CN114504003A - Disinfectant for in-vitro hatching of procambarus clarkia embryos and preparation method and application thereof - Google Patents

Disinfectant for in-vitro hatching of procambarus clarkia embryos and preparation method and application thereof Download PDF

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CN114504003A
CN114504003A CN202210118655.3A CN202210118655A CN114504003A CN 114504003 A CN114504003 A CN 114504003A CN 202210118655 A CN202210118655 A CN 202210118655A CN 114504003 A CN114504003 A CN 114504003A
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essential oil
disinfectant
hatching
embryo
embryos
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CN114504003B (en
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程顺
顾志敏
迟美丽
郑建波
贾永义
刘士立
李飞
刘一诺
韦永春
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Zhejiang Institute of Freshwater Fisheries
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/50—Culture of aquatic animals of shellfish
    • A01K61/59—Culture of aquatic animals of shellfish of crustaceans, e.g. lobsters or shrimps
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/22—Lamiaceae or Labiatae [Mint family], e.g. thyme, rosemary, skullcap, selfheal, lavender, perilla, pennyroyal, peppermint or spearmint
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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Abstract

The invention discloses a disinfectant for in-vitro hatching of a red swamp crayfish embryo, and a preparation method and application thereof. The invention comprises the following components: 500-1000ppm essential oil base liquid, 1000u/mg superoxide dismutase 6000-1000u/mg trehalose, 15-30mmol/L bovine serum albumin, 100-200mmol/L astaxanthin, 15-30mmol/L astaxanthin and the balance water; the essential oil base liquid consists of the following components in percentage by volume: 28-32% of thyme essential oil, 28-32% of sage essential oil, 18-22% of blue eucalyptus essential oil and 18-22% of mint essential oil; the invention also provides a preparation method of the disinfectant. The components supplement each other, cooperate with each other and promote each other, and the obtained disinfectant has no toxicity and harm, good disinfection effect, good protection effect on embryos, high hatching rate and good emergence rate, and can replace the existing formaldehyde disinfectant.

Description

Disinfectant for in-vitro hatching of procambarus clarkia embryos and preparation method and application thereof
Technical Field
The invention belongs to the technical field of cultivation of red swamp crayfish, and particularly relates to a disinfectant for in-vitro hatching of a red swamp crayfish embryo, and a preparation method and application thereof.
Background
Red crayfish (Cherax aquaticatus), also known as Australia freshwater crayfish, is freshwater crayfish in Australia and surrounding areas, belongs to Crustaoea, Decapoda, Paracradaceae, and Cherax, has the characteristics of rapid growth, strong stress resistance, tender meat quality, high meat yield and the like, and is one of the world famous and precious economic shrimps.
The embryo in-vitro incubation is a method for artificial incubation by stripping eggs, the incubation method can reduce the feeding and management cost of the egg-carrying shrimps and crabs, can also reduce the egg falling or collision damage probability in the egg-carrying process, is beneficial to improving the incubation rate, has higher incubation synchronism, is beneficial to realizing the intellectualization and automation of breeding, can develop 'order seedling raising' and 'anti-season seedling raising', and can finish the batch production of high-quality seedlings.
However, disinfectant treatment is generally considered to be one of the key factors for the success of in vitro hatching of freshwater crayfish embryos. In the prior art, a common disinfectant used in the process of in vitro hatching of embryos of freshwater crayfish is formaldehyde, and a disinfection method is to soak the freshwater crayfish in 3000ppm of formaldehyde for 15 min. Formaldehyde is harmful to the environment and human body, and the use of formaldehyde has been limited in many places. Therefore, the application of the formaldehyde disinfectant in the in vitro hatching of the freshwater crayfish embryo is greatly limited.
Disclosure of Invention
The invention aims to provide a disinfectant for in-vitro hatching of freshwater crayfish embryos, a preparation method and application thereof, and aims to solve the problem that in the prior art, the application of a formaldehyde disinfectant for in-vitro hatching of freshwater crayfish embryos is limited due to harm to the environment and human bodies.
