Application of combination of silibinin and chenodeoxycholic acid in improvement of quality reduction of procambarus clarkia caused by zero fish meal feed

Abstract

The invention provides application of silybin and chenodeoxycholic acid in improving the quality reduction of procambarus clarkia caused by zero-fish-meal feed, and belongs to the technical field of aquatic feed additive development. According to the present invention, silibinin and chenodeoxycholic acid are combined according to a mass ratio of (1-2): (1-2) so as to effectively improve the reduction of the quality of the procambarus clarkia caused by the zero fish meal feed, such that the zero fish meal use is achieved, and the procambarus clarkia breeding method is suitable for the procambarus clarkia breeding of procambarus clarkia breeding of procambarus clarkia breeding of procambarus clarkia breeding of procambarus clarkia. The feed cost can be effectively reduced, and the muscle quality of the procambarus clarkii cannot be reduced.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed additive development technology, specifically involving the application of silymarin combined with chenodeoxycholic acid in improving the quality decline of Procambarus clarkii caused by fishmeal-free feed. Background Technology

[0002] With increasingly scarce fishmeal resources and continuously rising prices, novel non-grain protein sources (such as soybean meal, rapeseed meal, cottonseed meal, fermented plant protein, insect protein, and single-cell protein) are widely used to replace fishmeal in aquatic feeds. However, numerous studies and production practices have shown that high proportions or improper use of non-grain protein sources can lead to a series of adverse consequences. Many plant-based proteins contain anti-nutritional factors such as saponins, phytic acid, tannins, and gossypol, which can easily damage the intestinal structure of aquatic animals, induce inflammatory responses, and reduce digestibility and absorption efficiency. Furthermore, the amino acid composition of non-grain proteins differs significantly from that of fishmeal, especially in the insufficient proportion of sulfur-containing amino acids and essential amino acids, which may limit protein deposition, leading to decreased growth performance and reduced feed conversion ratio. In addition, some non-grain proteins have weak support for lipid digestion and bile acid metabolism, easily causing limited energy utilization and metabolic imbalance. In species such as crustaceans that are more sensitive to feed adaptability, these problems may further manifest as impaired hepatopancreatic function, decreased stress resistance, and deterioration of muscle quality. Therefore, while replacing fishmeal with non-grain protein sources has sustainable significance, its potential negative impacts cannot be ignored, and corresponding nutritional regulation and functional additives are urgently needed to mitigate them.

[0003] The red swamp crayfish (Procambarus clarkii) has formed a highly integrated industrial chain encompassing aquaculture, processing, and catering / retail. In recent years, the overall scale of aquaculture has maintained growth, with continuous advancements in industrialization, large-scale production, and refined processing. However, issues such as differentiated aquaculture profitability and rising cost pressures have emerged. On the feed side, influenced by fluctuations in fishmeal prices and supply, the application of low-fishmeal or even zero-fishmeal formulations has accelerated. However, in red swamp crayfish, this often faces the problem of "unstable effects." Studies have observed that as the substitution level increases, growth, immune antioxidant indicators, and muscle texture / quality parameters are adversely affected. Therefore, the promotion of low / zero-fishmeal feeds in this species usually requires the use of functional additives and appropriate application methods to stabilize the intestinal-metabolic state and reduce the risk of decreased muscle quality. Exploring and developing effective alternative raw materials and their rational formulations is of significant theoretical and practical value for ensuring the growth performance and nutritional quality of red swamp crayfish. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first objective of the present invention is to provide the application of silymarin combined with chenodeoxycholic acid in the preparation of products that improve the quality of Procambarus clarkii caused by zero fishmeal feed.

[0005] A second objective of this invention is to provide a feed additive that improves the quality decline of red swamp crayfish caused by fishmeal-free feed.

[0006] A third objective of this invention is to provide a fishmeal-free feed for Procambarus clarkii.

