Aflatoxin B1 Exposure in Sheep: Insights into Hepatotoxicity Based on Oxidative Stress, Inflammatory Injury, Apoptosis, and Gut Microbiota Analysis

The widespread fungal toxin Aflatoxin B1 (AFB1) is an inevitable pollutant affecting the health of humans, poultry, and livestock. Although studies indicate that AFB1 is hepatotoxic, there are few studies on AFB1-induced hepatotoxicity in sheep. Thus, this study examined how AFB1 affected sheep liver function 24 h after the animals received 1 mg/kg bw of AFB1 orally (dissolved in 20 mL, 4% v/v ethanol). The acute AFB1 poisoning caused histopathological injuries to the liver and increased total bilirubin (TBIL) and alkaline phosphatase (AKP) levels. AFB1 also markedly elevated the levels of the pro-inflammatory cytokines TNF-α and IL-6 while considerably reducing the expression of antioxidation-related genes (SOD-1 and SOD-2) and the anti-inflammatory gene IL-10 in the liver. Additionally, it caused apoptosis by dramatically altering the expression of genes associated with apoptosis including Bax, Caspase-3, and Bcl-2/Bax. Notably, AFB1 exposure altered the gut microbiota composition, mainly manifested by BF311 spp. and Alistipes spp. abundance, which are associated with liver injury. In conclusion, AFB1 can cause liver injury and liver dysfunction in sheep via oxidative stress, inflammation, apoptosis, and gut-microbiota disturbance.


Introduction
Mycotoxins are widespread in all processes of agricultural production and can seriously endanger food and feed safety, threatening human and animal health [1]. Over 4.5 billion people are at high risk of exposure to these food contaminants, and it causes annual economic losses of hundreds of billions of dollars. According to the Food and Agriculture Organization of the United Nations (FAO), about 25% of the world's crops are polluted by mycotoxins in varying degrees [2,3]. Aflatoxin B 1 (AFB 1 ), the most toxic mycotoxin, is hepatotoxic, teratogenic, and mutagenic, and it is classified as a class I carcinogen by the World Health Organization [4,5]. The sensitivity of different livestock species to AFB 1 varies [6,7]. Chickens are highly sensitive to AFB 1 , and poisoning is caused mostly by long-term consumption of AFB 1 -contaminated feed [5,7]. Although sheep are typically thought to have significant tolerance to AFB 1 due to their status as ruminants, poisoning can, nonetheless, occur from prolonged or excessive consumption of an AFB 1 -containing diet [8].
As a hepatotoxic chemical substance, AFB1 is activated into AFB1 epoxide under the action of the cytochrome P450 (CYP450) enzyme, inducing liver cancer and hepatotoxicity [9]. AFB1 toxicity can lead to oxidative damage, apoptosis, and inflammation, resulting in liver congestion, pale coloration, enlargement, and necrosis, inducing chronic and acute  [7,[10][11][12]. Livestock and poultry can accumulate a certain level of AFB 1 through dietary exposure, leading to chronic and acute liver diseases in humans via biological accumulation in the food chain [13].
The liver is an important detoxification center as well as a target organ for the metabolic transformation of AFB 1 in the body, and it is crucial for the body's defense against xenobiotics [14]. Past studies on the hepatotoxic effects of AFB 1 exposure in sheep have not explored the association between changes in liver function indicators and oxidative stress, inflammation, apoptosis, or gut microbiota. Therefore, we sought to assess the liver function, oxidation, inflammation, and apoptosis indices and composition of the fecal microbial community to offer theoretical support and experimental data on liver damage caused by AFB 1 exposure in sheep. 1 Exposure on Clinical Symptoms, Body Temperature, Respiration, Heart Rate, and Conjunctival Color Sheep in the AFB 1 group showed poor mental status, foaming at the mouth, and reduced food intake after gavage compared to the control group. Before AFB 1 exposure, there were no significant differences between the control and AFB1 group body temperatures, respiration, or heart rates, as indicated in Table S2 (p = 0.280, 0.280, and 0.146, respectively). Nevertheless, 24 h after AFB 1 exposure, the respiration and heart rate of the AFB1 group were lower than those of the control group (p = 0.071 and 0.084, respectively), while the body temperature was higher (p = 0.519). The AFB 1 group had lower changes (slope) in respiration and heart rate than the control group (p = 0.488 and 0.022, respectively). Furthermore, the temperature change in the AFB 1 group was greater than in the control group (p = 0.043).

