Rice bran oil supplementation protects swine weanlings against diarrhea and lipopolysaccharide challenge Enhanced Publication

Rice bran oil supplementation protects swine weanlings against diarrhea and lipopolysaccharide challenge 日粮添加米糠油可增强断奶仔猪抵抗腹泻和脂多糖应激 1 2 3 first-author 1 2 3 1 2 3 4 4 1 2 3 1 2 3 1 2 3 1 2 3 corresp Libao MA, malibao@mail.hzau.edu.cn Libao MA, malibao@mail.hzau.edu.cn malibao@mail.hzau.edu.cn https://orcid.org/0000-0001-6806-6899 1 2 3 corresp Xianghua YAN, xhyan@mail.hzau.edu.cn Xianghua YAN, xhyan@mail.hzau.edu.cn xhyan@mail.hzau.edu.cn https://orcid.org/0000-0003-2238-6218 华中农业大学动物科学技术学院， 农业微生物学国家重点实验室， 湖北洪山实验室， 动物育种与健康养殖前沿科学 中心， 中国武汉市， 430070 华中农业大学动物科学技术学院， 农业微生物学国家重点实验室， 湖北洪山实验室， 动物育种与健康养殖前沿科学 中心， 中国武汉市， 430070 State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China 生猪健康养殖省部共建协同创新中心， 中国武汉市， 430070 生猪健康养殖省部共建协同创新中心， 中国武汉市， 430070 The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China 生猪精准饲养与饲料安全技术湖北省工程实验室， 中国武汉市， 430070 生猪精准饲养与饲料安全技术湖北省工程实验室， 中国武汉市， 430070 Hubei Provincial Engineering Laboratory for Pig Precision Feeding and Feed Safety, Wuhan 430070, China Hubei Provincial Engineering Laboratory for Pig Precision Feeding and Feed Safety, Wuhan 430070, China 宜春市大海龟生命科学有限公司， 中国宜春市， 336000 宜春市大海龟生命科学有限公司， 中国宜春市， 336000 Yichun Dahaigui Life Science Co., Ltd., Yichun 336000, China Yichun Dahaigui Life Science Co., Ltd., Yichun 336000, China Early weaned piglets suffer from oxidative stress and enteral infection, which usually results in gut microbial dysbiosis, serve diarrhea, and even death. Rice bran oil (RBO), a polyphenol-enriched by-product of rice processing, has been shown to have antioxidant and anti-inflammatory properties both in vivo and in vitro. Here, we ascertained the proper RBO supplementation level, and subsequently determined its effects on lipopolysaccharide (LPS)-induced intestinal dysfunction in weaned piglets. A total of 168 piglets were randomly allocated into four groups of seven replicates (42 piglets each group, (21±1) d of age, body weight (7.60±0.04) kg, and half males and half females) and were given basal diet (Ctrl) or basal diet supplemented with 0.01% (mass fraction) RBO (RBO1), 0.02% RBO (RBO2), or 0.03% RBO (RBO3) for 21 d. Then, seven piglets from the Ctrl and the RBO were treated with LPS (100 μg/kg body weight (BW)) as LPS group and RBO+LPS group, respectively. Meanwhile, seven piglets from the Ctrl were treated with the saline vehicle (Ctrl group). Four hours later, all treated piglets were sacrificed for taking samples of plasma, jejunum tissues, and feces. The results showed that 0.02% was the optimal dose of dietary RBO supplementation based on diarrhea, average daily gain, and average daily feed intake indices in early weaning piglets. Furthermore, RBO protected piglets against LPS-induced jejunal epithelium damage, which was indicated by the increases in villus height, villus height/crypt depth ratio, and Claudin-1 levels, as well as a decreased level of jejunal epithelium apoptosis. RBO also improved the antioxidant ability of LPS-challenged piglets, which was indicated by the elevated concentrations of catalase and superoxide dismutase, and increased total antioxidant capacity, as well as the decreased concentrations of diamine oxidase and malondialdehyde in plasma. Meanwhile, RBO improved the immune function of LPS-challenged weaned piglets, which was indicated by elevated immunoglobulin A (IgA), IgM, β‍‍-defensin-1, and lysozyme levels in the plasma. In addition, RBO supplementation improved the LPS challenge-induced dysbiosis of gut microbiota. Particularly, the indices of antioxidant capacity, intestinal damage, and immunity were significantly associated with the RBO-regulated gut microbiota. These findings suggested that 0.02% RBO is a suitable dose to protect against LPS-induced intestinal damage, oxidative stress, and jejunal microbiota dysbiosis in early weaned piglets. 