Impact of L-Arginine and L-Glutamine supplementation on growth performance and immune status in weanling pigs challenged with Escherichia coli F4

Abstract

Arginine (ARG) and Glutamine (GLN) have been reported to play significant roles in protein metabolism, immunity, and intestinal health in weanling pigs. The present study investigated the independent and interactive effect of supplementing ARG and GLN on pigs immune status and growth performance following an Escherichia coli F4 challenge. A total of 240 mixed-sex pigs (24 ± 2 d old; 7.3 ± 0.1 kg BW) were used in a 42-d experiment after selection for E. coli F4 susceptibility. The pigs were group-housed (3 pigs per pen), and pens were randomly assigned to five experimental treatments (N = 16 pens per treatment). Experimental treatments were: 1) a wheat–barley–soybean meal-based basal diet (CTRL), 2) a basal diet with 2500 mg/kg zinc oxide (ZnO), 3) a basal diet + 0.5% Glutamine (0.5% GLN), 4) basal diet + 0.5% Arginine (0.5% ARG), and 5) basal diet with 0.5% Glutamine + 0.5% Arginine (0.5% GLN + ARG). All Pigs were inoculated with E. coli F4 on days 7, 8, and 9 post-weaning. Rectal swabs were taken from each pig and plated on blood agar plates for E. coli F4 presence. Blood and fecal samples were taken to determine the acute phase response and selected fecal biomarkers for the immune response. Growth performance and fecal scores were recorded. Fecal swabs resulted in no positive pig for E. coli F4 before inoculation and 73.3% positive postinoculation. Diarrhea incidence during days 7 to 14 was significantly lower for the ZnO treatment (P < 0.05). The haptoglobin level on day 3 was lower than days 10 and 20, irrespective of treatment (P < 0.05). The albumin level was lower on day 20 compared to days 3 and 10 (P < 0.05). There was no treatment effect on albumin levels regardless of sampling day (P > 0.05). The PigMAP was lowest on day 3 and highest on day 10 (P < 0.05). We did not observe significant treatment differences (P > 0.05) in myeloperoxidase and calprotectin. Pancreatitis-associated protein was higher in the ZnO (P = 0.001) treatment than in the other treatments. Fecal IgA tended (P = 0.10) to be higher in the ZnO and 0.5% ARG treatments. There were no performance differences, except during days 0 to 7, where the ZnO treatment was lower in average daily gain and average daily feed intake (P < 0.001), while feed efficiency (G:F) FE was similar across treatments. In summary, no improved performance was observed with either ARG, glutamate, or both. The immune response results showed that the E. coli F4 challenge may have exacerbated the acute phase response; hence, the benefits of dietary treatments did not go beyond immune repair and reduction in inflammation.

Arginine and glutamine supplementation may improve the acute phase response and immune status in Escherichia coli-challenged weanling pigs.

Introduction

The concept of functional amino acids (AA) is related to the regulatory role of AA in body metabolism, gene expression, lactation, etc. (Wu, 2009, 2010). Broadly, these roles are typically not directly measured by current methods of AA requirement determination, such as growth performance or nitrogen balance (Lenis et al., 1999; Wellington et al., 2020). Notably, Arginine (ARG) and Glutamine (GLN) have been reported to play significant roles in the whole-body protein metabolism of the young pig (Wu et al., 2010; Yao et al., 2011a; Wang et al., 2012). Precisely, sow milk was reported to supply about 40% of the required ARG for protein accretion in suckling pigs, which leads to a reduced growth rate preweaning if lacking (Flynn and Wu, 1996; Wu et al., 2004). Plasma ARG levels decrease progressively from days 3 to 14 post-farrowing, affecting muscle growth and development (Flynn et al., 2000), suggesting that neonatal pigs are deficient in ARG. Furthermore, studies have reported that supplementation of ARG in early-weaned pigs improved immunity (Tan et al., 2009), enhanced intestinal development (Yao et al., 2011b), and improved growth (Kim et al., 2004). These studies confirm that ARG may be lacking in post-weaning pigs but also indicates that supplemental ARG could improve intestinal health and performance, as recently reviewed (Luise et al., 2023). Glutamine has been reported as a nutrient of the intestinal epithelium since about 70% of dietary GLN is catabolized in the small intestine of the young pig (Hou and Wu, 2018), acting as an important energy source for enterocytes (Cabrera et al., 2013). In addition to feeding the gut, dietary supplementation with 2% GLN in young pigs alleviated muscle protein losses by activating mTOR pathways for protein accretion (Kang et al., 2017). Following the inoculation of pigs with Escherichia coli K88 challenge, dietary supplementation with 2% GLN alleviated growth depression (Yi et al., 2005). When piglets were fed creep diets with GLN and then switched to post-weaning diets containing both GLN and glutamate (GLU), there was a 34% increase in feed efficiency which was related to 20% higher villi in pigs receiving the GLN and GLU supplemented diets, compared to the control diet (Cabrera et al., 2013). When GLN was supplemented to pigs at 1%, there was an increase in growth rate, feed efficiency, and increased jejunal villus height (He et al., 2016). From the above, there is an opportunity to improve the intestinal health and growth performance of weanling pigs by supplementing diets with ARG and GLN immediately post-weaning. Previously, using zinc oxide and antibiotics in weaner diets helped resolve the health challenges in the immediate post-weaning period. Since the regulatory ban on pharmacological zinc oxide use as prophylactic and in-feed antibiotics, it is important to investigate sustainable alternatives to support animal health without environmental impacts. The use of AA in different combinations and concentrations to enhance animal health and performance is one of the nutritional concepts reported to improve growth performance under enteric challenge conditions (Rodrigues et al., 2021, 2022).

