A method for culturing large-mouth bass gill explants and application thereof

Abstract

The present application relates to the field of aquaculture technology, and particularly relates to a culture method of bigmouth bass gill explants and application. The present application provides a culture method of bigmouth bass gill explants, which uses explant culture solution to culture the gill explants of bigmouth bass. The explant culture solution is formed by adding fetal bovine serum, fish serum, trinity, D-galactose, L-alanyl-L-glutamine, sodium pyruvate and melatonin into a basic culture medium. In combination with dynamic culture conditions, the natural three-dimensional structure and intercellular connection of the gill tissue can be preserved, and the metabolic activity and functional integrity thereof can be maintained. The present application can solve the technical problem that the prior art cannot provide an ideal research model which can both maintain the structural integrity of the gill tissue and accurately reflect the functional characteristics of the gill tissue. Through the culture method, research models such as ammonia nitrogen stress can be efficiently constructed, thereby providing technical support for the research of bigmouth bass gill tissue physiological mechanism, disease prevention and control, environmental risk assessment and the like.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for culturing and applying gill explants of largemouth bass. Background Technology

[0002] Largemouth bass ( Micropterus salmoides As a core species with significant economic value in my country's freshwater aquaculture industry, gill disease has seen continuous expansion in recent years due to its advantages such as tender meat and rapid growth, becoming one of the important pillars driving the development of aquaculture. However, behind the booming development of the industry, the lagging disease control technology has gradually become a key bottleneck restricting its sustainable development. Among them, the frequent occurrence of gill diseases is particularly prominent, which not only leads to a significant decrease in the survival rate of aquaculture but also causes serious economic losses, posing a severe challenge to the healthy development of the industry.

[0003] The gills, as vital multifunctional organs in fish, play an indispensable role in their physiological activities. They are both the core site of gas exchange and a "sensitive organ" for sensing changes in the external environment. When faced with environmental stresses such as fluctuations in dissolved oxygen and accumulation of ammonia nitrogen, the gills are the first to respond, and the stability of their structure and function directly affects the survival of fish. Simultaneously, due to their direct contact with water, the gills also become a primary gateway for pathogens (such as bacteria, viruses, and parasites) to invade the fish's body; the occurrence of many gill diseases is closely related to the colonization and proliferation of pathogens in this area. Therefore, in-depth research into the physiological mechanisms and pathological processes of the gills is of great significance for understanding the environmental adaptation patterns of fish and developing disease control technologies.

[0004] Currently, research on gill tissue mainly relies on two types of techniques, but both have significant limitations that are difficult to overcome:

[0005] Firstly, in vivo animal experiments ( In vivo While in vivo experiments can reflect the state of gill tissue within a complete organism, they suffer from long experimental periods and significant individual variability. Because fish growth and development are influenced by various factors such as environment and genetics, the responses of different individuals under the same treatment often fluctuate considerably, leading to poor experimental reproducibility. Furthermore, large-scale in vivo experiments require substantial amounts of aquaculture space and feed costs, resulting in high economic investment. More importantly, in vivo physiological activities of gill tissue are indirectly regulated by the neuroendocrine system, making it difficult to precisely pinpoint the independent functions of gill tissue itself, thus interfering with mechanistic studies.

[0006] Secondly, two-dimensional (2D) in vitro cell culture technology ( In vitroWhile 2D culture allows for precise manipulation of single cell types, it suffers from significant drawbacks due to its separation from the three-dimensional structure of tissues. In 2D culture systems, cell polarity is lost, and the intercellular connections and signal transduction pathways differ significantly from those in vivo. The tissue microenvironment is severely simplified, making it impossible to simulate the complex hierarchical structure (such as the spatial arrangement of gill filaments and gill lamellae) and physiological functions (such as ion exchange and mucus secretion) of gill tissue. This results in a substantial decrease in the correlation between experimental results and in vivo conditions, making it difficult to effectively guide practical research.

[0007] In contrast, three-dimensional (3D) explant culture technology ( Ex vivo As a research model between in vivo and in vitro cell culture, gill explant culture has unique advantages: it can preserve the in situ intercellular interactions and natural spatial conformation of tissues, more closely resembling the physiological microenvironment in vivo. However, current research on this technology is mostly focused on mammalian tissues and organs (such as liver and kidneys), and its application in the aquaculture field is still in its early stages, with very few related research reports. More importantly, the culture of gill explants in aquatic animals lacks standardized operating procedures. From tissue collection and culture medium formulation to the control of culture conditions, there is considerable arbitrariness in all aspects, leading to insufficient stability and comparability of experimental results. This severely restricts the promotion of this technology in academic research (such as the analysis of the physiological mechanisms of gill tissue) and industrial applications (such as the development of disease control technologies).

[0008] In summary, current technologies cannot provide an ideal research model that both maintains the structural integrity of gill tissue and accurately reflects its functional characteristics. This directly leads to slow progress in areas such as gill tissue-related physiological mechanisms, pollutant toxicity assessment, and efficient drug screening, making it difficult to meet the industry's urgent needs for disease control technologies. Therefore, establishing a stable, efficient, and in vivo-like method for largemouth bass gill explant culture is a key breakthrough to overcome current research bottlenecks. This method not only has significant scientific value, providing new technical support for fish physiology and pathology research, but also shows broad prospects in industrial applications such as aquaculture disease control and environmental risk assessment. Summary of the Invention

[0009] The present invention aims to provide a method for culturing gill explants of largemouth bass, in order to solve the technical problem that existing technologies cannot provide an ideal research model that can both maintain the integrity of gill tissue structure and accurately reflect its functional characteristics.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] A method for culturing gill explants of largemouth bass, wherein gill explants of largemouth bass are cultured using an explant culture medium; the explant culture medium is formed by adding fetal bovine serum, fish serum, three antibiotics, D-galactose, L-alanyl-L-glutamine, sodium pyruvate and melatonin to a basal culture medium.

[0012] Furthermore, the contents of fetal bovine serum, fish serum, triple antibodies, D-galactose, L-alanyl-L-glutamine, sodium pyruvate, and melatonin in the explant culture medium were 10-20%, 1-2%, 1-2%, 1-5mM, 1-5mM, 1-5mM, and 0.01-0.03mM, respectively.

[0013] Furthermore, the contents of fetal bovine serum, fish serum, three antibodies, D-galactose, L-alanyl-L-glutamine, sodium pyruvate, and melatonin in the explant culture medium were 15%, 1%, 1%, 5.0 mM, 2.5 mM, 5.0 mM, and 20 μM, respectively.

[0014] Furthermore, the three antibodies include penicillin, streptomycin, and amphotericin B, with final concentrations of 100 U / mL, 100 μg / mL, and 0.25 μg / mL, respectively.