In order to solve the technical problems, the invention is mainly realized by the following technical scheme:
in one aspect, the disinfectant for in-vitro hatching of the embryo of the red crayfish comprises the following components in concentration: 500-1000ppm essential oil base liquid, 1000u/mg superoxide dismutase 6000-1000u/mg trehalose, 15-30mmol/L bovine serum albumin, 100-200mmol/L astaxanthin, 15-30mmol/L astaxanthin and the balance water; the essential oil base liquid consists of the following components in percentage by volume: 28-32% of thyme essential oil, 28-32% of sage essential oil, 18-22% of eucalyptus globulus essential oil and 18-22% of mint essential oil.
The essential oil base liquid is composed of thyme essential oil, sage essential oil, eucalyptus globulus essential oil and mint essential oil, and has strong dissolving capacity and good disinfection effect. Adding superoxide dismutase (SOD) into the essential oil base solution is vital, because the stress reaction of the red crayfish embryo during disinfection can generate a large amount of oxygen free radicals which can influence the embryo hatching and the constitution of the hatched young shrimp, the damage of the oxygen free radicals to the embryo can be reduced by adding the antioxidant, and the SOD is the most effective antioxidant; therefore, the addition of SOD can reduce the oxidative damage of the embryo caused by disinfection and repair the damaged cells; therefore, the addition of SOD can protect the embryo of the red crayfish without affecting the disinfection effect of the essential oil base liquid. SOD has a fatal weakness, namely, the stability is poor, and the activity is easy to lose; the trehalose is added to protect SOD, the dried trehalose is in a glass state, and the SOD is supported and wrapped by a very compact grid structure, so that the change of a three-dimensional structure of SOD molecules is effectively prevented, and the inactivation of the SOD is avoided; in addition, trehalose also has the effects of maintaining the activity of embryonic cells, resisting stress, maintaining the integrity of membranes and the like. The protective effect of trehalose is limited, and particularly in adverse environments, bovine serum albumin as a type of albumin lacks unsaturated fatty acids rich in yolk, has a good function of adjusting colloid osmotic pressure, and also has the function of carrying substances such as hormones, growth factors, fatty vitamins, medicaments, trace elements and the like; and the bovine serum albumin is an enzyme stabilizer, and the addition of the bovine serum albumin can complement trehalose, so that the bovine serum albumin and the trehalose are cooperated with each other, and the degeneration caused by adverse environmental factors is reduced. Secondly, when SOD is added into the disinfectant, partial SOD can be inactivated, astaxanthin has the function of recovering antioxidant enzyme, and matching of astaxanthin and SOD can greatly increase the capability of resisting free radicals of embryos and accelerate the capability of removing the free radicals; therefore, the addition of astaxanthin can restore the activity of SOD. The essential oil base solution, the SOD, the trehalose, the bovine serum albumin and the astaxanthin supplement each other, cooperate with each other and promote each other, and the obtained disinfectant is non-toxic, harmless, green and environment-friendly, has good disinfection effect, good protection effect on embryos, good stability and convenient use, can replace the existing formaldehyde disinfectant, and has high hatching rate and good emergence rate.
As a preferred embodiment, the essential oil base solution is composed of the following components by volume percentage: thyme essential oil 30%, sage essential oil 30%, eucalyptus globulus essential oil 20%, and peppermint essential oil 20%. The thyme essential oil, the sage essential oil, the blue eucalyptus essential oil and the mint essential oil in the essential oil base liquid are all plant essential oils which are green and natural, have no side effect, do not harm embryos and do not harm the environment and human bodies; moreover, the dissolubility is good, and the disinfection and sterilization effect is good; the plant essential oils are mutually dissolved and mutually cooperated, and the using effect is good.
