Effects of Ilicis Chinensis folium extract on growth performance, immunity, antioxidant indicators, and Intestinal microbiota in weaned piglets

Effects of Ilicis Chinensis folium extract on growth performance, immunity, antioxidant indicators, and Intestinal microbiota in weaned piglets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Ilicis Chinensis folium extract on growth performance, immunity, antioxidant indicators, and Intestinal microbiota in weaned piglets Danni Liao, Bing Han, Shuowen Wang, Rihan Jiang, Xiang Li, Jiayi Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8730145/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract This study evaluated the effects of dietary supplementation with Ilicis chinensis folium extract (ICFE) on growth performance, immune function, antioxidant capacity, and gut microbiota composition in weaned piglets. A total of 224 healthy 21-day-old piglets were randomly assigned to four dietary treatments for 30 days: a control group receiving a basal diet and three experimental groups supplemented with 500 g/t, 1000 g/t, or 2000 g/t ICFE. Compared with the control group, the 1000 ICFE group exhibited significantly higher average daily feed intake ( P < 0.05), average daily gain, and final body weight, along with a lower feed conversion ratio and reduced diarrhea incidence ( P < 0.05). Serum globulin and aspartate aminotransferase levels were significantly reduced in the 1000 and 2000 ICFE groups ( P < 0.05), while the albumin-to-globulin ratio was significantly increased in the 2000 ICFE group ( P < 0.05). Immunoglobulin A, M, and G concentrations were significantly elevated in the 1000 and 2000 ICFE groups ( P < 0.05), whereas interleukin-1β and interleukin-6 levels were significantly decreased across all ICFE-treated groups ( P < 0.05). Antioxidant analysis showed significantly increased glutathione peroxidase and superoxide dismutase activities in the 500 and 1000 ICFE groups ( P < 0.05), accompanied by reduced malondialdehyde levels ( P < 0.05). Gut microbiota profiling revealed increased abundance of Actinobacteria in the 2000 ICFE group and enrichment of the genus SMB53 in the 1000 ICFE group ( P < 0.05). Correlation analysis indicated significant associations between microbial shifts and serum immune and antioxidant markers ( P < 0.05). Overall, ICFE supplementation improved growth performance, enhanced immune and antioxidant responses, reduced diarrhea incidence, and modulated gut microbiota in weaned piglets, with 1000 g/t identified as the optimal supplementation level. Ilicis Chinensis folium extract Weaned Piglets Growth Performance Serum Biochemistry Immunity Antioxidant Gut Microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction During the early stages of weaning, piglets experience substantial stress caused by changes in feed form, nutritional sources, and environment. This stress disrupts intestinal morphology, compromises barrier function, and alters the balance of the gut microbiota. As a result, immune function declines, adversely affecting piglet growth, development, and overall health (Tang et al. 2022 ). Traditional strategies to mitigate weaning stress focus on improving husbandry practices and supplementing diets with antibiotics. However, concerns regarding antibiotic resistance and drug residues arising from excessive antibiotic use increasingly restrict their practical application (Ventero et al. 2024 ). Consequently, natural plant extracts, characterised by multifunctional activity, safety, and environmental sustainability, have emerged as promising alternatives for alleviating weaning stress in piglets. Previous studies have shown that plant extracts can enhance growth performance in livestock and poultry, strengthen immune responses, increase antioxidant capacity, and stabilize gut microbiota (Ma et al. 2022 ; Che et al. 2024 ; Zhang et al. 2022 ). In addition, certain plant extracts exhibit antibacterial and antiviral effects and may modulate the animal nervous system (Seo et al. 2024 ; Liu et al. 2023 ). Ilicis chinensis folium extract (ICFE) is obtained from the dried leaves of the holly plant and contains several bioactive compounds, including pedunculoside, ursolic acid, protocatechuic acid, chlorogenic acid, and quercetin. Pedunculoside exhibits anti-inflammatory, hypolipidemic, and hepatoprotective effects (Liu et al. 2020 ; Wu et al. 2019 ), while ursolic acid demonstrates anti-inflammatory and immunomodulatory activity (Feng et al. 2025 ). Protocatechuic acid and chlorogenic acid possess strong antioxidant and anti-inflammatory properties (Albarakati 2022 ; Cheng et al. 2025 ), and quercetin has been widely reported to support intestinal health in poultry and livestock (Zou et al. 2016 ). Owing to these biological functions, ICFE has attracted growing interest for its potential role in animal nutrition and health regulation. Previous studies have shown that dietary ICFE supplementation improves duodenal and jejunal morphology in broilers, increases serum interleukin-4, immunoglobulin A, superoxide dismutase, and total antioxidant capacity levels, reduces malondialdehyde concentration, and does not induce organ toxicity, supporting its capacity to promote growth, enhance immune function, and strengthen antioxidant defenses in livestock and poultry (Zhong et al. 2023 ). However, evidence regarding the effects of ICFE in weaned piglets remains limited. Given the multifunctional properties of its bioactive components, this study aimed to evaluate the effects of dietary ICFE supplementation on growth performance, immune function, antioxidant capacity, and gut microbiota in weaned piglets. The findings are expected to provide a theoretical basis for the practical application of ICFE in weaned piglet production. Materials and methods Materials The ICFE used in the experiment was supplied by Hunan Jianong Zhenghe Biotechnology Co., Ltd. (Changsha, China); It appeared as a brownish-gray powder, with the following extract components: Pedunculoside(10.2 mg/g), Protocatechuic acid (5.9 mg/g), Chlorogenic acid (4.0 mg/g), Ursolic acid (0.9 mg/g), Caffeic acid (0.7 mg/g), Quercetin (0.6 mg/g), among others. Experimental design and animal management The experiment selected 224 weaned piglets, 21 days old, from a Duroc × Landrace × Yorkshire three-way cross. They were randomly divided into 4 groups, each with 4 replicates, and each replicate consisted of 14 piglets. The dietary treatments were as follows: a basal diet (Control); a basal diet + 500 g/t ICFE (500 ICFE); a basal diet + 1000 g/t ICFE (1000 ICFE); and a basal diet + 2000 g/t ICFE (2000 ICFE). All diets were formulated to meet or exceed all nutrient requirements according to the National Research Council (2012). The composition and nutritional levels of the base diet are shown in Table 1 . The experimental period lasted 30 days. The experiment was conducted at a pig farm in Jiangxi Province, China. Before the trial, the pig barns were cleaned and disinfected in accordance with the farm's relevant management regulations. During the experiment, piglets had free access to feed and water. Barn temperature and humidity were strictly controlled, and ventilation was maintained. Daily observations and records were made of the piglets' feeding and health status. Table 1 Composition and nutrient content of the basal diets (air-dry basis%) Ingredients Content% Nutritional level b Content% Corn 39.26 DE/(MJ/Kg) 13.39 Soybean meal 15.00 CP 19.50 Barley 39.50 EE 3.30 Fish meal 4.00 Ca 0.80 Salt 0.20 TP 0.72 Limestone 0.50 AP 0.51 Calcium hydrogenphosphate 1.00 Lys 1.20 Premis a 0.50 Met + Cys 0.75 Total 100.00 Thr 0.79 a The premix provides per kilogram of feed: Iron 100 mg, Copper 250 mg, Zinc 100 mg, Manganese 100 mg, Iodine 0.3 mg, Selenium mg, Vitamin A 13500 IU, Vitamin D3 2150 IU, Vitamin E 15 IU, Vitamin K 3 mg, Vitamin B1 1.8 mg, Vitamin B2 6 mg, Vitamin B3 24 mg, Vitamin B11 0.3 mg, Vitamin B12 0.024 mg, Calcium Pantothenate 20 mg, Choline 5000 mg, Biotin 4.5 mg, L-Lysine 3000 mg, DL-Methionine 1500 mg. b Values for nutrient level were calculated. DE, digestible energy; CP, crude protein; EE, ether extract; Ca, calcium; TP, total phosphorus; AP, available phosphorus; Lys, lysine; Met, methionine; Cys, cystine; Thr, threonine Growth performance assessment On Days 1 and 30 of the trial, weaned piglets were weighed on an empty stomach to calculate average daily gain (ADG). Feed intake was recorded to determine average daily feed intake (ADFI) for the weaned piglets, and the feed-to-gain ratio (F/G) and diarrhea rate were calculated throughout the trial period. Serum parameter analysis On day 30 of the trial, six piglets with similar average body weight were randomly selected from each group. Blood samples were collected from the anterior vena cava, and centrifuged to separate the serum which was then stored at -20°C. A fully automated biochemical analyzer (SMT-120 V) was used to determine 14 serum biochemical parameters. The commercial assay kits for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TC) were purchased from Wuhan Elarite Biotechnology Co., Ltd., with the experimental procedures strictly following the manufacturer's instructions. Assay kits for catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Biotechnology Co., Ltd. The concentrations of immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-2 (IL-2) in serum were measured by enzyme-linked immunosorbent assay (ELISA) kits supplied by Shanghai Enzyme-Linked Bio-Technology Co., Ltd. Fecal sample collection and sequencing On day 30 of the experiment, 10 fresh fecal samples were collected from each group and stored at -80°C for subsequent 16S rRNA sequencing (Paisenno Gene Cloud Company). Statistical analysis All data were analysed using SPSS 26.0 statistical software. One-way analysis of variance (ANOVA) was performed, followed by Duncan's multiple range test for post hoc comparisons. Differences were considered statistically significant at P < 0.05. Final experimental results for each group are presented as “mean ± standard deviation.” Spearman's correlation analysis was used to examine associations between gut microbiota and immune antioxidant capacity. Results Growth performance As shown in Table 2, no significant differences in initial body weight were observed among groups. Compared with the control group, the 1000 ICFE group showed significantly higher final body weight, average daily gain, and average daily feed intake at weaning, along with a significantly lower feed conversion ratio and reduced diarrhea