Exogenous Catalase Supplementation Alleviates Fusarium graminearum Mycotoxins-Induced Oxidative Stress in Weaned Piglets

Abstract

The objective of this study was to investigate the impact of exogenous catalase (CAT) on antioxidant properties, as well as on hepatic and intestinal health, in piglets exposed to Fusarium graminearum mycotoxins (FGM). Forty female weaned piglets were divided into five groups (eight replicates per group). The pre-feeding period was 3 days, followed by a 28-day experimental period. The piglets in the control (CON) group were fed a diet without FGM contamination, while those in the FGM-exposed (TOX) group were fed a diet with FGM contamination. The LCAT, MCAT, and HCAT groups received an FGM-contaminated diet supplemented with 100, 200, and 400 U/kg of CAT, respectively. The results indicated that 400 U/kg CAT supplementation inhibited (p < 0.05, linear p < 0.05, quadratic p < 0.05) the decreases in average daily gain and average daily feed intake of piglets exposed to FGM. Moreover, all doses of supplemental CAT suppressed (p < 0.05) the increases in diarrhea rate and diarrhea index of FGM-exposed piglets. Additionally, supplemental CAT reversed (p < 0.05, linear and quadratic p < 0.05 in ileal tissue, quadratic p < 0.05 in ileal chyme) the decrease in ileal tissue and increase in ileal chyme of reactive oxygen species of piglets exposed to FGM. Supplemental CAT also enhanced the activities of ileal CAT (p < 0.05, quadratic p < 0.05) coupled with hepatic superoxide dismutase and CAT (p < 0.05, linear p < 0.05, quadratic p < 0.05) and elevated (p < 0.05) the expression of ileal and hepatic antioxidation-related genes of FGM-exposed piglets. Furthermore, the CAT supplementation increased (p < 0.05) the expression of Occludin and Claudin-1 in the ileum and colon of piglets exposed to FGM. The FGM-induced increase in the genus Staphylococcus and decrease in the genus Lactobacillus in the ileum of piglets were inhibited (p < 0.05) by supplemental 400 U/kg CAT, which also modulated the metabolite profiles involved in the glycerophospholipid metabolism pathway in hepatic portal vein blood. Exogenous CAT mitigates oxidative stress induced by FGM, along with improving intestinal and hepatic health of piglets, which can be associated with its ability to enhance intestinal microbiota and regulate hepatic glycerophospholipid metabolism, aside from its direct ability to scavenge oxygen radicals. The appropriate amount of supplemental CAT was 400 U/kg.

1. Introduction

Fusarium graminearum, the most common pathogenic fungus infecting cereal crops, is capable of producing mycotoxins like deoxynivalenol (DON) and zearalenone (ZEA) during its growth and reproduction [1]. These mycotoxins exist in both free and modified forms [2]. Besides impairing the growth performance of animals, Fusarium graminearum mycotoxins (FGM) can accumulate in various organs and tissues of animals to endanger human health [3]. Due to fungal infection of cereal crops and the use of moldy grains and forage as ingredients in animal feed, FGM can appear in the feed chain and even exceed the limit range of the standard for feeds [3,4].

FGM-exposed animals may experience either acute or chronic poisoning, which is reflected in the decrease in feed intake and reduction in growth and nutrient absorption, accompanied by immune dysfunction [4,5]. Pigs have a high absorption rate and high bioavailability of FGM such as DON, alongside an absence of detoxifying gut microorganisms and a prolonged clearance time [6]. Thus, pigs are more susceptible to the toxicity of FGM. FGM are toxic to several organs (e.g., intestine, liver, and kidney), with oxidative stress being a key mechanism contributing to their toxicity [7]. Specifically, FGM results in dysfunction of the mitochondria and an elevation in the generation of reactive oxygen species (ROS) along with the reduction in antioxidants within the cells, which disrupts the body’s redox balance [7]. Therefore, inhibiting induced oxidative stress is an effective strategy for reducing the toxic effects of FGM on pigs.

Antioxidant enzymes are essential in supporting the antioxidant system’s ability to neutralize various free radicals produced in the body [8]. Hydrogen peroxide (H₂O₂) is a type of ROS that can induce oxidative stress by generating other ROS, such as hydroxyl radical, through the Fenton reaction [9]. Among the antioxidant enzymes, catalase (CAT) can neutralize H₂O₂ by decomposing it into molecular oxygen and H₂O [10]. Additionally, CAT can function in its peroxidatic mode to break down small substrates like methanol and ethanol, which can help reduce oxidative stress [11]. A previous study has demonstrated that the supplementation of CAT in the diet improved antioxidant properties, reduced peroxidation products, and simultaneously enhanced growth and health performance in pigs [12].