Therefore, the present study aimed to evaluate the independent and interactive effect of supplementing ARG and GLN on the immune status and growth performance of weanling pigs during an E. coli F4 challenge.

Materials and Methods

Ethical approval

The internal welfare body of Trouw Nutrition R&D reviewed and approved the experimental protocol and followed the Central Committee Animal Experimentation (CCD, The Hague, the Netherlands) approval with animal use protocol #AVD2040020184545 Appendix 1.

Animals, housing, and experimental design

A total of 240 mixed-sex pigs (Hypor Libra × Maxter, Hendrix Genetics B.V, Boxmeer, the Netherlands) were used in a 41 d growth experiment at the Trouw Nutrition R&D Swine Research Centre, St. Anthonis, the Netherlands. The pigs were weaned at 24 ± 2 d (7.3 ± SD = 0.08 kg body weight (BW)) after being previously selected for E. coli F4 susceptibility and randomly allocated to pens with dimensions 2.04 m × 1.2 m (3 pigs per pen). The BW at weaning was balanced in each pen, with males and females housed in separate pens. Pens were allocated to experimental treatments in a completely randomized design, with a total of 16 replicate pens per treatment (N = 16 pens/treatment). Five experimental diets were produced (Research Feed Plant, ForFarmers, Heijen, the Netherlands) consisting of 1) a wheat–barley–soybean meal-based diet (CTRL), which met the nutrient requirement of weanling pigs, 2) the basal diet with pharmacological ZnO inclusion at 2,500 mg/kg Zn (ZnO), 3) the CTRL diet + 0.5% GLN (0.5% GLN), 4) the CTRL diet + 0.5% ARG (0.5% ARG), and 5) the CTRL diet supplemented with 0.5% GLN + 0.5% ARG (0.5% GLN + ARG). The experimental diets (Table 1) were fed during days 0 to 21 post-weaning; thereafter (days 21 to 41), a common commercial diet was fed until the end of the study (MilkiWean link diet, Ghent, Belgium).

Table 1.

Composition of the experimental diets

	Dietary treatments1
Ingredients, %	CTRL	ZnO	0.5% GLN	0.5% ARG	0.5% ARG + GLN
Barley	20.0	20.0	20.0	20.0	20.0
Wheat	30.0	34.0	34.0	35.0	37.2
Maize	4.6	0.4	1.5	2.4	0.4
Wheat bran	3.2	3.2	3.2	3.2	3.2
Soybean meal	6.5	6.1	3.9	2.7	1.6
2 TN premix concentrate	30	30	30	30	30
DL-Methionine 99%	0.1	0.1	0.1	0.1	0.1
L-Lysine HCl 98%	0.2	0.2	0.2	0.3	0.3
L-Threonine 98%	0.1	0.1	0.1	0.1	0.1
L-Valine 96.5%	0.1	0.1	0.1	0.1	0.2
Sodium bicarbonate	0.2	0.2	0.5	0.4	0.5
Sugar	2.0	2.0	2.0	2.0	2.0
Soybean oil	2.6	2.8	2.8	2.4	2.6
Leucine	0.3	0.3	0.3	0.4	0.4
L-Histidine HCl 98%	0.1	0.1	0.5	0.2	0.2
Zinc oxide	–	0.3	–	–	–
Isoleucine 90%	0.1	0.1	0.2	0.2	0.2
L-Arginine 98%	–	–	–	0.5	0.5
L-Glutamic acid 98.5%	–	–	0.5	–	0.5
Calculated nutrient, %
ME, MJ/kg	13.9	13.9	13.8	13.8	13.8
NE, MJ/kg	10.2	10.2	10.2	10.2	10.2
DM	90.1	90.3	90.4	90.3	90.5
CP	17.6	17.6	17.6	17.6	17.6
Soluble dietary fiber	1.70	1.70	1.70	1.70	1.70
Insoluble dietary fiber	12.3	12.3	12.0	12.0	11.9
Total dietary fiber	14.8	14.8	14.4	14.4	14.2
3 SID Lys	1.25	1.25	1.25	1.25	1.25
SID Met	0.62	0.62	0.63	0.63	0.64
SID Met + Cys	0.88	0.88	0.88	0.88	0.88
SID Trp	0.26	0.26	0.26	0.26	0.26
SID Thr	0.88	0.88	0.88	0.88	0.88
SID Ile	0.65	0.65	0.65	0.65	0.65
SID Arg	0.77	0.76	0.69	1.16	1.13
SID Leu	1.25	1.25	1.25	1.25	1.25
SID Val	0.85	0.85	0.85	0.85	0.85
SID His	0.43	0.43	0.43	0.43	0.43
Ca, g/kg	5.34	5.34	5.29	5.28	5.27
P, g/kg	5.94	5.94	5.83	5.83	5.79
Na, g/kg	3.21	3.24	4.00	3.81	4.00
Cl, g/kg	5.67	5.68	6.43	5.96	6.04
K, g/kg	6.65	6.61	6.15	5.96	5.74
Zn, mg/kg	132	2500	131	131	131