incidence ( P < 0.05). Table 2 Effects of different doses of ICFE on growth performance of weaned piglets Item Control 500 ICFE 1000 ICFE 2000 ICFE P value Initial BW, kg 6.04 ± 0.39 5.75 ± 0.41 6.08 ± 0.38 5.82 ± 0.58 0.377 Final BW, kg 11.13 ± 0.65 a 10.27 ± 0.69 a 12.25 ± 0.46 b 10.17 ± 0.95 a 0.047 ADG, g 175.18 ± 14.16 a 164.66 ± 18.38 a 204.01 ± 16.56 b 167.51 ± 13.15 a 0.023 ADFI, g 282.33 ± 1.36 a 268.33 ± 9.34 a 317.00 ± 16.21 b 278.58 ± 13.79 a 0.001 F/G 1.67 ± 0.01 a 1.67 ± 0.06 a 1.62 ± 0.01 b 1.71 ± 0.01 a 0.039 Diarrhea rate, % 0.84 ± 0.30 a 0.73 ± 0.31 a 0.37 ± 0.23 b 0.59 ± 0.24 ab 0.006 ADFI, average daily feed intake; ADG, average daily gain; F/G, feed-to-gain ratio. Control group (fed basal diet), 500ICFE (basal diet + 500g/t ICFE), 1000ICFE (basal diet + 1000g/t ICFE), 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 6). Significant differences ( p < 0.05) are denoted by the distinct letters a and b Serum biochemical parameters As shown in Table 3, the 1000 and 2000 ICFE groups exhibited significantly lower globulin (GLB) and aspartate aminotransferase (AST) levels compared with the control group ( P < 0.05). The 2000 ICFE group also showed a significantly higher albumin-to-globulin ratio (AGR) ( P < 0.05), whereas no significant differences were observed in other biochemical parameters. Table 3 Effects of different doses of ICFEt on serum biochemical indicators in weaned piglets Item Control 500 ICFE 1000 ICFE 2000 ICFE P value ALB/(g/L) 29.70 ± 2.68 26.73 ± 1.57 26.63 ± 2.33 27.05 ± 1.38 0.063 TB/(g/L) 62.73 ± 6.93 58.20 ± 8.94 55.78 ± 3.10 53.03 ± 4.33 0.156 GLB/(g/L) 33.03 ± 2.03 a 31.50 ± 2.95 ab 29.18 ± 1.29 bc 25.83 ± 2.85 c 0.001 AGR 0.90 ± 0.05 a 0.85 ± 0.34 a 0.91 ± 0.08 a 1.05 ± 0.07 b 0.004 AST/(U/L) 128.25 ± 1.89 a 125.89 ± 7.83 a 102.00 ± 12.25 b 109.50 ± 8.22 b 0.001 ALT/(U/L) 101.50 ± 11.09 96.67 ± 9.03 106.67 ± 11.59 99.50 ± 11.39 0.538 AMY/(U/L) 2914.00 ± 395.15 3411.75 ± 401.27 3424.25 ± 316.96 3369.25 ± 437.39 0.089 CK/(U/L) 1201.25 ± 201.04 1138.33 ± 124.13 1338.33 ± 199.50 1123.00 ± 118.01 0.318 SCR/(U/L) 67.15 ± 15.09 51.55 ± 9.52 68.45 ± 2.66 52.85 ± 12.80 0.069 BUN/(µmol/L) 4.94 ± 1.14 3.80 ± 0.84 4.69 ± 0.86 4.24 ± 0.33 0.082 BUN/ SCR 73.47 ± 4.08 89.33 ± 7.89 66.82 ± 11.37 85.26 ± 12.08 0.052 GLU/(mmol/L) 5.38 ± 2.48 6.74 ± 1.12 6.38 ± 0.70 6.71 ± 1.02 0.223 Ca/(mmol/L) 2.79 ± 0.12 2.23 ± 0.42 2.82 ± 0.26 2.48 ± 0.26 0.074 P/(mmol/L) 3.75 ± 0.39 4.07 ± 1.00 3.31 ± 0.53 3.36 ± 0.30 0.335 TC/(mmol/L) 2.31 ± 0.70 2.45 ± 1.01 1.65 ± 0.33 2.28 ± 0.33 0.183 LDL-C/(mmol/L) 1.13 ± 0.43 1.56 ± 0.38 1.46 ± 0.36 1.59 ± 0.24 0.096 HDL-C/(mmol/L) 0.72 ± 0.16 0.74 ± 0.13 0.61 ± 0.07 0.80 ± 0.31 0.429 Control group (fed basal diet), 500ICFE (basal diet + 500g/t ICFE), 1000ICFE (basal diet + 1000g/t ICFE), 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 6). Significant differences ( p < 0.05) are denoted by the distinct letters a and b Serum immune capacity As shown in Fig. 1, serum IgA, IgM, and IgG levels were significantly higher in the 1000 and 2000 ICFE groups than in the control group ( P < 0.05). IL-1β and IL-6 levels were significantly reduced in all ICFE-treated groups ( P < 0.05), whereas no significant differences were detected in other immune parameters. Serum antioxidant capacity As shown in Fig. 2, GSH-Px and SOD levels were significantly higher in the 500 and 1000 ICFE groups compared with the control group ( P < 0.05), whereas MDA levels were significantly reduced ( P < 0.05). CAT and T-AOC did not differ significantly among groups. Analysis of gut microbiota This study evaluated the effects of dietary ICFE supplementation on the gut microbial community structure of weaned piglets using 16S ribosomal RNA gene amplification and sequencing. As shown in Fig. 3a, the dilution curve reached a plateau when the number of valid sequences exceeded 20,000, indicating adequate sequencing depth and sufficient sampling coverage. Figure 3b presents the Simpson diversity index, which reflects microbial diversity based on species richness and community evenness; higher values indicate greater diversity. No significant differences in microbial community diversity were observed among groups. Figure 3c illustrates β-diversity assessed by principal coordinate analysis. The 1000 ICFE group exhibited the most stable microbial community structure, with tightly clustered sample points that were clearly separated from those of the control group. The Venn diagram (Fig. 3d) shows the number of shared and unique operational taxonomic units (OTUs) across groups. Total OTU counts in the control, 500 ICFE, 1000 ICFE, and 2000 ICFE groups were 7582, 6482, 6921, and 8361, respectively. The control group contained 5254 unique OTUs, and the number of unique OTUs increased progressively with ICFE dose. Figure 4 presents the 10 most abundant microbial phyla and genera. At the phylum level, Firmicutes and Bacteroidetes dominated across all groups. Compared with the control group, the relative abundance of Actinobacteria was significantly increased in the 2000 ICFE group ( P 0.05). The relative abundance of Proteobacteria showed a decreasing trend with increasing ICFE dosage ( P = 0.06). At the genus level, the relative abundance of the probiotic genus SMB53 was significantly higher in the 1000 ICFE group ( P 0.05). Correlation analysis between gut microbiota abundance and serum antioxidant and immune markers To explore the relationship between gut microbiota composition and serum immune and antioxidant parameters in weaned piglets, LEfSe analysis was performed to identify taxa with significantly different abundances among groups. Using a threshold of linear discriminant analysis score > 3 and P < 0.05, 14 biomarker taxa were identified. Spearman’s rank correlation analysis was then conducted to evaluate associations between the relative abundance of these taxa and immune and antioxidant indicators (Fig. 5). Significant positive correlations were observed between serum IgA, IgG, and IgM levels and the abundance of Alphaproteobacteria , Ruminococcaceae , Lactobacillaceae , and the genus Lactobacillus ( P < 0.05). In contrast, these immunoglobulins were significantly negatively correlated with the abundance of Clostridiaceae and Paraeggerthella ( P < 0.05). SOD activity showed a significant positive correlation with Ruminococcaceae and Allobaculum abundance ( P < 0.05), and a significant negative correlation with Alphaproteobacteria ( P < 0.05). Both GSH-Px activity and T-AOC showed significant positive correlations with Ruminococcaceae abundance ( P < 0.05). Discussion In modern intensive pig production systems, early weaning at 3–4 weeks of age has become a common practice. However, early weaning can compromise digestive function, suppress immune responses, and impair growth, thereby exacerbating weaning stress in piglets (Upadhaya and Kim 2021 ). Plant extracts, which contain diverse bioactive compounds, offer promising advantages in mitigating stress-related physiological disturbances. ICFE is rich in active constituents such as protocatechuic acid, chlorogenic acid, quercetin, and pedunculoside, which exert anti-inflammatory, antioxidant, and gut microbiota-modulating effects (Jiang et al. 2023 ; Liu et al. 2020 ; Shabbir et al. 2021 ). In light of these multifunctional properties, this study evaluated the potential of ICFE as a dietary supplement for weaned piglets by assessing its impact on growth performance, immune function, antioxidant capacity, and gut microbiota composition. Growth performance and diarrhea incidence reflect feed utilization efficiency and overall health status in piglets. In this study, piglets in the 1000 ICFE group showed significantly higher final body weight, ADG, and ADFI, along with significantly lower F/G and reduced diarrhea incidence ( P < 0.05). Zhong et al. ( 2023 ) reported that ICFE enhances growth performance in broilers by improving intestinal morphology and antioxidant capacity. The growth-promoting effects of ICFE may arise from the synergistic actions of its bioactive monomers, including protocatechuic acid, chlorogenic acid, and quercetin. Hu et al. ( 2020 ) demonstrated that protocatechuic acid supports health and improves growth performance in lipopolysaccharide-induced piglet stress models by optimizing gut microbiota composition, strengthening intestinal barrier function, and enhancing antioxidant capacity. Chen et al. ( 2018 ) reported that chlorogenic acid increases ADG, reduces F/G, and lowers diarrhea incidence in piglets through mechanisms involving antioxidant regulation, improved intestinal absorptive function, and modulation of gut microbiota. Mao et al. ( 2024 ) further showed that quercetin promotes growth in weaned piglets through antioxidant and anti-inflammatory effects, as well as microbiota-regulating activity. Collectively, these findings suggest that ICFE enhances growth in weaned piglets by improving antioxidant capacity and supporting intestinal health through the combined effects of its active constituents. Serum biochemical indicators provide direct insight into metabolic activity and physiological function, serving as key markers of metabolic status and overall health. Serum GLB is closely linked to humoral immune responses and inflammatory activity, whereas an imbalanced AGR often indicates immune dysregulation. In this study, GLB levels in the 1000 and 2000 ICFE groups were significantly lower than those in the control group ( P < 0.05), while AGR was significantly higher in the 2000 ICFE group ( P < 0.05). These changes likely reflect the anti-inflammatory properties of ICFE. Under normal rearing conditions, piglets may experience mild inflammatory responses triggered by environmental stressors or microbial metabolites, resulting in moderate immune activation and increased GLB synthesis (Huang et al. 2021 ). Bioactive compounds in ICFE, such as pedunculoside and protocatechuic acid, can suppress the release of pro-inflammatory cytokines, including TNF-α and IL-1β. This attenuation of immune overactivation reduces GLB production and restores AGR balance, indirectly indicating alleviation of systemic inflammation and improved health status (Kan et al. 2021 ; Zhang et al. 2015 ). AST serves as a sensitive marker of hepatocellular injury, with lower concentrations often reflecting enhanced hepatic antioxidant protection and reduced inflammatory damage. In this study, AST levels were significantly decreased in the 1000 and 2000 ICFE groups ( P < 0.05). This finding is consistent with the hepatoprotective mechanism of ursolic acid