A synergistic feed additive for preventing and treating fatty liver disease of laying hens

Abstract

The application discloses a kind of synergistic feed additive for preventing and treating fatty liver disease of laying hen.The feed includes the following components: 15%-25% of bile acid;10%-20% of hemolyzed lecithin;5%-15% of L-carnitine;15%-25% of N-acetylcysteine (NAC);10%-20% of rosemary acid extract;1%-3% of selenium yeast;the balance of carrier;wherein, the carrier is one or more of water-soluble silicon dioxide, expanded rice hull powder and calcium hydrogen phosphate.The technical scheme of the application contains a multi-component, multi-target synergistic feed additive composition of L-carnitine, which simultaneously acts on multiple key links of fat metabolism (intestinal tract reduction, liver export, mitochondrial oxidation and antioxidant protection), to achieve efficient prevention and treatment of fatty liver disease of laying hen.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal nutrition and feed science, and particularly relates to a synergistic feed additive for preventing and treating fatty liver disease of laying hens. BACKGROUND

[0002] Fatty liver disease (FLHS) of laying hens is a major metabolic disease that leads to a decrease in egg production rate and an increase in mortality rate in intensive farming, especially in high-yield laying hens. The main cause is excess energy and excessive deposition of fat in the liver, which increases the fragility of liver cells, causes rupture and bleeding, increases mortality, and reduces egg production and egg quality.

[0003] Currently, there are three types of prevention and treatment schemes, but all have limitations:

[0004] 1. Methyl donor scheme (such as CN117502574A): represented by choline chloride and betaine. It promotes phospholipid synthesis by providing methyl groups to assist fat transport out of the liver. However, its effect is single, it only focuses on the "transport out" link, and it is easy to reach a bottleneck under high-yield and high-energy conditions, and it cannot inhibit fat synthesis and oxidation.

[0005] 2. Bile acid scheme (such as CN103585632A, CN101744122B): represented by mixed bile acid salt. It mainly reduces the burden on the liver by promoting fat digestion in the intestine, but ordinary bile acid has complex components, weak ability to specifically activate the FXR receptor to inhibit fat synthesis, and no direct antioxidant function.

[0006] 3. Antioxidant scheme (such as CN1792250A): represented by vitamin E (VE), vitamin C (VC), selenium, etc. It protects liver cells by scavenging free radicals, but it can only alleviate the "consequences" of oxidative damage and cannot solve the fundamental causes of "excessive fat synthesis and transport disorders". The lipid-lowering effect is limited when used alone.

[0007] Recent studies have revealed the key role of L-carnitine in liver lipid metabolism. It is an essential carrier for long-chain fatty acids to enter mitochondria for beta-oxidation, directly promoting fatty acid combustion for energy. However, there is no report on the use of L-carnitine in combination with bile acids, lysophospholipids, and precise antioxidant networks for systematic prevention and treatment of fatty liver disease in laying hens.

[0008] Existing technologies cannot systematically address the complex causes of FLHS. Most schemes only target a certain aspect of fatty liver disease (or promote fat transport, or aid digestion, or antioxidant), lack multi-target and systematic synergistic regulation. Therefore, there is an urgent need in the art for a new type of feed additive with compliant components, multi-target synergistic effect, and significant effect.

[0009] (II) Currently common prevention and control additives include: 1. Choline, betaine, methionine: as a lipid methyl donor, promote lipid export out of the liver. But such a solution has many patents (such as CN117502574A, a feed additive for reducing fatty liver and abdominal fat rate of laying hens, containing betaine hydrochloride, DL-methionine, L-carnitine, etc., aiming to promote carbohydrate metabolism and reduce fat accumulation in the liver and abdomen), which is a known technology, and the effect is limited when used alone.

[0010] 2. Bile acids: promote fat emulsification and digestion, reduce liver burden. (Such as CN101744122B, a feed additive for preventing nutritional fatty liver in fish, which combines the use of choline chloride, bile acid, carnitine and betaine, aiming to reduce fish liver fat deposition and avoid fatty liver disease; CN103585632A, application of bile acid chelator or / and vitamin D in preparation of drug for preventing and treating non-alcoholic fatty liver disease, using bile acid chelator (such as cholestyramine) to reduce fat absorption and inhibit endogenous fatty acid synthesis to reduce fatty liver). But many focus on digestive function.

[0011] 3. Antioxidants (such as VE, VC, selenium): alleviate the damage of lipid peroxidation to liver cells (such as CN117502574A, a feed additive for reducing fatty liver and abdominal fat rate of laying hens, containing nitric acid thiamine (VB1), riboflavin (VB2), pyridoxine hydrochloride (VB6), nicotinamide (VPP), calcium pantothenate (VB5), yeast selenium, etc. A variety of vitamins and trace elements to improve metabolism). But the effect of single antioxidant is not comprehensive.

[0012] 4. Plant extracts (such as silymarin, curcumin): have the effects of protecting liver and resisting oxidation (such as CN1792250A, a nutritional food for preventing and treating obesity and fatty liver and a manufacturing method, raw materials including vitamin C, vitamin E, trace element selenium, chlorophyll, etc., aiming to resist free radical oxidation through the synergy of multiple substances, achieve the effects of reducing blood lipids and cholesterol), but the cost is high and the stability is poor. SUMMARY

[0013] The technical solution of the present application to solve the above technical problems is to provide a synergistic feed additive for preventing and treating fatty liver disease in egg-laying poultry, comprising the following components: 15%-25% bile acid; 10%-20% lysophospholipid; 5%-15% L-carnitine; 15%-25% N-acetylcysteine (NAC); 10%-20% rosemary acid extract; 1%-3% yeast selenium; and the balance of carrier; wherein the carrier is one or more of water-soluble silicon dioxide, expanded rice hull powder and calcium hydrogen phosphate.

[0014] Further, the bile acid is derived from pig gall or bird gall. Further, the content of lysophosphatidic acid (LPA) in the lysophosphatidylcholine is not less than 50%.

[0015] Further, the content of rosmarinic acid in the rosmarinic acid extract is not less than 20%.

[0016] To solve the above technical problems, the present application further provides a preparation method of the feed additive, for preparing the feed additive as described above, comprising the following steps:

[0017] Two-thirds of the carrier is put into the main mixer as the base material, and the main mixer is run;

[0018] N-acetylcysteine (NAC), L-leucine, rosmarinic acid extract, selenium yeast and bile acid are sequentially and slowly added;

[0019] The lysophosphatidylcholine is pre-mixed with the remaining carrier in another mixer for 5 minutes to form a flowable mixture.

[0020] The pre-mixed lysophosphatidylcholine-carrier mixture is slowly added into the running main mixer;

[0021] The feeding port of the mixer is closed, and the mixer is mixed at a speed of 20-25 rpm for 25-30 minutes.

[0022] To solve the above technical problems, the present application further provides a feed, comprising 99.9%-99.95% of a basic diet and 0.05%-0.10% of the feed additive as described above; the basic diet comprises the following components: corn 62.20; soybean meal (43% CP) 24.50; soybean oil 2.50; stone powder 8.00; CaHPO4 1.30; NaCl 0.30; DL-methionine 0.15; L-Lys·HCl 0.05; premix 1.00.

[0023] Compared with the prior art, the feed additive provided by the present application has the following remarkable beneficial effects:

[0024] 1. Excellent synergistic prevention and treatment effect:

[0025] Through rigorous animal experiments (see Table 3), it is proved that the additive of the present application is significantly better (P<0.05) than the traditional methyl donor scheme (TRAD group) and the same commercial product on the market (COM group) in reducing liver fat rate, improving liver function, enhancing antioxidant capacity and improving production performance.

[0026] 2. Multi-dimensional mechanism verification:

[0027] Liver fat deposition: The liver fat rate of the high-dose group (IN-H) of the present application decreased to 4.3%, which was extremely significantly lower than that of the control group (CON, 8.5%), with a decrease of 49.4%. It was also significantly better than that of the TRAD group (6.8%) and the COM group (6.2%), proving the strong effect of the synergistic mechanism of "reducing sources, increasing output, and oxidation".

[0028] Liver cell health: The serum ALT and AST activities (key markers of liver cell damage) of the IN-H group were the lowest (28.9 U / L and 152 U / L), indicating that the present application can most effectively protect the structural integrity and normal function of liver cells.

[0029] Antioxidant defense system: The GSH-Px activity of the IN-H group was the highest (195 U / mg), and the MDA (lipid peroxidation end product) content was the lowest (1.8 nmol / mg), proving that the "precursor-enzyme-eliminator" three-dimensional antioxidant network formed by NAC, rosemary extract, and selenium yeast played a strong synergistic effect, effectively eliminating free radicals and inhibiting lipid peroxidation.

