BACILLUS STRAINS ALLEVIATE STRESS IN BROILERS Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 alleviate corticosterone-induced oxidative stress and intestinal injury in broilers

BACILLUS STRAINS ALLEVIATE STRESS IN BROILERS Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 alleviate corticosterone-induced oxidative stress and intestinal injury in broilers | 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 BACILLUS STRAINS ALLEVIATE STRESS IN BROILERS Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 alleviate corticosterone-induced oxidative stress and intestinal injury in broilers Zhuoying Chen, Jingjing Huang, Wanqing Liang, Shu Tang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8964671/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigated the protective effects of Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 on broilers subjected to corticosterone-induced oxidative stress. A total of 102 one-day-old Arbor Acres broilers were randomly assigned to six treatment groups. Birds received drinking water containing either TS1 or B64 for three weeks, followed by subcutaneous corticosterone administration (4 mg/kg) to induce oxidative stress. Growth performance, antioxidant capacity, intestinal morphology, microbial composition, and expression of intestinal barrier and antioxidant-related genes and proteins were evaluated. Supplementation with TS1 and B64 significantly improved body weight gain compared with corticosterone-treated controls. Both strains enhanced serum total antioxidant capacity and reduced malondialdehyde, lactate dehydrogenase, and creatine kinase levels, indicating mitigation of oxidative damage. Histological analysis revealed that TS1 and B64 protected duodenal and jejunal villus structure and preserved mucosal integrity. 16S rRNA sequencing showed that corticosterone disrupted intestinal microbial balance, while both Bacillus strains restored microbial diversity and increased beneficial genera such as Lactobacillus and Akkermansia . At the molecular level, TS1 and B64 upregulated the transcription and expression of intestinal tight junction proteins (Claudin-1, Claudin-3, ZO-1, and Mucin-2), thereby enhancing barrier function. Both strains activated the KEAP1/NRF2 signaling pathway, evidenced by increased expression of NRF2, HO-1, and NQO1, and suppressed KEAP1 expression, suggesting improved antioxidant defense. Among the two strains, B64 exhibited slightly stronger regulatory effects on antioxidant and tight junction markers. In conclusion, Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 effectively alleviate corticosterone-induced oxidative stress and intestinal injury in broilers by modulating the intestinal microbiota, enhancing antioxidant capacity, and activating the KEAP1/NRF2 pathway. Supplementation with these Bacillus strains can improve oxidative resilience and intestinal health in broilers under stress, providing a potential alternative to antibiotics for promoting performance and gut integrity in commercial poultry production. Bacillus pumilus Bacillus amyloliquefaciens corticosterone oxidative stress broiler Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In poultry production, various stressors such as noise, handling, and transportation can trigger complex pathophysiological responses in birds, including increased aggression, feather-pecking behavior, reduced growth rate, impaired feed conversion, immune dysfunction, and disturbances in the intestinal microbiota. These effects not only compromise animal welfare but also lead to substantial economic losses in the broiler industry and, in severe cases, even mortality (NAWAB A, 2018). Therefore, developing effective strategies to mitigate stress is essential, and the use of feed additives represents one promising approach. For decades, antibiotics have served as the cornerstone of poultry production (Seven P, 2008). However, their excessive and indiscriminate use has led to ecological and environmental pollution, antimicrobial resistance, and significant economic concerns. Consequently, many countries, including China, have moved toward restricting or phasing out antibiotic use in animal feeds. Probiotics have emerged as a safe and effective alternative, these beneficial microorganisms help maintain intestinal microecological balance, enhance nutrient digestion and absorption, inhibit pathogenic colonization, and strengthen intestinal barrier function, thereby improving overall performance and health in poultry (De Filippis F, 2020; Vemuri R, 2017; Suez J, 2019). According to previous studies, corticosterone ( CORT ) is commonly used to establish a chronic stress model in animals ( Dallman M F, 1973; Costantini David, 2008). Oxidative stress results in the excessive generation of reactive oxygen species ( ROS ), which damage cellular structures and impair physiological functions. The enzymatic antioxidant defense system plays a central role in maintaining redox balance and protecting the body from oxidative injury. Total antioxidant capacity ( T-AOC ) serves as a key indicator reflecting the overall efficiency of the organism’s defense mechanisms against free radicals. Classic markers of oxidative damage include malondialdehyde ( MDA ), which reflects the degree of lipid peroxidation, and catalase ( CAT ), a major peroxisomal enzyme responsible for hydrogen peroxide decomposition. In addition, lactate dehydrogenase ( LDH ), a key glycolytic enzyme that indicates cellular membrane integrity, and creatine kinase ( CK ), whose serum concentration correlates with intestinal mucosal injury and microbial imbalance, were also measured. Therefore, these biochemical indicators were analyzed in the serum of broilers to evaluate the protective effects of Bacillus supplementation (MOOLI R G R, 2022; CHENG D Y, 2024; Altan O, 2003). Our previous research identified two probiotic strains, Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64, with promising biological functions. Feeding medium to high doses of Bacillus pumilus TS1 has been shown to effectively regulate key proteins such as Nuclear factor E2 related factor 2 ( Nrf2 ), p38 mitogen-activated pro-tein kinase( p38 ), mitogen-activated protein kinase,༈ MAPK ༉, Nuclear factor-kappa B,NF-κB༈ NF-κB ༉, and heme oxygenase-1 ( HO-1 ) within inflammatory signaling pathways, thereby enhancing antioxidant and anti-inflammatory responses. Meanwhile, Bacillus amyloliquefaciens has been reported to improve growth performance, enhance digestive enzyme activity, and strengthen immune function in broiler chickens (Liu Y, 2024; Zhu Yongming, 2023; RAMLUCKEN U, 2020; Bampidis V, 2021; Bampidis V, 2022). Building on these findings, the present study aimed to further investigate whether supplementation with Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 could alleviate corticosterone (CORT)-induced oxidative stress through modulation of the KEAP1/NRF2 antioxidant signaling pathway. Previous studies have demonstrated that CORT induces oxidative stress and apoptosis both in vivo and in vitro , leading to excessive free radical generation and cellular injury. In this experiment, Bacillus strains were administered via drinking water. Specifically, Bacillus pumilus TS1 (1.4 × 10⁷ CFU/mL), isolated from yak, and Bacillus amyloliquefaciens B64 (5.0 × 10⁷ CFU/mL), isolated from cattle, were used to colonize the intestinal tract of broilers. The intestinal microbiota plays a central role in maintaining host health by regulating nutrient metabolism, immune function, and intestinal homeostasis. Disruption of microbial balance can lead to bacterial translocation, intestinal barrier dysfunction, epithelial injury, and increased permeability, which amplify stress responses and mucosal damage (HEZT, 2022; CUI M X, 2020). To evaluate whether TS1 and B64 could mitigate such intestinal injury, this study analyzed intestinal villus height and crypt depth, assessed mucin production, and quantified key tight junction proteins, including occludin, claudin, and zonula occludens ( ZO-1 ), which are essential for maintaining epithelial barrier integrity (PARADIS T, 2021). Additionally, the KEAP1/NRF2 pathway, a critical endogenous antioxidant defense system, was examined. Activation of NRF2 and suppression of its negative regulator KEAP1 promote the transcription of cytoprotective genes such as HO-1 and NAD(P)H quinone oxidoreductase 1 ( NQO1 ), enhancing cellular antioxidant capacity and reducing oxidative injury (Xin X, 2024; Niu B, 2024; Shen Y, 2019). In summary, this study investigates the protective effects of Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 on the intestinal health of broilers, focusing on their ability to mitigate CORT-induced oxidative stress and elucidating the underlying KEAP1/NRF2-mediated mechanisms.And evaluate which of Bacillus subtilis and Bacillus amyloliquefaciens has stronger stress relieving ability. Materials and Methods Experimental design and animal preparation A total of 102 one-day-old Arbor Acres ( AA ) broiler chicks were obtained from Nanjing Te Awesome Planting Cooperative (Nanjing, China). Upon arrival, all birds were individually weighed, wing-banded for identification, and each group is randomly assigned to 3–4 cages to ensure equal average initial body weight among groups. The chicks were allowed a 7-day acclimation period before the start of the experiment, during which body weights were recorded at predetermined intervals. Birds were housed in environmentally controlled rooms equipped with wire-floor cages. The ambient temperature was maintained at 30 ± 3°C during the first week and gradually reduced to 25 ± 3°C thereafter. Relative humidity was controlled at 60–70%, and natural ventilation was provided daily to ensure adequate air exchange. Feed and water were available ad libitum throughout the trial. All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Nanjing Agricultural University (Nanjing, China). Bacillus colonization and CORT treatment Table 1 Experimental Groups As shown in Fig. 1, bacterial suspensions were prepared by dissolving the Bacillus cultures in distilled water to achieve the desired concentrations. Following a one-week adaptation period, birds were provided drinking water containing the designated bacterial preparations according to their respective treatment groups. A total of six groups were established for administration, The following groups are respectively(CON group、CON+CORT group、TS1 group、TS1 + CORT group、B64 group and B64 + CORT group). After three weeks of Bacillus supplementation, birds received subcutaneous injections of CORT powder (Sigma-Aldrich, St. Louis, MO, USA) dissolved in physiological saline with anhydrous ethanol as a solvent at a dose of 4 mg/kg body weight. The injections were administered once daily for seven consecutive days to induce oxidative stress. At the end of the treatment period, birds were fasted for 8 hours before sample collection. Sample collection Broiler chickens were euthanized by neck breakage.Blood samples were immediately collected, and serum was separated by centrifugation and stored at − 80°C until analysis. Subsequently, portions of major organs, including intestinal tissues, were divided into two parts: one was snap-frozen in liquid nitrogen and stored at − 80°C for biochemical and molecular analyses, while the other was fixed in 10% neutral-buffered formalin (Sinopharm Chemical Reagent Co., Shanghai, China) for histological examination. Enzyme-linked immunosorbent assay ( ELISA ) Serum levels of creatine kinase-MB (CK-MB), malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and total antioxidant capacity (T-AOC) were determined using commercial ELISA kits (Beyotime Biotechnology, Nanjing, China). All assays were performed in accordance with the manufacturer’s protocols. Measurements were conducted at the School of Veterinary Medicine, Nanjing Agricultural University (Nanjing, China), and absorbance was read using a microplate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland). Hematoxylin and eosin (H&E) staining Following euthanasia by carotid artery exsanguination, intestinal tissue samples were immediately collected and processed according to standard histological procedures. Fresh tissues were fixed in 10% neutral-buffered formalin (Sinopharm Chemical Reagent Co.) for 6–12 h, then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections of 4–5 µm thickness were mounted on glass slides. After deparaffinization and rehydration, the sections were stained with hematoxylin for 5 min, differentiated in acid alcohol, and blued in alkaline solution. Cytoplasmic counterstaining was performed with eosin for 1 min, followed by dehydration in graded ethanol, clearing in xylene, and sealing with neutral resin. Under microscopic examination, nuclei appeared blue-purple, whereas cytoplasm and collagen fibers appeared pink, allowing clear visualization of histological features. This classical and widely applied staining method enabled evaluation of pathological alterations and provided morphological evidence supporting experimental results. High-throughput analysis of microorganisms Intestinal contents from the six experimental groups were collected under sterile conditions and subjected to 16S rRNA gene next-generation sequencing. Total microbial DNA was extracted from each sample using a commercial DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA quality and concentration were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified by PCR (Eppendorf™, Model # AG 22331, Hamburg, Germany), and the amplicons were purified, quantified, and normalized prior to library construction. Sequencing libraries were prepared using the Illumina TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina NovaSeq 6000 platform. Raw sequence data were processed through quality control and paired-end read assembly to remove low-quality and chimeric sequences. Operational taxonomic units ( OTUs ) were clustered based on 97% sequence similarity and annotated using the SILVA reference database. Taxonomic classification and inter-group community comparisons were performed at both the phylum and genus levels. α-diversity indices, including Shannon, ACE, and Chao1, were calculated to evaluate microbial diversity among samples, and visualization of relative abundance distributions was generated in bar and petal plots for comparative analysis. Immunohistochemical assay Paraffin-embedded intestinal tissue sections were first incubated at 60°C for 1 h to melt residual paraffin, followed by deparaffinization in xylene and rehydration through a graded ethanol series. Antigen retrieval was carried out in preheated sodium citrate buffer (pH 6.0) using a water bath, after which the sections were rinsed three times with phosphate-buffered saline ( PBS ) for 5 min each. A hydrophobic barrier was drawn around the tissue using an immunohistochemical pen. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature, followed by three additional PBS washes. Non-specific binding was blocked with normal goat serum (Beyotime Biotechnology) for 10 min at room temperature. Sections were then incubated overnight at 4°C with the appropriately diluted primary antibodies prepared in antibody dilution buffer (P0023A, Beyotime Biotechnology). After washing with PBS, the slides were incubated with a biotinylated secondary antibody for 30 min at room temperature, followed by treatment with avidin–biotin complex solution and visualization using 3,3′-diaminobenzidine ( DAB ) chromogen. Counterstaining was performed with hematoxylin, followed by differentiation, bluing, dehydration, and mounting with neutral resin. Immunostaining was examined under a fluorescence microscope (Imager A2, Zeiss, Oberkochen, Germany). Brownish-yellow staining was interpreted as a positive reaction, whereas blue staining indicated cell nuclei. Quantitative real-time PCR (qPCR) Total RNA was extracted from approximately 50 mg of intestinal tissue using FreeZol Reagent (Novozymes, Nanjing, China) according to the manufacturer’s protocol. Briefly, 500 µL of reagent was added to each sample for lysis, followed by a 5 min incubation at room temperature. After centrifugation for 5 min, the supernatant was mixed with Dilution Buffer (5:1, v/v) and centrifuged for 15 min. The resulting supernatant was transferred to a new microcentrifuge tube, and an equal volume of isopropanol was added to precipitate RNA. Following a 10 min incubation at room temperature, the mixture was centrifuged again, and the pellet was washed with 1 mL of 75% ethanol, centrifuged for 3 min, air-dried, and dissolved in 20–100 µL of DEPC-treated water. RNA purity and concentration were determined spectrophotometrically using a NanoDrop 2000 (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from total RNA using a reverse transcription kit (CW3360, Kangwei Century, Taizhou, China). Quantitative PCR was performed using SYBR Green Master Mix (Qingke Biotechnology, Beijing, China) with gene-specific primers (Table 1) on a real-time PCR system (Eppendorf AG). The amplification program consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. Gene expression levels were determined based on Ct (threshold cycle) values, and relative expression was calculated using the 2 −ΔΔCt method, with GAPDH serving as the internal reference. Statistical analysis of gene expression data was conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). RESULTS Effects of Bacillus supplementation on body weight and serum antioxidant indices Figure 2 (A) Body weight change trend (B-F) Serum stress factors detected by ELISA: T-AOC, MDA, CAT, LDH, CK As shown in Fig. 2A, the body weight of broilers increased linearly during the experimental period. On day 14, birds in the TS1 group exhibited a significant increase in body weight compared with the control (P < 0.05), and this difference became highly significant on day 21 (P < 0.01). By day 35, both the CORT and control groups showed pronounced weight loss (P < 0.001), reflecting the inhibitory effect of stress on growth performance. In contrast, birds supplemented with TS1 maintained higher body weights, although a slight reduction was still observed (P < 0.05). Similarly, the TS1 + CORT and CON+CORT groups exhibited mild but significant decreases in body weight (P < 0.05). As illustrated in Figs. 2B–F, CORT administration markedly elevated serum MDA levels (P < 0.001) and significantly increased CAT, LDH, and CK activities (P < 0.05), indicating enhanced oxidative stress and tissue damage. Conversely, supplementation with TS1 or B64 effectively restored antioxidant balance. The TS1 + CORT and CON+CORT groups displayed significant increases in T-AOC (P < 0.01), accompanied by marked reductions in MDA and CAT levels (P < 0.01) and a strong suppression of CK activity (P < 0.001). Similarly, birds in the B64 + CORT and CON+CORT groups exhibited a highly significant enhancement of T-AOC (P < 0.001) and reduced MDA and CAT expression (P < 0.05). These findings indicate that both Bacillus strains enhanced the systemic antioxidant defense, mitigated oxidative damage, and improved overall physiological resilience under CORT-induced stress. Effects of Bacillus supplementation on intestinal morphology in CORT-treated broilers Figure 3 (A-B) HE-stained sections of duodenum and jejunum (C) Quantitative analysis of intestinal villi (D) Quantitative analysis of intestinal crypts (E) Statistical analysis of villus-to-crypt ratio. As shown in Fig. 3, supplementation with TS1 or B64 effectively mitigated intestinal structural damage caused by CORT exposure. In the duodenum, villus height was significantly greater in the TS1 + CORT and CON+CORT groups compared with the control (P < 0.05), while the B64 + CORT group exhibited a highly significant increase in villus length (P < 0.001). No notable differences were observed in crypt depth or the villus-to-crypt ratio among the duodenal samples. In the jejunum, however, CORT treatment resulted in a pronounced shortening of villi (P < 0.001) and a reduction in crypt size (P < 0.05) compared with the CON group, indicating structural injury due to oxidative stress. Supplementation with Bacillus strains significantly reversed these changes: both the CON+CORT and B64 + CORT groups displayed a marked increase in crypt depth (P < 0.01), restoring intestinal morphology toward normal levels. These results suggest that TS1 and B64 supplementation contributes to maintaining villus architecture and mucosal integrity in broilers under CORT-induced stress. Effects of Bacillus supplementation on cecum microbial diversity and community composition Figure 4 Cecal microbiota α-diversity analysis (A-C) Shannon, ACE, and Chao indices (D) Total number of microbial communities in each group (E) Microbial community analysis at the phylum level (F) Microbial community analysis at the genus level. As shown in Figs. 4A–D, α-diversity analysis of the cecum microbiota revealed distinct differences among the treatment groups. According to the Shannon index, microbial diversity in the CON and B64 groups was significantly lower than that in the CORT-treated groups (P < 0.001), while both the TS1 and B64 groups showed a marked reduction in microbial abundance compared with the latter (P < 0.01). Consistent patterns were observed in the ACE and Chao1 indices, where the number of microbial species in the CON and CON+CORT groups decreased significantly compared with the other treatments (P < 0.01), and the B64 group exhibited the lowest richness (P < 0.001). At the phylum level (Figs. 4E–F), Proteobacteria , Bacteroidetes , and Actinobacteria were dominant taxa in the intestinal communities. Their relative abundances were notably reduced in the TS1 + CORT and B64 + CORT groups compared with the CORT group, suggesting that Bacillus supplementation helped restore microbial balance disrupted by oxidative stress. At the genus level, CORT exposure led to increased proportions of Akkermansia and Lactobacillus , accompanied by a reduction in Erysipelotrichaceae species. Treatment with TS1 and B64 partially reversed these changes, indicating that both strains promoted a more favorable microbial structure conducive to intestinal health and resilience against CORT-induced dysbiosis. Effects of Bacillus supplementation on tight junction and antioxidant-related gene expression Figure 5 (A-C) Detection of mRNA expression levels of Claudin-3, ZO-1, Mucin2, Occludin, NRF2, HO-1, NQO1, SOD, CAT, and GSH-PX in the duodenal intestine by qPCR (D-F) mRNA transcription levels of Claudin-1, Claudin-5, ZO-1, Mucin2, NRF2, HO-1, NQO1, SOD, CAT, and GSH-PX in the jejunal intestine. As shown in Fig. 5A, supplementation with TS1 and B64 significantly enhanced the transcription of tight junction and mucin-related genes in the duodenum. Compared with the CORT and CON groups, ZO-1 expression was significantly upregulated (P < 0.05). In the TS1 + CORT group, Claudin-3 transcription increased (P < 0.05), while ZO-1 and Mucin-2 levels rose markedly (P < 0.01). Similarly, in the B64 + CORT group, Claudin-3 and Mucin-2 were significantly upregulated (P < 0.01), ZO-1 expression showed a strong increase (P < 0.001), and Occludin expression also rose (P < 0.05). Moreover, when comparing TS1 + CORT and B64 + CORT, the latter exhibited higher Claudin-3 expression (P < 0.01), indicating that B64 more effectively preserved tight junction integrity. Figures 5B and 5C demonstrate that CORT exposure markedly suppressed NRF2 transcription (P < 0.01) and downregulated antioxidant genes SOD and CAT (P < 0.05) in the duodenum. However, TS1 and B64 supplementation effectively counteracted these effects. In the TS1 + CORT group, HO-1 (P < 0.05), CAT (P < 0.001), and GSH-PX (P < 0.05) transcription levels significantly increased compared with CORT alone. In the B64 + CORT group, NRF2 and HO-1 expression were strongly upregulated (P < 0.001), while NQO1 and CAT also increased (P < 0.05), and SOD and GSH-PX levels rose significantly (P < 0.01). Notably, B64 supplementation produced greater increases in NRF2 (P < 0.05), HO-1, SOD, and CAT (P < 0.01) compared with TS1, suggesting superior antioxidant activation. As shown in Fig. 5D, similar patterns were observed in the jejunum. The TS1 + CORT group exhibited significantly higher Claudin-1, Claudin-5, and Mucin-2 transcription (P < 0.01) and increased ZO-1 expression (P < 0.05) compared with CORT. The B64 + CORT group also displayed elevated Claudin-5 and Mucin-2 (P < 0.05) and enhanced ZO-1 (P < 0.01). In Figs. 5E and 5F, CORT markedly suppressed NRF2 expression in the jejunum (P < 0.01), but both Bacillus strains reversed this effect. TS1 + CORT increased NRF2 (P < 0.01), HO-1 (P < 0.001), and NQO1 (P < 0.05), whereas B64 + CORT induced robust upregulation of NRF2, HO-1, NQO1, and CAT (P < 0.001), with additional increases in SOD and GSH-PX (P < 0.05). Compared with TS1 + CORT, the B64 + CORT group showed higher SOD and GSH-PX expression (P < 0.05) and a markedly greater rise in CAT transcription (P < 0.001). These findings indicate that both Bacillus strains enhanced intestinal barrier function and activated KEAP1/NRF2-mediated antioxidant defense, with B64 exhibiting a stronger regulatory effect. Immunohistochemical analysis of intestinal oxidative stress and junction protein expression Figure 6 Immunohistochemistry detection of NRF2 (A-B), KEAP1 (C-D), NQO1 (E-F), Claudin-1 (G-H), and Mucin2 (I-J) expression levels in the duodenum and jejunum, and Image J statistical analysis of the duodenum (K-M) and jejunum (L-N). As shown in Figs. 6A–N, immunohistochemical staining revealed clear differences in the expression patterns of oxidative stress–related and intestinal barrier–associated proteins among the treatment groups. In both the duodenum and jejunum, the CORT and CON groups exhibited lighter positive staining for NRF2、KEAP1、NQO1、Claudin-1 and Mucin-2, whereas TS1 and B64 supplementation markedly enhanced the intensity of these signals, indicating increased protein expression. Conversely, the KEAP1-positive staining was more intense in the CORT group, reflecting elevated KEAP1 expression, while it was noticeably lighter in the TS1 and B64 groups, suggesting that both strains suppressed KEAP1 expression and mitigated oxidative stress. Quantitative analysis using ImageJ further supported these observations. In the duodenum, CORT treatment led to a downward trend in NRF2 expression, a significant increase in KEAP1 (P < 0.01), and NQO1 declined compared with CON. In the jejunum, NRF2 expression decreased markedly (P < 0.001), while KEAP1 increased (P < 0.05)and a significant reduction in NQO1 (P < 0.01) compared with CON. Supplementation with TS1 + CORT reversed these effects, showing elevated NRF2 and KEAP1 levels in the duodenum (P < 0.05) and a highly significant increase in NRF2 in the jejunum (P < 0.001). Compared with TS1 + CORT, the B64 + CORT group displayed lower NRF2 expression in the duodenum (P < 0.05) but higher NRF2 levels in the jejunum (P < 0.05). Regarding intestinal barrier proteins, Claudin-1 expression in the duodenum tended to decrease in the CORT group but significantly increased in B64 + CORT (P < 0.05), accompanied by a marked rise in Mucin-2 expression (P < 0.01). Similarly, in the jejunum, CORT treatment reduced both Claudin-1 and Mucin-2 expression (P < 0.05), whereas Bacillus supplementation restored their levels. These results confirm that TS1 and B64 alleviated CORT-induced oxidative stress by downregulating KEAP1 and enhancing NRF2-mediated antioxidant activity while simultaneously reinforcing intestinal barrier integrity through upregulation of Claudin-1 and Mucin-2. DISCUSSION Body weight gain or loss serves as a direct indicator of the physiological and health status of broilers during experimental evaluation. Under oxidative stress, broilers typically exhibit reduced feed intake, decreased water consumption, and a lowered metabolic rate, all of which contribute to inhibited growth performance (Zhao G L, 2022;YANG X, 2010). Previous studies have demonstrated that Bacillus supplementation can enhance growth performance by improving nutrient utilization and maintaining intestinal integrity. In this study, consistent with these findings, the B64 group showed a significant increase in body weight as early as day 7, while the TS1 group exhibited notable gains on days 14 and 21, indicating that both strains exerted growth-promoting effects at different stages. Conversely, broilers in the CORT group experienced a pronounced reduction in body weight by day 35, confirming that CORT exposure induced anorexia and growth suppression. These results suggest that TS1 and B64 supplementation effectively counteracted the adverse effects of oxidative stress on growth performance, thereby supporting their potential use as functional probiotics to maintain productivity in stressed broilers. Intestinal oxidative stress increases the production of reactive oxygen species, impairing cellular function, disrupting the body’s antioxidant defense balance, and leading to ROS-induced damage. This process also results in the release of toxic metabolites into circulation, which alters the activity of key antioxidant markers such as CAT, SOD, and GSH-Px (Altan O, 2003;BAI K, 2018༛PETERSON L W, 2014). In the present study, subcutaneous administration of CORT significantly elevated serum MDA, CAT, and LDH levels, accompanied by an upward trend in T-AOC, confirming the activation of oxidative stress pathways. Following probiotic colonization with TS1 and B64, the levels of LDH and MDA declined markedly, whereas T-AOC significantly increased, indicating mitigation of oxidative damage. Gene transcription analysis further supported these findings: SOD, CAT, and GSH-Px expression decreased notably in the duodenum and jejunum of the CORT group but showed a clear recovery or upward trend in the TS1 + CORT and B64 + CORT groups. These results demonstrate that both TS1 and B64 effectively suppressed oxidative stress and enhanced the antioxidant defense system in CORT-induced broilers. Meanwhile, the antioxidant capacity of TS1 is stronger than that of B64.The pronounced antioxidant effects observed in this study are consistent with previous reports confirming the protective role of Bacillus species in reducing oxidative injury and improving redox homeostasis (SZETO H H, 2006; WANG Y, 2018). The intestine functions as a critical barrier that protects the host from bacterial toxins and other harmful agents. Structural parameters such as villus height, crypt depth, and the villus-to-crypt ratio are key morphological indicators of intestinal health. Longer villi provide a larger absorptive surface area, thereby improving feed utilization efficiency and nutrient absorption, ultimately supporting growth performance in broilers (CASPARY W F, 1992; Seppi M, 2023). In the present study, histopathological observations revealed that the intestinal tissue of the CON group displayed a well-organized architecture with intact mucosa, orderly epithelial alignment, and no signs of cellular degeneration or necrosis. In contrast, the CORT group exhibited marked histological alterations, including villus disruption in both the duodenum and jejunum, crypt atrophy, mucosal defects, and distorted epithelial morphology, hallmarks of stress-induced intestinal injury. Supplementation with TS1 and B64 notably alleviated these pathological changes. Both TS1 + CORT and B64 + CORT groups showed significant increases in duodenal villus height compared with CORT, while jejunal villi were also lengthened, although crypt depth in the duodenum remained unchanged. In the jejunum, CORT exposure caused a significant reduction in crypt size, whereas TS1 + CORT and B64 + CORT treatments restored crypt depth to near-normal levels. No significant difference was observed in the villus-to-crypt ratio. These findings suggest that colonization with TS1 and B64 effectively mitigated CORT-induced mucosal injury and maintained intestinal structural integrity, consistent with previous reports that Bacillus supplementation enhances gut morphology and barrier resilience under stress conditions (Liu Y, 2023). The intestinal microbiota plays a vital role in maintaining host nutrition, immunity, metabolism, and disease resistance. A stable microbial community is essential for intestinal homeostasis and optimal growth performance in animals, while a reduction in microbial diversity disrupts this balance and compromises gut health. As a primary target of oxidative stress, alterations in the intestinal microbiota are key indicators of redox imbalance. The intestinal microbial community is predominantly composed of Actinobacteria , Bacteroidetes , Bacteroidota , and Verrucomicrobiota (Zhao Y, 2020; Yaklai K, 2021; XU ZR, 2003; Liu JH,2020). Under stress conditions, the equilibrium of this ecosystem is disturbed, leading to compositional changes that adversely affect host physiology. The ratio of Firmicutes to Bacteroidetes , a well-established marker of intestinal health, has dual pathological significance: an elevated ratio is associated with metabolic dysfunction, while a decreased ratio indicates inflammatory injury. Bacillus species can help reestablish microbial balance by modulating the intestinal environment, forming a biological barrier, suppressing pathogenic colonization, and restoring microbial homeostasis (ZHANG B, 2011; GREINER T B, 2011; AMOAH K, 2020;GU Y F, 2022). In this study, α-diversity analysis revealed that microbial richness decreased in both the TS1 and B64 groups, suggesting that single-strain colonization modestly influenced the native microbial structure. However, a marked reduction in microbial abundance was observed in the CORT group, indicating that CORT disrupted gut microbial stability. At the phylum level, the CORT group exhibited increased proportions of Actinobacteria , Bacteroidota , and Proteobacteria , which are associated with dysbiosis, metabolic disturbances, and compromised mucosal integrity, potentially heightening the risk of inflammatory bowel conditions. Conversely, in the TS1 + CORT and B64 + CORT groups, the relative abundance of Actinobacteria and Bacteroidota declined, suggesting partial restoration of microbial equilibrium. The elevated abundance of Proteobacteria in these groups may represent a compensatory mechanism supporting epithelial repair and crypt regeneration, while the enrichment of Bacteroides , Verrucomicrobiota , and Actinomycetes contributed to microbiota stabilization (Yang H, 2019). Notably, the increase in Akkermansia in the TS1 + CORT and B64 + CORT groups is indicative of improved digestive capacity, reduced intestinal permeability, and enhanced anti-inflammatory activity, thereby strengthening the mucosal barrier and nutrient absorption (Huck O, 2020; Keshavarz Azizi Raftar S, 2021). The concurrent rise in Lactobacillus , a well-known probiotic genus, further supports intestinal homeostasis by inhibiting pathogenic bacteria and promoting intestinal motility (WANG Y, 2017). Meanwhile, the decline of Erysipelotrichaceae , an opportunistic pathogen within Firmicutes , implies reduced harmful bacterial proliferation and alleviation of intestinal stress, consistent with earlier findings. The distinct microbial patterns observed between the TS1 and B64 treatments may be attributed to inherent differences in their spore-forming properties and colonization dynamics. The intestinal epithelial barrier plays a crucial role in maintaining cellular integrity and selective permeability, thereby preventing the invasion of harmful substances, preserving microbial balance, and sustaining a stable environment for bacterial symbiosis. Tight junctions form the first line of defense within the intestinal epithelium and consist of transmembrane proteins (Occludin, Claudin), peripheral membrane scaffolding proteins (ZO-1), and mucin glycoproteins (Mucin-2). Occludin is a multifunctional transmembrane protein involved in intercellular adhesion and permeability regulation, while ZO-1, a cytoplasmic member of the zonula occludens family, links Claudin proteins to the actin cytoskeleton and plays an essential role in maintaining epithelial integrity, permeability, and cell differentiation (Saitou M, 1997; MEI M, 2016). Members of the Claudin family are integral components of tight junctions: Claudin-1 regulates epithelial permeability, Claudin-3 forms a continuous sealing layer that limits paracellular transport, and Claudin-5 tightens intercellular junctions in epithelial and endothelial tissues (Gong Y, 2016; SUZUKI K, 2023; AMASHEH S, 2005). Mucin-2, secreted by goblet cells, serves as a protective mucosal layer that strengthens barrier function, suppresses inflammation, and prevents pathogen invasion. Disruption of these proteins can increase mucosal permeability, compromise barrier integrity, and exacerbate oxidative stress, leading to inflammatory injury and impaired intestinal homeostasis (HE L Q, 2017; PARADIS T, 2021). Oxidative stress has also been shown to inhibit intestinal stem cell proliferation, disrupt tight junction continuity, and heighten susceptibility to intestinal inflammation. Probiotics and their metabolites, however, can enhance immune cell activity, support epithelial regeneration, and mitigate stress-induced damage (XU ZR, 2003; GRONDIN J A,2020). In the present study, transcriptional analysis revealed that the expression of Claudin-3, ZO-1, Mucin-2, and Occludin decreased in the duodenal CORT group, with Claudin-1 and Mucin-2 showing a downward trend. Similarly, in the jejunum, Claudin-1, Claudin-3, ZO-1, and Mucin-2 were downregulated, with Claudin-1 and Mucin-2 significantly reduced, indicating compromised barrier integrity and enhanced inflammatory susceptibility. Conversely, TS1 + CORT treatment increased the expression of Claudin-1 and Mucin-2 in both the duodenum and jejunum, while B64 + CORT supplementation significantly upregulated Mucin-2 expression in the duodenum. The consistent transcriptional and protein-level results confirm that TS1 and B64 effectively repaired intestinal barrier damage and restored mucosal function, At the same time, The repair ability of B64 in the duodenum is stronger than TS1, while the repair ability of the two in the jejunum is similar, findings that align with previous reports on the barrier-protective roles of Bacillus probiotics under oxidative stress conditions. The Keap1/Nrf2 signaling pathway is a pivotal endogenous defense mechanism against oxidative stress. Nrf2, a key transcription factor within this pathway, regulates the expression of antioxidant genes and is closely associated with SIRT3-mediated redox balance in the nicotinamide adenine dinucleotide-dependent deacetylase family, while Keap1 serves as its upstream negative regulator (Wang L, 2022; Xin X, 2024; Niu B, 2024). Under physiological conditions, Nrf2 and Keap1 remain bound in the cytoplasm, maintaining redox homeostasis. When oxidative stress occurs, Nrf2 dissociates from Keap1 and translocates into the nucleus, where it activates the transcription of downstream antioxidant genes such as HO-1 and NQO1, thereby restoring the cellular oxidative–antioxidant equilibrium (Shen Y,2019;Xin X,2024;Niu B,2024). In this study, CORT administration suppressed the transcription and protein expression of Nrf2 and NQO1 in both the duodenum and jejunum, reduced HO-1 transcription, and increased Keap1 expression, indicating excessive oxidative stress and impaired antioxidant signaling. However, supplementation with TS1 + CORT and B64 + CORT reversed these effects. Both treatments upregulated the transcription and expression of Nrf2 and NQO1, elevated HO-1 levels, and inhibited Keap1 expression. These results demonstrate that colonization with TS1 and B64 effectively reactivated the Keap1/Nrf2 signaling pathway, thereby mitigating CORT-induced intestinal oxidative injury.,two types of Bacillus have comparable stress relieving abilities.The findings are consistent with previous studies highlighting the capacity of Bacillus species to modulate Nrf2-mediated antioxidant defenses and protect intestinal integrity under stress conditions. CONCLUSION In this study, both Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 promoted growth performance in broiler chickens, with TS1 exerting a more pronounced effect. Under CORT-induced stress, TS1 notably improved body weight gain, and both strains demonstrated strong antioxidant properties. Supplementation with TS1 and B64 positively influenced intestinal morphology by increasing villus height and crypt depth while maintaining a balanced villus-to-crypt ratio. Both strains also modulated the intestinal microbiota by suppressing harmful bacteria, enhancing the abundance of beneficial genera such as Lactobacillus, and optimizing the relative proportion of Bacteroidetes. Furthermore, TS1 and B64 enhanced the transcription and expression of key intestinal barrier proteins, including Claudin, ZO-1, Occludin, and Mucin-2, thereby reinforcing epithelial integrity. Under CORT-induced oxidative stress, the KEAP1/NRF2 signaling pathway was activated, and Bacillus supplementation significantly enhanced antioxidant defenses by upregulating NRF2 and its downstream targets (HO-1 and NQO1) while suppressing KEAP1 expression. Overall, Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 exerted marked protective and antioxidant effects against CORT-induced intestinal injury, overall, TS1 has stronger antioxidant capacity than B64suggesting their potential as effective probiotic feed additives for improving intestinal health and oxidative resilience in broiler production, and further research on related oxidative stress pathways can be conducted to improve its function Declarations Ethics Statement This study was approved by the Ethics Committee of the College of Arts, Univeristy of College of Veterinary Medicine, Nanjing Agricultural University.All procedures performed in this study involving human participants were conducted in accordance with the ethical standards of the institutional research committee . Consent to Publish All participants provided written consent for the publication of anonymised data collected during the study. No identifiable information about participants will be disclosed in this publication. Consent for publication of case study details, including anonymised narratives, was obtained from all individuals involved. Informed consent was obtained from all individual participants included in the study. Participants were provided with detailed information about the study objectives and procedures prior to their voluntary consent. Data availability statements The datasets generated and analysed during the current study are not publiclyavailable due to the experiment is not yet fully completed and is still in the confidential stage but are available fromthe corresponding author on reasonable request. Conflicts of Interest The authors declare that they have no conflict of interest regarding this research. Author Contribution Chen zhuoying wrote the main manuscript text and Hang jingjing and Liang wanqing prepared figures 1-3. All authors reviewed the manuscript Acknowledgments This work was supported by Natural Science Foundation of Ningxia Province (2023AAC05052);and National Natural Science Foundation of China (grant number 31602027). The funding body did not play a role in the design, analysis, and reporting of the study, but did provide financial support. Data Availability The datasets generated and analysed during the current study are not publiclyavailable due to the experiment is not yet fully completed and is still in the confidential stage but are available fromthe corresponding author on reasonable request. References AMASHEH, S,SCHMIDT T,MAHN, M et al (2005) Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells[J]. Cell Tissue Res 321(1):89–96 AMOAH K,DONG X H,TAN B, P et al (2020) Administration of probiotic Bacillus licheniformis induces growth,immune and antioxidant enzyme activities,gut microbiota assembly and resistance to Vibrio parahaemolyticus in Litopenaeus vannamei[J]. Aquaculture Nutr 26(5):1604–1622 Altan O, Pabuçcuoğlu A, Altan A, Konyalioğlu S, Bayraktar H (2003) Effect of heat stress on oxidative stress, lipid peroxidation and some stress parameters in broilers. Br Poult Sci 44:545–550 BAI K, FENG C, JIANG L et al (2018) Dietary effects of Bacillus subtilis fmbj on growth performance, small intestinal morphology, and its antioxidant capacity of broilers[J]. Poult Sci 97(7):2312–2321 Bampidis V, Azimonti G, Bastos ML et al (2021) Safety and efficacy of a feed additive consisting of Bacillus subtilis strains CNCM I-4606, CNCM I-5043 and CNCM I-4607 and Lactococcus lactis CNCM I-4609 for all animal species (Nolivade)[J]. EFSA J 19(11):e6907 Bampidis V, Azimonti G, Bastos ML et al (2022) Efficacy of the feed additive consisting of Bacillus velezensisNRRL B-67259 (Correlink ABS1781) as a feed additive for all growing poultry species (Elanco GmbH)[J]. EFSA J 20(12):e7711 CHENG D Y, ZHANG M, ZHENG Y Z et al α-Ketoglutarate prevents hyperlipidemia-induced fatty liver mitochondrial dysfunction and oxidative stress by activating the AMPK-pgc-1α/Nrf2 pathway[J]. Redox Biology 2024, 74: 103230 CASPARY W F Physiology and pathophysiology of intestinal absorption[J]. Am J Clin Nutr 1992, 55(Suppl.1):299S–308S CUI M X,ZHANG M,WU J, W et al Marine polysaccharides from Gelidium pacificum Okamura and Cereus sinensis reveal prebiotic functions[J]. Int J Biol Macromol 2020, 164:4 381–4390 Costantini David;;Fanfani Alberto;;Dell'omo Giacomo.Effects of corticosteroids on oxidative damage and circulating carotenoids in captive adult kestrels (Falco tinnunculus).