CN111802535B - Natural composition for improving anti-stress response of fish and application thereof - Google Patents
Natural composition for improving anti-stress response of fish and application thereof Download PDFInfo
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Abstract
The invention discloses a natural composition for improving anti-stress response of fish, 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 of red sage root organic solvent extract, perilla leaf organic solvent extract and angelica organic solvent extract; the natural compound anti-stress agent II comprises two or more of Saviae Miltiorrhizae radix water extract, folium Ginkgo water extract and folium Mori water extract. The natural composition provided by the invention is used for preparing the anti-hypoxia reoxygenation stress reaction additive or feed for fish, is a natural plant extract, and does not have other substances artificially added into the basic feed raw material. The natural functional feed can improve the tolerance of the fish to hypoxia and reoxygenation survival rate in cultivation, 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 response of fish and application thereof.
Background
At present, the current situation of aquaculture mainly comprises natural water aquaculture, and freshwater aquaculture mainly comprises pond aquaculture. The growth of farmed fish depends on the dissolved oxygen content in the body of water. However, natural water culture has the disadvantage that the dissolved oxygen of the water is greatly influenced by environmental factors. The dissolved oxygen content in natural bodies of water is affected by diurnal, weather and seasonal variations. Photosynthesis of green plants is very active in a sunny daytime water body, and the dissolved oxygen content of the water body can reach a supersaturated state at the moment; at night, due to stagnation of photosynthesis of plants, dissolved oxygen in water can be greatly reduced. Weather changes can affect illumination and photosynthesis of plants, thereby affecting dissolved oxygen in water. Seasonal variations can affect not only the illumination, but also the water temperature. In the physiological temperature range of 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 relief of dissolved oxygen in natural water can cause the fish body to suffer from hypoxia and reoxygenation stress, thereby reducing the production performance of the fish.
"high density, intensification" is an important feature of current aquaculture. But the high-density culture mode improves the fish carrying capacity of the water body; the relative living space of fish in the water body is reduced, competition and interference among different individuals are aggravated, so that the activity of the fish is improved, and the factors improve the oxygen demand of the fish body to different degrees. The high-density cultivation increases the feeding amount of the feed, increases the cultivation waste in the water body, aggravates the conversion of water body substances, and increases the oxygen consumption in the water body by the factors. The above situation further aggravates the anoxic stress of the farmed fish. The use of the current aerator can greatly relieve the anoxic state of the high-density aquaculture water body, but also aggravates the possibility that the fish body suffers from reoxygenation stress. The phenomenon of fish turning over and dying caused by the change of dissolved oxygen in water body still frequently occurs. The hypoxia and reoxygenation stress reaction can reduce the immunity of the fish and improve the morbidity and mortality of the fish, so that the use amount of antibiotics and chemical drugs in the cultivation is increased and the drug residue of the fish meat is increased. 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 fish without residue, has no toxic or side effect and does not pollute the environment, and is used for improving the culture benefit, reducing the drug residue and improving the fish quality.
The high-density intensive culture needs to use complete compound feed. The addition of antioxidants, mildewcides, growth promoters, phagostimulants and the like to compound feeds has been a common phenomenon. The antioxidants currently used in many cases are mainly synthetic antioxidants, including Ethoxyquinoline (EQ), butylated Hydroxyanisole (BHA), dibutylhydroxytoluene (BHT), etc. Fish meal and grease are important feed raw materials, and the addition amount of the synthetic antioxidant can reach 0.1% in the fish meal processing process; synthetic antioxidants are also added to the feed grade oils and fats. However, synthetic antioxidants have been reported to be carcinogenic and genotoxic to animals and to cause their residues and enrichment in fish products, causing food safety hazards and endangering human health. Month 2 of 2017, the european union promulgates 2017G/SPS/EU/190 bulletins: the use of EQ added to animal feed was stopped. Meanwhile, the cancer risk of adding BHT and BHA into animal feed is higher than EQ. Additives such as mould inhibitors, growth promoters, feeding attractants, etc. which have been reported to be chemically synthesized are also potentially toxic to animals and may cause residues in fish. The drawbacks of compound feed will therefore lead to an increased market demand for natural product compound feed.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the invention provides a natural composition for improving the anti-stress reaction of fish and application thereof, which solves the problems that the fish suffers from the aggravation of hypoxia reoxygenation stress due to high density and natural water body culture, and the existing feed additives have toxic and side effects and even cancerogenic effects on animals and possibly cause food safety hidden danger and endanger human health due to residues in fish meat. Is a natural compound prepared based on medicinal and edible Chinese herbal medicines, is used as an anti-hypoxia reoxygenation stress reaction additive for fish and a natural functional feed, has no toxic or side effect, and has important significance for improving economic and social benefits.
