Ursodeoxycholic acid preparation for inducing juvenile fish cluster behavior as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of aquaculture and nutrition regulation and control, and particularly relates to an ursodeoxycholic acid preparation for inducing cluster behaviors of juvenile fishes as well as a preparation method and application of the ursodeoxycholic acid preparation. The ursodeoxycholic acid preparation for inducing the cluster behavior of the juvenile fish consists of ursodeoxycholic acid and an additive which accounts for 0-2% of the mass of the ursodeoxycholic acid; the additive is cholic acid or a mixing agent, and the mixing agent is a mixture of taurine and tryptophan according to the mass ratio of 1: 1. After the ursodeoxycholic acid preparation for inducing the juvenile fish cluster behavior is used for feeding the larimichthys crocea juvenile fish, the olfaction of the larimichthys crocea juvenile fish can be stimulated, the juvenile fish cluster behavior is induced, the growth of the larimichthys crocea juvenile fish is promoted, the metabolism is promoted, and the immunity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture and nutritional regulation, and particularly relates to a ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, a preparation method thereof, and an application thereof. Background Art

[0002] In aquaculture, the survival rate, growth performance, and stress resistance of juvenile fish are key factors affecting aquaculture efficiency. In the natural environment, fish reduce the risk of predation, improve feeding efficiency, and optimize energy metabolism through schooling behavior. However, under artificial breeding conditions, the schooling behavior of juvenile fish is often weak, resulting in problems such as slow growth, metabolic disorders, and decreased immunity.

[0003] Currently, in aquaculture, optimizing the feed formula (such as adding attractants) or improving the breeding environment (such as water flow regulation) is often used to promote the growth of juvenile fish, but the effect is limited. Although chemical signal molecules (such as bile acids) have potential effects on fish olfactory perception and physiological regulation, there is still a lack of research on their role in inducing schooling behavior. Bile acids (such as cholic acid CA) can be detected by the fish olfactory system and trigger electrophysiological responses, but existing bile acid additives (such as tauroursodeoxycholic acid TUDCA) have problems such as poor stability in the prior art, and their optimal concentration and molecular mechanism have not been clarified.

[0004] Ursodeoxycholic acid (UDCA, also known as ursodiol) is a hydrophilic bile acid that is used in medicine for the treatment of liver and gall diseases, but its application in aquaculture is mostly limited to cholesterol metabolism regulation, and there is no research on its induction of the schooling behavior of juvenile fish through the olfactory pathway. In addition, traditional additives are mostly directly mixed into the feed, ignoring the immediate regulatory role of olfactory signals in fish behavior, and there is a lack of in-depth research on related receptors (such as GPCRs) and signal pathways.

[0005] Therefore, developing an ursodeoxycholic acid preparation based on the olfactory receptor perception mechanism that can induce the schooling behavior of juvenile fish is of great significance for improving aquaculture efficiency. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides an ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, a preparation method thereof, and an application thereof.

[0007] The first object of the present invention is to provide an ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, which is composed of ursodeoxycholic acid and an additive equivalent to 0% - 2% of the mass of ursodeoxycholic acid;

[0008] The additive is cholic acid (whose molecular formula is C 24 H 40 O5) or a mixture, and the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:1.

[0009] The present invention uses ursodeoxycholic acid as the core component to induce the schooling behavior of juvenile fish, promote the growth of juvenile large yellow croaker, enhance the metabolism of juvenile large yellow croaker, and improve immunity. The additive (cholic acid or mixture) can enhance the stability and application effect of the ursodeoxycholic acid preparation. In addition, the present invention combines electrophysiology, transcriptomics, and molecular biology techniques to reveal its mechanism of activating downstream signaling pathways through specific olfactory receptors (such as V2R1, OR11A1), filling the technical gap in this field.

[0010] Preferably, the ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish is composed of ursodeoxycholic acid and an additive equivalent to 1% - 1.5% of the mass of ursodeoxycholic acid.

[0011] The second object of the present invention is to provide a preparation method of an ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, including: preparing ursodeoxycholic acid and an additive equivalent to 0% - 2% of the mass of ursodeoxycholic acid, and mixing them evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish.

[0012] The third object of the present invention is to provide an application of an ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, and the application refers to stimulating the olfaction of juvenile fish to induce the schooling behavior.

[0013] Preferably, the juvenile fish are juvenile large yellow croaker.

[0014] Preferably, the application refers to at least one of promoting the growth of juvenile large yellow croaker, enhancing metabolism, and improving immunity.

