Dietary anti- Escherichia coli K88 IgY enhances immune response and intestinal barrier function in piglets challenged with Escherichia coli K88

Abstract

Background

Chicken egg yolk immunoglobulin Y (IgY) has shown potential in preventing pathogen infections. This study examined the effectiveness of dietary IgY supplementation at 6 g/kg in preventing weaned piglets from developing diarrhoea after a challenge with Escherichia coli K88 (E. coli K88). The experiment employed a 2 × 2 factorial design. Twenty-four healthy 17-day-old piglets with similar body weights (4.63 ± 0.39 kg) were selected and divided into 4 groups, each with 6 replicates (n = 6). The experimental period lasted 11 days. The control group (CON) and the E. coli K88 group (ECON) were fed a basal diet supplemented with 6 g/kg of common yolk powder. Meanwhile, the anti-E. coli K88 IgY group (IgY) and the E. coli K88 IgY group (EIgY) received a diet supplemented with 6 g/kg of anti-E. coli K88 IgY. From days 8 to 11 of the formal trial, piglets in the CON and IgY groups received an equal volume of normal saline (NS), while the ECON and EIgY groups were orally administered 10 mL of NS containing approximately 1 × 10¹⁰ CFU/mL of E. coli K88.

Results

Results showed that dietary 6 g/kg anti-E. coli K88 IgY supplementation increased the IL-10 protein expression level in the jejunum tissue, villus height (VH), the villus height to crypt depth ratio (V: C), sucrase activities, and reduced colonic E. coli counts, diarrhea incidence, and D-LA levels in E. coli K88-infected piglets (P < 0.05). Meanwhile, the IgY diet increased Claudin-1 protein levels and decreased IL-8 levels in the jejunum tissue of E. coli K88-challenged piglets (P < 0.05).

Conclusions

The dietary 6 g/kg anti-E. coli K88 IgY supplementation can reduce intestinal structural damage and inflammation, improving growth by enhancing passive immunity and intestinal barrier function in E coli K88-challenged piglets.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12917-026-05676-5.

Background

The weaning period represents a critical physiological stage in pig production [1]. Piglets are more susceptible to pathogens because their gastrointestinal and immune systems are not fully developed [2]. Enterotoxigenic Escherichia coli (ETEC) is the primary cause of post-weaning diarrhea, creating significant challenges for the global swine industry. The susceptibility is caused by K88⁺ fimbriae attaching to intestinal receptors, with K88⁺ ETEC strains being the most common in clinical cases [3, 4]. K88⁺ETEC invades host cells by binding to K88⁺-specific receptors, colonizes the small intestine, and releases enterotoxins, which disrupt normal peristalsis and the passive immune response of the mucosa, thereby causing diarrhea in piglets [5]. ETEC infection is a serious threat to piglet health. Currently, for piglet diarrhea caused by ETEC, production mainly relies on antibiotic treatment, but long-term use can easily lead to drug resistance, intestinal microecology imbalance, and drug residues. Safe and effective alternative strategies need to be developed.

Immunoglobulin Y (IgY), a natural antibody extracted from immunized avian yolk, shows promise as a solution. Studies indicated that IgY provided anti-infection effects by neutralizing viral antigens, regulating intestinal flora, and enhancing mucosal barrier passive immunity, thereby strengthening disease defense in both animals and humans [6]. IgY has demonstrated significant inhibitory effects against pathogenic diseases such as ETEC, porcine infectious gastroenteritis, porcine epidemic diarrhea virus, Salmonella, and coronavirus rotavirus [7, 8]. Laying hens produce high-quality polyclonal IgY, a non-invasive antibody source from egg yolk [9]. Preparing IgY provides advantages: non-invasive collection, no harm to animals, high yield, strong specificity, easy to prepare, safe, and cost-effective [10]. The integrity of the intestinal barrier is preserved by various components, such as tight junction proteins (ZO-1, Claudin-1, and Occludin), mucus secretion, and immune cells, forming the first line of defense against pathogen invasion. However, whether anti-E. coli K88 IgY can mitigate ETEC-induced intestinal barrier damage by synergistically regulating these mechanisms, which remains to be systematically clarified.

This study developed an E. coli K88-induced diarrhea model in weaned piglets to assess the effects of dietary anti-E. coli K88 IgY supplementation on immune function and intestinal barrier integrity. It evaluated jejunal mucosal immunity, histomorphology, tight junction protein expression, and E. coli colonization.

In this study, a diarrhea model induced by Escherichia coli K88 was successfully established in weaned piglets. For the first time, we systematically evaluated the practical application effects of an anti-E. coli K88 IgY product developed and mass‑produced against this bacterial strain. The protective effects of this anti-E. coli K88 IgY on piglets was comprehensively assessed across multiple dimensions, including growth performance, immune function, and intestinal barrier function. It evaluated jejunal mucosal immunity, histomorphology, tight junction protein expression, and E. coli colonization.

Materials and methods

The IgY source

Anti-E. coli K88 IgY was developed and produced by the team of Professor Pingli He of China Agricultural University [11]. As follows, the eggs laid by the homogenized hens were collected, and the yolk powder was prepared through cleaning and disinfection, yolk separation, filtration, homogenization, concentration, and spray drying. The IgY was extracted using the PEG-6000 method. The titer (1: 25600) of anti-E. coli K88 IgY was determined by indirect ELISA. The base diet was supplemented at 6 g/kg.

