Vitamin compound for prolonging survival time of sterile zebra fish and preparation method thereof

Abstract

The invention belongs to the technical field of experimental animal nutriology and sterile animal culture, and particularly relates to a vitamin compound for prolonging the survival time of sterile zebra fish and a preparation method of the vitamin compound. The vitamin compound is prepared from the following components in parts by mass: 600 to 1000 parts of vitamin A, 4000 to 6000 parts of vitamin C, 10 to 30 parts of vitamin D3, 1500 to 2200 parts of vitamin E, 80 to 120 parts of vitamin K3, 15000 to 25000 parts of compound B vitamins and sterile culture water. The preparation method comprises the following steps: dissolving the vitamin raw materials in sterile culture water, performing ultrasonic assisted dissolution, and then filtering and sterilizing to obtain the vitamin supplement. When the vitamin supplement is used for feeding the sterile zebra fish, the survival time of the sterile zebra fish can be obviously prolonged, and the expression level of growth hormone genes and insulin-like growth factor genes can be up-regulated. The application value of the sterile zebrafish in scientific research is further expanded.

Description

Technical Field

[0001] This invention belongs to the fields of experimental animal nutrition and sterile animal culture technology, specifically relating to a vitamin complex for prolonging the survival period of sterile zebrafish and its preparation method. Background Technology

[0002] Zebrafish (Danio rerio) are important model organisms with wide applications in developmental biology, genetics, toxicology, and disease models. Zebrafish possess advantages such as small size, short reproductive cycle, transparent embryos, and high genome similarity to humans, making them the second most common vertebrate model after mice. Germ-free zebrafish refer to zebrafish cultured under sterile conditions, free from any living microorganisms, both in vivo and in vitro. Germ-free zebrafish models eliminate interference from microbial factors, allowing for more accurate study of the host's physiological functions and responses to external factors. They are crucial tools for investigating cutting-edge life science issues such as host-microbe interactions, immune system development, and metabolic regulation.

[0003] The establishment and maintenance of germ-free animal models has always been a technical challenge in the field of laboratory animal science. Under normal physiological conditions, the gut microbiota plays an irreplaceable role in the host's nutrient metabolism, immune system development, and maintenance of overall physiological homeostasis. Gut symbiotic bacteria can endogenously synthesize various essential nutrients such as vitamin K and B vitamins, and participate in the digestion and decomposition of nutrients through various enzyme systems, significantly improving the host's bioavailability of nutrients. Vitamins are trace organic compounds necessary for maintaining normal growth and development in animals, but fish cannot synthesize most vitamins themselves and must obtain them from external sources. Under normal aquaculture conditions, fish mainly obtain vitamins through two pathways: one is through ingestion, and the other is through the synthesis of some B vitamins and vitamin K by gut microbiota. Vitamin A is involved in visual function, epithelial tissue maintenance, and immune regulation; vitamin C is an important antioxidant, involved in collagen synthesis and immune function; vitamin D3 regulates calcium and phosphorus metabolism and promotes bone development; vitamin E has antioxidant effects and protects cell membrane integrity; vitamin K3 is involved in blood clotting and bone metabolism; and B vitamins, as coenzymes, participate in various physiological processes such as energy metabolism and protein synthesis. A deficiency in these vitamins can lead to problems such as slow growth, increased deformity rate, and decreased immunity in zebrafish.