¹A basal wheat–barley–soybean meal diet (CTRL); CTRL + ZnO inclusion at 2,500 mg/kg Zn (ZnO); CTRL diet + 0.5% GLN (0.5% GLN); CTRL diet + 0.5% ARG (0.5% ARG), and CTRL + 0.5% GLN + 0.5% ARG (0.5% GLN + ARG).

²Trouw Nutrition premix concentrate; Supplied per kilogram of complete diet: Vitamin A, 8000 IU; Vitamin D3, 2000 IU; Vitamin E, 30 IU; Vitamin K3-menadione, 1.5 mg; Vitamin B12, 0.03 mg; Thiamin, 1.00 mg; Niacin, 20 mg; Riboflavin, 4 mg; Pantothenate, 13 mg; Folic acid, 0.30 mg; Pyridoxine, 1.0 mg; Iron sulphate, 100 mg; Zinc sulphate, 100 mg; Magnesium oxide, 30 mg; Copper sulphate, 20 mg; Copper chelate, 70 mg; Sodium selenite, 0.30 mg; Iodine, 1 mg.

³SID, Standardized ileal digestible.

E. coli challenge administration

All pigs used in the experiment were preselected before weaning based on their susceptibility to E. coli F4 based on their genotype, with 25% susceptible homozygote (SS) pigs and 75% susceptible heterozygote (SR) pigs (Roubos-van den Hil et al., 2017). The challenge model was applied by administration of the E. coli F4 to all pigs via a revolver syringe containing a 5 mL saline solution infected with 5 × 10⁸ CFU/mL of E. coli F4 on day 7 and repeated on days 8 and 9 postweaning.

Rectal swabs sampling for E. coli F4

On day 6 (before E. coli inoculation) and day 13 (after E. coli inoculation) post-weaning, rectal swabs were taken from each piglet and plated on Columbia blood agar base plates for molecular determination of E. coli F4 presence or absence. The first screen was done by checking for hemolysis of the colonies on the plate. The E. coli F4 strain showed clear hemolytic colonies. When no hemolytic colonies were observed, the sample was noted as negative (absence of this E. Coli F4 strain). Samples that showed hemolytic colonies were analyzed by qPCR assay to confirm E. coli F4 presence.

Growth performance and fecal scores

Individual BW and pen feed intake were recorded weekly (days 0 to 7, 7 to 14, 14 to 21, 21 to 28, 28 to 35, and 35 to 41), and feed efficiency (G:F) was calculated using the recorded average daily gain (ADG) and average daily feed intake (ADFI). In case of mortality or culling, the animal was weighed, and ADG and feed intake were adjusted accordingly. Fecal scores were scored daily by the trained technicians via visual inspection, and the evaluation was based on an internal protocol where scoring was done in the morning, with scores 0 = normal feces; 1 = shapeless feces, soft feces; 2 = thick liquid feces, mild diarrhea; 3 = thin liquid, watery, severe diarrhea; 9 = no feces. Diarrhea incidence was then calculated as the proportion of pens with an incidence score of 2 or 3 within a sampling period.

Blood and fecal sampling

Blood samples were taken from the same pig for each time point (representative pig; similar BW to the pen average) on days 3, 10, and 20 post-weaning. Samples were collected into non-heparinized vacutainer tubes from the jugular vein and allowed to coagulate at room temperature before centrifuging at 2500 × g for 15 min at 4 °C. Serum was collected and stored in 2 mL cryovials and stored at −80 °C until analysis for acute phase proteins (APP). Fecal samples were collected from each pig in a pen on day 10 post-weaning (1 d post-inoculation) via rectal palpation and pooled per pen. Fecal samples were stored at −80 °C until analysis for fecal biomarkers for immune status and health.

