Use of cholesterol in the preparation of feed additives, feed and method of preparation

Abstract

The application provides application of cholesterol in preparation of a feed additive, a feed and a preparation method, and comprises the application of cholesterol or a feed-acceptable ester thereof in preparation of a feed additive for crustaceans, is used for regulating lipid metabolism of the crustaceans, and improves the antioxidant capacity of the crustaceans. The content of animal protein source in the feed is not higher than 30%. In the application, the cholesterol is added in the feed of the crustaceans as a functional additive of sterols in a low dose, on the one hand, effective addition of the cholesterol can significantly improve the growth performance of the crustaceans under the condition of low fish meal feed, on the other hand, the lipid metabolism and the antioxidant capacity of the crustaceans can be regulated, and the physiological health is maintained.

Description

Technical Field

[0001] This invention relates to the technical field of feed additives, specifically to the application of cholesterol in the preparation of feed additives, feed, and preparation method. Background Technology

[0002] With the continuous expansion of aquaculture and the increasing depletion of marine fishery resources, the supply of traditional high-quality animal protein sources, such as fishmeal, is tight and prices are rising continuously. In recent years, in order to control the production cost of aquatic feed, the industry has widely adopted plant protein sources to replace traditional animal protein sources, such as fishmeal, making low-fishmeal feed an inevitable trend in the industry's development.

[0003] However, for crustaceans such as shrimp and crabs, long-term feeding with low-fishmeal diets often leads to decreased growth performance and impaired physiological health, severely impacting aquaculture efficiency. In particular, lipid metabolism disorders are a significant concern; low-fishmeal diets can easily cause impaired hemolymphatic lipid transport and utilization in crustaceans, resulting in serious health risks. Furthermore, low-fishmeal diets can weaken the antioxidant defenses of crustaceans, making them more susceptible to oxidative stress, environmental stress, and pathogen attack, thus threatening aquaculture production.

[0004] Currently, there is an urgent need for a technology that can effectively compensate for the breeding deficiencies caused by low fishmeal feed, solve lipid metabolism disorders in crustaceans, and maintain their physiological health. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies and provide a feed for crustaceans with low animal protein content, in which cholesterol, as a sterol functional additive, is applied. On the one hand, the effective addition of cholesterol significantly improves the growth performance of crustaceans under low fishmeal feed conditions; on the other hand, it regulates the lipid metabolism and antioxidant capacity of crustaceans, maintaining their physiological health.

[0006] The first aspect of the invention is to provide the use of cholesterol or a feed-acceptable ester thereof in the preparation of a crustacean feed additive for regulating lipid metabolism in crustaceans, wherein the content of animal protein source in the feed is not higher than 30%.

[0007] According to a preferred embodiment of the present invention, lipid metabolism in crustaceans is regulated, specifically by reducing the levels of total cholesterol, triglycerides, and / or low-density lipoprotein cholesterol in the hemolymph and / or increasing the levels of high-density lipoprotein cholesterol in the hemolymph, thereby promoting the transport and storage of lipids to tissues such as the liver and pancreas.

[0008] A second aspect of the invention is to provide the use of cholesterol or a feed-acceptable ester thereof in the preparation of a crustacean feed additive to improve the antioxidant capacity of crustaceans, wherein the content of animal protein source in the feed is not higher than 30%.

[0009] According to a preferred embodiment of the present invention, improving the antioxidant capacity of crustaceans enhances the body's antioxidant defense capabilities and alleviates oxidative stress damage. Specifically, this is manifested in the increase of superoxide dismutase activity in the hepatopancreas, the increase of total antioxidant capacity, and / or the decrease of malondialdehyde content in the hepatopancreas.

[0010] According to a preferred embodiment of the present invention, the crustacean is any one of the following: Litopenaeus vannamei, Penaeus monodon, Penaeus sinensis, Macrobrachium nipponense, Eriocheir sinensis, Procambarus clarkii, Muscovy crab, and Portunus trituberculatus.

