CN115316319A - Cultivation method for improving activity of antithrombin of leeches - Google Patents
Cultivation method for improving activity of antithrombin of leeches Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/40—Culture of aquatic animals of annelids, e.g. lugworms or Eunice
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/24—Animal feeding-stuffs from material of animal origin from blood
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
In order to solve the problem that the antithrombin activity of the existing artificial leech culture is lower than that of wild leech seriously, the invention provides a culture method for improving the antithrombin activity of leech, which comprises the following steps: s1, arranging a net cage; s2, putting seedlings; s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water; s4, bait feeding: mixing the nutrient solution and fresh pig blood proportionally, filling into cleaned pig intestine, and binding two ends to ensure no leakage; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; s5, controlling the illumination intensity: the environmental illumination intensity is controlled to be 20-50Lux at 8-20 points every day, and the environmental illumination intensity is controlled to be 0Lux at other times. The cultivation method greatly improves the antithrombin activity and growth speed of the leeches.
Description
Technical Field
The invention belongs to the technical field of leech culture, and particularly relates to a culture method for improving the antithrombin activity of leeches.
Background
Leeches have a long history of being used for treating diseases at home and abroad. Leeches, as a traditional Chinese medicine in China, are recorded in Shennong Ben Cao Jing, which means that the leeches can expel malignant blood and extravasated blood and break blood clots, and are the only leech medicinal material type which is recorded in the Chinese pharmacopoeia and takes animal blood as a food source at present. Along with the continuous and deep clinical research on leeches in recent years, leeches also have the effects of resisting thrombus, inflammation, tumors, atherosclerosis and the like, and particularly show great superiority and wide prospect in the aspect of treating cardiovascular and cerebrovascular diseases, cancers and other diseases seriously threatening the health of human beings, so that leech culture has good market prospect and economic benefit.
Due to the use of a large amount of pesticides and disinfectants and the man-made capturing, the wild leech resources are seriously damaged, and the ever-rising market demand cannot be met at all. Although the artificial breeding of the leeches relieves the contradiction between supply and demand of the leeches to a certain extent, the traditional breeding method and the traditional breeding mode generally have the problem of low survival rate, and meanwhile, the antithrombin activity of the leeches under the artificial breeding condition is lower than that of the wild leeches, so the utilization value in the pharmaceutical process is reduced or even the artificial breeding method cannot be used. The bloody water produced by the leech in the feeding process can cause certain pollution to the local environment. These problems seriously hinder the large-scale cultivation of the leeches, and can not fundamentally solve the contradiction between supply and demand of domestic and foreign markets for the leech demand.
Disclosure of Invention
The invention provides a cultivation method for improving the antithrombin activity of leeches, which aims to solve the problem that the antithrombin activity of the existing artificial cultivation of leeches is seriously lower than that of wild leeches.
The invention relates to a cultivation method for improving the antithrombin activity of leeches, which comprises the following steps:
s1, arranging a net cage;
s2, putting seedlings;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the proportion of 5-10:2-5, then pouring into the cleaned pig casing, and binding two ends to ensure no leakage; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days;
s5, controlling the illumination intensity: the environmental illumination intensity is controlled to be 20-50Lux at 8-20 points every day, and the environmental illumination intensity is controlled to be 0Lux at other times.
Further, in the step S1, the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm × 25cm drainage port is arranged in the middle of the bottom of the net cage, and a 20-mesh escape-preventing net is installed.
Further, in the step S2, the seedlings are thrown according to 1500 g/box, that is, 5000 leeches/box with the specification of 0.3 g/leech.
Further, in the step S4, the nutrient solution is an aqueous solution of nutrient substances consisting of proteins, complex vitamins, complex amino acids, complex enzymes and preventive drugs, and the concentration of the nutrient solution is 20-30mg/ml.
Further, in the step S4, the nutrient substances are composed of the following substances by mass ratio:
further, in the step S4, the nutrient substances are composed of the following substances by mass ratio:
further, in the step S4, the protein is soluble fish protein; the compound vitamin is compound of vitamin A, B, B2, C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Further, in the step S4, the preventive medicine is radix isatidis, coptis chinensis, golden cypress, scutellaria baicalensis and hawthorn in a mass ratio of 3-5:0.5-1.5:0.5-1.5:0.5-1.5:2-5, adding water, decocting with strong fire, and making into medicinal liquid with concentration of 120-180 mg/ml.
Further, in the step S4, the preventive medicine is radix isatidis, coptis chinensis, phellodendron, scutellaria baicalensis and hawthorn in a mass ratio of 4:1:1:1:3 adding water and decocting with strong fire to obtain 150mg/ml liquid medicine.