The intestinal microbiota is an emerging target for FGM, and changes in intestinal microbiota caused by FGM exposure have been found in pigs, broilers, and mice [13]. Through fecal microbiota transplantation and antibiotic treatment, it has been proved that DON and aflatoxin B₁ (AFB₁) exert their toxicity on the intestine, liver, and spleen of mice through intestinal microbiota, as manifested by promoted accumulation of hepatic ROS and pyroptosis in the liver and spleen [14,15]. Our previous study discovered that exogenous CAT reduces oxidative stress and intestinal injury in broilers exposed to DON, potentially due to its capacity to enhance intestinal microbiota, in addition to its direct effects in scavenging oxygen radicals [16]. However, the influence of CAT on antioxidant properties, intestinal microbiota, and blood metabolite profiles in FGM-exposed piglets is still unclear. Therefore, this study is intended to explore the potential effects of exogenous CAT on reducing oxidative stress, intestinal microbiota dysbiosis, and changes in metabolite profiles in piglets exposed to FGM. The results of this research may provide valuable insights into how exogenous CAT can be utilized in piglet farming, offering a practical strategy to prevent mycotoxin contamination.

2. Materials and Methods

2.1. Animals, Diet, and Experimental Design

The Animal Care and Use Committee of the South China Agricultural University (China) approved the experimental animal protocols for this study (Ethics Approval Number: 2024F227/15 March 2024).

Forty female weaned piglets (aged 28 ± 2 days; body weight [BW] = 7.90 ± 0.68 kg; Duroc × Landrace × Large White; weaned at 25 days of age, followed by a 3-day adaptation period to minimize weaning stress) were randomly allotted to five treatments in a completely block design with 8 replicates per treatment and 1 piglet per replicate. Piglets that received a basal diet served as the control (CON) group, while those receiving an FGM-contaminated diet formed the FGM-exposed (TOX) group. The other three groups received an FGM-contaminated diet supplemented with CAT at 100 U/kg (LCAT group), 200 U/kg (MCAT group), or 400 U/kg (HCAT group). The experiment lasted for a total of 28 days. The basal diet (

Table 1. Composition of the basal diet (air-dry basis).

Ingredients	Content (%)	Nutrient Levels	Content (%)
Corn	57.83	Analyzed nutrient level
Soybean meal	18.60	Crude protein	18.69
Soybean oil	1.40	Organic matter	80.94
Extruded soybean meal	8.00	Neutral detergent fiber	7.12
Whey powder	10.00	Acid detergent fiber	3.60
Salt	0.32	Calcium	0.61
Limestone	0.50	Total phosphorus	0.55
Dicalcium phosphate	1.23	Calculated nutrient level
Choline chloride (50%)	0.16	Metabolizable energy, MJ/kg	13.81
L-lysine (99%)	0.58	Total lysine	1.30
DL-methionine (98%)	0.17	Total methionine + cystine	0.73
L-threonine (98.5%)	0.17	Total tryptophan	0.23
L-tryptophan (98%)	0.05	Total threonine	0.83
Premix (1)	1.00
Total	100.00

⁽¹⁾ Supplied per kilogram of diet: vitamin B₁, 1.25 mg; vitamin B₂, 5 mg; vitamin B₆, 2.5 mg; vitamin B₁₂, 0.0125 mg; vitamin A, 5000 IU; vitamin D₃, 2000 IU; vitamin E, 37.5 mg; vitamin K₃, 3 mg; niacin, 25 mg; pantothenic acid, 12.5 mg; folic acid, 2.5 mg; biotin, 0.25 mg; Cu, 35 mg; Fe, 120 mg; Zn, 80 mg; Mn, 65 mg; I, 0.3 mg; Se, 0.3 mg; Co, 0.15 mg.

) was formulated to satisfy the specific nutritional needs of piglets, following the Chinese National Feeding Standard (GB/T 39235-2020) [17]. Briefly, values of metabolizable energy and amino acid nutrient levels were calculated with reference to the Chinese National Feeding Standard (GB/T 39235-2020) [17]. Nitrogen (N) was quantified using an automated Kjeldahl nitrogen analyzer (Foss Kjeltec 8400, Horsens, Denmark), according to the standard GB/T 6432-2018 [18], and the crude protein content was obtained by N × 6.25. The levels of calcium and phosphorus in the diet were evaluated based on the ammonium metavanadate colorimetric and ethylene diamine tetraacetic acid methods, as specified in the standards GB/T 6436-2018 [19] and GB/T 6437-2018 [20]. In addition, the dietary levels of organic matter, neutral detergent fiber, and acid detergent fiber were analyzed according to the standards GB/T 6438-2007 [21], GB/T 20806-2006 [22], and NY/T 1459-2022 [23], respectively. The basal diet was supplemented with FGM-enriched rice to fortify the diet with FGM. FGM-enriching rices were fabricated following the methods outlined in our previous study [16], with minor modifications.