reported by Zhou et al. ( 2024 ), whereby inhibition of lipid peroxidation and oxidative stress reduces inflammatory responses, limits hepatocellular injury, and decreases AST release. No significant differences were observed in serum alanine aminotransferase, creatinine, urea, blood urea nitrogen, total cholesterol, glucose, or other biochemical indices among ICFE-treated groups compared with the control group ( P > 0.05). These results indicate that ICFE does not adversely affect liver or kidney function, glucose homeostasis, or lipid metabolism, further supporting its safety as a dietary feed additive in piglets. Weaning stress can impair immune function in piglets, with immunoglobulins and cytokines serving as key indicators of immune status. In this study, serum IgA, IgM, and IgG levels were significantly higher in the 1000 and 2000 ICFE groups than in the control group ( P < 0.05), whereas IL-1β and IL-6 levels were significantly lower in all ICFE-treated groups ( P < 0.05). These findings indicate that ICFE enhances immune function and mitigates inflammatory responses in weaned piglets. The immune-enhancing effects observed in piglets are consistent with the findings reported by Zhong et al. ( 2023 ), who showed that ICFE increases IgA levels in broiler chickens. Moreover, the immunomodulatory activities of key ICFE monomer components have been independently validated. Chlorogenic acid has been shown to significantly elevate IgM and IgA levels in sows by inhibiting the TLR4/NF-κB signaling pathway (Ye et al. 2017 ; Zhang et al. 2025 ). In inflammatory models, chlorogenic acid also suppresses proinflammatory cytokines such as TNF-α and IL-6 while promoting immunoglobulin production, with mechanisms similarly linked to modulation of TLR4-mediated inflammatory signaling (Zhang et al. 2016 ; Liu et al. 2014 ). The antioxidant capacity of weaned piglets is closely linked to intestinal health, growth performance, and resistance to disease. Enhancing antioxidant defenses can strengthen resistance to oxidative stress, thereby reducing intestinal mucosal damage and inflammatory responses induced by weaning. In this study, serum SOD and GSH-Px activities were significantly increased in the 500 and 1000 ICFE groups ( P < 0.05), whereas MDA levels were significantly reduced in the 1000 ICFE group ( P < 0.05). These results indicate that appropriate ICFE supplementation effectively enhances antioxidant capacity in weaned piglets. Consistent with these findings, Zhong et al. ( 2023 ) reported that ICFE increased serum SOD and T-AOC while reducing MDA levels in broilers. The antioxidant effects of ICFE are largely attributable to its bioactive monomers, particularly protocatechuic acid and chlorogenic acid. Protocatechuic acid has been shown to significantly decrease serum MDA concentrations while increasing GSH-Px and SOD activity (Habib et al. 2021 ; Yüksel et al. 2017 ). Similarly, chlorogenic acid enhances antioxidant enzyme activity and reduces MDA levels in weaned piglets. These protective effects are mediated, at least in part, through activation of the Keap1/Nrf2 signaling pathway and upregulation of downstream antioxidant gene expression (Zhang et al. 2018 ; Shang et al. 2024 ).. The gut microbiota plays a central role in nutrient absorption, growth and development, and immune regulation in weaned piglets, while fecal microbiota composition serves as an indicator of intestinal health. Microbial diversity is essential for maintaining gut homeostasis. In this study, Simpson diversity indices showed no significant differences between ICFE-treated groups and the control group, indicating that ICFE did not markedly alter overall microbial diversity. However, β-diversity analysis revealed a clear separation trend between the 1000 ICFE group and the control group, suggesting a shift in microbial community structure. This finding aligns with those of Shi et al. ( 2020 ), who reported that quercetin improved gut microbial composition in antibiotic-induced dysbiosis in mice did not significantly affect α-diversity in healthy animals. These results suggest that ICFE may exert structural rather than diversity-driven effects by reshaping microbial composition without altering overall richness or evenness. At the phylum level, Firmicutes and Bacteroidetes were the dominant taxa in all groups, accounting for more than 85% of total relative abundance. No significant differences in the Firmicutes -to- Bacteroidetes ratio (F/B) were observed among groups, indicating that ICFE did not disrupt the balance of core gut microbiota and helped preserve fundamental intestinal function. The relative abundance of Actinobacteria was significantly increased in the 2000 ICFE group ( P < 0.05), whereas Proteobacteria showed a decreasing trend across all ICFE dosage groups ( P = 0.06). Wang et al. ( 2019 ) reported that protocatechuic acid supplementation increased beneficial Actinobacteria while reducing Proteobacteria containing opportunistic pathogens, thereby improving gut health through bidirectional microbiota regulation. At the genus level, the 1000 ICFE group exhibited a significant increase in the abundance of SMB53 ( P < 0.05), a genus capable of fermenting plant polysaccharides to produce butyrate, a key energy source for intestinal epithelial cells that supports barrier integrity (Sun et al. 2016 ). The relative abundance of Roseburia was also highest in the 1000 ICFE group ( P > 0.05). Roseburia is known to produce short-chain fatty acids that enhance nutrient absorption and feed efficiency, suggesting a potential mechanism underlying growth promotion at this dosage (Nie et al. 2021 ; Zhou et al. 2022 ). Overall, ICFE demonstrated dose-dependent effects on gut microbiota regulation. Supplementation at 1000 g/t appeared to promote intestinal homeostasis by enriching probiotic and short-chain fatty acid-producing bacteria while suppressing potentially pathogenic taxa. In contrast, supplementation at 2000 g/t increased Actinobacteria abundance but also reduced beneficial genera such as Lactobacillus . Therefore, a dietary inclusion rate of 1000 g/t ICFE appears to represent the optimal dose for balancing microbiota modulation and intestinal health in weaned piglets. Nevertheless, inter-individual variation and the relatively short experimental duration may have influenced microbiota stability. Future studies should include larger sample sizes and longer intervention periods to validate these findings. Weaned piglets represent a critical developmental window for the establishment of gut microbiota and the maturation of immune and antioxidant homeostasis, during which shifts in microbial community structure can directly influence growth and health (Yu et al. 2024 ). In this study, 14 differentially abundant microbial taxa were identified using LEfSe analysis, and their relationships with serum immune and antioxidant markers were examined to elucidate the mechanisms through which ICFE enhances growth performance via microbiota-mediated modulation of immune and antioxidant functions. Significant positive correlations were observed between the abundance of Lactobacillaceae and Lactobacillus and serum IgA, IgG, and IgM levels ( P < 0.01). Han et al. ( 2020 ) reported that these probiotic taxa promote immune tolerance and suppress inflammation by regulating intestinal butyrate concentrations. Kang et al. ( 2023 ) and Shi et al. ( 2023 ) further demonstrated that Lactobacillus and related strains significantly increase serum IgA, IgG, SOD, and T-AOC, while reducing MDA levels ( P < 0.05). Collectively, these findings suggest that ICFE supports host homeostasis by coordinating immune regulation and antioxidant defense through targeted modulation of beneficial gut microbiota, thereby establishing a physiological foundation for improved growth performance in weaned piglets. The Ruminococcaceae family showed significant positive correlations with IgA, IgG, IgM, T-AOC, GSH-Px, and SOD ( P < 0.05). As a major producer of short-chain fatty acids, increased abundance of Ruminococcaceae may simultaneously enhance immunoglobulin production and antioxidant activity, thereby strengthening intestinal barrier function (Xie et al. 2025 ). Cao et al. ( 2022 ) and Xu et al. ( 2022 ) reported that higher Ruminococcaceae abundance is associated with improved serum antioxidant indices, increased immunoglobulin secretion, and greater weight gain in piglets. Alphaproteobacteria exhibited positive correlations with IgA, IgG, and IgM, but a negative correlation with SOD activity ( P < 0.05). Members of this class may contribute to host homeostasis by activating innate immune responses, while potentially inducing mild oxidative stress that reduces host antioxidant enzyme activity (Corona-Cervantes et al. 2025 ; Lee et al. 2024 ). In contrast, the abundance of Clostridiaceae and Paraeggerthella was negatively correlated with IgA, IgG, and IgM ( P < 0.05). Clostridiaceae has been linked to impaired regulatory T cell differentiation, whereas Paraeggerthella is associated with intestinal inflammation and autoantibody interference (Cekanaviciute et al. 2018 ; Balakrishnan et al. 2023 ). Reduced abundance of these taxa may therefore alleviate immune dysregulation and intestinal inflammation, indirectly improving nutrient absorption. Overall, these findings indicate that ICFE promotes healthy growth in weaned piglets by enriching beneficial bacterial taxa such as Lactobacillaceae and Ruminococcaceae , suppressing potentially harmful microbes, enhancing immune function, reducing oxidative stress, and improving the intestinal digestive and absorptive environment. Conclusions In conclusion, dietary supplementation with ICFE in weaned piglets improves growth performance and overall health status, enhances immune and antioxidant functions, and optimizes gut microbiota composition. Under the experimental conditions of this study, an ICFE inclusion rate of 1000 g/t represents the optimal supplementation level. Declarations Acknowledgements We would like to thank Editage (www.editage.cn) for English language editing. Author contributions D.L., B.H., S.W. and R.J. conducted experiments, analyzed results, and wrote the draft. J.W., X.Z. and Z.L. assisted in experiment conduction and result analysis. D.L., B.H. and Y.W. revised the manuscript. X.L. supervised and offered resources. D.L., X.L. and Y.W. developed the experimental design, analyzed results, revised the manuscript, supervised and offered resources. All authors have read and agreed to the submitted version of the manuscript. Funding This work was financially supported by the Hunan Provincial College Student Innovation Training Program (Grant No.S202410537038) and the Hunan Provincial Graduate Student Research Innovation Project (Grant No.QL20230182). Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. 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Ulus Travma Acil Cerrahi Derg 23(1): 1–6. https://doi.org/10.5505/tjtes.2016.20165 Zhang, F., Jin, E., Liu, X., Ji, X., Hu, H. (2022) Effect of dietary Fructus mume and Scutellaria baicalensis Georgi on the fecal microbiota and its correlation with apparent nutrient digestibility in weaned piglets. Animals (Basel) 12(18): 2418. https://doi.org/10.3390/ani12182418 Zhang, X., Li, C., Li, J., Xu, Y., Guan, S., Zhao, M. (2015) Protective effects of protocatechuic acid on acute lung injury induced by lipopolysaccharide in mice via p38MAPK and NF-κB signal pathways. Int Immunopharmacol 26(1): 229–236. https://doi.org/10.1016/j.intimp.2015.03.031 Zhang, Y., Qu, H., Pan, H., Xiang, D., Choi, S., Liang, S. (2025) Effects of supplementation with chlorogenic acid-rich extract from Eucommia ulmoides Oliver during peri-implantation on the reproductive performance and gut microbiota of sows. Vet Sci 12(9): 857. https://doi.org/10.3390/vetsci12090857 Zhang, Y., Wang, Y., Chen, D., Yu, B., Zheng, P., Mao, X., He, J. (2018) Dietary chlorogenic acid supplementation affects gut morphology, antioxidant capacity and intestinal selected bacterial populations in weaned piglets. Food Funct 9(9): 4968–4978. https://doi.org/10.1039/c8fo01126e Zhang, Z., Wu, X., Cao, S., Wang, L., Wang, D., Yang, H., Li, L. (2016) Caffeic acid ameliorates colitis in association with increased Akkermansia population in the gut microbiota of mice. Oncotarget 7(22): 31790–31799. https://doi.org/10.18632/oncotarget.9306 Zhong, Y., Li, L., Chen, W., Xing, D., Wu, X. (2023) Effects of Ilicis Chinensis folium extract supplementation on growth performance, serum parameters, intestinal morphology, and antioxidant capacity of broiler chickens. BMC Vet Res 19(1): 94. https://doi.org/10.1186/s12917-023-03667-4 Zhou, J., Wang, Y., Wang, L., Tu, J., Yang, L., Yang, G., Qiao, S. (2022) Compromised hindgut microbial digestion, rather than chemical digestion in the foregut, leads to decreased nutrient digestibility in pigs fed low-protein diets. Nutrients 14(14): 2793. https://doi.org/10.3390/nu14142793 Zhou, L., Xiao, M., Li, Y., Chitrakar, B., Sheng, Q., Zhao, W. (2024) Ursolic acid ameliorates alcoholic liver injury through attenuating oxidative stress-mediated ferroptosis and modulating gut microbiota. J Agric Food Chem 72(38): 21181–21192. https://doi.org/10.1021/acs.jafc.4c04762 Zou, Y., Wei, H. K., Xiang, Q., Wang, J., Zhou, Y., Peng, J. (2016) Protective effect of quercetin on pig intestinal integrity after transport stress is associated with regulation oxidative status and inflammation. J Vet Med Sci 78(9): 1487–1494. https://doi.org/10.1292/jvms.16-0090 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 Mar, 2026 Reviewers agreed at journal 21 Feb, 2026 Reviewers invited by journal 18 Feb, 2026 Editor assigned by journal 03 Feb, 2026 Submission checks completed at journal 03 Feb, 2026 First submitted to journal 29 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8730145","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":593364991,"identity":"aeeb2095-4e6c-4df4-a265-285a5a3981a9","order_by":0,"name":"Danni Liao","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Danni","middleName":"","lastName":"Liao","suffix":""},{"id":593364998,"identity":"c28a4643-2aef-4bea-a165-d0b1d7143811","order_by":1,"name":"Bing Han","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Han","suffix":""},{"id":593364999,"identity":"653298b1-1bfb-42d2-b4f7-5789aaba8e91","order_by":2,"name":"Shuowen Wang","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shuowen","middleName":"","lastName":"Wang","suffix":""},{"id":593365000,"identity":"f14c1229-4756-4433-a485-04aebfc7cefb","order_by":3,"name":"Rihan Jiang","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rihan","middleName":"","lastName":"Jiang","suffix":""},{"id":593365004,"identity":"83000fab-1fad-447c-9cd6-e022136eaebe","order_by":4,"name":"Xiang Li","email":"","orcid":"","institution":"Hunan Canzoho Biological Technology Co. 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Control group (fed basal diet), 500ICFE (basal diet +500g/t ICFE), 1000ICFE (basal diet + 1000g/t ICFE), 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 6). Significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05) are denoted by the distinct letters a and b\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8730145/v1/7dce775916833b59e36cfccd.png"},{"id":103122915,"identity":"46a6ddbe-e488-4914-a245-24b446998c59","added_by":"auto","created_at":"2026-02-21 09:49:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130918,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different doses of ICFE on serum antioxidant capacity in weaned piglets. Control group (fed basal diet), 500ICFE (basal diet +500g/t ICFE), 1000ICFE (basal diet + 1000g/t ICFE), 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 6). Significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) are denoted by the distinct letters a and b\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8730145/v1/16248bed7dc6e480e16ecc41.png"},{"id":103504432,"identity":"e1d7342c-1a1b-4c1b-8acc-0c7147fcff24","added_by":"auto","created_at":"2026-02-26 13:19:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":618277,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ICFE the fecal-like flora of weaned piglets. (a) Sample dilution curve plot. (b) Grouped box plots of the alpha diversity index. (c) Two-dimensional ordination plot of samples analysed by beta diversity PCoA. (d) Venn diagram. Control group (fed basal diet); 500ICFE (basal diet +500g/t ICFE); 1000ICFE (basal diet + 1000g/t ICFE); 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 5).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8730145/v1/09c9e29306957862dec1c0eb.png"},{"id":103122918,"identity":"8f9b80f7-7d17-4756-91c3-e359d868648e","added_by":"auto","created_at":"2026-02-21 09:49:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":228932,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ICFE the fecal-like flora of weaned piglets. (a) Distribution of taxonomic composition at the phyla level. (b) Distribution of taxonomiccomposition at the genus level. Control group (fed basal diet); 500ICFE (basal diet +500g/t ICFE); 1000ICFE (basal diet + 1000g/t ICFE); 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 5).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8730145/v1/01152b32c892ff174a4efd8c.png"},{"id":103122919,"identity":"2e952461-e518-45f2-acda-b292558f0046","added_by":"auto","created_at":"2026-02-21 09:49:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":243082,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis chart. X-axis: Immune and antioxidant indicators. Y-axis: microbial mes. Redrepresents a positive correlation and blue represents a negative correlation. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8730145/v1/b10b403d6b5853d855280c31.png"},{"id":104397257,"identity":"5f25cc01-75cb-4c9a-8b64-634919a513f7","added_by":"auto","created_at":"2026-03-11 11:45:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2096554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8730145/v1/b27f4d79-79e9-4473-b51f-bb2efac8a520.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Ilicis Chinensis folium extract on growth performance, immunity, antioxidant indicators, and Intestinal microbiota in weaned piglets","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuring the early stages of weaning, piglets experience substantial stress caused by changes in feed form, nutritional sources, and environment. This stress disrupts intestinal morphology, compromises barrier function, and alters the balance of the gut microbiota. As a result, immune function declines, adversely affecting piglet growth, development, and overall health (Tang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Traditional strategies to mitigate weaning stress focus on improving husbandry practices and supplementing diets with antibiotics. However, concerns regarding antibiotic resistance and drug residues arising from excessive antibiotic use increasingly restrict their practical application (Ventero et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, natural plant extracts, characterised by multifunctional activity, safety, and environmental sustainability, have emerged as promising alternatives for alleviating weaning stress in piglets. Previous studies have shown that plant extracts can enhance growth performance in livestock and poultry, strengthen immune responses, increase antioxidant capacity, and stabilize gut microbiota (Ma et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Che et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, certain plant extracts exhibit antibacterial and antiviral effects and may modulate the animal nervous system (Seo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIlicis chinensis\u003c/em\u003e folium extract (ICFE) is obtained from the dried leaves of the holly plant and contains several bioactive compounds, including pedunculoside, ursolic acid, protocatechuic acid, chlorogenic acid, and quercetin. Pedunculoside exhibits anti-inflammatory, hypolipidemic, and hepatoprotective effects (Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), while ursolic acid demonstrates anti-inflammatory and immunomodulatory activity (Feng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Protocatechuic acid and chlorogenic acid possess strong antioxidant and anti-inflammatory properties (Albarakati \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and quercetin has been widely reported to support intestinal health in poultry and livestock (Zou et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Owing to these biological functions, ICFE has attracted growing interest for its potential role in animal nutrition and health regulation. Previous studies have shown that dietary ICFE supplementation improves duodenal and jejunal morphology in broilers, increases serum interleukin-4, immunoglobulin A, superoxide dismutase, and total antioxidant capacity levels, reduces malondialdehyde concentration, and does not induce organ toxicity, supporting its capacity to promote growth, enhance immune function, and strengthen antioxidant defenses in livestock and poultry (Zhong et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, evidence regarding the effects of ICFE in weaned piglets remains limited. Given the multifunctional properties of its bioactive components, this study aimed to evaluate the effects of dietary ICFE supplementation on growth performance, immune function, antioxidant capacity, and gut microbiota in weaned piglets. The findings are expected to provide a theoretical basis for the practical application of ICFE in weaned piglet production.