Analytical procedures

The dietary dry matter (DM) was measured according to method 930.15 (AOAC, 2007), and nitrogen (N) by the combustion method (method 990.03; LECO FP 528 MI, USA) using the LECO Nitrogen analyzer and crude protein calculated as N × 6.25. Dietary crude fat was determined as an extraction method 920.39 according to AOAC (AOAC, 2007). The ash content of the diets was measured according to method 942.05 (AOAC, 2007). The mineral analysis was completed according to NEN-EN 15550 specifications (2017). This method uses the inductively coupled plasma atomic emission spectroscopy (ICP-OES) method to determine zinc, calcium, and phosphorus content in animal feed after dry ashing. The amino acid analyzer was used to determine the total AA content of the experimental diets according to an in-house method (Nutreco-MasterLab, Boxmeer, the Netherlands) based on NEN-EN-ISO 13903. Briefly, for the determination of AA, the samples are hydrolyzed with hydrochloric acid solution and then evaporated into a film evaporator before being taken up by a buffer solution. Samples are then separated on a cation exchanger with a gradient step and postcolumn derivatization with ninhydrin; the AA are then measured at 440 and 570 nm using a flow colorimeter. All samples were analyzed in duplicate, and the results are presented in Table 2.

Table 2.

Analyzed nutrient content of the experimental diets

	Dietary treatments1
Nutrients, %	CTRL	ZnO	0.5% Gln	0.5% Arg	0.5% Gln + Arg
Crude protein	17.6	17.4	17.7	17.5	17.8
Crude fiber	3.0	2.7	2.7	2.8	2.6
Dry matter	89.5	90.2	89.8	89.6	89.6
Zinc, mg/kg	109	2300	98	99	100
Total amino acids, %
Arginine	0.86	0.85	0.77	1.19	1.17
Cysteine	0.29	0.28	0.27	0.26	0.26
Phenylalanine	0.74	0.78	0.73	0.71	0.69
Glycine	0.63	0.63	0.58	0.57	0.56
Glutamic acid	3.64	3.59	3.89	3.29	3.87
Histidine	0.45	0.43	0.66	0.44	0.44
Isoleucine	0.7	0.7	0.7	0.7	0.72
Leucine	1.36	1.34	1.34	1.33	1.33
Lysine	1.32	1.28	1.28	1.27	1.31
Methionine	0.6	0.57	0.59	0.6	0.62
Proline	1.24	1.25	1.17	1.15	1.15
Serine	0.74	0.72	0.66	0.64	0.64
Threonine	0.95	0.89	0.9	0.91	0.93
Tyrosine	0.54	0.57	0.53	0.52	0.5
Valine	0.92	0.89	0.88	0.88	0.91
Alanine	0.58	0.59	0.55	0.53	0.52
Aspartic acid	1.18	1.12	1.02	0.96	0.92

¹A basal wheat–barley–soybean meal diet (CTRL); CTRL + ZnO inclusion at 2,500 mg/kg Zn (ZnO); CTRL diet + 0.5% GLN (0.5% GLN); CTRL diet + 0.5% ARG (0.5% ARG), and CTRL + 0.5% GLN + 0.5% ARG (0.5% GLN + ARG).

Fecal and serum sample analyses

Fecal biomarkers were analyzed at a commercial laboratory (BaseClear, Leiden, the Netherlands). The analysis for the fecal calprotectin assay was based on the competitive inhibition enzyme immunoassay technique, according to the manufacturer’s instructions, using a plate reader to measure absorbance at 450 nm. Intra-assay CV is < 8%, and interassay CV is < 10%. The protocol was optimized for use with fecal samples, which were diluted 1:5 with sample diluent. The detection range of the assay is between 0.312 and 20 ng/mL. The assay for the pancreatitis-associated protein (REG3a/PAP) and myeloperoxidase (MPO) assay was based on sandwich enzyme-linked immunosorbent assay technology (ELISA). The detection range of MPO is 15.6 ng to 1000 ng/mL, and of PAP is 15.6 to 1000pg/mL. The sandwich ELISA is the technique used to detect Pig immunoglobulin A (IgA). When this protein is present in the test sample, it is captured by an antipig IgA antibody that has been pre-adsorbed on the surface of microtiter wells. After sample binding, unbound proteins and molecules are washed off, and a biotinylated detection antibody is added to the wells to bind to the captured IgA. A streptavidin-conjugated horseradish peroxidase was added to catalyze the colorimetric reaction with the chromogenic substrate 3,3ʹ,5,5ʹ-tetramethylbenzidine. The colorimetric reaction produced a blue product, which turned yellow when the reaction was terminated by adding dilute sulfuric acid. Serum samples were analyzed by the Royal GD (GD diergezondheid, Deventer, the Netherlands). Serum haptoglobin and albumin analyses were completed via a calorimetry method according to the internal protocol of the GD. The turbidimetric method was used to analyze pig-major acute phase protein (PigMAP).

Statistical analysis

All data were tested for normality of residuals using PROC UNIVARIATE in SAS (SAS Inst. Inc., Cary, NC). The PROC MIXED model was used to analyze growth performance data. The statistical model for growth performance and fecal biomarker data included the fixed effect of dietary treatment (N = 5) and block (initial BW and sex nested) as a random effect variable. The initial BW was modeled as a covariate. The statistical model for the blood samples included a fixed effect of treatment (N = 5) and time as the repeated variable (N = 3). Diarrhea incidence was analyzed using PROC GLIMMIX with beta distribution and logit link function. The P-values < 0.05 were considered significant for all data, and P ≤ 0.10 were considered trends. The mean separation was done by the PDIFF option in SAS and adjusted by the Tukey-Kramer test.

