Preparation method of nereis extract and application of nereis extract in litopenaeus vannamei feed

Abstract

The invention belongs to the field of aquatic nutritional feed, and particularly relates to a preparation method of nereis extract and application of the nereis extract in litopenaeus vannamei feed. Comprising the following steps: (1) selecting perinereis aibuhitensis with a proper nereis development period and a proper body color; (2) disinfecting the nereis; (3) extracting effective components of nereis; and (4) applying the effective components to the litopenaeus vannamei compound feed. According to the method disclosed by the invention, the nutritional ingredients of the bred litopenaeus vannamei parents can be changed, and the content of long-chain polyunsaturated fatty acids in the bred litopenaeus vannamei parents and gonads, especially the content of DHA, is increased. The method has the advantages that synthesis of sex steroid hormones of the litopenaeus vannamei is promoted, the egg laying amount is increased by 30.3%, the multiple egg laying rate is increased by 22.1%, the average egg laying frequency is increased by 27.3%, and the nauplius metamorphosis rate is increased by 21.8%. The nereis raw material adopted by the technology has the potential of low-cost large-scale production, and has very strong operability.

Description

Technical Field

[0001] The invention belongs to the field of aquatic nutritional feed, and particularly relates to a preparation method of a nereid extract and application of the extract in Litopenaeus vannamei feed. Background Art

[0002] The rapid development of aquaculture is inseparable from the solid support of the aquatic feed industry. The utilization of traditional raw materials such as plant meal and animal by-product protein has reached its limit, leaving limited room for supply growth. Furthermore, aquaculture animals have limited efficiency in utilizing alternative plant and animal-based raw materials, making further improvement difficult. Therefore, the discovery of locally sourced, non-conventional raw materials with promising efficacy and functionality is crucial to supporting the development of sustainable aquaculture.

[0003] Lugworms 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 lugworm species possess the ability to synthesize LC-PUFAs de novo, making them a valuable source of LC-PUFAs for aquaculture and a suitable alternative to fish oil. Furthermore, lugworms possess excellent feeding properties (e.g., they can be used as a good natural bait), and their inclusion in aquatic feed can promote feeding in farmed animals.

[0004] In large-scale shrimp and crab aquaculture, lugworms are a significant byproduct of pond production. Harvesting lugworms after catching is a crucial method for increasing aquaculture profitability. Lugworms are widely used as a biological feed in broodstock culture for crustaceans, particularly shrimp. The active ingredients they contain are believed to be essential for promoting gonadal development and maintaining high shrimp yield, fertilization, and post-larval survival. Currently, lugworms are primarily used as live bait, with small amounts added to feed through primary processing. However, feeding live lugworms carries a significant risk of pathogen infection. Numerous pathogens carried by lugworms can be transmitted through bait, posing a significant threat to farmed fish and shrimp, leading to reduced or even complete crop failure. Conventional high-temperature and high-pressure disinfection methods often destroy and deplete lugworm nutrients, necessitating the development of novel raw material processing technologies to extract and preserve their active nutrients.

[0005] Currently, there is still a lack of research and technological development on the exploration and application of functional active substances of nereids, which seriously hinders the efficient and high-value utilization of nereids in aquatic feed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to extract and screen active substances in nereids that can promote the growth and development of Penaeus vannamei by physical and chemical methods, and to develop a method for efficiently utilizing the active substances in Penaeus vannamei broodstock feed.

[0007] The preparation method of the nereid extract of the present invention is achieved by the following technical scheme:

[0008] (1) Select the suitable nereid developmental stage and the bilaterian nereid of suitable body color;

[0009] (2) disinfecting the nereid;

[0010] (3) Extraction of active ingredients from nereids: Using the sample prepared in step (2), nereid powder and ethanol solvent were mixed in a material-liquid 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 nereid powder was further added with solvent for extraction. The above operation was repeated 3 times and the extracts were combined to obtain nereid ethanol extract.

[0011] Neutral lipids were extracted using the Bligh and Dyer method. Nereis powder was fractionated into a methanol-water-soluble fraction and a chloroform layer. The chloroform layer was collected, the solvent evaporated, and then treated with chilled acetone to separate into an acetone-soluble and an acetone-insoluble fraction. The acetone-soluble fraction was the neutral lipids, and the nereis neutral lipid extract was obtained by evaporating the acetone on a rotary evaporator.

[0012] (4) In the formula feed of Penaeus vannamei, the above-mentioned nerei ethanol extract and neutral lipid extract were mixed in a ratio of 1:2 and added to the formula feed of Penaeus vannamei at a ratio of 1.2%. After adding 0.02% hemolytic phospholipids by weight, cholesterol was added at the same time at a level of 5% of the total fat level.

[0013] The developmental period of the sandworm 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: soak the fresh sandworms in 89 mg / L povidone iodine disinfectant for 3 hours, rinse with clean water and then soak in 21 mg / L bromine chloride disinfectant for 3 hours, freeze-dry after cleaning, and grind with a grinder until it passes through a 40-mesh sieve.