As a preferred embodiment, the essential oil base solution is prepared by: 1) drying thyme leaf in air, and distilling to obtain thyme essential oil; taking sage leaves, air-drying and distilling to obtain sage essential oil; taking stems of the eucalyptus globulus, air-drying and distilling to obtain eucalyptus globulus essential oil; taking mint leaves, air-drying and distilling to obtain mint essential oil; 2) mixing thyme essential oil, herba Salvia officinalis essential oil, blue eucalyptus essential oil and herba Menthae essential oil to obtain essential oil base solution.
The thyme essential oil is extracted from thyme leaves, the sage essential oil is extracted from sage leaves, the blue eucalyptus essential oil is extracted from the stem of the blue eucalyptus, the mint essential oil is extracted from mint leaves, and the essential oils have wide sources and are easy to obtain and convenient to extract.
Thyme (Thymus vulgaris) leaves, sage (Salvia officinalis) leaves, Eucalyptus globulus (Eucalyptus globulus) stems and mint (Mentha piperita) leaves are all natural plants, and the obtained thyme essential oil, sage essential oil, Eucalyptus globulus essential oil and mint essential oil are directly mixed into an essential oil base liquid, or an emulsifier can be added, and the essential oil base liquid is obtained after emulsification of the emulsifier. The obtained essential oil base liquid is analyzed by adopting GC/MS (gas chromatography/mass spectrometry), wherein the GC/MS analysis is that an Elmer-Clarus 500 capillary gas chromatograph is directly connected to a mass spectrometer system, an SGE nonpolar fused quartz capillary column is applied, and under the following conditions: the temperature program of the oven is from 60 ℃ (10min) to 250 ℃, the temperature is 4 ℃/min, and the final temperature is kept for 10 min; the temperature of the oil sprayer is 220 ℃; helium is taken as carrier gas, and the flow rate is 1.5 mL/min; the base solution was diluted with 1ul of hexane injected; non-shunt injection technology; ionization energy in Electron Ionization (EI) mode is 70 eV; the ion source temperature is 200 ℃; the scanning mass range is 35-425m/z, and the interface line temperature is 250 ℃. The components of the essential oil base fluid are identified, calculated and analyzed according to the retention time of a series of alkanes (C4-C28) serving as a reference substance and the similarity of the mass spectrum of the alkane with the reference substance.
As a preferred embodiment, the molar ratio of trehalose, bovine serum albumin and astaxanthin is 3:20: 3. The trehalose and the astaxanthin are matched with SOD, the bovine serum albumin is matched with the trehalose, the bovine serum albumin and the astaxanthin are in ring-to-ring buckling and close connection, and the proportion of the trehalose, the bovine serum albumin and the astaxanthin is controlled, so that the comprehensive effect of the disinfectant is improved.
In another aspect, the invention provides a method for preparing a disinfectant for hatching the embryos of the red crayfish in vitro, which comprises the following steps: 1) sequentially adding SOD, trehalose, bovine serum albumin and astaxanthin into water, stirring and mixing to obtain a mixed solution; 2) adding the essential oil base solution into the mixed solution, stirring, and mixing to obtain the disinfectant.
The preparation method of the disinfectant for in-vitro hatching of the embryos of the red swamp crayfish is simple to operate, has no special requirements on equipment, mild conditions, high dispersion speed and good dispersion effect, is easy to realize industrialization, forms a uniform solution, is non-toxic and harmless, is green and environment-friendly, has a good disinfection effect, and has a good protection effect on the embryos.
As a preferred embodiment, in said step 1), the temperature of the water is between 28 and 30 ℃. The SOD, the trehalose, the bovine serum albumin and the astaxanthin are dissolved in warm water, the dissolving speed is high, meanwhile, the water temperature is low, the effective components of the SOD, the trehalose, the bovine serum albumin and the astaxanthin cannot be damaged, and the using effect is good.
As a preferred embodiment, in the step 1), the stirring time is 4-6 min. According to the invention, the SOD, the trehalose, the bovine serum albumin and the astaxanthin are stirred in water clockwise under the action of the glass rod, and are stirred in the same direction, so that the stirring is mild, the energy consumption is low, and the materials can be prevented from splashing.
