Bear bile deoxycholic acid preparation for inducing juvenile fish shoaling behavior, and preparation method and application thereof

Abstract

The present application belongs to the field of aquaculture and nutrition regulation technology, and particularly relates to a ursodeoxycholic acid preparation for inducing the group behavior of juvenile fish, a preparation method and application. The ursodeoxycholic acid preparation for inducing the group behavior of juvenile fish 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 mixed agent, and the mixed agent is a mixture of taurine and tryptophan in a mass ratio of 1:1. After the ursodeoxycholic acid preparation for inducing the group behavior of juvenile fish is used to feed large yellow croaker juveniles, the juvenile fish olfaction can be stimulated, the group behavior of the juvenile fish can be induced, the growth of the large yellow croaker juveniles can be promoted, the metabolism can be promoted, and the immunity can be improved.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture and nutrition regulation technology, specifically relating to an ursodeoxycholic acid preparation for inducing juvenile fish to cluster, its preparation method, and its application. Background Technology

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

[0003] Currently, aquaculture often uses optimized feed formulations (such as adding attractants) or improved aquaculture environments (such as water flow control) to promote the growth of juvenile fish, but the effects are limited. While chemical signaling molecules (such as bile acids) have potential roles in fish olfactory perception and physiological regulation, research on their ability to induce schooling behavior is still lacking. Bile acids (such as cholic acid CA) can be detected by the fish olfactory system and elicit electrophysiological responses, but existing bile acid additives (such as tauroursodeoxycholic acid TUDCA) suffer from poor stability, and their optimal concentration and molecular mechanism of action remain unclear.

[0004] Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid used in medicine to treat hepatobiliary diseases. However, its application in aquaculture is mostly limited to cholesterol metabolism regulation, and there is a lack of research on its ability to induce schooling behavior in juvenile fish through the olfactory pathway. Furthermore, traditional additives are often directly mixed into feed, neglecting 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 signaling pathways.

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

[0006] To address the aforementioned technical problems, this invention provides an ursodeoxycholic acid preparation for inducing swarming behavior in juvenile fish, along with its preparation method and application.

[0007] The first objective of this invention is to provide an ursodeoxycholic acid preparation for inducing gregarious behavior in juvenile fish, comprising ursodeoxycholic acid and an additive of 0% to 2% by mass of ursodeoxycholic acid.

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

[0009] This invention uses ursodeoxycholic acid (UDCA) as its core component to induce gregarious behavior in juvenile large yellow croaker, promoting their growth, metabolism, and immunity. Additives (cholic acid or a mixture) enhance the stability and efficacy of the UDCA preparation. Furthermore, this invention combines electrophysiology, transcriptomics, and molecular biology techniques to reveal its mechanism of activating downstream signaling pathways through specific olfactory receptors (such as V2R1 and OR11A1), filling a technological gap in this field.

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

[0011] The second objective of this invention is to provide a method for preparing an ursodeoxycholic acid preparation that induces schooling behavior in juvenile fish, comprising: preparing ursodeoxycholic acid and an additive equivalent to 0% to 2% of the mass of ursodeoxycholic acid, mixing them evenly to obtain the ursodeoxycholic acid preparation that induces schooling behavior in juvenile fish.

[0012] The third objective of this invention is to provide an application of an ursodeoxycholic acid preparation that induces schooling behavior in juvenile fish, wherein the application refers to stimulating the olfactory sense of juvenile fish to induce schooling behavior.

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

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

[0015] Preferably, promoting the growth of juvenile large yellow croaker means increasing 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.

[0016] The aforementioned promotion of metabolism refers to the enhancement of intestinal lipase activity;

[0017] The aforementioned enhancement of immunity refers to strengthening antioxidant capacity and improving survival rate after fishing stress.