[0015] The present invention also includes a preparation method of the nerei extract and application of the extract in Litopenaeus vannamei feed.

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

[0017] (1) The method of the present invention can improve the nutritional composition 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) Promote the synthesis of sex steroid hormones in Penaeus vannamei, increase spawning volume by 30.3%, multiple spawning rate by 22.1%, average spawning times by 27.3%, and nauplii metamorphosis rate by 21.8%.

[0019] (3) The lugworm raw material used in this technology has the potential for low-cost large-scale production and is very operational. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] In the picture, the upper lugworm is cyan, and the lower one is orange-red;

[0022] Figure 2 Figure 2 shows the astaxanthin content of different types of nereids (farm 1).

[0023] Values ​​are presented as mean ± SD (n = 3);

[0024] Figure 3 Figure 2 shows the astaxanthin content of different body colors of sandworms (farm 2).

[0025] The values ​​are expressed as mean ± standard error (n = 3). Note: ** indicates significant differences between red and green lugworms, with significance levels of P < 0.01, respectively;

[0026] Figure 4 PCA principal component analysis of lipidomics of different colored sandworms (farm 2#)

[0027] The red dots in the picture are cyan lugworms, and the blue dots are orange-red lugworms;

[0028] Figure 5 Figure 2 shows the PLS-DA analysis of lipidomics of different colored nereids (farm 2).

[0029] The red dots in the picture are cyan lugworms, and the blue dots are orange-red lugworms;

[0030] Figure 6 Figure 2 shows the difference in lipids in lipid analysis of different colors of nereids (farm 2#)

[0031] The G1 / G2 / G3 samples in the picture are cyan sandworms, and the R1 / R2 / R3 are orange-red sandworms. DETAILED DESCRIPTION

[0032] The technical features of the present invention are further explained below through examples, but the protection scope of the present invention is not limited in any form by the examples.

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

[0034] 1. Sample preparation. This experiment took different batches and different colors of double-toothed nereids from Qingdao Jimo No. 1 breeding farm to analyze the nutritional components. A total of 5 batches were sampled, with 100g of each type of nereid in each batch. The samples from the 5 batches were mixed and analyzed for conventional nutritional components, and the values ​​were averaged. Each batch was analyzed for other components such as amino acids and fatty acids, and then statistical analysis was performed (SPSS25.0, analysis of variance). After sampling, it was identified that the development period was the "II" and "III" stages of gonadal development. The colors are divided into "orange-red" and "cyan" ( Figure 1 , in the picture, the upper sandworm is cyan and the lower one is orange-red).

[0035] 2. Nutrient composition analysis. Crude component analysis was performed according to the standard method of AOAC (2005). Samples were dried in a 105°C oven to constant weight for moisture analysis. The dried feed and shrimp samples were ground and pulverized 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 ignition method in a muffle furnace (Thermo F6000) after heating at 550°C for 8 hours.

[0036] Fatty acid composition was determined by gas chromatography. A 0.3 g sample was extracted using the chloroform-methanol method to extract total lipids. The sample was then cooled with cold water and 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 to mix thoroughly. The sample was then cooled in a water bath for 30 minutes. The sample was then extracted with 1 mL of n-hexane and 1 mL of pure water. The sample was then incubated on ice for 1 hour and centrifuged at 3000 rpm for 2 minutes. The supernatant was filtered through a membrane filter and added to a sample injection vial. The sample was then analyzed using a gas chromatograph (GC-2010 Pro, Shimadzu, Japan). Results are expressed as the percentage of each fatty acid relative to the total fatty acids.

[0037] Amino acids were analyzed using a fully automatic 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 diluted to 1350 mL, and 0.5 mL was taken and dried under nitrogen at 40°C. Dissolution was then added with 1 mL of 0.02 mol / L hydrochloric acid. After membrane filtration, the solution was added to a sample injection vial and analyzed. The analysis principle is to sequentially elute the amino acids in the sample using buffers of varying pH values ​​after ion exchange, based on their structure, acidity, polarity, and molecular size. The amino acids were then mixed with ninhydrin reagent and then transferred to a spiral reaction tube for a colorimetric reaction, resulting in a blue-purple product with a maximum absorption at 570 nm.

[0038] The astaxanthin content determination was entrusted to Qingdao Yuanxin Technology Testing Company for testing the astaxanthin content of red green sandworms.

[0039] 3. Test results.

[0040] From the perspective of conventional nutritional components (Table 1), the crude protein and crude fat contents of orange-red stage III lugworms were the highest, indicating the best nutritional composition.

[0041] Table 1 Nutritional analysis of lugworms at different developmental stages and colors (farm #1, % wet weight)

[0042] index Orange-red Phase II Orange-red stage III Cyan Phase II Cyan III 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 stage III showed the best performance in terms of total amino acid content and essential amino acid content, especially lysine, phenylalanine, and arginine, which are amino acids that play an important role in animal reproductive physiology.