A good correlation between the results obtained by high-performance liquid chromatography and the ELISA kit for the determination of mycotoxin content in feed indicated that the ELISA results were reliable [24]. Furthermore, our previous study [16] and that of Marquis et al. [25] have shown that the contents of modified DON in diets contaminated with mycotoxins were low, with free-form DON being the dominant form. Therefore, the contents of four primary FGM in the diet were quantified using the respective ELISA kit (Romer Labs, Getzersdorf, Austria), which are exhibited in Table S1. The estimated levels of DON and ZEA in the FGM-contaminated diet were 1.87 mg/kg and 1.91 mg/kg, respectively. The preparation of CAT was sourced from Vetland Bio-Technology Co., Ltd. (Shenyang, China), and the actual activity of the supplement was determined to be 520 U/g. All piglets had unrestricted access to water and food. The diarrhea severity of each piglet was visually evaluated every day based on the pig’s fecal score assessment (Table S2).

At the end of the experiment, all piglets underwent an overnight fasting period. Blood samples from the hepatic portal vein were collected in vacutainers after anesthesia by injecting sodium pentobarbital (40 mg/kg BW). The serum samples were then collected following centrifugation (3500× g for 10 min) and preserved at −80 °C. Subsequently, the visceral organs, including the liver, spleen, thymus, ileum, and colon, were separated. The central sections of the ileum, colon, and liver were gently rinsed with phosphate-buffered saline, then quickly frozen and preserved at −80 °C. Furthermore, the chyme from the middle section of the ileum was immediately collected, frozen, and kept at −80 °C for future microbial analysis.

2.2. Assessment of Growth Performance, Diarrhea Index and Organ Indexes

Piglets were weighed at days 1 and 28, and the feed intake per pen was recorded to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). The evaluation of diarrhea was evaluated using both diarrhea incidence and diarrhea index (a fecal score of 2 or 3 is classified as clinical diarrhea). The calculation of diarrhea incidence and diarrhea index followed the methods outlined by [26].

The harvested organs, including the liver, thymus, and spleen, were weighed to determine their organ indexes, which were determined by the ratio of organ weight (g) to BW (kg).

2.3. Detection of Oxidative Status

The activities of superoxide dismutase (SOD; Product No. A001-3-2) and CAT (Product No. A007-1-1), along with the content of H₂O₂ (Product No. A064-1-1) in the ileum and liver of piglets, were measured using the appropriate assay kits (Jiancheng Bioengineering Institute, Nanjing, China). The protein contents in the ileum and liver were measured using a BCA assay with the Pierce BCA Protein Assay Kit (Product No. 23225; Thermo Fisher Scientific, Waltham, MA, USA).

Following the kit’s guidelines (Product No. JL-T3037; JONLANBIO, Shanghai, China), the ileal tissue, ileal chyme, and liver were homogenized in extracting buffer. Subsequently, the homogenate was then centrifuged at 12,000× g for 10 min at 4 °C to collect the supernatant. Then the ROS production was assessed by treating the supernatant with 2′,7′-dichlorohydro fluorescein diacetate. The samples were incubated at 37 °C for 30 min, and fluorescence intensity was measured using a Synergy 2 Bio Tek microplate reader (Agilent, Santa Clara, CA, USA).

2.4. Analysis of Gene Expression

In short, total RNA from the ileum, colon, and liver was extracted and then reverse transcribed to cDNA following the methods outlined in our previous study [16]. Then the gene expression was analyzed via real-time PCR on a CFX96 Touch Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) with the 2×Polarsignal qPCR Mix (MIKX Co., Ltd., Shenzhen, China). The primer information of β-actin (used as a reference gene) and the target genes is presented in Table S3. The genes’ relative mRNA expression levels were calculated according to the 2^−△△Ct method.

2.5. Analysis of Ileal Microbiota and Metabolome Analysis of Hepatic Portal Vein Blood

Bacterial genomic DNA was extracted from ileum digesta and then analyzed for microbiota following the methods in our previous study [16]. The serum collected from the hepatic portal vein was used for metabolomics analysis according to the methods of [27]. Then Spearman correlation analysis was performed to evaluate the relationships between the bacterial composition and metabolite profiles of hepatic portal vein blood.