早期断奶仔猪遭受氧化应激和肠道感染，通常会导致肠道微生物失调、腹泻甚至死亡。米糠油（RBO）是一种富含多酚的大米加工副产品，在体内和体外都具有抗氧化和抗炎特性。本研究中，我们确定了早期断奶仔猪日粮中适当的RBO补充水平，随后确定了它对脂多糖（LPS）诱导的断奶仔猪肠道功能障碍的影响。将168头仔猪随机分4组，每组7个重复（42头/组，(21±1)日龄，体重(7.60±0.04) kg，公母各半），分别接受基础日粮（Ctrl）或补充基础日粮含0.01% RBO（RBO1）、0.02% RBO（RBO2）或0.03% RBO（RBO3）。饲喂21天后，Ctrl和RBO组的7头仔猪经LPS（100 μg/kg BW）处理（分别为LPS组和RBO+LPS组）。同时将Ctrl组的7头仔猪用载体盐水处理作为对照（Ctrl组）。四小时后，处死所有处理组仔猪并采集血浆、空肠组织和粪便。分别检测血浆中抗氧化和免疫指标，评估空肠组织形态和屏障功能以及通过16S rDNA测序分析肠道微生物组成、功能及多样性。结果表明，根据早期断奶仔猪的腹泻、平均日增重和平均日采食量指标，0.02%是日粮中添加RBO的最佳剂量。此外，RBO可以缓解LPS诱导的仔猪空肠上皮损伤，表现为绒毛高度、绒毛高度/隐窝深度比和Claudin-1水平的增加，以及空肠上皮细胞凋亡的改善。RBO还提高了LPS应激仔猪的抗氧化能力，表现为血浆中过氧化氢酶和超氧化物歧化酶浓度升高，总抗氧化能力提升，以及二胺氧化酶和丙二醛浓度降低。 同时，RBO提高了LPS应激的断奶仔猪的免疫功能，表现为血浆中IgA、IgM、β‍-防御素-1和溶菌酶升高。此外，补充RBO还改善了LPS应激引起的肠道菌群失调。相关性分析结果发现，仔猪抗氧化能力、肠道损伤和免疫力指标分别与RBO调节的肠道微生物群显着相关。综上所述，0.02% RBO是缓解LPS诱导的早期断奶仔猪肠道损伤、氧化应激和空肠微生物群失调的适当剂量。 米糠油 肠道微生物 早期断奶仔猪 氧化应激 脂多糖 Rice bran oil Gut microbiota Weaned piglets Oxidative stress Lipopolysaccharide 1 Introduction 1 The strategy of early weaning of piglets aims to improve the efficiency of modern swine production systems but usually results in weaning stress ( Campbell et al., 2013 Campbell et al., 2013 ). Early weaning stress usually induces severe intestinal bacterial infection, intestinal dysfunction, diarrhea, and ultimately high mortality among piglets ( Gresse et al., 2017 Gresse et al., 2017 ). High dietary zinc oxide and antibiotics have been widely used to improve pathologies in piglets induced by early weaning ( Bhandari et al., 2008 Bhandari et al., 2008 ; Starke et al., 2014 Starke et al., 2014 ). However, considering their side effects on the gut microbiota and the concerns of public health and environment, European nations and China have implemented bans on the use of antibiotics for non-therapeutic purposes and have also limited the dose of dietary zinc oxide ( Allen et al., 2013 Allen et al., 2013 ; Wang et al., 2017 Wang et al., 2017 ). Rice bran oil (RBO), a byproduct of the rice milling industry, contains tocotrienols, tocopherols, and many other phytosterols ( Kushwaha, 2018 Kushwaha, 2018 ). RBO has been reported to exhibit antioxidant and anti-inflammatory properties in vivo and in vitro ( Kushwaha, 2018 Kushwaha, 2018 ; Punia et al., 2021 Punia et al., 2021 ). Supplementation with RBO could improve oxidative stress in diabetic rats ( Posuwan et al., 2013 Posuwan et al., 2013 ). For early weaned piglets, weaning stress induces an increase in intestinal permeability and inflammation, facilitating reactive oxygen species (ROS) production and release into the intestinal lumen ( Gresse et al., 2017 Gresse et al., 2017 ; Zeng et al., 2017 Zeng et al., 2017 ). These high ROS levels favor the bloom of facultative anaerobes but lessen obligate anaerobes, and consequently induce gut microbiota dysbiosis in early weaned piglets ( Gresse et al., 2017 Gresse et al., 2017 ; Zeng et al., 2017 Zeng et al., 2017 ). Recent evidence suggests that gut microbiota dysbiosis is a crucial factor that accounts for these intestinal disorders in these animals ( Dou et al., 2017 Dou et al., 2017 ; Gresse et al., 2017 Gresse et al., 2017 ; Ren et al., 2022 Ren et al., 2022 ). We therefore assumed that RBO may serve as a cost-effective dietary supplementation to ameliorate weaning stress by improving gut microbiota in swine weanlings. To determine the appropriate dose of RBO supplementation, we evaluated the diarrhea scores and growth performance indices of piglets fed with three concentrations of dietary RBO supplementation, namely 0.01%, 0.02%, or 0.03% (mass fraction). Subsequently, we investigated the protective effects of dietary RBO supplementation against lipopolysaccharide (LPS) challenge in early weaned piglets. 