[0011] The above two aspects are applied in the preparation of crustacean feed additives, especially for Litopenaeus vannamei and Macrobrachium jinnense.

[0012] According to a preferred embodiment of the present invention, the content of animal protein source in the feed is not higher than 30%, preferably not higher than 26%. The application of the above two aspects in the preparation of crustacean feed additives is mainly for feeds where the animal protein source is added at a low content because plant protein sources or other protein sources replace part of the animal protein source. The content of animal protein source in the feed is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any value between them.

[0013] Preferably, the animal protein source in the feed includes fishmeal, with a fishmeal content not exceeding 25%, preferably not exceeding 20% ​​or 15%. The application of the above two aspects in the preparation of crustacean feed additives is mainly for feeds where fishmeal is added in low amounts due to the substitution of some fishmeal by plant protein sources or other protein sources. The fishmeal content in the feed is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or any value between them.

[0014] According to a preferred embodiment of the present invention, the structural formula of cholesterol is shown in formula (I).

[0015] (I)

[0016] According to a preferred embodiment of the invention, feed-acceptable esters of cholesterol include esterified products of cholesterol obtained by combining with any form of fatty acid, or mixtures of cholesterol and partially esterified products. The aforementioned any form of fatty acid includes mono-fatty acids or mixed fatty acids.

[0017] Cholesterol esters that are acceptable for feed include, but are not limited to, one or more of cholesterol formate, cholesterol acetate, cholesterol oleate, and cholesterol palmitate.

[0018] According to a preferred embodiment of the present invention, the amount of cholesterol or its feed-acceptable ester added to the feed is 0.02% to 0.4% or 0.05% to 0.2%, preferably 0.05% to 0.1% or 0.1% to 0.2%.

[0019] The amount of cholesterol or its feed-acceptable esters added to the feed is 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, or any value between them.

[0020] According to a preferred embodiment of the present invention, the use of cholesterol or its feed-acceptable esters in the preparation of crustacean feed additives can also be used to improve digestive enzyme activity and enhance growth performance.

[0021] According to a preferred embodiment of the present invention, improving the digestive enzyme activity of crustaceans involves specifically enhancing the activity of digestive enzymes and optimizing the digestion and absorption pattern of nutrients. Specifically, this is manifested in the enhancement of lipase and protease activity, which effectively improves the digestion and absorption of nutrients through a shift in energy metabolism preferences.

[0022] According to a preferred embodiment of the present invention, the growth performance of crustaceans is improved, specifically by increasing the final weight, weight gain rate, specific growth rate, and / or reducing the feed conversion ratio of crustaceans.

[0023] A third aspect of the invention is to provide a crustacean feed comprising the aforementioned cholesterol or a feed-acceptable ester thereof.

[0024] According to a preferred embodiment of the present invention, crustacean feed, based on dry weight, comprises the following components: (1) Animal protein source 10%~40%; (2) Plant protein source 20%~60%; (3) Carbohydrates 5%~30%; (4) Fat 0%~10%; (5) 1% to 10% of nutritional supplements and ingredients, including cholesterol or its feed-acceptable esters.

[0025] According to a preferred embodiment of the present invention, crustacean feed, based on dry weight, comprises the following components: (1) Animal protein source 10%~30%; (2) Plant protein source 30%~60%; (3) Carbohydrates 15%~25%; (4) Fat 4%~8%; (5) 5% to 10% of nutritional supplements and excipients, including cholesterol or its feed-acceptable esters, wherein the amount of cholesterol or its feed-acceptable esters added to the feed is 0.02% to 0.4% or 0.05% to 0.2%, preferably 0.05% to 0.1% or 0.1% to 0.2%.

[0026] A fourth aspect of the present invention is to provide a method for preparing the above-mentioned crustacean feed, comprising the following steps: (1) Using the equal incremental method, cholesterol or its feed-acceptable esters are mixed stepwise with other trace components in the formula to prepare compound premix I; (2) After the bulk raw materials are dry premixed, they are mixed evenly with composite premix I to obtain composite premix II; (3) After mixing the fatty components, mix them evenly with composite premix II to obtain composite premix III; (4) Spray water onto the composite premix III to adjust the moisture content of the material, and then process it into a finished product after conditioning, granulation and drying.

