CN111802535A - Natural composition for improving fish anti-stress response and application thereof - Google Patents
Natural composition for improving fish anti-stress response and application thereof Download PDFInfo
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Abstract
The invention discloses a natural composition for improving fish anti-stress response, which comprises a natural compound anti-stress agent I and/or a natural compound anti-stress agent II; the natural compound anti-stress agent I comprises two or more than two of salvia miltiorrhiza organic solvent extract, perilla leaf organic solvent extract and angelica sinensis organic solvent extract; the natural compound anti-stress agent II comprises two or more of water extract of Saviae Miltiorrhizae radix, water extract of folium Ginkgo and water extract of folium Mori. The natural composition provided by the invention is used for preparing the anti-hypoxia reoxygenation stress response additive or feed for the fish, is a natural plant extract, and other substances are not artificially added into the basic feed raw materials. The natural functional feed can improve the tolerance of the cultured fish to oxygen deficiency and the reoxygenation survival rate, and has no residue, no toxic or side effect and no environmental pollution.
Description
Technical Field
The invention relates to the technical field of freshwater fish culture, in particular to a natural composition for improving anti-stress reaction of fish and application thereof.
Background
The current situation of aquaculture is mainly natural water body culture, and freshwater culture is mainly pond culture. The growth of farmed fish depends on the dissolved oxygen content in the water body. However, natural water body cultivation has the disadvantage that dissolved oxygen in water body is greatly influenced by environmental factors. The dissolved oxygen content in natural water is affected by day and night, weather and seasonal changes. The photosynthesis of green plants in the water body is very active in sunny days, and the dissolved oxygen content of the water body may reach an oversaturated state; the dissolved oxygen in the water body is greatly reduced due to the stagnation of the photosynthesis of the plants at night. Weather changes can affect illumination and photosynthesis of plants, thereby affecting dissolved oxygen in water. Seasonal changes can affect not only the light but also the water temperature. In the physiological temperature range of the fish, the oxygen consumption of the fish body is increased by 2-3 times when the water temperature is increased by 10 ℃. It has been reported that the fluctuation of dissolved oxygen in natural water body can make fish body suffer from oxygen deficiency and reoxygenation stress, so that the production performance of fish is reduced.
"high density, intensification" is an important feature of current aquaculture. But the fish carrying amount of the water body is improved by the high-density culture mode; the relative living space of the fishes in the water body is reduced, the competition and interference among different individuals are intensified, the activity of the fishes is improved, and the factors improve the oxygen demand of the fishes to different degrees. The high-density culture improves the feed feeding amount, increases culture wastes in the water body, and aggravates the conversion of water body substances, and the factors improve the oxygen consumption in the water body. The above situation further aggravates the anaerobic stress of the cultured fish. The oxygen-increasing machine can greatly relieve the oxygen deficiency state of the high-density aquaculture water body, but also aggravates the possibility that the fish body suffers reoxygenation stress. The phenomenon of pond turning over and fish death caused by the change of dissolved oxygen in the water body still often occurs. The anoxia and reoxygenation stress reaction can reduce the immunity of the fish and improve the morbidity and mortality of the fish, thereby improving the use amount of antibiotics and chemical drugs in the culture and increasing the drug residue of fish meat. The defects of natural water body culture and high-density culture make the market urgently need a feed which can improve the anti-hypoxia and reoxygenation stress capability of the fish without residue, toxic or side effect and environmental pollution, and is used for improving the culture benefit, reducing the drug residue and improving the fish meat quality.
