Impact of a Phytogenic Feed Additive on Diarrhea Incidence, Intestinal Histomorphology and Fecal Excretion of F4-Fimbriated Enterotoxigenic Escherichia coli in Post-Weaning Piglets

Abstract

This study investigated the effects of a phytogenic feed additive (PFA) containing a blend of herbs, plant extracts and essential oils from the Lamiaceae, Schisandraceae, Zingiberaceae and Fabaceae families on the fecal score, intestinal histomorphology and fecal excretion of F4-fimbriated enterotoxigenic Escherichia coli (F4-ETEC) in post-weaning piglets. Thirty 31-day-old weaned piglets were randomly allocated to three treatment groups. The positive control (PC) group received colistin via drinking water from d 8 to 14 post-weaning and the same basal diet as the negative control (NC) group; the treatment group received the basal diet with PFA supplementation (1 g/kg of feed). The experiment lasted 21 days. At day 9 post-weaning, all piglets were orally administered 3.0 × 10¹⁰ CFU/piglet of the F4-ETEC strain. The PC piglets had higher fecal consistency than the NC and PFA piglets. PFA supplementation resulted in a lower percentage of piglets excreting F4-ETEC in the feces on days 4–7 post-challenge than in the NC group (p < 0.05) but a higher percentage versus the PC group on day 3–7 post-challenge (p < 0.05). The number of goblet cells (GCs) in the jejunum of the PFA piglets was higher than the NC and PC piglets (p < 0.01). The GC density in the jejunum of the PFA piglets was larger than in the PC piglets (p < 0.05) and similar to the NC piglets (p > 0.10). Mucus thickness in the jejunum of the PFA piglets was similar to the NC piglets and PC piglets (p > 0.10). In conclusion, PFA supplementation to the F4-ETEC-challenged piglets reduced the prevalence of fecal E. coli excretion and improved jejunal histomorphology.

1. Introduction

The weaning of young animals seems to be an everlasting challenge. It is a critical period of significant physiological and psychological stress, accompanied with a high susceptibility to enteric infections, gut disorders such as post-weaning diarrhea and a drop in performance [1]. A major factor contributing to these disorders is the shift from highly digestible sow milk to a solid diet based on plant proteins, starches and complex carbohydrates, which the still-maturing gut of the piglet struggles to adapt to during weaning [2]. The abrupt dietary shift can lead to reduced feed intake, weight loss and compromised immune function [3]. Additionally, the gut microbiota undergoes substantial changes, often resulting in bacterial dysbiosis, which exacerbates the risk of gastrointestinal issues [4].

In the past, bacterial dysbiosis could be controlled through in feed or therapeutic antibiotics or pharmaceutical levels of zinc oxide (ZnO). However, current regulations ban the use of these substances due to concerns over antibiotic resistance and environmental impacts. Consequently, other management and nutritional measures are needed to ease the weaning process. These include the use of different in-feed additives solutions designed to support gut health and enhance the piglets’ resilience to stress and disease [5].

One potential measure to support resilience and strengthen piglets to overcome weaning challenges are phytogenics [6]. The term “phytogenics” refers to ingredients naturally derived from or found in plants, such as herbs or essential oils. These components consist of bioactive molecules, for instance terpenes, polyphenols and glycosides. While their exact mechanisms of action remain not fully clear, research highlights several beneficial effects of phytogenics for livestock health and productivity. These include stimulating early feed intake, digestion, and the immune system, exhibiting anti-inflammatory and antimicrobial properties, modulating intestinal microbiota, and providing antioxidant benefits [7,8]. Given the variety of active compounds with various beneficial effects available, careful selection is essential. Extensive literature on carvacrol shows it can reduce inflammation by inhibiting the production of pro-inflammatory cytokine production [9], mitigate oxidative stress by free radical scavenging [10] and have antibacterial and antibiofilm properties against E. coli [11,12]. Other substances, such as trans-anethole, fenugreek and curcumin, being the main active component of turmeric, have also demonstrated improvements in immune function, gut inflammatory response and intestinal mucosal integrity [7,13,14,15,16,17,18]. Although specific piglet trials and in vivo evidence are less common, the literature suggests that there is potential for phytogenics like carvacrol to disrupt bacterial communication and inhibit virulent behaviors such as biofilm formation or bacterial adhesion [12,19,20]. This mechanism of action is known as quorum sensing and has been suggested as a potential attenuator of different veterinary pathogens [21]. Additionally, while the antibacterial effect of phytogenic substances, particularly essential oils, are well-documented in vitro, the required dosages often are not commercially viable, and there is growing interest in exploring the impact of selected phytogenic substances used below minimum inhibitory concentration levels on gut health parameters in piglets.

