River crab hepatopancreatic necrosis syndrome model, construction method and application

Abstract

The invention provides a river crab hepatopancreatic necrosis syndrome model, a construction method and application, and belongs to the technical field of aquaculture. According to the invention, aflatoxin B1 (AFB1) is utilized to construct a river crab hepatopancreatic necrosis syndrome model, and river crabs treated by the AFB1 have pathological characteristics such as severe hepatopancreatic atrophy, erosion, necrosis, color lightening, hepatopancreatic index reduction, body cavity effusion and the like, and are highly matched with typical symptoms of the river crab hepatopancreatic necrosis syndrome (commonly known as' Shrimp disease '). According to the river crab hepatopancreatic necrosis syndrome model, the blank of risk assessment of the river crab hepatopancreatic necrosis syndrome is filled, and theoretical support can be provided for multi-etiological mechanism analysis and early diagnosis marker screening of the river crab hepatopancreatic necrosis syndrome; a standard reference model is provided for development of drugs for preventing or treating the hepatopancreatic necrosis syndrome of the river crabs, and the method has important application value for promoting healthy culture of aquatic products.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and in particular to a river crab hepatopancreatic necrosis syndrome model, a construction method and an application thereof. Background Art

[0002] The Chinese mitten crab (Eriocheir sinensis) is an important economic aquaculture species in my country. Its hepatopancreas, which plays a crucial role in digestion, metabolism, and immune regulation, is crucial for overall health. However, in recent years, frequent cases of hepatopancreatic necrosis syndrome (HPNS), commonly known as "water-shrinking" disease, have severely impacted crab production and quality, and continue to threaten the stability of the crab aquaculture industry.

[0003] The onset of "water-shrunken crab" disease may be linked to a variety of factors, including drug abuse, environmental stress such as pH, metabolic imbalances, and long-term feeding of nutritionally unbalanced or bio-contaminated feeds. For example, long-term exposure to the pesticide deltamethrin can significantly damage the hepatopancreas, leading to thickening of the basement membrane, epithelial cell enlargement, and increased oxidative stress. Microcystins trigger apoptosis through oxidative stress, but the pathogenic mechanism of feed-derived toxins remains unclear. The hepatopancreas of diseased crabs exhibits abnormal coloration, such as pale yellow or white, and exhibits liquefaction and necrosis of the tissue structure. The mortality rate can reach over 50%, severely hindering the sustainable development of river crab aquaculture.

[0004] Current research on water-shriveled crab disease primarily involves introducing products that prevent or treat the disease under conventional rearing conditions, and then evaluating the reduction in the incidence of the disease. However, because the occurrence of water-shriveled crab disease is related to multiple factors and the conditions under which it occurs, the effectiveness of these methods in preventing or treating the disease is also affected. There is a lack of a standardized water-shriveled crab disease model to evaluate the effectiveness of products that prevent or treat the disease.

[0005] Therefore, constructing a standard river crab hepatopancreatic necrosis syndrome model can not only fill the gap in the risk assessment of river crab hepatopancreatic necrosis syndrome, but also provide theoretical support for the analysis of the multi-causal mechanisms of the disease and the screening of early diagnostic markers, which has important application value for promoting healthy aquaculture. Summary of the Invention

[0006] The purpose of the present invention is to provide a river crab hepatopancreatic necrosis syndrome model, construction method and application, and successfully construct a river crab hepatopancreatic necrosis syndrome model. The pathological characteristics of the river crab hepatopancreas in this model, such as white erosion, lighter color, decreased hepatopancreatic index and body cavity fluid accumulation, are highly consistent with the typical symptoms of river crab "water-shrinking disease".

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for constructing a crab hepatopancreatic necrosis syndrome model, comprising the following steps:

[0009] (1) Preparation of feed: prepare a basic feed and add aflatoxin B1 to the basic feed, mix well to obtain AFB1 feed;

[0010] (2) The crabs were raised in an aerated tap water environment and fed with the AFB1 feed of step (1) to obtain a crab hepatopancreatic necrosis syndrome model.

