Compound preparation for resisting fish stress and application thereof

Abstract

The invention relates to the technical field of fish stress resistance, in particular to a compound preparation for resisting fish stress and application of the compound preparation. The compound preparation is prepared from the following components in parts by weight: 1-100 parts of Chinese angelica, 1-100 parts of rosemary and 1-100 parts of perilla leaf extract. The compound preparation can improve the anti-stress capability of the pelteobagrus fulvidraco in an excessive feeding state after the temperature is rapidly changed, and alleviate the body resistance reduction of the pelteobagrus fulvidraco in a stress state, so as to improve the breeding survival rate.

Description

Technical Field

[0001] The present application relates to the technical field of fish stress resistance, and in particular to a fish stress resistance compound preparation and application thereof. Background Art

[0002] The stress response of fish refers to a non-specific physiological response of fish to abnormal stimulation of various environmental factors (also known as stressors). Excessive or prolonged stress response will harm the fish body, leading to slow growth and development, decreased reproductive capacity, low immune function, and increased morbidity and mortality. Common stressors include the first category of environmental factors, such as water temperature, salinity, dissolved oxygen, ammonia, pH, etc. The second category is physical interference factors, such as transportation, pool separation, sex sorting and other operations; the third category is biological factors, such as crowding and invasion of pathogenic microorganisms.

[0003] Common measures to reduce fish stress reactions mainly include the use of oxygenation facilities, feeding vitamins, adding zeolite and using microbial preparations. However, during large-scale fish diseases, diseased fish show extremely strong stress. For example, under the dual stress of rapid temperature changes and satiation, yellow catfish produces severe stress reactions, which affects the metabolic function of the fish and further leads to the imbalance of the fish's internal environmental homeostasis and the decline of disease resistance, making the fish more susceptible to bacteria, parasites and other diseases. Conventional measures are difficult to combat these stress reactions. Summary of the invention

[0004] In view of this, the inventors of the present application creatively disclosed a compound preparation for resisting fish stress, which can improve the anti-stress ability of yellow catfish under overfeeding state after rapid temperature change, alleviate the decreased resistance of yellow catfish under stress state caused by this, and improve the survival rate of breeding. To this end, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiment discloses a composite preparation for resisting fish stress, which comprises 1 to 100 parts by weight of angelica sinensis, 1 to 100 parts by weight of rosemary and 1 to 100 parts by weight of perilla leaf extract.

[0006] In some embodiments, the raw materials of the compound preparation include 30 to 80 parts by weight of angelica sinensis, 20 to 50 parts by weight of rosemary and 20 to 50 parts by weight of perilla leaf extract.

[0007] In some embodiments, the raw materials of the compound preparation include 40 parts of angelica sinensis, 30 parts of rosemary and 30 parts of perilla leaf extract by weight.

[0008] In some embodiments, the raw materials of the compound preparation include 40 parts of angelica sinensis, 20 parts of rosemary and 20 parts of perilla leaf extract by weight.

[0009] In some embodiments, the raw materials of the compound preparation include 40 parts of angelica, 30 parts of rosemary and 20 parts of perilla leaf extract by weight.

[0010] In some embodiments, the raw materials of the compound preparation include 40 parts of angelica, 20 parts of rosemary and 30 parts of perilla leaf extract by weight.

[0011] In the second aspect, the embodiment discloses a method for preparing a composite preparation for resisting fish stress, which comprises adding a formula amount of angelica, rosemary and perilla leaf extracts into water and boiling at 80-100° C. for 2-4 hours, filtering, concentrating and drying the filtrate, and obtaining the solid product as the composite preparation.

[0012] In a third aspect, the embodiments disclose the use of the compound preparation described in the first aspect in the preparation of an anti-stress preparation for fish.

[0013] In some embodiments, the application includes administering 0.3-1 ppm of the compound preparation to the water where the fish grow.

[0014] Chinese herbal medicine has the advantages of wide sources, multiple targets, and no toxic side effects, and is increasingly used in the prevention and treatment of aquatic diseases. In order to solve this problem, the inventor screened a variety of Chinese herbal medicines and formulated them. Finally, it was found that the combination of angelica, rosemary, and perilla leaf extracts in a ratio of 4:3:3 can play a good anti-stress role and has a good protective effect on yellow catfish under the dual stress of rapid temperature changes and satiation in early spring.

