A method for constructing a liver injury model in California bass and its application.

Abstract

This invention provides a method for constructing a liver injury model in largemouth bass and its application, belonging to the field of aquatic pharmaceuticals. The construction method includes the following steps: 1) Intraperitoneal injection of thioacetamide into largemouth bass at a dose of 100-150 mg / kg body weight, once; 2) Obtaining the model after 6-15 days of conventional rearing. This invention enables model creation with a short cycle, low cost, good reproducibility, and simple operation, solving the problem of high animal mortality in existing technologies for creating acute liver injury models.

Description

Technical Field

[0001] This invention relates to the field of aquatic drug research and development technology, specifically to a method for constructing a liver injury model in California bass and its application, for screening products (e.g., aquatic drugs) for the prevention and / or treatment of fish liver diseases. Background Technology

[0002] With increasing fish farming density, deteriorating aquatic environments, overuse of drugs, and imbalanced feed nutrition and harmful substances in feed, fish diseases are becoming increasingly frequent, causing significant losses to fisheries. Multiple factors can lead to fatty liver disease in farmed fish, including feed factors (nutritional imbalance and oxidized oils), mycotoxins (aflatoxin and vomitoxin), harmful drugs (sulfonamides and furans), and deteriorating aquatic environments (excessive ammonia and nitrite levels, oxygen deficiency, and heavy metal poisoning). If farmed fish develop liver disease, mild cases affect growth rate, production potential, and feed conversion efficiency, while also impacting normal immune function and metabolism. Severe cases lead to hepatobiliary syndromes, such as fatty hepatitis, fatty liver, hepatobiliary syndrome, and hepatorenal syndrome. When multiple syndromes occur, it indicates multiple organ failure syndrome in fish, ultimately causing mass mortality.

[0003] Liver and gallbladder problems are currently widespread in aquatic animals, affecting not only grass carp, common carp, yellow catfish, large yellow croaker, tilapia, spotted catfish, and snakehead, but also both juvenile and adult fish. In the aquaculture industry, some farmers, in pursuit of maximum profits, employ improper farming methods and overuse drugs and additives, which can severely damage the livers of aquatic animals.

[0004] The current difficulties in constructing liver injury models include: the choice of drug type and dosage significantly impacts model construction; for example, too low a dosage may result in insignificant liver damage, failing to meet experimental requirements; too high a dosage may cause acute liver failure, leading to mass mortality in experimental animals; and different animal species exhibit varying sensitivities to drugs. Inducing liver injury models with drugs requires a certain period of accumulation, typically involving continuous administration for several days to weeks. However, excessively long induction times may lead to decreased immunity in fish, increasing the risk of secondary infections; conversely, insufficient induction times may prevent the formation of a stable liver injury model. Furthermore, aquatic animals have lower metabolic enzyme activity and weaker detoxification abilities, further complicating the development of liver injury models in largemouth bass.

[0005] To explore and study the pathogenesis and process of liver lesions in farmed fish under experimental conditions, it is necessary to establish a liver injury model in largemouth bass. However, there is currently no method to replicate a large number of controllable largemouth bass liver lesion models with consistent lesion severity within a limited time. Summary of the Invention

[0006] Purpose of the invention

[0007] The purpose of this invention is to provide a method for constructing a liver injury model in California bass and its application. This invention enables model creation with a short cycle time, low cost, good reproducibility, and simple operation, solving the problem of high animal mortality rates in existing technologies for creating acute liver injury models. It can replicate a batch of controllable fish liver lesion samples with consistent lesion severity within a limited time (e.g., on the 3rd day). Through fish liver injury experiments, an experimental model of fish hepatopancreatic injury and its corresponding evaluation index system are established, providing an experimental platform for exploring and studying the occurrence, development mechanisms, and processes of fish liver lesions, and for screening preventive and therapeutic drugs or feed additives.

[0008] Solution

[0009] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0010] In a first aspect, the present invention provides a method for constructing a hepatopancreatic injury model in California bass, comprising the following steps:

[0011] 1) Intraperitoneal injection of thioacetamide (TAA) into California bass, at a dose of 100-150 mg / kg body weight, once.

[0012] 2) It can be obtained in 6 to 15 days with conventional feeding.