[0011] Preferably, in step (1), the concentration of AFB1 in the AFB1 feed is 25 to 3200 μg / kg.

[0012] Preferably, in step (1), the basic feed includes the following nutritional components:

[0013] Crude protein 40.0%, crude fat 3.5%, crude fiber 8.0%, crude ash 16.0%, lysine 2.0%, total phosphorus 0.9%, moisture 12.0%.

[0014] Preferably, in step (2), the conditions of the aerated tap water environment are: water temperature is maintained at 20-30°C, dissolved oxygen content is 5-8 mg / L, and pH is 7.2-8.5.

[0015] The present invention also provides a river crab hepatopancreatic necrosis syndrome model constructed by the above-mentioned river crab hepatopancreatic necrosis syndrome model construction method.

[0016] The present invention also provides an application of a river crab hepatopancreatic necrosis syndrome model in the research of river crab hepatopancreatic necrosis syndrome.

[0017] Preferably, the research includes developing or screening products that can treat or prevent river crab hepatopancreatic necrosis syndrome, analyzing the multiple etiological mechanisms of river crab hepatopancreatic necrosis syndrome, or screening diagnostic markers for river crab hepatopancreatic necrosis syndrome.

[0018] The beneficial effects of the present invention compared with the prior art are:

[0019] (1) The present invention uses aflatoxin B1 (AFB1) to construct a model of hepatopancreatic necrosis syndrome in river crabs. The hepatopancreatic index of river crabs treated with AFB1 significantly decreased. In terms of appearance, the color of the river crabs' hepatopancreas changed significantly. As the concentration of AFB1 in the feed increased, the river crabs' hepatopancreas changed from normal orange-yellow to orange-red, and then gradually to grayish white. The hepatopancreas was severely atrophied, eroded, and necrotic, and the color became lighter. Symptoms similar to the "water-shrunken" disease that occurs in production appeared. The degree of necrosis of the hepatopancreas was aggravated by the deformation of the stellate structure of the hepatic ducts, obvious damage to the undifferentiated cells at the edge, rupture of the B cell apical vesicles, and separation of the outer membrane from the epithelial cells. After analyzing the color changes of the hepatopancreas of Chinese mitten crabs using the International Commission on Illumination's L*a*b* color system, it was found that the L* value of the hepatopancreas increased significantly. Both the a* value and the b* value showed a trend of first decreasing and then increasing with the increase of the AFB1 addition level in the feed, confirming that the color changes of the hepatopancreas of Chinese mitten crabs were consistent with those observed with the naked eye. At the same time, AFB1 treatment significantly inhibited the growth performance, nonspecific immune ability and antioxidant capacity of river crabs, increased the accumulation level in the hepatopancreas, muscle and gill tissues of river crabs, and the levels of hepatopancreatic damage markers such as alanine aminotransferase and aspartate aminotransferase in the hepatopancreas, seriously damaged the tissue structure of the hepatopancreas, intestines and gills, and caused serious body damage to the Chinese mitten crab. These AFB1-induced pathological characteristics of river crabs, such as whitening and erosion of the hepatopancreas, decreased hepatopancreatic index and body cavity accumulation, are highly consistent with the typical symptoms of river crab hepatopancreatic necrosis syndrome (commonly known as "water-deficient crab disease").

[0020] (2) The present invention constructs a standard model of hepatopancreatic necrosis syndrome in river crabs, filling the gap in the risk assessment of hepatopancreatic necrosis syndrome in river crabs. It can also provide theoretical support for the analysis of the multi-cause mechanism of hepatopancreatic necrosis syndrome in river crabs and the screening of early diagnostic markers. It provides a standard reference model for the development of drugs for the prevention or treatment of hepatopancreatic necrosis syndrome in river crabs, and has important application value for promoting healthy aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a graph showing the effect of adding different levels of AFB1 on the hepatopancreas morphology in Example 1 of the present invention;

[0023] Figure 2This is a graph showing the effect of adding different levels of AFB1 on the antioxidant capacity of Chinese mitten crab serum in Example 1 of the present invention (n=6), wherein A is SOD (superoxide dismutase); B is CAT (catalase); C is GSH-Px (glutathione peroxidase); D is GSH (glutathione); E is T-AOC (total antioxidant capacity); F is MDA (malondialdehyde); and G is GST (glutathione-S-transferase);