The aim of this study was to evaluate the effect of a feeding strategy including a phytogenic feed additive (PFA) against an F4-fimbriae-bearing enterotoxigenic Escherichia coli (F4-ETEC) challenge in weaned piglets.

2. Results

The piglets from the three experimental treatments showed similar feed intake and growth performance over the experimental period. Feed intake and BW values are reported in Supplementary Table S1.

2.1. Fecal Consistency Score

Overall, the positive control (PC) piglets had significantly higher fecal consistency scores than the negative control (NC) or PFA treatments (6.4 vs. 5.7 vs. 5.9 ± 0.13, p < 0.001, respectively). The fecal consistency score overtime is represented in Figure 1. Numerically, piglets in the PFA treatment group had an intermediate fecal score between the PC and NC treatments, which was stable at the optimal level of the scale (6) during the entire experiment.

2.2. Prevalence of F4-ETEC Shedding in Piglets

The percentage of F4-ETEC shedding in the piglets over the post-weaning period is presented in Figure 2. In this challenge model, F4-ETEC shedding is expected to peak in the first 5 days post-inoculation. As expected, the inoculated F4-ETEC strain was not detected (below the LOD) at day 8 post-weaning, before inoculation. On the first day of post-inoculation (day 10), all piglets in the PFA group were shedding F4-ETEC; however, 4 piglets from both the PC and NC groups were not shedding F4-ETEC. All NC piglets were shedding F4-ETEC on days 11, 12, 13 and 14, while the PC piglets were not shedding F4-ETEC on days 13 and 14. The supplementation of the PFA to the piglets significantly reduced the shedding of F4-ETEC compared to the NC piglets on days 13, 14 and 16.

2.3. F4-ETEC Concentration in Feces

Overall, the PC piglets had a significantly lower concentration of F4-ETEC in feces than the NC or PFA treatments (2.84 vs. 4.90 vs. 4.47 ± 0.175 log₁₀ CFU/g feces p < 0.001, respectively). The concentration of F4-ETEC in feces overtime is presented in Figure 3. The concentration of F4-ETEC in PC piglets was consistently lower compared to NC piglets from d 12 to 16 (p < 0.05), indicating the success of the challenge model. The concentration of F4-ETEC in the PFA piglets was, numerically, between the PC and NC piglets from d 12 to 16, being significantly different on d 13 (p < 0.05).

2.4. Intestinal Histomorphometry

The histomorphometry analysis of the jejunum and colon in piglets at day 21 post-weaning is presented in Table 1. In the jejunum, the PFA group exhibited an increase in the number of GCs per villus compared to both the PC and NC groups (p < 0.01). The number of GCs per 100 µm of villi was higher in piglets fed the PFA compared to the PC treatment (p < 0.05). Additionally, mucus thickness in the jejunum was significantly greater in the NC group compared to the PC and PFA groups (p < 0.05). However, no significant differences were observed in CD or GC counts in the colon across treatments.

3. Discussion

The results of this study highlight the efficacy of PFAs in mitigating the adverse effects of weaning stress in piglets. Piglets receiving the PFA resulted in a numerical intermediate fecal score between the PC and NC groups, which was stable at the optimal score for the full trial period after challenge, significantly reduced the percentage of piglets shedding F4-ETEC, reduced the concentration of F4-ETEC in feces and increased GCs at the jejunum. Thus, the data reported in this study indicate the ability of PFA piglets to recover earlier than non-medicated piglets from an E. coli challenge. Furthermore, the model used in this experiment to challenge the piglets with F4-ETEC was successful in evaluating the PFA, as the NC piglets had lower fecal scores, a higher proportion of piglets excreting F4-ETEC and higher concentrations of F4-ETEC shedding than the PC piglets. This is in accordance with previous studies performed by the authors using this model [22].