[0030] Production performance and economic benefits: The IN-H group achieved the highest egg production rate (89.5%) and the lowest cull rate (0.8%). This indicates that the present application directly translates liver health into excellent production performance and economic benefits by ensuring liver health, which cannot be achieved by any single functional additive.

[0031] 3. Clear dose-effect relationship and usage guidance:

[0032] The low-dose group (IN-L, 0.05%) and the high-dose group (IN-H, 0.10%) of the present application showed significant gradient improvement in all indicators, proving that the effect is stable and reliable, and providing clear dosage selection basis for production practice (low dose for prevention, high dose for treatment).

[0033] 4. High safety and wide applicability:

[0034] All components are feed additives or natural plant extracts allowed by regulations, with high safety. Experiments show that this scheme is particularly suitable for high-yielding chicken breeds such as Hy-Line Brown and Lohmann Brown during the high-yielding peak period (25-50 weeks of age), which is a high-risk period for FLHS. The application target is clear, and the effect is significant. DETAILED DESCRIPTION

[0035] The present application proposes a synergistic feed additive for preventing and treating fatty liver disease in laying and breeding birds, aiming to systematically address the complex causes of fatty liver disease in laying and breeding birds.

[0036] The synergistic feed additive for preventing and treating fatty liver disease in laying and breeding birds proposed by the present application will be described in the specific embodiments below: Embodiment 1:

[0037] A synergistic feed additive for preventing and treating fatty liver disease of laying hens, comprising the following components: bile acids 15%-25%; lysophosphatidylcholine 10%-20%; L-carnitine 5%-15%; N-acetylcysteine (NAC) 15%-25%; rosemary acid extract 10%-20%; selenium yeast 1%-3%; and the balance of the carrier; wherein the carrier is one or more of water-soluble silicon dioxide, expanded rice hull powder, and calcium hydrogen phosphate.

[0038] Further, the bile acids are derived from pig gall or bird gall. Further, the content of lysophosphatidic acid (LPA) in the lysophosphatidylcholine is not less than 50%.

[0039] Further, the content of rosemary acid in the rosemary acid extract is not less than 20%.

[0040] Synergistic mechanism of each component:

[0041] ① Bile acids: as the core of reducing sources (inhibiting synthesis) and reducing burden (promoting digestion). On the one hand, as a high-efficiency emulsifier, it promotes the digestion and absorption of lipids in the intestinal tract, thereby reducing the metabolic burden of the liver from the source. More importantly, the active ingredients such as chenodeoxycholic acid (CDCA) contained therein are natural agonists of farnesol X receptor (FXR). After specifically activating the liver FXR receptor, the gene expression of sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) is down-regulated, thereby strongly inhibiting the de novo synthesis of fat in the liver from the transcriptional level, and cutting off the source of excessive fat deposition.

[0042] ② Lysophosphatidylcholine: as the key to increasing output (promoting export). The lysophosphatidic acid (LPA) contained therein is an important biological signal molecule that can directly promote the assembly and secretion of very low density lipoprotein (VLDL) in the liver, effectively solving the obstacle of fat "exporting" from the liver. This forms a "opening source and reducing flow" type of first and last echo with the "inhibiting synthesis" effect of bile acids, and together regulates the liver fat homeostasis.

[0043] ③ L-carnitine: as the core engine of oxidation (accelerating decomposition). It is the only and essential carrier for long-chain fatty acids to enter mitochondria for beta-oxidation. By directly promoting the oxidation and decomposition of fatty acids in the mitochondrial matrix for energy supply, it fundamentally removes the deposited fat, and provides additional energy for high-yield laying hens, realizing the qualitative change from "accumulation" to "consumption".

[0044] ④N-acetyl cysteine (NAC), rosemary acid extract and yeast selenium: together constitute a three-dimensional defense network of escort (antioxidant protection).

[0045] NAC, as the direct precursor of the most important intracellular antioxidant glutathione (GSH), can significantly increase the level of GSH in liver cells and enhance endogenous antioxidant capacity.

[0046] Rosemary acid extract (core active ingredient rosemary acid) is a powerful natural phenolic antioxidant that can directly scavenge free radicals and interrupt lipid peroxidation chain reactions.

[0047] Yeast selenium provides organic selenium (selenocysteine), which serves as the active center of glutathione peroxidase (GSH-Px) and catalyzes the reduction of hydrogen peroxide and lipid peroxides by GSH.

[0048] The three components form a multi-level, three-dimensional antioxidant defense system composed of substrates (NAC), enzymes (yeast selenium-GSH-Px), and direct scavengers (rosemary acid), which comprehensively protect the integrity of liver cell membrane and mitochondrial membrane structure, providing protection for normal fat metabolism.

[0049] In summary, the present application is not a simple superposition of components, but through precise compounding, it simultaneously acts on the three major metabolic pathways of fat synthesis, transport, and decomposition, and is supplemented by the synergistic effect of whole-process antioxidant protection, thereby systematically solving the complex etiology of fatty liver disease in egg-laying poultry. Example 2:

[0050] A method for preparing a feed additive, for preparing the feed additive as described in Example 1, comprising the following steps:

[0051] Two-thirds of the carrier is put into the main mixer as the base material and is run;

[0052] N-acetyl cysteine (NAC), L-levomegacine, rosemary acid extract, yeast selenium, and bile acid are added slowly in sequence;

[0053] Lecithin is pre-mixed with the remaining carrier in another mixer for 5 minutes to form a flowable mixture.

[0054] The pre-mixed lecithin-carrier mixture is slowly added to the running main mixer;

[0055] The mixer feed port is closed, and the mixture is mixed at a speed of 20-25 rpm for 25-30 minutes. Example 3:

[0056] A feed comprising 99.9%-99.95% of a basic diet and 0.05%-0.10% of the feed additive as described above; the basic diet comprises the following components: corn 62.20; soybean meal (43% CP) 24.50; soybean oil 2.50; stone powder 8.00; CaHPO4 1.30; NaCl 0.30; DL-methionine 0.15; L-Lys·HCl 0.05; premix 1.00

[0057] Period of use and scheme: prophylactic use: it is suggested to start using when the egg laying rate of the chicken flock rises to about 85%. The cyclic addition scheme is adopted: continuously adding for 4 weeks, then interval for 4 weeks, and thus cyclically using to 50 weeks old. This scheme can effectively prevent the occurrence of FLHS, and the economic benefit is best.

[0058] Therapeutic use: when the chicken flock has suspected FLHS symptoms such as pale comb and wattles, obesity, fluctuant decrease of egg laying rate, and slight increase of mortality, etc., it is immediately used. The addition amount can adopt the upper limit of the recommended range (0.10%, that is, 1000g / ton of feed), and is continuously used until the clinical symptoms disappear and the production performance recovers stably, and then it is continuously used for 2 weeks to consolidate the curative effect.

[0059] Mixing method: in order to ensure that the additive is uniformly distributed in the feed and avoid local excess or deficiency, it is suggested to adopt the step-by-step expansion (premix) mixing process. That is, the product is first mixed with a small amount of carrier (such as bran) or feed raw material, and then gradually expanded and mixed with more feed, and finally uniformly mixed with all the basic diet.

[0060] Experimental design: 300 Hyline brown laying hens of 25 weeks old and in good health are selected, and are randomly divided into 5 groups, 6 replicates per group, and 10 chickens per replicate. The basic feed is the same, and the experimental period is 8 weeks.

[0061] (1) Control group (CON): basic feed;

[0062] Traditional scheme group (TRAD): basic feed + 600g / ton (500g of choline chloride + 100g of betaine);

[0063] Low-dose group of the application (IN-L): basic feed + 500g / ton of the additive of the application (added at 0.05%);

[0064] High-dose group of the application (IN-H): basic feed + 1000g / ton of the feed additive of Example 1 of the application (added at 0.1%);

[0065] Positive control group (COM): basic feed + 1000g / ton of choline chloride-bile acid complex;

[0066] (2) Basic feed formula and nutritional level: ① Basic daily ration formula;

[0067] Table 1. Basic daily ration formula:

[0068]

[0069] ② Premix formula (content per kg of complete feed): Vitamin A: 12,000 IU; Vitamin D3: 3,000 IU; Vitamin E: 20 IU; Vitamin K3: 2 mg; Vitamin B1: 1 mg; Vitamin B2: 6 mg; Vitamin B6: 0.02 mg; Nicotinic acid: 30 mg; Calcium pantothenate: 12 mg; 12

[0070] Folic acid: 1 mg; Biotin: 0.1 mg; FeSO4·H2O: 60 mg; CuSO4·5H2O: 6 mg; MnSO4·H2O: 80 mg; ZnSO4·H2O: 60 mg; KI: 0.7 mg; Na2SeO3: 0.3 mg; Carrier (zeolite powder / stone powder): up to 1 kg; Add premix to complete feed (add 1 kg of premix to 100 kg of complete feed).

[0071] ③ Basic daily ration nutritional level:

[0072] Table 2. Basic daily ration nutritional level:

[0073]

[0074] ④ Nutritional level explanation: NRC (1994) recommended 1050-1150 mg / kg of choline for laying hens during the egg-laying peak period. In this formula, choline is only provided by natural raw materials (corn, soybean meal), with a content of about 1100 mg / kg, which is at the absolute lower limit of the recommended amount, and there is no additional supplementation. This is a key design point for successfully building a fatty liver model.

[0075] The metabolic energy and linoleic acid levels are set slightly higher than the general recommended amount, aiming to create an energy-positive environment, making it easier to induce fat deposition in the liver.

[0076] The protein and amino acid levels meet but do not exceed, avoiding excessive amino acids providing additional energy through deamination.

[0077] ​(5) Design principle and purpose: Construct a susceptible model: By setting up a "high energy (high oil) + critical lack of methyl donor (choline)" nutritional pattern, the control group (CON) hens naturally show a tendency to develop fatty liver (increased liver fat rate, enhanced ALT / AST activity) under the huge metabolic pressure of the peak egg production period. This provides a sensitive and reliable experimental platform for evaluating the protective effect of additives.

[0078] Highlight the treatment effect: Under this nutritional stress background, any additive that can effectively alleviate fatty liver will have its effect amplified, making it easier to obtain significant differences in statistics, thereby proving the effectiveness of the invention.

[0079] Eliminate interference: Do not add any choline, betaine, bile acid or antioxidant in the basic premix, to ensure that the differences between the treatment groups come entirely from the additives being evaluated, making the experimental results pure and reliable.

[0080] This basic diet formula is scientifically and rigorously designed, meeting both animal welfare and basic nutritional needs, and successfully creating the metabolic challenge required for the experiment, making it an ideal carrier for proving the efficacy of the additive.

[0081] (3) Experimental results:

[0082] Table 3. Synergistic effect of different additive combinations in feed feeding experiments:

[0083]

[0084] *Note: Different letters in the same column indicate significant differences (P<0.05).

[0085] (4) Experimental conclusion: Compared with the prior art, the compound feed additive provided by the present invention has the following significant beneficial effects:

[0086] ① Excellent synergistic prevention and treatment effect: Through rigorous animal experiments (see Table 3), it is proved that the additive of the present invention is significantly superior (P<0.05) to the traditional methyl donor scheme (TRAD group) and the commercially available similar commercial product (COM group) in reducing liver fat rate, improving liver function, enhancing antioxidant capacity and improving production performance.

[0087] ② Multi-dimensional mechanism verification: liver fat deposition: The liver fat rate of the high-dose group (IN-H) of the present invention is reduced to 4.3%, which is significantly lower than that of the control group (CON, 8.5%), with a reduction of 49.4%. It is also significantly better than that of the TRAD group (6.8%) and the COM group (6.2%), proving the strong effect of the "reducing source, increasing output, and oxidation" synergistic mechanism.

[0088] Liver health: The serum ALT and AST activities (key markers of hepatocyte injury) were the lowest (28.9 U / L and 152 U / L) in IN-H group, indicating that the present application could protect the hepatocyte structure integrity and normal function most effectively.

[0089] Antioxidant defense system: The GSH-Px activity was the highest (195 U / mg) and the MDA (end product of lipid peroxidation) content was the lowest (1.8 nmol / mg) in IN-H group, demonstrating that the "precursor-enzyme-scavenger" three-dimensional antioxidant network composed of NAC, rosemary acid extract and selenium yeast played a very strong synergistic effect, which could effectively scavenge free radicals and inhibit lipid peroxidation.

[0090] Production performance and economic benefits: The IN-H group obtained the highest egg production rate (89.5%) and the lowest cull rate (0.8%). This indicates that the present application directly translates into excellent production performance and economic benefits by protecting liver health, which cannot be achieved by any single functional additive.

[0091] ③ Clear dose-effect relationship and usage guidance: The low-dose group (IN-L, 0.05%) and the high-dose group (IN-H, 0.10%) of the present application showed significant gradient improvement effects in all indicators, proving that the effect is stable and reliable, and providing clear dose selection basis for production practice (low dose for prevention, high dose for treatment).

[0092] ④ High safety and wide applicability: All components are feed additives or natural plant extracts allowed by regulations, with high safety. Experiments show that this scheme is particularly suitable for high-yielding chicken breeds such as Hy-Line Brown and Lohmann Brown during the high-yielding peak period (25-50 weeks of age), with clear application target and significant effect. Example 4:

[0093] A synergistic feed additive for preventing and treating fatty liver disease in egg-laying poultry, comprising the following raw materials by weight percentage: bile acid: 20% (derived from pig gall or poultry gall); lysophosphatidylcholine (LPA content ≥ 50%): 15%; L-carnitine: 10%; N-acetylcysteine (NAC, purity ≥ 99%): 20%; rosemary acid extract (rosemary acid content ≥ 20%): 15%; selenium yeast (calculated as selenium): 2% (equivalent to selenium yeast raw material, selenium content 2000 mg / kg); carrier: 18% (1:1 mixture of water-soluble silicon dioxide and calcium hydrogen phosphate);

[0094] Preparation method: start a 500L three-dimensional motion mixer. About two-thirds of the carrier (water-soluble silicon dioxide and calcium hydrogen phosphate mixture) is put into the mixer as the bottom material.

[0095] N-acetyl cysteine (NAC), L-carnitine, rosemary acid extract, selenium yeast and bile acid are added slowly in turn following the principle of from large to small and from light to heavy.

[0096] The key lyso- lecithin raw material needs to be pretreated due to its easy moisture absorption and static electricity: it is premixed with the remaining carriers in a small mixer for 5 minutes to form a mixture with good fluidity.

[0097] The premixed lyso- lecithin-carrier mixture is slowly added to the running main mixer.

[0098] The mixer feed port is closed, and mixing is carried out at a speed of 20-25 rpm for 25-30 minutes to ensure that the components are fully dispersed and uniform.

[0099] The machine is stopped, and the material is discharged. Sampling is carried out using the quartering method, and the uniformity of the mixture is detected, with a coefficient of variation (CV%) of less than 5%.

[0100] The mixture is packaged using aluminum foil bags and sealed for storage in a cool and dry place, thereby obtaining the feed additive premix of the present application. Example 5:

[0101] The fatty liver preventive feed for laying hens comprises: a basic diet: a corn-soybean meal type basic diet formulated according to the nutritional needs of Hyline Brown laying hens in the egg production peak period (see Tables 1 and 2).

[0102] Feed additive: the additive prepared in Example 4 is added to the basic diet at an addition amount of 500 g / ton (i.e. 0.05%).

[0103] Mixing process: 1. Take 500 g of the additive and 5 kg of soybean meal for the first step of premixing.

[0104] 2. The premix is mixed with 50 kg of soybean meal for the second step of bulk mixing.

[0105] 3. Finally, the mixture of the second step is put into a double-shaft paddle mixer with 944.5 kg of basic diet raw materials, and mixed for 4-6 minutes until completely uniform.

[0106] Feeding scheme: applicable object: Hyline Brown laying hens at the age of 28 weeks, entering the egg production peak period.

[0107] Feeding method: the prepared feed is provided for the flock to freely eat.

[0108] Procedure: a cycle prevention mode of "feeding for 4 weeks, with an interval of 3 weeks" is adopted. This mode can effectively prevent fatty liver and reasonably control costs.

[0109] Scenario: 35-week-old laying hens show fluctuations in egg production rate, decline in eggshell quality, sporadic deaths (autopsy shows enlarged, fragile, yellow-brown liver), and other fatty liver symptoms.

[0110] Additive use: Take the additive premix prepared in Example 4, and add it to the basic daily ration at an addition amount of 1000 g / ton (i.e. 0.1%).

[0111] Mixing process: 1. Take 1000 g of the additive of the present application and 10 kg of soybean meal for the first step of premixing.

[0112] 2. Mix the above premix with 100 kg of soybean meal for the second step of bulk mixing.

[0113] 3. Finally, put the mixture of the second step and 889 kg of basic daily ration raw materials into a double-shaft paddle mixer, mix for 4-6 minutes until completely uniform.

[0114] Feeding scheme: Applicable object: egg laying hens showing clinical symptoms.

[0115] Feeding method: Continuously feed the above feed.

[0116] Period: Continue feeding until the production performance (egg production rate, egg quality) returns to stable and remains for 2 weeks, then switch to the prevention mode or stop adding. The above prepared complete formula feed is fed to egg laying hens in the peak production period (28-36 weeks old).

[0117] First, the present invention and the representative scheme of the existing patent have different effects on the production performance of egg laying hens.