Further, in the step S4, the nutrient solution and the fresh pig blood are mixed according to a ratio of 7:3, mixing uniformly according to the proportion.
Further, in step S5, the ambient light intensity is controlled as follows: 40Lux in the morning, 25Lux in the afternoon, and 0Lux in other times.
Compared with the prior art, the invention has the following advantages:
1. the illumination intensity of the living environment of the leech is accurately controlled, and the antithrombin activity and the growth speed of the leech are greatly improved.
2. The nutrient solution is added into the bait, so that the problems of insufficient nutrition and unbalanced nutrition caused by a single blood bait source are solved, simultaneously, the problems of dyspepsia caused by blood sucking of the leech or related diseases caused by bacterial infection are solved, and the antithrombin activity and the growth speed of the leech are improved.
3. Fresh pig blood is used as a phagostimulant, and the optimal proportion is determined, so that the problem of poor ingestion of the artificial compound feed is solved.
4. Greatly relieving the problem of blocking large-scale leech cultivation caused by insufficient fresh blood in partial areas.
Detailed Description
The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Those skilled in the art can make insubstantial modifications and adaptations to the embodiments described above while remaining within the scope of the invention.
Example 1
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged at the middle position of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 pieces/box of 0.3 g/piece leech of specification is thrown;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the proportion of 5:2, uniformly mixing the components in proportion, then filling the mixture into a clean pig casing, and binding two ends of the pig casing to ensure that the pig casing does not leak; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; the nutrient solution is a nutrient substance water solution consisting of protein, compound vitamin, compound amino acid, compound enzyme and preventive medicine, and the concentration of the nutrient solution is 20mg/ml.
S5, controlling the illumination intensity: the environmental illumination intensity is controlled to be 20Lux at 8-20 points every day, and the environmental illumination intensity is controlled to be 0Lux at other times.
In the step S4, the nutrient substances consist of the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is compound of vitamin A, B, B2, C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Wherein the preventive medicine is prepared from radix isatidis, goldthread root, phellodendron, scutellaria baicalensis and hawthorn according to the mass ratio of 3:1.5:0.5:1.5:2 adding water and decocting with strong fire to obtain a liquid medicine with the concentration of 120 mg/ml.
Example 2
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged in the middle of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 pieces/box of 0.3 g/piece leech of specification is thrown;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the proportion of 7:3, uniformly mixing the components in proportion, then filling the mixture into a clean pig casing, and binding two ends of the pig casing to ensure that the pig casing does not leak; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; the nutrient solution is a nutrient substance water solution consisting of protein, compound vitamin, compound amino acid, compound enzyme and preventive medicine, and the concentration of the nutrient solution is 25mg/ml.
S5, controlling the illumination intensity: controlling the ambient light intensity as follows: 40Lux in the morning, 25Lux in the afternoon, and 0Lux in other times.
In the step S4, the nutrient substances consist of the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is a compound of A, B, B2 and C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Wherein the preventive medicine is prepared from radix isatidis, goldthread root, phellodendron, scutellaria baicalensis and hawthorn in a mass ratio of 4:1:1:1:3 adding water and decocting with strong fire to obtain a liquid medicine with the concentration of 150 mg/ml.
Example 3
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged at the middle position of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 leeches/box with the specification of 0.3 g/leech;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the weight ratio of 10:5, uniformly mixing the components in proportion, then filling the mixture into a clean pig casing, and binding two ends of the pig casing to ensure that the pig casing does not leak; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; the nutrient solution is a nutrient substance water solution consisting of protein, compound vitamin, compound amino acid, compound enzyme and preventive medicine, and the concentration of the nutrient solution is 30mg/ml.
S5, controlling the illumination intensity: the environmental illumination intensity is controlled to be 50Lux at 8-20 points every day, and the environmental illumination intensity is controlled to be 0Lux at other times.
In the step S4, the nutrient substances consist of the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is compound of vitamin A, B, B2, C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Wherein the preventive medicine is prepared from isatis root, goldthread root, phellodendron bark, scutellaria and hawthorn fruit according to the mass ratio of 5:0.5:1.5:0.5:5 adding water, decocting with strong fire, and making into 180mg/ml liquid medicine.
Example 4
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged in the middle of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 pieces/box of 0.3 g/piece leech of specification is thrown;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the weight ratio of 10:2, uniformly mixing the components in proportion, then filling the mixture into a clean pig casing, and binding two ends of the pig casing to ensure that the pig casing does not leak; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; the nutrient solution is a nutrient substance water solution consisting of protein, compound vitamin, compound amino acid, compound enzyme and preventive medicine, and the concentration of the nutrient solution is 28mg/ml.
