Preparation method of a nereid polychaete extract and application thereof in feed of litopenaeus vannamei

Abstract

The present application belongs to the field of aquatic nutrition feed, and particularly relates to a preparation method of a nereid extract and application of the nereid extract in Litopenaeus vannamei feed. The method comprises the following steps: (1) selecting suitable nereid development period and suitable body color of Perinereis aibuhitensis; (2) disinfecting the nereid; (3) extracting effective components from the nereid; and (4) applying the nereid extract to the Litopenaeus vannamei compound feed. The method can improve the nutritional components of the cultured Litopenaeus vannamei parent, and improve the content of long-chain polyunsaturated fatty acids, especially the content of DHA, in the cultured Litopenaeus vannamei parent and gonad. The method promotes the synthesis of sex steroid hormones of the Litopenaeus vannamei, and the egg production is increased by 30.3%, the multiple spawning rate is increased by 22.1%, the average egg production frequency is increased by 27.3%, and the nauplius metamorphosis rate is increased by 21.8%. The nereid raw material used in the method has the potential of low-cost and large-scale production, and has very strong operability.

Description

Technical Field

[0001] This invention belongs to the field of aquatic nutritional feed, specifically relating to a method for preparing a sandworm extract and its application in Litopenaeus vannamei feed. Background Technology

[0002] The rapid development of aquaculture relies heavily on the robust support of the aquatic feed industry. The utilization of traditional raw materials such as plant-based meals and animal by-product proteins is nearing its limit, with very limited room for supply growth. Furthermore, the utilization efficiency of farmed aquatic animals for traditional plant and animal-based alternatives is limited and difficult to further improve. Therefore, exploring geographically sourced unconventional raw materials with good efficacy and functionality is particularly important for supporting the development of sustainable aquaculture.

[0003] Sandworms are a natural food source for aquatic animals in the wild. They contain essential amino acids for aquatic growth (phenylalanine, methionine, lysine, threonine, tryptophan, leucine, isoleucine, valine, arginine, and histidine) as well as long-chain polyunsaturated fatty acids (LC-PUFAs) such as docosahexaenoic acid (22:6n-3, DHA), eicosapentaenoic acid (20:5n-3, EPA), and arachidonic acid (20:4n-6, ARA). Notably, some sandworm species possess the ability to synthesize LC-PUFAs de novo, making them an excellent source of LC-PUFAs for aquaculture and a suitable alternative to fish oil. Furthermore, sandworms have good palatability (e.g., they can be used as good natural hook bait), and when used in aquatic feed, they can promote feeding in farmed animals.

[0004] Currently, in large-scale shrimp and crab farming, sandworms are an important byproduct of shrimp and crab ponds. Harvesting sandworms after the shrimp and crabs are harvested is an important way to increase farming efficiency. Sandworms are widely used as live feed in the broodstock culture of crustaceans, especially shrimp. Their active ingredients are considered essential for promoting shrimp gonadal development and maintaining high yields, fertilization rates, and larval survival rates. Currently, sandworms are mainly used as live feed, with a small amount added to feed through primary processing. However, feeding live sandworms carries a significant risk of pathogen infection. A range of pathogens carried by sandworms can spread through feed, posing a serious pathogen threat to farmed fish and shrimp, leading to reduced or even complete crop failure. Commonly used high-temperature, high-pressure sterilization methods often destroy and lose nutrients in sandworms, necessitating the development of new raw material processing technologies to extract and preserve their nutritional and active substances.

[0005] Research and technological development on the discovery and application of functional active substances in sandworms are still insufficient, which seriously hinders the efficient and high-value utilization of sandworms in aquatic feed. Summary of the Invention

[0006] The technical problem to be solved by this invention is to extract and screen active substances in sandworms that can promote the growth and development of Litopenaeus vannamei using physicochemical methods, and to develop a method for their efficient utilization in formulated feed for Litopenaeus vannamei broodstock.

[0007] The method for preparing the nereis extract of this invention is achieved through the following technical solution:

[0008] (1) Select the appropriate developmental stage of the sandworm and the appropriate body color of the double-toothed sandworm;

[0009] (2) Disinfect the sandworms;

[0010] (3) Extraction of active ingredients from sandworm: Using the sample obtained in step (2), the sandworm powder and ethanol solvent were mixed at a ratio of 8:1 and then extracted at 4°C for 12 hours. The supernatant was centrifuged and concentrated under reduced pressure in a rotary evaporator. The remaining sandworm powder was further extracted with solvent. The above operation was repeated 3 times and the extracts were combined to obtain the sandworm ethanol extract.

[0011] The extraction of neutral lipids followed the Bligh and Dyer methods. The *Nephropus nereis* powder was fractionated into a methanol-water soluble fraction and a chloroform layer. The chloroform layer was collected, and after evaporating the solvent, it was treated with acetone to separate it into acetone-soluble and acetone-insoluble fractions. The acetone-soluble fraction was the neutral lipid, which was then evaporated with acetone using a rotary evaporator to obtain the *Nephropus nereis* neutral lipid extract.

[0012] (4) In the compound feed of Litopenaeus vannamei, the above-mentioned ethanol extract of sandworm and neutral lipid extract were mixed at a ratio of 1:2 and added to the compound feed of Litopenaeus vannamei at an addition ratio of 1.2%. After adding 0.02% lysophospholipid by weight, cholesterol was added at 5% of the total fat level.

[0013] The sandworm development stage described in step (1) is gonadal development stage III, and the suitable body color is orange-red.

[0014] The disinfection method described in step (2) is as follows: soak live sandworms in 89 mg / L povidone-iodine disinfectant for 3 hours, rinse them with clean water, and then soak them in 21 mg / L bromochlorohydantoin disinfectant for 3 hours. After cleaning, freeze-dry them and then crush them into powder through a 40-mesh sieve.

[0015] The present invention also includes the above-mentioned method for preparing the nervous silkworm extract and its application in Litopenaeus vannamei feed.

[0016] The beneficial effects of this invention compared to the prior art are as follows:

[0017] (1) The method of the present invention can modify the nutritional components of cultured Litopenaeus vannamei broodstock, and increase the content of long-chain polyunsaturated fatty acids, especially DHA, in cultured Litopenaeus vannamei broodstock and gonads.

[0018] (2) Promotes the synthesis of sex steroid hormones in Litopenaeus vannamei, increases spawning by 30.3%, increases the rate of multiple spawning by 22.1%, increases the average number of spawnings by 27.3%, and increases the nauplius metamorphosis rate by 21.8%.

[0019] (3) The raw material of the sandworm used in this technology has the potential for low-cost large-scale production and is highly operable. Attached Figure Description

[0020] Figure 1 Examples of different colored sandworms in Example 1

[0021] The sandworms at the top of the picture are blue, and those at the bottom are orange-red;

[0022] Figure 2 Figure showing astaxanthin content in different types of sandworms (farm #1)

[0023] Numerical values ​​are expressed as mean ± standard error (n=3);

[0024] Figure 3 Figure showing astaxanthin content in different body colors of sandworms (farm #2)

[0025] Numerical values ​​are expressed as mean ± standard error (n=3). Note: ** indicates a significant difference between red and green silkworms, with a significance level of P<0.01.

[0026] Figure 4 Principal component analysis (PCA) of lipidomics in different colored sandworm organisms (farm #2)

[0027] The red dots in the image represent cyan sandworms, and the blue dots represent orange-red sandworms.

[0028] Figure 5 PLS-DA analysis of lipidomics in different colored sandworms (farm #2)

[0029] The red dots in the image represent cyan sandworms, and the blue dots represent orange-red sandworms.

[0030] Figure 6 Figure showing differential lipids in lipidological analysis of different colored sandworms (farm #2).

[0031] In the figure, samples G1 / G2 / G3 are cyan sandworms, and samples R1 / R2 / R3 are orange-red sandworms. Detailed Implementation

[0032] The technical features of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0033] Example 1: Comparison of nutritional components of sandworms at different developmental stages and of different colors in Farm No. 1

[0034] 1. Sample Preparation. This experiment used different batches and colors of *Pteris vittata* (a type of sandworm) from Farm No. 1 in Jimo, Qingdao, for nutritional analysis. A total of 5 batches were sampled, with 100g of each species per batch. The 5 batches were pooled for routine nutritional analysis, and the values ​​were averaged. Other components such as amino acids and fatty acids were analyzed separately for each batch, followed by statistical analysis (SPSS 25.0, ANOVA). After sampling, the developmental stages were identified as gonadal development stages "II" and "III". The colors were divided into "orange-red" and "cyan". Figure 1 (The sandworms at the top of the picture are blue, and those at the bottom are orange-red).

[0035] 2. Nutritional Composition Analysis. Crude component analysis was performed according to the standard method of AOAC (2005). Samples were dried in a 105℃ oven to constant weight for moisture analysis. The dried feed and shrimp samples were ground and used for crude protein, crude fat, and ash analysis. Crude protein content was determined using the Kjeldahl method (FOSS KJELTEC 2300, Hillerod, Denmark); crude fat content was determined using the chloroform-methanol method; and crude ash content was determined using the high-temperature ablation method in a muffle furnace (Thermo F6000), after heating at 550℃ for 8 hours.

[0036] Fatty acid composition was determined by gas chromatography. 0.3 g of sample was extracted with chloroform-methanol to obtain total lipids. 2 mL of KOH-methanol was added, and the mixture was incubated in a 75°C water bath for 30 minutes. After cooling with cold water, 1 mL of boron trifluoride-methanol solution was added and vortexed. The mixture was incubated in a water bath for 30 minutes and then cooled. 1 mL of n-hexane and 1 mL of pure water were added for further extraction, and the mixture was allowed to stand on ice for 1 hour. The mixture was then centrifuged at 3000 rpm for 2 minutes. The supernatant was filtered through a membrane and added to a sample vial. Analysis was performed using a gas chromatograph (GC-2010Pro, Shimadzu, Japan). The results are expressed as the percentage of each fatty acid relative to the total fatty acids.

