The protective role of a moderate protein diet in ETEC-infected piglets: Optimization of growth, immunity, and microbial balance

Abstract

Diarrhea is a common issue among weaned piglets and enterotoxigenic Escherichia coli (ETEC) is one of the causes. However, there is limited research on how dietary protein levels affect piglets in a diarrhea model. This study aimed to investigate the effects of dietary protein levels on growth performance, immunity and microbial balance of weaned piglets post-infection with ETEC. One hundred and ninety-two 24-d-old piglets (Duroc × Landrace × Yorkshire) with a body weight (BW) of 7.20 ± 0.12 kg were used in a 2 × 3 factorial treatment arrangement. The factors were ETEC challenge (oral 3 × 10¹¹ colony-forming unit (CFU) ETEC O149:K19:K88 on experimental d 1, 2, and 3 or not), and three dietary crude protein (CP) levels (high protein [HP]-20.5% CP, medium protein [MP]-17.5% CP, and low protein [LP]-14.5% CP). On d 7, eight piglets from each treatment were sampled, and the remaining piglets were raised for another 22 d. The trial lasted 28 d. Results showed that ETEC infection increased the diarrhea rate, serum lipolyaccharide (LPS) level, and the levels of pro-inflammatory cytokines (interleukin-1β [IL-1β], interferon-γ [IFN-γ], and interleukin-6 [IL-6]), as well as the number of E. coli in the cecal digesta of piglets (P < 0.05). The ETEC infection reduced piglets’ average daily gain (ADG):average daily feed intake (ADFI) (G:F) by 22.2% under the HP diet (P = 0.022) compared with the CHP group, but had no significant effect in MP and LP diets (P > 0.05). Moreover, the MP diet decreased serum diamine oxidase (DAO), D-lactic acid (D-LA), and IL-6 levels, compared with the LP diet on d 6 under ETEC infection, and the MP diet decreased gene expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in ileal mucosa and the number of E. coli in the cecal digesta compared with the HP and LP diets on d 6 under ETEC infection (P < 0.05). Additionally, the G:F was increased with the increase of dietary protein levels under normal conditions from 1 to 6 d (P = 0.040). Furthermore, as dietary protein levels increased, growth performance (body weight on d 28 [BW28], ADG, and G:F) of piglets improved from 7 to 28 d (P < 0.05) and serum IFN-γ and LPS levels decreased on d 28 (P < 0.05) with or without ETEC infection. These findings indicated that a MP diet can enhance growth performance, reduce the expression of apoptosis-related genes, and regulate intestinal flora structure under ETEC infection. However, a HP diet remained the optimal choice for promoting growth performance under normal conditions or during an ETEC infection repair period.

1. Introduction

The health and growth performance of weaned piglets are critical determinants of productivity in swine production systems. The post-weaning period, particularly the early stages, is a vulnerable period for piglets due to the abrupt transition from sow's milk to solid feed, coupled with environmental and social stressors (Campbell et al., 2013; Liu et al., 2021). These changes often lead to compromised gut health and immune function, making piglets highly susceptible to infections by pathogenic microorganisms such as enterotoxigenic Escherichia coli (ETEC) (Che et al., 2019; Pluske et al., 2018). The ETEC, especially serotype O149:K91:K88 (commonly known as K88), is a primary causative agent of post-weaning diarrhea, a condition characterized by severe diarrhea, growth retardation, and increased mortality rates (Fairbrother et al., 2005; Rhouma et al., 2017). This multifactorial challenge creates a vicious cycle: gut dysbiosis during weaning allows pathogenic colonization, leading to nutrient malabsorption that further impairs growth and immunity (Lallès et al., 2007).

Dietary protein plays a paradoxical role in this context. As a vital nutrient for muscle synthesis and immune function, higher crude protein (CP) levels (20%–23%) typically improve growth under normal conditions (Nyachoti et al., 2006; Wellock et al., 2006; Zhang et al., 2020). However, under pathogenic challenge, excessive undigested protein reaching the hindgut becomes a double-edged sword. Mechanistically, proteolytic fermentation of undigested protein elevates luminal ammonia and biogenic amines (Gao et al., 2022), which not only damage intestinal barrier integrity but also provide favorable conditions for ETEC proliferation (Heo et al., 2013). Conversely, low-protein diets have been shown to reduce the risk of diarrhea by minimizing undigested protein in the gut, but may limit methionine and lysine availability critical for mucosal repair (Opapeju et al., 2008; Wellock et al., 2008). Despite the importance of protein in piglet nutrition, the temporal dynamics of protein-pathogen interactions across infection phases are poorly understood.

To address this, the studyaimed to investigate the effects of dietary protein levels on growth performance, immunity and microbial balance of weaned piglets post-infection with ETEC. This will provide a scientific basis for optimizing dietary protein levels for weaned piglets under infection challenge. The study may also provide practical guidance for farmers to adjust dietary protein levels in high-risk environments.

2. Materials and methods

2.1. Animal ethics statement

This experiment was conducted at Sichuan Agricultural University in China. The Ethical Commission approved the experimental protocol (SAU20230906), and the animals were handled and killed humanely following the guidelines established by this Commission.

2.2. Experimental design

A total of 192 weaned 24-d-old (Duroc × Landrace × Yorkshire) piglets (96 males and 96 females) with an initial body weight (BW) of 7.20 ± 0.12 kg were assigned to a 2 × 3 factorial treatment arrangement. The factors included three dietary CP levels (high protein [HP], medium protein [MP], and low protein [LP] diets with protein contents of 20.5%, 17.5%, and 14.5% respectively), and ETEC challenge status (infected or noninfected). Piglets were stratified by initial BW and sex (half male and half female) and randomly allocated to six groups: 20.5% CP + ETET-noninfected group (CHP), 17.5% CP + ETEC-noninfected group (CMP), 14.5% CP + ETEC-noninfected group (CLP), 20.5% CP + ETEC-infected group (EHP), 17.5% CP + ETEC-infected group (EMP), and 14.5% CP + ETEC-infected group (ELP). There were eight replicates per group and four piglets per replicate. The ETEC-infected groups received 150 mL of ETEC suspension (serotype O149:K91:K88, 2 × 10⁹ colony-forming unit [CFU]/mL) via oral gavage on the morning of d 1 to 3, whereas control groups received an equivalent volume of sterile saline (Pu et al., 2018). On d 7, eight piglets from each group were sampled, and the remaining piglets were raised for another 22 d. The experiment lasted 28 d.