S5, controlling the illumination intensity: the environmental illumination intensity is controlled to be 30Lux at 8-20 points every day, and is controlled to be 0Lux at other times.
In the step S4, the nutrient substances consist of the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is compound of vitamin A, B, B2, C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Wherein the preventive medicine is prepared from radix isatidis, goldthread root, phellodendron, scutellaria baicalensis and hawthorn in a mass ratio of 4:1:1:1:2.5 adding water and decocting with strong fire to obtain a liquid medicine with the concentration of 160 mg/ml.
Comparative example 1 (example 2 without nutrient solution)
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged at the middle position of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 leeches/box with the specification of 0.3 g/leech;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: filling fresh pig blood into a cleaned pig casing, and binding two ends to ensure no leakage; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days;
s5, controlling the illumination intensity: controlling the ambient light intensity as follows: 40Lux in the morning, 25Lux in the afternoon, and 0Lux in other times.
Comparative example 2 (maintaining Low intensity illumination unlike example 2 illumination conditions)
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged in the middle of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 pieces/box of 0.3 g/piece leech of specification is thrown;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the weight ratio of 7:3, uniformly mixing the components in proportion, then filling the mixture into a clean pig casing, and binding two ends of the pig casing to ensure that the pig casing does not leak; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; the nutrient solution is a nutrient substance water solution consisting of protein, compound vitamin, compound amino acid, compound enzyme and preventive medicine, and the concentration of the nutrient solution is 25mg/ml.
S5, controlling the illumination intensity: 0-10Lux is kept at 8-20 points every day, and 0Lux is kept at other times.
In the step S4, the nutrient substances consist of the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is compound of vitamin A, B, B2, C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Wherein the preventive medicine is prepared from radix isatidis, goldthread root, phellodendron, scutellaria baicalensis and hawthorn in a mass ratio of 4:1:1:1:3 adding water and decocting with strong fire to obtain 150mg/ml liquid medicine.
Comparative example 3 (maintaining higher intensity illumination, different from the illumination conditions of example 2)
A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, setting a net cage: the length, width and height of the net cage are respectively 120cm, 60cm and 45cm, a 25cm multiplied by 25cm drainage port is arranged in the middle of the bottom of the net cage, and a 20-mesh escape-proof net is installed;
s2, seedling throwing: the seedling throwing density is 1500 g/box, namely 5000 leeches/box with the specification of 0.3 g/leech;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the proportion of 7:3, uniformly mixing the components in proportion, then filling the mixture into a clean pig casing, and binding two ends of the pig casing to ensure that the pig casing does not leak; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days; the nutrient solution is a nutrient substance water solution consisting of protein, compound vitamin, compound amino acid, compound enzyme and preventive medicine, and the concentration of the nutrient solution is 25mg/ml.
S5, controlling the illumination intensity: controlling the ambient light intensity as follows: keeping 150-200Lux at 8-20 points every day, and keeping 0Lux at other times.
In the step S4, the nutrient substances consist of the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is a compound of A, B, B2 and C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
Wherein the preventive medicine is prepared from isatis root, goldthread root, phellodendron bark, scutellaria and hawthorn in a mass ratio of 4:1:1:1:3 adding water and decocting with strong fire to obtain 150mg/ml liquid medicine.
Antithrombin activity, survival rate, and weight gain assays were performed on examples 1-4 and comparative examples 1 and 2:
the antithrombin activity detection method comprises the following steps:
directly beating the leech cultured for 60 days into leech slurry, accurately weighing about 1g of the leech slurry, accurately adding 5ml of 0.9% sodium chloride solution into a 5ml big tripe pipette, fully and uniformly stirring, covering a plug, leaching for 30 minutes, shaking all the time, putting into a centrifuge, and centrifuging for 5 minutes at the rotating speed of 2000 r/min. The solution was decanted into another 10ml centrifuge tube and placed in a centrifuge at 2000r/min for 5 minutes. 100. Mu.l of the supernatant was measured with a 100. Mu.l sample feeder, placed in a 9X 110mm cuvette, 200. Mu.l of tris (hydroxymethyl) aminomethane hydrochloride buffer (ready for use) containing 0.5% (bovine) fibrinogen (as coagulum) was added with a 500. Mu.l sample feeder, shaken, and immersed in a water bath (37 ℃ C. + -0.5 ℃ C.) of a disintegrator at medium temperature for 5 minutes. A100. Mu.l sample applicator was used to add 56 units or 43 units of thrombin solution per 1ml (5. Mu.l each) to the coagulation, and the number of volumes of thrombin solution consumed was recorded and calculated according to the following formula:
in the formula: u-per 1g antithrombin activity unit, U/g;
c1-concentration of thrombin solution, U/ml;
c2-concentration of the test solution, g/ml;
v1 — volume of thrombin solution consumed, μ l;
v2-amount of test solution added, μ l.