[0037] Amino acids were detected using a fully automated amino acid analyzer (Hitachi L-8900 automatic amino acid analyzer, Hitachi, Japan). 0.3 g of sample was weighed into a test tube, and 15 mL of 6 mol / L hydrochloric acid solution was added. The solution was hydrolyzed at 110 °C for 24 h. The solution was then brought to a final volume of 1350 mL. 0.5 mL of the solution was dried under nitrogen at 40 °C, and then 1 mL of 0.02 mol / L hydrochloric acid was added to dissolve the amino acid. The solution was filtered through a membrane and then added to a sample vial for analysis. The principle is based on the differences in structure, acidity / basicity, polarity, and molecular size of the amino acids in the sample. After ion exchange, each amino acid was sequentially eluted using buffers of different pH values. Each amino acid was then mixed with ninhydrin reagent and flowed together into a spiral reaction tube for a colorimetric reaction, forming a blue-violet product with maximum absorption at 570 nm.

[0038] The astaxanthin content was determined by Qingdao Yuanxin Technology Testing Company, which was commissioned to conduct the astaxanthin content test on red silkworms.

[0039] 3. Test results.

[0040] From the perspective of conventional nutritional components (Table 1), the orange-red stage III sandworms have the highest crude protein and crude fat content, indicating optimal nutritional composition.

[0041] Table 1. Nutritional composition analysis of sandworms at different developmental stages and of different colors (Farm No. 1, % wet weight)

[0042] Moisture 20.57 21.30 22.57 22.18 crude protein 12.12 13.22 12.75 12.63 Crude fat 4.01 5.84 4.33 4.30

[0043] From the perspective of amino acid composition (Table 2), the orange-red III phase showed the best performance in terms of total amino acid content and essential amino acid content, especially amino acids such as lysine, phenylalanine and arginine, which play an important role in animal reproductive physiology.

[0044] Table 2. Amino acid composition analysis of sandworms at different developmental stages and of different colors (farm #1, % dry weight)

[0045] Arginine (Arg) <![CDATA[3.26±0.03 b ]]> <![CDATA[3.77±0.06 a ]]> <![CDATA[3.38±0.07 ab ]]> <![CDATA[3.42±0.17 ab ]]> 0.131 Histidine <![CDATA[1.28±0.02 ab ]]> <![CDATA[1.32±0.01 a ]]> <![CDATA[1.24±0.01 b ]]> <![CDATA[1.29±0.02 ab ]]> 0.028 Isoleucine Ile <![CDATA[2.51±0.03 a ]]> <![CDATA[2.52±0.01 a ]]> <![CDATA[2.41±0.03 b ]]> <![CDATA[2.40±0.02 b ]]> 0.027 Leucine Leu <![CDATA[3.69±0.05 bc ]]> <![CDATA[3.90±0.03 a ]]> <![CDATA[3.82±0.04 ab ]]> <![CDATA[3.60±0.04 c ]]> 0.008 Lysine <![CDATA[4.10±0.08 b ]]> <![CDATA[4.42±0.02 a ]]> <![CDATA[4.17±0.05 b ]]> <![CDATA[4.14±0.03 b ]]> 0.020 Methionine (Met) 1.03±0.02 1.02±0.03 1.04±0.01 1.03±0.02 0.953 Phenylalanine (Phe) <![CDATA[2.11±0.03 c ]]> <![CDATA[2.40±0.02 a ]]> <![CDATA[2.30±0.01 b ]]> <![CDATA[2.02±0.02 d ]]> 0.000 Threonine Thr <![CDATA[2.34±0.02 a ]]> <![CDATA[2.19±0.02 bc ]]> <![CDATA[2.15±0.04 c ]]> <![CDATA[2.28±0.02 ab ]]> 0.016 Valine <![CDATA[2.64±0.03 ab ]]> <![CDATA[2.66±0.00 a ]]> <![CDATA[2.57±0.02 bc ]]> <![CDATA[2.55±0.02 c ]]> 0.033 EAA <![CDATA[22.96±0.31 b ]]> <![CDATA[24.21±0.16 a ]]> <![CDATA[23.08±0.23 b ]]> <![CDATA[22.73±0.25 b ]]> 0.030 Alanine (Ala) 3.98±0.07 4.21±0.02 4.15±0.09 4.03±0.05 0.185 Aspartic acid Asp <![CDATA[5.19±0.09 b ]]> <![CDATA[5.83±0.05 a ]]> <![CDATA[5.16±0.05 b ]]> <![CDATA[5.23±0.04 b ]]> 0.001 Cystine (Cys) 1.06±0.01 0.96±0.10 0.96±0.04 0.92±0.04 0.382 Glutamic acid (Glu) <![CDATA[7.68±0.11 b ]]> <![CDATA[8.35±0.13 a ]]> <![CDATA[8.01±0.16 ab ]]> <![CDATA[7.83±0.07 b ]]> 0.061 Glycine 2.83±0.01 2.97±0.10 2.83±0.10 2.97±0.05 0.408 Proline <![CDATA[2.82±0.02 b ]]> <![CDATA[3.52±0.06 a ]]> <![CDATA[2.90±0.10 b ]]> <![CDATA[2.85±0.03 b ]]> 0.002 Serine 2.06±0.03 2.05±0.03 1.97±0.03 2.07±0.02 0.113 Taurine <![CDATA[1.37±0.02 a ]]> <![CDATA[1.18±0.00 c ]]> <![CDATA[1.30±0.01 ab ]]> <![CDATA[1.25±0.03 bc ]]> 0.008 Tyrosine Tyr <![CDATA[1.84±0.02 b ]]> <![CDATA[2.16±0.05 a ]]> <![CDATA[2.12±0.01 a ]]> <![CDATA[1.71±0.04 c ]]> 0.000 NEAA <![CDATA[28.82±0.35 b ]]> <![CDATA[31.23±0.23 a ]]> <![CDATA[29.42±0.47 b ]]> <![CDATA[28.85±0.28 b ]]> 0.018 TAA <![CDATA[51.79±0.66 b ]]> <![CDATA[55.44±0.40 a ]]> <![CDATA[52.50±0.69 b ]]> <![CDATA[51.59±0.52 b ]]> 0.020

[0046] Note: EAA: Essential amino acids; NEAA: Non-essential amino acids; TAA: Total amino acids; Values ​​are expressed as mean ± standard error; One-way ANOVA and Duncan's method were performed using SPSS 25.0, and there were significant differences between values ​​with different superscript letters in the same data (P < 0.05).

[0047] Regarding fatty acid composition (Table 3), long-chain polyunsaturated fatty acids such as EPA and arachidonic acid, which play an important role in animal growth physiology, have the highest content in the orange-red phase III.

[0048] Table 3. Fatty acid composition analysis of polychaete at different developmental stages and colors (Farm No. 1, % total fatty acids)

[0049] C16:0 <![CDATA[17.42±0.01 c ]]> <![CDATA[19.91±0.35 a ]]> <![CDATA[18.99±0.15 b ]]> <![CDATA[19.86±0.21 a ]]> 0.004 C18:0 <![CDATA[4.63±0.31 b ]]> <![CDATA[5.36±0.12 a ]]> <![CDATA[4.64±0.08 b ]]> <![CDATA[5.53±0 a ]]> 0.039 C22:0 <![CDATA[0.64±0 ab ]]> <![CDATA[0.57±0.01 b ]]> <![CDATA[0.75±0.04 a ]]> <![CDATA[0.66±0.05 ab ]]> 0.065 C24:1n-9 <![CDATA[0.42±0.01 c ]]> <![CDATA[1.69±0.08 a ]]> <![CDATA[1.22±0.10 b ]]> <![CDATA[1.19±0.02 b ]]> 0.001 ΣSFA <![CDATA[23.11±0.30 c ]]> <![CDATA[27.53±0.57 a ]]> <![CDATA[25.60±0.21 b ]]> <![CDATA[27.24±0.23 a ]]> 0.003 C16:1n-7 <![CDATA[2.86±0.02 ab ]]> <![CDATA[3.45±0.30 ab ]]> <![CDATA[2.80±0.14 b ]]> <![CDATA[3.49±0.03 a ]]> 0.078 C18:1n-9t 2.07±1.84 5.20±0.26 3.64±0.07 4.72±0.03 0.228 C18:1n-9c <![CDATA[3.31±0.12 ab ]]> <![CDATA[3.63±0.09 a ]]> <![CDATA[3.26±0.07 b ]]> <![CDATA[3.15±0.02 b ]]> 0.057 C20:1n-9 <![CDATA[2.99±0.06 a ]]> <![CDATA[1.98±0.09 c ]]> <![CDATA[2.42±0.09 b ]]> <![CDATA[2.26±0.03 bc ]]> 0.002 C22:1n-9 <![CDATA[0.11±0.11 c ]]> <![CDATA[0.44±0.02 ab ]]> <![CDATA[0.37±0.05 b ]]> <![CDATA[0.53±0.04 a ]]> 0.009 ΣMUFA 11.34±1.94 14.69±0.57 12.48±0.18 14.14±0.11 0.221 C18:2n-6c <![CDATA[15.22±0.91 b ]]> <![CDATA[12.07±0.27 c ]]> <![CDATA[17.70±0.53 a ]]> <![CDATA[12.03±0.05 c ]]> 0.005 C18:3n-6 0.57±0.31 0.13±0.01 0.51±0.37 0.62±0.47 0.742 C20:2n-6 9.66±0.04 8.51±0.50 8.94±0.29 9.71±0.20 0.123 C20:3n-6 <![CDATA[0.54±0.01 c ]]> <![CDATA[1.29±0.08 a ]]> <![CDATA[0.62±0.05 c ]]> <![CDATA[0.94±0.03 b ]]> 0.002 C20:4n-6 1.42±1.23 3.06±0.12 2.44±0.06 2.47±0.03 0.416 Σn-6PUFA <![CDATA[27.41±2.41 ab ]]> <![CDATA[25.06±0.44 b ]]> <![CDATA[30.20±0.45 a ]]> <![CDATA[25.76±0.33 ab ]]> 0.139 C18:3n-3 <![CDATA[2.46±0.10 a ]]> <![CDATA[1.83±0.07 b ]]> <![CDATA[2.09±0.12 b ]]> <![CDATA[1.83±0.000 b ]]> 0.017 C20:5n-3 <![CDATA[6.25±0.26 b ]]> <![CDATA[8.89±0.21 a ]]> <![CDATA[5.40±0.08 c ]]> <![CDATA[8.59±0.13 a ]]> 0.000 C22:5n-3 <![CDATA[1.02±0.11 ab ]]> <![CDATA[1.47±0.09 a ]]> <![CDATA[0.69±0.19 b ]]> <![CDATA[1.33±0.05 a ]]> 0.036 C22:6n-3 1.94±0.02 1.88±0.90 1.87±0.81 2.20±0.07 0.975 Σn-3PUFA <![CDATA[11.67±0.29 ab ]]> <![CDATA[14.07±1.09 a ]]> <![CDATA[10.04±0.57 b ]]> <![CDATA[13.94±0.11 a ]]> 0.028 Σn-3 / Σn-6 <![CDATA[0.43±0.03 bc ]]> <![CDATA[0.56±0.05 a ]]> <![CDATA[0.33±0.02 c ]]> <![CDATA[0.54±0.01 ab ]]> 0.022

[0050] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; values ​​are expressed as mean ± standard error; one-way ANOVA and Duncan's method were performed using SPSS 25.0, and there were significant differences in values ​​with different superscript letters in the same data (P < 0.05).