Effects of AFB
As can be observed in Figure S1A-C, the conjunctival color L of the AFB1 group dropped considerably (p = 0.005) after acute exposure compared with the control group, although redness (a value) and yellowness (b value) did not differ significantly (p = 0.442 and 0.262, respectively).

Effects of AFB 1 Exposure on Liver Tissue Structure and Liver Tissue Function
As shown in Figure 1A-C, the ALT, AST, and AKP levels in the AFB 1 group increased when compared with those of the control group. Among them, the AKP level was considerably higher than that in the control group (p = 0.007). Figure 1D-G depicts liver tissue stained with H&E. The liver lobules of the control group had normal shape; the cords were arranged in an orderly, radial pattern; and the liver cells were complete and uniform in size and cytoplasm. The liver tissue of the AFB 1 group, in contrast, exhibited pathological changes, including abnormal hepatic lobule structure, disorganized hepatic cord arrangement, swollen and unevenly sized hepatocytes, severe infiltration of inflammatory cells, vacuolar degeneration, and necrosis in some hepatocytes. group, in contrast, exhibited pathological changes, including abnormal hepatic lobule structure, disorganized hepatic cord arrangement, swollen and unevenly sized hepatocytes, severe infiltration of inflammatory cells, vacuolar degeneration, and necrosis in some hepatocytes.

Effects of AFB1 Exposure on Liver Oxidative Damage
A decrease in liver function is related to oxidative stress [15]. Measurements were taken of the concentrations of MDA and T-AOC and the activities of antioxidant enzymes (CAT, SOD). There was no significant difference in MDA, SOD, CAT, and T-AOC levels between the two groups (p > 0.05; Figure 2A-D). Notably, MDA concentration and SOD activity showed an increasing and decreasing trend compared with the control group, respectively. Protein and mRNA levels typically show reasonable correlation [16], so we estimated the expression of SOD-1 and SOD-2 genes in liver tissues. SOD-1 and SOD-2 levels in the AFB1 group were considerably lower than those in the control group (p < 0.001; Figure 2E,F).

Effects of AFB 1 Exposure on Liver Oxidative Damage
A decrease in liver function is related to oxidative stress [15]. Measurements were taken of the concentrations of MDA and T-AOC and the activities of antioxidant enzymes (CAT, SOD). There was no significant difference in MDA, SOD, CAT, and T-AOC levels between the two groups (p > 0.05; Figure 2A-D). Notably, MDA concentration and SOD activity showed an increasing and decreasing trend compared with the control group, respectively. Protein and mRNA levels typically show reasonable correlation [16], so we estimated the expression of SOD-1 and SOD-2 genes in liver tissues. SOD-1 and SOD-2 levels in the AFB 1 group were considerably lower than those in the control group (p < 0.001; Figure 2E,F).

Effects of AFB1 Exposure on the Expression of Inflammation-Related Factors in the Liver
To further investigate the effects of AFB1 on liver inflammation, we examined the expression of the inflammatory cytokines TNF-α, IL-6, and IL-1β and anti-inflammatory cytokine IL-10 in the liver tissues of sheep in each group ( Figure 3A-D). The results indicate that the AFB1 group had significantly higher levels of IL-1β and IL-6 expression than those of the control group (p = 0.021 and 0.003, respectively). Furthermore, IL-10 expression was considerably lower in the AFB1 group (p = 0.034), although TNF-α expression did not change significantly (p = 0.641). Pearson correlation analysis revealed that IL-6 gene expression was positively correlated to serum TBIL levels (r = 0.798 and p = 0.01; Figure  3E).