2 Materials and methods 1 2.1 Animals and experimental treatments 2 We randomly allocated 168 early weaned piglets (Landrace×Yorkshire, aged (21±1) d, body weight of (7.60±0.04) kg, half males and half females) into four groups with seven replicates (42 piglets in each group, half males and half females), which received basal diet (Ctrl), or basal diet supplemented with 0.01% RBO (RBO1), 0.02% RBO (RBO2), or 0.03% RBO (RBO3) for 21 d. Then, seven piglets from the Ctrl and RBO groups were treated with an intraperitoneal injection of 200 μL LPS solution per piglet (100 μg/kg body weight (BW), Escherichia coli 0111:B4, Sigma, USA), as LPS group and RBO+LPS group, respectively. Meanwhile, seven piglets from the Ctrl were treated with the saline vehicle (Ctrl group). Four hours later, all treated piglets were sacrificed after the injection of sodium pentobarbital (50 mg/kg BW) for sampling ( Xu et al., 2021b Xu et al., 2021b ). The composition of the basal experimental diet (Table S1) was in accordance with the recommendations of the National Research Council ( NRC, 2012 NRC, 2012 ). The ultra-performance liquid chromatography (UPLC) chromatogram of RBO (10.08% tocopherols; and 7.56% tocotrienols; Yichun Dahaigui Life Science Co., Ltd., Yichun, China) was presented in Fig. S1. 2.2 Piglet growth performance and fecal scores 2 The growth performance indicators of piglets, including average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR), were also determined. The fecal scores of piglets during trial were established using the following scale: 1 for normal, solid feces; 2 for soft, looser than normal feces, slight diarrhea; 3 for moderate diarrheic feces; 4 for liquid, severe diarrheic feces for all pigs daily ( Yi et al., 2005 Yi et al., 2005 ). 2.3 Plasma and intestinal sample collection 2 Blood samples were collected from the anterior cava using an anticoagulant tube, and then centrifuged at 4 ℃, 3000 g for 10 min to obtain plasma samples. We sampled the jejunal stools and then took three 2-cm segments of jejunal tissues (one was fixed in 4% (mass fraction) paraformaldehyde solution and the others were frozen in dry ice after slight wash with ice-cold phosphate-buffered saline (PBS)). All frozen samples were then stored at -80 ℃ until further use. 2.4 Histomorphology examination of the jejunum tissue 2 After fixation in 4% paraformaldehyde for 48 h, segments of jejunal tissues were embedded in paraffin and then sectioned at 5 µm for hematoxylin and eosin (H&E) staining. The villus height, crypt depth, and villus height/crypt depth ratio were determined using an Olympus BX51 microscope with Integrated Digital Imaging Analysis System (Olympus Co., Tokyo, Japan). 2.5 Apoptosis assessment of jejunal epithelium 2 The apoptosis of jejunal epithelium was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay ( Gavrieli et al., 1992 Gavrieli et al., 1992 ). The average optical density (AOD) of TUNEL (green) was identified using ImageJ to assess the apoptosis level of the jejunal epithelium ( Schneider et al., 2012 Schneider et al., 2012 ). 2.6 Determination of plasma antioxidant indices 2 The antioxidant indices of plasma were determined using commercial kits (Solarbio Science & Technology, Beijing, China) including total antioxidant capacity (T-AOC, BC1315), malondialdehyde (MDA, BC0025), catalase (CAT, BC0205), superoxide dismutase (SOD, BC0175), diamine oxidase (DAO, BC1285), and nitric oxide (NO, BC1475) according to the corresponding manufacturer’s instructions. 2.7 Determination of plasma immune indices 2 The plasma levels of immunoglobulin A (IgA), IgM, IgG, β‍‍-defensin-1, and lysozyme were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu Meimian Industrial Co., Ltd., China) according to the manufacturer’s instructions. 2.8 Tight junction protein determination of the jejunum tissue 2 The total protein of jejunal tissue was extracted using radio immunoprecipitation assay (RIPA) buffer containing protease inhibitor buffer (1:100, volume ratio, the same below), and the protein concentration was quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, USA). Rabbit polyclonal antibodies Occludin (91131S, 1:1000 dilution, Cell Signaling Technology Beverly, MA, USA) and Claudin-1 (13050-1-AP, 1:1000 dilution, Proteintech, Wuhan, China) were used as primary antibodies. The western blot results were analyzed using ImageJ software. 