[0051] Regarding astaxanthin content ( Figure 2 The orange-red stage III sandworm shrimp had a significantly higher oxalool content (0.52 mg / kg) than the other groups. The cyan stage II shrimp had a content of only 0.32 mg / kg.

[0052] Example 2: Comparison of nutritional components of different colored sandworms in Farm No. 2

[0053] 1. Sample acquisition, detection, and analysis methods.

[0054] A batch of samples was taken from Farm No. 2 in Jimo, Qingdao, yielding only two colors (orange-red and blue) of sandworms in stage III of gonadal development. The detection methods for each nutrient component were consistent with those in Example 1. Lipidomics analysis was also performed by Shanghai Meiji Biomedical Technology Co., Ltd.

[0055] 2. Experimental Results

[0056] The results of this batch show that the crude protein and crude fat content of orange-red sandworms is much higher than that of blue sandworms (Table 4).

[0057] Table 4. Composition of two colors of *Nematocera bidentata* body (% wet weight)

[0058] Orange-red sandworms 79.04±1.02 <![CDATA[13.24±0.61 ** ]]> <![CDATA[2.55±0.18 ** ]]> <![CDATA[1.39±0.07 ** ]]> Blue sandworm <![CDATA[87.60±0.71 ** ]]> 8.16±0.48 1.58±0.11 0.93±0.06

[0059] Note: * The values ​​, **, and *** represent significant differences between red and green silkworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

[0060] Amino acid composition analysis showed that the difference in amino acid composition between the two colors in this batch was not as great as that in farm No. 1, but the content of lysine, arginine and essential amino acids in orange-red sandworms was still significantly higher than that in blue sandworms (Table 5).

[0061] Table 5. Complete amino acid composition (% dry matter) of two colored bidentate sandworms.

[0062] Essential amino acid EAA Threonine Thr 2.42±0.16 2.17±0.09 Valine 2.26±0.12 2.26±0.09 Methionine (Met) 1.17±0.09 1.16±0.03 Isoleucine Ile 2.20±0.15 2.12±0.08 Leucine Leu 3.70±0.25 3.62±0.14 Phenylalanine (Phe) 2.05±0.13 2.01±0.09 Lysine <![CDATA[5.40±0.30 * ]]> 4.32±0.18 Histidine 1.19±0.09 1.27±0.02 Arginine (Arg) <![CDATA[4.57±0.23 * ]]> 3.56±0.13 ΣEAA <![CDATA[24.96±1.49 * ]]> 22.48±0.85 Non-essential amino acid ΣNEAA Taurine 1.38±0.11 1.03±0.07 Aspartic acid Asp 5.77±0.39 5.15±0.16 Serine 2.16±0.16 2.08±0.09 Glutamic acid (Glu) 8.36±0.54 8.20±0.26 Glycine 2.52±0.13 2.59±0.05 Alanine (Ala) 3.68±0.26 4.04±0.11 Cysteine 1.00±0.09 0.89±0.02 Tyrosine Tyr 1.69±0.08 1.71±0.05 Proline 6.74±0.46 6.46±0.24 ΣNEAA 33.3±2.21 32.16±1.04 ΣTAA 56.25±3.70 54.64±1.89

[0063] Note: * The values ​​, **, and *** represent significant differences between red and green silkworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

[0064] Regarding free amino acids, the free amino acid composition of red and blue *Pteris vittata* is shown in Table 6. The Thr content of blue *Pteris vittata* is significantly higher than that of green *Pteris vittata* (P<0.05), but the Val, Ile, Leu, Phe, His, Glu, Ala, and Pro content of orange-red *Pteris vittata* is significantly higher than that of red *Pteris vittata* (P<0.05).

[0065] Table 6. Free amino acid composition of *Nematocystis rubrum* (mg / 100g)

[0066] Essential amino acid EAA Threonine Thr 402.62±54.65 <![CDATA[726.99±20.56 ** ]]> Valine <![CDATA[204.08±11.47 * ]]> 125.99±17.02 Methionine (Met) 29.58±4.46 26.40±8.19 Isoleucine Ile <![CDATA[133.43±6.84 ** ]]> 63.47±6.31 Leucine Leu <![CDATA[181.59±7.49 *** ]]> 82.1±6.48 Phenylalanine (Phe) <![CDATA[201.96±22.04 * ]]> 108.52±7.53 Lysine 1103.47±101.41 942.23±46.06 Histidine <![CDATA[337.11±43.32 * ]]> 197.20±12.41 Arginine (Arg) 145.50±11.76 137.54±5.35 ΣEAA 2739.34±228.67 2410.43±55.24 Non-essential amino acid ΣNEAA Taurine 2581.14±176.03 2738.7±14.51 Aspartic acid Asp 384.78±23.33 402.38±13.31 Serine 31.09±31.09 0.00±0.00 Glutamic acid (Glu) <![CDATA[955.27±67.33 ** ]]> 545.13±26.51 Glycine 454.79±33.33 493.80±7.32 Alanine (Ala) <![CDATA[2555.81±166.60 ** ]]> 1548.03±28.03 Cysteine 147.68±13.51 88.81±31.09 Tyrosine Tyr 164.29±16.25 135.17±9.10 Proline <![CDATA[732.70±52.11 ** ]]> 422.69±17.53 ΣNEAA 2858.76±215.38 <![CDATA[6374.71±84.95 * ]]> ΣTAA 10746.89±746.85 8785.14±127.33

[0067] Note: * The values ​​, **, and *** represent significant differences between red and green silkworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

[0068] The fatty acid composition of red and blue dipterine sandworms is shown in Table 7. The C16:0, C24:1n-9, and SFA contents of red sandworms are significantly higher than those of blue sandworms (P<0.05), while the contents of C16:1n-7, C18:2n-6, and C20:3n-6 are significantly lower than those of blue sandworms (P<0.05). These fatty acid compositions show the advantage of orange-red sandworms in terms of the fatty acid composition required for energy supply.

[0069] Table 7. Fatty acid composition of *Nematocystis rubrum* (% of total fatty acids)

[0070]

[0071]

[0072] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; *, **, and *** represent significant differences between red and green silkworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

[0073] The oxalool content of the red-green double-toothed sand shrimp is as follows: Figure 3 The results showed that the astaxanthin content in the red sandworm was significantly higher than that in the green sandworm (P<0.05).

[0074] Lipidomics analysis of different body-colored sandworms (farm #2) showed that 578 lipid molecule types were successfully identified in the red and blue groups. Principal component analysis (PCA) of the two groups of sandworms revealed... Figure 4 The clustering patterns of the two groups of *Nematocystis jirovecii* showed significant differences, indicating substantial variations in their lipid metabolism patterns and lipid composition, and also suggesting potential differences in functional lipid components. Similar results were obtained in the validation of the PLS-DA model. Figure 5 ).

[0075] In terms of specific differential lipids ( Figure 6 The orange-red sandworm has higher lipid content in TG (19:1 / 18:2 / 18:1), PS (20:3e / 20:5), TG (16:0 / 16:0 / 20:1), TG (18:1e / 16:0 / 20:5), TG (18:1e / 16:0 / 22:5), and PS (20:3e / 22:6) than the blue sandworm, but lower lipid content in Cer (d18:1 / 17:0) and AEA (18:1).

[0076] In summary, the orange-red sandworms in gonadal development stage III are significantly superior to the blue sandworms in terms of overall nutritional composition, especially in terms of lipids that are functionally active in animal gonadal development.

[0077] Example 3: Sandworm extracts prepared by different extraction processes and their application in compound feed for Litopenaeus vannamei broodstock. Experiment 1: Preparation of Sandworm extract.

[0078] Fresh *Nematostella bidentata* were washed, freeze-dried, and pulverized. *Nematostella bidentata* extracts were prepared using solvents such as pure water (double-distilled water), ethanol, and petroleum ether. *Nematostella bidentata* powder and ethanol solvent were mixed at a material-to-liquid ratio of 8:1 and extracted at 4°C for 12 hours. The supernatant was collected, centrifuged, and concentrated under reduced pressure in a rotary evaporator. The remaining *Nematostella bidentata* powder was further extracted with solvent. The above operation was repeated 3 times, and the extracts were combined to obtain *Nematostella bidentata* aqueous extract, ethanol extract, and petroleum ether extract.

[0079] The extraction of neutral lipids followed the Bligh and Dyer method. The *Nephropus nereis* powder was fractionated into a methanol-water soluble fraction and a chloroform layer. The chloroform layer was collected, and after evaporating the solvent, it was treated with acetone to separate it into acetone-soluble and acetone-insoluble fractions. The acetone-soluble fraction was the neutral lipid, which was then evaporated in a rotary evaporator to obtain the *Nephropus nereis* neutral lipid extract.

[0080] All extracts were stored at 4°C until they were mixed with other ingredients to prepare experimental feed.

[0081] 2. Experimental design and feed preparation

[0082] A basal diet containing approximately 52% crude protein and 13% crude fat was prepared using fishmeal as the main protein source (its composition and nutritional components are shown in Table 8). The control group received the basal diet (CON), while the experimental groups were supplemented with 1.2% of nereis sericulture water extract (WE), ethanol extract (AE), petroleum ether extract (PE), neutral lipid extract (NLF), and a mixture of ethanol water extract and neutral lipids (1:1 group, 1A1N group; or 1:2, 1A2N group). The experimental diets were prepared according to standard laboratory procedures. The raw materials were pulverized, thoroughly mixed, and made into strips with a diameter of 1 mm. After steaming for 15 minutes, the strips were dried at 55°C, crushed and sieved using a mixer, and then stored in a -20°C cold storage for later use.

[0083] Table 8 Experimental feed formulation and conventional nutrient composition (% dry matter)

[0084] fish meal 47 47 47 47 47 47 47 Krill powder 10 10 10 10 10 10 10 gluten 8 8 8 8 8 8 8 soybean meal 5 5 5 5 5 5 5 high gluten flour 16.93 15.73 15.73 15.73 15.73 15.73 15.73 fish oil 4.8 4.8 4.8 4.8 4.8 4.8 4.8 Soy lecithin 2 2 2 2 2 2 2 Arachidonic acid 0.65 0.65 0.65 0.65 0.65 0.65 0.65 calcium dihydrogen phosphate 1.5 1.5 1.5 1.5 1.5 1.5 1.5 mineral mixture 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Vitamin Mixture 1 1 1 1 1 1 1 Vitamin C 0.5 0.5 0.5 0.5 0.5 0.5 0.5 choline chloride 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Calcium propionate 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Lysophospholipids 0.02 0.02 0.02 0.02 0.02 0.02 0.02 cholesterol 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Cane Su Pink 0.4 0.4 0.4 0.4 0.4 0.4 0.4 Nereis aqueous extract WE 0 1.2 0 0 0 0.6 0.4 AE of Silene oleracea ethanol extract 0 0 1.2 0 0 0 0 PE (petroleum ether extract of Nematodes) 0 0 0 1.2 0 0 0 NLF (Neutral Lipid Extract of Nephrolepis cordifolia) 0 0 0 0 1.2 0.6 0.8 Standard composition Moisture 4.68 4.73 4.25 3.79 4.49 4.43 4.44 crude protein 52.68 53.07 52.59 52.43 52.63 52.53 52.43 Crude fat 13.49 13.30 14.06 13.77 13.50 13.40 13.30 Ash 12.15 12.21 12.27 12.16 11.98 11.58 11.22

[0085] Note: Astaxanthin is provided by Canex Pink. The effective content of astaxanthin is 10%, and the remaining 90% are excipients, including lignin sulfonate, corn starch, and dextrin.

[0086] The fatty acid composition of the experimental diets is shown in Table 9. As can be seen from the table, the saturated fatty acid content in each treatment group was low. The C14:0 content in the control group was significantly higher than that in the PE, 1A2N, and NLF groups, and the C22:0 content was significantly higher than that in the NLF and 1A2N groups. The SFA content in the control group was significantly higher than that in the PE, 1A2N, and NLF groups (P<0.05). There was no significant difference in the total monounsaturated fatty acid content between the treatment groups and the control group (P>0.05). The n-6 series polyunsaturated fatty acid content in the NLF group was significantly higher than that in the control group. Furthermore, the C18:3n-6 content in the AE, 1A1N, and PE groups was significantly higher than that in the control group, and the C22:5n-3 content was significantly higher than that in the control group and other treatment groups. The DHA content in the WE, 1A1N, and 1A2N groups was significantly higher than that in the control group and other treatment groups. The n-3 / n-6 content was highest in the AE and 1A1N groups (P<0.05).

[0087] Table 9. Fatty acid content (% of total fatty acids) of each group of feeds

[0088]

[0089] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; values ​​are expressed as mean ± standard error; one-way ANOVA and Duncan's method were performed using SPSS 25.0, and there were significant differences in values ​​with different superscript letters in the same data (P < 0.05).

[0090] The amino acid composition of the experimental diets is shown in Table 10. As can be seen from the table, the WE, 1A1N, and 1A2N groups had the highest total essential amino acid content. The Ile content in the control group was significantly higher than that in the PE and NLF groups, and the Lys content was significantly higher than that in the NLF group (P<0.05). The Phe, Lys, and total amino acid contents were highest in the WE, 1A1N, and 1A2N groups, but there were no significant differences compared to the control group (P>0.05).

[0091] Table 10 Amino acid composition of each group of feeds (% dry matter)

[0092]

[0093] Note: One-way ANOVA and Duncan's method were used in SPSS 25.0. There were significant differences in the values ​​of different superscript letters in the same row data (P<0.05).

[0094] 3. Experimental shrimp and their husbandry management

[0095] The shrimp used in the experiment were purchased from Bangpu Seed Industry Technology Co., Ltd., Changyi City, Weifang City, Shandong Province. The male shrimp were a highly resistant strain, and the female shrimp were both highly resistant and fast-growing strains. The initial weight was 56.11±0.95g. The culture ponds were 2m in diameter and 1.3m in height. The broodstock shrimp were randomly divided into 15 culture ponds, with 22 shrimp per pond (16 males and 6 females). Each group was fed in three replicates. Still water culture was used, with deep well seawater at a temperature of (26.5±0.5)℃, dissolved oxygen above 8mg / L, salinity 31, pH 7.9~8.2, ammonia nitrogen ≤0.55mg / L, and nitrite ≤0.045mg / L. The water was changed once daily, with a replacement volume of 70-75%. The shrimp were fed five times daily at 6:00, 10:00, 14:00, 18:00, and 22:00.

[0096] 4. Sample Collection

[0097] After a 24-hour fasting period following the end of the rearing period, the number of surviving broodstock shrimp in the rearing ponds was recorded and their weight was recorded. All remaining female shrimp were sampled, and two male shrimp were randomly selected from each rearing pond. Surface moisture was absorbed with absorbent paper, and the weight was recorded using an electronic balance. Hemolymph was extracted from the abdomen using a sterile syringe and aliquoted into centrifuge tubes containing 1 mL of anticoagulant. The tubes were allowed to stand for 4 hours, then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and stored at -20°C. The broodstock shrimp were dissected live on an ice tray, and the hepatopancreas and gonads were dissected using surgical scissors. The samples were accurately weighed, and the gonads and hepatopancreas were flash-frozen in liquid nitrogen and then stored at -80°C.

[0098] 5. Analytical Methods

[0099] Crude composition determination of feed and broodstock shrimp muscle: The crude composition analysis of feed and shrimp muscle was performed according to the standard method of AOAC (2005). Samples were dried in an oven at 105℃ to constant weight for moisture analysis. The dried feed and shrimp samples were ground and used for crude protein, crude fat, and ash analysis. Crude protein content was determined using the Kjeldahl method (FOSS KJELTEC2300, Hillerod, Denmark); crude fat content was determined using the chloroform-methanol method; and crude ash content was determined using a muffle furnace (Thermo F6000) high-temperature agitation method, after heating at 550℃ for 8 hours.

[0100] Fatty acid determination of hepatopancreatic and gonadal fatty acids: Fatty acids in the samples were determined by gas chromatography. 0.3 g of sample was extracted with chloroform-methanol to obtain total lipids. 2 mL of KOH-methanol was added, and the mixture was incubated in a 75°C water bath for 30 minutes. After cooling with cold water, 1 mL of boron trifluoride-methanol solution was added and vortexed. The mixture was incubated in a water bath for 30 minutes and then cooled. 1 mL of n-hexane and 1 mL of pure water were added for further extraction, and the mixture was allowed to stand on ice for 1 hour. The mixture was then centrifuged at 3000 rpm for 2 minutes. The supernatant was filtered through a membrane and added to a sample vial. Analysis was performed using a gas chromatograph (GC-2010Pro, Shimadzu, Japan). The results are expressed as the percentage of each fatty acid relative to the total fatty acids.

[0101] Amino acid determination of hepatocellular carcinoma, pancreas, and gonads: The amino acid composition of fertilized eggs and nauplii was determined using a fully automated amino acid analyzer (Hitachi L-8900 automatic amino acid analyzer, Hitachi, Japan). 0.3 g of sample was weighed into a test tube, and 15 mL of 6 mol / L hydrochloric acid solution was added. The solution was hydrolyzed at 110 °C for 24 h. The solution was then brought to a final volume of 50 mL. 0.5 mL of the solution was dried under nitrogen at 40 °C, and then 1 mL of 0.02 mol / L hydrochloric acid was added to dissolve the solution. The solution was filtered through a membrane and then added to a sample vial for analysis. The principle is based on the differences in structure, acidity, polarity, and molecular size of the amino acids in the sample. After ion exchange, each amino acid was sequentially eluted using buffers of different pH values. Each amino acid was then mixed with ninhydrin reagent and flowed together into a spiral reaction tube for a colorimetric reaction, forming a blue-violet product with maximum absorption at 570 nm.

[0102] Serum biochemical indicators were measured using kits for triglycerides (A110-1-1), estradiol, progesterone, and testosterone produced by Nanjing Jiancheng Bioengineering Institute.

[0103] 6. Data Processing and Analysis

[0104] SPSS 25.0 statistical analysis software was used to perform ANOVA univariate analysis and Duncan multiple comparison analysis on the data. P < 0.05 was considered significant. Data values ​​are expressed as mean ± standard error.

[0105] Calculation formula:

[0106] Survival rate (%) = Total number of shrimp in the pond after the end of the breeding period / Total number of shrimp initially put into the pond in the experiment × 100;

[0107] Weight gain rate (%) = (final weight - initial weight) / initial weight × 100;

[0108] Average daily molting count (times / day) = Total molting count per pool during the intensive period / Number of intensive days

[0109] Hepatopancreas Index = Hepatopancreas weight / Female broodstock shrimp body weight × 100%

[0110] Gonadal index = Gonadal weight / Female broodstock shrimp body weight × 100%

[0111] 7. Experimental Results

[0112] Survival rate, average daily molting count, hepatopancreatic index, and gonadal index of broodstock shrimp: The growth performance results of broodstock shrimp are shown in Table 11. The survival rate of the 1A2N and NFL groups was significantly higher than that of the control group (P<0.05), while there were no significant differences between the other treatment groups and the control group (P>0.05). The average daily molting count of the 1A2N and AE groups was significantly higher than that of the control group (P>0.05). The hepatopancreatic index of the WE, AE, PE, and 1A1N groups was significantly higher than that of the control group (P<0.05). The hepatopancreatic index of the NFL group was higher than that of the control group, but the difference was not significant (P>0.05). The gonadal index of the 1A2N group was significantly higher than that of the other groups except PE (P<0.05).

[0113] Table 11 Survival rate, average daily number of molts, and hepatopancreas and gonadal indices of female Litopenaeus vannamei broodstock in each experimental group

[0114]

[0115] Note: Different letters in the superscript of data from the same row indicate significant differences (P<0.05); the same letter or no letter in the superscript indicates no significant differences (P>0.05).

[0116] Because the performance of AE and PE was below expectations, these two groups were removed from subsequent tests. Only WE, NLF, 1A1N, 1A2N, and the control group were tested, and the indicators were tested separately for males and females.

[0117] The crude composition of broodstock muscle is shown in Table 12. The table shows that the moisture content of female shrimp in group CON was significantly higher than that of groups 1A1N, NFL, and 1A2N (P<0.05), but not significantly different from group 1A1N (P>0.05). The crude protein and crude fat contents of female shrimp in all treatment groups were significantly higher than those in the control group (P<0.05). The crude protein content of male shrimp in group 1A2N was significantly higher than that in the control group, while the crude fat content was not significantly different from the control group (P<0.05). The ash content of both male and female broodstock shrimp was not significantly different from the control group (P>0.05).

[0118] Table 12. Composition of crude shrimp muscle components

[0119] Moisture (%) female shrimp <![CDATA[79.89±0.03 a ]]> <![CDATA[78.75±0.33 ab ]]> <![CDATA[77.18±0.30 c ]]> <![CDATA[78.34±0.14 bc ]]> <![CDATA[77.15±0.56 bc ]]> male shrimp 75.40±1.00 76.43±1.43 74.26±0.47 72.78±2.11 74.69±0.37 Crude protein (% wet weight) female shrimp <![CDATA[18.59±0.06 d ]]> <![CDATA[19.41±0.16 c ]]> <![CDATA[20.63±0.17 a ]]> <![CDATA[19.9±0.04 b ]]> <![CDATA[19.5±0.20 bc ]]> male shrimp <![CDATA[22.22±0.08 b ]]> <![CDATA[22.34±0.12 ab ]]> <![CDATA[22.72±0.11 ab ]]> <![CDATA[22.52±0.00 ab ]]> <![CDATA[22.80±0.24 a ]]> Crude fat (% wet weight) female shrimp <![CDATA[1.99±0.01 c ]]> <![CDATA[2.48±0.08 ab ]]> <![CDATA[2.65±0.06 a ]]> <![CDATA[2.31±0.04 b ]]> <![CDATA[2.34±0.08 b ]]> male shrimp 2.80±0.12 3.07±0.36 3.21±0.10 3.24±0.21 3.22±0.20 Ash content (% wet weight) female shrimp 1.53±0.04 1.48±0.01 1.47±0.04 1.57±0.09 1.45±0.02 male shrimp 1.42±0.08 1.32±0.07 1.44±0.05 1.51±0.04 1.45±0.01

[0120] Note: Different letters in the superscript of data from the same row indicate significant differences (P<0.05); the same letter or no letter in the superscript indicates no significant differences (P>0.05).

[0121] The fat content of hepatopancreas and gonads in each treatment group is shown in Table 13. As can be seen from the table, compared with the control group, there was no significant difference in fat content of hepatopancreas and gonads in female shrimp (P>0.05). The fat content of hepatopancreas in groups CON, NLF, and 1A2N in male shrimp was significantly higher than that in groups WE and 1A1N. The fat content of gonads in groups 1A2N and NLF in male shrimp was significantly higher than that in the control group (P<0.05).

[0122] Table 13. Fat content (%) of hepatopancreas and gonads in broodstock shrimp of different treatment groups

[0123]

[0124] Note: Different letters in the superscript of data from the same row indicate significant differences (P<0.05); the same letter or no letter in the superscript indicates no significant differences (P>0.05).

[0125] The fatty acid composition of the hepatopancreas of female shrimp is shown in Table 14. As can be seen from the table, the content of 22:5n-3 in the WE group was significantly higher than that in the control group and the 1A1N group (P<0.05). The 1A2N group had the highest EPA and DHA content.

[0126] Table 14 Fatty acid composition of female shrimp hepatopancreas (% of total fatty acids)

[0127] 14:0 0.53±0.01 0.55±0.01 0.97±0.46 0.63±0.15 0.63±0.18 14:1n-5 1.58±0.05 1.13±0.31 0.89±0.33 1.44±0.23 1.64±0.15 16:0 15.36±0.00 17.87±1.98 18.44±1.38 16.85±0.57 16.01±1.7 16:1n-7 3.28±1.58 2.41±0.71 2.19±0.56 2.88±1.05 1.93±0.41 17:0 3.04±0.14 2.16±0.88 2.5±0.64 3.11±0.46 3.03±0.13 17:1n-7 0.50±0.05 0.41±0.05 0.40±0.00 0.51±0.01 0.47±0.06 18:0 10.57±0.33 9.4±1.22 10.41±2.18 10.28±0.66 10.94±0.33 18:1n-9c 11.79±1.09 13.44±1.01 12.21±1.3 12.56±1.99 10.56±1.25 18:2n-6c 5.99±0.36 8.16±0.63 6.39±1.09 6.07±0.49 6.16±0.36 18:3n-3 1.41±0.07 1.54±0.24 1.71±0.14 1.70±0.14 1.49±0.42 20:2n-6 0.88±0.01 0.77±0.39 1.32±0.04 1.14±0.03 1.36±0.11 20:4n-6 6.86±0.18 5.57±0.80 5.87±1.03 6.08±0.32 7.68±0.66 20:5n-3 13.08±0.76 10.29±1.85 11.39±1.57 13.24±2.36 14.16±0.58 22:5n-3 <![CDATA[0.47±0.04 b ]]> <![CDATA[0.73±0.08 a ]]> <![CDATA[0.67±0.05 ab ]]> <![CDATA[0.49±0.04 b ]]> <![CDATA[0.54±0.07 ab ]]> 22:6n-3 16.17±1.61 14.34±2.36 13.93±2.30 15.35±0.75 18.92±3.45 SFA 29.49±0.47 30.98±0.86 32.32±1.57 30.87±0.56 30.62±1.64 MUFA 17.15±2.67 17.39±1.36 15.69±1.51 17.39±2.77 14.60±1.70 PUFA 47.86±1.78 41.40±4.91 41.29±3.56 44.07±3.09 47.31±3.85 n-6PUFA 13.73±0.56 14.50±1.04 13.58±0.11 13.29±0.18 15.21±0.62 n-3PUFA 34.13±2.34 26.90±3.89 27.70±3.51 30.78±3.23 32.10±3.57 n-3 / n-6 1.61±1.16 0.85±0.34 1.02±0.51 1.25±0.77 1.03±0.55 PUFA / SFA 1.62±0.03 1.35±0.19 1.29±0.15 1.42±0.07 1.56±0.19

[0128] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; Values ​​are expressed as mean ± standard error; Different letters in the superscript of data from the same period indicate significant differences (P<0.05); The same letter in the superscript or no letter indicates no significant differences (P>0.05).

[0129] The fatty acid composition of male shrimp hepatopancreas is shown in Table 15. The table shows that the 17:1n-7 content in the 1A1N group was significantly lower than that in other treatment groups. The 20:4n-6 content in the 1A1N and WE groups was higher than that in the control group, but the difference was not significant (P>0.05). The MUFA content in the 1A2N and NLF groups was higher than that in the control group. The PUFA content in the NLF group and the n-6 content in the 1A1N and NFL groups were higher than those in the control group, but the differences were not significant (P>0.05). The 1A2N group had the highest EPA and DHA content.

[0130] Table 15 Fatty acid composition of male shrimp hepatopancreas (% of total fatty acids)

[0131] 14:0 2.56±0.29 2.74±0.31 3.06±0.09 3.11±0.13 2.53±0.26 14:1n-5 0.30±0.08 0.33±0.08 0.49±0.12 0.45±0.05 0.32±0.15 16:0 24.29±1.15 24.19±0.43 23.76±0.56 23.06±0.44 24.4±0.61 16:1n-7 4.24±0.41 3.84±0.25 4.44±0.43 4.79±0.47 4.90±0.44 17:0 0.37±0.10 0.49±0.11 0.29±0.05 0.33±0.15 0.23±0.01 17:1n-7 <![CDATA[0.32±0.02 a ]]> <![CDATA[0.31±0.02 a ]]> <![CDATA[0.31±0.02 a ]]> <![CDATA[0.16±0.09 b ]]> <![CDATA[0.31±0.03 a ]]> 18:0 3.81±0.76 4.00±0.51 3.00±0.44 3.88±0.63 2.76±0.32 18:1n-9c 18.38±0.47 18.15±0.37 18.16±0.55 17.79±1.04 18.22±0.31 18:2n-6c 9.9±0.49 8.91±0.40 10.13±0.47 9.6±0.31 10.08±0.11 18:3n-3 2.02±0.03 2.23±0.08 2.20±0.15 2.26±0.22 2.19±0.04 20:2n-6 1.21±0.30 1.30±0.19 1.46±0.10 1.21±0.07 1.31±0.21 20:4n-6 <![CDATA[2.32±0.23 ab ]]> <![CDATA[2.45±0.24 ab ]]> <![CDATA[2.11±0.17 ab ]]> <![CDATA[2.7±0.24 a ]]> <![CDATA[2.01±0.06 b ]]> 20:5n-3 5.51±0.51 5.22±0.33 5.33±0.55 5.85±0.18 6.38±0.20 22:5n-3 1.01±0.09 1.09±0.16 1.02±0.09 1.33±0.09 1.06±0.01 22:6n-3 10.2±0.38 10.16±0.5 9.85±0.69 9.87±0.39 10.37±0.41 SFA 31.03±1.2 31.42±0.65 30.11±0.71 30.37±1.29 29.92±0.60 MUFA <![CDATA[23.24±0.22 ab ]]> <![CDATA[22.64±0.42 ab ]]> <![CDATA[23.39±0.41 a ]]> <![CDATA[21.76±0.54 b ]]> <![CDATA[23.75±0.59 a ]]> PUFA <![CDATA[33.39±0.24 a ]]> <![CDATA[31.36±0.22 b ]]> <![CDATA[33.46±0.7 a ]]> <![CDATA[32.82±0.49 ab ]]> <![CDATA[32.61±0.47 ab ]]> n-6PUFA <![CDATA[13.43±0.44 ab ]]> <![CDATA[12.65±0.12 b ]]> <![CDATA[13.69±0.31 a ]]> <![CDATA[13.51±0.19 ab ]]> <![CDATA[13.40±0.15 ab ]]> n-3PUFA 18.74±0.79 18.71±0.24 18.4±1.13 19.31±0.36 19.21±0.59 n-3 / n-6 1.39±0.01 1.48±0.03 1.34±0.06 1.43±0.02 1.43±0.06 PUFA / SFA 1.04±0.08 1.00±0.03 1.07±0.07 1.09±0.06 1.09±0.04

[0132] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; Values ​​are expressed as mean ± standard error; Different letters in the superscript of data from the same period indicate significant differences (P<0.05); The same letter in the superscript or no letter indicates no significant differences (P>0.05).

[0133] The fatty acid composition of female shrimp gonads is shown in Table 16. The table shows that the 18:1n-9 content in the 1A1N, WE, and NFL groups was significantly higher than that in the CON group. The 18:3n-3 content in the 1A1N group was higher than that in other treatment groups, and the 22:5n-3 content in the NFL group was significantly higher than that in the control group (P<0.05). The 1A2N group had the highest ARA and DHA content.

[0134] Table 16 Fatty acid composition of female shrimp gonads (% of total fatty acids)

[0135] 14:0 0.86±0.10 1.46±0.11 1.29±0.39 1.15±0.36 1.49±0.40 14:1n-5 <![CDATA[2.42±0.06 a ]]> <![CDATA[0.14±0.01 c ]]> <![CDATA[0.76±0.17 bc ]]> <![CDATA[1.35±0.37 bc ]]> <![CDATA[1.71±0.27 ab ]]> 16:0 21.78±1.82 22.76±0.29 22.27±0.55 22.39±0.67 21.55±1.41 16:1n-7 2.83±0.23 3.48±0.23 3.52±0.68 3.16±0.80 4.12±0.76 17:0 <![CDATA[2.87±0.39 a ]]> <![CDATA[1.19±0.4 b ]]> <![CDATA[1.07±0.34 b ]]> <![CDATA[1.65±0.64 ab ]]> <![CDATA[2.01±0.12 ab ]]> 17:1n-7 <![CDATA[0.66±0.02 a ]]> <![CDATA[0.47±0.04 bc ]]> <![CDATA[0.37±0.04 c ]]> <![CDATA[0.55±0.05 ab ]]> <![CDATA[0.48±0.01 bc ]]> 18:0 <![CDATA[10.42±1.23 a ]]> <![CDATA[7.03±0.06 b ]]> <![CDATA[7.51±0.22 b ]]> <![CDATA[8.52±1.41 ab ]]> <![CDATA[9.08±0.30 ab ]]> 18:1n-9c <![CDATA[15.70±0.72 c ]]> <![CDATA[19.3±0.45 a ]]> <![CDATA[18.55±0 ab ]]> <![CDATA[18.71±0.67 ab ]]> <![CDATA[16.73±0.67 bc <!-- 15 -->]]> 18:2n-6c 5.34±0.40 5.90±0.21 9.37±2.32 5.77±0.25 7.50±2.43 18:3n-3 <![CDATA[0.99±0.03 c ]]> <![CDATA[1.13±0.04 b ]]> <![CDATA[1.10±0.00 bc ]]> <![CDATA[1.24±0.04 a ]]> <![CDATA[1.11±0.01 bc ]]> 20:2n-6 0.87±0.01 0.89±0.06 0.78±0.09 0.95±0.13 1.04±0.13 20:4n-6 <![CDATA[5.69±0.02 a ]]> <![CDATA[5.38±0.15 ab ]]> <![CDATA[3.52±0.44 b ]]> <![CDATA[6.07±1.13 a ]]> <![CDATA[6.38±0.37 ab ]]> 20:5n-3 <![CDATA[13.43±0.33 a ]]> <![CDATA[9.11±0.28 b ]]> <![CDATA[9.32±0.14 b ]]> <![CDATA[9.65±0.67 b ]]> <![CDATA[10.61±0.59 b ]]> 22:5n-3 <![CDATA[0.54±0.07 b ]]> <![CDATA[0.72±0.05 ab ]]> <![CDATA[0.83±0.04 a ]]> <![CDATA[0.64±0.04 ab ]]> <![CDATA[0.54±0.07 b ]]> 22:6n-3 9.46±1.12 10.72±0.24 10.48±0.41 9.58±0.95 11.06±0.70 SFA 35.92±2.77 32.44±0.63 32.15±0.33 33.71±1.49 34.13±1.64 MUFA 21.60±1.02 23.79±0.39 23.74±0.35 23.77±1.06 23.03±0.93 PUFA 37.33±1.73 33.85±0.19 32.06±2.45 33.91±1.47 35.23±2.05 n-6PUFA 12.90±0.38 12.18±0.33 13.68±2.13 12.80±1.40 13.92±2.80 n-3PUFA 24.43±1.35 21.67±0.51 22.72±0.51 21.11±1.31 21.31±0.75 n-3 / n-6 1.89±0.05 1.78±0.09 1.97±0.29 1.70±0.26 1.66±0.32 PUFA / SFA 1.05±0.13 1.04±0.02 1.08±0.02 1.01±0.06 1.04±0.11

[0136] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; Values ​​are expressed as mean ± standard error; Different letters in the superscript of data from the same period indicate significant differences (P<0.05); The same letter in the superscript or no letter indicates no significant differences (P>0.05).

[0137] The fatty acid composition of male shrimp gonads is shown in Table 17. As can be seen from the table, the 16:0 content in the WE, 1A1N, and 1A2N groups was significantly higher than that in the control group (P<0.05). The 18:1n-9 and 18:2n-6c contents in each treatment group were significantly higher than those in the control group (P<0.05). The 20:2n-6 content in the WE, NFL, and 1A2N groups was significantly higher than that in the control group (P<0.05). The EPA content in the 1A2N group was significantly higher than that in the control group (P<0.05). The MUFA and n-6 ​​contents in each treatment group were significantly higher than those in the control group (P<0.05).

[0138] Table 17 Fatty acid composition of male shrimp gonads (% of total fatty acids)

[0139]

[0140]

[0141] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids; Values ​​are expressed as mean ± standard error; Different letters in the superscript of data from the same period indicate significant differences (P<0.05); The same letter in the superscript or no letter indicates no significant differences (P>0.05).

[0142] The amino acid composition of male shrimp hepatopancreas is shown in Table 18. As can be seen from the table, the Arg content of the WE and 1A1N groups was higher than that of the control group, but there was no significant difference (P>0.05).

[0143] Table 18 Amino acid composition of female shrimp hepatopancreas (% dry matter)

[0144]

[0145]

[0146] Note: Values ​​are expressed as mean ± standard error; different letters in the same row indicate significant differences (P<0.05); the same letter or no letter in the same row indicates no significant differences (P>0.05).

[0147] The amino acid composition of the hepatopancreas of male shrimp is shown in Table 19. As can be seen from the table, the Cys content in each treatment group was significantly higher than that in the control group, and the Pro content in the NFL group was significantly higher than that in the control group (P<0.05). The 1A2N group had the highest arginine content.

[0148] Table 19 Amino acid composition of male shrimp hepatopancreas (% dry matter)

[0149] Essential amino acid EAA Threonine Thr 0.46±0.02 0.44±0.04 0.47±0.06 0.50±0.03 0.43±0.02 Valine 0.54±0.03 0.57±0.05 0.61±0.06 0.51±0.10 0.56±0.02 Methionine (Met) 0.01±0.01 0.10±0.04 0.06±0.05 0.08±0.03 0.06±0.03 Isoleucine Ile 0.42±0.03 0.42±0.04 0.46±0.05 0.41±0.09 0.41±0.01 Leucine Leu 0.62±0.04 0.64±0.06 0.69±0.08 0.59±0.14 0.61±0.02 Phenylalanine (Phe) 0.39±0.06 0.36±0.04 0.41±0.05 0.34±0.11 0.37±0.03 Lysine 0.68±0.01 0.70±0.06 0.74±0.09 0.56±0.10 0.61±0.05 Histidine 0.28±0.02 0.28±0.02 0.31±0.03 0.26±0.06 0.28±0.02 Arginine (Arg) 0.46±0.04 0.47±0.04 0.61±0.10 0.44±0.14 0.63±0.04 ΣEAA 3.87±0.24 3.97±0.36 4.37±0.46 3.60±0.81 3.76±0.16 Non-essential amino acid ΣNEAA Taurine 0.41±0.13 0.39±0.09 0.56±0.11 0.47±0.08 0.43±0.03 Aspartic acid Asp 1.06±0.08 1.04±0.09 1.13±0.13 1.00±0.21 1.00±0.05 Serine 0.41±0.02 0.41±0.03 0.44±0.05 0.47±0.04 0.4±0.02 Glutamic acid (Glu) 1.19±0.03 1.23±0.11 1.28±0.15 1.38±0.14 1.16±0.05 Glycine 0.54±0.03 0.62±0.05 0.59±0.08 0.55±0.12 0.53±0.02 Alanine (Ala) 0.54±0.02 0.54±0.04 0.54±0.06 0.47±0.10 0.50±0.03 Cysteine <![CDATA[0.01±0.00 b ]]> <![CDATA[0.24±0.02 a ]]> <![CDATA[0.16±0.09 a ]]> <![CDATA[0.16±0.03 a ]]> <![CDATA[0.28±0.02 a ]]> Tyrosine Tyr 0.17±0.09 0.27±0.06 0.31±0.09 0.24±0.13 0.29±0.04 Proline <![CDATA[0.57±0.02 b ]]> <![CDATA[0.61±0.03 ab ]]> <![CDATA[0.66±0.02 a ]]> <![CDATA[0.61±0.04 ab ]]> <![CDATA[0.57±0.01 b ]]> ΣNEAA 4.89±0.38 5.29±0.41 5.56±0.70 4.96±1.10 4.99±0.21 ΣTAA 8.76±0.61 9.26±0.77 9.93±1.12 8.56±1.91 8.75±0.32

[0150] Note: Values ​​are expressed as mean ± standard error; different letters in the same row indicate significant differences (P<0.05); the same letter or no letter in the same row indicates no significant differences (P>0.05).

[0151] The amino acid composition of the female shrimp gonads is shown in Table 20. The table shows that the Tyr content in the NFL group was higher than that in the control group, but the difference was not statistically significant (P>0.05). The Pro content in the 1A1N group was significantly higher than that in the control group (P<0.05). The 1A2N group had the highest arginine content.

[0152] Table 20 Amino acid composition of female shrimp gonads (% dry matter)

[0153] Essential amino acid EAA Threonine Thr 0.46±0.02 0.46±0.06 0.53±0.05 0.52±0.03 0.52±0.04 Valine 0.53±0.03 0.56±0.05 0.63±0.07 0.59±0.05 0.62±0.04 Methionine (Met) <![CDATA[0.16±0.01 a ]]> <![CDATA[0.05±0.03 b ]]> <![CDATA[0.17±0.02 a ]]> <![CDATA[0.04±0.01 b ]]> <![CDATA[0.06±0.03 b ]]> Isoleucine Ile 0.44±0.02 0.44±0.05 0.49±0.04 0.48±0.03 0.50±0.04 Leucine Leu 0.70±0.05 0.72±0.08 0.76±0.05 0.76±0.04 0.80±0.07 Phenylalanine (Phe) 0.42±0.02 0.42±0.07 0.44±0.04 0.46±0.03 0.48±0.05 Lysine 0.73±0.04 0.75±0.04 0.85±0.04 0.78±0.08 0.9±0.06 Histidine 0.28±0.02 0.29±0.04 0.29±0.02 0.3±0.01 0.32±0.04 Arginine (Arg) 0.76±0.07 0.66±0.11 0.67±0.01 0.85±0.08 0.89±0.08 ΣEAA 4.46±0.23 4.35±0.47 4.83±0.31 4.78±0.35 5.05±0.34 Non-essential amino acid ΣNEAA Taurine 0.63±0.06 0.6±0.09 0.5±0.13 0.49±0.04 0.63±0.07 Aspartic acid Asp 1.03±0.05 1.04±0.17 1.11±0.08 1.14±0.06 1.18±0.11 Serine 0.43±0.02 0.42±0.04 0.47±0.03 0.46±0.02 0.47±0.03 Glutamic acid (Glu) 1.45±0.09 1.52±0.14 1.64±0.06 1.62±0.10 1.76±0.16 Glycine 0.84±0.05 0.77±0.11 0.74±0.05 0.9±0.07 0.82±0.07 Alanine (Ala) 0.55±0.04 0.59±0.04 0.66±0.05 0.64±0.05 0.68±0.05 Cysteine <![CDATA[0.25±0.02 a ]]> <![CDATA[0.08±0.05 b ]]> <![CDATA[0.24±0.02 a ]]> <![CDATA[0.07±0.04 b ]]> <![CDATA[0.07±0.05 b ]]> Tyrosine Tyr <![CDATA[0.37±0.03 ab ]]> <![CDATA[0.31±0.03 b ]]> <![CDATA[0.41±0.02 a ]]> <![CDATA[0.34±0.02 ab ]]> <![CDATA[0.37±0.02 ab ]]> Proline <![CDATA[0.57±0.04 b ]]> <![CDATA[0.66±0.11 ab ]]> <![CDATA[0.62±0.04 ab ]]> <![CDATA[0.70±0.03 a ]]> <![CDATA[0.58±0.03 ab ]]> ΣNEAA 6.10±0.35 5.92±0.55 6.37±0.07 6.37±0.21 6.56±0.44 ΣTAA 10.57±0.58 10.27±1.02 11.21±0.38 11.15±0.56 11.62±0.78

[0154] Note: Values ​​are expressed as mean ± standard error; different letters in the same row indicate significant differences (P<0.05); the same letter or no letter in the same row indicates no significant differences (P>0.05).

[0155] The amino acid composition of male shrimp gonads is shown in Table 21. As can be seen from the table, the Tau content in groups 1A2N and 1A1N was significantly higher than that in the control group (P<0.05), and the Tau content in group 1A2N was the highest.

[0156] Table 21 Amino acid composition of male shrimp gonads (% dry matter)

[0157]

[0158]

[0159] Note: Values ​​are expressed as mean ± standard error; different letters in the same row indicate significant differences (P<0.05); the same letter or no letter in the same row indicates no significant differences (P>0.05).

[0160] Regarding serum biochemical indicators: The effects of adding *Nematostella spp.* extract to the feed on serum biochemical indicators of Litopenaeus vannamei are shown in Table 22. In female shrimp, the progesterone content in group 1A2N was significantly higher than in other groups except 1A1N (P<0.05). Group 1A2N had the highest estradiol content, significantly higher than the WE and NFL groups (P<0.05). Group 1A2N also had the highest vitellin content. The total protein content in group 1A1N was significantly higher than in other treatment groups (P<0.05). The free fatty acid content in groups 1A1N and 1A2N was higher than in the control group, but there was no significant difference (P>0.05). In male shrimp, the estradiol content in the NFL group was higher than in the control group, but there was no significant difference (P>0.05). The total protein content in male shrimp in groups 1A1N and 1A2N was significantly higher than in the control group (P<0.05).

[0161] Table 22 Effects of Adding Nereid Extract to Feed on Serum Biochemical Indicators

[0162]

[0163] Note: Values ​​are expressed as mean ± standard error; different letters in the same row indicate significant differences (P<0.05); the same letter or no letter in the same row indicates no significant differences (P>0.05).

[0164] Regarding the gene expression of sex steroid hormone synthesis, the effects of adding nereis extract to the feed on the gene expression of the hepatopancreas of male Litopenaeus vannamei are shown in Table 23. The expression level of STAR gene in the 1A2N group was significantly higher than that in the CON, WE and NFL groups (P<0.05), and the expression level of 17β-HSD gene in the 1A1N group was significantly higher than that in the control group and other treatment groups (P<0.05).

[0165] Table 23 Effects of adding *Nematostella spp.* extract to feed on gene expression in the hepatopancreas of male shrimp.

[0166] PCNA 1.00±0.26 0.92±0.26 0.93±0.15 1.75±0.7 1.02±0.32 ECR 1.00±0.26 0.41±0.11 0.87±0.61 0.52±0.43 0.98±0.30 HMGCR 1.00±0.07 1.24±0.17 1.13±0.17 1.17±0.1 1.29±0.16 STAR <![CDATA[1.00±0.04 b ]]> <![CDATA[0.92±0.12 b ]]> <![CDATA[1.03±0.11 b ]]> <![CDATA[1.27±0.21 ab ]]> <![CDATA[1.64±0.06 a ]]> RXRA 1.00±0.08 1.05±0.15 1.13±0.21 0.87±0.10 1.10±0.22 17β-HSD <![CDATA[1.00±0.23 b ]]> <![CDATA[1.51±0.69 b ]]> <![CDATA[1.04±0.29 b ]]> <![CDATA[4.14±0.28 a ]]> <![CDATA[1.01±0.35 b ]]> MF 1.00±0.37 0.62±0.11 1.26±0.43 1.04±0.11 1.31±0.23 VGR 1.00±0.52 2.24±0.68 1.45±0.64 1.76±0.47 2.06±0.49

[0167] Note: PCNA: Proliferating cell nuclear antigen; ECR: Ecdysone receptor; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; STAR: Acute regulatory protein of steroid synthesis; RXRA: Retinoid X receptor α; 17β-HSD: 17β-hydroxysteroid dehydrogenase 1; MF: Cell maturation factor; VGR: Vitellogenin receptor. Values ​​are expressed as mean ± standard error; different letters in the superscript of data from the same row indicate significant differences (P<0.05); the same letter in the superscript or no letter indicates no significant difference (P>0.05).

[0168] The effects of adding *Nematostella spp.* extract to the feed on gonadal gene expression in female *Litopenaeus vannamei* are shown in Table 24. The expression of the PCNA gene in the WE and NFL groups was significantly higher than that in the control group (P<0.05). The expression level of the STAR gene in the 1A2N group was significantly higher than that in other groups (P<0.05).

[0169] Table 24 Effects of adding worm extract to feed on gonadal gene expression in female shrimp

[0170] PCNA <![CDATA[1.00±0.10 b ]]> <![CDATA[1.85±0.10 b ]]> <![CDATA[3.36±0.53 a ]]> <![CDATA[1.85±0.1 b ]]> <![CDATA[2.2±0.25 ab ]]> ECR 1.00±0.02 0.82±0.15 2.3±0.96 0.82±0.15 3.28±0.64 HMGCR 1.00±0.07 1.33±0.45 2.04±0.33 1.33±0.45 2.08±0.4 STAR <![CDATA[1.00±0.07 b ]]> <![CDATA[1.27±0.48 b ]]> <![CDATA[2.15±0.54 b ]]> <![CDATA[1.27±0.48 b ]]> <![CDATA[4.23±0.41 a ]]> RXRA 1.00±0.24 1.43±0.69 2.11±0.27 1.43±0.69 2.54±0.27 VGR 1.00±0.31 1.35±0.79 0.79±0.12 1.35±0.79 2.06±0.44

[0171] Note: PCNA: Proliferating cell nuclear antigen; ECR: Ecdysone receptor; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; STAR: Acute regulatory protein of steroidogenesis; RXRA: Retinol X receptor α; VGR: Vitellogeninogen receptor. Values ​​are expressed as mean ± standard error; different letters in the superscript of data from the same line indicate significant differences (P<0.05); the same letter or no letter in the superscript indicates no significant difference (P>0.05).

[0172] Example 4: Experiment 2 (Pilot-scale) of Sandworm Extracts Prepared Using Different Extraction Processes and Their Application in Litopenaeus vannamei Broodstock Formula Feed

[0173] 1. Sources of experimental feed and broodstock shrimp

[0174] Two groups, NFL and 1A2N (plus a control group), which showed the best performance in the above results, were selected for a pilot-scale broodstock feeding experiment. The feed preparation process was the same as in Example 3. The feed ingredients were crushed and passed through a 60-mesh sieve. After being mixed using a step-by-step scaling-up method, water was added and stirred. The mixture was then fed into a pellet mill and pressed into broodstock feed with a specification of 1.5mm × 3.0mm. The pellets were dried at 50℃ for 12 hours and then stored in a cool, dark place for later use. Specific details are not repeated here.

[0175] Source of broodstock: The Litopenaeus vannamei broodstock used in the experiment were obtained from a shrimp farm in Weifang, Shandong Province. They were 11 months old and had not undergone any nutritional fortification treatment in the early stages. Female shrimp had a body length of (12.10±0.58) cm, a weight of (32.61±3.91) g, a hepatopancreas index of (3.14±0.42)%, and no gonadal development. Male shrimp had a body length of (11.88±0.72) cm and a weight of (27.25±3.35) g. Nutritional fortification was initiated three months before spawning, with a fortification period of three months. Each treatment had three replicates, with 20 shrimp per replicate (female to male ratio of 1:1). After gonadal development, the eyestalks were removed for maturation, artificial insemination, and hatching according to standard production procedures. These processes were completed with the assistance of skilled workers at the shrimp farm in Weifang, Shandong Province. An eye tag was attached to one side of the eyestalk of each broodstock to distinguish individuals. All female broodstock underwent unilateral eyestalk removal surgery using a forceps-scalding method. Data on reproduction and larval development were collected using artificial insemination.

[0176] 2. Sample collection and analysis methods

[0177] After artificial insemination, the number of eggs laid, the hatching rate of fertilized eggs, and the metamorphosis rate of nauplius larvae were recorded in each culture tank. The egg-laying rate, multiple egg-laying rate, and average number of egg-laying times of female broodstock shrimp in each replicate tank were calculated. Approximately 50 fertilized eggs were collected from each of the first ten egg-laying cycles and preserved in a 4% formaldehyde solution for subsequent measurement of egg diameter. Simultaneously, approximately 0.2g of fertilized eggs were collected from each of the first ten egg-laying cycles in each treatment group and preserved in liquid nitrogen for determination of the fatty acid composition of the eggs.

[0178] Routine feed composition analysis: Dry matter content was determined according to national standard GB / T 6435-1986; crude protein content was determined according to national standard GB / T 6432-1994 (Kjeldahl method, VELP-UDK129); crude fat content was determined according to national standard GB / T 6433-1994 (Denmark FOSS SOXTEC 2050 - petroleum ether); ash content was determined according to national standard GB / T 6438-1992 (calcined in a muffle furnace at 550℃ to constant weight). The fatty acid content in the feed and fertilized eggs was determined using gas chromatography.

[0179] Breeding-related indicators are calculated using the following formula:

[0180] Female broodstock shrimp spawning rate = (Number of female broodstock shrimp that spawned / Total number of female broodstock shrimp surviving during fertilization and spawning period) × 100%

[0181] Multiple spawning rate = (Number of female broodstock shrimp that spawned multiple times (twice or more) / Number of female broodstock shrimp that spawned) × 100%

[0182] Average number of spawnings = total number of spawnings in each parallel group during fertilization and spawning period / number of female parent shrimp that spawned; egg diameter = (long diameter + short diameter) / 2.

[0183] The data were analyzed using SPSS 19.0 statistical software through ANOVA univariate analysis and Duncan's multiple comparisons (P < 0.05).

[0184] 3. Experimental Results

[0185] In terms of growth rate (Table 25), there were no significant differences among the groups. However, the hepatopancreatic index was significantly lower in the 1A2N group than in the CON and NFL groups. The gonadal index was significantly higher in the 1A2N and NFL groups than in the control group. This indicates the transfer of nutrients from the hepatopancreas to the gonads in the 1A2N group.

[0186] Table 25 Hepatopancreas and gonad indices of female Litopenaeus vannamei broodstock in each experimental group

[0187] Weight gain rate of female broodstock shrimp (%) 48.62±3.73 49.63±5.62 58.10±2.63 Weight gain rate (%) of male broodstock shrimp 39.65±1.93 42.32±3.31 45.65±3.74 Hepatopancreatic index (%) <![CDATA[2.56±0.14 a ]]> <![CDATA[3.21±0.11 a ]]> <![CDATA[2.31±0.12 b ]]> Gonadal index (%) <![CDATA[4.30±0.10 a ]]> <![CDATA[5.20±0.21 b ]]> <![CDATA[5.22±0.07 b ]]>

[0188] Note: Different superscript letters in the same row indicate significant differences (P<0.05).

[0189] From the perspective of fatty acid composition of fertilized eggs (Table 26), the 1A2N group has higher contents of EPA, DHA and PUFA.

[0190] Table 26 Fatty acid composition (% total fatty acids) of fertilized eggs in each treatment group

[0191]

[0192]

[0193] Note: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids. Fatty acid content is expressed as the proportion of each fatty acid methyl ester to the total fatty acid methyl esters. Different superscript letters in the same row indicate significant differences (P<0.05).

[0194] From the perspective of reproductive indicators such as egg production (Table 27), the 1A2N group had the highest egg production, significantly higher than the control group, increasing by 30.3%. The multiple egg-laying rate, average number of egg-laying cycles, and nauplius metamorphosis rate of the NFL and 1A2N groups were significantly higher than those of the control group. The multiple egg-laying rate, average number of egg-laying cycles, and nauplius metamorphosis rate of the 1A2N group were 22.1%, 27.3%, and 21.8% higher than those of the control group, respectively. The number of fertilized eggs in the NFL group was significantly higher than that in the control group and the 1A2N group. There were no significant differences in egg production rate and fertilized egg hatching rate among the groups.

[0195] Table 27. Egg production, fertilized egg hatching rate, and nauplius metamorphosis rate for each treatment group

[0196] Egg production (in ten thousand) <![CDATA[17.06±0.48 b ]]> <![CDATA[20.85±2.55 ab ]]> <![CDATA[22.23±1.61 a ]]> Egg laying rate (%) 70.11±1.19 69.22±3.46 69.36±1.13 Multiple spawning rate (%) <![CDATA[37.15±2.47 b ]]> <![CDATA[55.22±7.32 a ]]> <![CDATA[45.35±3.16 a ]]> Average number of spawnings (times) <![CDATA[1.32±0.71 b ]]> <![CDATA[1.81±0.28 a ]]> <![CDATA[1.68±0.23 a ]]> fertilized egg diameter (μm) <![CDATA[246.71±4.41 b ]]> <![CDATA[275.82±7.39 a ]]> <![CDATA[254.20±8.11 b ]]> Hatching rate of fertilized eggs (%) 47.65±3.44 46.21±1.21 48.33±0.96 Nauplius metamorphosis rate (%) <![CDATA[60.27±2.32 b ]]> <![CDATA[71.32±2.24 a ]]> <![CDATA[73.41±3.11 a ]]>

[0197] Note: Different superscript letters indicate significant differences (P<0.05).

Claims

1. A method for preparing a sandworm extract and its application in Litopenaeus vannamei feed, characterized by comprising the following steps: (1) Select the *Pteris vittata* species whose developmental stage is gonadal development stage III and whose body color is orange-red. (2) Disinfecting the sandworms, the disinfection method is as follows: soaking the live sandworms in 89mg / L povidone-iodine disinfectant for 3 hours, rinsing them with clean water, then soaking them in 21mg / L bromochlorohydantoin disinfectant for 3 hours, and then freeze-drying them after cleaning, and then crushing them into 40-mesh sieves. (3) Extraction of active ingredients from *Nematocystis jirovecii*: Using the sample obtained in step (2), *Nematocystis jirovecii* powder and ethanol solvent were mixed at a ratio of 8:1 and extracted at 4°C for 12 hours. The supernatant was centrifuged and concentrated under reduced pressure in a rotary evaporator. The remaining *Nematocystis jirovecii* powder was further extracted with solvent. The above operation was repeated 3 times, and the extracts were combined to obtain *Nematocystis jirovecii* ethanol extract. The extraction of neutral lipids was carried out according to the Bligh and Dyer methods. The nematodes powder was fractionated into a methanol-water soluble fraction and a chloroform layer. The chloroform layer was collected, and after the solvent in the chloroform layer was evaporated, it was treated with acetone and separated into two parts: acetone-soluble and acetone-insoluble. The acetone-soluble fraction was the neutral lipids. After evaporating the acetone in a rotary evaporator, the nematodes neutral lipid extract was obtained. (4) In the compound feed of Litopenaeus vannamei, the above-mentioned ethanol extract of sandworm and neutral lipid extract were mixed at a ratio of 1:2 and added to the compound feed of Litopenaeus vannamei at an addition ratio of 1.2%. 0.02% lysophospholipids were added by weight, and cholesterol was added at 5% of the total fat level.

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