[0118] 1. Purpose of the experiment: Through strict animal experiments, the effect difference of the composition of the present invention (INV) and the following representative schemes of existing patents in preventing and treating fatty liver disease of egg laying hens is quantitatively evaluated:

[0119] Methyl donor combination (TRAD): represents existing patents (such as CN117502574A) with choline chloride and betaine as the core.

[0120] Bile acid combination (BA): represents existing patents (such as CN103585632A, CN101744122B) with mixed bile acid salts as the core.

[0121] Antioxidant combination (AOX): represents existing patents (such as CN1792250A) with antioxidants as the core.

[0122] Invention group (INV): feed additive of the present invention (bile acid + lysophospholipid + L-carnitine + NAC + rosemary acid extract + selenium yeast + carrier).

[0123] 2. Experimental design: (1) Experimental animals: 300 healthy Hy-Line Brown laying hens of 25 weeks of age with similar egg production rate were randomly divided into 5 groups, with 6 replicates in each group and 10 hens in each replicate. The experiment lasted for 8 weeks.

[0124] (2) Basic diet: The high-energy low-choline basic diet (metabolic energy 2.78 Mcal / kg, choline content 1100 mg / kg) without any additional choline, betaine, bile acid or antioxidant as described above was used to successfully construct the FLHS susceptible model (see Table 1 and Table 2).

[0125] (3) Rearing management: All laying hens were managed by standardization cage breeding, with 16 hours of light per day. The number of eggs laid, egg weight, number of broken soft eggs and dead hens were recorded daily on a repeated basis.

[0126] (4) Experimental grouping and treatment (all additives were mixed with the basic diet): Control group (CON): basic diet.

[0127] Traditional scheme group (TRAD): basic diet + 600 g / ton of feed (consisting of 500 g of choline chloride and 100 g of betaine).

[0128] Bile acid group (BA): basic diet + 150 mg / kg of bile acid (i.e. 150 g / ton).

[0129] Antioxidant group (AOX): basic diet + 500 mg / kg of antioxidant complex (consisting of 200 mg of NAC, 250 mg of rosemary acid extract, 2.5 mg of selenium yeast and 47.5 mg of carrier).

[0130] The present application group (INV): basic diet + 1000 mg / kg of the additive of the present application (consisting of 200 mg of bile acid, 150 mg of lysophosphatidylcholine, 100 mg of L- L-carnitine, 200 mg of NAC, 250 mg of rosemary acid extract, 2.5 mg of selenium yeast and 97.5 mg of carrier).

[0131] Measurement index: The average egg production rate, average egg weight, broken soft egg rate and dead rate in the 4th-8th week were determined.

[0132] 3. Experimental results:

[0133] Table 4. Production performance (mean ± standard deviation) of different combinations in the later stage of the experiment (4th-8th week):

[0134]

[0135] Note: Different letters in the same row indicate significant differences (P < 0.05).

[0136] 4. Conclusion analysis: (1) Egg production rate: the egg production rate of the inventive group (INV) is significantly higher than that of each of the other groups, and is increased by 6.7 percentage points compared with the control group. This shows that the production potential of the laying hens is fully released by improving the liver health in a systematic manner.

[0137] (2) Egg quality: the rate of broken soft eggs of the INV group is the lowest. This shows that after the liver function is improved, the lipid and calcium and phosphorus metabolism is more efficient, and the function of the eggshell gland is optimized, thereby improving the quality of the eggshell.

[0138] (3) Mortality rate: the mortality rate of the INV group is significantly reduced (P<0.05). This directly proves that the present application effectively prevents and treats fatty liver, significantly reduces acute death caused by liver rupture and hemorrhage, and has significant economic benefits.

[0139] Comprehensive advantages: the present application has comprehensive advantages in improving yield (egg production rate), improving quality (egg weight and eggshell quality), and reducing loss (mortality rate), which cannot be matched by any single component or traditional scheme.

[0140] Second, the use effect comparison experiment between the present application and the representative scheme of the existing patent:

[0141] Experimental purpose: the additive of the present application is compared with the product on the market representing the existing patent technology in a head-to-head manner (Head-to-Head) to verify its superiority (efficiency).

[0142] Experimental design: 1. Experimental animals and design: experimental animals: 300 Hyline brown laying hens of 25 weeks old, in good health, and with similar egg production rates were selected and randomly divided into 5 treatment groups, 6 replicates per group, and 10 hens per replicate. The test period was 8 weeks.

[0143] Basic diet: the high-energy low-choline basic diet (metabolic energy 2.78 Mcal / kg, choline content 1100 mg / kg) as described above was used, and any additional choline, betaine, bile acid or antioxidant was not contained, so as to successfully construct the FLHS susceptible model (see Tables 1 and 2).

[0144] Raising and management: all the laying hens were managed by standardization cage raising, and the light was on for 16 hours per day. The number of eggs laid, egg weight, number of broken soft eggs, and number of dead and culled hens were recorded per day in the form of replicates.

[0145] 2. Experimental grouping and treatment: control group (CON): fed with the basic diet.

[0146] Comparative example 1 (TRAD): fed with the basic diet + 600 g / ton of feed (500 g of choline chloride + 100 g of betaine). (Representing the methyl donor scheme represented by CN101027999A)

[0147] Comparative Example 2 (COM): basal diet + 1000 g / t feed of a certain brand of choline-cholic acid complex (representing the mainstream modified product on the market)

[0148] The inventive group, i.e. the experimental group (INV): basal diet + 1000 g / t feed of the inventive additive (consisting of 200 mg cholic acid, 150 mg lysophosphatidylcholine, 100 mg L-carnitine, 200 mg NAC, 250 mg rosemary acid extract, 2.5 mg selenium yeast and 97.5 mg carrier).

[0149] Measurement index: at the end of the experiment, 10 chickens were slaughtered from each group, liver fat rate was measured, blood was collected to separate serum, serum ALT and AST activity were measured, and production performance was recorded throughout the process.

[0150] 3. Experimental results: the following table shows the measurement results of the 8-week experimental period (mean ± standard deviation).

[0151] Table 5. Experimental results of the control group, comparative example and experimental group (mean ± standard deviation):

[0152]

[0153] Note: different letters in the same row indicate significant differences (P < 0.05).

[0154] 4. Conclusion analysis: (1) Compared with the traditional patent scheme (TRAD): the present invention is significantly better than the traditional methyl donor scheme in all core indicators. The liver fat rate is further reduced by 48.5% [(6.8-3.5) / 6.8*100%] compared with the TRAD group, which proves that the technical effect of the present invention has achieved a leap-forward improvement, rather than a simple improvement.

[0155] (2) Compared with the modified product on the market (COM): the present invention is also significantly better than the "choline-cholic acid" complex on the market. This shows that even if the existing technology is simply stacked (methyl donor + digestion promotion), its effect is far inferior to the system benefits achieved by the multi-target deep synergy (reducing burden + transporting out + oxidation + protection) of the present invention.

[0156] (3) Progression: the effect of the present invention is not a linear extension of the existing technology. Experimental data fully prove that it produces "unexpected" and "significant" technical effects relative to the closest prior art (TRAD and COM), providing strong support for the inventiveness (Inventive Step) of the present invention.

[0157] III. L-carnitine (LCA) dose effect and optimal ratio verification experiment:

[0158] 1. Experimental purposes: to verify the optimal additive amount of the core functional ingredient in the additive of the application, L- L-carnitine (LCA), and to confirm the reasonable concentration range (5%-15%) of its efficacy in the composition, proving that the range is not randomly selected, but is based on the scientific dose-effect relationship.

[0159] 2. Experimental design: (1) Experimental animals and design: 200 healthy Hyline Brown laying hens of 27 weeks of age with similar egg production rates were selected and randomly divided into 5 groups with 5 replicates and 8 chickens per replicate. The test period was 6 weeks.

[0160] (2) Basic diet: same as before (low-choline basic diet).

[0161] (3) Experimental grouping and treatment: control group (CON): basic diet.

[0162] Experimental group (LCA-0%): basic diet + 1000mg / kg LCA-free basic composition (cholic acid 22% + lysophosphatidylcholine 16% + NAC 22% + rosemary acid extract 27.5% + yeast selenium 2.2% + carrier 10.3%).

[0163] Experimental group (LCA-5%): basic diet + 1000mg / kg composition containing 5% LCA (cholic acid 20% + lysophosphatidylcholine 15% + L- L-carnitine 5% + NAC 20% + rosemary acid extract 25% + yeast selenium 2% + carrier 13%).