High-density intensive culture requires the use of complete formula feed. The addition of antioxidants, mildewcides, growth promoters, phagostimulants and the like to compound feeds is a common phenomenon. The major antioxidants currently used are mainly synthetic antioxidants, including Ethoxyquinoline (EQ), Butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), and the like. The fish meal and the grease are important feed raw materials, and the adding amount of the synthetic antioxidant in the fish meal processing process can reach 0.1 percent; synthetic antioxidants are also added to feed-grade oils. However, synthetic antioxidants have been reported to be carcinogenic and genotoxic to animals and to cause residues and enrichment in fish products, creating a food safety hazard and endangering human health. In 2 months in 2017, the European Union issues the G/SPS/EU/190 announcement in 2017: the addition of EQ to the animal feed was stopped. Also, BHT and BHA added to animal feed have been reported to have higher carcinogenic risk than EQ. Chemically synthesized additives such as mildewcides, growth promoters, phagostimulants and the like have also been reported to be potentially toxic to animals and may cause residues in fish meat. The disadvantages of compound feeds will therefore lead to an increased market demand for natural product compound feeds.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides a natural composition for improving fish anti-stress response and an application thereof, wherein the natural composition can solve the problems that fish are subjected to hypoxia reoxygenation stress aggravated by high-density and natural water body culture, the existing feed additives have toxic and side effects and even carcinogenic effects on animals, and can cause food safety hidden danger and human health danger caused by residue in fish meat. The compound is a natural compound prepared based on medicinal and edible Chinese herbal medicines, is used as an anti-hypoxia reoxygenation stress response additive for fish and for preparing natural functional feed, has no toxic or side effect, and has important significance for improving the economic and social benefits of the fish.
The invention is realized by the following technical scheme:
a natural composition for improving fish anti-stress response comprises natural compound anti-stress agent I and/or natural compound anti-stress agent II; the natural compound anti-stress agent I comprises two or more than two of salvia miltiorrhiza organic solvent extract, perilla leaf organic solvent extract and angelica sinensis organic solvent extract; the natural compound anti-stress agent II comprises two or more of water extract of Saviae Miltiorrhizae radix, water extract of folium Ginkgo and water extract of folium Mori.
The fish anti-stress reaction compound provided by the invention is a natural plant extract, and other substances are not artificially added. The natural functional feed prepared by taking the active substance can improve the tolerance of the cultured fish to oxygen deficiency and the reoxygenation survival rate, and has no residue, no toxic or side effect and no environmental pollution.
Preferably, the salvia miltiorrhiza organic solvent extract is salvia miltiorrhiza ethyl acetate extract, the perilla leaf organic solvent extract is perilla leaf ethyl acetate extract, and the angelica sinensis organic solvent extract is angelica sinensis ethyl acetate extract.
Preferably, the content of flavonoids in the salvia miltiorrhiza ethyl acetate extract is 82.53mg/g-97.60 mg/g; the content of flavonoids in the perilla leaf ethyl acetate extract is 64.45mg/g-70.28 mg/g; the content of polyphenols in the radix Angelicae sinensis ethyl acetate extract is 46.76mg/g-55.31 mg/g.
The red sage root ethyl acetate extract is obtained by extracting analytical grade ethyl acetate and is a brownish red extract, wherein the content of flavonoids is 82.53mg/g-97.60 mg/g. The folium Perillae ethyl acetate extract is obtained by extracting with analytical grade ethyl acetate, and is dark brown extract, wherein the content of flavonoids is 64.45mg/g-70.28 mg/g. The angelica ethyl acetate extract is obtained by extracting analytical grade ethyl acetate, and the extract is a brownish red extract, wherein the content of polyphenol substances is 46.76mg/g-55.31 mg/g.
Further preferably, in the salvia miltiorrhiza water extract, the mass percentage of crude protein is 14.23% -16.08%, and the mass percentage of soluble sugar is 38.89% -46.30%; in the ginkgo leaf water extract, the mass percentage of crude protein is 5.76-7.14%, and the mass percentage of soluble sugar is 51.32-58.29%; in the mulberry leaf water extract, the mass percentage of crude protein is 13.07-15.64%, and the mass percentage of total sugar is 35.67-42.54%.
The red sage root water extract is obtained by extracting with distilled water and is brownish red powder, the mass percentage of crude protein is 14.23-16.08 percent, and the mass percentage of soluble sugar is 38.89-46.30 percent. The water extract of mulberry leaves is obtained by extracting with distilled water and is dark brown powder, the mass percentage of crude protein is 13.07-15.64 percent, and the mass percentage of total sugar is 35.67-42.54 percent. The ginkgo leaf water extract is obtained by extracting through distilled water and is light brown powder, the mass percentage of crude protein is 5.76-7.14%, and the mass percentage of soluble sugar is 51.32-58.29%.