[0027] Trace components are those that are present in less than 5% of the formulation and are non-fatty. Other trace components in the above formulation refer to those that are present in less than 5% of the formulation and are non-fatty, excluding cholesterol or its feed-acceptable esters.

[0028] The main raw materials are components that make up no less than 5% of the formula and are non-fatty.

[0029] According to a preferred embodiment of the present invention, before step (1) of mixing cholesterol or its feed-acceptable esters with other trace components step by step, cholesterol or its feed-acceptable esters are first premixed with a porous carrier using a carrier dilution method. The porous structure and adsorption properties of the porous carrier are used to uniformly fix the trace amounts of cholesterol or its feed-acceptable esters to obtain a cholesterol premix, which is then mixed with other trace components. Preferably, the porous carrier comprises bentonite.

[0030] The present invention has the following beneficial effects: The application of cholesterol or its feed-acceptable esters in crustacean feed additives in this invention can significantly improve the physiological health of crustaceans, especially by regulating lipid metabolism and alleviating lipid metabolism disorders induced by low-fishmeal feed, even at low dosage levels.

[0031] Meanwhile, the application of cholesterol or its feed-acceptable esters in crustacean feed additives can significantly enhance the antioxidant defense function of crustaceans and reduce oxidative stress even at low dosages. Attached Figure Description

[0032] Figure 1 These are hepatopancreatic tissue sections from each group of Litopenaeus vannamei in Example 1. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0034] Unless otherwise specified, the raw materials and equipment used in the following embodiments of the present invention are commercially available.

[0035] Unless otherwise specified, all percentages, parts, ratios, etc. mentioned in this invention are based on weight, unless being based on weight would not be in accordance with the common understanding of those skilled in the art.

[0036] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that the ranges of 60-110 and 80-120 will also be understood. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers.

[0037] Unless otherwise specified in this application, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0038] Unless otherwise specified, all technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0039] In this application, unless otherwise specified, the terms "comprising" and "including" as used herein are open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0040] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more.

[0041] This invention discloses a method for applying cholesterol in low-fishmeal feed for crustaceans, such as Litopenaeus vannamei and Macrobrachium chinense. Specifically, by exogenously adding cholesterol within a specific dosage range to the low-fishmeal feed, a series of growth and health problems caused by reduced fishmeal are systematically solved.

[0042] The overall implementation path of the plan is as follows: Formulate a low-fishmeal basic feed with soybean meal, peanut meal and other protein sources as the main sources; through a step-by-step mixing feed processing technology, precisely measured cholesterol or feed-acceptable esters are uniformly added to the feed to make pelleted feed; finally, by feeding this specific feed, the growth performance, lipid metabolism, antioxidant defense system and digestive function of crustaceans are measured and analyzed.

[0043] Example 1

[0044] 1.1 Test Materials

[0045] Experimental animals: Litopenaeus vannamei ( Litopenaeus vannamei Juvenile shrimp (also known as whiteleg shrimp) have an initial weight of about 0.80g.

[0046] Test substance: Cholesterol (purity ≥97.5%), provided by Zhejiang Garden Biomedical Co., Ltd.

[0047] Feed formulation: An isonitrogenous and isolipid-based feed is used, with the basal feed being a low-fishmeal feed (fishmeal content 15%), and a portion of the fishmeal is replaced by a mixed plant protein source (soybean meal, peanut meal, and wheat gluten). First, accurately weighed cholesterol and bentonite are premixed using a carrier dilution method, utilizing the porous structure and adsorption properties of bentonite to uniformly fix trace amounts of cholesterol, thus obtaining a cholesterol-bentonite premix. Second, using an equal-incremental method, the cholesterol-bentonite premix is ​​gradually mixed with other trace components in the formulation to obtain a composite premix. Then, bulk raw materials such as fishmeal, soybean meal, and peanut meal are put into a mixer for dry premixing, followed by the addition of the composite premix and continued mixing until homogeneous. Subsequently, fish oil, soybean oil, and soybean lecithin are mixed in proportion according to the formulation and added, and mixing continues until homogeneous. Finally, water is sprayed evenly to adjust the moisture content of the material, and after conditioning, granulation, and drying, the finished product is obtained.

