A method for promoting ovary development of pelteobagrus vachlii

Abstract

The application discloses a method for promoting ovary development of Pelteobagrus vishnui, belongs to the technical field of fish culture, and feeds a functional additive during an ovary development preparation period and a strengthening period in the process of culturing Pelteobagrus vishnui, wherein the functional additive is a microcapsule prepared by mixing epigallocatechin gallate and a composite unsaturated fatty acid, embedding the inside layer by using beta-cyclodextrin, and coating the outside layer by using chitosan; and the composite unsaturated fatty acid is composed of monounsaturated fatty acid and polyunsaturated fatty acid at a volume ratio of 0.8-1:1-1.2. In the application, the EGCG and the composite unsaturated fatty acid are compounded, and then embedded to prepare the microcapsule, which is added into feed for feeding, so that the ovary development of Pelteobagrus vishnui is effectively promoted, the number of oocytes is increased, the proportion of III stage oocytes is increased, the fecundity of Pelteobagrus vishnui is effectively improved, and the spawning quantity, fertilization rate and hatching rate are increased.

Description

Technical Field

[0001] This invention relates to the field of fish breeding technology, specifically to a method for promoting ovarian development in yellow catfish. Background Technology

[0002] Yellow catfish (Pelteobagrus vachelli) Pelteobagrus vachellii The yellow catfish (Pelteobagrus vachelli) is an important freshwater economic fish in my country, characterized by its tender flesh, rich nutrition, and high market demand. In recent years, with the rapid development of large-scale aquaculture of this species, artificial breeding technology has become a key bottleneck restricting industrial upgrading, mainly focusing on two major problems: poor ovarian development, leading to low reproductive efficiency and degraded offspring quality. Due to frequent high temperatures, the ovarian development of juvenile yellow catfish is affected by high water temperatures, resulting in slow or even stagnant development, often exhibiting poor developmental synchronicity, low oocyte maturation rate, insufficient egg production, and decreased egg quality. While existing control methods, such as inducing ovulation with chemical hormones (e.g., luteinizing hormone-releasing hormone analogs), can promote ovarian development in the short term, improper long-term use of exogenous hormones can accelerate ovarian development disorders, inhibit the HPG axis feedback in parent fish, and degrade their own hormone secretion capacity, resulting in "false maturity" and preventing normal embryonic development after fertilization. Furthermore, there are potential risks such as drug residues, impacts on fish health, and food safety.

[0003] Unsaturated fatty acids are essential substances involved in biological metabolic processes. Insufficient intake of exogenous unsaturated fatty acids can lead to slow growth and increased mortality in fish. Unsaturated fatty acids also play a crucial role in fish reproductive capacity. Polyunsaturated fatty acids (PUFAs) are core components of phospholipids and precursors to steroid hormones. Adding unsaturated fatty acids to feed can promote estradiol secretion, thereby increasing spawning volume and hatching rate.

[0004] However, in practical applications, it has been found that the promoting efficiency of PUFA is limited, and when it is added directly to feed, PUFA is easily oxidized, producing MDA that damages oocytes. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a method for promoting ovarian development in yellow catfish. This method involves adding functional components composed of epigallocatechin gallate (EGCG) and complex unsaturated fatty acids to the basic aquaculture feed. This effectively solves the problems of easy oxidation of the active ingredient PUFA and the fact that high concentrations of catechins can disrupt the body's oxidation-antioxidant balance, reduce the fish's absorption efficiency of nutrients, and inhibit the growth of yellow catfish.

[0006] Another objective of this invention is to provide a functional additive that promotes the development of fish ovarian cells.

[0007] A third objective of this invention is to provide a method for preparing the aforementioned functional additives.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for promoting ovarian development in yellow catfish (Pelteobagrus vachelli), characterized in that: during the breeding of yellow catfish, functional additives are fed during the preparatory and intensive stages of ovarian development. The functional additives are prepared by mixing epigallocatechin gallate and complex unsaturated fatty acids as functional components, first using β-cyclodextrin (β-CD) for inner layer encapsulation, and then using chitosan for outer layer encapsulation to prepare microcapsules.

[0010] Furthermore, the mass ratio of EGCG to complex unsaturated fatty acids in the functional components is 0.15~0.2:8~10.

[0011] Furthermore, complex unsaturated fatty acids are composed of monounsaturated fatty acids and polyunsaturated fatty acids in a volume ratio of 0.8~1:1~1.2.

[0012] Furthermore, in the complex unsaturated fatty acids, the monounsaturated fatty acids are selected from oleic acid, palmitic acid, or erucic acid, and the polyunsaturated fatty acids are selected from linoleic acid, α-linolenic acid, or γ-linolenic acid.