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eThe ICFE used in the experiment was supplied by Hunan Jianong Zhenghe Biotechnology Co., Ltd. (Changsha, China); It appeared as a brownish-gray powder, with the following extract components: Pedunculoside(10.2 mg/g), Protocatechuic acid (5.9 mg/g), Chlorogenic acid (4.0 mg/g), Ursolic acid (0.9 mg/g), Caffeic acid (0.7 mg/g), Quercetin (0.6 mg/g), among others.\u003c/p\u003e \u003cp\u003eExperimental design and animal management\u003c/p\u003e \u003cp\u003eThe experiment selected 224 weaned piglets, 21 days old, from a Duroc \u0026times; Landrace \u0026times; Yorkshire three-way cross. They were randomly divided into 4 groups, each with 4 replicates, and each replicate consisted of 14 piglets. The dietary treatments were as follows: a basal diet (Control); a basal diet\u0026thinsp;+\u0026thinsp;500 g/t ICFE (500 ICFE); a basal diet\u0026thinsp;+\u0026thinsp;1000 g/t ICFE (1000 ICFE); and a basal diet\u0026thinsp;+\u0026thinsp;2000 g/t ICFE (2000 ICFE). All diets were formulated to meet or exceed all nutrient requirements according to the National Research Council (2012). The composition and nutritional levels of the base diet are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The experimental period lasted 30 days.\u003c/p\u003e \u003cp\u003eThe experiment was conducted at a pig farm in Jiangxi Province, China. Before the trial, the pig barns were cleaned and disinfected in accordance with the farm's relevant management regulations. During the experiment, piglets had free access to feed and water. Barn temperature and humidity were strictly controlled, and ventilation was maintained. Daily observations and records were made of the piglets' feeding and health status.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition and nutrient content of the basal diets (air-dry basis%)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContent%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNutritional level\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContent%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDE/(MJ/Kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBarley\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimestone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium hydrogenphosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremis\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMet\u0026thinsp;+\u0026thinsp;Cys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e The premix provides per kilogram of feed: Iron 100 mg, Copper 250 mg, Zinc 100 mg, Manganese 100 mg, Iodine 0.3 mg, Selenium mg, Vitamin A 13500 IU, Vitamin D3 2150 IU, Vitamin E 15 IU, Vitamin K 3 mg, Vitamin B1 1.8 mg, Vitamin B2 6 mg, Vitamin B3 24 mg, Vitamin B11 0.3 mg, Vitamin B12 0.024 mg, Calcium Pantothenate 20 mg, Choline 5000 mg, Biotin 4.5 mg, L-Lysine 3000 mg, DL-Methionine 1500 mg. \u003csup\u003eb\u003c/sup\u003e Values for nutrient level were calculated. DE, digestible energy; CP, crude protein; EE, ether extract; Ca, calcium; TP, total phosphorus; AP, available phosphorus; Lys, lysine; Met, methionine; Cys, cystine; Thr, threonine\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eGrowth performance assessment\u003c/p\u003e \u003cp\u003eOn Days 1 and 30 of the trial, weaned piglets were weighed on an empty stomach to calculate average daily gain (ADG). Feed intake was recorded to determine average daily feed intake (ADFI) for the weaned piglets, and the feed-to-gain ratio (F/G) and diarrhea rate were calculated throughout the trial period.\u003c/p\u003e \u003cp\u003eSerum parameter analysis\u003c/p\u003e \u003cp\u003eOn day 30 of the trial, six piglets with similar average body weight were randomly selected from each group. Blood samples were collected from the anterior vena cava, and centrifuged to separate the serum which was then stored at -20\u0026deg;C. A fully automated biochemical analyzer (SMT-120 V) was used to determine 14 serum biochemical parameters. The commercial assay kits for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TC) were purchased from Wuhan Elarite Biotechnology Co., Ltd., with the experimental procedures strictly following the manufacturer's instructions. Assay kits for catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Biotechnology Co., Ltd. The concentrations of immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-2 (IL-2) in serum were measured by enzyme-linked immunosorbent assay (ELISA) kits supplied by Shanghai Enzyme-Linked Bio-Technology Co., Ltd.\u003c/p\u003e \u003cp\u003eFecal sample collection and sequencing\u003c/p\u003e \u003cp\u003eOn day 30 of the experiment, 10 fresh fecal samples were collected from each group and stored at -80\u0026deg;C for subsequent 16S rRNA sequencing (Paisenno Gene Cloud Company).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were analysed using SPSS 26.0 statistical software. One-way analysis of variance (ANOVA) was performed, followed by Duncan's multiple range test for post hoc comparisons. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Final experimental results for each group are presented as \u0026ldquo;mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u0026rdquo; Spearman's correlation analysis was used to examine associations between gut microbiota and immune antioxidant capacity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eGrowth performance\u003c/p\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;2, no significant differences in initial body weight were observed among groups. Compared with the control group, the 1000 ICFE group showed significantly higher final body weight, average daily gain, and average daily feed intake at weaning, along with a significantly lower feed conversion ratio and reduced diarrhea incidence (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffects of different doses of ICFE on growth performance of weaned piglets\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e500 ICFE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1000 ICFE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2000 ICFE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInitial BW, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.04 ± 0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.75 ± 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.08 ± 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.82 ± 0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.377\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinal BW, kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.13 ± 0.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.27 ± 0.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.25 ± 0.46\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.17 ± 0.95\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADG, g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175.18 ± 14.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e164.66 ± 18.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e204.01 ± 16.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e167.51 ± 13.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADFI, g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e282.33 ± 1.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e268.33 ± 9.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e317.00 ± 16.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e278.58 ± 13.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF/G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.67 ± 0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.67 ± 0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.62 ± 0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.71 ± 0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiarrhea rate, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.84 ± 0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.73 ± 0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37 ± 0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.59 ± 0.24\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eADFI, average daily feed intake; ADG, average daily gain; F/G, feed-to-gain ratio. Control group (fed basal diet), 500ICFE (basal diet + 500g/t ICFE), 1000ICFE (basal diet + 1000g/t ICFE), 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 6). Significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) are denoted by the distinct letters a and b\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eSerum biochemical parameters\u003c/p\u003e\n\u003cp\u003eAs shown in Table 3, the 1000 and 2000 ICFE groups exhibited significantly lower globulin (GLB) and aspartate aminotransferase (AST) levels compared with the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The 2000 ICFE group also showed a significantly higher albumin-to-globulin ratio (AGR) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), whereas no significant differences were observed in other biochemical parameters.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffects of different doses of ICFEt on serum biochemical indicators in weaned piglets\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e500 ICFE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1000 ICFE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2000 ICFE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eALB/(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.70 ± 2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.73 ± 1.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.63 ± 2.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.05 ± 1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTB/(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.73 ± 6.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.20 ± 8.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.78 ± 3.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.03 ± 4.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGLB/(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.03 ± 2.