Tryptophan-added bullfrog feed composition and application thereof

Abstract

The invention provides a bullfrog feed composition added with tryptophan and application of the bullfrog feed composition. The bullfrog feed composition comprises the following components in percentage by weight: 40-60% of a protein source, 20-30% of a carbon water source, 6-15% of a fat source and the balance of an additive, in the additives, a mineral supplement accounts for 1-7% of the total weight of the bullfrog feed composition, a vitamin supplement accounts for 0.1-5% of the total weight of the bullfrog feed composition, and an amino acid supplement accounts for 1-15% of the total weight of the bullfrog feed composition; the tryptophan level of the bullfrog feed composition is 0.20%-0.60%. The bullfrog feed composition is used for bullfrog breeding, can accelerate growth and metamorphosis development of bullfrog tadpoles, shorten the breeding period and improve the uniformity and yield of seedlings on the premise of guaranteeing basic nutrition supply and health maintenance, so that the overall production efficiency and economic benefits are improved, and the bullfrog feed composition has definite practical value and wide popularization prospects.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and feed science, specifically relating to a bullfrog feed composition and its application, and more particularly to a bullfrog feed composition with finely regulated tryptophan levels and its method for promoting the growth and metamorphosis of bullfrog tadpoles. Background Technology

[0002] As an important economically farmed frog species, the length of the bullfrog's farming cycle directly affects its economic benefits. Within the bullfrog farming cycle, the growth rate of the tadpole stage and the synchronicity and uniformity of metamorphosis are crucial in determining overall farming efficiency. Slow tadpole metamorphosis and a prolonged cycle not only increase farming costs and risks but also lead to uneven adult frog sizes, impacting market returns.

[0003] Currently, there are few practices in the industry that precisely regulate tryptophan levels in feed and apply them to frog farming. Existing conventional feeds typically supplement tryptophan indirectly by increasing dietary protein levels. For example, literature CN105851679A adds silkworm pupa powder to the feed for spiny-breasted frog tadpoles. This ingredient contains high levels of methionine, lysine, tryptophan, unsaturated fatty acids, antimicrobial peptides, and various trace elements, and can partially replace corn gluten meal and fish meal to promote spiny-breasted frog tadpole growth and improve their immune function. Using rationally proportioned amino acid compositions to replace part of the dietary protein is becoming an important direction in feed nutrition formulation. Mansano et al. determined the digestible lysine requirement (2.71% by dry weight) of adult bullfrogs (post-metamorphosis stage) and estimated the requirements of other digestible amino acids (arginine 2.16%, histidine 0.94%, isoleucine 1.34%, leucine 2.39%, methionine 0.79%, phenylalanine 1.31%, threonine 1.34%, tryptophan 0.23%, valine 1.58%, cystine 0.36%, tyrosine 1.07%) based on the ideal essential amino acid ratio. This information was used to guide the formulation of diets containing appropriate amino acid levels to improve the growth efficiency and protein utilization of bullfrogs.

[0004] It is noteworthy that tryptophan metabolism exhibits significant species differences. Unlike most species, including mammals, aquatic animals such as frogs contain almost no albumin in their plasma, making tryptophan more readily available in the body and thus more sensitive to excess tryptophan (Int. J. Tryptophan. Res. 2022, 15, 11786469221122511). This also means that excessively high tryptophan levels in feed may trigger toxic reactions. Therefore, precise regulation of tryptophan levels in frog feed is particularly important for feed nutrient formulation design. However, optimization and regulation strategies for tryptophan levels in bullfrog tadpole feed are still lacking.

[0005] To solve the above problems, it is necessary to develop a novel bullfrog feed composition containing tryptophan. Summary of the Invention

[0006] This application aims to provide a bullfrog feed composition with finely regulated tryptophan levels, which can promote the healthy and rapid growth of bullfrog tadpoles and significantly accelerate their metamorphosis.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] A bullfrog feed composition, the formula of which is as follows: Protein source: 40-60%, carbohydrate source: 20-30%, fat source: 6-15%, and the balance being additives; of the additives, mineral supplements account for 1-7% of the total weight of the bullfrog feed composition, vitamin supplements account for 0.1-5% of the total weight of the bullfrog feed composition, and amino acid supplements account for 1-15% of the total weight of the bullfrog feed composition; the tryptophan level of the bullfrog feed composition is 0.20-0.60%.

[0009] This application also provides the following technical solutions: A bullfrog feed, using the aforementioned bullfrog feed composition as dry matter.

[0010] This application also provides the following technical solutions: The aforementioned application of bullfrog feed in bullfrog farming.

[0011] The technical solution provided in this application has the following beneficial effects: (1) Based on the unique nutritional needs and physiological characteristics of bullfrog tadpoles, this application systematically proportions and synergistically designs proteins, fats, carbohydrates, minerals, vitamins and amino acids to provide a high-performance feed composition that has extremely high safety and nutritional utilization efficiency for bullfrog tadpoles, can fully meet the metabolic needs of bullfrog tadpoles, and promote the rapid and healthy growth of bullfrog tadpoles.

[0012] (2) This application utilizes the mechanism of tryptophan's effect on the growth and metamorphosis of bullfrog tadpoles to design a safe, economical, and efficient optimal addition level. The feed composition provided by this application can efficiently promote the growth and metamorphosis of bullfrog tadpoles by precisely controlling the tryptophan level.

[0013] (3) Applying the feed composition provided in this application to bullfrog farming can significantly shorten the breeding cycle and improve the uniformity of hatching and yield by accelerating the growth and metamorphosis of bullfrog tadpoles, while ensuring basic nutrition supply and health maintenance. This is beneficial to improving overall production efficiency and economic benefits, and has clear practical value and broad prospects for promotion. Attached Figure Description

[0014] Figure 1 The effect of dietary tryptophan levels on liver thyroid hormone metabolism in bullfrog tadpoles.