Animals and diets

A total of 24 healthy castrated male pigs (Duroc × Landrace × Yorkshire) weaned at 17 days of age with an initial BW of 4.63 ± 0.39 kg provided by a commercial farm (Yige family farm, Hunan, Liuyang, China) were used in this study. Piglets were assigned to the following groups: (1) CON group, 6 g/kg common yolk powder added to the basic diet, n = 6; (2) IgY group, 6 g/kg anti-E. coli K88 IgY added to the basic diet, n = 6; (3) ECON group, common yolk powder challenge group, n = 6; (4) EIgY group, anti-E. coli K88 IgY challenge group, n = 6. The experiment was carried out on the first floor of the animal facility at the Institute of Subtropical Agriculture, Chinese Academy of Sciences. A total of 24 individual pens were used, with one pig housed per pen; the temperature in the pigsty is 25 ± 2 ℃, and the humidity is between 65% and 70%. All pigs were fed ad libitum throughout the experiment.

The trial consisted of a 4-day pre-feeding period followed by an 11-day formal period. On the 8th day of the experiment, each piglet in the ECON and EIgY groups was given 10 mL of 1 × 10¹⁰ CFU/mL E. coli K88 solution every day for 3 consecutive days, and the CON and IgY groups were used as controls to given 10 mL of 0.9% normal saline solution. The composition and nutrient level of the diet (Table S1), which was prepared according to NRC (2012) for 5–7 kg weaned piglets. The feed samples were crushed (RS-FS2201, Royalstar) and sieved through a 0.42-mm mesh before nutrient analysis. The dry matter (DM), crude ash (Ash), ether extract (EE), and crude protein (CP) contents were determined using the method described by AOAC [12].

Animal performance and diarrhea

Average daily gain (ADG) and average daily feed intake (ADFI) were derived from body weight and feed intake measurements taken on days 1, 7, and 12. Body weight was recorded on the morning of each sampling day, following a 12-h fast to minimize variability. ADG was calculated for the intervals of days 1–7 and days 7–12, with ADFI and feed‑to‑gain ratio (F: G) determined over the same intervals.

Diarrhea incidence was assessed daily throughout the experimental period. Diarrhea scores refer to [13]. A stool consistency scoring system was used (0 = normal stool; 1 = soft stool; 2 = mild diarrhea; 3 = severe diarrhea) to judge each piglet’s diarrhea frequency and severity. Fecal consistency was scored uniformly, with ≤ 1 indicating diarrhea-free status. The diarrhea incidence (%) for each group was calculated using the following formula:

E. coli K88 preservation and cultivation conditions

E. coli K88 (BNCC 125988) was purchased from Beijing Beina Chuanglian Biotechnology Institute (Beijing, China). It was cultured in Luria–Bertani (LB) broth at 37 ℃ until it reached 1 × 10⁹ CFU/mL. Following centrifugation, the bacterial pellet was resuspended in a reduced volume of LB broth to achieve a final concentration of 1 × 10¹⁰ CFU/mL.

Sample collection

All pigs were killed by exsanguination after electrical stunning on the morning of the 12th day of the experiment. The 2-cm mid-jejunal segments were excised from piglets, gently rinsed with pre-cooled saline to remove contents, and immersed in 4% paraformaldehyde for fixation and storage to analyze histomorphology. Another jejunal intestinal segment was dissected with scissors. After being rinsed with saline and dried with filter paper, mucosal scrapings were wrapped in foil, quickly frozen in liquid nitrogen, and stored at -80 ℃.

Jejunal mucosal digestive enzymes and oxidoreductase activity detection

Jejunal mucosa samples (0.1 g) were weighed and homogenised using an ultrasonic homogeniser in 0.9 mL of PBS (4 ℃, pre-cooled). The homogenate was then subjected to centrifugation (3000 × g, 15 min at 4 ℃). The protein concentration of the supernatant was initially determined for calibration purposes using a commercial BCA kit (Biyuntian, Shanghai, China).

The levels of cytokines in the jejunal mucosa, including total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), and catalase (CAT), D-lactate (D-LA), and diamine oxidase (DAO), were measured using suitable commercially available biochemical kits (Suzhou Grace Biotechnology Co., Ltd) according to the manufacturer’s instructions. Amylase (AMS), sucrase, lactase (LCT), trypsin, lipase (LPS), and alkaline phosphatase (AKP) in the jejunum were analysed using appropriate commercial biochemical kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) following the manufacturer’s instructions. ELISA was employed to analyse jejunal mucosal immune factors. Protein concentrations were determined by the BCA assay. The mucosal cytokines were quantified using commercial ELISA kits (Wuhan Huamei Bioengineering Co., Ltd., Wuhan, China) following the manufacturer’s instructions, including Interleukin-6 (IL-6, CSB-E06786p), Interleukin-8 (IL-8, CSB-E06787p), Interleukin-10 (IL-10, CSB-E06779p-lS), Interleukin-1β (IL-1β, CSB-E06782p), Interferon-γ (IFN-γ, CSB-E06794p), immunoglobulin A (IgA, CSB-E13234p), immunoglobulin G (IgG, CSB-E06804p) and immunoglobulin M (IgM, CSB-E06805p).