[J].Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology.2008,178(7). Dallman M F;;Jones M T.Corticosteroid feedback control of ACTH secretion: effect of stress-induced corticosterone ssecretion on subsequent stress responses in the rat.[J].Endocrinology.1973(5). De Filippis F, Pasolli E, Ercolini D (2020) The food-gut axis: lactic acid bacteria and their link to food, the gut microbiome and human health[J]. FEMS Microbiol Rev 44(4):454–489 GU Y F,CHEN Y P,JIN, R et al Dietary chitooligosaccharide supplementation alleviates intestinal barrier damage,and oxidative and immunological stress in lipopolysaccharide-challenged laying hens[J]. Poult Sci 2022, 101(4):101701 Gong Y, Li H, Li Y (2016) Effects of Bacillus subtilis on Epithelial Tight Junctions of Mice with Inflammatory Bowel Disease[J]. J Interferon Cytokine Res 36(2):75–85 GRONDIN J A,KWON Y H,FAR P, M et al (2020) Mucins in intestinal mucosal defense and inflammation:learning from clinical and experimental studies[J]. Front Immunol 11:2054 GREINER T, BCKHED F (2011) Effects of the gut microbiota on obesity and glucose homeostasis[J].Trends in Endocrinology and Metabolism,. 22:117–123 HE L Q,ZHOU X H,HUANG, N et al Administration of alpha-ketoglutarate improves epithelial restitution under stress injury in early-weaning piglets[J].Oncotarget.2017,8(54):91965–91978 Huck O, Mulhall H, Rubin G et al (2020) Akkermansia muciniphila reduces Porphyromonas gingivalis-induced inflammation and periodontal bone destruction [. J] J Clin Periodontol 47(2):202–212 HEZT,LIYJ,XIONG T D etal.Effectofdietary resveratrolsupplementationongrowthperformance,antioxidant capacity,intestinal immunity and gut microbiota in Yellow-feathered broilers challenged with lipopolysaccharide[J]. Front Microbiol 2022, 13:977087 Keshavarz Azizi Raftar S, Hoseini Tavassol Z, Amiri M et al (2021) Assessment of fecal Akkermansia muciniphila in patients with osteoporosis and osteopenia:a pilot study [J]. J Diabetes Metab Disord 20(1):279–284 Liu JH, Yue T, Luo ZW et al (2020) Akkermansia muciniphila promotes type H vessel formation and bone fracture healing by reducing gut permeability and inflammation [J]. Dis Model Mech 13(11):dmm043620 Liu Y, Liu S, Wan S, Li Z, Li H, Tang S (2024) Anti-inflammatory properties of Bacillus pumilus TS1 in lipopolysaccharide-induced inflammatory damage in broilers. Animal Biotechnology, 35(1). doi.10.1080/10495398.2024.2418516 Liu Y, Xie TT, Yin LT et al (2023) Effect of canthaxanthin on antioxidant capacity and small intestinal mucosal structure of Huaixiang chickens under heat stress[J]. Chin J Veterinary Sci 43(10):2171–2180 MEI M,XIANG R L,YU G, Y et al (2016) Research progress in regulation of tight junction of pro-inflammatory factors [J]. Progress Physiological Sci 47(1):27–32 MOOLI R G R, MUKHI D, RAMAKRISHNAN S K (2022) Oxidative stress and redox signaling in the pathophysiology of liver diseases [J]. Compr Physiol 12(2):3167–3192 Niu B, Feng YX,Cheng X et al (2024) The alleviative effects of viable and inactive Lactobacillus paracasei CCFM1120 against alcoholic liver disease via modulation of gut microbiota and the Nrf2/HO-1 and TLR4/MyD88/NF-κB pathways[J]. Food Funct 15(17):8797–8809 NAWAB A,IBTISHAM F,LI, G et al Heat stress in poultry production:Mitigation strategies to overcome the future challenges facing the global poultry industry[J]. J Therm Biology 2018, 78:131–139 PETERSON L W ARTISD (2014) Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J]. Nat Rev Immunol 14(3):141–153 PARADIS, T,BÈGUE H,BASMACIYAN, L et al (2021) Tight junctions as a key for pathogens invasion in intestinal epithelial cells[J]. Int J Mol Sci 22(5):2506 RAMLUCKEN U,RAMCHURAN S O,MOONSAMY, G et al Production and stability of a multi-strain Bacillus based probiotic product for commercial use in poultry[J].Biotechnology Reports.2020,29(prepublish):e00575 SaitouM AndoAkatsukaY, ItohM, etal.Mammalianoccludininepithelialcells:itsexpressionandsubcellulardistribution.EurJCellBiol.1997,73:222231 Seppi M, Pasqualini J, Facchin S et al (2023) Emergent functional organization of gut microbiomes in health and diseases[J].Biomolecules. 14(1):5 Seven PT (2008) Sevenb I.Effect of dietary turkish propolis as alternative to antibiotic on performance and digestibility in broilers exposed to heat stress[J]. J Appl Anim Res 34(2):193–196 Suez J, Zmora N, Segal E et al The pros, cons, and many unknowns of probiotics[J]. Nat Med 2019, 25(5):716–729 Shen Y, Liu X, Shi J et al (2019) Involvement of Nrf2 in myocardialischemia and reperfusion injury[J]. Int J Biol Macromol 125(1):496–502 SZETO H H Mitochondria-targeted peptide antioxidants:novel neuroprotective agents [J].AAPS J.2006,8(3):E521–E531 SUZUKI K, YAMAGA K, TOKUMASU R et al Double mutation of claudin-1 and claudin-3 causes alopecia in infant mice[J]. Ann N Y Acad Sci 2023, 1523(1): 51–61 Vemuri R, Gundamaraju R, Eri R (2017) Role of Lactic Acid Probiotic Bacteria in IBD[J]. Curr Pharm Design 23(16):2352–2355 WANG, Y,WU Y P,WANG Y, Y et al Antioxidant properties of probiotic bacteria[J].Nutrients.2017,9(5):521 WANG, Y, BRANICKY R,NOEA et al Superoxide dismutases:dual roles in controlling Ros damage and regulating ROssignalingUl. Cell Biol 2018, 217(6):1915–1928 Wang L (2022) He C.Nrf2-mediated anti-inflammatory polarization of macrophages as therapeutic targets for osteoarthritisU].Front lmmunol.13:967193 XU, ZR,HU CH,XIA, MS et al (2003) Effects of dietary fructooligosaccharide on digestive enzyme activities,intestinal microflora and morphology of male broilers[J]. Poult Sci 82(6):1030–1036 XU, ZR,HU CH,XIA, MS et al (2003) Effects of dietary fructooligosaccharide on digestive enzyme activities,intestinal microflora and morphology of male broilers[J]. Poult Sci 82(6):1030–1036 Xin X, Liu J, Liu X et al (2024) Melatonin-Derived Carbon Dots with Free Radical Scavenging Property for Effective Periodontitis Treatment via the Nrf2/HO-1 Pathway[J]. ACS Nano 18(11):8307–8324 Yaklai K, Pattanakuhar S, Chattipakorn N et al (2021) The role of acupuncture on the gut-brain-microbiota axis in irritable bowel syndrome[J]. Am J Chin Med 49(2):285–314 Yang H, Yu HB, Bhinder G et al TLR9 limits enteric antimicrobial responses and promotes microbiota-based colonisation resistance during Citrobacter rodentium infection[J]. Cell Microbiol 2019, 21(7):e13026 YANG X, ZHANG B, GUO Y et al (2010) Effects of dietary lipids and Clostridium butyricum on fat deposition and meat quality of broiler chickens[J]. Poult Sci 89:254–260 ZHANG B, YANG X, GUO Y et al Effects of dietary lipids and Clostridium butyricum on serum lipids and lipid-related gene expression in broiler chickens[J].Animal.2011, 5:1909–1915 Zhu Yongming F, Jinmin S, Yuzhuo (2023) etc The effect of fermentation broth of Bacillus amyloliquefaciens B-4M-6 from chickens on growth performance, immune function, and intestinal health of broiler chickens [J]. Feed Research. 46 (03): 36–41. 10.13557/j.cnki.issn1002-2813.2023.008 Zhao Y Zhang X.Interactions of tea polyphenols with intestinal microbiota and their implication for anti-obesity [. J] J Sci Food Agric 2020, 100(3):897–903. 10.1002/jsfa.10049 Zhao GL, Niu Y, Zhang RQ et al Effects of yeast culture on growth performance, immune function and cecal microbiota of yellow-feathered broilers[J]. Chin J Anim Nutr 2022, 34(12): 7701–7710 (in Chinese with English abstract). Tables Table 1 is not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-8964671","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597783966,"identity":"f691658b-03ec-446a-b232-ec6a84ab14fb","order_by":0,"name":"Zhuoying Chen","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhuoying","middleName":"","lastName":"Chen","suffix":""},{"id":597783967,"identity":"4583d6d8-cc30-4d5c-80b1-47db3ca59272","order_by":1,"name":"Jingjing Huang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Huang","suffix":""},{"id":597783968,"identity":"34e12bd6-1cec-45c1-8123-847124e4c172","order_by":2,"name":"Wanqing Liang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wanqing","middleName":"","lastName":"Liang","suffix":""},{"id":597783969,"identity":"83a201c6-1983-4f81-9c88-4d0cd4bf0110","order_by":3,"name":"Shu Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYLACxgYGOQaGAyAmM2HVPFAtxqRrSWyA8InQYs9+9vDLnzsOp29nPPxMgqHCOrGB/ewB/Lbw5KVZ8545nLuz4ZiZBMOZ9MQGnrwEAg7LMTNmbDucu+HAATMJICOxQYLHAL8W/jdmhj/bDqcbHDj+TYLxHzFaJHKMH/C2HU4wOHAGaEsDMVpuvDFj5m1LN9xw4EyxRcKxdOM2nhz8Wtj7c4w//myzlje4cXzjjQ811rL97GfwawECNgkwJXGAgSEBxCWkHgiYP4Ap/gYi1I6CUTAKRsGIBAAVmEfVicmmkQAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shu","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2026-02-25 07:40:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8964671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8964671/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104402840,"identity":"d9cc26bc-bde4-4986-8be9-8d5b201e1496","added_by":"auto","created_at":"2026-03-11 12:16:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1005773,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/e07af906e409ac6715d7b625.png"},{"id":104071715,"identity":"bacdb4bf-76b1-4b5b-81ce-491372768ea1","added_by":"auto","created_at":"2026-03-06 11:57:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3193686,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/645d26b3b535bcc041d62299.png"},{"id":104071718,"identity":"23218d0a-4ceb-42e5-b206-96f29f26cd9c","added_by":"auto","created_at":"2026-03-06 11:57:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55126718,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/9b711e05fd4d53f942da6e46.png"},{"id":104403401,"identity":"5465d26e-a1ba-4ca2-9c3d-0a281302247e","added_by":"auto","created_at":"2026-03-11 12:18:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4330287,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/e6397ef8b1b315cc31e37417.png"},{"id":104071712,"identity":"c0959a1f-256d-4ea0-903a-74a55746efa7","added_by":"auto","created_at":"2026-03-06 11:57:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6458680,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/45eaa35fa8bc086ab9ed77a2.png"},{"id":104071716,"identity":"fb03c3ee-f9cd-4b28-ae0e-1ae24656bc99","added_by":"auto","created_at":"2026-03-06 11:57:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9449150,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/57be67c7007c2dfb7a2de8d1.png"},{"id":104403738,"identity":"c2b3957e-2243-4d69-920f-4100fd461cb3","added_by":"auto","created_at":"2026-03-11 12:18:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5028998,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/f29a090797543078cdb60320.png"},{"id":104409498,"identity":"44a95083-0ca4-46cf-a5ac-43c8b8a0ca8c","added_by":"auto","created_at":"2026-03-11 12:45:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":84707699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8964671/v1/239ca0e4-acff-43b6-a95a-f7bbf18e1827.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"BACILLUS STRAINS ALLEVIATE STRESS IN BROILERS Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 alleviate corticosterone-induced oxidative stress and intestinal injury in broilers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn poultry production, various stressors such as noise, handling, and transportation can trigger complex pathophysiological responses in birds, including increased aggression, feather-pecking behavior, reduced growth rate, impaired feed conversion, immune dysfunction, and disturbances in the intestinal microbiota. These effects not only compromise animal welfare but also lead to substantial economic losses in the broiler industry and, in severe cases, even mortality (NAWAB A, 2018). Therefore, developing effective strategies to mitigate stress is essential, and the use of feed additives represents one promising approach. For decades, antibiotics have served as the cornerstone of poultry production (Seven P, 2008). However, their excessive and indiscriminate use has led to ecological and environmental pollution, antimicrobial resistance, and significant economic concerns. Consequently, many countries, including China, have moved toward restricting or phasing out antibiotic use in animal feeds. Probiotics have emerged as a safe and effective alternative, these beneficial microorganisms help maintain intestinal microecological balance, enhance nutrient digestion and absorption, inhibit pathogenic colonization, and strengthen intestinal barrier function, thereby improving overall performance and health in poultry (De Filippis F, 2020; Vemuri R, 2017; Suez J, 2019).\u003c/p\u003e \u003cp\u003eAccording to previous studies, corticosterone (\u003cb\u003eCORT\u003c/b\u003e) is commonly used to establish a chronic stress model in animals ( Dallman M F, 1973; Costantini David, 2008). Oxidative stress results in the excessive generation of reactive oxygen species (\u003cb\u003eROS\u003c/b\u003e), which damage cellular structures and impair physiological functions. The enzymatic antioxidant defense system plays a central role in maintaining redox balance and protecting the body from oxidative injury. Total antioxidant capacity (\u003cb\u003eT-AOC\u003c/b\u003e) serves as a key indicator reflecting the overall efficiency of the organism\u0026rsquo;s defense mechanisms against free radicals. Classic markers of oxidative damage include malondialdehyde (\u003cb\u003eMDA\u003c/b\u003e), which reflects the degree of lipid peroxidation, and catalase (\u003cb\u003eCAT\u003c/b\u003e), a major peroxisomal enzyme responsible for hydrogen peroxide decomposition. In addition, lactate dehydrogenase (\u003cb\u003eLDH\u003c/b\u003e), a key glycolytic enzyme that indicates cellular membrane integrity, and creatine kinase (\u003cb\u003eCK\u003c/b\u003e), whose serum concentration correlates with intestinal mucosal injury and microbial imbalance, were also measured. Therefore, these biochemical indicators were analyzed in the serum of broilers to evaluate the protective effects of Bacillus supplementation (MOOLI R G R, 2022; CHENG D Y, 2024; Altan O, 2003).