Preferably, the natural compound anti-stress agent I comprises three kinds of salvia miltiorrhiza organic solvent extract, perilla leaf organic solvent extract and angelica sinensis organic solvent extract, and the mass ratio of the salvia miltiorrhiza organic solvent extract, the perilla leaf organic solvent extract and the angelica sinensis organic solvent extract is 2:1:1 or 1:2:1 or 1:1:2 or 2:2:1 or 1:2:2 or 2:1: 2.
Further preferably, the natural compound anti-stress agent II comprises three of a salvia miltiorrhiza water extract, a ginkgo biloba leaf water extract and a mulberry leaf water extract, and the mass ratio of the salvia miltiorrhiza water extract, the ginkgo biloba leaf water extract and the mulberry leaf water extract is 2:1:1 or 1:2:1 or 1:1:2 or 2:2:1 or 1:2:2 or 2:1: 2.
Further preferably, the mass ratio of the natural compound anti-stress agent I to the natural compound anti-stress agent II is 0.3-5.
The application of the natural composition for improving the anti-stress response of the fish is used for preparing an anti-stress response additive or feed for freshwater fish; the stress response includes an anoxic reoxygenation stress response. The feed herein is preferably a natural functional feed.
A fish feed comprises a basal feed and the natural composition for improving the anti-stress response of fish.
Further preferably, the adding amount of the natural compound anti-stress agent I is 0.3 to 0.5 percent of the weight of the basic feed; the addition amount of the natural compound anti-stress agent II is 0.5 to 1.0 percent of the weight of the basic feed.
Further preferably, the basic feed comprises the following components in parts by weight: 15-19 parts of fish meal, 20-25 parts of soybean meal, 15-17 parts of peanut meal, 3-7 parts of cottonseed meal, 0.3-0.7 part of DL-methionine, 32-36 parts of wheat flour, 1.0-1.5 parts of rapeseed oil, 0.5-1.0 part of vitamin compound and 0.5-1.0 part of mineral compound. Preferably, the basic feed comprises the following components in parts by weight: 17 parts of fish meal, 23 parts of soybean meal, 16 parts of peanut meal, 5 parts of cottonseed meal, 0.5 part of DL-methionine, 35 parts of wheat flour, 1.5 parts of rapeseed oil, 1 part of vitamin compound and 1 part of mineral compound.
The main raw materials of the anti-fish hypoxia reoxygenation feed are all natural products, and other substances are not artificially added; the additive components meet the requirements of national standard NY/T471-2018; the functional components of the feed additive are natural plant extracts, and the feed additive has certain functions of resisting feed oxidation and preventing mildew. The natural functional feed can improve the tolerance of the fish to oxygen deficiency and the reoxygenation survival rate in the culture.
The preparation method of the fish feed comprises the steps of dissolving the natural compound anti-stress agent I in grease, mixing, adding into the basic feed, and mixing; and/or dissolving natural compound anti-stress agent II in water, mixing, adding into basic feed, and mixing.
For example, in preparing a feed containing natural compound anti-stress agent I and natural compound anti-stress agent II: firstly, calculating the mass of oil required by unit mass of basic feed according to a feed formula, adding a natural compound anti-stress agent I accounting for 0.3-0.5% of the weight of the basic feed into the oil, and then uniformly mixing the oil and the basic feed; and dissolving the natural compound anti-stress agent II with the weight of 0.5-1.0% of the weight of the basic feed into drinkable water, and uniformly mixing the dissolved natural compound anti-stress agent II into the basic feed to prepare pellet feed for inhibiting the anoxic reoxygenation stress reaction of the freshwater fish.
The invention has the following advantages and beneficial effects:
1. the fish anti-stress reaction compound provided by the invention is a natural plant extract, and other substances are not artificially added. The natural functional feed prepared by taking the active substance can improve the tolerance of the cultured fish to oxygen deficiency and the reoxygenation survival rate, and has no residue, no toxic or side effect and no environmental pollution.