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.50 ± 2.95\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.18 ± 1.29\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.83 ± 2.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90 ± 0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.85 ± 0.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91 ± 0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05 ± 0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAST/(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.25 ± 1.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.89 ± 7.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.00 ± 12.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.50 ± 8.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eALT/(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.50 ± 11.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.67 ± 9.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e106.67 ± 11.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.50 ± 11.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAMY/(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2914.00 ± 395.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3411.75 ± 401.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3424.25 ± 316.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3369.25 ± 437.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.089\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK/(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1201.25 ± 201.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1138.33 ± 124.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1338.33 ± 199.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1123.00 ± 118.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.318\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSCR/(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.15 ± 15.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.55 ± 9.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.45 ± 2.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.85 ± 12.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBUN/(µmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.94 ± 1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.80 ± 0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.69 ± 0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.24 ± 0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBUN/ SCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.47 ± 4.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.33 ± 7.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.82 ± 11.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.26 ± 12.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGLU/(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.38 ± 2.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.74 ± 1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.38 ± 0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.71 ± 1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.223\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCa/(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.79 ± 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.23 ± 0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.82 ± 0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.48 ± 0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP/(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.75 ± 0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.07 ± 1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.31 ± 0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.36 ± 0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.335\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTC/(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.31 ± 0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.45 ± 1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65 ± 0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.28 ± 0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.183\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDL-C/(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.13 ± 0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.56 ± 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.46 ± 0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.59 ± 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.096\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHDL-C/(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72 ± 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74 ± 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.61 ± 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.80 ± 0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.429\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eControl group (fed basal diet), 500ICFE (basal diet + 500g/t ICFE), 1000ICFE (basal diet + 1000g/t ICFE), 2000ICFE(basal diet + 2000g/t ICFE). Mean ± SEM are shown (n = 6). Significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) are denoted by the distinct letters a and b\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eSerum immune capacity\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;1, serum IgA, IgM, and IgG levels were significantly higher in the 1000 and 2000 ICFE groups than in the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). IL-1β and IL-6 levels were significantly reduced in all ICFE-treated groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), whereas no significant differences were detected in other immune parameters.\u003c/p\u003e\n\u003cp\u003eSerum antioxidant capacity\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;2, GSH-Px and SOD levels were significantly higher in the 500 and 1000 ICFE groups compared with the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), whereas MDA levels were significantly reduced (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). CAT and T-AOC did not differ significantly among groups.\u003c/p\u003e\n\u003cp\u003eAnalysis of gut microbiota\u003c/p\u003e\n\u003cp\u003eThis study evaluated the effects of dietary ICFE supplementation on the gut microbial community structure of weaned piglets using 16S ribosomal RNA gene amplification and sequencing. As shown in Fig.\u0026nbsp;3a, the dilution curve reached a plateau when the number of valid sequences exceeded 20,000, indicating adequate sequencing depth and sufficient sampling coverage. Figure\u0026nbsp;3b presents the Simpson diversity index, which reflects microbial diversity based on species richness and community evenness; higher values indicate greater diversity. No significant differences in microbial community diversity were observed among groups. Figure\u0026nbsp;3c illustrates β-diversity assessed by principal coordinate analysis. The 1000 ICFE group exhibited the most stable microbial community structure, with tightly clustered sample points that were clearly separated from those of the control group. The Venn diagram (Fig.\u0026nbsp;3d) shows the number of shared and unique operational taxonomic units (OTUs) across groups. Total OTU counts in the control, 500 ICFE, 1000 ICFE, and 2000 ICFE groups were 7582, 6482, 6921, and 8361, respectively. The control group contained 5254 unique OTUs, and the number of unique OTUs increased progressively with ICFE dose.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;4 presents the 10 most abundant microbial phyla and genera. At the phylum level, \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e dominated across all groups. Compared with the control group, the relative abundance of \u003cem\u003eActinobacteria\u003c/em\u003e was significantly increased in the 2000 ICFE group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), whereas \u003cem\u003eLactobacillus\u003c/em\u003e abundance was the lowest among the four groups (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). The relative abundance of \u003cem\u003eProteobacteria\u003c/em\u003e showed a decreasing trend with increasing ICFE dosage (\u003cem\u003eP\u003c/em\u003e = 0.06). At the genus level, the relative abundance of the probiotic genus \u003cem\u003eSMB53\u003c/em\u003e was significantly higher in the 1000 ICFE group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while \u003cem\u003eRoseburia\u003c/em\u003e exhibited the highest abundance among the four groups (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eCorrelation analysis between gut microbiota abundance and serum antioxidant and immune markers\u003c/p\u003e\n\u003cp\u003eTo explore the relationship between gut microbiota composition and serum immune and antioxidant parameters in weaned piglets, LEfSe analysis was performed to identify taxa with significantly different abundances among groups. Using a threshold of linear discriminant analysis score \u0026gt; 3 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, 14 biomarker taxa were identified. Spearman’s rank correlation analysis was then conducted to evaluate associations between the relative abundance of these taxa and immune and antioxidant indicators (Fig.\u0026nbsp;5). Significant positive correlations were observed between serum IgA, IgG, and IgM levels and the abundance of \u003cem\u003eAlphaproteobacteria\u003c/em\u003e, \u003cem\u003eRuminococcaceae\u003c/em\u003e, \u003cem\u003eLactobacillaceae\u003c/em\u003e, and the genus \u003cem\u003eLactobacillus\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). In contrast, these immunoglobulins were significantly negatively correlated with the abundance of \u003cem\u003eClostridiaceae\u003c/em\u003e and \u003cem\u003eParaeggerthella\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). SOD activity showed a significant positive correlation with \u003cem\u003eRuminococcaceae\u003c/em\u003e and \u003cem\u003eAllobaculum\u003c/em\u003e abundance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and a significant negative correlation with \u003cem\u003eAlphaproteobacteria\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Both GSH-Px activity and T-AOC showed significant positive correlations with \u003cem\u003eRuminococcaceae\u003c/em\u003e abundance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn modern intensive pig production systems, early weaning at 3\u0026ndash;4 weeks of age has become a common practice. However, early weaning can compromise digestive function, suppress immune responses, and impair growth, thereby exacerbating weaning stress in piglets (Upadhaya and Kim \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plant extracts, which contain diverse bioactive compounds, offer promising advantages in mitigating stress-related physiological disturbances. ICFE is rich in active constituents such as protocatechuic acid, chlorogenic acid, quercetin, and pedunculoside, which exert anti-inflammatory, antioxidant, and gut microbiota-modulating effects (Jiang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shabbir et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In light of these multifunctional properties, this study evaluated the potential of ICFE as a dietary supplement for weaned piglets by assessing its impact on growth performance, immune function, antioxidant capacity, and gut microbiota composition.