Chicken fatty liver syndrome grading method based on oil red O staining
By combining paraffin section H&E staining and frozen section oil red O staining, the problem of low grading efficiency of chicken fatty liver syndrome was solved, rapid and accurate quantitative grading was achieved, and a grading standard based on oil red O staining was established.
Patent Information
- Application Number
- CN202510414147.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art cannot efficiently and quickly quantify the severity of chicken fatty liver syndrome. The H&E staining method for paraffin sections is cumbersome and time-consuming, and the oil-red O staining method lacks corresponding grading standards.
The combined paraffin section H&E staining method and frozen section oil red O staining method were used to collect images through a microscope imaging system to quantify the degree of liver steatosis, establish a grading standard for chicken fatty liver syndrome based on oil red O staining, and use the oil red O staining method to identify the degree of liver steatosis.
The rapid and accurate quantitative grading of chicken fatty liver syndrome has been achieved, and the efficiency and accuracy have been improved, providing reference for related research.
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Figure CN120253390A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of poultry genetic breeding, and particularly relates to a grading method for chicken fatty liver syndrome based on Oil Red O staining. Background Art
[0002] Chicken fatty liver syndrome (FLS) is a disease caused by abnormal accumulation of fat due to disordered lipid metabolism in the liver. Chicken fatty liver syndrome affects the egg-laying performance of hens, and in severe cases, it can lead to sudden death of chickens, causing huge economic losses to the poultry industry. The histological and pathological characteristics of chicken fatty liver syndrome are that more than 5% of hepatocytes show hepatic steatosis (excessive accumulation of lipid droplets in hepatocytes). At present, the evaluation of the severity of chicken fatty liver syndrome mainly refers to the identification method of human non-alcoholic fatty liver disease (NAFLD), that is, the degree of hepatic steatosis (the percentage of the area of lipid droplets in the section field accounting for the entire field area) is quantitatively analyzed and graded by the method of H&E staining of paraffin sections of liver tissue: FLS-0 represents a healthy liver without lipid droplet accumulation (<5% hepatic steatosis); FLS-1 represents mild lipid droplet accumulation in the liver (5%-33% hepatic steatosis); FLS-2 represents moderate lipid droplet accumulation in the liver (33%-66% hepatic steatosis); FLS-3 represents severe lipid droplet accumulation in the liver (>66% hepatic steatosis). However, there are certain defects in grading chicken fatty liver syndrome by H&E staining of paraffin sections. For example, the preparation process of paraffin sections is cumbersome, and it takes a long time to quantitatively analyze the degree of hepatic steatosis. Therefore, it is necessary to establish an efficient method for quantitatively grading the severity of chicken fatty liver syndrome. Compared with paraffin sections, the preparation process of frozen sections is simple. When quantitatively analyzing the degree of hepatic steatosis by Oil Red O staining, it is not necessary to identify each lipid droplet (the white vacuoles in hepatocytes in H&E staining) one by one, and only the whole lipid droplets (the red-stained area of liver tissue in Oil Red O staining) need to be identified as a whole. Therefore, compared with the paraffin section H&E method, the efficiency of identifying chicken fatty liver syndrome by Oil Red O staining of frozen sections is higher. However, so far, due to the lack of corresponding grading criteria, the research on identifying chicken fatty liver syndrome by Oil Red O staining only makes a preliminary qualitative evaluation of whether the detected chickens suffer from fatty liver syndrome, and cannot quantitatively grade the severity of chicken fatty liver syndrome. Summary of the Invention
[0003] To solve the above problems, the present invention provides a grading method for chicken fatty liver syndrome based on Oil Red O staining, which solves the problem that the prior art cannot efficiently and quickly quantitatively grade the severity of chicken fatty liver.