[0018] Preferably, the method of application is:

[0019] The application method is as follows: Ursodeoxycholic acid (UDCA) preparations that induce juvenile fish schooling behavior are mixed with a basal feed, with 50-100 mg of UDCA preparations added per kilogram of basal feed. The basal feed, on a dry weight basis, is a mixture of the following components in the following mass ratio: fish meal: soybean meal: soybean protein concentrate: gluten meal: wheat flour: fish oil: palm oil: calcium dihydrogen phosphate: soybean phospholipids: multivitamin and mineral mixture: antifungal agent: antioxidant: microcrystalline cellulose, in a ratio of 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 juvenile fish schooling behavior of the present invention consists of ursodeoxycholic acid and an additive equivalent to 0% to 2% by mass of ursodeoxycholic acid; the additive is cholic acid or a mixture, wherein the mixture is a mixture of taurine and tryptophan in a 1:1 mass ratio. Feeding juvenile large yellow croaker with the ursodeoxycholic acid preparation for inducing juvenile fish schooling behavior of the present invention can stimulate the juvenile fish's sense of smell, induce schooling behavior, promote the growth of juvenile large yellow croaker, promote metabolism, and enhance immunity. Promoting the growth of juvenile large yellow croaker refers to increasing survival rate, body weight, and condition factor, and increasing the crude fat and crude protein content of the whole fish and muscle tissue; promoting metabolism refers to increasing the activity of intestinal lipase; enhancing immunity refers to enhancing antioxidant capacity and improving survival rate after fishing stress. Attached Figure Description

[0022] Figure 1 Y-shaped maze apparatus and plume observation; where A, the apparatus is filled with seawater; B, the plume after adding potassium permanganate to one side of the selection arm for 20 seconds; C, the plume after adding potassium permanganate to one side of the selection arm for 60 seconds.

[0023] Figure 2 This study investigated the underwater electro-olfactogram (EOG) response and behavioral behavior of large yellow croaker to different bile acids (CA, UDCA, TUDCA, GUDCA, DCA, LCA, and HDCA). A represents the EOG concentration-response relationship of bile acids (mean ± standard error, n = 5); B represents the response of large yellow croaker to a concentration of 10... -8 Behavioral responses to different concentrations of bile acids (mol / L) are expressed as preference indices (mean ± standard error, n = 20); C, preference index of large yellow croaker's behavioral responses 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 ursodeoxycholic acid treatment; A, Pearson correlation coefficient comparison; B, principal component analysis of samples, with the ellipse representing the 95% confidence interval; C, gene expression volcano plot, where CON represents the olfactory epithelium sample group of large yellow croaker before ursodeoxycholic acid treatment, Day 3 represents the olfactory epithelium sample group of large yellow croaker 3 days after ursodeoxycholic acid treatment, and Day 7 represents the olfactory epithelium sample group of large yellow croaker 7 days after ursodeoxycholic acid treatment. P-value indicates whether the expression difference of a gene between the comparison groups is sufficiently significant, with P-value < 0.05 set as significant.

[0025] Figure 4 The results of the expression pattern analysis of the sensory receptor gene in the olfactory epithelium of large yellow croaker on the control group (CON), day 3 and day 7 after ursodeoxycholic acid treatment, are mors.

[0026] Figure 5 The results of the expression pattern analysis of the sensory receptor gene 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 ursodeoxycholic acid treatment are as follows: taars.

[0027] Figure 6 The results of the expression pattern analysis of the sensory receptor gene 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 ursodeoxycholic acid treatment, are shown in vrs.

[0028] Figures 4-6 In the diagram, gene expression levels are represented by FPKM, with red indicating high expression levels and blue indicating low expression levels.

[0029] Figure 7 The effect of ursodeoxycholic acid treatment on the expression levels of four olfactory receptor genes in large yellow croaker; where A is v2r1, B is or11a1, C is taar13c-10, and D is or52l2.

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

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

[0032] Figure 10 Functional characteristics of four olfactory receptors in large yellow croaker. A. Detection of calcium ion mobilization in HEK293T cells stimulated with different concentrations of ursodeoxycholic acid, and calcium response value (Ca). 2+ %) at 10 -4 mol·L -1 The calcium concentration used as a baseline for ursodeoxycholic acid activation of V2R1 was used for standardization; B, the luciferase activity of HEK293T cells stimulated with different concentrations of ursodeoxycholic acid was detected, with luciferase activity values ​​set at 10⁻⁶. -4 mol·L -1 Normalized based on the response of ursodeoxycholic acid to activate OR11A1 (mean ± standard error, n = 3); C, the regulatory effect of calcium mobilization response U-73122 (PLC inhibitor) on V2R1 olfactory receptor function; D, the regulatory effect of cAMP response SQ22536 (AC inhibitor) on Rho-OR11A1 olfactory receptor function; E, the regulatory effect of cAMP response SQ22536 on TAAR13C-10 olfactory receptor function; F, the regulatory effect of cAMP response SQ22536 on Rho-OR52L2 olfactory receptor function; among which, the calcium response (fold relative to control) level and the relative cAMP activation level are both normalized based on 10. -4 mol·L -1 DMSO-treated receptor-expressing cells were used as the baseline (mean ± standard error, n = 3). Detailed Implementation

[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 accompanying drawings.