[0004] Germ-free zebrafish exhibit a series of severe physiological defects and developmental abnormalities due to the complete absence of their gut microbiota. Experimental results from Rawls et al.'s research team indicate significant impairments in the intestinal development and function of germ-free zebrafish. Morphologically, they exhibit typical germ-free animal phenotypes such as thinned epithelial cell layer, incomplete development of intestinal villi, and reduced intestinal wall thickness. Functionally, intestinal epithelial differentiation is impaired, alkaline phosphatase activity is significantly reduced, goblet cells and enteroendocrine cells show developmental defects, and glycosylation levels are abnormal. More seriously, the nutrient absorption capacity of germ-free zebrafish is severely impaired, with significant declines in both lipid and protein absorption. Of particular concern is that, under current standardized culture systems, germ-free zebrafish exhibit a 100% mortality rate at 20 days post-fertilization (20 dpf), severely limiting the application prospects of the germ-free zebrafish model in long-term follow-up experiments and chronic disease research. This phenomenon suggests that germ-free zebrafish may experience key nutrient deficiencies or disruptions in key metabolic pathways during embryonic development and larval stages. Given that germ-free zebrafish completely lack endogenous vitamins synthesized by gut microbiota, a scientifically sound exogenous vitamin supplementation strategy may be the key to solving this problem. However, current research both domestically and internationally on improving the survival rate and extending the lifespan of germ-free zebrafish remains relatively scarce. Existing studies mainly focus on short-term applications within 7 days post-flop (dpf) and optimization of preparation processes, while exploration of nutritional intervention techniques for long-term stable culture is still insufficient. Therefore, there is an urgent need to develop targeted nutritional supplementation strategies or physiological regulation methods to extend their lifespan, thereby expanding their application value in the scientific research field.

[0005] Beyond their direct nutritional functions, the impact of vitamin supplementation on the growth regulation mechanisms of germ-free zebrafish requires further investigation. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) play crucial roles in coordinating the physiological functions of bony fish. GH, secreted by the anterior pituitary gland, stimulates the liver to produce IGF-1 by binding to the growth hormone receptor (GHR), thereby promoting protein synthesis, cell proliferation, and skeletal development. Normal function of the GH / IGF-1 axis is essential for fish growth and development, and its expression level is positively correlated with growth performance. However, a systematic evaluation of the effects of vitamin supplementation on the GH / IGF-1 system in germ-free zebrafish is currently lacking, and this knowledge gap urgently needs to be filled.

[0006] In summary, existing sterile zebrafish culture techniques suffer from the following problems: (1) short survival time, with most dying within 20 days post-flop (dpf); (2) lack of vitamin supplementation programs specifically addressing the unique nutritional needs of early-stage sterile zebrafish; (3) limited effectiveness of single-vitamin supplementation; and (4) insufficient understanding of the regulatory mechanisms of growth factor expression in zebrafish under sterile conditions. These problems severely restrict the application value of sterile zebrafish in scientific research. Therefore, given the unique physiological state of sterile zebrafish, it is urgent to develop a vitamin complex formulation system specifically for the juvenile stage of sterile zebrafish. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a vitamin complex for prolonging the survival period of germ-free zebrafish.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A vitamin complex for extending the survival period of sterile zebrafish, the vitamin complex comprising the following components in parts by weight: 600-1000 parts of vitamin A, 4000-6000 parts of vitamin C, 10-30 parts of vitamin D3, 1500-2200 parts of vitamin E, 80-120 parts of vitamin K3, 15000-25000 parts of B complex vitamins, and sterile culture water.

[0010] Preferably, the complex B vitamins contain vitamin B1, nicotinamide, vitamin B2, vitamin B6, and calcium pantothenate.

[0011] Preferably, the mass-to-volume ratio of vitamin A to the sterile culture water is 600-1000 mg: 150-200 mL, more preferably 800 mg: 200 mL.

[0012] Preferably, the vitamin complex comprises the following components in parts by weight: 700-900 parts of vitamin A, 4500-5500 parts of vitamin C, 15-25 parts of vitamin D3, 1700-1900 parts of vitamin E, 90-110 parts of vitamin K3, 18000-22000 parts of B complex vitamins, and sterile aquaculture water.

[0013] More preferably, the vitamin complex comprises the following components in parts by weight: 800 parts of vitamin A, 5000 parts of vitamin C, 320 parts of vitamin D, 1800 parts of vitamin E, 100 parts of vitamin K, 20000 parts of B complex vitamins, and sterile culture water.

