Modern swine production systems rely on early weaning to optimize productivity, but this practice consistently exposes piglets to high physiological and nutritional stress (1–3). Early weaning is universally recognized as a critical period in a pig’s life, marked by detrimental impacts on health and performance (4). The abrupt dietary transition from highly digestible, liquid sow milk to a more complex solid feed compromises gastrointestinal health. This stress, combined with the piglet’s immature digestive and immune systems, results in reduced feed intake (anorexia), compromised nutrient absorption, disorganized epithelial architecture, and an imbalanced gut microbiota homeostasis (dysbiosis) (4–6). These effects frequently lead to diarrhea, slow growth, and mortality, causing significant economic losses (5, 7).

Historically, pig farmers and veterinarians extensively used prophylactic antibiotics, including antibiotic growth promoters (AGPs), to control post-weaning infections and mitigate the detrimental effects of stress (8). However, the irrational and widespread application of these antimicrobials has triggered therapeutic failures due to the global threat of antimicrobial resistance and marked intestinal dysbiosis (9, 10). In response to rising public health concerns, stringent governmental regulations, such as the 2006 European Union ban on AGPs (11) and the 2019/4 regulation restricting the prophylactic use of antimicrobials in medicated feed (12), have mandated a search for effective alternatives.

This global restriction has spurred intense research into safe, sustainable, and natural nutritional strategies capable of supporting intestinal health and replacing antibiotics in swine diets. Promising alternatives include acidifiers, prebiotics, probiotics and phytobiotics (plant extracts and essential oils, EOs) (13–15).

Current strategies focus on incorporating specific compounds with established efficacy in enhancing gut health. Fructooligosaccharides (FOS) are non-digestible carbohydrates that resist hydrolysis by porcine intestinal enzymes. As established prebiotics, short-chain fructooligosaccharides (scFOSs) selectively stimulate the growth of beneficial bacteria, such as Lactobacillus spp. and Bifidobacterium spp. (16). Their fermentation by gut microbes yields beneficial short-chain fatty acids (VFAs), notably acetic and butyric acid, which are critical for colonocyte energy and intestinal barrier function maintenance. Dietary scFOSs consumption was shown to enhance intestinal integrity, reduce diarrhea index, and attenuate mucosal inflammation in weaned piglets (17). EOs derived from aromatic plants, such as oregano (Lippia origanoides; Lo) and clove (Eugenia caryophyllata; Ec), are rich in phenolic compounds like thymol, carvacrol, and eugenol. These compounds exert antibacterial and antioxidant effects, helping to dampen the negative metabolic impact of weaning stress on enterocytes (as evidenced by increased plasma citrulline concentrations). These formulations, particularly those combining EOs, have demonstrated improvements in intestinal architecture, as increased villus height (Vh) and ratio to crypt depth (Cd), digestive enzyme activity (13, 17) and microbiota modulation (17). Moreover, sodium humates (SH), components sometimes included in these formulations, possess high adsorptive capacity, potentially assisting in reducing diarrhea incidence and influencing gut ecology (17–19).

Research indicates that combining different alternatives to antibiotics, often encapsulated for targeted release, is a more robust strategy than using single additives to replace antimicrobial use entirely. Technologies such as microencapsulation and additive combinations have been shown to be more effective in enhancing immuno-antioxidant capacity, improving intestinal barrier function, and modulating hindgut microbiota structure (13, 20). Given the potential demonstrated by the functional ingredients previously mentioned, the objective of the present study was to evaluate the effects of three additives: scFOSs, a combination of EOs of Lo and Ec with SH, and a combination of scFOSs and EOLo, focusing on key indicators of intestinal health, morphology, metabolism, and microbiota modulation in weaned piglets.

Animals remained healthy, with consistent food and water consumption and no evidence of diarrhea throughout the trial.

Early weaning imposes a multifactorial stress response in piglets with pronounced alterations in gastrointestinal morphology and immune function (1, 2). Since the global intensification of swine production systems made early weaning a standard practice, numerous studies have focused on optimizing post-weaning diets to mitigate these detrimental effects. Without appropriate nutritional and management strategies, the acute post-weaning phase consistently results in impaired performance and significant economic losses (4, 40).