[0015] Furthermore, the gill explant includes 10-15 gill filaments and does not contain gill arches; the gill explant is rinsed sequentially with alcohol, pre-cooled PBS buffer, PBS buffer containing three antibodies, and explant culture medium.

[0016] Furthermore, after inoculating the gill explants into the explant culture medium, the culture conditions were set as follows: gas environment 4-6% CO2, 20-25% O2, 70-75% N2; culture temperature 20-30℃; and the explant culture medium inoculated with gill explants was intermittently shaken by shaking for 5-15 minutes and then standing for 20-30 minutes; the shaking speed was 3-7 rpm.

[0017] Furthermore, after inoculating the gill explants into the explant culture medium, the culture conditions were set as follows: gas environment 5% CO2, 20% O2, 75% N2; culture temperature 25℃; and the explant culture medium inoculated with gill explants was intermittently shaken with a shaking time of 10 minutes and a settling time of 20 minutes; the shaking speed was 5 rpm.

[0018] This technical solution also provides a method for culturing largemouth bass gill explants, and the application of the obtained largemouth bass gill explants in establishing a largemouth bass gill explant model under ammonia nitrogen stress. The largemouth bass gill explants are cultured in a nitrogen-containing explant culture medium, and the culture conditions are controlled as follows: gas environment 4-6% CO2, 20-25% O2, 70-75% N2; culture temperature 20-30℃, and the explant culture medium inoculated with gill explants is intermittently shaken by shaking for 5-15 minutes, 3-7 rpm and then standing for 20-30 minutes; after culturing for 4-8 hours, a largemouth bass gill explant model under ammonia nitrogen stress is obtained.

[0019] This technical solution also provides an explant culture medium for culturing gill explants of largemouth bass, which is formed by adding fetal bovine serum, fish serum, three antibiotics, D-galactose, L-alanyl-L-glutamine, sodium pyruvate and melatonin to the basal culture medium.

[0020] Furthermore, the basal culture medium is Leibovitz's L-15; the contents of fetal bovine serum, fish serum, three antibiotics, D-galactose, L-alanyl-L-glutamine, sodium pyruvate, and melatonin in the explant culture medium are 10-20%, 1-2%, 1-2%, 1-5mM, 1-5mM, 1-5mM, and 0.01-0.03mM, respectively.

[0021] In summary, based on the core advantages of three-dimensional explant culture technology, this invention constructs a stable culture system that simulates the in vivo physiological state of largemouth bass gill tissue. The technical principle is as follows:

[0022] (1) Design of biomimetic cultivation system

[0023] Based on a basal culture medium suitable for aquatic animal cell culture, a variety of functional components are added in a scientifically proportioned manner to form a synergistic explant culture medium. Each component has a significant and synergistic effect on the technical efficacy. This formulation system, through the synergistic effect of its components, can preserve the natural three-dimensional structure of gill tissue and the intercellular connections to the greatest extent, solving the problems of simplified tissue microenvironment and disconnect between cell function and in vivo state in traditional two-dimensional cell culture.

[0024] Xenobiotic serum (such as fetal bovine serum) is rich in growth factors, albumin, and other essential nutrients, providing broad-spectrum nutritional support for gill explants, promoting cell metabolism and proliferation, and ensuring tissue survival. Homologous fish serum precisely mimics the species-specific microenvironment of largemouth bass. Its fish-specific hormones and cytokines enhance recognition and communication between gill tissue cells, maintaining the natural spatial conformation of gill filaments and lamellae, and preventing tissue structural disorder caused by xenobiotic environments. Antibacterial components (such as penicillin, streptomycin, and amphotericin B) effectively reduce the risk of explant contamination by inhibiting the growth of bacteria, fungi, and other microorganisms, providing a clean environment for tissue survival and solving the problem of tissue necrosis caused by microbial growth in in vitro culture. Energy metabolism-related substances (such as D-galactose, L-alanyl-L-glutamine, and sodium pyruvate) are key precursors of cell metabolism, directly participating in processes such as glycolysis and protein synthesis, maintaining energy supply and metabolic homeostasis of gill cells, and preventing functional decline due to nutrient deficiency after in vitro culture. Antioxidant and anti-apoptotic components (such as melatonin) can remove excess reactive oxygen species in the in vitro environment, inhibit the activation of apoptosis pathways, delay the aging and degradation of gill tissue, significantly prolong the survival time of explants and maintain their physiological activity.

[0025] (2) Optimization of dynamic culture conditions

[0026] To address the physiological characteristics of gill tissue, a gas composition simulating its in vivo environment was designed. By adjusting the gas ratios, suitable oxygen partial pressure and acid-base balance were maintained, preventing tissue dysfunction caused by gas imbalance. Simultaneously, an intermittent shaking culture mode was employed to ensure uniform distribution of nutrients in the culture medium while minimizing damage to the gill explants from continuous mechanical stimulation, providing the tissue with a dynamic survival environment close to its natural state. This optimized culture condition significantly extended the survival time of ex vivo gill tissue, maintained its metabolic activity and structural integrity, and provided a stable research subject for subsequent experiments.

[0027] (3) Verification of the equivalence of the ammonia nitrogen stress model

[0028] Based on the characteristic of gill explants being directly exposed to the stress environment, stress concentrations with biological relevance to the in vivo model were designed. Through short-term culture, the explants rapidly exhibited pathological changes similar to those observed under long-term in vivo stress, including abnormal tissue morphology, altered cell proliferation activity, and cell cycle disorders. The core principle lies in the fact that the explant model eliminates the indirect influence of the body's overall regulatory systems (such as the neuroendocrine network) under in vivo conditions, allowing gill tissue to directly respond to external stress. This enables highly efficient simulation of in vivo pathological processes and provides a reliable model basis for rapidly elucidating stress mechanisms.

[0029] The beneficial effects of this technical solution are as follows:

[0030] (1) Break through traditional technical bottlenecks and provide an ideal research model

[0031] Compared to in vivo experiments, this method shortens the ammonia nitrogen stress study period from 30 days to 6 hours, reduces the number of animal samples required from 60 to 3, reduces experimental costs by 90%, and avoids interference from individual differences in the results. Compared to 2D cell culture, it preserves the natural structure and cellular heterogeneity of gill tissue, significantly improves the correlation between experimental results and in vivo conditions, and solves the problem that in vitro results cannot reflect the real physiological processes in vivo.

[0032] (2) Standardize operating procedures to improve experimental stability and repeatability.