[0014] Preferably, the vitamin complex comprises the following components: vitamin A 700-900mg, vitamin C 4500-5500mg, vitamin D3 15-25mg, vitamin E 1700-1900mg, vitamin K3 90-110mg, and B complex vitamins 18000-22000mg, supplemented to 150-300mL of sterile aquaculture water.

[0015] As a preferred embodiment, the vitamin complex comprises the following components: 800 mg of vitamin A, 5000 mg of vitamin C, 20 mg of vitamin D3, 1800 mg of vitamin E, 100 mg of vitamin K3, 20 g of B complex vitamins, and 200 mL of sterile culture water.

[0016] The second objective of this invention is to provide a method for preparing vitamin supplements using the aforementioned vitamin complex.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] The method for preparing vitamin supplements using the aforementioned vitamin complex involves taking vitamin raw materials according to the aforementioned formula, adding them to sterile culture water, sonicating them to dissolve them, filtering them, and obtaining the vitamin supplements.

[0019] Preferably, the ultrasound time is 10-30 minutes, more preferably 20 minutes.

[0020] As a preferred embodiment, the method includes the following steps:

[0021] 1) Accurately weigh each vitamin ingredient according to the formula;

[0022] 2) Add the weighed vitamin ingredients to 150mL of sterile zebrafish culture water;

[0023] 3) Use ultrasound to aid dissolution for 10-30 minutes to ensure all components are fully dissolved;

[0024] 4) Adjust the volume to 200mL using sterile zebrafish culture water;

[0025] 5) Use a 0.22μm filter membrane for sterilization;

[0026] 6) Dispense into sterile containers and store at 4°C away from light.

[0027] A third objective of this invention is to provide a vitamin supplement prepared using the aforementioned method.

[0028] Preferably, the vitamin supplement is a concentrated mother liquor.

[0029] The fourth objective of this invention is to provide a method for culturing sterile zebrafish.

[0030] To achieve the above objectives, the present invention adopts the following technical solution:

[0031] The method for culturing sterile zebrafish involves placing sterile zebrafish embryos in a sterile culture system and adding the aforementioned vitamin complex and / or the aforementioned vitamin supplements to the culture water.

[0032] Different types of aseptic feed exhibit significant differences in vitamin content. Aseptic egg yolks have relatively low vitamin content, requiring more frequent exogenous vitamin supplementation; while commercial aseptic feeds are inherently rich in vitamins, and the irradiation sterilization process primarily affects irradiation-sensitive B vitamins, while other vitamins remain relatively stable. Therefore, the frequency of vitamin supplementation should be adjusted according to the type of feed used to avoid adverse reactions caused by vitamin overdose (such as edema, deformities, etc.) or growth and development problems caused by vitamin deficiency.

[0033] Preferably, the vitamin supplement is first added on the day of 3dpf cell rupture, and the frequency of addition is adjusted according to the feed type: when using sterile egg yolk feed, the liquid is completely changed and vitamin supplement is added every other day; when using sterile commercial feed, the liquid is completely changed and vitamin supplement is added weekly.

[0034] More preferably, the vitamin supplement is added for the first time on the 3rd day; when using sterile egg yolk feed, the liquid is completely changed and vitamin supplement is added on odd-numbered days such as the 5th and 7th days; when using sterile commercial feed, the liquid is completely changed and vitamin supplement is added every 7 days.

[0035] Preferably, the volume ratio of the vitamin supplement to the culture water is 1:500-2000; the culture conditions include: temperature 15-30℃, pH 6.5-8.

[0036] More preferably, the volume ratio of the vitamin supplement to the culture water is 1:1000. For example, it is added at a ratio of 50 μL vitamin supplement / 50 mL water.

[0037] As a preferred option, the culture conditions include: a temperature of 28.5°C and a pH of 7.0-7.5.