[0015] Figure 2 The effect of dietary tryptophan levels on the synthesis of thyroid hormones in the brains of bullfrog tadpoles.

[0016] Figure 3 This diagram illustrates the serotonin synthesis pathway, serotonin degradation pathway, and melatonin synthesis pathway.

[0017] Figure 4 The study investigated the effects of dietary tryptophan levels on the levels of TRP and five metabolites (5-HTP, 5-HT, 5-HIAA, NAS, and MT) in the brains of bullfrog tadpoles.

[0018] Figure 5 The effect of dietary tryptophan levels on the activities of five metabolic enzymes (TPH, AAAD, MAO, AANAT, and ASMT) in the brain of bullfrog tadpoles was investigated. Invention Details 1. Terminology Explanation All patents and other publications cited herein are incorporated herein in their entirety. In the event of any conflict between any description of terminology herein and any document incorporated herein by reference, this document shall prevail.

[0019] Numerical ranges can be represented by a hyphen "-" or a tilde "~", and their endpoints are included by default. Unless otherwise specified, the numerical types within the range include, but are not limited to, integers, non-integers, percentages, fractions, etc., and the numerical types are not limited by the specific representation of the endpoints.

[0020] The terms “including,” “containing,” and similar expressions have a non-restrictive meaning.

[0021] The term "combination" of an enumeration item means any two or more of the enumeration items used together, unless the context explicitly excludes it or the combination is technically impossible.

[0022] A “composition” refers to a substance consisting of two or more components.

[0023] "Balance" refers to the amount used to bring the composition to 100%.

[0024] In feed, "additives" refer to primary and / or auxiliary functional components other than basic nutrients (such as protein sources, carbohydrate sources, and fat sources). Types of additives in feed compositions include, but are not limited to, mineral supplements, vitamin supplements, amino acid supplements, antimicrobial agents, and antioxidants.

[0025] "Mineral supplements" refer to inorganic salts and / or organic chelates that provide essential mineral elements to target animals. They are designed to supplement macroelements (such as calcium, phosphorus, magnesium, potassium, and sodium) and microelements (such as iron, zinc, copper, manganese, selenium, iodine, and cobalt) to maintain the body's structure, electrolyte balance, and normal function of enzyme systems.

[0026] "Vitamin supplements" refer to compounds or preparations used to provide essential vitamins to target animals. Their main function is to supplement vitamins in feed that the target animal cannot synthesize on its own or whose synthesis is insufficient to meet its physiological needs. In animal nutrition, vitamins can be classified according to their solubility into fat-soluble vitamins (e.g., vitamins A, D, E, and K) and water-soluble vitamins (e.g., B vitamins and vitamin C). For the purposes of this application, vitamins also include their derivatives or precursors, which can be converted into vitamin active substances through metabolism in the target animal.

[0027] "Amino acid supplements" refer to single or compound amino acid products used to supplement the amino acid content in feed. The amino acids in amino acid supplements can be in free form or their derived salts. The amino acids can be of natural or non-natural origin, including 20 standard amino acids and other amino acids (such as citrulline, ornithine, hydroxyproline, etc.).

[0028] "Antioxidants" refer to additives that can delay or prevent oxidation reactions, used to improve the antioxidant stability of feed or enhance the stability of nutrients. These include, but are not limited to, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ethoxyquin, and natural antioxidants. Antioxidants can also be added as components of other additives (such as vitamin supplements).

[0029] Some raw materials possess two or more additive functions. Their classification depends first on this application, and secondly on the common understanding of their main role in feed compositions by those skilled in the art.

[0030] In this application, the nutritional level of the feed (including protein level, fat level, and amino acid level) is assumed to be the actual content (measured value), unless otherwise specified (e.g., predicted value or theoretical value).

[0031] Fishmeal refers to a high-protein animal feed ingredient prepared from fish or fish processing by-products through processes such as cooking, pressing, drying, and grinding. Fishmeal can be whole fishmeal, by-product fishmeal, or a mixture thereof. Whole fishmeal is made from small, whole-caught fish, such as anchovy meal and sardine meal. By-product fishmeal is made from fish processing residues such as fish heads, bones, and viscera. Fishmeal can be white fishmeal, red fishmeal, or a mixture thereof. White fishmeal comes from white-fleshed fish (such as cod), while red fishmeal comes from red-fleshed fish (such as anchovies, sardines, and bonito). Fishmeal can be processed into whole-fat fishmeal, defatted fishmeal, steam-dried fishmeal, and roasted fishmeal, depending on the processing method.

[0032] "Fish solubles" refers to liquid byproducts rich in water-soluble nutrients (such as soluble proteins, amino acids, small molecule peptides, minerals, etc.) obtained during the production of fishmeal or related fish raw materials through processing techniques including but not limited to cooking, enzymatic hydrolysis, pressing, centrifugation, and concentration.

[0033] 2. Implementation Plan One embodiment of this application is as follows: A bullfrog feed composition, the formula of which is as follows: Protein source: 40-60%, carbohydrate source: 20-30%, fat source: 6-15%, and the balance being additives; of the additives, mineral supplements account for 1-7% of the total weight of the bullfrog feed composition, vitamin supplements account for 0.1-5% of the total weight of the bullfrog feed composition, and amino acid supplements account for 1-15% of the total weight of the bullfrog feed composition; the tryptophan level of the bullfrog feed composition is 0.20-0.60%.

[0034] In some specific implementation schemes, the protein source is selected from any one of animal protein raw materials, plant protein raw materials, and combinations thereof.

[0035] In some specific implementation schemes, the protein source components in the bullfrog feed composition are as follows: fish meal, corn gluten powder, gelatin, and fish solubles.