Effects of AFB 1 Exposure on the Expression of Inflammation-Related Factors in the Liver
To further investigate the effects of AFB 1 on liver inflammation, we examined the expression of the inflammatory cytokines TNF-α, IL-6, and IL-1β and anti-inflammatory cytokine IL-10 in the liver tissues of sheep in each group ( Figure 3A-D). The results indicate that the AFB 1 group had significantly higher levels of IL-1β and IL-6 expression than those of the control group (p = 0.021 and 0.003, respectively). Furthermore, IL-10 expression was considerably lower in the AFB 1 group (p = 0.034), although TNF-α expression did not change significantly (p = 0.641). Pearson correlation analysis revealed that IL-6 gene expression was positively correlated to serum TBIL levels (r = 0.798 and p = 0.01; Figure 3E).

Effects of AFB1 Exposure on the Expression of Inflammation-Related Factors in the Liver
To further investigate the effects of AFB1 on liver inflammation, we examined the expression of the inflammatory cytokines TNF-α, IL-6, and IL-1β and anti-inflammatory cytokine IL-10 in the liver tissues of sheep in each group ( Figure 3A-D). The results indicate that the AFB1 group had significantly higher levels of IL-1β and IL-6 expression than those of the control group (p = 0.021 and 0.003, respectively). Furthermore, IL-10 expression was considerably lower in the AFB1 group (p = 0.034), although TNF-α expression did not change significantly (p = 0.641). Pearson correlation analysis revealed that IL-6 gene expression was positively correlated to serum TBIL levels (r = 0.798 and p = 0.01; Figure  3E).

Effects of AFB 1 Exposure on Liver Cell Apoptosis and Apoptosis-Related Gene Expression
TUNEL staining in the liver of sheep in each group is shown in Figure 4A,B. The AFB 1 group had a considerably higher positive rate of TUNEL than the control group. We also detected the mRNA expression of Caspase-3, Bax, and Bcl-2 in the hepatocytes. As seen in Figure 4C,D, the Bcl-2/Bax ratio was reduced dramatically (p < 0.001) in the AFB 1 group compared with the control group, whereas Caspase-3 gene expression increased significantly (p = 0.002).
IL-6, and IL-10 mRNA levels detected in liver. (E) Heat map showing the correlation between indicators of liver function and levels of inflammatory gene expression. * p < 0.05, ** p < 0.01.

Effects of AFB1 Exposure on Liver Cell Apoptosis and Apoptosis-Related Gene Expression.
TUNEL staining in the liver of sheep in each group is shown in Figure 4A,B. The AFB1 group had a considerably higher positive rate of TUNEL than the control group. We also detected the mRNA expression of Caspase-3, Bax, and Bcl-2 in the hepatocytes. As seen in Figure 4C,D, the Bcl-2/Bax ratio was reduced dramatically (p < 0.001) in the AFB1 group compared with the control group, whereas Caspase-3 gene expression increased significantly (p = 0.002).

Changes in Gut Microbiota Induced by AFB1 Exposure
An increasing number of studies show that changes in gut microbiota composition and function are crucial for liver health [17]. To study how the composition of gut microbiota changed after AFB1 exposure, 16S rRNA gene sequencing was performed. The AFB1 and control groups had coverage rates of 98.39% and 96.46%, respectively, indicating that the majority of the gut microbiota diversity was detected ( Figure 5A). In comparison to the control group, the observed species, Shannon, Simpson, and Good's coverage indexes in the AFB1 group were reduced (p = 0.39, 0.021, 0.021, and 0.25, respectively; Figure 5A). The gut microbiota composition showed a trend of relative separation (p = 0.06) for Beta According to the Pearson correlation analysis, AKP (tissue) was negatively correlated with the Bcl-2/Bax ratio (r = −0.762 and p = 0.004) and positively correlated with the Caspase-3 gene (r = 0.730 and p = 0.07; Figure 4E). The Bcl-2/Bax ratio and serum TBIL level had a negative correlation (r = −0.883 and p = 0.002; Figure 4E).