[0004] The technical solution adopted by the present invention is as follows: a chicken fatty liver syndrome grading method based on Oil Red O staining, characterized in that the method is as follows:
[0005] S1. Preparation of paraffin sections of chicken liver tissue, H&E staining, observation of lipid droplets and quantification of liver fatty degeneration: Three paraffin sections were prepared for each chicken and H&E staining was performed. Three fields of view were randomly selected for each paraffin section, and images were collected using a microscope imaging system. The degree of liver fatty degeneration was quantified for each field of view, that is, the percentage of lipid droplet area to the field of view of the paraffin section. With reference to the identification method and grading standard of human NAFLD, the grade of chicken fatty liver syndrome was defined according to the degree of liver fatty degeneration: FLS-0 represents a healthy liver without lipid accumulation, that is, <5% liver fatty degeneration; FLS-1 represents mild liver fat accumulation, that is, 5%-33% liver fatty degeneration; FLS-2 represents moderate liver fat accumulation, that is, 33%-66% liver fatty degeneration;
[0006] S2. Preparation of frozen sections of chicken liver tissue and Oil Red O staining: The liver tissue was fixed on the sample holder with OCT embedding glue, sliced after freezing, and stored in cryopreservation; the liver tissue was stained with Oil Red O working solution in the dark, then washed, and then stained in hematoxylin; then the frozen Oil Red O stained sections of the chicken liver tissue were observed for lipid droplets and the degree of liver fatty degeneration was quantified: 3 frozen sections were prepared for each chicken, 3 fields of view were randomly selected for each frozen section, and images were collected using a microscope imaging system. The degree of liver fatty degeneration was quantified for each field of view, that is, the ratio of the pixel value of the red area to the pixel value of the entire image was the degree of liver fatty degeneration;
[0007] S3. Based on the grading results of fatty liver syndrome in paraffin sections stained with H&E as the benchmark, the ranges of quantitative results of frozen sections stained with Oil Red O corresponding to different FLS grades of high- and low-fat hens were counted, and the FLS grading standard based on Oil Red O staining was established, as shown in Table 1: Oil Red O staining quantitative results less than 10% were defined as no fatty liver syndrome, i.e., FLS-0; Oil Red O staining quantitative results between 10% and 34% were defined as mild fatty liver syndrome, i.e., FLS-1; Oil Red O staining quantitative results greater than 34% were defined as moderate fatty liver syndrome and above, i.e., FLS-2;
[0008] Table 1 Grading criteria for chicken fatty liver syndrome based on Oil Red O-stained sections
[0009]
[0010] Advantages and beneficial effects of the present invention: The present invention is more efficient and more accurate in quantifying the degree of fatty degeneration of the liver based on frozen section Oil Red O staining, can quickly quantitatively grade the severity of chicken fatty liver syndrome, and provide a reference for related research. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 These are representative pictures of the identification of high- and low-fat line hens (FLS) at 27 weeks, 35 weeks, and 53 weeks of age by the paraffin section H&E staining method in the present invention; among them, A) Representative pictures of H&E staining of liver tissue sections of high- and low-fat lines at 27 weeks of age, scale bar: 50 μm. B) Representative pictures of H&E staining of liver tissue sections of high- and low-fat lines at 35 weeks of age, scale bar: 50 μm. C) Representative pictures of H&E staining of liver tissue sections of high- and low-fat lines at 53 weeks of age, scale bar: 50 μm. 27W, 27 weeks of age; 35W, 35 weeks of age; 53W, 53 weeks of age. L-FLS-0, low-fat line hens without FLS; L-FLS-1, low-fat line hens with mild FLS; L-FLS-2, low-fat line hens with moderate FLS; F-FLS-0, high-fat line hens without FLS; F-FLS-1, high-fat line hens with mild FLS; F-FLS-2, high-fat line hens with moderate FLS;