[0015] Preferably, promoting the growth of juvenile large yellow croaker refers to increasing the survival rate, body weight, and condition factor, increasing the crude fat content in the whole fish and muscle tissue, and increasing the crude protein content in the whole fish and muscle tissue;

[0016] Enhancing metabolism refers to increasing the intestinal lipase activity;

[0017] Improving immunity refers to enhancing the antioxidant capacity and increasing the survival rate after fishing stress.

[0018] Preferably, the method of the application is:

[0019] The method of the application is as follows: Mix the ursodeoxycholic acid preparation that induces the schooling behavior of juvenile fish with the basic feed. The mass of the ursodeoxycholic acid preparation added to each kilogram of the basic feed is 50 mg to 100 mg. The basic feed, calculated on a dry weight basis, is composed of the following substances mixed in the following mass ratios: fish meal: soybean meal: soy protein concentrate: wheat gluten: flour: fish oil: palm oil: calcium dihydrogen phosphate: soy lecithin: multi-vitamin and multi-mineral mixture: mold inhibitor: antioxidant: microcrystalline cellulose = 200:400:80:50:70:40:30:5:20:15:1:0.5:83.5. The antioxidant is tert-butylhydroquinone.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish in the present invention is composed of ursodeoxycholic acid and additives equivalent to 0% to 2% of the mass of ursodeoxycholic acid; the additives are cholic acid or a mixture, and the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:1. After feeding juvenile large yellow croakers with the ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish in the present invention, it can stimulate the olfaction of juvenile fish, induce the schooling behavior of juvenile fish, promote the growth, metabolism and immunity improvement of juvenile large yellow croakers. The promotion of the growth of juvenile large yellow croakers refers to the improvement of survival rate, body weight and condition factor, and the increase of the crude fat and crude protein contents in whole fish and muscle tissues; the promotion of metabolism refers to the improvement of intestinal lipase activity; the improvement of immunity refers to the enhancement of antioxidant capacity and the increase of the survival rate after fishing stress. Description of the Drawings

[0022] Figure 1 For the Y-maze device and plume observation; wherein, A, the device is filled with seawater; B, the plume after adding potassium permanganate to one of the choice arms for 20 seconds; C, the plume after adding potassium permanganate to one of the choice arms for 60 seconds.

[0023] Figure 2 For the underwater electro-olfactogram (EOG) response and behavioral response of large yellow croakers to different bile acids (CA, UDCA, TUDCA, GUDCA, DCA, LCA and HDCA); wherein, A, the EOG concentration-response relationship of bile acids (mean ± standard error, n = 5); B, the behavioral response of large yellow croakers to different bile acids with a concentration of 10 -8 mol / L, expressed as the preference index (mean ± standard error, n = 20); C, the preference index of the behavioral response of large yellow croakers to different concentrations of ursodeoxycholic acid (mean ± standard error, n = 20).

[0024] Figure 3Transcriptome analysis of the olfactory epithelium of large yellow croaker before and after treatment with ursodeoxycholic acid; among them, A, Pearson correlation coefficient comparison; B, principal component analysis of samples, with the ellipse representing the 95% confidence interval; C, gene expression volcano plot, CON represents the group of large yellow croaker olfactory epithelium samples before treatment with ursodeoxycholic acid, Day 3 represents the group of large yellow croaker olfactory epithelium samples 3 days after treatment with ursodeoxycholic acid, Day 7 represents the group of large yellow croaker olfactory epithelium samples 7 days after treatment with ursodeoxycholic acid, where P-value indicates whether the expression difference of a certain gene between the comparison groups is significant enough, and P-value < 0.05 is set as significant.

[0025] Figure 4 Analysis results of the expression patterns of olfactory receptor genes in the olfactory epithelium of large yellow croaker in the control group (CON), the third day (Day 3) and the seventh day (Day 7) after treatment with ursodeoxycholic acid, mors.

[0026] Figure 5 Analysis results of the expression patterns of olfactory receptor genes in the olfactory epithelium of large yellow croaker in the control group (CON), the third day (Day 3) and the seventh day (Day 7) after treatment with ursodeoxycholic acid, taars.

[0027] Figure 6 Analysis results of the expression patterns of olfactory receptor genes in the olfactory epithelium of large yellow croaker in the control group (CON), the third day (Day 3) and the seventh day (Day 7) after treatment with ursodeoxycholic acid, vrs.

[0028] Figures 4 to 6 Among them, the expression level of the gene is represented by FPKM, red indicates a high expression level, and blue indicates a low expression level.

[0029] Figure 7 Effect of ursodeoxycholic acid treatment on the expression levels of 4 olfactory receptor genes in large yellow croaker; among them, A is v2r1, B is or11a1, C is taar13c - 10, D is or52l2.

