A method for detecting metabolites in different developmental stages of fertilized eggs of Megalobrama triangularis

Abstract

The present invention relates to a method for detecting and analyzing metabolites during the hatching process of fertilized eggs of fish, and in particular to a method for detecting metabolites at different developmental stages of the hatching of fertilized eggs of a triangular bream. The method uses a non-targeted metabolomics method to study fertilized egg samples during the hatching process of a triangular bream, analyzes the types, abundances and metabolic pathways of the main differential metabolites during the embryonic development of the triangular bream, and clarifies the regulatory mechanism of the differential metabolites affecting the embryonic development of the triangular bream, thereby providing theoretical support for the optimization of efficient hatching technology facilities and systems for triangular bream, and also providing basic data for the breeding and breeding of excellent germplasm of triangular bream.

Description

Technical Field

[0001] The invention relates to a method for detecting and analyzing metabolites in a fish fertilized egg hatching process, and in particular to a method for detecting metabolites in different developmental stages of a bream fertilized egg hatching process. Background Art

[0002] Triangle bream ( Megalobrama amblycephala ) belongs to the genus Megalobrama, family Cypriniformes, order Cypriniformes Cypriniformes ), widely distributed in most rivers and lakes in my country. In recent years, with the continuous expansion of the breeding area of ​​triangular bream, the demand for fry has also increased. However, at this stage, the artificial breeding of triangular bream in my country mostly adopts the traditional method of artificial induction of spawning and running water hatching under natural open conditions, while the circulating water breeding technology under fully artificial control conditions indoors is not yet mature, resulting in poor controllability, consumption of a large amount of water resources, unstable breeding, and seedlings are prone to pathogens. The breeding and hatching technology is relatively weak and cannot meet the rapid growth needs of the industry. In addition, the research foundation of fish fertilized egg metabolites is weak, and the types and abundance of metabolites during the hatching process are still unknown. This seriously affects the facility optimization of the indoor fish fertilized egg fully automatic circulating water hatching system, and it is impossible to effectively remove specific metabolites, resulting in the deterioration of water quality at specific stages of the hatching process, which seriously affects the hatching rate of fertilized eggs and is not conducive to the healthy development of the industry. Therefore, improving the hatching rate of fertilized eggs is of great significance to the sustainable and healthy development of its industry.

[0003] The embryonic development of the triangular bream goes through the pre-cleavage stage, cleavage stage, blastocyst stage, gastrula stage, neurula stage, organogenesis stage, and exocyst stage. Liu Juan et al. found that in the embryonic development of the triangular bream, the TET1 gene is expressed from the fertilized egg, and the expression level increases significantly with the continuous development of the embryo, significantly increasing at 20hpf and maintaining a high level at 20-44hpf. The TET1 gene may be involved in the embryonic development process and play an important role in the embryonic development process. "Analysis of the expression of leptin gene in various tissues and early development of adult amblycephala bream [J]. Journal of Huazhong Agricultural University, 2016, 35 (01): 92-98." Zhao Honghao et al. found that the expression trends of leptin, leptin-R and leprotl-1 genes in various stages of early embryonic development of amblycephala bream were basically consistent and these three genes may be involved in energy regulation in the early stages of embryonic development. "Correlation between water temperature, dissolved oxygen, flow rate, and egg density and the emergence rate of amblycephala bream [J]. Fisheries Science and Technology Information, 1997, (02): 25-27." Hao Siping et al. found that the hatching temperature of amblycephala bream was 20℃-26℃, the dissolved oxygen content of the water was above 4mg / L, the flow rate of the water in the loop was 0.25-0.49m / sec, and the egg density was 150-200×104 / m 3 So far, there are no reports on the study of exogenous toxicity in the embryos of the triangular bream, but in the model fish zebrafish ( Daniorerio) in "Study on the Effect of Trichlorfon on Embryonic Development, Larval Growth and Neurotoxicity of Zebrafish [D]. Henan Normal University, 2022." Chen Yangli found that after exposure to trichlorfon, the survival rate of zebrafish embryos decreased, the hatching rate decreased, the deformity rate increased, the heart rate slowed down, the weight decreased, and the body length shortened, affecting their normal growth and development. "Toxicity and Oxidative Damage Mechanism of Phosalone on Zebrafish Heart Development [J]. Chinese Environmental Science, 2023, 43 (04): 1957-1966." Zhong Shengzhong et al. found that exposure to phosalone can reduce the hatching rate, body length and heart rate of zebrafish embryos, cause pericardial edema and abnormal atrial and ventricular differentiation, affect the normal expression of genes related to cardiac development, and have obvious cardiac developmental toxicity. Metabolomics analysis can more intuitively analyze the main metabolites in each developmental period and their role in the embryonic development and growth process. "Study on the combined toxic effects of different forms of microplastics and mercury on zebrafish embryonic development [J]. Chinese Environmental Science, 2023, 43 (04): 1978-1984." Shi Yansong, Wang Jing, et al. explored the mechanism by which microplastics reduce the toxic effects of Hg on zebrafish embryonic development from the perspective of metabolomics. "Study on the environmental exposure characteristics of organophosphates in typical industrial areas and their effects on zebrafish embryo metabolism [D]. China University of Geosciences, 2022." Liu Yunlang used metabolomics to find that exposure to TCEP (tris(2-chloroethyl) phosphate) caused the downregulation of 5 metabolites such as retinal and 11-cis-retinal and the upregulation of 16 metabolites such as citrulline, histamine, docosahexaenoic acid, and glutamine, affecting the gene expression and metabolic activity, redox defense, energy charge state improvement, and retinol metabolism regulation of zebrafish embryos. Metabolic regulation analysis showed that TCEP at environmental equivalent concentrations had the potential to destroy redox homeostasis and embryonic development of zebrafish embryos. "Effects of replacing fish meal with American cockroach meal on biochemical indices, disease resistance and metabolomics of rainbow trout juveniles [J]. Southern Fisheries Science, 2023, 19 (04): 86-97" Chen Li et al. used metabolomics analysis results to find that replacing fish meal with American cockroach meal mainly affects phospholipid and amino acid metabolism in rainbow trout juveniles. With the development of metabolomics technology, metabolomics technology can detect the changes in the relative content of metabolites in the embryonic development of fertilized eggs of triangular bream and understand their functions and metabolic pathways, but no research on fertilized eggs of triangular bream in this regard has been reported. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a method for detecting metabolites in different developmental stages of the hatching of fertilized eggs of triangular bream. The method studies the fertilized egg samples taken by a non-targeted metabolomics method, analyzes the types, abundance and metabolic pathways of the main differential metabolites in the embryonic development of triangular bream, and clarifies the mechanism by which differential metabolites affect the embryonic development of triangular bream, in order to analyze the main metabolic mechanisms in the hatching of fertilized eggs of triangular bream, thereby providing theoretical support for the optimization of the efficient hatching technology system of triangular bream, and also providing basic data for the breeding and breeding of excellent germplasm of triangular bream.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] A method for detecting metabolites in different developmental stages of fertilized eggs of Megalobrama triangularis, the method comprising the following steps:

[0007] 1) Place artificially induced fertilized eggs of Megalobrama triangularis in a culture dish for static water incubation, pick out dead eggs every 3-5 hours, replace the aerated water, and take fertilized egg samples at different stages of incubation and development;

[0008] 2) Weigh the sample into a centrifuge tube and add grinding beads; add the extract containing the internal standard, grind in a -20~-5℃ frozen tissue grinder, and perform low-temperature ultrasonic extraction at 1-10℃;

[0009] 3) Place the sample at -25-15℃ for 20-40min; centrifuge at 10000-15000g for 10-20min at 1-10℃, transfer the supernatant into a vial with an inner insert and analyze on the analyzer;

[0010] 4) Analyze using ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry system to obtain total ion chromatogram;

[0011] 5) The raw data were imported into the metabolomics processing software for baseline filtering, peak identification, integration, retention time correction, and peak alignment to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information; then, a characteristic peak search library was performed to match the MS and MS / MS mass spectrometry information with the metabolic database, and the MS mass error was set to less than 10 ppm. At the same time, metabolites were identified based on the secondary mass spectrometry matching score, and metabolic set analysis was performed using differential metabolic sets. The screened differential metabolites were annotated, classified, clustered, enriched, and analyzed for overall changes in metabolic pathways using the KEGG database.

[0012] Preferably, in step 1), the sperm-egg mixture is placed in a culture dish with a diameter of 15 cm, and then oxygenated and aerated clean water is added and shaken to make it evenly distributed in the culture dish, and about 300 fertilized eggs are placed in each culture dish; during the incubation process, the water is changed every four hours, dead eggs and moldy eggs are cleaned, and samples are taken at 17h, 25h, 32h, 41h, 50h, and 58h respectively.

[0013] Preferably, the extract in step 2) is a methanol-water solution, methanol: water = 4:1 (v:v), and the internal standard includes L-2-chlorophenylalanine, 0.02 mg / mL.

[0014] Preferably, 20 µL of supernatant is taken from each sample in step 3), and the mixture is used as a quality control sample. The volume of each quality control sample is the same as that of the sample, and the sample is processed and tested in the same way as the analytical sample. During the instrument analysis, a quality control sample is inserted into every 5-15 analytical samples to examine the stability of the entire detection process.

[0015] Preferably, in the step 4), the ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry system adopts the ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system of Thermo Fisher Scientific; the chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC HSS T3; the mobile phase A is 95% water + 5% acetonitrile by volume, and 0.1% formic acid is added to the total volume; the mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water by volume, and 0.1% formic acid is added to the total volume, the injection volume is 3 μL, and the column temperature is 40°C; the mass spectrometry conditions are as follows: the sample is ionized by electrospray, and the mass spectrometry signals are collected in positive and negative ion scanning modes respectively.

[0016] Preferably, in step 5), according to the correlation of the metabolic pathways of the screened differential metabolites, a KEGG main metabolic pathway map - nucleotide metabolism is drawn, and a total of 14 key differential metabolites are identified, among which the relative contents of dIMP, IMP, hypoxanthine, inosine, uracil, guanine, guanosine, deoxyuridine and thymine are up-regulated, and the relative contents of adenine), UMP, CMP and dTMP are down-regulated.

[0017] Preferably, the KEGG enrichment in step 5) found that compared with the control group, a total of 23 differential metabolites were screened out: the relative contents of inosine, L-tryptophan, thymine, phosphorylcholine, hypoxanthine, glycerophosphatidylcholine, aspartic acid, ornithine, uracil, guanosine, uridine, uric acid, 5 (S) -HETE, prostaglandin H2, retinoic acid, L-leucine and L-isoleucine were significantly upregulated, while the relative contents of adenine, adenosine, creatine, thymidine deoxynucleotide, ADP ribose and maltotriose were significantly downregulated.

[0018] Furthermore, the present invention also discloses the application of the method in improving the hatching process of fertilized eggs of Megalobrama triangularis, increasing the hatching rate and / or increasing the stability of embryonic development.

[0019] Furthermore, the present invention also discloses the application of the metabolome obtained by the method as a marker for detecting the hatching process of fertilized eggs of Megalobrama triangularis.

[0020] The present invention adopts the above-mentioned technical scheme, takes the fertilized eggs of the triangular bream gastrula stage (17h), neurula stage (25h), myometrium and eye base formation stage (32h), tail bud stage (41h), muscle effect stage (50h) and heartbeat stage (58h) as experimental objects, and uses non-targeted metabolomics method to study and analyze the fertilized egg samples. The results show that compared with the control group, all experimental groups screened out 946 differential metabolites, mainly including differential metabolites such as inosine, purine, L-valine, and L-isoleucine. Compared with the control group at 17 h, the expression levels of differential metabolites such as prostaglandin H2, sorbitol, tyrosine and glutamate were up-regulated in the experimental group at 25 h; the expression levels of differential metabolites such as prostaglandin H2, glycyltyrosine and pregnenolone were up-regulated in the experimental group at 32 h; the expression levels of differential metabolites such as imidazole propionic acid, dynorphin B (6-9) and glycyl-phenylalanine were up-regulated, and the expression level of ADP ribose was down-regulated in the experimental group at 41 h; the expression levels of differential metabolites such as neuromedin B, alanylproline and α-hederosaponin were up-regulated, and the expression level of ADP ribose was down-regulated in the experimental group at 50 h; the expression levels of differential metabolites such as uridine, hydroxyconopteris and neopterin were up-regulated in the experimental group at 58 h. KEGG pathway enrichment analysis showed that these differential metabolites were significantly enriched in pathways such as ABC transporter, steroid hormone biosynthesis, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, and arachidonic acid metabolism. Among them, in H25VS.H17, the nucleotide metabolism, ABC transporter, and pyrimidine metabolism pathways were more enriched; in H32VS.H17, the nucleotide metabolism, ABC transporter, and arachidonic acid metabolism pathways were more enriched; in H41VS.H17, the nucleotide metabolism, purine metabolism, and arachidonic acid metabolism pathways were more enriched; in H50VS.H17, the nucleotide metabolism, purine metabolism, and arachidonic acid metabolism pathways were more enriched; in H58VS.H17, the nucleotide metabolism, purine metabolism, and purine metabolism pathways were more enriched. According to the functions of differential metabolites and the metabolic pathways involved, the hatching and development of fertilized eggs of G. triangularis may be related to the increased secretion of metabolites such as retinol, guanine and aspartic acid. It is speculated that the embryonic development is mainly affected by pathways such as glycerophospholipid metabolism, pyrimidine metabolism, arachidonic acid metabolism, purine metabolism, ABC transporters, and retinol metabolism, thus providing a theoretical basis for an efficient hatching system for G. triangularis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The PCA diagram and Venn diagram for comparative analysis of samples in six periods; A is the PCA diagram, and B is the Venn diagram; the abscissa PC1 represents the first principal component, and the ordinate PC2 represents the second principal component; H17, H25, H32, H41, H50, H58, and QC are samples incubated at 17h (gastrula stage), 25h (neurula stage), 32h (myotome and eye base formation stage), 41h (tail bud stage), 50h (muscle effect stage), 58h (heartbeat stage), and quality control stage, respectively.

[0022] Figure 2 Pie chart showing the classification statistics of HMDB compounds.

[0023] Figure 3 This is the volcano plot of the 25h group and the 17h group under the anion-cation binding mode.

[0024] Figure 4 This is the volcano plot of the 32h group and the 17h group under the anion-cation binding mode.

[0025] Figure 5 This is the volcano plot of the 41h group and the 17h group under the anion-cation binding mode.

[0026] Figure 6 This is the volcano plot of the 50h group and the 17h group under the anion-cation binding mode.

[0027] Figure 7 This is the volcano plot of the 58h group and the 17h group under the anion-cation binding mode.

[0028] Figure 8 Heat map of differential metabolites cluster analysis for each sample in the six periods.

[0029] Fig. 9 VIP diagram of significantly different metabolites between the 25h group and the 17h group.

[0030] Fig.10 VIP diagram of significantly different metabolites between the 32h group and the 17h group.

[0031] Fig.11 VIP diagram of significantly different metabolites between the 41h group and the 17h group.

[0032] Fig.12 VIP diagram of significantly different metabolites between the 50h group and the 17h group.

[0033] Fig.13 VIP diagram of significantly different metabolites between the 58h group and the 17h group.

[0034] Fig.14 Bubble diagram of pathway enrichment in 25h group and 17h group.

[0035] Fig.15 Bubble diagram of pathway enrichment in the 32h group and the 17h group.

[0036] Fig.16 This is the pathway enrichment bubble diagram of the 41h group and the 17h group.

[0037] Fig.17 Bubble diagram of pathway enrichment in the 50h group and the 17h group.

[0038] Fig.18 This is the pathway enrichment bubble diagram of the 58h group and the 17h group.

[0039] Fig.19 This is a diagram for the analysis of the nucleotide metabolic pathway. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0041] 1 Materials and methods

[0042] 1.1 Materials

[0043] The fish species used in the present invention are from Huzhou Deqing Haoyuan Aquatic Seed Co., Ltd., and the fertilized eggs are obtained by artificial induction of spawning of parents with strong physique, no disease or injury, and good maturity (female fish weighing 1500g, 3-year-old fish; male fish weighing 1398g, 2-year-old fish).

[0044] 1.2 Methods

[0045] 1.2.1 Artificial induction of labor and embryo hatching

[0046] Select parents with strong physique, no disease or injury, and good maturity for artificial induction of labor. The injection dose of oxytocin is 2000IUHCG+2ugLRH-A3 / kg, and the dose for male fish is halved. When the water temperature is about 25℃, the effect time is 8-10h. After induction of labor, artificial insemination is used. First, dry the triangular bream parents with a towel, squeeze the eggs of the female fish into a dry and smooth basin, and spread 2-3 layers flat. Then add physiological saline to the semen of the triangular bream to dilute it, mix the diluted semen and eggs thoroughly, and stir for about 2 minutes. Put the sperm and egg mixture into a culture dish with a diameter of 15cm, then add oxygenated and aerated clean water to shake it so that it is evenly distributed in the culture dish, and place about 300 fertilized eggs in each culture dish. During the incubation process, the water was changed every four hours, and dead and moldy eggs were cleaned up. An equal number of samples were taken at 17h, 25h, 32h, 41h, 50h, and 58h, with six replicates in each period. The samples were sent to Meiji Biotechnology for metabolome sequencing.

[0047] 1.2.2 Metabolomics experimental methods

[0048] 1) Sample processing

[0049] Accurately weigh 65±5 mg of sample into a 2 mL centrifuge tube and add a 6 mm diameter grinding bead; add 400 µL of extraction solution (methanol: water = 4:1 (v:v)) containing internal standard (L-2-chlorophenylalanine (0.02 mg / mL) etc.); grind with a frozen tissue grinder for 6 min (-10°C, 50 Hz); perform low temperature ultrasonic extraction for 30 min (5°C, 40 KHz); place the sample at -20°C for 30 min; centrifuge for 15 min (13000 g, 4°C), transfer the supernatant to an injection vial with an inner tube for analysis; in addition, transfer 20 µL of supernatant from each sample and mix them as quality control samples.

[0050] 2) LC-MS detection

[0051] The instrument platform for LC-MS analysis of the present invention is an ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system from Thermo Fisher Scientific.

[0052] Chromatographic conditions: The chromatographic column was ACQUITY UPLC HSS T3 (100 mm × 2.1 mmi.d., 1.8 µm; Waters, Milford, USA); mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), the injection volume was 3 μL, and the column temperature was 40 °C.

[0053] Mass spectrometry conditions: The samples were ionized by electrospray ionization, and the mass spectrometry signals were collected in positive and negative ion scanning modes, respectively.

[0054] Quality Control

[0055] Quality control samples (QC) are prepared by mixing equal volumes of extracts from all samples. The volume of each QC is the same as that of the sample and is processed and tested in the same way as the analytical samples. During instrument analysis, a QC sample is inserted into every 5-15 analytical samples to examine the stability of the entire detection process.

[0056] Data Results

[0057] The components of the sample separated by chromatography continuously enter the mass spectrometer, and the mass spectrometer is continuously scanned for data acquisition. Each scan obtains a mass spectrum. All ion intensities in each mass spectrum are added to obtain a total ion current intensity. The total ion chromatogram is drawn with time as the horizontal axis and the sum of ion intensities as the vertical axis. The raw data are imported into the metabolomics processing software ProgenesisQI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, peak alignment, etc., and finally a data matrix containing information such as retention time, mass-to-charge ratio and peak intensity is obtained. The software is then used to search the library for characteristic peaks, match the MS and MS / MS mass spectrometry information with the metabolic database, and the MS mass error is set to less than 10ppm. At the same time, metabolites are identified according to the secondary mass spectrometry matching score, and the differential metabolic set is used for metabolic set analysis. The differential metabolites screened out are annotated, classified, clustered, enriched and analyzed for overall changes in metabolic pathways through the KEGG database.

[0058] 2 Results and analysis

[0059] 2.1 Sample Comparative Analysis

[0060] Metabolite analysis was performed after sampling 17h (gastrula stage), 25h (neurula stage), 32h (myotome and eye base formation stage), 41h (tail bud stage), 50h (muscle effect stage) and 58h (heartbeat stage) of bream fertilized eggs. Six replicate samples were sent for each sampling period. In the anion and cation mode, the results showed that the aggregation within the group was good, and there were differences between the groups ( Figure 1 A in the figure).

[0061] The metabolites of bream fertilized eggs at six different developmental stages were compared, and a total of 530 metabolites were screened in the anion and cation modes. According to the Venn diagram analysis of sample comparison, it was found that there were 790 metabolites in common at 17h, 25h, 32h, 41h, 50h, and 58h of incubation of bream fertilized eggs, of which there were two metabolites unique to 32h and three metabolites unique to 58h ( Figure 1 B in the figure).

[0062] 2.2 Metabolite analysis of HMDB database

[0063] The metabolites were classified and counted using the HMDB4.0 database. The results showed that ( Figure 2 ), lipid and lipid molecular metabolites accounted for the largest number, accounting for 35.97%; followed by organic acids and their derivatives accounting for 20.14%, organic heterocyclic compounds accounting for 11.54%, organic oxygen compounds accounting for 9.28%, etc.; the least metabolites were hydrocarbon derivatives accounting for 0.11%, organic phosphides accounting for 0.11% and organic sulfides accounting for 0.11%.

[0064] 2.3 Analysis of differential metabolites

[0065] According to the volcano plot of differential metabolites, the differential metabolites between groups were further screened and classified. Figure 3-Figure 7 It can be seen that in the anion-cation binding mode, a total of 952 metabolites were detected with H25 VS. H17, and the contents of 286 differential metabolites showed significant changes ( P <0.05 or VIP>1), of which 247 were significantly upregulated and 39 were downregulated; H32 VS.H17, a total of 952 metabolites were detected, and the contents of 330 differential metabolites showed significant changes ( P <0.05 or VIP>1), of which 309 were significantly upregulated and 21 were downregulated; H41 VS. H17, a total of 952 metabolites were detected, and the contents of 351 differential metabolites showed significant changes ( P <0.05 or VIP>1), of which 304 were significantly upregulated and 47 were downregulated; H50VS.H17, a total of 952 metabolites were detected, and the contents of 353 differential metabolites showed significant changes ( P <0.05 or VIP>1), of which 305 were significantly upregulated and 48 were downregulated; H58 VS.H17, a total of 952 metabolites were detected, and the contents of 362 differential metabolites showed significant changes ( P <0.05 or VIP>1), of which 296 were significantly up-regulated and 66 were down-regulated.

[0066] In the anion-cation binding mode, multiple groups were compared and analyzed, and it was found that there were very significant differential metabolites such as hydroxyvalermomorphine, glycyltyrosine, 1-hexylarabinoside, ichthyocide B2, L-phenylalanyl-L-proline, astramycin, N-acetyl-7-O-acetylneuraminic acid, dyclonine, 6-epi-7-isocucurbitacin glucoside, uridine, α-hedera saponin, and notoginseng saponin R10. The main biological functions of these differential metabolites include amino sugar and nucleotide sugar metabolism, ABC transporter, pyrimidine metabolism, glycerophospholipid metabolism, α-linolenic acid metabolism, arachidonic acid metabolism, linoleic acid metabolism, nucleotide metabolism, purine metabolism, etc.

[0067] 2.4 Cluster analysis of differential metabolites

[0068] In order to more intuitively display the changing trends of metabolites in different groups, Figure 8 It can be seen that the expression levels of inosine, 9H-purine-9-Ol, 10-HDoHE, (+ / -)17-HDoHE, L-proline, betaine, L-valine, L-isoleucine and indoleacrylic acid gradually increased with the developmental period of the selected fertilized eggs, and the expression levels of palmitoylcarnitine, L-carnitine, 1-cosose and maltotriose gradually decreased with the developmental period of the selected fertilized eggs.

[0069] 2.5 VIP map of differential metabolites

[0070] In order to more intuitively see the importance of differential metabolites and the trend of expression, Figure 9-13, H25VSH17, dynorphin B (6-9), 3b, 8b-dihydroxy-6b-(3-chloro-2-hydroxy-2-methylbutyryloxy)-7 (11)-thermobacterium-12,8-lactone), 3,3-bis (carboxymethyl) hexadecanoic acid, prostaglandin H2, Nα-acetyl-L-lysine, N2-acetyl-L-ornithine, tyrosine glutamate (Tyr Glu) and other expression levels were significantly upregulated; H32 VS The expression levels of H17, 3b, 8b-dihydroxy-6b-(3-chloro-2-hydroxy-2-methylbutyryloxy)-7(11)-thermobacterium-12,8-lactone, dynorphin B(6-9), 6-epi-7-isocucurbitacin glucoside, glycyltyrosine, imidazole propionic acid, PS (monomer (11,5) / monomer (13,5), N2-acetyl-L-ornithine were significantly upregulated; H41 VS H17, the expression levels of estradiol-17Alpha3-D-glucuronide, N2-acetyl-L-ornithine, glycyl-phenylalanine, Nα-acetyl-L-lysine, imidazole propionic acid, alanylproline, Panax notoginseng saponin R10 were significantly upregulated, but the expression of ADP ribose was downregulated; H50VSH17, uridine, (2S)-3-(1H-indol-3-yl)-N-[[1-(4-methoxyphenyl)cyclohexyl]methyl ]-2-[(4-nitrophenyl)carbamoyl]propylamine, estradiol-17Alpha3-D-glucuronide, hydroxypterygium, alanylproline, α-hederaponin, etc. were significantly upregulated, but the expression of ADP ribose was downregulated; H58VSH17, the expression levels of uridine, hydroxypterygium, estradiol-17Alpha3-D-glucuronide, neopterin, alanylproline, α-hederaponin, etc. were significantly upregulated.

[0071] 2.6 Analysis of metabolite differences at different times

[0072] According to the non-targeted metabolomics analysis (see Tables 1-6), in the gastrula stage (17h), the relative content of 1-palmitoylphosphatidylcholine was the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), cyclohexene, etc., and the relative content of propylene was relatively low; in the neurula stage (25h), the relative content of 1-palmitoylphosphatidylcholine was the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), cyclohexene, etc. C (18:1 (11Z) / 0:0), PC (18:3 (9Z, 12Z, 15Z) / 18:0), etc., the relative content of propylene is low; in the myometrium and eye base formation period (32h), the relative content of 1-palmitoylphosphatidylcholine is the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), LysoPC (18:2 (9Z, 12Z) / 0:0), etc., propylene The relative content of olefins was low, and tetradeca-9,11-dienoylcarnitine was a unique metabolic species at 32h. At the tail bud stage (41h), the relative content of species was the same as that of the myometrium and eye base formation stages (32h). At the muscle effect stage (50h), PC (22:6 / 0:0) had the highest relative content, followed by 1-palmitoylphosphatidylcholine, LysoPC (18:1 (11Z) / 0:0), LysoPC (18:2 (9Z,12Z ) / 0:0), 2-lysophosphatidylcholine had the lowest relative content; during the cardiac period (58h), 1-palmitoylphosphatidylcholine had the highest relative content, followed by PC (22:6 / 0:0), LysoPC (18:1(11Z) / 0:0), PC (18:3(9Z,12Z,15Z) / 18:0), etc., 2-lysophosphatidylcholine had the lowest relative content, and septopterin and β-nitropropionic acid were the unique metabolic species at 58h.

[0073] Table 1 is the content of metabolites in gastrula stage (17h) (unit: %)

[0074] Metabolites Relative content 1-Palmitoylphosphatidylcholine 9.24 PC(22:6 / 0:0) 9.1 LysoPC(18:1(11Z) / 0:0) 9.03 Cyclohexane 8.69 PC(18:3(9Z,12Z,15Z) / 18:0) 8.63 LysoPC(18:2(9Z,12Z) / 0:0) 8.62 PC(16:0 / 0:0) 8.53 Propylene 8.49

[0075] Table 2 shows the content of metabolites in the neurula stage (25h) (unit: %)

[0076] Metabolites Relative content 1-Palmitoylphosphatidylcholine 9.21 PC(22:6 / 0:0) 9.02 LysoPC(18:1(11Z) / 0:0) 8.99 PC(18:3(9Z,12Z,15Z) / 18:0 8.79 Cyclohexane 8.69 LysoPC(18:2(9Z,12Z) / 0:0) 8.54 PC(16:0 / 0:0) 8.49 Propylene 8.49

[0077] Table 3 shows the content of metabolites during the myometrium and eye base formation period (32h) (in %)

[0078] Metabolites Relative content 1-Palmitoylphosphatidylcholine 9.24 PC(22:6 / 0:0) 9.11 LysoPC(18:1(11Z) / 0:0) 9.05 LysoPC(18:2(9Z,12Z) / 0:0) 8.64 PC(18:3(9Z,12Z,15Z) / 18:0) 8.6 Cyclohexane 8.6 PC(16:0 / 0:0) 8.5 Propylene 8.4 Tetradeca-9,11-dienoylcarnitine 84477.5

[0079] Table 4 shows the content of metabolites in the tail bud stage (41h) (unit: %)

[0080] Metabolites Relative content 1-Palmitoylphosphatidylcholine 9.2 PC(22:6 / 0:0) 9.2 LysoPC(18:1(11Z) / 0:0) 9.07 LysoPC(18:2(9Z,12Z) / 0:0) 8.7 PC(18:3(9Z,12Z,15Z) / 18:0) 8.65 Cyclohexane 8.53 PC(16:0 / 0:0) 8.45 Propylene 8.35

[0081] Table 5 shows the content of metabolites in muscle effect period (50h) (unit: %)

[0082] Metabolites Relative content PC(22:6 / 0:0) 9.22 1-Palmitoylphosphatidylcholine 9.22 LysoPC(18:1(11Z) / 0:0) 9.1 LysoPC(18:2(9Z,12Z) / 0:0) 8.75 Cyclohexane 8.47 PC(18:3(9Z,12Z,15Z) / 18:0) 8.45 PC(16:0 / 0:0) 8.44 2-Lysophosphatidylcholine 8.34

[0083] Table 6 shows the content of metabolic substances during the heartbeat period (58h) (unit: %)

[0084] Metabolites Relative content 1-Palmitoylphosphatidylcholine 9.27 PC(22:6 / 0:0) 9.2 LysoPC(18:1(11Z) / 0:0) 9.09 LysoPC(18:2(9Z,12Z) / 0:0) 8.76 PC(18:3(9Z,12Z,15Z) / 18:0) 8.5 PC(16:0 / 0:0) 8.49 Cyclohexane 8.4 2-Lysophosphatidylcholine 8.38 Sepiaterin 159233.3 β-Nitropropionic acid 64105

[0085] 2.7 KEGG database differential metabolite analysis

[0086] 2.7.1 KEGG database differential metabolite pathway enrichment

[0087] Analysis of KEGG functional pathways revealed that the metabolic pathways related to differentially expressed metabolites were divided into five categories, namely metabolism, genetic information processing, environmental information processing, organism system, and cell processing. Among them, the number of differential metabolites in the metabolic pathway and biosynthesis cofactor pathway in the metabolism category was relatively large, at 38 and 12, respectively.

[0088] According to the KEGG database, pathway enrichment was performed on 276 differential metabolites, and it was found that the differential metabolites were significantly enriched in metabolic pathways such as ABC transporters, steroid hormone biosynthesis, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, arachidonic acid metabolism, pyrimidine metabolism, purine metabolism, arginine biosynthesis, histidine metabolism, glycine, serine and threonine metabolism, valine, leucine and isoleucine biosynthesis, β-alanine metabolism, valine, leucine and isoleucine degradation, mTOR signaling pathway, and GnRH signaling pathway. Among them, in H25 VS H17, the nucleotide metabolism, ABC transporter and pyrimidine metabolism pathways were highly enriched; in H32VSH17, the nucleotide metabolism, ABC transporter and arachidonic acid metabolism pathways were highly enriched; in H41VSH17, the nucleotide metabolism, purine metabolism and arachidonic acid metabolism pathways were highly enriched; in H50VSH17, the nucleotide metabolism, purine metabolism and arachidonic acid metabolism pathways were highly enriched; in H58VSH17, the nucleotide metabolism, purine metabolism and purine metabolism pathways were highly enriched. The five signaling pathways related to metabolism, namely nucleotide metabolism, pyrimidine metabolism, purine metabolism, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism and arginine biosynthesis, were highly enriched ( Figure 14-18 ).

[0089] 2.7.2 KEGG database differential metabolite pathway analysis

[0090] According to the correlation of the metabolic pathways of the screened differential metabolites, the KEGG main metabolic pathway map - nucleotide metabolism was drawn, and a total of 14 key differential metabolites were identified, among which the relative contents of dIMP, IMP, hypoxanthine, inosine, uracil, guanine, guanosine, deoxyuridine and thymine were upregulated, and the relative contents of adenine, UMP, CMP and dTMP were downregulated. The relative content of thymine is upregulated. The upregulation of thymine can accelerate the generation of succinyl-CoA to enter TCA and gluconeogenesis. TCA can provide energy for the body, and gluconeogenesis can synthesize glucose to provide energy reserves for growth and development. The increase in the relative content of hypoxanthine, guanine and guanosine can accelerate the decomposition to produce uric acid. Uric acid oxidase exists in the body of fish, which can decompose uric acid into highly water-soluble allantoin, and excrete toxic substances after growth ( Fig.19 ).

[0091] 3 Discussions

[0092] The present invention conducts metabolomics analysis on fertilized eggs of Megalobrama triangularis at different hatching periods, and finds a series of differential metabolites in pairwise comparisons: under the anion-cation binding mode, compared with the control group H17, in the experimental group 25h, 247 metabolites are upregulated, and 39 metabolites are downregulated; in the experimental group H32, 309 metabolites are upregulated, and 21 metabolites are downregulated; in the experimental group H41, 304 metabolites are upregulated, and 47 metabolites are downregulated; in the experimental group H50, 305 metabolites are upregulated, and 48 metabolites are downregulated; in the experimental group H58, 296 metabolites are upregulated, and 66 metabolites are downregulated. KEGG enrichment revealed that compared with the control group, a total of 23 differential metabolites were screened out: the relative contents of inosine, L-tryptophan, thymine, phosphorylcholine, hypoxanthine, glycerylphosphatidylcholine, aspartic acid, ornithine, uracil, guanosine, uridine, uric acid, 5 (S) -HETE, prostaglandin H2, retinoic acid, L-leucine, L-isoleucine, etc. were significantly upregulated, while the relative contents of adenine, adenosine, creatine, thymidine deoxynucleotide, ADP ribose, maltotriose, etc. were significantly downregulated. These differential metabolites mainly affect the embryonic development of fertilized eggs through pathways such as the intestinal immune network for the production of IgA, linoleic acid metabolism, pantothenic acid and coenzyme A biosynthesis, arginine biosynthesis, β-alanine metabolism, glycine, serine and threonine metabolism, histidine metabolism, aminoacyl tRNA biosynthesis, glycerophospholipid metabolism, pyrimidine metabolism, arachidonic acid metabolism, purine metabolism, and ABC transporters.

[0093] Retinol metabolism plays a major role in maintaining normal visual responses as well as cell proliferation and differentiation. The relative content of retinol is increased, which enhances the retinol metabolic pathway and thus ensures normal embryonic development. Retinol is not only involved in the retinol metabolic pathway, but also in the intestinal immune network pathway that produces IgA. The relative contents of urinary purine, guanine, guanosine and hypoxanthine were significantly increased, and the relative contents of adenine and adenosine were significantly decreased. They all belong to purine substances. Purine substances are important components in cells, participate in the synthesis of DNA and RNA, and play an important role in energy transfer and metabolic regulation. Changes in their content will strengthen the purine metabolic pathway, which may promote protein synthesis and energy metabolism. In addition, the purine metabolic pathway is involved in various inflammatory processes. Uric acid is the final product of purine metabolism. The synthesis of uric acid has two aspects. The main pathway is the synthesis of inosylic acid from non-purine precursors, which is then converted into adenylic acid. The salvage pathway is that purine bases or purine nucleosides in the liver first synthesize inosine nucleotide (IMP or inosinic acid), which is then converted into adenylic acid and guanylic acid. The significant increase in uric acid may be related to the enhancement of purine metabolism. Arachidonic acid is a typical polyunsaturated fatty acid and an essential nutrient for the normal growth, development and reproduction of fish. The arachidonic acid metabolic pathway produces a variety of derivatives with different physiological activities through the catalysis of multiple enzymes. For example, arachidonic acid metabolism in fish produces prostaglandins, leukotrienes and oxidation products such as 5-HETE and prostaglandin H2. Arachidonic acid and its metabolites are involved in the formation and shaping of bones in bony fish. In this study, the relative content of prostaglandin H2 and 5-HETE was upregulated, which may be related to the formation of body shape during the development of fertilized eggs. The significant upregulation of glycerophosphatidylcholine levels is also related to the metabolism of linoleic acid, α-linolenic acid and arachidonic acid. As the main essential fatty acids of freshwater fish such as eels, linoleic acid and α-linolenic acid can maintain the normal structure and function of biological membranes and play an important role in the metabolism of lipids such as cholesterol. Amino acids play a vital role in various physiological processes, and branched-chain amino acids (BCAA)-leucine, isoleucine and valine are of vital importance because these three branched-chain amino acids provide energy through the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-ketoacid dehydrogenase (BCKDH) pathways. Aspartate, whether in the D form or the L form, uses the aspartate malate shuttle (MAS) to transfer electrons from the cytoplasm to the mitochondria, thereby maintaining the cytoplasmic NAD / NADH redox balance to provide energy. The relative contents of aspartic acid, L-leucine, and L-isoleucine were all significantly increased, which may be due to the increase in energy required during the development of the fertilized egg. Valine is related to the metabolism of glucose, and L-valine is a major form of valine. The significant increase in L-valine indicates that energy metabolism during the development of the fertilized egg is improved.Creatine is synthesized from three amino acids: arginine, glycine and methionine. The relative content of creatine is significantly reduced, which increases the energy supply in the fertilized egg and may lead to enhanced energy metabolism in the tricarboxylic acid cycle.

[0094] 4 Conclusion

[0095] In the anion-cation binding mode, 17h was used as the control group, and the experimental groups at 25h, 32h, 41h, 50h, and 58h were compared with the control group, and a total of 946 metabolic differences were detected.

[0096] Cluster analysis was performed on the top 20 differential metabolites, which mainly included inosine, purine, proline, betaine, L-valine, L-isoleucine, indoleacetic acid, glyceride, acetylcarnitine, phosphocholine, glycerophosphocholine, dioxoglutaric acid, palmitoylcarnitine, L-carnitine and maltotriose.

[0097] According to the KEGG database, pathway enrichment was performed on 276 differential metabolites, and it was found that the differential metabolites were significantly enriched in metabolic pathways such as ABC transporters, steroid hormone biosynthesis, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, arachidonic acid metabolism, pyrimidine metabolism, purine metabolism, arginine biosynthesis, histidine metabolism, glycine, serine and threonine metabolism, valine, leucine and isoleucine biosynthesis, β-alanine metabolism, valine, leucine and isoleucine degradation, mTOR signaling pathway, GnRH signaling pathway and melanogenesis.

[0098] 4) The metabolite results of fertilized eggs at different hatching stages showed that in the gastrula stage (17h), the relative content of 1-palmitoylphosphatidylcholine was the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), cyclohexene, etc., and the relative content of propylene was relatively low; in the neurula stage (25h), the relative content of 1-palmitoylphosphatidylcholine was the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), PC (18:3 (9Z, 12Z, 15Z) / 18:0), etc., and the relative content of propylene was relatively low; in the myotome and eye base formation stage (32h) and the coccygeal stage (41h), the relative content of 1-palmitoylphosphatidylcholine was the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), LysoPC (1 8:2 (9Z, 12Z) / 0:0), etc. The relative content of propylene is low. Tetradeca-9,11-dienoylcarnitine is a 32h-specific metabolite. During the muscle effect period (50h), PC (22:6 / 0:0) has the highest relative content, followed by 1-palmitoylphosphatidylcholine, LysoPC (18:1 (11Z) / 0:0), LysoPC (18:2 (9Z, 12Z) / 0:0) , the relative content of 2-lysophosphatidylcholine was the lowest; during the cardiac period (58h), the relative content of 1-palmitoylphosphatidylcholine was the highest, followed by PC (22:6 / 0:0), LysoPC (18:1 (11Z) / 0:0), PC (18:3 (9Z, 12Z, 15Z) / 18:0), etc., the relative content of 2-lysophosphatidylcholine was the lowest, and septopterin and β-nitropropionic acid were the unique metabolites of 58h.

[0099] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A method for detecting metabolites of fertilized eggs of Megalobrama triangularis at different developmental stages during hatching, characterized in that: The method comprises the following steps: 1) The artificially induced fertilized eggs of the triangular bream were placed in a culture dish for static water incubation, dead eggs were picked out every 3-5 hours, and the aerated water was replaced. Fertilized egg samples were taken at different stages of incubation and development; the different stages of incubation and development were the gastrula stage, neurula stage, myometrium and eye base formation stage, tail bud stage, muscle effect stage and heartbeat stage of the triangular bream; 2) Select the same amount of samples into centrifuge tubes, add grinding beads; add the extract containing the internal standard, grind in a -20~-5℃ frozen tissue grinder, and extract with low temperature ultrasound at 1-10℃; the extract is a methanol-water solution, with a volume ratio of methanol: water = 4:1; the internal standard includes L-2-chlorophenylalanine, 0.02mg / mL; 3) Place the sample at -25-15℃ for 20-40min; centrifuge at 10000-15000g for 10-20min at 1-10℃, transfer the supernatant into a vial with an inner insert and analyze on the analyzer; 4) Ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry system was used for analysis to obtain total ion chromatograms; the ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry system used the Thermo Fisher Scientific's ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system; chromatographic conditions: the chromatographic column was ACQUITY UPLC H ST3; the mobile phase A was 95% water + 5% acetonitrile by volume, and 0.1% formic acid was added to the total volume; the mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water by volume, and 0.1% formic acid was added to the total volume, the injection volume was 3 μL, and the column temperature was 40°C; mass spectrometry conditions: the sample was ionized by electrospray, and the mass spectrometry signals were collected in positive and negative ion scanning modes respectively; 5) The raw data were imported into the metabolomics processing software for baseline filtering, peak identification, integration, retention time correction, and peak alignment to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information; then, a characteristic peak search library was performed to match the MS and MS / MS mass spectrometry information with the metabolic database, and the MS mass error was set to less than 10 ppm. At the same time, metabolites were identified based on the secondary mass spectrometry matching score, and differential metabolic sets were used for metabolic set analysis. The KEGG database was used to annotate, classify, cluster, enrichment analyze, and analyze the overall changes in metabolic pathways for the screened differential metabolites; A total of 23 differential metabolites were screened out by KEGG enrichment: the contents of inosine, L-tryptophan, thymine, choline phosphate, hypoxanthine, glycerophosphatidylcholine, aspartic acid, ornithine, uracil, guanosine, uridine, uric acid, 5(S)-HETE, prostaglandin H2, retinoic acid, L-leucine and L-isoleucine were significantly up-regulated in the neurula stage, myometrium and eye base formation stage, tail bud stage, muscle effect stage and heartbeat stage compared with the gastrula stage, while the contents of adenine, adenosine, creatine, thymidine deoxynucleotide, ADP ribose and maltotriose were significantly down-regulated in the neurula stage, myometrium and eye base formation stage, tail bud stage, muscle effect stage and heartbeat stage compared with the gastrula stage. The obtained differential metabolites were used as markers to detect the hatching process of fertilized eggs of Triceratops triangularis.

2. The method according to claim 1, characterized in that In step 1), the sperm-egg mixture is placed in a culture dish with a diameter of 15 cm, and then oxygenated and aerated clean water is added and shaken to make it evenly distributed in the culture dish. About 300 fertilized eggs are placed in each culture dish. During the incubation process, the water is changed every four hours, and dead and moldy eggs are cleaned up. Samples are taken at 17h, 25h, 32h, 41h, 50h, and 58h respectively.

3. The method according to claim 1, characterized in that In step 3), 20 µL of supernatant was taken from each sample and mixed as quality control samples. The volume of each quality control sample was the same as that of the sample and it was processed and tested in the same way as the analytical samples. During the instrument analysis, a quality control sample was inserted into every 5-15 analytical samples to examine the stability of the entire detection process.

4. The method according to claim 1, characterized in that: Step 4) The components of the sample separated by chromatography continuously enter the mass spectrometer, and the mass spectrometer continuously scans for data acquisition. Each scan obtains a mass spectrum. All ion intensities in each mass spectrum are added together to obtain a total ion current intensity; the total ion chromatogram is drawn with time as the horizontal axis and the total ion intensity as the vertical axis.

5. Use of the method according to any one of claims 1 to 4 in improving the hatching process of fertilized eggs of Megalobrama bream, increasing the hatching rate and / or increasing the stability of embryonic development.

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