[0024] Figure 3 This is a graph showing the effect of adding different levels of AFB1 on the antioxidant capacity of the hepatopancreas of Chinese mitten crab in Example 1 of the present invention (n=6), wherein A is SOD (superoxide dismutase); B is CAT (catalase); C is GSH-Px (glutathione peroxidase); D is GSH (glutathione); E is T-AOC (total antioxidant capacity); F is MDA (malondialdehyde); G is GST (glutathione-S-transferase); H is AST (aspartate aminotransferase); and I is ALT (alanine aminotransferase).

[0025] Figure 4 This is a graph showing the effect of adding different levels of AFB1 on the AFB1 content in different parts of the Chinese velvet claw in Example 1 of the present invention, where A is the hepatopancreas, B is the gill, and C is the muscle (n=3);

[0026] Figure 5 Figure 1 shows the effect of different levels of AFB1 added to the hepatopancreas, intestine, and gill tissue structures in Example 1 of the present invention (n=6). Lu represents the lumen of the hepatic duct / intestinal cavity; B represents B cells; S represents the adventitia; Sb represents the striated border; Ch represents the stratum corneum; Ep represents the epithelial cell layer; Mf represents the circular muscle layer; Lb represents the longitudinal muscle bundle; Ga represents the gill axis; Gl represents the gill filament; H represents the gill cavity; and P represents the columnar cell. (Scale bar = 200 μm)

[0027] Figure 6 This is a diagram showing the degree of damage to different tissues of Eriocheir sinensis caused by adding different levels of AFB1 in Example 1 of the present invention, where A represents the damage rate of the hepatopancreatic ductules, B represents the gill axis, and C represents the gill filament thickness. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1

[0034] Example 1 of the present invention provides a method for constructing a crab hepatopancreatic necrosis syndrome model. The specific steps are as follows:

[0035] 1. Experimental Methods

[0036] (1) Feed preparation

[0037] The experiment used the Chinese mitten crab compound feed produced by Shanghai Yixin Feed Development Co., Ltd. as the basic feed. The basic nutritional components of the feed were guaranteed to be 40.0% crude protein, 3.5% crude fat, 8.0% crude fiber, 16.0% crude ash, 2.0% lysine, 0.9% total phosphorus, and 12.0% moisture.

[0038] Six experimental feeds were prepared according to the concentration of AFB1 in feed at 0, 5, 25, 125, 625, and 3125 μg / kg, respectively recorded as: T0, T5, T 25 、T 125 、T 625 、T 3125The added AFB1 standard was purchased from Shanghai Boer Chemical Reagent Co., Ltd. The standard was first dissolved in 70% methanol and water to a stock solution concentration of 500 μg / mL. The standard was then diluted with water to 5% of the feed mass, added to the feed, and mixed thoroughly. The solution was then dried in an electric blast oven at 55°C for 24 hours to essentially evaporate the trace amount of methanol, and then stored in a refrigerator at -20°C. Using an enzyme-linked immunosorbent assay (ELISA), the actual AFB1 content of the feed in each group was: 0, 3, 27, 121, 619, and 3139 μg / kg.

[0039] (2) Grouping and feeding management

[0040] After two weeks of rearing, 270 experimental crabs weighing 20.73 ± 0.29 g were randomly assigned to 12 white rectangular plastic boxes (93 cm × 60 cm × 25 cm, 100 L volume). Each crab was housed individually in a polyethylene plastic box measuring 21 cm × 14 cm × 7 cm (1500 mL volume). Six groups were set up, each with three replicates, and each replicate contained 15 crabs. The culture period lasted eight weeks.