\u003c/p\u003e \u003cp\u003eOur previous research identified two probiotic strains, \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64, with promising biological functions. Feeding medium to high doses of \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 has been shown to effectively regulate key proteins such as Nuclear factor E2 related factor 2 (\u003cb\u003eNrf2\u003c/b\u003e), p38 mitogen-activated pro-tein kinase(\u003cb\u003ep38\u003c/b\u003e), mitogen-activated protein kinase,༈\u003cb\u003eMAPK\u003c/b\u003e༉, Nuclear factor-kappa B,NF-κB༈\u003cb\u003eNF-κB\u003c/b\u003e༉, and heme oxygenase-1 (\u003cb\u003eHO-1\u003c/b\u003e) within inflammatory signaling pathways, thereby enhancing antioxidant and anti-inflammatory responses. Meanwhile, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e has been reported to improve growth performance, enhance digestive enzyme activity, and strengthen immune function in broiler chickens (Liu Y, 2024; Zhu Yongming, 2023; RAMLUCKEN U, 2020; Bampidis V, 2021; Bampidis V, 2022). Building on these findings, the present study aimed to further investigate whether supplementation with \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64 could alleviate corticosterone (CORT)-induced oxidative stress through modulation of the KEAP1/NRF2 antioxidant signaling pathway.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that CORT induces oxidative stress and apoptosis both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, leading to excessive free radical generation and cellular injury. In this experiment, Bacillus strains were administered via drinking water. Specifically, \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 (1.4 \u0026times; 10⁷ CFU/mL), isolated from yak, and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64 (5.0 \u0026times; 10⁷ CFU/mL), isolated from cattle, were used to colonize the intestinal tract of broilers. The intestinal microbiota plays a central role in maintaining host health by regulating nutrient metabolism, immune function, and intestinal homeostasis. Disruption of microbial balance can lead to bacterial translocation, intestinal barrier dysfunction, epithelial injury, and increased permeability, which amplify stress responses and mucosal damage (HEZT, 2022; CUI M X, 2020).\u003c/p\u003e \u003cp\u003eTo evaluate whether TS1 and B64 could mitigate such intestinal injury, this study analyzed intestinal villus height and crypt depth, assessed mucin production, and quantified key tight junction proteins, including occludin, claudin, and zonula occludens (\u003cb\u003eZO-1\u003c/b\u003e), which are essential for maintaining epithelial barrier integrity (PARADIS T, 2021). Additionally, the KEAP1/NRF2 pathway, a critical endogenous antioxidant defense system, was examined. Activation of NRF2 and suppression of its negative regulator KEAP1 promote the transcription of cytoprotective genes such as HO-1 and NAD(P)H quinone oxidoreductase 1 (\u003cb\u003eNQO1\u003c/b\u003e), enhancing cellular antioxidant capacity and reducing oxidative injury (Xin X, 2024; Niu B, 2024; Shen Y, 2019).\u003c/p\u003e \u003cp\u003eIn summary, this study investigates the protective effects of \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64 on the intestinal health of broilers, focusing on their ability to mitigate CORT-induced oxidative stress and elucidating the underlying KEAP1/NRF2-mediated mechanisms.And evaluate which of Bacillus subtilis and Bacillus amyloliquefaciens has stronger stress relieving ability.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and animal preparation\u003c/h2\u003e \u003cp\u003eA total of 102 one-day-old Arbor Acres (\u003cb\u003eAA\u003c/b\u003e) broiler chicks were obtained from Nanjing Te Awesome Planting Cooperative (Nanjing, China). Upon arrival, all birds were individually weighed, wing-banded for identification, and each group is randomly assigned to 3\u0026ndash;4 cages to ensure equal average initial body weight among groups. The chicks were allowed a 7-day acclimation period before the start of the experiment, during which body weights were recorded at predetermined intervals. Birds were housed in environmentally controlled rooms equipped with wire-floor cages. The ambient temperature was maintained at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C during the first week and gradually reduced to 25\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C thereafter. Relative humidity was controlled at 60\u0026ndash;70%, and natural ventilation was provided daily to ensure adequate air exchange. Feed and water were available ad libitum throughout the trial. All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Nanjing Agricultural University (Nanjing, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacillus colonization and CORT treatment\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;1 Experimental Groups\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;1, bacterial suspensions were prepared by dissolving the Bacillus cultures in distilled water to achieve the desired concentrations. Following a one-week adaptation period, birds were provided drinking water containing the designated bacterial preparations according to their respective treatment groups. A total of six groups were established for administration, The following groups are respectively(CON group、CON+CORT group、TS1 group、TS1\u0026thinsp;+\u0026thinsp;CORT group、B64 group and B64\u0026thinsp;+\u0026thinsp;CORT group). After three weeks of Bacillus supplementation, birds received subcutaneous injections of CORT powder (Sigma-Aldrich, St. Louis, MO, USA) dissolved in physiological saline with anhydrous ethanol as a solvent at a dose of 4 mg/kg body weight. The injections were administered once daily for seven consecutive days to induce oxidative stress. At the end of the treatment period, birds were fasted for 8 hours before sample collection.\u003c/p\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eBroiler chickens were euthanized by neck breakage.Blood samples were immediately collected, and serum was separated by centrifugation and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Subsequently, portions of major organs, including intestinal tissues, were divided into two parts: one was snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for biochemical and molecular analyses, while the other was fixed in 10% neutral-buffered formalin (Sinopharm Chemical Reagent Co., Shanghai, China) for histological examination.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnzyme-linked immunosorbent assay (\u003c/em\u003e \u003cb\u003eELISA\u003c/b\u003e \u003cem\u003e)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSerum levels of creatine kinase-MB (CK-MB), malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and total antioxidant capacity (T-AOC) were determined using commercial ELISA kits (Beyotime Biotechnology, Nanjing, China). All assays were performed in accordance with the manufacturer\u0026rsquo;s protocols. Measurements were conducted at the School of Veterinary Medicine, Nanjing Agricultural University (Nanjing, China), and absorbance was read using a microplate reader (Infinite M200 Pro, Tecan, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n\u003ch3\u003eHematoxylin and eosin (H\u0026E) staining\u003c/h3\u003e\n\u003cp\u003eFollowing euthanasia by carotid artery exsanguination, intestinal tissue samples were immediately collected and processed according to standard histological procedures. Fresh tissues were fixed in 10% neutral-buffered formalin (Sinopharm Chemical Reagent Co.) for 6\u0026ndash;12 h, then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections of 4\u0026ndash;5 \u0026micro;m thickness were mounted on glass slides. After deparaffinization and rehydration, the sections were stained with hematoxylin for 5 min, differentiated in acid alcohol, and blued in alkaline solution. Cytoplasmic counterstaining was performed with eosin for 1 min, followed by dehydration in graded ethanol, clearing in xylene, and sealing with neutral resin. Under microscopic examination, nuclei appeared blue-purple, whereas cytoplasm and collagen fibers appeared pink, allowing clear visualization of histological features. This classical and widely applied staining method enabled evaluation of pathological alterations and provided morphological evidence supporting experimental results.\u003c/p\u003e\n\u003ch3\u003eHigh-throughput analysis of microorganisms\u003c/h3\u003e\n\u003cp\u003eIntestinal contents from the six experimental groups were collected under sterile conditions and subjected to 16S rRNA gene next-generation sequencing. Total microbial DNA was extracted from each sample using a commercial DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer\u0026rsquo;s instructions. DNA quality and concentration were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The V3\u0026ndash;V4 hypervariable regions of the bacterial 16S rRNA gene were amplified by PCR (Eppendorf\u0026trade;, Model # AG 22331, Hamburg, Germany), and the amplicons were purified, quantified, and normalized prior to library construction. Sequencing libraries were prepared using the Illumina TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina NovaSeq 6000 platform. Raw sequence data were processed through quality control and paired-end read assembly to remove low-quality and chimeric sequences. Operational taxonomic units (\u003cb\u003eOTUs\u003c/b\u003e) were clustered based on 97% sequence similarity and annotated using the SILVA reference database. Taxonomic classification and inter-group community comparisons were performed at both the phylum and genus levels. α-diversity indices, including Shannon, ACE, and Chao1, were calculated to evaluate microbial diversity among samples, and visualization of relative abundance distributions was generated in bar and petal plots for comparative analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical assay\u003c/h2\u003e \u003cp\u003eParaffin-embedded intestinal tissue sections were first incubated at 60\u0026deg;C for 1 h to melt residual paraffin, followed by deparaffinization in xylene and rehydration through a graded ethanol series. Antigen retrieval was carried out in preheated sodium citrate buffer (pH 6.0) using a water bath, after which the sections were rinsed three times with phosphate-buffered saline (\u003cb\u003ePBS\u003c/b\u003e) for 5 min each. A hydrophobic barrier was drawn around the tissue using an immunohistochemical pen. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature, followed by three additional PBS washes. Non-specific binding was blocked with normal goat serum (Beyotime Biotechnology) for 10 min at room temperature. Sections were then incubated overnight at 4\u0026deg;C with the appropriately diluted primary antibodies prepared in antibody dilution buffer (P0023A, Beyotime Biotechnology). After washing with PBS, the slides were incubated with a biotinylated secondary antibody for 30 min at room temperature, followed by treatment with avidin\u0026ndash;biotin complex solution and visualization using 3,3\u0026prime;-diaminobenzidine (\u003cb\u003eDAB\u003c/b\u003e) chromogen. Counterstaining was performed with hematoxylin, followed by differentiation, bluing, dehydration, and mounting with neutral resin. Immunostaining was examined under a fluorescence microscope (Imager A2, Zeiss, Oberkochen, Germany). Brownish-yellow staining was interpreted as a positive reaction, whereas blue staining indicated cell nuclei.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative real-time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from approximately 50 mg of intestinal tissue using FreeZol Reagent (Novozymes, Nanjing, China) according to the manufacturer\u0026rsquo;s protocol. Briefly, 500 \u0026micro;L of reagent was added to each sample for lysis, followed by a 5 min incubation at room temperature. After centrifugation for 5 min, the supernatant was mixed with Dilution Buffer (5:1, v/v) and centrifuged for 15 min. The resulting supernatant was transferred to a new microcentrifuge tube, and an equal volume of isopropanol was added to precipitate RNA. Following a 10 min incubation at room temperature, the mixture was centrifuged again, and the pellet was washed with 1 mL of 75% ethanol, centrifuged for 3 min, air-dried, and dissolved in 20\u0026ndash;100 \u0026micro;L of DEPC-treated water. RNA purity and concentration were determined spectrophotometrically using a NanoDrop 2000 (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from total RNA using a reverse transcription kit (CW3360, Kangwei Century, Taizhou, China). Quantitative PCR was performed using SYBR Green Master Mix (Qingke Biotechnology, Beijing, China) with gene-specific primers (Table\u0026nbsp;1) on a real-time PCR system (Eppendorf AG). The amplification program consisted of an initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of denaturation at 95\u0026deg;C for 5 s and annealing/extension at 60\u0026deg;C for 30 s. Gene expression levels were determined based on Ct (threshold cycle) values, and relative expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, with GAPDH serving as the internal reference. Statistical analysis of gene expression data was conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Bacillus supplementation on body weight and serum antioxidant indices\u003c/h2\u003e \u003cp\u003eFigure 2 (A) Body weight change trend (B-F) Serum stress factors detected by ELISA: T-AOC, MDA, CAT, LDH, CK\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;2A, the body weight of broilers increased linearly during the experimental period. On day 14, birds in the TS1 group exhibited a significant increase in body weight compared with the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and this difference became highly significant on day 21 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). By day 35, both the CORT and control groups showed pronounced weight loss (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), reflecting the inhibitory effect of stress on growth performance. In contrast, birds supplemented with TS1 maintained higher body weights, although a slight reduction was still observed (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, the TS1\u0026thinsp;+\u0026thinsp;CORT and CON+CORT groups exhibited mild but significant decreases in body weight (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs illustrated in Figs.\u0026nbsp;2B\u0026ndash;F, CORT administration markedly elevated serum MDA levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and significantly increased CAT, LDH, and CK activities (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating enhanced oxidative stress and tissue damage. Conversely, supplementation with TS1 or B64 effectively restored antioxidant balance. The TS1\u0026thinsp;+\u0026thinsp;CORT and CON+CORT groups displayed significant increases in T-AOC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), accompanied by marked reductions in MDA and CAT levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a strong suppression of CK activity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, birds in the B64\u0026thinsp;+\u0026thinsp;CORT and CON+CORT groups exhibited a highly significant enhancement of T-AOC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and reduced MDA and CAT expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that both Bacillus strains enhanced the systemic antioxidant defense, mitigated oxidative damage, and improved overall physiological resilience under CORT-induced stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Bacillus supplementation on intestinal morphology in CORT-treated broilers\u003c/h2\u003e \u003cp\u003eFigure 3 (A-B) HE-stained sections of duodenum and jejunum (C) Quantitative analysis of intestinal villi (D) Quantitative analysis of intestinal crypts (E) Statistical analysis of villus-to-crypt ratio.