Disinfectant for in-vitro incubation of procambarus clarkii embryos as well as preparation method and application of disinfectant
By providing a disinfectant for in vitro incubation of the Chlorella embryos including sodium chloride, trehalose, sodium dodecyl benzenesulfonate, essential oil mixture, six-grass mixture, six-flower mixture, polysorbate-80, astaxanthin and glycine, the harm of formaldehyde disinfectant to the environment and the human body is solved, and an efficient and safe embryo disinfection and hatching effect is achieved.
Patent Information
- Application Number
- CN202411704611.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-03-11
AI Technical Summary
In the prior art, the formaldehyde disinfectant used when incubating embryos of pro-Chazard Crayfish in vitro is harmful to the environment and the human body, and limits its application.
A disinfectant for incubation of Crayfish embryos including a first disinfectant, a second disinfectant and a third disinfectant is provided. The first disinfectant consists of sodium chloride, trehalose, sodium dodecylbenzenesulfonate and water, the second disinfectant includes essential oil mixture, six-grass mixture and six-flower mixture, and the third disinfectant consists of polysorbate-80, astaxanthin and glycine. These disinfectants promote each other and synergize to form non-toxic, harmless, green and environmentally friendly disinfectants.
The disinfectant is non-toxic and harmless, green and environmentally friendly, has a good disinfection effect, has a good protective effect on the embryo, is good instability and is easy to use. It can replace the existing formaldehyde disinfectant and improve the hatching rate and seedling emergence rate.
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of Procambarus clarkii breeding, and particularly relates to a disinfectant for in vitro hatching of Procambarus clarkii embryos, and a preparation method and application thereof. Background Art
[0002] Procambarus clarkii is an arthropod of the family Amphiprionidae, commonly known as crayfish or freshwater lobster. Adult Procambarus clarkii is generally 6-13cm long, with a stout body, slightly flat dorsally and ventrally, and the body is divided into the cephalothorax and abdomen, and the body surface is covered with a carapace. The body color of mature individuals is dark red or dark red, while the body color of immature individuals can be light brown, reddish brown, and a few individuals are blue.
[0003] In the breeding process of Procambarus clarkii, the parent shrimps have to be cultivated in cement ponds for a long time before the brooding shrimps can be obtained. During the cultivation of these brooding shrimps in the floating tanks, the embryos are prone to infection, mold or death during hatching. The dead, moldy or infected embryos may fall off from the mother, resulting in a low hatching rate of shrimp fry and insufficient seedlings, which hinders the development of the industry.
[0004] In the prior art, the disinfectants commonly used in the breeding process of freshwater crayfish include formaldehyde, hydrogen peroxide, potassium permanganate, copper sulfate, povidone iodine or isopropyl alcohol, among which formaldehyde is the most effective disinfectant, and other chemical reagents such as hydrogen peroxide, potassium permanganate, copper sulfate, povidone iodine or isopropyl alcohol have poor disinfection effects; however, formaldehyde is harmful to the environment and human body, and the use of formaldehyde has been restricted in many places. Therefore, it is urgent to develop an effective disinfectant widely used in the breeding process of Procambarus clarkii. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a disinfectant for in vitro hatching of crayfish embryos and a preparation method and application thereof, aiming to solve the problem that formaldehyde disinfectants used for in vitro hatching of crayfish embryos in the prior art are harmful to the environment and human body, thus limiting their application.
[0006] To this end, the present invention provides a disinfectant for in vitro hatching of Procambarus clarkii embryos, the disinfectant comprising at least one of a first disinfectant, a second disinfectant and a third disinfectant; the first disinfectant comprising sodium chloride, trehalose, sodium dodecylbenzene sulfonate and water; the second disinfectant comprising at least one of an essential oil mixture, a six-herb mixture and a six-flower mixture; the essential oil mixture comprising thyme essential oil, sage essential oil, eucalyptus globulus essential oil, peppermint essential oil and water; the six-herb mixture comprising gentian, houttuynia cordata, orchid vanilla, ground ivy, liquorice, lavender and an ethanol solution; the six-flower mixture comprising honeysuckle, marigold, calendula, safflower, lilac, rose and an ethanol solution; the third disinfectant comprising polysorbate 80, astaxanthin, glycine and water.