2.3. Experimental diet

The experimental HP diet was formulated to meet or exceed nutrient requirements based on the National Research Council (NRC, 2012). The MP and LP diets were derived from the HP diet by sequentially reducing the CP content by three percentage points. To maintain balanced amino acid (AA) profiles, the MP and LP diets were supplemented with L-trysine, L-arginine, and L-isoleucine, and the LP diet was further supplemented with L-phenylalanine. The ingredient composition and nutritional values of the experimental diets are presented in Table 1. The digestible energy (DE), Ca, total P, available P, acid detergent fiber (ADF), and neutral detergent fiber (NDF) were calculated according to the China Feed Database (2020). The CP were determined using a Kjeldahl apparatus (Model OLB9870A, Olabo Scientific Co., Ltd., Guangzhou, Guangdong, China) according to GB/T 6432-2018 (China National Standard, 2018). The gross energy (GE) was determined using a Parr 6400 Automatic Oxygen Bomb Calorimeter (Parr Instrument Co., Moline, IL, USA). The dry matter (DM) was measured using an oven (Model DHG-9140 A, Shanghai Jinghong Experimental Equipment Co., Ltd., Shanghai, China) according to the GB/T 6435-2014 (China National Standard, 2014). The ash was measured using a muffle furnace (Model SRJX-3-9, Shenyang Electric Furnace Factory, Shenyang, Liaoning, China) according to the GB/T 6438-2007 (China National Standard, 2007). The organic matter (OM) was calculated according to DM and ash. The lysine, methionine, methionine + cystine, threonine, valine, phenylalanine, leucine, and isoleucine levels were analyzed using Hitachi L-8900 amino acid analyzer (Hitachi Ltd., Tokyo, Japan) in accordance with GB/T 18246-2019 (China National Standard, 2019), and the standardized ileal disgestible (SID)-tryptophan levels were calculated in accordance with the China Feed Database (2020).

Table 1.

Composition and nutrient levels of experimental diet (air-dry basis, %).

Ingredients	Diets			Items	Diets
	HP	MP	LP		HP	MP	LP
Corn	39.96	46.3	52.63	Analyzed nutrients
Extruded corn	15.00	15.00	15.00	GE, MJ/kg	16.40	15.57	15.57
Soybean meal	15.00	10.00	5.00	CP	20.25	16.91	14.48
Extruded soybean	5.00	5.00	5.00	DM	90.08	90.54	90.31
Fish meal	2.00	2.00	2.00	OM	84.38	85.63	85.66
Whey powder	5.00	5.00	5.00	Lysine	1.26	1.04	0.91
Soy protein concentrate	9.00	7.00	5.00	Methionine	0.33	0.23	0.21
Soybean oil	1.00	1.50	2.00	Methionine + cystine	0.49	0.38	0.35
Glucose	2.00	2.00	2.00	Threonine	0.74	0.61	0.51
Sucrose	3.00	3.00	3.00	Valine	0.98	0.82	0.74
Limestone	0.70	0.71	0.72	Phenylalanine	1.12	0.91	0.75
Dicalcium phosphate	1.00	1.10	1.20	Leucine	1.81	1.53	1.35
NaCl	0.40	0.40	0.40	Isoleucine	0.94	0.77	0.66
L-Lysine·HCl	0.19	0.21	0.23	Calculated nutrients
DL-Methionine	0.05	0.02		DE, MJ/kg	14.81	14.77	14.77
L-Tryptophan		0.01	0.01	Ca	0.80	0.80	0.80
L-Arginine		0.04	0.07	Total P	0.66	0.65	0.64
L-Isoleucine		0.01	0.03	Available P	0.42	0.42	0.42
L-Phenylalanine			0.01	ADF	3.22	3.06	2.90
Chloride choline	0.10	0.10	0.10	NDF	8.04	8.07	8.09
Vitamin premix1	0.05	0.05	0.05	SID-Tryptophan	0.26	0.22	0.18
Mineral premix2	0.20	0.20	0.20
Acidifier	0.30	0.30	0.30
Zymin	0.05	0.05	0.05
Total	100.00	100.00	100.00

HP = high protein; LP = low protein; MP = medium protein; GE = gross energy; CP = crude protein; DM = dry matter; OM = organic matter; DE = digestible energy; ADF = acid detergent fiber; NDF = neutral detergent fiber; SID = standardized ileal disgestible lysine.

¹The vitamin premix provided following per kg of diets: biotin, 0.265 mg; folic acid, 2.65 mg; niacin, 52 mg; pantothenic, 24.1 mg; vitamin A, 15,500 IU; vitamin B₁, 5.175 mg; vitamin B₂, 13.2 mg; vitamin B₆, 6.3 mg; vitamin B₁₂, 0.0625 mg; vitamin D₃, 5250 IU; vitamin E, 42 mg; vitamin K₃, 2.26 mg.

²The mineral premix provided following per kg of diets: Cu (CuSO₄·5H₂O), 6 mg; Fe (FeSO₄·H₂O), 100 mg; I (KI), 0.14 mg; Mn (MnSO₄·H₂O), 4 mg; Se (Na₂SeO₃), 0.3 mg; Zn (ZnSO₄·H₂O), 100 mg.

2.4. Animal management and growth performance

All weaned piglets were fed the experimental diets four times daily at 08:00, 12:00, 16:00, and 20:00, with ad libitum access to clean drinking water. Body weight was measured in the mornings of d 1, 7, and 29 after a 12-h fasting period to minimize variability. The daily feed intake was recorded per pen throughout the trial, and these data were used to calculate average daily gain (ADG), average daily feed intake (ADFI), and ADG:ADFI (G:F).

Health status and diarrhea incidence were monitored daily for all piglets. Fecal consistency was scored by a single trained observer using the following scoring system to ensure scoring uniformity: 0 = normal, firm feces; 1 = soft feces, possible slight diarrhea; 2 = formless, semifluid feces, moderate diarrhea; 3 = very watery and frothy feces, severe diarrhea. The fecal score ≥2 was defined as diarrhea. Diarrhea incidence (%) was calculated as follows:

Diarrhea incidence (%) = (Total number of pigs per pen with diarrhea)/(Number of pigs per pen × Test period) × 100.