[0030] Figure 8 Secondary protein structures of V2R1, OR11A1, TAAR13C - 10 and OR52L2 predicted by PROTTER.

[0031] Figure 9Heterologous expression and immunocytochemical detection of V2R1, Rho-OR11A1, TAAR13C-10 and Rho-OR52L2 in HEK293T cells. The experimental groups were transfected with pEGFP-N1 vector, pCI-mRTP1s helper plasmid, empty vector pcDNA3.1 (control group), or recombinant plasmid of target gene containing 1D4 tag. Blue tracer is DAPI (cell nucleus); red is 1D4 antibody labeling (receptor protein); green is EGFP (cytoplasm) and DiO (cell membrane). Scale bar: 20 μm.

[0032] Figure 10 Functional characterization analysis of four olfactory receptors in Larimichthys crocea. A, Detection of calcium mobilization in HEK293T cells stimulated with different concentrations of ursodeoxycholic acid, and the calcium response value (Ca 2+ %) was normalized based on the calcium concentration that activates V2R1 by 10 -4 mol·L -1 ursodeoxycholic acid; B, Detection of luciferase activity in HEK293T cells stimulated with different concentrations of ursodeoxycholic acid, and the luciferase activity value was normalized based on the response of 10 -4 mol·L -1 ursodeoxycholic acid that activates OR11A1 (mean ± standard error, n = 3); C, Detection of the regulatory effect of U-73122 (PLC inhibitor) on the olfactory receptor function of V2R1 in the calcium mobilization assay; D, Detection of the regulatory effect of SQ22536 (AC inhibitor) on the olfactory receptor function of Rho-OR11A1 in the cAMP assay; E, Detection of the regulatory effect of SQ22536 on the olfactory receptor function of TAAR13C-10 in the cAMP assay; F, Detection of the regulatory effect of SQ22536 on the olfactory receptor function of Rho-OR52L2 in the cAMP assay; among them, the calcium response (relative fold of control) level and the cAMP relative activation level were both based on the receptor-expressing cells treated with 10 -4 mol·L -1 DMSO (mean ± standard error, n = 3). Detailed implementation manners

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0034] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0035] Mechanism research 1. Analysis of the behavior and olfactory function of ursodeoxycholic acid

[0036] 1. Experimental method:

[0037] (1) Cholic acid (CA), Ursodeoxycholic acid (UDCA), Tauroursodeoxycholic acid (TUDCA), Glycoursodeoxycholic acid (GUDCA), Deoxycholic acid (DCA), Lithocholic acid (LCA), and Hyodeoxycholic acid (HDCA) (Macklin Biochemical Co., Ltd., China) were all dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 10 -2 mol / L and stored at -20 °C. On the day of the experiment, the stock solution was diluted to the required concentration with filtered seawater.

[0038] (2) The Y-maze device is a Y-shaped water tank made of polypropylene material, including two choice arms and a mixing arm (both with dimensions of 100 cm × 40 cm × 30 cm). Each of the two choice arms is connected to an inlet pipe and a water pump, and the mixing arm is connected to an outlet pipe to ensure that the Y-maze device is filled with flowing seawater. The seawater depth during the experiment was 5 cm. As Figure 1 shown, when the flow rate in the inlet pipe was maintained at 600 mL / min, a solution containing potassium permanganate dye was injected into one of the choice arms, and the red dye gradually diffused into the mixing arm but did not enter the other choice arm.

[0039] (3) EOG detection

[0040] At the start of the experiment, the fish were first anesthetized by oral perfusion with 20 mg·L -1 of MS-222 (Sigma-Aldrich, St. Louis, USA), and then about 5 mg·kg -1 body weight of gallamine triethiodide was intramuscularly injected to further relax the muscles. The fish were fixed in the experimental container, the body position was stabilized with clamps, and seawater was continuously introduced through a silicone tube inserted into the mouth at a flow rate of 0.5 mL·s -1 to maintain gill respiration. During the experiment, the fish body was covered with a wet towel to ensure that its body surface remained moist. For easy electrode placement, microsurgery was performed to remove the skin and connective tissue on the surface of the olfactory epithelium. EOG recording was carried out using two Ag / AgCl electrodes (World Precision Instruments, Sarasota, USA). The electrode capillary was filled with an electrolyte solution containing 3 mol·L - 1 KCl, 0.15 mol·L -1 NaCl, and 0.5 g / 100 g agar (tip diameter 150 μm). Among them, the recording electrode was placed on the surface of the olfactory epithelium, and the reference electrode was fixed on the skin between the two eyes. The test solution was at a flow rate of 6 mL·min -1The flow rate is transported through a catheter, and each stimulation lasts for 5 seconds (ensuring that the EOG signal reaches its peak), with a stimulation interval of 120 seconds to avoid olfactory adaptation. Each fish is sequentially stimulated with reagents of different concentrations from low to high, with a maximum of 10 stimulations. The EOG signal is amplified, digitized, and displayed in real-time on a computer by the BL-420F data acquisition and analysis system (designed by Taimeng Software, Chengdu, China).