[0033] The clearly defined culture medium formula and culture conditions (such as gas ratio and shaking mode) form a standardized system, which overcomes the shortcomings of the large degree of arbitrariness in the existing aquatic explant culture, makes the results of different laboratories comparable, and provides reliable technical support for the study of gill tissue physiological mechanisms and the assessment of pollutant toxicity.

[0034] Specifically, the gill explant model and corresponding culture method established in this protocol have significant application value in ammonia nitrogen stress research. This model, through in vitro culture of fish gill tissue, can maximally preserve the original structure and physiological function of the gill organs (such as the integrity of gill filaments and gill lamellae) while eliminating interference from other organs or systems in vivo, enabling precise and targeted research on ammonia nitrogen stress. In practical applications, considering the fluctuations in ammonia nitrogen exposure concentrations faced by fish in natural water bodies, studies can be conducted on different concentrations of ammonia nitrogen stress (for the same batch of fish, ammonia nitrogen stress concentration in the gill explants of 1 / 20th of the in vivo concentration can be simulated). By comparing and analyzing the changes in various indicators of the gill explants under different concentrations, the dose-response relationship of ammonia nitrogen stress can be systematically explored. Specific detection contents may include: tissue morphological changes, histopathological changes, oxidative stress response, cell apoptosis level, gene expression differences, etc.

[0035] (3) Broad industrial application prospects

[0036] It can be rapidly applied to research on the mechanism of gill tissue diseases in aquaculture, toxicity screening of environmental pollutants (such as ammonia nitrogen and heavy metals), and screening of fish drugs. It provides an efficient in vitro validation platform for the development of gill damage repair drugs, helps solve the industry pain points of disease prevention and control in largemouth bass farming, and has significant economic value and social benefits.

[0037] (4) Synergistic effect of the formula and outstanding technological innovation

[0038] The combination of fish serum and melatonin produces a synergistic effect, which not only enhances the utilization of species-specific nutrients by explants, but also reduces oxidative damage to isolated tissues through the antioxidant properties of melatonin, significantly reducing the contamination rate and improving tissue activity. This optimized formula provides a technical paradigm that can be referenced for the culture of explants from other aquatic animals. Attached Figure Description

[0039] Figure 1 This is a flowchart of Example 1.

[0040] Figure 2 The results of superoxide dismutase (SOD, left) and catalase (CAT, right) activity assays in Example 2 are shown below (I: control group; II: saline culture group; III: gill cell culture medium group; IV: gill explant culture medium group; Duncan's test was used for significance analysis, and the data in the statistical bar chart are marked with lowercase letters above them; in the same statistical bar chart, different lowercase letters indicate significant differences between the data, p < 0.05).

[0041] Figure 3 The results of superoxide dismutase (SOD, left) and catalase (CAT, right) activity assays in Example 2 are shown below (I: control group; II: gill explant culture medium group; III: gill explant culture medium lacking melatonin group (FS added alone); IV: gill explant culture medium lacking fish serum group (MT added alone); V: gill explant culture medium lacking both fish serum and melatonin group; Duncan's test was used for significance analysis, and the data in the statistical bar chart are marked with lowercase letters above them; in the same statistical bar chart, different lowercase letters indicate significant differences between the data, p < 0.05).

[0042] Figure 4 The results of cell death rate (left) and Caspase-3 expression level (right) in Example 2 are shown below (Ⅰ: control group; Ⅱ: saline culture group; Ⅲ: gill cell culture medium group; Ⅳ: gill explant culture medium group; Duncan's test was used to analyze the significance of differences, and the data in the statistical bar chart are marked with lowercase letters above them; in the same statistical bar chart, different lowercase letters indicate significant differences between the data, p < 0.05).

[0043] Figure 5The results of cell mortality (left) and Caspase-3 expression level (right) in Example 2 are shown below (Ⅰ: control group; Ⅱ: gill explant culture medium group; Ⅲ: gill explant culture medium lacking melatonin group (FS added alone); Ⅳ: gill explant culture medium lacking fish serum group (MT added alone); Ⅴ: gill explant culture medium lacking both fish serum and melatonin group; the Duncan test was used to analyze the significance of the differences, and the data in the statistical bar chart are marked with lowercase letters above them; in the same statistical bar chart, different lowercase letters indicate that there are significant differences between the data, p < 0.05).

[0044] Figure 6 Typical morphological photographs of gill explants under different culture conditions in Example 3 and statistical results of contamination rate (Ⅰ, Ⅱ, Ⅲ: representing the saline culture group, gill cell culture medium group, and gill explant culture medium group, respectively; Duncan's test was used for significance analysis of differences, and the data in the statistical bar chart are marked with lowercase letters; in the same statistical bar chart, different lowercase letters indicate significant differences between the data, p < 0.05).

[0045] Figure 7 The statistical results of the study on the effect of shaking culture in Example 4 are shown in the following figure (Duncan's test was used to analyze the significance of the difference, and the data in the statistical bar chart are marked with lowercase letters above them; in the same statistical bar chart, different lowercase letters indicate that there is a significant difference between the data, p < 0.05).

[0046] Figure 8 This is a flowchart illustrating the application of the gill explant model from Example 5 in ammonia nitrogen stress research.

[0047] Figure 9 The images show typical H&E staining results of the in vivo and gill explant models in Example 5 (A and C are in vivo experiments; A is the structure of the gill tissue in the control group, which is neatly arranged; C is the histopathological changes of the gill in the ammonia nitrogen stress group, which shows obvious gill lamella hyperplasia (★), gill lamella adhesion (black arrow), and gill lamella edema; B and D are gill explant culture experiments; B is the histological structure of the gill explant in the control group, which shows thin gill filament epithelium (black double arrow); D is the histological structure of the gill explant in the ammonia nitrogen stress group, which shows epithelial cell shedding (white arrow) and obvious gill lamella hyperplasia (☆), forming a thick interlamellar cell cluster (black double arrow)).

[0048] Figure 10Typical PCNA immunofluorescence staining images of the in vivo and gill explant models of Example 5 (AB are in vivo experiments; A is PCNA immunofluorescence of the control group gills, with positive signals (green) mainly distributed at the base of the gill lamellae; B is PCNA immunofluorescence of the ammonia nitrogen stress group gills, showing a large number of positive signals (green) scattered in the interlamellar cell clusters (white dashed lines); CD is the gill explant culture experiment; C is PCNA immunofluorescence staining of the control group gills, with undifferentiated cells mainly located at the base of the gill filaments and mitochondrial-rich cells mainly located in the gill filament epithelium; D is PCNA immunofluorescence staining of the ammonia nitrogen stress group gills, with interlamellar cell clusters formed between the gill lamellae (white dashed lines), showing a large number of undifferentiated cells; PCNA: proliferating cell nuclear antigen; DAPI: 4',6-diamidinyl-2-phenylindole) used for nuclear staining).