[0007] A fourth objective of this invention is to provide the application of the above-mentioned feed additives or Procambarus clarkii feed in Procambarus clarkii farming.

[0008] A fifth objective of this invention is to provide a method for farming red swamp crayfish.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of silymarin combined with chenodeoxycholic acid in the preparation of products that improve the quality of red swamp crayfish caused by zero fishmeal feed.

[0010] Preferably, the mass ratio of silymarin to chenodeoxycholic acid is (1~2):(1~2).

[0011] This invention provides a feed additive to improve the quality decline of Procambarus clarkii caused by fishmeal-free feed, the feed additive being composed of silymarin and chenodeoxycholic acid.

[0012] Preferably, the mass ratio of silymarin to chenodeoxycholic acid is (1~2):(1~2).

[0013] This invention provides a fishmeal-free feed for Procambarus clarkii, wherein the feed does not contain fishmeal and includes the aforementioned feed additives.

[0014] Preferably, the amount of the feed additive added is 1~1.5g / kg based on the feed weight.

[0015] Preferably, the feed consists of the following components: low-erucic acid rapeseed meal, corn starch, soybean oil, soybean lecithin oil, sodium alginate, calcium dihydrogen phosphate, spray-dried yeast powder, cholesterol, multivitamins and minerals, sodium chloride, choline chloride, vitamin C, chitosan, carboxymethyl cellulose, and the above-mentioned feed additives.

[0016] Preferably, the feed, by weight parts, comprises the following components: 72-75 parts of low-erucic acid rapeseed meal, 8-8.5 parts of corn starch, 4-4.6 parts of soybean oil, 1-2 parts of soybean lecithin oil, 2-3 parts of sodium alginate, 2-3 parts of calcium dihydrogen phosphate, 2.5-3.5 parts of spray-dried yeast powder, 0.4-0.6 parts of cholesterol, 1.5-2.5 parts of multivitamins and minerals, 0.1-0.3 parts of sodium chloride, 0.2-0.3 parts of choline chloride, 0.4-0.6 parts of vitamin C, 0.2-0.4 parts of chitosan, 0.05-0.1 parts of carboxymethyl cellulose, and 0.1-0.15 parts of the above feed additives.

[0017] This invention provides the application of the above-mentioned feed additives or Procambarus clarkii feed in Procambarus clarkii farming.

[0018] This invention provides a method for farming red swamp crayfish, comprising the following steps: feeding the red swamp crayfish with the above-mentioned feed.

[0019] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention discovers that a combination of silymarin and chenodeoxycholic acid in a specific mass ratio can effectively improve the decline in muscle quality of Procambarus clarkii caused by fishmeal-free feed. It can significantly improve the water retention and texture properties of Procambarus clarkii muscle, improve the amino acid composition of muscle, increase the content of umami and sweet amino acids, and enhance the level of functional fatty acids in muscle. This effectively avoids the impact of declining muscle quality of Procambarus clarkii caused by fishmeal-free feed, thus eliminating the need for fishmeal and effectively reducing feed and farming costs. Attached Figure Description

[0020] Figure 1 Results of muscle texture in each group of Procambarus clarkii. Detailed Implementation

[0021] This invention provides the application of silymarin combined with chenodeoxycholic acid in the preparation of products that improve the quality decline of Procambarus clarkii caused by fishmeal-free feed. The quality of Procambarus clarkii includes the muscle quality, which includes the cooking loss rate, freezing loss rate, hardness, adhesiveness, chewiness, and resilience of the muscle, as well as the amino acid content (such as umami amino acids, sweet amino acids, and flavor amino acids) and fatty acid content (such as polyunsaturated fatty acids) in the muscle. The mass ratio of silymarin to chenodeoxycholic acid in this invention is (1~2):(1~2), more preferably 1:1.