[0041] (4) Behavioral test

[0042] After 24 hours of starvation treatment, the experimental fish are subjected to behavioral tests. Five experimental fish are randomly selected and placed in a Y-maze device to adapt to the environment for 20 minutes. A camera is installed above the Y-maze device to record the swimming behavior and movement path of the experimental fish throughout the process. During the experiment, the experimental fish can swim freely in the choice arm and the mixing arm. Five minutes after the start of the experiment, the water inlet pipe connected to the choice arm with fewer experimental fish is switched from filtered seawater to the test solution prepared in advance, while the other choice arm continues to pump in filtered seawater. The camera video is captured every 5 seconds to record and count the number of experimental fish in the choice arm.

[0043] The preference index (PI) characterizes the attractiveness of the test solution to the experimental fish, and the calculation formula is PI = [Ae÷(Ae + Be) - Ac÷(Ac + Bc)], where Be represents the number of experimental fish in the experimental group before the introduction of the test solution, Bc represents the number of experimental fish in the choice arm of the control group before the introduction of the test solution, Ae represents the number of experimental fish in the choice arm of the experimental group after the introduction of the test solution, and Ac represents the number of experimental fish in the choice arm of the control group after the introduction of the test solution. The PI value ranges from -1 to 1. The higher the PI value, the stronger the attractiveness, and the lower the PI value, the weaker the attractiveness. The behavioral test time is from 10:00 to 16:00. After the experiment, the experimental device is thoroughly rinsed successively with 5% mass fraction bleach, tap water, and filtered seawater. Each experiment uses experimental fish that have not participated in the experiment, and each test substance in the behavioral test is repeated more than 20 times.

[0044] (5) Transcriptome analysis of the olfactory epithelium tissue of large yellow croaker after treatment with ursodeoxycholic acid

[0045] The experimental fish came from Xixuan Fishery Science and Technology Island, Zhoushan City, Zhejiang Province. They were healthy large yellow croakers from the same batch, weighing 51.2±7.5g and 16.4±2.9cm in total length. Before the experiment, 360 experimental fish were randomly divided into 24 tanks, 15 in each tank, for two weeks to adapt to the experimental environment. The experimental fish were in a flow-through system, with a water volume of 400L in each tank, a water temperature of 25.0℃, a pH of 8.2, dissolved oxygen greater than 7.0mg / L, a salinity of 26.0, and ammonia nitrogen of 0.04mg / L. During the experiment, Tongwei commercial pellet feed was used to feed the fish at 8:00 am and 18:00 pm. After each feeding for one hour, about 75% of the seawater in each tank was replaced. To carry out a 7-day ursodeoxycholic acid treatment experiment. Before the experiment, ursodeoxycholic acid was dissolved in DMSO to prepare 4×10 -4 mol / L ursodeoxycholic acid solution was stored in a 4°C refrigerator. During the experiment, 4 mL of ursodeoxycholic acid solution was injected into the experimental group water tanks (a total of 12) along the inner wall twice a day (5 min before feeding) under static water conditions. The final concentration of ursodeoxycholic acid in the breeding tank was 1×10 -8 mol / L; 4mL DMSO was injected into 12 water tanks in the control group.

[0046] Total RNA was extracted from tissues using the TRIzol (Thermo Fisher Scientific, USA) method. After the cDNA library was constructed, it was sequenced using the Illumina NovaSeq 6000 platform (Illumina, USA), and bioinformatics analysis was performed. β-actin was selected as the internal reference gene, and qRT-PCR verification was performed using an ABI Real-Time PCR instrument (Thermo Fisher Scientific, USA).