Jejunal morphology analysis

Jejunal samples were fixed overnight in 4% paraformaldehyde, then dehydrated through a graded ethanol series and embedded in paraffin. The paraffin blocks were cut into 5‑µm thick sections, which were subsequently stained with hematoxylin and eosin. Images of jejunal morphology were captured using a light microscope (Nikon ECLIPSE 80i, Tokyo, Japan). From each image, fifteen well‑oriented jejunal villi and their contiguous crypts were randomly selected. Villus height (VH) and crypt depth (CD) were measured using Image‑Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) [14]. The villus height-to-crypt depth ratio (VH/CD) was then calculated.

The real-time qPCR analyses

Total RNA was extracted from the jejunum using an RNA extraction kit (cat. no. CW3711; Beijing CoWin Biotech Co., Ltd., Beijing, China). RNA concentration and purity were measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Only RNA samples with an A260/A280 ratio between 1.8 and 2.1 were used for cDNA synthesis. All quantitative real-time polymerase chain reaction (qPCR) reactions were performed in duplicate on the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with TB Green Premix Ex Taq II (Tli RNaseH Plus; Takara Biotechnology Co., Ltd.). A 20-µL reaction mixture (Takara Biotechnology, Dalian, China) was prepared according to the manufacturer’s guidelines, and each sample was analyzed in triplicate. PCR amplification conditions involved incubation at 95 ℃ for 30 s, followed by 45 cycles of 5 s at 95 ℃ and 30 s at 62 ℃. All primers and their sequences are listed in Table S2. The expression levels of target mRNA were normalized to β-actin and calculated using the 2^−ΔΔCt method.

Western blotting

Total protein is extracted from the jejunal mucosa, the tissue is lysed with RIPA lysate, and after the protein is released, the protein concentration is measured using the BCA method. Subsequently, the protein samples were analyzed using SDS-PAGE electrophoresis to separate proteins based on their molecular weight. After transferring to PVDF membranes (0.45 μm), nonspecific binding was blocked with 5% skim milk powder. Specific rabbit primary antibodies—GAPDH (SA30-01), ZO-1 (PSH07-09), Occludin (bs-1495R), and Claudin-1 (JE30-33) (1:1,000 dilution)—were added to an incubator box (Huaan Biotechnology Co., Ltd., Hangzhou, China) and incubated overnight on a 4 ℃ shaker. Finally, fluorescently labeled rabbit secondary antibodies that bind to the primary antibodies were added. The expression of the target protein was detected using chemiluminescence and normalized for presentation.

Intestinal microbial community assessment

A 0.5 g aliquot of thawed colonic chyme (n = 6) was used for microbial genomic cDNA extraction. DNA integrity was verified using agarose gel electrophoresis, and DNA concentration was adjusted to 10 ng/µL (total DNA ≥ 500 ng). High-fidelity PCR was performed to pre-amplify the DNA template (without spike-in reference DNA), with a standard bacterial genomic DNA mix as the positive control. The V3-V4 hypervariable regions of the 16S rRNA gene and spike-ins were amplified with the primers 341 F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’) and then sequenced using Illumina NovaSeq 6000 sequencer. The raw reads were processed using QIIME2, with adapters and primers trimmed by cutadapt, followed by quality control and ASV inference via DADA2. Taxonomic assignment was performed against Greengenes (v13.8) using a Naive Bayes classifier (confidence threshold 0.8). Spike‑in sequences were identified and counted, and a standard curve was constructed for each sample to calculate absolute ASV copy numbers; spike‑ins were then removed from subsequent analyses.

Statistical analysis

All data were analyzed using SPSS 26.0 (IBM Corp., Armonk, NY, USA). Before conducting parametric analyses, the assumptions of normality and homogeneity of variances were verified. Normality was assessed with the Shapiro–Wilk test (Shapiro & Wilk, 1965), performed separately for each experimental group. Pre-challenge data were analyzed with an independent-samples t-test. Homogeneity of variances was evaluated with Levene’s test (Levene, 1960). Variables that met both assumptions (Shapiro–Wilk: P > 0.05; Levene’s test: P > 0.05) were subjected to two‑way ANOVA with IgY supplementation and ETEC challenge as fixed factors, followed by Tukey’s multiple-comparison test. The interaction between ETEC and IgY was also evaluated. Significance was established at P < 0.05, with a trend toward significance for 0.05 ≤ P < 0.10. All data are shown as the LSmeans ± SEM.

Results

Growth performance of piglets

Compared to adding 6 g/kg of common egg yolk powder, anti-E. coli K88 IgY supplementation did not affect weaned piglets’ growth performance from 1 to 7 days (P > 0.05) (Tables 1 and 2). Compared with the ECON group, E. coli K88 infection increased the diarrhea rate in weaned piglets (P < 0.05); adding 6 g/kg of anti-E. coli K88 IgY reduced the diarrhea rate of E. coli K88-infected weaned piglets (P < 0.05) (Table 2).

Table 1.