[0015] The main components of angelica are volatile oils, flavonoids, amino acids, organic acids and polysaccharides. Its nature and flavor are sweet, warm and pungent. It belongs to the heart, spleen and liver meridians. It has the effects of nourishing blood and promoting blood circulation, anti-inflammatory, anti-tumor, regulating menstruation and relieving pain, and enhancing immunity. The main components of rosemary are acid phenols, flavonoids, terpenes and essential oil compounds, which have multiple pharmacological effects such as antioxidant, antibacterial, anti-tumor, anti-inflammatory, and anti-depression. Perilla leaves contain a variety of biologically active substances, which are mainly divided into volatile oils, flavonoids, phenolic acids, anthocyanins, polysaccharides, triterpenes, steroid compounds, etc., and have good therapeutic effects such as anti-tumor, antibacterial, anti-inflammatory, anti-influenza, anti-allergic, antioxidant, anti-depressant, anti-cancer, detoxification, sedation and analgesia. With angelica as the main drug and rosemary and perilla leaf extracts as auxiliary drugs, a good anti-stress effect can be exerted through optimizing the ratio. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0017] 1. Compound preparations

[0018] Add the formula amount of angelica, rosemary and perilla leaf extracts to water and boil at 80-100°C for 2-4 hours, filter, concentrate and dry the filtrate, and the obtained solid is the compound preparation. The preparation of the perilla leaf extract includes weighing 200g of dried perilla leaves and extracting them with 50% ethanol reflux for 3 times, each time for 3 hours, filtering the residue, combining the extracts and concentrating under reduced pressure, and refrigerating for use. The term "portion" refers to any weight as one portion, which is only used to limit the weight ratio, and does not limit the specific absolute weight.

[0019] Table 1 Raw materials of compound preparations

[0020] Serial number Angelica (portion) Rosemary (part) Perilla leaf extract (part) Preparation 1 40 20 10 Preparation 2 40 20 20 Preparation 3 40 20 30 Preparation 4 40 30 10 Preparation 5 40 30 20 Preparation 6 40 30 30

[0021] Test 1

[0022] 1. Test materials and methods

[0023] The yellow catfish, with a size of about 10 g, was purchased from a farmer in Jiangxia District, Wuhan City.

[0024] The experiment was conducted in a glass aquarium (57.8cm×38.2cm×22.7cm). The test fish were randomly divided into 8 groups, including a control group (no constant temperature and no medication), a negative control group (variable temperature and no medication) and 6 test groups (variable temperature and medication), with 3 parallel groups in each group and 10 fish in each tank. The water temperature during the temporary culture period was 15℃. After the start of the experiment, the corresponding compound preparation was added to the water at 0.5ppm in each test group, and the water temperature was controlled to increase by 1 degree every 4h and rise to 25℃ within 48h. The water temperature of the control group remained unchanged, and the water temperature of the negative control group rose to 25℃ within 48h. Sampling was taken at 48h, and 3 fish were randomly selected from each tank. Blood was collected from the tail vein, and after standing for 30min, it was placed at 4℃ overnight, and centrifuged at 3000r / min for 10min to separate the serum sample, and the cortisol content in the serum was determined.

[0025] 2. Test results and analysis

[0026] As shown in Table 2, the content of cortisol was as follows: negative control group > preparation group 1 > preparation group 4 > preparation group 2 > preparation group 3 > preparation group 5 > preparation group 6 > control group. Among them, there was no significant difference between preparation group 1 and preparation group 4 and the negative control group (P>0.05), there was a significant difference between preparation group 2, preparation group 3, preparation group 5 and preparation group 6 and the negative control group (P<0.05), and there was no significant difference between preparation group 6 and the control group (P>0.05).

[0027] Table 2 Serum cortisol levels of yellow catfish in each group, shoulder marks indicate significant differences

[0028] Serial number Cortisol content (ng / mL) Preparation 1 <![CDATA[149.35±12.49 a ]]> Preparation 2 <![CDATA[127.36±14.27 b ]]> Preparation 3 <![CDATA[120.94±10.54 b ]]> Preparation 4 <![CDATA[136.31±9.93 a ]]> Preparation 5 <![CDATA[114.28±17.64 c ]]> Preparation 6 <![CDATA[106.53±13.58 d ]]> Control group <![CDATA[98.35±6.27 d ]]> Negative control group <![CDATA[156.42±10.54 a ]]>

[0029] From a physiological point of view, fish stress response is generally divided into primary, secondary and tertiary stress response according to its occurrence process. Primary stress response mainly includes the recognition of stressors and the initiation of biological defense. It is the body's response at the level of the nervous and endocrine systems, activating two stress response systems mainly based on the fish sympathetic-chromaffin system and the hypothalamus-pituitary-interrenal axis, and promoting the increase of catecholamines, cortisol and other stress hormone levels in the fish blood. Cortisol hormone is recognized as a good indicator for evaluating fish stress response because of its good stability, and the positive correlation between the increase amplitude and duration with the intensity and duration of the stressor. In this test, affected by the rapid increase in temperature, the serum cortisol content of yellow catfish in the negative control group increased significantly (P < 0.05), and after medication, the serum cortisol content of yellow catfish in the preparation 2, preparation 3, preparation 5 and preparation 6 groups decreased significantly (P < 0.05), indicating that it has the effect of improving the stress response of yellow catfish, among which the serum cortisol content of yellow catfish in the preparation 6 group has no significant difference with the control group (P > 0.05), and the anti-stress effect is the best. It can be seen that the compound preparation provided by the present application has an obvious anti-stress effect.

[0030] Test 2

[0031] 1. Test materials and methods

[0032] Pelteobagrus fulvidraco, with a size of about 10 g, was purchased from a farmer in Jiangxia District, Wuhan. To simulate the sudden change of temperature in spring, two groups of rapid temperature change groups were designed, one group was a rapid temperature increase group (rapidly increased from 15°C to 25°C), and the other group was a rapid temperature decrease group (rapidly decreased from 25°C to 15°C). The rapid temperature increase group and the rapid temperature decrease group were divided into a normal feeding group, an overfeeding group, and an overfeeding medication group (adding compound preparation 6 to the water body at 0.5ppm). Each group had three parallels, with 20 fish in each parallel. The fish in the rapid temperature decrease group were temporarily kept at a water temperature of 25°C for 7 days, without feeding during the temporary holding period, and the water temperature was cooled to 15°C within 48h after the temporary holding; the fish in the rapid temperature increase group were temporarily kept at a water temperature of 15°C for 7 days, without feeding during the temporary holding period, and the water temperature was heated to 25°C within 48h. Normal feeding amount: 1.5% of the body weight of the fish fed at low temperature (15°C), 3% of the body weight of the fish fed at suitable temperature (25°C); overfeeding: 2.5% of the body weight of the fish fed at low temperature, 4.5% of the body weight of the fish fed at suitable temperature. Sampling was carried out at 0h, 24h and 48h after the start of the experiment. Three experimental fish were randomly selected from each breeding tank, blood was collected from the tail vein, and after standing for 30min, the serum samples were separated by centrifugation at 3000r / min for 10min. The lysozyme activity, alkaline phosphatase activity and cortisol concentration in the serum were detected by conventional methods.

[0033] 2. Test results and analysis

[0034] As shown in Table 3, in the rapidly warming group, the cortisol content showed an upward trend in all groups, the medication group was slightly higher than the normal feeding group, and lower than the overfeeding group; the lysozyme activity increased with time, the overfeeding group was higher than the normal feeding group, the medication group was slightly higher than the normal group, and lower than the overfeeding group; the alkaline phosphatase activity in the normal feeding group first increased and then decreased, the overfeeding group showed a downward trend, and the medication group first increased and then decreased, but there was no significant difference in each time period (P>0.05).

[0035] In the rapid cooling group, cortisol showed an upward trend in all groups, and the medication group was slightly higher than the normal feeding group and lower than the overfeeding group; lysozyme activity first increased and then decreased with time, and the medication group was higher than the normal feeding group at 24 hours and lower than the overfeeding group, and at 48 hours, the medication group was higher than the normal feeding group; alkaline phosphatase activity in the normal feeding group first increased and then decreased, and the overfeeding group showed a downward trend, and the medication group first increased and then decreased, but there was no significant difference in each time period (P>0.05).

[0036] Studies have shown that the cortisol level in fish increases under stress conditions, and the severity of stress can be expressed by the degree and duration of this increase. Therefore, the cortisol content can be an important physiological indicator of stress levels. In different temperature-changing groups, overfeeding led to an increase in cortisol content. The cortisol content in the medication group was lower than that in the overfeeding group, indicating that the compound preparation can alleviate the stress caused by overfeeding after a sharp change in temperature.

[0037] Lysozyme is a non-toxic alkaline protein that is widely present in fish mucus, liver, digestive tract secretions, serum and lysosomes. It is an important non-specific immune factor. Alkaline phosphatase can catalyze the hydrolysis of phosphate monoesters and the conversion of phosphate groups, and is of great significance in the immune defense response of biological organisms. Therefore, lysozyme and alkaline phosphatase can be used to evaluate the body's ability to resist disease. From the results, it can be seen that overfeeding leads to an increase in the activity of lysozyme in the warming group, and the activity of lysozyme in the cooling group first increases and then decreases. Regardless of the warming or cooling group, alkaline phosphatase shows a downward trend, indicating that the stress response of overfeeding after a sharp change in temperature will cause the body resistance of yellow catfish to decrease. The activities of lysozyme and alkaline phosphatase in the medication group are higher than those in the overfeeding group, indicating that the compound preparation can improve the anti-stress ability of yellow catfish and alleviate the decrease in resistance under the stress state of overfeeding after a sharp change in temperature.