[0007] Preferably, the disinfectant includes the first disinfectant, the second disinfectant and the third disinfectant, and the volume ratio of the first disinfectant, the second disinfectant and the third disinfectant is (0.8-1.2):(1-1.5):(0.9-1.2).
[0008] Preferably, the second disinfectant comprises an essential oil mixture, a six-herb mixture and a six-flower mixture, and the volume ratio of the essential oil mixture, the six-herb mixture and the six-flower mixture is (12-15):(0.5-2):(0.5-2).
[0009] Preferably, the concentration of sodium chloride is 65-75 g / L, the concentration of trehalose is 2-10 g / L, and the concentration of sodium dodecylbenzene sulfonate is 5-15 g / L.
[0010] Preferably, the volume percentage of each component in the essential oil mixture is: thyme essential oil 0.02%-0.03%, sage essential oil 0.02%-0.03%, eucalyptus globulus essential oil 0.02%-0.03%, and peppermint essential oil 0.02%-0.03%.
[0011] Preferably, the concentration of gentian is 40%-60%, the concentration of houttuynia cordata is 40%-60%, the concentration of vanilla is 40%-60%, the concentration of euphorbia pulegium is 40%-60%, the concentration of licorice is 40%-60%, and the concentration of lavender is 40%-60%.
[0012] Preferably, the concentration of honeysuckle is 40%-60%, the concentration of marigold is 40%-60%, the concentration of calendula is 40%-60%, the concentration of safflower is 40%-60%, the concentration of lilac is 40%-60%, and the concentration of rose is 40%-60%.
[0013] Preferably, the concentration of polysorbate 80 is 15-25 g / L, the concentration of astaxanthin is 0.5-1.5 g / L, and the concentration of glycine is 0.5-1.5 g / L.
[0014] The invention discloses a method for preparing a disinfectant for in vitro hatching of Procambarus clarkii embryos. The method for preparing the first disinfectant comprises: mixing sodium chloride, trehalose and sodium dodecylbenzene sulfonate, adding pure water and stirring evenly; the method for preparing the essential oil mixture comprises: mixing thyme essential oil, sage essential oil, eucalyptus globulus essential oil and peppermint essential oil, adding pure water and stirring evenly; the method for preparing the six-herb mixture comprises: mixing gentian, houttuynia cordata, orchid, herbaceous sedge, licorice and lavender, adding ethanol solution, filtering the drug residues with a rotary filter, and stirring evenly after the filtrate is decolorized by activated carbon; the method for preparing the six-flower mixture comprises: mixing honeysuckle, calendula, calendula, safflower, lilac and rose, adding 70% ethanol solution, filtering the drug residues with a rotary filter, and stirring evenly after the filtrate is decolorized by activated carbon; the method for preparing the third disinfectant comprises: mixing polysorbate 80, astaxanthin and glycine, and adding pure water and stirring evenly.
[0015] The application of the disinfectant for in vitro hatching of Procambarus clarkii embryos of the present invention is used as a disinfectant in the process of in vitro hatching of Procambarus clarkii embryos, and the temperature of the disinfectant when used is 28-30°C.
[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: the present invention provides a disinfectant for in vitro hatching of Procambarus clarkii embryos and its application, the disinfectant comprising at least one of a first disinfectant, a second disinfectant and a third disinfectant; the first disinfectant comprising sodium chloride, trehalose, sodium dodecylbenzene sulfonate and water; the second disinfectant comprising at least one of an essential oil mixture, a six-herb mixture and a six-flower mixture; the essential oil mixture comprising thyme essential oil, sage essential oil, eucalyptus globulus essential oil, peppermint essential oil and water; the six-herb mixture comprising gentian, houttuynia cordata, orchid vanilla, ground ivy, licorice, lavender and an ethanol solution; the six-flower mixture comprising honeysuckle, marigold, calendula, safflower, lilac, rose and an ethanol solution; the third disinfectant comprising polysorbate 80, astaxanthin, glycine and water. The disinfectant of the present invention comprises a first disinfectant, a second disinfectant and a third disinfectant, the three of which promote each other and synergize to enhance the effectiveness. The obtained disinfectant is non-toxic and harmless, green and environmentally friendly, has a good disinfection effect, has a good protective effect on embryos, has good stability, is easy to use, can replace the existing formaldehyde disinfectant, has a high hatching rate and a good germination rate. DETAILED DESCRIPTION
[0017] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Meanwhile, after reading the present invention, those skilled in the art may make various modifications or changes to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.