[0048] 1.2 Experimental Design

[0049] Experimental group: HF group: High fish meal control group (25% fish meal, no added cholesterol).

[0050] LF group: Low fish meal control group (15% fish meal, no added cholesterol).

[0051] Group C0.1: Low fish meal + 0.1% cholesterol.

[0052] Group C0.2: Low fish meal + 0.2% cholesterol.

[0053] The specific feed formulations for each experimental group, including those with high fishmeal and low fishmeal content, are shown in Table 1.

[0054] Table 1 Feed Formulation

[0055] Culture conditions: Indoor recirculating aquaculture system, culture cycle 56 days. Water temperature (28±0.5)℃, salinity (32±0.5)‰.

[0056] Indicator measurements: growth indicators (final average weight, weight gain rate, specific growth rate, feed conversion ratio), body composition, blood and lymph physiological and biochemical indicators, lipid metabolism indicators, antioxidant indicators, and digestive enzyme indicators.

[0057] 1.3 Results and Analysis

[0058] 1.3.1 Growth performance

[0059] The final average weight, weight gain rate, specific growth rate and feed conversion ratio of Litopenaeus vannamei in each group were measured.

[0060]

[0061] W t The average body weight of Litopenaeus vannamei at the end of the experiment (unit: g / tail). W0 represents the average body weight of Litopenaeus vannamei at the start of the experiment (unit: g / tail). t is the test period (unit: days); M represents the total weight of the feed given (in g). n is the number of Litopenaeus vannamei shrimp used in the experiment (unit: shrimp).

[0062] As shown in Table 2, the low fishmeal group resulted in slow growth, significantly reduced weight gain and specific growth rate of Litopenaeus vannamei, and low feed utilization efficiency (increased feed conversion ratio).

[0063] Adding a small amount of cholesterol (0.1%-0.2%) to low-fishmeal feed significantly improved the average final weight, weight gain, and specific growth rate of Litopenaeus vannamei. Specifically, adding 0.1% cholesterol to the low-fishmeal feed brought these values ​​to levels indistinguishable from the high-fishmeal group (25% fishmeal). The addition of cholesterol to the low-fishmeal feed improved growth performance and feed conversion efficiency.

[0064] Meanwhile, adding a small amount of cholesterol (0.1%-0.2%) to low-fishmeal feed significantly reduced the feed conversion ratio. A lower feed conversion ratio means less undigested nutrient discharge, which helps reduce the pollution load on aquaculture water bodies, aligns with environmentally friendly sustainable development principles, and contributes to improving the aquaculture environment.

[0065] Table 2. Effects of cholesterol on the growth performance of Litopenaeus vannamei.

[0066] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05). IW, initial average weight; FW, final average weight; SR, survival rate; WGR, weight gain rate; SGR, specific growth rate; FCR, feed conversion ratio; feed intake, FI; protein efficiency, PER.

[0067] 1.3.2 Lipid Metabolism

[0068] As shown in Table 3, the low fishmeal group resulted in increased levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the hemolymph of Litopenaeus vannamei. Compared to the LF group, adding a small amount of cholesterol to the diet significantly reduced TG and LDL-C levels in the hemolymph. When 0.1% cholesterol was added to the low fishmeal diet, TG and LDL-C levels returned to the levels of the HF group. Cholesterol addition did not significantly alter HDL-C levels in the hemolymph.

[0069] This indicates that adding cholesterol to low-fishmeal feed can alleviate lipid metabolism disorders.