[0013] Further, in step 1), thioacetamide is injected in solution form. Optionally, the amount of thioacetamide solution injected is 0.05-0.2 mL, or optionally 0.05-0.1 mL.

[0014] Furthermore, thioacetamide is dissolved in sterile ultrapure water.

[0015] Further, in step 1), select juvenile California bass weighing 15-100g, or preferably juvenile fish weighing 25-30g each.

[0016] Furthermore, a scoring system related to liver injury symptoms is established, and a grading assessment method is developed. Optional grading assessment methods include liver scoring, surface scoring, and abdominal scoring. Optional scoring methods include:

[0017]

[0018] Optionally, the sum of the liver score, body surface score, and abdominal score can be used as a comprehensive liver injury score.

[0019] Furthermore, in step 2), the model is considered successfully constructed when the experimental animal exhibits one of the liver damage symptoms described in the scoring table and the sampled damage rate is ≥80%.

[0020] Furthermore, in step 2), the regular feeding period is 6 to 8 days.

[0021] Furthermore, the constructed model is used to screen products for the prevention and / or treatment of liver diseases in fish; optionally, the fish include one or more of the following: California bass, grass carp, spotted catfish, yellow catfish, carp, large yellow croaker, tilapia, and snakehead.

[0022] Furthermore, in step 2), the feed is conventional feed, fed twice a day, with a daily feed amount of 1-5% of body weight, fed to satiety, and optionally fed continuously for 1-2 weeks, with an optional feeding period of 1 week.

[0023] Furthermore, the feed comprises the following components by weight: 8-12 parts soybean meal, 0.5-1.5 parts wheat gluten, 5-10 parts fermented soybean meal, 15-25 parts domestic defatted fish meal, 25-35 parts super imported steamed fish meal, 10-15 parts imported chicken meal, 1-3 parts plasma protein powder, 1-3 parts seaweed powder, 2-6 parts tapioca starch, 5-10 parts high-gluten flour, 4-8 parts fish oil, 1-3 parts aquatic multivitamins, and 2-4 parts aquatic multiminerals.

[0024] Secondly, a grading method for a California bass liver injury model is provided. The grading method includes liver scoring, body surface scoring, and abdominal scoring. Optional scoring methods include:

[0025]

[0026] Optionally, the sum of the liver score, body surface score, and abdominal score can be used as a comprehensive liver injury score.

[0027] Thirdly, the method described in the first aspect provides an application of the model constructed in screening products for the prevention and / or treatment of fish liver diseases; optionally, the fish include one or more of the following: California bass, grass carp, channel catfish, yellow catfish, carp, large yellow croaker, tilapia, and snakehead.

[0028] Fourthly, a fish liver injury model for screening the prevention and / or treatment of fish liver diseases is provided, the model being constructed by the method described in the first aspect;

[0029] Optional fish species include one or more of the following: California bass, grass carp, spotted catfish, yellow catfish, carp, large yellow croaker, tilapia, and snakehead.

[0030] Beneficial effects

[0031] (1) This invention, through intraperitoneal injection of a certain dose of TAA into California bass, creates a model that not only induces hepatopancreatic injury in fish but also maintains a certain survival rate. Furthermore, it achieves a short cycle, low cost, good reproducibility, and simple operation, solving the problem of high animal mortality rates in existing technologies for creating acute liver injury models.

[0032] (2) This invention induces oxidative damage to the liver by injecting TAA into California bass, thereby achieving a rapid and efficient induction effect of liver damage, while reducing the mortality rate of experimental animals. It constructs an experimental model of liver and pancreas damage in fish and its corresponding evaluation index system, providing an experimental platform for exploring and studying the occurrence, development mechanism and process of liver lesions in fish, and screening preventive drugs or feed additives. Attached Figure Description

[0033] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0034] Figure 1 The liver color changes in different symptoms of liver damage in California bass.

[0035] Figure 2 The change in feeding rate in test groups A to E in test example 1.

[0036] Figure 3 The changes in feeding rate in Examples 1 to 7 of Test Example 2.

[0037] Figure 4 The change in feeding rate in Examples 8 to 13 of Test Example 3. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0040] Due to differences in drug sensitivity and adaptability to different challenge methods among animals, obtaining a large bass liver injury model that has a certain survival rate and meets the basic requirements of a liver injury experimental model presents a great challenge.