Changes in Gut Microbiota Induced by AFB 1 Exposure
An increasing number of studies show that changes in gut microbiota composition and function are crucial for liver health [17]. To study how the composition of gut microbiota changed after AFB 1 exposure, 16S rRNA gene sequencing was performed. The AFB1 and control groups had coverage rates of 98.39% and 96.46%, respectively, indicating that the majority of the gut microbiota diversity was detected ( Figure 5A). In comparison to the control group, the observed species, Shannon, Simpson, and Good's coverage indexes in the AFB1 group were reduced (p = 0.39, 0.021, 0.021, and 0.25, respectively; Figure 5A). The gut microbiota composition showed a trend of relative separation (p = 0.06) for Beta diversity between the control and AFB 1 groups (PCo1 contribution of 27.80%, PCo2 contribution of 22.80%; Figure 5B). A total of 2204 ASVs were found in both groups, with 9768 and 6632 ASVs found in the control and AFB 1 groups, respectively ( Figure 5C). Firmicutes, Bacteroidetes, Spirochaetes, Verrucomicrobia, and Proteobacteria were the top five abundant phyla in both groups, as shown in Figure 5D. Firmicutes, Spirochaetes, Verrucomicrobia, and Proteobacteria were more abundant in the AFB 1 group (73.49%, 0.96%, 0.99%, and 0.61%, respectively) than in the control group (72.23%, 0.56%, 0.49%, and 0.56%, respectively), while Bacteroidetes were more abundant in the control group (24.32%) than in the AFB 1 group (23.01%; Figure 5D-I). The Firmicutes/Bacteroides ratio (F/B ratio) was higher in the AFB 1 group than in the control group, but the difference was not significant (p = 0.564; Figure 5J).
The top 30 most important genera were screened using random forest analysis, and the most important species at the genus level were BF311 spp., Roseburia spp., and Butyrivibrio spp. (Figure 6A). Thirty-three characteristic species were identified using a Venn diagram analysis of the LEfSe results (LDA score > 2; p < 0.05), and 10 genera were screened out from the top 30 genera based on significance scores ( Figure 6B).

Discussion
Body temperature, breathing rate, and heart rate are important indicators of physical health [18]. In this study, the body temperatures of sheep increased and respiration and heart rates decreased after intake of AFB1. AFB1 exposure-induced gastrointestinal and neurological symptoms were also observed in this study. These results suggest that AFB1 exposure affects the health of sheep.
Jaundice is an important sign of liver dysfunction [19]. As AFB1 targets the liver, exposure to AFB1 may cause jaundice and change the color of the conjunctiva. Therefore, we measured conjunctival color and noted that AFB1 exposure significantly reduced the L values. However, no significant difference in b and a values was detected between the two groups, which could be attributed to the short-term exposure of the animals to AFB1 in this study.