[0022] This invention provides a feed additive to improve the quality decline of *Procambarus clarkii* caused by fishmeal-free feed. The feed additive is composed of silymarin and chenodeoxycholic acid. The mass ratio of silymarin to chenodeoxycholic acid is (1~2):(1~2), more preferably 1:1. The quality of *Procambarus clarkii* includes the muscle quality, which includes the cooking loss rate, freezing loss rate, hardness, adhesiveness, chewiness, and resilience of the muscle, as well as the amino acid content (such as umami amino acids, sweet amino acids, and flavor amino acids) and fatty acid content (such as polyunsaturated fatty acids) in the muscle.

[0023] This invention provides a fishmeal-free feed for Procambarus clarkii, wherein the feed does not contain fishmeal and includes the aforementioned feed additives. Preferably, the amount of the feed additives added is 1-1.5 g / kg by weight of the feed, more preferably 1.1 g / kg, 1.2 g / kg, 1.3 g / kg, or 1.4 g / kg. The fishmeal-free Procambarus clarkii feed of this invention may use low-erucic acid rapeseed meal as the protein source.

[0024] Preferably, the feed of the present invention is composed of the following components: low-erucic acid rapeseed meal, corn starch, soybean oil, soybean lecithin oil, sodium alginate, calcium dihydrogen phosphate, spray-dried yeast powder, cholesterol, multivitamins and minerals, sodium chloride, choline chloride, vitamin C, chitosan, carboxymethyl cellulose, and the feed additives mentioned above.

[0025] Preferably, the feed, by weight parts, comprises the following components: 72-75 parts of low-erucic acid rapeseed meal, 8-8.5 parts of corn starch, 4-4.6 parts of soybean oil, 1-2 parts of soybean lecithin oil, 2-3 parts of sodium alginate, 2-3 parts of calcium dihydrogen phosphate, 2.5-3.5 parts of spray-dried yeast powder, 0.4-0.6 parts of cholesterol, 1.5-2.5 parts of multivitamins and minerals, 0.1-0.3 parts of sodium chloride, 0.2-0.3 parts of choline chloride, 0.4-0.6 parts of vitamin C, 0.2-0.4 parts of chitosan, 0.05-0.1 parts of carboxymethyl cellulose, and 0.1-0.15 parts of the aforementioned feed additives. More preferably, the feed contains 73-74 parts of low-erucic acid rapeseed meal, 8.15-8.3 parts of corn starch, 4.2-4.4 parts of soybean oil, 1.5-1.8 parts of soybean lecithin oil, 2.5-2.8 parts of sodium alginate, 2.5-2.6 parts of calcium dihydrogen phosphate, 2.8-3 parts of spray-dried yeast powder, 0.5 parts of cholesterol, 1.8-2 parts of multivitamins and minerals, 0.15-0.2 parts of sodium chloride, 0.25 parts of choline chloride, 0.5 parts of vitamin C, 0.3 parts of chitosan, 0.06-0.08 parts of carboxymethyl cellulose, and 0.12-0.14 parts of the aforementioned feed additives.

[0026] The multivitamin and mineral premix described in this invention refers to a premix of vitamins and minerals, which may include vitamin A acetate, vitamin D, dl-α-tocopherol acetate, menadione, thiamine nitrate, riboflavin (vitamin B2), pyridoxine hydrochloride, cyanocobalamin, calcium D-hexanoate, nicotinamide, folic acid, D-biotin, L-ascorbic acid 2-phosphate, inositol, magnesium, iron, zinc, manganese, copper, cobalt, selenium, and iodine. The raw material components of the feed described in this invention are conventional commercially available products.

[0027] This invention provides the application of the above-mentioned feed additives or Procambarus clarkii feed in Procambarus clarkii farming, which can effectively improve the decline in muscle quality of Procambarus clarkii caused by fishmeal-free feed, improve the water retention and texture properties of Procambarus clarkii muscle, improve the amino acid composition of muscle, increase the content of umami amino acids and sweet amino acids, and enhance the level of functional fatty acids in muscle, thereby avoiding the use of fishmeal and effectively reducing feed costs and farming costs.