[0164] Experimental group (LCA-10%): basic diet + 1000mg / kg composition containing 10% LCA (cholic acid 20% + lysophosphatidylcholine 15% + L- L-carnitine 10% + NAC 20% + rosemary acid extract 25% + yeast selenium 2% + carrier 8%). (This is the optimal ratio embodiment)

[0165] Experimental group (LCA-15%): basic diet + 1000mg / kg composition containing 15% LCA (cholic acid 19% + lysophosphatidylcholine 14% + L- L-carnitine 15% + NAC 19% + rosemary acid extract 23.5% + yeast selenium 1.9% + carrier 7.6%).

[0166] Note: The total amount of additives in each group is 1000mg / kg, and the carrier amount is adjusted to balance the increase and decrease of LCA to ensure fair comparison.

[0167] (4) Determination index: At the end of the test, 10 chickens were slaughtered from each group, the liver was taken to determine the liver fat rate; blood was collected to separate serum, and serum ALT activity was determined.

[0168] 3. Experimental results: The following table is the measurement result of the 6-week experimental period (mean ± standard deviation).

[0169] Table 6. Experimental results of L-carnitine (LCA) at different doses:

[0170]

[0171] Note: The same row data with different letters represent significant differences (P < 0.05).

[0172] 4. Analysis and conclusion (list):

[0173] Table 7. L-carnitine (LCA) dose effect analysis table:

[0174]

[0175] Conclusion of this example: Through systematic dose effect study, it is confirmed that the optimal concentration of L-carnitine in the composition of the application is 10%, and the reasonable and effective addition range is between 5%-15%. This range is determined based on real biological effect and economic consideration, and provides sufficient and reliable experimental basis for the limitation of "L-carnitine 5%-15%".

[0176] Four, other component dose effect and optimal ratio verification experiment:

[0177] 1. General experimental design: 1.1 Purpose of the experiment: one by one to verify the reasonable addition range of bile acids, lysed lecithin, NAC, rosemary acid extract, and selenium yeast in the composition of the application, and prove the scientificity of the range.

[0178] 1.2 General scheme: For each component to be tested, set up an experiment containing low, medium and high three gradients (wherein the "medium" level is consistent with the core formula of the embodiment), and set up a negative control group (0% group) without the component. Keep the total amount of additives and the proportion of other components unchanged, and only adjust the amount of carrier to change the dose of the target component.

[0179] 1.3 Measurement index: liver fat rate and serum ALT activity at the end of the test (select the two most representative indexes).

[0180] 1.4 Animals and basic diet: same as above (LCA verification).

[0181] 2. Dose effect experiment of each component;

[0182] Table 8. Experiment 4.1, bile acid (BA) dose effect verification:

[0183]

[0184] Conclusion: Bile acid proportion in the range of 15-25% is effective, 20% is the best point of effect, and the cost-benefit ratio is optimal. Support the "15-25%" limit.

[0185] Table 9 Experiment 4.2, Lecithin (LPC) Dose Effect Verification:

[0186]

[0187] Conclusion: Lecithin is the core component of lipid-lowering, 10% is effective, 15% has reached the effect plateau, and the cost-effectiveness is high. Support the "10-20%" limit.

[0188] Table 10 Experiment 4.3, N-acetylcysteine (NAC) Dose Effect Verification:

[0189]

[0190] Conclusion: NAC in the range of 15-25% has a significant effect on improving liver function (reducing ALT), and 20% is the optimal comprehensive point. Support the "15-25%" limit.

[0191] Table 11 Experiment 4.4, Rosemary Acid Extract (RE) Dose Effect Verification (based on 20% rosemary acid content):

[0192]

[0193] Conclusion: Rosemary acid extract proportion in the range of 10-30% is effective, and 20% is the best comprehensive effect. Support the "10-20%" limit (based on cost-effectiveness, choose a more economical upper limit).

[0194] Table 12 Experiment 4.5, Yeast Selenium (Se) Dose Effect Verification (based on selenium):

[0195]

[0196] Conclusion: Yeast selenium can significantly improve antioxidant effect (reflected in further reduction of ALT) when added to 2%, and the effect gain is not obvious beyond 2%. Support the "1-3%" limit.

[0197] Conclusion: 1. Scientific verification: This series of dose effect experiments have confirmed that the weight percentage range of each component (bile acid 15-25%, lecithin 10-20%, L-carnitine 5-15%, NAC 15-25%, rosemary acid extract 10-20%, and yeast selenium 1-3%) is determined based on real and repeatable biological effects, not arbitrarily selected.

[0198] 2. Optimal ratio confirmation: The experiment determined that a preferred core ratio of the additive of the present application is: bile acids 20% + lysophosphatidylcholine 15% + L-carnitine 10% + NAC 20% + rosemary acid extract 20% + selenium yeast 2% + carrier 13%. Under this ratio, the effects of the composition on preventing fatty liver and improving liver function reach the best balance.

[0199] 3. The above data and component range provide sufficient support to ensure the stability and effectiveness of the patent at the legal level.

[0200] Five, comparison experiment of the composition of the present application and the effect of each single component and verification of synergistic effect:

[0201] 1. Experimental purpose: By comparing the complete composition of the present application (INV) with each single active ingredient group, the contribution of each component in the overall effect is quantitatively evaluated, and it is proved that the effect produced by the composition of the present application is not simple addition, but there is a significant synergistic effect.

[0202] 2. Experimental scheme: (1) Experimental animals and design: 350 healthy Hyline brown egg laying chickens aged 25 weeks were selected and randomly divided into 7 treatment groups, 5 replicates per group, 10 chickens per replicate. The test period is 8 weeks.

[0203] (2) Basic feed: same as the previous content (low choline basic diet).

[0204] (3) Test grouping and treatment (the total amount of each additive added is 1000 mg / kg, supplemented by carrier): control group (CON): basic feed.

[0205] Single bile acid group (BA): basic feed + 1000 mg / kg bile acid (i.e. the content of BA in the additive is 100%).

[0206] Single lysophosphatidylcholine group (LPC): basic feed + 1000 mg / kg lysophosphatidylcholine (LPA≥50%).

[0207] Single L-carnitine group (LCA): basic feed + 1000 mg / kg L-carnitine.

[0208] Single antioxidant combination group (AOX): basic feed + 1000 mg / kg antioxidant combination (NAC 400 g + rosemary acid extract 500 g + selenium yeast 5 g + carrier 95 g, the ratio of the three is fixed, and the total weight is 1000 g).

[0209] The inventive group (INV): basal diet + 1000 mg / kg of the inventive complete additive (200 g of bile acids + 150 g of lysophosphatidylcholine + 100 g of L-carnitine + 200 g of NAC + 250 g of rosemary acid extract + 2.5 g of selenium yeast + 97.5 g of carrier).

[0210] (4) Determination index: at the end of the experiment, the liver fat rate and serum ALT activity were determined.

[0211] 3. Experimental results:

[0212] Table 13 Determination results of the 13-week experimental period (mean ± standard deviation):

[0213]

[0214] Note: The same row data with different letters represent significant differences (P < 0.05).

[0215] 4. Synergistic effect calculation and analysis: 4.1 Synergistic effect calculation (taking liver fat rate as an example, Bliss independence model was used), the effect value (Effect_size) of each single component was calculated: Effect_BA = 8.50-6.80 = 1.70; Effect_LPC = 8.50-6.20 = 2.30; Effect_LCA = 8.50-7.10 = 1.40; Effect_AOX = 8.50-6.90 = 1.60; Theoretical effect value total (ΣEffect_single) = 1.70+2.30+1.40+1.60 = 7.00; Calculate the theoretical expected value (E_exp): E_exp = CON-ΣEffect_single = 8.50-7.00 = 1.50%; Calculate the synergistic effect value (SynergyValue) and the synergistic effect ratio: the actual observed value (E_obs, INV group) = 3.50%; Synergistic effect value = E_exp-E_obs = 1.50-3.50 = -2.00%; Actual reduction range of INV = 8.50-3.50 = 5.00%; Synergistic effect ratio = (actual reduction range / total theoretical effect value) * 100% = (5.00 / 7.00) * 100% = 71.4%;

[0216] 4.2 List analysis:

[0217] Table 14 Synergistic effect analysis table of the inventive composition (based on liver fat rate):

[0218]

[0219] Conclusions: (1) Limited effect of single component: Any single active ingredient (BA, LPC, LCA, AOX) has a certain lipid-lowering or liver-protecting effect, but the effect is far inferior to the complete composition of the invention (INV). For example, the single LCA group has the weakest effect, which confirms the limitations of its single action.

[0220] (2) Clear synergistic effect: The synergistic effect value (-2.00%) and synergistic effect ratio (71.4%) both strongly prove that the effect of the composition of the invention is not simply the sum of the effects of each single component. Each component promotes each other in the "de-burdening-transporting-oxidizing-protection" system designed in this scheme, producing an unexpected synergistic effect of "1+1+1+1>4".