[0070] Table 3. Effects of cholesterol on lipid metabolism in Litopenaeus vannamei.

[0071] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P<0.05). TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.

[0072] 1.3.3 Antioxidant

[0073] As shown in Table 4, compared with the high fishmeal group, the low fishmeal group resulted in a significant decrease in the antioxidant capacity of Litopenaeus vannamei. Compared with the LF group, the addition of a small amount of cholesterol to the diet significantly reduced the MDA content in the hepatopancreas, increased SOD activity, and significantly increased T-AOC. When 0.1% cholesterol was added to the low fishmeal diet, the MDA content, SOD activity, and T-AOC content returned to the levels of the HF group.

[0074] This indicates that adding cholesterol to low-fishmeal feed can enhance the antioxidant capacity of Litopenaeus vannamei.

[0075] Table 4. Effects of cholesterol on the antioxidant capacity of Litopenaeus vannamei.

[0076] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05%. MDA, malondialdehyde; SOD, superoxide dismutase; T-AOC, total antioxidant capacity.

[0077] 1.3.4 Digestive enzyme activity

[0078] As shown in Table 5, low-fishmeal diets significantly reduced the activities of lipase and trypsin in Litopenaeus vannamei, resulting in low efficiency in the digestion and absorption of nutrients, especially fats and proteins. Adding a small amount of cholesterol to low-fishmeal diets significantly increased the activities of trypsin and lipase in Litopenaeus vannamei and significantly reduced amylase activity. When 0.1%-0.2% cholesterol was added to low-fishmeal diets, the activities of lipase, amylase, and trypsin recovered to the levels of the HF group, indicating that it effectively improved the digestibility and absorption of nutrients. This suggests that adding cholesterol to low-fishmeal diets encourages the use of fat for energy, while cholesterol effectively improves the digestibility of protein in the feed.

[0079] Specifically, the addition of cholesterol to low-fishmeal feed induces a shift in energy metabolism preferences, leading to increased activity of lipases and proteases. This means the body's ability to digest fats and proteins is enhanced, allowing them to be used more efficiently as energy and growth raw materials. Correspondingly, the body's dependence on carbohydrates decreases, manifested as a decline in amylase activity.

[0080] Table 5. Effects of cholesterol on digestive enzymes in Litopenaeus vannamei.

[0081] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05).

[0082] 1.3.5 Body Composition

[0083] Table 6 shows that the addition of cholesterol had no significant effect on the moisture, crude fat, and crude ash content of whole Litopenaeus vannamei. Under low fishmeal feed conditions, the addition of a small amount of cholesterol significantly increased the crude protein content of whole shrimp, and there was no significant difference compared with the HF group.

[0084] Table 6. Effect of cholesterol on body composition (%, wet weight) of Litopenaeus vannamei

[0085] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05).

[0086] 1.3.6 Liver and pancreas tissue sections

[0087] like Figure 1 As shown, morphological studies of hepatopancreatic tissue revealed that in the HF group, the hepatic tubules were tightly packed, the cell shapes were more regular, the basement membrane structure was more intact, and the lumen exhibited a regular star shape. In the LF group, the hepatic tubules were more loosely packed; however, after adding cholesterol to the feed, the hepatic tubules became more tightly packed, and the basement membrane became clearer. Overall, adding cholesterol to the feed did not cause significant liver morphological changes and could effectively improve the liver morphological changes caused by low fishmeal diets.

[0088] Example 2

[0089] 2.1 Test Materials

[0090] Experimental animals: Japanese giant freshwater prawn (Macrobrachium nipponense) Macrobrachium nipponense Juvenile shrimp, with an initial weight of 0.12±0.003g.

[0091] Test substance: Cholesterol (purity ≥96.5%), produced by Zhejiang Garden Biomedical Co., Ltd.

[0092] Feed formulation: An isonitrogenous and lipid-based feed is used, with the basal feed being a low-fishmeal feed (fishmeal content of 11%), and a portion of the fishmeal is replaced by a mixture of plant protein sources (soybean meal, peanut meal, rapeseed meal, etc.).