Effects of IgY supplementation on piglet performance before challenge^a

Item	CON	IgY	SEM	P-value
1–7 d
IBW, kg	4.64	4.62	0.162	0.879
FBW, kg	5.49	5.43	0.207	0.750
ADG, g/d	120.83	114.88	20.33	0.772
ADFI, g/d	217.02	210.32	25.81	0.798
F: G	1.93	1.99	0.21	0.770
Diarrhea rate, %	53.57	36.90	13.91	0.244

IBW initial body weight, FBW final body weight, ADG average daily gain, ADFI average daily feed intake, F: G feed-to-gain ratio

^aCON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge. Noteworthy differences (P < 0.05) among the four groups are indicated by different lowercase letters, n = 12 piglets/group

Table 2.

Effects of IgY supplementation on growth performance in weaned piglets during E. coli K88 challenge

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
8–11 d
IBW, kg	5.38	5.43	5.60	5.42	0.107	0.758	0.618	0.565
FBW, kg	5.79	5.93	5.85	5.75	0.119	0.931	0.796	0.618
ADG, g/d	104.17ab	120.83a	60.42b	83.33ab	7.457	0.199	0.013	0.836
ADFI, g/d	204.75	234.04	157.46	189.00	9.707	0.133	0.027	0.954
F: G	2.28	1.88	3.06	2.52	0.165	0.171	0.045	0.845
Diarrhea rate, %	25.00b	8.33b	66.67a	20.83b	3.870	0.001	0.002	0.074

IBW initial body weight, FBW final body weight, ADG average daily gain, ADFI average daily feed intake, F: G feed-to-gain ratio

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Morphologic structure of the jejunum

As shown in Fig. 1, the E. coli K88 challenge reduced the villus height (VH) and the villus height to crypt depth ratio (V: C) while increasing crypt depth (CD) (P < 0.05) (Table 3). Compared to adding 6 g/kg of common egg yolk powder, adding 6 g/kg of anti-E. coli K88 IgY to the diet increased VH and the V: C in weaned piglets infected with E. coli K88, while reducing CD (P < 0.05) (Fig. 1).

Fig. 1.

Effects of IgY supplementation on jejunal morphology of E. coli K88-challenged weaned piglets. Representative histological micrographs of the jejunum in weaned piglets (hematoxylin and eosin staining; magnification 50 ×, scale bar = 100 μm). Histological parameters of the jejunum. The arrow indicated the normal structure of the villus. The circle indicated the broken brush border of the villus. CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group. P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Table 3.

Effect of dietary IgY supplementation on the morphological structure of the jejunum in weaned piglets during E. coli K88 challenge

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
VH, µm	297.15a	319.26a	251.74b	297.55a	4.865	0.002	0.003	0.237
CD, µm	124.88b	105.83c	141.47a	113.48bc	1.562	< 0.001	0.001	0.168
V: C	2.41b	3.04c	1.79a	2.63bc	0.056	< 0.001	< 0.001	0.375

VH = villus height, CD = crypt depth, V: C = villus height to crypt depth ratio

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Antioxidant function of jejunal mucosa

Regarding antioxidant status, there was no significant interaction between the two factors on the indices of antioxidant enzymes. Anti-E. coli K88 IgY supplementation had no significant effect on the levels of T-AOC, SOD, GSH-Px, CAT, and MDA in jejunal mucosa (P > 0.05). In contrast, E. coli K88 reduced MDA expression (P < 0.05) (Table 4).

Table 4.

Effect of dietary supplementation with IgY on the antioxidant function of jejunal mucosa of weaned piglets challenged by E. coli K88

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
T-AOC, µmol/mg prot	5.78	5.81	5.64	5.73	0.128	0.807	0.659	0.903
SOD, U/mg prot	0.62	0.65	0.59	0.65	0.015	0.191	0.631	0.709
GSH-Px, mmol/mg prot	0.38	0.41	0.36	0.39	0.014	0.331	0.455	0.908
CAT, mmol/mg prot	2.60	2.96	2.44	2.51	0.131	0.415	0.258	0.578
MDA, nmol/mg prot	0.11b	0.10b	0.17a	0.15ab	0.006	0.586	0.001	0.586

T-AOC the total antioxidant capacity, SOD superoxide dismutase, GSH-Px  glutathione peroxidase, CAT catalase, MDA malondialdehyde

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Expression of inflammatory factors in the jejunal mucosa

Compared to CON group, E. coli K88 infection increased the levels of IL-8 in the jejunal mucosa tissue of weaned piglets and decreased IL-10 levels (P < 0.05) (Table 5). Under E. coli K88 infection conditions, compared to adding 6 g/kg of common egg yolk powder, supplementing with 6 g/kg anti-E. coli K88 IgY in the diet reduced IL-8 levels in the jejunal mucosa tissue of ETEC-infected piglets and increased IL-10 levels (P < 0.05). Furthermore, E. coli K88 infection and IgY supplementation significantly interacted in modulating IL‑8 levels (P < 0.05).

Table 5.

Effect of dietary addition of IgY on the expression of inflammatory factors in the jejunal mucosa of piglets challenged by E. coli K88

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
IL-6, pg/mg prot	25.74	25.39	27.72	25.80	1.007	0.579	0.560	0.700
IL-8, ng/mg prot	0.72b	0.70b	1.05a	0.74b	0.035	0.027	0.016	0.041
IL-10, pg/mg prot	17.34a	18.46a	12.47b	16.76a	0.442	0.006	0.001	0.088
IL-1β, pg/mg prot	48.42	47.68	54.57	49.44	1.325	0.282	0.152	0.417
IFN-γ, ng/mg prot	0.40ab	0.34b	0.47a	0.38ab	0.016	0.036	0.080	0.601

IL interleukin, IFN-γ interferon-γ

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

As shown in Fig. 2, compared to adding 6 g/kg of common egg yolk powder, supplementing with 6 g/kg anti-E. coli K88 IgY to the diet has no effect on the mRNA expression levels of inflammatory factors (P > 0.05).