2.5. Sample collection

On d 7 and 29 of the experiment, eight piglets close to the average BW from each treatment were selected after 12 h of fasting. Blood samples were collected from the anterior vena cava using sterile vacuum tubes. Plasma were separated by centrifugation at 3500 × g for 10 min at −4 °C and stored at −20 °C for further analysis.

On d 7 of the experiment, the same eight piglets per treatment used for blood collection were euthanized via intravenous injection of sodium pentobarbital (200 mg/kg BW) for sampling. Immediately after euthanasia, the abdominal cavity was opened, and the jejunum, ileum, and cecum were carefully isolated. The luminal contents of the jejunum and ileum were gently flushed with ice-cold normal saline. Mucosal samples from the jejunum and ileum were collected by scraping the luminal surface with sterile glass slides. Cecal contents were flash-frozen in liquid nitrogen and stored at −80 °C for subsequent analysis. Additionally, approximately 3 cm segments from the mid-ileum were excised and fixed in 4% paraformaldehyde solution for intestinal morphological analysis.

2.6. Biochemical analysis of the serum

Total antioxidant capacity (T-AOC; A015-1), malondialdehyde (MDA; A003-1), and blood urea nitrogen (BUN; C013-2) levels in the serum were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) and with a SpectraMax 190 microplate reader (Molecular Devices Corp., San Jose, CA, USA) according to the manufacturer's instructions.

2.7. Enzyme-linked immunosorbent assays (ELISA)

Interferon-γ (IFN-γ; 2412003), interleukin 1β (IL-1β; 2412009), interleukin 6 (IL-6; 2412012), immunoglobulin A (IgA; 2412030), immunoglobulin G (IgG; 2412035), immunoglobulin M (IgM; 2412032), diamine oxidase (DAO; 2412028), D-lactic acid (D-LA; 2412039) and lipolyaccharide (LPS; 2412042) levels in the serum were measured via ELISA kits (Jiangsu Meimian Industry Co., Ltd., Yancheng, Jiangsu, China) and with a SpectraMax 190 microplate reader according to the manufacturer’s instructions.

2.8. Intestinal morphology analysis

After fixation with 4% paraformaldehyde, ileal tissue samples were dehydrated through a graded ethanol series, embedded in paraffin, sectioned at 5-μm thickness using a microtome, and stained with hematoxylin and eosin (H&E). Histological images were captured using an Eclipse Ci-L camera microscope (Nikon Instruments Inc., Tokyo, Japan) under 40 × magnification. For morphometric analysis, five intact and well-oriented villi and their corresponding crypts were randomly selected from each tissue section. Villus height and crypt depth were measured using Image-Pro Plus 6.0 analysis software (version 6.0, Media Cybernetics Inc., Rockville, MD, USA). The villus height-to-crypt depth ratio (V:C) was then calculated.

2.9. Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from jejunum and ileal mucosa using the Trizol Reagent (TaKaRa Biotechnology Co., Ltd., Dalian, Liaoning, China), and the concentration and purity of total RNA were assayed by spectrophotometer (NanoDrop, Gene Co., Ltd., Guangzhou, Guangdong, China) at 260 and 280 nm according to the manufacturer’s instructions. Reverse transcription was carried out using the Prime Script RT reagent kit (TaKaRa Biotechnology Co., Ltd., Dalian, Liaoning, China) according to the manufacturer’s instructions.

Quantitative real-time PCR was performed to analyze the expression levels of IL-1β, IFN-γ, IL-6, Bax, Bcl-2, and Caspase-3 using TB Green Premix Ex Taq II Fast qPCR reagent (TaKaRa Biotechnology Co., Ltd., Dalian, Liaoning, China) and the QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems Inc., Foster City, CA, USA). The experiment was carried out in a 10 μL reaction mixture, which included 5 μL of TB Green Premix Ex Taq II Fast qPCR reagent (2 × ), 0.4 μL each of reverse and forward primers, 3.2 μL of nuclease-free water, and 1 μL of cDNA template. The thermal cycling parameters were as follows: 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Table S2 displays the sequence of primers that was used to amplify target genes. The target gene expression normalized by housekeeping gene of β-actin was calculated and analyzed by the 2^-△△Ct method (Fleige et al., 2006).

2.10. Selected microbiota analysis of the cecum

Total DNA from the cecal digesta was extracted using the E.Z.N.A Stool DNA kit (Omega Bio-Tek, Doraville, CA, USA) according to the manufacturer’s instructions. Total bacteria were identified in a final volume of 25 μL with 1 μL forward primer, 1 μL reverse primer, 12.5 μL TB Green Premix EX Taq II Fast (TaKaRa Biotechnology Co., Ltd., Dalian, Liaoning, China), 1 μL template DNA, and 9.5 μL nuclease-free water. The thermal cycling parameters were as follows: pre-denaturation step at 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s. For the quantification of Lactobacillus, E. coli, Bacillus, and Bifidobacterium, real-time quantificative PCR was conducted in a 20 μL volume with 0.4 μL probe enhancer solution, 0.2 μL ROX Reference Dcye, 0.6 μL forward and 0.6 μL reverse primer, 10 μL SuperReal Premix (Tiangen Biotech Co., Ltd., Beijing, China), 1 μL template DNA, and 7.2 μL nuclease-free water. The PCR protocol involved 15 m at 95 °C, 49 cycles of 5 s at 95 °C and 25 s at 53 °C, and 10 s at 95 °C. The primers and probe sequences of bacteria are listed in Table S3. Copies per sample were calculated with the threshold cycle values and standard curve from the previous work by Qi et al. (2011).

2.11. Short chain fatty acid (SCFA) assay in cecal digesta

The concentrations of SCFA were quantified using a gas chromatograph system (VARIAN CP-3800, Varian Inc., Palo Alto, CA, USA) according to the method (Franklin et al., 2002). Briefly, approximately 0.5 g of digesta samples were centrifuged at 10,000 × g for 15 min after adding 1.2 mL distilled water. Then 1 mL of superserum was removed to a new sterile tube to which 0.2 mL of 25% metaphosphoric acid and 23.3 μL of crotonic acid solution (210 mmol/L) were added, left for 30 min at 4 °C and centrifuged at 8000 × g for 10 min. The supernatant (0.3 mL) was transferred to a new 1.5 mL EP tube and 0.9 mL of chromatographic methanol reagent was added, mixed and centrifuged at 5000 × g for 5 min. Then the SCFA were separated and quantified.