Discussion
Body temperature, breathing rate, and heart rate are important indicators of physical health [18]. In this study, the body temperatures of sheep increased and respiration and heart rates decreased after intake of AFB 1 . AFB 1 exposure-induced gastrointestinal and neurological symptoms were also observed in this study. These results suggest that AFB 1 exposure affects the health of sheep.
Jaundice is an important sign of liver dysfunction [19]. As AFB 1 targets the liver, exposure to AFB 1 may cause jaundice and change the color of the conjunctiva. Therefore, we measured conjunctival color and noted that AFB 1 exposure significantly reduced the L values. However, no significant difference in b and a values was detected between the two groups, which could be attributed to the short-term exposure of the animals to AFB 1 in this study. The activities of serum enzymes such as ALT and AST, as well as TP, ALB, TBIL, and GLO concentration, have been defined as critical indicators of liver injury and function [20][21][22]. In this investigation, AFB 1 exposure significantly increased the serum TBIL content of sheep, while the levels of serum TP, ALB, GLO, and A/G tended to decrease, and AST and ALT contents tended to increase. These phenomena suggest that exposure to AFB 1 may harm the liver and impair liver function in sheep [23]. BUN and CRE are the main biochemical indexes of renal function, which increase with the aggravation of renal injury [24]. In this study, BUN and CRE levels tended to increase slightly, which may have been caused by the short acting time of AFB 1 in this study. Ca 2+ is an important signaling molecule involved in numerous cellular processes, and its content decreases with the aggravation of liver and kidney diseases [25]. AFB 1 exposure significantly decreased the serum Ca content in sheep, indicating the possibility of liver damage. We next assessed liver tissue to explore whether AFB 1 exposure would negatively affect sheep liver tissue. AFB 1 exposure resulted in hepatocyte degeneration and considerable inflammatory cell infiltration, suggesting liver tissue damage and corroborating the results of Tsiouris et al. (2021) [26]. In general, elevated ALT, AST, and AKP levels indicate liver dysfunction, which is critical for the differential diagnosis of liver diseases [27]. In the present study, AFB 1 exposure increased ALT, AST, and AKP levels in the liver tissue of sheep compared with those of the control group, reflecting the negative impact of AFB 1 exposure on liver function.
Numerous studies have demonstrated that AFB 1 increases lipid peroxidation and induces the formation of high levels of reactive oxygen species and free radicals, damaging body organs through oxidative stress [28,29]. Since MDA is a byproduct of lipid peroxidation, the level of lipid peroxidation can be inferred from the MDA content in living things [30,31]. In this study, the MDA content increased slightly, indicating liver tissue injury. Antioxidant markers, including SOD, CAT, and T-AOC, are known to play key roles in attenuating oxidative stress by scavenging reactive oxygen species [32]. Although SOD and T-AOC levels were both reduced in the liver of sheep exposed to AFB 1 compared to those of the control group, the difference was not significant. The activity of SOD, a particular antioxidant enzyme that neutralizes superoxide anions in ROS free radicals, indirectly reflects the capacity to neutralize oxygen free radicals and is crucial in liver injury [33]. Further examination of gene expression revealed that SOD-1 and SOD-2 levels were significantly decreased. However, an apparent increase in the CAT content was observed in the present study, which may have resulted from the defense mechanism of the body against AFB 1 toxicity [8,34]. In this context, Cao et al. (2021) discovered that AFB 1 intoxication significantly increases CAT activity in sheep exposed with AFB 1 [8]. In addition, the correlation between liver function and antioxidant enzyme activity revealed that AFB 1 exposure causes a decrease in liver function related to oxidative damage.
The inflammatory response is an important mechanism in AFB 1 toxicity [35]. Oxidative stress can increase the production of different types of ROS in the body, and ROS can activate the nuclear factor-kappa B (NF-κB) pathway, leading to the production of inflammatory cytokines [36]. TNF-α is the most prominent pro-inflammatory cytokine involved in the activation of NF-κB, which induces the expression of IL-1β, IL-6, and other downstream inflammatory mediators [37]. AFB 1 significantly increased the expression of the inflammatory factors IL-1β and IL-6, according to our findings. Furthermore, the anti-inflammatory IL-10 gene, which is linked to liver function, was drastically downregulated. The changes in the inflammatory factor expression levels in the liver tissue imply that exposure to AFB 1 causes inflammatory injury to the liver.
It has been reported that AFB 1 destroys the integrity of the cell membrane by stimulating phospholipids and inducing ROS formation [38]. When excessive ROS is produced and the scavenging capacity of the body decreases, it can lead to protein, DNA, and mitochondrial damage, thus inducing apoptosis [39]. Excessive apoptosis can lead to organ damage, which is considered one of the mechanisms of AFB 1 -induced toxicity [40]. Our findings showed that when compared with the control group, the AFB 1 group had a significantly higher rate of hepatocyte apoptosis detected using the TUNEL method. These results are similar with the findings of a prior study by Xu et al. (2021) [41]. The release of mitochondrial cytochrome c is affected by changes in the ratio of Bcl-2 to Bax expression in cells, and a decrease in this ratio results in apoptosis, according to studies conducted on mammals [42,43]. At the same time, caspase-3 is a common effector of apoptosis [44]. In this study, we discovered that after AFB 1 exposure, Bcl-2/Bax gene expression decreased while caspase-3 gene expression increased, indicating that AFB 1 exposure can promote liver cell apoptosis. In addition, the correlation between liver function and apoptosis showed that the decline in liver function caused by AFB 1 exposure is related to apoptosis.
Like most mycotoxins, AFB 1 not only directly damages body organs but also interferes with the normal activities of animal intestinal flora via enterohepatic circulation [7,8]. For example, long-term feeding of AFB 1 can significantly reduce most intestinal microbiota in mice [45], and microbiota decline was observed in the acute AFB 1 poisoning experiment in this study. Moreover, intestinal flora can combine, transform, degrade, and transfer mycotoxins; promote the healthy growth of livestock and poultry; and participate in material metabolism [46][47][48]. Through amplicon sequencing, we discovered that Firmicutes and Bacteroidetes were the two largest phyla that made up the sheep intestinal flora, which is consistent with earlier research on mammalian intestinal flora [49]. Firmicutes/Bacteroidetes ratios are typically correlated with inflammatory marker levels and pathological conditions of intestinal metabolic homeostasis [49]. AFB 1 exposure significantly raised the F/B ratio in this study, implying that AFB 1 exposure may disrupt gut metabolic homeostasis and alter inflammatory metabolite levels. At the same time, by screening at the genus level, we identified two genera that deserve attention: BF311 spp. and Alistipes spp. Although there are few studies on the function of BF311 spp., some suggest that BF311 spp. may play a crucial role in the rumen ecosystem and even in rumen synchronization [50]. As a relatively newly identified bacterial genus, Alistipes spp. has been shown to be associated with liver fibrosis, cardiovascular disease, cancer immunotherapy, cardiovascular disease, colitis, and depression [51]. The decrease of Alistipes spp. causes a reduction in short-chain fatty acids (SCFA), which further leads to a decrease in the levels of anti-inflammatory cytokines and decreased inhibition of Th17 cells, resulting in liver fibrosis and hepatocellular carcinoma [51]. Our results suggest that exposure to AFB 1 may lead to an increase in BF311 spp. and a decrease in Alistipes spp. populations, and the changes in these bacteria are related to liver dysfunction in sheep.