[0038] The fifth objective of this invention is to provide the application of the aforementioned vitamin complex and / or the aforementioned vitamin supplement in the preparation of products for prolonging the survival period of germ-free zebrafish and / or promoting the growth and development of germ-free zebrafish.

[0039] To achieve the above objectives, the present invention adopts the following technical solution:

[0040] The aforementioned vitamin complex and / or the aforementioned vitamin supplements are used in the preparation of products for prolonging the survival period of germ-free zebrafish and / or promoting the growth and development of germ-free zebrafish.

[0041] Preferably, promoting the growth and development of germ-free zebrafish includes promoting the expression of growth factor genes in germ-free zebrafish; the growth factor genes include any one or more of gh1, gh1a, gh1b, igf1, igf1a, and igf1b.

[0042] Our research team previously conducted exploratory experiments on various nutritional intervention strategies. First, we attempted to supplement with single vitamins by adding them to the culture water. However, due to the complex synergistic effects and metabolic interactions between vitamins, single-component supplementation strategies failed to achieve the expected physiological regulatory effects. Second, we evaluated the application potential of commercially available fish vitamin premixes, but found that these products are mainly formulated to meet the production needs of economically important fish species. Their nutrient ratios and dosages differed significantly from the specific physiological needs of germ-free zebrafish, resulting in unsatisfactory application effects. Third, we explored the feasibility of vitamin supplementation via feed carriers, but this method has a significant time window limitation: germ-free zebrafish rely primarily on endogenous yolk nutrition for growth during the critical early developmental stage (3-7 dpf) and have not yet established exogenous feeding behavior; therefore, feed supplementation is ineffective during this crucial developmental window. Thus, direct addition to the water provides a more effective approach for nutritional intervention in the early developmental stage.

[0043] The vitamin complex of this invention, through scientific component ratios and water addition methods, not only effectively compensates for vitamin deficiencies caused by the absence of intestinal microorganisms and promotes normal growth and development under sterile conditions, but also precisely controls the added concentration of each nutrient component to avoid nutrient overdose or toxic reactions caused by potential changes in the body's requirements for certain vitamins under sterile conditions. This significantly extends the life cycle of sterile zebrafish, improves their survival rate and development quality, and provides key technical support for the long-term application of the sterile zebrafish model.

[0044] The beneficial effects of this invention are as follows:

[0045] 1) The vitamin complex of this invention can effectively and significantly prolong the survival period of germ-free zebrafish. Data shows that using the vitamin complex of this invention, the survival rate of germ-free zebrafish can reach 100% at 10 dpf, 63-100% at 14 dpf, 18-35 individuals can still survive at 21 dpf, 9-16 individuals at 30 dpf, and 1-3 individuals at 40 dpf, with the longest survival period reaching 46 dpf. In contrast, all germ-free zebrafish in existing technologies die within 20 dpf. Observations revealed that the mortality of juvenile fish is mainly related to their own feeding ability, rather than the limitation of the vitamin complex's effect. This result breaks through the limitation of the survival period of germ-free zebrafish in existing technologies, providing a possibility for long-term experimental research.

[0046] 2) The vitamin complex of the present invention effectively promotes the growth and development of germ-free zebrafish. qPCR analysis revealed that the expression levels of growth hormone genes (gh1, gh1a, gh1b) and insulin-like growth factor genes (igf1, igf1a, igf1b) in the germ-free zebrafish group supplemented with vitamins (GF+V group) were significantly higher than those in the unsupplemented group (GF group), indicating that the vitamin complex of the present invention can effectively promote the expression of growth-related genes in germ-free zebrafish.

[0047] 3) The formula is scientifically sound and the preparation method is simple. This invention meets the special nutritional needs of sterile zebrafish under sterile conditions by rationally proportioning fat-soluble vitamins (A, D3, E, K3), water-soluble vitamins (C), and B vitamins. The preparation process is simple and easy to standardize for production.