In this context, the search for natural feed additives capable of sustaining intestinal integrity, enhancing digestive functionality, and reducing reliance on antibiotics has intensified. Among these, scFOSs have been widely evaluated as prebiotic ingredients capable of promoting beneficial bacterial populations, improving enterocyte maturation, and strengthening mucosal barrier function. Their fermentation by commensal microbiota resulted in production of VFA, modulation of intestinal pH, and competitive exclusion of pathogens, collectively contributing to improved gut health in weaned piglets (41, 42). Plant-derived EOs, particularly those rich in phenolic compounds such as thymol, carvacrol, and eugenol, including extracts from Lo and Ec, have gained substantial interest as natural antimicrobial and anti-inflammatory tools for swine nutrition.

When included as dietary additives, EOs were shown to improve gastrointestinal function and mitigate the adverse consequences of weaning stress (10, 43, 44). Their bioactive constituents modulated intestinal redox homeostasis, suppressed opportunistic pathogens, and favorably influenced the microbial community structure in weaned pigs (13). Nonetheless, EOs vary widely in biological activity depending on botanical source, extraction method, degree of microencapsulation, and administration strategy (45). Consequently, each formulation must be specifically assessed under real production conditions to determine its efficacy and applicability. Humic substances, including SH, have emerged as promising functional additives in swine nutrition. Their physicochemical properties enable toxin binding, modulation of oxidative stress, and immunomodulatory activity, thereby enhancing gastrointestinal epithelial resilience (46, 47). In young pigs, SH supported mucosal recovery during the post-weaning transition by increasing antioxidant defenses and attenuating inflammatory signaling, notably through reductions in pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β (18, 19). Concomitantly, SH contributed to improved intestinal barrier integrity via the upregulation of tight junction proteins, including occludin and claudins. These effects were accompanied by a favorable modulation of gut microbiota, characterized by increased abundance of beneficial genera and reduced proliferation of enteropathogenic bacteria, collectively reinforcing intestinal homeostasis (19).

Together, these additive classes represent distinct but potentially complementary strategies to mitigate the digestive and immune challenges experienced by piglets during weaning. The present study evaluated the effects of three functional dietary strategies, compared with a basal diet (control group): scFOSs, a blend of EOs with SH (EOs-SH), and a combination of scFOSs and EOLo, on physiological, biochemical, microbiological, and mucosal parameters in piglets during the early post-weaning phase.

The post-weaning phase is characterized by pronounced neuroendocrine activation, with cortisol serving as a key biomarker of the systemic response to environmental, nutritional, and social challenges (25, 29). In the present study, all piglet groups exhibited similar plasma cortisol concentrations throughout the sampling period, indicating that none of the supplementation strategies noticeably modulated the systemic stress response. Notably, when sampling days were considered, the highest cortisol values were observed at weaning (Table 2), highlighting the abrupt transition from sow-dependent to independent feeding as the period of greatest physiological stress. Thereafter, cortisol concentrations showed a clear decline by day 8 post-weaning, which likely reflects the natural attenuation of hypothalamic–pituitary–adrenal axis activation during the adaptation period (48).

Plasma citrulline dynamics clearly reflected the transient impairment and subsequent recovery of enterocyte functional mass typically associated with the post-weaning period. Across days, citrulline concentrations declined markedly on days 4 and 8, mirroring the acute suppression of enterocyte function widely documented during early weaning stress (13, 27). From day 12 onward, citrulline levels progressively increased and returned to baseline by day 15, indicating recovery of mucosal functionality. These findings support the use of citrulline as a sensitive indicator of mucosal integrity and enterocyte mass in swine (29).

The enhanced citrulline concentrations observed in EOs-SH-supplemented piglets may be biologically explained by known properties of the EO components. Molecules such as carvacrol and thymol (present in Lo) and eugenol (derived from Ec) have been shown to modulate key transcriptional pathways involved in oxidative and inflammatory responses, including NF-κB and Nrf2 signaling (49, 50). Through the suppression of oxidative stress, scavenging of reactive oxygen species, and attenuation of inflammation and apoptosis (10, 49), these compounds help preserve enterocyte viability and metabolic performance during the critical early post-weaning window. Collectively, the consistent elevation of citrulline in EOs-SH group suggests that this formulation was particularly effective at mitigating the early decline in enterocyte function and supporting intestinal mucosal resilience.