2.12. Statistical analysis

Statistical analyses were performed using IBM SPSS 27.0 (IBM Corp., Armonk, NY, USA). Dietary CP levels and ETEC challenge were analyzed using a general linear model (GLM) to analyze main and interaction effect. As follows:

$$Y_{ijk}=\mu +{\alpha }_i+{\beta }_j+{\left(\alpha \beta \right)}_{ij}+{ϵ}_{ijk}\text{,}$$

where Y_ijk is the dependent variable; μ is the overall mean; α_i is the effect of the ETEC; β_j is the effect of the dietary protein levels; (αβ)_ijk is the interaction effect of ETEC × CP; ϵ_ijk is random error. When the interaction effect is significant, differences among the six groups were compared using Duncan’s multiple comparison tests. Polynomial contrasts within the GLM framework were employed to evaluate linear and quadratic trends in CP. Meanwhile, pairwise comparisons between ETEC-infected and noninfected groups were conducted within each dietary protein level. The results of data analysis were expressed by mean and standard error. P < 0.05 was considered statistically significant, and 0.05 ≤ P < 0.10 was considered a tendency.

3. Results

3.1. Growth performance and diarrhea rate of piglets from 1 to 6 d

As shown in Table 2, ETEC infection decreased the ADFI (P = 0.020) and showed a tendency to decrease the ADG (P = 0.096) of piglets from 1 to 6 d, as well as increased diarrhea rate (P < 0.001) of piglets from 1 to 6 d. With increasing dietary protein levels, piglets showed a tendency toward higher ADG (P = 0.054) during 1 to 6 d and improved G:F (P = 0.049). There was an interaction between ETEC challenge and dietary protein levels on G:F (P = 0.040). Compared with the HP group, G:F was decreased (P < 0.05) in the MP and LP groups under normal condition; however, under infected condition, the EMP group displayed the numerically highest G:F value, representing increases of 9.26% and 11.11% compared with the EHP and ELP groups, respectively, though no differences were observed. Meanwhile, ADG and G:F of piglets from 1 to 6 d increased (P < 0.05) linearly with increasing dietary protein levels while diarrhea rate (P = 0.065) showed a quadratic trend, initially increasing then decreasing as protein levels rose. The pairwise comparisons of growth performance and diarrhea rate in piglets with and without ETEC infection are presented in Table S4. Under the 20.5% CP level, ETEC infection reduced (P < 0.05) piglets' body weight on d 6 (BW6) by 3.1%, ADG by 29.5%, and G:F by 22.2% compared with the ETEC non-infection group.

Table 2.

Effects of dietary protein levels and ETEC challenge on growth performance and diarrhea rate of piglets from 1 to 6 d of experiment.

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
BW1, kg	7.20	7.20	7.20	7.20	7.20	7.20	0.047	0.987	0.999	1.000	0.998	0.960
BW6, kg	8.05	7.83	7.77	7.80	7.83	7.74	0.056	0.412	0.465	0.629	0.220	0.952
ADFI, g/d	227.43	215.00	212.71	197.97	197.24	183.74	5.366	0.020	0.533	0.874	0.265	0.953
ADG, g/d	141.89	106.25	94.95	100.00	105.73	88.96	5.115	0.096	0.054	0.165	0.016	0.964
G:F	0.63a	0.48b	0.44b	0.49b	0.54ab	0.48b	0.018	0.700	0.049	0.040	0.015	0.986
Diarrhea rate, %	10.94	13.54	11.98	28.65	36.46	27.08	1.897	<0.001	0.178	0.480	0.936	0.065

ETEC = enterotoxigenic Escherichia coli; BW1 = body weight on d 1; BW6 = body weight on d 6; ADFI = average daily feed intake; ADG = average daily gain; G:F = ADG:ADFI; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

In the same row, different superscript letters denote significant difference (P_{ETEC × CP} < 0.05, n = 8).

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group.

3.2. Serum immunoglobulins, inflammatory factors, and antioxidant capacity of piglets on d 6

As shown in Table 3, the ETEC infection elevated serum LPS, immunoglobulins (IgG, IgM, and IgA), and pro-inflammatory cytokines (IL-1β, IFN-γ, and IL-6) levels (P < 0.05), while reducing T-AOC and BUN levels (P < 0.05). The main effect of CP levels showed that compared with the 20.5% CP and 17.5% CP diets, piglets fed the 14.5% CP diet increased LPS and IFN-γ levels (P < 0.05), but decreased BUN levels (P < 0.05) in serum.

Table 3.

Effects of dietary protein levels and ETEC challenge on serum immunoglobulins, antioxidant capacity and inflammatory factors in piglets on d 6.

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
LPS, ng/L	240.04	250.95	306.95	386.71	410.65	524.17	16.092	<0.001	<0.001	0.133	<0.001	0.039
IgG, μg/mL	174.17c	202.19c	278.91b	287.99b	386.17a	370.28a	12.624	<0.001	<0.001	0.005	<0.001	0.186
IgM, μg/mL	33.29c	31.93c	46.31b	63.16a	42.55b	43.93b	1.779	<0.001	<0.001	<0.001	0.157	<0.001
IgA, μg/mL	35.13b	34.53b	40.13ab	41.38ab	43.04a	37.36ab	0.995	0.038	0.967	0.045	0.832	0.886
IL-1β, ng/L	13.59	10.74	11.17	17.88	16.41	21.51	0.837	<0.001	0.201	0.141	0.698	0.079
IFN-γ, pg/μL	2.71	2.80	3.05	3.83	3.69	4.39	0.122	<0.001	0.077	0.616	0.052	0.201
IL-6, ng/L	467.31d	468.73d	547.12cd	637.55bc	686.68b	1081.08a	33.657	<0.001	<0.001	<0.001	0.002	0.007
T-AOC, U/mL	0.73bc	1.25a	1.05a	0.71bc	0.53bc	0.45c	0.066	<0.001	0.387	0.026	0.822	0.182
MDA, nmol/mL	3.58a	2.44c	2.63bc	3.09abc	3.48a	3.24ab	0.112	0.058	0.109	0.005	0.104	0.425
BUN, mmol/L	6.23	6.51	5.19	5.37	5.64	4.70	0.191	0.045	0.034	0.884	0.057	0.069

ETEC = enterotoxigenic Escherichia coli; LPS = lipolyaccharide; Ig = immunoglobulin; IL = interleukin; IFN-γ = Interferon-gamma; T-AOC = total antioxidant capacity; MDA = malondialdehyde; BUN = blood urea nitrogen; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

In the same row, different superscript letters denote significant difference (P_{ETEC × CP} < 0.05, n = 8).