[0048] 4) Convenient to use and stable in effect. The vitamin complex of the present invention is formulated as a concentrated mother liquor vitamin supplement. It only needs to be diluted and added according to the ratio when used, which is simple to operate and has a stable and reliable effect. Attached Figure Description

[0049] Figure 1 The survival rate of zebrafish juveniles in the CONR, GF, and GF+V groups within 14 dpf is shown in the graph.

[0050] Figure 2 A graph showing the survival rate range of GF+V group zebrafish juveniles at different time points during long-term culture;

[0051] Figure 3 A graph showing the effects of CONR, GF, and GF+V on the expression of the growth hormone gene gh1 in zebrafish juveniles;

[0052] Figure 4 A graph showing the effects of CONR, GF, and GF+V on the expression of the growth hormone gene gh1a in zebrafish juveniles;

[0053] Figure 5 A graph showing the effects of CONR, GF, and GF+V on the expression of the growth hormone gene gh1b in zebrafish juveniles;

[0054] Figure 6 A graph showing the effects of CONR, GF, and GF+V on the expression of the insulin-like growth factor gene igf1 in zebrafish juveniles;

[0055] Figure 7 A graph showing the effects of CONR, GF, and GF+V on the expression of the insulin-like growth factor gene igf1ra in zebrafish juveniles;

[0056] Figure 8 The figure shows the effects of CONR, GF, and GF+V on the expression of the insulin-like growth factor gene igf1rb in zebrafish juveniles. Detailed Implementation

[0057] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0058] In this invention, it should be understood that the terminology used is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0060] In the embodiments of this invention, the materials, reagents, animals, or instruments involved, unless otherwise specified, can all be purchased commercially. The related experimental methods, unless otherwise specified, are conventional techniques in the art. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention are still within the scope of protection of this invention.

[0061] In this embodiment of the invention, different types of aseptic feed exhibit significant differences in vitamin content. Aseptic egg yolks have relatively low vitamin content, requiring more frequent exogenous vitamin supplementation; while commercial aseptic feeds are inherently rich in vitamins, and the irradiation sterilization process primarily affects irradiation-sensitive B vitamins, while other vitamins remain relatively stable. Therefore, the frequency of vitamin supplementation should be adjusted according to the actual type of feed used to avoid adverse reactions caused by vitamin overdose (such as edema, deformities, etc.) or growth and development problems caused by vitamin deficiency.

[0062] In some embodiments, vitamin supplements are first added on the day of 3dpf cell rupture, and the frequency of addition is adjusted according to the feed type: when using sterile egg yolk feed, the liquid is completely changed and vitamin supplements are added every other day; when using sterile commercial feed, the liquid is completely changed and vitamin supplements are added weekly.

[0063] In this embodiment of the invention, the compound B vitamins are selected from compound vitamin B soluble powder, purchased from Hebei Hangsen Biotechnology Co., Ltd., Hengxin brand, veterinary drug registration number 031886071; the compound B vitamins contain vitamin B1, nicotinamide, vitamin B2, vitamin B6 and calcium pantothenate.

[0064] Example 1

[0065] A vitamin complex comprising the following components in parts by weight:

[0066] Vitamin A 800 parts, Vitamin C 5000 parts, Vitamin D 320 parts, Vitamin E 1800 parts, Vitamin K 3100 parts, B-complex vitamins 20000 parts, and sterile aquaculture water. The B-complex vitamins include Vitamin B1, niacinamide, Vitamin B2, Vitamin B6, and calcium pantothenate.