[0012] Figure 2 These are the result pictures of quantifying the degree of liver steatosis by H&E staining in the present invention; among them, A) Quantitative results of H&E staining of the low-fat line at 27 weeks of age. FLS-0, low-fat line hens without FLS (n = 33); FLS-1, low-fat line hens with mild FLS (n = 5). B) Quantitative results of H&E staining of the high-fat line at 27 weeks of age. FLS-0, high-fat line hens without FLS (n = 13); FLS-1, high-fat line hens with mild FLS (n = 22); FLS-2, high-fat line hens with moderate FLS (n = 4). C) Quantitative results of H&E staining of the low-fat line at 35 weeks of age. FLS-0, low-fat line hens without FLS (n = 33); FLS-1, low-fat line hens with mild FLS (n = 5). D) Quantitative results of H&E staining of the high-fat line at 35 weeks of age. FLS-0, high-fat line hens without FLS (n = 10); FLS-1, high-fat line hens with mild FLS (n = 43); FLS-2, high-fat line hens with moderate FLS (n = 3). E) Quantitative results of H&E staining of the low-fat line at 53 weeks of age. FLS-0, low-fat line hens without FLS (n = 24); FLS-1, low-fat line hens with mild FLS (n = 14); FLS-2, low-fat line hens with moderate FLS (n = 2). F) Quantitative results of H&E staining of the high-fat line at 53 weeks of age. FLS-1, high-fat line hens with mild FLS (n = 24); FLS-2, high-fat line hens with moderate FLS (n = 15). Different capital letters indicate significant differences between different grades of FLS (P < 0.01). The results are expressed as least squares means ± standard error. 27W, 27 weeks of age; 35W, 35 weeks of age; 53W, 53 weeks of age;
[0013] Figure 3These are representative pictures of the frozen sections of the liver tissues of high- and low-fat line hens at 27 weeks, 35 weeks, and 53 weeks of age stained by the Oil Red O staining method of the present invention; among them, A) Representative pictures of the frozen sections of the liver tissues of high- and low-fat lines at 27 weeks of age stained by Oil Red O, scale bar: 50 μm. B) Representative pictures of the frozen sections of the liver tissues of high- and low-fat lines at 35 weeks of age stained by Oil Red O, scale bar: 50 μm. C) Representative pictures of the frozen sections of the liver tissues of high- and low-fat lines at 53 weeks of age stained by Oil Red O, scale bar: 50 μm. 27W, 27 weeks of age; 35W, 35 weeks of age; 53W, 53 weeks of age. L-FLS-0, low-fat line hens without FLS; L-FLS-1, low-fat line hens with mild FLS; L-FLS-2, low-fat line hens with moderate FLS; F-FLS-0, high-fat line hens without FLS; F-FLS-1, high-fat line hens with mild FLS; F-FLS-2, high-fat line hens with moderate FLS;
[0014] Figure 4 These are the result graphs of the present invention for quantifying the degree of hepatic steatosis by Oil Red O staining; among them, A) Quantitative results of Oil Red O staining for the low-fat line at 27 weeks of age. FLS-0, low-fat line hens without FLS (n = 32); FLS-1, low-fat line hens with mild FLS (n = 5). B) Quantitative results of Oil Red O staining for the high-fat line at 27 weeks of age. FLS-0, high-fat line hens without FLS (n = 13); FLS-1, high-fat line hens with mild FLS (n = 22); FLS-2, high-fat line hens with moderate FLS (n = 4). C) Quantitative results of Oil Red O staining for the low-fat line at 35 weeks of age. FLS-0, low-fat line hens without FLS (n = 33); FLS-1, low-fat line hens with mild FLS (n = 5). D) Quantitative results of Oil Red O staining for the high-fat line at 35 weeks of age. FLS-0, high-fat line hens without FLS (n = 10); FLS-1, high-fat line hens with mild FLS (n = 43); FLS-2, high-fat line hens with moderate FLS (n = 3). E) Quantitative results of Oil Red O staining for the low-fat line at 53 weeks of age. FLS-0, low-fat line hens without FLS (n = 24); FLS-1, low-fat line hens with mild FLS (n = 14); FLS-2, low-fat line hens with moderate FLS (n = 2). F) Quantitative results of Oil Red O staining for the high-fat line at 53 weeks of age. FLS-1, high-fat line hens with mild FLS (n = 24); FLS-2, high-fat line hens with moderate FLS (n = 15). Different capital letters indicate significant differences between different grades of FLS (P < 0.01). The results are expressed as least squares means ± standard error. 27W, 27 weeks of age; 35W, 35 weeks of age; 53W, 53 weeks of age;
[0015] Figure 5 These are the result graphs of the correlation analysis between the degree of hepatic steatosis quantified by H&E staining and the degree of hepatic steatosis quantified by Oil Red O staining. Detailed implementation manners
[0016] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments. The present invention uses AA broilers as the basic population, and selects bidirectionally with the abdominal fat percentage (AFP) at 7 weeks of age and plasma very low density lipoprotein (VLDL) as the selection indicators to cultivate a broiler abdominal fat bidirectional selection line (hereinafter referred to as "high- and low-fat lines"). The high- and low-fat line broilers are a genetic model. After being selected to the 28th generation, starting from the 4th generation, there is a significant difference in AFP between the two lines. The AFP of the high-fat line at 7 weeks of age in the 28th generation is 17.32 times that of the low-fat line. The long-term selection of AFP will change the liver fat content, and the liver fat content of the high-fat line broilers is significantly higher than that of the low-fat line broilers. Since the increase in liver fat content is one of the main clinical features of FLS, the high- and low-fat line broilers can be used as an animal model for conducting research related to FLS.