[0018] The disinfectant for in vitro hatching of Procambarus clarkii embryos of the present invention comprises at least one of a first disinfectant, a second disinfectant and a third disinfectant.
[0019] Preferably, the disinfectant of the present invention comprises a first disinfectant, a second disinfectant and a third disinfectant, and the volume ratio of the first disinfectant, the second disinfectant and the third disinfectant is (0.8-1.2):(1-1.5):(0.9-1.2).
[0020] Further preferably, the volume ratio of the first disinfectant, the second disinfectant and the third disinfectant is 1:1:1.
[0021] The disinfectant of the present invention comprises a first disinfectant, a second disinfectant and a third disinfectant, the three of which promote each other and synergize to enhance the effectiveness. The obtained disinfectant is non-toxic and harmless, green and environmentally friendly, has a good disinfection effect, has a good protective effect on embryos, has good stability, is easy to use, can replace the existing formaldehyde disinfectant, has a high hatching rate and a good germination rate.
[0022] The disinfectant of the present invention overcomes the shortcomings of existing disinfectants such as high cost, rapid volatilization, poor stability and slow effect, avoids the problem that existing disinfectants are not suitable for actual production applications due to too short disinfection time, and also overcomes the shortcomings of existing disinfectants such as poor stability, easy volatility, easy oxidation and poor bactericidal effect.
[0023] The first disinfectant includes sodium chloride, trehalose, sodium dodecylbenzene sulfonate and water. The crayfish embryos are immersed in the first disinfectant for disinfection. Sodium chloride can increase the density of the first disinfectant, so that the dead eggs, bad eggs and moldy eggs in the crayfish embryos can float away, leaving the normal crayfish embryos. Trehalose can play the role of anti-stress and anti-dehydration, and protect the crayfish embryos without affecting the disinfection effect, and prevent the crayfish embryos from being damaged; secondly, trehalose also has the functions of maintaining embryo cell activity, anti-stress, and maintaining membrane integrity, maintaining the cell membrane stability and permeability of the embryos in the disinfectant, protecting the embryos, preventing the embryos from being damaged, and thus improving the emergence rate. Sodium dodecylbenzene sulfonate mainly plays the role of cleaning and disinfection.
[0024] Among them, the concentration of sodium chloride is 65-75g / L. Within this concentration range, the density of the first disinfectant can be reasonably increased to ensure that dead eggs, bad eggs, and moldy eggs in the crayfish embryos float away while leaving normal crayfish embryos.
[0025] The concentration of trehalose is 2-10g / L. Within this concentration range, trehalose can effectively resist stress and dehydration, and can effectively protect the crayfish embryos and effectively prevent the crayfish embryos from being damaged.
[0026] The concentration of sodium dodecylbenzene sulfonate is 5-15g / L. Within this concentration range, it can play an effective role in cleaning and disinfection.
[0027] Preferably, the concentration of sodium chloride is 67-72 g / L, the concentration of trehalose is 4-7 g / L, and the concentration of sodium dodecylbenzene sulfonate is 8-12 g / L.
[0028] The second disinfectant includes at least one of an essential oil mixture, a six-herb mixture, and a six-flower mixture. In this embodiment, the second disinfectant includes an essential oil mixture, a six-herb mixture, and a six-flower mixture, and the volume ratio of the essential oil mixture, the six-herb mixture, and the six-flower mixture is (12-15): (0.5-2): (0.5-2).
[0029] Preferably, the volume ratio of the essential oil mixture, the six-herb mixture and the six-flower mixture is (13-14): (0.8-1.5): (0.8-1.5). The three promote each other and synergize to enhance the effectiveness. The obtained disinfectant is non-toxic and harmless, green and environmentally friendly, has a good disinfection effect, has a good protective effect on embryos, has good stability, is easy to use, can replace existing formaldehyde disinfectants, has a high hatching rate and a good germination rate.