[0013] Furthermore, the staged feeding involves mixing the microcapsules with the basic aquaculture feed at a mass ratio of 5-8%, feeding them twice daily during the initial ovarian development preparation period (days 1-28) and four times daily during the ovarian development enhancement period (days 29-56).

[0014] Furthermore, the feeding phases are as follows: during the preparatory stage of ovarian development, the fish are fed twice a day at 8:00 and 20:00, with a daily feed amount of 4-6% of their body weight; during the intensive stage of ovarian development, the fish are fed twice a day at 8:00, 12:00, 17:00, and 22:00, with a daily feed amount of 4-6% of their body weight, of which the feed at 22:00 accounts for 35% of the total daily feed amount.

[0015] During the breeding period, dissolved oxygen should be ≥5mg / L and pH should be 7.2-7.5.

[0016] Furthermore, the inner layer encapsulation is prepared by mixing EGCG solution and complex unsaturated fatty acids, then adding soybean lecithin to prepare an emulsion. The emulsion is then added to a β-CD aqueous solution, stirred, and allowed to stand at 4-6°C for 12-16 hours. The precipitate is then collected by centrifugation and dried to obtain a solid complex.

[0017] Furthermore, the ratio of soybean lecithin, complex unsaturated fatty acids, EGCG solution, and β-CD aqueous solution is 0.4~0.5g:8~12g:15~20mL:100mL, the concentration of β-CD aqueous solution is 50~60mg / L, and the concentration of EGCG solution is 12~16mg / L.

[0018] Furthermore, the outer coating is prepared by mixing chitosan solution and pectin solution, adding solid complex, ultrasonically dispersing, adding glycerol, stirring continuously to obtain a suspension, and finally adding sodium tripolyphosphate (TPP) solution for cross-linking reaction.

[0019] When PUFA is used as a functional component, its effect on promoting ovarian development in yellow catfish is not ideal, and direct addition will cause oxidation, generating MDA that damages oocytes.

[0020] In this invention, a composite unsaturated fatty acid composed of monounsaturated and polyunsaturated fatty acids is used, and epigallocatechin gallate (EGCG) is added to form a functional core material. EGCG protects the unsaturated fatty acids from oxidation, while its estrogen-like activity accelerates the conversion of unsaturated fatty acids into estrogen, forming a protective-promoting synergistic effect with the lipid system.

[0021] However, the above-mentioned components have the following problems that need to be addressed during use: Firstly, excessively high initial concentrations of epigallocatechin gallate (EGCG) in fish can inhibit the activity of lipases and proteases, hindering the decomposition and absorption of proteins and lipids in feed and suppressing fish growth. Secondly, EGCG is metabolized rapidly in fish, while the metabolism of unsaturated fatty acids in fish is a long-term process. This leads to asynchronous action between EGCG and unsaturated fatty acids, resulting in an unsatisfactory synergistic effect.

[0022] In this invention, the functional components undergo a special encapsulation process. First, β-cyclodextrin is used for molecular anchoring to prevent direct contact between fatty acids and chitosan, which could lead to emulsification and stratification. The inner β-CD hydrophobic cavity is embedded, with the hydrophobic chains of fatty acids embedded in the cavity. The hydrophobic groups of EGCG are bonded to the cavity through van der Waals forces. Then, the outer chitosan layer is encapsulated, with pectin added during this process. Pectin and chitosan form a composite gel network through polyelectrolyte complexation, reducing the large porosity of the single chitosan layer. Glycerol is embedded in the network, reducing network rigidity, increasing the fluidity of molecular chains, and enhancing the flexibility of the encapsulation structure. This allows for precise control of the release process, altering the release kinetics of EGCG, enabling its slow release and sustained effect in fish. It inhibits the initial formation of high concentrations of EGCG and maintains a stable low concentration in the intestine, avoiding the inhibitory effect of high concentrations while maintaining a sustained synergistic effect. Simultaneously, it reduces the damage to the intestine caused by fatty acid oxidation products (such as MDA), achieving synchronization of EGCG and unsaturated fatty acid release and enhancing their synergistic effect on promoting ovarian development.

[0023] Furthermore, the pectin solution is prepared by dissolving pectin in deionized water to form a solution with a mass ratio of 1~1.5%, and then gelatinizing it at 50~60℃ for 15~25 minutes.

[0024] Furthermore, the chitosan solution is prepared by adding chitosan to an aqueous solution of glacial acetic acid, stirring at 50-60°C until dissolved, and adjusting the pH to 5.2-5.5 to form a chitosan solution with a mass concentration of 1.5-2%.

[0025] Furthermore, the mass ratio of the solid complex, chitosan solution, pectin solution, glycerol and TPP solution is 1g:50~60mL:15~25mL:0.8~0.9mL:100mL, and the mass concentration of the TPP solution is 0.8~1.2%.