[0017] Example 1
[0018] Identification of fatty liver syndrome in high- and low-fat line hens
[0019] I. Experimental materials
[0020] Using 249 hens of the broiler abdominal fat bidirectional selection line cultivated by Northeast Agricultural University as materials, among which the number of hens used at 27 weeks of age is 76 (39 in the high-fat line and 37 in the low-fat line), the number of hens used at 35 weeks of age is 94 (56 in the high-fat line and 38 in the low-fat line), and the number of hens used at 53 weeks of age is 79 (39 in the high-fat line and 40 in the low-fat line).
[0021] II. Preparation of paraffin sections of liver tissue and H&E staining
[0022] (1) Treatment and sectioning of liver tissue samples: The collected liver tissue was washed in physiological saline and then fixed in 4% paraformaldehyde solution. After 7 days, the fixed tissue was taken out, trimmed and placed in an embedding cassette, and rinsed under running tap water for 12 h. After the water rinsing was completed, the water on the surface of the tissue was blotted dry with filter paper, and the tissue was successively dehydrated in 30% (1 h), 50% (1 h), 70% (1 h), 80% (1 h), 95% (1 h), and 100% (2 h) alcohol. After dehydration, the tissue was successively placed in anhydrous ethanol / xylene (1:1) and xylene solution for 30 min each. After clearing, the tissue was placed in paraffin melted at 60 °C and allowed to stand for 2 h, then embedded and sectioned (5 μm). The sections were fully spread on a spreading machine at 42 °C, picked up on glass slides and dried on a baking machine at 37 °C, and stored in a section box.
[0023] (2) Staining and mounting: Place the prepared sections on a slide rack and put them in an oven at 60 °C for 1 h, and stain them using an H&E (hematoxylin-eosin) staining kit. The specific steps are as follows: First, dewax and hydrate the paraffin sections, and sequentially place them in xylene (20 min), absolute ethanol (3 min), 95% ethanol (3 min), 80% ethanol (3 min), 70% ethanol (3 min), and distilled water (2 min); stain with hematoxylin solution for 4 min and rinse with tap water for 1 min; differentiate with the differentiating solution for 3 min; after soaking in tap water for 4 min, place in eosin solution for 2 min, rinse with tap water for 2 - 3 s, and sequentially place in 70% ethanol I (2 - 3 s), 80% ethanol II (2 - 3 s), 95% ethanol (2 - 3 s), absolute ethanol (2 - 3 s), and xylene (2 min) for dehydration and clearing; drop neutral resin on the cleared sections, cover with a coverslip for mounting.
[0024] (3) Observation of lipid droplets and quantification of liver steatosis degree: Prepare 3 sections for each chicken, randomly select 3 fields of view for each section, and collect images using a Nikon microscope imaging system. Calculate the liver steatosis degree for each field of view using Photoshop software, that is, the percentage of the area of lipid droplets (white vacuoles) in the area of the section field of view. Specifically, use the magic wand tool in Photoshop software to continuously select the area where the white vacuoles are located. After all selections for each field of view are completed, the ratio of the pixel value of the area of the white vacuoles to the pixel value of the entire section field of view is the liver steatosis degree. The present invention refers to the identification method and grading standard of human NAFLD, and defines the grades of chicken FLS according to the liver steatosis degree: FLS-0 represents a healthy liver without lipid accumulation (<5% liver steatosis); FLS-1 represents mild fat accumulation in the liver (5% - 33% liver steatosis); FLS-2 represents moderate fat accumulation in the liver (33% - 66% liver steatosis); FLS-3 represents severe fat accumulation in the liver (>66% liver steatosis).