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

[0045] amino acids Orange-red Phase II Orange-red stage III Cyan Phase II Cyan III P-value Arginine <![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 <![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 MethionineMet 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 <![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 Glutamate <![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 Gly 2.83±0.01 2.97±0.10 2.83±0.10 2.97±0.05 0.408 Proline Pro <![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 Tau <![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 <![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 acid; NEAA: non-essential amino acid; TAA: total amino acid; values ​​are expressed as "mean ± standard error"; one-way analysis of variance was performed using SPSS 25.0 version ANOVA and Duncan's method. There were significant differences between values ​​with different superscript letters in the same data (P < 0.05).

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

[0048] Table 3 Analysis of fatty acid composition of nereids at different developmental stages and colors (farm 1#, % total fatty acids)

[0049] fatty acid Orange-red Phase II Orange-red stage III Cyan Phase II Cyan III P-value 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-6 PUFA <![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-3 PUFA <![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 acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 series polyunsaturated fatty acid; n-3PUFA: n-3 series polyunsaturated fatty acid; values ​​are expressed as "mean ± standard error"; one-way analysis of variance was performed using SPSS 25.0 version ANOVA and Duncan's method. There were significant differences between the values ​​with different superscript letters in the same data (P < 0.05).

[0051] In terms of astaxanthin content ( Figure 2 ), the astaxanthin content of orange-red stage III nerei (0.52 mg / kg) was much higher than that of other groups, while the content of cyan stage II was only 0.32 mg / kg.

[0052] Example 2: Comparison of nutritional components of different colored lugworms from farm No. 2

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

[0054] A batch of samples was collected from the No. 2 breeding farm in Jimo, Qingdao. Only two colors (i.e., orange-red and cyan) of nereids in the "III" stage of gonadal development were obtained. The detection methods for each nutrient component were consistent with those in Example 1. Separate lipidomics analysis was commissioned by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0055] 2. Experimental results

[0056] The results of this batch showed that the crude protein and crude fat contents of orange-red lugworms were much higher than those of cyan lugworms (Table 4).

[0057] Table 4 Body composition of two colors of Perineuridae bicolor (% wet weight)

[0058] crude ingredient Moisture crude protein crude fat ash content Orange-red sandworm 79.04±1.02 <![CDATA[13.24±0.61 ** ]]> <![CDATA[2.55±0.18 ** ]]> <![CDATA[1.39±0.07 ** ]]> Cyan sandworm <![CDATA[87.60±0.71 ** ]]> 8.16±0.48 1.58±0.11 0.93±0.06

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

[0060] The 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 1#, but the content of lysine, arginine and essential amino acids in orange-red lugworms was still significantly higher than that in cyan lugworms (Table 5).

[0061] Table 5 Total amino acid composition of two colors of Perineuridae bicolor (% dry matter)

[0062] Complete amino acid composition Orange-red sandworm Cyan sandworm Essential amino acids (EAAs) Threonine 2.42±0.16 2.17±0.09 Valine 2.26±0.12 2.26±0.09 MethionineMet 1.17±0.09 1.16±0.03 Isoleucine Ile 2.20±0.15 2.12±0.08 Leucine 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 <![CDATA[4.57±0.23 * ]]> 3.56±0.13 ΣEAA <![CDATA[24.96±1.49 * ]]> 22.48±0.85 Non-essential amino acids ΣNEAA Taurine Tau 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 Glutamate 8.36±0.54 8.20±0.26 Glycine Gly 2.52±0.13 2.59±0.05 Alanine Ala 3.68±0.26 4.04±0.11 Cysteine ​​Cys 1.00±0.09 0.89±0.02 Tyrosine 1.69±0.08 1.71±0.05 Proline Pro 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: * , **, *** represent significant differences between red and green lugworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

[0064] In terms of free amino acids, the free amino acid composition of red and green double-toothed perinecrosis is shown in Table 6. The Thr content of green nereid was significantly higher than that of green nereid (P<0.05), but the Val, Ile, Leu, Phe, His, Glu, Ala, and Pro contents of orange-red nereid were significantly higher than those of red nereid (P<0.05).

[0065] Table 6 Free amino acid composition of red and green double-toothed perineri (mg / 100g)

[0066] Free amino acids Orange-red sandworm Cyan sandworm Essential amino acids (EAAs) Threonine 402.62±54.65 <![CDATA[726.99±20.56 ** ]]> Valine <![CDATA[204.08±11.47 * ]]> 125.99±17.02 MethionineMet 29.58±4.46 26.40±8.19 Isoleucine Ile <![CDATA[133.43±6.84 ** ]]> 63.47±6.31 Leucine <![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 145.50±11.76 137.54±5.35 ΣEAA 2739.34±228.67 2410.43±55.24 Non-essential amino acids ΣNEAA Taurine Tau 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 Glutamate <![CDATA[955.27±67.33 ** ]]> 545.13±26.51 Glycine Gly 454.79±33.33 493.80±7.32 Alanine Ala <![CDATA[2555.81±166.60 ** ]]> 1548.03±28.03 Cysteine ​​Cys 147.68±13.51 88.81±31.09 Tyrosine 164.29±16.25 135.17±9.10 Proline Pro <![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: * , **, *** represent significant differences between red and green lugworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

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

[0069] Table 7 Fatty acid composition of Perinereilia rubripes (% total fatty acids)

[0070]

[0071]

[0072] Note: SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 series polyunsaturated fatty acid; n-3PUFA: n-3 series polyunsaturated fatty acid; *, **, *** indicate significant differences between red and green lugworms, with significance levels of P<0.05, P<0.01, and P<0.001, respectively.

[0073] Astaxanthin content of red green double-toothed Perinereworm Figure 3 The results showed that the astaxanthin content in red nerei was significantly higher than that in green nerei (P<0.05).

[0074] The results of lipidomics analysis of different colored nereids (farm 2#) showed that a total of 578 lipid molecular types were successfully identified in the red and green groups of nereids. The results of PCA principal component analysis of the two groups of nereids showed that ( Figure 4 ), the clustering patterns of the two groups of nereids were very different, indicating significant differences in their fat metabolism patterns and lipid compositions, and also indicating differences in functional lipid composition. Similar results were obtained in the PLS-DA model validation ( Figure 5 ).

[0075] In terms of specific differential lipids ( Figure 6 ), the orange-red nereid had higher lipid contents 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 cyan nereid, but lower lipid contents in Cer (d18:1 / 17:0) and AEA (18:1) than the cyan nereid.

[0076] In summary, the comprehensive nutritional components of the orange-red sandworm at the gonad development stage III are much better than those of the cyan sandworm, especially in terms of lipids that are functionally active for animal gonad development.

[0077] Example 3. Different extraction and preparation processes for nereid extracts and their application in formula feeds for Litopenaeus vannamei broodstock Experiment 1. Preparation of nereid extracts.

[0078] Fresh Peri-necrotizing Sandworms were washed, freeze-dried and crushed, and sandworm extracts were prepared using pure water (double-distilled water), ethanol, petroleum ether and other solvents. Sandworm powder and ethanol solvent were mixed in a material-liquid 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 sandworm powder was further added with solvent for extraction. The above operation was repeated 3 times and the extracts were combined to obtain sandworm water extract, ethanol extract and petroleum ether extract.

[0079] Neutral lipids were extracted according to the Bligh and Dyer method. Nereis powder was fractionated into a methanol-water-soluble fraction and a chloroform layer. The chloroform layer was collected, the solvent evaporated, and then treated with chilled acetone to separate into an acetone-soluble and an acetone-insoluble fraction. The acetone-soluble fraction was the neutral lipids, and the acetone was evaporated on a rotary evaporator to obtain the nereis neutral lipid extract.

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

[0081] 2. Experimental design and feed preparation

[0082] A basal diet containing approximately 52% crude protein and 13% crude fat was prepared with fish meal as the primary protein source (see Table 8 for its composition and nutritional content). The control group received the basal diet (CON), while the experimental groups received 1.2% of a water extract (WE) of Nereis wormwood, an ethanol extract (AE), a petroleum ether extract (PE), a neutral lipid extract (NLF), or a mixture of an ethanol extract and neutral lipids (1:1, 1A1N group; or 1:2, 1A2N group). The experimental diets were prepared according to standard laboratory procedures. The raw materials were pulverized and thoroughly mixed to form 1 mm diameter strips. The strips were then steamed in a steamer for 15 minutes, dried at 55°C, crushed and sieved using a blender, and stored in a -20°C freezer until ready for use.

[0083] Table 8 Experimental feed formula and general nutritional composition (% dry matter)

[0084] Raw material ingredients CON WE AE PE NLF 1A1N 1A2N fishmeal 47 47 47 47 47 47 47 Krill meal 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 Mix 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 Calix 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 Nereis ethanol extract AE 0 0 1.2 0 0 0 0 Nereis petroleum ether extract PE 0 0 0 1.2 0 0 0 Neutral lipid extract NLF 0 0 0 0 1.2 0.6 0.8 General 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 content 12.15 12.21 12.27 12.16 11.98 11.58 11.22

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

[0086] The fatty acid composition of the experimental feed is shown in Table 9. As can be seen from the table, the saturated fatty acid content of each treatment group was low. The C14:0 content of the control group was significantly higher than that of the PE, 1A2N, and NLF groups, and the C22:0 content was significantly higher than that of the NLF and 1A2N groups. The SFA content of the control group was significantly higher than that of 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 of the NLF group was significantly higher than that of the control group. The C18:3n-6 content of the AE, 1A1N, and PE groups was significantly higher than that of the control group, and the C22:5n-3 content was significantly higher than that of the control group and the other treatment groups. The DHA content of the WE, 1A1N, and 1A2N groups was significantly higher than that of the control group and the other treatment groups. The n-3 / n-6 ratio of the AE and 1A1N groups was the highest (P<0.05).

[0087] Table 9 Fatty acids in each group of feed (% total fatty acids)

[0088]

[0089] Note: SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 series polyunsaturated fatty acid; n-3PUFA: n-3 series polyunsaturated fatty acid; values ​​are expressed as "mean ± standard error"; one-way analysis of variance was performed using SPSS 25.0 version ANOVA and Duncan's method. There were significant differences between the values ​​with different superscript letters in the same data (P < 0.05).

[0090] The amino acid composition of the experimental feed is shown in Table 10. As can be seen, the WE, 1A1N, and 1A2N groups had the highest total essential amino acid content. The control group had significantly higher Ile content than the PE and NLF groups, and significantly higher Lys content than the NLF group (P < 0.05). The WE, 1A1N, and 1A2N groups had the highest Phe, Lys, and total amino acid contents, but these did not differ significantly from the control group (P > 0.05).

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

[0092]

[0093] Note: One-way analysis of variance was performed using SPSS 25.0 version ANOVA and Duncan's method. The values ​​with different superscript letters in the same data group showed significant differences (P<0.05).

[0094] 3. Experimental shrimp and breeding management

[0095] Experimental shrimp were purchased from Bangpu Seed Technology Co., Ltd., Changyi, Weifang, Shandong Province. Male shrimp were a highly resistant strain, while female shrimp were both highly resistant and fast-growing strains. Initial body weight was 56.11 ± 0.95 g. The broodstock were randomly distributed into 15 ponds, each containing 22 shrimp (16 males and 6 females). Each group was fed three replicates of the diet. The shrimp were cultured in a static aquaculture system using deep-well seawater at a temperature of (26.5 ± 0.5)°C, a dissolved oxygen concentration of greater than 8 mg / L, a salinity of 31°C, a pH of 7.9–8.2, ammonia nitrogen ≤ 0.55 mg / L, and nitrite ≤ 0.045 mg / L. Water was changed daily, with a 70–75% water exchange rate. 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 fast at the end of the culture period, the number of surviving broodstock in the culture pond was recorded and weighed. All remaining female shrimp were sampled, and two male shrimp were randomly selected from each culture pond. The surface moisture was blotted with absorbent paper, and the weight was recorded using an electronic balance. Hemolymph was extracted from the abdomen using a sterile syringe and divided into centrifuge tubes containing 1 mL of anticoagulant. The tubes were allowed to stand for 4 hours, centrifuged at 4000 rpm for 10 minutes, and the supernatant was stored in a -20°C refrigerator. The broodstock were dissected alive on an ice tray, and the hepatopancreas and gonads were dissected out with surgical scissors and accurately weighed. The gonads and hepatopancreas were quickly frozen in liquid nitrogen and then stored in a -80°C refrigerator.

[0098] 5. Analysis methods

[0099] Crude composition analysis of feed and broodstock muscle: Crude composition analysis of feed and prawn muscle was performed according to the standard method of AOAC (2005). Samples were oven-dried at 105°C to constant weight for moisture analysis. Dried feed and prawn samples were ground and pulverized for crude protein, crude fat, and ash analysis. Crude protein was determined using the Kjeldahl method (FOSS KJELTEC 2300, Hillerod, Denmark); crude fat was determined using the chloroform-methanol method; and crude ash was determined using the high-temperature ignition method in a muffle furnace (Thermo F6000) after heating at 550°C for 8 hours.

[0100] Hepatopancreas and gonad fatty acid analysis: Fatty acid content in the samples was determined using gas chromatography. A 0.3 g sample was extracted using the chloroform-methanol method. The total lipid content was extracted using 2 mL of KOH-methanol and 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 to mix thoroughly. The sample was then cooled in a water bath for 30 minutes. Extraction was then performed with 1 mL of n-hexane and 1 mL of pure water. The sample was then incubated on ice for 1 hour and centrifuged at 3000 rpm for 2 minutes. The supernatant was filtered through a membrane and added to a sample injection vial. The sample was then analyzed using a gas chromatograph (GC-2010 Pro, Shimadzu, Japan). Results are expressed as the percentage of each fatty acid relative to the total fatty acids.

[0101] Amino acid analysis of hepatopancreas and gonads: The amino acid composition of fertilized eggs and nauplii was determined using an automatic amino acid analyzer (Hitachi L-8900 automatic amino acid analyzer, Hitachi, Japan). 0.3 g of sample was weighed into a test tube and added to 15 mL of 6 mol / L hydrochloric acid solution. The solution was then acid-hydrolyzed at 110°C for 24 h. The solution was then diluted to 50 mL, and 0.5 mL was taken and dried under nitrogen at 40°C. The solution was then dissolved in 1 mL of 0.02 mol / L hydrochloric acid. The sample was filtered through a filter and transferred to a vial for analysis. The analyzer uses an ion exchange column, followed by elution using buffers of varying pH values, based on the structure, acidity, polarity, and molecular size of the amino acids in the sample. The amino acids were then eluted sequentially using ninhydrin reagent, mixed individually, and then transferred to a spiral reaction tube for a colorimetric reaction, resulting in a blue-purple product with a maximum absorption at 570 nm.

[0102] Serum biochemical indexes were determined using triglyceride (A110-1-1), estradiol, progesterone, and testosterone kits produced by Nanjing Jiancheng Bioengineering Research 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. P < 0.05 was considered significant. The data values ​​were expressed as mean ± standard error.

[0105] Calculation formula:

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

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

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

[0109] Hepatopancreas index = hepatopancreas weight / female broodstock weight × 100%

[0110] Gonad index = gonad weight / female broodstock weight × 100%

[0111] 7. Experimental Results

[0112] Broodstock survival rate, average daily molt number, hepatopancreatic index, and gonadal index: Broodstock growth performance results 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). There were no significant differences between the other treatment groups and the control group (P > 0.05). The average daily molt number 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 there was no significant difference (P > 0.05). The gonadal index of the 1A2N group was significantly higher than that of the other groups except the PE group (P < 0.05).

[0113] Table 11 Survival rate, average daily molting number and hepatopancreas and gonadal index of female broodstock of each experimental group

[0114]

[0115] Note: Data in the same row with different letters indicate significant differences (P<0.05); data with the same letters or no letters indicate no significant differences (P>0.05).

[0116] Because the aforementioned indicators for AE and PE did not perform as expected, these two groups were removed from subsequent testing. Only WE, NLF, 1A1N, 1A2N, and the control group were tested, and the indicators were tested separately for males and females.

[0117] The crude component composition of broodstock muscle is shown in Table 12. As can be seen from the table, the moisture content of female shrimp in the CON group was significantly higher than that in the 1A1N, NFL, and 1A2N groups (P < 0.05), but did not differ significantly from the 1A1N group (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 the 1A2N group was significantly higher than that in the control group, while the crude fat content did not differ significantly from that in the control group (P < 0.05). There was no significant difference in ash content between male and female broodstock and the control group (P > 0.05).

[0118] Table 12 Crude component composition of broodstock muscle

[0119] crude ingredient CON WE NFL 1A1N 1A2N 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: Data in the same row with different letters indicate significant differences (P<0.05); data with the same letters or no letters indicate no significant differences (P>0.05).

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

[0122] Table 13 Fat content of hepatopancreas and gonads of broodstock in each treatment group (%)

[0123]

[0124] Note: Data in the same row with different letters indicate significant differences (P<0.05); data with the same letters or no letters indicate 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 22:5n-3 content 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 contents.

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

[0127] Fatty acid composition CON WE NFL 1A1N 1A2N 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 acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 polyunsaturated fatty acid; n-3PUFA: n-3 polyunsaturated fatty acid; Values ​​are expressed as "mean ± standard error"; Data in the same row with different letters in the shoulder indicate significant differences (P<0.05); Data in the same row with the same letter in the shoulder or no letter in the shoulder indicate no significant differences (P>0.05).

[0129] The fatty acid composition of the hepatopancreas of male shrimp is shown in Table 15. As can be seen from the table, the 17:1n-7 content in the 1A1N group was significantly lower than in the other treatment groups. The 20:4n-6 content in the 1A1N and WE groups was higher than in the control group, but there was no significant difference (P>0.05). The MUFA content in the 1A2N and NLF groups was higher than 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 in the control group, but there was no significant difference (P>0.05). The 1A2N group had the highest EPA and DHA content.

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

[0131] Fatty acid composition CON WE NFL 1A1N 1A2N 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 acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 polyunsaturated fatty acid; n-3PUFA: n-3 polyunsaturated fatty acid; Values ​​are expressed as "mean ± standard error"; Data in the same row with different letters in the shoulder indicate significant differences (P<0.05); Data in the same row with the same letter in the shoulder or no letter in the shoulder indicate no significant differences (P>0.05).

[0133] The fatty acid composition of the gonads of female shrimp is shown in Table 16. As can be seen from the table, 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 the other treatment groups. 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 contents.

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

[0135] Fatty acid composition CON WE NFL 1A1N 1A2N 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 acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 polyunsaturated fatty acid; n-3PUFA: n-3 polyunsaturated fatty acid; Values ​​are expressed as "mean ± standard error"; Data in the same row with different letters in the shoulder indicate significant differences (P<0.05); Data in the same row with the same letter in the shoulder or no letter in the shoulder indicate no significant differences (P>0.05).

[0137] The fatty acid composition of the male shrimp gonad is shown in Table 17. It can be seen from the table that 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), and 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 gonad (% total fatty acids)

[0139]

[0140]

[0141] Note: SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6PUFA: n-6 polyunsaturated fatty acid; n-3PUFA: n-3 polyunsaturated fatty acid; Values ​​are expressed as "mean ± standard error"; Data in the same row with different letters in the shoulder indicate significant differences (P<0.05); Data in the same row with the same letter in the shoulder or no letter in the shoulder indicate no significant differences (P>0.05).

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

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

[0144]

[0145]

[0146] Note: The values ​​are expressed as "mean ± standard error"; data in the same row with different letters in the shoulder indicate significant differences (P<0.05); data in the same row with the same letter or no letter in the shoulder indicate 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 of each treatment group was significantly higher than that of the control group, and the Pro content of the NFL group was significantly higher than that of 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] Amino acid composition CON WE NFL 1A1N 1A2N Essential amino acids (EAAs) Threonine 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 MethionineMet 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 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 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 acids ΣNEAA Taurine Tau 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 Glutamate 1.19±0.03 1.23±0.11 1.28±0.15 1.38±0.14 1.16±0.05 Glycine Gly 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 ​​Cys <![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 0.17±0.09 0.27±0.06 0.31±0.09 0.24±0.13 0.29±0.04 Proline Pro <![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: The values ​​are expressed as "mean ± standard error"; data in the same row with different letters in the shoulder indicate significant differences (P<0.05); data in the same row with the same letter or no letter in the shoulder indicate no significant differences (P>0.05).

[0151] The amino acid composition of the gonads of female shrimp is shown in Table 20. As can be seen from the table, the Tyr content in the NFL group was higher than that in the control group, but the difference was not 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] Amino acid composition CON WE NFL 1A1N 1A2N Essential amino acids (EAAs) Threonine 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 MethionineMet <![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 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 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 acids ΣNEAA Taurine Tau 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 Glutamate 1.45±0.09 1.52±0.14 1.64±0.06 1.62±0.10 1.76±0.16 Glycine Gly 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 ​​Cys <![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 <![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 Pro <![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: The values ​​are expressed as "mean ± standard error"; data in the same row with different letters in the shoulder indicate significant differences (P<0.05); data in the same row with the same letter or no letter in the shoulder indicate no significant differences (P>0.05).

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

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

[0157]

[0158]

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

[0160] Serum biochemical indices: The effects of adding nereid extract to the feed on serum biochemical indices of Litopenaeus vannamei are shown in Table 22. The progesterone content in female shrimp fed the 1A2N group was significantly higher than in the other groups except the 1A1N group (P < 0.05). The 1A2N group had the highest estradiol content, significantly higher than the WE and NFL groups (P < 0.05). Female shrimp fed the 1A2N group had the highest yolk protein content. The 1A1N group had a significantly higher total protein content than the other treatment groups (P < 0.05). The free fatty acid content in the 1A1N and 1A2N groups was higher than in the control group, but there was no significant difference (P > 0.05). The estradiol content in male shrimp fed 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 fed the 1A1N and 1A2N groups 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: The values ​​are expressed as "mean ± standard error"; data in the same row with different letters in the shoulder indicate significant differences (P<0.05); data in the same row with the same letter or no letter in the shoulder indicate no significant differences (P>0.05).

[0164] In terms of gene expression of sex steroid hormone synthesis, the effect of adding nereid extract to the feed on the hepatopancreas gene expression of male Litopenaeus vannamei is 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 nereid extract to feed on hepatopancreas gene expression in male shrimp

[0166] Gene CON WE NFL 1A1N 1A2N 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: steroidogenic acute regulatory protein; RXRA: retinoid X receptor α; 17β-HSD: 17-β-hydroxysteroid dehydrogenase 1; MF: maturation-stimulating factor; VGR: vitellogenin receptor. Values ​​are expressed as mean ± standard error. Data in the same row with different letters indicate significant differences (P < 0.05); data with the same letter or no letter indicate no significant differences (P > 0.05).

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

[0169] Table 24 Effect of adding nereid extract to feed on gene expression in female shrimp gonads

[0170] Gene CON WE NFL 1A1N 1A2N 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: steroidogenic acute regulatory protein; RXRA: retinoid X receptor α; VGR: vitellogenin receptor. Values ​​are expressed as mean ± standard error. Data in the same row with different letters indicate significant differences (P < 0.05); data with the same letter or no letter indicate no significant differences (P > 0.05).

[0172] Example 4. Experiment 2 on the application of nereid extracts prepared by different extraction processes and their application in formula feeds for Litopenaeus vannamei broodstock (pilot scale)

[0173] 1. Experimental feed and broodstock sources

[0174] The two groups, NFL and 1A2N, which performed best in the above results (plus a control group), were selected for a pilot-scale broodstock fortification 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 mixing using a step-by-step amplification method, water was added and stirred. The feed was then pressed into a pelletizer to form broodstock feed with a size of 1.5 mm x 3.0 mm. The feed was then dried at 50°C for 12 hours. After drying, it was stored in a cool, dark place until ready for use. The specific details are not repeated here.

[0175] Broodstock Source: 11-month-old Litopenaeus vannamei broodstock were obtained from a shrimp farm in Weifang, Shandong Province. No nutritional enrichment was performed. Females measured (12.10±0.58) cm in length, (32.61±3.91) g in weight, and had a hepatopancreatic index of (3.14±0.42)%. Gonadal development was absent in males. Three months prior to spawning, males measured (11.88±0.72) cm in length and (27.25±3.35) g in weight. Nutritional enrichment was initiated for both male and female broodstock for three months. Three replicates were used for each treatment, with 20 shrimp per replicate (a 1:1 ratio of male to female). After gonadal development, eyestalk clipping, artificial insemination, and hatching were performed according to standard production procedures. All procedures were performed at a farm in Weifang, Shandong Province, with the assistance of skilled labor. An eye tag was placed on one eye stalk of each broodstock for individual identification. All female broodstock underwent unilateral eyestalk removal using forceps and scalding. Artificial insemination was used to collect reproductive and larval development data.

[0176] 2. Sample collection and analysis methods

[0177] After artificial insemination, the egg production, hatchability, and nauplii metamorphosis rates of each culture tank were recorded. The spawning rate, multi-spawning rate, and average number of spawnings of female broodstock were calculated for each replicate tank. Approximately 50 fertilized eggs were collected from each of the first ten spawnings and stored in a 4% formaldehyde solution. The diameter of the fertilized eggs was subsequently measured. Approximately 0.2 g of fertilized eggs were collected from each of the first ten spawnings in each treatment group and stored in liquid nitrogen for determination of the fatty acid composition of the eggs.

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

[0179] Reproduction-related indicators are calculated according to the following formula:

[0180] Female broodstock spawning rate = number of broodstock that lay eggs / total number of broodstock that survived the fertilization and spawning period × 100%

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

[0182] Average number of spawning times = total number of spawning times of each parallel group during fertilization and spawning / number of spawning female broodstock, egg diameter = (long diameter + short diameter) / 2.

[0183] SPSS 19.0 statistical analysis software was used to perform ANOVA univariate analysis and Duncan's multiple comparison (P < 0.05) analysis on the data.

[0184] 3. Experimental results

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

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

[0187] index CON NFL 1A2N Weight gain rate of female broodstock (%) 48.62±3.73 49.63±5.62 58.10±2.63 Weight gain rate of male broodstock (%) 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 fatty acid composition of fertilized eggs (Table 26), the 1A2N group had higher EPA, DHA and PUFA contents.

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

[0191]

[0192]

[0193] Note: SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids. Fatty acid content is expressed as the ratio of each fatty acid methyl ester to total fatty acid methyl esters. Different superscript letters within the same group indicate significant differences (P < 0.05).

[0194] Judging from reproductive indicators such as egg production (Table 27), the 1A2N group had the highest egg production, significantly exceeding the control group by 30.3%. The NFL and 1A2N groups had significantly higher rates of multiple spawning, average number of eggs laid, and nauplii metamorphosis than the control group. The 1A2N group had significantly higher rates of multiple spawning, average number of eggs laid, and nauplii metamorphosis than the control group, respectively, by 22.1%, 27.3%, and 21.8%. The egg diameter of fertilized eggs in the NFL group was significantly higher than that in the control and 1A2N groups. There were no significant differences in egg production or hatchability among the groups.

[0195] Table 27 Egg production, fertilized egg hatching rate and nauplii metamorphosis rate of each treatment group

[0196] index CON NFL 1A2N Egg production (10,000) <![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 spawning 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 ]]> Fertilized egg hatching rate (%) 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 nereid extract, comprising the following steps: (1) Select the suitable nereid developmental stage and the bilaterian nereid of suitable body color; (2) disinfecting the nereid; (3) Extraction of active ingredients from nereid: Using the sample prepared in step (2), nereid powder and ethanol solvent were mixed in a material-liquid ratio of 8:1 and then extracted at 4°C for 12h. The supernatant was centrifuged and concentrated under reduced pressure in a rotary evaporator. The remaining nereid powder was further added with solvent for extraction. The above operation was repeated 3 times and the extracts were combined to obtain nereid ethanol extract. Neutral lipids were extracted using the Bligh and Dyer method. Nereis powder was fractionated into a methanol-water-soluble fraction and a chloroform layer. The chloroform layer was collected, the solvent evaporated, and then treated with chilled acetone to separate into an acetone-soluble and an acetone-insoluble fraction. The acetone-soluble fraction was the neutral lipids, and the nereis neutral lipid extract was obtained by evaporating the acetone on a rotary evaporator. (4) In the formula feed of Penaeus vannamei, the above-mentioned nerei ethanol extract and neutral lipid extract were mixed in a ratio of 1:2 and added to the formula feed of Penaeus vannamei at a ratio of 1.2%. After adding 0.02% hemolytic phospholipids by weight, cholesterol was added at the same time at a level of 5% of the total fat level.

2. The method for preparing the lugworm extract according to claim 1, wherein: The developmental period of the sandworm in step (1) is gonadal development stage III, and the suitable body color is orange-red.

3. The method for preparing the lugworm extract according to claim 1, wherein: The disinfection method described in step (2) is: soak the fresh sandworms in 89 mg / L povidone iodine disinfectant for 3 hours, rinse with clean water and then soak in 21 mg / L bromine chloride disinfectant for 3 hours, freeze-dry after cleaning, and grind with a grinder until it passes through a 40-mesh sieve.

4. Use of the preparation method of the lugworm extract according to any one of claims 1 to 3 in Litopenaeus vannamei feed.

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