Fig. 2.

Effects of IgY on messenger RNA (mRNA) expression of inflammatory factor in the jejunum of weaned piglets challenged by E. coli K88. IL = interleukin, IFN-γ = interferon-γ. CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group. P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Jejunal mucosal immunoglobulin content

E. coli K88 infection decreased the levels of IgA and IgM in the jejunal mucosa tissue of weaned piglets (P < 0.05) (Table 6). Compared to adding 6 g/kg of common egg yolk powder, supplementing with 6 g/kg anti-E. coli K88 IgY to the diet had no difference in immunoglobulin levels in piglets infected with E. coli K88 (P > 0.05). However, the EIgY group had higher levels of IgA, IgM, and IgG than the ECON group.

Table 6.

Effect of IgY supplementation on jejunal mucosal immunoglobulin expression in weaned piglets challenged by E. coli K88

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
IgA, pg/mg prot	11.07a	14.06a	5.72b	10.65ab	0.669	0.008	0.004	0.476
IgM, ng/mg prot	2.71a	2.81a	1.64b	2.45ab	0.127	0.089	0.010	0.169
IgG, ng/mg prot	172.89	178.87	137.37	167.31	6.620	0.190	0.091	0.377

Ig immunoglobulin

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Digestive enzyme activity of the jejunum mucosa

The two factors had no significant interaction with each index. However, IgY showed independent principal effects. Supplementing with 6 g/kg anti-E. coli K88 IgY has a main effect on jejunal sucrase and lactase (LCT) activities (P < 0.05) (Table 7). Compared to adding 6 g/kg of common egg yolk powder, supplementing with 6 g/kg anti-E. coli K88 IgY increased the activity of sucrase in the jejunal mucosa of weaned piglets infected with E. coli K88 (P < 0.05). However, dietary anti-E. coli K88 IgY supplementation had no significant effect on LCT activity in E. coli K88‑infected piglets (P > 0.05).

Table 7.

Effect of adding IgY to the diet on the activity of digestive enzymes of the jejunal mucosa of weaned piglets challenged by E. coli K88

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
AMS, U/g prot	116.67	119.11	112.41	114.27	2.289	0.643	0.333	0.951
Sucrase, U/mg prot	60.61ab	71.83a	44.80b	68.78a	2.756	0.005	0.103	0.261
LCT, U/mg prot	7.74ab	9.04a	5.51b	8.66ab	0.427	0.017	0.142	0.294
Trypsin, U/mg prot	46.82	48.67	43.58	47.16	1.334	0.322	0.385	0.748
LPS, U/g prot	36.33	39.95	35.51	36.73	1.504	0.430	0.509	0.695
AKP, U/g prot	78.57	86.76	74.95	84.67	3.402	0.203	0.679	0.912

AMS amylase, LCT lactase, LPS lipase, AKP alkaline phosphatase

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Jejunal permeability

IgY has a main effect on DAO activity and D-LA levels (P < 0.05) (Table 8); ETEC has a significant main effect on DAO activity (P = 0.008). Under ETEC infection conditions, compared to adding 6 g/kg of common egg yolk powder, supplementing with 6 g/kg anti-E. coli K88 IgY reduced the level of D-LA in the jejunal mucosa of weaned piglets infected with E. coli K88 (P < 0.05).

Table 8.

Effect of adding IgY to the diet on the DAO and D-lactate levels of the jejunal mucosa of weaned piglets challenged by E. coli K88

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
DAO, U/mg	113.90ab	107.40b	120.25a	114.27ab	1.129	0.012	0.008	0.911
D-LA, µmol/g	7.07ab	6.21b	8.72a	6.39b	0.232	0.003	0.063	0.129

DAO Diamine Oxidase, D-LA D-Lactic Acid

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Jejunal tight junction protein expression

As shown in Fig. 4, IgY and ETEC demonstrated individual primary effects on the expression of ZO-1, Occludin, and Claudin-1 mRNA (P < 0.05) (Fig. 3A). Although there was no difference between the groups (P > 0.05). However, the EIgY group showed higher mRNA expression of ZO-1, Occludin, and Claudin-1 than the ECON group.

Fig. 4.

Effects of anti-E. coli K88 IgY supplementation on the diversity of intestinal microbiota in pigs during E. coli K88 challenge. A-C Chao, ACE, and Shannon of colon microbiota. D PCA at OUT level of colon microbiota. E The absolute abundance of colon microbiota at phylum and genus levels. E, H The absolute abundance of colon microbiota at phylum and genus levels. F, G Differences in the absolute abundance of colon microbiota at the phylum level. I, J Differences in the absolute abundance of colon microbiota at the genus level. CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group. P₁: P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P₂: P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P₁ × P₂: P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

Fig. 3.

