Procambarus clarkii parent feed and preparation method thereof

Abstract

The invention relates to the technical field of aquaculture, and discloses a procambarus clarkii parent feed and a preparation method thereof. According to the procambarus clarkii parent feed provided by the invention, by systematically optimizing the amino acid composition mode of the feed and reconstructing the proportion of key functional amino acids such as methionine, lysine and the like, the precise regulation and control and human intervention on the parent gonad development process are realized, the parent gonad development tends to be synchronous, the parent mature period is concentrated, and the procambarus clarkii parent feed can be used for breeding procambarus clarkii. The technical problems that in a traditional breeding mode, individual development is not synchronous, and the breeding cycle is scattered are effectively solved. After the female juvenile shrimps are fed with the feed for 80 days, the gonad development synchronism is remarkably improved, the follicle proportion, the maturation coefficient and the gonad weight in the maturation period are remarkably increased, the egg carrying rate can reach 95% or above, the single-tail seedling emergence amount exceeds 330, the parent reproductive performance is remarkably improved, and the survival rate of the female juvenile shrimps is remarkably increased. And a stable and high-quality parent guarantee is provided for facility, factory and large-scale artificial breeding of the procambarus clarkii water spray seedlings.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a broodstock feed for Procambarus clarkii and its preparation method. Background Technology

[0002] Red swamp crayfish (Procambarus clarkii) Procambarus clarkii As an important aquaculture species in my country, the scale of its cultivation has continued to expand, resulting in significant industrial benefits. However, with the advancement of intensive and large-scale farming models, problems such as declining broodstock reproductive performance, unstable seedling supply, and insufficient supply security have become increasingly prominent, becoming key obstacles restricting the healthy development of the industry. The "self-breeding and self-raising" model upon which the production of *Procambarus clarkii* seedlings relies, lacking systematic breeding and nutritional regulation, will further exacerbate these problems. Particularly noteworthy is the significant individual asynchrony in gonadal development among broodstock, leading to dispersed reproductive cycles and a wide range of maturity periods. This makes it impossible to provide a large quantity of mature broodstock of suitable age for factory breeding, resulting in low seedling production efficiency, inconsistent sizes, and difficulty in meeting the seedling demands of large-scale farming.

[0003] Promoting the facility-based, factory-style, and standardized breeding of swamp crayfish larvae is a crucial measure to solidify the foundation of the industry's development. The core advantage of facility-based, factory-style breeding lies in its ability to standardize and scale up the production process under controllable conditions, thereby ensuring a stable supply of larvae. However, in factory-style breeding practices, the parent stock cultivation stage still faces key technological bottlenecks: how to achieve controlled and synchronized intervention in parent gonadal development through technological means, enabling parents to reach gonadal maturity within a specific timeframe, has become a critical issue for improving the stability of larval quality and ensuring the supply of breeding stock.

[0004] In the process of broodstock breeding, nutritional fortification is the core approach to regulating reproductive performance. However, research on specialized feeds for the critical stages of gonadal development in prespawning swamp crayfish is still in its early stages. On the one hand, existing technologies mostly follow or slightly modify conventional growth feeds, or generally adopt a simple approach of increasing the total crude protein content of the feed to try to meet the nutritional needs of the broodstock. For example, some studies have shown that feeding feeds with protein levels as high as 40%-45% can improve the relative reproductive capacity of the broodstock, but this high-protein strategy not only significantly increases the cost of feed ingredients, but also easily leads to excessive nitrogen emissions in the form of ammonia nitrogen, which damages the breeding environment of the broodstock. On the other hand, most related studies focus on adding single-category functional substances, such as using arachidonic acid (ARA) to promote lipid metabolism, using methylfarnesyl ester (MF) to regulate endocrine function, or exploring the specific effects of single amino acids (such as histidine and threonine). Although these approaches can achieve certain effects in specific aspects, they do not address the essential nutritional needs of gonadal development to achieve precise regulation and synchronous induction of the gonadal development process of the broodstock, and their application effects are limited and unstable.

[0005] In addition, in the breeding of crustacean aquatic animals, some species (such as Litopenaeus vannamei) have long relied on live feed. This method has problems such as high cost and high biosafety risks. This indicates that it is particularly important to develop special artificial compound feed that is precisely adapted to the needs of industrialized breeding and realizes the synchronous development of the gonads of the parent animals in the breeding of aquatic animals.