[0041] By trying various drugs and methods of attacking the virus, the inventors finally achieved the ability to replicate a large number of controllable California bass liver lesion models with consistent lesion severity within a limited time.

[0042] In the following experiments, the feeding rate = daily food intake per tank / number of experimental animals per tank / average body weight * 100.

[0043] In the following experiment, the mortality rate = number of deaths / initial number * 100.

[0044] Animal grouping: The experimental feed pellets were approximately 5 mm in diameter. 1620 juvenile California bass (25-30 g in weight, purchased from Beijing Shengyuan Fuze Technology Co., Ltd.) were randomly divided into 18 groups, with 3 replicates per group and 30 fish per replicate. The fish were temporarily held for one week before the experiment, and the experiment began when the feeding rate reached 3%. The 18 groups were used for experimental groups A-E and Examples 1-13, respectively.

[0045] The feed used is conventional California bass feed. The following is the composition of the feed ingredients, which can be composed of the following components by weight (by weight of air-dried matter): 8 parts soybean meal, 1.5 parts wheat gluten, 6 parts fermented soybean meal, 17 parts domestic defatted fish meal, 25 parts super imported steam fish meal, 10 parts imported chicken meal, 3 parts plasma protein, 2 parts seaweed powder, 3 parts tapioca starch, 7 parts high-gluten flour, 6 parts fish oil, 2 parts aquatic multivitamins, and 1.5 parts aquatic multiminerals. The feed contains 8% moisture, 10% crude fat, 13% crude ash, and 48% crude protein.

[0046] Challenge and experimental recording methods: After the experiment began, animals were challenged by intraperitoneal injection according to the concentration and dosage of the challenge reagent, once. The control group was injected with physiological saline. After challenge, animals were fed normally twice a day, at 08:30 and 17:30. The feeding amount was adjusted according to the feeding situation. The feeding amount of each group of experimental animals was recorded. The experimental period was 7 days. The number of dead fish in each tank was observed and the mortality rate of each group was recorded. On the 3rd day of the experiment, 3 fish were taken from each tank from the surviving fish. On the 7th day of the experiment, all surviving experimental animals were dissected and sampled. Changes in body surface and liver color were recorded according to Table 1 (liver color changes can be referenced). Figure 1 The water was discharged daily and the water was changed every 2 days, with about 1 / 3 of the water replaced. During the experiment, the water temperature was 25-30℃, ammonia nitrogen concentration <2mg / L, nitrite concentration <0.01mg / L, dissolved oxygen content >5.0mg / L, and pH 7.5-8.0.

[0047] Table 1 Liver Injury Symptom Scores

[0048]

[0049] Test Example 1

[0050] To screen for suitable agents as a model of liver injury in largemouth bass, the inventors conducted extensive research and ultimately selected thioacetamide (TAA) as the inducing agent. Some representative screening experiments are as follows:

[0051] Experimental group A (control): Intraperitoneal injection of physiological saline, dose of 0.1 mL.

[0052] Experimental Group B (TAA Group): The challenge reagent was TAA. The TAA solution was prepared by dissolving the TAA in sterile ultrapure water and injected intraperitoneally. The challenge dosage was 0.1 mL and the challenge concentration was 100 mg TAA / kg fish body weight.

[0053] Experimental group C (CCl4 group): The challenge agent was carbon tetrachloride, which was diluted with olive oil to make a carbon tetrachloride solution. It was injected intraperitoneally, with a challenge concentration of 0.5 mL CCl4 / kg fish body weight and a challenge dosage of 0.1 mL.

[0054] Experimental group D (D-GalN group): The challenge agent was D-galactosamine, which was prepared by dissolving in sterile ultrapure water and injected intraperitoneally. The challenge concentration was 400 mg GalN / kg fish body weight, and the challenge dosage was 0.1 mL.

[0055] Experimental group E (DMN group): The challenge agent was dimethylnitrosamine, which was prepared by dissolving D-galactosamine in sterile ultrapure water and injected intraperitoneally. The challenge concentration was 10 mg DMN / kg fish body weight, and the challenge volume was 0.1 mL.