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5 % CP diet + ETEC-infection group.

Interaction effects between ETEC challenge and dietary protein levels were observed for serum IgG, IgM, IgA, IL-6, T-AOC, and MDA levels (P < 0.05). Under normal conditions, the CLP group showed the highest serum IgG and IgM, while the CHP group exhibited the highest serum MDA and lowest T-AOC (P < 0.05); under infected conditions, compared with the EHP group, IgG levels increased in the EMP and ELP groups, but IgM levels decreased (P < 0.05). Compared with the EHP and EMP groups, IL-6 levels increased in the ELP group (P < 0.05). Meanwhile, LPS, IgG, and IL-6 decreased linearly with increasing dietary CP levels (P < 0.05), and IFN-γ tended to decrease linearly (P = 0.052); while IgM showed a quadratic response (P < 0.001), and IL-1β exhibited a quadratic trend (P = 0.079), initially decreasing and then increasing as protein levels rose.

The pairwise comparisons of serum immunoglobulins, antioxidant capacity, and inflammatory factors in piglets with or without ETEC infection on d 6 are presented in Table S5. Under the 20.5% CP level, ETEC challenge elevated serum IgA and MDA levels in piglets (P < 0.05). When piglets were fed the 17.5% CP level, ETEC infection increased serum MDA concentrations (P = 0.032), reduced T-AOC levels (P < 0.001), and tended to elevate IgA content (P = 0.050). In piglets receiving the 14.5% CP level, ETEC exposure decreased serum T-AOC (P = 0.038) and showed a tendency toward elevated MDA content (P = 0.078).

3.3. Intestinal health of piglets

The intestinal morphology was investigated in the section of ileum by using H&E staining (Fig. 1A). A tendency for interaction effects between dietary protein level and ETEC infection on ileal crypt depth (P = 0.065) and V:C (P = 0.060) in piglets was observed (Table 4). In the noninfected groups, the V:C was lower in the CHP group compared to the CLP group; in the infected groups, ileal crypt depth was lower in the ELP group compared to the EMP group (Table 4).

Fig. 1.

Effects of dietary protein levels and enterotoxigenic Escherichia coli (ETEC) infection on intestinal health of piglets. (A) Hematoxylin and eosin staining of ileum on d 6. (B) Inflammatory cytokines genes in jejunum and ileum. (C) Expression of intestinal apoptosis-related genes in jejunal and ileum mucosa. P₁, the effects of the ETEC infection; P₂, the effects of the dietary protein levels; P₃, the interaction effect of the ETEC infection and dietary protein levels; P₄, the linear effect of CP level; P₅, the quadratic effect of CP level. CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group. Scale bar, 500 μm. HP = high protein; LP = low protein; MP = medium protein; CP = crude protein. Different lowercase letters above columns represent significant differences among treatments at P₃ < 0.05 (n = 8).

Table 4.

Effect of dietary protein levels and ETEC challenge on intestinal morphology and barrier in piglets on d 6.

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
Intestinal morphology
Villus height, μm	337.85	304.13	253.02	285.66	316.68	299.67	9.621	0.901	0.218	0.101	0.145	0.527
Crypt depth, μm	208.66	195.89	210.64	208.64	226.73	192.83	4.130	0.597	0.608	0.065	0.927	0.262
V:C	1.67	1.74	1.22	1.39	1.38	1.57	0.053	0.842	0.606	0.060	0.203	0.982
Intestinal barrier
DAO, ng/mL	250.45c	267.00c	261.13c	348.83b	391.03b	476.01a	14.390	<0.001	0.003	0.009	<0.001	0.760
D-LA, ng/L	1.45c	1.49c	1.60c	2.00b	2.05b	2.73a	0.072	<0.001	<0.001	<0.001	<0.001	0.006

ETEC = enterotoxigenic Escherichia coli; DAO = diamine oxidase; D-LA = D-lactic acid; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

In the same row, different superscript letters denote significant difference (P_{ETEC × CP} < 0.05, n = 8).

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group.

The ETEC infection increased (P < 0.05) intestinal barrier markers (DAO and D-LA) in serum and upregulated (P < 0.05) the expression of Caspase-3 in the jejunal mucosa and Bax in the ileal mucosa, as well as had a tendency to upregulate the expression of the IL-1β (P = 0.065) and Bax (P = 0.091) in the jejunal mucosa (Table 4; Fig. 1B and C). Compared with the 14.5% CP diet, feeding the 17.5% CP diet reduced the gene expression of IL-1β, Bcl-2, Bax, and Caspase-3 in the jejunal mucosa of piglets (P < 0.05).

Interaction effects (P < 0.05) between ETEC infection and dietary protein levels were observed for serum DAO, D-LA levels, IL-6, Bax, Bcl-2, and Caspase-3 in the ileal (Table 4; Fig. 1B and C). In the noninfected groups, the mRNA expression levels of Bcl-2 and Caspase-3 genes in the ileal mucosa were lower (P < 0.05) in the CHP group compared to the CLP and CMP groups; in the infected groups, the mRNA expression levels of Bcl-2 and Bax genes in the ileal mucosa were lower (P < 0.05) in the EMP group compared with the EHP and ELP groups. Meanwhile, serum DAO content on d 6 decreased (P < 0.05) linearly with increasing dietary protein levels (Table 4). The relative mRNA expression of IL-1β, Bax, and Bcl-2 in jejunal and ileal mucosa showed quadratic responses (P < 0.05), initially decreasing then increasing with rising protein levels (Fig. 1B and C).

The pairwise comparisons of intestinal health are presented in Table S6. At the 20.5% CP level, ETEC infection upregulated the relative mRNA expression of Bcl-2 (P = 0.029) and tended to increase the relative expression of IL-6 (P = 0.085) and Caspase-3 (P = 0.084) in the ileal mucosa. At the 17.5% CP level, ETEC infection increased Bax and Caspase-3 mRNA expression in the jejunal mucosa (P < 0.05), while downregulating Bcl-2 and Caspase-3 mRNA expression in the ileum (P < 0.05), and tended to downregulate IFN-γ (P = 0.058) in the ileal mucosa. At the 14.5% CP level, ETEC infection increased V:C and Bax mRNA expression in the ileal mucosa (P < 0.05).