[0041] During the experiment, the crabs were fed a daily feed intake of 3%-5% of their body weight, twice daily at 8:00 AM and 6:00 PM (40% in the morning and 60% in the afternoon). Feed intake, mortality, and water quality were recorded daily. The next morning, residual feed and dead crabs were removed and the water was replaced with aerated freshwater, which accounted for 50% of the total aquaculture water supply. The water was continuously oxygenated. During the experiment, the water temperature was maintained at 22.06±0.30°C, the dissolved oxygen content was 7.70±0.20 mg / L, and the pH was 7.3-7.6.

[0042] (3) Sample collection

[0043] After 8 weeks of the experiment, the crabs were counted in groups, their bodies were wiped dry and weighed, and the length, width, and thickness of the carapace were measured. After the crabs were anesthetized in an ice bath, hemolymph was extracted from the joints of their walking legs. Part of it was stored in a 4°C refrigerator for serum collection. The other part was diluted with twice the volume of anticoagulant solution (ACD) pre-cooled at 4°C to prepare anticoagulated blood.

[0044] Secondly, the complete hepatopancreas was isolated and weighed to calculate the hepatopancreas index. The hepatopancreas and carapace were placed in a culture dish (90 mm in diameter) and the color of the hepatopancreas and carapace heart was measured using a Trixon-R18 colorimeter. Then, the gills, hepatopancreas, leg muscles, and intestines were rinsed in saline and placed in a 5 mL centrifuge tube filled with Davidson's fixative for fixation. After 24 hours, the fixative in the tube was replaced with 90% ethanol solution for paraffin section preparation. The remaining gills, hepatopancreas, and muscles were placed in a 5 mL centrifuge tube, snap-frozen in liquid nitrogen, and stored in a -80°C freezer for the determination of AFB1 residue and antioxidant enzyme activity.

[0045] (4) Growth index calculation formula

[0046] Carapace thickness-to-body weight ratio (CT / BW, μm / g) = carapace center thickness / crab body weight

[0047] Weight gain rate (WG,%) = (final weight - initial weight) / initial weight × 100%

[0048] Specific growth rate (SGR, % / d) = [Ln (final mass) - Ln (initial mass)] / number of culture days × 100%

[0049] Fatness (CF, g / cm3) = (crab body mass / carapace length cubed) × 100

[0050] Hepatopancreatic index (HSI,%) = hepatopancreatic mass / crab body mass × 100%

[0051] Survival rate (SR, %) = (final number of crabs / initial number of crabs) × 100%

[0052] After 8 weeks of feeding with different levels of AFB1, the final body weight, weight gain rate, specific growth rate, carapace thickness-body weight ratio, survival rate, hepatopancreas index and fatness of Chinese mitten crabs showed a downward trend with the increase of AFB1 supplementation level. 25 The final body weight, weight gain rate, specific growth rate, survival rate, hepatopancreas index and fatness of each group were significantly reduced (p < 0.05) in a concentration-dependent manner; 625 The carapace thickness-body weight ratio of each group was significantly reduced (p<0.05).

[0053] Table 1 Effects of different levels of AFB1 in feed on the growth performance of Chinese mitten crab

[0054]

[0055]

[0056] (5) Carapace and hepatopancreas color

[0057] The color of the crab carapace and hepatopancreas was measured using a Trix-R18 colorimeter. The color of the crab was expressed using the International Commission on Illumination (CIE 1976) L*a*b* system. The measurement parameters included brightness (L* value), red-green value (a* value), and yellow-blue value (b* value). The incidence of "water bug" disease in each group was statistically analyzed. The results are as follows: Figure 1 As shown in Tables 2 and 3.

[0058] Figure 1 The results showed that the addition of AFB1 to feed changed the color of the carapace and hepatopancreas of Chinese mitten crab, and the changes became more obvious with the increase of the addition amount. The shell color gradually changed from normal blue to grayish white. The hepatopancreas showed obvious lightening and atrophy, and the color of T0, T5, T 25 、T 125 、T 625 、T 3125 The incidence rates of hepatopancreatic necrosis syndrome in the three groups were 2.50±2.50%, 2.50±2.50%, 15.00±2.50%, 26.67±2.89%, 40.00±2.50% and 45.83±1.44%, respectively. The addition of AFB1 significantly increased the incidence rate of hepatopancreatic necrosis syndrome in river crabs (p<0.05).