The invention is realized by the following technical scheme:
a natural composition for improving fish anti-stress reaction 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 of red sage root organic solvent extract, perilla leaf organic solvent extract and angelica organic solvent extract; the natural compound anti-stress agent II comprises two or more of Saviae Miltiorrhizae radix water extract, folium Ginkgo water extract and folium Mori water extract.
The fish anti-stress complex provided by the invention is a natural plant extract, and other substances are not added artificially. The natural functional feed prepared by using the active substances can improve the tolerance of the fish to hypoxia and the reoxygenation survival rate in cultivation, and has no residue, no toxic or side effect and no environmental pollution.
Further preferably, the red sage root organic solvent extract is a red sage root ethyl acetate extract, the perilla leaf organic solvent extract is a perilla leaf ethyl acetate extract, and the angelica organic solvent extract is an angelica ethyl acetate extract.
Further preferably, the content of flavonoids in the ethyl acetate extract of salvia miltiorrhiza is 82.53mg/g-97.60mg/g; the content of flavonoids in the perilla leaf ethyl acetate extract is 64.45mg/g-70.28mg/g; the content of polyphenols in the angelica sinensis ethyl acetate extract is 46.76mg/g-55.31mg/g.
The Saviae Miltiorrhizae radix ethyl acetate extract is obtained by extracting with analytical grade ethyl acetate, and is brownish red extract, wherein the content of flavonoids is 82.53mg/g-97.60mg/g. The perilla leaf ethyl acetate extract is obtained by extracting with analytical grade ethyl acetate, and the extract is dark brown extract, wherein the content of flavonoid substances is 64.45mg/g-70.28mg/g. The Angelica sinensis ethyl acetate extract is obtained by extracting with analytical grade ethyl acetate, and the extract is brownish red extract, wherein the content of polyphenols is 46.76mg/g-55.31mg/g.
Further preferably, in the salvia miltiorrhiza bunge 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%, and the mass percentage of soluble sugar is 38.89% -46.30%. The mulberry leaf water extract is obtained by extracting with distilled water, and is dark brown powder, the mass percentage of crude protein is 13.07% -15.64%, and the mass percentage of total sugar is 35.67% -42.54%. The ginkgo leaf water extract is obtained by extracting with distilled water, is light tan powder, contains 5.76 to 7.14 mass percent of crude protein and 51.32 to 58.29 mass percent of soluble sugar.
Further preferably, the natural compound anti-stress agent I comprises three of a red sage root organic solvent extract, a perilla leaf organic solvent extract and an angelica organic solvent extract, and the mass ratio of the red sage root organic solvent extract, the perilla leaf organic solvent extract and the angelica 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 red sage root water extract, a ginkgo leaf water extract and a mulberry leaf water extract, and the mass ratio of the red sage root water extract, the ginkgo 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 basic feed and a natural composition for improving anti-stress response of fish.
Further preferably, the adding amount of the natural compound anti-stress agent I is 0.3-0.5% of the weight of the basic feed; the addition amount of the natural compound anti-stress agent II is 0.5% -1.0% 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 cotton 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 bean pulp, 16 parts of peanut meal, 5 parts of cotton seed meal, 0.5 part of DL-methionine, 35 parts of wheat flour, 1.5 parts of rapeseed oil, 1 part of vitamin complex and 1 part of mineral complex.
