Anti-stress experimental mouse compound feed and preparation method thereof

Abstract

This invention relates to a compound feed for anti-stress laboratory mice and its preparation method. The compound feed comprises 0.1-0.3 parts feed additives, 20-40 parts corn, 10-40 parts wheat, 1-10 parts wheat middlings, 2-10 parts soybean meal, 1-10 parts fish meal, 1-10 parts chicken meal, 1-10 parts soybean hulls, 1-5 parts soybean oil, 0.5-2 parts limestone powder, 0.5-1.5 parts dicalcium phosphate, and 2-5 parts premix. The feed additives include moringa flavonoids, vitamin C, and quercetin in a weight ratio of 1-6:1-3:0.5-2. Moringa flavonoids have strong antioxidant properties to reduce stress responses caused by transportation, experimental operations, etc.; vitamin C is an active substance that maintains the growth and metabolism of bone tissue and cells in the body and promotes the body's anti-stress function; quercetin can effectively scavenge free radicals and has a strong anti-stress ability. The above-mentioned feed additives, when added to a base feed with a specific composition and processed through an extrusion process, enhance the laboratory mice's ability to digest and absorb nutrients and improve their stress resistance.

Description

Technical Field

[0001] This invention relates to the field of laboratory animal feed technology, and in particular to an anti-stress compound feed for laboratory mice and its preparation method. Background Technology

[0002] With the rapid development of laboratory animal science in my country, the importance of laboratory animals has become increasingly prominent, and the quality of laboratory animals and animal experiments is constantly improving. Mice are one of the most common laboratory animals, and they frequently experience stress responses during their rearing. Stress refers to the non-specific response of an animal's body to various stressors, specifically manifested as increased secretion of pituitary and adrenal cortex hormones, sympathetic nerve excitation, elevated blood sugar, increased blood pressure, increased heart rate, and accelerated respiration. Under stress, the metabolic state of laboratory mice is disrupted, and their immune system exhibits an inhibitory effect, seriously endangering their health. Many factors can cause stress in mice, such as experimental procedures, transportation, and changes in the rearing environment. Conducting experiments with mice under stress usually affects the experimental results, thus impacting the accuracy of research findings.

[0003] Stress can enhance the activity of the hypothalamic-pituitary-adrenal axis, exerting its effects through glucocorticoids, leading to a series of metabolic and functional changes. Under prolonged stress, the small intestine morphology of laboratory mice may be damaged, the expression of related genes may change, and the immune function may be severely impaired, thus affecting the quality of the mice and seriously impacting animal production and experimental work. Under stress, laboratory mice have weakened resistance and increased nutritional needs.

[0004] Chinese invention patent (application number 200810116768.X), "An Anti-Stress Feed Additive," discloses a comprehensive feed additive composed of traditional Chinese medicine, amino acids, minerals, and vitamins, which promotes various physiological and dynamic changes in the body under anti-stress conditions and improves the anti-stress effect of animals. Chinese invention patent (application number 20111014531.2), "An Anti-Stress Humic Acid Added Feed," discloses a compound anti-stress feed additive composed of humic acid and traditional Chinese medicine such as Schisandra chinensis, which reduces the stress caused by changes such as weaning, grouping, and transportation. However, the above-mentioned feeds still have certain shortcomings in terms of nutritional composition and anti-stress effect in laboratory mice. Therefore, there is an urgent need for an optimized feed that is nutritionally complete and can effectively address the health hazards of stress responses in laboratory mice. Summary of the Invention

[0005] To overcome at least one problem existing in the prior art, the present invention provides a feed additive and a feed composition containing the above feed additive, and provides a method for preparing the above feed composition and its application in stress resistance in laboratory mice. The above feed composition is nutritionally complete and can solve the harm caused to the health of laboratory mice by stress response in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention is to provide a feed additive comprising moringa flavonoids, vitamin C, and quercetin, wherein the weight ratio of moringa flavonoids, vitamin C, and quercetin is 1-6:1-3:0.5-2, preferably 2-4:2:1, and more preferably 3:2:1. In one specific embodiment, the feed additive consists only of moringa flavonoids, vitamin C, and quercetin.