[0093] 2.2 Experimental Design

[0094] Experimental group: LF group: Low fish meal control group (11% fish meal, no added cholesterol).

[0095] Group L0.05: Low fish meal + 0.05% cholesterol.

[0096] Group L0.1: Low fish meal + 0.1% cholesterol.

[0097] Group L0.2: Low fish meal + 0.2% cholesterol.

[0098] The specific feed formulations for each experimental group are shown in Table 7.

[0099] Table 7 Feed Formulation

[0100] Culture conditions: Culture tank with purified water system, culture cycle 56 days. During the culture period, dissolved oxygen ≥ 5.5 mg / L, pH 7.8 ± 0.4, ammonia nitrogen < 0.1 mg / L, nitrite < 0.005 mg / L.

[0101] Indicator measurements: growth indicators (final average weight, weight gain rate, specific growth rate, feed conversion ratio), hemolymph physiological and biochemical indicators, lipid metabolism indicators, antioxidant indicators, and digestive enzyme indicators.

[0102] 2.3 Results and Analysis

[0103] 2.3.1 Growth performance

[0104] The final average weight, weight gain rate, specific growth rate and feed conversion ratio of each group of Japanese freshwater prawns were measured. The calculation formula was consistent with that of Litopenaeus vannamei in Example 1.

[0105] As shown in Table 8, the addition of a small amount of cholesterol (0.05%-0.2%) to low-fishmeal feed improved the average final weight, weight gain rate, and specific growth rate of *Macrobrachium nipponense*. Furthermore, the feed conversion ratio decreased, indicating improved feed utilization efficiency. This demonstrates that the addition of cholesterol to low-fishmeal feed enhances the growth performance of *Macrobrachium nipponense* and improves feed utilization efficiency.

[0106] Table 8. Effects of cholesterol on the growth performance of Japanese giant freshwater prawns

[0107] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05). IW, Initial Average Weight; FW, Final Average Weight; SR, Survival Rate; WGR, Weight Gain Rate; SGR, Specific Growth Rate; FCR, Feed Conversion Ratio.

[0108] 2.3.2 Lipid Metabolism

[0109] As shown in Table 9, compared with the LF group, the addition of a small amount of cholesterol to the feed affected the levels of TC, TG, HDL-C, and LDL-C in the hemolymph. In particular, when 0.05%-0.1% cholesterol was added to the low-fishmeal feed, the HDL-C level increased significantly, indicating that the addition of trace amounts of cholesterol to the low-fishmeal feed can regulate the HDL-C level and has the function of regulating lipid metabolism and alleviating lipid metabolism disorders.

[0110] Table 9. Effects of cholesterol on lipid metabolism in Japanese marsh shrimp.

[0111] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05). TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.

[0112] 2.3.3 Antioxidant

[0113] As shown in Table 10, compared with the LF group, the addition of a small amount of cholesterol to the feed significantly reduced the MDA content in the hepatopancreas; SOD activity also showed an upward trend. This indicates that the addition of a small amount of cholesterol to the low fishmeal feed has the function of improving the antioxidant capacity of Litopenaeus vannamei.

[0114] Table 10 Effects of cholesterol on the antioxidant capacity of Japanese freshwater prawns

[0115] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P <0.05%. MDA, malondialdehyde; SOD, superoxide dismutase.

[0116] 2.3.4 Digestive enzyme activity

[0117] As shown in Table 11, the addition of a small amount of cholesterol to low-fishmeal feed significantly increased the activity of neutral protease and lipase in the hepatopancreas of Japanese freshwater prawns. This indicates that the addition of a small amount of cholesterol to low-fishmeal feed can effectively improve the digestibility and absorption of nutrients (especially fat and protein), making the feed more likely to utilize fat for energy, while also effectively improving the digestibility of protein in the feed.