[0036] In some specific implementations, the fishmeal is any one of anchovy meal, bonito meal, basa fish meal, sardine meal, tuna meal, and combinations thereof.

[0037] In some specific embodiments, the fishmeal content in the bullfrog feed composition is 2-15%, preferably 7%.

[0038] In some specific embodiments, the content of corn gluten meal in the bullfrog feed composition is 5-40%, preferably 35%.

[0039] In some specific embodiments, the content of gelatin in the bullfrog feed composition is 0-10%, preferably 5%.

[0040] In some specific embodiments, the fish slurry content in the bullfrog feed composition is 2-5%, preferably 3%.

[0041] In some specific implementations, the carbohydrate source is selected from any one of corn flour, wheat flour, wheat bran, brown rice flour, microcrystalline cellulose, and combinations thereof.

[0042] In some specific implementation schemes, the carbohydrate source components of the bullfrog feed composition are as follows: high-gluten flour and microcrystalline cellulose.

[0043] In some specific embodiments, the content of high-gluten flour in the bullfrog feed composition is 20-25%, preferably 22%.

[0044] In some specific embodiments, the content of microcrystalline cellulose in the bullfrog feed composition is 0-5%, preferably 2.43%.

[0045] In some specific implementations, the fat source is selected from any one of vegetable oils, animal fats, lecithin, and combinations thereof.

[0046] In some specific implementations, the fat source components in the bullfrog feed composition are as follows: soybean oil and lecithin.

[0047] In some specific embodiments, the content of soybean oil in the bullfrog feed composition is 1-10%, preferably 4.3%.

[0048] In some specific embodiments, the content of lecithin in the bullfrog feed composition is 1-10%, preferably 4.3%.

[0049] In some specific implementations, the additive is selected from any one of mineral supplements, vitamin supplements, amino acid supplements, antimicrobial agents, antioxidants, and combinations thereof.

[0050] In some specific implementations, the additive consists of mineral supplements, vitamin supplements, and amino acid supplements.

[0051] In some specific implementations, the mineral supplement contains inorganic salts that provide mineral elements, and optionally also contains a carrier.

[0052] In some specific implementations, the inorganic salt is selected from any one of calcium salts, phosphate salts, potassium salts, sodium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, cobalt salts, iodine salts, selenium salts, and combinations thereof.

[0053] In some specific implementations, the carrier in the mineral supplement is zeolite powder.

[0054] In some specific implementation schemes, the mineral supplement components in the bullfrog feed composition are as follows: multi-mineral premix, calcium dihydrogen phosphate, and calcium carbonate; the multi-mineral premix components are as follows: potassium chloride, potassium iodide, cobalt sulfate, copper sulfate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate, magnesium sulfate heptahydrate, sodium selenite, and zeolite powder.

[0055] In some specific implementations, the content of the polymineral premix in the bullfrog feed composition is 0.1-1%, preferably 0.5%.

[0056] In some specific embodiments, the content of calcium dihydrogen phosphate in the bullfrog feed composition is 1-5%, preferably 4%.

[0057] In some specific embodiments, the content of calcium carbonate in the bullfrog feed composition is 1-5%, preferably 1.8%.

[0058] In some specific embodiments, the multi-mineral premix components in the bullfrog feed composition are as follows: 100-300 parts by weight of potassium chloride, 20-100 parts by weight of potassium iodide, 50-150 parts by weight of cobalt sulfate, 10-40 parts by weight of copper sulfate, 300-500 parts by weight of ferrous sulfate monohydrate, 100-300 parts by weight of zinc sulfate monohydrate, 50-100 parts by weight of manganese sulfate monohydrate, 500-1000 parts by weight of magnesium sulfate heptahydrate, 20-80 parts by weight of sodium selenite, and 2000-4000 parts by weight of zeolite powder.

[0059] In some specific embodiments, the vitamin supplement contains vitamins, and optionally also contains antioxidants and / or carriers; the vitamins are selected from any one of vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, B vitamins, and combinations thereof.

[0060] In some specific implementations, the carrier in the vitamin supplement is flour.

[0061] In some specific implementations, the antioxidant in the vitamin supplement is ethoxyquinoline.

[0062] In some specific implementation schemes, the vitamin supplement components in the bullfrog feed composition are as follows: multivitamin premix, choline chloride, and vitamin C; wherein, the multivitamin premix components are as follows: vitamin B1, riboflavin, pyridoxine hydrochloride, vitamin B12, vitamin K3, inositol, calcium pantothenate, niacin, folic acid, biotin, vitamin A, vitamin D3, vitamin E, ethoxyquinoline, and flour.

[0063] In some specific embodiments, the vitamin A is selected from any one of retinol, retinol propionate, retinol acetate, retinol palmitate, and combinations thereof, preferably retinol acetate.

[0064] In some specific implementations, the content of the multi-dimensional premix in the bullfrog feed composition is 0.1-1%, preferably 0.28%.

[0065] In some specific embodiments, the content of choline chloride in the bullfrog feed composition is 0.1-1%, preferably 0.5%.

[0066] In some specific embodiments, the content of vitamin C in the bullfrog feed composition is 0.1-1%, preferably 0.1%.

[0067] In some specific embodiments, the multivitamin premix components in the bullfrog feed composition are as follows: 5-15 parts by weight of vitamin B1, 5-15 parts by weight of riboflavin, 5-15 parts by weight of pyridoxine hydrochloride, 0.1-0.5 parts by weight of vitamin B12, 5-15 parts by weight of vitamin K3, 50-150 parts by weight of inositol, 10-30 parts by weight of calcium pantothenate, 30-80 parts by weight of niacin, 1-5 parts by weight of folic acid, 1-5 parts by weight of biotin, 10-15 parts by weight of vitamin A, 2-8 parts by weight of vitamin D3, 50-150 parts by weight of vitamin E, 100-200 parts by weight of ethoxyquinoline, and 8000-12000 parts by weight of flour.