[0056] The concentrations and dosages of the challenge reagents in experiments B through E above were obtained through multiple experiments and were the most effective data in their respective reagent groups. For the sake of intuitive comparison, only the above experimental groups are listed.

[0057] Observe the changes in feed intake in experimental groups A through E and calculate the feeding rate.

[0058] The changes in feeding rates in experimental groups A through E are as follows: Figure 2 It can be seen that there were significant differences in the changes in feeding rate among the groups 7 days after the challenge. The feeding rate of group A remained at around 2% on the second day after the challenge. The feeding rate of group B gradually increased with the extension of the breeding time. The feeding rate was lower than that of the control group, but higher than that of groups C, D and E. Among them, group C had the lowest feeding rate, which was less than 1% in the later stage.

[0059] Liver injury rate and mortality

[0060] In experimental groups A to E, liver samples were taken by dissection on the 3rd and 7th days after the viral challenge, and the liver condition was recorded. At the same time, the liver was scored according to Table 1, and the percentage of liver damage and mortality rate were calculated.

[0061] The percentage of liver damage is calculated by including any abnormal liver symptoms other than a reddish liver as liver damage.

[0062] Largemouth bass in experimental groups A to E were dissected and sampled on days 3 and 7 after being challenged with the drug. The severity of liver damage at different stages is shown in Table 2. It can be seen that the proportion of liver damage was high in all groups after being challenged with different reagents on days 3 and 7, all above 90%. Group E had the highest proportion of damage, but there was no significant difference between the reagent groups (P>0.05). The mortality rate of each group was observed within 7 days. The mortality rate of groups C, D, and E was significantly higher than that of groups A and B (P<0.05), and the mortality rate was above 60%. This indicates that carbon tetrachloride, D-galactosamine, and dimethylnitrosamine are not suitable as experimental models for liver damage repair in largemouth bass.

[0063] Table 2. Percentage of liver injury and mortality rate at each stage in experimental groups A through E.

[0064]

[0065]

[0066] Note: Different letters in the subheadings in Table 2 represent significant differences between groups (P < 0.05), while the same letter represents no significant difference.

[0067] Liver score

[0068] The liver color, body surface, and abdominal cavity changes of the experimental animals were scored at various time points, and the results are shown in Table 3. It can be seen that the liver samples taken from the experimental animals in Group A on the 3rd and 7th days after the challenge were normal with no abnormal changes, which was significantly lower than that of other experimental groups (P<0.05). All other experimental groups showed obvious liver damage symptoms after the challenge. Among them, the disease activity index score of Group B was significantly lower than that of Groups C, D, and E on the 3rd and 7th days (P<0.05). There was no significant difference among the groups in Groups C, D, and E on the 3rd day (P>0.05). On the 7th day, there was no significant difference between Groups D and C (P>0.05), but Group D was significantly lower than Group E (P<0.05).

[0069] Table 3 Liver Injury Disease Activity Index Scores

[0070] Grouping Day 3 Disease Activity Index Score Day 7 Disease Activity Index Score A (Control) <![CDATA[0.00±0.00 a ]]> <![CDATA[0.00±0.00 a ]]> B <![CDATA[3.27±0.20 b ]]> <![CDATA[3.36±0.22 b ]]> C <![CDATA[5.37±0.12 c ]]> <![CDATA[5.08±0.25 cd ]]> D <![CDATA[5.39±0.12 c ]]> <![CDATA[4.71±0.12 c ]]> E <![CDATA[5.07±0.31 c ]]> <![CDATA[5.19±0.18 d ]]>

[0071] Note: Different letters in the subheadings in Table 3 represent significant differences between groups (P < 0.05), while the same letter represents no significant difference.

[0072] The comparative experiments above show that under different reagent challenge conditions, all reagent groups can show obvious liver damage, but the effects of liver damage vary significantly. Among them, the CCl4 group, D-GalN group, and DMN group have more severe liver damage symptoms and higher mortality rates, making them unsuitable for subsequent experiments. In contrast, the TAA group has obvious liver damage symptoms and a significantly lower mortality rate, making it an effective platform for subsequent research on liver damage.