2. The invention adopts the extract of natural plants with homology of medicine and food as the feed additive, removes most ineffective components to enhance the activity, and the comprehensive functions of the medicinal plants are decomposed after extraction, and the specific functions are concentrated, thereby improving the effect of a certain function. The plant extract has reduced volume compared with Chinese herbal medicine, and is convenient for storage, transportation and transaction.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a graph showing the effect of feeding compound ester extract on the duration of hypoxia tolerance of crucian; in the figure, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper case letters in the same column data indicates significant difference (P < 0.05);
fig. 2 shows the effect of feeding compound ester extract on the reoxygenation recovery of crucian after oxygen deficiency and side turning; in the figure, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper case letters in the same column data indicates significant difference (P < 0.05);
FIG. 3 is the effect of the feed of the compound water extract on the duration of hypoxia tolerance of crucian; in the figure, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper case letters in the same column data indicates significant difference (P < 0.05);
FIG. 4 shows the effect of feeding the feed with the compound water extract on the reoxygenation recovery of crucian after oxygen deficiency and rollover; in the figure, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper case letters in the same column data indicates significant difference (P < 0.05);
FIG. 5 is the effect of feeding compound anti-stress agent feed on the duration of hypoxia tolerance of crucian; in the figure, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper case letters in the same column data indicates significant difference (P < 0.05);
fig. 6 shows the effect of feeding a compound anti-stress agent on the reoxygenation recovery of crucian after oxygen deficiency and side turning; in the figure, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Examples
The embodiment provides a natural composition for improving the anti-stress response of fish and application thereof.
Natural composition for improving fish anti-stress response
1) Organic solvent ester extract
The red sage root ethyl acetate extract is obtained by extracting analytical grade ethyl acetate and is a brownish red extract, wherein the content of flavonoids is 82.53mg/g-97.60 mg/g.
The folium Perillae ethyl acetate is obtained by extracting with analytical grade ethyl acetate, and is dark brown extract, wherein the content of flavonoids is 64.45mg/g-70.28 mg/g.
The angelica ethyl acetate is obtained by extracting analytical grade ethyl acetate and is a brownish red extract, wherein the content of polyphenol substances is 46.76mg/g-55.31 mg/g.
The mulberry leaf ethyl acetate extract is obtained by extracting analytical grade ethyl acetate, is dark brown extract, and has polyphenol substance content of 71.62mg/g-76.20 mg/g.
The folium Ginkgo ethyl acetate extract is obtained by analytical grade ethyl acetate extraction, is brown extract, and has flavonoid content of 67.43g/kg-72.71 g/kg.
2) Aqueous extracts
The red sage root water extract is obtained by extracting with distilled water and is brownish red powder, the mass percentage of crude protein is 14.23-16.08 percent, and the mass percentage of total sugar is 38.89-46.30 percent.
The water extract of the perilla leaf is obtained by extracting with distilled water and is dark brown powder, the mass percentage of crude protein is 15.52 to 17.90 percent, and the mass percentage of total sugar is 31.36 to 35.49 percent.
The water extract of angelica is obtained by extracting with distilled water, and is light red yellow powder, the mass percentage of crude protein is 19.05-22.56%, and the mass percentage of total sugar is 31.64-35.15%.
The water extract of mulberry leaves is obtained by extracting with distilled water and is dark brown powder, the mass percentage of crude protein is 13.07-15.64 percent, and the mass percentage of total sugar is 35.67-42.54 percent.
The ginkgo leaf water extract is obtained by extracting through distilled water and is light brown powder, the mass percentage of crude protein is 5.76-7.14%, and the mass percentage of total sugar is 51.32-58.29%.
Second, basic feed
The used raw material sources are as follows: the fish meal, the bean pulp, the peanut meal, the cottonseed meal, the wheat flour and the rapeseed oil are all self-made, and other substances are not added artificially except the nutrients of the materials. The specific formulation is shown in table 1:
TABLE 1 basic feed formulation
Remarking:
1the fish meal contains 89.88% of dry matter, 51.70% of crude protein, 15.01% of crude fat and 18.63% of crude ash, and is prepared from low-cost silver carp, crucian carp and grass carp by air-blast drying at 80 deg.C.