\u003c/p\u003e \u003cp\u003eGrowth performance and diarrhea incidence reflect feed utilization efficiency and overall health status in piglets. In this study, piglets in the 1000 ICFE group showed significantly higher final body weight, ADG, and ADFI, along with significantly lower F/G and reduced diarrhea incidence (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Zhong et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that ICFE enhances growth performance in broilers by improving intestinal morphology and antioxidant capacity. The growth-promoting effects of ICFE may arise from the synergistic actions of its bioactive monomers, including protocatechuic acid, chlorogenic acid, and quercetin. Hu et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) demonstrated that protocatechuic acid supports health and improves growth performance in lipopolysaccharide-induced piglet stress models by optimizing gut microbiota composition, strengthening intestinal barrier function, and enhancing antioxidant capacity. Chen et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that chlorogenic acid increases ADG, reduces F/G, and lowers diarrhea incidence in piglets through mechanisms involving antioxidant regulation, improved intestinal absorptive function, and modulation of gut microbiota. Mao et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) further showed that quercetin promotes growth in weaned piglets through antioxidant and anti-inflammatory effects, as well as microbiota-regulating activity. Collectively, these findings suggest that ICFE enhances growth in weaned piglets by improving antioxidant capacity and supporting intestinal health through the combined effects of its active constituents.\u003c/p\u003e \u003cp\u003eSerum biochemical indicators provide direct insight into metabolic activity and physiological function, serving as key markers of metabolic status and overall health. Serum GLB is closely linked to humoral immune responses and inflammatory activity, whereas an imbalanced AGR often indicates immune dysregulation. In this study, GLB levels in the 1000 and 2000 ICFE groups were significantly lower than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while AGR was significantly higher in the 2000 ICFE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These changes likely reflect the anti-inflammatory properties of ICFE. Under normal rearing conditions, piglets may experience mild inflammatory responses triggered by environmental stressors or microbial metabolites, resulting in moderate immune activation and increased GLB synthesis (Huang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bioactive compounds in ICFE, such as pedunculoside and protocatechuic acid, can suppress the release of pro-inflammatory cytokines, including TNF-α and IL-1β. This attenuation of immune overactivation reduces GLB production and restores AGR balance, indirectly indicating alleviation of systemic inflammation and improved health status (Kan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). AST serves as a sensitive marker of hepatocellular injury, with lower concentrations often reflecting enhanced hepatic antioxidant protection and reduced inflammatory damage. In this study, AST levels were significantly decreased in the 1000 and 2000 ICFE groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This finding is consistent with the hepatoprotective mechanism of ursolic acid reported by Zhou et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), whereby inhibition of lipid peroxidation and oxidative stress reduces inflammatory responses, limits hepatocellular injury, and decreases AST release. No significant differences were observed in serum alanine aminotransferase, creatinine, urea, blood urea nitrogen, total cholesterol, glucose, or other biochemical indices among ICFE-treated groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results indicate that ICFE does not adversely affect liver or kidney function, glucose homeostasis, or lipid metabolism, further supporting its safety as a dietary feed additive in piglets.\u003c/p\u003e \u003cp\u003eWeaning stress can impair immune function in piglets, with immunoglobulins and cytokines serving as key indicators of immune status. In this study, serum IgA, IgM, and IgG levels were significantly higher in the 1000 and 2000 ICFE groups than in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas IL-1β and IL-6 levels were significantly lower in all ICFE-treated groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that ICFE enhances immune function and mitigates inflammatory responses in weaned piglets. The immune-enhancing effects observed in piglets are consistent with the findings reported by Zhong et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who showed that ICFE increases IgA levels in broiler chickens. Moreover, the immunomodulatory activities of key ICFE monomer components have been independently validated. Chlorogenic acid has been shown to significantly elevate IgM and IgA levels in sows by inhibiting the TLR4/NF-κB signaling pathway (Ye et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In inflammatory models, chlorogenic acid also suppresses proinflammatory cytokines such as TNF-α and IL-6 while promoting immunoglobulin production, with mechanisms similarly linked to modulation of TLR4-mediated inflammatory signaling (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe antioxidant capacity of weaned piglets is closely linked to intestinal health, growth performance, and resistance to disease. Enhancing antioxidant defenses can strengthen resistance to oxidative stress, thereby reducing intestinal mucosal damage and inflammatory responses induced by weaning. In this study, serum SOD and GSH-Px activities were significantly increased in the 500 and 1000 ICFE groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas MDA levels were significantly reduced in the 1000 ICFE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that appropriate ICFE supplementation effectively enhances antioxidant capacity in weaned piglets. Consistent with these findings, Zhong et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that ICFE increased serum SOD and T-AOC while reducing MDA levels in broilers. The antioxidant effects of ICFE are largely attributable to its bioactive monomers, particularly protocatechuic acid and chlorogenic acid. Protocatechuic acid has been shown to significantly decrease serum MDA concentrations while increasing GSH-Px and SOD activity (Habib et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Y\u0026uuml;ksel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similarly, chlorogenic acid enhances antioxidant enzyme activity and reduces MDA levels in weaned piglets. These protective effects are mediated, at least in part, through activation of the Keap1/Nrf2 signaling pathway and upregulation of downstream antioxidant gene expression (Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)..\u003c/p\u003e \u003cp\u003eThe gut microbiota plays a central role in nutrient absorption, growth and development, and immune regulation in weaned piglets, while fecal microbiota composition serves as an indicator of intestinal health. Microbial diversity is essential for maintaining gut homeostasis. In this study, Simpson diversity indices showed no significant differences between ICFE-treated groups and the control group, indicating that ICFE did not markedly alter overall microbial diversity. However, β-diversity analysis revealed a clear separation trend between the 1000 ICFE group and the control group, suggesting a shift in microbial community structure. This finding aligns with those of Shi et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who reported that quercetin improved gut microbial composition in antibiotic-induced dysbiosis in mice did not significantly affect α-diversity in healthy animals. These results suggest that ICFE may exert structural rather than diversity-driven effects by reshaping microbial composition without altering overall richness or evenness. At the phylum level, \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e were the dominant taxa in all groups, accounting for more than 85% of total relative abundance. No significant differences in the \u003cem\u003eFirmicutes\u003c/em\u003e-to-\u003cem\u003eBacteroidetes\u003c/em\u003e ratio (F/B) were observed among groups, indicating that ICFE did not disrupt the balance of core gut microbiota and helped preserve fundamental intestinal function. The relative abundance of \u003cem\u003eActinobacteria\u003c/em\u003e was significantly increased in the 2000 ICFE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas \u003cem\u003eProteobacteria\u003c/em\u003e showed a decreasing trend across all ICFE dosage groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06). Wang et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that protocatechuic acid supplementation increased beneficial \u003cem\u003eActinobacteria\u003c/em\u003e while reducing \u003cem\u003eProteobacteria\u003c/em\u003e containing opportunistic pathogens, thereby improving gut health through bidirectional microbiota regulation. At the genus level, the 1000 ICFE group exhibited a significant increase in the abundance of \u003cem\u003eSMB53\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), a genus capable of fermenting plant polysaccharides to produce butyrate, a key energy source for intestinal epithelial cells that supports barrier integrity (Sun et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The relative abundance of \u003cem\u003eRoseburia\u003c/em\u003e was also highest in the 1000 ICFE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). \u003cem\u003eRoseburia\u003c/em\u003e is known to produce short-chain fatty acids that enhance nutrient absorption and feed efficiency, suggesting a potential mechanism underlying growth promotion at this dosage (Nie et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overall, ICFE demonstrated dose-dependent effects on gut microbiota regulation. Supplementation at 1000 g/t appeared to promote intestinal homeostasis by enriching probiotic and short-chain fatty acid-producing bacteria while suppressing potentially pathogenic taxa. In contrast, supplementation at 2000 g/t increased \u003cem\u003eActinobacteria\u003c/em\u003e abundance but also reduced beneficial genera such as \u003cem\u003eLactobacillus\u003c/em\u003e. Therefore, a dietary inclusion rate of 1000 g/t ICFE appears to represent the optimal dose for balancing microbiota modulation and intestinal health in weaned piglets. Nevertheless, inter-individual variation and the relatively short experimental duration may have influenced microbiota stability. Future studies should include larger sample sizes and longer intervention periods to validate these findings.