[0068] In some specific implementations, the amino acid supplement contains amino acids, and optionally also contains antioxidants and / or carriers.

[0069] In some specific embodiments, the amino acid is selected from any one of tryptophan, alanine, lysine, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine, cystine, tyrosine, and combinations thereof.

[0070] In some specific implementation schemes, the amino acid supplement components in the bullfrog feed composition are as follows: amino acid premix, alanine, and L-tryptophan; wherein, the amino acid premix components are as follows: valine, isoleucine, threonine, lysine, histidine, arginine, and tyrosine.

[0071] In some specific embodiments, the content of amino acid premix in the bullfrog feed composition is 1-15%, preferably 9.39%.

[0072] In some specific embodiments, the content of alanine in the bullfrog feed composition is 0-1%, preferably 0-0.3%.

[0073] In some specific embodiments, the content of L-tryptophan in the bullfrog feed composition is 0-1%, preferably 0.1-0.4%.

[0074] In some specific embodiments, the amino acid premix composition of the bullfrog feed composition includes the following components: 0.5-1 parts by weight of valine, 0.5-1 parts by weight of isoleucine, 0.5-1 parts by weight of threonine, 1-5 parts by weight of lysine hydrochloride, 0.5-1 parts by weight of histidine hydrochloride, 0.5-3 parts by weight of arginine hydrochloride, and 0.5-3 parts by weight of tyrosine.

[0075] In some specific embodiments, the bullfrog feed composition comprises the following components: Anchovy meal: 7%, corn gluten meal: 35%, gelatin: 5%, high-gluten flour: 22%, microcrystalline cellulose: 2.51%, fish solubles: 3%, soybean oil: 4.3%, lecithin: 4.3%, calcium dihydrogen phosphate: 4%, calcium carbonate: 1.8%, choline chloride: 0.5%, multi-mineral premix: 0.5%, multi-vitamin premix: 0.2%, vitamin C: 0.1%, amino acid premix: 9.39%, alanine: 0-0.3%, L-tryptophan: 0.1-0.4%; The combined content of alanine and L-tryptophan is 0.4%.

[0076] In some specific embodiments, the tryptophan level of the bullfrog feed composition is 0.29%-0.50%.

[0077] In some specific embodiments, the tryptophan level of the bullfrog feed composition is 0.36%-0.43%.

[0078] In some specific embodiments, the protein level of the bullfrog feed composition is 38-50%.

[0079] In some specific embodiments, the fat level of the bullfrog feed composition is 7-12%.

[0080] One embodiment of this application is as follows: A bullfrog feed, wherein any of the aforementioned bullfrog feed compositions is used as the dry matter.

[0081] In some specific implementations, the bullfrog feed is obtained by the following preparation method: a) Weigh each ingredient according to the formula of the bullfrog feed composition, and mix all ingredients except for the fat source ingredients; b) Add water to the mixture obtained in step a) for conditioning; c) The conditioned material is then expanded and granulated; d) The expanded granules are then dried; e) Apply the fat source material to the surface of the particles to obtain the bullfrog feed.

[0082] In some specific implementation schemes, the lumpy raw materials are individually crushed and sieved before use to obtain powdered raw materials.

[0083] In some specific implementations, the particle size of the powdered raw material does not exceed 60 mesh, preferably not exceeding 80 mesh.

[0084] In some specific implementation plans, the amount of water used for conditioning is 30-35% of the dry matter in the feed.

[0085] In some specific implementations, the puffing is carried out under conditions of 2-4 MPa, preferably under conditions of 3 MPa.

[0086] In some specific implementations, the puffing is carried out at 130-150°C.

[0087] In some specific implementations, the puffing time is 20-40 seconds, preferably 30 seconds.

[0088] In some specific implementations, the drying process is a baking process.

[0089] In some specific implementations, the drying process is carried out at 100-110°C, preferably at 105°C.

[0090] In some specific implementations, the drying process lasts for 12-36 hours, preferably 24 hours.

[0091] In some specific implementations, the fat source material is mixed and then sprayed onto the surface of the particles.

[0092] One embodiment of this application is as follows: The application of any of the aforementioned bullfrog feeds in bullfrog farming.

[0093] In some specific implementations, the bullfrog feed is used to feed bullfrog tadpoles.

[0094] In some specific implementations, the daily feeding amount of bullfrog feed is 5-8% of the tadpole's body weight, preferably 6.5%. Detailed Implementation The raw materials used in this application can be purchased or synthesized in-house. The following specific embodiments are used to further describe the implementation of the present invention and do not limit the scope of the invention.

[0095] Example 1 This embodiment uses anchovy meal, corn gluten meal, gelatin, and fish lysate as protein sources, and soybean oil and lecithin as fat sources to formulate five groups of feeds with tryptophan levels ranging from 0.21% to 0.50%. The raw material composition and nutrient levels of each feed formulation are shown in Table 1. The measured values ​​of tryptophan levels are as follows: 0.21% (Formula I), 0.29% (Formula II), 0.36% (Formula III), 0.43% (Formula IV), and 0.50% (Formula V).

[0096] Anchovy powder and corn gluten powder are separately ground in a grinder to about 80 mesh. The ground materials are then sieved to remove unground coarse particles, and the qualified powder is collected for later use.

[0097] Weigh each ingredient according to the formula and add them to the mixing container in sequence. First, add high-gluten flour, anchovy powder, corn gluten powder, and gelatin and premix. Then add microcrystalline cellulose, calcium dihydrogen phosphate, calcium carbonate, choline chloride, multi-mineral premix, multi-vitamin premix, amino acid premix, fish solubles, vitamin C, alanine, and L-tryptophan and mix thoroughly. Next, add an appropriate amount of water (approximately 30-35% of the dry matter) and continue mixing until the material has a uniform color and no local color differences.