Effects of IgY on the intestinal barrier function in the jejunal mucosa of weaned piglets challenged by E. coli K88. A Gene expression of tight junction proteins in the jejunum mucosa. B The Western blot of zonula occludens-1 (ZO-1), Occludin, claudin-1, and β-actin of the jejunal mucosa. CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group. P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

The two factors did not interact with each index. However, IgY and ETEC demonstrated individual principal effects. Compared to adding 6 g/kg of common egg yolk powder, supplementing with 6 g/kg anti-E. coli K88 IgY increased the level of Claudin-1 in the jejunal mucosa of weaned piglets (P < 0.05) (Table 9) (Fig. 3B).

Table 9.

Effects of IgY on the intestinal barrier function in the jejunal mucosa of weaned piglets challenged by E. coli K88

Item	Treatments1				SEM	P-value2
	CON	IgY	ECON	EIgY		P IgY	P ETEC	P IgY*ETEC
ZO-1	1.00a	1.10a	0.16b	0.82ab	0.020	0.036	0.006	0.099
Occludin	1.00ab	1.25a	0.62b	0.91ab	0.028	0.022	0.006	0.829
Claudin-1	1.00b	1.31a	0.52c	0.66c	0.014	0.001	0.001	0.065

ZO-1 zonula occludin 1

¹CON, basal diet with 6 g/kg of common egg yolk powder without E. coli K88 challenge; IgY, basal diet with 6 g/kg of anti-E. coli K88 IgY without E. coli K88 challenge; ECON, basal diet with 6 g/kg of common egg yolk powder and challenge E. coli K88 challenge, EIgY, basal diet with 6 g/kg of anti-E. coli K88 IgY and challenge E. coli K88 challenge. n = 6 piglets/group

²P_IgY, A two-way ANOVA evaluated the significance of the main effect of IgY. P_ETEC, A two-way ANOVA evaluated the significance of the main effect of ETEC. P_IgY*ETEC, A two-way ANOVA of variance evaluated the significance of the interaction between IgY and ETEC. Different superscript letters within a row indicate a significant difference (P < 0.05)

16S rDNA analysis of the colon microbiota

Dietary anti-E. coli K88 IgY had no main effect on gut microbial α-diversity (P > 0.05), while E. coli K88 challenge had a primary impact on Chao1 and ACE index (P < 0.05) (Fig. 4A, B). A significant interaction between IgY and ETEC was observed for the Shannon index (P < 0.05) (Fig. 4C). No differences in β-diversity were observed among groups (P > 0.05) (Fig. 4D). At the phylum level (Fig. 4E), the colon microbiota were mainly composed of Bacilliota, Pseudomonadota, and Themodesulfobacteriota. Anti-E. coli K88 IgY supplementation increased the colon contents abundance of Bacillota and Pseudomonadota in E. coli K88-infected weanling piglets (P < 0.05) (Fig. 4F, G). A significant interaction between IgY and ETEC was observed for Bacillota and Pseudomonadota (P < 0.05). At the genus level (Fig. 4H), E. coli K88 challenge had main effects on the absolute abundance of Lactobacillus (P < 0.05) (Fig. 4I). Anti-E. coli K88 IgY supplementation reduced the number of Escherichia-Shigella in the colon of E. coli K88-challenged piglets (P < 0.05). A trend toward an interaction between IgY and ETEC was observed for Escherichia-Shigella (0.05 < P < 0.10) (Fig. 4J).

Discussion

E. coli K88 infection is a leading cause of diarrhoea and piglet growth impairment [15, 16]. Studies have shown that ETEC infections can cause typical pathological changes like intestinal VH and CD in piglets, impairing nutrient digestion and absorption, ultimately resulting in diarrhea [17]. Adding 6 g/kg of anti-E. coli K88 IgY reduced the diarrhea rate of E. coli K88-infected weaned piglets. Indicating that its growth performance and intestinal health were severely impaired. VH and CD are essential indicators of intestinal barrier function [18]. Reduced VH and increased CD indicate a physical barrier and impaired digestion [19]. The jejunal VH and CD observed in this study provide direct morphological evidence of intestinal damage caused by the challenge. Notably, anti-E. coli K88 IgY supplementation effectively reversed these negative morphological changes, significantly increasing VH and decreasing CD. This improvement demonstrates that anti-E. coli K88 IgY boosted the intestine’s absorption ability by repairing its morphological structure, which alleviates diarrhoea and thereby improves piglet growth performance.