Gastrointestinal pH values remained within the expected physiological range for piglets of this age and intestinal segment, in agreement with previous reports in weaned pigs (13, 51). No treatment effects were observed in the ileum, caecum, or colon, indicating that the evaluated supplements did not induce substantial shifts in luminal acidity in the distal gut. The only significant difference was detected in the stomach, where piglets receiving the combined scFOSs-EOLo supplementation exhibited a moderately higher gastric pH compared with the control group. Functionally, a slight reduction in gastric acidity may help preserve mucosal integrity and enhance the survival of beneficial bacteria transiting through the stomach. However, excessive increases in gastric pH could theoretically impair the activation of pepsin and early protein digestion, as pepsin requires an acidic environment to function optimally (52). In the present study, the magnitude of the change was modest and unlikely to compromise digestive physiology. Overall, the data indicate that none of the supplementation strategies produced disruptive shifts in gastrointestinal pH, supporting their safety and compatibility with normal digestive function in early-weaned piglets.

Digestive enzyme activity displayed clear segment-specific patterns, indicating that the supplements differentially influence brush-border function along the small intestine. Compared with the control group, scFOSs supplementation increased lactase activity in the proximal jejunum, whereas both scFOSs and the EOs-SH formulation enhanced duodenal maltase activity. These responses suggest improved brush-border maturation or modulation of enterocyte turnover, consistent with the well-described trophic effects of prebiotics and phytogenic compounds on the intestinal epithelium (10, 53–55).

The increased maltase activity observed in the EOs-SH group aligned with the higher plasma citrulline concentrations recorded in these animals, supporting the interpretation that this supplement favored greater preservation of enterocyte functional mass during the acute post-weaning phase. A similar enhanced brush-border enzymatic activity with Lo and Ec EOs has been previously reported by Diéguez et al. (13). In contrast, the combination of scFOSs-EOLo did not enhance digestive enzyme activity and consistently showed lower values than those obtained with individual supplements. This lack of additivity suggests potential interference at the metabolic or microbial level, such as competition between fermentable substrates and antimicrobial phytochemicals, that may limit epithelial adaptation rather than promote synergistic effects. Taken together, the enzymatic profile indicates that scFOSs and EOs-SH exert beneficial but non-synergistic effects on mucosal maturation, and that combining both supplements may attenuate rather than potentiate their functional benefits.

Histomorphometric parameters exhibited limited variation in dietary treatments in this study. No significant effects were detected for IAA or for the Vh: Cd ratio, and most values remained within the expected physiological range for piglets of this age. A reduction in ileal Cd was observed in EOs-SH and scFOSs-EOLo groups compared with the control group, which may reflect decreased proliferative turnover or attenuated inflammatory remodeling of the mucosa.

Phytogenic feed additives, particularly formulations containing thymol, carvacrol, and eugenol, can promote beneficial architectural changes such as reduced Cd, increased Vh, and improved Vh: Cd ratios (53, 54). Similar reductions in Cd and improvements in villus morphology have been reported by Diéguez et al. (13) in piglets supplemented with Lo and Ec EOs. However, unlike those studies, our results did not reveal a consistent treatment-driven improvement across all histomorphological indices. The decrease in Cd in EOs-SH and scFOSs-EOLo animals may suggest a mild trophic or anti-inflammatory effect of these additives, but the overall mucosal architecture remained largely unchanged. This limited response could be related to the duration of supplementation or the intensity of the post-weaning challenge.

Across all groups, IEL counts remained within the physiological range for healthy piglets (0–10 IELs/100 enterocytes), yet meaningful differences emerged within this normal window. The lowest IEL densities in the ileum were observed in piglets supplemented with scFOSs, while the EOs-SH group exhibited comparably low values, although these did not differ significantly from the control group. Lower IEL counts are widely interpreted as indicative of reduced basal immune activation, improved epithelial stability, and diminished inflammatory signaling (56, 57). These findings align with the known anti-inflammatory and antioxidative properties of scFOSs (58) and phytogenic compounds such as thymol, carvacrol, and eugenol, which attenuated NF-κB-mediated responses and mitigated epithelial stress (10, 55). The scFOSs-EOLo formulation did not reproduce these benefits, suggesting that the immunomodulatory effects observed with scFOSs or EOs-SH were not additive and that co-administration of distinct bioactive compounds does not necessarily enhance mucosal immune regulation.

A significant treatment effect was detected for bacterial adherence to intestinal mucus, with the EOs-SH group exhibiting a higher percentage of E. coli O157: H7 adhesion compared with the control. Although in vitro adhesion assays do not necessarily predict pathogen colonization in vivo, they are sensitive indicators of biochemical modifications in the mucus layer, such as changes in glycan composition, mucin sulphation, or cross-linking. Similar increases in pathogen adhesion have been described in piglets supplemented with EOs, where phytogenic compounds stimulated mucin biosynthesis and altered mucus chemistry (59–62). Thus, the increased adhesion observed in EOs-SH piglets may reflect enhanced mucus complexity or secretion rather than a compromised barrier.

