Method for judging vitality of young crabs and application

Abstract

The invention discloses a method for judging the vitality of young crabs, which comprises the following steps of: overturning the young crabs to enable the back shells of the young crabs to face downwards, and observing and recording the time required by the young crabs from the beginning of the overturning action to the complete recovery of the young crabs to the normal posture; when the use time is less than or equal to 1s, the juvenile crabs have excellent vitality; when the use time is more than 1s and less than or equal to 3s, the juvenile crabs have good vitality; when the use time is more than 3 seconds and less than or equal to 15 seconds, the juvenile crab vitality is general; and when the use time is longer than 15s, the juvenile crabs are poor in vigor. The judgment method is free of damage, easy and convenient to operate, accurate, reliable and high in practicability, overcomes the defects of individual damage, accuracy and low efficiency of a traditional method, and provides a new method for rapidly screening the juvenile crabs with good vigor and high vigor and improving culture benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular relates to a method for judging the vitality of young crabs and an application thereof. Background Art

[0002] Chinese mitten crab (Eriocheir sinensis) is an important economic aquatic species in China. It is delicious and nutritious, and is widely loved by consumers. In its breeding process, the vitality assessment of young crabs is a crucial link, which directly affects the breeding benefits.

[0003] Traditional methods of evaluating vitality, such as visually observing the physical characteristics and feeding ability of young crabs, are highly subjective and have low accuracy. Some methods based on physiological indicators, such as measuring enzyme activity and metabolic rate, can cause damage to young crabs, and are complex to operate and costly. These methods are difficult to quickly, effectively, and non-destructively screen out young crabs with strong vitality. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for quickly, accurately and reliably judging the vitality of young crabs; another purpose of the present invention is to provide an application of the method for judging the vitality of young crabs.

[0005] Technical solution: The method for judging the vitality of young crabs of the present invention comprises the following steps: turning the young crabs over so that their back shells face downwards, and observing and recording the time required for the young crabs to completely recover to a normal posture from the beginning of the turning action;

[0006] When the time is ≤1s, the crab juveniles have excellent vitality;

[0007] When the time is >1s and ≤3s, the juvenile crabs are in good vitality;

[0008] When the time is >3s and ≤15s, the vitality of the juvenile crab is average;

[0009] When the time is greater than 15s, the vitality of the young crabs is poor.

[0010] Preferably, the juvenile crab is a juvenile Chinese mitten crab.

[0011] Preferably, the young crabs are of similar size, have sound appendages, and have no obvious damage on the exterior.

[0012] Preferably, the size of the young crab is 2.35±0.16g.

[0013] Preferably, the reliability of the method is verified by measuring physiological parameters related to the evaluation of the health status of juvenile crabs.

[0014] Preferably, hemolymph and hepatopancreas samples are collected from the juvenile crabs after the vitality is judged by the resetting reflex, and the relevant physiological parameters are evaluated by measuring the health status of the juvenile crabs, and the vitality of the juvenile crabs is comprehensively evaluated to verify the reliability of the method for judging the vitality of the juvenile crabs.

[0015] Preferably, the collected hemolymph is used to measure the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0016] Preferably, the harvested hepatopancreas is used to measure ACP and AKP activities.

[0017] The method for judging the vitality of young crabs of the present invention is used in judging the vitality of young crabs.

[0018] Preferably, the method of judging the vitality of young crabs is to quickly judge the Chinese mitten crab seedlings with good vitality and strong survival ability.

[0019] The method for judging the vitality of young crabs of the present invention is obtained by the following experimental steps:

[0020] Step 1: Conduct random sampling surveys on juvenile crabs collected from breeding bases to ensure that the samples are representative;

[0021] Step 2: Conduct a resetting reflex experiment on the sampled juvenile crabs, specifically, gently flip the juvenile crabs so that their back shells face downwards, observe and record the time it takes for the juvenile crabs to completely return to their normal posture from the beginning of the flipping action, and record the entire process on video;

[0022] Step 3: Through accurate statistical analysis of the recorded videos, the sampled juvenile crabs are divided into four groups: excellent, good, average, and poor according to the predetermined evaluation criteria based on the time taken for the resetting reflex of the sampled juvenile crabs;

[0023] Step 4: Collect the hepatopancreas and hemolymph of 10 crab larvae in each group to measure the nonspecific immune characteristic parameters and health status evaluation parameters of the crab larvae, and combine the physiological parameters to evaluate the vitality of the crab larvae.

[0024] Among them, the evaluation criteria for grouping in step 2 are that if the reduction reflex is completed within 1 second, it is considered excellent; if it takes more than 1 second and ≤ 3 seconds, it is considered good; if it takes more than 3 seconds and ≤ 15 seconds, it is considered fair; and if it takes more than 15 seconds, it is considered poor.

[0025] Furthermore, the physiological parameters in step three include the activities of ACP and AKP in the hepatopancreas, which are used to reflect the immune status of the body, and the activities of ACP, AKP, ALT and AST in serum, which are used to reflect the health of the hepatopancreas.

[0026] Furthermore, the physiological parameter analysis in step 4 is used to assist in verifying the reliability of the results of the reset reflex in judging the vitality of juvenile crabs.

[0027] Principle of the invention: The righting reflex refers to the physiological reaction of an animal that can quickly return to its normal physiological posture after being turned over or inverted. This behavior not only reflects the health of the animal's nervous system, but also reflects the strength and coordination of its muscles. In the cultivation of juvenile Chinese mitten crabs, the righting reflex ability of the juvenile crabs is closely related to their vitality. A healthy juvenile crab has the ability to quickly restore its posture, which indicates that its nervous system and muscle system are in good working condition. On the contrary, juvenile crabs with poor vitality show a slow righting reflex or are unable to complete the righting reflex, which is a precursor to their poor health or the presence of potential diseases.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: Based on the characteristics of animal neurobiology and behavior, the present invention proposes for the first time a simple, rapid, scientific, reliable, operable and practical method for judging the vitality of young crabs by observing, recording and analyzing the time taken by young crabs to complete the reset reflex. The judgment method of the present invention is non-destructive, easy to operate, accurate and reliable, and highly practical. It overcomes the defects of traditional methods such as damage to individuals, low accuracy and low efficiency, and provides a new method for quickly screening crab seedlings with good vitality and strong survival ability and improving breeding benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the difference analysis of the survival rate of juvenile crabs in different activity groups in Example 2 (Note: different letters on the column indicate significant differences P<0.05);

[0030] Figure 2 This is the difference analysis of hepatopancreatic immune enzyme activity of crab juveniles in different activity groups in Example 3 (Note: different letters on the column indicate significant difference P<0.05);

[0031] Figure 3 This is the difference analysis of serum immune enzyme activity of crab larvae in different vitality groups in Example 3 (Note: different letters on the columns indicate significant differences P<0.05). DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with embodiments and drawings.

[0033] Example 1

[0034] Juvenile Chinese mitten crabs were collected from the river crab breeding base in Gaochun, Nanjing. Juvenile crabs of similar size, intact appendages and no obvious damage on the body surface were selected, and the vitality of the juvenile crabs was determined by the repositioning reflex experiment. The specific judgment method was: turn the juvenile crab over so that its back shell was facing down, and observe and record the time required for the juvenile crab to completely recover from the beginning of the flipping action to the normal posture; when the time is ≤1s, the juvenile crab has excellent vitality; when the time is >1s and ≤3s, the juvenile crab has good vitality; when the time is >3s and ≤15s, the juvenile crab has average vitality; when the time is >15s, the juvenile crab has poor vitality.

[0035] Example 2

[0036] The young crabs of the four groups of excellent, good, average and poor vitality determined in Example 1 were further divided into groups for a survival rate experiment.