[0098] The conditioned material is poured into an extruder for extrusion (approximately 3 MPa, 130-150℃, 30 seconds) and pelleted. The extruded pellets are promptly loaded into trays and sent to a drying device for drying (105℃, 24 hours). The dried feed is then transferred to a cool, dry place for storage. A predetermined amount of soybean oil and lecithin are mixed and sprayed onto the surface of the feed pellets to obtain the finished feed.

[0099] Table 1. Raw material composition and nutrient levels of feed formulation IV (on dry weight)

[0100] Multi-mineral premix 1 The ingredients are as follows: 200 parts by weight of potassium chloride, 60 parts by weight of potassium iodide, 100 parts by weight of cobalt sulfate, 24 parts by weight of copper sulfate, 400 parts by weight of ferrous sulfate monohydrate, 174 parts by weight of zinc sulfate monohydrate, 78 parts by weight of manganese sulfate monohydrate, 800 parts by weight of magnesium sulfate heptahydrate, 50 parts by weight of sodium selenite, and 3114 parts by weight of zeolite powder. Multi-dimensional premix 2The ingredients are as follows: 10 parts by weight of vitamin B1, 8 parts by weight of riboflavin, 10 parts by weight of pyridoxine hydrochloride, 0.2 parts by weight of vitamin B12, 10 parts by weight of vitamin K3, 100 parts by weight of inositol, 20 parts by weight of calcium pantothenate, 50 parts by weight of niacin, 2 parts by weight of folic acid, 2 parts by weight of biotin, 12.5 parts by weight of vitamin A, 5 parts by weight of vitamin D3, 100 parts by weight of vitamin E, 150 parts by weight of ethoxyquinoline, and 9520.3 parts by weight of flour; amino acid premix 3 The ingredients are as follows: 0.69 parts by weight of valine, 0.67 parts by weight of isoleucine, 0.86 parts by weight of threonine, 3.94 parts by weight of lysine hydrochloride, 0.52 parts by weight of histidine hydrochloride, 1.56 parts by weight of arginine hydrochloride, and 1.16 parts by weight of tyrosine.

[0101] Example 2 This embodiment involves a bullfrog tadpole feeding experiment.

[0102] Bullfrog tadpoles with an initial average weight of approximately 0.004 g were randomly divided into 5 groups and fed with five different experimental diets (Formula IV) with different tryptophan levels. Each group had 3 replicates, with each replicate containing 35 tadpoles.

[0103] The experimental feed was given to the tadpoles for 84 consecutive days, with a daily feed amount of 6.5% of their body weight. The tadpoles were weighed weekly, and the feed amount was adjusted accordingly for the following week. In the early stages of the experiment, if the tadpoles were too small to be fed pelleted feed, the pelleted feed was ground into powder using a grinder before feeding.

[0104] Survival status was recorded during the experiment, and the weight of each tadpole was measured at the end of the experiment. The final average weight (FBW), weight gain (WG), survival rate (SR), specific growth rate (SGR), and feed conversion ratio (FCR) of each replicate group were calculated according to the following formulas, and the results were recorded as “mean ± standard deviation” in Table 2.

[0105]

[0106] Among them, W f The final weight (g) of a single tadpole, N f The number of surviving tadpoles at the end of the experiment;

[0107] Wherein, IBW is the initial average weight (0.004 g);

[0108] Where, N i The number of surviving tadpoles at the start of the experiment (35);

[0109] Where T is the number of days of the experiment (84 days);

[0110] Where FI is the total feeding amount (g) during the experiment, and BW is the total feeding amount (g) during the experiment. g The total weight gain of the tadpole group (g).

[0111] Table 2. Effects of dietary tryptophan levels on the growth performance of bullfrog tadpoles

[0112] Note: In the same row of data, different superscript letters indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0113] The results showed that the survival rate of bullfrog tadpoles in all groups was greater than 99%, and the feed conversion ratio was approximately 1.3, indicating that the feed composition provided in this application has extremely high safety and utilization rate. Specifically, the specific growth rate (8.71-8.80%) of the experimental groups fed with formula II-V (tryptophan level 0.29-0.50%) was significantly higher than that of the experimental group fed with formula I (8.39%). The weight gain rate of bullfrog tadpoles showed a trend of first increasing and then decreasing with increasing tryptophan level in the feed. When the tryptophan level was 0.29% (formula II), the weight gain rate (15.34%) was not significantly different from the higher tryptophan level groups (formula III, 16.27%; formula IV, 14.99%), indicating that this dosage could basically meet the normal growth requirements of bullfrog tadpoles. The weight gain rate of tadpoles reached its maximum when the tryptophan level in the feed was 0.36% (formula III). These results demonstrate that the feed composition provided in this application can achieve rapid and healthy growth of bullfrog tadpoles.

[0114] Example 3 This embodiment evaluates the effects of dietary tryptophan levels on the metamorphosis rate and thyroid hormone levels of bullfrog tadpoles.

[0115] In Example 2, after the feeding experiment, bullfrog tadpoles were fasted for 24 hours and anesthetized with MS-222 (200 mg / kg, Aladdin, Shanghai, China). Developmental stages of all bullfrog tadpoles in each tank were statistically analyzed. Blood samples were collected from the tails of 15 bullfrog tadpoles (randomly selected from 35 tadpoles) in each tank using a 75 μL anticoagulant capillary micropipette. The samples were transferred to sterile 1.5 mL tubes, centrifuged (3000 rpm, 10 minutes, 4°C), and the plasma was separated and stored at -80°C for the determination of plasma T3 and T4 levels. The levels of triiodothyronine (T3) and thyroid hormone (T4) were determined using a competitive assay with an amphibian-specific enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute).

