Freeze-dried Aspergillus ochraceus powder was purchased from the Guangdong Microbial Culture Collection Center (CGMCC 3.3876, Guangzhou, China). According to the instructions, strain activation culture was carried out in CDM and PDA medium. Feed was treated with a conidial suspension of Aspergillus ochraceus and maintained at 29–30°C for 14 days. OTA concentration was quantified using high-performance liquid chromatography with ultraviolet detection (HPLC-UVD) for subsequent experiments. Selenomethionine (SeMet) was procured from Zhejiang MinSheng Biotechnology Co, Ltd. (205,135 T, Zhejiang, China). The OTA dosage (1.0 mg/kg feed) was standardized at tenfold the maximum permissible threshold (100 μg/kg) defined by Chinese feed safety guidelines (GB 13078–2017) (24). The SeMet dosage (0.5 mg/kg feed) was based on previous studies demonstrating its efficacy in mitigating renal injury without adverse effects on broiler health (25). Therefore, 1.0 mg/kg OTA and 0.5 mg/kg SeMet were selected as the experimental doses.

All animal experiments were performed in compliance with the ethical protocols approved by Shenyang Agricultural University (No. 201806014) and followed the national legislative framework governing animal model research in China. The temperature of the chicken house was steadily maintained at 32°C (± 5°C) and the relative humidity was kept at 40% (±5%). During the feeding period, broilers had free access to feed and water, and a 12-h continuous light cycle was maintained. The broilers received a nutritionally balanced basal diet designed to comply with the National Research Council (NRC) guidelines for poultry growth.

One-day-old white-feathered broilers were purchased from the original chicken farm company of Shenyang Agricultural University. Sixty 1-day-old healthy white feather broilers were randomly allocated into four experimental groups after 1 week of adaptive feeding, with 15 broiler chicken in each group: Control group: maintained on a basal diet, OTA group: the diet was supplemented with 1.0 mg/kg OTA, SeMet group: the diet was supplemented with 0.5 mg/kg SeMet and OTA + SeMet group: the diet was supplemented with 1.0 mg/kg OTA and 0.5 mg/kg SeMet. The broiler chicken’ mental status and body weight were recorded every day for 21 days. After the last feeding, the broilers’ fasting body weight was measured without food and water for 24 h. Blood specimens were collected via cardiac puncture, and broilers were euthanized by cervical dislocation, and their kidneys were harvested for further experiments.

The tissue samples underwent immersion fixation in 4% paraformaldehyde (PFA; Sinopharm Chemical, Shanghai) followed by paraffin embedding. Serial sections of 5 μm thickness were prepared using a microtome, subjected to ethanol gradient hydration, and xylene-based dewaxing (2 h). Histochemical staining was performed with hematoxylin (Merck, Darmstadt) and eosin (Sigma-Aldrich), with subsequent dehydration through a graded ethanol series and xylene clearing. Permanent mounting was achieved using neutral resin. Morphological evaluation of renal tissue sections was conducted under bright-field illumination using a Leica DM750 microscope (Leica Microsystems, Beijing).

After the slaughter of broilers, samples should be taken within 1–3 min, a petri dish containing electron microscope fixative solution (2.5% glutaraldehyde) was prepared in advance before the sample. The small renal tissue blocks were removed from the body and immediately put into the culture dish, then cut into small pieces with a scalpel in the fixed liquid of the culture dish. The volume of the sampled tissue was controlled in the following sizes (1 mm × 1 mm × 1 mm cube, 1 mm × 1 mm × 3 mm rectangle, 1 mm × 2 mm × 3 mm flake). Mechanical damage such as squeezing of tweezers should be avoided, and the blade should be sharp to avoid contusion tissue. After the tissue was removed, it was put into the electron microscope fixation liquid immediately at room temperature and fixed without the light for 2 h, then transferred to 4°C preservation and transportation. The follow-up test was completed by Wuhan Sevier Biotechnology Co., Ltd., and the fixation liquid should not be frozen during storage and transportation.

After blood collection, the blood was placed at room temperature for 2 h, centrifuged at 4000 rpm for 10 min, and the supernatant was sucked out. Creatinine (CRE), urea nitrogen (BUN), uric acid (UA), malondialdehyde (MDA), reduced glutathione/oxidized glutathione (GSH/GSSG), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), reactive oxygen species (ROS) and ferrous ion (Fe²⁺) were conducted by biochemical kits from Nanjing Jiancheng Bioengineering Institute.