Conclusions
This study confirmed that oxidative stress, inflammatory injury, apoptosis, and gut microbiota are involved in the liver injury and liver dysfunction caused by AFB 1 exposure in sheep. The study also offers a valuable reference for future research into the mechanism underlying the hepatotoxic effects of aflatoxin on sheep.

Animals, Exposure Experiment
The study was mainly conducted at Henan Agricultural University's Xuchang practical teaching base, China. Twelve Dorper RAMS with an average body weight of 22.34 ± 5.07 kg were individually identified by ear tag and randomly divided into two groups of three replicates and two sheep each. The sheep were immunized according to routine procedures. The control group received 4% ethanol (20 mL) through gavage, while the AFB 1 group received AFB 1 (1 mg/kg, dissolved in 20 mL 4% ethanol) (half of LD50) orally [8]. Following the gavage of AFB 1 , there was a 24 h fast from food and water, and surgical sampling was carried out 24 h later. The animal care and experimental procedures were approved by the Institutional Animal Welfare and Research Ethics Committee of Henan Agricultural University's College of Veterinary Medicine (Zhengzhou, China) (Permit No: 17-0126, Year of approval: 2017). The experimental animals were kept under anesthesia during surgery and every effort was made to minimize their pain, suffering, and death.

The Color of Conjunctiva
A handheld colorimeter (#SR-62; Shenzhen 3nh Technology Co., Ltd., Shenzhen, China) was used to assess conjunctival color prior to before intragastric administration and surgical sampling. Reflectance spectrometry was used for the color determination using the CIELab method.

Sample Collection
Temperature, respiration, and heart rate were measured before and 24 h after intragastric administration. Rectal temperature and heart rate were measured using a mercuryin-glass thermometer (range 35-42 • C; accuracy ± 0.3) and stethoscope, respectively. By counting the movement of the abdominal muscles on both sides while breathing, the sheep's breathing rate per minute was calculated. Blood samples were drawn from the jugular vein; serum was isolated through centrifugation at 4 • C for 15 min at 3000× g and stored at −20 • C. Fecal samples were collected from the sheep rectum before surgery and kept at −80 • C. The anterior abdominal midline of umbilicus was selected as the surgical incision. The liver was exposed by laparotomy, about 2 cm × 3 cm × 3 cm hepatic lobules were collected for liver evaluation, and clamp hemostasis or electrocautery hemostasis was used. The wound was strictly aseptic debridement; each layer of abdominal wall was closed and wrapped with elastic bandage. Part of the liver tissue was cut into 1 cm 3 pieces and transferred in liquid nitrogen to the laboratory, where it was preserved at −80 • C for further analysis. The other portion was fixed for 24 h with 4% paraformaldehyde.