Results

General observation

In general, the animals remained healthy and consumed feed allocated ad-lib as expected, except during the E. coli inoculation period, where we observed increased loose feces, reduced appetite, and increased listlessness typical for this experimental model. The symptoms subsided within a few days after the inoculation. To validate the challenge model, fecal swabs were taken before (day 6 post-weaning) the E. coli inoculation and then 4 d after the E. coli inoculation (day 13 post-weaning). These samples were tested for the presence or absence of the inoculated E. coli F4 strain. The results indicated that for the samples collected on day 6, none of the pigs (0%) tested positive for E. coli F4, which indicated that the pigs selected for the trial were not positive for E. coli F4 before inoculation. Samples collected 4 d after the last inoculation showed that 176/240 (73.3%) of the piglets still tested positive for the inoculated strain.

We analyzed the experimental diets to check for consistency with the formulated nutrients. As shown in Table 2, the crude protein levels were similar across the dietary treatments (17.4% to 17.8%). Also, the Zn level in the ZnO treatment was confirmed to be 2,300 ppm, while the other treatments had Zn levels ranging 98 to 100 ppm. The dietary total AA levels analyzed were consistent in the dietary treatments, except for Arg and Glutamic acid (including glutamine), which were higher in the treatments formulated with higher ARG or GLN.

Diarrhea incidence

As shown in Figure 1, there was an interactive effect between dietary treatment and period on diarrhea incidence (P < 0.0001). During days 7 to 14, ZnO showed a significantly lower diarrhea incidence than the rest of the treatments (P < 0.05). This observation is relevant because the E. coli inoculation was done during this period, which signifies a higher chance of a diarrhea response in the pigs. The incidence of diarrhea remains lower for ZnO during days 14 to 21, while the rest of the treatments also show a decline in diarrhea incidence scores to similar levels of ZnO, except the 0.5% GLN + ARG group showed a numerically higher diarrhea incidence (P > 0.10). After the feed switch on day 21, the incidence of diarrhea appeared to be higher for all treatments until the last week of the trial (day 34 to 41), where the 0.5% GLN + ARG showed the highest diarrhea incidence, with CTRL and 0.5% ARG having the lowest, and intermediate for ZnO and 0.5% GLN.

Figure 1.

The treatment × day interaction on piglet diarrhea incidence when fed the experimental diets. Within each period (d7 = days 0 to 7; d14 = days 6 to 14; d21 = days 14 to 21; d28 = days 21to 28; d34 = days 28 to 24, and d41 = days 34 to 41), we present the dietary treatment effects on the proportion of time in a period that pens where observed to have diarrhea (fecal score 2 or 3). A basal wheat–barley–soybean meal diet (CTRL); CTRL + ZnO inclusion at 2,500 mg/kg Zn (ZnO); CTRL diet + 0.5% GLN (0.5% GLN); CTRL diet + 0.5% ARG (0.5% ARG), and CTRL + 0.5% GLN + 0.5% ARG (0.5% GLN + ARG).

Acute phase response parameters (APP)

The results of the APP analyzed in serum are shown in ­Figure 2A–C. In the present study, we observed that both the time (day of sampling) and dietary treatment independently had significant effects (P < 0.05) on serum haptoglobin levels. Before the challenge (d3), haptoglobin levels were lower than on day 10 (1 d postinoculation) and day 20, irrespective of treatment (P < 0.05). Haptoglobin levels were significantly higher on day 10 than on day 3 (P < 0.05) and remained high on day 20. The ZnO treatment reduced haptoglobin levels compared to the rest of the dietary treatments, regardless of sampling day (P < 0.0001). A significant day effect was observed, with the lowest level of PigMAP on day 3 and the highest on day 10 (P < 0.05). On day 20, levels of PigMAP returned to the pre-inoculation level of day 3 (P > 0.05). There was no significant dietary effect observed (P > 0.05) for PigMAP. In the present study, we observed the lowest albumin levels on day 20 compared to days 3 and 10 (P < 0.05), but the albumin level on day 10 was lower than on day 3 (P < 0.05). Regardless of the sampling day, we observed no treatment effect on albumin levels (P > 0.05).

Figure 2.

Serum acute phase protein analysis for PigMAP (A), Albumin (B), and Haptoglobin (C) from blood samples taken on days 3, 10, and 20 postweaning from 16 pigs per treatment at each time point, using the same representative pigs per pen. A basal wheat–barley–soybean meal diet (CTRL); CTRL + ZnO inclusion at 2,500 mg/kg Zn (ZnO); CTRL diet + 0.5% GLN (0.5% GLN); CTRL diet + 0.5% ARG (0.5% ARG), and CTRL + 0.5% GLN + 0.5% ARG (0.5% GLN + ARG). Superscript letters ^a,b,c are used to separate means different at P < 0.05 for the main effect of ‘day’ only.