[0034] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0035] Mechanistic Study 1: Analysis of Ursodeoxycholic Acid Behavior and Olfactory Function

[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 porcine deoxycholic acid (HDCA) (Maclean Biochemical Technology Co., Ltd., China) were all dissolved in dimethyl sulfoxide (DMSO) to prepare 10 -2 A stock solution of mol / L was prepared 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-shaped maze device is a Y-shaped water tank made of polypropylene, consisting of two selection arms and one mixing arm (each measuring 100cm × 40cm × 30cm). Each selection arm is connected to an inlet pipe and a water pump, respectively, while the mixing arm is connected to an outlet pipe to ensure the Y-shaped maze device is filled with flowing seawater. The seawater depth was 5cm during the experiment. Figure 1 As shown, when the flow rate in the inlet pipe is maintained at 600 mL / min, a solution containing potassium permanganate dye is injected into one of the selector arms. The red dye gradually diffuses into the mixing arm, but does not enter the other selector arm.

[0039] (3) EOG detection

[0040] At the start of the experiment, 20 mg / L was first administered orally. -1 The fish were anesthetized with MS-222 (Sigma-Aldrich, St. Louis, USA), followed by an intramuscular injection of approximately 5 mg / kg. -1 Triiodoquamidine was administered by body weight to further relax the muscles. The fish were secured in the experimental container with clamps, and administered 0.5 mL / s via a silicone tube inserted into their oral cavity. -1 A continuous flow of seawater was introduced to maintain gill respiration. During the experiment, the fish were covered with a damp towel to ensure their body surface remained moist. Microsurgery was performed to remove the skin and connective tissue from the olfactory epithelium for electrode placement. EOG recording was performed using two Ag / AgCl electrodes (World Precision Instruments, Sarasota, USA), with the electrode capillaries filled with 3 mol·L⁻¹ solution. - 1 KCl, 0.15 mol·L -1 An electrolyte solution of NaCl and 0.5 g / 100 g agar (tip diameter 150 μm) was used, with the recording electrode placed on the olfactory epithelium and the reference electrode fixed to the skin between the eyes. The test solution was dispensed at 6 mL / min. -1The reagent was delivered via a catheter at a high flow rate, with each stimulation lasting 5 seconds (to ensure the EOG signal reaches its peak), and a 120-second interval between stimulations to avoid olfactory adaptation. Each fish received stimulation with reagents of varying concentrations, from low to high, up to a maximum of 10 stimulations. The EOG signals were amplified, digitized, and displayed in real-time on a computer using the BL-420F data acquisition and analysis system (designed by Taimeng Software, Chengdu, China).

[0041] (4) Behavioral testing

[0042] After a 24-hour starvation period, the experimental fish underwent behavioral testing. Five fish were randomly selected and placed in a Y-shaped maze apparatus for 20 minutes to acclimatize. A camera was mounted above the Y-shaped maze apparatus to record the swimming behavior and movement paths of the fish throughout the experiment. During the experiment, the fish could swim freely in the selection arm and the mixing arm. Five minutes after the start of the experiment, the inlet pipe connected to the selection arm with fewer fish was switched from filtered seawater to the pre-prepared test solution, while the other selection arm continued to pump filtered seawater. The camera recorded a screenshot every 5 seconds to record and count the number of fish in the selection arm.

[0043] The preference index (PI) characterizes the attractiveness of the test solution to the experimental fish. The 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 control group's selection arm before the introduction of the test solution, Ae represents the number of experimental fish in the experimental group's selection arm after the introduction of the test solution, and Ac represents the number of experimental fish in the control group's selection arm after the introduction of the test solution. The PI value ranges from -1 to 1; a higher PI value indicates a stronger attractiveness, and a lower PI value indicates a weaker attractiveness. The behavioral test was conducted from 10:00 to 16:00. After the experiment, the experimental apparatus was thoroughly rinsed sequentially with 5% bleach, tap water, and filtered seawater. Experimental fish not previously used in the experiment were used in each experiment, and each test substance was repeated at least 20 times.