[0047] (6) Heterologous expression and functional verification of ursodeoxycholic acid olfactory receptor

[0048] First, collect the olfactory epithelial tissues of healthy large yellow croakers (weight 28.7 ± 4.6 g) from Zhoushan aquaculture base. After RNA extraction (using Solarbio kit) and cDNA synthesis, use the RACE technique to clone the full-length genes of 4 receptors such as V2R1 and Rho-OR11A1, and obtain the correct sequence through construction of pMD19-T vector and sequencing verification. Subsequently, construct the pcDNA3.1-Rho-1D4 expression vector (containing rhodopsin signal peptide), and ligate it with the receptor gene by double digestion with EcoRI / Xho I. At the same time, construct the pCI-mRTP1s and pCI-Gαolf co-expression vectors. In HEK293T cells, co-transfect the receptor expression plasmid and the co-expression plasmid using Lipofectamine 3000, and verify the localization by immunofluorescence (1D4 antibody / AlexaFluor 555 secondary antibody). Use the Fluo-4AM probe to detect the calcium response stimulated by bile acids such as ursodeoxycholic acid (using Tecan Spark microplate reader), and establish a CRE-luciferase reporter system (pGL4.29 vector) to detect the activation of cAMP signal (using Tecan Spark microplate reader).

[0049] 2. Experimental results

[0050] (1) Results of electrophysiological response

[0051] Detect the EOG responses of large yellow croakers induced by CA, UDCA, TUDCA, GUDCA, DCA, LCA, and HDCA. Figure 2 Figure A shows the EOG response amplitude normalized by the percentage of L-serine. The results show that with the increase in concentration, the EOG responses induced by bile acids gradually increase. Among them, the EOG responses induced by bile acids such as ursodeoxycholic acid, DCA, and TUDCA are the most significant, showing a concentration-response relationship curve with rapid growth, and the detection threshold is as low as 10 -12 mol / L.

[0052] (2) Results of Y-maze apparatus behavior test

[0053] An increase or decrease in the number of experimental fish on the side where the test substance is added is regarded as attraction or avoidance. At a concentration of 10 -8 mol / L, CA and ursodeoxycholic acid significantly induced the preference behavior of experimental fish (P < 0.001)( Figure 2B). However, the attracting effects of TUDCA, GUDCA, DCA, LCA, HDCA, and DMSO on the experimental fish were not significant (P>0.05). It is worth noting that the preference index of ursodeoxycholic acid was 43.84% higher than that of CA, indicating that ursodeoxycholic acid has a stronger attracting ability for large yellow croaker. Further research found that the preference index of ursodeoxycholic acid was dose-dependent, that is, as the concentration of ursodeoxycholic acid increased, the preference index increased and reached the maximum at a concentration of 10 -8 mol / L; at higher concentrations, the preference index of ursodeoxycholic acid decreased with the increase in concentration ( Figure 2 C).

[0054] (3) Results of transcriptome analysis of olfactory epithelium after treatment with ursodeoxycholic acid

[0055] To explore the olfactory receptors of large yellow croaker that sense ursodeoxycholic acid and its olfactory signal transduction mechanism, cDNA libraries of olfactory epithelium tissues of large yellow croaker were constructed for the control group (CON) before treatment with ursodeoxycholic acid, Day 3 and Day 7 after treatment with ursodeoxycholic acid, and a total of 414,416,420 raw reads were obtained. 91.14% of the clean reads were successfully mapped to the reference genome of large yellow croaker, and a total of 23,842 genes were annotated. Compared with the control group (CON), the gene expression differences in the ursodeoxycholic acid treatment groups (Day 3 and Day 7) were significant ( Figure 3 ).

[0056] Four types of olfactory receptor genes were successfully obtained in the olfactory epithelium tissue of large yellow croaker, namely 96 mor genes, 37 taar genes, 2 v1r genes, and 13 v2r genes ( Figures 4 to 6 ). According to the criteria of |log 2fold change|>1.3 and adjusted-P(q)value<0.05, 15 DEGs were screened. Among them, in Day 3 vs. CON, it was found that the expression of 1 v2r1 gene was up-regulated; while in Day 7 vs. CON, it was found that the expression of 11 olfactory receptor genes was up-regulated and 3 were down-regulated. No differential expression of olfactory receptor genes was observed in Day 7 vs. Day 3. Further research found that after treatment with ursodeoxycholic acid, the expression levels of four olfactory receptors, namely v2r1, or11a1, taar13c-10, and or52l2, were significantly increased ( Figure 7 ).