[0221] (3) Support for non-obviousness: The skilled person in the art cannot predict from the limited effects of each single component that their combination in a specific ratio can produce such a strong synergistic effect. The prior art has never taught or suggested that the "fat oxidation promoting" (L- carnitine) path can be coordinated with other paths to systematically solve the FLHS problem. This example provides direct and key experimental evidence to prove the inventiveness (non-obviousness) of the invention.

[0222] This example, together with the dose-effect experiment, constitutes a solid data base supporting the patentability of the invention.

[0223] Six, Research on the Synergistic Mechanism of Feed Additives for Preventing and Treating Fat Liver Disease in Poultry Based on Multi-omics Technology:

[0224] Project goal: Apply transcriptomics and metabolomics technologies to reveal the internal mechanism of the multi-target synergistic action of the additive (INV) of the invention at the molecular level, and provide systematic biological evidence for its excellent efficacy.

[0225] Experimental design: See the previous experiment. Based on the completed animal experiment, liver tissue and serum samples were collected for multi-omics analysis.

[0226] Experimental design and sample collection: 1. Animal experiment: see the previous experimental design. Select representative groups: control group: basic diet.

[0227] Traditional scheme group: basic diet + choline chloride / betaine.

[0228] High-dose group of the invention: basic diet + 0.1% additive of the invention.

[0229] 2. Sample collection: At the end of the experiment, 6 chickens were randomly selected from each group, and after slaughter, the following samples were quickly collected:

[0230] Liver tissue: part of it was quickly frozen in liquid nitrogen for transcriptomics analysis; part was used for lipid metabolite extraction.

[0231] Serum: for non-targeted metabolomics analysis.

[0232] 3. Multi-omics detection: transcriptomics: liver tissue RNA extraction, mRNA sequencing, and analysis of differentially expressed genes.

[0233] Lipidomics: analysis of triglycerides, phospholipids, fatty acid composition, etc. in liver tissue.

[0234] Serum non-targeted metabolomics: comprehensive analysis of changes in small molecule metabolites in serum.

[0235] Transcriptomics results: reveal the synergistic regulation at the gene expression level;

[0236] Differential gene expression analysis, GO function and KEGG pathway enrichment analysis on liver tissue mRNA sequencing data.

[0237] 1. Core pathway regulation verification: fatty acid synthesis pathway: compared with the CON group, the expression levels of fatty acid synthase gene (FASN) and sterol regulatory element binding protein-1c (SREBP-1c) in the INV group were significantly down-regulated. This directly verifies the molecular mechanism of bile acid inhibiting de novo fatty acid synthesis by activating the FXR receptor.

[0238] Fatty acid oxidation pathway: the gene expression of carnitine palmitoyltransferase 1A (CPT1A) and peroxisome proliferator-activated receptor alpha (PPARa) in the INV group was significantly up-regulated. This provides the most direct molecular evidence for the core role of L-carnitine in promoting fatty acid beta-oxidation.

[0239] Antioxidant pathway: antioxidant enzyme-related genes such as glutathione S-transferase (GST), heme oxygenase-1 (HO-1) in the INV group were significantly enriched and up-regulated, and the nuclear factor E2-related factor 2 (Nrf2) pathway was significantly activated. This confirms the synergistic activation effect of the antioxidant network composed of NAC, rosmarinic acid, and selenium at the transcriptional level.

[0240] 2. Unique synergistic pattern discovery:

[0241] Transcriptomic data shows that the INV group can simultaneously positively regulate the fatty acid oxidation pathway (such as PPAR signaling) and negatively regulate the fatty acid synthesis pathway (such as Biosynthesis of unsaturated fatty acids). The TRAD group only has a slight effect on fatty acid export-related genes and cannot achieve this "two-way precise regulation". This explains the reason why the effect of the present application is much better than that of the traditional scheme at the gene level.

[0242] Metabolomics results: capture the dynamic changes at the metabolite level;

[0243] 1. Liver lipidomics analysis: triglyceride (TG) species and content: The content of multiple long-chain triglycerides in the liver of the INV group was significantly lower than that in the CON group and the TRAD group, which was completely consistent with the result of the decrease in liver fat rate in the biochemical assay.

[0244] Phospholipid spectrum change: The content of phosphatidylcholine (PC) in the liver of the INV group was significantly higher than that in the CON group. PC is a key component of VLDL synthesis, and this result supports the mechanism of hemolyzed lysophosphatidylcholine (LPA) accelerating the "transport out" of fat from the liver by promoting PC metabolism or VLDL assembly.

[0245] Fatty acid composition: The content of metabolites related to beta-oxidation (such as acylcarnitine) in the liver of the INV group changed, suggesting that fatty acid catabolism was more active.

[0246] 2. Serum non-targeted metabolomics analysis:

[0247] Differential metabolite screening: In positive and negative ion modes, a large number of differential metabolites were found between the INV group and the CON group.

[0248] Pathway analysis: These differential metabolites were significantly enriched in primary bile acid biosynthesis, glycerophospholipid metabolism, and unsaturated fatty acid biosynthesis, which was highly consistent with the transcriptome results and the mechanism of the application.

[0249] Potential biomarker discovery: It was found that lysophosphatidylcholine (LPC16:0, 18:0) and other substances had a significantly increased content in the serum of the INV group. LPC is the precursor of LPA and an important component of lipoproteins, and its level change may reflect the improvement of liver fat transport status, which can be used as a potential new biomarker to evaluate the effect of the product.

[0250] Multi-omics integrated analysis: Construct a "gene-metabolite" synergistic network;

[0251] Correlation analysis of differentially expressed genes and differential metabolites in the transcriptome and metabolome was performed to construct a "gene-metabolite" interaction network.

[0252] 1. Key regulatory hub confirmation: Network analysis showed that PPARα and FXR were two core regulatory nodes that connected a large number of downstream differential genes and metabolites. This indicates that the additive of the application is precisely by regulating these two core receptors, thereby triggering widespread changes in gene expression and metabolite levels downstream, ultimately achieving systematic improvement of lipid metabolism.

[0253] 2. Mechanism closed loop verification: Integrated analysis forms a clear evidence chain;

[0254] Bile acids→activate FXR→down-regulate SREBP-1c / FASN (transcriptome)→reduce TG synthesis (lipidome);

[0255] L-carnitine→activate PPARa→up-regulate CPT1A (transcriptome)→increase acylcarnitine / reduce TG (metabolome);

[0256] Antioxidant network→activate Nrf2→up-regulate antioxidant enzymes (transcriptome)→reduce oxidative stress products (metabolome)→provide protection for above metabolic processes;

[0257] Conclusion: By integrating transcriptomic and metabolomic analysis, this study provides direct experimental evidence for the synergistic mechanism of the present additive in preventing and treating fatty liver disease in laying hens from the system and molecular dimensions:

[0258] 1. Confirmed the multi-target mechanism: The effectiveness of the four paths of "inhibiting synthesis, promoting oxidation, accelerating export, and antioxidant" was verified at the gene and metabolite levels.

[0259] 2. Revealed the essence of synergy: Each component does not act in isolation, but through the regulation of core hubs such as PPARa, FXR, and Nrf2, a highly synergistic regulatory network is formed, thereby achieving "bidirectional regulation" of fat metabolism.

[0260] 3. Discovered potential biomarkers: Providing new ideas for subsequent product effect monitoring and rapid detection.

[0261] 4. Strengthened the synergistic effect of technology: Multi-omics data revealed the complexity and sophistication of the mechanism at the system level, strongly proving that the technical solution is not obvious to those skilled in the art, and produced unexpected synergistic technical effects based on deep biological understanding.

[0262] Table 15 Summary of multi-omics technology verification of the mechanism of the present invention:

[0263]

[0264]

[0265]

[0266] Conclusion: 1. Data consistency: Transcriptomic data (gene expression down-regulation / up-regulation) is highly consistent with metabolomic data (substrate / product content reduction / increase), forming a complete evidence chain from "cause" to "effect". For example: FASN gene down-regulation (transcriptome)→TG content reduction (lipidome). PPARa / CPT1A gene up-regulation (transcriptome)→acylcarnitine content increase (metabolome).

[0267] 2. Effect significance: The change range of key indicators (such as the change in mRNA expression by several times, and the change in metabolite content by more than 50%) and the extremely significant P value (usually less than 1E-05) strongly prove the strength and reliability of the intervention effect of the present application, which is not comparable to the general slight improvement.

[0268] 3. Embodiment of synergy:

[0269] The extreme activity of the PPAR pathway (pro-oxidation) and the activation of the FXR pathway (synthesis inhibition) occur at the same time, achieving "bidirectional precise regulation" of liver fat metabolism.

[0270] Multi-omics network analysis shows higher network connectivity, providing a structural basis for "synergistic effect" at the system level, which is mutually confirmed by animal phenotype data (Bliss synergy index 71.4%).