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

[0045] The experimental fish were healthy large yellow croakers from the same batch, sourced from Xixuan Fishery Science and Technology Island, Zhoushan City, Zhejiang Province. They weighed 51.2±7.5g and had a total length of 16.4±2.9cm. Before the experiment, 360 fish were randomly assigned to 24 tanks (15 fish per tank) for a two-week acclimatization period. The fish were housed in a flowing water system with each tank containing 400L of water at a temperature of 25.0℃, pH 8.2, dissolved oxygen greater than 7.0mg / L, salinity 26.0, and ammonia nitrogen 0.04mg / L. During the experiment, the fish were fed a full meal of Tongwei commercial pellet feed at 8:00 AM and 6:00 PM. Approximately 75% of the seawater in each tank was replaced one hour after each feeding. A 7-day ursodeoxycholic acid (UDCA) treatment experiment was also conducted. Before the experiment, UDCA was dissolved in DMSO to prepare 4×10⁻⁶ ursodeoxycholic acid (UDCA) solutions. -4 A ursodeoxycholic acid (URC) solution of mol / L was stored at 4°C. During the experiment, under static water conditions, 4 mL of URCC solution was injected twice daily (5 minutes before feed feeding) along the inner wall into the experimental group's water tanks (a total of 12 tanks). The final concentration of URCC in the breeding tanks was 1 × 10⁻⁶ mol / L. -8 mol / L; the control group of 12 water tanks was injected with 4 mL DMSO.

[0046] Total RNA was extracted from tissues using the TRIzol method (Thermo Fisher Scientific, USA). After constructing cDNA libraries, sequencing was performed using an Illumina NovaSeq 6000 platform (Inmena, USA), followed by bioinformatics analysis. β-actin was selected as an internal control gene and validated by qRT-PCR 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, olfactory epithelial tissue was collected from healthy large yellow croakers (weighing 28.7±4.6g) at a Zhoushan aquaculture base. After RNA extraction (Solarbio kit) and cDNA synthesis, the full-length genes of four receptors, including V2R1 and Rho-OR11A1, were cloned using RACE technology. The correct sequences were obtained through pMD19-T vector construction and sequencing verification. Subsequently, the pcDNA3.1-Rho-1D4 expression vector (containing rhodopsin signal peptide) was constructed and ligated to the receptor gene via EcoRI / Xho I double digestion. Simultaneously, pCI-mRTP1s and pCI-Gαolf helper expression vectors were constructed. In HEK293T cells, the receptor expression plasmid and helper plasmid were co-transfected using Lipofectamine 3000, and localization was verified by immunofluorescence (1D4 antibody / Alexa Fluor 555 secondary antibody). The calcium response to bile acid stimulation, such as ursodeoxycholic acid, was detected using the Fluo-4AM probe (Tecan Spark microplate reader), and a CRE-luciferase reporter system (pGL4.29 vector) was established to detect cAMP signal activation (Tecan Spark microplate reader).

[0049] 2. Experimental Results

[0050] (1) Electrophysiological response results

[0051] The EOG response induced by CA, UDCA, TUDCA, GUDCA, DCA, LCA, and HDCA in large yellow croaker was detected. Figure 2 Figure A shows the EOG response amplitude after L-serine percentage normalization. The results indicate that the EOG response amplitude induced by bile acids gradually increases with increasing concentration. Among them, ursodeoxycholic acid, DCA, and TUDCA induced the most significant EOG responses, exhibiting rapidly increasing concentration-response curves, and detection thresholds as low as 10. -12 mol / L.