[0038] Table 3 Effects of the compound preparation on improving serum biochemical indices of yellow catfish under dual stress of temperature and satiation

[0039]

[0040] It can be seen from this that the compound preparation provided in the present application can improve the anti-stress ability of yellow catfish in an overfed state after a sudden change in temperature, and alleviate the decreased body resistance of yellow catfish in a stress state caused by this.

[0041] Test 3

[0042] 1. Materials and Methods

[0043] The experiment was conducted at a yellow catfish farmer in Jiangxia District, Wuhan City. During the spring temperature change period (April-May), the test pond was sprayed with 0.5ppm preparation 6 every other day for one month, and the control pond was not medicated. The survival of yellow catfish was observed during the period. At the end of the experiment, 10 fish were taken from each group, blood was collected from the tail vein, and serum samples were separated after standing for 30 minutes and centrifuged at 3000r / min for 10 minutes to measure relevant indicators.

[0044] 2. Test results and analysis

[0045] During the trial, a total of 32 fish died in the test group, and a total of 211 fish died in the control group. The effective rate of reducing spring mortality of farmed yellow catfish reached 84.8% ((211-32)÷211=84.8%).

[0046] Table 3 Changes in serum biochemical indicators

[0047] Cortisol (ng / mL) Lysozyme (U / mL) Alkaline phosphatase (U / L) Control group 134.26±1.33 243.48±12.47 18.39±0.52 Test Group 104.53±5.68* 324.72±16.2* 27.41±3.85*

[0048] Note: “*” indicates significant difference, P < 0.05

[0049] As shown in Table 3, the cortisol content was significantly lower than that of the control group (P < 0.05), and the lysozyme and alkaline phosphatase activities of the test group were significantly higher than those of the control group (P < 0.05). If fish are in a state of stress for a long time, changes such as reduced growth rate, reproductive capacity and disease resistance will occur at the individual or group level of fish. Studies have shown that the cortisol level in fish increases under stress conditions, and the severity of stress can be expressed by the degree and duration of this increase. Therefore, the cortisol content can be an important physiological indicator of stress level. The decrease in cortisol levels in the test group indicates that the stress pressure was relieved after medication.

[0050] Lysozyme is a non-toxic alkaline protein that is widely present in fish mucus, liver, digestive tract secretions, serum and lysosomes. It is an important non-specific immune factor. Alkaline phosphatase can catalyze the hydrolysis of phosphate monoesters and the conversion of phosphate groups, and is of great significance in the immune defense response of biological organisms. Therefore, lysozyme and alkaline phosphatase can be used to evaluate the body's ability to resist disease. The lysozyme activity and alkaline phosphatase activity in the serum of the yellow catfish in the test group were higher than those in the control group, indicating that the disease resistance of the yellow catfish treated with medication has been enhanced.

[0051] It can be seen from this that the compound preparation provided in the present application can improve the stress resistance of cultured yellow catfish and increase the survival rate of culture.

[0052] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A composite preparation for resisting fish stress, comprising 1 to 100 parts by weight of angelica, 1 to 100 parts by weight of rosemary and 1 to 100 parts by weight of perilla leaf extract.

2. The compound preparation according to claim 1, wherein the raw materials of the compound preparation include 30 to 80 parts by weight of angelica sinensis, 20 to 50 parts by weight of rosemary and 20 to 50 parts by weight of perilla leaf extract.

3. The compound preparation according to claim 1, wherein the raw materials of the compound preparation include 40 parts by weight of angelica sinensis, 30 parts by weight of rosemary and 30 parts by weight of perilla leaf extract.

4. The compound preparation according to claim 1, wherein the raw materials of the compound preparation include 40 parts by weight of angelica, 20 parts by weight of rosemary and 20 parts by weight of perilla leaf extract.

5. The compound preparation according to claim 1, wherein the raw materials of the compound preparation include 40 parts by weight of angelica sinensis, 30 parts by weight of rosemary and 20 parts by weight of perilla leaf extract.

6. The compound preparation according to claim 1, wherein the raw materials of the compound preparation include 40 parts by weight of angelica sinensis, 20 parts by weight of rosemary and 30 parts by weight of perilla leaf extract.

7. A method for preparing a composite preparation for resisting fish stress, comprising adding a formula amount of angelica, rosemary and perilla leaf extracts into water and boiling at 80-100° C. for 2-4 hours, filtering, concentrating and drying the filtrate, and obtaining the solid product as the composite preparation.

8. Use of the compound preparation according to any one of claims 1 to 6 in the preparation of an anti-stress preparation for fish.

9. The use according to claim 8, comprising administering 0.3-1 ppm of the compound preparation to the water body where the fish grow.

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