2.6. Statistical Analysis

Data analysis was conducted utilizing the one-way ANOVA in the SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Differences among groups were assessed using Duncan’s multiple comparisons. Orthogonal polynomial contrasts facilitated the estimation of both linear and quadratic effects of the increasing supplemental CAT levels. The chi-square test was applied to analyze the diarrhea rate and diarrhea index. Results are presented as mean ± SEM, with statistical significance set at p < 0.05, while a p-value between 0.05 and 0.10 was interpreted as a trend toward significance.

3. Results

3.1. Effects of Exogenous CAT on Growth Performance and Organ Indexes of FGM-Exposed Piglets

As presented in

Table 2. Effects of exogenous CAT on the growth performance of piglets exposed to FGM (n = 8).

Items	CON (1)	TOX	LCAT	MCAT	HCAT	SEM	p-Value (2)
							Treatment	Linear	Quadratic
IBW (kg)	7.93	7.89	7.89	7.91	7.91	0.113	1.000	0.943	0.997
FBW (kg)	17.07	15.54	16.95	17.08	18.85	0.312	0.067	0.009	0.035
ADG (g)	332.37 ab	268.36 b	314.22 ab	327.05 ab	381.96 a	9.945	0.039	0.004	0.016
ADFI (g)	559.08 ab	462.29 b	555.74 ab	549.03 ab	695.91 a	14.146	0.002	0.001	0.003
FCR	1.69	1.75	1.77	1.68	1.83	0.026	0.387	0.762	0.445
Diarrhea rate (%)	12.95 c	30.61 a	24.49 ab	16.96 b	16.07 b	2.533	<0.001	/	/
Diarrhea index	0.50 c	0.99 a	0.76 b	0.65 b	0.58 b	0.059	<0.001	/	/

SEM = pooled standard error of the mean; IBW = initial body weight; FBW = final body weight; ADG = average daily gain; ADFI = average daily feed intake; FCR = feed conversion ratio. ⁽¹⁾ CON = basal diet; TOX = FGM-contaminated diet; LCAT = FGM-contaminated diet + 100 U/kg CAT; MCAT = FGM-contaminated diet + 200 U/kg CAT; HCAT = FGM-contaminated diet + 400 U/kg CAT. ⁽²⁾ Treatment: treatment effect according to Duncan’s test; Linear and Quadratic: linear and quadratic dose trend-effects of exogenous CAT at 0, 100, 200, and 400 U/kg according to polynomial contrasts. ^a–c Means within a row with different superscripts differ (p < 0.05).

, the TOX group exhibited a decreasing trend (p < 0.10) of FBW compared with the CON group, while the supplementation of CAT tended to (p < 0.10, linear p < 0.05, quadratic p < 0.05) increase FBW compared with the TOX group. The supplementation of 400 U/kg CAT increased (p < 0.05, linear p < 0.05, quadratic p < 0.05) both ADG and ADFI of piglets exposed to FGM. Supplemental 200 U/kg and 400 U/kg CAT inhibited (p < 0.05) the increase in diarrhea rate of FGM-exposed piglets. In addition, all doses of CAT supplementation mitigated (p < 0.05) the increase in diarrhea index due to FGM exposure. Supplemental CAT had no impact on FCR of piglets affected by FGM exposure. The liver index increased (p < 0.05) linearly with increasing CAT level in the diet (

Table 3. Effects of exogenous CAT on the organ indexes of piglets exposed to FGM (n = 8).

Items	CON (1)	TOX	LCAT	MCAT	HCAT	SEM	p-Value (2)
							Treatment	Linear	Quadratic
Liver index (g/kg)	27.76	26.82	25.68	27.83	29.14	0.349	0.110	0.044	0.066
Thymus index (g/kg)	2.59	2.54	2.08	2.97	2.55	0.099	0.170	0.462	0.760
Spleen index (g/kg)	2.09	2.07	1.83	2.09	2.14	0.038	0.460	0.227	0.214

SEM = pooled standard error of the mean. ⁽¹⁾ CON = basal diet; TOX = FGM-contaminated diet; LCAT = FGM-contaminated diet + 100 U/kg CAT; MCAT = FGM-contaminated diet + 200 U/kg CAT; HCAT = FGM-contaminated diet + 400 U/kg CAT. ⁽²⁾ Treatment: treatment effect according to Duncan’s test; Linear and Quadratic: linear and quadratic dose-trend effects of exogenous CAT at 0, 100, 200, and 400 U/kg according to polynomial contrasts.

).

3.2. Effects of Exogenous CAT on the Oxidative Status of the Intestine and Liver of FGM-Exposed Piglets

As presented in

Table 4. Effects of exogenous CAT on antioxidant parameters of piglets exposed to FGM (n = 8).