[0073] This invention also investigated the effects of different doses and concentrations of the antiviral agent on a liver injury model in largemouth bass, as detailed below:

[0074] Example 1

[0075] The challenge agent was TAA (dissolved in ultrapure water), administered via intraperitoneal injection. The amount of challenge agent used was 0.05 mL, and the concentration of challenge agent was 300 mg TAA / kg fish body weight.

[0076] Example 2

[0077] In this embodiment, the TAA challenge concentration is the same as in Example 1, and the challenge dosage is 0.1 mL.

[0078] Example 3

[0079] In this embodiment, the TAA challenge concentration is the same as in Example 1, and the challenge volume is 0.2 mL.

[0080] Example 4

[0081] In this embodiment, the TAA challenge concentration is the same as in Example 1, and the challenge dosage is 0.3 mL.

[0082] Example 5

[0083] In this embodiment, the TAA challenge concentration is the same as in Example 1, and the challenge volume is 0.4 mL.

[0084] Example 6

[0085] In this embodiment, the TAA challenge concentration is the same as in Example 1, and the challenge dosage is 0.5 mL.

[0086] Example 7 (Control)

[0087] This example serves as the control group, with intraperitoneal injection of physiological saline at a dose of 0.3 mL.

[0088] Example 8

[0089] The challenge agent was TAA (thioacetamide), administered via intraperitoneal injection. The dosage was 0.1 mL, and the concentration was 50 mg TAA / kg fish body weight.

[0090] Example 9

[0091] The challenge agent was TAA (thioacetamide), administered via intraperitoneal injection at a dose of 0.1 mL and a concentration of 100 mg TAA / kg fish body weight.

[0092] Example 10

[0093] The challenge agent was TAA (thioacetamide), administered via intraperitoneal injection. The dosage was 0.1 mL, and the concentration was 150 mg TAA / kg fish body weight.

[0094] Example 11

[0095] The challenge agent was TAA (thioacetamide), administered via intraperitoneal injection at a dose of 0.1 mL and a concentration of 200 mg TAA / kg fish body weight.

[0096] Example 12

[0097] The challenge agent was TAA (thioacetamide), administered via intraperitoneal injection at a dose of 0.1 mL and a concentration of 250 mg TAA / kg fish body weight.

[0098] Example 13 (Control)

[0099] This example serves as the control group, which receives an intraperitoneal injection of 0.1 mL of physiological saline.

[0100] Test Example 2

[0101] The changes in feed were observed in Examples 1 to 7. Samples were taken from the liver on the 3rd and 7th day after the challenge in each example. The liver condition was recorded, and the liver damage rate and mortality rate were calculated according to Table 1.

[0102] The percentage of liver damage is calculated by including any abnormal liver symptoms other than a reddish liver as liver damage.

[0103] The changes in feeding rate in Examples 1-7 are as follows: Figure 3 It can be seen that after the challenge experiment, the feeding rate of Example 7, which served as the control, showed an upward trend, indicating that the injection of physiological saline had no effect on the food intake of largemouth bass. Examples 1-6, which served as the experimental groups, showed similar trends, with the feeding rate showing a significant downward trend starting on the 3rd day. The feeding rates of Examples 1 and 2 were higher than those of the other examples, demonstrating that at the same injection concentration, the injection dose can significantly affect the food intake of experimental animals.

[0104] The percentage of liver injury and mortality rates in Examples 1–7 are shown in Table 4. The results indicate that:

[0105] On the 3rd day after the challenge, the livers of the experimental animals in Example 7, which served as the control, were all healthy, significantly lower than those in other examples (P<0.05). The proportion of liver damage in Examples 1-6 was relatively high, with no significant difference among the examples (P>0.05), except that in Example 1, the liver damage was relatively low.

[0106] On day 7 post-infection, samples were taken from all experimental animals. The liver injury rate in Example 7, serving as a control, remained at 0%, significantly lower than in other examples (P<0.05). Specifically, the liver injury rate in Example 1 was 85.96%, significantly lower than in Examples 3-6 (P<0.05). Mortality and liver injury showed similar changes across examples. The mortality rate in Example 7, serving as a control, was 0%, significantly lower than in other examples (P<0.05). The mortality rates in Examples 1 and 2 were 45.33% and 44%, respectively, significantly lower than in Examples 3-6 (P<0.05). There were no significant differences in Examples 3-5 (P>0.05), but they were significantly lower than in Example 6 (P<0.05). There were no significant differences in Examples 4-6 (P>0.05).