[0028] This invention provides a method for cultivating *Procambarus clarkii*, comprising the following steps: feeding *Procambarus clarkii* feed as described above. As an optional implementation, the feed is preferably given twice daily, at 7:00 AM and 7:00 PM, with a daily feeding amount of 3-5% of the *Procambarus clarkii*'s weight. The specific feeding amount can be adjusted according to the *Procambarus clarkii*'s feeding behavior and weather conditions.

[0029] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] In a specific embodiment of the present invention, the multivitamin and mineral premix refers to a premix of vitamins and minerals (per kg of feed) comprising the following: Vitamin A acetate, 450,000 IU; Vitamin D, 1,000,000 IU; dl-α-tocopherol acetate, 5 g; menadione, 0.5 g; thiamine nitrate, 0.5 g; riboflavin (vitamin B2), 0.7 g; pyridoxine hydrochloride, 0.6 g; cyanocobalamin, 0.002 g; calcium D-hexanoate, 2 g; nicotinamide, 3.5 g; folic acid, 0.15 g; D-biotin, 0.006 g; L-ascorbic acid 2-phosphate (calculated as L-ascorbic acid), 10 g; inositol, 8 g; magnesium, 20 g; iron, 2 g; zinc, 7.5 g; manganese, 2 g; copper, 1.5 g; cobalt, 0.08 g; selenium, 0.01 g; and iodine, 0.1 g.

[0031] In a specific embodiment of the present invention, silymarin and chenodeoxycholic acid were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0032] Unless otherwise specified, the following embodiments are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1 A feed additive to improve the quality decline of red swamp crayfish caused by fishmeal-free feed: a mixture of silymarin and chenodeoxycholic acid in a mass ratio of 1:1.

[0035] Example 2 A feed additive to improve the quality decline of red swamp crayfish caused by fishmeal-free feed: a mixture of silymarin and chenodeoxycholic acid in a mass ratio of 1:2.

[0036] Example 3 A feed additive to improve the quality decline of red swamp crayfish caused by fishmeal-free feed: a mixture of silymarin and chenodeoxycholic acid in a mass ratio of 2:1.

[0037] Example 4 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 0%, low-erucic acid rapeseed meal 74%, corn starch 8.15%, soybean oil 4.4%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 0.08%, silymarin 0.06%, chenodeoxycholic acid 0.06%.

[0038] Preparation method: Crush all feed ingredients, pass them through a 40-mesh sieve, add them one by one in order of increasing formula ratio and mix them evenly. Then use a feed mill (SLP-45, Shanghai Huaxia Fishery Machinery & Instrument Co., Ltd.) to make them into 1mm diameter pellets, dry them at 60℃, seal them and store them in a -20℃ refrigerator for later use.

[0039] Example 5 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 0%, low-erucic acid rapeseed meal 74%, corn starch 8.15%, soybean oil 4.4%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 0.08%, silymarin 0.04%, chenodeoxycholic acid 0.08%.

[0040] Preparation method: Same as in Example 4.

[0041] Example 6 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 0%, low-erucic acid rapeseed meal 74%, corn starch 8.15%, soybean oil 4.4%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 0.08%, silymarin 0.08%, chenodeoxycholic acid 0.04%.

[0042] Preparation method: Same as in Example 4.

[0043] Comparative Example 1 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 46%, low-erucic acid rapeseed meal 0%, corn starch 32%, soybean oil 1.5%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 7.25%, silymarin 0%, chenodeoxycholic acid 0%.

[0044] Preparation method: Same as in Example 4.

[0045] Comparative Example 2 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 0%, low-erucic acid rapeseed meal 74%, corn starch 8.15%, soybean oil 4.4%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 0.2%, silymarin 0%, chenodeoxycholic acid 0%.

[0046] Preparation method: Same as in Example 4.