Items	CON (1)	TOX	LCAT	MCAT	HCAT	SEM	p-Value (2)
							Treatment	Linear	Quadratic
ileal tissue
ROS	1.00 a	0.74 b	0.93 a	0.90 a	0.93 a	0.018	0.006	0.026	0.021
H2O2 (mmol/g prot)	8.69	10.47	8.35	9.41	9.43	0.315	0.301	0.633	0.349
SOD (U/mg prot)	23.27	22.34	22.49	20.56	21.88	0.272	0.057	0.208	0.288
CAT (U/mg prot)	7.35 a	3.44 c	4.21 bc	4.90 b	3.65 c	0.115	<0.001	0.280	0.002
ileal chyme
ROS	1.00 b	1.95 a	0.98 b	1.17 b	1.17 b	0.086	0.018	0.068	0.026
H2O2 (mmol/g prot)	1.11	2.61	1.93	2.41	1.97	0.175	0.072	0.408	0.661
liver
ROS	1.00	1.06	0.94	1.00	1.00	0.040	0.940	0.907	0.944
H2O2 (mmol/g prot)	2.83	4.28	3.93	3.55	3.22	0.204	0.214	0.086	0.237
SOD (U/mg prot)	80.61 c	88.56 bc	98.14 ab	108.04 a	105.10 a	1.600	<0.001	0.005	0.006
CAT (U/mg prot)	52.80 c	48.20 c	42.03 c	127.95 b	154.94 a	2.930	<0.001	<0.001	<0.001

SEM = pooled standard error of the mean; ROS = reactive oxygen species; H₂O₂ = hydrogen peroxide; SOD = superoxide dismutase; CAT = catalase. ⁽¹⁾ CON = basal diet; TOX = FGM-contaminated diet; LCAT = FGM-contaminated diet + 100 U/kg CAT; MCAT = FGM-contaminated diet + 200 U/kg CAT; HCAT = FGM-contaminated diet + 400 U/kg CAT. ⁽²⁾ Treatment: treatment effect according to Duncan’s test; Linear and Quadratic: linear and quadratic dose trend-effects of exogenous CAT at 0, 100, 200, and 400 U/kg according to polynomial contrasts. ^a–c Means within a row with different superscripts differ (p < 0.05).

, the relative ROS level of the TOX group showed a decrease (p < 0.05) in ileal tissue and an increase (p < 0.05) in ileal chyme, relative to the CON group, whereas all doses of CAT supplementation reversed (p < 0.05, linear and quadratic p < 0.05 in ileal tissue, quadratic p < 0.05 in ileal chyme) those effects. The relative ROS level in the liver and the content of H₂O₂ in the ileum and liver did not differ (p > 0.05) among groups. Regarding the antioxidant enzymes, compared with the TOX group, 200 U/kg and 400 U/kg CAT supplementation increased (p < 0.05, linear p < 0.05, quadratic p < 0.05) the hepatic SOD and CAT activities. The activity of ileal CAT in the TOX group was lower (p < 0.05) than that in the CON group while supplemental 200 U/kg CAT elevated (p < 0.05, quadratic p < 0.05) the activity of CAT.

3.3. Effects of Exogenous CAT on the Relative mRNA Expression of Antioxidation-Related Genes of FGM-Exposed Piglets

As depicted in Figure 1, in contrast to the CON group, the TOX group exhibited decreases (p < 0.05) in the expression of NAD(P)H oxidoreductase 1 (NQO1), SOD1, SOD2, CAT, heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1(Keap1) and nuclear factor erythroid-2-related factor (Nrf2) in the ileum of piglets. Supplemental 100 U/kg CAT elevated (p < 0.05) ileal NQO1, HO-1, and Nrf2 expression, which was the group with the highest expression level among the three intervention groups. However, FGM did not affect (p > 0.05) the expression of hepatic antioxidation-related genes, relative to the CON group. Compared with the TOX group, 100 U/kg CAT supplementation elevated (p < 0.05) all these antioxidation-related genes in the liver, while 200 U/kg CAT supplementation elevated (p < 0.05) hepatic NQO1, SOD2, CAT, HO-1, Keap1, and Nrf2 expression.