The main raw materials of the anti-fish anoxia reoxygenation feed are natural products, and other substances are not artificially added; the additive components of the additive meet the requirements of national standard NY/T471-2018; the functional components of the feed are natural plant extracts, and the feed has certain functions of resisting feed oxidation and preventing mildew. The natural functional feed can improve the tolerance of fish to hypoxia and reoxygenation survival rate in cultivation.
The preparation method of the fish feed comprises the steps of dissolving the natural compound anti-stress agent I in grease, mixing, and then adding the mixture into basic feed for mixing; and/or dissolving the natural compound anti-stress agent II in water for mixing, and then adding the mixture into basic feed for mixing.
Such as in the preparation of a feed comprising a natural complex anti-stress agent I and a natural complex anti-stress agent II: firstly, calculating the mass of oil required by basic feed per unit mass according to a feed formula, adding natural compound anti-stress agent I accounting for 0.3% -0.5% of the weight of the basic feed into the oil, and uniformly mixing the oil into the basic feed; and then dissolving the natural compound anti-stress agent II accounting for 0.5% -1.0% of the weight of the basic feed into the potable water, and uniformly mixing the solution into the basic feed to prepare the pellet feed for inhibiting the hypoxia reoxygenation stress reaction of the freshwater fish.
The invention has the following advantages and beneficial effects:
1. the fish anti-stress complex provided by the invention is a natural plant extract, and other substances are not added artificially. The natural functional feed prepared by using the active substances can improve the tolerance of the fish to hypoxia and the reoxygenation survival rate in cultivation, and has no residue, no toxic or side effect and no environmental pollution.
2. The invention adopts the extracts of natural plants with homology of medicine and food as feed additives, removes most of ineffective components to enhance the activity, and the medicinal plants are decomposed by extracting the comprehensive functions of the medicinal plants, and the specific functions are concentrated, thereby improving the effect of a certain function of the medicinal plants. The plant extract has reduced volume relative to the Chinese herbal medicine, and is convenient for storage, transportation and transaction.
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:
FIG. 1 is the effect of feed fed complex ester extract on duration of hypoxia tolerance of crucian carp; in the figure, data are expressed as mean ± standard deviation of 3 replicates; the different superscripts in the same column data represent significant differences (P < 0.05);
FIG. 2 is the effect of feed fed complex ester extract on reoxygenation recovery after hypoxia rollover of crucian carp; in the figure, data are expressed as mean ± standard deviation of 3 replicates; the different superscripts in the same column data represent significant differences (P < 0.05);
FIG. 3 is the effect of feed fed a compound aqueous extract on the duration of hypoxia tolerance of crucian carp; in the figure, data are expressed as mean ± standard deviation of 3 replicates; the different superscripts in the same column data represent significant differences (P < 0.05);
FIG. 4 is the effect of feed fed with a compound water extract on recovery of reoxygenation after hypoxia rollover of crucian carp; in the figure, data are expressed as mean ± standard deviation of 3 replicates; the different superscripts in the same column data represent significant differences (P < 0.05);
FIG. 5 is the effect of feed fed with a composite anti-stress agent on the duration of hypoxia tolerance of crucian carp; in the figure, data are expressed as mean ± standard deviation of 3 replicates; the different superscripts in the same column data represent significant differences (P < 0.05);
FIG. 6 is the effect of feeding a compound anti-stress agent on reoxygenation recovery after hypoxia rollover of crucian carp; in the figure, data are expressed as mean ± standard deviation of 3 replicates; the different superscript letters in the same column of data indicate significant differences (P < 0.05).
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
Examples
The embodiment provides a natural composition for improving the anti-stress response of fish and application thereof.