[0052] (2) Behavioral test results of the Y-shaped maze device

[0053] An increase or decrease in the number of experimental fish on the side where the test substance was added was interpreted as attraction or avoidance. 10 -8 At a concentration of mol / L, CA and ursodeoxycholic acid significantly induced the preferred behaviors of the experimental fish (P<0.001). Figure 2(B). TUDCA, GUDCA, DCA, LCA, HDCA, and DMSO showed no significant attraction effects on the experimental fish (P>0.05). Notably, the preference index of ursodeoxycholic acid (UDCA) was 43.84% higher than that of CA, indicating that UDCA had a stronger attraction for large yellow croaker. Further research revealed that the preference index of UDCA was dose-dependent, meaning that the preference index increased with increasing UDCA concentration, and reached a peak around 10. -8 The preference index for ursodeoxycholic acid reaches its maximum at a concentration of mol / L; at higher concentrations, the preference index decreases with increasing concentration. Figure 2 (C).

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

[0055] To investigate the olfactory receptors in large yellow croaker (Cynodon dactylis) and their olfactory signal transduction mechanisms, cDNA libraries were constructed from olfactory epithelial tissues of large yellow croaker before ursodeoxycholic acid treatment (CON), and on days 3 and 7 after ursodeoxycholic acid treatment. A total of 414,416,420 raw reads were obtained. 91.14% of clean reads were successfully mapped to the large yellow croaker reference genome, and 23,842 genes were annotated. Significant differences in gene expression were observed between the ursodeoxycholic acid treatment groups (Day 3 and Day 7) and the control group (CON). Figure 3 ).

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

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

[0058] This invention successfully cloned the full-length sequences of the above four olfactory receptor genes using RACE technology. Analysis showed that the proteins encoded by the four genes have the typical seven-transmembrane structure characteristics of GPCRs. Figure 8 The olfactory receptor gene expression vectors pcDNA3.1-Rho-OR-1D4 and pcDNA3.1-OR-1D4 were constructed from large yellow croaker and then co-transfected into HEK293T cells with the pCI-mRTP1s gene expression vector and the pEGFP-N1 plasmid. Experimental results showed that all four olfactory receptor proteins were successfully expressed on the HEK293T cell membrane, with the Rho-tag signal peptide enhancing the expression of OR11A1 and OR52L2 proteins on the cell membrane. (The last sentence appears to be incomplete and possibly refers to intracellular Ca2+ expression.) 2+ Detection and CRE-firefly luciferase reporter gene assays revealed that V2R1 participates in the transduction of ursodeoxycholic acid olfactory signals via the PLC pathway, while TAAR13C-10, OR11A1, and OR52L2 participate via the AC-cAMP pathway. Figures 9-10 ).

[0059] Based on the above mechanistic analysis results, this invention focuses on the effects of ursodeoxycholic acid on olfactory receptors and their signal transduction pathways in juvenile large yellow croaker.

[0060] Example 1

[0061] A ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish, namely ursodeoxycholic acid.

[0062] Example 2

[0063] A ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish comprises ursodeoxycholic acid and an additive equivalent to 1% by mass of ursodeoxycholic acid; the additive is cholic acid. The raw materials are mixed evenly to obtain the ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish.

[0064] Example 3

[0065] A ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish comprises ursodeoxycholic acid and an additive equivalent to 1.5% by mass of ursodeoxycholic acid; the additive is cholic acid. The raw materials are mixed evenly to obtain the ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish.

[0066] Example 4

[0067] A ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish comprises ursodeoxycholic acid and an additive equivalent to 2% by mass of ursodeoxycholic acid; the additive is cholic acid. The raw materials are mixed evenly to obtain the ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish.

[0068] Example 5

[0069] An ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish comprises ursodeoxycholic acid and an additive equivalent to 1% by mass of ursodeoxycholic acid; the additive is a mixture of taurine and tryptophan in a 1:1 mass ratio. All raw materials are mixed thoroughly to obtain the ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish.

[0070] Example 6

[0071] A ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish comprises ursodeoxycholic acid and an additive equivalent to 1.5% by mass of ursodeoxycholic acid; the additive is a mixture of taurine and tryptophan in a 1:1 mass ratio. All raw materials are mixed thoroughly to obtain the ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish.

[0072] Example 7

[0073] An ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish comprises ursodeoxycholic acid and an additive equivalent to 2% by mass of ursodeoxycholic acid; the additive is a mixture of taurine and tryptophan in a 1:1 mass ratio. All raw materials are mixed thoroughly to obtain the ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish.