As a preferred embodiment, in the step 2), the stirring time is 4-6 min. The essential oil base liquid is stirred clockwise in the mixed liquid under the action of the glass rod, the stirring time is controlled, and the convenience of operation is further improved.
In a further aspect, the invention relates to the use of a disinfectant for the in vitro hatching of a procambarus clarkia embryo, which is used as a disinfectant during the in vitro hatching of a procambarus clarkia embryo.
When the disinfectant is used for in-vitro hatching of the embryos of the red swamp crayfish, the embryos of the red swamp crayfish are disinfected firstly, and are disinfected every other day in the hatching process; the procambarus clarkia embryos which are disinfected and hatched by the disinfectant have higher hatchability and emergence rate, and can replace the traditional formaldehyde disinfectant.
As a preferred embodiment, the disinfectant is used at a temperature of 28-30 ℃. The disinfectant is usually stored in a brown bottle, stored in a dry place at room temperature, and heated to 28-30 ℃ by adopting water bath before use. The disinfectant has proper use temperature and does not influence the normal hatching of the embryos of the red swamp crayfish.
Compared with the prior art, the invention has the beneficial effects that: the essential oil base liquid is composed of thyme essential oil, sage essential oil, eucalyptus globulus essential oil and mint essential oil, and has strong dissolving capacity and good disinfection effect. Adding superoxide dismutase into the essential oil base solution to reduce oxidative damage to the embryo during disinfection and repair damaged cells, thereby protecting the embryo of the red crayfish; the dried trehalose is in a glass state, and the superoxide dismutase is supported and wrapped by a very compact grid structure, so that the change of the three-dimensional structure of the superoxide dismutase molecule is effectively prevented, and the inactivation of the superoxide dismutase is avoided; bovine serum albumin, as a type of albumin, lacks unsaturated fatty acids rich in yolk, has a good function of adjusting colloid osmotic pressure, and is complementary with trehalose, so that denaturation caused by adverse environmental factors is reduced; the matching of the astaxanthin and the superoxide dismutase can greatly increase the capability of resisting free radicals of the embryo, accelerate the capability of removing the free radicals and recover the activity of the superoxide dismutase; the essential oil base solution, the superoxide dismutase, the trehalose, the bovine serum albumin and the astaxanthin supplement each other, cooperate with each other and promote each other, and the obtained disinfectant is non-toxic, harmless, green and environment-friendly, has a good disinfection effect, a good protection effect on embryos, good stability and convenient use, can replace the existing formaldehyde disinfectant, and has high hatching rate and good emergence rate.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The disinfectant for in-vitro hatching of the embryos of the red swamp crayfish comprises the following components in concentration: 500-1000ppm essential oil base liquid, 1000u/mg superoxide dismutase 6000-1000u/mg trehalose, 15-30mmol/L bovine serum albumin, 100-200mmol/L astaxanthin, 15-30mmol/L astaxanthin and the balance water; the essential oil base solution comprises the following components in percentage by volume: 28-32% of thyme essential oil, 28-32% of sage essential oil, 18-22% of eucalyptus globulus essential oil and 18-22% of mint essential oil.
Preferably, the essential oil base solution consists of the following components in percentage by volume: thyme essential oil 30%, sage essential oil 30%, eucalyptus globulus essential oil 20%, and peppermint essential oil 20%.
Further, the preparation method of the essential oil base liquid comprises the following steps: 1) drying thyme leaf in air, and distilling to obtain thyme essential oil; taking sage leaves, air-drying and distilling to obtain sage essential oil; taking stems of the eucalyptus globulus, air-drying and distilling to obtain eucalyptus globulus essential oil; taking mint leaves, air-drying and distilling to obtain mint essential oil; 2) mixing thyme essential oil, herba Salvia officinalis essential oil, blue eucalyptus essential oil and herba Menthae essential oil to obtain essential oil base solution.
Preferably, the molar ratio of the trehalose, the bovine serum albumin and the astaxanthin is 3:20: 3.