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;3, supplementation with TS1 or B64 effectively mitigated intestinal structural damage caused by CORT exposure. In the duodenum, villus height was significantly greater in the TS1\u0026thinsp;+\u0026thinsp;CORT and CON+CORT groups compared with the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the B64\u0026thinsp;+\u0026thinsp;CORT group exhibited a highly significant increase in villus length (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No notable differences were observed in crypt depth or the villus-to-crypt ratio among the duodenal samples. In the jejunum, however, CORT treatment resulted in a pronounced shortening of villi (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a reduction in crypt size (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the CON group, indicating structural injury due to oxidative stress. Supplementation with Bacillus strains significantly reversed these changes: both the CON+CORT and B64\u0026thinsp;+\u0026thinsp;CORT groups displayed a marked increase in crypt depth (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), restoring intestinal morphology toward normal levels. These results suggest that TS1 and B64 supplementation contributes to maintaining villus architecture and mucosal integrity in broilers under CORT-induced stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Bacillus supplementation on cecum microbial diversity and community composition\u003c/h2\u003e \u003cp\u003eFigure 4 Cecal microbiota α-diversity analysis (A-C) Shannon, ACE, and Chao indices (D) Total number of microbial communities in each group (E) Microbial community analysis at the phylum level (F) Microbial community analysis at the genus level.\u003c/p\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;4A\u0026ndash;D, α-diversity analysis of the cecum microbiota revealed distinct differences among the treatment groups. According to the Shannon index, microbial diversity in the CON and B64 groups was significantly lower than that in the CORT-treated groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while both the TS1 and B64 groups showed a marked reduction in microbial abundance compared with the latter (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Consistent patterns were observed in the ACE and Chao1 indices, where the number of microbial species in the CON and CON+CORT groups decreased significantly compared with the other treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the B64 group exhibited the lowest richness (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eAt the phylum level (Figs.\u0026nbsp;4E\u0026ndash;F), \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, and \u003cem\u003eActinobacteria\u003c/em\u003e were dominant taxa in the intestinal communities. Their relative abundances were notably reduced in the TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT groups compared with the CORT group, suggesting that Bacillus supplementation helped restore microbial balance disrupted by oxidative stress. At the genus level, CORT exposure led to increased proportions of \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e, accompanied by a reduction in \u003cem\u003eErysipelotrichaceae\u003c/em\u003e species. Treatment with TS1 and B64 partially reversed these changes, indicating that both strains promoted a more favorable microbial structure conducive to intestinal health and resilience against CORT-induced dysbiosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Bacillus supplementation on tight junction and antioxidant-related gene expression\u003c/h2\u003e \u003cp\u003eFigure 5 (A-C) Detection of mRNA expression levels of Claudin-3, ZO-1, Mucin2, Occludin, NRF2, HO-1, NQO1, SOD, CAT, and GSH-PX in the duodenal intestine by qPCR (D-F) mRNA transcription levels of Claudin-1, Claudin-5, ZO-1, Mucin2, NRF2, HO-1, NQO1, SOD, CAT, and GSH-PX in the jejunal intestine.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;5A, supplementation with TS1 and B64 significantly enhanced the transcription of tight junction and mucin-related genes in the duodenum. Compared with the CORT and CON groups, ZO-1 expression was significantly upregulated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the TS1\u0026thinsp;+\u0026thinsp;CORT group, Claudin-3 transcription increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while ZO-1 and Mucin-2 levels rose markedly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, in the B64\u0026thinsp;+\u0026thinsp;CORT group, Claudin-3 and Mucin-2 were significantly upregulated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), ZO-1 expression showed a strong increase (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and Occludin expression also rose (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, when comparing TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT, the latter exhibited higher Claudin-3 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that B64 more effectively preserved tight junction integrity.\u003c/p\u003e \u003cp\u003eFigures 5B and 5C demonstrate that CORT exposure markedly suppressed NRF2 transcription (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and downregulated antioxidant genes SOD and CAT (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the duodenum. However, TS1 and B64 supplementation effectively counteracted these effects. In the TS1\u0026thinsp;+\u0026thinsp;CORT group, HO-1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), CAT (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and GSH-PX (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) transcription levels significantly increased compared with CORT alone. In the B64\u0026thinsp;+\u0026thinsp;CORT group, NRF2 and HO-1 expression were strongly upregulated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while NQO1 and CAT also increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and SOD and GSH-PX levels rose significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, B64 supplementation produced greater increases in NRF2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), HO-1, SOD, and CAT (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with TS1, suggesting superior antioxidant activation.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;5D, similar patterns were observed in the jejunum. The TS1\u0026thinsp;+\u0026thinsp;CORT group exhibited significantly higher Claudin-1, Claudin-5, and Mucin-2 transcription (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and increased ZO-1 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with CORT. The B64\u0026thinsp;+\u0026thinsp;CORT group also displayed elevated Claudin-5 and Mucin-2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and enhanced ZO-1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In Figs.\u0026nbsp;5E and 5F, CORT markedly suppressed NRF2 expression in the jejunum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but both Bacillus strains reversed this effect. TS1\u0026thinsp;+\u0026thinsp;CORT increased NRF2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), HO-1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and NQO1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas B64\u0026thinsp;+\u0026thinsp;CORT induced robust upregulation of NRF2, HO-1, NQO1, and CAT (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with additional increases in SOD and GSH-PX (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with TS1\u0026thinsp;+\u0026thinsp;CORT, the B64\u0026thinsp;+\u0026thinsp;CORT group showed higher SOD and GSH-PX expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a markedly greater rise in CAT transcription (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings indicate that both Bacillus strains enhanced intestinal barrier function and activated KEAP1/NRF2-mediated antioxidant defense, with B64 exhibiting a stronger regulatory effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical analysis of intestinal oxidative stress and junction protein expression\u003c/h2\u003e \u003cp\u003eFigure 6 Immunohistochemistry detection of NRF2 (A-B), KEAP1 (C-D), NQO1 (E-F), Claudin-1 (G-H), and Mucin2 (I-J) expression levels in the duodenum and jejunum, and Image J statistical analysis of the duodenum (K-M) and jejunum (L-N).\u003c/p\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;6A\u0026ndash;N, immunohistochemical staining revealed clear differences in the expression patterns of oxidative stress\u0026ndash;related and intestinal barrier\u0026ndash;associated proteins among the treatment groups. In both the duodenum and jejunum, the CORT and CON groups exhibited lighter positive staining for NRF2、KEAP1、NQO1、Claudin-1 and Mucin-2, whereas TS1 and B64 supplementation markedly enhanced the intensity of these signals, indicating increased protein expression. Conversely, the KEAP1-positive staining was more intense in the CORT group, reflecting elevated KEAP1 expression, while it was noticeably lighter in the TS1 and B64 groups, suggesting that both strains suppressed KEAP1 expression and mitigated oxidative stress.\u003c/p\u003e \u003cp\u003eQuantitative analysis using ImageJ further supported these observations. In the duodenum, CORT treatment led to a downward trend in NRF2 expression, a significant increase in KEAP1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and NQO1 declined compared with CON. In the jejunum, NRF2 expression decreased markedly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while KEAP1 increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)and a significant reduction in NQO1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with CON. Supplementation with TS1\u0026thinsp;+\u0026thinsp;CORT reversed these effects, showing elevated NRF2 and KEAP1 levels in the duodenum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a highly significant increase in NRF2 in the jejunum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with TS1\u0026thinsp;+\u0026thinsp;CORT, the B64\u0026thinsp;+\u0026thinsp;CORT group displayed lower NRF2 expression in the duodenum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but higher NRF2 levels in the jejunum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eRegarding intestinal barrier proteins, Claudin-1 expression in the duodenum tended to decrease in the CORT group but significantly increased in B64\u0026thinsp;+\u0026thinsp;CORT (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), accompanied by a marked rise in Mucin-2 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, in the jejunum, CORT treatment reduced both Claudin-1 and Mucin-2 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas Bacillus supplementation restored their levels. These results confirm that TS1 and B64 alleviated CORT-induced oxidative stress by downregulating KEAP1 and enhancing NRF2-mediated antioxidant activity while simultaneously reinforcing intestinal barrier integrity through upregulation of Claudin-1 and Mucin-2.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBody weight gain or loss serves as a direct indicator of the physiological and health status of broilers during experimental evaluation. Under oxidative stress, broilers typically exhibit reduced feed intake, decreased water consumption, and a lowered metabolic rate, all of which contribute to inhibited growth performance (Zhao G L, 2022;YANG X, 2010). Previous studies have demonstrated that \u003cem\u003eBacillus\u003c/em\u003e supplementation can enhance growth performance by improving nutrient utilization and maintaining intestinal integrity. In this study, consistent with these findings, the B64 group showed a significant increase in body weight as early as day 7, while the TS1 group exhibited notable gains on days 14 and 21, indicating that both strains exerted growth-promoting effects at different stages. Conversely, broilers in the CORT group experienced a pronounced reduction in body weight by day 35, confirming that CORT exposure induced anorexia and growth suppression. These results suggest that TS1 and B64 supplementation effectively counteracted the adverse effects of oxidative stress on growth performance, thereby supporting their potential use as functional probiotics to maintain productivity in stressed broilers.\u003c/p\u003e \u003cp\u003eIntestinal oxidative stress increases the production of reactive oxygen species, impairing cellular function, disrupting the body\u0026rsquo;s antioxidant defense balance, and leading to ROS-induced damage. This process also results in the release of toxic metabolites into circulation, which alters the activity of key antioxidant markers such as CAT, SOD, and GSH-Px (Altan O, 2003;BAI K, 2018༛PETERSON L W, 2014). In the present study, subcutaneous administration of CORT significantly elevated serum MDA, CAT, and LDH levels, accompanied by an upward trend in T-AOC, confirming the activation of oxidative stress pathways. Following probiotic colonization with TS1 and B64, the levels of LDH and MDA declined markedly, whereas T-AOC significantly increased, indicating mitigation of oxidative damage. Gene transcription analysis further supported these findings: SOD, CAT, and GSH-Px expression decreased notably in the duodenum and jejunum of the CORT group but showed a clear recovery or upward trend in the TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT groups. These results demonstrate that both TS1 and B64 effectively suppressed oxidative stress and enhanced the antioxidant defense system in CORT-induced broilers. Meanwhile, the antioxidant capacity of TS1 is stronger than that of B64.The pronounced antioxidant effects observed in this study are consistent with previous reports confirming the protective role of Bacillus species in reducing oxidative injury and improving redox homeostasis (SZETO H H, 2006; WANG Y, 2018).