[0006] Therefore, there is an urgent need to develop a special feed for broodstock that meets the needs of industrialized artificial breeding of Procambarus clarkii. Through precise nutritional pattern design, the development of broodstock gonads can be synchronized and controlled, providing a large number of broodstock with consistent maturity for industrialized artificial breeding. This will break through the technical bottlenecks of existing breeding models, promote the standardization and industrialization of Procambarus clarkii seedling production, and provide core technical support for the high-quality development of the industry. Summary of the Invention

[0007] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a broodstock feed for the red swamp crayfish.

[0008] The second objective of this invention is to provide a method for preparing this broodstock feed for Procambarus clarkii.

[0009] The third objective of this invention is to provide the application of this Procambarus clarkii parent feed.

[0010] The fourth objective of this invention is to provide another application for this Procambarus clarkii parent feed.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a broodstock feed for Procambarus clarkii, wherein the amino acid composition of the feed, on a dry weight basis, satisfies the following parameters: ∑TAA=32.0%-33.5%; ∑EAA=14.5%-16.0%; ∑EAA / ∑NEAA=0.83-0.91; Met=0.90%-1.45%; Lys=2.35%-2.90%; Met+Lys=3.30%-4.30%; Met / Lys=0.38-0.50; (Met+Lys) / ∑TAA=0.10-0.15; (Met+Lys) / ∑EAA=0.22-0.31; ∑GAA=10.50%-11.50%; ∑GAA / ∑TAA=0.33-0.37; Wherein, ∑TAA is the total amino acid content; ∑EAA is the essential amino acid content; ∑NEAA is the non-essential amino acid content; Met is the methionine content; Lys is the lysine content; and ∑GAA is the total content of seven gonadal development-related amino acids, namely methionine, lysine, arginine, threonine, histidine, leucine, and tryptophan.

[0012] In some embodiments of the present invention, the amino acid composition pattern of the feed satisfies the following parameters: ∑TAA=32.5%-33.5%; ∑EAA=15.1%-16.0%; ∑EAA / ∑NEAA=0.86-0.90; Met=1.10%-1.32%; Lys=2.50%-2.81%; Met+Lys=3.50%-4.12%; Met / Lys=0.40-0.48; (Met+Lys) / ∑TAA=0.10-0.12; (Met+Lys) / ∑EAA=0.23-0.26; ∑GAA=10.70%-11.50%; ∑GAA / ∑TAA=0.33-0.35.

[0013] In some embodiments of the present invention, the feed has a crude protein content of 37.5%-40.5% and a crude fat content of 6.0%-6.5% on a dry weight basis.

[0014] In some preferred embodiments of the present invention, the feed has a crude protein content of 38.5%-40.0% and a crude fat content of 6.0%-6.4% by dry weight.

[0015] In some embodiments of the present invention, the feed comprises, by weight percentage: 40%-56% plant protein source, 10%-18% animal protein source, 22%-33% starch source, 3%-5% fat source, 0.4%-1.0% crystalline DL-methionine, and 0.7%-1.6% crystalline L-lysine hydrochloride.

[0016] In some preferred embodiments of the present invention, the feed comprises the following components by weight percentage: 44%-54% plant protein source, 13%-17% animal protein source, 23%-28% starch source, 3.5%-4.5% fat source, 0.6%-0.8% crystalline DL-methionine, and 1.0%-1.3% crystalline L-lysine hydrochloride.

[0017] In some embodiments of the present invention, the composition of the plant protein source, based on feed dry weight, is: 18%-25% soybean meal, 15%-20% rapeseed meal, 3%-5% dephenolized cottonseed meal, and 4%-6% soybean protein concentrate.

[0018] In some preferred embodiments of the present invention, the composition of the plant protein source, based on feed dry weight, is: 20%-24% soybean meal, 16%-20% rapeseed meal, 3.5%-4.5% dephenolized cottonseed meal, and 4.5%-5.5% soybean protein concentrate.

[0019] In some embodiments of the present invention, the particle size of the soybean meal, rapeseed meal, and dephenolized cottonseed meal is less than or equal to 60 mesh.

[0020] In some embodiments of the present invention, the animal protein source includes chicken meal.

[0021] In some embodiments of the present invention, the particle size of the animal protein source is less than or equal to 60 mesh.

[0022] In some embodiments of the present invention, the starch source includes high-gluten flour.

[0023] In some embodiments of the present invention, the fat source includes soybean oil.

[0024] In some embodiments of the present invention, the feed further comprises, by weight percentage: 0.8%-1.2% vitamin premix, 1.6%-2.4% mineral premix, 0.008%-0.012% antioxidant, 0.024%-0.036% mold inhibitor, 0.08%-0.12% palatability enhancer, and 0.16%-0.24% choline.