The invention discloses a preparation method of a disinfectant for in vitro hatching of a procambarus clarkia embryo, which comprises the following steps of:
1) sequentially adding superoxide dismutase, trehalose, bovine serum albumin and astaxanthin into water, stirring, and mixing to obtain a mixed solution;
2) adding the essential oil base solution into the mixed solution, stirring, and mixing to obtain the disinfectant.
Preferably, in the step 1), the temperature of the water is 28-30 ℃.
Preferably, in the step 1), the stirring time is 4-6 min.
Preferably, in the step 2), the stirring time is 4-6 min.
The disinfectant is used as the disinfectant in the in-vitro hatching process of the embryos of the red swamp crayfish.
Preferably, the disinfectant is used at a temperature of 28-30 ℃.
Example one
The invention discloses a preparation method of a disinfectant for in-vitro hatching of a red swamp crayfish embryo, which comprises the following steps:
1) weighing the following raw materials in percentage by volume: 28% of thyme essential oil, 32% of sage essential oil, 18% of eucalyptus globulus essential oil and 22% of mint essential oil;
2) mixing the thyme essential oil, the sage essential oil, the blue eucalyptus essential oil and the mint essential oil obtained in the step 1) to obtain essential oil base liquid;
3) sequentially adding superoxide dismutase, trehalose, bovine serum albumin and astaxanthin into water, stirring, and mixing to obtain a mixed solution;
4) adding the essential oil base liquid obtained in the step 2) into the mixed liquid, stirring and mixing to obtain a disinfectant, wherein the concentration of the essential oil base liquid is 500ppm, the concentration of superoxide dismutase is 6000u/mg, the concentration of trehalose is 15-30mmol/L, the concentration of bovine serum albumin is 100mmol/L, and the concentration of astaxanthin is 15 mmol/L.
Comparative example 1
A preparation method of a disinfectant for in vitro hatching of a red swamp crayfish embryo comprises the following steps:
adding the essential oil base liquid obtained in the step 2) in the embodiment I into water, stirring and mixing to obtain the control disinfectant.
Wherein the concentration of the essential oil base solution in the control disinfectant is 500 ppm.
Example two
The invention discloses a preparation method of a disinfectant for in-vitro hatching of a red swamp crayfish embryo, which comprises the following steps:
1) taking 90g of thyme leaves, air-drying, distilling for 4h to obtain thyme essential oil; taking 90g of salvia leaves, air-drying, distilling for 4h to obtain salvia essential oil; taking 90g of eucalyptus globulus stems, air-drying, distilling for 4h to obtain eucalyptus globulus essential oil; taking 90g of mint leaves, air-drying, distilling for 4 hours to obtain mint essential oil;
2) weighing the following raw materials in percentage by volume: thyme essential oil 30%, sage essential oil 30%, eucalyptus globulus essential oil 20%, peppermint essential oil 20%;
3) mixing the thyme essential oil, the sage essential oil, the blue eucalyptus essential oil and the mint essential oil obtained in the step 2) to obtain essential oil base liquid;
4) sequentially adding superoxide dismutase, trehalose, bovine serum albumin and astaxanthin into water of 28 deg.C, clockwise stirring with glass rod for 5min, and mixing to obtain mixed solution;
5) adding the essential oil base liquid obtained in the step 3) into the mixed liquid, stirring clockwise for 5min by using a glass rod, and mixing to obtain a disinfectant, wherein the concentration of the essential oil base liquid in the disinfectant is 1000ppm, the concentration of superoxide dismutase is 10000u/mg, the concentration of trehalose is 30mmol/L, the concentration of bovine serum albumin is 200mmol/L, and the concentration of astaxanthin is 30 mmol/L.
Comparative example No. two
A preparation method of a disinfectant for in vitro hatching of a red swamp crayfish embryo comprises the following steps:
adding the essential oil base liquid obtained in the step 3) in the second embodiment into water, stirring and mixing to obtain the control disinfectant.
Wherein the concentration of the essential oil base solution in the control disinfectant is 1000 ppm.