[0025] (4) Analyzed the liver steatosis degree of high- and low-fat line hens at 27, 35, and 53 weeks of age using the H&E staining method for paraffin sections of liver tissue, and identified the FLS grade of each chicken according to the human NAFLD grading standard. The results showed that at 27 and 35 weeks of age, the low-fat line only had individuals with FLS-0 (not suffering from FLS) and FLS-1 (mild FLS), and in addition to individuals with FLS-0 and FLS-1, the high-fat line also had individuals with FLS-2 (moderate FLS); at 53 weeks of age, the low-fat line had individuals with FLS-0, FLS-1, and FLS-2, and the high-fat line only had individuals with FLS-1 and FLS-2 (Table 2). The H&E staining results showed that the hepatic cords of FLS-0 hens were arranged regularly and there were no obvious lipid droplets in the cytoplasm (Figure 1 ) In hens with FLS, the hepatic cords were disordered, there were lipid droplets of different sizes in the cytoplasm, the cell nucleus was squeezed to one side, and the lipid droplets in the hepatocytes of hens with FLS grade 2 were larger and more numerous than those in hens with FLS grade 1. Figure 1 ) The results of quantitative analysis by H&E staining showed that in both the low-fat line and the high-fat line, at each week of age, the degree of hepatic steatosis increased significantly with the increase of FLS grade (P<0.01, Figure 2 ) These results indicate that we successfully quantified the severity of FLS in high- and low-fat line hens using the H&E staining method.
[0026] Table 2 Identification of fatty liver syndrome in high- and low-fat line hens at 27, 35, and 53 weeks of age
[0027]
[0028]
[0029] Example 2
[0030] Establishment of a grading standard for chicken fatty liver syndrome based on Oil Red O staining
[0031] I. Experimental materials
[0032] Using 249 hens from the bidirectional selected line of abdominal fat in broilers cultivated by Northeast Agricultural University as materials, among which the number of hens used at 27 weeks of age was 76 (39 in the high-fat line and 37 in the low-fat line), the number of hens used at 35 weeks of age was 94 (56 in the high-fat line and 38 in the low-fat line), and the number of hens used at 53 weeks of age was 79 (39 in the high-fat line and 40 in the low-fat line).
[0033] II. Preparation of frozen sections of liver tissue and Oil Red O staining
[0034] (1) Treatment, sectioning and staining of liver tissue samples: Fix the liver tissue on the sample holder with OCT embedding medium, section (10 μm) after equilibrating at -20°C for 10 min, and store the frozen sections at -20°C. Let the sections stand at room temperature for 5 min before staining, fix with 10% formalin for 15 min, rinse with water for 2 s, and then place in 60% isopropanol and let stand for 2 min. Stain with Oil Red O working solution (prepared by mixing Oil Red O stock solution and distilled water at a ratio of 3:2 and filtering) in the dark for 10 min, wash with 60% isopropanol for 2 - 3 s, rinse with water for 2 s, and then stain the sections with hematoxylin for 4 min.
[0035] (2) Observation of lipid droplets and quantification of hepatic steatosis: Three sections were prepared from each chicken, and three fields of view were randomly selected from each section. Images were acquired using a Nikon microscope imaging system. The degree of hepatic steatosis (percentage of the red area in the section field of view) was quantified using Photoshop software for each field of view. The magic wand tool was used to select the area stained with Oil Red O, and the ratio of the pixel value of the red area to the pixel value of the entire image was the degree of hepatic steatosis.