[0037] The specific grouping rules are:

[0038] Fast group: The crabs have excellent vitality (the time taken for the crabs to fully recover to their normal posture from the beginning of the flipping action is ≤ 1s);

[0039] General group: the juvenile crabs have good vitality and are normal (the time taken for the juvenile crabs to fully recover to the normal posture from the beginning of the flipping action is greater than 1s and less than 15s);

[0040] Slow group: The juvenile crabs had poor vitality (the time taken for the juvenile crabs to fully recover to the normal posture from the beginning of the flipping movement was >15s).

[0041] Five replicates were set up in each group, with 15 young crabs in each replicate, for the breeding experiment to calculate the survival rate of each group, and the results were presented as mean ± standard error.

[0042] The specific implementation method of statistical survival rate is as follows: a breeding experiment is carried out on the crab juveniles whose vitality is judged based on the reset reflex experiment, and the water temperature is maintained at 24℃±1℃, and no oxygen is added during the breeding period. Dead crabs are removed in time during the breeding period to avoid water pollution, and the death of crab juveniles in each group is recorded at 24h, 48h and 72h after the start of breeding, and the survival rate of each group is finally calculated. The results are as follows Figure 1 The data showed that at 24 hours, the survival rate of the juvenile crabs in the fast group was 100%, the survival rate of the general group was about 96%, and the survival rate of the slow group was only about 78.67%; at 48 hours, the survival rate of the juvenile crabs in the fast group was 100%, the survival rate of the general group was about 89.33%, and the survival rate of the slow group was only about 73.33%; at 72 hours, the survival rate of the juvenile crabs in the fast group was about 98.67%, the survival rate of the general group was about 76%, and the survival rate of the slow group was only about 69.33%.

[0043] In summary, the vitality of juvenile crabs was evaluated based on the time taken to complete the reset reflex. The longer the time taken to complete the reset reflex, the lower the survival rate of juvenile crabs in culture. The survival rates of juvenile crabs in different groups differed significantly. This example proves that the method of judging the vitality of juvenile crabs based on the reset reflex is reliable.

[0044] Example 3

[0045] Samples were collected from the four groups of crabs with excellent, good, average, and poor vitality as determined in Example 1, and relevant physiological parameter verification experiments were performed. The hepatopancreas was collected and quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for subsequent enzyme activity determination. Hemolymph was collected, and the supernatant was obtained after centrifugation and stored in a -80°C refrigerator for subsequent enzyme activity determination. The results were presented using mean ± standard error.

[0046] The specific implementation method of measuring the enzyme activity in the hepatopancreas and serum is as follows:

[0047] The activities of ACP and AKP in the hepatopancreas and serum were determined using an ACP assay kit (microplate method) and an AKP assay kit (microplate method), respectively. The kits were purchased from Nanjing Jiancheng Bioengineering Institute. ACP or AKP decomposes disodium phenyl phosphate to produce free phenol and phosphoric acid. Phenol reacts with 4-aminoantipyrine in an alkaline solution and is oxidized by potassium ferrocyanide to produce a red quinone derivative. The enzyme activity can be determined based on the depth of the red color. The absorbance was measured at a wavelength of 520 nm using an enzyme reader to calculate the activity of ACP or AKP in the sample. The results are shown in Figure 2. Figure 2 and Figure 3 As shown in , the activities of ACP and AKP in the hepatopancreas decreased significantly as the vitality of the juvenile crabs deteriorated, while the activities of ACP and AKP in serum showed an opposite trend, with significant differences between different groups. When the vitality of river crabs is poor, their physiological functions will be affected, including the metabolism and immune function of the hepatopancreas, which may lead to a decrease in the synthesis or release of ACP and AKP in the hepatopancreas. When the hepatopancreas of juvenile crabs is damaged or the body is in poor health, ACP and AKP may be released from damaged hepatopancreatic cells into the hemolymph, resulting in increased ACP and AKP activities in serum. In addition, excessive ACP and AKP activities in serum may indicate that the body is in an immune response or disease state.