[0116] Table 3. Effects of dietary tryptophan levels on metamorphosis of bullfrog tadpoles

[0117] Note: In the same column of data, different superscript letters indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0118] The results (Table 3) showed that with increasing dietary tryptophan levels, the metamorphosis rates at stages 40 and 41, as well as plasma T4 and T3 levels in bullfrog tadpoles, exhibited a trend of first increasing and then decreasing. Specifically, the stage 41 metamorphosis rate reached its maximum at a dietary tryptophan level of 0.36%, while the stage 40 metamorphosis rate, plasma T3, and T4 levels reached their maximum at a dietary tryptophan level of 0.43%. This indicates that the feed composition of this application can more efficiently promote the synthesis and release of thyroid hormones (T4 and T3) in bullfrog tadpoles under tryptophan levels of 0.36-0.43%. T3 is a key active hormone regulating tadpole metamorphosis, mainly derived from T4 by deiodinase in peripheral tissues. At a tryptophan level of 0.43%, the T4 / T3 ratio was lowest while the T3 level was highest, indicating that deiodinase activity may be optimally regulated under these conditions, significantly improving the conversion efficiency of T4 to T3, thereby efficiently promoting the metamorphosis process of bullfrog tadpoles.

[0119] Example 4 This embodiment evaluates the effect of dietary tryptophan levels on the metabolism of thyroid hormones in the liver of bullfrog tadpoles.

[0120] After the blood collection step in Example 3 was completed, tadpole livers were collected and quickly transferred to 2 mL cryovials. These were then flash-frozen in liquid nitrogen and stored at -80°C until subsequent analysis of liver biochemical parameters. The enzyme activities of deiodinase I (DIO-1), deiodinase II (DIO-2), and deiodinase III (DIO-3) in the liver, as well as the content of thyroxine-binding globulin (TBG), were determined using an amphibian-specific enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu Enzyme Immunosorbent Assay Co., Ltd.) via a double-antibody sandwich method.

[0121] The liver is the core organ for thyroid hormone metabolism. Deiodinases (DIOs) are a class of enzymes closely related to thyroid hormone metabolism, including DIO-1, DIO-2, and DIO-3. DIO-1 and DIO-2 can convert T4 to T3 through outer ring deiodination, promoting thyroid hyperplasia; DIO-3 inactivates both T3 and T4, inhibiting thyroid hyperplasia. Thyroxine-binding globulin (TBG) is the main protein in plasma that transports thyroid hormones and has an extremely high affinity for T4.

[0122] Experimental results ( Figure 1 The results showed that with increasing tryptophan levels in the diet, the levels of DIO-1, DIO-2, and TBG in the liver of bullfrog tadpoles generally exhibited a trend of first increasing and then decreasing, while the DIO-3 level showed a trend of first decreasing and then increasing. Specifically, DIO-1 and TBG levels reached their maximum values ​​when tryptophan levels were 0.36-0.43%, DIO-2 levels reached their maximum values ​​when tryptophan levels were 0.29-0.36%, and DIO-3 reached its minimum values ​​when tryptophan levels were 0.36-0.43%. These results indicate that when tryptophan levels are 0.36%-0.43%, the bullfrog feed composition provided in this application can synergistically upregulate DIO-1, DIO-2, and TBG levels and downregulate DIO-3 levels, significantly promoting the conversion of T4 to T3 while increasing the transport capacity of thyroid hormones, thereby efficiently driving the metamorphosis of bullfrog tadpoles.

[0123] Example 5 This embodiment evaluates the effect of dietary tryptophan levels on the synthesis of thyroid hormones in the brains of bullfrog tadpoles.

[0124] After the blood collection step in Example 3 was completed, tadpole brains were collected and rapidly transferred to 2 mL cryovials. These were then flash-frozen in liquid nitrogen and stored at -80°C until subsequent analysis of brain biochemical indicators. The levels of tryptophan (TRP), 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), thyroglobulin (TG), sodium-iodine cotransporter (NIS), and thyroid-stimulating hormone receptor (TSHR) in the brain, as well as the enzyme activities of tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AAAD), monoamine oxidase (MAO), and thyroid peroxidase (TPO), were all measured using an amphibian-specific enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu Enzyme Immunosorbent Assay Co., Ltd.) via a double-antibody sandwich method. The levels of thyroid hormone receptors α and β (TR-α, TR-β), N-acetylserotonin (NAS), and the enzyme activities of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) in the brain were measured using an amphibian-specific enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.) via a double-antibody sandwich method. Melatonin (MT) levels were measured using an amphibian ELISA kit (Nanjing Jiancheng Bioengineering Institute) via a competitive assay. All procedures were performed strictly according to the manufacturer's instructions.

[0125] TG is a precursor substrate for thyroid hormone synthesis, and changes in its content reflect the consumption and accumulation of raw materials for thyroid hormone synthesis. TPO is a key enzyme that catalyzes the binding of tyrosine residues on TG with iodine to complete thyroid hormone synthesis, and its content reflects the enzymatic capacity for thyroid hormone synthesis. TR-α and TR-β are the main nuclear receptors that mediate gene expression regulation after thyroid hormone enters target cells, and their content reflects the thyroid hormone signaling potential. The levels of TSHR and NIS reflect the tissue sensitivity to thyroid-stimulating hormone and the thyroid cell's ability to take up iodine, respectively.