[0057] (4) Results of heterologous expression and functional verification of ursodeoxycholic acid olfactory receptors

[0058] The full-length sequences of the above 4 olfactory receptor genes were successfully cloned by RACE technology in the present invention. Analysis showed that the proteins encoded by the 4 genes have the typical seven-transmembrane structural characteristics of GPCRFigure 8 ) After constructing the expression vectors pcDNA3.1-Rho-OR-1D4 and pcDNA3.1-OR-1D4 of large yellow croaker olfactory receptor genes, they were co-transfected into HEK293T cells together with the pCI-mRTP1s gene expression vector and the pEGFP-N1 plasmid. The experimental results showed that the four olfactory receptor proteins were successfully expressed on the HEK293T cell membrane, and the Rho-tag signal peptide enhanced the expression of OR11A1 and OR52L2 proteins on the cell membrane. Through intracellular Ca 2+ detection and CRE-firefly luciferase reporter gene detection, it was found that V2R1 participated in the transduction of the olfactory signal of ursodeoxycholic acid through the PLC pathway, while TAAR13C-10, OR11A1, and OR52L2 participated in the transduction of the olfactory signal of ursodeoxycholic acid through the AC-cAMP pathway ( Figures 9 to 10 ).

[0059] Based on the above results of mechanism analysis, the present invention focused on studying the effects of ursodeoxycholic acid on the olfactory receptors and their signal transduction pathways of large yellow croaker larvae.

[0060] Example 1

[0061] An ursodeoxycholic acid preparation for inducing the schooling behavior of larvae is ursodeoxycholic acid.

[0062] Example 2

[0063] An ursodeoxycholic acid preparation for inducing the schooling behavior of larvae consists of ursodeoxycholic acid and an additive equivalent to 1% of the mass of ursodeoxycholic acid; the additive is cholic acid. The raw materials are mixed evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of larvae.

[0064] Example 3

[0065] An ursodeoxycholic acid preparation for inducing the schooling behavior of larvae consists of ursodeoxycholic acid and an additive equivalent to 1.5% of the mass of ursodeoxycholic acid; the additive is cholic acid. The raw materials are mixed evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of larvae.

[0066] Example 4

[0067] An ursodeoxycholic acid preparation for inducing the schooling behavior of larvae consists of ursodeoxycholic acid and an additive equivalent to 2% of the mass of ursodeoxycholic acid; the additive is cholic acid. The raw materials are mixed evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of larvae.

[0068] Example 5

[0069] A ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, which is composed of ursodeoxycholic acid and an additive equivalent to 1% of the mass of ursodeoxycholic acid; the additive is a mixture, and the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:1. Each raw material is mixed evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish.

[0070] Example 6

[0071] A ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, which is composed of ursodeoxycholic acid and an additive equivalent to 1.5% of the mass of ursodeoxycholic acid; the additive is a mixture, and the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:1. Each raw material is mixed evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish.

[0072] Example 7

[0073] A ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish, which is composed of ursodeoxycholic acid and an additive equivalent to 2% of the mass of ursodeoxycholic acid; the additive is a mixture, and the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:1. Each raw material is mixed evenly to obtain the ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish.

[0074] Application experiment 1, the effect of ursodeoxycholic acid preparation on juvenile large yellow croaker

[0075] 1. Preparation of breeding feed

[0076] For different breeding feed formulas, see Table 2. The preparation process is to first mix the powdered ursodeoxycholic acid preparation for inducing the schooling behavior of juvenile fish with the basal feed evenly, and then perform extrusion, granulation and drying operations. Ursodeoxycholic acid: Macklin Biochemical Technology Co., Ltd. (China), purity 99%, CAS (Chemical Abstract Service) registration number 128-13-2.

[0077] The basic feed formula is shown in Table 1. In Table 1, fish meal: Kodiak Fish Meal Company (USA); soybean meal: Yihai Kerry Co., Ltd. (China); soy protein concentrate: Yihai Kerry Co., Ltd. (China); vital gluten flour, flour (high-gluten flour): commercially available; fish oil: Rongcheng Ails Marine Biotechnology Co., Ltd. (China); palm oil: COFCO Corporation (China); soy lecithin: Cargill (Germany). The composition of the multi-vitamin and multi-mineral mixture and the mold inhibitor refers to the previous published literature "TANP, LI X, XIANG X, et al. Adipose tissue contributes to hepatic pro-inflammatory response when dietary fish oil is replaced by vegetable oil in large yellow croaker (Larimichthys crocea): An ex vivo study [J]. Fish & Shellfish Immunology, 2019, 84: 955-961". Antioxidant: tert-butylhydroquinone.

[0078] Table 1 Basic Feed Formula

[0079] Raw material Mass concentration (g, dry weight basis) Fish meal 200 Soybean meal 400 Soybean protein concentrate 80 Vital gluten 50 Flour 70 Fish oil 40 Palm oil 30 Calcium dihydrogen phosphate 5 Soybean phospholipid 20 Multi-vitamin and multi-mineral mixture 15 Mildew preventive 1 Antioxidant 0.5 Microcrystalline cellulose 83.5

[0080] The aquaculture feed formula for the experimental group is shown in Table 2.

