villi and larger villus-to-crypt ratios are important for greater absorption of nutrients from the intestinal lumen, with villus atrophy associated with malabsorption of nutrients, contributing to stunted growth [145,146]. While there is no published evidence of XOS supplementation modulating GC expression in the small intestine, recent studies have shown XOS supplementation is associated with increased GC expression in the caecum [72,136] and colon [134].

Table 6. Xylo-oligosaccharide effects on gut architecture.

Growth Stage	XOS Purity, % (w/w)	In-Feed XOS, %	Effect of XOS, % Difference to Control †				Reference
			Jejunum		Ileum
			VH	VCR	VH	VCR
Weaning	95	0.01	-	-	6.2	7.2	Chen, et al. [72]
		0.05	5.1	7.5	9.2	11.4
		0.1	-	-	-	-
Weaning	≥35	0.01	-	-	-	-	Su et al. [138]
		0.025	-	-	-	-
		0.05	-	14.6	17.6	-
Weaning	95	0.05			↑	↑	Chen et al. [129]
Weaning	70	0.1			↑	↑	Gao et al. [134]
Weaning	70	1.0			↑	-	Sun et al. [128]
Weaning	35	0.02	-	12.4	19.8	8.7	Wang et al. [131]
Weaning	≥35	0.025			12.2	10	Ding et al. [135]
Weaning	50	0.02	-	11.3	-	-	Liu et al. [73]

^† Percentage increases in the histomorphometric measurement of the intestinal section of animals on prebiotic oligosaccharide supplements are indicated compared to controls, where VH is villus height and VCR villus-to-crypt ratio. - indicates metric measured but no significant difference compared to controls; blank cells denote no measurement; denotes a significant increase but the metric was not reported. ↑ denotes a significant increase in the taxa abundance.

Table 6. Xylo-oligosaccharide effects on gut architecture.

Growth Stage	XOS Purity, % (w/w)	In-Feed XOS, %	Effect of XOS, % Difference to Control †				Reference
			Jejunum		Ileum
			VH	VCR	VH	VCR
Weaning	95	0.01	-	-	6.2	7.2	Chen, et al. [72]
		0.05	5.1	7.5	9.2	11.4
		0.1	-	-	-	-
Weaning	≥35	0.01	-	-	-	-	Su et al. [138]
		0.025	-	-	-	-
		0.05	-	14.6	17.6	-
Weaning	95	0.05			↑	↑	Chen et al. [129]
Weaning	70	0.1			↑	↑	Gao et al. [134]
Weaning	70	1.0			↑	-	Sun et al. [128]
Weaning	35	0.02	-	12.4	19.8	8.7	Wang et al. [131]
Weaning	≥35	0.025			12.2	10	Ding et al. [135]
Weaning	50	0.02	-	11.3	-	-	Liu et al. [73]

^† Percentage increases in the histomorphometric measurement of the intestinal section of animals on prebiotic oligosaccharide supplements are indicated compared to controls, where VH is villus height and VCR villus-to-crypt ratio. - indicates metric measured but no significant difference compared to controls; blank cells denote no measurement; denotes a significant increase but the metric was not reported. ↑ denotes a significant increase in the taxa abundance.

3.5. Immunomodulatory Effects of Galacto- and Xylo-Oligosaccharides

The immunomodulatory effects of GOS and XOS on the immune responses and expression of cytokines and chemokines in intestinal tissues have been a focus for research. In the porcine gastrointestinal tract, tight junction proteins (TJPs) such as claudins, zona occludens (ZO) and occludin are crucial for maintaining epithelial barrier integrity [147]. Transmembrane claudins form the primary barrier against the paracellular movement of ions and solutes by sealing adjacent epithelial cells [147]. ZO-1 and ZO-2 link claudins to the actin cytoskeleton, providing structural support to tight junctions, and influence the assembly and disassembly of tight junctions in response to physiological changes in the gut [148,149]. Occludin also contributes to sealing the paracellular space and interacts with claudins and ZO proteins to anchor all TJPs to the cytoskeleton [150]. GOS and XOS directly upregulate claudin 1, zona occludens 1 and 2 and occludin in the porcine GIT, thereby improving gut epithelial barrier integrity [67,72,138,151,152]. GOS also directly upregulates secretory product genes linked with high-molecular-weight glycoprotein mucins and intestinal factors that stabilize the integrity of the mucus layer (mucin 2, mucin 4 and trefoil factor 3) as well as antimicrobial proteins and peptides such as resistin-like molecule beta and porcine beta defensin 2 [47,67,153,154]. Although mucin expression is less studied with respect to XOS supplementation, mucin 2 expression was significantly increased in weaning pigs fed 0.1% w/w XOS for 28 days compared to pigs receiving a basal diet [134]. The secretory goblet cell lineage gene Atonal bHLH transcription factor 1 (ATOH1) is upregulated by prebiotics, while the suppressor of the transcription factor ATOH1, HES1, is downregulated by probiotics [155].