EXAMPLE III
The invention discloses a preparation method of a disinfectant for in-vitro hatching of a red swamp crayfish embryo, which comprises the following steps:
1) drying thyme leaf in air, and distilling to obtain thyme essential oil; taking sage leaves, air-drying and distilling to obtain sage essential oil; taking stems of the eucalyptus globulus, air-drying and distilling to obtain eucalyptus globulus essential oil; taking mint leaves, air-drying and distilling to obtain mint essential oil;
2) weighing the following raw materials in percentage by volume: 32% of thyme essential oil, 28% of sage essential oil, 22% of blue eucalyptus essential oil and 18% of mint essential oil;
3) mixing the thyme essential oil, the sage essential oil, the blue eucalyptus essential oil and the mint essential oil obtained in the step 2) to obtain essential oil base liquid;
4) sequentially adding superoxide dismutase, trehalose, bovine serum albumin and astaxanthin into water of 30 deg.C, clockwise stirring with glass rod for 4min, and mixing to obtain mixed solution;
5) adding the essential oil base liquid obtained in the step 3) into the mixed liquid, adopting a glass rod to stir for 6min along the clockwise direction, and mixing to obtain a disinfectant, wherein the concentration of the essential oil base liquid is 800ppm, the concentration of superoxide dismutase is 9000u/mg, the concentration of trehalose is 25mmol/L, the concentration of bovine serum albumin is 150mmol/L, and the concentration of astaxanthin is 25 mmol/L.
Comparative example No. three
A preparation method of a disinfectant for in vitro hatching of a red swamp crayfish embryo comprises the following steps:
adding the essential oil base liquid obtained in the step 3) in the third embodiment into water, stirring and mixing to obtain the control disinfectant.
Wherein the concentration of the essential oil base solution in the control disinfectant is 800 ppm.
Control sample
A preparation method of a disinfectant for in vitro hatching of a red swamp crayfish embryo comprises the following steps:
adding commercially available formaldehyde into water, stirring, and mixing to obtain control sample.
Wherein the concentration of formaldehyde in the control sample was 3000 ppm.
Experiment 1
Three disinfectants obtained in the first to third examples and four disinfectants obtained in the first, second, third and control examples were subjected to disinfection effect test experiments, respectively.
Selecting 7 pairs of the (orange) red crayfish embryos in the period of appendage, peeling off the embryos from the attached bristles on the abdomen and feet through a comb, dividing the embryos into 7 groups on average, and disinfecting by adopting the seven disinfectants respectively, wherein the disinfectants obtained in the first to third examples of the invention and the disinfectants obtained in the first, second and third comparative examples have the disinfection time of 10s, and the disinfection time of the comparative example is 15 min.
Putting the disinfected procambarus clarkia embryos into an incubation box at the density of 500 granules/box, and putting the incubation box into an incubation device for incubation; during embryo incubation, corresponding disinfector is adopted to disinfect every other day, no disinfection treatment is carried out after incubation, and the incubation water temperature is 29 ℃; after hatching, the number of the juvenile shrimps in the 1 st stage is counted, and the hatching rate is determined.
And respectively inserting fine sponge strips into the hatching boxes of each group to enable the young shrimps which are hatched for the first time to be attached, and counting the rate of emergence after the young shrimps grow to the 3 rd stage and can be scattered and move freely. During the cultivation period, dead eggs and dead shrimps were periodically removed.
10 replicates of each disinfectant were taken and the mean value was calculated and shown in Table 1.
TABLE 1 incubation results of embryos of red swamp crayfish sterilized with different disinfectants
Sample name Hatching rate (%) Percentage of emergence (%)
Example one 89.24±2.17b 59.28±4.42bc
Comparative example 1 83.72±4.82a 55.72±2.86a
Example two 89.54±4.22b 62.22±3.55c
Comparative example No. two 84.84±5.59a 58.50±2.73b
EXAMPLE III 89.52±5.08b 60.52±2.09bc
Comparative example No. three 84.52±2.74a 58.28±3.17b
Control sample 89.68±4.99b 61.94±5.01c
Note: the data are analyzed by adopting a sps independent sample, and the same upper mark and the same letter represent that the difference is not significant (P > 0.05); the same superscript different letters indicate significant differences (P < 0.05).