[0107] Table 4. Percentage of Liver Injury and Mortality at Each Stage

[0108] Grouping Percentage of liver damage on day 3 Percentage of liver damage on day 7 mortality rate% Example 1 <![CDATA[86.67±32.09 b ]]> <![CDATA[85.96±4.83 b ]]> <![CDATA[45.33±10.07 b ]]> Example 2 <![CDATA[93.33±11.55 b ]]> <![CDATA[93.17±6.67 bc ]]> <![CDATA[44.00±4.00 b ]]> Example 3 <![CDATA[100.00±0.00 b ]]> <![CDATA[93.17±4.44 c ]]> <![CDATA[53.33±6.11 bc ]]> Example 4 <![CDATA[100.00±0.00 b ]]> <![CDATA[95.24±4.76 c ]]> <![CDATA[62.67±8.33 cd ]]> Example 5 <![CDATA[100.00±0.00 b ]]> <![CDATA[100.00±0.00 c ]]> <![CDATA[64.00±4.00 cd ]]> Example 6 <![CDATA[100.00±0.00 b ]]> <![CDATA[100.00±0.00 c ]]> <![CDATA[69.33±2.31 d ]]> Example 7 (Control) <![CDATA[0.00±0.00 a ]]> <![CDATA[0.00±0.00 a ]]> <![CDATA[0.00±0.00 a ]]>

[0109] Note: Different letters in the subheadings in Table 4 represent significant differences between groups (P < 0.05), while the same letter represents no significant difference.

[0110] In Examples 1-7, California bass were dissected and sampled on days 3 and 7 after being challenged with the virus. Table 5 shows the severity of liver damage at different stages. As a control, Example 7 had liver disease activity index scores of 0.07 and 0 on days 3 and 7, respectively, significantly lower than the other examples (P<0.05). Examples 1-6 showed the same trend in liver disease activity index scores on days 3 and 7, all increasing with increasing virus dosage. On day 3, Example 1 was significantly lower than Examples 3-6 (P<0.05), but not significantly different from Example 2 (P>0.05). Example 2 showed no significant difference from Examples 3 and 4 (P>0.05), but significantly lower than Examples 5 and 6 (P<0.05). On day 7 of the experiment, there was no significant difference between Example 1 and Example 2 (P>0.05), but the difference was significantly lower than that between Examples 3-6 (P<0.05). There was no significant difference between Examples 3-5 (P>0.05), but the difference was significantly lower than that between Examples 6 (P<0.05).

[0111] Table 5 Liver Injury Disease Activity Index Scores in Examples 1-7

[0112] Grouping Day 3 Disease Activity Index Score Day 7 Disease Activity Index Score Example 1 <![CDATA[3.07±0.70 b ]]> <![CDATA[3.41±0.42 b ]]> Example 2 <![CDATA[4.00±0.40 bc ]]> <![CDATA[3.64±0.59 b ]]> Example 3 <![CDATA[4.40±1.31 cd ]]> <![CDATA[4.88±0.14 c ]]> Example 4 <![CDATA[4.67±0.64 cd ]]> <![CDATA[4.70±0.15 c ]]> Example 5 <![CDATA[5.40±0.87 d ]]> <![CDATA[5.08±0.41 c ]]> Example 6 <![CDATA[5.40±0.35 d ]]> <![CDATA[5.68±0.17 d ]]> Example 7 (Control) <![CDATA[0.07±0.12 a ]]> <![CDATA[0.00±0.00 a ]]>

[0113] Note: In Table 5, different letters on the shoulder labels represent significant differences between groups (P < 0.05), while the same letter represents no significant difference.

[0114] The above experimental results show that, under the condition of 300 mg / kg injection drug concentration, the injection dose has a significant effect on the feeding effect, liver damage and mortality of largemouth bass. In this experiment, a liver lesion sample of fish with controllable mortality and basically consistent lesion degree can be established when the injection dose is 0.05-0.1 ml.