[0025] In some preferred embodiments of the present invention, the feed further comprises, by weight percentage: 0.9%-1.1% vitamin premix, 1.8%-2.2% mineral premix, 0.009%-0.011% antioxidant, 0.027%-0.033% mold inhibitor, 0.09%-0.11% palatability enhancer, and 0.18%-0.22% choline.

[0026] In some embodiments of the present invention, the feed further includes 0.8%-1.2% of an amino acid coating carrier based on the dry weight of the feed; the amino acid coating carrier includes carboxymethyl cellulose (CMC).

[0027] In some preferred embodiments of the present invention, the feed further includes 0.9%-1.1% amino acid coating carrier based on the dry weight of the feed.

[0028] In some embodiments of the present invention, the particle size of the broodstock feed for Procambarus clarkii is 1-3 mm.

[0029] The second aspect of the present invention provides a method for preparing the broodstock feed for *Procambarus clarkii* as described in the first aspect of the present invention, comprising the following steps: S1. The vitamin premix, mineral premix, antioxidant, antifungal agent, palatability enhancer and choline are initially mixed to obtain a small mixture; the small mixture is then mixed with plant protein source, animal protein source and starch source in a secondary process to obtain a main mixture; S2. Crystalline DL-methionine and crystallized L-lysine hydrochloride are mixed with a fat source to obtain an amino acid-oil mixture; the main mixture, the amino acid-oil mixture, and the amino acid coating carrier are dry-mixed to obtain a mixed dry material. S3. Add water to the mixed dry material, stir until plastic, granulate and dry to obtain the Procambarus clarkii parent feed; In step S3, the amount of water added is 5%-15% of the mass of the mixed dry material.

[0030] In some embodiments of the present invention, step S2, after the main mixture, the amino acid-oil mixture and the amino acid coating carrier are dry-mixed, also includes a sieving operation.

[0031] In some embodiments of the present invention, in step S3, the amount of water added is 8%-12% of the mass of the mixed dry material.

[0032] Specifically, crystalline DL-methionine and crystalline L-lysine hydrochloride are water-soluble amino acids. During feed processing (including granulation and water addition) and feeding into the aquaculture water, they are easily lost from the feed due to water solubility. This significantly reduces the amount of amino acids actually consumed and utilized by the broodstock of *Procambarus clarkii*, failing to ensure the precise implementation of the designed amino acid composition pattern in the feed and consequently affecting the regulation of gonadal development in the broodstock. This invention addresses the problem of water loss of crystalline amino acids by designing a dual protection mechanism based on a step-by-step mixing preparation process. It utilizes an amino acid coating carrier to achieve efficient fixation and retention of crystalline amino acids. First, crystalline DL-methionine and crystalline L-lysine hydrochloride are premixed with a fat source to form an amino acid-oil mixture. The hydrophobicity of the fat source forms an oil-isolation film on the surface of the crystalline amino acids, achieving primary hydrophobic encapsulation and initially preventing direct contact between water and the crystalline amino acids, thus reducing dissolution during subsequent granulation with added water. Then, this amino acid-oil mixture is dry-mixed with the main mixture and the amino acid coating carrier. During this process, the amino acid coating carrier selectively adsorbs onto the surface of the amino acid-oil mixture in micropowder form and disperses in the main mixture and the amino acid-oil mixture. In the contact gaps of the lipid mixture, during granulation with added water and stirring, the amino acid-coated carrier rapidly dissolves and hydrates upon contact with water, forming a high-viscosity microgel layer. This microgel layer tightly coats the outer layer of the amino acid-lipid mixture particles, forming a double-layer targeted protection structure with the inner lipid film. This firmly fixes the crystalline amino acids within the microstructure composed of the microgel and the lipid film. Simultaneously, a small amount of the amino acid-coated carrier forms a gel micronetwork in the gaps inside the feed particles, further anchoring the protected crystalline amino acid particles in the feed matrix, preventing them from falling out of the feed or dissolving in water during granulation, transportation, and feeding.

[0033] The third aspect of the present invention provides the application of the broodstock feed for Procambarus clarkii described in the first aspect of the present invention in the breeding of broodstock of Procambarus clarkii.

[0034] In some embodiments of the present invention, the parent crayfish is a female juvenile crayfish with an initial weight of 7-13g.

[0035] In some embodiments of the present invention, the application includes the phased feeding of broodstock feed for red swamp crayfish, completely replacing conventional commercial feed.

[0036] In some embodiments of the present invention, the cultivation period is 60-100 days.

[0037] In some preferred embodiments of the present invention, the cultivation period is 70-90 days.