2The soybean meal contains 89.13% of dry matter, 46.92% of crude protein, 1.53% of crude fat and 5.46% of crude ash, and is a byproduct of soybean after heating, crushing, oil extraction and solvent evaporation.
3The peanut meal contains 88.21% of dry matter, 47.54% of crude protein, 1.42% of crude fat and 5.33% of crude ash, and is a byproduct of peanut kernels after heating, crushing, oil extraction and solvent evaporation.
4The cottonseed meal contains 89.46% of dry matter, 44.37% of crude protein, 0.54% of crude fat and 5.82% of crude ash, and is a byproduct of cottonseed kernels after heating, crushing, oil extraction and solvent evaporation.
5DL-methionine meets the requirement of NY/T471-2018.
6Wheat flour is obtained by pulverizing wheat, and contains dry matter 87.60%, crude protein 13.32%, crude fat 2.03%, and crude ash 1.84%.
7Rapeseed oil contained 99.03% dry matter, 0.00% crude protein, 98.00% crude fat and 0% crude ash53% from rapeseed by pressing.
8The vitamin supplement comprises per kilogram: 500,000IU of vitamin A acetate, 20.00g of vitamin D3240,000IU, 20.00g of DL-alpha-tocopherol, K30.10g of vitamin, 10.10g of vitamin B, 0.50g of riboflavin, 0.92g of pyridoxine hydrochloride, 0.001g of cyanocobalamine, 2.46g of D-calcium pantothenate, 2.80g of nicotinic acid, 0.10g of D-biotin, 51.81g of inositol and 0.50g of folic acid, and the carrier is defatted rice bran. The components meet the requirements of NY/T471-2018.
9The mineral supplement comprises per kilogram: FeSO437.90g, 1.69g of copper methionine, 23.71g of zinc methionine, 7.15g of manganese methionine, 0.06g of selenium methionine and 0.134g of KI, and the diluent is limestone powder. The components meet the requirements of NY/T471-2018.
Screening of anti-stress combination
1. Natural composite anti-stress agent I
According to the formula of the basic feed, 5 kinds of extracts are respectively added into the grease of the basic feed at the concentration of 1g/kg according to the following table to prepare 6 kinds of corresponding pellet feeds. The experimental design is shown in table 2.
TABLE 2 organic solvent extract anti-Fish stress test design
Remarking: the basal feed without any extract is marked as 'blank', the feed with all 5 extracts is marked as 'control', and compared with the 'control', the salvia miltiorrhiza ethyl acetate extract, the perilla leaf ethyl acetate extract, the angelica sinensis ethyl acetate extract, the mulberry leaf ethyl acetate extract and the ginkgo leaf ethyl acetate extract are respectively marked as '1', '2', '3', '4' and '5'.
After crucian fries are purchased from the Yong Zhen in the city of Nejiang province in Sichuan province, the crucian fries are temporarily cultured in a culture room for one week. Selecting crucian carp fries 420 with equivalent weight (the difference is not more than 1%) and randomly dividing the crucian carp fries into 7 treatment groups, wherein each group comprises 3 fish tanks. The shape, size and color of each fish tank are the same, the size is 30 multiplied by 40cm, and an 800L/h flow aerator is arranged in each fish tank. The water in the fish tank takes drinkable tap water as a water source, and the volume of the water in the fish tank is 30L. The temperature of the culture room is controlled to be 22 +/-1 ℃, and the oxygen is continuously supplied by the oxygen increasing machine. The 7 treatment groups were fed with the feeds labeled "blank", "control", "1", "2", "3", "4" and "5", respectively, 4 times per day of satiation. And 7 days later, measuring the hypoxia tolerance of the crucian.
The determination method comprises the following steps: and (3) an airless sealing method, namely selecting 13 tails of crucian carps with the same weight of each treatment group from each fish tank, preparing 30mL of saturated dissolved oxygen water per g of fish body, and sealing in a plastic bottle in an airless manner. The duration of time from normal to loss of balance (rollover) and oxygen consumption rate were recorded and calculated for 10 fish among them. Rapidly transferring 10-tail crucian carp turned on side into saturated dissolved oxygen water, and recording the tail number of the crucian carp recovering to the normal swimming posture within 2 hours. The results of the tests are shown in table 2 and fig. 1 and 2.