\u003c/p\u003e \u003cp\u003eThe intestine functions as a critical barrier that protects the host from bacterial toxins and other harmful agents. Structural parameters such as villus height, crypt depth, and the villus-to-crypt ratio are key morphological indicators of intestinal health. Longer villi provide a larger absorptive surface area, thereby improving feed utilization efficiency and nutrient absorption, ultimately supporting growth performance in broilers (CASPARY W F, 1992; Seppi M, 2023). In the present study, histopathological observations revealed that the intestinal tissue of the CON group displayed a well-organized architecture with intact mucosa, orderly epithelial alignment, and no signs of cellular degeneration or necrosis. In contrast, the CORT group exhibited marked histological alterations, including villus disruption in both the duodenum and jejunum, crypt atrophy, mucosal defects, and distorted epithelial morphology, hallmarks of stress-induced intestinal injury.\u003c/p\u003e \u003cp\u003eSupplementation with TS1 and B64 notably alleviated these pathological changes. Both TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT groups showed significant increases in duodenal villus height compared with CORT, while jejunal villi were also lengthened, although crypt depth in the duodenum remained unchanged. In the jejunum, CORT exposure caused a significant reduction in crypt size, whereas TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT treatments restored crypt depth to near-normal levels. No significant difference was observed in the villus-to-crypt ratio. These findings suggest that colonization with TS1 and B64 effectively mitigated CORT-induced mucosal injury and maintained intestinal structural integrity, consistent with previous reports that \u003cem\u003eBacillus\u003c/em\u003e supplementation enhances gut morphology and barrier resilience under stress conditions (Liu Y, 2023).\u003c/p\u003e \u003cp\u003eThe intestinal microbiota plays a vital role in maintaining host nutrition, immunity, metabolism, and disease resistance. A stable microbial community is essential for intestinal homeostasis and optimal growth performance in animals, while a reduction in microbial diversity disrupts this balance and compromises gut health. As a primary target of oxidative stress, alterations in the intestinal microbiota are key indicators of redox imbalance. The intestinal microbial community is predominantly composed of \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e (Zhao Y, 2020; Yaklai K, 2021; XU ZR, 2003; Liu JH,2020). Under stress conditions, the equilibrium of this ecosystem is disturbed, leading to compositional changes that adversely affect host physiology. The ratio of \u003cem\u003eFirmicutes\u003c/em\u003e to \u003cem\u003eBacteroidetes\u003c/em\u003e, a well-established marker of intestinal health, has dual pathological significance: an elevated ratio is associated with metabolic dysfunction, while a decreased ratio indicates inflammatory injury. \u003cem\u003eBacillus\u003c/em\u003e species can help reestablish microbial balance by modulating the intestinal environment, forming a biological barrier, suppressing pathogenic colonization, and restoring microbial homeostasis (ZHANG B, 2011; GREINER T B, 2011; AMOAH K, 2020;GU Y F, 2022).\u003c/p\u003e \u003cp\u003eIn this study, α-diversity analysis revealed that microbial richness decreased in both the TS1 and B64 groups, suggesting that single-strain colonization modestly influenced the native microbial structure. However, a marked reduction in microbial abundance was observed in the CORT group, indicating that CORT disrupted gut microbial stability. At the phylum level, the CORT group exhibited increased proportions of \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, and \u003cem\u003eProteobacteria\u003c/em\u003e, which are associated with dysbiosis, metabolic disturbances, and compromised mucosal integrity, potentially heightening the risk of inflammatory bowel conditions. Conversely, in the TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT groups, the relative abundance of \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eBacteroidota\u003c/em\u003e declined, suggesting partial restoration of microbial equilibrium. The elevated abundance of \u003cem\u003eProteobacteria\u003c/em\u003e in these groups may represent a compensatory mechanism supporting epithelial repair and crypt regeneration, while the enrichment of \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eVerrucomicrobiota\u003c/em\u003e, and \u003cem\u003eActinomycetes\u003c/em\u003e contributed to microbiota stabilization (Yang H, 2019).\u003c/p\u003e \u003cp\u003eNotably, the increase in \u003cem\u003eAkkermansia\u003c/em\u003e in the TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT groups is indicative of improved digestive capacity, reduced intestinal permeability, and enhanced anti-inflammatory activity, thereby strengthening the mucosal barrier and nutrient absorption (Huck O, 2020; Keshavarz Azizi Raftar S, 2021). The concurrent rise in \u003cem\u003eLactobacillus\u003c/em\u003e, a well-known probiotic genus, further supports intestinal homeostasis by inhibiting pathogenic bacteria and promoting intestinal motility (WANG Y, 2017). Meanwhile, the decline of \u003cem\u003eErysipelotrichaceae\u003c/em\u003e, an opportunistic pathogen within \u003cem\u003eFirmicutes\u003c/em\u003e, implies reduced harmful bacterial proliferation and alleviation of intestinal stress, consistent with earlier findings. The distinct microbial patterns observed between the TS1 and B64 treatments may be attributed to inherent differences in their spore-forming properties and colonization dynamics.\u003c/p\u003e \u003cp\u003eThe intestinal epithelial barrier plays a crucial role in maintaining cellular integrity and selective permeability, thereby preventing the invasion of harmful substances, preserving microbial balance, and sustaining a stable environment for bacterial symbiosis. Tight junctions form the first line of defense within the intestinal epithelium and consist of transmembrane proteins (Occludin, Claudin), peripheral membrane scaffolding proteins (ZO-1), and mucin glycoproteins (Mucin-2). Occludin is a multifunctional transmembrane protein involved in intercellular adhesion and permeability regulation, while ZO-1, a cytoplasmic member of the zonula occludens family, links Claudin proteins to the actin cytoskeleton and plays an essential role in maintaining epithelial integrity, permeability, and cell differentiation (Saitou M, 1997; MEI M, 2016). Members of the Claudin family are integral components of tight junctions: Claudin-1 regulates epithelial permeability, Claudin-3 forms a continuous sealing layer that limits paracellular transport, and Claudin-5 tightens intercellular junctions in epithelial and endothelial tissues (Gong Y, 2016; SUZUKI K, 2023; AMASHEH S, 2005). Mucin-2, secreted by goblet cells, serves as a protective mucosal layer that strengthens barrier function, suppresses inflammation, and prevents pathogen invasion.\u003c/p\u003e \u003cp\u003eDisruption of these proteins can increase mucosal permeability, compromise barrier integrity, and exacerbate oxidative stress, leading to inflammatory injury and impaired intestinal homeostasis (HE L Q, 2017; PARADIS T, 2021). Oxidative stress has also been shown to inhibit intestinal stem cell proliferation, disrupt tight junction continuity, and heighten susceptibility to intestinal inflammation. Probiotics and their metabolites, however, can enhance immune cell activity, support epithelial regeneration, and mitigate stress-induced damage (XU ZR, 2003; GRONDIN J A,2020).\u003c/p\u003e \u003cp\u003eIn the present study, transcriptional analysis revealed that the expression of Claudin-3, ZO-1, Mucin-2, and Occludin decreased in the duodenal CORT group, with Claudin-1 and Mucin-2 showing a downward trend. Similarly, in the jejunum, Claudin-1, Claudin-3, ZO-1, and Mucin-2 were downregulated, with Claudin-1 and Mucin-2 significantly reduced, indicating compromised barrier integrity and enhanced inflammatory susceptibility. Conversely, TS1\u0026thinsp;+\u0026thinsp;CORT treatment increased the expression of Claudin-1 and Mucin-2 in both the duodenum and jejunum, while B64\u0026thinsp;+\u0026thinsp;CORT supplementation significantly upregulated Mucin-2 expression in the duodenum. The consistent transcriptional and protein-level results confirm that TS1 and B64 effectively repaired intestinal barrier damage and restored mucosal function, At the same time, The repair ability of B64 in the duodenum is stronger than TS1, while the repair ability of the two in the jejunum is similar, findings that align with previous reports on the barrier-protective roles of \u003cem\u003eBacillus\u003c/em\u003e probiotics under oxidative stress conditions.\u003c/p\u003e \u003cp\u003eThe Keap1/Nrf2 signaling pathway is a pivotal endogenous defense mechanism against oxidative stress. Nrf2, a key transcription factor within this pathway, regulates the expression of antioxidant genes and is closely associated with SIRT3-mediated redox balance in the nicotinamide adenine dinucleotide-dependent deacetylase family, while Keap1 serves as its upstream negative regulator (Wang L, 2022; Xin X, 2024; Niu B, 2024). Under physiological conditions, Nrf2 and Keap1 remain bound in the cytoplasm, maintaining redox homeostasis. When oxidative stress occurs, Nrf2 dissociates from Keap1 and translocates into the nucleus, where it activates the transcription of downstream antioxidant genes such as HO-1 and NQO1, thereby restoring the cellular oxidative\u0026ndash;antioxidant equilibrium (Shen Y,2019;Xin X,2024;Niu B,2024).\u003c/p\u003e \u003cp\u003eIn this study, CORT administration suppressed the transcription and protein expression of Nrf2 and NQO1 in both the duodenum and jejunum, reduced HO-1 transcription, and increased Keap1 expression, indicating excessive oxidative stress and impaired antioxidant signaling. However, supplementation with TS1\u0026thinsp;+\u0026thinsp;CORT and B64\u0026thinsp;+\u0026thinsp;CORT reversed these effects. Both treatments upregulated the transcription and expression of Nrf2 and NQO1, elevated HO-1 levels, and inhibited Keap1 expression. These results demonstrate that colonization with TS1 and B64 effectively reactivated the Keap1/Nrf2 signaling pathway, thereby mitigating CORT-induced intestinal oxidative injury.,two types of Bacillus have comparable stress relieving abilities.The findings are consistent with previous studies highlighting the capacity of \u003cem\u003eBacillus\u003c/em\u003e species to modulate Nrf2-mediated antioxidant defenses and protect intestinal integrity under stress conditions.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn this study, both Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 promoted growth performance in broiler chickens, with TS1 exerting a more pronounced effect. Under CORT-induced stress, TS1 notably improved body weight gain, and both strains demonstrated strong antioxidant properties. Supplementation with TS1 and B64 positively influenced intestinal morphology by increasing villus height and crypt depth while maintaining a balanced villus-to-crypt ratio. Both strains also modulated the intestinal microbiota by suppressing harmful bacteria, enhancing the abundance of beneficial genera such as Lactobacillus, and optimizing the relative proportion of Bacteroidetes.\u003c/p\u003e \u003cp\u003eFurthermore, TS1 and B64 enhanced the transcription and expression of key intestinal barrier proteins, including Claudin, ZO-1, Occludin, and Mucin-2, thereby reinforcing epithelial integrity. Under CORT-induced oxidative stress, the KEAP1/NRF2 signaling pathway was activated, and Bacillus supplementation significantly enhanced antioxidant defenses by upregulating NRF2 and its downstream targets (HO-1 and NQO1) while suppressing KEAP1 expression.\u003c/p\u003e \u003cp\u003eOverall, \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64 exerted marked protective and antioxidant effects against CORT-induced intestinal injury, overall, TS1 has stronger antioxidant capacity than B64suggesting their potential as effective probiotic feed additives for improving intestinal health and oxidative resilience in broiler production, and further research on related oxidative stress pathways can be conducted to improve its function\u003c/p\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eThis study was approved by the Ethics Committee of the College of Arts, Univeristy of College of Veterinary Medicine, Nanjing Agricultural University.All procedures performed in this study involving human participants were conducted in accordance with the ethical standards of the institutional research committee .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eConsent to Publish\u003c/h2\u003e \u003cp\u003eAll participants provided written consent for the publication of anonymised data collected during the study. No identifiable information about participants will be disclosed in this publication.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eof case study details, including anonymised narratives, was obtained from all individuals involved.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003ewas obtained from all individual participants included in the study. Participants were provided with detailed information about the study objectives and procedures prior to their voluntary consent.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eData availability statements\u003c/h2\u003e \u003cp\u003eThe datasets generated and analysed during the current study are not publiclyavailable due to the experiment is not yet fully completed and is still in the confidential stage but are available fromthe corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest regarding this research.