[0036] (3) To establish a grading standard for FLS based on Oil Red O staining, we first compared whether there were differences in Oil Red O staining of frozen sections of liver tissue among different grades of FLS based on H&E staining. The results showed that at 27, 35, and 53 weeks of age, in both the low-fat and high-fat lines, there were almost no red-stained areas in the liver tissue of FLS-0 hens; there were red-stained areas in the liver tissue of hens with FLS, and the red-stained area in the liver tissue of FLS-2 hens was larger than that of FLS-1 hens ( Figure 3 ). Subsequently, the degree of hepatic steatosis was quantified based on Oil Red O staining. The results showed that at 27, 35, and 53 weeks of age, in both the low-fat and high-fat lines, the quantitative results of ORO staining increased significantly with the increase in FLS grade (P<0.01, Figure 4 ), which was consistent with the quantitative results of H&E staining. To further verify the consistency between the quantitative results of Oil Red O staining and H&E staining, we performed a Spearman correlation analysis on the quantitative results of Oil Red O staining and H&E staining. The results showed that the degree of hepatic steatosis quantified by Oil Red O staining was extremely significantly positively correlated with the degree of hepatic steatosis quantified by H&E staining (r = 0.9507, P<0.0001, Figure 5 ). The above results indicate that it is reliable to quantitatively grade the severity of chicken FLS based on Oil Red O staining.
[0037] Subsequently, based on the grading results of chicken fatty liver syndrome by H&E staining, the ranges of the quantitative results of Oil Red O staining corresponding to different grades of fatty liver syndrome in high- and low-fat line hens were respectively statistically analyzed. As can be seen from Table 3, the consistency of the ranges of the quantitative results of Oil Red O staining between the low-fat and high-fat lines for the same grade of chicken fatty liver syndrome was good. Therefore, the results of the two lines were combined to establish a grading standard for chicken fatty liver syndrome based on Oil Red O staining: a quantitative result of Oil Red O staining less than 10% was defined as not having chicken fatty liver syndrome (FLS-0), a quantitative result of Oil Red O staining between 10% and 34% was defined as mild chicken fatty liver syndrome (FLS-1), and a quantitative result of Oil Red O staining greater than 34% was defined as moderate chicken fatty liver syndrome and above (FLS-2) (Table 3).
[0038] Table 3 Grading standard for chicken fatty liver syndrome based on Oil Red O staining
[0039]
[0040] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. A grading method for chicken fatty liver syndrome based on Oil Red O staining, characterized in that, The method is as follows: S1. Preparation of paraffin sections of chicken liver tissue, H&E staining, observation of lipid droplets and quantification of the degree of hepatic steatosis: Three paraffin sections were prepared for each chicken and subjected to H&E staining. Three fields of view were randomly selected from each paraffin section, and images were collected using a microscope imaging system. The degree of hepatic steatosis in each field of view was quantified, that is, the percentage of the lipid droplet area in the area of the paraffin section field of view. Referring to the identification method and grading standard of human NAFLD, the grade of chicken fatty liver syndrome was defined according to the degree of hepatic steatosis: FLS-0 represents a healthy liver without lipid accumulation, that is, <5% hepatic steatosis; FLS-1 represents mild fat accumulation in the liver, that is, 5%-33% hepatic steatosis; FLS-2 represents moderate fat accumulation in the liver, that is, 33%-66% hepatic steatosis; S2. Preparation of frozen sections of chicken liver tissue and Oil Red O staining: The liver tissue was fixed on a sample holder with OCT embedding medium, sectioned after freezing, and stored frozen. The Oil Red O working solution was used for staining in the dark and then washed, and then stained with hematoxylin. Then, the lipid droplets in the frozen sections of chicken liver tissue stained with Oil Red O were observed and the degree of hepatic steatosis was quantified: Three frozen sections were prepared for each chicken. Three fields of view were randomly selected from each frozen section, and images were collected using a microscope imaging system. The degree of hepatic steatosis in each field of view was quantified, that is, the ratio of the pixel value of the red area to the pixel value of the whole picture was the degree of hepatic steatosis; S3. Based on the grading results of chicken fatty liver syndrome by H&E staining of paraffin sections, the ranges of the quantitative results of Oil Red O staining of frozen sections corresponding to different FLS grades of high- and low-fat line hens were statistically analyzed respectively, and a FLS grading standard based on Oil Red O staining was established, as shown in Table 1: The quantitative result of Oil Red O staining less than 10% was defined as not suffering from chicken fatty liver syndrome, that is, FLS-0; the quantitative result of Oil Red O staining between 10% and 34% was defined as mild chicken fatty liver syndrome, that is, FLS-1; the quantitative result of Oil Red O staining greater than 34% was defined as moderate chicken fatty liver syndrome and above, that is, FLS-2, Table 1 Grading standard of chicken fatty liver syndrome based on Oil Red O-stained sections