[0126] Experimental results ( Figure 2The results showed that as the tryptophan level in the feed increased, the TG level in the bullfrog tadpole brain exhibited a trend of first decreasing and then increasing, reaching its minimum at a tryptophan level of 0.36-0.43%, indicating that the consumption of raw materials for thyroid hormone synthesis was relatively significant at this level. In contrast, the TPO and TR-β levels showed a trend of first increasing and then decreasing, with TPO reaching its maximum at a tryptophan level of 0.36%, and TR-β reaching a relatively large value at a tryptophan level of 0.29-0.43%. This indicates that at a tryptophan level of 0.36-0.43%, the enzymatic capacity for thyroid hormone synthesis reached its maximum, and the thyroid hormone signal transduction capacity was also high. The levels of TR-α, TSHR, and NIS were relatively stable within the tested tryptophan level range. These results indicate that the feed composition provided in this application, at a tryptophan level of 0.36%-0.43%, can enhance local hormone production by consuming substrate TG and upregulating TPO, while simultaneously upregulating TR-β levels to improve the efficiency of metamorphosis-driven development, thereby promoting metamorphosis in bullfrog tadpoles.

[0127] Example 6 Serotonin is crucial for the neurodevelopment of bullfrog tadpoles. This example assesses the effects of dietary tryptophan levels on tryptophan-serotonin metabolism in the brains of bullfrog tadpoles, involving the activities of TRP, five metabolites (5-HTP, 5-HT, 5-HIAA, NAS, MT), and five metabolic enzymes (TPH, AAAD, MAO, AANAT, ASMT). Relevant metabolic pathways (serotonin synthesis pathway, serotonin degradation pathway, and melatonin synthesis pathway) are as follows: Figure 3 As shown.

[0128] The experimental method was the same as in Example 5. The test results for TRP and the content of five metabolites ( Figure 4 The study showed that the levels of TRP, 5-HIAA, and NAS in the brains of bullfrog tadpoles increased with increasing dietary tryptophan levels, reaching a maximum at a tryptophan level of 0.50%. P <0.05%. 5-HT content reached its maximum at a tryptophan level of 0.29% ( P <0.05%, and remained relatively stable with continued increases in tryptophan levels. However, the contents of 5-HTP and MT did not show significant differences with changes in feed tryptophan levels ( P >0.05), which may be because the metabolic pathway of melatonin may be influenced by more external factors such as photoperiod and circadian rhythm, rather than being regulated by changes in tryptophan concentration. This suggests that under the conditions of this study, tryptophan metabolism may be more inclined to flow toward serotonin and its degradation products.

[0129] Results of the test of the activities of 5 metabolic enzymes ( Figure 5The results showed that the activity of TPH enzyme in the brain of bullfrog tadpoles increased with increasing dietary tryptophan levels, reaching a maximum at a tryptophan level of 0.50% (P<0.05). The activity of AAAD enzyme was relatively high at tryptophan levels of 0.36-0.50% (P<0.05). The activities of MAO and AANAT enzymes did not show significant differences with changes in dietary tryptophan levels (P>0.05). The activity of ASMT enzyme showed a trend of first decreasing and then increasing with increasing dietary tryptophan levels, reaching a minimum at a tryptophan level of 0.29% (P<0.05).

[0130] The above results indicate that the feed composition provided in this application can most effectively optimize tryptophan-serotonin metabolism in the brain of bullfrog tadpoles at a tryptophan level of 0.29%-0.50%. In particular, the synthesis of the core neurotransmitter 5-HT reaches the optimal level (highest at 0.29%), while the key synthases TPH and AAAD also maintain high activity, which can promote the neural development and health of tadpoles and lay a good neuroendocrine foundation for subsequent metamorphosis.

Claims

1. A bullfrog feed composition, characterized in that, Its formula is as follows: Protein source: 40-60%, carbohydrate source: 20-30%, fat source: 6-15%, and the balance being additives; of the additives, mineral supplements account for 1-7% of the total weight of the bullfrog feed composition, vitamin supplements account for 0.1-5% of the total weight of the bullfrog feed composition, and amino acid supplements account for 1-15% of the total weight of the bullfrog feed composition; the tryptophan level of the bullfrog feed composition is 0.20-0.60%.

2. The bullfrog feed composition according to claim 1, characterized in that, The protein source is selected from any one of animal protein raw materials, plant protein raw materials, and combinations thereof; Preferably, the protein source components in the bullfrog feed composition are as follows: fish meal, corn gluten meal, gelatin, and fish solubles; wherein the fish meal is any one of anchovy meal, bonito meal, basa meal, sardine meal, tuna meal, and combinations thereof; wherein the content of fish meal in the bullfrog feed composition is preferably 2-15%, more preferably 7%; the content of corn gluten meal in the bullfrog feed composition is preferably 5-40%, more preferably 35%; the content of gelatin in the bullfrog feed composition is preferably 0-10%, more preferably 5%; and the content of fish solubles in the bullfrog feed composition is preferably 2-5%, more preferably 3%.

3. The bullfrog feed composition according to claim 1, characterized in that, The carbohydrate source is selected from any one of corn flour, wheat flour, wheat bran, brown rice flour, microcrystalline cellulose, and combinations thereof; Preferably, the carbon source components in the bullfrog feed composition are as follows: high-gluten flour and microcrystalline cellulose; wherein, the content of high-gluten flour in the bullfrog feed composition is preferably 20-25%, more preferably 22%; and the content of microcrystalline cellulose in the bullfrog feed composition is preferably 0-5%, more preferably 2.43%.

4. The bullfrog feed composition according to claim 1, characterized in that, The fat source is selected from any one of vegetable oils, animal fats, lecithin, and combinations thereof; Preferably, the fat source components in the bullfrog feed composition are as follows: soybean oil and lecithin; wherein, the content of soybean oil in the bullfrog feed composition is preferably 1-10%, more preferably 4.3%; and the content of lecithin in the bullfrog feed composition is preferably 1-10%, more preferably 4.3%.