2.9 Gut microbiota profiling 2 The total microbial genomic DNA in jejunal stool was extracted using a commercial kit (TGuide S96 Magnetic Soil/Stool, TIANGEN, Beijing, China). Qualified genomic DNA was used as a template for polymerase chain reaction (PCR) amplification of the V4 hypervariable regions of 16S ribosomal RNA (rRNA) genes with universal primers 515F and 806R2. The purified amplicons were pooled in equimolar quantities and paired-end sequenced (2×250) on a NovaSeq 6000 platform (Illumina, San Diego, USA) at Biomarker Technologies Co., Ltd. (Beijing, China). The generated sequencing raw data were analyzed using Quantitative Insights into Microbial Ecology software package (Version 2021.6) ( Bolyen et al., 2019 Bolyen et al., 2019 ; Xu et al., 2021a Xu et al., 2021a ). Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA, with 9999 Monte Carlo permutations) based on Bray–Curtis distances were performed using the package “vegan” in R software (Version 4.3.1) ( Wang et al., 2019 Wang et al., 2019 ). The marker genera were identified by linear discriminant analysis (LDA) effect size (LEfSe) ( Segata et al., 2011 Segata et al., 2011 ). 2.10 Analysis of gut microbial function and metabolites 2 The function of gut microbiota was determined using PICRUSt2 based on the genes of 16S rRNA data and reference genomes ( Douglas et al., 2020 Douglas et al., 2020 ). Then, gas chromatography was employed to determine certain gut microbial metabolites, namely short-chain fatty acids (SCFAs) of acetate, propionate, and butyrate in colonic stools ( Yan et al., 2020 Yan et al., 2020 ). 2.11 Data processing and statistic analysis 2 The experimental data were analyzed by one-way analysis of variance (ANOVA) tests, followed by Fisher’s least significant difference and the Duncan multiple comparison test with GraphPad 8.0 software. All results were presented as mean±standard error of the mean (SEM) and the significance was set as * P < 0.05, ** P < 0.01, and *** P < 0.001. P values between 0.05 and 0.10 were considered as indicative of a trend. 3 Results 1 3.1 Effects of dietary RBO supplementation on the diarrhea and growth performance of early weaned piglets 2 In order to identify the appropriate supplementation level of RBO, the effects of dietary supplementation of 0.01%, 0.02%, and 0.03% RBO on the diarrhea and growth performance of early weaned piglets were determined. As shown in Figs. 1 1 a and 1 1 b, compared with the Ctrl group, dietary RBO supplementation at different levels was able to decrease the fecal scores at 4, 8, and 12 d post-weaning. At 16 d post-weaning, only RBO2 decreased the fecal scores compared with the Ctrl group. For growth performance, compared with the Ctrl, RBO2 and RBO3 both increased ADG and ADFI, but only RBO2 improved FCR ( Figs. 2 Figs. 2 c‍–‍2e). According to the above indices, 0.02% was the optimal supplementation level of RBO to improve diarrhea and growth performance in early weaned piglets. 3.2 Protective effect of dietary RBO supplementation against LPS-induced jejunal damage in early weaned piglets 2 In order to determine the effect of dietary RBO supplementation on improving early weaning stress, an LPS challenge was conducted to simulate the enteral infections of post-weaning piglets ( Fig. 2 Fig. 2 a). As presented in Figs. 2 2 b and 2 2 c, LPS crushed jejunal mucosa and reduced the villus height/crypt depth ratio compared to the Ctrl group. LPS also increased the apoptosis of jejunal epithelium (Figs. 2 2 d and 2 2 e) and decreased the Claudin-1 level ( Figs. 2 Figs. 2 f‍–‍2h). Compared with the LPS group, RBO improved the jejunal mucosa morphology and villus height/crypt depth ratio (Figs. 2 2 a– 2 2 c), as well as the apoptosis of the jejunal epithelium (Figs. 2 2 d and 2 2 e), while it increased the Claudin-1 level (Figs. 2 2 f– 2 2 h). 