3.4. Effects of Exogenous CAT on the Relative mRNA Expression of Tight Junction Proteins of FGM-Exposed Piglets

The expression of ileal Occludin and Claudin-1 was lower (p < 0.05) in the TOX group in contrast to the CON group (Figure 2A), whereas ileal Occludin expression in the LCAT group was higher (p < 0.05) than that in the TOX, MCAT, and HCAT groups. In addition, ileal Claudin-1 expression in all doses of CAT supplementation groups was higher (p < 0.05) compared with the TOX group. Similarly, the expression of colonic Occludin was lower (p < 0.001) in the TOX group compared with the CON group (Figure 2B), while colonic Occludin expression in the MCAT group was higher (p = 0.001) than that in the TOX group. Colonic Claudin-1 expression was higher (p < 0.05) in all doses of CAT supplementation groups than that in the TOX group.

3.5. Effects of Exogenous CAT on the Ileal Microbiota of FGM-Exposed Piglets

Based on the results of growth performance, the piglets of the CON, TOX, and HCAT groups were selected for ileal microbiota analysis. No difference (p > 0.05) was observed in the α-diversity indices and β-diversity of ileal microbiota among the groups (Figure 3).

The ileum of piglets predominantly hosted the phyla Firmicutes, Actinobacteria, and Proteobacteria (Figure 4A), with Firmicutes being the most prevalent. At the genus level, the CON group had a higher relative abundance of Staphylococcus with a lower relative abundance of Lactobacillus than those in the TOX group (Figure 4B). On the contrary, the relative abundance of Staphylococcus was lower, whilst Lactobacillus was higher in the HCAT group as compared with the TOX group.

Bacterial richness (p < 0.05, LDA > 2.0) was identified by LDA combined effect size measurements (LEfSe) analysis. As depicted in Figure 5, specific bacterial members, including the order Clostridia, the family Peptostreptococcaceae, and the species Glaesserella parasuis and Enterococcus faecalis, were found to be more abundant in the CON group. Comparatively, the genus Methanobrevibacter was enriched in the TOX group, and the species Streptococcus pluranimalium was enriched in the HCAT group.

3.6. Effects of Exogenous CAT on the Metabolome of Hepatic Portal Vein Blood of FGM-Exposed Piglets

Based on the results of growth performance, the hepatic portal vein blood of piglets from the CON, TOX, and HCAT groups was selected for metabolomic analysis. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were conducted on samples from the CON, TOX, and HCAT groups to assess the differences in metabolite profiles among groups. As shown in Figure 6, the samples from three groups were clustered separately, while samples from the HCAT group were closer to those from the CON group than those from the TOX group.

A volcano plot displays the relative differences in metabolite content between two groups and the statistically significant differences. As presented in Figure 7, the TOX group showed an increase (p < 0.05) in 42 metabolites and a decrease (p < 0.05) in 82 metabolites when contrasted with the CON group. However, the HCAT group upregulated (p < 0.05) 8 metabolites and downregulated (p < 0.05) 10 metabolites, relative to the CON group. Among them, as shown in Table S4, the levels of 6 metabolites were lower (p < 0.05) in the TOX group than those in the CON group and higher (p < 0.05) in the HCAT group than those in the TOX group. The TOX group exhibited a higher level of allolithocholate (p = 0.022) when compared to the CON group, whereas the HCAT group showed a lower (p = 0.019) in comparison to the TOX group.

As illustrated in Figure 8, in the TOX–CON comparison, the differential metabolites were primarily enriched in the choline metabolism in cancer as well as glycerophospholipid metabolism. Similarly, in the HCAT–TOX comparison, the differential metabolites were predominantly enriched in glycerophospholipid metabolism.

3.7. Effects of Exogenous CAT on Correlations Between Ileal Microbiota and Metabolome of FGM-Exposed Piglets

Spearman’s correlation analysis was employed to identify the relationships between ileal microbiota and metabolite profiles of hepatic portal vein blood among groups. As shown in Figure 9A, in the TOX–CON comparison, the family Peptostreptococcaceae had a significant positive correlation with 23 different metabolites such as Tyr-Tyr-Leu (p = 0.018), Damascenone (p = 0.019), Tigecycline (p = 0.013), BIX 01294 Trihydrochloride (p = 0.004), and Geldanamycin (p = 0.004), but showed significant negative correlations (p < 0.05) with 18 different metabolites. Similarly, in the HCAT–TOX comparison, the family Peptostreptococcaceae was significantly positively correlated with Tyr-Tyr-Leu (p = 0.003), (±)15-HEPE (p = 0.012), Damascenone (p = 0.015), Tigecycline (p = 0.003), BIX 01294 Trihydrochloride (p = 0.033), and Geldanamycin (p = 0.001), but displayed significant negative correlations (p < 0.05) with seven different metabolites (Figure 9B). The metabolites listed above are common differential metabolites in the TOX–CON comparison and the HCAT–TOX comparison (Table S4). The genus Staphylococcus, known as a potential pathogen, showed a significant negative correlation with Tyr-Tyr-Leu (p = 0.047) and (±)15-HEPE (p = 0.045).