The levels of the cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum were measured by ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). All assay results were in line with the manufacturer’s recommended protocol.

RNA from chicken kidney tissue was extracted by RNA extraction kit (Nanjing Noviacan Biotechnology Co., Ltd., Nanjing, China), and cDNA was obtained by reverse transcription of the extracted RNA by qRT-PCR kit (Nanjing Noviacan Biotechnology Co., Ltd., Nanjing, China). Specific primer sequences are listed in Supplementary Table S1 and quantified by SYBR green real-time PCR. This PCR was performed using a CFX ConnectTM real-time system (Bio-Rad, CA, United States) and AG™ SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology Co., Ltd., Hunan, China). qRT-PCR system: 2 × SYBRGREEN MIX 10 μL (Nanjing Noviacan Biotechnology Co., Ltd., Nanjing, China), upstream and downstream primers (10 μmol/L) 0.4 μL each, 2.0 μL of cDNA template, and 6.2 μL of ddH₂O. We employed the 2^−ΔΔCt formula to assess mRNA expression levels.

A protein extraction kit (Solarbio Science & Technology, Beijing) was employed to isolate total protein from preserved renal tissue. The resulting lysates were then analyzed for protein concentration using the manufacturer’s BCA kit. Proteins were placed onto SDS-PAGE gels (Shanghai Yazyme Biotechnology Co, Ltd., Shanghai, China) and PVDF membranes were employed for protein transfer following electrophoresis. Membranes were blocked with 5% skim milk for 2 h and incubated overnight with their corresponding primary antibodies which were appropriately diluted: SLC7A11 (1:1000) (A2413, ABclonal Technology, United States), TFR1 (1:1000) (WL03500, Wanlei Technology, China), GPX4 (1:1000) (WL05406, Wanlei Technology, China), FTH1 (1:1000) (WL05360, Wanlei Technology, China), Nrf2 (1:1000) (WL02135, Wanlei Technology, China), NQO1 (1:1000) (WL04860, Wanlei Technology, China), Keap1 (1:1000) (WL03285, Wanlei Technology, China), HO-1 (1:1000) (WL02400, Wanlei Technology, China), and β-actin (1:1000) (YM8010, ImmunoWay, China). Subsequently, the membrane was incubated with enzyme-labeled secondary antibody (1:10000) (WLA023, Wanjiao, China) for 1 h at room temperature. Chemiluminescent substrates (Azure Biosystems, United States) were used to detect protein signals, with imaging performed on a chemiluminescence detection system. Quantitative analysis was performed using ImageJ (Version 1.46), with β-actin as the internal control. Target protein expression levels were normalized to β-actin OD values.

Data analysis was conducted with IBM SPSS Statistics 25 (SPSS Inc., Chicago, IL, United States), and outcomes were expressed as mean ± standard deviation (mean ± SD). For comparisons across experimental groups (control, OTA, SeMet, and OTA + SeMet), normalized data were subjected to one-way ANOVA. Graphical representations were generated via GraphPad Prism 8 (GraphPad Software, United States). Statistical significance thresholds were set at p < 0.05 (significant) and p < 0.01 (highly significant). Results surpassing these thresholds were deemed statistically reliable.

The weight and organ index of broilers in each group are shown in Figures 1A,B separately. Relative to the control group, the body weight and kidney coefficient of the broilers in the OTA group were substantially reduced (p < 0.01), while the changes in the SeMet group and OTA + SeMet group were not significant. At the same time, relative to the OTA group, the body weight and kidney coefficient of broilers in the OTA + SeMet group increased (p < 0.05).

HE staining results of renal histopathological sections are shown in Figure 2, and the morphological structure of the kidneys in the control group (Figure 2A) and SeMet group (Figure 2C) was normal, and no significant pathological changes were observed. The glomeruli exhibited no structural abnormalities, the size of the renal sac cavity was normal and symmetrical, the tubular epithelial cells were arranged in a regular manner, the boundary was clear, the cytoplasm exhibited intense staining, and the nucleus was large and round. The distal convoluted lumen was large and clear, the nucleus was located on the surface of the proximal lumen, and the cytoplasmic staining was shallow. Relative to the control group, the OTA group (Figure 2B) showed a significant increase in the renal capsule cavity, renal tubular swelling, obvious inflammatory cell infiltration and bleeding. Relative to the OTA group, the space size of renal vesicles was substantially improved in the OTA + SeMet group (Figure 2D), and the inflammatory cell infiltration and hemorrhage were substantially restored.