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChen zhuoying wrote the main manuscript text and Hang jingjing and Liang wanqing prepared figures 1-3. All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by Natural Science Foundation of Ningxia Province (2023AAC05052);and National Natural Science Foundation of China (grant number 31602027). The funding body did not play a role in the design, analysis, and reporting of the study, but did provide financial support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analysed during the current study are not publiclyavailable due to the experiment is not yet fully completed and is still in the confidential stage but are available fromthe corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAMASHEH, S,SCHMIDT T,MAHN, M et al (2005) Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells[J]. Cell Tissue Res 321(1):89\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAMOAH K,DONG X H,TAN B, P et al (2020) Administration of probiotic Bacillus licheniformis induces growth,immune and antioxidant enzyme activities,gut microbiota assembly and resistance to Vibrio parahaemolyticus in Litopenaeus vannamei[J]. Aquaculture Nutr 26(5):1604\u0026ndash;1622\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltan O, Pabu\u0026ccedil;cuoğlu A, Altan A, Konyalioğlu S, Bayraktar H (2003) Effect of heat stress on oxidative stress, lipid peroxidation and some stress parameters in broilers. Br Poult Sci 44:545\u0026ndash;550\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBAI K, FENG C, JIANG L et al (2018) Dietary effects of Bacillus subtilis fmbj on growth performance, small intestinal morphology, and its antioxidant capacity of broilers[J]. Poult Sci 97(7):2312\u0026ndash;2321\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBampidis V, Azimonti G, Bastos ML et al (2021) Safety and efficacy of a feed additive consisting of Bacillus subtilis strains CNCM I-4606, CNCM I-5043 and CNCM I-4607 and Lactococcus lactis CNCM I-4609 for all animal species (Nolivade)[J]. EFSA J 19(11):e6907\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBampidis V, Azimonti G, Bastos ML et al (2022) Efficacy of the feed additive consisting of Bacillus velezensisNRRL B-67259 (Correlink ABS1781) as a feed additive for all growing poultry species (Elanco GmbH)[J]. EFSA J 20(12):e7711\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHENG D Y, ZHANG M, ZHENG Y Z et al α-Ketoglutarate prevents hyperlipidemia-induced fatty liver mitochondrial dysfunction and oxidative stress by activating the AMPK-pgc-1α/Nrf2 pathway[J]. Redox Biology 2024, 74: 103230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCASPARY W F Physiology and pathophysiology of intestinal absorption[J]. Am J Clin Nutr 1992, 55(Suppl.1):299S\u0026ndash;308S\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCUI M X,ZHANG M,WU J, W et al Marine polysaccharides from Gelidium pacificum Okamura and Cereus sinensis reveal prebiotic functions[J]. Int J Biol Macromol 2020, 164:4 381\u0026ndash;4390\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCostantini David;;Fanfani Alberto;;Dell'omo Giacomo.Effects of corticosteroids on oxidative damage and circulating carotenoids in captive adult kestrels (Falco tinnunculus).[J].Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology.2008,178(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDallman M F;;Jones M T.Corticosteroid feedback control of ACTH secretion: effect of stress-induced corticosterone ssecretion on subsequent stress responses in the rat.[J].Endocrinology.1973(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Filippis F, Pasolli E, Ercolini D (2020) The food-gut axis: lactic acid bacteria and their link to food, the gut microbiome and human health[J]. FEMS Microbiol Rev 44(4):454\u0026ndash;489\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGU Y F,CHEN Y P,JIN, R et al Dietary chitooligosaccharide supplementation alleviates intestinal barrier damage,and oxidative and immunological stress in lipopolysaccharide-challenged laying hens[J]. Poult Sci 2022, 101(4):101701\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong Y, Li H, Li Y (2016) Effects of Bacillus subtilis on Epithelial Tight Junctions of Mice with Inflammatory Bowel Disease[J]. J Interferon Cytokine Res 36(2):75\u0026ndash;85\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGRONDIN J A,KWON Y H,FAR P, M et al (2020) Mucins in intestinal mucosal defense and inflammation:learning from clinical and experimental studies[J]. Front Immunol 11:2054\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGREINER T, BCKHED F (2011) Effects of the gut microbiota on obesity and glucose homeostasis[J].Trends in Endocrinology and Metabolism,. 22:117\u0026ndash;123\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHE L Q,ZHOU X H,HUANG, N et al Administration of alpha-ketoglutarate improves epithelial restitution under stress injury in early-weaning piglets[J].Oncotarget.2017,8(54):91965\u0026ndash;91978\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuck O, Mulhall H, Rubin G et al (2020) Akkermansia muciniphila reduces Porphyromonas gingivalis-induced inflammation and periodontal bone destruction [. J] J Clin Periodontol 47(2):202\u0026ndash;212\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHEZT,LIYJ,XIONG T D etal.Effectofdietary resveratrolsupplementationongrowthperformance,antioxidant capacity,intestinal immunity and gut microbiota in Yellow-feathered broilers challenged with lipopolysaccharide[J]. Front Microbiol 2022, 13:977087\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeshavarz Azizi Raftar S, Hoseini Tavassol Z, Amiri M et al (2021) Assessment of fecal Akkermansia muciniphila in patients with osteoporosis and osteopenia:a pilot study [J]. J Diabetes Metab Disord 20(1):279\u0026ndash;284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu JH, Yue T, Luo ZW et al (2020) Akkermansia muciniphila promotes type H vessel formation and bone fracture healing by reducing gut permeability and inflammation [J]. Dis Model Mech 13(11):dmm043620\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Liu S, Wan S, Li Z, Li H, Tang S (2024) Anti-inflammatory properties of Bacillus pumilus TS1 in lipopolysaccharide-induced inflammatory damage in broilers. Animal Biotechnology, 35(1). doi.10.1080/10495398.2024.2418516\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Xie TT, Yin LT et al (2023) Effect of canthaxanthin on antioxidant capacity and small intestinal mucosal structure of Huaixiang chickens under heat stress[J]. Chin J Veterinary Sci 43(10):2171\u0026ndash;2180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMEI M,XIANG R L,YU G, Y et al (2016) Research progress in regulation of tight junction of pro-inflammatory factors [J]. Progress Physiological Sci 47(1):27\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMOOLI R G R, MUKHI D, RAMAKRISHNAN S K (2022) Oxidative stress and redox signaling in the pathophysiology of liver diseases [J]. Compr Physiol 12(2):3167\u0026ndash;3192\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu B, Feng YX,Cheng X et al (2024) The alleviative effects of viable and inactive Lactobacillus paracasei CCFM1120 against alcoholic liver disease via modulation of gut microbiota and the Nrf2/HO-1 and TLR4/MyD88/NF-κB pathways[J]. Food Funct 15(17):8797\u0026ndash;8809\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNAWAB A,IBTISHAM F,LI, G et al Heat stress in poultry production:Mitigation strategies to overcome the future challenges facing the global poultry industry[J]. J Therm Biology 2018, 78:131\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePETERSON L W ARTISD (2014) Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J]. Nat Rev Immunol 14(3):141\u0026ndash;153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePARADIS, T,B\u0026Egrave;GUE H,BASMACIYAN, L et al (2021) Tight junctions as a key for pathogens invasion in intestinal epithelial cells[J]. Int J Mol Sci 22(5):2506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRAMLUCKEN U,RAMCHURAN S O,MOONSAMY, G et al Production and stability of a multi-strain Bacillus based probiotic product for commercial use in poultry[J].Biotechnology Reports.2020,29(prepublish):e00575\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaitouM AndoAkatsukaY, ItohM, etal.Mammalianoccludininepithelialcells:itsexpressionandsubcellulardistribution.EurJCellBiol.1997,73:222231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeppi M, Pasqualini J, Facchin S et al (2023) Emergent functional organization of gut microbiomes in health and diseases[J].Biomolecules. 14(1):5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeven PT (2008) Sevenb I.Effect of dietary turkish propolis as alternative to antibiotic on performance and digestibility in broilers exposed to heat stress[J]. J Appl Anim Res 34(2):193\u0026ndash;196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuez J, Zmora N, Segal E et al The pros, cons, and many unknowns of probiotics[J]. Nat Med 2019, 25(5):716\u0026ndash;729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Y, Liu X, Shi J et al (2019) Involvement of Nrf2 in myocardialischemia and reperfusion injury[J]. Int J Biol Macromol 125(1):496\u0026ndash;502\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSZETO H H Mitochondria-targeted peptide antioxidants:novel neuroprotective agents [J].AAPS J.2006,8(3):E521\u0026ndash;E531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSUZUKI K, YAMAGA K, TOKUMASU R et al Double mutation of claudin-1 and claudin-3 causes alopecia in infant mice[J]. Ann N Y Acad Sci 2023, 1523(1): 51\u0026ndash;61\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVemuri R, Gundamaraju R, Eri R (2017) Role of Lactic Acid Probiotic Bacteria in IBD[J]. Curr Pharm Design 23(16):2352\u0026ndash;2355\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWANG, Y,WU Y P,WANG Y, Y et al Antioxidant properties of probiotic bacteria[J].Nutrients.2017,9(5):521\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWANG, Y, BRANICKY R,NOEA et al Superoxide dismutases:dual roles in controlling Ros damage and regulating ROssignalingUl. Cell Biol 2018, 217(6):1915\u0026ndash;1928\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L (2022) He C.Nrf2-mediated anti-inflammatory polarization of macrophages as therapeutic targets for osteoarthritisU].Front lmmunol.13:967193\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXU, ZR,HU CH,XIA, MS et al (2003) Effects of dietary fructooligosaccharide on digestive enzyme activities,intestinal microflora and morphology of male broilers[J]. Poult Sci 82(6):1030\u0026ndash;1036\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXU, ZR,HU CH,XIA, MS et al (2003) Effects of dietary fructooligosaccharide on digestive enzyme activities,intestinal microflora and morphology of male broilers[J]. Poult Sci 82(6):1030\u0026ndash;1036\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXin X, Liu J, Liu X et al (2024) Melatonin-Derived Carbon Dots with Free Radical Scavenging Property for Effective Periodontitis Treatment via the Nrf2/HO-1 Pathway[J]. ACS Nano 18(11):8307\u0026ndash;8324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYaklai K, Pattanakuhar S, Chattipakorn N et al (2021) The role of acupuncture on the gut-brain-microbiota axis in irritable bowel syndrome[J]. Am J Chin Med 49(2):285\u0026ndash;314\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Yu HB, Bhinder G et al TLR9 limits enteric antimicrobial responses and promotes microbiota-based colonisation resistance during Citrobacter rodentium infection[J]. Cell Microbiol 2019, 21(7):e13026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYANG X, ZHANG B, GUO Y et al (2010) Effects of dietary lipids and Clostridium butyricum on fat deposition and meat quality of broiler chickens[J]. Poult Sci 89:254\u0026ndash;260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHANG B, YANG X, GUO Y et al Effects of dietary lipids and Clostridium butyricum on serum lipids and lipid-related gene expression in broiler chickens[J].Animal.2011, 5:1909\u0026ndash;1915\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Yongming F, Jinmin S, Yuzhuo (2023) etc The effect of fermentation broth of Bacillus amyloliquefaciens B-4M-6 from chickens on growth performance, immune function, and intestinal health of broiler chickens [J]. Feed Research. 46 (03): 36\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13557/j.cnki.issn1002-2813.2023.008\u003c/span\u003e\u003cspan address=\"10.13557/j.cnki.issn1002-2813.2023.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y Zhang X.Interactions of tea polyphenols with intestinal microbiota and their implication for anti-obesity [. J] J Sci Food Agric 2020, 100(3):897\u0026ndash;903.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jsfa.10049\u003c/span\u003e\u003cspan address=\"10.1002/jsfa.10049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao GL, Niu Y, Zhang RQ et al Effects of yeast culture on growth performance, immune function and cecal microbiota of yellow-feathered broilers[J]. Chin J Anim Nutr 2022, 34(12): 7701\u0026ndash;7710 (in Chinese with English abstract).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacillus pumilus, Bacillus amyloliquefaciens, corticosterone, oxidative stress, broiler","lastPublishedDoi":"10.21203/rs.3.rs-8964671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8964671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the protective effects of \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64 on broilers subjected to corticosterone-induced oxidative stress. A total of 102 one-day-old Arbor Acres broilers were randomly assigned to six treatment groups. Birds received drinking water containing either TS1 or B64 for three weeks, followed by subcutaneous corticosterone administration (4 mg/kg) to induce oxidative stress. Growth performance, antioxidant capacity, intestinal morphology, microbial composition, and expression of intestinal barrier and antioxidant-related genes and proteins were evaluated. Supplementation with TS1 and B64 significantly improved body weight gain compared with corticosterone-treated controls. Both strains enhanced serum total antioxidant capacity and reduced malondialdehyde, lactate dehydrogenase, and creatine kinase levels, indicating mitigation of oxidative damage. Histological analysis revealed that TS1 and B64 protected duodenal and jejunal villus structure and preserved mucosal integrity. 16S rRNA sequencing showed that corticosterone disrupted intestinal microbial balance, while both Bacillus strains restored microbial diversity and increased beneficial genera such as \u003cem\u003eLactobacillus and Akkermansia\u003c/em\u003e. At the molecular level, TS1 and B64 upregulated the transcription and expression of intestinal tight junction proteins (Claudin-1, Claudin-3, ZO-1, and Mucin-2), thereby enhancing barrier function. Both strains activated the KEAP1/NRF2 signaling pathway, evidenced by increased expression of NRF2, HO-1, and NQO1, and suppressed KEAP1 expression, suggesting improved antioxidant defense. Among the two strains, B64 exhibited slightly stronger regulatory effects on antioxidant and tight junction markers. In conclusion, \u003cem\u003eBacillus pumilus\u003c/em\u003e TS1 and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e B64 effectively alleviate corticosterone-induced oxidative stress and intestinal injury in broilers by modulating the intestinal microbiota, enhancing antioxidant capacity, and activating the KEAP1/NRF2 pathway. Supplementation with these Bacillus strains can improve oxidative resilience and intestinal health in broilers under stress, providing a potential alternative to antibiotics for promoting performance and gut integrity in commercial poultry production.\u003c/p\u003e","manuscriptTitle":"BACILLUS STRAINS ALLEVIATE STRESS IN BROILERS Bacillus pumilus TS1 and Bacillus amyloliquefaciens B64 alleviate corticosterone-induced oxidative stress and intestinal injury in broilers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 11:57:47","doi":"10.21203/rs.3.rs-8964671/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69576878-4240-4014-aa98-410cea7c3482","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T20:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 11:57:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8964671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8964671","identity":"rs-8964671","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}