3.4. Microbial populations and metabolites of piglets

As shown in Table 5, ETEC infection increased the numbers of total bacteria, E. coli, and Bacillus in cecal digesta of piglets (P < 0.05). An interaction effect between ETEC infection and dietary protein levels was observed for the number of E. coli (P = 0.016). Under normal condition, compared with the CMP and CLP groups, the CHP group showed lower the number of E. coli in cecal digesta (P < 0.05); under infected condition, the EMP group had the lowest the number of E. coli. Meanwhile, the number of E. coli decreased linearly with increasing dietary protein levels (P = 0.026). The pairwise comparisons of microbial populations in cecal digesta of weaned piglets are presented in Table S7. At the 20.5% CP level, ETEC infection upregulated the number of E. coli (P = 0.002). At the 14.5% CP level, ETEC infection upregulated the numbers of total bacteria, E. coli, and Bacillus (P < 0.05).

Table 5.

Effects of dietary protein levels and ETEC infection on microbial populations in cecal digesta of weaned piglets [log (copies/g)].

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
Total bacteria	9.38	9.52	9.61	9.82	9.81	10.22	0.078	0.003	0.181	0.650	0.083	0.531
Escherichia coli	8.04c	9.08b	8.88b	9.49ab	8.91b	9.94a	0.137	0.001	0.072	0.016	0.026	0.690
Bifidobacterium	4.00	4.09	4.46	4.73	4.12	4.39	0.103	0.268	0.397	0.239	0.833	0.184
Lactobacillus	6.69	6.93	6.98	6.69	7.46	7.37	0.144	0.299	0.279	0.743	0.177	0.394
Bacillus	7.48	7.75	7.74	7.95	7.95	8.23	0.072	0.007	0.281	0.640	0.113	0.995

ETEC = enterotoxigenic Escherichia coli; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

In the same row, different superscript letters denote significant difference (P_{ETEC × CP} < 0.05, n = 8).

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group.

Table 6 presents the differences in intestinal microbial metabolites. The ETEC infection exhibited no impact on the concentrations of acetic acid, propionic acid, and butyric acid (P > 0.05); however, ETEC infection and dietary protein levels had an interaction effect on the concentrations of butyric acid (P = 0.038). The pairwise comparisons of SCFAs in cecal digesta of weaned piglets are presented in Table S8. At the 20.5% CP level, ETEC infection decreased the concentrations of acetic acid and propionic acid (P < 0.05) while tending to decrease butyrate concentration (P = 0.074). At the 14.5% CP level, ETEC infection increased the concentration of propionic acid (P = 0.030) and tended to increase the concentration of butyric acid (P = 0.084).

Table 6.

Effects of dietary protein levels and ETEC infection on short chain fatty acids in cecal digesta of weaned piglets (μmol/g).

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
Acetic acid	24.71	16.93	17.58	18.38	19.23	21.43	1.104	0.978	0.439	0.131	0.445	0.304
Propionic acid	13.64	9.63	9.15	10.16	11.43	14.27	0.721	0.416	0.690	0.051	0.915	0.369
Butyric acid	4.42	4.72	2.12	2.20	3.69	4.60	0.387	0.729	0.557	0.038	0.957	0.285

ETEC = enterotoxigenic Escherichia coli; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group; n = 8.

3.5. Growth performance and diarrhea rate of piglets in 7 to 28 d

As shown in Table 7, ETEC infection did not affect piglet growth performance at 7 to 28 d. As protein levels increased, BW28, ADG, and G:F increased (P < 0.05). Compared with the 20.5% CP diet, diarrhea rate was lower in the 14.5% CP diet (P < 0.05). Meanwhile, BW28, ADFI, ADG, G:F, and diarrhea rate increased linearly to increasing dietary protein levels (P < 0.05).

Table 7.

Effects of dietary protein levels and ETEC challenge on growth performance and diarrhea rate of piglets from 7 to 28 d of experiment.

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
BW7, kg	8.05	7.82	7.81	7.81	7.90	7.81	0.054	0.652	0.661	0.487	0.367	0.942
BW28, kg	16.27	14.33	13.53	15.70	14.35	13.02	0.232	0.315	<0.001	0.737	<0.001	0.429
ADFI, g	680.79	629.43	633.47	674.86	625.22	574.64	12.587	0.352	0.052	0.595	0.019	0.600
ADG, g	373.70	295.79	260.24	358.54	293.33	240.00	9.565	0.361	<0.001	0.861	<0.001	0.352
G:F, g	0.55	0.47	0.41	0.53	0.47	0.42	0.010	0.694	<0.001	0.708	<0.001	0.598
Diarrhea rate, %	24.30	18.94	9.46	26.47	19.27	16.23	1.564	0.278	0.003	0.636	<0.001	0.997

ETEC = enterotoxigenic Escherichia coli; BW7 = body weight on d 7; BW28 = body weight on d 28; ADFI = average daily feed intake; ADG = average daily gain; G:F = ADG:ADFI; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group; n = 8.

3.6. Serum immunoglobulins, antioxidant capacity and inflammatory factors of piglets on d 28

As shown in Table 8, ETEC infection elevated serum LPS, IgG, IgM, IgA, IL-1β, IFN-γ, and IL-6 levels in piglets on d 28 (P < 0.05). The main effect of CP levels showed that 14.5% CP diet exhibited the highest LPS and IFN-γ levels in serum, however the 20.5% CP diet exhibited the higher BUN levels in serum (P < 0.05). Interaction effects between ETEC infection and dietary protein levels were observed for serum IgG, IgM, IgA, IL-1β, IL-6, and T-AOC levels (P < 0.05). Among the noninfected groups, compared with the CLP group, serum immunoglobulin (IgG, IgM, and IgA) levels decreased in the CHP and CMP groups (P < 0.05). Compared with the CMP group, serum IL-1β, IL-6, and T-AOC levels decreased in the CHP and CLP groups (P < 0.05); among the infected groups, serum IL-1β and IL-6 levels were increased on d 28 as dietary protein levels decreased (P < 0.05). EMP group had the lowest immunoglobulin (IgG, IgM, and IgA) levels compared with the EHP and ELP groups (P < 0.05). Meanwhile, serum LPS and IFN-γ decreased linearly with increasing dietary protein levels, while IgG, IgM, IgA, IL-6, and T-AOC exhibited quadratic responses, initially decreasing then increasing with rising protein levels (P < 0.05).