1. Natural composition for improving anti-stress response of fish
1) Organic solvent ester extracts
The Saviae Miltiorrhizae radix ethyl acetate extract is obtained by extracting with analytical grade ethyl acetate, and is brownish red extract, wherein the content of flavonoids is 82.53mg/g-97.60mg/g.
The perilla leaf ethyl acetate is obtained by extraction of analytical grade ethyl acetate and is dark brown extract, wherein the content of flavonoid substances is 64.45mg/g-70.28mg/g.
The angelica sinensis ethyl acetate is obtained by extraction of analysis-grade ethyl acetate and is a brownish red extract, wherein the content of polyphenols is 46.76mg/g-55.31mg/g.
The mulberry leaf ethyl acetate extract is obtained by extraction of analysis grade ethyl acetate, and is dark brown extract, and the content of polyphenols is 71.62mg/g-76.20mg/g.
The ginkgo leaf ethyl acetate extract is obtained by extraction of analytical grade ethyl acetate, and is a yellow brown extract with the flavonoid content of 67.43g/kg-72.71g/kg.
2) Aqueous extract
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%, and the mass percentage of total sugar is 38.89% -46.30%.
The perilla leaf water extract is obtained by extracting with distilled water, is dark brown powder, contains 15.52 to 17.90 mass percent of crude protein and 31.36 to 35.49 mass percent of total sugar.
The angelica water extract is obtained by extracting with distilled water, is light red and 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 mulberry leaf water extract is obtained by extracting with distilled water, and is dark brown powder, the mass percentage of crude protein is 13.07% -15.64%, and the mass percentage of total sugar is 35.67% -42.54%.
The ginkgo leaf water extract is obtained by extracting with distilled water, is light tan powder, contains 5.76 to 7.14 mass percent of crude protein and contains 51.32 to 58.29 mass percent of total sugar.
2. Basal feed
The sources of the raw materials are as follows: the fish meal, the bean pulp, the peanut meal, the cotton meal, the wheat flour and the rapeseed oil are self-made, and other substances are not added manually except the nutrients of the rapeseed oil. The specific formulation is shown in table 1:
table 1 basal feed formulation
Remarks:
1 the fish meal contains dry matter 89.88%, crude protein 51.70%, crude fat 15.01% and crude ash 18.63%, and is prepared from cheap mixed fish of silver carp, crucian carp and grass carp by air drying at 80deg.C.
2 The 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, extracting oil and fat and volatilizing a dry solvent.
3 The peanut meal contains 88.21% of dry matters, 47.54% of crude proteins, 1.42% of crude fats and 5.33% of crude ash, and is a byproduct of peanut kernels after heating, crushing, extracting grease and volatilizing a dry solvent.
4 The cotton seed 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 cotton seed kernel after heating, crushing, extracting grease and volatilizing a dry solvent.
5 DL-methionine meets the requirements of NY/T471-2018.
6 Wheat flour is prepared by pulverizing wheat, and contains dry matter 87.60%, crude protein 13.32%, crude fat 2.03% and crude ash 1.84%.
7 Rapeseed oil contains 99.03% of dry matter, 0.00% of crude protein, 98.00% of crude fat and 0.53% of crude ash, and is obtained by squeezing rapeseed.
8 Each kilogram of vitamin additive comprises: 500,000IU of vitamin A acetate, D3240,000IU of vitamin, 20.00g of DL-alpha-tocopherol, 0.10g of vitamin K, 0.10g of vitamin B, 0.50g of riboflavin, 0.92g of pyridoxine hydrochloride, 0.001g of cyanocobalamin, 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.
9 Each kilogram of mineral additive comprises: feSO 4 37.90g, 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.
3. Screening of anti-stress combinations
1. Natural composite anti-stress agent I
According to the basic feed formulation, 5 extracts were added to the grease of the basic feed at a concentration of 1g/kg, respectively, to prepare 6 corresponding pellet feeds. The experimental design is shown in table 2.
TABLE 2 design of organic solvent extract anti-fish stress test
Remarks: the basic feed without any extract is marked as a blank, the feed with all 5 extracts is marked as a control, and the ethyl acetate extract of salvia miltiorrhiza, the ethyl acetate extract of perilla leaf, the ethyl acetate extract of angelica sinensis, the ethyl acetate extract of mulberry leaf and the ethyl acetate extract of ginkgo leaf are respectively marked as 1, 2, 3, 4 and 5 after one by one compared with the control.
After crucian fries are purchased from the Yongzhen in the city of the inner river of Sichuan province, the crucian fries are temporarily cultivated in a cultivation room for one week. The crucian fries 420 with equivalent weight (the difference is not more than 1%) are randomly divided into 7 treatment groups, and 3 fish tanks are selected from each group. The shape, the size and the color of each fish tank are the same, the size is 30 multiplied by 40cm, and one 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 water in the tank is 30L. The temperature of the culture room is controlled to be 22+/-1 ℃, and the oxygen-increasing machine continuously supplies oxygen. The 7 treatment groups were fed feed labeled "blank", "control", "1", "2", "3", "4" and "5", respectively, 4 satiety feeds per day. And measuring the hypoxia tolerance of the crucian carp after 7 days.
The measuring method comprises the following steps: and selecting 13 crucian carp with the same weight of each treatment group from each fish tank, preparing 30mL of saturated oxygen-dissolved water for each g of fish weight, and sealing the fish in a plastic bottle without air. The time and oxygen consumption rate were recorded and calculated for 10 fish from normal to out of balance (rollover). And (3) rapidly transferring the 10-tail side-turned crucian to a saturated dissolved oxygen water body, and recording the mantissa of the fish recovering the normal swimming posture within 2 hours. The test results are shown in Table 2 and FIGS. 1 and 2.
As shown in table 2, and fig. 1 and 2, the results demonstrate that feeding the composite extract to a different extent increases the duration of hypoxia tolerance of fish compared to the blank. Hypoxia tolerance duration sequence: 4=5 > control=2 > 3=1, 14.02h, 13.18h, 12.04h, 11.75h, 10.55h, 10.51h, respectively. Reoxygenation and recovery of fish mantissa sequence after anoxic rollover: control=4=5 > 2=1=3, 8.00 tail, 7.67 tail, 6.33 tail, 6.00 tail, respectively. Therefore, in the components of the composite extract, the ethyl acetate extract of mulberry leaves and the ethyl acetate extract of ginkgo leaves play a role in inhibiting the anti-stress effect of other components of the composite extract; and 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 preliminary screening composite extract are salvia miltiorrhiza ethyl acetate extract, angelica ethyl acetate extract and perilla ethyl acetate extract, and the natural composite anti-stress agent I is marked.
TABLE 2 Effect of feeding complex ester extracts on the hypoxia tolerance of Carassius auratus
Remarks: data are expressed as mean ± standard deviation of 3 replicates; the different superscript letters in the same column of data indicate significant differences (P < 0.05). Oxygen consumption rate = (water-insoluble oxygen-water-primary soluble oxygen) ×water volume/fish weight/duration.
2. Natural composite anti-stress agent II
According to the basic feed formulation, 5 extracts were added to the basic feed at a concentration of 2g/kg, respectively, by dissolving in potable water, to prepare 6 corresponding pellet feeds. The experimental design is shown in table 3 below.
TABLE 3 design of anti-fish stress test for complex aqueous extracts
| Additives | | Control | 1 | 2 | 3 | 4 | 5 | |
| Water extract of red sage root | × | ○ | × | ○ | ○ | ○ | ○ | |
| Perilla leaf water extract | × | ○ | ○ | × | ○ | ○ | ○ | |
| Angelica sinensis water extract | × | ○ | ○ | ○ | × | ○ | ○ | |
| Mulberry leaf water extract | × | ○ | ○ | ○ | ○ | × | ○ | |
| Aqueous extract of ginkgo leaf | × | ○ | ○ | ○ | ○ | ○ | × |
Remarks: the basic feed without any extract is marked as a blank, the feed with all 5 extracts is marked as a control, and the feeds with the red sage root water extract, the perilla leaf water extract, the angelica water extract, the mulberry leaf water extract and the ginkgo leaf water extract 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 Yongzhen in the city of the inner river of Sichuan province, the crucian fries are temporarily cultivated in a cultivation room for one week. The crucian fries 420 with equivalent weight (the difference is not more than 1%) are randomly divided into 7 treatment groups, and 3 fish tanks are selected from each group. The shape, the size and the color of each fish tank are the same, the size is 30 multiplied by 40cm, and one 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 water in the tank is 30L. The temperature of the culture room is controlled to be 22+/-1 ℃, and the oxygen-increasing machine continuously supplies oxygen. The 7 treatment groups were fed feed labeled "blank", "control", "1", "2", "3", "4" and "5", respectively, 4 satiety feeds per day. And measuring the hypoxia tolerance of the crucian carp after 7 days.
The measuring method comprises the following steps: and selecting 13 crucian carp with the same weight of each treatment group from each fish tank, preparing 30mL of saturated oxygen-dissolved water for each g of fish weight, and sealing the fish in a plastic bottle without air. The time and oxygen consumption rate were recorded and calculated for 10 fish from normal to out of balance (rollover). And (3) rapidly transferring the 10-tail side-turned crucian to a saturated dissolved oxygen water body, and recording the mantissa of the fish recovering the normal swimming posture within 2 hours. The test results are shown in Table 4 and FIGS. 3 and 4.
As shown in table 4, and in figures 3 and 4, the results demonstrate that feeding the composite extract increases the duration of hypoxia tolerance of fish to varying degrees compared to the blank. Hypoxia tolerance duration sequence: 3 > 2=control > 5=1 > 4, respectively 12.23h, 11.31h, 11.24h, 10.26h, 10.21h, 9.27h. Reoxygenation and recovery of fish mantissa sequence 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 composite extract, the water extracts of the perilla leaves and the angelica have an inhibiting effect on the anti-stress effect of other components of the composite extract; the water extract of red sage root, mulberry leaf and gingko leaf has the promoting effect. Therefore, the components of the preliminary screening composite water extract are red sage root water extract, mulberry leaf water extract and ginkgo leaf water extract, and the composite water extract is marked as composite anti-stress agent II.
TABLE 4 Effect of feeding a Compound Water extract on the hypoxia tolerance of Carassius auratus
Remarks: data are expressed as mean ± standard deviation of 3 replicates; the different superscript letters in the same column of data indicate significant differences (P < 0.05). Oxygen consumption rate = (water-insoluble oxygen-water-primary soluble oxygen) ×water volume/fish weight/duration.
3. Compounding and verification of compound anti-stress agent
In order to verify the anti-stress effect, a compound anti-stress agent I (abbreviated as 'I') is added into basic feed, and the compound anti-stress agent I is compared with feed No. 4 (abbreviated as 'control I') with the best effect in the component screening test I under the same culture environment and conditions. The compound anti-stress agent II (abbreviated as "II") is added into basic feed, and the feed is compared with 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 (content of 0.3%) and the feed containing the compound anti-stress agent II (content of 0.6%) were mixed in equal amounts (average content of 0.45%) (abbreviated as "(I+II)/2"), and compared with the feed containing the compound anti-stress agent I and the feed containing the compound anti-stress agent II under the same cultivation environments and conditions as above. The blank groups were also fed basal feed. The experimental design is shown in table 5.
Table 5 test design of composite anti-stress agent
Feeding crucian under the same culture environment and conditions, and measuring the hypoxia tolerance of the crucian according to the same method. The experimental results (as shown in table 6, and fig. 5 and 6) demonstrate that feeding the composite extract to varying degrees increases the duration of hypoxia tolerance of fish compared to the blank. Hypoxia tolerance duration sequence: i > control I; II > control II; (i+ii)/2 > i=ii; reoxygenation and recovery of fish mantissa sequence after anoxic rollover: i = control I; II > control II; (I+II)/2 > II > I. It can be seen that the anti-stress effect of the compound anti-stress agent II is higher than that of the control, and the effect of mixing the compound anti-stress agent II and the control is better.