[0030] The essential oil mixture comprises thyme essential oil, sage essential oil, eucalyptus globulus essential oil, peppermint essential oil and water; thyme essential oil, sage essential oil, eucalyptus globulus essential oil and peppermint essential oil are all plant essential oils, which are green and natural, have no side effects, are harmless to embryos, and are also harmless to the environment and human body; moreover, they have strong dissolving ability and good disinfection and sterilization effects; these plant essential oils dissolve in each other, act synergistically with each other, and have good use effects.
[0031] The thyme essential oil of the invention is extracted from thyme leaves, the sage essential oil is extracted from sage leaves, the eucalyptus globulus essential oil is extracted from eucalyptus globulus stems, and the mint essential oil is extracted from mint leaves. The raw materials of these essential oils are widely available, easy to obtain, and convenient to extract.
[0032] The volume percentage of each component in the essential oil mixture is: thyme essential oil 0.02%-0.03%, sage essential oil 0.02%-0.03%, eucalyptus globulus essential oil 0.02%-0.03%, peppermint essential oil 0.02%-0.03%. The essential oils of the above volume contents dissolve in each other, act in synergy with each other, and have the best use effect.
[0033] Preferably, the volume percentage of each component in the essential oil mixture is: thyme essential oil 0.025%, sage essential oil 0.025%, eucalyptus globulus essential oil 0.025%, and peppermint essential oil 0.025%.
[0034] The six-herb mixture includes gentian, houttuynia, orchid vanilla, ground ivy, licorice, lavender and ethanol solution; gentian, houttuynia, orchid vanilla, ground ivy, licorice and lavender are all plant herbs, green and natural, with no side effects, no harm to the embryo, and no harm to the environment and human body; the six plant herbs cooperate and work synergistically with each other to effectively kill bacteria.
[0035] The concentration of gentian is 40%-60%, the concentration of houttuynia cordata is 40%-60%, the concentration of vanilla is 40%-60%, the concentration of ground ivy is 40%-60%, the concentration of licorice is 40%-60%, and the concentration of lavender is 40%-60%.
[0036] Preferably, the concentration of gentian is 45%-55%, the concentration of houttuynia cordata is 45%-55%, the concentration of vanilla is 45%-55%, the concentration of euphorbia pulegium is 45%-55%, the concentration of licorice is 45%-55%, and the concentration of lavender is 45%-55%.
[0037] The six-flower mixture includes honeysuckle, calendula, safflower, lilac, rose and ethanol solution; honeysuckle, calendula, safflower, lilac and rose are all natural plants, green and natural, without side effects, no harm to embryos, and no harm to the environment and human body. The six flowers cooperate with each other and work synergistically to effectively kill bacteria, with good sterilization effect, fast action speed, good stability, low toxicity, no residue, no corrosion, no irritation, easy to dissolve in water, safe for humans and animals, cheap and easy to obtain, and low pollution to the environment.
[0038] The concentration of honeysuckle is 40%-60%, the concentration of marigold is 40%-60%, the concentration of calendula is 40%-60%, the concentration of safflower is 40%-60%, the concentration of lilac is 40%-60%, and the concentration of rose is 40%-60%.
[0039] Preferably, the concentration of honeysuckle is 45%-55%, the concentration of marigold is 45%-55%, the concentration of calendula is 45%-55%, the concentration of safflower is 45%-55%, the concentration of lilac is 45%-55%, and the concentration of rose is 45%-55%.
[0040] The third disinfectant includes polysorbate-80, astaxanthin, glycine and water.
[0041] Because the stress response of crayfish embryos during disinfection will produce a large number of oxygen free radicals, these oxygen free radicals will affect the hatching of the embryos and the physical condition of the hatched shrimps.
[0042] Polysorbate 80 can neutralize residual disinfectant and prevent the sterilized embryos from continuing to be stressed.
[0043] Astaxanthin can significantly increase the embryo's ability to resist free radicals and accelerate the ability to eliminate free radicals.
[0044] Glycine can play an antioxidant and anti-stress role, reducing the damage of oxygen free radicals to the embryo.
[0045] The concentration of polysorbate 80 is 15-25 g / L, the concentration of astaxanthin is 0.5-1.5 g / L, and the concentration of glycine is 0.5-1.5 g / L.
[0046] Preferably, the concentration of polysorbate 80 is 20 g / L, the concentration of astaxanthin is 1 g / L, and the concentration of glycine is 1 g / L.
[0047] The preparation method of the disinfectant for in vitro hatching of Procambarus clarkii embryos of the present invention comprises:
[0048] The preparation method of the first disinfectant comprises: mixing sodium chloride, trehalose and sodium dodecylbenzene sulfonate, adding pure water and stirring evenly;
[0049] The preparation method of the essential oil mixture comprises: mixing thyme essential oil, sage essential oil, eucalyptus globulus essential oil and peppermint essential oil, adding pure water and stirring evenly;
[0050] The preparation method of the six-herb mixture includes: mixing gentian, houttuynia cordata, orchid vanilla, euphorbia pulegosa, licorice and lavender, adding ethanol solution, filtering the drug residues with a rotary filter, decolorizing the filtrate with activated carbon and stirring evenly;
[0051] The preparation method of the six-flower mixed solution comprises: mixing honeysuckle, marigold, calendula, safflower, lilac and rose, adding 70% ethanol solution, filtering the drug residue with a rotary filter, decolorizing the filtrate with activated carbon and stirring evenly;
[0052] The preparation method of the third disinfectant includes: mixing polysorbate 80, astaxanthin, and glycine, adding pure water and stirring evenly.
[0053] The disinfectant of the invention is used as a disinfectant in the process of in vitro hatching of Procambarus clarkii embryos, and the temperature of the disinfectant when used is 28-30°C.
[0054] Example 1
[0055] The disinfectant of this embodiment includes a first disinfectant, a second disinfectant and a third disinfectant in a volume ratio of 1:1:1, and the second disinfectant includes an essential oil mixture, a six-herb mixture and a six-flower mixture in a volume ratio of 10:1:1.
[0056] The preparation method of the disinfectant of this embodiment comprises:
[0057] (1) The preparation method of the first disinfectant comprises: mixing 70 g of sodium chloride, 5 g of trehalose, and 10 g of sodium dodecylbenzene sulfonate, adding 1 liter of pure water, and stirring evenly.
[0058] (2) The preparation method of the second disinfectant comprises:
[0059] Essential oil mixture: 0.25 ml of thyme essential oil, sage essential oil, eucalyptus globulus essential oil, and peppermint essential oil were mixed in equal proportions, and dissolved in 1 L of pure water to prepare an essential oil mixture;
[0060] Six-herb mixture: 5g of gentian, 5g of houttuynia, 5g of orchid vanilla, 5g of euphorbia pulegia, 5g of licorice, and 5g of lavender are mixed and added with 100ml of 70% ethanol solution. The residue is filtered through a rotary filter, and the filtrate is decolorized by activated carbon and stirred evenly to obtain a six-herb mixture;
[0061] Six-flower mixture: Mix 5g of honeysuckle, 5g of calendula, 5g of iris, 5g of safflower, 5g of clove and 5g of rose, add 100ml of 70% ethanol solution, use a rotary filter to filter the residue, decolorize the filtrate through activated carbon and stir evenly to obtain the six-flower mixture.
[0062] (3) The preparation method of the third disinfectant comprises:
[0063] 2 g of polysorbate 80, 0.1 g of astaxanthin, and 0.1 g of glycine were dissolved in 100 ml of pure water to obtain a third disinfectant.
[0064] Example 2
[0065] The disinfectant of this embodiment 2 is substantially the same as the disinfectant of embodiment 1, except that the second disinfectant of this embodiment 2 includes a mixture of essential oils.
[0066] Example 3
[0067] The disinfectant of the present embodiment 3 is substantially the same as the disinfectant of the present embodiment 1, except that the second disinfectant of the present embodiment 3 comprises a mixture of essential oils and a mixture of six herbs in a volume ratio of 10:1.
[0068] Example 4
[0069] The disinfectant of this embodiment 4 is basically the same as the disinfectant of embodiment 1, except that the second disinfectant of this embodiment 4 includes an essential oil mixture and a six-flower mixture in a volume ratio of 10:1.