[0026] A method for promoting ovarian development in the yellow catfish (Pelteobagrus vachelli), characterized by comprising the following steps:

[0027] S1. Functional component pretreatment

[0028] Add EGCG to an ethanol solution with a volume concentration of 50-60% at 40-50℃. After complete dissolution, adjust the pH to 5.0-5.5 to prepare an EGCG solution with a concentration of 12-16 mg / L.

[0029] The functional component is obtained by mixing EGCG solution and complex unsaturated fatty acids. The mass ratio of EGCG to complex unsaturated fatty acids is 0.15~0.2:8~10. The complex unsaturated fatty acids are composed of monounsaturated fatty acids and polyunsaturated fatty acids in a mass ratio of 0.8~1:1~1.2. Among the complex unsaturated fatty acids, the monounsaturated fatty acids are selected from oleic acid, palmitic acid, or erucic acid, and the polyunsaturated fatty acids are selected from linoleic acid, α-linolenic acid, or γ-linolenic acid.

[0030] S2. Microcapsule preparation

[0031] (1) Dissolve β-CD in deionized water, adjust the pH to 6~6.5, and prepare a β-CD aqueous solution of 50~60mg / L. Mix the functional component and soybean lecithin solution, stir and homogenize at 5000~6000rpm for 3~5min to obtain an emulsion. Add the emulsion dropwise to the β-CD aqueous solution, stir at 500~600rpm for 2~3h, let stand at 4~6℃ for 12~16h, then centrifuge at 4000~5000rpm for 12~15min, collect the precipitate, and dry to obtain a solid complex. The ratio of soybean lecithin, EGCG solution in the functional component and β-CD aqueous solution is 0.4~0.5g: 15~20mL: 100mL.

[0032] (2) Chitosan (degree of deacetylation ≥ 85%) is added to an aqueous solution of glacial acetic acid and stirred at 50-60℃ until dissolved. The pH is adjusted to 5.2-5.5 to form a chitosan solution with a mass concentration of 1.5-2%. The solid complex is added to the mixture of chitosan solution and pectin solution and dispersed by ultrasonication. Glycerol is then added and stirring is continued to obtain a suspension. Finally, sodium tripolyphosphate (TPP) is added and stirred at 80-100 rpm for 20-30 min for crosslinking. The mixture is then centrifuged at 3500-4000 rpm for 10-15 min, washed with deionized water, and dried to obtain microcapsules. The mass ratio of the solid complex, chitosan solution, pectin solution, glycerol, and TPP solution is 1g:50-60mL:15-25mL:0.8-0.9mL:100mL. The mass concentration of the TPP solution is 0.8-1.2%.

[0033] S3. Stage Feeding

[0034] Microcapsules were mixed with basic aquaculture feed at a ratio of 5-8% by weight to form functional feed. Healthy juvenile yellow catfish with a body length of 4-6 cm and a weight of 3-4 g were randomly grouped and placed in outdoor cement ponds. The functional feed was then fed according to the following strategy:

[0035] During the initial 1-28 day ovarian development preparation period: feed twice daily at 8:00 and 20:00, with a daily feed amount of 4-6% of the fish's body weight;

[0036] During the ovarian development enhancement period of 29-56 days: feed once each at 8:00, 12:00, 17:00 and 22:00 daily, with the daily feed amount being 4-6% of the fish's body weight, of which the feed at 22:00 accounts for 35% of the total daily feed amount;

[0037] During the breeding period, dissolved oxygen should be ≥5mg / L and pH should be 7.2-7.5.

[0038] A functional additive for fish ovarian cell development, characterized in that: the functional additive is composed of a mixture of epigallocatechin gallate (EGCG) and complex unsaturated fatty acids, which are first encapsulated in the inner layer with β-cyclodextrin and then coated in the outer layer with chitosan, wherein the complex unsaturated fatty acids are composed of monounsaturated fatty acids and polyunsaturated fatty acids in a volume ratio of 0.8~1:1~1.2.

[0039] Furthermore, the mass ratio of EGCG to complex unsaturated fatty acids in the functional components is 0.15~0.2:8~10.

[0040] Furthermore, in the complex unsaturated fatty acids, the monounsaturated fatty acids are selected from oleic acid, palmitic acid, or erucic acid, and the polyunsaturated fatty acids are selected from linoleic acid, α-linolenic acid, or γ-linolenic acid.

[0041] The aforementioned functional additives are made by forming functional components into microcapsules. The specific preparation method is to first use β-cyclodextrin for inner layer encapsulation, and then use chitosan for outer layer coating.

[0042] Furthermore, the inner layer encapsulation is prepared by mixing EGCG solution and complex unsaturated fatty acids, then adding soybean lecithin to prepare an emulsion. The emulsion is then added to a β-CD aqueous solution, stirred, and allowed to stand at 4-6°C for 12-16 hours. The precipitate is then collected by centrifugation and dried to obtain a solid complex.