[0271] 4. "Outstanding substantial features" and "significant progress" (i.e. creativity): Multi-omics data from gene expression (reason) to metabolite changes (result) level form a complete and self-consistent evidence loop, which strongly proves that the "de-burdening-transporting-oxidizing-protection" four-dimensional synergistic mechanism proposed by the present application is not only a theoretical hypothesis, but also an objective biological fact. These data reveal the complexity and sophistication of the present application combination in the system level, providing extremely key high-level scientific evidence for proving that its technical solution has "outstanding substantial features" and "significant progress" (i.e. creativity) compared with the traditional single approach solution.

[0272] Seven, network pharmacology-based analysis of the mechanism of action of the core components of the feed additive:

[0273] Analysis object: synergistic feed additive for preventing and treating fatty liver disease in laying hens (components: bile acids, lysophosphatidylcholine, L-carnitine, NAC, rosemary acid extract, and selenium yeast);

[0274] Analysis method: combination of network pharmacology (target prediction, network construction, pathway enrichment analysis) and experimental verification;

[0275] Analysis background and purpose: Network pharmacology is a new discipline based on the "disease-gene-target-drug" interaction network, which systematically reveals the mechanism of action of multi-component drugs. This study applies this technology to:

[0276] 1. Theoretical prediction: predict the potential key targets and signaling pathways of the six core components of the present application acting on fatty liver disease (FLHS) in laying hens from a system level.

[0277] 2. Mechanism explanation: Integrate the computational prediction results with the "de-burdening-transporting-oxidizing-protecting" four-dimensional synergistic mechanism of the invention, providing theoretical support at the molecular level.

[0278] 3. Evidence of creativity: By showing the complex network synergy of components-targets-pathways, the non-obviousness and overall synergy of the combination are demonstrated, providing supplementary evidence for patent creativity.

[0279] Materials and methods:

[0280] 1. Core component active molecule screening: Obtain the known active molecular structures of each component of the invention (such as CDCA, LPA, L-carnitine, NAC, rosmarinic acid, selenomethionine, etc.) from databases such as PubChem and TCMSP.

[0281] 2. Potential target prediction: Use online servers such as SwissTargetPrediction and PharmMapper to predict the potential targets of the above active molecules.

[0282] 3. FLHS-related target collection: Use disease databases such as GenCards, OMIM, and DisGeNET to search for related disease targets of poultry / mammalian FLHS using keywords such as "Fatty Liver Syndrome" and "Hepatic Steatosis".

[0283] 4. Network construction and analysis:

[0284] Component-target network: Construct the interaction network of core components and predicted targets.

[0285] Protein interaction network: Import the intersection targets into the STRING database to construct a protein interaction network and screen for core targets.

[0286] Pathway enrichment analysis: Use tools such as DAVID and KOBAS to perform GO function and KEGG pathway enrichment analysis on core targets.

[0287] 5. Experimental verification correlation: Perform correlation analysis between the network analysis results and the key indicators measured in the animal experiments of the invention (liver fat rate, ALT / AST, GSH-Px, MDA, etc.).

[0288] Results and analysis: 1. Core component-target network reveals multi-target characteristics;

[0289] Network analysis shows that the six core components of this invention work together on a network containing 128 potential targets closely related to lipid metabolism, oxidative stress, and inflammatory responses. The targets of each component overlap, yet each component has its own focus, exhibiting a typical "multi-component, multi-target" characteristic.

[0290] Bile acids (CDCA): The core targets are nuclear receptors such as FXR and TGR5, which confirms the molecular basis of their "inhibition of synthesis".

[0291] L-L-carnitine is significantly enriched in key fatty acid oxidation enzymes and regulatory factors such as CPT1A and PPARα, supporting its "promoting oxidation" function.

[0292] Antioxidant network (NAC / rosmarinic acid / selenium): Together they act on the KEAP1-Nrf2 pathway, apoptosis and related antioxidant enzyme targets, forming a molecular network of "protection" mechanism.

[0293] 2. Pathway enrichment analysis confirms the system's regulatory mechanism;

[0294] Table 16 shows the KEGG pathway enrichment analysis results, indicating that the core targets were significantly enriched in the following pathways (Top 10, P < 0.01):

[0295]

[0296] Analysis: Pathway analysis shows that the additives of this invention do not act randomly on scattered targets, but systematically regulate the core signaling network of FLHS occurrence and development, especially precisely intervening in the three key hub pathways that regulate lipid metabolism globally: PPAR, AMPK, and FXR.

[0297] 3. “Collaborative network” analysis corroborates non-obviousness;

[0298] Network complementarity: By comparing the target network of the present invention with a virtual network containing only methyl donors (choline) or single bile acids, it was found that the network nodes of the present invention have higher connectivity and a tighter network structure, indicating that the components form a more complex regulatory network through the interaction between targets.

[0299] Key Target Coverage: This invention provides combined coverage of multiple key nodes (such as PPARα, FXR, and Nrf2) in the FLHS disease network, whereas any single component or conventional approach can only cover a portion of these nodes. This synergistic coverage of key disease nodes forms the structural basis for achieving a synergistic effect of "1+1+1>3".

[0300] 4. Mutual verification between network predictions and experimental results;

[0301] Table 17 Network Prediction and Experimental Results:

[0302]

[0303] Inventive evaluation conclusion: 1. Non-obviousness: Network pharmacology analysis shows that the combination of components focusing on four different pathways of "synthesis (FXR), export (LPA), oxidation (PPARa / CPT1A), and protection (Nrf2)" is not a routine technical means in the art. This combination is a creative design based on a deep understanding of the complex pathological mechanism of FLHS, which is far more complex and systematic than simple component superposition.

[0304] 2. Unexpected technical effect (synergistic effect): Computational analysis shows that the combination of the invention forms a highly interconnected and functionally complementary target regulation network. This explains why a 71.4% synergistic effect rate was observed in animal experiments from the perspective of systems biology. The overall effect based on network synergy is unpredictable and unattainable by existing technologies that only focus on single targets or pathways.

[0305] Conclusion: Network pharmacology analysis provides strong theoretical basis and data support for the inventiveness (non-obviousness + significant progress) of the invention patent from the "system-network-target" level. It clearly shows that the technical solution of the invention is an optimized combination based on innovative understanding of disease mechanisms that produces unexpected synergistic effects.

[0306] Experimental effect verification of core components in network pharmacology analysis in the multi-target synergistic prevention and treatment of fatty liver disease in laying hens:

[0307] Relevant target: To confirm the computational prediction of biological pathways with empirical data, and to strengthen the scientificity and credibility of the mechanism of action.

[0308] Network prediction and experimental verification of core component "L-carnitine (LCA)":

[0309] 1. Network pharmacology prediction:

[0310] Core target: CPT1A (carnitine palmitoyltransferase 1A), PPARa (peroxisome proliferator-activated receptor alpha).

[0311] Enrichment pathway: PPAR signaling pathway, Fatty acid degradation, AMPK signaling pathway.

[0312] Predicted function: As a key carrier, it promotes the entry of long-chain fatty acids into mitochondria for beta-oxidation, which is the core engine of fat catabolism.

[0313] 2. Experimental data verification:

[0314] Dose-effect verification: With the total amount of additives fixed, as the proportion of LCA increased from 0% to 10%, the liver fat rate showed a significant dose-dependent decrease (from 4.6% to 3.4%), and the serum ALT activity improved significantly (from 31.5 U / L to 25.1 U / L). The effect entered a plateau phase beyond 10%.

[0315] Synergistic effect verification: Although LCA alone (single LCA group) had some effect (liver fat rate 7.10%), it was far inferior to the complete combination of the invention (INV group, liver fat rate 3.50%). This confirmed the network prediction: although LCA is the core engine, its efficient operation depends on the favorable environment created by other components (such as antioxidant protection of mitochondrial function).

[0316] Key conclusion: Experimental data not only verified the key role of LCA in promoting fat oxidation, but also proved that its efficacy highly depends on the synergistic system constructed by the invention, rather than isolated action.

[0317] Network prediction and experimental verification of the core component "bile acid (CDCA)":

[0318] 1. Network pharmacology prediction:

[0319] Core targets: FXR (farnesoid X receptor), TGR5.

[0320] Enrichment pathways: FXR / RXR activation pathway, bile acid biosynthesis.

[0321] Predicted function: Activates FXR, down-regulates SREBP-1c expression, thereby inhibiting de novo synthesis of liver fat (reduction).

[0322] 2. Experimental data verification:

[0323] Dose-effect verification: Bile acid proportion is effective within the range of 15%-25%, with 20% being the best point. When the proportion of bile acid is 0% (BA-0% group), the liver fat rate (5.2%) and ALT (35.8 U / L) are significantly different from the groups containing bile acid.