As shown in table 2, and figures 1 and 2, the results show that feeding the complex extract increased the duration of hypoxia tolerance in the fish to varying degrees compared to the blank group. Duration sequence of hypoxia tolerance: 4-5 > control-2-3-1, 14.02h, 13.18h, 12.04h, 11.75h, 10.55h and 10.51h respectively. Restoring the fish tail number sequence by reoxygenation after anoxic rollover: the control is 8.00, 7.67, 6.33, 6.00, 3 with 5 > 2 ═ 1 ═ 3. Therefore, in the components of the composite extract, the ethyl acetate extracts of the mulberry leaves and the ethyl acetate extracts of the ginkgo leaves have an inhibiting effect on the anti-stress effect of other components of the composite extract; the perilla ethyl acetate extract, the salvia miltiorrhiza ethyl acetate extract and the angelica ethyl acetate extract play a promoting role. Therefore, the components of the primarily screened compound extract are salvia miltiorrhiza ethyl acetate extract, angelica sinensis ethyl acetate extract and perilla ethyl acetate extract, and are marked as natural compound anti-stress agent I.
TABLE 2 Effect of feeding Complex ester extracts on the hypoxia tolerance of Carassius auratus
Remarking: data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05). Oxygen consumption rate (end dissolved oxygen in water-initial dissolved oxygen in water) x water volume/fish body weight/duration.
2. Natural composite anti-stress agent II
According to the basal feed formulation, 5 extracts were added to the basal feed in a concentration of 2g/kg, respectively, by dissolving in drinkable water, according to the following table, to make 6 kinds of corresponding pellet feeds. The experimental design is shown in table 3 below.
TABLE 3 design of fish stress resistance test with complex water extract
| Additive material | | Control | 1 | 2 | 3 | 4 | 5 | |
| Water extract of red sage root | × | ○ | × | ○ | ○ | ○ | ○ | |
| Perilla leaf water extract | × | ○ | ○ | × | ○ | ○ | ○ | |
| Aqueous extract of Angelica sinensis | × | ○ | ○ | ○ | × | ○ | ○ | |
| Aqueous extract of mulberry leaves | × | ○ | ○ | ○ | ○ | × | ○ | |
| Aqueous extract of ginkgo leaf | × | ○ | ○ | ○ | ○ | ○ | × |
Remarking: the basal feed without any extract is marked as 'blank', the feed with all 5 kinds of extracts is marked as 'control', and the feeds with the water extracts of radix salviae miltiorrhizae, perilla leaf, angelica, mulberry leaf and ginkgo leaf subtracted one by one are respectively marked as '1', '2', '3', '4' and '5' compared with the 'control'.
After crucian fries are purchased from the Yong Zhen in the city of Nejiang province in Sichuan province, the crucian fries are temporarily cultured in a culture room for one week. Selecting crucian carp fries 420 with equivalent weight (the difference is not more than 1%) and randomly dividing the crucian carp fries into 7 treatment groups, wherein each group comprises 3 fish tanks. The shape, size and color of each fish tank are the same, the size is 30 multiplied by 40cm, and an 800L/h flow aerator is arranged in each fish tank. The water in the fish tank takes drinkable tap water as a water source, and the volume of the water in the fish tank is 30L. The temperature of the culture room is controlled to be 22 +/-1 ℃, and the oxygen is continuously supplied by the oxygen increasing machine. The 7 treatment groups were fed with the feeds labeled "blank", "control", "1", "2", "3", "4" and "5", respectively, 4 times per day of satiation. And 7 days later, measuring the hypoxia tolerance of the crucian.
The determination method comprises the following steps: and (3) an airless sealing method, namely selecting 13 tails of crucian carps with the same weight of each treatment group from each fish tank, preparing 30mL of saturated dissolved oxygen water per g of fish body, and sealing in a plastic bottle in an airless manner. The duration of time from normal to loss of balance (rollover) and oxygen consumption rate were recorded and calculated for 10 fish among them. Rapidly transferring 10-tail crucian carp turned on side into saturated dissolved oxygen water, and recording the tail number of the crucian carp recovering to the normal swimming posture within 2 hours. The test results are shown in table 4 and fig. 3 and 4.