[0059] The carapace color index measured by the color difference tool showed that compared with the control group T0, the carapace L* value of the crabs in the AFB1 stress group increased significantly (p<0.05); the a* value showed a trend of first increasing and then decreasing with the increase of AFB1 addition level in the feed, especially in T5 and T 25 、T 125 The carapace a* value of the group was significantly higher than that of the control group (p<0.05); the carapace b* value increased with the increase of AFB1 supplementation level in the feed, among which T 25 、T 125 、T 625 、T 3125 The carapace b* value of the group was significantly higher than that of the control group (p<0.05).

[0060] After opening the carapace, it was found that the color of the hepatopancreas in the normal group was orange-yellow, and it turned orange-red with the increase of AFB1 addition level (T 25 ) and then turned milky white (T 125 –T 3125 ), and from T 125 The hepatopancreas is pasty and watery, and the hepatopancreas is obviously atrophied. 25 The hepatopancreatic index of the T0 group was significantly decreased (p < 0.05). 125 、T 625 、T 3125The L* value of hepatopancreas in the group was significantly increased (p<0.05); the a* value showed a trend of first decreasing and then increasing with the increase of AFB1 supplementation level in the feed, among which T5, T 25 、T 125 The hepatopancreas a* value of group T was significantly decreased (p<0.05). 625 、T 3125 The b* value showed a trend of decreasing first and then increasing with the increase of AFB1 supplementation level in feed. 125 、T 625 、T 3125 The hepatopancreas b* value of group A was significantly increased (p<0.05), T5, T 25 The b* value of hepatopancreas in group A was not significant (p>0.05). The changes in these values ​​were consistent with the results observed with the naked eye.

[0061] Table 2 Incidence of “Water Tripod” disease in each group

[0062]

[0063] Note: p is less than 0.05 level

[0064] Table 3 Effects of different levels of AFB1 in feed on the color of carapace and hepatopancreas (n=6)

[0065]

[0066]

[0067] (6) Determination of biochemical indicators in serum and hepatopancreas

[0068] After the hemolymph stored at 4°C in step (3) has been allowed to stand for 24 hours, the thawed hemolymph is shaken and mixed using a vortex mixer, and then centrifuged in a low-temperature high-speed centrifuge at 4°C and 13371×g for 20 minutes to collect the serum, which is then aliquoted and stored at -80°C for detection of enzyme activity;

[0069] Changes in nonspecific immune function and antioxidant indices of hemolymph and hepatopancreas of Chinese mitten crab after feeding different levels of AFB1:

[0070] Total hemocyte count (THC): Use a micropipette to transfer 20 μL of anticoagulated blood into a hemocytometer. After the blood is allowed to stand for 1 min, quantitative cell analysis is performed under an upright microscope.

[0071] Respiratory burst (RB): Following the method described by Miyazaki, 100 μL of anticoagulated hemolymph was placed in a 1.5 mL centrifuge tube. An equal volume of 0.2% nitro blue tetrazolium (NBT) solution was added, mixed, and incubated at 25°C for 30 min. After incubation, 50 μL of the suspension was placed in a new 1.5 mL centrifuge tube, 1.0 mL of N,N-dimethylformamide (DMF) was added, mixed, and centrifuged at 3000 g for 5 min. The supernatant was then collected and the optical density (OD) was measured at 540 nm using DMF as a blank.

[0072] Phenoloxidase (PO) activity was measured using a modified method of Ashida et al.

[131] . 300 μL of 0.1 mol / L pH 6.0 sodium phosphate buffer, 0.01 mol / L L-dopa, and 10 μL of the test solution, pre-incubated at 28°C, were added to each microwell. After mixing, the initial OD value at 490 nm was measured. The experimental reaction cycle was set to 20 min, and the OD value was read every 2 min. An increase of 0.001 in the OD490 nm value per minute was considered 1 unit of activity.

