Animals
A total of 180 1-day-old AA broilers weighed 55.67 ± 1.43 g was purchased from Shanxi Elephant Agriculture and Animal Husbandry Group Co., Ltd, and randomly divided into two groups, control group and DON groups. Control group received basal diet and DON group received 10 mg/kg DON contaminated basal diet respectively, the ingredients and composition of the basal diets were as follows (Table 5). All broilers were free drinking, immunized with routine vaccines and had routinely feeding management.
Table 5
Ingredients and nutrient composition of the basal diets
Ingredients | Percentage (%) | Nutrient composition | Percentage (%) |
Corn | 61.17 | Metabolism energy, (MJ/kg) | 12.97 |
Soybean meal | 29.50 | Crude protein (%) | 20.8 |
Fishmeal | 6.50 | Available P (%) | 0.45 |
DL-Met | 0.19 | Ca (%) | 1.02 |
L-Lys•HCl | 0.05 | Lys (%) | 1.20 |
Bone Meal | 1.22 | Met + Cys (%) | 0.86 |
Sodium chloride | 0.37 | | |
Microelement and Vitamin Compound Premix | 1.00 | | |
Total | 100 | | |
Premix can provide per kilogram of basal diet, Vitamin A 9500 IU, Vitamin E 30 IU, Vitamin D3 62.5 µg, Vitamin K3 2.65 mg, Vitamin B1 2 mg, Vitamin B2 6 mg, Vitamin B12 0.025 mg, Biological Element C 0.0325 mg, folic acid 1.25 mg, pantothenic acid 12 mg, niacin 50 mg, copper 8 mg, zinc 80 mg, manganese 80 mg, iodine 0.35 mg, selenium 0.3 mg.
Reagents And Antibodies
Amylase (C016-1-1), Trypsin (A080-2-2) and Lipase (A054-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent was obtained from Tiangen Biotech Co., Ltd. (Beijing, China). Reverse transcription kit from TaKaRa (Japan), 2×SYBR Green qPCR Master Mix from Bimake (Houston, USA), RIPA cell lysate, protease inhibitor and phosphatase inhibitor were purchased from Solarbio (Beijing, China). β-actin Monoclonal Antibody was purchased from Immunoway Bitechnology company (Jiangsu, China). ZO-1(ab96587) was obtained from Abcam (Cambridge, MA, USA). Occludin (bs-10011R) was obtained from Bioss (Wuhan, China). Claudin-1 (13050-1-AP) was obtained from Proteintech (Wuhan, China) and DAB kit was purchased from CWBIO (Beijing, China). Fusarium graminearum ACCC 37687 was provided by Professor Yang Xiaojun, College of Animal Science and Technology, Northwest A&F University. AA broilers were purchased from Shanxi Elephant Farming and animal husbandry group (China).
Preparation Of Don Contaminated Feed
According to the methods described by Yu [10] and Yang [11], Fusarium graminearum ACCC 37687 was inoculated into potato dextrose agar medium and cultured at 27°C for 7 d to obtain a solid culture of Fusarium graminearum. Post inoculation, the medium was incubated at 27°C and 180 rpm for 7 d to obtain a liquid culture of Fusarium graminearum. 25 g of solid culture of Fusarium graminearum was inoculated into 100 mL of liquid culture and mixed with 200 g of rice, then further cultivated for 28 d to obtain DON-contaminated fragrant rice. The crushed DON contaminated rice was added to normal feed in a ratio of 10 mg/kg.
Intestinal Injury Score Of Broilers
According to the method of the previous studies [36, 37], the small intestine of broilers was scored by gross morphology and histomorphology.
Scanning Electron Microscopy
The tissue samples were fixed in glutaraldehyde by conventional method [38], and observed under scanning electron microscope (JEM-6490LV, JEOL, Japan).
Histopathological Observation Of Small Intestine
The duodenum, jejunum, and ileum were fixed in Bouin’s solution for 24 hours, tissues were sectioned, stained with H&E, and mounted with neutral gum. The histological changes in intestinal tissue were observed with a microscope. Image J software (National Institutes of Health, USA) was used to measure the villus height (V) and crypt depth (C) and the villi/crypt ratio (V/C) were calculated.
Detection Of Small Intestine Tight Junction Proteins
The total RNA and proteins of the duodenum, jejunum and ileum samples were extracted, qRT-PCR and WB were used to detect the expression of ZO-1, Occludin and Claudin-1 in each intestinal segment, and the 2-△△ct method and Image J software were used to calculate the relative expression of ZO-1, Occludin and Claudin-1 mRNA and protein level, respectively [39–41]. Immunohistochemical methods were used to detect the localization of ZO-1, Occludin and Claudin-1 in the duodenum, jejunum and ileum. The optical density was measured by Image Pro Plus software.
Determination of Lipase, Amylase and Trypsin of the small intestine
The collected duodenum, jejunum and ileum were ground, and then the lipase, amylase, and trypsin of mucosal homogenates in the duodenum, jejunum, and ileum were measured using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
High-throughput Sequencing Of Cecal Intestinal Flora
The 16S rDNA high-throughput sequencing method was used to detect the bacterial flora in the cecum by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).
Growth Performance
Broilers in both groups were fed for 7 d and weighed on fasting basis. The average daily gain (ADG, G), the average daily feed intake (ADFI, F) and F/G were calculated.
Statistical analysis
All data were expressed as mean standard error of the mean (Mean SEM). The differences among groups were analyzed by t-test using Graphpad Prism 5 (Graphpad Software, USA), *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant.