Preparation and application of silymarin in feed to alleviate fatty liver in Taihe black-boned chickens

Abstract

This invention discloses the preparation and application of silymarin in feed for alleviating fatty liver in Taihe black-boned chickens. Silymarin alleviates and / or prevents fatty liver in Taihe black-boned chickens by reducing liver damage. Silymarin also alleviates and / or prevents fatty liver in Taihe black-boned chickens by reducing oxidative damage to liver tissue. Silymarin works by upregulating... PPAR‑α , FXR downregulate mRNA expression levels SCP2 , ApoB , ApoVLDL , CYP7A1 , SREBP‑1c This invention aims to alleviate and / or prevent fatty liver in Taihe black-bone chickens by regulating the mRNA expression level. A feed additive, the active ingredient of which includes silymarin, is also included. A laying hen feed comprises a basal feed and the aforementioned feed additive. This invention establishes a fatty liver model in Taihe black-bone chickens through in vivo experiments, demonstrating that the application of silymarin can significantly increase egg production in laying hens and also has good effects in preventing and / or alleviating fatty liver, which is of significant value to the Taihe black-bone chicken industry.

Description

Technical Field

[0001] This invention belongs to the fields of poultry farming technology and animal nutrition, specifically relating to the preparation and application of silymarin in the preparation of feed additives that improve lipid metabolism in Taihe black-bone chickens. Background Technology

[0002] Taihe Black-boned Chicken, a Chinese national geographical indication product, integrates meat, egg, medicinal, and ornamental uses. It is famous for its ten characteristics: "comb, tassel head, green ears, beard, silky feathers, feathered feet, five claws, black skin, black meat, and black bones," as well as its high nutritional and medicinal value.

[0003] With the rapid development of my country's egg-laying hen industry and the continuous expansion of intensive farming, the intensive farming model leads to less space for laying hens to move around, resulting in lipid metabolism disorders and making them more susceptible to fatty liver hemorrhagic syndrome (FLHS). FLHS is a common nutritional metabolic disease in laying hens, also known as non-alcoholic fatty liver disease. Currently, there are no drugs specifically for FLHS; prevention and control mainly rely on adjusting feed formulation and husbandry management. FLHS not only affects the egg production performance of laying hens, reducing economic benefits, but also seriously impacts the development of the poultry industry. Compared to common commercial laying hens, Taihe Silkie chickens exhibit more pronounced abdominal fat accumulation and more severe fatty liver disease, thus necessitating the search for feed additives to prevent FLHS.

[0004] Silymarin is a class of natural flavonoid lignans extracted and isolated from the dried fruit and seeds of milk thistle (Silybum marianum), a plant in the Asteraceae family. It mainly consists of five types of flavonoid lignans: silybin (A and B), isosilybin (A and B), silybinine, silybinin, and silybinin tincture. Among these, silybinine is the most abundant, accounting for 50%–70% of slymarin, and also exhibits the strongest activity. Silymarin possesses a wide range of biological activities, including hepatoprotective, antioxidant, anti-inflammatory, immunomodulatory, lipid metabolism-regulating, anti-tumor, and nervous system-protective functions. Studies have confirmed that slymarin optimizes the intestinal flora structure of animals, improves animal growth performance, promotes animal growth, significantly enhances antioxidant stress capacity, and strengthens animal immunity.

[0005] Currently, the application of silymarin in livestock and poultry production is limited. While silymarin has hepatoprotective effects and can alleviate fatty liver disease clinically, its application in laying hens, especially Taihe Silkie chickens, and in cases of fatty liver disease has not been reported. In particular, the specific molecular mechanisms by which silymarin improves lipid deposition by regulating bile acid metabolism and influencing gut microbiota composition in a high-energy, low-protein diet-induced fatty liver model remain unclear. Therefore, feed development and efficacy research of silymarin in alleviating fatty liver hemorrhage syndrome are of paramount importance. Summary of the Invention

[0006] The purpose of this invention is to provide the application of silymarin in alleviating fatty liver in Taihe black-boned chickens and improving egg production performance, thereby solving the problems existing in the prior art. This invention comprehensively explores the preventive effect of different doses of silymarin on fatty liver in laying hens induced by high-energy, low-protein diets through in vivo experimental modeling and detection of relevant indicators. Furthermore, it reduces hepatic lipid deposition by regulating hepatic lipid synthesis and metabolism and bile acid metabolism signaling pathways, thereby effectively preventing fatty liver hemorrhage syndrome induced by high-energy, low-protein diets.

[0007] To achieve the above objectives, the present invention provides the following solution: Technical Solution 1: Application of silymarin in the preparation of feed additives for the prevention and / or relief of fatty liver in Taihe black-bone chickens.

[0008] Furthermore, the silymarin can prevent and / or alleviate fatty liver in Taihe black-boned chickens by improving lipid metabolism in the liver and reducing liver damage.

[0009] Furthermore, the silymarin can prevent and / or alleviate fatty liver in Taihe black-boned chickens by reducing oxidative damage to the liver tissue.

[0010] Furthermore, the silymarin is obtained by upregulating... PPAR-α , FXR downregulate mRNA expression levels SCP2 , ApoB , ApoVLDL , CYP7A1 , SREBP-1c The mRNA expression level of Taihe black-bone chicken can be used to alleviate and / or prevent fatty liver.

[0011] Technical Solution 2: A feed additive, the active ingredient of which includes silymarin.

[0012] Technical Solution 3: A layer hen feed, comprising a basic feed and the aforementioned feed additives.

[0013] Technical Solution 4: Application of silymarin as a feed additive in alleviating fatty liver and associated liver damage in Taihe black-bone chickens induced by high-energy, low-protein diets.

[0014] Technical Solution 5: Application of silymarin, the aforementioned feed additive, or the aforementioned laying hen feed in improving fatty liver in Taihe black-bone chickens during the late laying period and enhancing egg production performance.

[0015] Furthermore, the Taihe black-bone chicken fatty liver includes Taihe black-bone chicken fatty liver induced in the late laying period by a high-energy, low-protein diet.

[0016] The present invention discloses the following technical effects: This invention establishes a fatty liver model in Taihe black-boned chickens through in vivo experiments, demonstrating that silymarin can significantly increase egg production in laying hens and also has good effects in preventing and / or alleviating fatty liver. This invention studies the effects of silymarin on egg production and the prevention and / or alleviation of fatty liver in Taihe black-boned chickens, concluding that silymarin can increase egg production and improve the chicken's production performance. Furthermore, by increasing the activity of hepatic antioxidant enzymes, reducing hepatic lipid oxidative stress, and enhancing the expression of hepatic lipid metabolism PPAR-α and FXR signaling pathways, it effectively improves the level of lipid deposition in the liver of FLHS Taihe black-boned chickens, thus providing a certain degree of liver protection. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without additional creative effort.

[0018] Figure 1 The effects of high-energy, low-protein diets and silymarin on abdominal fat percentage and liver index in Taihe black-boned chickens; Figure 2 The changes in the liver structure of Taihe black-bone chickens in each group were shown in the image; HE staining was performed; black boxes represent magnified areas with enlarged intercellular spaces; red boxes represent hemorrhage points in the liver; Figure 2 Part A is the control group (CON). Figure 2 Part B is the High Energy Low Protein Model Group (HELP). Figure 2 Part C is the silymarin treatment group (SIL). Figure 3 The study investigated the changes in serum ALT, AST, T-CHO, TG, HDL-c, LDL-c, LPL, and LPS levels in each group of Taihe black-boned chickens. ALT represents alanine aminotransferase (ALT), AST represents aspartate aminotransferase (AST), T-CHO represents total cholesterol, TG represents triglycerides, HDL-c represents high-density lipoprotein (HDL-c), LDL-c represents low-density lipoprotein (LDL-c), LPL represents lipoprotein lipase, and LPS represents lipase. Figure 4The antioxidant indicators CAT, GSH-Px, MDA, T-AOC and T-SOD in the serum of Taihe black-bone chickens in each group were determined. Among them, CAT is catalase, GSH-Px is glutathione peroxidase, MDA is malondialdehyde, T-AOC is total antioxidant capacity and T-SOD is total superoxide dismutase. Figure 5 The results of RT-qPCR for lipid synthesis-related genes and bile acid metabolism pathway-related genes in the liver tissue of Taihe black-boned chicken; where AH correspond to... PPAR-α , SCP2 , ApoB , ApoVLDL , FXR , CYP7A1 , MRP2 , SREBP-1c mRNA expression level of a gene. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0019] Figure 6 Differences in species and functions among the three groups of microorganisms: CON, HELP, and SIL; among which Figure 6 Part A: Differences in community abundance at the phylum level; Figure 6 Part B: Differences in community abundance at the genus level; Figure 6 Part C: Heatmap of microbial community similarity; Figure 6 Part D: LEfSe analysis of microbial abundance; Figure 6 Part E: Microbial community Circos analysis; Figure 6 Part F: Analysis of differential microbial communities in three groups; Figure 7 A bile acid metabolism profile of the cecal contents of Taihe black-boned chicken; among which Figure 7 Part A: Heatmap of similarity in secondary bile acid metabolism; Figure 7 Part B: MSEA enrichment analysis; Figure 7 Part C: KEGG enrichment pathway. Detailed Implementation

[0020] Various improvements and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, which will be obvious to those skilled in the art. Those skilled in the art should understand that any modifications or equivalent substitutions made without departing from the principles of the invention fall within the protection scope of this invention.

[0021] Example: In vivo animal experiments A fatty liver model of laying hens with full-blown hematopoietic stem dysplasia (FLHS) was established using a high-energy, low-protein diet (13.25 MJ / kg metabolizable energy, 13.27% crude protein). The experiment lasted for 10 weeks. Different doses of silymarin (SIL) were added to both the basal diet (11.95 MJ / kg metabolizable energy, 16.37% crude protein) and the high-energy, low-protein diet. 432 healthy late-laying Taihe black-boned chickens at 33 weeks of age with similar body weight (1.2 kg ± 0.2 kg) and laying rate (45.31% ± 2.74%) were selected and housed in three-tiered cages at the Taihe Aoxin Black-boned Chicken Development Co., Ltd. The poultry house was cleaned and disinfected with hypochlorous acid three days before pre-feeding, and the chickens were fed for 7 days according to the daily feeding and management practices of the Taihe Aoxin Black-boned Chicken Development Co., Ltd. breeding farm. The hens were randomly divided into six groups: the CON group (control group, fed a basal diet); the HELP group (high-energy, low-protein diet group, fed a high-energy, low-protein diet); the SIL-0.025 group (high-energy, low-protein diet + 0.025% silymarin); the SIL-0.05 group (high-energy, low-protein diet + 0.05% silymarin); the SIL-0.075 group (high-energy, low-protein diet + 0.075% silymarin); and the SIL-0.1% group (high-energy, low-protein diet + 0.1% silymarin). During the experiment, the hens were fed twice daily (8:00 and 17:00), with free access to water. Eggs were collected daily at 16:30. The hens were housed in an environment with a 16+8 hour light-dark cycle, maintained at a temperature of 20℃-25℃ and a humidity of 50%-70%. The growth and production performance of the hens were observed and recorded. The experiment lasted for 10 weeks, with samples collected for relevant experiments.

[0022] Feed intake was measured weekly, and egg production rate, average daily feed intake, and feed conversion ratio were calculated. The results showed that silymarin had the following effects on average daily feed intake, egg production, and feed conversion ratio in a high-energy, low-protein diet-induced hepatic steatosis model in Taihe black-boned chickens, as shown in Table 1. Compared with the CON group, the HELP group showed a significantly lower egg production rate (…). P < 0.05, feed conversion ratio increased significantly ( P <0.05). Compared with the HELP group, the SIL levels in the four groups were significantly higher in the SIL group, except for the SIL 0.025 group which showed no significant difference. P < 0.05) Egg production rate and decrease ( P < 0.05) Feed conversion ratio. Furthermore, the egg production rate and feed conversion ratio showed a quadratic regression relationship with the silymarin dosage, with the addition of 0.05% silymarin showing the best effect.

[0023] Table 1. Effects of high-energy, low-protein diets and / or silymarin on average daily feed intake, egg production, and feed conversion ratio in Taihe black-boned chickens. Average daily feed intake (g / d) 59.47 56.37 53.77 56.58 56.12 57.95 0.49 0.073 0.348 0.471 Egg production rate (%) <![CDATA[40.92 a ]]> <![CDATA[36.49 b ]]> <![CDATA[37.11 b ]]> <![CDATA[39.32 a ]]> <![CDATA[38.72 a ]]> <![CDATA[38.36 a ]]> 0.71 0.003 0.129 0.041 Feed conversion ratio (%) <![CDATA[4.00 b ]]> <![CDATA[4.22 a ]]> <![CDATA[4.17 a ]]> <![CDATA[3.88 c ]]> <![CDATA[4.00 b ]]> <![CDATA[4.13 b ]]> 0.01 0.035 0.178 0.048 Based on the results in Table 1, after 10 weeks of experimentation, samples were collected from the CON group, HELP group, and SIL-0.05 group after a 12-hour fast. Blood was collected via the wing vein into centrifuge tubes, incubated at room temperature for 2 hours, and then centrifuged for 10 minutes (3500 rpm / min). Serum was aliquoted into 0.5 mL centrifuge tubes and stored at -80°C. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total triglycerides (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in serum were measured according to the instructions of the Shanghai Enzyme-Linked Biological Products Kit. The levels of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in serum were measured according to the instructions of the kit purchased from Nanjing Jiancheng Biotechnology Institute. Liver tissue samples were collected according to groups. Chickens were anesthetized and immediately dissected. The livers were carefully removed, and the weight of the laying hens, liver, and abdominal fat were weighed and recorded. Liver tissue samples were quickly aliquoted onto ice into cryovials and stored at -80°C for subsequent analysis. Routine histopathological sections were used for observation of liver tissue structure.

[0024] Abdominal fat percentage and liver index are shown in Figure 1 Compared to the CON group, the HELP group had a lower abdominal fat percentage ( P < 0.01) and liver index ( P < 0.05) all significantly increased; significantly decreased after adding 0.05% SIL to the diet. The effect of SIL on the histological characteristics of Taihe black-boned chicken liver induced by a high-energy, low-protein diet is as follows: Figure 2 Compared to the CON group, the HELP group showed multiple hemorrhages in the liver under 10x magnification and enlarged intercellular spaces under 20x magnification. Adding 0.05% SIL to the diet reduced the number of hemorrhages and the intercellular spaces in the liver. These results indicate that SIL can alleviate HELP-induced liver damage in Taihe black-boned chickens.

[0025] Serum biochemical parameters: Effects of SIL on serum ALT, AST, TG, T-CHO, LDL-c, HDL-c, LPL, and LPS levels in a high-energy, low-protein diet-induced hepatic steatosis model in laying hens. Figure 3 As shown. Compared with the CON group, the HELP group showed significantly increased serum levels of ALT, AST, TG, T-CHO, HDL-c, and LPS. P < 0.05), LPL level decreased significantly ( P < 0.05); After adding SIL, the levels of ALT, AST, TG, T-CHO, HDL-c and LPS decreased significantly (P < 0.05), LPL levels increased significantly ( P < 0.05). The results indicate that high-energy, low-protein diets induce hepatic steatosis, which causes liver damage and disordered hepatic lipid metabolism, while SIL effectively reduces liver damage and regulates hepatic lipid metabolism.

[0026] Serum antioxidant markers: The effects of serum CAT, GSH-Px, MDA, T-AOC, and T-SOD on a high-energy, low-protein diet-induced hepatic steatosis model in laying hens are as follows: Figure 4 Compared with the CON group, the serum CAT, GSH-Px, and SOD levels of Taihe black-boned chicken in the HELP group were significantly reduced. P < 0.05%, MDA content increased significantly ( P < 0.05); After the addition of SIL, the levels of CAT, GSH-Px and SOD in serum decreased ( P < 0.05) MDA content. The results indicate that high-energy, low-protein diet-induced hepatic steatosis causes hepatic lipid oxidative stress, while SIL can effectively alleviate lipid oxidative stress caused by lipid metabolism disorders and protect the liver.

[0027] The effects of SIL on lipid metabolism and synthesis-related genes in a high-energy, low-protein diet-induced hepatic steatosis model in laying hens, such as... Figure 5 As shown, compared with the CON group, the HELP group had higher levels of liver lipid metabolism genes. PPAR-α The relative expression level of mRNA was significantly downregulated ( P < 0.05); SCP2 ( P < 0.01) ApoB ( P < 0.05) and ApoVLDL ( P The relative expression level of mRNA (< 0.001) was significantly upregulated; the addition of SIL effectively upregulated the expression level of mRNA. PPAR-α relative mRNA expression levels ( P < 0.05); lowered SCP2 , ApoB and ApoVLDL relative mRNA expression levels ( P < 0.05). The results indicate that SIL can alleviate lipid deposition by regulating lipid synthesis-related genes. The effect of SIL on genes related to bile acid metabolism pathways in the liver of Taihe black-boned chickens was compared with that in the CON group; the HELP group showed a greater effect. CYP7A1 The relative expression level of mRNA was significantly upregulated ( P < 0.01); After adding SIL, the effect was effectively increased. FXR relative mRNA expression levels ( P< 0.05), significantly reduced CYP7A1 relative mRNA expression levels ( P < 0.01). This indicates that SIL can regulate bile acid metabolism and may have a significant alleviating effect on lipid metabolism.

[0028] Mechanism analysis: PPAR-α is a key regulator of fatty acid β-oxidation, and its upregulation helps promote the oxidative breakdown of fatty acids in the liver. ApoB and ApoVLDL are involved in lipoprotein synthesis and transport; SIL may reduce excessive lipid output from the liver to the bloodstream or improve lipid transport efficiency by downregulating these genes. Furthermore, FXR (farnesol X receptor), as a bile acid nuclear receptor, was significantly increased in the SIL group, suggesting that SIL may regulate lipid metabolism by activating the FXR signaling pathway.

[0029] The effects of SIL on cecal microbiota in a high-energy, low-protein diet-induced model of fatty liver degeneration in laying hens, such as... Figure 6 As shown. At the phylum level, the most abundant phyla in the gut microbiota are Bacteroides and Firmicutes. The average relative abundance of Bacteroides in each group exceeds 70%, and the average relative abundance of Bacteroides and Firmicutes in each group exceeds 95%. Figure 6 Part A). At the genus level, *Bacteroides*, *Alistipes*, *Phocaeicola*, and *Phascolarctobacterim* were the dominant genera, with relative abundance exceeding 20% ​​in all groups. Figure 6 (Part B of the study). At the species level, the three groups of microorganisms have similar compositions, only... Figure 6 The eight microbial species in the F part showed significant differences. Therefore, although the richness of the cecal microbiota did not vary much among the groups, there were still differences in the composition of the gut microbiota among the three groups.

[0030] Discovered through similarity heatmaps ( Figure 6 Part C), only Phocaeicola_plebeius Abundance decreased due to the HELP diet, but increased after SIL treatment. Therefore, it is judged that... Phocaeicola_plebeius These were the main metabolic bacteria of SIL. LEfSe analysis was used to determine the differences in microbial abundance among the three groups. Linear discriminant analysis was used as the criterion to identify differentially abundant gut microbes in each group whose relative abundance was significantly higher than that in the other groups, serving as biomarkers. Results ( Figure 6Part D indicates that at the genus level, *Ruminiclostridium* and *Escherichia* are enriched in the CON group, *Desulfovibrio* in the HELP group, and *Muribaculum* in the SIL group. At the species level, Bacteroidetes_bacterium_RIFOXYB2_FULL_39_7 and Alistipes_inops Three types of microorganisms were enriched in the CON group. Alistipes_communis Enriched in the HELP group Phocaeicola_salanitronis and Lachnolostridium_edouardi Four microorganisms were enriched in the SIL group. These analyses support the different conclusions regarding the microbial community structure of the three groups of samples. In this invention, the harmful genus *Desulfovibrio* was enriched in the HELP group, while the beneficial genus *Muribaculum* was enriched in the SIL group, and... Phocaeicola_salanitronis and Lachnolostridium_ edouardi Beneficial bacteria were significantly enriched in the SIL group, indicating that SIL treatment can effectively enhance the lipid metabolism capacity of beneficial bacteria, which is beneficial for treating fatty liver in Taihe black-bone chickens during the late egg-laying period.

[0031] To understand the differences in bile acid profiles among different groups of Taihe black-boned chicken and to reveal the regulatory mechanism of SIL on HELP-induced lipid metabolism problems, bile acid profiles in cecal contents were analyzed based on bile acid-targeted metabolomics. This was achieved through sample clustering heatmaps (…). Figure 7 Part A of the study observed the trends in metabolite changes. Compared with the HELP group, the SIL group showed increased levels of four secondary bile acids (ω-mouse cholic acid, 23-nordeoxycholic acid, 12-ketolithocholic acid, and glycodeoxycholic acid), consistent with the trend in the CON group. MSEA enrichment analysis was used to compare the identified secondary bile acids with data from The Small Molecule Pathway Database. Figure 7 Part B of the study revealed that the enriched pathways, including those related to hepatobiliary diseases, portal vein obstruction, and acute liver failure, are largely associated with liver diseases. KEGG pathway enrichment analysis (…) Figure 7(Part C of the study) The detected bile acids were significantly correlated with the primary bile acid biosynthesis pathway and taurine and taurine metabolism stimulation pathway. This indicates that SIL can affect bile acid metabolism, regulate lipid metabolism in Taihe black-boned chickens, improve lipid metabolism disorders induced by HELP diet, and alleviate fatty liver.

[0032] The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents.

Claims

1. Application of silymarin in the preparation of feed additives for alleviating and / or preventing fatty liver in laying Taihe black-boned chickens.

2. The application according to claim 1, characterized in that, The silymarin mentioned above alleviates and / or prevents fatty liver in Taihe black-boned chickens by reducing liver damage.

3. The application according to claim 1, characterized in that, The silymarin mentioned above alleviates and / or prevents fatty liver in Taihe black-boned chickens by reducing oxidative damage to liver tissue.

4. The application according to claim 1, characterized in that, The silymarin is upregulated PPAR-α , FXR downregulate mRNA expression levels SCP2 , ApoB , ApoVLDL , CYP7A1 , SREBP-1c The mRNA expression level of Taihe black-bone chicken can be used to alleviate and / or prevent fatty liver.

5. A feed additive, characterized in that, The active ingredient includes silymarin.

6. A type of laying hen feed, characterized in that, Includes basic feed and feed additives as described in claim 5.

7. Application of silymarin as a feed additive in alleviating fatty liver and associated liver damage in Taihe black-bone chickens induced by high-energy, low-protein diets.

8. The application of silymarin, the feed additive of claim 5, or the laying hen feed of claim 6 in alleviating fatty liver in Taihe black-boned chickens during the late laying period and improving laying performance.

9. The application according to claim 8, characterized in that, The Taihe black-bone chicken fatty liver refers to Taihe black-bone chicken fatty liver induced by a high-energy, low-protein diet.

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