[0073] Commercial biochemical kits purchased from Nanjing Jiancheng Bioengineering Institute were used to determine the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione-S transferase (GST), aspartate aminotransferase (AST), acid phosphatase (ACP), alanine aminotransferase (ALT), and alkaline phosphatase (AKP), as well as the total antioxidant capacity (T-AOC) and the contents of malondialdehyde (MDA) and glutathione (GSH) in serum and hepatopancreas samples.

[0074] The serum antioxidant index of Chinese mitten crab is as follows Figure 2 shown. Figure 2 The results showed that the activities of superoxide dismutase, glutathione peroxidase and glutathione S-transferase in serum showed a decreasing trend with the increase of AFB1 supplementation level in feed. Compared with the T0 group, the T 125 、T 625 and T 3125 The activities of superoxide dismutase, glutathione peroxidase and glutathione S-transferase in the T0 group were significantly decreased (p<0.05). The malondialdehyde, glutathione content, total antioxidant capacity and catalase activity all showed a trend of increasing first and then decreasing with the increase of AFB1 supplementation level. Compared with the T0 group, the T 25 、T 125 、T 625 and T 3125 The catalase activity of group T was significantly increased (p<0.05);25 、T 125 、T 625 The glutathione content in the T group was significantly increased (p<0.05). 3125 There was no significant difference in glutathione content between the T0 group and the T1 group (p>0.05); 125 、T 625 and T 3125 The total antioxidant capacity and malondialdehyde content in the two groups were significantly increased (P<0.05).

[0075] Antioxidant indexes of the hepatopancreas of Chinese mitten crab Figure 3 shown. Figure 3 The results showed that the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase in the hepatopancreas showed a decreasing trend with the increase of AFB1 supplementation level in the feed. Compared with the T0 group, the T 25 、T 125 、T 625 and T 3125 The activities of superoxide dismutase and glutathione S-transferase in the T group were significantly decreased (p<0.05). 25 、T 125 and T 625 There was no significant difference among the three groups (p>0.05); 125 、T 625 and T 3125 The catalase activity and glutathione peroxidase activity of the control group were significantly decreased (p<0.05), and there was no significant difference among the three groups (p>0.05). The total antioxidant capacity, alanine aminotransferase activity, glutathione and malondialdehyde content all showed an increasing trend with the increase of AFB1 supplementation level in the feed, and compared with the control group, T 125 、T 625 、T 3125 The levels of T 125 and T 625 There was no significant difference between the two groups (p>0.05). The activity of aspartate aminotransferase increased first and then decreased with the increase of AFB1 supplementation level in the feed. Compared with the T0 group, the T 125 、T 625 The difference between the two groups was not significant (p<0.05). 25 、T 3125 There was no significant difference among the groups (p>0.05).

[0076] The changes in the nonspecific immune function of the hemolymph and hepatopancreas of Chinese mitten crab are shown in Table 3. Compared with T0, the total number of blood cells and respiratory burst activity in the hemolymph decreased with the increase of AFB1 supplementation level in the feed. 125 Group, T 625Group, T 3125 The total blood cell count in the AFB1-treated group was significantly decreased (p<0.05), and the respiratory burst activity in the AFB1-treated group was significantly decreased (p<0.05). The activity of phenoloxidase in the serum of the AFB1-treated group was significantly increased (p<0.05), and showed an increasing trend with the increase of AFB1 supplementation level, among which T5 and T 25 Between groups, T 125 、T 625 、T 3125 There was no significant difference between the groups (p>0.05); the activity of phenoloxidase in the hepatopancreas decreased with the increase of AFB1 supplementation level. Compared with the T0 group, the activity of phenoloxidase in T5 and T 25 There was no significant difference between the two groups (p>0.05). 125 、T 625 and T 3125 The activity of acid phosphatase in serum and hepatopancreas showed a trend of increasing first and then decreasing with the increase of AFB1 supplementation level. Compared with T0 group, T 125 and T 625 The T5 and T 25 and T 3125 There was no significant difference in the activity of the two groups (p>0.05). The activity of alkaline phosphatase in serum and hepatopancreas also showed a trend of first increasing and then decreasing with the increase of AFB1 supplementation level. Compared with the T0 group, there was no significant difference in the activity of serum alkaline phosphatase in the AFB1 treatment group (p>0.05); T 125 、T 625 and T 3125 The alkaline phosphatase activity of the hepatopancreas in the AFB1-treated group was significantly increased (p<0.05), while there were no significant differences in the hepatopancreas among the other AFB1-treated groups (p>0.05).