[0049] Figure 11 The results of flow cytometry analysis of the in vivo model and gill explant model of Example 5 are shown below (AB represents the in vivo experiment, which represents the DNA content distribution in the control group and the ammonia nitrogen stress group, respectively; CD represents the gill explant culture experiment, which represents the DNA content distribution in the control group and the ammonia nitrogen stress group, respectively). Detailed Implementation

[0050] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0051] Example 1: Establishment of a gill explant model

[0052] (1) Obtaining gill explants

[0053] The obtained whole gill tissue from largemouth bass was processed as follows: rinsing once each with 75% alcohol, pre-cooled PBS buffer, PBS buffer containing three antibiotics, and explant culture medium. Gill tissue is an important respiratory organ in fish, and its structure mainly includes gill arches, gill filaments, and gill lamellae. The gill arch is the supporting structure of the gill, arc-shaped, and serves to support the gill filaments. Gill filaments are located on the outer side of the gill arch, while gill rakers are located on the inner side. Gill filaments are the main respiratory structure of the gill, located on the outer side of the gill arch, and are filamentous. Numerous capillaries are distributed within the gill filaments, serving as the main site of gas exchange. Gill lamellae are thin, lamellar structures on the gill filaments, distributed radially in an orderly manner. Chloride-secreting cell clusters are located at the ends of the gill lamellae. The gill lamellae are densely covered with capillaries; when water flows through the gill lamellae, oxygen enters the capillaries through diffusion, while carbon dioxide is expelled into the water. For the whole gill tissue, the gill arch was cut along its base, removing the gill arch portion while preserving the gill filament structure, maintaining the connection between the gill filaments and the end near the gill arch, thus obtaining approximately 2 mm of the tissue. 3The gill explants consist of 10-15 individual gill filaments, with each filament connected near the gill arch. The gill explants are approximately 1 mg of wet tissue. Fresh gill explants are immediately transferred into six-well plates (1 explant / well, 5 mL explant culture medium / well).

[0054] The explant culture medium is prepared as follows:

[0055] The explant culture medium was prepared using Leibovitz's L-15 commercially available medium as the basal medium. The explant culture medium contained 10-20% (v / v, preferably 15%) fetal bovine serum (FBS), 1-2% (v / v, preferably 1%) fish serum (FS, Nanjing Shenghang Biotechnology Co., Ltd., BioChannel, SBJ-SE-F001), 1-2% (v / v, preferably 1%) triple antibiotics (penicillin-streptomycin-amphotericidal; stock solution concentration: penicillin: 10000 U / mL, streptomycin: 10000 μg / mL; amphotericin B: 25 μg / mL; preferred working final concentration: penicillin: 100 U / mL, streptomycin: 100 μg / mL; amphotericin B: 0.25 μg / mL), and 1-5 mM (preferably 5.0 mM). D-galactose, 1-5 mM (preferably 2.5 mM) L-alanyl-L-glutamine, 1-5 mM (preferably 5.0 mM) sodium pyruvate, 10-30 μM (preferably 20 μM) melatonin (MT). The pH of the explant culture medium is 7.0-7.4.

[0056] The PBS buffer containing triple antibodies is specifically a PBS buffer containing 1-2% (preferably 1%) of triple antibodies (penicillin-streptomycin-amphotericidal), with the concentrations of penicillin, streptomycin, and amphotericin B in the stock solution being 10000 U / mL, 10000 μg / mL, and 25 μg / mL, respectively. The working concentrations of penicillin, streptomycin, and amphotericin B are 100-200 U / mL, 100-200 μg / mL, and 0.25-0.50 μg / mL, respectively.

[0057] It is important to emphasize that this technical solution only involves the cultivation and processing of gill tissue and gill explants after obtaining them, and does not involve the process of removing gill tissue from the fish or separating gill explants. The process of removing gill tissue from the fish and separating gill explants is a conventional method in existing technology and will not be elaborated upon here. The purpose of this solution is to establish a cultivation method for gill explants and a 3D explant (…). Ex vivo )Model.

[0058] (2) Gill explant culture

[0059] Place the six-well plate inoculated with explants on the platform of a mini orbital shaker (3-7 rpm, preferably 5 rpm), ensuring the plate is stable and fixed (use suitable clamps or silicone pads to prevent slippage). Select intermittent shaking (shake for 5-15 minutes, rest for 20-30 minutes, and repeat this cycle; preferably shake for 10 minutes, rest for 20 minutes) to reduce the damage to the explant structure caused by continuous mechanical stimulation. Place the shaker in a three-gas incubator, connect the power supply, and set the appropriate gas environment (4-6% CO2 + 20-25% O2 + 70-75% N2; preferably 5% CO2 + 20% O2 + 75% N2) to meet the high oxygen consumption characteristics of gill tissue. Incubate at 20-30℃ (preferably 25℃) for 4-8 hours (preferably 6 hours).

[0060] (3) Vitality test

[0061] After culture, harvest the gill explants from the six-well plates promptly. Add an appropriate amount of 0.25% trypsin using a Pasteur pipette for digestion, agitating for 2 seconds while occasionally shaking the six-well plate to observe the digestion. Then add a few drops of FBS and shake well to stop digestion. Gently and evenly agitate the flocculent material to form individual suspended cell clusters. Filter several times through 70μm and 40μm cell filters, collecting the filtrate in a 50mL centrifuge tube. Perform viable cell counting using trypan blue staining. Mix 0.4% trypan blue solution with an appropriately diluted cell suspension at a 1:1 volume ratio in a 1.5mL centrifuge tube. Add an appropriate amount of the mixture to the edge of the coverslip of the cell counting plate, allowing it to diffuse and fill the counting area through surface tension. Observe under a microscope to determine if the cells are stained, and calculate the viability value based on the percentage of live (or dead) cells.

[0062] The above flowchart can be found here. Figure 1 .

[0063] Example 2: Study on the effects of adding fish serum and melatonin

[0064] The experimental groups are as follows:

[0065] Control group (gill explants collected at 0h, directly subjected to various tests);

[0066] The saline culture group (conducted according to the optimal method of Example 1, except that the explant culture medium was replaced with saline, and all other operations were the same as the optimal method of Example 1).

[0067] The gill cell culture medium group (conducted according to the optimal method of Example 1, except that the explant culture medium was replaced with conventional cell culture medium: the basal culture medium was prepared using commercially available Leibovitz's L-15 medium, and the conventional cell culture medium was supplemented with 15% FBS, 200 U / mL penicillin, 200 μg / mL streptomycin, 2.5 mmol / L glutamine and 2.5 mmol / L sodium pyruvate, and all other operations were the same as the optimal method of Example 1).