[0067] The vitamin complex is prepared into a concentrated mother liquor form as a vitamin supplement. The specific preparation method is as follows:

[0068] Accurately weigh 800mg of Vitamin A, 5000mg of Vitamin C, 20mg of Vitamin D3, 1800mg of Vitamin E, 100mg of Vitamin K3, and 20g of a complex of B vitamins (containing Vitamin B1, nicotinamide, Vitamin B2, Vitamin B6, and calcium pantothenate). Add the above ingredients to 150mL of sterile culture water and sonicate for 20 minutes (200W power, 40kHz frequency) to ensure complete dissolution of all components. Adjust the volume to 200mL with sterile zebrafish culture water, mix well, and filter sterilize using a 0.22μm PES membrane. Aliquot the prepared vitamin complex stock solution into 10mL sterile centrifuge tubes and store at 4℃ protected from light. Shelf life is 3 months.

[0069] Example 2. Effects of vitamin complex on survival rate and lifespan of germ-free zebrafish

[0070] 1. Experimental grouping and survival rate detection

[0071] AB strain zebrafish were selected, and embryos obtained from in vitro fertilization were collected under normal conditions. The embryos were then sterilized in a sterile environment to obtain sterile zebrafish embryos, which were then cultured under sterile conditions. Standardized screening was performed on the hatched zebrafish: (1) individuals that hatched normally were selected; (2) individuals with abnormal morphological development were removed; (3) healthy individuals with normal swimming activity and no obvious defects were selected as experimental subjects.

[0072] The selected sterile zebrafish were randomly divided into three groups: the CONR group (conventionally fed zebrafish), the GF group (sterile zebrafish), and the GF+V group (sterile zebrafish with vitamin supplementation). Each group used 200 individuals in each experiment, with a total of three biological replicates. Both groups were cultured in a sterile culture system at 28.5℃ and pH 7.0-7.5. The GF group used sterile culture water; the GF+V group received vitamin supplementation starting from the day of 3-day post-flop (dpf) thawing, at a ratio of 50 μL stock solution / 50 mL water. The frequency of vitamin supplementation was adjusted according to the feed type, with regular medium changes and replenishment. The number of surviving zebrafish in each group was recorded daily, and the survival rate was calculated.

[0073] Survival data are expressed as mean ± standard error (Mean ± SEM, n = 3), with an initial sample size of 200 individuals per group. Survival was monitored from 0 dpf to 14 dpf, with the number of surviving individuals recorded daily. Survival-time curves were plotted using an XY scatter plot, and data processing and plotting were performed using GraphPad Prism 10.0 software. Each data point is based on the mean of three independent experiments, and the error bar represents the standard error.

[0074] 2. Effects of vitamin complex on the survival rate of short-term cultured germ-free zebrafish

[0075] like Figure 1Survival curves showed significant differences among the three experimental groups during the 14-day observation period (0-14 dpf). In the GF group, mortality began to appear around 8 dpf, with the survival rate continuously declining, reaching 15-28.5% by 14 dpf. The CONR group showed relatively stable performance, with a small number of deaths starting at 9-10 dpf, and a survival rate maintained at 72-100% by 14 dpf. The GF+V group performed best, with mortality occurring latest (11-12 dpf), and a survival rate still reaching 63-100% at 14 dpf, significantly higher than the GF group. These results indicate that the addition of a vitamin complex can effectively delay mortality in germ-free zebrafish juveniles and significantly improve their survival status.

[0076] 3. Effects of vitamin complex on the survival period of long-term cultured sterile zebrafish

[0077] like Figure 2 As shown, in long-term culture experiments, the number of surviving individuals in the GF+V group was typically 18-35 at 21 dpf, maintained at 9-16 at 30 dpf, and decreased to 1-3 at 40 dpf. Through multiple independent batches of experiments, individuals with a survival period of up to 46 dpf were successfully cultured, far exceeding the survival period reported in existing technologies. The results indicate that the vitamin complex of this invention can significantly prolong the survival period of germ-free zebrafish, effectively overcoming the technical bottleneck of existing technologies where all germ-free zebrafish typically die within 20 dpf.