[0118] Table 11 Effects of cholesterol on digestive enzymes in Japanese freshwater prawns

[0119] Note: Data are mean ± standard error. Different letters in the same row indicate significant differences. P<0.05). NPA, neutral protease; LPS, lipase; α-AMS, α-amylase.

[0120] In summary, cholesterol, as a functional additive in low-fishmeal feed, has a significant promoting effect on improving the growth performance and maintaining the physiological health of Litopenaeus vannamei and Macrobrachium nipponense. It can improve the health of shrimp by optimizing lipid metabolism, enhancing antioxidant capacity, and improving digestion. The optimal dosage of cholesterol in low-fishmeal feed is 0.05%-0.4%, preferably 0.05%-0.2%, more preferably 0.05%-0.1% or 0.1%-0.2%.

[0121] Adding cholesterol to low-fishmeal feed can effectively regulate blood lipids and lipid metabolism disorders by reducing triglyceride and low-density lipoprotein cholesterol levels in the hemolymph or increasing high-density lipoprotein cholesterol levels and promoting cholesterol transport to the liver and pancreas.

[0122] Adding cholesterol to low-fishmeal feeds may enhance the body's antioxidant capacity by increasing the activity of antioxidant enzymes. Due to the enhanced antioxidant capacity and improved liver and pancreatic health, feeding the feed of this invention improves the body's ability to cope with environmental stresses, such as sudden temperature changes, salinity variations, or hypoxia, contributing to a potential increase in stress resistance.

[0123] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The use of cholesterol or its feed-acceptable esters in the preparation of crustacean feed additives, characterized in that, To regulate lipid metabolism in crustaceans, the content of animal protein sources in the feed should not exceed 30%.

2. The use of cholesterol or its feed-acceptable esters in the preparation of crustacean feed additives, characterized in that, To enhance the antioxidant capacity of crustaceans, the content of animal protein sources in the feed should not exceed 30%.

3. The application according to any one of claims 1-2, characterized in that, The crustaceans mentioned are any one of the following: Litopenaeus vannamei, Penaeus monodon, Penaeus sinensis, Macrobrachium nipponense, Eriocheir sinensis, Procambarus clarkii, Muscovy crab, and Portunus trituberculatus.

4. The application according to any one of claims 1-2, characterized in that, The animal protein content in the feed shall not exceed 26%.

5. The application according to any one of claims 1-2, characterized in that, The animal protein source includes fish meal, and the content of fish meal is no more than 20%, preferably no more than 15%.

6. The application according to any one of claims 1-2, characterized in that, The cholesterol or its feed-acceptable esters are added to the feed at a rate of 0.02% to 0.4%. Preferably, the cholesterol or its feed-acceptable ester is added to the feed at an amount of 0.05% to 0.1% or 0.1% to 0.2%.

7. A crustacean feed, characterized in that, Includes cholesterol or a feed-acceptable ester thereof as described in any one of claims 1-2.

8. The feed according to claim 7, characterized in that, The feed, based on its dry weight, comprises the following components: (1) Animal protein source 10%~40%; (2) Plant protein source 20%~60%; (3) Carbohydrates 5%~30%; (4) Fat 0%~10%; (5) 1% to 10% of nutritional supplements and ingredients, including the cholesterol or its feed-acceptable esters.

9. The feed according to claim 8, characterized in that, The cholesterol or its feed-acceptable ester is added to the feed at a rate of 0.02% to 0.4%, preferably 0.05% to 0.1% or 0.1% to 0.2%.

10. A method for preparing crustacean feed, characterized in that, Includes the following steps: (1) Using the equal incremental method, the cholesterol or feed-acceptable ester of any one of claims 1-2 is mixed stepwise with other trace components in the formula to obtain compound premix I; (2) After the bulk raw materials are dry premixed, they are mixed evenly with the composite premix I to obtain composite premix II; (3) After mixing the fatty components, mix them evenly with the composite premix II to obtain composite premix III; (4) The composite premix III is processed into a finished product after conditioning, granulation and drying.

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