Table 8.

Effect of dietary protein levels and ETEC challenge on serum immunoglobulins, antioxidant capacity and inflammatory factors in piglets on d 28.

Items	Treatments1						SEM	P-value
	CHP	CMP	CLP	EHP	EMP	ELP		ETEC	CP	ETEC × CP	CP linear	CP quadratic
LPS, ng/L	273.21	279.64	336.26	391.02	424.00	455.38	13.247	<0.001	0.010	0.759	0.004	0.489
IgG, μg/mL	181.18c	196.00c	244.44b	311.43a	214.95b	320.86a	9.445	<0.001	<0.001	<0.001	0.010	<0.001
IgM, μg/mL	33.14c	38.65bc	43.57ab	47.55a	39.37b	49.52a	1.110	<0.001	<0.001	0.006	0.004	0.013
IgA, μg/mL	58.29c	57.38c	72.54b	94.34a	51.82c	73.91b	2.452	<0.001	<0.001	<0.001	0.342	<0.001
IL-1β, ng/L	20.28d	14.49c	24.13cd	29.15bc	34.59b	40.64a	1.469	<0.001	<0.001	0.017	<0.001	0.021
IFN-γ, pg/μL	5.44	5.65	6.22	7.31	7.25	8.59	0.205	<0.001	0.004	0.492	0.003	0.130
IL-6, ng/L	413.49d	305.22e	460.55d	607.45c	711.68b	873.32a	31.818	<0.001	<0.001	0.002	<0.001	0.006
T-AOC, U/mL	1.40a	0.49c	0.82b	1.03b	0.86b	0.74bc	0.055	0.774	<0.001	0.002	<0.001	<0.001
MDA, nmol/mL	4.63	4.06	3.00	5.08	4.14	4.75	0.222	0.080	0.151	0.258	0.066	0.567
BUN, mmol/L	5.91	4.17	3.95	5.05	3.91	3.58	0.186	0.111	<0.001	0.705	<0.001	0.085

ETEC = enterotoxigenic Escherichia coli; LPS = lipolyaccharide; Ig = immunoglobulin; IL = interleukin; IFN-γ = Interferon-gamma; T-AOC = total antioxidant capacity; MDA = malondialdehyde; BUN = blood urea nitrogen; HP = high protein; LP = low protein; MP = medium protein; CP = crude protein; SEM = standard error of the mean.

In the same row, different superscript letters denote significant difference (P_{ETEC × CP} < 0.05, n = 8).

¹CHP, 20.5% CP diet + ETEC-noninfection group; CMP, 17.5% CP diet + ETEC-noninfection group; CLP, 14.5% CP diet + ETEC-noninfection group; EHP, 20.5% CP diet + ETEC-infection group; EMP, 17.5% CP diet + ETEC-infection group; ELP, 14.5% CP diet + ETEC-infection group.

The pairwise comparisons of serum immunoglobulins, antioxidant capacity, and inflammatory factors are presented in Table S9. In piglets receiving the 20.5% CP level, ETEC infection elevated serum IgM, IgA, and T-AOC levels on d 28 (P < 0.05). At the 17.5% CP level, infection increased serum T-AOC content (P = 0.022). At the 14.5% CP level, ETEC challenge raised serum IgM and MDA levels (P < 0.05).

4. Discussion

In this study, ETEC infection increased diarrhea rate, serum LPS, immunoglobulins (IgG, IgM, and IgA) and pro-inflammatory cytokines (IL-1β, IFN-γ, and IL-6) levels, and ETEC infection also increased the number of E. coli in the cecal digesta of piglets, which is consistent with previous studies (Wang et al., 2020; Zhen et al., 2022).

Dietary CP levels and ETEC infection affect growth performance of weaned piglets. Under noninfected condition, the study found that HP diet exhibited higher ADG compared to MP and LP diets from 1 to 6 d, with this advantage persisting through 7 to 28 d. This aligns with previous studies, indicating that protein levels in the diet should not be reduced for healthy weaned piglets to better achieve their growth potential (Heo et al., 2009; Tybirk et al., 2020). However, ETEC infection fundamentally altered this dynamic. This study found that the MP diet demonstrated unique advantages under ETEC challenge. This difference may stem from differential protein utilization pathways: while high protein diets (>20%) maximize anabolism in healthy piglets, their undigested excess protein might promote proteolytic fermentation during infection, exacerbating intestinal inflammation (Bikker et al., 2006; Gao et al., 2022). Medium protein diets (17%–18% CP) appear to strike a balance between maintaining essential AA supply while minimizing substrates for pathogenic proliferation (Heo et al., 2013; Nyachoti et al., 2006). This suggested that MP diet might be better for supporting growth performance of piglets in ETEC-infected environments. The observed disparity in growth performance of piglets by dietary protein levels under different conditions may be related to organismal immunity, intestinal inflammation and microbial homeostasis. Based on the experimental findings, the potential reasons for piglets fed the MP diet to have optimal G:F after ETEC infection are as follows.

Firstly, reducing dietary protein level under ETEC infection condition helps enhance piglets’ immune responses and maintain immune homeostasis. This study found that reducing the dietary protein level increases the IgG levels, but decreases the IgM levels in piglets during ETEC infection. The IgG provides protection through pathogen neutralization, phagocytosis enhancement, and T-cell regulation (Bournazos and Ravetch, 2017). The IgM, as the first line of defense, functions as a polymeric antibody that rapidly generates high-affinity antibodies against invading pathogens, playing a critical role in initial pathogen clearance (Khwaja et al., 2022). Additionaly, piglets fed HP and LP diets did not show a significant increase in IgA upon ETEC infection on d 6, whereas piglets on MP diets exhibited a significant rise in IgA levels after ETEC infection. The IgA is primarily localized to mucosal barriers and exocrine systems, where it plays a central role in establishing mucosal immune defenses (Brabdtzaeg, 2009). Therefore, it can be speculated that MP diet during ETEC infection is beneficial for maintaining immune homeostasis. Pro-inflammatory cytokines regulate the inflammatory response and reflect the inflammatory state of the host organism (Ljuca et al., 2010; Xiong et al., 2019). The study found that serum IL-1β and IL-6 levels were significantly higher in the LP diet compared to the MP diet under infected conditions at the d 6 and 28. These results indicated that LP diet might exacerbate the inflammatory response in piglets under infected conditions.