The results of transmission electron microscopy were shown in Figure 2. The mitochondrial morphology of CON group (Figure 2E) and SeMet group (Figure 2G) was normal and complete. In the OTA group (Figure 2F), the mitochondrial volume decreased, the mitochondrial membrane ruptured, the membrane density condensed, and the mitochondrial ridge decreased, which was a typical feature of mitochondria when ferroptosis occurred in the cells. In the OTA + SeMet group, the mitochondria returned to normal and the morphology was relatively complete (Figure 2H).

The renal function indexes of broilers in each group are shown in Figure 3. Relative to the control group, there was no significant change in renal function in the SeMet group. However, the serum concentration of CRE, BUN and UA in the OTA group and OTA + SeMet group were substantially increased (p < 0.01). Relative to the OTA group, the concentration of CRE, BUN and UA in the OTA + SeMet combined group were substantially lower (p < 0.01). Therefore, the data reveal that SeMet can alleviate the damage of OTA on kidney function in broilers.

As shown in Figure 4, the qRT-PCR and ELISA test results of IL-1β and IL-6 in the kidney tissues of broilers in each group are shown. Relative to the control group, a significant upregulation of IL-1β and IL-6 was observed in the OTA group (p < 0.01), and the results of the SeMet group were comparable to those of the control group except for the ELISA kit detection results of IL-6, and the mRNA expression levels of IL-1β and IL-6 in the OTA + SeMet group were not substantially different from those in the control group. The concentrations of IL-1β and IL-6, as measured by ELISA, were substantially elevated compared to the control group (p < 0.01). Relative to the OTA group, the expression levels of the OTA + SeMet group were substantially reduced (p < 0.01). In summary, SeMet was able to attenuate OTA-induced increases in the expression of pro-inflammatory factors IL-1β and IL-6.

As shown in Figure 5, relative to the control group, the levels of antioxidant enzymes SOD, T-AOC and GSSH/GSSG in the OTA group were substantially decreased (p < 0.01), while the levels of ROS and MDA were substantially increased (p < 0.01), and the levels of SOD, T-AOC, ROS and MDA in the SeMet group were not substantially different from those in the control group, and it is worth noting that the levels of GSH/GSSG in the SeMet group were substantially higher than those in the control group. Relative to the OTA group, the levels of SOD, T-AOC, and GSSH/GSSG in the OTA + SeMet group were substantially increased (p < 0.01), while the levels of ROS and MDA were substantially decreased (p < 0.01). In summary, SeMet was able to alleviate the generation of ROS, the accumulation of MDA, and the decrease in antioxidant enzyme activity caused by OTA.

As shown in Figure 6, relative to the control group (p < 0.01), the ferrous ion content of the OTA group was substantially increased. In addition, OTA group substantially decreased the mRNA and protein expression levels of FTH1 and increased TFR1 (p < 0.01), and relative to the OTA group, the expression levels of FTH1 in the OTA + SeMet group were substantially increased (p < 0.01), while the expression levels of Fe²⁺ and TFR1 were substantially decreased (p < 0.01).

As shown in Figure 7, relative to the control group, the mRNA and protein expression levels of Nrf2, GPX4, NQO1, HO-1 and SLC7A11 were substantially reduced in the OTA group (p < 0.01), and the mRNA and protein expression levels of Keap1 were increased (p < 0.01). In addition, we found that the mRNA and protein levels of GPX4 in the SeMet group were substantially higher than those in the control group (p < 0.05), indicating that SeMet substantially increased the level of GPX4. Relative to the OTA group, the expression levels of Nrf2, GPX4, NQO1, HO-1 and SLC7A11 in the OTA + SeMet group were substantially increased (p < 0.01), while the expression level of Keap1 was substantially decreased (p < 0.01). Our results suggest that OTA can induce ferroptosis by regulating iron metabolism and inhibiting the Nrf2/GPX4 pathway, while SeMet can inhibit ferroptosis by activating the Nrf2/GPX4 pathway.