[0038] The fourth aspect of the present invention provides the application of the broodstock feed for *Procambarus clarkii* described in the first aspect of the present invention in the preparation of products that promote the synchronization of gonadal development in broodstock *Procambarus clarkii*.

[0039] Compared with the prior art, the beneficial effects of the present invention are: The broodstock feed for Procambarus clarkii provided by this invention achieves precise regulation and artificial intervention in the gonadal development process of broodstock by systematically optimizing the amino acid composition pattern and reconstructing the ratio of key functional amino acids such as methionine and lysine. This promotes the synchronization of gonadal development and concentrates the maturation period of broodstock, effectively solving the technical problems of asynchronous gonadal development and dispersed reproductive cycles in traditional breeding models. After feeding female juvenile shrimp with this feed for 80 days, the synchronicity of gonadal development is significantly improved, the proportion of mature follicles, the maturity coefficient, and gonadal weight are all significantly increased, the egg-carrying rate can reach over 95%, and the number of larvae per shrimp exceeds 330. The reproductive performance of the broodstock is significantly improved, providing a stable and high-quality broodstock guarantee for the facility-based, factory-based, and large-scale artificial breeding of Procambarus clarkii larvae. Attached Figure Description

[0040] Figure 1 HE staining comparison of ovarian tissues of Procambarus clarkii after 40 days of feeding with different parent diets in the application example; Figure 2 HE staining comparison of ovarian tissues of Procambarus clarkii after 80 days of feeding with different broodstock diets in the application example; Figure 3 The effects of different broodstock diets of Procambarus clarkii on the follicle morphology and developmental stage of Procambarus clarkii in the application examples; Figure 4 The maturity coefficient (a) and gonad weight (b) of Procambarus clarkii fed with different parent diets in the application example are shown. Detailed Implementation

[0041] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0042] Example 1 This embodiment prepares a broodstock feed for Procambarus clarkii, the components of which are shown in Table 1: Table 1. Components and content of the feed for broodstock crayfish in Example 1

[0043] The steps for preparing broodstock feed for Procambarus clarkii are as follows: S11. The vitamin premix, mineral premix, antioxidant, antifungal agent, palatability enhancer and choline are initially mixed to obtain a small mixture; the small mixture is then mixed with 60-mesh soybean meal, 60-mesh rapeseed meal, 60-mesh dephenolized cottonseed meal, soybean protein concentrate, 60-mesh chicken powder and high-gluten flour in a secondary process to obtain the main mixture. S21. Mix crystalline DL-methionine and crystalline L-lysine hydrochloride with soybean oil to obtain an amino acid-oil mixture; dry mix the main mixture, the amino acid-oil mixture, and carboxymethyl cellulose, and sieve to obtain a mixed dry material; S31. Add 10wt% water to the mixed dry feed and stir until it becomes plastic. Use a feed forming machine to form the feed into pellets of about 2mm in size. Place them in a cool place to air dry to obtain the broodstock feed for Procambarus clarkii. Seal and store at -20℃ until used.

[0044] Example 2 This embodiment prepares a broodstock feed for Procambarus clarkii, using the same preparation method as in Example 1. The components are shown in Table 2. Table 2. Components and content of the feed for the broodstock of Procambarus clarkii in Example 2

[0045] Example 3 This embodiment prepares a broodstock feed for Procambarus clarkii, using the same preparation method as in Example 1. The components are shown in Table 3. Table 3. Components and content of the feed for the broodstock crayfish in Example 3

[0046] Example 4 This embodiment prepares a broodstock feed for Procambarus clarkii, using the same preparation method as in Example 1. The components are shown in Table 4. Table 4. Components and content of the feed for the broodstock of Procambarus clarkii in Example 4

[0047] Comparative Example 1 A comparative feed for broodstock of Procambarus clarkii was prepared, and its components are shown in Table 5. Table 5. Components and content of the feed for the broodstock of Procambarus clarkii in Comparative Example 1

[0048] The steps for preparing broodstock feed for Procambarus clarkii are as follows: S11. The vitamin premix, mineral premix, antioxidant, antifungal agent, palatability enhancer and choline are initially mixed to obtain a small mixture; the small mixture is then mixed with 60-mesh soybean meal, 60-mesh rapeseed meal, 60-mesh dephenolized cottonseed meal, soybean protein concentrate, 60-mesh chicken powder and high-gluten flour in a secondary process to obtain the main mixture. S31. Add soybean oil and 10wt% water to the main mixed dry feed, stir thoroughly into a paste, use a feed forming machine to form the feed into pellets of about 2mm in size, place in a cool place to air dry, and obtain the broodstock feed for Procambarus clarkii. Seal and store at -20℃ until used.