[0043] Furthermore, the ratio of soybean lecithin, EGCG solution, and β-CD aqueous solution is 0.4~0.5g:15~20mL:100mL, the concentration of β-CD aqueous solution is 50~60mg / L, and the concentration of EGCG solution is 12~16mg / L.

[0044] More specifically, EGCG solution and complex unsaturated fatty acids are mixed, and soybean lecithin is added. The mixture is stirred and homogenized at 5000-6000 rpm for 3-5 min to obtain an emulsion. The emulsion is added dropwise to β-CD aqueous solution and stirred at 500-600 rpm for 2-3 h. The mixture is then allowed to stand at 4-6℃ for 12-16 h, and then centrifuged at 4000-5000 rpm for 12-15 min. The precipitate is collected and dried to obtain a solid complex.

[0045] Furthermore, the outer coating is prepared by mixing chitosan solution and pectin solution, adding solid complex, ultrasonically dispersing, adding glycerol, stirring continuously to obtain a suspension, and finally adding sodium tripolyphosphate (TPP) solution for cross-linking reaction.

[0046] Furthermore, the pectin solution is prepared by dissolving pectin in deionized water to form a solution with a mass ratio of 1~1.5%, and then gelatinizing it at 50~60℃ for 15~25 minutes.

[0047] Furthermore, the chitosan solution is prepared by adding chitosan to an aqueous solution of glacial acetic acid, stirring at 50-60°C until dissolved, and adjusting the pH to 5.2-5.5 to form a chitosan solution with a mass concentration of 1.5-2%.

[0048] Furthermore, the mass ratio of the solid complex, chitosan solution, pectin solution, glycerol and TPP solution is 1g:50~60mL:15~25mL:0.8~0.9mL:100mL, and the mass concentration of the TPP solution is 0.8~1.2%.

[0049] The present invention has the following technical effects:

[0050] In this invention, a microcapsule-type functional additive is prepared by combining epigallocatechin gallate (EGCG) and complex unsaturated fatty acids and then encapsulating them. This functional additive is added to the feed of yellow catfish (Pelteobagrus vachelli) to effectively promote ovarian development, increase the number of oocytes, and increase the proportion of stage III oocytes, thereby effectively improving the reproductive capacity of yellow catfish and increasing the number of eggs laid, fertilization rate, and hatching rate. Attached Figure Description

[0051] Figure 1 Statistical graph of mid-segment ovarian oocyte count in the control group and Example 1 group.

[0052] Figure 2 HE-stained tissue sections of the ovaries of yellow catfish (Pelteobagrus vachelli) after 8 weeks of feeding in the control group and Example 1.

[0053] Figure 3 : Sustained-release curves of functional components in microcapsules prepared in Example 1 and each comparative example. Detailed Implementation

[0054] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0055] The basic aquaculture feed formula used in this invention is as follows:

[0056]

[0057] Example 1

[0058] A method for promoting ovarian development in yellow catfish (Pelteobagrus vachelli) includes the following steps:

[0059] S1. Functional component pretreatment

[0060] Add EGCG to a 55% ethanol solution at 45℃, and after it is completely dissolved, adjust the pH to 5.2 to prepare an EGCG solution with a concentration of 15 mg / L.

[0061] The functional component is obtained by mixing EGCG solution and complex unsaturated fatty acids, wherein the mass ratio of EGCG to complex unsaturated fatty acids is 0.18:10, and the complex unsaturated fatty acids are composed of palmitoleic acid and linoleic acid in a mass ratio of 0.8:1.

[0062] S2. Microcapsule preparation

[0063] (1) Dissolve β-CD in deionized water, adjust the pH to 6.3, and prepare a 55 mg / L β-CD aqueous solution. Mix the functional component and soybean lecithin solution, stir and homogenize at 5500 rpm for 4 min to obtain an emulsion. Add the emulsion dropwise to the β-CD aqueous solution, stir at 550 rpm for 2.5 h, let stand at 4℃ for 12 h, then centrifuge at 4500 rpm for 14 min, collect the precipitate, and dry to obtain a solid complex. The ratio of soybean lecithin, EGCG solution in the functional component and β-CD aqueous solution is 0.45 g: 18 mL: 100 mL.

[0064] (2) Chitosan (degree of deacetylation ≥ 85%) was added to an aqueous solution of glacial acetic acid and stirred at 55°C until dissolved. The pH was adjusted to 5.4 to form a chitosan solution with a mass concentration of 1.6%. The solid complex was added to the mixture of chitosan solution and pectin solution and ultrasonically dispersed. Glycerin was then added and stirring was continued to obtain a suspension. Finally, sodium tripolyphosphate (TPP) was added and stirred at 90 rpm for 25 min for crosslinking. The mixture was then centrifuged at 3800 rpm for 12 min, washed with deionized water, and dried to obtain microcapsules. The mass ratio of the solid complex, chitosan solution, pectin solution, glycerin, and TPP solution was 1 g: 55 mL: 20 mL: 0.8 mL: 100 mL, and the mass concentration of the TPP solution was 1%.