As can be seen from Table 1, when the disinfectant obtained by the method of the invention is used for disinfecting the embryos of the crayfish, the hatchability of the embryos of the crayfish is higher than that of the embryos of the crayfish when pure essential oil base liquid is used as the disinfectant under the same concentration (namely, a comparative example); when the disinfectant prepared by the method is used for disinfecting the red swamp crayfish embryos, the emergence rate of the obtained red swamp crayfish embryos is obviously greater than that of the red swamp crayfish embryos when pure essential oil base liquid is used as the disinfectant under the same concentration (namely a comparative example). When the disinfectant obtained by the method of the present invention is used for disinfecting the embryos of the red swamp crayfish, the hatchability of the embryos of the red swamp crayfish is 84 to 89%, which is basically the same as the hatchability of the existing commercial formaldehyde disinfectant (i.e., the control). When the disinfectant obtained by the method is used for disinfecting the red crayfish embryos, the emergence rate of the obtained red crayfish embryos is 58-62 percent, which is basically the same as the emergence rate of the existing commercial formaldehyde disinfectant (namely the control). Therefore, the disinfectant obtained by the method can replace the traditional formaldehyde disinfectant.
Therefore, compared with the prior art, the invention has the beneficial effects that: the essential oil base liquid is composed of thyme essential oil, sage essential oil, eucalyptus globulus essential oil and mint essential oil, and has strong dissolving capacity and good disinfection effect. Adding superoxide dismutase into the essential oil base solution to reduce oxidative damage to the embryo during disinfection and repair damaged cells, thereby protecting the embryo of the red crayfish; the dried trehalose is in a glass state, and the superoxide dismutase is supported and wrapped by a very compact grid structure, so that the change of the three-dimensional structure of the superoxide dismutase molecule is effectively prevented, and the inactivation of the superoxide dismutase is avoided; bovine serum albumin, as a type of albumin, lacks unsaturated fatty acids rich in yolk, has a good function of adjusting colloid osmotic pressure, and is complementary with trehalose, so that denaturation caused by adverse environmental factors is reduced; the matching of the astaxanthin and the superoxide dismutase can greatly increase the capability of resisting free radicals of the embryo, accelerate the capability of removing the free radicals and recover the activity of the superoxide dismutase; the essential oil base solution, the superoxide dismutase, the trehalose, the bovine serum albumin and the astaxanthin supplement each other, cooperate with each other and promote each other, and the obtained disinfectant is non-toxic, harmless, green and environment-friendly, has a good disinfection effect, a good protection effect on embryos, good stability and convenient use, can replace the existing formaldehyde disinfectant, and has high hatching rate and good emergence rate.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. The disinfectant for in-vitro hatching of the embryos of the red swamp crayfish is characterized by comprising the following components in concentration:
500-1000ppm essential oil base liquid, 1000u/mg superoxide dismutase 6000-1000u/mg trehalose, 15-30mmol/L bovine serum albumin, 100-200mmol/L astaxanthin, 15-30mmol/L astaxanthin and the balance water;
the essential oil base liquid consists of the following components in percentage by volume: 28-32% of thyme essential oil, 28-32% of sage essential oil, 18-22% of eucalyptus globulus essential oil and 18-22% of mint essential oil.
2. The disinfectant for in vitro hatching of a procambarus clarkia embryo according to claim 1, wherein:
the essential oil base liquid consists of the following components in percentage by volume: thyme essential oil 30%, sage essential oil 30%, eucalyptus globulus essential oil 20%, and peppermint essential oil 20%.