TABLE 6 influence of feeding composite anti-stress agent on the hypoxia tolerance of crucian carp
Note that: data are expressed as mean ± standard deviation of 3 replicates; the different superscript letters in the same column of data indicate significant differences (P < 0.05). Oxygen consumption rate = (water-insoluble oxygen-water-primary soluble oxygen) ×water volume/fish 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 verifying of the compound anti-stress agent) of the fish not participating in the hypoxia tolerance assay, 15 fish were randomly selected for each treatment group, and equally divided into 3 fish tanks (5 fish per tank); 0.6mg Cu/L copper sulfate is dissolved in each fish tank water body. Each fish tank is fed with the natural compound anti-stress agent I, the natural compound anti-stress agent I and the compound anti-stress agent for 4 times per day.
From the above (compounding and verifying of the compound anti-stress agent) of the fish not participating in the hypoxia tolerance measurement, 15 fish were randomly selected for each treatment group, and evenly distributed to 3 fish tanks (5 fish per tank); 2.2mg/L trichlorfon is dissolved in each fish tank water body. Each fish tank is fed with the natural compound anti-stress agent I, the natural compound anti-stress agent I and the compound anti-stress agent for 4 times per day.
After 3 days, tail vein blood is taken from each fish, red blood cells are separated, and the apoptosis rate of the red blood cells is measured by using an Annexin V-FITC kit and a flow cytometer.
(2) Verification result:
as shown in table 7, the results show that feeding the composite extract inhibits the copper sulfate or trichlorfon-induced apoptosis of the red blood cells of the crucian; the anti-erythrocyte apoptosis effect of the compound anti-stress agent (I+II)/2 is higher than that of the compound anti-stress agent (I and II).
Note that: data are expressed as mean ± standard deviation of 3 replicates, 5 fish per replicate; the different superscript letters in the same column of data indicate significant differences (P < 0.05).
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (6)
1. The natural composition for improving the anti-stress response of the fish is characterized by comprising a natural composite anti-stress agent I and a natural composite anti-stress agent II, wherein the mass ratio of the natural composite anti-stress agent I to the natural composite anti-stress agent II is 1:2;
the natural compound anti-stress agent I comprises an ethyl acetate extract of salvia miltiorrhiza, an ethyl acetate extract of perilla leaves and an ethyl acetate extract of angelica sinensis; the mass ratio of the salvia miltiorrhiza ethyl acetate extract, the perilla leaf ethyl acetate extract and the angelica ethyl acetate 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;
the natural compound anti-stress agent II comprises a red sage root water extract, a ginkgo leaf water extract and a mulberry leaf water extract; the mass ratio of the red sage root water extract, the ginkgo 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.
2. The use of a natural composition for increasing anti-stress response in fish as claimed in claim 1, for preparing additive or feed for anti-stress response in crucian; the stress reaction is anoxic reoxygenation stress reaction.
3. A fish feed comprising a basal feed and a natural composition for improving the anti-stress response of fish according to claim 1.
4. A fish feed according to claim 3, wherein the natural compound anti-stress agent I is added in an amount of 0.3% to 0.5% by weight of the base feed; the addition amount of the natural compound anti-stress agent II is 0.5-1.0% of the weight of the basic feed.
5. A fish feed according to claim 3, wherein the basal 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 cotton 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.
6. A method for preparing a fish feed according to any one of claims 3 to 5, characterized in that the natural compound anti-stress agent I is dissolved in grease and mixed, and then added into the basic feed for mixing; and dissolving the natural compound anti-stress agent II in water, mixing, and adding into basic feed for mixing.
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