3.3 Effects of dietary RBO supplementation on the antioxidant capacity and immunity function of LPS-challenged early weaned piglets 2 As shown in Table S2, the LPS challenge increased the plasma levels of DAO and MDA, whereas it decreased the activity of CAT, SOD, and T-AOC. Compared to the LPS group, RBO supplementation significantly increased CAT, SOD, and T-AOC, whereas it decreased the level of DAO and MDA. As shown in Table S3, for specific immunity indices, LPS challenge decreased IgM, but had no significant effect on IgG or IgA when compared with the Ctrl group. Compared with the LPS, dietary RBO supplementation increased IgA and IgM. For the innate immunity indices, LPS challenge decreased plasma lysozyme, but had no significant effect on β‍-defensin-1 compared with the Ctrl group. Compared with the LPS, dietary RBO supplementation increased the levels of both β‍-defensin-1 and lysozyme. 3.4 Effect of dietary RBO supplementation on LPS-induced jejunal microbiota dysbiosis in early weaned piglets 2 The Venn diagram of operational taxonomic unit (OTU) distribution showed that the RBO+LPS group shared 124 OTUs and the LPS group shared 58 OTUs with the Ctrl ( Fig. 3 Fig. 3 a). The PCoA of β‍‍-diversity indicated that the LPS group formed a markedly distinct cluster from those of the Ctrl and RBO+LPS groups ( Fig. 3 Fig. 3 b). For a single sample, alpha diversity analysis showed that LPS decreased the indicators of observed species, Chao1, and abundance-based coverage estimator (ACE), while it had no significant effect on Shannon, Simpson, or coverage compared with the Ctrl group ( Fig. 3 Fig. 3 c). Compared with the LPS group, dietary RBO supplementation increased Chao1 ( Fig. 3 Fig. 3 c). 3.5 Effects of dietary RBO supplementation on the relative abundance of jejunal microbiota in early weaned piglets 2 As shown in Figs. 4 4 a and 4 4 b, LPS shifted the relative abundance of bacteria at both the phylum level ( Fig. 4 Fig. 4 a) and family level ( Fig. 4 Fig. 4 b). The LEfSe results showed that f_Ruminococcaceae, f_Erysipelotrichaceae, c_Erysipelotrichi, and o_Erysipelotrichales were enriched in the Ctrl group, whereas g_ Staphylococcus , f_Staphylococcaceae, f_Brevibacteriaceae, g_ Ochrobactrum , o_Actinomycetales, and f_Bacillaceae were enriched in the LPS group ( Fig. 4 Fig. 4 c). In terms of RBO+LPS vs. LPS treatment, the LEfSe results showed that f_Clostridiaceae and s_ Collinsella aerofaciens were enriched in the RBO+LPS, while f_Staphylococcaceae, f_Brevibacteriaceae, g_ Ochrobactrum , o_Actinomycetales, and o_Bacillales were enriched in the LPS group ( Fig. 4 Fig. 4 d). 3.6 Effects of dietary RBO supplementation on the function and metabolites of jejunal microbiota in early weaned piglets 2 The analysis of gut microbial function showed that, compared with the Ctrl, LPS challenge increased the functions of amino acid metabolism, cellular community, digestive system, lipid metabolism, metabolism of terpenoids and polyketides, substance dependence, transport and catabolism, and xenobiotics biodegradation and metabolism, whereas it decreased the function of carbohydrate metabolism ( Fig. 5 Fig. 5 a). Compared with the LPS group, dietary RBO supplementation increased the functions of carbohydrate metabolism, excretory system, and xenobiotics biodegradation and metabolism, whereas it decreased the functions of folding, sorting and degradation, biosynthesis of other secondary metabolites, energy metabolism, environmental adaptation, and glycan biosynthesis and metabolism ( Fig. 5 Fig. 5 b). Correspondingly, LPS decreased the concentrations of fecal SCFAs including propionate and butyrate, compared with the Ctrl group (Table S4). Compared with the LPS group, dietary RBO supplementation increased the concentrations of fecal SCFAs including acetic acid, propionic acid, and butyric acid (Table S4). 