[0047] Comparative Example 3 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 0%, low-erucic acid rapeseed meal 74%, corn starch 8.15%, soybean oil 4.4%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 0.08%, silymarin 0.12%, chenodeoxycholic acid 0%.

[0048] Preparation method: Same as in Example 4.

[0049] Comparative Example 4 A zero-fishmeal feed for Procambarus clarkii (by weight percentage): fishmeal 0%, low-erucic acid rapeseed meal 74%, corn starch 8.15%, soybean oil 4.4%, soybean lecithin oil 1.5%, sodium alginate 2.5%, calcium dihydrogen phosphate 2.5%, spray-dried yeast powder 3%, cholesterol 0.5%, multivitamins and minerals 2%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.5%, chitosan 0.3%, carboxymethyl cellulose 0.08%, silymarin 0%, chenodeoxycholic acid 0.12%.

[0050] Preparation method: Same as in Example 4.

[0051] Experimental Example 1 Aquaculture trials were conducted using the feed formulations of Examples 4-6 and Comparative Examples 1-4, respectively. The crude protein and crude fat content of each feed were tested, and the results are shown in Table 1.

[0052] Table 1. Nutritional levels (%) of each feed group

[0053] The results showed that the crude protein and crude fat contents of the feeds in each group were similar.

[0054] Crawfish were fed different diets for 6 weeks, with 3 replicates per group and 30 crawfish per replicate. After 6 weeks of rearing, the crawfish were fasted for 24 hours, and muscle tissue was collected to analyze its texture, amino acid and fatty acid composition. This study verified and evaluated the effect of adding silymarin and chenodeoxycholic acid to the diet on improving the decline in muscle quality of crawfish caused by a fishmeal-free diet.

[0055] Muscle texture results as follows Figure 1As shown in the figure, (A) represents cooking loss rate, (B) represents freezing loss rate, (C) represents hardness, (D) represents adhesiveness, (E) represents chewiness, and (F) represents resilience. The results showed that compared with the positive control group (FM) using fishmeal as the protein source, the negative control group (DL) using low-erucic acid rapeseed meal as the sole protein source exhibited significant adverse changes in all muscle texture indicators. The cooking and freezing loss rates in the DL group were significantly higher, reaching the highest levels among all treatment groups, indicating that the zero-fishmeal rapeseed meal diet significantly weakened the water-holding capacity of the shrimp meat. Simultaneously, the muscle hardness, chewiness, and resilience in the DL group were significantly reduced, while the absolute value of adhesiveness was significantly increased, indicating that the muscle structure became loose, and elasticity and structural integrity were compromised. Adding different additives to the DL diet alleviated these adverse changes to varying degrees. Adding silymarin (SB) or chenodeoxycholic acid (CA) alone significantly reduced cooking and freezing loss rates and improved hardness, chewiness, and resilience, but the overall improvement was limited, with most indicators still significantly lower than the FM group. In comparison, the compound treatments (SC1, SC2, and SC3) showed more significant improvements in most textural parameters. Among them, SC1 (600 mg / kg silymarin + 600 mg / kg chenodeoxycholic acid) was the most effective in reducing cooking and freezing losses, demonstrating good water retention. SC2 (400 mg / kg silymarin + 800 mg / kg chenodeoxycholic acid) showed outstanding improvement in chewiness. SC3 (800 mg / kg silymarin + 400 mg / kg chenodeoxycholic acid) showed advantages in hardness and overall textural recovery. Furthermore, the resilience of all three compound groups was significantly higher than that of the DL group, recovering to a level comparable to the FM group. Overall, the low-erucic acid rapeseed meal and zero-fishmeal feed significantly reduced the textural quality of Procambarus clarkii muscle, while the compound addition of silymarin and chenodeoxycholic acid was superior to either alone in improving muscle water retention and textural properties, exhibiting a certain degree of ratio dependence.

[0056] The results of the amino acid composition analysis are shown in Table 2.

