Abstract Porcine epidemic diarrhea virus (PEDV) causes severe intestinal damage, posing significant threats to the swine industry. Fucoidan (FUC), a biologically active compound, exhibits antiviral activity against multiple viruses. This study aimed to investigate the protective effects of FUC on PEDV-induced intestinal injury in piglets and explore its underlying mechanisms. A total of 28 healthy crossbred piglets were randomly allocated into four experimental groups using a 2 × 2 factorial design: (1) a control group, (2) an FUC group, (3) a PEDV group, and (4) an FUC+PEDV group. From day 4 to day 10, the piglets in the FUC and FUC+PEDV groups were orally administered fucoidan at a dosage of 20 mg/kg body weight (BW) each day. On day 8, the piglets in the PEDV and FUC+PEDV groups were orally administered PEDV at a dose of 3 × 10^5.5 TCID[50]. The results show that FUC supplementation significantly decreased plasma DAO activity (p < 0.05) and increased the villus height, villus area, as well as the villus height/crypt depth (p < 0.05) in the intestine when compared to the PEDV-infected piglets. This indicates that FUC could alleviate the disruption of intestinal morphology and function caused by PEDV infection. FUC enhanced the antioxidant capacity of the piglets by increasing SOD and GSH-Px activity. Transcriptional profiling combined with quantitative analysis revealed that FUC regulates immune responses, substance transport, and arginine metabolism. Notably, FUC downregulated arginase 1 expression, which may redirect arginine toward nitric oxide synthesis, thereby establishing an antiviral state in the host. These findings highlight the potential application of FUC as a natural agent for mitigating PEDV-induced intestinal damage and improving gut health. Additionally, monitoring the health status of piglets is necessary when FUC is applied in practical applications. 1. Introduction The animal intestinal tract plays a pivotal role in nutrient absorption and barrier function, which are essential for maintaining adequate nutrition. Beyond its role in nutrient regulation, the intestinal tract also serves as a critical barrier, preventing pathogen invasion and preserving intestinal integrity [[34]1]. In practice, numerous factors can contribute to intestinal damage in piglets, among which the porcine epidemic diarrhea virus (PEDV) is a prominent causative agent. PEDV causes porcine epidemic diarrhea (PED), which is an acute, highly contagious enteric disease. This virus exhibits high pathogenicity and can infect pigs of all ages and breeds, with piglets being the most severely affected, often presenting with pronounced clinical symptoms, including vomiting, diarrhea, and lethargy [[35]2]. PEDV primarily targets intestinal epithelial cells, causing rapid cell lysis, necrosis, and villous atrophy [[36]3]. Additionally, the enzymatic activity and content of damaged cells are significantly diminished, leading to a compromise of the gut’s nutrient absorption capacity [[37]4]. Despite widespread vaccination, the infection rate of PEDV has risen significantly due to the highly mutated rates of amino acids within the neutralizing epitopes of the spike gene [[38]5,[39]6]. This poses a substantial threat to the swine industry nationwide. Polysaccharides derived from animals, plants, and microorganisms are natural products known for their ability to regulate various physiological functions. They are characterized by their safety, low toxicity, and minimal side effects [[40]7]. Fucoidan (FUC), a sulfated polysaccharide primarily composed of L-fucose and sulfate groups, also contains trace amounts of xylose, mannose, arabinose, galactose, and glucuronic acid [[41]8]. FUC is predominantly found in the cell walls and intercellular spaces of brown algae, such as kelp and wakame [[42]9]. FUC does not cause significant toxicity within reasonable dosage ranges. Recent studies have demonstrated that FUC exhibits a wide range of biological activities, including anticoagulant, antibacterial, antiviral, antitumor, and immunomodulatory effects [[43]10,[44]11]. FUC modulates the intestinal mucosal barrier, thereby stabilizing the microecological environment and mitigating inflammatory responses [[45]12]. FUC promotes intestinal health by regulating gut microbiota, repairing the intestinal mucosa, enhancing digestive enzyme activity, and increasing the number of goblet cells [[46]13]. Moreover, it attenuates pro-inflammatory responses, such as IL-6 and TNF-α, in porcine intestinal epithelial cells stimulated by E. coli through the NF-κB signaling pathway, while also reducing bacterial adhesion and invasion [[47]14]. Numerous studies have demonstrated that both naturally extracted and synthetic sulfated polysaccharides exhibit inhibitory activity against a variety of DNA and RNA viruses in vitro and in vivo [[48]15]. Research indicates that FUC can inhibit the entry of SARS-CoV-2 into host cells by binding to the spike glycoprotein [[49]16,[50]17]. Additionally, FUC has been shown to effectively block influenza A virus infection in vitro, with low toxicity and minimal propensity for inducing viral resistance [[51]18]. Although FUC has been shown to protect the intestinal tract and exhibit antiviral effects, there is a lack of research on its ability to defend against viral infections in the intestines of pigs and/or other animal species. Therefore, we conducted this study to evaluate the effects of FUC on intestinal injury induced by PEDV in piglets. 