The antithrombin activity, survival rate and weight gain rate tests carried out in examples 1 to 4 and comparative examples 1 and 2 gave the following results:
| antithrombin Activity (u/g) | Survival rate (%) | Percentage of increase (%) | |
| Example 1 | 129 | 95 | 433 |
| Example 2 | 152 | 98 | 526 |
| Example 3 | 133 | 91 | 439 |
| Example 4 | 145 | 96 | 502 |
| Comparative example 1 | 90 | 65 | 133 |
| Comparative example 2 | 80 | 78 | 166 |
| Comparative example 3 | 92 | 80 | 172 |
Compared with experimental data, in the culture period of 60 days, the antithrombin for culturing the leeches reaches 129-152u/g, the survival rate reaches 91-98%, the weight gain rate reaches 433-526%, and all data are obviously superior to those of the traditional culture method.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements, etc. made by those skilled in the art within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A cultivation method for improving the antithrombin activity of leeches comprises the following steps:
s1, arranging a net cage;
s2, putting seedlings;
s3, water quality management: the culture water adopts well water, the water temperature is kept within the range of 22-28 ℃, the PH value is kept within the range of 6.8-7.2, and the water body in the culture tank adopts micro-flowing water;
s4, bait feeding: mixing the nutrient solution and fresh pig blood according to the proportion of 5-10:2-5, then pouring into the clean pig casing, and binding the two ends to ensure no leakage; putting the leech into a leech culturing box, sucking leeches, and feeding again at intervals of 7 days;
s5, controlling the illumination intensity: the environmental illumination intensity is controlled to be 20-50Lux at 8-20 points every day, and the environmental illumination intensity is controlled to be 0Lux at other times.
2. The cultivation method for improving the antithrombin activity of leeches according to claim 1, wherein in the step S1, the net cage is arranged to be 120cm, 60cm and 45cm in length, width and height respectively, a 25 cm-by-25 cm drainage port is arranged in the middle of the bottom of the net cage, and a 20-mesh escape-preventing net is installed.
3. The cultivation method for improving the activity of leech antithrombin enzyme according to claim 1, wherein in the step S2, the seeding throwing density is 1500 g/box, namely 5000 pieces/box of leech with the specification of 0.3 g/piece.
4. The cultivation method for improving the antithrombin activity of leeches according to claim 1, wherein in the step S4, the nutrient solution is a nutrient solution consisting of protein, compound vitamins, compound amino acids, compound enzymes and preventive medicines, and the concentration of the nutrient solution is 20-30mg/ml.
5. The cultivation method for improving the antithrombin activity of leeches as claimed in claim 4, wherein in the step S4, the nutrient substances comprise the following substances in percentage by mass:
6. the cultivation method for improving the antithrombin activity of leeches as claimed in claim 5, wherein in the step S4, the nutrient substances comprise the following substances in percentage by mass:
wherein the protein is soluble fish protein; the compound vitamin is a compound of A, B, B2 and C, D, E; the compound amino acid is a compound of lysine, cysteine and histidine; the complex enzyme is a complex of cellulase, amylase, pectinase, xylanase, mannase, neutral protease and a carrier.
7. The cultivation method for improving the activity of leech antithrombin enzyme according to claim 1, wherein in the step S4, the preventive medicine is radix isatidis, coptis chinensis, phellodendron amurense, scutellaria baicalensis and hawthorn in a mass ratio of 3-5:0.5-1.5:0.5-1.5:0.5-1.5:2-5, adding water, decocting with strong fire, and making into medicinal liquid with concentration of 120-180 mg/ml.
8. The cultivation method for improving the activity of leech antithrombin enzyme according to claim 7, wherein in the step S4, the preventive medicine is radix isatidis, coptis chinensis, phellodendron amurense, scutellaria baicalensis and hawthorn in a mass ratio of 4:1:1:1:3 adding water and decocting with strong fire to obtain 150mg/ml liquid medicine.
9. The cultivation method for improving leech antithrombin activity according to any one of claims 1 to 8, wherein in the step S4, the nutrient solution and the fresh pig blood are mixed according to the ratio of 7:3, mixing uniformly according to the proportion.
10. The cultivation method for improving leech antithrombin activity according to any one of claims 1 to 8, wherein in the step S5, the environmental light intensity is controlled as follows: 40Lux in the morning, 25Lux in the afternoon, and 0Lux in other times.
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