In addition to the physical barrier (TJP) against infection, the porcine GIT harbours a delicate balance of pro- and anti-inflammatory cytokines to maintain intestinal homeostasis and protection against pathogens. During acute intestinal inflammation, toll-like receptor-2 (TLR-2) and TLR-4 are upregulated on the surface of macrophages, which stimulates the production of the pro-inflammatory cytokines interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) [156]. While these cytokines are essential for pathogen clearance, they cause localized tissue damage through the release of reactive oxygen species (ROS) by recruited neutrophils [157]. Several pro-inflammatory cytokines, such as TNFα, IL1ß, IL6, IL8, IL12A, Il17A, IL17F, IL22 and IL33, are downregulated by GOS as well as the immune-regulatory and pro-inflammatory cytokines IFNγ and NF-κB [68,158,159,160,161,162]. XOS exerts similar effects on the pro-inflammatory cytokine profile of pig intestinal tissue and mucosa, with significant decreases in IL-1ß, IL-6, IL-12 and IFN-y throughout the GIT arising as a result of XOS supplementation [72,134,138]. To maintain intestinal homeostasis, interleukin-10 (IL-10) suppresses the production of pro-inflammatory IL-6 and TNF-α and modulates macrophage activity [163,164]. Additionally, transforming growth factor-beta (TGF-β) aids in tissue repair and restores mucosal barrier integrity [165,166]. GOS and XOS upregulate the cytokine synthesis inhibitory factor IL10 to produce anti-inflammatory effects [72,134,159], while TGFß, an anti-inflammatory cytokine which regulates cell proliferation and growth, is upregulated only by GOS [68]. Generally, prebiotics including GOS have both direct and indirect immunomodulatory effects. Prebiotics can directly act on GIT epithelial cells through toll-like receptors, which leads to cytokine production through NF- κB activation that eventually leads to IL-2, IL-4 and IL-10 production. Indirect effects are mediated by GIT bacteria, which ferment GOS and XOS to SCFAs. These can bind to G-protein-coupled receptors located on GIT epithelial cells and induce the production of IL10 and tissue growth factor-β [167]. In suckling piglets, it has been demonstrated that GOS increases the abundance of SCFA-producing bacteria such as Prevotella, Barnesiella and Parabacteroides, which is accompanied by increasing SCFA concentrations in the colon. Furthermore, the higher colonic SCFA concentration of GOS piglets can alter the gene expression of inflammatory factors through the regulation of NF-κB and protein-kinase signalling pathways, demonstrating microbial mediation of immune function [119,152].

XOS has been reported to decrease lactate dehydrogenase as an indicator of cell damage in IPEC-J2 cells (a porcine jejunal epithelial cell line) challenged with Salmonella enterica serovar Typhimurium. The pro-inflammatory cytokines IL-1β and TNF-α were significantly downregulated, while the tight junction proteins occludin and ZO-1 were significantly upregulated in IPEC-J2 cells treated with S. Typhimurium and XOS compared to cells treated with S. Typhimurium alone [103]. Also, using IPEC-J2 cells, Tian et al. demonstrated that GOS increased the expression of tight junction proteins and was able to relieve LPS-induced cell damage, inflammation and oxidative stress [168]. These data provide evidence that XOS and GOS can alleviate the cellular inflammatory responses of pig gastrointestinal-epithelial-derived cells independently of the presence of the intestinal microbiota.

3.6. Late Gestational Effects of Galacto-Oligosaccharides on Sows and Piglets

Whilst GOS can inhibit rotavirus infection [92], little is known about the effects of supplementing sows’ diets with GOS during late gestation and if there are effects on neonates. Recent studies have shown that direct GOS supplementation is able to reduce the incidence and severity of RV-associated diarrhoea and influence the immune response against RV infections in suckling rats [99]. GOS was fed to non-RV-challenged and RV-challenged neonates, and it was concluded that RV infection could be ameliorated by nutritional intervention with bioactive compounds, such as prebiotics.