3. The disinfectant for in vitro hatching of the embryos of the red crayfish according to claim 1 or 2, wherein the essential oil base solution is prepared by a method comprising:
1) drying thyme leaf in air, and distilling to obtain thyme essential oil; taking sage leaves, air-drying and distilling to obtain sage essential oil; taking stems of the eucalyptus globulus, air-drying and distilling to obtain eucalyptus globulus essential oil; taking mint leaves, air-drying and distilling to obtain mint essential oil;
2) mixing thyme essential oil, herba Salvia officinalis essential oil, blue eucalyptus essential oil and herba Menthae essential oil to obtain essential oil base solution.
4. The disinfectant for in vitro hatching of a procambarus clarkia embryo according to claim 1, wherein:
the molar ratio of the trehalose to the bovine serum albumin to the astaxanthin is 3:20: 3.
5. A method for preparing a disinfectant for hatching an embryo of a red crayfish in vitro according to any one of claims 1 to 4, comprising the steps of:
1) sequentially adding superoxide dismutase, trehalose, bovine serum albumin and astaxanthin into water, stirring, and mixing to obtain a mixed solution;
2) adding the essential oil base solution into the mixed solution, stirring, and mixing to obtain the disinfectant.
6. The method for preparing a disinfectant for in vitro hatching of a procambarus clarkia embryo according to claim 5, wherein the disinfectant comprises:
in the step 1), the temperature of the water is 28-30 ℃.
7. The method for preparing a disinfectant for in vitro hatching of a procambarus clarkia embryo according to claim 5, wherein the disinfectant comprises:
in the step 1), the stirring time is 4-6 min.
8. The method for preparing a disinfectant for in vitro hatching of a procambarus clarkia embryo according to claim 5, wherein the disinfectant comprises:
in the step 2), the stirring time is 4-6 min.
9. Use of a disinfectant for the ex vivo hatching of the embryo of the red crayfish according to any one of claims 1 to 4, characterized in that:
the disinfectant is used as a disinfectant in the in-vitro hatching process of the embryo of the red crayfish.
10. The use of a disinfectant for the ex vivo incubation of the embryos of red crayfish according to claim 9, characterized in that:
the disinfectant is used at 28-30 deg.C.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2636642A1 (en) * 1988-09-20 1990-03-23 Millou Yves Apparatus and method intended for producing terpenes derivatives of the oxonium type, initiators of superoxide dismutase
TW200830991A (en) * 2006-04-20 2008-08-01 Biomor Israel Ltd TTO-based disinfectants & anesthetics for use in aquaculture
CN103494038A (en) * 2013-09-30 2014-01-08 江苏省淡水水产研究所 Chinese herbal medicine immunopotentiator for resisting white spot syndrome virus (WSSV) of procambarus clarkia, as well as preparation method and application thereof
CN106107997A (en) * 2016-08-17 2016-11-16 陈石良 A kind of antioxidation hypertension and hyperlipemia SOD Semen Lini oil soft capsule and preparation method thereof
CN106259115A (en) * 2016-08-23 2017-01-04 合肥申仁养殖有限公司 A kind of high-efficiency breeding method of Lobster juvenile prawn

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2636642A1 (en) * 1988-09-20 1990-03-23 Millou Yves Apparatus and method intended for producing terpenes derivatives of the oxonium type, initiators of superoxide dismutase
TW200830991A (en) * 2006-04-20 2008-08-01 Biomor Israel Ltd TTO-based disinfectants & anesthetics for use in aquaculture
CN103494038A (en) * 2013-09-30 2014-01-08 江苏省淡水水产研究所 Chinese herbal medicine immunopotentiator for resisting white spot syndrome virus (WSSV) of procambarus clarkia, as well as preparation method and application thereof
CN106107997A (en) * 2016-08-17 2016-11-16 陈石良 A kind of antioxidation hypertension and hyperlipemia SOD Semen Lini oil soft capsule and preparation method thereof
CN106259115A (en) * 2016-08-23 2017-01-04 合肥申仁养殖有限公司 A kind of high-efficiency breeding method of Lobster juvenile prawn

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
宋光同;丁凤琴;武松;陈静;汪翔;侯冠军;: "维生素C、E及高度不饱和脂肪酸交互作用对克氏原螯虾繁殖性能的影响" *

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