[0077] Table 4 Effects of different levels of AFB1 on nonspecific immune indicators of Chinese mitten crab (n=3)

[0078]

[0079] (7) Determination of AFB1 content in experimental feed and crab hepatopancreas, gills, and muscles

[0080] 4.0 g of crushed corn and crab tissue samples were extracted with a methanol-water solution (volume ratio 7:3) by oscillation, and the test solution was filtered to obtain a test solution. Aflatoxin B1 enzyme-linked immunosorbent assay kit purchased from Shanghai Youlong Biotechnology Co., Ltd. was used for quantitative determination.

[0081] After 8 weeks of feeding the experimental feed of Chinese mitten crab with AFB1, the AFB1 content in the hepatopancreas, gills and muscles of the experimental crabs increased with the increase of AFB1 supplementation level in the feed (p<0.05), and there was a dose-dependent effect. Moreover, the AFB1 content in the hepatopancreas, muscle and gills of all crabs fed with the feed containing AFB1 was significantly higher than that in the T0 group (p<0.05). Figure 4 shown.

[0082] (8) Pathological examination of hepatopancreas, intestine and gill tissues

[0083] After the crab tissue samples were fixed, Nanjing Miledy Biotechnology Co., Ltd. was commissioned to prepare paraffin sections and HE staining of pathological tissues.

[0084] Determination of the rate of hepatic duct injury per unit area: Five 2 mm × 2 mm rectangular areas were randomly selected from the hepatopancreatic slice views of each parallel sample in each group, and the number of structurally intact and damaged hepatic ducts within the area was counted. The criteria for hepatic duct injury were mainly: epithelial cell damage, separation of epithelial cells from the striated border or adventitia, and the proportion of damaged hepatic ducts in the statistical area to the total number of hepatic ducts was calculated.

[0085] Determination of gill filament and gill axis thickness: Five gill filaments were randomly selected from the gill section view of each parallel sample in each group, and then the gill filament thickness was measured at 10 randomly selected sites from the gill axis to the end of the gill filament; the gill axis thickness was measured at 10 randomly selected points from the gill axis of each parallel sample in each group.

[0086] The pathological changes of Chinese mitten crab tissues after feeding with feeds containing different concentrations of AFB1 for 8 weeks are shown in Figure 2. Figure 5 、 Figure 6 As shown in the figure, the hepatopancreas of the T0 group showed a clear stellate structure in the hepatopancreas, neatly arranged hepatopancreatic epithelial cells, and tight connections between the striated border, epithelial cells, and adventitia. Intestinal epithelial cells were tightly connected to the cuticle, with well-developed intestinal folds and abundant longitudinal muscle bundles. The gill filaments and gill axis were tightly connected, and the gill septa were abundant. Eriocheir sinensis fed AFB1 showed varying degrees of lesions in the hepatopancreas, intestine, and gills, with more severe lesions observed with higher AFB1 supplementation.

[0087] Compared with the T0 group, the T5 group showed stellate structures in the hepatic ducts, with the intraluminal striated borders tightly connected to the epithelial cells, and separation of the adventitia from the epithelial cells in some areas. The rate of hepatic duct injury per unit area was increased (p>0.05). The intestines were similar to those in the T0 group, with neatly arranged epithelial cells, a tightly connected stratum corneum, well-developed folds, and reduced longitudinal muscle bundles beneath some folds. The gills were generally normal.

[0088] T 25 In the group A, the hepatic tubules still maintained a stellate structure, the epithelial cells were fragmented, the B cell apical vesicles were fragmented, and the outer membrane and epithelial cells were separated; Figure 6 The rate of hepatic duct injury per unit area was significantly higher than that in the T0 group (p<0.05). Intestinal epithelial cells were arranged more neatly, with some cells shedding. The stratum corneum remained tightly connected to the epithelial cells. Some longitudinal muscle bundles were further reduced, and the circular muscle layer began to thin. The branchial septum within the gill axis was partially opened.