Post-weaning diarrhea is often associated with the fecal shedding of a large number of β-hemolytic enterotoxigenic E. coli serotypes that colonize the small intestine after weaning [1,2,4,23]. In this study, the PFA group had a smaller proportion of piglets shedding F4-ETEC during the experiment and excreted lower concentrations of F4-ETEC than the non-medicated piglets. This is in accordance with previous research where phytogenic compounds were able to reduce fecal E. coli shedding [24]. Additionally, in this study, the fecal score of piglets receiving the PFA was numerically stable at the optimal level, with an intermediate value between the PC and NC. In previous research, some studies [13,25] also found numerical differences in the fecal scores only when PFAs were evaluated using similar challenge studies, whereas other studies were able to demonstrate significant improvements in fecal scores [7,14,26,27,28]. This study highlights the consistency in fecal scores across treatment groups even under challenged conditions, with the majority of piglets ranking between 5 and 7 on a scale of 2 to 9. This suggests that the extreme ends of the scale did not accurately represent the pigs’ conditions, as most were likely in acceptable health. As noted by other authors [29], this underscores the importance of reporting additional biological parameters in challenge models to better assess health outcomes.

Goblet cells in the intestinal epithelium produce and maintain the mucus layer in the lumen, bringing protection to the host against pathogens and harmful substances [2,30,31]. In this study, the significant increase in the GC absolute number and its density in the jejunum of piglets receiving the PFA suggests improved mucosal protection when PFAs are supplemented to post-weaning piglets. This aligns with previous research reporting that phytogenic compounds can support gut health through various mechanisms, including the promotion of intestinal GC proliferation in piglets [14,15,32] and other monogastric species as broilers [33,34]. While the PFA group showed improvements in goblet cell parameters, no significant differences were noted in the villus height or crypt depth compared to controls. This may suggest that while PFAs enhance certain aspects of intestinal morphology, other factors, such as the duration of supplementation or the specific composition of the diet, may also play critical roles in influencing these parameters. Future studies could explore the long-term effects of PFAs on gut morphology and function, as well as the optimal timing for supplementation.

The PFA used in this study contains several active compounds, each contributing unique benefits. Carvacrol can be found in certain plants of the Lamiaceae family and is well known for its antimicrobial [35,36,37,38], anti-inflammatory [39,40] and antioxidant properties [41,42]. In line with our findings, it has been demonstrated that PFAs containing carvacrol among other phytogenic compounds improved the fecal score, gastrointestinal integrity and immune status of challenged piglets [13,14,28,43]. Trans-anethole, from the Schisandraceae family, has been shown to promote the secretion of digestive enzymes and improving nutrient absorption, antioxidant status and immune function in broilers [44,45]. Turmeric, a member of the Zingiberaceae family, is renowned for its anti-inflammatory and antioxidant properties, primarily attributed to its active compound, curcumin. Curcumin has been shown to reduce inflammation in the gut, which is crucial during the stressful weaning period [16,17,18,46,47]. In line with our results, some authors [7,13,15,48] reported that PFAs containing curcumin among other phytogenic compounds positively improved the intestinal mucosal barrier integrity, morphology and immune status of weaned piglets challenged with enterotoxigenic E. coli. Lastly, fenugreek, from the Fabaceae family, has been recognized for its antidiabetic, anti-inflammatory and cholesterol-lowering effects [49]. The galactomannans present in fenugreek can potentially act as prebiotics to influence the intestinal adhesion of pathogenic bacteria [50]. In previous studies, fenugreek seed extract supplementation improved intestinal histomorphology and reduced diarrhea scores in weanling piglets [24,25,26,51,52]. In summary, post-weaning diarrhea is caused by multiple factors, with pathogenic strains of E. coli often associated with its incidence. Literature evidence demonstrates the potential of phytogenic compounds to support piglets during these critical post-weaning periods. However, knowledge of their in vivo mode of action is scarce and cannot be addressed mechanistically in this work.

Considering the components and inclusion rates of their individual molecules, a direct antibacterial effect against specific pathogens is unlikely due to the concentration of active compounds being below the reported minimum inhibitory concentrations for most bacterial species. There are likely several benefits for the host due to the above cited effects from the phytogenic compounds within the PFA. However, it could be speculated that the virulent behavior of bacteria such as pathogenic E. coli may be influenced by different mechanisms, as shown for example with the production of F4 fimbriae in F4-fimbriated E. coli being regulated by quorum sensing [53]. Quorum sensing has been suggested as a novel strategy for combating veterinary infections by the attenuation of bacterial pathogenesis [21], and the ability of phytogenic compounds such as carvacrol to interfere with bacterial quorum sensing at sublethal levels has been previously reported in in vitro models [19,20]. Therefore, one potential target of at least parts of the specific phytogenic formulation used in this study could be within such virulence factor regulation mechanisms of pathogens. To further advance our understanding of this mechanism of action, future research using in vivo models is needed to confirm or reject this hypothesis.

In conclusion, the incorporation of a specifically formulated PFA in the diets of weaned piglets challenged with F4-ETEC appears to offer significant benefits in terms of reducing pathogen shedding and improving intestinal histomorphometry. This is particularly important in the context of the ban on traditional antimicrobial growth promoters, highlighting the need for alternative strategies to maintain animal health and productivity. Future research will need to evaluate the hypothesis that PFAs may influence virulence factor regulation mechanisms of pathogens in vivo to optimize such approaches.

4. Materials and Methods

4.1. Experimental Design

The experiment was conducted at the Schothorst Feed Research (SFR) laboratory facilities for pigs (Lelystad, The Netherlands). The study was approved by the Dutch Central Authority for Scientific Procedures on Animals (license AVD24600202114588, 18 January 2022) and the Animal Care and Use Committee of SFR. The protocol of the experiment was carried out in compliance with the ARRIVE guidelines and in accordance with the Dutch law on animal experimentation, which complies with European Directive 2010/63/EU on the protection of animals used for scientific purposes. A total of 30 weaned piglets [Tempo × TN70 (Large White × Norsvin Landrace)] aged 30.7 ± 0.70 days, F4ac-ETEC susceptible and with an initial live weight of 9.4 ± 1.28 kg (mean ± SEM) were used in a completely randomized design. The experiment consisted of 3 experimental treatments; piglets of both sexes were blocked based on gender, weaning weight, weaning age, litter of origin and sow parity. The experimental treatments were as follows: (1) positive control (PC, n = 10), where piglets were fed a basal diet and provided with colistin (COLISOL^® 250 00 I.E./mL; Dopharma; REG NL 2182/UDD) via drinking water between d 8 and 14 post-weaning; (2) negative control (NC, n = 10), where piglets were fed a basal diet and provided with regular drinking water; (3) phytogenic feed additive (PFA, n = 10), where piglets were fed a basal diet supplemented with a PFA at 1 g/kg of feed (Cinergy^® Protect, Cargill Incorporated, Engerwitzdorf, Austria). The experimental diets were given from weaning until the end of the experiment at day 21 post-weaning. For the first three days of the experiment, all piglets were treated with colistin via drinking water to standardize and reduce ETEC presence in the intestine before the start of the experiment. At day 9 post-weaning, all piglets were orally administered 3.0 × 10¹⁰ CFU/piglet of an F4ac enterotoxigenic ETEC strain (F4+, Lt+ and STb+). The ETEC strain was orally administered via syringe in a 5 mL solution (2.8 × 10¹⁰ CFU before the first inoculated piglet and 3.2 × 10¹⁰ CFU after the last inoculated piglet).

4.2. Animal Inclusion and Exclusion Criteria

Only litters that would be 28 days or older at weaning were selected for blood sampling during the suckling period to determine F4ac susceptibility via an SNP2 gene expression analysis [22]. Out of these litters, 140 piglets were selected based on birth weight and average body weight of the litter by eye at the day of blood sampling. Out of the 140 blood-sampled piglets, 80 turned out to be F4ac-ETEC susceptible. F4ac-ETEC-resistant piglets were excluded from selection at weaning. Moreover, piglets showing signs of injury or illness at the time of blood sampling or weaning were excluded from selection, as well as piglets that were medically treated during the suckling period. At weaning, 30 out of 140 piglets were selected based on F4ac susceptibility (only F4-susceptible piglets were selected). After inclusion in the study, piglets were excluded when a humane endpoint, approved by the Animal Care and Use Committee of SFR, was met.

4.3. Animal Housing and Management

Animals were housed in pens with a size of 2.00 × 1.00 m equipped with slatted floors and a rubber mat in a climate-controlled room. The rubber mat covered 1.2 m² of the slatted floor. Piglets were housed in groups of 5 piglets per pen. Each pen contained 2 feeding troughs and 2 drinking nipples. Piglets were provided with non-edible enrichment and a cotton rope. The non-edible enrichment was alternated daily, and the cotton rope was replaced at day 8, 11, 13, 15 and 17 post-weaning to keep the infection pressure low during the F4-ETEC challenge. Room temperature was automatically regulated by a climate computer following a temperature curve starting at 28 °C on the day of weaning to 23 °C at 21 days post-weaning. The room was ventilated using outdoor air. Humidity in the room was dependent on the outdoor humidity and ventilation rate. Artificial lights were provided from 7.00 to 17.00 h. Animals had ad libitum access to feed and water.

4.4. Feed Preparation and Diet Composition

The experimental diets were manufactured in 3 mm pellet form at Research Diet Services B.V. (Wijk bij Duurstede, The Netherlands). Diets were prepared by double mixing. First, a sufficient amount of the basal diet was produced for the three experimental treatments. Thereafter, the basal diet was split into three portions: (1) the basal diet that was administered to piglets under the PC treatment, (2) the basal diet that was administered in the NC treatment and (3) the basal diet supplemented with the PFA. The PFA used in this experiment is a blend of herbs (fenugreek and turmeric) and essential oils (with main compounds trans-anethole and carvacrol). The diets were formulated to meet the requirements for all essential nutrients for piglets according to the CVB (Centraal Veevoeder Bureau, Dutch feed table). The detailed ingredient content and nutrient composition of the basal diet is given in Supplementary Table S2.

4.5. Recordings and Sample Collection

Pigs were inspected daily for general health and findings were recorded. In the first five days after inoculation (days 10–14 post-weaning), the piglets were closely monitored three times a day, and thereafter between days 15 and 21 post-weaning two times a day. The rectal temperature of the pigs was monitored at day 8, 10, 11, 12, 13 and 14 post-weaning. Individual body weight (BW) was recorded at the start of the experiment (weaning, day 0), as well as at day 8, 14 and 21 post-weaning. Feed allowance and refusal were recorded per pen. Fresh fecal samples were collected from individual pigs after rectal stimulation at day 1, 8, 10, 11, 12, 13, 14, 16, 18 and 21 post-weaning. The fecal consistency of the samples was determined on an 8-point scale [54] from severely watery, thin diarrhea to hard, dry and lumpy feces. A fecal score of 6 was considered the optimal fecal score. After scoring the fecal consistency, the fresh samples were put on ice and handed over to the lab. Per fecal sample, 200–300 mg was taken at day 8, 10, 11, 12, 13, 14, 16 and 18 post-weaning, and the samples were stored at −80 °C until shipment to the microbiology laboratory for F4-ETEC shedding (BaseClear, Leiden, The Netherlands). At day 21 post-weaning, all piglets were euthanized to collect tissue from the jejunum and ileum for histomorphometry analysis. The samples were collected as soon as possible (<15 min) after the dissection of the piglets. Sections about 5 cm long were collected, opened lengthwise and fixed into 10% neutral-buffered formalin. Jejunum samples were collected at the ½ part of the whole small intestine, and colon samples were collected at 50 cm distal from the ileocecal valve.

4.6. Laboratory Analysis

Blood samples (1 mL whole blood in an EDTA tube per piglet) collected 17 days prior weaning were analyzed to determine SNP2 gene expression by the Animal Genetics Lab, University of Gent, Belgium. All experimental diets were analyzed for moisture [55], crude protein [56], crude ash [57], crude fat [58], crude fiber [59] and enzymatic starch [60]. DNA was extracted from each fecal sample and used to quantify fecal F4-ETEC shedding using quantitative PCR (qPCR targeting the inoculated F4-ETEC strain) [22]. Intestinal tissues samples for the morphometric study were dehydrated and embedded in paraffin wax, sectioned at 4 µm and stained with the Periodic acid–Schiff method (Leica Autostainer CV 5030, Leica Biosystems, Illinois, United States). Morphometric measurements were performed with a light microscope (BHS, Olympus Corporation, Pennsylvania, United States) using a linear ocular micrometer (Olympus, Ref. 209-35040, Microplanet, Barcelona, Spain). Villus height (VH) and crypt depth (CD) were measured in 10 well-oriented complexes of villi and crypts from intestinal sections. The VH:CD ratio was calculated in the jejunum. The number of goblet cells (GCs) was counted and expressed as total per villi (jejunum) or per crypt (colon), and per 100 µm. Also, the thickness of the mucus was determined. Morphometric measurements were performed by the same person who was blinded to the treatments.

4.7. Statistical Analysis

The experimental data were analyzed using GenStat^® Version 23 for Windows^TM (VSN International Ltd., Hemel Hempstead, UK). The main response parameters are the fecal consistency score, the prevalence and concentration of fecal F4-ETEC shedding and intestinal histomorphometry. Significant differences are declared at p < 0.05, with near-significant trends as 0.05 < p < 0.10. Multiple comparisons between treatments groups were tested with the Tukey adjustment. Data are presented as absolute means, adjusted for covariates if were any included. The p-value and standard error of the mean (SEM) are reported per response parameter. Piglet was the experimental unit for fecal consistency, F4-ETEC shedding and intestinal histomorphology measures. Treatment was used as the fixed effect and replicate was used as the random effect. Fecal consistency and the concentration of F4-ETEC shedding over time were analyzed in an ANOVA using treatment, day and their interaction as fixed effects and replicate as the random effect. In addition, the concentration of F4-ETEC shedding per day was analyzed in an ANOVA using treatment as fixed effects and replicate as the random effect. Chi-square tests were used to analyze the prevalence of F4-ETEC shedding per day.

Author Contributions

Conceptualization, M.L.G. and A.B.; data curation, X.G, A.M. and M.N.; formal analysis, X.G., E.J.-M. and M.N.; funding acquisition, G.M.; investigation, F.M., X.G. and A.M.; visualization, T.-P.A.; writing—original draft, T.-P.A. and A.K.; writing—review and editing, M.L.G., F.M., E.J.-M., A.B., G.M., M.N. and T.A. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

Alberto T., A.B. and E.J. are employers of Cargill S.L.U. (Spain), which is a wholly owned subsidiary of Cargill Incorporated. Anja K. is an employee of Cargill GmbH (Germany), which is a wholly owned subsidiary of Cargill Incorporated. Tobias A. is an employee of Delacon Biotechnik GmbH (Austria), which is a wholly owned subsidiary of Cargill Incorporated. Maud L.G. is an employee of Provimi-France S.A.S. (France), which is a wholly owned subsidiary of Cargill Incorporated. Graziano M. is an employee of Cargill S.R.L. (Italy), which is a wholly owned subsidiary of Cargill Incorporated.

Footnotes

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Figures and Table

Figure 1. The effect of treatment and its interaction with day on the fecal consistency score over the experimental period. Inoculation took place at day 9 post-weaning. PC = positive control, NC = negative control, PFA = phytogenic feed additive.

Figure 2. The effect of treatment and its interaction with day on the percentage of piglets shedding F4-ETEC. Different letters in each day mean significant differences (p [less than] 0.05). PC = positive control, NC = negative control, PFA = phytogenic feed additive.

Figure 3. The effect of treatment and its interaction with day on the F4-ETEC concentration in feces. Different letters in each day mean significant differences (p [less than] 0.05). PC = positive control, NC = negative control, PFA = phytogenic feed additive.

Table 1

The effect of treatment on intestinal histomorphometry of the piglets at day 21 post-weaning ¹.

	PC	NC	PFA	SEM	p-Value
Jejunum
Villus height (VH), µm	543	538	558	39.5	0.78
Crypt depth (CD), µm	189	185	203	15.4	0.54
VH:CD ratio	3.1	3.3	3.0	0.44	0.57
Goblet cells (GC), n/villi	3.7 b	5.0 b	7.4 a	0.68	0.002
GC, n/100 µm	0.69 b	0.99 ab	1.37 a	0.186	0.010
Mucus thickness, µm	1.617 b	2.111 a	1.652 ab	0.205	0.035
Colon
CD, µm	480	489	475	30.2	0.90
GC, n/crypt	31.7	29.6	34.7	2.74	0.40
GC, n/100 µm	6.70	5.94	7.28	0.512	0.11
Mucus thickness, µm	1.185	1.204	1.142	0.095	0.72

¹ PC = positive control, NC = negative control, PFA = phytogenic feed additive. Different letters mean significant differences (p < 0.05).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/stresses5010008/s1, Table S1: The effect of treatment on piglet body weight (BW) and average daily feed intake (ADFI); Table S2: Diet composition and calculated nutrient values of basal diet.

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56. ISO 16634-2:2016 Food Products. Determination of the Total Nitrogen Content by Combustion According to the Dumas Principle and Calculation of the Crude Protein Content. Part 2: Cereals, Pulses and Milled Cereal Products; Nederlands Normalisatie-Instituut: Delft, The Netherlands, 2016.

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58. ISO 6492:1999 Animal Feeding Stuffs-Determination of Fat Content; Nederlands Normalisatie-instituut: Delft, The Netherlands, 1999.

59. ISO 6865:2001 Animal Feeding Stuffs-Determination of Crude Fibre Content-Method with Intermediate Filtration; Nederlands Normalisatie-instituut: Delft, The Netherlands, 2001.

60. ISO 15914:2004 Animal Feeding Stuffs-Enzymatic Determination of Total Starch Content; Nederlands Normalisatie-Instituut: Delft, The Netherlands, 2004.