Goblet cell density is commonly considered an indicator of mucus production and has been associated with mucus barrier function and bacterial adhesion (63, 64). However, in the present study, no treatment-related differences were observed in goblet cell numbers, indicating that the increased bacterial adhesion to mucus in EOs-SH piglets was not associated with changes in goblet cell abundance. These findings reinforce the notion that mucus quality and functionality are not solely determined by goblet cell density, but are strongly influenced by qualitative, biochemical, and structural properties of the mucus layer, including mucin composition, organization, and epithelial secretory responses, as previously mentioned (65, 66). Indeed, diet-induced alterations in mucin composition, glycosylation, viscosity, or secretion dynamics may occur independently of goblet cell numbers and can modulate bacterial-mucus interactions and intestinal permeability (67–70).

When viewed alongside the other mucosal parameters, a coherent pattern emerges. Piglets supplemented with scFOSs exhibited the lowest ileal IEL counts, indicating reduced baseline epithelial immune activation. Similar values were achieved with EOs-SH supplementation, although no statistically significant differences from the control group were observed. Overall, the combined findings suggest that the additive-induced changes in mucus properties, particularly in the EOs-SH group, occurred in the context of low immune activation and preserved mucosal integrity. In this regard, previous studies highlight that improvements in mucosal barrier function induced by phytogenic compounds are often associated with broader regulatory effects on the intestinal ecosystem, including modulation of host–microbe interactions (17).

Microbiota diversity and composition were interpreted in relation to the intestinal and immunological outcomes observed in the present study. At the community level, the absence of significant differences in alpha diversity and the overlapping patterns observed in beta diversity analyses indicate that the dietary interventions did not induce broad restructuring of the microbial ecosystem. Instead, the treatments promoted selective shifts in specific bacterial taxa with functional relevance. Such targeted modulation is particularly expected during the early post-weaning period, which is characterized by high microbiota instability and marked inter-individual variability.

In line with this conceptual framework, microbiome analysis revealed clear, treatment-specific shifts that aligned with the functional and immunological patterns observed in the present study. Supplementation with EOs-SH produced the most pronounced ecological modulation, significantly reducing several genera commonly associated with proinflammatory or dysbiotic states, including Clostridium sensu stricto 1, Terrisporobacter, Prevotella 115, and Subdoligranulum. These taxa have been described as markers of intestinal dysbiosis or mucosal stress in pigs, often enriched under fermentative imbalance, epithelial perturbation, or immune activation. Their reduction therefore suggests a microbiota configuration less conducive to inflammatory signaling. These findings are consistent with previous reports describing the modulatory effects of phytogenic compounds on gut microbial communities. Similar shifts in microbial profiles, characterized by a reduction of Firmicutes groups linked to fermentative by-products, epithelial stress, and mucosal inflammation, were reported by Ángel-Isaza et al. (17), together with improvements in gut barrier function and immune indices. In line with this framework, the microbial changes observed in the EOs-SH group coincided with lower ileal IEL counts and preserved mucosal architecture, supporting a coordinated reduction in epithelial immune activation.

In contrast, scFOSs supplementation induced a robust enrichment of Lactobacillus, a hallmark response to prebiotic oligosaccharides. Expansion of Lactobacillus populations has been consistently associated with enhanced epithelial integrity, improved mucin metabolism, increased immune tolerance, and the production of beneficial metabolites in weaned piglets (16, 71, 72). Accordingly, the increased abundance of Lactobacillus observed in scFOSs-fed piglets in the present study was accompanied by reduced IEL density, supporting an improved mucosal immune equilibrium during the post-weaning period.