It has now been established that GOS supplementation of late gestational sows on a commercial farm with natural endemic rotavirus challenge could improve maternal and neonatal immunity, reduce rotavirus infection, modulate the intestinal microbiota through entero-mammary pathways and confer immunity to neonates. A major finding was that GOS supplementation to late gestational sows significantly increased RVA-specific IgG and IgA in the colostrum [169]. This is probably due to the existence of entero-mammary pathways that programme the mammary gland to serve the nutritional, microbiological and immunological requirements of the neonate [170]. This would account for the second major finding that 65% of non-GOS piglet faecal samples tested positive for RVA as opposed to 45% for GOS-fed piglet faecal samples, representing a significant reduction in the infectivity of RVA in the maternal GOS-fed group. This is the first time that late gestational GOS feeding of sows has been shown to reduce RVA in neonates. Whilst studies have shown that GOS administered to neonatal mammals directly reduced RV infection [121,171], this study has shown that late gestational feeding of sows with GOS significantly affects pathogen (RVA) infection and modulates the microbiome of both nursing sows and neonatal piglets. These effects likely manifest through entero-programming immune mammary pathways, as described by Rodríguez et al. [170], whereby colostrum and milk provide infants with gut microbes, immune cells and stem cells from the mother. Recent studies strongly suggest the existence of an endogenous entero-mammary pathway for some bacteria, including Lactobacillus, during lactation in the sow [172,173]. Given the prebiotic effect of GOS, as established by Lee et al. [169], it is not unreasonable to assume that late gestational feeding of GOS to sows would increase the abundance of probiotic bacteria such as Lactobacillus and Bifidobacterium in the sow GIT, and that these could be translocated from the GIT to the mammary gland and then to suckling neonates via colostrum. This could improve intestinal epithelial and mucus barrier development, antimicrobial peptide expression and innate immune cell expression in the neonate [174] and plausibly reduce RVA infection. However, the mechanism by which GOS increases IgG and IgA in sow colostrum remains to be elucidated. It could be speculated that GOS acting as a soluble cell surface decoy [32,38,39] reduces the RVA burden in the sow and bolsters RVA antigen sequestration by the immune system. A possible mechanism is the modulation of immunoglobulin secretion by the maternal microbiome. In murine models, gut-microbiome-induced maternal IgG is transferred to the neonatal intestine through milk via neonatal Fc receptors, directly inhibiting pathogen colonization [175]. For IgA, the gut microbiota induces Peyer’s-patch-dependent secretion of maternal IgA into milk. Antigen sampling by M cells in Peyer’s patches is the major source of migratory IgA plasma cells in mammary glands that produce maternal IgA found in milk [176]. Similar mechanisms are found in sows with IgA secreted by mammary-gland-recruited plasma cells exhibiting specificity for antigens in the maternal GIT, e.g., RVA. This entero-mammary link is due to the migration of lymphocytes originating in gut-associated lymphoid tissue via the bloodstream to the mammary gland [177]. Other mechanisms may include the viral triggering of GC-associated pathways, which presents antigens to the immune system and serves as a mechanism of tolerance or translocation outside the GIT [178]. GOS is known to upregulate secretory goblet cell lineage gene transcription factor 1, ATOH1 [155], and significantly increases GC expression in the GIT of suckling pigs [71]. Therefore, it is not unreasonable to speculate that GOS fed to sows also increases GC expression in the maternal GIT. Given the endemic nature of RVA in pigs and the presence of RVA and/or RVA antigens in sows, albeit the majority remain immune and asymptomatic, it may be plausible that increased GC expression in sows following GOS feeding increases RVA antigen sequestration and presentation to the immune system, thereby increasing RVA-specific IgG and IgA in colostrum. Thus, it may be speculated that there are at least two mechanisms whereby GOS fed to late gestational sows significantly reduces RVA infection in neonates, these being the translocation of GOS-induced probiotic bacteria to the neonate via entero-mammary pathways and the induction of colostral RVA-specific IgG and IgA via increased immunomodulatory GC expression due to GOS. Whether or not this is true for other viral pathogens remains to be observed.

There are few reports of XOS as a dietary intervention for gestational sows. However, Guo et al. [179] reported investigations with XOS and active yeast as dietary supplements that improved sow milk production and weight gained by the piglets. By 7 and 14 days of age, the piglet serum IgG contents were significantly increased by 19.42% and 13.57% on the combined XOS and active yeast treatment.

4. Conclusions

Alternatives to the use of antimicrobial growth promoters are of key interest to animal agriculture and notably in pig production, given the usage of AGPs. Environmental concerns have led to the abandonment of zinc oxide as an alternative to control diarrhoeal episodes and promote the growth of weaning pigs in various territories. Prebiotic galacto-oligosaccharides and xylo-oligosaccharides are not mechanistically direct replacements for these measures but have emerged as sustainable dietary supplements that can improve gut health and immunity to promote lifelong welfare and productivity. A summary of the inter-connected actions of GOS and XOS in the developing pig is presented in Figure 2.

Although GOS and XOS have been demonstrated to have effects at the cellular level with respect to the suppression of inflammatory cytokine/chemokine responses and reinforcing the expression of proteins required to maintain tight junctions, the majority of the biological effects are mediated through interactions with the intestinal microbiota. Not least is the increase in the abundance of SCFA-producing bacteria in the hind gut, notably the LAB species Lactobacillus, Bifidobacterium and Streptococcus, and for GOS, these include Prevotella, Barnesiella and Parabacteroides. Increases in the abundance of members of these genera accompany increasing SCFA concentrations, which lower the pH of the gut lumen, provide nutritional support for gut epithelial cells and modify the expression of genes associated with inflammatory responses. Which species become dominant will depend upon the available diversity within the microbiota, but the major benefit of using prebiotic supplements is that these effects can be achieved by modifying the resident microbiota, with subsequent positive benefits for pathogen suppression, immunomodulation and GIT physiology. In commercial pig production, pathogen suppression by antimicrobial treatments has become necessary due to persistent pathogen challenge in intensive rearing environments. The benefits of such treatments have been realized at the expense of rising antimicrobial resistance and the development of fragile and non-productive intestinal microbiomes. The use of antibiotics to suppress the entire microbiome versus prebiotic administration to enhance the microbiome needs to be considered in future production strategies. Overall, the administration of GOS and XOS prebiotics demonstrates positive outcomes for animal welfare as opposed to the traditional use of growth-promoting antibiotics.