3.7 Correlations among the jejunal microbiota and the indices of immunity, oxidative stress, and jejunum morphology 2 As shown in Fig. 6 Fig. 6 , Collinsella aerofaciens was positively correlated with Claudin-1 and villus height but negatively correlated with MDA. Clostridiaceae were positively correlated with villus height but negatively correlated with MDA. The alpha diversity index ACE was positively correlated with Claudin-1. The acetate was positively correlated with villus height and villus height/crypt depth but negatively correlated with DAO. The propionate was positively correlated with T-AOC, villus height, and villus height/crypt depth, but was negatively correlated with DAO. The butyrate was positively correlated with Occludin, Claudin-1, villus height, and villus height/crypt depth, but was negatively correlated with DAO and MDA. 4 Discussion 1 Early weaned piglets suffer from oxidative stress and enteral infection, which usually leads to gut microbial dysbiosis, serve diarrhea, and even death ( Gresse et al., 2017 Gresse et al., 2017 ). RBO, a polyphenol-enriched byproduct of the rice milling industry, has been reported to present antioxidant and anti-inflammatory properties both in vivo and in vitro ( Kushwaha, 2018 Kushwaha, 2018 ; Punia et al., 2021 Punia et al., 2021 ). In this work, we identified the appropriate dose and effect of dietary RBO supplementation on improving weaning stress. Firstly, we determined the effects of dietary 0.01%, 0.02%, and 0.03% RBO supplementation on the diarrhea and growth performance of early weaned piglets and found that 0.02% RBO was a cost-effective supplementation level. Dietary RBO supplementation for 21 d promoted early weaned piglets’ growth performance by improving ADG. Compared with these results, supplementation with tocopherols as the main components of RBO could improve the growth performance of both weaned piglets and finishing pigs ( Boler et al., 2012 Boler et al., 2012 ; Lu et al., 2014 Lu et al., 2014 ). However, another study found that supplementation with 200 IU/kg tocopherols had no significant effects on improving piglets’ growth performance ( Silva-Guillen et al., 2020 Silva-Guillen et al., 2020 ). We therefore conducted an LPS challenge to simulate enteral infections that post-weaning piglets generally suffer from ( Xu et al., 2021b Xu et al., 2021b ). Given that piglets’ small intestine is the main target of enteral infections and pathogenic bacteria-derived LPS ( Xu et al., 2018 Xu et al., 2018 , 2020 2020 ; Hu et al., 2022 Hu et al., 2022 ), we analyzed the morphology of jejunum, and the results demonstrated that dietary RBO enhanced piglets’ capability to prevent LPS-induced intestinal damage. Specifically, dietary RBO supplementation improved the villus height and villus height/crypt depth ratio in LPS-challenged piglets. Similarly, Chen et al. (2019) Chen et al. (2019) reported that dietary tocopherols decreased the crypt depth and increased the villus width by inhibiting the proliferation of intestinal epithelial cells within the jejunum in weaned piglets. We also showed an extraordinary antioxidant activity of RBO regarding the antioxidant capacity indices in the plasma of early weaned piglets. RBO supplementation significantly increased CAT, SOD, and T-AOC, whereas it decreased the levels of DAO and MDA in the plasma of LPS-challenged piglets. These anti-inflammation and anti-oxidative stress functions of RBO had been widely evidenced in human beings and rodent animal studies ( Punia et al., 2021 Punia et al., 2021 ). The LPS challenge could induce intestinal barrier function damage and apoptosis of the small intestinal epithelial cells ( Sun et al., 2021 Sun et al., 2021 ; Zhang et al., 