[0008] This invention combines moringa flavonoids, vitamin C, and quercetin. Vitamin C is an essential nutrient for the normal functioning of the animal immune system. External stimuli in laboratory mice can affect the synthesis and utilization of ascorbic acid; supplementing with vitamin C can inhibit the release of corticosterone and enhance stress resistance. Moringa flavonoids, the main flavonoid compounds in moringa, and quercetin, a natural antioxidant extracted from plants, both possess strong antioxidant properties. They can reduce oxidative damage in tissues by scavenging free radicals, protecting the body from free radical damage. The above-mentioned feed additive, by mixing moringa flavonoids, vitamin C, and quercetin, yields a safe feed additive that improves the stress resistance and immunity of laboratory mice, with few side effects. Compared with ordinary feed additives, the above-mentioned feed additive has a simple preparation method, reasonable formulation, and can significantly reduce cortisol levels in laboratory mice, significantly improve their antioxidant capacity, and contains no Western medicine components, making it safe and without toxic side effects.

[0009] A second aspect of the invention is to provide a feed composition comprising any of the feed additives described in the first aspect of the invention. It is understood that the feed composition may contain any suitable base feed to suit different animals and different nutritional needs. The feed composition is preferably used to prepare an anti-stress laboratory mouse feed.

[0010] Furthermore, in the feed composition, based on the mass of the compound feed (basic feed) as 100%, the weight percentage of the feed additive is 0.1-0.3%. If the added amount of moringa flavonoids, vitamin C, and quercetin in the above feed composition exceeds 0.3%, the stress resistance of the experimental mice will decrease and the cost will increase. Concentrations below this level cannot effectively improve the health of the experimental mice, and the technical effect of any one or two components of the above feed additive is weaker than the combination of the three components.

[0011] Further, by weight, the feed composition comprises: 0.1-0.3 parts feed additive, 20-40 parts corn, 10-40 parts wheat, 1-10 parts wheat middlings, 2-10 parts soybean meal, 1-10 parts fish meal, 1-10 parts chicken meal, 1-10 parts soybean hulls, 1-5 parts soybean oil, 0.5-2 parts limestone powder, 0.5-1.5 parts dicalcium phosphate, and 2-5 parts premix. Preferably, the feed composition comprises: 0.1-0.3 parts feed additive, 30-40 parts corn, 30-40 parts wheat, 1-6 parts wheat middlings, 2-5 parts soybean meal, 1-5 parts fish meal, 5-10 parts chicken meal, 1-6 parts soybean hulls, 1-5 parts soybean oil, 1-2 parts limestone powder, 0.8-1.5 parts dicalcium phosphate, and 2-5 parts premix. More preferably, the feed composition comprises: 0.2 parts feed additive, 37 parts corn, 32 parts wheat, 4 parts wheat middlings, 3 parts soybean meal, 4 parts fish meal, 6 parts chicken meal, 4 parts soybean hulls, 3 parts soybean oil, 1.8 parts limestone powder, 1.0 part dicalcium phosphate, and 4 parts premix. In one specific embodiment, the above feed composition consists only of the above ingredients.

[0012] Further, each kilogram of the premix comprises vitamin A 350,000-750,000 IU, vitamin D 40,000-100,000 IU, vitamin E 3,000-6,000 IU, vitamin K 300-650 mg, vitamin B1 2,000-3,000 mg, vitamin B2 500-750 mg, vitamin B6 500-700 mg, vitamin B12 0.5-1.5 mg, niacin 1,500-3,500 mg, pantothenic acid 600-1,200 mg, folic acid 150-300 mg, biotin 10-20 mg, choline 31,000-70,000 mg, iron 5,000-10,000 mg, copper 250-500 mg, manganese 2,000-4,000 mg, zinc 3,000-5,000 mg, and selenium 5-15 mg. Preferably, each kilogram of premix contains 550,000 IU of vitamin A, 60,000 IU of vitamin D, 5,200 IU of vitamin E, 500 mg of vitamin K, 2,600 mg of vitamin B1, 600 mg of vitamin B2, 600 mg of vitamin B6, 1.2 mg of vitamin B12, 3,000 mg of niacin, 980 mg of pantothenic acid, 270 mg of folic acid, 15 mg of biotin, 50,000 mg of choline, 6,000 mg of iron, 350 mg of copper, 3,000 mg of manganese, 3,100 mg of zinc, and 10 mg of selenium.

[0013] In the above feed composition, corn, wheat, and wheat middlings are mainly energy sources; soybean meal, fish meal, and chicken meal are mainly protein sources; soybean oil is mainly fat source; soybean hulls are mainly crude fiber source; limestone and dicalcium phosphate are mainly mineral sources; and the premix is ​​mainly a source of trace elements and vitamins. Substances of similar nature can be replaced by other substances; for example, soybean hulls can be replaced by wheat bran or beet meal.

[0014] A third aspect of the invention is to provide the use of any of the feed additives described in the first aspect of the invention, or any of the feed compositions described in the second aspect of the invention, in the preparation of animal feed.

[0015] Furthermore, the feed is an anti-stress experimental mouse feed. It is understood that the above feed additives can also be combined with different basal feeds to suit different animals.

[0016] Furthermore, the method for preparing the animal feed includes the following steps: crushing and sieving corn, wheat, soybean meal, and soybean hulls to obtain powder; mixing wheat middlings, fish meal, chicken meal, soybean oil, and the powder to obtain a first mixture; crushing and sieving the first mixture to obtain a mixed powder; mixing limestone powder, dicalcium phosphate, multi-vitamin and multi-mineral premix, and feed additives with the mixed powder to obtain a second mixture; and sequentially puffing, drying, and cooling the second mixture to obtain the animal feed.

[0017] Furthermore, the method for preparing the animal feed also includes the step of: subjecting the animal feed to high-pressure sterilization or irradiation sterilization to obtain SPF-grade, sterile-grade animal feed.

[0018] Further, the method for preparing the animal feed specifically includes the following steps: S1) Corn, wheat, soybean meal, and soybean hulls are first pulverized through a 1.5mm sieve to obtain powder; S2) Wheat bran, fish meal, chicken meal, soybean oil, and the powder obtained in step S1 are first mixed for 3 minutes; S3) The mixture obtained in S2 is pulverized a second time through a 1.5mm sieve; S4) Limestone powder, dicalcium phosphate, premix, and feed additives are added to the mixture obtained in S3 for a second mixing for 3 minutes; S5) The mixture obtained in S4 is puffed at a temperature of 90-100℃ for 2 minutes, with a feed diameter of 1.8-2.2 mm and a length of 1.7-1.9 mm. S6) The puffed feed obtained in S5 is dried for 25 minutes. S7) The dried feed from S6 is transferred to a cooling chamber for 15 minutes, maintaining a feed temperature below 5℃ to obtain an anti-stress experimental mouse feed. S8) SPF-grade and sterile-grade experimental mouse feeds require the compound feed obtained in S7 to be sterilized by autoclaving or irradiation.

[0019] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0020] When laboratory mice experience stress, excessive free radicals are produced in their bodies and cannot be processed in time, leading to lipid peroxidation, which causes diseases in tissues and organs and harms the animals' health. The anti-stress laboratory mouse feed described in this invention uses feed additives including moringa flavonoids, vitamin C, and quercetin. Moringa flavonoids alleviate stress responses caused by transportation and experimental procedures through multiple mechanisms, such as inhibiting oxidative stress, improving energy metabolism, and enhancing mitochondrial function. Vitamin C is an active substance that maintains the growth and metabolism of bone tissue and cells, and promotes the body's anti-stress function. Quercetin, as a nutritional additive, contains five phenolic hydroxyl groups, which can effectively scavenge free radicals and has a strong anti-stress ability. The combined use of moringa flavonoids, vitamin C, and quercetin in an optimized basic compound feed provides comprehensive nutrition to meet the growth and development needs of laboratory mice. Furthermore, the extrusion process enhances the mice's ability to digest and absorb nutrients, while also strengthening their anti-stress ability. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to specific embodiments, but this is not intended to limit the scope of the invention.

[0023] Example 1 - Feed Additives

[0024] This example provides a feed additive. Moringa flavonoids, vitamin C, and quercetin are mixed thoroughly according to the mass ratio shown in the table. Moringa flavonoids are marketed as "Moringa Leaf Extract Moringa Flavonoids," with a particle size of 80 mesh; vitamin C is marketed as "Jinweixi-97," with a particle size of 60-80 mesh; quercetin is marketed as "Quercetin," with a particle size of 80 mesh. All three are in powder form. Usage: Add 0.1%-0.3% by weight directly to the compound feed (basal feed).

[0025]

[0026] Example 2 – Feed Composition and its Preparation

[0027] This example provides a feed composition comprising a base feed and feed additives. The specific raw material composition is shown in the table below. The feed additives consist of Moringa flavonoids, Vitamin C, and Quercetin in a 3:2:1 ratio. Each kilogram of premix contains the following: Vitamin A 550,000 IU, Vitamin D 60,000 IU, Vitamin E 5,200 IU, Vitamin K 500 mg, Vitamin B12 600 mg, Vitamin B2 600 mg, Vitamin B6 600 mg, Vitamin B12 1.2 mg, Niacin 3,000 mg, Pantothenic Acid 980 mg, Folic Acid 270 mg, Biotin 15 mg, Choline 50,000 mg, Iron 6,000 mg, Copper 350 mg, Manganese 3,000 mg, Zinc 3,100 mg, and Selenium 10 mg.