4. Discussion

Mycotoxins produced by Fusarium graminearum have been shown to cause acute and chronic toxicity in animals [5]. In the present study, the contents of AFB₁ and fumonisins were lower than the limit of the Chinese hygienical standard for feeds (GB 13078-2017) [28]. Thus, piglets were mainly poisoned by DON and ZEA in this study. Research on performance and toxicological effects in pigs has primarily concentrated on medium to high levels of DON, specifically ranging from 2 to 10 mg/kg of feed [5]. In contrast, the content of DON and ZEA in the FGM-contaminated diet in this study was 1.87 mg/kg and 1.91 mg/kg, respectively, belonging to the low-dose treatment.

Similarly to a previous study [29], the present study found that FGM caused growth retardation in piglets, as evidenced by the decrease in ADG, accompanied by the increased diarrhea rate and diarrhea index. It was reported that CAT can enhance intestinal antioxidant capacity and change the microbiota composition to ameliorate intestinal mucosal damage induced by lipopolysaccharide [12]. However, limited research has been carried out to investigate the impact of CAT on pigs exposed to FGM. In this study, the CAT supplementation attenuated the damage of FGM on the growth performance of piglets, which showed that 400 U/kg CAT supplementation increased ADG and ADFI, and all doses of CAT supplementation alleviated diarrhea. The organ index, which refers to the relative organ weight, serves as a common indicator for assessing health condition in piglets. According to Xiao et al. [30], dietary 4 mg/kg DON increased the indexes of kidney and spleen of piglets. However, in this study, we found no changes in the indexes of piglets, which may be related to the relatively low dose of FGM in the diet.

The resultant redox imbalance in the organs of the piglet, particularly the liver and intestine, due to FGM exposure, has been well-established [31]. It has been proposed that oxidative stress is associated with increased production of ROS in the intestine and liver [32]. On the contrary, in this study, FGM decreased the level of ROS in ileal tissue of piglets, while CAT supplementation reversed this change. Interestingly, the subsequent analysis indicated that FGM contamination increased the level of ROS in ileal chyme. It was possible that FGM resulted in the intestinal barrier damage along with cell death and rupture, subsequently allowing ROS to move from epithelial cells into the intestinal lumen. The decreased gene expression of intestinal tight junction proteins caused by FGM could lend support to this conjecture. SOD and CAT help prevent redox imbalance by specifically neutralizing superoxide anion and H₂O₂, respectively [10]. In this study, FGM decreased ROS in ileal tissue, increased ROS in ileal chyme, and impaired antioxidant property, which indicated an oxidative stress in piglets [4,7]. However, FGM-induced changes in ROS level in both ileal tissue and chyme were normalized by CAT supplementation, which also improved the activities of ileal SOD and CAT along with hepatic CAT of piglets exposed to FGM. These findings underscored the efficacy of exogenous CAT in mitigating oxidative stress induced by FGM in piglets.

The Nrf2/Keap1/HO-1 pathway is one of the essential mechanisms for cells to fight against oxidative stress [33]. Upon exposure to excessive free radicals, Nrf2 separates from Keap1 and translocates into the nucleus, ultimately activating the transcription of antioxidant enzymes such as CAT, HO-1, and SOD [34]. This study, like Qiu et al. [35], demonstrated that FGM suppressed the expression of antioxidation-related genes (e.g., CAT, SOD1) in the ileum of piglets. There were no notable changes in the expression of antioxidation-related genes in the liver of FGM-exposed piglets, which did not agree with the study of Ji et al. [36], who found that 3 mg/kg DON reduced the expression of hepatic CAT and SOD1 of piglets. The discrepancy might be due to the difference in the content of FGM in the diet. The CAT supplementation elevated the expression of antioxidation-related genes in the ileum and liver of piglets exposed to FGM, with higher dose of CAT being less efficient. These results demonstrated that a high dose of CAT probably mitigated FGM-induced oxidative stress by directly eliminating radical accumulation, thereby decreasing the consumption of antioxidant enzymes, rather than enhancing the expression of antioxidant enzyme genes via the Nrf2/Keap1/HO-1 pathway.

The intestine represents the first target organ of FGM [37]. As the crucial barrier structure of the intestine, tight junctions comprise various structurally unique proteins, including Occludin, Claudins, and Zonula occluden, which form a paracellular permeability barrier and preserve intestinal integrity [38]. Oxidative damage induced by FGM contamination has been proved to reduce the expression of intestinal tight junction proteins in piglets [35]. As expected, this study showed that supplemental CAT alleviated the decreases in gene expression of Occludin and Claudin-1 in the ileum and colon of piglets exposed to FGM, demonstrating that the improvement of intestinal physical barrier function by CAT addition was probably related to the observed enhancement of intestinal antioxidant capacity.