[0065] S3. Stage Feeding

[0066] Microcapsules were mixed with basic aquaculture feed at a ratio of 6% by weight to form functional feed. Healthy juvenile yellow catfish with a body length of 4-6 cm and a weight of 3-4 g were selected, randomly grouped, and placed in outdoor cement ponds. The functional feed was then fed according to the following strategy:

[0067] During the initial 1-28 day ovarian development preparation period: feed twice daily at 8:00 and 20:00, with a daily feed amount of 4-6% of the fish's body weight;

[0068] During the ovarian development enhancement period of 29-56 days: feed once each at 8:00, 12:00, 17:00 and 22:00 daily, with the daily feed amount being 4-6% of the fish's body weight, of which the feed at 22:00 accounts for 35% of the total daily feed amount;

[0069] During the breeding period, the dissolved oxygen was maintained at ≥5mg / L and the pH was 7.2-7.5. After 8 weeks of continuous feeding during the preparatory and intensive periods, the relevant indicators of ovarian development of yellow catfish were detected.

[0070] Figure 1 The chart shows the mid-segment ovarian oocyte counts for the control group and Example 1 group (the control group was fed directly with basic aquaculture feed, while the EGCG + complex unsaturated fatty acid group is Example 1). Figure 2 The images show HE-stained tissue sections. Image A is an HE-stained section of the mid-section ovary from the control group (basic feed group), and Image B is an HE-stained section of the mid-section ovary from the EGCG+ complex unsaturated fatty acid group corresponding to Example 1. It can be seen that the EGCG+ complex unsaturated fatty acid group has a greater number of oocytes than the control group, and is mainly composed of stage III oocytes; while in the control group, oocytes develop slowly, and are mainly stage I and stage II oocytes.

[0071] During the experiment, different functional components (including single catechins, monounsaturated fatty acid groups, different combinations of catechins and different unsaturated fatty acids, etc.) were added to the basic aquaculture feed and fed in the same manner. The effects of the functional components on the ovarian development of *Pelteobagrus vachelli* were detected by measuring gonadal index, egg production, and egg survival rate. (In the experiment, the functional components were directly added to the basic aquaculture feed for preliminary effect verification. The amount of catechins, monounsaturated fatty acids, and polyunsaturated fatty acids added in each group was the same as the corresponding components in Example 1. The basic aquaculture feed was used as a blank control group.) The results are shown in Table 1.

[0072] Table 1:

[0073]

[0074] It can be seen that when single catechins (EC, EGC, ECG, EGCG) are added as functional ingredients, their effect on improving the gonadal index (GSI) of *Pelteobagrus vachelli* is EC > EGC > ECG > EGCG. However, when each catechin is combined with complex unsaturated fatty acids, the efficiency of improving GSI shows a new trend: EGCG > ECG > EC > EGC. This change is because some catechin components (EC / EGC) have strong cell penetration and metabolic stability, so they are effective when used alone. However, compared with EGCG and ECG, their estrogen-like activity is weak, while EGCG and ECG have stronger estrogen-like activity. When combined with complex unsaturated fatty acids, they accelerate the conversion of unsaturated fatty acids into estrogen, producing a stronger synergistic effect. Moreover, the high penetrability of EC / EGC allows it to quickly enter the cell. Due to insufficient antioxidant capacity, it cannot cope with lipid oxidation, and the formation of high concentrations of catechins intracellularly inhibits the expression of fatty acid transporter proteins, hindering the transport of unsaturated fatty acids to the ovary, thereby weakening the synergistic effect.

[0075] Comparative Example 1

[0076] Compared with Example 1, the difference is that only β-CD inner layer embedding was performed in step S2 microcapsule preparation, and chitosan embedding was not performed. The remaining steps are the same as in Example 1.

[0077] Comparative Example 2

[0078] Compared with Example 1, the difference is that the β-CD inner layer embedding in step (1) was not carried out in step S2 microcapsule preparation. Instead, the chitosan embedding in step (2) was carried out directly. The remaining steps are the same as in Example 1.

[0079] Comparative Example 3

[0080] The difference from the example is that no pectin and glycerol were added during chitosan encapsulation in step S2, while the remaining steps are the same as in example 1.

[0081] The difference compared to Example 1 is that,

[0082] Example 1: The encapsulation efficiency, sustained-release performance, and antioxidant performance of the microcapsules prepared in Comparative Examples 1, 2, and 3 were determined.