[0074] Application Experiment 1: Effects of Ursodeoxycholic Acid Preparations on Juvenile Large Yellow Croaker

[0075] 1. Preparation of livestock feed

[0076] See Table 2 for different aquaculture feed formulations. The preparation process involves first mixing the powdered ursodeoxycholic acid preparation that induces juvenile fish herding behavior evenly with the basic feed, followed by extrusion, granulation, and drying. Ursodeoxycholic acid: Maclean Biotech Co., Ltd. (China), 99% purity, CAS (Chemical Abstract Service) Registry No. 128-13-2.

[0077] The basic feed formulation is shown in Table 1. In Table 1, fishmeal is from Kodiak Fishmeal Company (USA); soybean meal is from Yihai Kerry Corporation (China); soybean protein concentrate is from Yihai Kerry Corporation (China); wheat flour and high-gluten flour are commercially available; fish oil is from Rongcheng Ayers Marine Biotechnology Co., Ltd. (China); palm oil is from COFCO Group Co., Ltd. (China); soybean lecithin is from Cargill (Germany); the composition of the multivitamin and mineral mixture and antifungal agent is referenced from the previously 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 yellowcroaker (Larimichthys crocea): An ex vivo study[J]. Fish & Shellfish Immunology, 2019, 84:955-961"; and the antioxidant is tert-butylhydroquinone.

[0078] Table 1 Basic Feed Formulation

[0079] raw material Mass concentration (g, on dry weight) fish meal 200 soybean meal 400 Soy protein concentrate 80 Gu Ruanfen 50 flour 70 fish oil 40 Palm oil 30 calcium dihydrogen phosphate 5 Soybean phospholipids 20 Multidimensional and multi-mineral mixture 15 Antifungal agent 1 antioxidants 0.5 microcrystalline cellulose 83.5

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

[0081] Table 2 Feed Formulation for Animal Husbandry

[0082]

[0083]

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

[0085] 2. Aquaculture

[0086] The aquaculture experiment was conducted in the flow-through aquaculture system of Xixuan Fishery Science and Technology Island, Zhoushan City, Zhejiang Province. Prior to the experiment, healthy and uniformly sized juvenile large yellow croakers from the same batch underwent a two-week acclimatization period to ensure they adapted to the aquaculture conditions. Four hundred juvenile large yellow croakers with an initial weight of 15.55 ± 0.02 g were randomly assigned to rearing tanks, 25 fish per tank, with four replicates per treatment. Experimental feed was randomly distributed to each tank. The rearing period was 70 days, with feeding twice daily at 6:00 AM and 5:00 PM. The tank capacity was 1000 L, and the seawater flow rate was approximately 3 L / min. During the experiment, 400 L of seawater was replaced twice daily, and feces were removed using a siphon during water changes. Residual feed was counted and tallied. The number of dead fish was recorded twice daily, and the weight of the deceased fish was also recorded. No diseases or other unforeseen events occurred during the rearing period.

[0087] 3. Sample collection and testing

[0088] After the culture experiment, the experimental fish were fasted for 12 hours and anesthetized with MS-222 before being weighed individually. Five fish were randomly selected from each culture tank for nutrient composition analysis. Ten fish were randomly selected from each culture tank to measure their body length and total length, and to determine the mass of their visceral mass and liver to calculate condition factor, visceral-to-body ratio, and liver-to-body ratio. Liver and intestinal tissue samples were collected for physiological and biochemical parameter determination. Blood samples were obtained using the tail vein method, allowed to stand for 4 hours, centrifuged, and serum was separated for subsequent physiological, biochemical, and immunological parameter determination. Samples used for physiological and biochemical parameter determination were stored at -80°C.

[0089] The main steps of the fishing stress test were as follows: Seawater was drained from the culture tank to 30 cm from the bottom; a fishing net was used to agitate the fish in the tank to simulate a fishing environment, with an agitation speed of 6 seconds per revolution for 5 minutes. Agitation was repeated every 30 minutes for a total of 4 times. The number of fish deaths was recorded every 2 hours over the following 48 hours. The fishing stress test used a semi-lethal condition for the experimental fish.

[0090] Refer to the following formulas to calculate the relevant indicators:

[0091] Survival rate (SR, %) = Number of fish in the final experiment per tank × 100 / Number of fish in the initial experiment per tank;

[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 weight of fish (g);

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

[0095] Where t is the experimental period, in days.

[0096] 4. Experimental Results

[0097] 4.1 Results of inducing schooling behavior in juvenile fish

[0098] Table 3 shows that, compared to the control group, the addition of ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce juvenile schooling behavior increased the preference index for juvenile schooling behavior, and the increase was more significant with higher doses (P < 0.05). The preference indices of controls 1 and 2 also increased compared to the control group (P < 0.05), indicating that cholic acid or its mixtures, used alone, can also induce juvenile schooling behavior.

[0099] Table 3. Results of the preference index test for inducing schooling behavior in 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 Tables 4 and 5. Compared with the control group, the survival rate, final body weight, and condition factor of fish supplemented with ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce juvenile schooling behavior were significantly increased (P < 0.05), while the feeding rate did not differ significantly. The final body weight and condition factor of controls 1 and 2 were also increased compared with the control group (P < 0.05).

[0104] Table 4. Results of survival, growth performance, and body composition tests of large yellow croaker (low dose).

[0105]

[0106] Table 5. Results of survival, growth performance, and body composition tests of large yellow croaker (high dose).

[0107]

[0108]

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

[0110] The results are shown in Tables 6 and 7. Compared with the control group, the addition of ursodeoxycholic acid or other ursodeoxycholic acid preparations that induce juvenile fish schooling behavior significantly increased the crude protein, crude fat, and muscle fat of the whole fish (P < 0.05), while the difference in liver crude fat was not significant. The crude protein, crude fat, and muscle fat of controls 1 and 2 were also increased compared with the control group (P < 0.05).

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

[0112]

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

[0114]

[0115]

[0116] 4.3 Intestinal lipase activity

[0117] Table 8 shows that the addition of ursodeoxycholic acid, which induces swarming behavior in juvenile fish, significantly increased the intestinal lipase activity in large yellow croaker (P < 0.05), indicating an improvement in its metabolic function. The intestinal lipase activity in control 1 and control 2 large yellow croakers also increased compared to the control group (P < 0.05).

[0118] Table 8 Results of intestinal lipase activity test

[0119]

[0120] 4.4 Results of Immune-Related Indicator Tests

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

[0122] Table 9 Results of Immune-Related Indicators Tests

[0123]

[0124] The above experimental results show that when ursodeoxycholic acid is combined with cholic acid or a mixture, it is most beneficial to induce schooling behavior and growth in juvenile large yellow croaker.

[0125] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0126] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A ursodeoxycholic acid preparation for inducing schooling behavior in juvenile fish, characterized in that, It consists of ursodeoxycholic acid and additives at a mass equivalent to 1% to 1.5% of ursodeoxycholic acid; The additive is cholic acid or a mixture, wherein the mixture is a mixture of taurine and tryptophan in a mass ratio of 1:

1.

2. The method for preparing the ursodeoxycholic acid preparation for inducing juvenile fish schooling behavior according to claim 1, characterized in that, include: Prepare ursodeoxycholic acid and an additive of 1% to 1.5% by mass of ursodeoxycholic acid, mix them evenly to obtain an ursodeoxycholic acid preparation that induces schooling behavior in juvenile fish.

3. The application of the ursodeoxycholic acid preparation for inducing juvenile fish schooling behavior according to claim 1, characterized in that, The application refers to stimulating the sense of smell of juvenile fish to induce their schooling behavior.

4. The application of the ursodeoxycholic acid preparation for inducing juvenile fish schooling behavior according to claim 3, characterized in that, The juvenile fish mentioned are juvenile large yellow croaker.

5. The application of the ursodeoxycholic acid preparation for inducing juvenile fish schooling behavior according to claim 4, characterized in that, The application refers to at least one of the following: promoting the growth of juvenile large yellow croaker, promoting metabolism, and enhancing immunity; The promotion of growth in juvenile large yellow croaker refers to improving survival rate, weight and fatness, increasing crude fat content in whole fish and muscle tissue, and increasing crude protein content in whole fish and muscle tissue. The aforementioned promotion of metabolism refers to the enhancement of intestinal lipase activity; The aforementioned enhancement of immunity refers to strengthening antioxidant capacity and improving survival rate after fishing stress.

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