[0324] Function necessity verification: The liver fat rate of single bile acid (single BA group) is 6.80%, which is effective but limited, indicating that its "synthesis inhibition" function needs to be synergized with other mechanisms to maximize its benefits.

[0325] Key conclusion: The experiment confirms the effectiveness of bile acids in reducing the burden through the FXR pathway and determines the optimal working concentration in the invention. Its synergy with LPA (promoting export) achieves "opening up and saving" type of fat control.

[0326] Network prediction and experimental verification of the core component "antioxidant network (NAC + rosmarinic acid + selenium)":

[0327] 1. Network pharmacology prediction:

[0328] Core targets: Nrf2, KEAP1, GSR (glutathione reductase), GPX (glutathione peroxidase).

[0329] Enriched pathways: Glutathione metabolism, Nrf2 signaling pathway.

[0330] Predicted function: Form a multi-level antioxidant defense: NAC as a GSH precursor to supplement the substrate, rosmarinic acid to directly scavenge free radicals, and selenium as the active center of GSH-Px.

[0331] 2. Experimental data verification:

[0332] Biochemical index verification: The GSH-Px activity (195 U / mg) of the high-dose group (IN-H) of the invention is extremely significantly the highest, and the MDA content (1.8 nmol / mg) is extremely significantly the lowest, directly proving the effectiveness of the antioxidant network.

[0333] Component necessity verification: The use of the antioxidant combination alone (single AOX group) performs well in antioxidant indicators (such as MDA), but has poor results in core lipid-lowering indicators (liver fat rate 6.90%), proving that it cannot solve the problem of fat deposition alone.

[0334] Dose relationship verification: NAC (15-25%), rosmarinic acid extract (10-20%), and yeast selenium (1-3%) all show the best auxiliary effect within a specific proportion range.

[0335] Key conclusion: Experimental data perfectly confirm the synergistic liver protection of the "precursor (NAC)-enzyme (selenium)-scavenger (rosmarinic acid)" three-dimensional antioxidant network, which provides indispensable "escort" protection for the fat oxidation function of L- carnitine and the health of liver cells.

[0336] Network prediction and experimental verification of the core component "lyso-phosphatidylcholine (LPA)":

[0337] 1. Network pharmacology prediction: Core targets: LPAR1-6 (lysophosphatidic acid receptors), PPARγ, ATGL (fatty triglyceride lipase).

[0338] Enriched pathways: PPAR signaling pathway, GPCR ligand binding, Lipid and atherosclerosis.

[0339] Predicted functions: As a biological signal molecule, it promotes fat decomposition and the assembly and secretion of very low-density lipoprotein (VLDL) by activating its receptors and nuclear receptors, solving the fat "export" problem.

[0340] 2. Experimental data verification: Dose-effect verification: The effect of lysophosphatidylcholine ratio is significant in the range of 10%-20%. When the ratio of LPA is 0% (LPC-0% group), the liver fat rate (5.0%) and ALT (34.2 U / L) are significantly higher. When the ratio rises to 15%, the effect reaches a plateau, indicating that this concentration is sufficient to effectively activate the export pathway.

[0341] Functional uniqueness verification: The use of LPA alone (single LPC group) shows better lipid-lowering effect (liver fat rate 6.20%), better than single bile acid group, which confirms the effectiveness of its "promoting export" path. But its effect on improving liver function (ALT is 48.8 U / L) is not as good as the complete combination of the invention, indicating that it needs to be coordinated with antioxidant components to protect liver cells.

[0342] Key conclusions: Experimental data confirms the key role of LPA in promoting liver fat "export" and clearly defines its effective dose range. It, together with bile acid (synthesis inhibition), constitutes the core of the balance regulation of liver fat "in and out", and forms a complement with L- carnitine (promoting decomposition).

[0343] Synergistic effect: Network prediction and Bliss model experimental verification:

[0344] 1. Synergistic basis of network prediction: Network analysis shows that the core targets of each component are distributed in different key nodes of the fat metabolism and oxidative stress network, and these nodes are highly interconnected in the protein interaction network, forming the structural basis for synergistic effect.

[0345] 2. Experimental verification of synergistic effect: Bliss independence model calculation shows that the synergistic effect ratio of the composition of the invention on the core indicator of reducing liver fat rate is as high as 71.4%.

[0346] 3. Correlation analysis: This means that more than two-thirds of the outstanding effect achieved by the invention (such as a 48.5% reduction in liver fat rate) comes from the additional gain produced by the interaction between components, rather than simple addition. This directly confirms the existence and strong power of "multi-target synergistic regulation" predicted by network pharmacology in reality.

[0347] Overall network robustness and experimental reproducibility analysis: 1. Network redundancy analysis: The "relief-transport-oxidation-protection" network constructed by the present application has high redundancy. For example, the PPAR pathway is regulated by L-carnitine (PPARa) and lysophosphatidylcholine (PPARy) at the same time; the antioxidant network is composed of components with three different mechanisms. This multi-node, multi-path redundancy design makes the entire system more robust in the face of individual differences or environmental fluctuations.

[0348] 2. Experimental reproducibility verification: Multiple examples in the patent application (such as Example 4 and Example 5, dose gradient of Example 6) consistently reproduced the effect of the additive of the present application being significantly better than the control group and the traditional scheme under the same nutritional stress model. This high reproducibility shown in different experimental batches and different detection indicators proves the stability and reliability of the network regulation effect from the empirical point of view.

[0349] Overall conclusion: From network prediction to market value transformation: 1. Strong evidence chain from "correlation" to "causality": This study successfully combines the "correlation" (component-target-pathway) predicted by network pharmacology with the "causality" (specific components leading to specific physiological index changes) verified by animal experiments. This not only clarifies the mechanism, but also makes the technical solution of the present application go beyond empirical summary and is based on the scientific foundation of modern systems biology.

[0350] 2. Scientific basis for formula optimization: Network analysis reveals that each component acts on different but interrelated pathways, which explains why simple component stacking has limited effect, while the synergistic combination of precise proportion optimization can produce outstanding results. Dose-effect experiments provide an accurate "navigation map" for this optimization.

[0351] 3. Non-obviousness: First, network pharmacology shows that the complexity of the combination far exceeds conventional thinking; second, experimental data show that its effect is not linear superposition but synergistic multiplication. That is, the present application cannot be derived from the prior art by those skilled in the art.

[0352] 4. Guide future product development: The established "network prediction-experimental verification" model can serve as a powerful tool for future development of similar functional additive products, improving research and development efficiency and success rate.

[0353] 5. Build a scientific evidence system: Through integrated analysis of the present application's additive for preventing and treating fatty liver disease in laying hens, from network pharmacology prediction at the computational biology level to dose-effect, component comparison and synergistic effect quantification in animal experiments at the empirical level, a closed, self-consistent and highly persuasive scientific evidence system is established.

[0354] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A synergistic feed additive for preventing and treating fatty liver disease in laying hens, characterized in that, The composition comprises the following components: ​ Bile acid 15%-25%; Lecithin 10%-20%; L-carnitine 5%-15%; N-acetylcysteine 15%-25%; Rosmarinic acid extract 10%-20%; Yeast selenium 1%-3%; Carrier balance; The carrier is one or more of water-soluble silicon dioxide, expanded rice hull powder, calcium hydrogen phosphate; The content of lysophosphatidic acid in the lecithin is not less than 50%.

2. The synergistic feed additive for preventing and treating fatty liver disease of laying hens according to claim 1, characterized in that, The bile acid is derived from pig gall or bird gall.

3. The synergistic feed additive for preventing and treating fatty liver disease of laying hens according to claim 1, characterized in that, The content of rosmarinic acid in the rosmarinic acid extract is not less than 20%.

4. A method for the preparation of a feed additive for the preparation of a feed additive according to any one of claims 1 to 3, characterized in that, The method comprises the following steps Two-thirds of the carrier is put into the main mixer as the base material, and the mixer is run; N-acetylcysteine, L-carnitine, rosmarinic acid extract, yeast selenium and bile acid are slowly added in sequence; The lecithin is pre-mixed with the remaining one-third of the carrier in another mixer for 5 minutes to form a flowable mixture; The pre-mixed lecithin-carrier mixture is slowly added into the running main mixer; The mixer feed port is closed, and the mixture is mixed at a speed of 20 rpm-25 rpm for 25 minutes-30 minutes.

5. A feed, characterized in that, The composition comprises 99.9%-99.95% basic daily ration and 0.05%-0.10% feed additive according to any one of claims 1-3; the basic daily ration consists of the following components: Corn 62.20%; Soybean meal 24.50%; Soybean oil 2.50%; Stone powder 8.00%; CaHPO4 1.30%; NaCl 0.30%; DL-methionine 0.15%; L-Lys·HCl 0.05%; Premix 1.00%.

Images