[0070] Example 5
[0071] The disinfectant of this embodiment 5 is basically the same as the disinfectant of embodiment 1, except that the disinfectant of this embodiment 5 includes a first disinfectant and a second disinfectant in a volume ratio of 1:1, and the second disinfectant includes an essential oil mixture, a six-herb mixture, and a six-flower mixture in a volume ratio of 10:1:1.
[0072] Example 6
[0073] The disinfectant of this embodiment 6 is basically the same as the disinfectant of embodiment 1, except that the disinfectant of this embodiment 6 includes a second disinfectant, and the second disinfectant includes an essential oil mixture, a six-herb mixture, and a six-flower mixture in a volume ratio of 10:1:1.
[0074] Comparative Example 1
[0075] The disinfectant of Comparative Example 1 includes a first disinfectant, a third disinfectant and a 3000 ppm formaldehyde solution in a volume ratio of 1:1:1.
[0076] Comparative Example 2
[0077] The disinfectant of Comparative Example 2 includes a first disinfectant and a 3000 ppm formaldehyde solution in a volume ratio of 1:1.
[0078] Comparative Example 3
[0079] The disinfectant of this comparative example 3 includes 3000 ppm formaldehyde solution.
[0080] Experiment 1
[0081] The six disinfectants obtained from Examples 1 to 6 and the three disinfectants from Comparative Examples 1 to 3 were respectively subjected to disinfection effect test experiments.
[0082] The mid-term embryos (nauplii embryos, brown) of Procambarus clarkii were selected, and they were gently peeled off from the attached ventral foot bristles by combing, and after being treated with disinfectants, they were placed in an incubation box at a density of 400 grains / box, and the incubation box was placed in an incubation device for incubation, and divided into 10 groups on average, and the above 9 disinfectants were used for disinfection respectively, and the remaining group of embryos was not disinfected. Among them, the disinfection time of the disinfectants in Examples 1 to 6 of the present invention was 10s respectively; the disinfection time of the first disinfectant and the third disinfectant in Comparative Example 1 was 10s, and the disinfection time of formaldehyde was 15min; the disinfection time of the first disinfectant in Comparative Example 2 was 10s, and the disinfection time of formaldehyde was 15min; the disinfection time of formaldehyde in Comparative Example 3 was 15min
[0083] The sterilized crayfish embryos were placed in an incubation box at a density of 400 per box, and the incubation box was placed in an incubation device for incubation; during the embryo incubation period, the embryos were disinfected once every other day with a corresponding disinfectant, and no disinfection was performed after incubation. The incubation water temperature was 28-29°C; after incubation, the number of the first-stage juvenile shrimps was counted and the hatching rate was determined.
[0084] Thin sponge strips were inserted into the hatching boxes of each group to allow the newly hatched shrimp to attach. When the shrimp developed to the third stage and could disperse and swim freely, the emergence rate was counted. Dead eggs and dead shrimp were removed regularly during the incubation period.
[0085] Six parallel samples were made for each disinfectant, and the average values were calculated and listed in Table 1.