[0078] Example 3. Analysis of the effect of vitamin complex on growth factor gene expression in sterile zebrafish

[0079] 1. Experimental grouping and growth factor gene expression detection methods

[0080] The preparation, standardized screening, experimental grouping, culture conditions, and addition of the GF+V vitamin complex for sterile zebrafish embryos were all performed according to Example 2. At 7 days post-exposure (dpf), six biological replicates (each containing 100 fish) were randomly selected from each group, and total RNA was extracted. Reverse transcription was performed using the PrimeScript RT reagent Kit (TaKaRa) to obtain cDNA. The expression levels of growth hormone genes (gh1, ghra, ghrb) and insulin-like growth factor genes (igf1, igf1ra, igf1rb) were detected using SYBR Green real-time quantitative PCR and standardized.

[0081] Experimental data are expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using GraphPadPrism 10.0 software. One-way ANOVA was used to compare differences among multiple groups, followed by Tukey's multiple comparison test. p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0082] 2. The regulatory effect of vitamin complexes on growth factor gene expression

[0083] like Figure 3-8 As shown, qPCR results indicated that, compared to the CONR group, the expression levels of growth hormone genes (gh1, ghra, ghrb) and insulin-like growth factor genes (igf1, igf1ra, igf1rb) in juvenile zebrafish in the GF group were significantly downregulated. The GF+V group significantly upregulated the expression levels of these genes, with a statistically significant difference compared to the GF group, but still showed varying degrees of downregulation compared to the CONR group. These results demonstrate that the vitamin complex of the present invention not only prolongs the survival time of germ-free zebrafish but also effectively promotes the expression of growth-related genes, improving the growth and development status of germ-free zebrafish.

Claims

1. A vitamin complex for prolonging the survival of germ-free zebrafish, characterized in that, The vitamin complex comprises the following components in parts by weight: 600-1000 parts of vitamin A, 4000-6000 parts of vitamin C, 10-30 parts of vitamin D3, 1500-2200 parts of vitamin E, 80-120 parts of vitamin K3, 15000-25000 parts of B complex vitamins, and sterile aquaculture water.

2. The vitamin complex according to claim 1, characterized in that, The complex B vitamins contain vitamin B1, nicotinamide, vitamin B2, vitamin B6, and calcium pantothenate.

3. The vitamin complex according to claim 1, characterized in that, The vitamin complex comprises the following components in parts by weight: 700-900 parts of vitamin A, 4500-5500 parts of vitamin C, 15-25 parts of vitamin D3, 1700-1900 parts of vitamin E, 90-110 parts of vitamin K3, 18000-22000 parts of B complex vitamins, and sterile aquaculture water.

4. A method for preparing vitamin supplements using the vitamin complex according to any one of claims 1 to 3, characterized in that, Vitamin raw materials are taken according to the formula of any one of claims 1 to 3, added to sterile culture water, sonicated to dissolve, filtered, and vitamin supplements are obtained.

5. The method according to claim 4, characterized in that, The ultrasound session lasts 10-30 minutes.

6. A vitamin supplement prepared by the method according to any one of claims 4 to 5.

7. A method for culturing sterile zebrafish, characterized in that, Sterile zebrafish embryos are placed in a sterile culture system, and the vitamin complex of any one of claims 1 to 3 and / or the vitamin supplement of claim 6 are added to the culture water.

8. The cultivation method according to claim 7, characterized in that, The volume ratio of the vitamin supplement to the culture water is 1:500-2000; the culture conditions include: temperature 15-30℃, pH 6.5-8.

9. The use of the vitamin complex according to any one of claims 1 to 3 and / or the vitamin supplement according to claim 6 in the preparation of products for prolonging the survival period of germ-free zebrafish and / or promoting the growth and development of germ-free zebrafish.

10. The application according to claim 9, characterized in that, The promotion of growth and development in germ-free zebrafish includes promoting the expression of growth factor genes in germ-free zebrafish; the growth factor genes include any one or more of gh1, gh1a, gh1b, igf1, igf1a, and igf1b.

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