[0081] Table 2 Aquaculture Feed Formula

[0082]

[0083]

[0084] Note: "-" indicates that this item is not added.

[0085] 2. Aquaculture

[0086] The aquaculture experiment was carried out in the flow-through aquaculture system on Xixuan Fishery Science and Technology Island, Zhoushan City, Zhejiang Province. Before the aquaculture experiment, juvenile large yellow croakers of the same batch, healthy and of uniform size were acclimated to commercial feed for two weeks to ensure that the experimental fish adapted to the aquaculture conditions. 400 juvenile large yellow croakers with an initial body weight of 15.55 ± 0.02 g were randomly assigned to the aquaculture barrels, 25 fish per barrel, and 4 replicates were set for each treatment group in the experiment. The experimental feeds were randomly distributed to each aquaculture barrel. The aquaculture cycle was 70 days, and the fish were fed to satiation once at 6:00 am and 17:00 pm every day during the experiment. The capacity of the aquaculture barrel was 1000 L, and the seawater flow rate was about 3 L / min. During the experiment, the seawater was changed twice a day, 400 L of seawater was replaced each time, and the feces were removed by siphon during the water change, and the residual feeds were counted and statistically analyzed. The number of dead experimental fish was recorded twice a day, and the dead experimental fish were weighed and recorded. During the aquaculture period, no diseases or other emergencies occurred.

[0087] 3. Sample Collection and Testing

[0088] After the aquaculture experiment ended, the experimental fish were fasted for 12 hours and anesthetized with MS-222, and then weighed one by one. 5 experimental fish were randomly selected from each aquaculture barrel for analysis of the nutritional composition of the fish body. 10 experimental fish were randomly selected from each aquaculture barrel to measure their body length and total length, and the visceral mass and liver mass were measured to calculate the condition factor, viscerosomatic index and hepatosomatic index. Liver and intestinal tissue samples were collected for determination of physiological and biochemical indexes. Blood samples were obtained by caudal vein blood sampling, left to stand for 4 hours and then centrifuged to separate the serum for subsequent determination of physiological, biochemical and immune indexes. The samples for determination of physiological and biochemical indexes were stored in a -80 °C refrigerator.

[0089] The main steps of the capture stress test were as follows: The seawater in the aquaculture barrel was drained to 30 cm from the bottom of the barrel; a fishing net was used to stir in the aquaculture barrel to simulate the capture environment, the stirring speed was 6 s / week, and the duration was 5 min. The stirring was repeated every 30 min for a total of 4 times. The number of dead experimental fish was recorded every 2 hours within the subsequent 48 hours. The semi-lethal condition of the experimental fish was used in the capture stress test.

[0090] The following formulas were referred to for calculation of relevant indexes:

[0091] Survival rate (SR, %) = number of final experimental fish in each aquaculture barrel × 100 / number of initial experimental fish in that aquaculture barrel;

[0092] Feed intake (FI, % / day) = 100 × total feed consumption (g) / [(initial body weight of experimental fish + final body weight of experimental fish) / 2] / t;

[0093] Feed conversion rate (FCR) = Dry feed intake (g) / Increase in wet body weight of fish (g);

[0094] Condition factor (CF, %) = 100 × (Final weight of experimental fish, g) / (Body length, cm) 3 ;

[0095] where t is the experimental period, unit: days.

[0096] 4. Experimental results

[0097] 4.1. Results of inducing the schooling behavior response of juvenile fish

[0098] The results in Table 3 show that compared with the blank group, for those with ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce the schooling behavior of juvenile fish added, the preference index for inducing the schooling behavior response of juvenile fish has increased, and the higher the dose, the more significant the increase, P < 0.05. The preference indices of Control 1 and Control 2 have also increased compared with the blank group, P < 0.05, indicating that bile acid or the mixture used alone can also induce the schooling behavior response of juvenile fish.

[0099] Table 3 Test results of the preference index for inducing the schooling behavior response of juvenile fish

[0100]

[0101]

[0102] 4.2. Effects on the survival, growth performance and body composition of large yellow croaker

[0103] The results are shown in Table 4 and Table 5. Compared with the blank group, for those with ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce the schooling behavior of juvenile fish added, the survival rate, final weight and condition factor have all increased, with significant differences (P < 0.05), and the feeding rate has no significant difference. The final weight and condition factor of Control 1 and Control 2 have also increased compared with the blank group, P < 0.05.