[0057] Table 2. Amino acid composition results of each group of feeds

[0058] Note: # represents umami amino acids. It is a sweet-tasting amino acid.

[0059] The results showed that different feed treatments significantly affected the amino acid composition of *Procambarus clarkii* muscle. Compared with the fishmeal control group (FM), the content of multiple amino acids in the muscle of the low-erucic acid rapeseed meal and zero-fishmeal group (DL) was significantly reduced, especially umami and sweet-tasting amino acids such as aspartic acid, glutamic acid, glycine, tyrosine, and phenylalanine. This resulted in the lowest levels of total umami amino acids (UAA), total sweet amino acids (SAA), and total flavor amino acids (DAA), indicating that the zero-fishmeal feed significantly weakened the flavor quality of the shrimp meat. Adding different additives to the DL group improved the muscle amino acid composition to varying degrees. Adding silymarin (SB) or chenodeoxycholic acid (CA) alone significantly increased the content of key flavor amino acids such as aspartic acid, glutamic acid, and glycine, causing UAA, SAA, and DAA to rebound significantly compared to the DL group, but the overall levels were still slightly lower than or close to those of the FM group. In contrast, the combined additive treatments (SC1, SC2, SC3) were more effective in increasing the amino acid content of the muscle. Group SC1 showed significantly higher levels of various amino acids, including aspartic acid, glutamic acid, glycine, and tyrosine, than Group FM. Its UAA, SAA, and DAA levels were the highest among all treatments, demonstrating the best flavor improvement effect. Groups SC2 and SC3 also significantly increased the content of most flavor-related amino acids, outperforming the single-addition groups overall. Alanine levels did not differ significantly among groups, indicating minimal influence from feed. Overall, zero-fishmeal and rapeseed meal diets reduced the accumulation of flavor amino acids in the muscle of Procambarus clarkii, while the combination of silymarin and chenodeoxycholic acid effectively improved the muscle amino acid composition, exhibiting a more pronounced synergistic effect at certain ratios.

[0060] The results of fatty acid composition analysis are shown in Table 3.

[0061] Table 3. Fatty acid composition results of each group of feeds

[0062] The results showed that compared with the fishmeal control group (FM), the low-fat rapeseed meal zero-fishmeal group (DL) had significantly lower levels of polyunsaturated fatty acids, especially linoleic acid (C18:2n6), α-linolenic acid (C18:3n3), arachidonic acid (C20:4n6), and long-chain polyunsaturated fatty acids EPA (C20:5n3) and DHA (C22:6n3), which were significantly lower than those in the FM group. This resulted in a significant decrease in the total EPA+DHA and total polyunsaturated fatty acids (ΣPUFA), indicating that the zero-fishmeal rapeseed meal diet is not conducive to the deposition of functional fatty acids in muscle. Adding different additives to the DL diet improved the fatty acid composition of each treatment group to varying degrees. Adding silymarin (SB) or chenodeoxycholic acid (CA) alone significantly increased the contents of linoleic acid, arachidonic acid, EPA, and DHA, resulting in a significant rebound in EPA+DHA and ΣPUFA levels compared to the DL group, but still slightly lower overall than the FM group. In contrast, the combined additive treatment showed more significant improvement in fatty acid composition. The SC1 group (600 mg / kg silymarin + 600 mg / kg chenodeoxycholic acid) approached or reached the levels of the FM group in most fatty acid indicators. Its EPA, DHA, and EPA+DHA contents were significantly higher than the DL group and significantly higher than some single-addition groups. Simultaneously, its ΣPUFA reached 3.93 mg / g, second only to the FM group. The SC2 and SC3 groups also significantly increased EPA, DHA, and total polyunsaturated fatty acids, but the improvement was slightly less than that of the SC1 group. Overall, the zero-fishmeal and rapeseed meal feed significantly reduced the deposition of polyunsaturated fatty acids, especially n-3 series fatty acids, in the muscle of *Procambarus clarkii*. The combined addition of silymarin and chenodeoxycholic acid effectively mitigated this adverse effect, with the equal-proportion combination showing a more significant advantage in increasing the level of functional fatty acids in the muscle.