As shown in table 4, and figures 3 and 4, the results show that feeding the complex extract increased the duration of hypoxia tolerance in the fish to varying degrees compared to the blank group. Duration sequence of hypoxia tolerance: 3 > 2 ═ control > 5 ═ 1 > 4, 12.23h, 11.31h, 11.24h, 10.26h, 10.21h, 9.27h, respectively. Restoring the fish tail number sequence by reoxygenation after anoxic rollover: 3-control > 2 > 1-5 > 4, 8.00, 7.67, 7.33, 6.33, 6.00 tails, respectively. Therefore, in the components of the compound extract, the water extract of the perilla leaf and the water extract of the angelica play a role in inhibiting the anti-stress effect of other components of the compound extract; the red sage root water extract, mulberry leaf water extract and ginkgo leaf water extract play a promoting role. Therefore, the components of the primarily screened compound water extract comprise a red sage root water extract, a mulberry leaf water extract and a ginkgo leaf water extract which are marked as compound anti-stress agents II.
TABLE 4 influence of Compound Water extract on the hypoxia tolerance of Carassius auratus
Remarking: data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05). Oxygen consumption rate (end dissolved oxygen in water-initial dissolved oxygen in water) x water volume/fish body weight/duration.
3. Compounding and verification of compound anti-stress agent
To verify the anti-stress effect, the compound anti-stress agent I (abbreviated as 'I') was added to the basal feed and compared with the feed No. 4 (abbreviated as 'control I') having the best effect in the component screening test I in the same breeding environment and conditions. The compound anti-stress agent II (abbreviated as 'II') is added into the basal feed and compared with the feed No. 3 (abbreviated as 'control II') with the best effect in the component screening test under the same culture environment and conditions. The feed containing the compound anti-stress agent I (the content of which is 0.3%) and the feed containing the compound anti-stress agent II (the content of which is 0.6%) are mixed in equal amount (the average content is 0.45%) (abbreviated as "(I + II)/2"), and the mixture is compared with the feed containing the compound anti-stress agent I and the feed containing the compound anti-stress agent II under the same culture environment and conditions. The blank group was fed with basal diet as well. The experimental design is shown in table 5.
TABLE 5 composite anti-stress agent test design
The crucian is fed under the same culture environment and conditions, and the hypoxia tolerance of the crucian is measured according to the same method. The results of the experiment (as shown in table 6, and fig. 5 and 6) show that feeding the complex extract improves the duration of hypoxia tolerance of the fish to a different extent compared to the blank group. Duration sequence of hypoxia tolerance: i > control I; II > control II; (I + II)/2 > I ═ II; restoring the fish tail number sequence by reoxygenation after anoxic rollover: i ═ control I; II > control II; (I + II)/2 > II > I. Therefore, the anti-stress effect of the compound anti-stress agent II is higher than that of the control, and the mixed effect of the two is better.
TABLE 6 influence of feeding compound anti-stress agent on the anoxia endurance of crucian carp
Note: data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05). Oxygen consumption rate (end dissolved oxygen in water-initial dissolved oxygen in water) x water volume/fish body weight/duration.
4. Verification of anti-erythrocyte apoptosis ability of composite anti-stress agent
(1) The verification method comprises the following steps:
from the above (compounding and verification of the compound anti-stress agent) fish not participating in the hypoxia tolerance determination, 15 fish were randomly selected for each treatment group and evenly distributed to 3 fish tanks (5 fish per tank); 0.6mg of Cu/L copper sulfate is dissolved in the water body of each fish tank. Feeding the natural compound anti-stress agent I, the natural compound anti-stress agent I and the compound anti-stress agent marked as above to each fish tank respectively, and feeding 4 times per day after satiation.
From the fish which do not participate in the hypoxia tolerance determination (compounding and verification of the compound anti-stress agent), 15 fish are randomly selected from each treatment group and are averagely distributed to 3 fish tanks (5 fish in each tank); 2.2mg/L of trichlorfon is dissolved in the water body of each fish tank. Feeding the natural compound anti-stress agent I, the natural compound anti-stress agent I and the compound anti-stress agent marked as above to each fish tank respectively, and feeding 4 times per day after satiation.