[0104] Table 4 Test results of the survival, growth performance and body composition of large yellow croaker (low dose)

[0105]

[0106] Table 5 Test results of the survival, growth performance and body composition of large yellow croaker (high dose)

[0107]

[0108]

[0109] 4.2 Crude fat and crude protein contents of whole fish, liver and muscle

[0110] The results are shown in Tables 6 - 7. Compared with the blank group, for those supplemented with ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce schooling behavior in juvenile fish, the crude protein in whole fish, crude fat in whole fish and crude fat in muscle all increased, with significant differences (P < 0.05), while there was no significant difference in the crude fat in the liver. The crude protein in whole fish, crude fat in whole fish and crude fat in muscle of Control 1 and Control 2 also increased compared with the blank group, with P < 0.05.

[0111] Table 6 Crude fat and crude protein contents of whole fish, liver and muscle (low dose)

[0112]

[0113] Table 7 Crude fat and crude protein contents of whole fish, liver and muscle (high dose)

[0114]

[0115]

[0116] 4.3 Intestinal lipase activity

[0117] As can be seen from Table 8, after adding ursodeoxycholic acid preparations that induce schooling behavior in juvenile fish, the intestinal lipase activity of large yellow croaker increased significantly, with P < 0.05, indicating an improvement in its metabolic function. The intestinal lipase activity of large yellow croaker in Control 1 and Control 2 also increased compared with the blank group, with P < 0.05.

[0118] Table 8 Test results of intestinal lipase activity

[0119]

[0120] 4.4 Test results of immune - related indicators

[0121] In Table 9, compared with the blank group, for those supplemented with ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce schooling behavior in juvenile fish, both the antioxidant capacity and the survival rate after fishing stress increased, with P < 0.05. The antioxidant capacity and the survival rate after fishing stress of Control 1 and Control 2 also increased compared with the blank group, with P < 0.05.

[0122] Table 9 Test results of immune - related indicators

[0123]

[0124] The above application experiment results show that when ursodeoxycholic acid is combined with cholalic acid or a mixture, it is most beneficial for inducing schooling behavior, growth, etc. of juvenile large yellow croaker.

[0125] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the step methods adopted are the same as those in the embodiments, in order to prevent repetition, the present invention describes preferred embodiments. Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the creative concept of the present invention, they can make additional changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0126] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of equivalent technologies of the present invention, the present invention also intends to include these changes and modifications.

Claims

1. An ursodeoxycholic acid preparation for inducing swarming behavior of young fish, characterized in that: It is composed of ursodeoxycholic acid and an additive equivalent to 0% to 2% of the mass of ursodeoxycholic acid; The additive is bile acid or a mixture, and the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:

1.

2. The ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 1, characterized in that: The invention is composed of ursodeoxycholic acid and additives equivalent to 1% to 1.5% of the mass of the ursodeoxycholic acid.

3. The method for preparing the ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 1, characterized in that: include: Ursodeoxycholic acid and an additive equivalent to 0% to 2% of the mass of ursodeoxycholic acid are prepared and mixed evenly to obtain an ursodeoxycholic acid preparation that induces clustering behavior of young fish.

4. The use of the ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 1, characterized in that: The application refers to stimulating the sense of smell of young fish and inducing clustering behavior of young fish.

5. The use of the ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 4, characterized in that: The young fish is a young large yellow croaker.

6. The use of the ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 5, characterized in that: The application refers to at least one of promoting the growth of young large yellow croaker, promoting metabolism and enhancing immunity.

7. The use of the ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 6, characterized in that: Promoting the growth of juvenile large yellow croaker refers to improving the survival rate, weight and fatness, increasing the crude fat content of the whole fish and muscle tissue, and increasing the crude protein content of the whole fish and muscle tissue; The metabolism promotion refers to the enhancement of intestinal lipase activity; The said enhancement of immunity refers to the enhancement of antioxidant capacity and the improvement of survival rate after fishing stress.

8. The use of the ursodeoxycholic acid preparation for inducing swarming behavior of young fish according to claim 4, characterized in that: The application method is: mixing the ursodeoxycholic acid preparation inducing the swarming behavior of young fish with the basic feed, with the mass of the added preparation being 50mg to 100mg per kilogram of the basic feed.

9. The use according to claim 8, characterized in that: The basic feed is composed of the following materials in the following mass proportions based on dry weight: Fish meal: soybean meal: soybean protein concentrate: cereal powder: flour: fish oil: palm oil: calcium dihydrogen phosphate: soybean lecithin: multivitamin mineral mixture: mold inhibitor: antioxidant: microcrystalline cellulose 200:400:80:50: 70：40：30：5：20：15：1：0.5：83.5。 10. The use according to claim 9, characterized in that: The antioxidant is tert-butylhydroquinone.

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