Fecal biomarkers of immune status

Data for fecal biomarkers are presented in Figure 3A–D. Pancreatitis-associated protein (REG3A/PAP) showed significantly higher levels of ZnO (P = 0.001) than the other treatments. We did not observe any significant treatment differences (P > 0.05) in MPO levels; however, numerically, the ZnO group appeared to have the lowest MPO levels. The treatment effects on IgA showed a tendency (P = 0.10) for higher IgA per gram of feces for ZnO and 0.5% ARG treatments compared to the rest of the treatments. Results for the fecal calprotectin showed no significant treatment effects (P > 0.05).

Figure 3.

Fecal analyses of immune biomarkers REG3a/PAP (A), Myeloperoxidase (B), Calprotectin (C), and Immunoglobulin A (D) from fecal samples pooled per pen and taken on day 10 postweaning (1 d after the E. coli F4 inoculation). A total of 16 pen per /treatment was used in the analysis. A basal wheat–barley–soybean meal diet (CTRL); CTRL + ZnO inclusion at 2,500 mg/kg Zn (ZnO); CTRL diet + 0.5% GLN (0.5% GLN); CTRL diet + 0.5% ARG (0.5% ARG), and CTRL + 0.5% GLN + 0.5% ARG (0.5% GLN + ARG).

Growth performance response

Data for the growth performance is shown in Table 3. The ADG during the period days 0 to 7 post-weaning was significantly lower in the ZnO group (P = 0.0004) compared to the rest of the treatments. Among the rest of the treatments, there were no significant differences (P > 0.05) in ADG days 0 to 7. Similarly, the ADFI (days 0 to 7) was observed to be significantly lower (P = 0.0002) for the ZnO treatment compared to the rest of the treatments. However, G:F was not significantly different (P > 0.05) between treatments during days 0 to 7. Due to the differences in ADG, during period day 0 to 7, we observed a lower BW (P = 0.001) on day 7 for ZnO treatment compared to the rest of the treatments. During the period days 7 to 14, we observed no treatment differences for ADG or ADFI (P > 0.05), but for GF, ZnO treatment was significantly higher (P = 0.02) than the rest of the treatments. This observation did not affect BW at day 14. During the period day 14 to 21, we did not observe any significant treatment differences for any of the growth parameters (P > 0.05). After the switch to a common diet for all treatments, we observed no significant differences during days 21 to 28, 28 to 34, and 34 to 41 (P > 0.05).

Table 3.

Dietary treatments effect on piglet growth performance¹

	CTRL	ZnO	0.5% Gln	0.5%Arg	0.5% Gln + Arg	SEM2	P-value
Bodyweight, kg
Day 0	7.3a,b	7.4a	7.2b	7.4a	7.3a	0.26	< 0.05
Day 7	8.7a	8.3b	8.7a	8.7a	8.7a	0.08	< 0.01
Day 14	10.2	9.9	10.1	10.2	10.1	0.17	0.712
Day 21	13.3	12.7	12.9	13.2	13.0	0.24	0.417
Day 28	17.1	16.1	16.8	16.8	16.6	0.33	0.216
Day 34	21.1	19.9	20.8	21.0	20.4	0.41	0.206
Day 41	26.2	24.8	26.2	26.2	25.7	0.52	0.116
Average daily gain, g/d
Day 0–7	169a	125b	180a	183a	171a	10.2	< 0.01
Day 7–14	217	234	194	206	200	17.5	0.499
Day 14–21	448	398	404	427	418	16.9	0.263
Day 21–28	539	482	553	517	505	27.2	0.318
Day 28–34	664	638	660	702	644	27.8	0.519
Day 34–41	737	692	766	737	745	28.6	0.237
Day 0–41	462	426	462	461	448	12.7	0.107
Average daily feed intake, g/d
Day 0–7	173a	134b	182a	189a	177a	8.8	< 0.01
Day 7–14	266	250	244	265	257	13.9	0.759
Day 14–21	518	486	463	504	472	18.2	0.210
Day 21–28	668	599	660	658	636	21.2	0.130
Day 28–34	745a,b	681a	753b	775b	743a,b	24.1	0.065
Day 34–41	972	939	1024	979	964	32.3	0.146
Day 0–41	552a	511b	549a	556a	537a,b	14.5	0.089
Gain: feed
Day 0–7	0.98	0.92	0.99	0.96	0.96	0.03	0.532
Day 7–14	0.80b	0.94a	0.77b	0.75b	0.78b	0.04	< 0.05
Day 14–21	0.87	0.83	0.88	0.85	0.89	0.02	0.276
Day 21–28	0.81	0.79	0.84	0.79	0.79	0.03	0.650
Day 28–34	0.89	0.94	0.88	0.90	0.87	0.02	0.226
Day 34–41	0.76	0.74	0.75	0.75	0.77	0.02	0.488
Day 0–41	0.84	0.84	0.84	0.83	0.83	0.01	0.717

¹Data represent LSMEANS of 16 replicate pens per treatment. A basal wheat–barley–soybean meal diet (CTRL); CTRL + ZnO inclusion at 2,500 mg/kg Zn (ZnO); CTRL diet + 0.5% GLN (0.5% GLN); CTRL diet + 0.5% ARG (0.5% ARG), and CTRL + 0.5% GLN + 0.5% ARG (0.5% GLN + ARG) and the initial bodyweight was used as a covariate in the statistical model.