[0089] T 125 In the group A, the stellate structure of some hepatic tubules was deformed or even completely disappeared, the undifferentiated cells at the edge were obviously damaged, the rupture of B cell apical vesicles increased, the outer membrane was separated from the epithelial cells, and a small amount of cell debris and contents leaked out; Figure 6 The results showed that the rate of hepatic duct injury per unit area was further significantly increased (p<0.05); the epithelial cells in some parts of the intestine were loosely arranged, the stratum corneum and epithelial cell layers were detached on a large scale, the longitudinal muscle bundles were significantly less, the circular muscle layer was significantly thinner and fractured; the gill axis gaps were enlarged, and the number of branchial septum fractures increased.

[0090] T 625 The stellate structure of the hepatic tubules in the T group was almost gone, the epithelial cells were loosely arranged and severely broken, the outer membrane was completely separated from the epithelial cell layer, the striated border structure was destroyed, and a large amount of cell fragments and contents leaked out. The hepatic tubule injury rate per unit area was second only to that of T 3125 Group: The epithelial cells were completely detached, part of the stratum corneum was completely separated from the epithelial cell layer, the longitudinal muscle bundles were greatly reduced, and the circular muscle layer became further thinned and irregular in shape with multiple breaks; Figure 5 The results showed that the large-scale rupture of the internal septum of the gill axis caused the abnormal and significant expansion of the gill axis (p<0.05), but the internal septum of the gill filaments was not broken.

[0091] T 3125 In the group A, the stellate structure of the hepatic tubules completely disappeared, the epithelial cells were completely broken, the outer membrane and striated border were completely separated from the epithelial cell fragments and were irregularly distributed, the background was filled with a large number of cell fragments, and the hepatic tubule damage rate per unit area was the highest; the intestinal epithelial cells were arranged in a disorderly manner, most of the stratum corneum fell off the epithelial cell layer, only a small amount of residual longitudinal muscle bundles were visible, and the circular muscle layer was irregular and broken as the intestine twisted; the gill axis was about to break into two parts, and the thickness and inner cavity septum of the gill filaments were also broken.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing a model of hepatopancreatic necrosis syndrome in river crabs, characterized in that: The steps include: (1) Preparation of feed: prepare a basic feed and add aflatoxin B1 to the basic feed, mix well to obtain AFB1 feed; (2) The crabs were raised in an aerated tap water environment and fed with the AFB1 feed of step (1) to obtain a crab hepatopancreatic necrosis syndrome model.

2. The method for constructing the crab hepatopancreatic necrosis syndrome model according to claim 1, characterized in that: In step (1), the concentration of AFB1 in the AFB1 feed is 25 to 3200 μg / kg.

3. The method for constructing the crab hepatopancreatic necrosis syndrome model according to claim 1, characterized in that: In step (1), the basic feed includes the following nutritional components: Crude protein 40.0%, crude fat 3.5%, crude fiber 8.0%, crude ash 16.0%, lysine 2.0%, total phosphorus 0.9%, moisture 12.0%.

4. The method for constructing the crab hepatopancreatic necrosis syndrome model according to claim 1, characterized in that: In step (2), the conditions of the aerated tap water environment are: the water temperature is maintained at 20-30° C., the dissolved oxygen content is 5-8 mg / L, and the pH is 7.2-8.

5.

5. A river crab hepatopancreatic necrosis syndrome model constructed according to the method for constructing a river crab hepatopancreatic necrosis syndrome model according to any one of claims 1 to 4.

6. Use of the crab hepatopancreatic necrosis syndrome model according to any one of claims 1 to 4 in the study of crab hepatopancreatic necrosis syndrome, characterized in that: The research includes developing or screening products that can treat or prevent river crab hepatopancreatic necrosis syndrome, analyzing the multiple etiological mechanisms of river crab hepatopancreatic necrosis syndrome, or screening diagnostic markers for river crab hepatopancreatic necrosis syndrome.

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