2022 Zhang et al., 2022 ). Here, we found that dietary RBO supplementation not only reduced the apoptosis of jejunal epithelium but also ameliorated the intestinal barrier function damage by elevating the Claudin-1 level in LPS-challenged piglets. In vitro evidence suggested that tocopherols were involved in the modulation of oxidative stress, proliferation, inflammation, and apoptosis by interacting with related genes ( Mocchegiani et al., 2014 Mocchegiani et al., 2014 ). The protective effect of tocotrienol against oxidative insults was more potent than that of tocopherol ( Mocchegiani et al., 2014 Mocchegiani et al., 2014 ). α-Tocopherol succinate is synthesized by the esterification of α-tocopherol, which could protect mice from radiation-induced gastrointestinal damage by inhibiting apoptosis, promoting the regeneration of crypt cells, and inhibiting the translocation of gut bacteria ( Singh et al., 2011 Singh et al., 2011 ). Therefore, tocotrienols and tocopherols, the main biological active components of RBO, may play a vital role in ameliorating apoptosis and barrier function damage of jejunal epithelium in LPS-challenged piglets. LPS challenge is able to decrease the serum concentrations of IgG and IgM in piglets ( Prates et al., 2021 Prates et al., 2021 ). Comparably, we found that LPS challenge decreased the plasma concentrations of IgM and lysozyme. Dramatically, dietary RBO supplementation could improve the immunological function against LPS challenge by increasing the plasma concentrations of IgA, IgM, β‍-defensin-1, and lysozyme in LPS-challenged piglets. Fragou et al. (2004) Fragou et al. (2004) also found that the tocopherol supplementation could boost the immune system development of piglets in the first two weeks after weaning. Gut microbiota dysbiosis can facilitate enteral bacterial infections and intestinal barrier function damage during the weaning transition of piglets ( Dou et al., 2017 Dou et al., 2017 ; Gresse et al., 2017 Gresse et al., 2017 ; Ren et al., 2022 Ren et al., 2022 ). The increased intestinal permeability and inflammation of early weaned piglets facilitate ROS accumulation in the intestinal lumen, which triggers gut microbiota dysbiosis ( Gresse et al., 2017 Gresse et al., 2017 ; Zeng et al., 2017 Zeng et al., 2017 ). Liu et al. (2021) Liu et al. (2021) found that tocopherols modulated the gut microbiota during mitigating colitis and protecting the intestinal barrier function in mice. Bao et al. (2021) Bao et al. (2021) found phenolic profile of jujube fruit subjected to gut microbiota fermentation and its antioxidant potential against ethyl carbamate-induced oxidative damage in vivo. In this study, we found that LPS challenge resulted in jejunal microbiota dysbiosis by reducing the alpha diversity and shifting the microbial structure, which was improved by dietary RBO supplementation in early weaned piglets. Trillions of microbes that inhabit the mammalian intestine are extremely oxygen-sensitive and therefore occupy separate ecological niches along the intestine lumen ( Albenberg et al., 2014 Albenberg et al., 2014 ; Frese et al., 2015 Frese et al., 2015 ). The accumulation of nitrate and ROS confers aerobic and facultative anaerobic microbes with growth superiority over obligate anaerobic bacteria in the inflamed intestine ( Winter et al., 2013 Winter et al., 2013 ; Yardeni et al., 2019 Yardeni et al., 2019 ). In this study, the intestinal oxidative stress induced by LPS challenge decreased the alpha diversity and shifted the structure of the jejunal microbiota, which were restored at least partly by dietary RBO supplementation. Taken together, dietary RBO supplementation may prevent the dysbiosis of jejunal microbiota by improving the intestinal inflammation and oxidative stress induced by LPS challenge. Ruminococcaceae as extremely oxygen-sensitive bacteria are able to degrade indigestible polysaccharides