[0086] Table 1
[0087] Group No. Hatching rate (%) Seedling rate (%) Example 1 <![CDATA[91.33±5.05 a ]]> <![CDATA[61.04±4.00 a ]]> Example 2 <![CDATA[85.75±3.53 b ]]> <![CDATA[56.88±2.00 b ]]> Example 3 <![CDATA[85.17±1.55 b ]]> <![CDATA[56.46±6.67 b ]]> Example 4 <![CDATA[85.38±3.57 b ]]> <![CDATA[56.17±7.80 b ]]> Example 5 <![CDATA[81.08±3.05 c ]]> <![CDATA[53.38±5.32 c ]]> Example 6 <![CDATA[78.96±5.92 c ]]> <![CDATA[52.96±6.22 c ]]> Comparative Example 1 <![CDATA[91.08±3.04 a ]]> <![CDATA[61.38±4.98 a ]]> Comparative Example 2 <![CDATA[86.92±4.59 ab ]]> <![CDATA[58.08±6.88 ab ]]> Comparative Example 3 <![CDATA[84.50±5.59 b ]]> <![CDATA[57.25±8.83 ab ]]> Not sterile <![CDATA[60.25±8.03 d ]]> <![CDATA[37.67±7.83 d ]]>
[0088] As can be seen from Table 1, when the disinfectant of the embodiment of the present invention is used to disinfect crayfish embryos, the hatching rate of the obtained crayfish embryos is greater than the hatching rate of the crayfish embryos when the disinfectant of the comparative example is used at the same concentration. When the disinfectant obtained by the method of the present invention is used to disinfect crayfish embryos, the emergence rate of the obtained crayfish embryos is significantly greater than the emergence rate of the crayfish embryos when the disinfectant of the comparative example is used at the same concentration. Moreover, when the disinfectant of the present invention is used to disinfect crayfish embryos, the hatching rate of the obtained crayfish embryos is 78-91%, which is basically the same as the hatching rate of the existing commercially available formaldehyde disinfectants. When the disinfectant obtained by the method of the present invention is used to disinfect crayfish embryos, the emergence rate of the obtained crayfish embryos is 52-61%, which is also basically the same as the emergence rate of the existing commercially available formaldehyde disinfectants. Therefore, the disinfectant obtained by the method of the present invention can replace the traditional formaldehyde disinfectant.
[0089] Compared with the prior art, the beneficial effects of the present invention are: the disinfectant of the present invention comprises a first disinfectant, a second disinfectant and a third disinfectant, the three of which promote each other and synergize to enhance the effectiveness, the obtained disinfectant is non-toxic and harmless, green and environmentally friendly, has a good disinfection effect, has a good protective effect on embryos, has good stability, is easy to use, can replace the existing formaldehyde disinfectant, has a high hatching rate and a good germination rate.
[0090] The first disinfectant includes sodium chloride, trehalose, sodium dodecylbenzene sulfonate and water. Sodium chloride can increase the density of the first disinfectant, so that dead eggs, bad eggs and moldy eggs in the crayfish embryos can float away, leaving normal crayfish embryos. Trehalose can play an anti-stress and anti-dehydration role, and protect the crayfish embryos without affecting the disinfection effect, and prevent the crayfish embryos from being damaged; secondly, trehalose also has the functions of maintaining embryo cell activity, anti-stress, and maintaining membrane integrity, maintaining the cell membrane stability and permeability of the embryo in the disinfectant, protecting the embryo, preventing the embryo from being damaged, and thus improving the germination rate. Sodium dodecylbenzene sulfonate mainly plays the role of cleaning and disinfection.
[0091] The essential oil mixture comprises thyme essential oil, sage essential oil, eucalyptus globulus essential oil, peppermint essential oil and water; thyme essential oil, sage essential oil, eucalyptus globulus essential oil and peppermint essential oil are all plant essential oils, which are green and natural, have no side effects, are harmless to embryos, and are also harmless to the environment and human body; moreover, they have strong dissolving ability and good disinfection and sterilization effects; these plant essential oils dissolve in each other, act synergistically with each other, and have good use effects.
[0092] The six-flower mixture includes honeysuckle, calendula, safflower, lilac, rose and ethanol solution; honeysuckle, calendula, safflower, lilac and rose are all natural plants, green and natural, without side effects, no harm to embryos, and no harm to the environment and human body. The six flowers cooperate with each other and work synergistically to effectively kill bacteria, with good sterilization effect, fast action speed, good stability, low toxicity, no residue, no corrosion, no irritation, easy to dissolve in water, safe for humans and animals, cheap and easy to obtain, and low pollution to the environment.
[0093] The six-herb mixture includes gentian, houttuynia, orchid vanilla, ground ivy, licorice, lavender and ethanol solution; gentian, houttuynia, orchid vanilla, ground ivy, licorice and lavender are all plant herbs, green and natural, with no side effects, no harm to the embryo, and no harm to the environment and human body; the six plant herbs cooperate and work synergistically with each other to effectively kill bacteria.
[0094] Since the stress response of crayfish embryos during disinfection will produce a large number of oxygen free radicals, these oxygen free radicals will affect the hatching of embryos and the physical condition of hatched shrimps. Polysorbate-80 can neutralize the residual disinfectant and protect the embryos from continued stress after disinfection. Astaxanthin can significantly increase the embryo's ability to resist free radicals and accelerate the ability to remove free radicals. Glycine can play an antioxidant and anti-stress role, reducing the damage of oxygen free radicals to embryos.