[0049] The conventional nutrient components and amino acid composition of the broodstock feeds for *Procambarus clarkii* prepared in Examples 1-4 and Comparative Example 1 were analyzed, and the results were as follows: The crude protein content was determined according to GB / T 6432-2018 "Determination of Crude Protein in Feed - Kjeldahl Method", using a fully automated Kjeldahl nitrogen analyzer. The crude fat content was determined by ether extraction, referring to GB / T 6433-2025 "Determination of Crude Fat in Feed"; The amino acid composition and content were determined in accordance with GB / T 18246-2019 "Determination of Amino Acids in Feed". Tryptophan was detected by alkaline hydrolysis, and the remaining amino acids were determined by an amino acid analyzer using hydrochloric acid hydrolysis.

[0050] Table 6. Conventional nutrient composition and amino acid composition of the feed for broodstock crayfish in Examples 1-4 and Comparative Example 1

[0051] Wherein, ∑TAA is the total amino acid content; ∑EAA is the essential amino acid content; ∑NEAA is the non-essential amino acid content; Met is the methionine content; Lys is the lysine content; and ∑GAA is the total content of seven gonadal development-related amino acids (methionine, lysine, arginine, threonine, histidine, leucine, and tryptophan).

[0052] Table 6 shows the conventional nutritional components and amino acid composition of the broodstock feed for *Procambarus clarkii* in Examples 1-4 and Comparative Example 1. As shown in Table 6, the crude protein (37.67%-40.49%) and crude fat (6.08%-6.50%) contents of the broodstock feed for *Procambarus clarkii* in Examples 1-4 and Comparative Example 1 are similar, but the amino acid patterns are different. The feeds in Examples 1-4 contain different amounts of methionine (Met), lysine (Lys), total methionine and lysine (Met+Lys), the ratio of methionine to lysine (Met / Lys), and the ratio of methionine and lysine to total amino acids ((Met+Lys) / ∑). TAA The ratio of methionine and lysine to essential amino acids ((Met+Lys) / ∑) EAA All of these indicators were higher than those in Comparative Example 1, and from Example 1 to Example 4, the above indicators all showed a gradual upward trend, indicating that the present invention has finely controlled the ratio of key functional amino acids in the broodstock feed of Procambarus clarkii.

[0053] Application examples The broodstock feeds for Procambarus clarkii prepared in Examples 1-4 and Comparative Example 1 were applied to the breeding of broodstock Procambarus clarkii, and the steps were as follows: Healthy female juvenile Procambarus clarkii with an initial weight of 10±3g were selected. After being domesticated for 15 days by feeding them the Procambarus clarkii broodstock feed prepared in Comparative Example 1, they were randomly divided into 5 groups and cultured in cement ponds with a slight flow of water and continuous oxygenation. The water source was filtered pond water. They were fed the Procambarus clarkii broodstock feed prepared in Examples 1-4 and Comparative Example 1, respectively, for broodstock cultivation. The cultivation period was 80 days, and each group was fed the corresponding feed at regular intervals every day.