and produce SCFAs ( Shang et al., 2016 Shang et al., 2016 ). Erysipelotrichaceae, Erysipelotrichia, and Erysipelotrichales are positively correlated with fat/lipid metabolism ( Greiner and Bäckhed, 2011 Greiner and Bäckhed, 2011 ; Kaakoush, 2015 Kaakoush, 2015 ). Therefore, shifts in the relative abundance of these bacteria may be due to the oxidation of dietary fat/lipid by ROS in the inflamed jejunal lumen induced by LPS challenge ( Gresse et al., 2017 Gresse et al., 2017 ). Dietary RBO supplementation increased the functions of carbohydrate metabolism, excretory system, and xenobiotics biodegradation and metabolism, whereas it decreased the functions of folding, sorting and degradation, biosynthesis of other secondary metabolites, energy metabolism, environmental adaptation, and glycan biosynthesis and metabolism. Correspondingly, LPS challenge decreased the concentrations of fecal SCFAs including propionate and butyrate. On the contrary, compared with the LPS group, dietary RBO supplementation increased the concentrations of fecal SCFAs including acetate, propionate, and butyrate. There were significant associations between the characteristics of gut microbiota (taxon, diversity indices, and metabolites) shifted by dietary RBO supplementation and the indices of inflammation, immunity, oxidative stress, and jejunum morphology. Comparably, dietary antioxidants including resveratrol, quercetin, ferulic acid, vanillic acid, and pyrroloquinoline quinone could attenuate intestinal inflammation and oxidative damage in association with the modification of gut microbiota ( Huang et al., 2021 Huang et al., 2021 ; Qiu et al., 2021 Qiu et al., 2021 ; Xu et al., 2021a Xu et al., 2021a ; Hu et al., 2022 Hu et al., 2022 ). Overall, we demonstrated that dietary 0.02% RBO supplementation improves antioxidant capacity, intestinal damage, and immunity in association with the gut microbiota in LPS-challenged piglets. 5 Conclusions 1 Taken together, our findings suggested that dietary supplementation with RBO, the byproduct of rice processing, could improve growth performance and reduce LPS-induced intestinal damage, oxidative stress, and inflammation in early weaned piglets. These beneficial effects of dietary RBO supplementation occurred in association with the simultaneous modification of gut microbiota. 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This work was supported by the National Natural Science Foundation of China (Nos. 32230099 and 31925037), the Hubei Hongshan Laboratory (No. 2021hszd018), and the Yichun Dahaigui Life Science Co., Ltd. (No. 20222ZDH04093). We thank Yan lab’s members for helpful discussion and critical reading of the manuscript. 10.1631/jzus.B2200565 1673-1581（2023）05-0430-12 黄俊程,秦文峡,徐保阳等.日粮添加米糠油可增强断奶仔猪抵抗腹泻和脂多糖应激[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(05):430-441. 黄俊程,秦文峡,徐保阳等.日粮添加米糠油可增强断奶仔猪抵抗腹泻和脂多糖应激[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(05):430-441. This work was supported by the National Natural Science Foundation of China (Nos. 32230099 and 31925037), the Hubei Hongshan Laboratory (No. 2021hszd018), and the Yichun Dahaigui Life Science Co., Ltd. (No. 20222ZDH04093). 2022-11-07 2023-01-27 2023-05-15 2023-05-15 2023-04-14 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 37538393 Juncheng HUANG, Wenxia QIN, Baoyang XU, Haihui SUN, Fanghua JING, Yunzheng XU, Jianan ZHAO, Yuwen CHEN, Libao MA, and Xianghua YAN declare that they have no conflict of interest. Our animal experiments were conducted in accordance with the Animal Experimental Ethical Inspection of Laboratory Animal Centre, Huazhong Agriculture University, Wuhan, China (No. HZAUSW20210012). All institutional and national guidelines for the care and use of laboratory animals were followed. Compliance with ethics guidelines Supplementary information 1 Tables S1‒S4; Fig. S1 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 24