5. The bullfrog feed composition according to claim 1, characterized in that, The mineral supplement contains inorganic salts that provide mineral elements, and optionally also contains a carrier; preferably, the inorganic salts are selected from any one of calcium salts, phosphate salts, potassium salts, sodium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, cobalt salts, iodine salts, selenium salts, and combinations thereof; the carrier in the mineral supplement is preferably zeolite powder; Preferably, the mineral supplement components in the bullfrog feed composition are as follows: multi-mineral premix, calcium dihydrogen phosphate, and calcium carbonate; wherein the multi-mineral premix comprises: potassium chloride, potassium iodide, cobalt sulfate, copper sulfate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate, magnesium sulfate heptahydrate, sodium selenite, and zeolite powder; the content of the multi-mineral premix in the bullfrog feed composition is preferably 0.1-1%, more preferably 0.5%; the content of calcium dihydrogen phosphate in the bullfrog feed composition is preferably 1-5%, more preferably 4%; the content of calcium carbonate in the bullfrog feed composition is preferably... The content is selected as 1-5%, more preferably 1.8%; preferably, the multi-mineral premix composition of the bullfrog feed composition is as follows: 100-300 parts by weight of potassium chloride, 20-100 parts by weight of potassium iodide, 50-150 parts by weight of cobalt sulfate, 10-40 parts by weight of copper sulfate, 300-500 parts by weight of ferrous sulfate monohydrate, 100-300 parts by weight of zinc sulfate monohydrate, 50-100 parts by weight of manganese sulfate monohydrate, 500-1000 parts by weight of magnesium sulfate heptahydrate, 20-80 parts by weight of sodium selenite, and 2000-4000 parts by weight of zeolite powder.

6. The bullfrog feed composition according to claim 1, characterized in that, The vitamin supplement contains vitamins, and optionally also contains antioxidants and / or a carrier; the vitamins are selected from any one of vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, B vitamins, and combinations thereof; the carrier in the vitamin supplement is preferably flour; the antioxidant in the vitamin supplement is preferably ethoxyquinoline; Preferably, the vitamin supplement components in the bullfrog feed composition are as follows: multivitamin premix, choline chloride, and vitamin C; wherein the multivitamin premix components are as follows: vitamin B1, riboflavin, pyridoxine hydrochloride, vitamin B12, vitamin K3, inositol, calcium pantothenate, niacin, folic acid, biotin, vitamin A, vitamin D3, vitamin E, ethoxyquinoline, and flour; the vitamin A is selected from any one of retinol, retinol propionate, retinol acetate, retinol palmitate, and combinations thereof, preferably retinol acetate; the content of the multivitamin premix in the bullfrog feed composition is preferably 0.1-1%, more preferably 0.28%; the content of choline chloride in the bullfrog feed composition is preferably 0.1-1%, more preferably 0.5%; the content of vitamin C is as follows: The preferred content of the bullfrog feed composition is 0.1-1%, more preferably 0.1%; preferably, the multivitamin premix components in the bullfrog feed composition are as follows: 5-15 parts by weight of vitamin B1, 5-15 parts by weight of riboflavin, 5-15 parts by weight of pyridoxine hydrochloride, 0.1-0.5 parts by weight of vitamin B12, 5-15 parts by weight of vitamin K3, 50-150 parts by weight of inositol, 10-30 parts by weight of calcium pantothenate, 30-80 parts by weight of niacin, 1-5 parts by weight of folic acid, 1-5 parts by weight of biotin, 10-15 parts by weight of vitamin A, 2-8 parts by weight of vitamin D3, 50-150 parts by weight of vitamin E, 100-200 parts by weight of ethoxyquinoline, and 8000-12000 parts by weight of flour.

7. The bullfrog feed composition according to claim 1, characterized in that, The amino acid supplement contains amino acids, and optionally also contains antioxidants and / or carriers; the amino acids are selected from any one of tryptophan, alanine, lysine, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine, cystine, tyrosine, and combinations thereof. Preferably, the amino acid supplement components in the bullfrog feed composition are as follows: amino acid premix, alanine, and L-tryptophan; wherein, the amino acid premix components are as follows: valine, isoleucine, threonine, lysine, histidine, arginine, and tyrosine; the content of the amino acid premix in the bullfrog feed composition is preferably 1-15%, more preferably 9.39%; the content of alanine in the bullfrog feed composition is preferably 0-1%, more preferably 0-0.3%; the content of L-tryptophan in the bullfrog feed composition is preferably 0-1%, more preferably 0.1-0.4%; preferably, the amino acid premix components in the bullfrog feed composition are as follows: 0.5-1 parts by weight of valine, 0.5-1 parts by weight of isoleucine, 0.5-1 parts by weight of threonine, 1-5 parts by weight of lysine hydrochloride, 0.5-1 parts by weight of histidine hydrochloride, 0.5-3 parts by weight of arginine hydrochloride, and 0.5-3 parts by weight of tyrosine.

8. The bullfrog feed composition according to claim 1, characterized in that, The bullfrog feed composition has a tryptophan level of 0.29%-0.50%, more preferably 0.36%-0.43%; Optionally, the protein level of the bullfrog feed composition is 38-50%; Optionally, the fat level of the bullfrog feed composition is 7-12%.

9. A bullfrog feed, characterized in that, Using the bullfrog feed composition of claim 1 as the dry matter; the bullfrog feed is preferably obtained by the following preparation method: a) Weigh each raw material according to the formula of the bullfrog feed composition according to claim 1, and mix all raw materials except for the fat source raw material; b) Add water to the mixture obtained in step a) for conditioning; c) The conditioned material is then expanded and granulated; d) The expanded granules are then dried; e) Apply the fat source material to the surface of the particles to obtain the bullfrog feed.

10. The application of the bullfrog feed according to claim 9 in bullfrog farming, characterized in that, The bullfrog feed is used to feed bullfrog tadpoles; Preferably, the daily feeding amount of the bullfrog feed is 5-8% of the tadpole's body weight, more preferably 6.5%.

Images