Secondly, dietary protein levels critically modulate the intestinal barrier-apoptosis-inflammation triad during ETEC challenge, with MP (17.5% CP) diets demonstrating optimal protective effects. The D-LA and DAO are mainly expressed and stored in intestinal villous epithelial cells, and when intestinal barrier function is impaired, DAO and D-LA are released from the intestinal epithelium into the blood (Anke and Spector, 1975; Kettner et al., 2022). Intestinal mucosal barrier function can be assessed by measuring the levels of D-LA and DAO (Meng et al., 2016). Under infected condition, serum DAO and D-LA levels were significantly elevated in the ELP group compared with the EHP and EMP group, indicating that the intestinal barrier integrity in the ELP group of piglets was more severely compromised. Pro-inflammatory cytokine overproduction in the intestinal mucosa induces barrier dysfunction via dysregulated inflammatory cascades (Al-Sadi et al., 2009). Mechanistically, ETEC infection-mediated IL-6 and IL-1β release drives intestinal barrier disruption (Che et al., 2017; Li et al., 2018; Pu et al., 2018). The study demonstrated that, compared with other infected groups, the EMP group under ETEC challenge showed significantly attenuated expression of IL-1β and IL-6 mRNA in the ileal mucosa. Furthermore, apoptosis critically regulates intestinal homeostasis, where dysregulated apoptosis induces dysfunction (Günther et al., 2013; Wong, 2004). The activation of Bax and Caspase-3 serves as biochemical hallmarks of mitochondrial apoptosis (Zheng et al., 2023). The study revealed significant downregulation of ileum Bax and Bcl-2 expression in the EMP group compared with the ELP group, indicating MP diet modulation of apoptotic pathways. The results indicated the MP (17.5% CP) diet preserved intestinal barrier integrity during ETEC challenge through downregulation of apoptotic effectors and attenuation of inflammatory mediators, whereas the LP diet exacerbated barrier deterioration under infectious conditions.

Thirdly, our data demonstrated that MP diets significantly inhibited ETEC-induced E. coli expansion compared to LP diets. The MP diet helps to inhibit the proliferation of pathogens and regulate the production of intestinal microbial metabolites. Previous study found that dietary protein modulates gut microbiota dynamics through nutrient provisioning for microbial fermentation, generating bioactive metabolites that influence host physiology (Zhang et al., 2020). Protein concentration critically determines commensal microbiota composition and metabolic functionality (Spring et al., 2020), with LP regimens suppressing E. coli proliferation (Wang et al., 2019). However, excessive protein restriction compromised disease resistance, facilitating enteropathogenic colonization (Lin et al., 2024). Potential explanations include impaired mucosal barrier function, reduced synthesis of antimicrobial peptides or mucus, or alterations in the gut microbiota composition that reduce competitive exclusion of pathogens (Chen et al., 2018; Wang et al., 2021). Critically, this increase in cecal E. coli load was associated with a concomitant elevation in serum LPS levels. This correlation strongly indicates heightened translocation of bacterial endotoxin across the intestinal barrier into the systemic circulation (Li et al., 2024). SCFAs are mainly derived from the fermentation of carbohydrates and AAs by intestinal bacteria and consist of acetic acid, propionic acid, and butyric acid (Louis and Flint, 2009; Wang et al., 2004). It has been found that moderate protein restriction modulates SCFA production, crucial for intestinal homeostasis and immunoregulation (Parada Venegas et al., 2019). Meanwhile, SCFA may strengthen the intestinal barrier by promoting the expression of tight junction proteins and inhibiting the growth of pathogenic bacteria (Peng et al., 2009). This study found the content of SCFA was decreasing as the dietary protein level decreased when piglets was infected. The results indicated that the MP (17.5% CP) diet restored microbial homeostasis through selective suppression of E. coli. In contrast, LP diet induced microflora dysbiosis, leading to E. coli overgrowth.

Additionally, according to Gao et al. (2020), piglets that were given a diet containing 29% CP exhibited notably elevated levels of TNF-α, IL-6, and IL-8 in the ileum compared to those on a 17% low-protein diet. Similarly, Pieper et al. (2012) demonstrated that piglets consuming a high-protein diet experienced a marked upregulation in the expression of IL-1β and IL-6 mRNA within their colonic epithelial cells. A HP diet (CP > 20%) can trigger intestinal inflammation. This reaction is linked to the activation of the NF-κB signaling pathway caused by antigenic proteins such as globulin and β-conglycinin, leading to an increase in cytokine production and damage to the intestinal epithelial cells (Peng et al., 2017). The study showed that mRNA expression of IL-1β and IL-6 genes in the ileal mucosa was significantly reduced in MP group in the ETEC-infected group. These results indicated that the MP (17.5% CP) diet might inhibit the expression of inflammatory factors by inhibiting the NK-κB pathway under ETEC-infected condition.

5. Conclusion

In conclusion, this study has found that a MP diet (17.5% CP) effectively maintained the growth performance, intestinal health, and immune homeostasis of piglets, while mitigating ETEC infection-induced inflammatory responses and microbial dysbiosis. Therefore, under conditions of high pathogenic bacterial exposure, a MP diet (17.5% CP) is recommended to optimize the health and growth performance of piglets. However, under normal conditions, a HP diet (20.5% CP) remains the optimal formulation for maximizing growth performance.

Credit Author Statement

Minmin Li: Writing – original draft, Visualization, Validation, Supervision. Mingyu Wang: Visualization, Validation, Conceptualization. Yuhan Chen: Visualization, Validation, Supervision. Daiwen Chen: Methodology. Bing Yu: Resources, Investigation. Jun He: Resources, Investigation. Jie Yu: Formal analysis, Data curation. Xiangbing Mao: Formal analysis, Data curation. Zhiqing Huang: Software, Formal analysis, Data curation. Yuheng Luo: Formal analysis, Data curation. Junqiu Luo: Writing – review & editing, Formal analysis. Hui Yan: Writing – review & editing. Ping Zheng: Project administration, Methodology, Funding acquisition.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper. Daiwen Chen is an Associate Editors for Animal Nutrition and was not involved in the editorial review or the decision to publish this article. Jun He is a Youth Editorial Board Member for Animal Nutrition and was not involved in the editorial review or the decision to publish this article.

Appendix A. Supplementary data

The following is the Supplementary data to this article: