Application of bile acid and cholesterol additives in promoting growth performance and regulating lipid metabolism of Litopenaeus vannamei

Abstract

The present invention discloses an application of an additive containing bile acid and cholesterol in promoting the growth performance of Penaeus vannamei and regulating lipid metabolism, and relates to the technical field of application of biological feed additives. The application is specifically: bile acid and cholesterol are added to the basic feed of Penaeus vannamei alone or together as feed additives. Beneficial effect: The present invention significantly improves the weight gain rate, specific growth rate, and feed conversion rate of Penaeus vannamei by adding lipid substances such as bile acid and cholesterol to the feed. The problem that the existing feed is easy to cause hepatopancreatic metabolic disorders in Penaeus vannamei is solved, and the yield of Penaeus vannamei is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological feed additives and their application, and in particular to the application of an additive containing bile acid and cholesterol in promoting the growth performance of Litopenaeus vannamei and regulating lipid metabolism. Background Art

[0002] In recent years, my country's shrimp farming industry has developed rapidly. According to the data of "China Fisheries Statistical Yearbook 2023", my country's shrimp production reached 1.66 million tons in 2022, of which the production of Penaeus vannamei reached 1.34 million tons. While pursuing high shrimp production, there are also prominent problems such as difficult prevention and control of diseases, drug abuse, and excessive and unbalanced nutrients in feed, which seriously restrict the green and healthy development of shrimp farming. At present, due to the excessive addition of protein and fat in shrimp compound feed and the replacement of plant protein, liver and pancreas metabolism disorders, fat accumulation, endotoxin increase and damage have been caused, which seriously damage the physiological health of shrimp and the sustainable development of the industry. It has become a research topic that needs to be urgently solved in the shrimp farming industry.

[0003] The Chinese patent application document with publication number CN108576495A discloses a breeding additive for broodstock of Penaeus vannamei and its application. Active ingredients such as dextran, coated sodium butyrate, nucleotides, antimicrobial peptides, and bacillus are used to improve the intestinal health and stress resistance of shrimp, improve the vitality and mating rate of shrimp, and reduce the frequency and management of disinfection drugs; emulsifiers and proteases are used to improve the digestion and absorption of high-protein and high-fat feeds by the liver and intestines to meet the broodstock's sharp demand for high nutrients during breeding; yeast, spirulina powder, carotene and other ingredients are used to improve the development quality of the hepatopancreas and gonads of broodstock by using their rich related pigment components and rich cell contents; and vitamin C, vitamin E, cholesterol and other ingredients are matched to improve the quality of gonads, shorten the maturation time of gonads, and improve the hatching rate of fertilized eggs and the survival rate of larvae. However, the patent does not involve the use of a certain proportion of lipid substances to promote the growth of Penaeus vannamei. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to solve the problem that the existing shrimp compound feed easily causes lipid metabolism disorder in Penaeus vannamei and affects the shrimp yield.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] The invention provides an application of an additive containing bile acid and / or cholesterol in promoting the growth performance of Penaeus vannamei and regulating lipid metabolism. The invention is characterized in that the bile acid and / or cholesterol are added to a basic feed of Penaeus vannamei as feed additives, the added amount of the bile acid is 0.04% of the total mass of the basic feed of Penaeus vannamei, the added amount of the cholesterol is 0.2-2.0% of the total mass of the basic feed of Penaeus vannamei, and the bile acid comprises the following main components: hyodeoxycholic acid, chenodeoxycholic acid, cholic acid and hyocholic acid.

[0007] Beneficial effects: The invention significantly improves the weight gain rate, specific growth rate and feed conversion rate of Penaeus vannamei by adding bile acid and / or cholesterol in a specific proportion to the feed. It solves the problem that the existing feed is prone to cause hepatopancreatic metabolic disorders in Penaeus vannamei and increases the yield of Penaeus vannamei.

[0008] Preferably, the mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, cholic acid and hyodeoxycholic acid is 9-12:19-22:3-5:3-5.

[0009] Preferably, the mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, cholic acid and hyodeoxycholic acid is 68-70:2-5:8-10:1-3.

[0010] Preferably, the added amount of cholesterol is 0.5-2.0% of the total mass of the basic feed of Penaeus vannamei.

[0011] Preferably, the added amount of cholesterol is 0.2-1.0% of the total mass of the basic feed of Penaeus vannamei.

[0012] Preferably, the Penaeus vannamei is juvenile Penaeus vannamei.

[0013] Preferably, the initial average weight of the juvenile Penaeus vannamei shrimp is 3.46 g / prawn.

[0014] Preferably, the basic feed for Penaeus vannamei is composed of the following ingredients in percentage by mass:

[0015]

[0016] Preferably, the daily feeding amount is 5% of the body weight of the vannamei shrimp, and the feeding is done twice a day.

[0017] The third aspect of the present invention provides a compound feed for Penaeus vannamei, which contains one or more of the above-mentioned bile acid A, bile acid B and cholesterol.

[0018] The advantages of the present invention are:

[0019] Beneficial effects: The invention significantly improves the weight gain rate, specific growth rate and feed conversion rate of Penaeus vannamei by adding bile acid, cholesterol and other lipid substances to the feed. It solves the problem that the existing feed is easy to cause hepatopancreatic metabolic disorders in Penaeus vannamei, and improves the yield of Penaeus vannamei. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparative diagram of the effect of adding bile acid and / or cholesterol to the feed on the hepatopancreas tissue sections of Penaeus vannamei in this Example 1, Figure A is a positive control group (commercially available bile acid); Figure BF is a bile acid A group (A1-A5); Figure GK is a bile acid B group (B1-B5); Figure LP is a cholesterol group (C1-C5); Figure QU is a bile acid A and cholesterol mixed group (A3+(C1-C5)); Figure V is a negative control group (no addition);

[0021] Figure 2 This is a comparative diagram of the effect of adding bile acid and / or cholesterol to the feed on the intestinal tissue structure of Penaeus vannamei in this Example 1, Figure A is a positive control group (commercially available bile acid); Figure BF is a bile acid A group (A1-A5); Figure GK is a bile acid B group (B1-B5); Figure LP is a cholesterol group (C1-C5); Figure QU is a bile acid A and cholesterol mixed group (A3+(C1-C5)); Figure V is a negative control group (no addition);

[0022] Figure 3 This is a statistical diagram of the number of taxa of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0023] Figure 4 This is a taxonomic composition analysis of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0024] Figure 5 This is a classification hierarchy tree diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0025] Figure 6 This is a phylogenetic tree diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0026] Figure 7 This is an Alpha diversity index diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0027] Figure 8 It is a sparse curve diagram of the effects of different feed additives on the intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0028] Fig. 9 This is a species accumulation curve diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0029] Fig.10 This is a richness grade curve diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0030] Fig.11 The distance matrix and PCOA analysis diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0031] Fig.12 This is an NMDS analysis diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0032] Fig.13 This is a partial clustering hierarchical analysis diagram of the effects of different feed additives on intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention;

[0033] Fig.14 This is a partial inter-group difference analysis diagram of the effects of different feed additives on the intestinal microorganisms of Penaeus vannamei in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0036] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0037] Bile acid A, bile acid B and cholesterol used in the following examples were all provided by Anhui Kebao Biotechnology Co., Ltd. (the components of bile acid A and bile acid B are shown in Table 1 below). The basic feed for Penaeus vannamei was produced by New Hope Group Co., Ltd. (the basic feed components are shown in Table 2 below).

[0038] Table 1: Composition of bile acids A and B

[0039]

[0040] The mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, cholic acid and hyodeoxycholic acid in bile acid A can be 9:19:3:3, 12:22:5:5, 10:21:4:4 or 10.5:20.8:3.96:3.64, etc.

[0041] The mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, cholic acid and hyodeoxycholic acid in bile acid B can be 68:2:8:1, 70:5:10:3, 69:4:9:2 or 69.88:3.14:9.58:2.04, etc.

[0042] Table 2: Formula composition of basic feed for Penaeus vannamei (by mass percentage 100%)

[0043]

[0044] Embodiment 1:

[0045] 1. Materials

[0046] 1.1 Feed preparation

[0047] The basic feed of Penaeus vannamei was crushed by a grinder and then passed through an 80-mesh sieve. An appropriate amount of bile acid A, bile acid B, cholesterol or a mixture of bile acid A and cholesterol (the specific amount added is shown in Table 3 below) was added to the basic feed. After being mixed evenly, the feed particles of appropriate size were made by a twin-screw extruder. After drying at 60°C, the particles were sealed in a sealing bag and stored in a refrigerator at -20°C. (The bile acid A and bile acid B used were produced from the same batch. The components were tested. The main components and mass percentages of bile acid A were: 10.5% hyodeoxycholic acid, 20.8% chenodeoxycholic acid, 3.96% cholic acid and 3.64% hyodeoxycholic acid; the main components and mass percentages of bile acid B were: 69.88% hyodeoxycholic acid, 3.14% chenodeoxycholic acid, 9.58% cholic acid and 2.04% hyodeoxycholic acid)

[0048] 1.2 Juvenile Penaeus vannamei

[0049] The juvenile Penaeus vannamei shrimp used in the experiment were provided by the aquaculture base of Shandong Agricultural University. The average initial weight of the juvenile shrimp was 3.46g / each. 3 In the cage (100cm×100cm×140cm), the water temperature and pH are 23-31°C and 8.1 respectively. The daily feed amount is 5% of the shrimp body weight, and feeding is done twice a day (6 am and 6 pm).

[0050] 2. Methods

[0051] 2.1 Experimental group design

[0052] 6600 healthy young Penaeus vannamei were randomly divided into 22 groups [5 groups of bile acid A, 5 groups of bile acid B, 5 groups of cholesterol, 5 groups of bile acid A + cholesterol, positive control group (commercially available bile acid products), negative control group (no bile acid, cholesterol added, only basic feed)], 3 replicates per group, 100 Penaeus vannamei in each replicate (the results were averaged). The basic feed was used for two weeks before the start of the experiment, and the formal feeding experiment began after stabilization. The test period was 60 days, and samples were collected on the 30th and 60th days of feeding.

[0053] Table 3: Addition amount of each group (calculated as a percentage of the total mass of the basic feed)

[0054]

[0055] 3. Data processing and statistical methods

[0056] SPSS24.0 software was used for statistical calculations. All data were expressed as mean ± standard deviation (Mea ± SE). One-way ANOVA was used for analysis, with P < 0.05 as the significant level of difference.

[0057] The physiological health index data were normalized by z-score to make the same index in each treatment group at the same comparison level, and heatmap was drawn using R software (Version 3.5.3).

[0058] Survival rate calculation: Survival rate = final number of survivors / total number released initially.

[0059] Weight gain rate (WGR, %) = (Wt-W0) / W0×100; W0 (g) is the initial average weight of juvenile shrimps, and Wt (g) is the final average weight of shrimps.

[0060] 4. Results

[0061] 4.1 Effects of different feed additives on the survival rate and growth performance of Penaeus vannamei

[0062] As shown in Table 4 below, the addition of bile acid A, B, and cholesterol had a significant effect on the weight gain rate of Penaeus vannamei (p<0.05), but had no significant effect on the survival rate (p>0.05).

[0063] Table 4: Effects of different feed additives on the survival and growth performance of Penaeus vannamei

[0064]

[0065]

[0066] As can be seen from Table 4, the 60-day weight gain rate of Penaeus vannamei in groups B3, C2, C3, C4, C5 and A3+(C1-C5) was significantly higher than that in the control group.

[0067] Sample collection and preparation:

[0068] Fifteen shrimps were randomly selected from each group. Hemolymph was sampled by using a syringe to draw 1 ml of anticoagulant (10 mM EDTA-Na2, 0.34 M NaCl and 30 mM trisodium citrate, pH 7.0, and the osmotic pressure was adjusted to 780 mOsm kg-1 with 0.115 M glucose) in advance, and hemolymph was extracted from the shrimp abdominal blood sinus at a ratio of 1:1. A part of the hemolymph sample was used for the determination of blood cell count and phagocytosis index, and the remaining samples were centrifuged at 4 ° C and 800 × g for 10 min, and the supernatant samples were collected. The hepatopancreas was dissected and ground in a mortar with liquid nitrogen. All samples were stored in a -80 ° C refrigerator.

[0069] 4.2 Effects of different feed additives on the activities of enzymes related to protein and fat metabolism in Penaeus vannamei

[0070] Table 5: Effects of different feed additives (30 days) on the activities of enzymes related to protein and fat metabolism in Penaeus vannamei

[0071]

[0072] Table 5 shows that after feeding with different feed additives for 30 days, no conclusions were drawn on the indicators of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C);

[0073] The results of alkaline phosphatase showed that the addition of bile acid A (A1-A5) and bile acid and cholesterol mixture (A3+(C1-C5)) to feed increased the activity of AKP in shrimp serum, among which bile acid (A1-A5) had the best effect, while the positive control group (commercial bile acid) reduced the activity of AKP, and the bile acid B group (B1-B5) and cholesterol group (C1-C5) had no obvious effect.

[0074] The results of acid phosphatase showed that the activity of ACP in the serum of shrimps was enhanced after the bile acid B group (B1-B5) was added to the feed, while the cholesterol group (C1-C5), the bile acid and cholesterol mixed group (A3+(C1-C5)), and the positive control group (commercial bile acid) reduced the activity of ACP. The result of adding bile acid A alone was not obvious.

[0075] The results of triglyceride (TG) showed that the addition of different ingredients to feed reduced the serum triglyceride content of shrimp, and the positive control (commercial bile acid) had the strongest inhibitory effect;

[0076] The results of α-amylase (AMS) activity showed that the negative control group (no addition) had the highest activity, and the addition of ingredients reduced the AMS activity. Bile acid A, bile acid B, cholesterol (bile acid and cholesterol mixed), and positive control (commercial bile acid) all reduced the AMS activity in shrimp serum, among which the positive control (commercial bile acid) group had the strongest inhibitory effect.

[0077] The results of aspartate aminotransferase (AST / GOT) showed that the addition of different ingredients to the feed reduced the activity of aspartate aminotransferase, with bile acid B having the strongest inhibitory effect and (bile acid and cholesterol mixed) group having the weakest inhibitory effect;

[0078] The results of alanine aminotransferase (ALT / GPT) showed that adding different ingredients to the feed reduced the activity of alanine aminotransferase, and bile acid B had the strongest inhibitory effect.

[0079] Table 6 shows that after 60 days of feeding with different feed additives, the results of high-density lipoprotein cholesterol (HDL-C) showed that the addition of different ingredients to the feed reduced the serum HDL-C content of shrimp, and the (bile acid and cholesterol mixed) group (A3+(C1-C5)) had a stronger inhibitory effect;

[0080] No results were obtained for the low-density lipoprotein cholesterol (LDL-C) index;

[0081] The results of alkaline phosphatase showed that adding different ingredients to the feed increased the activity of AKP in the serum of shrimp, among which bile acid A had the best effect, while the positive control (commercial bile acid) reduced the activity of AKP.

[0082] The results of acid phosphatase showed that the ACP activity in the serum of shrimp did not change significantly after adding different ingredients to the feed, among which the cholesterol group increased the ACP activity, while the positive control (commercial bile acid) group decreased the ACP activity;

[0083] The triglyceride (TG) results showed that bile acid A and cholesterol increased the serum triglyceride content of shrimp, while bile acid B and the positive control (commercial bile acid) reduced the serum triglyceride content of shrimp. When bile acid and cholesterol were mixed and fed, the effect was not much different from that of the negative control group (no addition).

[0084] The results of α-amylase (AMS) activity showed that the negative control group (no addition) had the highest activity, and the addition of ingredients reduced the AMS activity. Bile acid A, bile acid B, cholesterol, (bile acid mixed with cholesterol), and positive control (commercial bile acid) all reduced the AMS activity in shrimp serum, among which the (bile acid mixed with cholesterol) group had the strongest inhibitory effect.

[0085] The results of aspartate aminotransferase (AST / GOT) showed that the activity of aspartate aminotransferase was reduced after adding different ingredients to the feed, the positive control (commercial bile acid) had the strongest inhibitory effect, and the addition of cholesterol alone had the weakest inhibitory effect;

[0086] The results of alanine aminotransferase (ALT / GPT) showed that the activity of alanine aminotransferase was reduced after adding different ingredients to the feed, Control1 had the strongest inhibitory effect, and adding cholesterol alone had the weakest inhibitory effect.

[0087] Table 6: Effects of different feed additives (60 days) on the activities of enzymes related to protein and fat metabolism in Penaeus vannamei

[0088]

[0089] 4.3 Effects of bile acid on immune defense parameters of Litopenaeus vannamei

[0090] As shown in Figure 7, the addition of bile acid or cholesterol to feed had a significant effect on the number of blood cells, phagocytosis rate and the control group (P<0.05), while there was no significant change (P>0.05). After 30 days of feeding, the number of blood cells, phagocytosis rate and plasma antibacterial and lytic activity of shrimp were significantly higher than those of the control group. After 60 days of feeding, the number of blood cells and phagocytosis rate of shrimp were significantly higher than those of the control group.

[0091] Table 7: Statistics of cell number and phagocytosis rate

[0092] Group <![CDATA[Original cell count X10 6 > <![CDATA[Cell count in 30 days X10 6 > <![CDATA[Cell count in 60 days X10 6 > Original phagocytosis rate% 30-day phagocytosis rate% 60-day phagocytosis rate% A1 7.733±2.203 8.933±0.924 8.000±0.693 20.667±2.517 25.667±3.512 26.333±1.528 A2 7.733±2.203 9.467±1.222 7.600±1.442 20.667±2.517 24.333±0.577 26.667±2.517 A3 7.733±2.203 9.733±0.833 7.467±0.833 20.667±2.517 25.667±3.055 27.333±4.163 A4 7.733±2.203 10.270±0.462 9.067±0.231 20.667±2.517 26.000±3.000 27.000±1.732 A5 7.733±2.203 9.733±1.405 8.800±1.442 20.667±2.517 26.667±2.517 27.333±4.163 B1 7.733±2.203 8.533±1.155 7.733±1.155 20.667±2.517 25.333±3.786 24.667±0.577 B2 7.733±2.203 8.933±1.222 7.600±1.833 20.667±2.517 25.667±0.577 27.000±2.646 B3 7.733±2.203 9.200±0.800 8.533±1.155 20.667±2.517 26.000±2.646 26.333±2.517 B4 7.733±2.203 9.333±0.833 8.400±1.058 20.667±2.517 25.667±2.887 27.000±1.732 B5 7.733±2.203 9.467±1.665 8.533±1.155 20.667±2.517 27.000±2.000 27.333±4.163 C1 7.733±2.203 8.933±0.833 8.133±1.286 20.667±2.517 25.000±1.732 25.667±4.041 C2 7.733±2.203 9.333±1.155 8.267±0.833 20.667±2.517 26.333±3.055 26.333±2.517 C3 7.733±2.203 9.467±1.222 8.133±1.665 20.667±2.517 25.333±2.517 27.333±3.512 C4 7.733±2.203 9.867±1.007 8.400±1.744 20.667±2.517 25.667±2.082 27.000±1.000 C5 7.733±2.203 9.06 / ±1.804 8.533±1.665 20.66 / ±2.517 26.333±3.512 27.66 / ±1.155 A3+C1 7.733±2.203 8.533±1.405 8.267±1.007 20.667±2.517 25.000±2.646 26.000±4.000 A3+C2 7.733±2.203 9.733±0.611 8.400±1.744 20.667±2.517 25.667±3.786 27.667±2.517 A3+C3 7.733±2.203 9.867±0.833 9.067±0.611 20.667±2.517 26.667±3.215 28.000±4.583 A3+C4 7.733±2.203 9.067±1.007 8.800±1.058 20.667±2.517 27.333±4.933 28.667±1.528 A3+C5 7.733±2.203 9.600±1.442 9.200±1.058 20.667±2.517 28.333±4.933 28.333±3.512 D1 7.733±2.203 9.200±1.442 8.533±2.203 20.667±2.517 26.333±2.082 27.333±3.512 E1 7.733±2.203 8.667±1.007 7.467±1.222 20.667±2.517 24.667±2.517 25.333±3.512

[0093] 4.4 Effects of bile acid on the hepatopancreas structure of Litopenaeus vannamei

[0094] At 60 days of culture, microscopic observations were performed on the hepatopancreas of each group of Penaeus vannamei (e.g. Figure 1As shown in Figure A, the hepatopancreas histological structure of the positive control group (commercially available bile acid) was basically intact, and the rest of the structures were basically consistent with the characteristics of a healthy state. Figure B (bile acid A1 group) showed irregular arrangement of the hepatic ducts, loose arrangement of the connective tissue in the interstitium, and a small amount of granulocyte infiltration; there were many round vacuoles of different sizes in the hepatic duct epithelium; Figure C (bile acid A2 group) the hepatopancreas parenchyma was composed of well-developed and multi-branched hepatic ducts, the hepatic duct wall was composed of basement membrane and epithelium, the epithelium was a single layer of columnar cells, a small amount of epithelial cytoplasm vacuolation was visible, and inflammatory cell infiltration was occasionally seen in the interstitium. Figure D (bile acid A3 group) the hepatopancreas parenchyma was composed of well-developed and multi-branched hepatic ducts, the hepatic duct wall was composed of basement membrane and epithelium, some hepatic duct epithelium was flattened, and occasionally the hepatic duct epithelium cytoplasm vacuolation was seen, and a small amount of inflammatory cell infiltration was seen in the interstitium. Figure E (bile acid A4 group) The hepatopancreas is composed of well-developed and multi-branched hepatic ducts, with occasional hepatic duct epithelial shedding, a large number of epithelial cytoplasmic vacuoles, and occasional inflammatory cell infiltration in the liver interstitium. Figure F (bile acid A5 group) The hepatopancreas is irregularly arranged in the local hepatic ducts, with a small amount of hepatic duct necrosis, pyknosis and deep staining of the nuclei, and a large number of inflammatory cell infiltration in the interstitium. Figure G (bile acid B1 group) The hepatopancreas is composed of well-developed and multi-branched hepatic ducts, and the hepatic duct wall is composed of basement membrane and epithelium. The epithelium is a single layer of columnar cells, and a large number of hepatic duct epithelial cytoplasm vacuoles, the lumen is pentagonal or quadrangular star-shaped, and the connective tissue is rich in blood sinusoids. No obvious abnormalities are observed. Figure H (bile acid B2 group) The hepatic tubules in the hepatopancreas are disordered, with many hepatic tubules necrosis, nuclear fragmentation, increased eosinophilia, many irregular hepatic tubules, flattened epithelium, mild hyperplasia of local connective tissue in the interstitium, and more inflammatory cell infiltration. Figure I (bile acid B3 group) The hepatic tubules in the hepatopancreas are irregularly arranged, with occasional necrosis and shedding of hepatic tubular epithelium, nuclear fragmentation, cytoplasmic vacuolization of a small number of hepatic tubular epithelium, and a small amount of inflammatory cell infiltration in the interstitium. Figure J (bile acid B4 group) The hepatic tubules in the hepatopancreas in the bile acid group are irregularly arranged, with a small amount of necrosis and shedding of hepatic tubular epithelium, nuclear fragmentation, occasional cytoplasmic vacuolization of hepatic tubular epithelium, and a small amount of inflammatory cell infiltration in the interstitium. Figure K (bile acid B5 group) shows that the local hepatic ducts of the hepatopancreas are irregularly arranged, a small amount of hepatic tubular epithelial necrosis and detachment, nuclear fragmentation, occasional hepatic tubular epithelial cytoplasm vacuolation, and a large number of inflammatory cell infiltration can be seen in the interstitium. Figure L (cholesterol C1 group) shows that the hepatopancreas is composed of well-developed and multi-branched hepatic ducts. The hepatic duct wall is composed of basement membrane and epithelium. The epithelium is a single layer of columnar cells. A large number of hepatic tubular epithelial cytoplasm vacuolation, the lumen is pentagonal or quadrangular star-shaped, and occasional inflammatory cells are scattered in the interstitium. Figure M (cholesterol C2 group) shows that the local hepatic ducts of the hepatopancreas are irregularly arranged, occasional hepatic tubular epithelial detachment, occasional hepatic tubular epithelial cytoplasm vacuolation, and a small amount of inflammatory cell infiltration can be seen in the interstitium. Figure N (cholesterol C3 group) shows that the hepatopancreas occasionally has hepatic tubular epithelial detachment, a small amount of hepatic tubular epithelial cytoplasm vacuolation, and a small amount of inflammatory cell infiltration can be seen in the interstitium.Figure O (cholesterol C4 group) shows that the local hepatic tubules of the hepatopancreas are irregularly arranged, with occasional necrosis and shedding of hepatic tubule epithelium, nuclear fragmentation, and a large number of hepatic tubule epithelial cytoplasm vacuolation, and occasional inflammatory cell infiltration in the interstitium. Figure P (cholesterol C5 group) shows that the hepatopancreas is composed of well-developed and multi-branched hepatic tubules. The walls of the hepatic tubules are composed of basement membrane and epithelium. The epithelium is a single layer of columnar cells. Vacuolation of the cytoplasm of the tubule epithelium is widely seen, and the lumen is pentagonal or quadrangular. The connective tissue is rich in blood sinusoids, and no obvious abnormalities are found. Figure Q (A3+C1 group) shows that the local hepatic tubules of the hepatopancreas are irregularly arranged, with a large number of necrosis and shedding of hepatic tubule epithelium, nuclear fragmentation, and a large number of inflammatory cell infiltration in the interstitium. Figure R (A3+C2 group) shows that the hepatopancreas is composed of well-developed and multi-branched hepatic ducts. The hepatic duct wall is composed of basement membrane and epithelium. The epithelium is a single layer of columnar cells. A large number of hepatic duct epithelial cytoplasm vacuolation can be seen, and inflammatory cell infiltration can be seen occasionally in the interstitium. Figure S (A3+C3 group) shows that the hepatopancreas is composed of well-developed and multi-branched hepatic ducts. The hepatic duct wall is composed of basement membrane and epithelium. The epithelium is a single layer of columnar cells. A large number of hepatic duct epithelial cytoplasm vacuolation can be seen, and inflammatory cell infiltration can be seen occasionally in the interstitium. Figure T (A3+C4 group) shows that hepatopancreas occasionally has hepatic duct epithelial shedding, a small amount of hepatic duct epithelial cytoplasm vacuolation can be seen (yellow arrow), and a small amount of inflammatory cell infiltration can be seen in the interstitium. Figure U (A3+C5 group) shows that the hepatopancreas occasionally has necrosis and shedding of hepatic tubule epithelium, nuclear fragmentation, and a large number of hepatic tubule epithelial cytoplasm vacuolation, and a small amount of inflammatory cell infiltration can be seen in the interstitium (red arrow). Figure W (negative control) shows that the hepatopancreas is composed of well-developed and multi-branched hepatic tubules, and the hepatic tubule wall is composed of basement membrane and epithelium. The epithelium is a single layer of columnar cells, with occasional epithelial cytoplasm vacuolation, and a small amount of inflammatory cell infiltration can be seen in the interstitium.

[0095] 4.5 Effects of bile acid on the intestinal tissue structure of Penaeus vannamei

[0096] At 60 days of the culture experiment, the intestinal microstructure of Penaeus vannamei was observed (e.g. Figure 2As shown), it was found that no pathological changes were found in the positive control group of Figure A, and Figure B showed that the mucosal layer was widely defoliated in the intestinal tissue, the muscle fibers of the muscular layer were arranged irregularly, and a small amount of inflammatory cell infiltration was visible. Figure C showed that a small amount of mucosal epithelium was defoliated in the intestinal tissue, and the remaining mucosal epithelium was a single-layer columnar epithelium. The local striated border structure of the mucosal surface was unclear, no obvious goblet cells were found, the muscle fibers of the muscular layer were arranged regularly, and occasional inflammatory cell infiltration was seen. Figure D showed that mucosal epithelial cells were widely defoliated in the intestinal tissue, the muscle fibers of the muscular layer were arranged irregularly, and a small amount of inflammatory cell infiltration was seen. Figure E showed that the necrosis of the mucosal epithelium disappeared and was replaced by connective tissue hyperplasia in the intestinal tissue, the muscle fibers of the muscular layer were arranged irregularly, and a large amount of inflammatory cell infiltration was seen. Figure F showed that the mucosal layer of the intestinal tissue was significantly thickened, and the necrosis of the mucosal epithelium disappeared and was replaced by connective tissue hyperplasia, the muscle fibers of the muscular layer were arranged irregularly, and a large amount of inflammatory cell infiltration was seen. Figure G shows that a large number of mucosal epithelial cells have fallen off in the intestinal tissue, and the rest of the mucosal epithelium is a single layer of columnar epithelium. The local striated border structure of the mucosal surface is unclear, no obvious goblet cells are seen, the mucosal layer folds into the intestinal cavity to form intestinal villi, the muscle fibers of the muscular layer are arranged irregularly, and inflammatory cell infiltration is occasionally seen. Figure H shows that the mucosal epithelium has widely disappeared and replaced by connective tissue hyperplasia, the muscle fibers of the muscular layer are arranged irregularly, and a large number of inflammatory cell infiltrations can be seen. Figure I shows that a large number of mucosal epithelial cells have fallen off in the intestinal tissue, and the rest of the mucosal epithelium is a single layer of columnar epithelium. The striated border structure of the mucosal surface is unclear, a small amount of mucosal epithelial degeneration can be seen, the cytoplasm is loose and lightly stained, no obvious goblet cells are seen, the muscle fibers of the muscular layer are arranged irregularly, and a small amount of inflammatory cell infiltration can be seen. Figure J shows that the intestinal tissue mucosal epithelial structure is intact, which is a single layer of columnar epithelium. The striated border structure of the mucosal surface is unclear, no obvious goblet cells are seen, the muscle fibers of the muscular layer are arranged irregularly, and a small amount of inflammatory cell infiltration can be seen. Figure K shows that the intestinal tissue is widely visible that the mucosal epithelium has disappeared and is replaced by connective tissue hyperplasia. The muscle fibers of the muscular layer are arranged irregularly, and a large amount of inflammatory cell infiltration can be seen. Figure L shows that a large number of mucosal epithelial cells have fallen off in the intestinal tissue, and the rest of the mucosal epithelium is a single layer of columnar epithelium. The striated border structure of the mucosal surface is unclear, no obvious goblet cells are seen, the muscle fibers of the muscular layer are arranged irregularly, and a small amount of inflammatory cell infiltration can be seen. Figure M shows that the mucosal layer has fallen off widely in the intestinal tissue, the muscle fibers of the muscular layer are arranged irregularly, and a small amount of inflammatory cell infiltration can be seen. Figure N shows that the mucosal layer has fallen off widely in the intestinal tissue, the muscle fibers of the muscular layer are arranged irregularly, and a small amount of inflammatory cell infiltration can be seen. Figure O shows that the intestinal tissue mucosal epithelial structure is intact, which is a single-layer columnar epithelium, with a clear striated border structure on the mucosal surface, and the mucosal layer folds into the intestinal cavity to form intestinal villi. No obvious goblet cells are seen, and inflammatory cell infiltration is occasionally seen. Figure P shows that a small amount of mucosal epithelial cells can be seen falling off in the intestinal tissue, and the remaining mucosal epithelium is a single-layer columnar epithelium with a clear striated border structure. More mucosal epithelial degeneration can be seen, the cytoplasm is loose and lightly stained, the mucosal layer folds into the intestinal cavity to form intestinal villi, no obvious goblet cells are seen, and the muscle fibers of the muscular layer are irregularly arranged, and more inflammatory cell infiltration can be seen.Figure Q shows that the intestinal tissue mucosal epithelial structure is intact, the mucosal epithelium is a single layer of columnar epithelium, the striated border structure is clear, the mucosal layer folds into the intestinal cavity to form intestinal villi, no obvious goblet cells are seen, the muscle fibers of the muscular layer are arranged regularly, and a small amount of inflammatory cell infiltration can be seen. Figure R shows that a large number of mucosal cells can be seen falling off in the intestinal tissue, the remaining mucosal epithelium is flattened, the muscle fibers of the muscular layer are arranged irregularly, and no obvious inflammatory cell infiltration is seen. Figure S shows that a small number of mucosal epithelial cells can be seen falling off in the intestinal tissue, the remaining mucosal epithelium is a single layer of columnar epithelium, the striated border structure is clear, the mucosal layer folds into the intestinal cavity to form intestinal villi, no obvious goblet cells are seen, the muscle fibers of the muscular layer are arranged irregularly, and a small amount of inflammatory cell infiltration can be seen. Figure T shows that the intestinal tissue mucosal epithelial structure is intact, the epithelium is flattened, a large number of epithelial cells are degenerated, the cytoplasm is loose and lightly stained, the muscle fibers of the muscular layer are arranged irregularly, and occasional inflammatory cell infiltration is seen. Figure U shows that a small amount of mucosal epithelial cells can be seen falling off in the intestinal tissue, and the rest of the mucosal epithelium is a single layer of columnar epithelium. The structure of the local striated border on the mucosal surface is unclear, and the mucosal layer folds into the intestinal cavity to form intestinal villi. No obvious goblet cells are seen, and the muscle fibers of the muscular layer are arranged irregularly. A small amount of inflammatory cell infiltration can be seen. Figure U shows that the structure of the striated border on the mucosal surface is unclear, and some inflammatory cells are infiltrated.

[0097] 4.6 Effects of different feed additives on intestinal microorganisms of Penaeus vannamei

[0098] Figure 3 to Figure 14 In the above, XC1~XC22 correspond to A1~A5, B1~B5, C1~C5, A3+(C1~C5), D1, and E1 respectively. Figure 3 to Figure 14 C1 in the figure is a sample of Penaeus vannamei before the start of the rearing experiment.

[0099] 4.6.1 Species composition analysis

[0100] By statistically analyzing the ASV / OTU table after sampling, we can obtain the specific composition table of the microbial community in each sample at each classification level. Through this table, we can first calculate the number of taxonomic units contained in different samples at each classification level. Figure 3 As shown, there were significant differences between the control group and each treatment group at the order, family and genus classification levels (P < 0.05).

[0101] The stacked bar chart of species composition, or simply the bar chart, is the most commonly used method to characterize the species composition of multiple samples. By statistically analyzing the feature table after removing the singleton, the composition distribution of each sample at the six classification levels of phylum, class, order, family, genus, and species can be visualized, and the bar chart (such as Figure 4 The analysis results are presented as shown.

[0102] In the form of a treemap (a circle stacking diagram is one of the specific forms), a microbial taxonomic hierarchy tree is drawn and the abundance data of each ASV or OTU group is added to the figure in the form of a pie chart (e.g. Figure 5 In addition, a specific taxonomic level can be selected to emphasize the taxonomic composition of microorganisms at that level by coloring the ASV or OTU dots differently.

[0103] In addition to displaying the taxonomic composition through tree diagrams, ggtree can also be used to create phylogenetic trees to display the positions of each ASV / OTU in the phylogenetic tree, as well as the evolutionary distances between them, and to reflect their composition, abundance, taxonomy, and other information through heat maps and bar graphs. (e.g. Figure 6 (shown)

[0104] 4.6.2 Alpha Diversity Analysis

[0105] In order to comprehensively evaluate the alpha diversity of microbial communities, this process uses Chao1 and Observedspecies indices to represent richness, Shannon and Simpson indices to represent diversity, Faith's PD index to represent evolution-based diversity, Pielou's evenness index to represent uniformity, and Good's coverage index to represent coverage. (e.g. Figure 7 (shown)

[0106] In order to explore the changing trend of sample alpha diversity with sampling depth, a rarefaction curve can be drawn. Rarefaction curve is a commonly used method in the field of ecology. By randomly sampling a certain number of sequences from each sample (that is, resampling at a depth that does not exceed the sequencing amount of the existing samples), the total number of species that the sample may contain and the relative abundance of each species in it can be predicted at a given series of sequencing depths. Therefore, by drawing a rarefaction curve, the number of ASV / OTUs in different samples can also be compared at the same sequencing depth, thereby measuring the diversity of each sample to a certain extent. (such as Figure 8 (shown)

[0107] Use R scripts to plot the Specaccum species accumulation curve for the total number of ASV / OTUs corresponding to each sample in the ASV / OTU abundance matrix. Fig. 9 (shown)

[0108] Arrange the ASV / OTU of each sample / group in descending order along the horizontal axis according to their abundance, and use the abundance value after Log2 logarithmic transformation (Log10 transformation, percentage transformation or no transformation) as the vertical axis. Write a script in R software to draw the abundance level curve of each sample or group. (e.g. Fig.10 (shown)

[0109] 4.6.3 Beta diversity analysis

[0110] The Beta diversity index focuses on the comparison of diversity between different habitats, that is, the difference between samples. In fact, the difference between each species (ASV / OTU) between two samples is a dimension that reflects the difference in the community between the two samples. However, since the number of species in a community is often very large, the difference between the communities of samples is often multidimensional, making it difficult to make a direct comparison. (e.g. Fig.11 (shown)

[0111] Unlike PCoA analysis, NMDS analysis does not rely on the calculation of eigenvalues ​​and eigenvectors. Instead, it ranks sample distances so that the order of samples in low-dimensional space is as consistent as possible with the distance relationship between similar distances (rather than the exact distance value). Therefore, NMDS analysis is not affected by the numerical value of sample distances and only considers the size relationship between each other. For data with complex structures, the sorting results may be more stable. The smaller the stress value (Stress) of the NMDS result, the better. It is generally believed that when the value is less than 0.2, the results of NMDS analysis are more reliable. (e.g. Fig.12 (shown)

[0112] like Fig.13 As shown, the panel on the left is a hierarchical clustering tree diagram, in which samples are clustered according to their similarity to each other. The shorter the branch length between samples, the more similar the two samples are; the panel on the right (drawn by default) is a stacked bar chart of the top 10 genera in abundance.

[0113] Anosim (Analysis of similarities) analysis. It is a non-parametric test method to determine whether there are significant differences between two or more groups of samples. This method believes that if two groups of samples are indeed different in species composition, then the composition differences between these groups should be greater than the differences within the groups. The test statistic R takes values ​​between -1 and 1. When it is less than 0, it means that the difference within the group is greater than the difference between the groups. When it is greater than 0, it means that the difference between the groups is greater than the difference within the group. (such as Fig.14 (shown)

[0114] 5. Research Conclusion

[0115] 5.1. Effects of bile acid on promoting growth of Litopenaeus vannamei

[0116] Adding bile acid, cholesterol, etc. to feed can significantly promote the weight gain rate of Penaeus vannamei.

[0117] 5.2. Effects of bile acid on the physiological health of Penaeus vannamei

[0118] 1) Digestive capacity: Adding bile acid, cholesterol, etc. to feed can significantly enhance the activity of hepatopancreatic protease and lipase in Penaeus vannamei; adding bile acid, cholesterol, etc. to feed can improve the plasma biochemical composition of Penaeus vannamei, and the plasma triglyceride content is significantly reduced.

[0119] 2) Hepatopancreas microstructure: Adding bile acid, cholesterol, etc. to the feed can significantly affect the histological structure of the hepatopancreas of Penaeus vannamei. The hepatic corpuscles are intact and tightly arranged, the number of B cells (secretory cells) and R cells (absorptive cells) increases significantly, and there is no blood cell infiltration in the lumen.

[0120] 3) Immune defense ability: Adding bile acid, cholesterol, etc. to the feed can significantly enhance the number of blood cells, phagocytosis rate, and plasma antibacterial and lytic activity of Penaeus vannamei.

[0121] 4) Intestinal flora structure: Adding bile acid, cholesterol, etc. to feed can significantly change the intestinal flora structure of Penaeus vannamei and maintain intestinal health. In terms of flora function, the intestinal flora of each bile acid, cholesterol, etc. treatment group has increased exogenous substances and amino acid metabolism capabilities.

[0122] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A use of bile acid in preparing a feed for promoting the growth performance of Penaeus vannamei and regulating lipid metabolism, characterized in that: The bile acid is added to the basic feed of Penaeus vannamei as a feed additive, and the added amount of the bile acid is 0.04% of the total mass of the basic feed of Penaeus vannamei. The bile acid includes the following components: hyodeoxycholic acid, chenodeoxycholic acid, bile acid and hyocholic acid; the mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, bile acid and hyocholic acid is 68-70:2-5:8-10:1-3.

2. An application of bile acid and cholesterol in preparing a feed for promoting the growth performance of Penaeus vannamei and regulating lipid metabolism, characterized in that: The bile acid and cholesterol are added to the basic feed of Penaeus vannamei as feed additives, the added amount of the bile acid is 0.04% of the total mass of the basic feed of Penaeus vannamei, the added amount of the cholesterol is 0.2-2.0% of the total mass of the basic feed of Penaeus vannamei, the bile acid comprises the following components: hyodeoxycholic acid, chenodeoxycholic acid, bile acid and hyocholic acid; the mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, bile acid and hyocholic acid is 68-70:2-5:8-10:1-3.

3. The use according to claim 2, characterized in that: The mass ratio of hyodeoxycholic acid, chenodeoxycholic acid, cholic acid and hyodeoxycholic acid is 69.88:3.14:9.58:2.

04.

4. The use according to claim 2, characterized in that: The added amount of cholesterol is 0.5-2.0% of the total mass of the basic feed of Penaeus vannamei.

5. The use according to claim 2, characterized in that: The added amount of cholesterol is 0.2-1.0% of the total mass of the basic feed of Penaeus vannamei.

6. The use according to claim 2, characterized in that: The added amount of cholesterol is 1.0% of the total mass of the basic feed of Penaeus vannamei.

7. The use according to any one of claims 1 to 6, characterized in that: The vannamei shrimp is juvenile vannamei shrimp.

8. The use according to claim 7, characterized in that: The initial average weight of the juvenile Penaeus vannamei shrimp was 3.46 g / prawn.

9. The use according to claim 7, characterized in that: The basic feed for Penaeus vannamei is composed of the following components in percentage by mass: crude protein ≥46%, crude fiber ≤11%, crude fat ≥5%, crude ash ≤17%, lysine ≥2.1%, sodium chloride ≤3.8%, moisture ≤12%, calcium ≤5%, total phosphorus ≥0.5%, and the sum of all components is 100%.

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