2. Materials and Methods 2.1. Experimental Materials and Diets Fucoidan (purity > 85%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). A PEDV strain (Yunnan Province strain 8) was provided by the Hubei Key Laboratory of Animal Nutrition and Feed Science at Wuhan Polytechnic University, Wuhan, China. 2.2. Animals and Experiment Design A total of 28 healthy 7-day-old crossbred piglets (initial body weight: 2.49 ± 0.38 kg) were selected for this experiment. All the piglets were negative for PEDV by the experiment, which was confirmed by real-time PCR. They were randomly allocated into four experimental groups using a 2 × 2 factorial design: (1) a control group, (2) an FUC group, (3) a PEDV group, and (4) an FUC+PEDV group. The experimental period lasted for 11 days, with the first three days allocated for adaptation. From day 4 to day 10, piglets in the FUC and FUC+PEDV groups were orally administered fucoidan (dissolved in milk) at a dosage of 20 mg/kg body weight (BW) each day. The same volume of milk was provided to the piglets in the control and PEDV groups. On day 8, piglets in the PEDV and FUC+PEDV groups were orally administered PEDV at a dose of 3 × 10^5.5 TCID[50]. This dosage was determined according to our previous study, with slight adjustments [[52]19]. Piglets in the control and FUC groups received an equivalent DMEM solution. Fasting was initiated at 22:00 on day 10, with water and feed withdrawn for 8 h. At 06:00 on day 11, fasting body weights were recorded, followed by sequential oral administration of d-xylose (0.1 g/kg body weight) via gavage. Peripheral blood samples were collected from the jugular vein using EDTA-coated vacutainers. Terminal procedures commenced with intramuscular anesthesia (sodium pentobarbital, 50 mg/kg BW). Tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C for subsequent analysis. Piglets were weighed on an empty stomach each morning on days 0, 4, 8, and 11, with weights recorded to calculate the average daily gain (ADG). Throughout the experimental period, diarrhea and morbidity were observed both before and after each feeding session, and fecal morphology was assessed using a standardized scoring system: normal, dry feces were scored as 0; feces in a pasty state were scored as 1; feces in a semi-liquid state were scored as 2; and feces in a liquid state were scored as 3. 2.3. Biochemical Measurements in Plasma Blood samples were gently inverted several times to ensure thorough mixing of the blood with the anticoagulant and to prevent coagulation. Plasma and serum were subsequently separated by centrifugation. And the fresh plasma samples were immediately used for biochemical parameter analysis with a Hitachi 7100 Automatic Biochemical Analyzer (Hitachi, Tokyo, Japan). 2.4. Determination of D-Xylose and Diamine Oxidase Activity in Plasma Colorimetric methods were employed to measure d-xylose content and diamine oxidase (DAO) activity in plasma using kits obtained from the Nanjing Jiancheng Bioengineering Institute (d-xylose: catalog number A035-1-1; DAO: catalog number A088-1-1). All assays were conducted in accordance with the manufacturer’s instructions. 2.5. Antioxidant Capacity in Serum and Intestinal Mucosa The antioxidant enzymes and their associated products were analyzed using serum and intestinal mucosa samples from piglets. The activities and concentrations of glutathione peroxidase (GSH-Px), superoxide dismutase, total superoxide dismutase (T-SOD), myeloperoxidase (MPO), malondialdehyde (MDA), and hydrogen peroxide (H[2]O[2]) were measured using commercially available reagent kits from the Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). 2.6. Intestinal Histomorphology Briefly, 1 cm intestinal segments were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections with a thickness of 6 µm were cut, deparaffinized with xylene, and stained with hematoxylin and eosin. Morphometric parameters, including the villus height (VH), crypt depth (CD), villus width (VW), and the ratio of the villus height to crypt depth (VH/CD), were measured using an Olympus BX41 microscope (Tokyo, Japan) and analyzed with the ImageProPlus 6.0 software (Media Cybernetics, Rockville, MD, USA). 2.7. Real-Time PCR The total RNA was extracted from approximately 100 mg of intestinal tissue using RNAiso Plus (Takara, Dalian, China). RNA concentration was quantified using a NanoDrop^® ND-2000 UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was verified by 1% agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized using the PrimeScript^® RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Gene expression levels were analyzed by quantitative real-time PCR (qPCR) using SYBR^® Premix Ex Taq™ (Tli RNaseHPlus) (Takara, Dalian, China) on an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). The ribosomal protein L19 (RPL19) gene served as the internal reference, and relative gene expressions were calculated and statistically analyzed using the 2^−ΔΔCt method. Primer sequences for the target genes are provided in [53]Table 1. Table 1. Primer sequences used for qPCR analysis. Gene Forward (5′-3′) Reverse (5′-3′) Size (bp) References