In addition to Lactobacillus enrichment, scFOSs supplementation was associated with increased relative abundance of Streptococcus and Methanosphaera. These taxa are frequently linked to enhanced carbohydrate fermentation, cross-feeding interactions, and hydrogen utilization within the intestinal ecosystem (71, 73). Although their functional roles in early-weaned piglets remain less clearly defined, such microbial shifts are consistent with a transition toward a more active saccharolytic fermentative environment rather than a pro-inflammatory microbial profile, as previously described in prebiotic-supplemented piglets (16, 74). Taken together, these treatment-specific microbial shifts-characterized by a reduction of taxa associated with proinflammatory or dysbiotic states in the EOs-SH group and the enrichment of Lactobacillus in scFOSs-supplemented piglets-provide a biologically coherent link with the observed improvements in mucosal immune tone, digestive enzyme activity, and markers of enterocyte functionality. These findings reinforce the concept that targeted microbial modulation, rather than large-scale diversity shifts, can meaningfully support intestinal maturation during the early post-weaning transition.

Notably, the combined supplementation of scFOSs and EOs-Lo did not result in significant microbial modulation, despite the clear effects observed when each additive was administered individually. This lack of response parallels the attenuated effects detected for digestive enzymes, mucus-related parameters, and mucosal immune markers in the combination group. Non-additive or antagonistic interactions between prebiotic-driven saccharolytic niches and the antimicrobial or regulatory effects of phytogenic compounds may underlie this outcome. Similar constraints have been described when functional feed components target overlapping metabolic pathways or compete for ecological niches (17).

In the present study, no statistically significant differences were detected among treatments for any individual or total VFAs, although the EOs-SH group consistently showed numerically higher concentrations compared with the other groups. These findings suggest that, under the conditions evaluated, dietary modulation of microbial fermentation was limited. Importantly, the absence of statistical significance is consistent with previous short-duration post-weaning studies. Consistent with the present findings, no differences in cecal VFAs concentrations between supplemented and control piglets were observed by Decundo et al. (51) under a 15-day experimental period similar to that applied in this study, suggesting that early post-weaning dynamics and high inter-individual variability may mask treatment effects during this acute window. By contrast, longer supplementation periods have demonstrated clearer responses. For instance, Diéguez et al. (13) observed higher acetate and total VFAs concentrations in piglets receiving EOs-based formulations. The numerical increases seen in our EOs-SH group may reflect a shift toward enhanced fermentative output.

As growth performance data from the same experimental system have been previously published, the physiological and intestinal findings reported here provide mechanistic support for the productivity improvements described in our previous study (23) in post-weaning piglets supplemented with a prebiotic formulation, EOs of oregano and clove plus SH and a combination of prebiotics and EOs. In that large-scale study (two replicates of 400 animals each), the group supplemented with EOs of oregano and clove plus SH achieved the highest average daily gain (0.46 ± 0.08 kg/day per animal; p < 0.001) and the lowest feed conversion ratio (1.75 ± 0.29 kg feed/kg weight gain; p < 0.001), as well as the highest body weight at day 70 (28.99 ± 4.20 kg; p < 0.001). At day 40, both the EOs-SH and the prebiotic groups showed similarly higher body weights compared with the control, with the prebiotic group presenting the numerically highest value (11.05 ± 1.56 kg), although no significant differences were detected between these two treatments. The combined treatment did not differ from the control at any time point. That work lends external validity to our observations of enterocyte preservation, enzymatic enhancement, and microbial modulation in the EOs-SH group. The fact that scFOSs alone improved certain functional markers, but that the combined treatment did not surpass the individual additives, mirrors the productivity results and supports the hypothesis of functional interference in combined supplementation. Taken together, these observations suggest a coherent mechanistic pathway: supplementation with EOs-SH enhances intestinal function and microbiota balance, which in turn may translate into improved growth performance, as previously evidenced in Mozo et al. (23).

In summary, the present study shows that scFOSs and the EOs-SH formulation exerted distinct and biologically meaningful effects on intestinal maturation, mucosal immunity, and microbial ecology during the early post-weaning period, whereas their combination did not provide additive benefits. The capacity of EOs-SH to preserve enterocyte functional mass, enhance brush-border activity, and modulate potentially proinflammatory taxa, together with the prebiotic-driven expansion of Lactobacillus and attenuation of mucosal immune activation in scFOSs-fed piglets, underscores their value as early nutritional interventions. Importantly, these findings align with a growing body of evidence supporting the prophylactic inclusion of functional feed additives as a means to stabilize gastrointestinal physiology and reduce vulnerability during the critical weaning window. In an era marked by increasing restrictions on antimicrobial use and the global imperative to curb antimicrobial resistance, such dietary strategies represent promising tools to enhance resilience in young pigs while reducing reliance on conventional antibiotics. Future studies with longer supplementation periods and integrative functional endpoints will help define the durability, scalability, and practical relevance of these interventions in animal production.