[0068] Gill explant culture medium group (conducted according to the optimal method of Example 1, using explant culture medium).

[0069] The gill explant culture medium lacking melatonin was used in the optimal method of Example 1 (the optimal method of Example 1 was followed, using melatonin-MT-deficient explant culture medium instead of complete explant culture medium, and all other operations were the same as the optimal method of Example 1).

[0070] Fish serum-deficient explant culture medium (conducted according to the optimal method of Example 1, using explant culture medium deficient in fish serum FS instead of complete explant culture medium, and all other operations were the same as the optimal method of Example 1).

[0071] The gill explant culture medium lacking fish serum and melatonin was used in the optimal method of Example 1, using explant culture medium lacking melatonin MT and fish serum FS instead of complete explant culture medium, and all other operations were the same as the optimal method of Example 1.

[0072] After 6 hours of culture, various tests were performed on the gill explants, as follows:

[0073] (1) Antioxidant capacity

[0074] Remove the cultured gill explants and gently rinse three times with pre-chilled PBS buffer to remove any residual culture medium. Add pre-chilled physiological saline at a ratio of 1:9 (w / v) and transfer the gill explants to a glass homogenizer. Homogenize on ice to ensure thorough tissue disruption. Transfer the homogenate to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 10 minutes at 4°C. Collect the supernatant (the crude enzyme extract) and store it on ice for later use.

[0075] Protein concentration was determined to standardize enzyme activity, using bovine serum albumin as the standard. Following the BCA protein quantification kit instructions, a standard gradient and sample wells were set up, and the corresponding reagents were added before incubation at 37°C for 30 minutes. The absorbance at 562 nm was measured using a microplate reader, and the total protein concentration (mg / mL) in the sample was calculated.

[0076] SOD (superoxide dismutase) activity assay: SOD activity was measured using a kit (WST-1 method, Nanjing Jiancheng, A001-3-2) following the kit's operating procedures. SOD activity was characterized as enzyme activity U per mg of protein (U / mg).

[0077] CAT (catalase) activity assay: CAT activity was assayed using a kit (visible light, ammonium molybdate method, Nanjing Jiancheng, A007-1-1) following the kit's operating procedures. CAT activity was characterized as enzyme activity U per mg of protein (U / mg).

[0078] The experimental results are detailed in Table 1 and Figure 2 and Figure 3 .

[0079] Table 1: Results of SOD and CAT activity detection

[0080]

[0081] Compared with the control group, the antioxidant capacity of the saline culture group and the gill cell culture medium group was significantly reduced. However, the addition of melatonin to the explant culture medium can act as an endogenous antioxidant, upregulate the activity of SOD and CAT enzymes, improve the antioxidant capacity of the explant tissue, and promote its survival in vitro.

[0082] Analysis of the synergistic effect between MT and FS:

[0083] A: The synergistic effect of MT and FS in enhancing SOD activity

[0084] A-1: Preliminary assessment

[0085] If the combined effect of two substances is greater than the sum of their individual effects, then there is a synergistic effect; if the combined effect is approximately equal to the sum of their individual effects, then it is an additive effect; if the combined effect is less than the sum of their individual effects, then it is an antagonistic effect. This method is mainly used to quickly determine the nature of the combined effect of two substances, directly through the arithmetic summation of the effect values.

[0086] Gill explants were cultured using gill explant culture medium lacking MT and FS (gill explant culture medium lacking fish serum and melatonin). The average SOD activity was 40.0 U / mg, and data were standardized based on this.

[0087] The increase in SOD activity resulting from FS supplementation alone (gill explant culture medium lacking melatonin) was: 46.7 U / mg - 40.0 U / mg = 6.7 U / mg;

[0088] The increase in SOD activity resulting from the addition of MT alone (to the fish serum group lacking gill explant culture medium) was: 51.7 U / mg - 40.0 U / mg = 11.7 U / mg;

[0089] The increase in SOD activity resulting from the combined addition of MT+FS (gill explant culture medium group) was: 64.0 U / mg - 40.0 U / mg = 24.0 U / mg.

[0090] The sum of the individual added values ​​is (expected value) 6.7 U / mg + 11.7 U / mg = 18.4 U / mg < 24.0 U / mg. The actual value is much higher than the expected value. This shows that the combined use of MT and FS has a synergistic effect in improving antioxidant capacity.

[0091] A-2: Bliss Independent Model

[0092] The Bliss Independence Model is a pharmacological model used to evaluate the combined effects of two or more drugs (or bioactive substances). It is mainly used to determine whether the drug combination exhibits synergy, additive activity, or antagonism.

[0093] From an application perspective, the Bliss independent model is particularly suitable for evaluating combinations of substances with significantly different mechanisms of action. In this protocol, the combined effect assessment of fish serum FS and melatonin MT perfectly aligns with the applicable conditions of the Bliss independent model. Existing technical reports clearly show that their mechanisms of action differ significantly: fish serum FS, as a complex biological fluid component, may have effects related to basic physiological processes such as providing nutritional support, regulating osmotic pressure balance, and participating in local immune regulation, relying more on the overall regulation of the body fluid environment; while melatonin MT, as a classic neuroendocrine hormone, mainly functions through specific receptor-mediated pathways, participating in precise signaling pathways such as circadian rhythm regulation, oxidative stress response, and apoptosis regulation. This significant difference in mechanism of action means that when FS and MT act together, they are more likely to follow the principle of "independent action without interference": FS works by maintaining the basic physiological microenvironment of the gill explant, while MT regulates the activity of antioxidant enzymes through specific signaling pathways, with no significant overlap in their targets and pathways. The Bliss-independent model is not applicable when two drugs act on the same receptor simultaneously (e.g., drug A is an agonist that binds to receptor R and activates downstream signaling pathways, promoting cellular responses; drug B is another structurally similar agonist that also acts on the same receptor R, producing a similar effect). FS and MT clearly do not fall into this inapplicable situation, as their action pathways are relatively independent. Therefore, using the Bliss-independent model to assess their combined effect can more accurately reflect the individual contributions of the two substances and the nature of their combined effect, providing a scientific theoretical basis for determining whether synergistic effects exist.

[0094] The Bliss independent model is calculated using the following formula:

[0095] E AB =E A +E B -E A E B

[0096] E A E B The relative effect values ​​of drugs A and B acting alone are expressed as "the ratio of drug effect to control group" (range 0-1, reflecting the proportion of the effect intensity of a single drug on the control group). Here, "control group" refers to the group whose gill explant culture medium lacks fish serum and melatonin; E AB This refers to the expected effect when the two work together.