[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.
Claims
1. A disinfectant for in vitro hatching of Procambarus clarkii embryos, characterized in that: The disinfectant includes at least one of a first disinfectant, a second disinfectant, and a third disinfectant; The first disinfectant comprises sodium chloride, trehalose, sodium dodecylbenzene sulfonate and water; The second disinfectant includes at least one of an essential oil mixture, a six-herb mixture, and a six-flower mixture; the essential oil mixture includes thyme essential oil, sage essential oil, eucalyptus globulus essential oil, peppermint essential oil, and water; the six-herb mixture includes gentian, houttuynia cordata, orchid, eucalyptus, liquorice, lavender, and an ethanol solution; the six-flower mixture includes honeysuckle, calendula, calendula, safflower, lilac, rose, and an ethanol solution; The third disinfectant includes polysorbate-80, astaxanthin, glycine and water.
2. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The disinfectant includes the first disinfectant, the second disinfectant and the third disinfectant, The volume ratio of the first disinfectant, the second disinfectant and the third disinfectant is (0.8-1.2): (1-1.5): (0.9-1.2).
3. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The second disinfectant includes an essential oil mixture, a six-herb mixture, and a six-flower mixture. The volume ratio of the essential oil mixture, the six-herb mixture and the six-flower mixture is (12-15): (0.5-2): (0.5-2).
4. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The concentration of sodium chloride is 65-75 g / L, the concentration of trehalose is 2-10 g / L, and the concentration of sodium dodecylbenzene sulfonate is 5-15 g / L.
5. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The volume percentage content of each component in the essential oil mixture is: thyme essential oil 0.02%-0.03%, sage essential oil 0.02%-0.03%, eucalyptus globulus essential oil 0.02%-0.03%, and peppermint essential oil 0.02%-0.03%.
6. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The concentration of gentian is 40%-60%, the concentration of Houttuynia cordata is 40%-60%, the concentration of vanilla is 40%-60%, the concentration of ground ivy is 40%-60%, the concentration of licorice is 40%-60%, and the concentration of lavender is 40%-60%.
7. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The concentration of honeysuckle is 40%-60%, the concentration of marigold is 40%-60%, the concentration of calendula is 40%-60%, the concentration of safflower is 40%-60%, the concentration of lilac is 40%-60%, and the concentration of rose is 40%-60%.
8. The disinfectant for in vitro hatching of Procambarus clarkii embryos according to claim 1, characterized in that: The concentration of polysorbate 80 is 15-25 g / L, the concentration of astaxanthin is 0.5-1.5 g / L, and the concentration of glycine is 0.5-1.5 g / L.
9. The method for preparing a disinfectant for in vitro hatching of Procambarus clarkii embryos according to any one of claims 1 to 8, characterized in that: The preparation method of the first disinfectant comprises: mixing sodium chloride, trehalose and sodium dodecylbenzene sulfonate, adding pure water and stirring evenly; The preparation method of the essential oil mixture comprises: mixing thyme essential oil, sage essential oil, eucalyptus globulus essential oil and peppermint essential oil, adding pure water and stirring evenly; The preparation method of the six-herb mixture comprises: mixing gentian, houttuynia cordata, orchid vanilla, euphorbia pulegosa, licorice and lavender, adding ethanol solution, filtering the drug residues with a rotary filter, decolorizing the filtrate with activated carbon and stirring evenly; The preparation method of the six-flower mixed solution comprises: mixing honeysuckle, calendula, calendula, safflower, lilac and rose, adding 70% ethanol solution, filtering the drug residues with a rotary filter, decolorizing the filtrate with activated carbon and stirring evenly; The preparation method of the third disinfectant comprises: mixing polysorbate 80, astaxanthin and glycine, adding pure water and stirring evenly.
10. A use of the disinfectant for in vitro hatching of Procambarus clarkii embryos according to any one of claims 1 to 8, characterized in that: The disinfectant is used as a disinfectant in the process of in vitro hatching of Procambarus clarkii embryos, and the temperature of the disinfectant when used is 28-30°C.