[0115] Test Example 3

[0116] Examples 8-13 were challenge experiments conducted after selecting a dose of 0.1 mL. The challenge concentration was studied, and the changes in feed in Examples 8-13 were observed. Samples were taken from the liver on the 3rd and 7th day after challenge in each example. The liver status was recorded, and scores were calculated according to Table 1. The percentage of liver damage and the mortality rate were also calculated.

[0117] The percentage of liver damage is calculated by including any abnormal liver symptoms other than a reddish liver as liver damage.

[0118] The changes in feeding rate in Examples 8-13 are as follows: Figure 4 It can be seen that after the experiment, except for Example 13 which served as a control, the feeding rates of the other examples showed similar trends, with a sharp drop in feeding rate on the third day of rearing. The feeding rates of Examples 8, 9, and 10 were significantly higher than those of Examples 11 and 12, with Examples 8 and 9 showing an upward trend on the sixth day of rearing.

[0119] Table 6 shows the percentage of liver injury and mortality rates in Examples 8–13. The results indicate that:

[0120] After the experimental challenge, Example 13, serving as a control, showed similar results to Example 7, which also served as a control, after injection of saline. The liver remained largely healthy. Furthermore, the proportion of liver damage on days 3 and 7 after the challenge was significantly lower than that of Examples 8-12 (P<0.05). Under the same challenge dosage, the proportion of liver damage in Examples 8-12 increased with increasing challenge reagent concentration. On day 3, Example 8 showed a significantly lower proportion than Examples 9-12 (P<0.05), while other examples showed no significant difference (P>0.05). On day 7, there was no significant difference among Examples 8-10 (P>0.05), and the proportions were significantly lower than those in Examples 11 and 12 (P<0.05). The mortality rate of each example increased with increasing challenge reagent concentration. Specifically, the mortality rates of Examples 9, 10, and 13 showed no significant difference (P>0.05), and were significantly lower than those in Examples 10-12 (P<0.05).

[0121] Table 6. Percentage of liver injury and mortality rate at each stage of Examples 8-13.

[0122] Experimental grouping Percentage of liver damage on day 3 Percentage of liver damage on day 7 mortality rate% Example 8 <![CDATA[66.67±11.55 b ]]> <![CDATA[79.67±4.51 b ]]> <![CDATA[1.33±2.31 a ]]> Example 9 <![CDATA[93.33±11.55 c ]]> <![CDATA[94.61±2.26 bc ]]> <![CDATA[1.33±2.31 a ]]> Example 10 <![CDATA[93.33±11.55 c ]]> <![CDATA[90.76±4.97 b ]]> <![CDATA[12.00±4.00 b ]]> Example 11 <![CDATA[100.00±0.00 c ]]> <![CDATA[98.33±2.89 c ]]> <![CDATA[26.67±6.11 c ]]> Example 12 <![CDATA[100.00±0.00 c ]]> <![CDATA[100.00±0.00 c ]]> <![CDATA[60.00±10.58 d ]]> Example 13 (Control) <![CDATA[100.00±0.02 a ]]> <![CDATA[0.00±0.00 a ]]> <![CDATA[0.00±0.00 a ]]>

[0123] Note: Different letters in the subheadings in Table 6 represent significant differences between groups (P < 0.05), while the same letter represents no significant difference.

[0124] Although the dosage and concentration of the virus were the same in experimental group B and Example 9, the proportion of liver damage and the mortality rate differed slightly because they were different experimental batches. However, the differences were not significant, which also indicates that the model construction method of the present invention has good reproducibility.

[0125] In Examples 8-13, California bass were dissected and sampled on days 3 and 7 after viral challenge. Table 7 shows the severity of liver damage at different stages. It can be seen that the disease activity index score gradually increased with increasing viral concentration in each example. Example 13 showed no liver damage, with a disease activity index score of 0. Example 8 showed mild liver damage, with the main symptom being uneven reddish liver color; its liver activity index score was significantly lower than that of Examples 9-12 (P<0.05). In Example 9, the main symptoms of liver damage in the experimental animals were yellow liver, mottled liver, and variegated liver; the symptoms were uniform, and its disease activity index score was significantly lower than that of Examples 10-12 (P<0.05). In Examples 10-12, the main symptoms of liver damage in the experimental animals were yellow liver, variegated liver, yellow body, and ascites; the symptoms were relatively severe. There was no significant difference in the disease activity index scores between Examples 10 and 11 (P>0.05), and both were significantly lower than that of Example 12 (P<0.05).