[0028]

[0029] The preparation method of the above feed composition includes the following steps:

[0030] Step 1: Corn, wheat, soybean meal, and soybean hulls are automatically fed into the central control computer and crushed for the first time through a 1.5mm sieve to obtain powder.

[0031] Step 2: Automatically mix the wheat starch, fish meal, chicken meal, soybean oil and the powder from Step 1, and then perform the first mixing, which takes 3 minutes.

[0032] Step 3: The mixture obtained in step 2 is crushed a second time, with the sieve size being 1.5mm.

[0033] Step 4: Manually add stone powder, dicalcium phosphate, premix and feed additives to the mixture obtained in step 3, and then mix for a second time for 3 minutes.

[0034] Step 5: Extrude the mixture obtained in Step 4 at a temperature of 90-100℃ for 2 minutes. The feed should have a diameter of 1.8-2.2 mm and a length of 1.7-1.9 mm.

[0035] Step 6: Transfer the extruded feed obtained in step 5 to an oven for drying. The upper temperature of the oven is 55℃ and the lower temperature is 90℃. The drying time is 25 minutes.

[0036] Step 7: Transfer the dried feed from Step 6 to the cooling chamber and cool for 15 minutes. Keep the feed temperature below 5°C to obtain the anti-stress experimental mouse compound feed.

[0037] Step 8: SPF-grade and sterile laboratory mouse feed requires the compound feed obtained in step 7 to be sterilized by autoclaving or irradiation.

[0038] Example 3 - Application of Feed Composition

[0039] In this embodiment, feed composition 1, feed composition 2, and feed composition 3 prepared in Example 2 were used as compound feed for experimental mice, and their effects on resisting stress in experimental mice were evaluated through animal experiments.

[0040] One hundred 8-week-old, uniformly weighted SPF-grade Balb / C male rats were selected and subjected to a 5Hz stress model on a simulated transport stress vibration test bench. The 100 stress-model male rats were randomly divided into 5 groups of 20 mice each and fed the following diets:

[0041] Group A: Standard SPF grade maintenance diet for laboratory mice, including 36 parts corn, 15 parts wheat middlings, 20 parts wheat, 11 parts soybean meal, 2 parts fish meal, 3 parts chicken meal, 5.5 parts soybean hulls, 1.5 parts soybean oil, 1 part limestone powder, 1 part dicalcium phosphate, and 4 parts premix.

[0042] Group B: Based on feed composition 2, without feed additives, including 37.2 parts corn, 32 parts wheat, 4 parts wheat middlings, 3 parts soybean meal, 4 parts soybean hulls, 4 parts fish meal, 6 parts chicken meal, 3 parts soybean oil, 1.8 parts limestone powder, 1 part dicalcium phosphate, and 4 parts premix.

[0043] Group C: Feed composition 1 prepared in Example 2;

[0044] Group D: Feed composition 2 prepared in Example 2;

[0045] Group E: Feed composition 3 prepared in Example 2.

[0046] The experiment lasted for two weeks. A fixed amount of feed was administered at the same time each week, and the remaining feed was weighed at the same time the following week to record the weekly feed consumption. The body weight of all experimental mice was measured using an electronic balance at the same time each week. On the last day of the experiment, all experimental mice were fasted for 12 hours, and 2 ml of blood was collected from the orbital sinus. The blood was centrifuged at 3000 rpm for 15 minutes to separate the serum, which was then placed in EP tubes to measure glucose, white blood cell count, IgG, cortisol, total antioxidant capacity, superoxide dismutase, and malondialdehyde (MDA) levels.

[0047] The experimental results are shown in the table below:

[0048]

[0049]

[0050] Note: Different superscript letters indicate significant differences at P<0.05.

[0051] In the table above, groups A and B are experimental control groups, while groups C, D, and E are formulated diets for stress-resistant experimental mice containing feed additives. The results in the table show that:

[0052] (1) Mice fed with anti-stress experimental mouse compound diet (groups C, D, and E) for two weeks showed significantly higher weight gain than control group A and control group B, with group D containing 0.2% feed additive showing the most significant weight gain.

[0053] (2) Compared with groups A and B, the average weekly feed consumption of mice in groups C, D and E all increased. Among them, the average weekly feed consumption of mice in group D increased by 14.2%. This indicates that the feed additive has excellent anti-stress effect, can resist the effect of stress on digestive capacity, and enhance nutrient absorption.

[0054] (3) The survival rate of mice fed with anti-stress experimental mouse compound diet (groups C, D and E) was higher than that of control group A and group B.