[0054] 1. On days 40 and 80 after feeding different broodstock of Procambarus clarkii, after fasting for 24 hours, nine shrimp were randomly selected from each group. Gonadal tissue was collected and fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining to analyze the gonadal development of the broodstock of Procambarus clarkii in each group. The long and short diameters of 100 follicles in each group were measured and the follicles were classified into stages. Figure 1 HE staining comparison of ovarian tissues of Procambarus clarkii after 40 days of feeding with different parent diets in the application example; Figure 2 HE staining comparison of ovarian tissues of Procambarus clarkii after 80 days of feeding with different broodstock diets in the application example; Figure 3 To illustrate the effects of different broodstock diets of *Procambarus clarkii* on the follicle morphology and developmental stages of *Procambarus clarkii* eggs in the application examples, the following were observed. Figure 3 (a) shows the long and short axes of the follicles of the red swamp crayfish after 40 days of feeding. Figure 3 (b) shows the proportion of follicles at each developmental stage after 40 days of feeding. Figure 3 (c) in the figure represents the long and short diameters of the follicles of the red swamp crayfish after 80 days of feeding. Figure 3 (d) represents the proportion of follicles at each developmental stage after 80 days of feeding; Table 7 shows the effects of different broodstock diets of *Procambarus clarkii* on the length and short diameter of *Procambarus clarkii* follicle groups in the application examples. Figure 1 , Figure 2 , Figure 3 As can be seen from the data in Table 7: After 40 days of feeding, in the group fed with the Procambarus clarkii broodstock diet of Example 3 (methionine 1.31 wt%, lysine 2.81 wt%), the ovaries of the Procambarus clarkii developed the fastest, with the longest and shortest follicle diameters, and stage III follicles accounted for 64%, indicating that this amino acid pattern can accelerate early gonadal development. After 80 days of feeding, compared with the 40-day feeding, the follicle size of Procambarus clarkii in all groups further increased. Among them, in the group fed with the Procambarus clarkii broodstock diet of Example 2, the ovaries of the Procambarus clarkii developed faster, with the longest and shortest follicle diameters being 1443.30±122.96 μm and 1308.11±110.59 μm, respectively, significantly. The groups fed with the feed for broodstock Procambarus clarkii in Example 2 (follicle length 925.44±63.06μm, short diameter 833.58±83.58μm) showed higher follicle development than those fed with the feed for Comparative Example 1 (follicle length 925.44±63.06μm, short diameter 833.58±83.58μm), and 3% of the follicles reached stage V (mature stage). In contrast, the follicles in the groups fed with the feed for Comparative Example 1 were still in stage III. Furthermore, the follicles in the groups fed with the feeds for Examples 3 and 4 reached stage IV, while only 36% of the follicles in the group fed with the feed for Example 1 reached stage IV. This indicates that among the different broodstock Procambarus clarkii feeds, the amino acid pattern of the feed for Example 2 (methionine 1.10wt%, lysine 2.60wt%) was the most effective in promoting final gonadal maturation.

[0055] Table 7. Effects of different broodstock diets of Procambarus clarkii on the long and short axes of Procambarus clarkii follicle colonies in application examples.

[0056] All data are expressed as mean ± standard deviation. At the same feeding time, there were significant differences in the long / short diameter of follicles with different lowercase letter markings. p <0.05.

[0057] 2. On days 40 and 80 of feeding different broodstock diets of Procambarus clarkii, after fasting for 24 hours, 9 shrimp were randomly selected from each group. The surface moisture of the shrimp was dried with absorbent paper, and their weight was measured using a precision electronic balance. Subsequently, gonadal tissue was collected, and after the surface body fluid was removed, the gonadal weight was measured using the same precision electronic balance, and the maturity coefficient was calculated. The formula is: Maturity coefficient = Gonadal weight / Body weight × 100%.

[0058] Figure 4 The maturity coefficient (a) and gonad weight (b) of *Procambarus clarkii* fed different parent diets in the application example are shown. Under the same comparison dimension, different lowercase letters represent significant differences. p <0.05), by Figure 4It can be seen that after 80 days of feeding, the maturity coefficient of the swamp crayfish fed with the broodstock feed of Example 2 was significantly higher than that of the groups fed with the broodstock feed of Example 1, Example 4 and Comparative Example 1, and the gonad weight was significantly higher than that of the group fed with the broodstock feed of Comparative Example 1. The biological indicators quantitatively confirmed that the present invention can significantly increase the gonad quality of broodstock crayfish by optimizing the amino acid pattern.

[0059] 3. On the 80th day of feeding different broodstock diets of Procambarus clarkii, after fasting for 24 hours, 9 shrimp were randomly selected from each group, euthanized, and the tail muscle from the intersegment of the second to sixth abdominal segments was collected for muscle amino acid determination. The muscle amino acid determination was carried out in accordance with GB / T 18246-2019 "Determination of Amino Acids in Feed". Tryptophan was detected by alkaline hydrolysis method, and the remaining amino acids were determined by hydrochloric acid hydrolysis method using an amino acid analyzer.

[0060] Table 8. Effects of different broodstock diets of Procambarus clarkii on the amino acid composition and content in the muscle of Procambarus clarkii in application examples.

[0061] Wherein, T1 is the total amino acid content; T2 is the total essential amino acid content; T3 is the total non-essential amino acid content; T4 is the total umami amino acid (glutamic acid, aspartic acid); and T5 is the total flavor amino acid (glutamic acid, aspartic acid, glycine, alanine, serine, proline, tyrosine). All data are expressed as mean ± standard deviation. Data with different lowercase letter notations in the same row indicate significant differences. p <0.05.