²SEM, standard error of means.

^a,bLSMEANS within a row without a common superscript differ at (P < 0.05).

Discussion

Previously, the AA considered essential (e.g., Thr, Lys, Trp, Val, etc.) have received lots of attention because the lack of these AA usually led to poor animal growth performance and, in some cases, poor health. Currently, the use of AA beyond meeting requirements for protein synthesis and growth performance is receiving much research attention (Wu et al., 2004; Wu, 2010; Hou and Wu, 2018). A recent review (Le Floc’h et al., 2018), explained how some key AA could be used to enhance intestinal barrier function, immune defense proteins, and antioxidants.

The present study explored the independent and combined use of GLN and ARG supplementation in post-weaned pigs on growth performance and immune status after an E. coli F4 challenge. The use of the E. coli F4 challenge is quite popular in controlled piglet studies to mimic the incidence of post-weaning diarrhea in weanling pigs. The E. coli F4 model used in the present study has been validated (Roubos-van den Hil et al., 2017) and used extensively (Nyachoti et al., 2012; Adewole et al., 2016). While we observed that all pigs tested negative for the presence of E. coli F4 pre-inoculation, the post-inoculation measurement showed that only about 75% of the pigs were E. coli F4 positive. It may be assumed that all piglets were positive for E. coli F4 immediately post-inoculation and that some piglets may have shed the bacteria quickly enough, or the inoculations in this 25% of the pigs were unsuccessful. Since most of the piglets tested positive for E. coli F4, we can determine that the challenge was successful and that the E. coli F4 model was sufficient to test the hypothesis. In the present study and other studies, fecal scoring or diarrhea incidence has been used to evaluate the success of the E. coli F4 model, where a higher fecal score or diarrhea incidence is attributed to a successful challenge (Fairbrother et al., 2005; Nyachoti et al., 2012). Our observations show that the ZnO treatment significantly reduced the incidence of diarrhea during the second-week post-weaning (days 7 to 14), which was the peak of the E. coli F4 challenge, while the other treatments showed a higher diarrhea incidence. Previous studies have reported higher diarrhea incidence following an E. coli challenge (Carstensen et al., 2005; Sun and Kim, 2017; Becker et al., 2020; Hansen et al., 2022). But also in post-weaned piglets, high diarrhea is usually observed during the first 14 d post-weaning. The use of pharmacological levels of ZnO has been extensively reported to alleviate post-weaning and E. coli-induced diarrhea in piglets (Owusu-Asiedu et al., 2003; Khafipour et al., 2014; Lei and Kim, 2020). Therefore, our observations in the present work in relation to a higher incidence of diarrhea during period day 7 to 14 and reduction of diarrhea incidence with ZnO concur with the previous studies cited.

Although diarrhea incidence is a validated method of evaluating the impact of E. coli challenge in pigs, we also measured changes in acute phase response to evaluate the immune status of the pigs based on the dietary treatments over the study period. The acute phase response is an innate and nonspecific response to inflammation (Heegaard et al., 1998; Pomorska-Mól et al., 2014). APP such as haptoglobin, albumin, and PigMAP are important indicators of inflammation in pigs. Both haptoglobin and PigMAP are positive APP, where serum levels increase with inflammation, while albumin is a negative APP, where inflammation causes a reduction in serum albumin levels. Haptoglobin concentration in serum increased post-inoculation relative to pre-inoculation levels, indicating an inflammatory response. This observation confirms previous reports of high serum haptoglobin levels in weanling pigs following E. coli lipopolysaccharide injection and salmonella inoculation (Litvak et al., 2013; Wellington et al., 2019; Rodrigues et al., 2021). We observed that the ZnO treatment independently reduced haptoglobin levels post-inoculation, which could be related to acute capillary extravasation due to the pre-exposure of the ZnO treatment to the pigs. Supplementation of ZnO was reported to improve the adaptive immunity in piglets, evidenced by increased numbers in CD3 + and CD4 + regulatory T cells and increased interleukin production (Kloubert et al., 2018). Zinc may have caused an immune modulatory effect due to the exposure of piglets to ZnO diets (Guan et al., 2021); as such further immune stimulation with E. coli F4 inoculation only had a minimal effect on the immune response threshold; hence no change in haptoglobin levels with the ZnO treatment. The PigMAP, which is a positive APP, did not show any dietary effect; the observations were confirmatory of a time effect. Immediately post-inoculation (day 10), the serum PigMAP levels were higher than pre-inoculation levels. This observation indicated that there was an acute inflammation regardless of dietary treatment; however, this response was not long-term, as serum PigMAP levels dropped back to pre-inoculation levels on day 20. Previous studies show that the concentration of these APP may remain elevated up to 24 h post-inoculation (Heegaard et al., 1998; Gruys et al., 2005; Wellington et al., 2018). Serum albumin levels were also not affected by dietary treatment. As a negative APP, serum albumin was decreased immediately post-inoculation and even further on day 20. This observation indicated a successful immune stimulation and concurred with previous reports (Heegaard et al., 1998; Gruys et al., 2005). Taken together, the marked clinical observations, increased diarrhea incidence, increased haptoglobin, and PigMAP serum concentrations, and decreased albumin concentration following the E. coli F4 inoculation which was all indicative of effective stimulation of the acute phase response.