Intestinal microbiota are widely recognized for their crucial roles in regulating host growth and overall health. An increasing number of studies disclosed that oxidative damage induced by FGM contributes to the disruption of intestinal microbiota in various animal models [35,39]. Previous studies have suggested the improvement of intestinal microbiota by CAT supplementation in both piglets [12] and broilers [16]. In this study, based on the results from the assessment of growth performance, the CON, TOX, and HCAT group were selected for ileal microbiota analysis, which showed that the CAT supplementation did not cause significant changes in intestinal microbial diversity of piglets. However, significant changes were observed in ileal microbial composition, as manifested by the inhibition effect of CAT on the increased abundance of the genus Staphylococcus and the decreased abundance of the genus Lactobacillus induced by FGM. As a part of the typical cutaneous and mucosal microbiota of mammals, the genus Staphylococcus and its affiliated members (such as Staphylococcus aureus and Staphylococcus hycius) frequently serve as primary causative pathogens of infections in animals [40]. In contrast, the genus Lactobacillus, a well-established source of probiotics in the intestine, is involved in regulating intestinal epithelial barrier integrity and oxidative stress as well as improving host immune responses [41]. It was indicated that supplemental CAT improved the intestinal microbial composition in this study. Similarly to the current study, Xu et al. [42] reported the decreased abundance of genus the Lactobacillus in the intestine of pigs exposed to DON.

Alterations in the intestinal microbiota inevitably lead to corresponding changes in the metabolic profiles of the host, which have been extensively documented in a previous study [43]. The intestine and liver are linked through the portal vein and interact via several pathways [44]. Venous blood from the small and large intestine flows into the portal vein, which supplies 75% of the blood of the liver, carrying enterally absorbed nutrients as well as microbial metabolites [45]. In turn, the liver regulates the intestine by releasing bile containing bile acids and antimicrobial molecules directly into the small intestine, thereby completing the cycle by modulating the microbiota [46]. Microbial metabolites like secondary bile acids and ethanol may play a role in the onset of non-alcoholic fatty liver disease [45]. Therefore, we intended to understand the changes in metabolites from the intestine into the liver after FGM and exogenous CAT intervention. The CON, TOX, and HCAT groups were selected for metabolome analysis of hepatic portal vein blood. The results showed that samples from the HCAT group were closer to those from the CON group than those from the TOX group. Moreover, the differential metabolites were primarily enriched in glycerophospholipid metabolism due to the intervention of FGM and exogenous CAT. Among them, seven metabolites were screened from the volcano plot, including Tyr-Tyr-Leu, (±)15-HEPE, Damascenone, etc. Tyr-Tyr-Leu is a tripeptide with antioxidant activity [47]. (±)15-HEPE, which has anti-inflammatory actions [48], exhibits a negative correlation with MDA production [49]. Damascenone has been proved to benefit resistance against oxidative stress [50]. Accordingly, the above metabolites elevated by CAT addition could be deduced to improve the antioxidant capacity and improve the hepatic redox status of piglets.

Then Spearman’s correlation analysis was conducted to identify the relationships between ileal microbiota and metabolite profiles of hepatic portal vein blood among CON, TOX, and HCAT groups. The results showed that the increases in the six differential metabolites (Tyr-Tyr-Leu, (±)15-HEPE, Damascenone, Tigecycline, BIX 01294 Trihydrochloride, and Geldanamycin) were related to the increased abundance of the family Peptostreptococcaceae, whereas the increases in Tyr-Tyr-Leu and (±)15-HEPE levels were related to the decreased abundance of the genus Staphylococcus. The above results indicated that exogenous CAT changed the composition and abundance of intestinal microbiota, which could change the metabolite profiles of hepatic portal vein blood, probably alleviating the redox imbalance in the intestine and liver of piglets. However, further research regarding which metabolites are essential in the above processes deserves to be conducted.

5. Conclusions

Exogenous CAT enhanced the antioxidant capacity in both the ileum and the liver of piglets. Furthermore, supplemental 400 U/kg CAT improved the composition of intestinal microbiota and altered the metabolite profiles in hepatic portal vein blood (mainly through glycerophospholipid metabolism). These could explain the observed mitigating effects of CAT supplementation on FGM-induced impairments of piglets in growth performance and redox balance. The appropriate amount of supplemental CAT was 400 U/kg. Our findings offered a strategy to minimize the negative impacts of dietary FGM contamination on pigs.