KR100760583B1 - A Health Assessment Program of Cultured Fish - Google Patents

Abstract

The present invention relates to a health evaluation method for aquaculture fish, comprising: a sample size selection step (a1) and a sample size selection step (a1) for selecting 6 to 10 individuals having a uniform size among individuals from a target group of fish to be evaluated for health; A preparation step (A) comprising a transporting and storage step (a2) for transporting and storing the selected sample;

The optical microscopic biopsy of the gills and skin of the sample selected in the preparation step (A) was carried out to evaluate the contamination of the biological agent, and the systemic contamination of the bacteria was evaluated through the medium smear of blood and the blood cut of the kidney. Contamination evaluation step (b1) of the pathogenic factors, and blood anemia content evaluation step (b2) to measure the hematocrit (Ht), hemoglobin (Hb) and hemolytic propensity measurements of red blood cells by measuring biochemical values of blood (b2) And then, hepatic function impairment indicators were measured in plasma total protein (TP), ALT and AST, blood was dispensed in heparinized Eppendorf tubes, refrigerated at 4 ° C for one day, and centrifuged at 4,000 rpm for 5 minutes. After that, the supernatant is separated and measured by a biochemical automatic analyzer soybean health evaluation step (b3); Pathological histological evaluation step (b4), which evaluates the activity of the narrowing tissue for the spleen, the activity of the hematopoiesis for the kidneys, the health of the new unit, the presence of pericarditis or myocardial degeneration in the heart, and the activity of the gastric epithelium for the stomach. Health parameter evaluation step (B) made;

Cross-assessment of the evaluation results for each parameter in the health parameter evaluation stage (B) is very good (grade A), good (grade B), alternative good (grade C), poor (grade D) and very poor (grade E) It is characterized in that it consists of a comprehensive evaluation step (C) to determine a five-level grade, such as the improper salary of feed for farmed fish, indiscriminate use of drugs, screening of damaged fish By presenting a method to properly assess the state of health, it is possible to maintain and improve the anti-pathogenicity, to prevent and minimize the occurrence of farmed fish diseases, and to secure the safety of food from the production process by restricting the use of various therapeutic agents including antibiotics. At the same time, it provides health assessment methods for farmed fish that can lead to stabilization and maximize productivity.

Aquaculture Fish, Health Assessment Method, Health Parameter, Individual Assessment, Fish Rating, Comprehensive Assessment

Description

A Health Assessment Program of Cultured Fish

1 is an evaluation flow chart regarding the fish health evaluation method of the present invention

     Fish farming means to artificially cultivate and benefit from fish that should be raised in the natural world, and has recently given great attention to the health of farmed fish in relation to the development of diseases, but has no clinical problem. There are no examples of health assessments on fish at home and abroad. Until now, it has been limited to the identification of biological and pathogens such as bacteria, viruses, parasites, and chemical toxic substances in and outside the body, and overlooks the health status of the fish itself as a host. In order to perform a predictive health evaluation on disease occurrence, the health evaluation method is separated from the individual level passive health evaluation method for the observation of clinical findings and the diagnosis of disease centering on the cause factor detection (bacteria culture, PCR, etc.). It is at the point where the presentation of the The fish fish health assessment method focuses on the fact that a small number of representative samples can be used to evaluate the health of fish groups through active testing of the invasiveness (anatomy) in the accuracy of the evaluation results. As a result, it is a health assessment technique that can assess the health status of the farmed fish group that has not yet been able to cope with the disease before the onset.

The present invention relates to a health evaluation method of aquaculture fish developed in this purpose, the contents can be seen as one of the health evaluation techniques applied to fish among aquaculture fish. In aquaculture conditions that require overcrowding, once the disease occurs, the morbidity and mortality is likely to occur collectively. The morbidity and mortality of this type of disease is very high if the health status of farmed fish is not checked beforehand. The conventional method, which mainly focuses on infection, mainly due to the detection of causative agents, is difficult to be called a health evaluation method in a strict sense, and has a problem that can be referred to as a disease diagnosis level detection of causative agents.

An object of the present invention has been made to improve the problems with the conventional health evaluation form that is vulnerable to the above-mentioned disease occurrence, the sample size selection step (a1) of selecting a sample size of the fish for evaluation, and the sample Preparation step (A) consisting of transport and storage steps (a2) for transport and storage, contamination level evaluation step (b1) of pathogenic factors to evaluate the contamination of biological pathogens, hematocrit levels, hemoglobin levels and hemolytic propensity of red blood cells Anemia content evaluation step (b2) through the liver, to examine the color tone and weight ratio of the liver, to observe the morphology of the liver cells and to measure the hypertrophy index of the liver cells, and to measure and evaluate the biochemical values related to functional damage of the liver Histopathological evaluation of the health level (b3) and histological findings on the four major organs of the spleen, kidney, heart and stomach. The health parameter evaluation step (B) consisting of the four evaluation items in the provisional step (b4) and the evaluation results of the health parameter evaluation step (B) are synthesized and cross-analyzed based on the health status of the liver. Comprehensive evaluation step (C) is characterized in that, by providing a method for properly assessing the health of the damaged fish body due to various hazards, it is a task of the present invention to minimize the incidence and mortality of various diseases can do.

     The configuration of the present invention comprises a total of three steps, the first step of the sample size selection step (a1) of the evaluation fish, and the preparation step (A) consisting of a transport and storage step (a2) for transporting and storing the sample; Contamination assessment step (b1) evaluating detection of biological pathogens, assessment of anemia based on hematocrit, hemoglobin levels and hemolytic hemolysis tendency (b2), gross tones and weight ratio of liver, findings of hepatocyte nucleus and cytoplasm, Hepatic hypertrophy index and hepatic health level (b3), which is measured and cross-assessed by measuring hepatic damage-related biochemistry, and histological findings on the four major organs of the spleen, kidney, heart and stomach Based on the health parameter evaluation step (B) consisting of four evaluation items in the pathological histological evaluation step (b4) and the evaluation parameter in the health parameter evaluation step, a comprehensive evaluation focusing on the health status of the liver in particular Characterized in that it comprises a step (C).

    Therefore, the present invention is a health evaluation method for evaluating the health status of a fish as an infectious host based on the health status, minimizing the sensitivity according to exposure of various causative agents, normalizing or maximizing the natural immune function of the fish, and the effects of various feeds and drugs. Maximization and prevention of various accidents are the ultimate goals. In addition, since the desirable result of fish farming is obtained by managing fish in fish groups rather than individual management, it is in this sense that the fish farming health evaluation method is an evaluation method with very high application and expected value.

     If more detailed description of the configuration of the present invention through the accompanying drawings as follows. 1 is an evaluation flow chart of the health evaluation method for aquaculture fish of the present invention, the preparation step A, the sample size selection step (a1) and the transport and storage step (a2), the sample size selection step (a1), Selecting the sample size of the fish for evaluation, the fish group can be represented for the target group to be subjected to the health evaluation, the step of selecting the appropriate number of evenly sized individuals, the transportation and storage step (a2), In the step of transporting and storing the sample selected in the sample size selection step (a1) above, in case of a long distance, the sample is fasted for a predetermined time before oxygen packing, then transported by a vehicle or an airplane, and the autopsy is carried out at a certain time after transportation Is carried out.

     Health parameter evaluation step (B) is composed of the contamination level evaluation step (b1), anemia content evaluation step (b2), liver health evaluation step (b3), pathological histological evaluation step (b4).

      Contamination level evaluation of pathogenic factors (b1) is a step of evaluating the degree of biological pathogen contamination, by performing optical microscopic biopsy of gills and skin to evaluate the contamination of biological agents (bacteria or parasites), Medium smear (general nutrient medium) and kidney cross section stamps are used to evaluate the systemic contamination of bacteria, and if the parasite and bacteria or none of them are checked, the fish group will be `` very good ''. Evaluate.

      In the evaluation step (b2) of the anemia content, hematocrit levels, hemoglobin levels, and hemolytic tendencies of erythrocytes are measured, and the determination of anemia is made of hemoglobin levels with reference to hematocrit levels, and the hemolytic tendency of erythrocytes is associated with hematocrit levels. Evaluate the relevant content of the It is evaluated as "very good" when it falls within the setting criteria.

      Hepatic health evaluation step (b3) is performed by visual inspection of the visual tones and weight ratio of the liver, histological evaluation based on histologic findings and hypertrophy index of hepatic cells, and total protein (TP) and ALT indices of impaired hepatic function. And biochemical evaluation of blood to measure AST levels. If the range is satisfied, it is determined as "very good". The visual tones should be chosen as a whole, and the weight-to-weight ratio should not exceed the specified range. The histological findings of the liver should be within the hypertrophy index, the hepatic cell findings should be based on the location of the nucleus and the cytoplasm, the nucleus should be centrally located, and the cytoplasm should not be fat. Histopathological evaluation (b4) is mainly histological evaluation of the four major organs, namely, liver, spleen, kidney, heart, and stomach. If there is no significant lesion in each organ, it is evaluated as "very good." Significant lesions for each organ include the activity of the narrowing tissues for the spleen, the activity of the hematopoiesis and the health of the new unit for the kidneys, the presence of pericarditis (or myocardial degeneration) in the heart, and the activity of the gastric epithelium for the stomach. do.

       Comprehensive evaluation step (C) is achieved by cross-assessing the evaluation results for each parameter of the health parameter evaluation step (B). In the cross-valuation, the final assessment of the entire fish group is made, focusing on the health status of the liver. If there is no problem, it is judged as "very good."

[ Example 1 ]

Preparation stage (A)

  end. Fish Selection for Health Assessment

  Fish for health assessment are, in principle, limited to fish that are clinically benign. The selection of fish can select from 6 to 10 fish of the most representative size in aquaculture tanks and the number can be reduced within the same range as the population grows.

  I. Transport of Evaluation Laboratory of Fish

  The transport of fish may be by courier or direct transport, fasted a day in advance, packed in oxygen-filled packaging, and respiratory failure should not occur during the transport period.

  All. Necropsy timing and preparation

  An autopsy can start at any time, but record the start time and the end time (the autopsy time may affect hematocrit, but rarely occurs with hemoglobin). Anesthesia is performed just before the autopsy.

Health parameter  Evaluation stage (B)

  end. Contamination Assessment of Parasites and Bacteria

  Parasite contamination is based on wet mount test, gill and skin biopsy, bacterial contamination using spleen and blood sample, medium using BHIA (Brain Heart Infusion Agar), and culture time is 24 hours.

  I. Assessment of the Content of Anemia

  Hematocrit (Ht), hemoglobin (Hb) and the hemolytic propensity (%) is evaluated based on the specific evaluation method is as follows.

  1) Measurement of Ht and Hb

  In the anesthesia state, the blood collected from the arrhythmia is inhaled into the capillary for measuring Ht treated with anticoagulant, and then centrifuged to precipitate the blood cells using a centrifuge for Ht, and the percentage of the volume occupied by the blood cells is expressed as Ht value (%). , Hb is measured in 5ml of Drabkin solution in the test tube, 20μl of blood is added with a micropipette, shake the test tube up and down two or three times to mix well, and leave it at room temperature for 5-10 minutes, Measure the absorbance of the standard solution and the sample at a wavelength of 540 nm with a blank. The concentration of Hb is calculated as follows.

Hb Concentration (g / 100ml) = (Sample Absorbance ÷ Standard Absorbance) × Standard Liquid Concentration (16.0 g / 100ml)

  2) Red blood cell hemolysis propensity

  First, 1.5 ml of NaCl is taken in test tubes by concentration from 0% to 0.9%, 15 μl of heparin-treated blood is dispensed into each test tube, mixed, and left to stand at room temperature for 1 hour. After standing for 1 hour, centrifuged for 5 minutes at 4,000rpm, using a spectrophotometer to measure the upper layer of the test tube with physiological saline blank at 540nm, to obtain 50% hemolytic concentration by obtaining the hemolytic properties of NaCl concentration. The calculation method is as follows.

% Hemolysis = absorbance of each NaCl concentration / absorption of complete blood × 100

  All. Soy Health Assessment

  The evaluation of the liver and optical microscopic morphology of the liver and the three biochemical parameters that may suggest the damage of the liver are evaluated. Finally, the health of the liver is assessed in relation to the presence of anemia.

  1) Visual evaluation of soy sauce

  Measure the tone and tone of the liver, weight ratio of the liver (HSI (%), hepatosomatic index = (weight of liver ÷ fish weight) x 100). The weight ratio determines the presence and extent of macroscopic hypertrophy. The naked eye color evaluates the nutritional status of the liver, and the uniformity of blood circulation in the liver by the selection of the color tone.