[0083] (1) The content of beneficial EGCG and unsaturated fatty acids in the microcapsule product was detected by high performance liquid chromatography (HPLC), and the encapsulation efficiency was calculated:

[0084] Encapsulation rate = (Total added - Free content) / Total added × 100%

[0085] (2) Simulate the intestinal environment of fish (pH=7.4, 37℃) and measure the cumulative release rate at different time points within 48h.

[0086] (3) The microcapsules were stored at room temperature, normal pressure, and under light for 30 days. The peroxide value (POV) of the fatty acids inside the microcapsules was determined by iodometric titration, referring to GB 5009.227-2016 "National Food Safety Standard - Determination of Peroxide Value in Food". The change in malondialdehyde (MDA) content was determined by TBA colorimetric method, referring to GB 5009.181-2016 "National Food Safety Standard - Determination of Malondialdehyde in Food". The test results are shown in Table 2.

[0087] Table 2:

[0088]

[0089] As can be seen, the encapsulation efficiency of the functional components in Example 1 is excellent. The release curves of EGCG and unsaturated fatty acids in Example 1 and each comparative example are shown below. Figure 3 As shown, during slow release, EGCG and unsaturated fatty acids exhibited high synchronicity in release, with unsaturated fatty acids demonstrating excellent antioxidant properties and relatively low POV and MDA contents. In each comparative example, the encapsulation efficiency of EGCG and unsaturated fatty acids decreased to varying degrees, the synchronicity of EGCG and unsaturated fatty acid release decreased, the antioxidant protective effect of EGCG on unsaturated fatty acids decreased, and both POV and MDA contents significantly increased.

[0090] The study investigated the changes in ovarian development in *Pelteobagrus vachelli* after feeding in Examples 1, 1, 2, and 3, and the impact on the quality of subsequent fertilized egg rearing. A control group (CK) was used, consisting of feed with functional components added directly without encapsulation and only undergoing pretreatment (same formula as in Example 1). Hormone-induced spawning was employed. Spawns were collected from females immediately after spawning and fertilized. Fertilized eggs were observed under a microscope; fertilized eggs showed clear blastomeres, while unfertilized eggs showed no blasting and were either transparent or cloudy. The fertilization rate was calculated as (number of fertilized eggs / total number of observed eggs) × 100%. After all eggs hatched, the number of hatched fry was counted, and the hatching rate was calculated as (number of hatched fry / total number of fertilized eggs) × 100%. 24 hours after hatching, the fry were observed under a microscope, and the deformity rate was calculated as (number of deformed fry / total number of observed fry) × 100%. The survival rate of the fry was observed after 30 days of feeding. After 8 weeks of feeding, the body length and weight of the fish were tested. Specifically, the fry were placed on damp gauze, and the length from the snout to the base of the caudal fin was measured with calipers (accurate to 0.01 cm). The average value was calculated. After measuring the body length, the fry were dried with filter paper and weighed on an electronic balance (accurate to 0.01 g). The average value was also calculated. The results are shown in Table 3.

[0091] Table 3:

[0092]

[0093] Note: Data are the mean ± standard deviation of 3 parallel experiments. Statistical analysis was performed using SPSS 26.0. Different lowercase letters are used to indicate statistically significant differences in the same column. P <0.05).

[0094] It can be seen that the gonad index, egg production, and fertilization rate of the yellow catfish fed with the microcapsules prepared by encapsulation in Example 1 were significantly better than those of the CK group, which was directly fed with the microcapsules. Compared with Example 1, the yellow catfish cultured in Comparative Examples 1-3 also showed significant decreases in various indicators. This indicates that the specific encapsulation treatment regulated the release process of EGCG and complex unsaturated fatty acids, effectively improving the synergistic effect between EGCG and unsaturated fatty acids, thereby enhancing their promoting effect on the ovarian development of yellow catfish.

[0095] Example 2

[0096] A method for promoting ovarian development in yellow catfish (Pelteobagrus vachelli) includes the following steps:

[0097] S1. Functional component pretreatment

[0098] Add EGCG to a 50% ethanol solution at 50°C, and after it is completely dissolved, adjust the pH to 5.5 to prepare an EGCG solution with a concentration of 16 mg / L.

[0099] The functional component is obtained by mixing EGCG solution and complex unsaturated fatty acids, wherein the mass ratio of EGCG to complex unsaturated fatty acids is 0.15:8, and the complex unsaturated fatty acids are composed of oleic acid and γ-linolenic acid in a mass ratio of 0.9:1.2.

[0100] S2. Microcapsule preparation

[0101] (1) Dissolve β-CD in deionized water, adjust the pH to 6.5, and prepare a 60 mg / L β-CD aqueous solution. Mix the functional component and soybean lecithin solution, stir and homogenize at 6000 rpm for 5 min to obtain an emulsion. Add the emulsion dropwise to the β-CD aqueous solution, stir at 600 rpm for 3 h, let stand at 6℃ for 16 h, then centrifuge at 5000 rpm for 15 min, collect the precipitate, and dry to obtain a solid complex. The ratio of soybean lecithin, EGCG solution in the functional component and β-CD aqueous solution is 0.5 g: 20 mL: 100 mL.

[0102] (2) Chitosan (degree of deacetylation ≥ 85%) was added to an aqueous solution of glacial acetic acid and stirred at 60°C until dissolved. The pH was adjusted to 5.5 to form a chitosan solution with a mass concentration of 2%. The solid complex was added to the mixture of chitosan solution and pectin solution and ultrasonically dispersed. Glycerin was then added and stirring was continued to obtain a suspension. Finally, sodium tripolyphosphate (TPP) was added and stirred at 100 rpm for 30 min for crosslinking. The mixture was then centrifuged at 4000 rpm for 15 min, washed with deionized water, and dried to obtain microcapsules. The mass ratio of the solid complex, chitosan solution, pectin solution, glycerin, and TPP solution was 1 g: 60 mL: 25 mL: 0.9 mL: 100 mL, and the mass concentration of the TPP solution was 1.2%.

[0103] S3. Stage Feeding

[0104] Microcapsules were mixed with basic aquaculture feed at a ratio of 8% by weight to form functional feed. Healthy juvenile yellow catfish with a body length of 4-6 cm and a weight of 3-4 g were selected, randomly grouped, and placed in outdoor cement ponds. The functional feed was then fed according to the following strategy:

[0105] During the initial 1-28 day ovarian development preparation period: feed twice daily at 8:00 and 20:00, with a daily feed amount of 4-6% of the fish's body weight;

[0106] During the ovarian development enhancement period of 29-56 days: feed once each at 8:00, 12:00, 17:00 and 22:00 daily, with the daily feed amount being 4-6% of the fish's body weight, of which the feed at 22:00 accounts for 35% of the total daily feed amount;

[0107] During the breeding period, maintain dissolved oxygen ≥5mg / L and pH 7.2-7.5.

[0108] In this embodiment, the gonad index of the yellow catfish cultured for 8 weeks was 0.0076, and the number of eggs laid per fish was 7664.

[0109] Example 3

[0110] A method for promoting ovarian development in yellow catfish (Pelteobagrus vachelli) includes the following steps:

[0111] S1. Functional component pretreatment

[0112] Add EGCG to a 60% ethanol solution at 40℃, and after it is completely dissolved, adjust the pH to 5.0 to prepare an EGCG solution with a concentration of 12 mg / L.

[0113] An EGCG solution and a complex unsaturated fatty acid are mixed, wherein the mass ratio of EGCG to the complex unsaturated fatty acid is 0.2:10, and the complex unsaturated fatty acid is composed of erucic acid and α-linolenic acid in a mass ratio of 1:1.

[0114] S2. Microcapsule preparation

[0115] (1) Dissolve β-CD in deionized water, adjust the pH to 6, and prepare a 50 mg / L β-CD aqueous solution. Mix the functional component and soybean lecithin solution, stir and homogenize at 5000 rpm for 3 min to obtain an emulsion. Add the emulsion dropwise to the β-CD aqueous solution, stir at 500 rpm for 2 h, let stand at 4℃ for 12 h, then centrifuge at 4000 rpm for 12 min, collect the precipitate, and dry to obtain a solid complex. The ratio of soybean lecithin, EGCG solution in the functional component and β-CD aqueous solution is 0.4 g: 15 mL: 100 mL.

[0116] (2) Chitosan (degree of deacetylation ≥ 85%) was added to an aqueous solution of glacial acetic acid and stirred at 50°C until dissolved. The pH was adjusted to 5.2 to form a chitosan solution with a mass concentration of 1.5%. The solid complex was added to the mixture of chitosan solution and pectin solution and ultrasonically dispersed. Glycerin was then added and stirring was continued to obtain a suspension. Finally, sodium tripolyphosphate (TPP) was added and stirred at 80 rpm for 20 min for crosslinking. The mixture was then centrifuged at 3500 rpm for 10 min, washed with deionized water, and dried to obtain microcapsules. The mass ratio of the solid complex, chitosan solution, pectin solution, glycerin, and TPP solution was 1 g: 50 mL: 15 mL: 0.8 mL: 100 mL, and the mass concentration of the TPP solution was 0.8%.

[0117] S3. Stage Feeding

[0118] Microcapsules were mixed with basic aquaculture feed at a ratio of 5% by weight to form functional feed. Healthy juvenile yellow catfish with a body length of 4-6 cm and a weight of 3-4 g were selected, randomly grouped, and placed in outdoor cement ponds. The functional feed was then fed according to the following strategy:

[0119] During the initial 1-28 day ovarian development preparation period: feed twice daily at 8:00 and 20:00, with a daily feed amount of 4-6% of the fish's body weight;

[0120] During the ovarian development enhancement period of 29-56 days: feed once each at 8:00, 12:00, 17:00 and 22:00 daily, with the daily feed amount being 4-6% of the fish's body weight, of which the feed at 22:00 accounts for 35% of the total daily feed amount;

[0121] During the breeding period, maintain dissolved oxygen ≥5mg / L and pH 7.2-7.5.

[0122] In this embodiment, the gonad index of the yellow catfish cultured for 8 weeks was 0.0073, and the number of eggs laid per fish was 7698.

[0123] It should be noted that the functional additives prepared by combining EGCG and complex unsaturated fatty acids in this invention can be applied to other basic feeds and can also form functional feeds to exert corresponding effects.

Claims

1. A method for promoting ovarian development in *Pelteobagrus vachelli*, characterized in that: This refers to a functional additive administered to farmed yellow catfish during the pre-ovarian development and strengthening stages. The functional additive is a microcapsule prepared by mixing epigallocatechin gallate and complex unsaturated fatty acids as functional components, first encapsulating the inner layer with β-cyclodextrin, and then coating the outer layer with chitosan. The complex unsaturated fatty acids are composed of monounsaturated fatty acids and polyunsaturated fatty acids in a volume ratio of 0.8~1:1~1.

2. Among the complex unsaturated fatty acids, the monounsaturated fatty acids are selected from oleic acid, palmitoleic acid, or erucic acid, and the polyunsaturated fatty acids are selected from linoleic acid, α-linolenic acid, or γ-linolenic acid.

2. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 1, characterized in that: The staged feeding involves mixing the microcapsules with the basic aquaculture feed at a mass ratio of 5-8%, feeding twice daily during the initial ovarian development preparation period (days 1-28), and feeding four times daily during the ovarian development enhancement period (days 29-56).

3. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 2, characterized in that: During the preparatory stage of ovarian development, fish are fed twice daily at 8:00 and 20:00, with a daily feed amount of 4-6% of their body weight. During the intensive stage of ovarian development, fish are fed twice daily at 8:00, 12:00, 17:00, and 22:00, with a daily feed amount of 4-6% of their body weight, of which the feed at 22:00 accounts for 35% of the total daily feed amount.

4. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 3, characterized in that: The mass ratio of epigallocatechin gallate and complex unsaturated fatty acids in the functional components is 0.15~0.2:8~10.

5. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 4, characterized in that: The inner layer encapsulation is prepared by mixing epigallocatechin gallate solution and complex unsaturated fatty acids, then adding soybean lecithin to prepare an emulsion. The emulsion is then added to β-cyclodextrin aqueous solution, stirred, and allowed to stand at 4-6℃ for 12-16 hours. The precipitate is then collected by centrifugation and dried to obtain a solid complex.

6. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 5, characterized in that: The ratio of soybean lecithin, complex unsaturated fatty acids, epigallocatechin gallate solution, and β-cyclodextrin aqueous solution is 0.4~0.5g:8~12g:15~20mL:100mL, the concentration of β-cyclodextrin aqueous solution is 50~60mg / L, and the concentration of epigallocatechin gallate solution is 12~16mg / L.

7. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 6, characterized in that: The outer coating is prepared by mixing chitosan solution and pectin solution, adding solid complex, ultrasonically dispersing, adding glycerol, stirring continuously to obtain a suspension, and finally adding sodium tripolyphosphate solution to carry out a cross-linking reaction.

8. The method for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 7, characterized in that: The mass ratio of the solid complex, chitosan solution, pectin solution, glycerol and sodium tripolyphosphate solution is 1g:50~60mL:15~25mL:0.8~0.9mL:100mL, and the mass concentration of the sodium tripolyphosphate solution is 0.8~1.2%.

9. A functional additive for promoting ovarian development in yellow catfish (Pelteobagrus vachelli), characterized in that: The functional additive is composed of epigallocatechin gallate and complex unsaturated fatty acids. It is prepared by first embedding β-cyclodextrin in the inner layer and then coating it with chitosan in the outer layer. The complex unsaturated fatty acids are composed of monounsaturated fatty acids and polyunsaturated fatty acids in a volume ratio of 0.8~1:1~1.

2. Among the complex unsaturated fatty acids, the monounsaturated fatty acids are selected from oleic acid, palmitoleic acid, or erucic acid, and the polyunsaturated fatty acids are selected from linoleic acid, α-linolenic acid, or γ-linolenic acid.

10. The functional additive for promoting ovarian development in *Pelteobagrus vachelli* as described in claim 9, characterized in that: The mass ratio of epigallocatechin gallate and complex unsaturated fatty acids in the functional components is 0.15~0.2:8~10.

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