[0048] The activity of AST in serum was determined using the AST test kit (microplate method), which was purchased from Nanjing Jiancheng Bioengineering Research Institute. AST can transfer the amino and keto groups of α-ketoglutarate and aspartic acid to generate glutamic acid and oxaloacetic acid. Oxaloacetic acid can decarboxylate to pyruvic acid during the reaction. Pyruvic acid reacts with 2,4-dinitrophenylhydrazine to generate 2,4-dinitrophenylhydrazone, which appears reddish brown in alkaline solution. After measuring the absorbance at a wavelength of 510nm using an enzyme reader, the enzyme activity unit can be obtained according to the standard curve. The activity of ALT in serum was determined using the ALT test kit (microplate method), which was purchased from Nanjing Jiancheng Bioengineering Research Institute. ALT acts on a substrate composed of alanine and α-ketoglutarate at 37°C and pH 7.4 to generate pyruvic acid and glutamic acid. After 30 minutes of reaction, 2,4-dinitrophenylhydrazine hydrochloride solution was added, and 2,4-dinitrophenylhydrazine reacted with the hydroxyl group in the keto acid to generate pyruvate phenylhydrazone. Phenylhydrazone is reddish brown under alkaline conditions. The absorbance was measured at a wavelength of 505 nm using an enzyme reader and the enzyme activity was calculated. The results are as follows Figure 3 As shown in the figure, there are significant differences in AST and ALT activities in different vitality groups. The lower the vitality, the higher the AST and ALT activities in the serum of young crabs. AST and ALT are important indicators of hepatopancreatic damage. The increase in AST and ALT activities in serum indicates that the lower the vitality of young crabs, the more serious their hepatopancreatic damage.

[0049] In summary, the vitality of juvenile crabs was evaluated based on the time taken to complete the resetting reflex. The worse the vitality of the juvenile crabs, the lower the ACP and AKP activities in their hepatopancreas, and the higher the ACP, AKP, AST and ALT activities in their serum. This shows that the results of the physiological parameters of juvenile crabs are consistent with the results of the resetting reflex, proving that the method of judging the vitality of juvenile crabs based on the resetting reflex is extremely reliable.

Claims

1. A method for judging the vitality of young crabs, characterized in that: The method comprises the following steps: turning over the young crab so that its back shell faces downward, and observing and recording the time required for the young crab to completely recover to a normal posture from the beginning of the turning action; When the time is ≤1s, the crab juveniles have excellent vitality; When the time is >1s and ≤3s, the juvenile crabs are in good vitality; When the time is >3s and ≤15s, the vitality of the juvenile crab is average; When the time is greater than 15s, the vitality of the young crabs is poor.

2. The method for judging the vitality of young crabs according to claim 1, characterized in that: The young crabs are young Chinese mitten crabs.

3. The method for judging the vitality of young crabs according to claim 1, characterized in that: The juvenile crabs are of similar size, have sound appendages, and have no obvious damage on the exterior.

4. The method for judging the vitality of young crabs according to claim 3, characterized in that: The size of the juvenile crab is 2.35±0.16g.

5. The method for judging the vitality of young crabs according to claim 1, characterized in that: The reliability of the method was verified by measuring the physiological parameters related to the evaluation of the health status of juvenile crabs.

6. The method for judging the vitality of young crabs according to claim 5, characterized in that: The specific verification method is to collect hemolymph and hepatopancreas samples from juvenile crabs after the vitality is judged by resetting reflex, evaluate relevant physiological parameters by measuring the health status of juvenile crabs, and comprehensively evaluate the vitality of juvenile crabs to verify the reliability of the method for judging the vitality of juvenile crabs.

7. The method for judging the vitality of young crabs according to claim 6, characterized in that: The collected hemolymph was used to measure the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

8. The method for judging the vitality of young crabs according to claim 6, characterized in that: The collected hepatopancreas was used to measure the activities of ACP and AKP.

9. Use of the method for judging the vitality of young crabs according to claim 1 in judging the vitality of young crabs.

10. The use according to claim 9, characterized in that: The method for judging the vitality of young crabs is to quickly judge the Chinese mitten crab seedlings with good vitality and strong survival ability.

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