[0063] Synergy effect assessment, the synergy index calculation method is as follows: Calculate the expected results: Record the effects of adding SB(x) and CA(y) separately, and calculate the mean, standard deviation and other statistical indicators for each group.

[0064] Calculate the combined effect: Calculate the actual effect (Mx) produced by adding SB and CA together.

[0065] Calculate the Synergy Index (SI): using the formula for synergy effect: in, M x The effect observed after adding SB and CA; M x expected The expected effect, predicted based on the effects added individually, is calculated using the following formula: .

[0066] Criteria for judging synergistic effect: If SI>1, it indicates that there is a synergistic effect; if SI=1, it indicates that there is no synergistic effect; if SI<1, it indicates that there is an antagonistic effect.

[0067] The results of the synergistic effect assessment are shown in Table 4.

[0068] Table 4 Synergistic Index of Silymarin and Goodeoxycholic Acid

[0069] The results showed that silymarin combined with chenodeoxycholic acid had a synergistic effect in improving the amino acid composition of Procambarus clarkii muscle, increasing the content of umami and sweet amino acids, and enhancing the level of functional fatty acids in muscle.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of silymarin combined with chenodeoxycholic acid in the preparation of products to improve the quality of red swamp crayfish that have declined due to fishmeal-free feed.

2. The application according to claim 1, characterized in that, The mass ratio of silymarin to chenodeoxycholic acid is (1~2):(1~2).

3. A feed additive for improving the quality decline of red swamp crayfish caused by fishmeal-free feed, characterized in that, The feed additive consists of silymarin and chenodeoxycholic acid.

4. The feed additive according to claim 3, characterized in that, The mass ratio of silymarin to chenodeoxycholic acid is (1~2):(1~2).

5. A zero-fishmeal feed for Procambarus clarkii, characterized in that, The feed does not contain fishmeal, and the feed includes the feed additives described in any one of claims 3 to 4.

6. The Procambarus clarkii feed according to claim 5, characterized in that, The amount of the feed additive added is 1~1.5g / kg based on feed weight.

7. The Procambarus clarkii feed according to claim 5, characterized in that, The feed is composed of the following components: low-erucic acid rapeseed meal, corn starch, soybean oil, soybean lecithin oil, sodium alginate, calcium dihydrogen phosphate, spray-dried yeast powder, cholesterol, multivitamins and minerals, sodium chloride, choline chloride, vitamin C, chitosan, carboxymethyl cellulose, and the feed additives described in any one of claims 3 to 4.

8. The Procambarus clarkii feed according to claim 5, characterized in that, The feed, by weight, comprises the following components: 72-75 parts of low-erucic acid rapeseed meal, 8-8.5 parts of corn starch, 4-4.6 parts of soybean oil, 1-2 parts of soybean lecithin oil, 2-3 parts of sodium alginate, 2-3 parts of calcium dihydrogen phosphate, 2.5-3.5 parts of spray-dried yeast powder, 0.4-0.6 parts of cholesterol, 1.5-2.5 parts of multivitamins and minerals, 0.1-0.3 parts of sodium chloride, 0.2-0.3 parts of choline chloride, 0.4-0.6 parts of vitamin C, 0.2-0.4 parts of chitosan, 0.05-0.1 parts of carboxymethyl cellulose, and 0.1-0.15 parts of the feed additives described in any one of claims 3-4.

9. The application of the feed additive according to any one of claims 3 to 4 or the Procambarus clarkii feed according to any one of claims 5 to 8 in the cultivation of Procambarus clarkii.

10. A method for farming Procambarus clarkii, characterized in that, Includes the following steps: Feeding the red swamp crayfish with the feed described in any one of claims 5 to 8.

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