[0097] This model is commonly used for drug screening and combination optimization. By comparing the actual combined effect with the expected effect, it determines whether a synergistic effect exists. If the actual effect exceeds the expectation, a synergistic effect is considered to exist.

[0098] The specific calculations are as follows:

[0099] SOD activity in the control group (gill explant culture medium without FS and MT) was 40.0 U / mg (as the baseline for effect).

[0100] Increase in SOD activity by FS alone: ​​6.7 U / mg (the increase in SOD activity by FS alone relative to the control group);

[0101] Increase in SOD activity by MT alone: ​​11.7 U / mg (the increase in SOD activity by MT alone relative to the control group);

[0102] Actual increase in SOD activity due to the combined action of FS and MT: 24.0 U / mg (actual increase in SOD activity relative to the control group after the two drugs were used together).

[0103] E A (Relative effect of FS monotherapy) = 6.7 / 40 = 0.1675 (that is, FS monotherapy increased SOD activity by 16.75% relative to the control group).

[0104] E B (MMT monotherapy relative effect) = 11.7 / 40 = 0.2925 (that is, MT monotherapy increased SOD activity by 29.25% relative to the control group);

[0105] E AB =0.1675+0.2925-0.1675×0.2925=0.411 (Theoretically, the combined use of FS and MT can increase SOD activity by 41.1% relative to the control group).

[0106] The actual improvement in SOD from the combined use of FS and MT is: (64.0 - 40.0) / 40.0 = 60% > 41.1%. The actual value is much higher than the expected value, indicating a significant synergistic effect.

[0107] Since the actual relative effect (60%) was significantly higher than the model's expected relative effect (41.1%), it can be determined that the combined use of FS and MT has a synergistic enhancing effect on SOD activity.

[0108] Preferably, FS and MT exhibit a significant synergistic effect at a concentration of 1% (volume percentage): 20 μM. That is, FS is 1 mL FS / 100 mL culture medium, and MT is 20 μmol MT / 1 L culture medium. This translates to a 1 mL: 2 μmol ratio of FS to MT that produces the synergistic effect.

[0109] B: The synergistic effect of MT and FS in enhancing CAT activity

[0110] B-1: Preliminary Assessment

[0111] Gill explants were cultured using gill explant medium lacking MT and FS (gill explant medium lacking fish serum and melatonin). The average CAT activity was 62 U / mg, and data were standardized based on this.

[0112] The increase in CAT activity resulting from FS supplementation alone (gill explant culture medium lacking melatonin) was: 66.7 U / mg - 62 U / mg = 4.7 U / mg

[0113] The increase in CAT activity resulting from the addition of MT alone (to the fish serum group lacking gill explant culture medium) was: 75.3 U / mg - 62 U / mg = 13.3 U / mg;

[0114] The increase in CAT activity resulting from the combined addition of MT+FS (gill explant culture medium group) was 87.7 U / mg - 62 U / mg = 25.7 U / mg.

[0115] The sum of the individual increases is: 4.7 U / mg + 13.3 U / mg = 18 U / mg < 25.7 U / mg. This shows that the combined use of MT and FS produces a synergistic effect in enhancing antioxidant capacity.

[0116] B-2: Bliss Independent Model

[0117] The calculation method is the same as above. The actual improvement effect of using FS and MT together on CAT is 41.4%, while the expected effect of the model is 27.2%. It can be determined that when FS and MT are used together, there is a synergistic enhancement effect on the improvement of CAT activity.

[0118] (2) Mortality rate and expression of Caspase-3, a gene related to apoptosis.

[0119] The percentage of dead cells was determined according to the method described in Example 1 (approximately 5000 cells were present in 10 μl of diluted cell suspension, and this was used as the basis for determining the percentage of dead cells). Total RNA was extracted from gill explant samples, and the expression level of the apoptosis-related gene Caspase-3 was detected using conventional qPCR (with the control group as baseline "1"). Detailed experimental results are shown in Table 2 and... Figure 4 , Figure 5 .

[0120] Table 2: Results of Caspase-3 expression detection, a gene related to cell death rate and apoptosis.

[0121]

[0122] Analysis of the synergistic effect between MT and FS:

[0123] Comparing the "gill explant culture medium lacking fish serum and melatonin group" and the "gill explant culture medium lacking fish serum group," there was no significant difference between the two groups, and the mortality rates were similar. This indicates that adding MT alone (without FS) basically does not positively improve cell survival, meaning that MT itself does not have the effect of reducing gill cell mortality. Comparing the "gill explant culture medium lacking fish serum and melatonin group" and the "gill explant culture medium lacking melatonin group," it is evident that using FS alone has a certain effect on reducing mortality. Based on the above phenomena, theoretically, using MT and FS in combination (with their respective dosages consistent with those used individually) should result in a cell mortality rate that is similar to that of the "gill explant culture medium lacking melatonin group" using FS alone (theoretically, it should be slightly lower than 24.7%). However, actual experimental data show that the mortality rate of the "gill explant culture medium group" (using MT and FS in combination) can be reduced to 17.7%, which is lower than the theoretical mortality rate, indicating a synergistic effect between MT and FS. Specific analysis is as follows:

[0124] Gill explants were cultured using gill explant medium lacking MT and FS (gill explant medium lacking fish serum and melatonin). The average cell mortality rate was 32.7%, and data were standardized based on this baseline.

[0125] The reduction in cell death rate resulting from FS supplementation alone (gill explant culture medium lacking melatonin) was: 32.7% - 24.7% = 8%.

[0126] The reduction in cell death rate resulting from the addition of MT alone (in the group with gill explant culture medium lacking fish serum) was: 32.7% - 30.0% = 2.7%.

[0127] The reduction in cell death rate resulting from the combined addition of MT and FS (gill explant culture medium group) was: 32.7% - 17.7% = 15%.

[0128] The sum of the reduction values ​​added individually (expected value): 2.7% + 8% = 10.7% < 15%. The expected value is less than the actual value. This shows that the combined use of MT and FS has a synergistic effect in reducing cell death rate.

[0129] Example 3: Study on the effect of adding three types of antibiotics

[0130] The experimental groups are as follows:

[0131] The saline culture group (conducted according to the optimal method of Example 1, except that the explant culture medium was replaced with saline, and all other operations were the same as the optimal method of Example 1).

[0132] The gill cell culture medium group (conducted according to the optimal method of Example 1, except that the explant culture medium was replaced with conventional cell culture medium: the basal culture medium was prepared using commercially available Leibovitz's L-15 medium, and the conventional cell culture medium was supplemented with 15% fetal FBS, 200 U / mL penicillin, 200 μg / mL streptomycin, 2.5 mmol / L glutamine and 2.5 mmol / L sodium pyruvate, and all other operations were the same as the optimal method of Example 1).