Oxidative stress, caused by an imbalance between oxidants and antioxidants, produces free radicals that harm intestinal cells and is closely related to the inflammatory response of the pig intestine [20, 21]. MDA is an oxidative stress marker [22]. In this study, E. coli K88 significantly increased the level of MDA in the jejunum of piglets. At the same time, anti-E. coli K88 IgY supplementation had a main effect on MDA levels, indicating that anti-E. coli K88 IgY could reduce the oxidative stress caused by E. coli K88 infection. This antioxidant effect is likely indirect, mediated by neutralization of E. coli K88 fimbriae and subsequent reduction in bacterial adhesion and colonization in the small intestine. Regarding inflammatory response, studies showed that E. coli K88 could adhere to the intestinal mucosa, increase the expression of pro-inflammatory cytokines IL-8 and IFN-γ, and decrease the production of the anti-inflammatory factor IL-10, thus worsening intestinal inflammation [23]. IL-8 primarily plays a role in the chronic inflammatory process by activating and recruiting neutrophils [24], while IFN-γ plays a key role in antiviral defense and immune regulation [25]. In contrast, IL-10, a crucial anti-inflammatory cytokine, can effectively suppress excessive immune activation and help maintain intestinal immune balance [26]. The results showed that anti-E. coli K88 IgY reduced the levels of IL-8 in the jejunum of E. coli K88-infected piglets and increased the expression of IL-10. The NF‑κB pathway is a master regulator of pro‑inflammatory cytokine transcription. The observed decrease in IL‑8 is consistent with this interpretation, as IL‑8 is a well‑known NF‑κB target gene [27]. The increase in IL‑10 probably arises from a negative feedback loop that limits TLR4‑mediated inflammation and from enhanced regulatory T‑cell activity [28]. This finding aligns with existing research suggesting that anti-E. coli K88 IgY could decrease inflammation by modulating cytokine expression and immunoglobulin production [29]. This reduced the risk of inflammation in the jejunum caused by ETEC infection [7]. Humoral immunity is a vital defense against pathogen infection, mainly mediated by immunoglobulins IgA, IgM, and IgG [30]. IgG is key in defending against pathogens [31]. This study showed that E. coli K88 significantly reduced IgA, IgM, and IgG levels. It indicated that its humoral immune function is suppressed. Based on the findings of the studies above, anti-E. coli K88 IgY has the potential to enhance the passive immunity of weaned piglets through various comprehensive mechanisms. Specifically, it balanced pro-inflammatory and anti-inflammatory cytokines, increases mucosal and systemic immunoglobulin levels, and reduces oxidative stress and intestinal inflammation caused by E. coli K88 infection. These combined actions maintain intestinal immune homeostasis and boost the organism’s defense capabilities.

The intestinal mucosal barrier is essential for piglets’ digestion, absorption, and immune defense, acting as a key foundation for maintaining overall health [18]. ETEC infection not only disrupts the intestinal morphological structure and barrier integrity but also inhibits digestive enzyme activity, thereby impeding nutrient absorption and utilization. Typically, alterations in intestinal tissue morphology are associated with changes in the activity of various brush-border digestive enzymes to digestion, such as lactase and sucrase [32]. Increased digestive enzyme activity often results from improvements in gut structure, such as higher VH and lower CD, which help expand the absorption area and enhance nutrient digestion [33]. The study showed that adding anti-E. coli K88 IgY to the diet increased the activities of sucrase in the jejunal mucosa of weaned piglets infected with E. coli K88, which aligns with previous studies indicating that IgY can boost digestion by improving intestinal morphology and function [34]. This suggests that anti-E. coli K88 IgY might boost the activity of brush-border digestive enzymes by repairing intestinal structural damage caused by E. coli K88, ultimately leading to better nutrient absorption and gut health.

The results of this study indicated that E. coli K88 infection could cause chronic inflammatory responses in piglets’ intestines, which in turn increased intestinal permeability and led to immune overactivation [19]. The level of DAO in the intestinal mucosa is closely linked to intestinal development and can serve as a potential biomarker for assessing intestinal dysfunction caused by parenteral nutrition [35]. D-LA, a bacterial fermentation product, enters circulation during mucosal injury and is a biomarker [36]. In this study, ETEC infection markedly increased the levels of DAO and D-LA in the jejunum, signaling a compromised intestinal barrier. Anti-E. coli K88 IgY supplementation has a main effect on DAO activity and D-LA levels. These findings demonstrated that anti-E. coli K88 IgY could improve intestinal barrier function by decreasing E. coli K88 invasion of the intestinal mucosa and by enhancing intestinal permeability, a key mechanism for reducing intestinal inflammation and promoting gut health.