\u003c/p\u003e \u003cp\u003eWeaned piglets represent a critical developmental window for the establishment of gut microbiota and the maturation of immune and antioxidant homeostasis, during which shifts in microbial community structure can directly influence growth and health (Yu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, 14 differentially abundant microbial taxa were identified using LEfSe analysis, and their relationships with serum immune and antioxidant markers were examined to elucidate the mechanisms through which ICFE enhances growth performance via microbiota-mediated modulation of immune and antioxidant functions. Significant positive correlations were observed between the abundance of \u003cem\u003eLactobacillaceae\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e and serum IgA, IgG, and IgM levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Han et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that these probiotic taxa promote immune tolerance and suppress inflammation by regulating intestinal butyrate concentrations. Kang et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Shi et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) further demonstrated that \u003cem\u003eLactobacillus\u003c/em\u003e and related strains significantly increase serum IgA, IgG, SOD, and T-AOC, while reducing MDA levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Collectively, these findings suggest that ICFE supports host homeostasis by coordinating immune regulation and antioxidant defense through targeted modulation of beneficial gut microbiota, thereby establishing a physiological foundation for improved growth performance in weaned piglets. The \u003cem\u003eRuminococcaceae\u003c/em\u003e family showed significant positive correlations with IgA, IgG, IgM, T-AOC, GSH-Px, and SOD (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As a major producer of short-chain fatty acids, increased abundance of \u003cem\u003eRuminococcaceae\u003c/em\u003e may simultaneously enhance immunoglobulin production and antioxidant activity, thereby strengthening intestinal barrier function (Xie et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cao et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Xu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that higher \u003cem\u003eRuminococcaceae\u003c/em\u003e abundance is associated with improved serum antioxidant indices, increased immunoglobulin secretion, and greater weight gain in piglets. \u003cem\u003eAlphaproteobacteria\u003c/em\u003e exhibited positive correlations with IgA, IgG, and IgM, but a negative correlation with SOD activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Members of this class may contribute to host homeostasis by activating innate immune responses, while potentially inducing mild oxidative stress that reduces host antioxidant enzyme activity (Corona-Cervantes et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, the abundance of \u003cem\u003eClostridiaceae\u003c/em\u003e and \u003cem\u003eParaeggerthella\u003c/em\u003e was negatively correlated with IgA, IgG, and IgM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eClostridiaceae\u003c/em\u003e has been linked to impaired regulatory T cell differentiation, whereas \u003cem\u003eParaeggerthella\u003c/em\u003e is associated with intestinal inflammation and autoantibody interference (Cekanaviciute et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Balakrishnan et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Reduced abundance of these taxa may therefore alleviate immune dysregulation and intestinal inflammation, indirectly improving nutrient absorption. Overall, these findings indicate that ICFE promotes healthy growth in weaned piglets by enriching beneficial bacterial taxa such as \u003cem\u003eLactobacillaceae\u003c/em\u003e and \u003cem\u003eRuminococcaceae\u003c/em\u003e, suppressing potentially harmful microbes, enhancing immune function, reducing oxidative stress, and improving the intestinal digestive and absorptive environment.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, dietary supplementation with ICFE in weaned piglets improves growth performance and overall health status, enhances immune and antioxidant functions, and optimizes gut microbiota composition. Under the experimental conditions of this study, an ICFE inclusion rate of 1000 g/t represents the optimal supplementation level.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp; We would like to thank Editage (www.editage.cn) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eD.L., B.H., S.W. and R.J. conducted experiments, analyzed results, and wrote the draft. J.W., X.Z. and Z.L. assisted in experiment conduction and result analysis. D.L., B.H. and Y.W. revised the manuscript. X.L. supervised and offered resources. D.L., X.L. and Y.W. developed the experimental design, analyzed results, revised the manuscript, supervised and offered resources. All authors have read and agreed to the submitted version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp; This work was financially supported by the Hunan Provincial College Student Innovation Training Program (Grant No.S202410537038) and the Hunan Provincial Graduate Student Research Innovation Project (Grant No.QL20230182).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp; The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp; This study was performed in line with the principles of the Declaration of Helsinki. 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J Vet Med Sci 78(9): 1487\u0026ndash;1494. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1292/jvms.16-0090\u003c/span\u003e\u003cspan address=\"10.1292/jvms.16-0090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ilicis Chinensis folium extract, Weaned Piglets, Growth Performance, Serum Biochemistry, Immunity, Antioxidant, Gut Microbiota","lastPublishedDoi":"10.21203/rs.3.rs-8730145/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8730145/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the effects of dietary supplementation with \u003cem\u003eIlicis chinensis\u003c/em\u003e folium extract (ICFE) on growth performance, immune function, antioxidant capacity, and gut microbiota composition in weaned piglets. A total of 224 healthy 21-day-old piglets were randomly assigned to four dietary treatments for 30 days: a control group receiving a basal diet and three experimental groups supplemented with 500 g/t, 1000 g/t, or 2000 g/t ICFE. Compared with the control group, the 1000 ICFE group exhibited significantly higher average daily feed intake (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), average daily gain, and final body weight, along with a lower feed conversion ratio and reduced diarrhea incidence (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum globulin and aspartate aminotransferase levels were significantly reduced in the 1000 and 2000 ICFE groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the albumin-to-globulin ratio was significantly increased in the 2000 ICFE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Immunoglobulin A, M, and G concentrations were significantly elevated in the 1000 and 2000 ICFE groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas interleukin-1β and interleukin-6 levels were significantly decreased across all ICFE-treated groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Antioxidant analysis showed significantly increased glutathione peroxidase and superoxide dismutase activities in the 500 and 1000 ICFE groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), accompanied by reduced malondialdehyde levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gut microbiota profiling revealed increased abundance of \u003cem\u003eActinobacteria\u003c/em\u003e in the 2000 ICFE group and enrichment of the genus \u003cem\u003eSMB53\u003c/em\u003e in the 1000 ICFE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Correlation analysis indicated significant associations between microbial shifts and serum immune and antioxidant markers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Overall, ICFE supplementation improved growth performance, enhanced immune and antioxidant responses, reduced diarrhea incidence, and modulated gut microbiota in weaned piglets, with 1000 g/t identified as the optimal supplementation level.\u003c/p\u003e","manuscriptTitle":"Effects of Ilicis Chinensis folium extract on growth performance, immunity, antioxidant indicators, and Intestinal microbiota in weaned piglets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-21 09:49:21","doi":"10.21203/rs.3.rs-8730145/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-12T21:54:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168167183663983937144542874677272797821","date":"2026-02-21T21:52:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-18T12:06:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-03T08:45:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-03T08:41:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Antonie van Leeuwenhoek","date":"2026-01-29T09:30:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8de0407f-b717-4e29-91f1-dc3a491a5045","owner":[],"postedDate":"February 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-21T09:49:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-21 09:49:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8730145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8730145","identity":"rs-8730145","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}