[0062] Table 8 shows the effects of different broodstock diets of *Procambarus clarkii* on the amino acid composition and content in *Procambarus clarkii* muscle in the application examples. As shown in Table 8, 17 amino acids were detected in all samples fed with different broodstock diets, including 9 essential amino acids and 8 non-essential amino acids. Compared with the samples fed with the diet of Comparative Example 1, the total amino acid content (T1) in the muscle of *Procambarus clarkii* fed with the diets of Examples 1-4 all showed a decreasing trend. Among them, the total amino acid content in the muscle of *Procambarus clarkii* fed with the diet of Example 4 was the lowest, while the total amino acid levels in the muscle of *Procambarus clarkii* fed with the diets of Examples 1 and 3 were relatively high. This suggests that the addition of a high proportion of exogenous amino acids may affect the accumulation of amino acids in the muscle. The diet exhibited an inhibitory effect; regarding essential amino acids, the total essential amino acid content (T2) in the muscle of *Procambarus clarkii* fed with the diet in Example 4 was the lowest. The methionine content in the muscle of *Procambarus clarkii* fed with the diets in Example 2 and Comparative Example 1 was similar. The methionine content in the muscle of *Procambarus clarkii* fed with the diet in Example 3 was slightly increased, while the methionine content in the muscle of *Procambarus clarkii* fed with the diet in Example 1 was significantly decreased. The lysine content in the muscle of *Procambarus clarkii* fed with the diets in Examples 1-4 was lower than that in the sample fed with the diet in Comparative Example 1, with the difference being particularly pronounced in the sample fed with the diet in Example 4. This suggests that an excessively high proportion of exogenous amino acids may be detrimental to the deposition of some key essential amino acids in the muscle. Nevertheless, feeding... In all groups of the same feed, the proportion of essential amino acids in the total amino acid content (T2 / T1) remained around 54%, and the ratio of essential amino acids to non-essential amino acids (T2 / T3) remained stable between 1.16 and 1.23, indicating that although different levels of addition affected the absolute content of amino acids, they did not significantly change the balance of their nutritional structure. Regarding non-essential amino acids, the sample fed with Comparative Example 1 had the highest total non-essential amino acid content, followed by the samples fed with Example 3, Example 1, and Example 2. The cystine content in the muscle of *Procambarus clarkii* fed with the feeds of Examples 1-4 was higher than that of the sample fed with Comparative Example 1, with the sample fed with Example 3 being particularly prominent. Umami amino acids (… The total amount of T4 was highest in the sample fed with Comparative Example 1, followed by the sample fed with Example 3. The total amount of flavor amino acids was highest in the sample fed with Comparative Example 1, followed by the samples fed with Example 3, Example 1, and Example 2, indicating that moderate levels of exogenous amino acid addition help maintain the potential flavor characteristics of the muscle. In terms of proportion structure, the ratio of essential amino acids to total amino acid content (T2 / T1) in the muscle of Procambarus clarkii fed with the diets of Example 1, Example 2, and Example 4 was higher than that in the sample fed with Comparative Example 1, while the ratio of umami amino acids to total amino acid content (T4 / T1) was still highest in the sample fed with the diet of Example 3, followed by the samples fed with the diets of Comparative Example 1 and Example 2.

[0063] In summary, the broodstock feeds for Procambarus clarkii in Examples 2 and 3 performed better in maintaining the overall nutritional and edible quality of Procambarus clarkii muscle. However, the excessively high ratio of crystalline DL-methionine and crystalline L-lysine hydrochloride in Example 4 may inhibit the deposition of most amino acids, which is not conducive to maintaining muscle quality.

[0064] 4. After 80 days of feeding, the parent female shrimp that were not sampled in each group were paired with healthy male shrimp in a 1:1 ratio. They were fed normally every day, and the water was circulated and oxygenated. When the female shrimp carried eggs, the male shrimp were removed and the female shrimp were raised separately. During this period, the number of egg-carrying shrimp was recorded, the egg-carrying rate was calculated, and the number of hatching shrimp per shrimp (stage III larvae) was counted. The egg-carrying rate = egg-carrying female shrimp / total number of female shrimp.

[0065] Table 9. Effects of different broodstock diets on the reproductive performance of Procambarus clarkii in application examples.

[0066] All data are expressed as mean ± standard deviation. Significant differences exist between brood rates / hatching rates indicated by different lowercase letters within the same row. p <0.05.

[0067] Table 9 shows the effects of different broodstock feeds on the reproductive performance of *Procambarus clarkii* in the application examples. As can be seen from Table 9, the egg-carrying rate of *Procambarus clarkii* fed with the broodstock feeds of Examples 1-4 was significantly higher than that of *Procambarus clarkii* fed with the feed of Comparative Example 1. The hatching rate of *Procambarus clarkii* fed with the feeds of Examples 2 and 3 was significantly higher than that of *Procambarus clarkii* fed with the feed of Comparative Example 1. Among them, the egg-carrying rate of *Procambarus clarkii* fed with the feed of Example 2 was as high as 95.83%, and the hatching rate was as high as 331 individuals. This indicates that the present invention has realized the transformation of nutritional regulation into quantifiable and excellent reproductive output, and significantly improved the egg-carrying rate and hatching rate of broodstock *Procambarus clarkii*.