During bacterial invasion, tight junction proteins are essential in maintaining the integrity of the gut barrier. Reduced levels of proteins such as ZO-1, Occludin, and Claudin-1 weaken the barrier structure and increase intestinal permeability [37]. Studies have shown that E. coli K88 infection could lower the expression level of ZO-1 mRNA in the jejunal tissue of piglets [38]. It also reduced the expression level of tight junction proteins Occludin and Claudin-1 in piglets and impaired the integrity of the intestinal barrier [39, 40]. The results demonstrated that dietary supplementation with anti-E. coli K88 IgY could counteract the inhibition of tight junction protein expression, which was caused by E. coli K88 infection, and effectively raised the protein levels of key proteins such as Claudin-1 in jejunal tissue. This regulatory effect indicates that anti-E. coli K88 IgY can enhance the structural strength of the intestinal mucosal barrier through a specific passive immune mechanism, thereby strengthening the host’s defenses against E. coli K88 invasion. The gut microbiota is essential for health, functioning as the host’s metabolic organ. It supports intestinal homeostasis, including nutrient absorption, barrier integrity, and immune responses [41]. The gut microbiota composition and diversity in weaned piglets are closely linked to health [42]. E. coli K88 infection compromises intestinal structure and triggers immune dysfunction [43]. The E. coli K88 challenge had a primary impact on Chao1 and ACE index, which did not alter overall α-diversity. Pathogenic infections could destabilize communities, causing overgrowth of conditionally pathogenic bacteria. Notably, IgY and ETEC showed an antagonistic effect on the Shannon index, indicating anti-E. coli K88 IgY reduced ecological fluctuations caused by E. coli K88 by preserving dominant flora. Notable changes at the phylum level were in Bacillota and Pseudomonadota. Bacillota, the dominant gut phylum, is often linked to intestinal dysfunction. ETEC challenge reduced Bacillota, a typical marker of post-infection inflammation [44]. Adding anti-E. coli K88 IgY to the diet increased Bacillota and reduced Pseudomonadota counts in the colon of E. coli K88-challenged piglets, indicating it helped restore bacterial balance. At the genus level, more Escherichia-Shigella markers indicated increased pathogen invasion and colonization [45]. In this study, anti-E. coli K88 IgY supplementation reduced the number of Escherichia-Shigella in the colon of E. coli K88-challenged piglets, showing that anti-E. coli K88 IgY could inhibit pathogen colonization by binding to ETEC antigens. Lactobacillus, as a vital probiotic, is closely related to gut health [46]. It has various functions, such as strengthening the barrier, regulating immunity and metabolism [47]. This study showed that the anti-E. coli K88 group had the highest Lactobacillus levels. This suggests that anti-E. coli K88 IgY helps restore Lactobacillus by neutralizing pathogens, reducing inflammation, and supporting a healthy microenvironment, thereby improving intestinal health. In conclusion, anti-E. coli K88 IgY not only directly inhibited the colonization of pathogenic bacteria but also indirectly improved the growth environment of the bacterial flora by regulating the host immune system, thereby both directly and indirectly aiding in restoring homeostasis in the intestinal microbiota.

There are several limitations to this study. First, the experimental period is short. Although this short-term observation window aligns with the conventional design of the acute ETEC infection model, it limits our evaluation of the potential long-term effects of IgY, including its impact on piglets’ long-term growth performance, intestinal structural and functional adaptation, and durable establishment of immune function. Secondly, this study found that although the addition of 6 g/kg anti-E. coli K88 IgY was effective in preventing diarrhea in piglets, this dose level was still relatively high. From the perspective of industrial application, it is necessary to optimize the processing of IgY to improve its utilization efficiency, reduce the amount added and breeding cost. As an oral biologic, IgY is constrained by the acidic environment of the gastrointestinal tract, degradation by digestive enzymes, and insufficient long-term storage stability [48]. Chitosan-sodium kept IgY stable in gastric juice. The contents are released upon reaching the neutral pH of the small intestine, thereby achieving targeted intestinal delivery [49]. Kovacs-Nolan confirmed in the ETEC model of piglets that microencapsulated IgY can advance the antidiarrheal onset time from 72 h to 24 h in ETEC piglets [50]. Studies in broilers have also confirmed that microencapsulated IgY significantly improves daily weight gain and feed conversion while reducing the number of E. coli in the gut [51]. Envelope technology can improve anti-E. coli K88 IgY stability, enhance the efficiency of intestinal targeted delivery, accelerate onset, and reduce the effective dose. In the future, it is necessary to extend the experimental cycle, optimize the process, conduct dose-effect studies, and explore lower addition levels to achieve precise, low-cost application of IgY as a functional feed additive.

Conclusion

In summary, this study showed that dietary supplementation with 6 g/kg anti-E. coli K88 IgY improved intestinal barrier function, reduced intestinal inflammation, enhanced immune responses, and supported growth performance in weaned piglets challenged with E. coli K88. These results provide preliminary theoretical support for the potential use of anti-E. coli K88 IgY as a functional feed additive. However, given the limited number of animals in this study, the reproducibility and broader applicability of these findings still need to be confirmed through larger-scale experiments.

Abbreviations

ETEC

Enterotoxigenic Escherichia coli

Ig

Immunoglobulin

IL

Interleukin

ZO-1

Zonula Occludin 1

VH

Villus height

CD

Crypt depth

V: C

Villus height to crypt depth ratio

T-AOC

The total antioxidant capacity

SOD

Superoxide dismutase

GSH-Px

Glutathione peroxidase

CAT

Catalase

MDA

Malondialdehyde

AMS

Amylase

LCT

Lactase

AKP

Alkaline phosphatase

LPS

Lipase

Authors’ contributions

**XYY** and **BMM** : Writing – review & editing, Writing – original draft, Software, Formal analysis, Data curation. **LJH** : Data curation, Software, Writing – review & editing. **HRX** : Formal analysis, Data curation, Writing – review & editing. **HPL** : Supervision, Conceptualization, Writing – review & editing. **TBE** : Writing -review & editing, Supervision, Project administration, Conceptualization. **LHN** : Writing- review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Funding

This work was supported partly by the National Key Research and Development Program of China (2022YFD1300704, 2022YFD1300904), the Special Research Funding for the Natural Science Foundation of Hunan Province (2023JJ40643), and the National Natural Science Foundation of China (32402793).

Data availability

The repository names and accession numbers can be found in the NCBI SRA database under the identifier PRJNA1402760. [https://www.ncbi.nlm.nih.gov/sra/PRJNA1402760].

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in compliance with the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines (https://arriveguidelines.org) and were approved by the protocol management and review committee of the institute of subtropical agriculture, Chinese academy of sciences (ISA20240079).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.