In the present study, we observed no significant improvement in growth performance with the supplementation of either GLN, ARG, or both after the E. coli F4 challenge. Although some authors have reported improved performance of weanling pigs fed supplemental GLN and/or ARG (Shan et al., 2012; Cabrera et al., 2013), those studies were conducted without an enteric disease challenge. In a few studies where ARG and GLN have been investigated under a disease challenge, improved intestinal barrier function, reduced mucosal injury, and improved immune health have been reported without any significant effects on growth performance (Liu et al. 2008, 2009; Ewaschuk et al., 2011; Cabrera et al., 2013 ). The absence of a performance response could be related to many factors, including but not limited to the dose of the ARG and GLN used in the present study as well as the composition of the basal diet. In cases where the basal diet was of high quality, hence the additional effect of ARG or GLN may not be observed.

Since improved growth performance response was not observed in the present study, we used fecal samples collected during the peak of infection to investigate the treatment impact in reducing E. coli F4-induced inflammation. We analyzed the samples for biomarkers of the intestinal mucosal immune response. Calprotectin and myeloperoxidase are biomarkers reported to be highly secreted by neutrophils; quantification in feces is a good indicator of intestinal inflammation (Celi et al., 2019). No significant treatment differences were observed for both fecal MPO or calprotectin. The time of sampling at the peak of the E. coli F4 infection and/or a lack of a pre-challenge baseline makes it difficult to assess the impact of the dietary treatments in reducing the impact of the challenge. Nevertheless, the fecal IgA response, showing a tendency for higher fecal IgA in both ZnO and ARG, may reflect a positive impact of ZnO and 0.5% ARG in improving IgA secretion. The secretion of IgA in the mucosa is critical to maintaining intestinal health because, as the most abundant antibody, IgA binds to antigens, ensuring a reduced load of foreign objects in the gut to maintain intestinal equilibrium and immune response control. The observation of ZnO on increased fecal REG3a/PAP response confirms that ZnO presents an immune stimulatory effect (Guan et al., 2021); as such further stimulation by E. coli F4 inoculation in the present study significantly increased the response to REG3a/PAP, an intestinal epithelium gatekeeper secreted in the intestine and pancreas, where high levels in biological samples indicate high severity of infection (van Ampting et al., 2009; Celi et al., 2019). Other reports suggest that the PAP levels may increase substantially during E. coli infections due to the continued secretion of the REG3a/PAP protein in the gut as an antimicrobial response (Soler et al., 2015). In summary, no improvement in growth performance was reported in the present study. However, the immune response measured by fecal biomarkers showed evidence, especially with the fecal IgA response to the E. coli infection instigated by the ZnO and ARG treatments.

Conclusion

Although others have seen a positive improvement in performance with GLN and/ or ARG supplementation under non-challenge conditions, we did not observe the same response in the present study. This was because of the E. coli F4 challenge, which may have exacerbated the acute phase response; as observed, the benefits of dietary treatments did not go beyond immune repair and reduction in inflammation, as shown by the tendency for a higher fecal IgA content with 0.5% ARG and ZnO, but also with the REG3a/PAP protein response. Further studies should investigate changes in the biomarker response during the E. coli infection to mark the changes in biomarker response relative to the dietary treatments over time.

Glossary

Abbreviations:

AA

amino acid

ADFI

average daily feed intake

ADG

average daily gain

APP

acute phase protein

Arg

arginine

DM

dry matter

ELISA

enzyme-linked immunosorbent assay

ETEC

Escherichia coli K88

G:F

gain to feed

GLN

glutamine

GLU

glutamate

IgA

immunoglobin A

MPO

myeloperoxidase

N

nitrogen

PigMAP

pig major acute phase protein

REG3a/PAP

pancreatic associated protein

SR

susceptible heterozygote

SS

susceptible homozygous

ZnO

zinc oxide

Funding

Funding for this research was provided internally by Trouw Nutrition R&D, Netherlands.

Conflict of Interest Statement

All authors are Trouw Nutrition R&D employees and declare no actual or potential conflict of interest, financial or otherwise. All authors have read and approved the manuscript for submission.