  2) Histological evaluation of liver

  Based on the histological changes of hepatic parenchyma, the morphological abnormality of hepatocytes is first observed, and the ubiquitous presence of hepatocyte nuclei and the composition of the cytoplasm are evaluated. For cytoplasm, observe the content of sugar and lipids and the distribution of lipids. Evaluating the integrity of hepatocytes and the closure of flow, at the same time, yields histological hypertrophy (number / unit of visible nucleus).

  3) Measurement of TP, ALT and AST

  Total protein (TP), alanine aminotransferase (ALT), and asparate aminotransferase (AST) are measured to assess liver function and damage of hepatic biochemical parameters, and blood is dispensed in heparin-treated Eppendorf tubes. After refrigerated at 4 ℃ for one day and centrifuged for 5 minutes at 4,000rpm, the supernatant was separated and measured by a biochemical automatic analyzer.

  la. Histopathological evaluation of other organs

  The four major organs of the spleen, kidney, heart, and stomach, except for the liver, are examined visually and histologically. The spleen is characterized by changes in the degeneration including the activity of the narrow tissue and hematopoietic findings of the parenchyma. For the corpus callosum, the histological composition and hematopoietic activity of hematopoietic tissues of the head and body epilepsy, the degeneration of glomeruli and renal tubules, the presence and absence of parasites, and the pericarditis (including myocardial degeneration) in the heart; Analyze histological changes in the gastrointestinal epithelium.

Comprehensive Evaluation Step (C)

  The overall parameters including the causative agent contamination, anemia, liver health, and histological changes of organs and tissues were evaluated for each parameter by good and poor results. Comprehensive evaluation is performed through the evaluation method. The overall evaluation grade is determined in five stages: very good (grade A), good (grade B), alternative good (grade C), poor (grade D) and very poor (grade E). Grade A shall be within the range or approach of all health parameters.

As described above, although the present invention has been described with reference to the above embodiments, it is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention.

Referring to the effects of the above-described configuration and operation in detail as follows. By providing a method for properly evaluating the health status of damaged fish due to various hazards caused by artificial manipulation such as inappropriate feeding of feed, indiscriminate use of drugs, and screening for farmed fish of the present invention, At the same time, it can maintain and improve anti-pathogenicity, block and minimize the occurrence of disease, minimize the use of various therapeutic agents including antibiotics, and ensure the safety of food from the production process, It provides effects such as stabilization and maximization.

Claims (4)

In the health evaluation method of farmed fish, A sample size selection step (a1) for selecting 6-10 individuals of equal size among individuals from a target group of fish to be evaluated for health, and a transportation and storage step for transporting and storing the sample selected in the sample size selection step (a1). a preparation step (A) consisting of (a2); The optical microscopic biopsy of the gills and skin of the sample selected in the preparation step (A) was carried out to evaluate the contamination of the biological agent, and the systemic contamination of the bacteria was evaluated through the medium smear of blood and the blood cut of the kidney. Contamination level evaluation step (b1) of the pathogenic agent, Anemia content evaluation step (b2) in the blood, which is a combination of the hematocrit (Ht) value, hemoglobin (Hb) value and hemolytic tendency measurement of red blood cells by measuring the biochemical level of blood, Measure total hepatic protein (TP), ALT and AST, hepatic function impairment indicators, dispense blood into heparinized Eppendorf tubes, refrigerated at 4 ° C for one day, and centrifuge at 4,000 rpm for 5 minutes. Soy health evaluation step (b3) to separate the supernatant and measure it biochemically by measuring with a biochemical automatic analyzer, Pathological histological evaluation step (b4), which evaluates the activity of the narrowing tissue for the spleen, the activity of the hematopoiesis for the kidneys, the health of the new unit, the presence of pericarditis or myocardial degeneration in the heart, and the activity of the gastric epithelium for the stomach. Health parameter evaluation step (B) made; Cross-assessment of the evaluation results for each parameter in the health parameter evaluation stage (B) is very good (grade A), good (grade B), alternative good (grade C), poor (grade D) and very poor (grade E) Health evaluation method of aquaculture fish, characterized in that it consists of a comprehensive evaluation step (C) to determine a five-level grade as delete delete delete

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