[0133] The gill explant culture medium group (conducted according to the optimal method of Example 1, using explant culture medium, wherein the final concentrations of the three antibodies were: penicillin 100 U / mL, streptomycin 100 μg / mL, and amphotericin B 0.25 μg / mL).

[0134] After 6 hours of incubation, the contamination rate (bacterial and fungal contamination) was calculated. Each group was replicated 60 times (12 six-well plates per group, with one well in each plate left uninoculated as a negative control. Therefore, 12 plates × 5 effective wells = 60). The contamination rate was calculated by dividing the number of contaminated plates by 60 and multiplying by 100%. See the detailed experimental results below. Figure 6 The contamination rate in the saline culture group was 45% (50%, 45%, and 40% in three replicates), the contamination rate in the gill cell culture medium group was 19.3% (20%, 18%, and 20% in three replicates), and the contamination rate in the gill explant culture medium group was 10.3% (10%, 9%, and 12% in three replicates). The experiment showed that the gill lamellae in the saline culture group secreted a large amount of mucus, which aggregated, resulting in a high contamination rate, mainly due to bacterial and fungal contamination. In the gill cell culture medium group (with only antibiotics), the mucus secretion and aggregation of gill lamellae were improved, with a contamination rate of approximately 20%, and bacterial contamination was reduced, but some fungal contamination remained. In the explant culture medium group (with triple antibiotics), the gill lamellae were more dispersed, mucus secretion was less, and the contamination rate was approximately 10%, with reduced bacterial and fungal contamination. Therefore, adding triple antibiotics (penicillin 100 U / mL + streptomycin 100 μg / mL + amphotericin B 0.25 μg / mL) to the explant culture medium can effectively inhibit bacterial / fungal contamination.

[0135] Example 4: Study on the effect of shaking culture

[0136] The experimental groups are as follows:

[0137] Control group (gill explants collected at 0h, directly subjected to various tests);

[0138] Static culture group (conducted according to the optimal method of Example 1, the difference being in the gill explant culture process: the six-well plate inoculated with explants was placed directly in a three-gas incubator for culture without intermittent shaking, and all other operations were the same as the optimal method of Example 1).

[0139] The shaking culture group (conducted according to the optimal method of Example 1).

[0140] After 6 hours of culture, gill explants were harvested for ATP detection. A commercially available ATP extraction kit and a bioluminescent (luciferin-luciferase) ATP detection kit were used to measure ATP activity in the samples (results are expressed as pmol / mg protein). The highest ATP activity in the control group (three replicates) was defined as 100%. Based on this baseline, static culture led to gill cell hypoxia and necrosis, with ATP decreasing to an average of 47.3% after 6 hours (results in three replicates: 50%, 45%, and 46%). This protocol, through shaking culture, simulated the mechanical stimulation of blood flowing through the gill filaments, maintaining cell polarity distribution. The average ATP maintenance rate was approximately 77% (results in three replicates: 75%, 80%, and 78%), an improvement of 30% compared to static culture. See the experimental data statistics chart for details. Figure 7 .

[0141] In summary, after treating gill explants with the explant culture medium of this patented scheme for 6 hours, the apoptosis rate, ATP content, and antioxidant capacity of gill explant cells were significantly better than those of the conventional cell culture medium group (p < 0.01), and the contamination rate was significantly reduced. This demonstrates that it can maintain the structural and functional integrity of isolated tissues for a long time, providing a stable model for aquatic pathogen / toxicology research.

[0142] Example 5: Application of the gill explant model in ammonia nitrogen stress research

[0143] Ammonia nitrogen stress in largemouth bass (in vivo model): Largemouth bass were divided into a control group (Control) and an ammonia nitrogen stress group (TAN). Stress concentrations were based on the 96-hour median lethal concentration (LC50). 50 The concentration was set to 1 / 4 of the 96-hour median lethal concentration, i.e., NH4Cl 8 mg / L (150 μM), and the ammonia nitrogen concentration was adjusted using the NH4Cl stock solution. Largemouth bass were cultured in transparent glass tanks (1.1 × 0.7 × 0.8 m²). 3 Ammonia nitrogen stress was applied to the cells for 30 days. During the rearing period, dissolved oxygen was controlled at 7.05±0.23 mg / L, pH at 8.01±0.14, and water temperature at 23±1℃. After the ammonia nitrogen stress was completed, gill tissue was collected for flow cytometry cell cycle detection, histopathological analysis, and molecular biological analysis.

[0144] Ammonia nitrogen stress gill explants: This experiment was divided into two groups: a control group and an ammonia nitrogen stress group (TAN). The gill explant model was prepared according to the optimal method described in Example 1. During the gill explant culture, the ammonia nitrogen stress group had 7.5 μM total nitrogen (TAN) (1 / 20 of in vivo total nitrogen, 0.4 mg / L) added to the explant culture medium. The control group used explant culture medium without TAN. Each well of the six-well plates contained 5 mL of culture medium. The gill explants were transferred into the corresponding six-well plates of each group (1 explant / well) and cultured according to the optimal method in Example 1. After 6 hours (culturing for 4-8 hours is acceptable), the gill explants in the six-well plates were harvested promptly. The gill explants collected from each group were used for flow cytometry cell cycle detection, histopathological analysis, and molecular biological analysis.

[0145] The effectiveness of the gill explant model was investigated using histopathological and molecular biological analyses, with the in vivo model as a control. The in vivo model required a 30-day experimental period and 60 fish samples, resulting in high costs per experiment. The gill explant model, on the other hand, required a 6-hour experimental period and 3 fish samples, resulting in low costs per experiment, representing a 90% reduction in overall cost. A schematic diagram of the experimental procedure for applying the gill explant model in ammonia nitrogen stress research can be found here. Figure 8 .

[0146] Typical H&E staining images of ammonia-stressed largemouth bass (in vivo model) and gill explants under ammonia-stressed conditions are shown below. Figure 9 After subjecting largemouth bass to ammonia nitrogen stress, H&E staining revealed that the gill tissue in the control group was neatly arranged and structurally intact. In contrast, the gill tissue in the ammonia nitrogen stress group showed severe proliferation and fusion of gill lamellae. The cells in the proliferating areas were loosely arranged, and numerous mucus cells were observed. Furthermore, pathological changes such as gill lamellae epithelial cell detachment, edema, epithelial cell shedding, and gill lamellae adhesion were also observed in the ammonia nitrogen stress group. Using a gill explant model for ammonia nitrogen stress, H&E staining revealed that the gill lamellae in the control group were neatly arranged and structurally intact, with thin gill filament epithelium tightly adhering to the base of the gill filaments. In the ammonia nitrogen stress group, the gill explants mainly showed severe proliferation and fusion of gill lamellae, with thick interlayered cell clusters forming in the proliferating areas. Furthermore, pathological changes such as gill lamellae epithelial cell shedding, edema, and inflammatory cell infiltration were also observed. Therefore, the gill explant culture model can simulate the effect of 30 days of chronic ammonia nitrogen stress in vivo within 6 hours, and successfully establish a gill proliferation model.