[0055] (4) When stress occurs, the body usually resists stress by regulating hormone secretion. Cortisol is a steroid hormone secreted by the adrenal cortex and is an important indicator of whether the body is under stress. Compared with groups A and B, cortisol levels in groups C, D and E were effectively reduced. Among them, the cortisol content in the serum of mice in group D was significantly reduced (P < 0.05).

[0056] (5) The glucose content of mice fed with anti-stress experimental mice compound diet (group C, group D, group E) increased with the increase of feed additive, indicating that the body’s ability to produce glucose was enhanced to resist stress response; compared with the other four groups, group D diet significantly increased the number of white blood cells and IgG content in the serum of mice (P<0.05), which indicates that feed additive improved the resistance of mice under stress.

[0057] (6) Compared with groups C and E, mice in group D had significantly higher levels of superoxide dismutase (P < 0.05) and lower levels of malondialdehyde (MDA) than mice in groups A and B, indicating that the formulated diet for anti-stress experimental mice can improve the antioxidant capacity of mice.

[0058] In the above experimental groups, both Group B and Group A were control groups without feed additives. Group B showed a slightly better anti-stress effect than Group A, indicating that the basic feed in Group B had a better composition and more comprehensive nutritional components. Its combination with a 0.2% feed additive resulted in the best effect on the anti-stress ability of the mice. The combination of the feed additive and the basic feed comprehensively provided the nutrients required for mouse growth, enhanced the mice's digestive capacity, significantly increased their body weight, significantly reduced serum cortisol levels, increased glucose levels, significantly increased serum white blood cell count and IgG levels, significantly increased superoxide dismutase levels, and reduced malondialdehyde levels, effectively enhancing the mice's ability to digest and absorb nutrients and improving their anti-stress ability.

[0059] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A feed composition for preparing compound feed for stress-resistant laboratory mice, characterized in that, The total amount of the feed composition is 100 parts by weight, which consists of 0.2 parts feed additive, 20-40 parts corn, 10-40 parts wheat, 1-10 parts wheat middlings, 2-10 parts soybean meal, 1-10 parts fish meal, 1-10 parts chicken meal, 1-10 parts soybean hulls, 1-5 parts soybean oil, 0.5-2 parts limestone powder, 0.5-1.5 parts dicalcium phosphate, and 2-5 parts premix. The feed additive is composed of moringa flavonoids, vitamin C and quercetin, and the weight ratio of moringa flavonoids, vitamin C and quercetin is 3:2:

1.

2. The feed composition according to claim 1, characterized in that, The total amount of the feed composition is 100 parts by weight, which consists of 0.2 parts feed additive, 30-40 parts corn, 30-40 parts wheat, 1-6 parts wheat middlings, 2-5 parts soybean meal, 1-5 parts fish meal, 5-10 parts chicken meal, 1-6 parts soybean hulls, 1-5 parts soybean oil, 1-2 parts limestone powder, 0.8-1.5 parts dicalcium phosphate, and 2-5 parts premix.

3. The feed composition according to claim 2, characterized in that, Each kilogram of the premix comprises 350,000-750,000 IU of vitamin A, 40,000-100,000 IU of vitamin D, 3,000-6,000 IU of vitamin E, 300-650 mg of vitamin K, 2,000-3,000 mg of vitamin B1, 500-750 mg of vitamin B2, 500-700 mg of vitamin B6, and vitamin B... 12 0.5-1.5 mg, niacin 1500-3500 mg, pantothenic acid 600-1200 mg, folic acid 150-300 mg, biotin 10-20 mg, choline 31000-70000 mg, iron 5000-10000 mg, copper 250-500 mg, manganese 2000-4000 mg, zinc 3000-5000 mg, selenium 5-15 mg.

4. The use of a feed composition as described in any one of claims 1 to 3 in the preparation of animal feed.

5. The application according to claim 4, characterized in that, The feed was for stress-resistant experimental mice.

6. The application according to claim 4, characterized in that, The method for preparing the animal feed includes the following steps: crushing and sieving corn, wheat, soybean meal, and soybean hulls to obtain powder; mixing wheat middlings, fish meal, chicken meal, soybean oil, and the powder to obtain a first mixture; crushing and sieving the first mixture to obtain a mixed powder; mixing limestone powder, dicalcium phosphate, premix, and feed additives with the mixed powder to obtain a second mixture; and sequentially puffing, drying, and cooling the second mixture to obtain the animal feed.

7. The application according to claim 6, characterized in that, It also includes the step of: sterilizing the animal feed by high pressure or irradiation to obtain SPF grade or sterile grade animal feed.

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