After 3 days, the tail vein of each fish is subjected to blood sampling, red blood cells are separated, and the apoptosis rate of the red blood cells is determined by utilizing an Annexin V-FITC kit and a flow cytometer.
(2) And (4) verification result:
as shown in table 7, the results showed that the feeding of the complex extract inhibited the copper sulfate or dipterex-induced apoptosis of red blood cells of crucian; the anti-erythrocyte apoptosis effect of the compound anti-stress agent (I + II)/2 is higher than that of I and II.
Note: data are presented as mean ± standard deviation of 3 replicates, 5 fish per replicate; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05).
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A natural composition for improving fish anti-stress response is characterized by comprising a natural compound anti-stress agent I and/or a natural compound anti-stress agent II; the natural compound anti-stress agent I comprises two or more than two of salvia miltiorrhiza organic solvent extract, perilla leaf organic solvent extract and angelica sinensis organic solvent extract; the natural compound anti-stress agent II comprises two or more of water extract of Saviae Miltiorrhizae radix, water extract of folium Ginkgo and water extract of folium Mori.
2. The natural composition for improving fish anti-stress response as claimed in claim 1, wherein the organic solvent extract of red sage root is an ethyl acetate extract of red sage root, the organic solvent extract of perilla leaf is an ethyl acetate extract of perilla leaf, and the organic solvent extract of angelica is an ethyl acetate extract of angelica.
3. The natural composition for improving the anti-stress response of fish as claimed in claim 1, wherein the natural compound anti-stress agent I comprises three kinds of organic solvent extracts of radix salviae miltiorrhizae, folium perillae and radix angelicae sinensis, and the mass ratio of the organic solvent extracts of radix salviae miltiorrhizae, folium perillae and radix angelicae sinensis is 2:1:1 or 1:2:1 or 1:1:2 or 2:2:1 or 1:2:2 or 2:1: 2.
4. The natural composition for improving the anti-stress response of fish as claimed in claim 1, wherein the natural compound anti-stress agent II comprises three kinds of water extracts of radix salviae miltiorrhizae, folium ginkgo and folium mori, and the mass ratio of the water extracts of radix salviae miltiorrhizae, folium ginkgo and folium mori is 2:1:1 or 1:2:1 or 1:1:2 or 2:2:1 or 1:2:2 or 2:1: 2.
5. The natural composition for improving fish anti-stress response as claimed in claim 1, wherein the mass ratio of the natural compound anti-stress agent I to the natural compound anti-stress agent II is 0.5-2.0.
6. Use of a natural composition for enhancing the anti-stress response of fish as claimed in any one of claims 1 to 5, for the preparation of an anti-stress response additive or feed for freshwater fish; the stress response includes an anoxic reoxygenation stress response.
7. A fish feed comprising a basal feed and a natural composition as claimed in any one of claims 1 to 7 for enhancing the anti-stress response of fish.
8. The fish feed as claimed in claim 7, wherein the natural compound anti-stress agent I is added in an amount of 0.3-0.5% by weight based on the weight of the basic feed; the addition amount of the natural compound anti-stress agent II is 0.5 to 1.0 percent of the weight of the basic feed.
9. The fish feed of claim 8, wherein the basic feed comprises the following components in parts by weight: the basic feed comprises the following components in parts by weight: 15-19 parts of fish meal, 20-25 parts of soybean meal, 15-17 parts of peanut meal, 3-7 parts of cottonseed meal, 0.3-0.7 part of DL-methionine, 32-36 parts of wheat flour, 1.0-1.5 parts of rapeseed oil, 0.5-1.0 part of vitamin compound and 0.5-1.0 part of mineral compound.
10. A process for preparing a fish feed as claimed in any one of claims 7 to 9, wherein the natural complex anti-stress agent I is dissolved in fat and mixed, and then added to the basal feed and mixed; and/or dissolving natural compound anti-stress agent II in water, mixing, adding into basic feed, and mixing.
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