Serum Biochemistry
To determine the 16 biochemical indexes of the serum, a full-automatic biochemical analyzer (#SMT-120 V, Seamaty technology Co., Ltd., Chengdu, China) was used, and the reagent plate was obtained from Chengdu Polytech biological technology Co., Ltd., China. The following parameters were measured within 2 h: ALB, TP, CK, GLU, AMY, A/G, Ca, TBIL, AST, ALT, CRE, TG, P, BUN, and GLO.

Liver Histopathology
Paraformaldehyde-fixed liver tissue samples were washed and dehydrated in ethanol before being extracted with toluene and embedded in paraffin. For qualitative histological analysis, tissues were sectioned (5 µm) and stained with hematoxylin and eosin (H&E). Motic BA600-4 microscope was used to photograph the tissues (Motic, Xiamen, China).

Detection of Apoptosis
Paraffin slices of liver were dewaxed using xylene and anhydrous ethanol, then incubated in a 37 • C incubator for 22 min with protease K. TDT enzyme and dUTP were used to incubate in incubator at 37 • C for 2 h before dropping DAPI dye and incubating for 10 min at room temperature. A fluorescent microscope was used for photo imaging (Nikon Eclipse C1, Nikon, Japan). Apoptosis was indicated by green fluorescence.

Liver Function of Tissue
The liver tissue was homogenized to 10% by the dilution solution, and the activity of general marker enzymes like ALT, AST, and alkaline phosphatase (AKP) enzymes in liver tissue were assessed using kits (Nanjing Jiancheng Bioengineering Factory, Nanjing, China).

Liver Antioxidant Abilities
The activities of superoxide dismutase (SOD) and catalase (CAT), and the concentration of total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in the liver tissue supernatant were detected by the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Extraction of Total RNA and Quantitative Real-Time PCR Analysis
The CFX96 real-time PCR detection system was used for real-time quantitative PCR (Bio-Rad, Munich, Germany). TRIZOL reagent (Takara Biotechnology Co., Ltd., Dalian, China) was used to extract total RNA from liver tissue, and Superscript II reverse transcriptase was used to prepare first strand cDNA (Roche, Basel, Switzerland). SYBR Green I PCR Master Mix (Vazyme Biotech Co.,Ltd, Nanjing, China) was used to determine mRNA levels, which were then calculated using the 2 −∆∆CT method. Sangon Biotech (Shanghai, China) designed specific primers (Table S1) for inflammatory genes (TNF-α, IL-1β, IL-6, and IL-10), antioxidant genes (SOD-1, SOD-2), and apoptosis genes (Bcl-2, Bax, and Caspase-3) based on the NCBI database sequence (Table S1).

Extraction of Faecal DNA, PCR Amplification, and Illumina Sequencing
Following the manufacturer's instructions, total fecal DNA was extracted using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA). To amplify the V3-V4 region of the 16S rRNA gene, forward primer 338F and reverse primer 806R were used. QIIME2 dada2 and R package (v3.2.0) were primarily used for sequence data analysis. The National Center for Biotechnology Information Sequence Read Archive now contains the sequence information for the 16S rRNA gene obtained in our study (PRJNA844551).

Statistical Analysis
To ensure accuracy and reproducibility, every experiment was performed at least three times. Graphpad Prism (version 7.0) was used to analyze all of the data, which was expressed as means ± standard deviation (SD). The significance of any differences between the experimental groups was assessed using Student's t-test. Statistics were considered significant at p < 0.05.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/toxins14120840/s1, Table S1: Primer sequences for qPCR analysis; Table S2: Effects of AFB 1 exposure on body temperature, breathing, and heart rate of sheep; Table S3: Effects of AFB 1 exposure on serum biochemistry of sheep. Figure S1: Effects of AFB 1 exposure on conjunctival color.

Informed Consent Statement: Not applicable.
Data Availability Statement: The National Center for Biotechnology Information Sequence Read Archive now contains the sequence information for the 16S rRNA gene obtained in our study (PRJNA844551).

Conflicts of Interest:
The authors declare no conflict of interest.