[0068] This invention optimizes the amino acid composition of feed by scientifically increasing the levels and proportions of key amino acids such as methionine and lysine. This significantly promotes the synchronicity of gonadal development in broodstock Procambarus clarkii, shortens the pre-spawning rearing period, improves reproductive performance, and substantially increases egg-carrying rate and hatching volume, while reducing breeding costs. It should be noted that the broodstock feed for Procambarus clarkii provided by this invention is also applicable to other crustacean aquatic economic animals, or at least can provide a reference solution for the precise nutritional regulation of their broodstock.

Claims

1. A broodstock feed for Procambarus clarkii, characterized in that, On a dry weight basis, the amino acid composition pattern of the feed satisfies the following parameters: ∑TAA=32.0%-33.5%; ∑EAA=14.5%-16.0%; ∑EAA / ∑NEAA=0.83-0.91; Met=0.90%-1.45%; Lys=2.35%-2.90%; Met+Lys=3.30%-4.30%; Met / Lys=0.38-0.50; (Met+Lys) / ∑TAA=0.10-0.15; (Met+Lys) / ∑EAA=0.22-0.31; ∑GAA=10.50%-11.50%; ∑GAA / ∑TAA=0.33-0.37; Wherein, ∑TAA is the total amino acid content; ∑EAA is the essential amino acid content; ∑NEAA is the non-essential amino acid content; Met is the methionine content; Lys is the lysine content; and ∑GAA is the total content of seven gonadal development-related amino acids, namely methionine, lysine, arginine, threonine, histidine, leucine, and tryptophan.

2. The broodstock feed for Procambarus clarkii according to claim 1, characterized in that, On a dry weight basis, the feed contains 37.5%-40.5% crude protein and 6.0%-6.5% crude fat.

3. The broodstock feed for Procambarus clarkii according to claim 2, characterized in that, The feed comprises the following components by weight percentage: 40%-56% plant protein source, 10%-18% animal protein source, 22%-33% starch source, 3%-5% fat source, 0.4%-1.0% crystalline DL-methionine, and 0.7%-1.6% crystalline L-lysine hydrochloride.

4. The broodstock feed for Procambarus clarkii according to claim 3, characterized in that, Based on feed dry weight, the composition of the plant protein source is: 18%-25% soybean meal, 15%-20% rapeseed meal, 3%-5% dephenolized cottonseed meal, and 4%-6% soybean protein concentrate.

5. The broodstock feed for Procambarus clarkii according to claim 3, characterized in that, The animal protein source includes chicken meal.

6. The broodstock feed for Procambarus clarkii according to claim 3, characterized in that, The feed, by weight percentage, also includes the following components: 0.8%-1.2% vitamin premix, 1.6%-2.4% mineral premix, 0.008%-0.012% antioxidant, 0.024%-0.036% mold inhibitor, 0.08%-0.12% palatability enhancer, and 0.16%-0.24% choline.

7. The broodstock feed for Procambarus clarkii according to any one of claims 3-6, characterized in that, The feed, based on dry weight, also includes 0.8%-1.2% of an amino acid coating carrier; the amino acid coating carrier includes carboxymethyl cellulose.

8. The method for preparing the broodstock feed for Procambarus clarkii according to claim 7, characterized in that, Includes the following steps: S1. The vitamin premix, mineral premix, antioxidant, antifungal agent, palatability enhancer and choline are initially mixed to obtain a small mixture; the small mixture is then mixed with plant protein source, animal protein source and starch source in a secondary process to obtain a main mixture; S2. Crystalline DL-methionine and crystallized L-lysine hydrochloride are mixed with a fat source to obtain an amino acid-oil mixture; the main mixture, the amino acid-oil mixture, and the amino acid coating carrier are dry-mixed to obtain a mixed dry material. S3. Add water to the mixed dry material, stir until plastic, granulate and dry to obtain the Procambarus clarkii parent feed; In step S3, the amount of water added is 5%-15% of the mass of the mixed dry material.

9. The application of the broodstock feed for Procambarus clarkii according to any one of claims 1-7 in the breeding of broodstock of Procambarus clarkii.

10. The use of the Procambarus clarkii parent feed according to any one of claims 1-7 in the preparation of products that promote the synchronization of gonadal development in Procambarus clarkii parents.

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