[0147] See immunofluorescence assay targeting PCNA. Figure 10PCNA, or Proliferating Cell Nuclear Antigen, is a protein closely related to cell proliferation. Its expression level increases significantly during the DNA synthesis phase (S phase) of the cell cycle, participating in DNA replication, repair, and cell cycle regulation. PCNA is often used as an indicator of cell proliferation activity. In ammonia stress on largemouth bass, gill tissue was collected, and immunofluorescence experiments targeting PCNA revealed that after ammonia stress, the gill tissue of largemouth bass showed significant hyperplasia, losing its original orderly arrangement. The positive signal (PCNA) on the gill tissue of the control group largemouth bass was significantly lower. + PCNA is mainly distributed at the base of the gill filaments, while it is almost absent on the gill lamellae. + Cells. Largemouth bass gill tissue in the ammonia nitrogen stress group showed more positive signals, mainly distributed at the base of the gill filaments and in the interlayer cell clusters. Immunofluorescence staining was performed on paraffin sections of the explants. Based on the PCNA of the gill explants... + Immunofluorescence results showed that, compared with the control group, the ammonia nitrogen stress group had significantly higher rates of undifferentiated cells (PCNA). + The number of gill cells increased significantly, mainly distributed at the base of the gill filaments and in the interlayer cell clusters. Therefore, the gill explant culture model can simulate the effect of 30 days of in vivo chronic ammonia nitrogen stress within 6 hours. Both models showed a significantly enhanced proliferative and dividing capacity of gill tissue cells, thus forming the phenomenon of gill hyperplasia pathology.

[0148] Histological observation revealed that ammonia nitrogen stress induced severe proliferative lesions in the gill tissue and gill explants of largemouth bass, with a significant increase in cells capable of proliferation and division, indicating significant cell proliferation in the gill tissue and explants. To further investigate cell cycle changes under these two modes, flow cytometry was performed. The experimental results are detailed below. Figure 11 The results showed that the in vivo model and the gill explant model exhibited a similar trend: compared to the control group, the ammonia nitrogen stress group had significantly higher DNA content during the DNA synthesis (S) phase. Therefore, the gill explant culture model could simulate the effects of 30 days of chronic ammonia nitrogen stress in vivo within 6 hours. Both models enhanced the DNA synthesis capacity of gill cells and induced cell proliferation.

[0149] Explants / organoids possess self-renewal and self-organization capabilities, while exhibiting organ functions similar to their tissues of origin. They allow for further exploration of real-world interactions between tissue cells while maintaining relative tissue integrity. Furthermore, the isolation and different treatments of tissues from the same animal sample not only conform to the 3R (Replacement, Reduction, Refinement) principles of animal experiments but also largely avoid individual variability in experimental animals, making them a significant in vitro experimental model / in vitro validation model.

[0150] This technical solution utilizes explants ( Ex vivo A gill explant model for largemouth bass was constructed using a culture modality that includes all types of gill cells, potentially mimicking fish gill tissue. Furthermore, this model exhibits consistent tissue structure and molecular response trends with the in vivo model under ammonia nitrogen stress, thus it can be used as an alternative to gill cell culture models to validate the potential molecular mechanisms of in vivo responses.

[0151] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for culturing gill explants of largemouth bass, characterized in that: Largemouth bass gill explants were cultured using an explant culture medium. The explant culture medium was prepared by adding 10-20% fetal bovine serum, 1-2% fish serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B, 1-5 mM D-galactose, 1-5 mM L-alanyl-L-glutamine, 1-5 mM sodium pyruvate, and 10-30 μM melatonin to a basal medium. The basal culture medium was Leibovitz's L-15; the fish serum was SBJ-SE-F001 product provided by BioChannel. After inoculating the gill explants into the explant culture medium, the culture conditions were set as follows: gas environment 4-6% CO2, 20-25% O2, 70-75% N2; culture temperature 20-30℃; and the explant culture medium inoculated with gill explants was intermittently shaken by shaking for 5-15 minutes and then standing for 20-30 minutes; the shaking speed was 3-7 rpm.

2. The method for culturing gill explants of largemouth bass according to claim 1, characterized in that: Fetal bovine serum, fish serum, D-galactose, L-alanyl-L-glutamine, sodium pyruvate, and melatonin were present in explant culture medium at concentrations of 15%, 1%, 5.0 mM, 2.5 mM, 5.0 mM, and 20 μM, respectively.

3. The method for culturing gill explants of largemouth bass according to claim 1, characterized in that: The gill explants consist of 10-15 gill filaments and do not contain gill arches; the gill explants are rinsed sequentially with alcohol, pre-cooled PBS buffer, PBS buffer containing penicillin, streptomycin and amphotericin B, and explant culture medium.

4. A method for culturing gill explants of largemouth bass according to claim 1, characterized in that: After inoculating the gill explants into the explant culture medium, the culture conditions were set as follows: gas environment 5% CO2, 20% O2, 75% N2; culture temperature 25℃; and the explant culture medium containing the gill explants was intermittently shaken with a shaking speed of 5 rpm after 10 minutes of shaking and 20 minutes of resting.

5. The application of the largemouth bass gill explants obtained by the culture method of largemouth bass gill explants according to any one of claims 1-4 in establishing a largemouth bass gill explant model under ammonia nitrogen stress, characterized in that: Largemouth bass gill explants were cultured in a nitrogen-containing explant culture medium. The culture conditions were controlled as follows: gas environment 4-6% CO2, 20-25% O2, 70-75% N2; culture temperature 20-30℃; and the explant culture medium inoculated with gill explants was intermittently shaken with shaking for 5-15 minutes, 3-7 rpm and then allowed to stand for 20-30 minutes. After 4-8 hours of culture, a largemouth bass gill explant model under ammonia nitrogen stress was obtained.

6. An explant culture medium for culturing gill explants of largemouth bass, characterized in that: It is formed by adding 10-20% fetal bovine serum, 1-2% fish serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B, 1-5 mM D-galactose, 1-5 mM L-alanyl-L-glutamine, 1-5 mM sodium pyruvate, and 10-30 μM melatonin to the basal culture medium. The basal culture medium was Leibovitz's L-15; the fish serum was SBJ-SE-F001 product provided by BioChannel.

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