[0126] Table 7. Liver Injury Disease Activity Index Score

[0127]

[0128]

[0129] Note: Different letters in the subscripts in Table 7 represent significant differences between groups (P < 0.05), while the same letter represents no significant difference.

[0130] The above experimental results indicate that, under conditions of intraperitoneal injection of 0.1 mL of TAA at concentrations of 50-150 mg / kg, the feeding effect of largemouth bass was relatively good, showing an upward trend. At injection doses of 100-150 mg / kg, the proportion of liver damage was over 90%, and the mortality rate was below 20%. Liver damage mainly manifested as jaundice, and a damage rate ≥80% was used as the marker for successful model establishment. This experiment can establish fish liver lesion samples with controllable mortality and basically consistent lesion severity at injection doses of 0.05-0.1 mL.

[0131] The liver injury induced by TAA used in this invention is reversible to some extent, especially with low-dose or short-term administration.

[0132] Based on the above experimental results, when the TAA challenge dosage is 0.5-0.2 mL and the challenge concentration is 50-150 mg / kg, significant liver damage can be caused in largemouth bass. The main symptoms are yellow liver, yellowing of the liver, and mottled liver. Feed intake gradually recovers, and mortality is controllable. This can serve as an experimental platform for exploring and studying the occurrence, development mechanism and process of liver lesions in fish, and for screening preventive and therapeutic drugs or feed additives.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a hepatopancreatic injury model in California bass, characterized in that, The hepatopancreatic injury model is an acute liver injury model, and includes the following steps: 1) Intraperitoneal injection of thioacetamide into California bass, the injection dose is 100-150 mg / kg body weight, one injection; the amount of thioacetamide solution injected is 0.05-0.1 mL; California bass should be selected from juvenile fish weighing 15-100g / fish. 2) It takes 6-8 days of normal feeding to obtain; A scoring system was established to assess liver injury symptoms, and a grading system was developed. This system includes liver score, surface score, and abdominal score. The scoring methods include: The sum of the liver score, body surface score, and abdominal score is used as the comprehensive liver injury score. The model is considered successfully constructed when the experimental animal exhibits one of the liver damage symptoms described in the scoring table and the sampled damage rate is ≥80%.

2. The construction method according to claim 1, characterized in that, In step 1), thioacetamide is injected in solution form.

3. The construction method according to claim 1, characterized in that, In step 1), thioacetamide is dissolved in sterile ultrapure water.

4. The construction method according to claim 1, characterized in that, In step 1), select juvenile California bass weighing 25-30g each.

5. The construction method according to claim 1, characterized in that, In step 2), the model is constructed to screen for products that prevent and / or treat liver disease in fish, specifically California bass.

6. The construction method according to claim 1, characterized in that, In step 2), the feed is conventional feed, fed twice a day, with a daily feed amount of 1-5% of body weight, fed to satiety, and fed continuously for 1-2 weeks.

7. The construction method according to any one of claims 1 to 6, characterized in that, The feed consists of the following components by weight: 8-12 parts soybean meal, 0.5-1.5 parts wheat gluten, 5-10 parts fermented soybean meal, 15-25 parts domestic defatted fish meal, 25-35 parts super imported steamed fish meal, 10-15 parts imported chicken meal, 1-3 parts plasma protein powder, 1-3 parts seaweed powder, 2-6 parts tapioca starch, 5-10 parts high-gluten flour, 4-8 parts fish oil, 1-3 parts aquatic multivitamins, and 2-4 parts aquatic multiminerals.

8. A grading and assessment method for a hepatopancreatic injury model in California bass, characterized in that, The grading assessment methods include liver scoring, body surface scoring, and abdominal scoring. The scoring methods include: The liver score, body surface score, and abdominal score are summed to form a comprehensive liver injury score.

9. The use of a model constructed by the method of any one of claims 1 to 7 in screening products for the prevention and / or treatment of fish liver diseases; in, Fish include one or more of the following: California bass, grass carp, spotted catfish, yellow catfish, carp, large yellow croaker, tilapia, and snakehead.

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